Note: Descriptions are shown in the official language in which they were submitted.
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
METHODS OF USING KRILL OIL TO TREAT RISK FACTORS
FOR METABOLIC, CARDIOVASCULAR, AND INFLAMMATORY DISORDERS
[0001] This application claims the benefit of U.S. Prov. Appl. 61/181,743,
filed
May 28, 2009, and is a continuation-in-part of U.S. Appl. No. 12/057,775,
filed March
28, 2008, which claims the benefit of U.S. Prov. Appl. 60/920,483, filed Mar.
28,
2007, U.S. Prov. Appl. 60/975,058, filed Sep. 25, 2007, U.S. Prov. Appl.
60/983,446,
filed Oct. 29, 2007, and U.S. Prov. Appl. No. 61/024,072, filed Jan. 28, 2008,
all of
which are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] 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.
1
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
BACKGROUND OF THE INVENTION
[0003] 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). Krill 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.
During
winter, however, its food supply is limited to ice algae, bacteria, marine
detritus as
well as depleting body protein for energy. Virtue et al., Mar. Biol. 126, 521-
527. For
this reason, the nutritional values of krill vary during the season and to
some extent
annually. Phleger et al., Comp. Biochem. Physiol. 131B (2002) 733. In order to
accommodate variations in food supply, krill has developed an efficient
enzymatic
digestive apparatus resulting in a rapid breakdown of the proteins into amino
acids.
Ellingsen et al., Biochem. J. (1987) 246, 295-305. 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.
[0004] During the 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. In addition, 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. This operation is energy demanding and may also result in a
degradation of unstable bioactive components in the krill oil, such as omega-3
fatty
acids, phospholipids and astaxanthin. The presence of these compounds make
krill
oil an attractive source as a food supplement, a functional food product, and
a
pharmaceutical for the animal and human applications.
2
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[0005] 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.
[0006] 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. 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
pg/g astaxanthin, preferably 90 pg/g astaxanthin. Another krill lipid extract
disclosed
contained at least 250 pg/g canastaxanthin, preferably 270 pg/g
canastaxanthin.
[0007] Published PCT Application No. 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 cholesterol:HDL
ratio
decrease of about 14%
[0008] Published PCT Application No. WO 2007/080515 discloses a marine
lipid extract derived from krill. The extract can be used in methods for
preventing or
treating thrombosis.
[0009] 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.
[00010] 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
3
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
effective amount of a composition including conjugated linoleic acid and a
krill extract
comprising krill oil.
[00011] However, there remains a need in the art for methods of using
compositions comprising krill oil to treat risk factors for metabolic,
cardiovascular,
and inflammatory disorders.
SUMMARY OF THE INVENTION
[00012] 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.
[00013] In some embodiments, 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
4
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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.
[00014] In some embodiments, 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. These biological
processes
may be modulated by altering the expression of one or more genes, including,
but
not limited to, reduced or decreased expression of Ppargcla (peroxisome
proliferator-activated receptor gamma coactivator 1a), Hnf4a (hepatocyte
nuclear
factor 4 alpha), Pck1 (phosphoenolpyruvate carboxykinase 1), G6pc (glucose-6-
phosphatase, catalytic), Cptla (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 factor
2),
Ppargcl b (peroxisome proliferator-activated receptor gamma coactivator 1b),
and
Sod2 (superoxide dismutase 2). These biological processes may also be affected
by
enhanced or increased expression of NADH (nicotinamide adenine dinucleotide)
dehydrogenase and subunits thereof. The biological processes are also affected
by
factors including reduced hepatic glucose production, reduced hepatic
gluconeogenesis, and reduced hepatic lipid synthesis.
[00015] In some embodiments, 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. In some embodiments,
the
human subject is clinically obese.
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[00016] In certain embodiments, 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. In
some
embodiments, 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. In some
embodiments,
the human subject is clinically obese.
[00017] in certain embodiments, the present invention provides methods
comprising providing a krill oil composition to a human subject under
conditions such
that cannabinoid receptor signaling is reduced. In some embodiments,
inhibition of
the endocannabinoid system of the subject comprises lowering the levels of
arachidonylethanolamide (AEA) and/or 2-arachidonyl glycerol (2-AG). In some
embodiments, the human subject is clinically obese.
[00018] 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 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.
[00020] In certain embodiments, 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.
[00021] In certain embodiments, 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.
6
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[00022] 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 the
reproductive
performance is increased. In some embodiments, reproductive performance is
improved chance of ovulation in females. In some embodiments, reproductive
performance is spermatogenesis, sperm motility and/or acreosome reaction.
[00023] 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 the liver
and/or
kidney functions are improved.
[00024] Other novel features and advantages of the present invention will
become apparent to those skilled in the art upon examination of the following
or
upon learning by practice of the invention.
DETAILED DESCRIPTION OF THE DRAWINGS
[00025] FIG. 1. 31P NMR analysis of polar lipids in krill oil.
[00026] FIG. 2. Blood lipid profiles in Zucker rats fed different forms of
omega-3
fatty acids (TAG=FO, PLI=NKO and PL2=Superba).
[00027] FIG. 3. Plasma glucose concentration in Zucker rats fed different
forms
of omega-3 fatty acids.
[00028] FIG. 4. Plasma insulin concentration in Zucker rats fed different
forms
of omega-3 fatty acids.
[00029] FIG. 5. Estimated HOMA-IR values in Zucker rats fed different forms of
omega-3 fatty acids.
[00030] FIG. 6. The effect of dietary omega-3 fatty acids on TNF-a production
by peritoneal macrophages.
[00031] FIG. 7. The effect of dietary omega-3 fatty acids on lipid
accumulation
in the liver.
[00032] FIG. 8. The effect of dietary omega-3 fatty acids on lipid
accumulation
in the muscle.
[00033] FIG. 9. The effect of dietary omega-3 fatty acids on lipid
accumulation
in the heart.
[00034] FIG. 10. Relative concentrations of DHA in the brain in Zucker rats
supplemented with omega-3 fatty acids.
7
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[00035] FIG. 11. Mean group body weights (g) in the collagen-induced male
DBAl1 arthritic mice. B-PL2 is the krill oil group. * p<0.05, significantly
different from
Group A (Positive Control--Fish Oil) and Group C (Control).
[00036] FIG. 12. Body weight for the various treatment groups.
[00037] FIG. 13. Muscle weight for the various treatment groups.
[00038] FIG. 14. Muscle to body weight ratio for the various treatment groups.
[00039] FIG. 15. Serum adiponectin levels (ng/ml) for the various treatment
groups.
[00040] FIG. 16. Serum insulin levels for the various treatment groups.
[00041] FIG. 17. Blood glucose (mmol/l) levels in the various treatment
groups.
[00042] FIG. 18. HOMA-IR values for the various treatment groups.
[00043] FIG. 19. Liver triglyceride levels (pmol/g) for the various treatment
groups.
[00044] FIG. 20A-B. Levels of anandamide (arachidonoyl ethanolamide) and 2-
arachidonoyl glycerol in visceral adipose tissue in Zucker rats.
[00045] FIG. 21A-B. Levels of anandamide (arachidonoyl ethanolamide) and 2-
arachidonoyl glycerol in subcutaneous adipose tissue in Zucker rats.
[00046] FIG. 22A-B. Levels of anandamide (arachidonoyl ethanolamide) and
2-arachidonoyl glycerol in liver tissue in Zucker rats.
[00047] FIG. 23A-B. Levels of anandamide (arachidonoyl ethanolamide) and 2-
arachidonoyl glycerol in heart tissue in Zucker rats.
[00048] FIG. 24. Triacylglyceride content in liver.
[00049] FIG. 25. Triacylglyceride content in heart.
[00050] FIG. 26. Cholesterol profile in plasma.
[00051] FIG. 27. Fatty acid analyses of monocytes.
[00052] FIG. 28. TNF alpha release in peritoneal monocytes after ex vivo
challenge with LPS.
[00053] FIG. 29A-B. Liver (A) and heart (B) triacylglycerol concentrations of
obese Zucker 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 letter differ,
P <
0.05.
[00054] FIG. 30A-B. Visceral AEA (A) and 2-AG (B) concentrations in obese
Zucker rats fed control, fish oil, or krill oil diets for four weeks. Values
are expressed
as mean +l- SD, n=6. Means that do not have a common letter differ, P < 0.05.
8
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[00055] FIG. 31A-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.
[00056] FIG. 32A-B. Cholesterol (A) and TAG (B) concentrations in plasma
from rats fed control (C), fish oil (FO), or krill oil (KO) diets. Error bars
depict S.D.,
n=6. Different letters denote significant differences (p<0.05)
[00057] 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.
[00058] FIG. 34. Genes suggesting decreased glucose uptake and increased
fructose metabolism. KO diet showed a trend for increased Aldob expression
(p=0.022)
[00059] FIG. 35. Key genes regulating hepatic glucose production
[00060] FIG. 36. Key genes involved in fatty acid metabolism.
[00061] FIG. 37. Key genes regulating cholesterol biosynthesis in the liver 3-
hydroxy-3-methylglutaryl-Coenzyme A
[00062] FIG. 38. Transcriptional cofactors and gene targets proposed to
mediate the effect of krill-supplements on hepatic glucose metabolism and
lipid
biosynthesis.
DEFINITIONS
[00063] An "ether phospholipid" as used herein preferably refers to a
phospholipid having an ether bond at position 1 of the glycerol backbone.
Examples
of 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.
[00064] As used herein, the term "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),
9
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
4,7,10,13,16,19-docosahexaenoic acid (DHA) and 7,10,13,16,19-docosapentaenoic
acid (DPA).
[00065] As used herein, the term "w/w (weight/weight)" refers to the amount of
a given substance in a composition on weight basis. For example, 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).
DETAILED DESCRIPTION OF THE INVENTION
[00066] 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 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.
[00067] 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.
[00068] 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.
A. Methods of Using Krill Oil
[00069] The present invention relates to methods of using krill oil and
compositions comprising krill oil to treat one or more risk factors for
metabolic,
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
cardiovascular, and inflammatory disorders. There are a variety of risk
factors
associated with metabolic, cardiovascular, inflammatory, and other disorders,
and it
has been found in accordance with the present invention that krill oil
significantly
modulates a substantial number of risk factors associated with metabolic,
cardiovascular, inflammatory and other disorders. These disorders may include,
but
are not limited to, obesity, type II diabetes, type I diabetes, gestational
diabetes,
metabolic syndrome, dyslipidemia, hypercholesterolemia, hypertension, coronary
artery disease, atherosclerosis, stroke, rheumatoid arthritis, and
osteoarthritis.
[00070] 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 level(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.
[00071] 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. According to certain aspects of the invention, 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. According to other aspects of the
invention, 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.
According to some aspects of the invention, 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
11
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
or activity seen in an individual or population not suffering from a
metabolic,
cardiovascular, inflammatory disorder.
[00072] Other 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. According to certain aspects of the invention, the
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. According to other aspects of the invention, 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.
According to some aspects of the invention, 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.
[00073] 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. These
biological
processes are affected by expression of a number of genes, including, but not
limited
to, reduced or decreased expression of Ppargcla (peroxisome proliferator-
activated
receptor gamma coactivator 1a), Hnf4a (hepatocyte nuclear factor 4 alpha),
Pck1
(phosphoenolpyruvate carboxykinase 1), G6pc (glucose-6-phosphatase,
catalytic),
Cptl a (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), Ppargcl b (peroxisome proliferator-
activated
receptor gamma coactivator 1b), and Sod2 (superoxide dismutase 2). These
biological processes may also be affected by enhanced or increased expression
of
NADH (nicotinamide adenine dinucleotide) dehydrogenase and subunits thereof.
12
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
The biological processes are also affected by factors including reduced
hepatic
glucose production, reduced hepatic gluconeogenesis, and reduced hepatic lipid
synthesis.
[00074] These 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. Alternatively, 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.
[00075] 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.
In these aspects, 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. According to some aspects, 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. According to further aspects,
the
increase/decrease is at least 5% relative to the baseline level. Preferably
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. In some embodiments, 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.
13
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Cardiovascular Disease
[00076] In certain embodiments, the present invention can be used in methods
of decreasing cardiovascular disease risk factors of a subject. In some
embodiments, 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. In some
embodiments,
the subject is a human, and in other embodiments, the subject is clinically
obese.
[00077] In some embodiments, the krill oil composition of the present
invention
find use in the treatment of fatty heart disease, alcoholic fatty liver
disease, and non-
alcoholic fatty liver disease. Thus, the krill oil compositions are useful for
decreasing
the lipid content of the heart and/or liver of a subject. In other
embodiments, 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. In some embodiments, the subject
is a
human, and in other embodiments, the subject is clinically obese.
Obesity
[00078] Excess adipose tissue mass (overweight and obesity) is associated
with low grade inflammation in adipose tissue and in the whole body reflecting
the
inflammatory mediators "spilling over" from fat tissue. Inflammation appears
to be an
important link between obesity and metabolic syndrome/type-II diabetes as well
as
cardiovascular disease. Thus, excess adipose tissue is an unhealthy condition.
[00079] Weight reduction will improve the inflammatory condition, but
persistent
weight reduction is difficult to achieve. Omega-3 fatty acid supplementation
may
alleviate the inflammatory condition in adipose tissue and thus ideally
complement
the principal strategies of weight reduction, i.e., low calorie diet and
exercise.
Although 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.
14
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[00080] 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. Accordingly, 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.
[00081] In certain embodiments, 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. In some embodiments, the subject is a human, and in other
embodiments,
the subject is clinically obese.
[00082] In certain embodiments, 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. In some embodiments, the subject is a human, and in other
embodiments, the subject is clinically obese.
[00083] In still other embodiments, 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 and Metabolic Disorder
[00084] Type 2 diabetes is a metabolic disorder characterized by impaired
glycemic control (high blood glucose levels). In type 2 diabetes, 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. In further embodiments, krill oil is
effective in
reducing risk factors of type 2 diabetes such as hyperinsulinemia and insulin
resistance.
[00085] The methods of the present invention may be used to treat and/or
prevent type II diabetes and metabolic syndrome in a subject, or reduce the
incidence of type II 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
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
improving the blood lipid profile by increasing the HDL cholesterol levels,
decreasing
the LDL cholesterol and triglyceride levels. Hence 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. In another embodiment of the invention, krill oil compositions
are
provided that are effective and safe for the treatment of type II diabetes and
metabolic syndrome in humans.
Endocannabinoid Modulation
[00086] In certain embodiments, the present invention provides methods
comprising providing a krill oil composition to a subject under conditions
such that
cannabinoid receptor signaling is reduced. In some embodiments, inhibition of
the
endocannabinoid system of the subject comprises lowering the levels of
arachidonylethanolamide (AEA) and/or 2-arachidonyl glycerol (2-AG). In some
embodiments, the subject is a human, and in other embodiments, the subject is
clinically obese.
[00087] 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.
[00088] 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.
[00089] 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
16
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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.
[00090] 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). However, 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.
[00091] The various methods of the present invention demonstrate that krill
oil
is effective in changing endocannabinoid receptor signaling by modulating the
level
of the cannabinoid receptor ligands. The levels of endocannabinoid precursors,
i.e.,
arachidonic acid attached to phospholipids, are reduced as well. It might be
that the
high level of omega-3 phospholipids play a role in the effective modulation of
the
endocannabinoid system, however the mechanism of action by which krill oil
works
remains unknown at this stage.
[00092] The present invention also relates to modulation of the
endocannabinoid system in tissues such as kidney, testis, different brain
areas,
17
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
intestines, pancreas, thyroids glands, etc. A preferred embodiment of this
invention
is the use of krill 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.
B. Krill Oil
[00093] According to some aspects, the various methods of the present
invention may be carried out using krill 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.
[00094] Krill 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.
[00095] In preferred embodiments, the krill oil compositions are made as
described in co-pending application PCT/GB2008/001080, which is incorporated
herein by reference. In other preferred embodiments, compositions for use in
accordance with the invention may include, but are not limited to, SuperbaTM
krill oil
(Aker Biomarine, Norway). In other preferred embodiments, the compositions for
use in accordance with the invention comprise krill oil that is obtained from
krill meal
by ethanol extraction and/or CO2 extraction. However, any suitable methods for
extracting oil from krill may be used in accordance with the present
invention.
[00096] 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 03/011873 and Neptune brand krill oil (NKO , Neptune Technologies &
18
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Bioressources, Laval, Quebec, Canada), by having substantially higher levels
of
non-ether phospholipids, ether phospholipids, and astaxanthin.
[00097] 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.
[00098] In some preferred embodiments, 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. In some embodiments the ether phospholipids are
preferably
alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
alkylacylphosphatidyl-ethanolamine or combinations thereof. In some
embodiments,
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%. One of skill in the art will recognize that 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.
[00099] In further embodiments, the compositions comprise from about 20% to
45% w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin. In
some embodiments, 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.
[000100] 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. In some embodiments, 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
19
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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. In
some preferred embodiments, the krill oil comprises EPA and DHA residues. In
some embodiments, the krill oil compositions comprise less than about 5%, 4%,
3%
or preferably 2% free fatty acids on a weight/weight (w/w) basis. In some
embodiments, the krill oil compositions comprise less than about 25%, 20%,
15%,
10% or 5% triglycerides (w/w). In some embodiments, 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
krill
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. In some
embodiments, 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.
[000101] In some embodiments, the present invention is carried out using a
neutral krill oil extract comprising greater than about 70%, 75% 80%, 85% or
90%
triglycerides. In some embodiments, the krill oil compositions comprise from
about
50 to about 2500 mg/kg astaxanthin esters. In some embodiments, the krill oil
compositions comprise from about 50, 100, 200, or 500 to about 750, 1000, 1500
or
2500 mg/kg astaxanthin esters. In some embodiments, 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. In some embodiments, the krill oil
compositions comprise less than about 20%, 15%, 10% or 5% phospholipids.
[000102] In some embodiments, 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. In some embodiments, the krill oil is clear or only
has a
pale red color. In some embodiments, 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. In some embodiments, the krill material is then subjected to
supercritical
fluid extraction with carbon dioxide and a polar entrainer such as ethanol,
preferably
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
about 20% ethanol. The oil extracted during this step is characterized in
containing
low amounts of astaxanthin. In other embodiments, krill oil comprising
astaxanthin is
extracted by countercurrent supercritical fluid extraction with neat carbon
dioxide to
provide a low-astaxanthin krill oil.
[000103] In some embodiments, the present invention is carried out using krill
oil
that is substantially odorless. By substantially odorless it is meant that the
krill oil
lacks an appreciable odor as determined by a test panel. In some embodiments,
the
substantially odorless krill oil comprises less than about 10, 5 or 1
milligrams/kilogram trimethylamine. In some preferred embodiments, 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.
C. Krill Oil-Based Compositions
[000104] In some embodiments, the present invention provides encapsulated
Euphausia superba krill oil compositions. In some embodiments, 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.
[000105] In some embodiments, 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. In other
embodiments,
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 CO2
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 C02 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.
21
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[000106] In some embodiments, 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 7Ø 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.).
[000107] 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. For example, 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.
For
example, the 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. Such optional
ingredients may
be either naturally occurring or concentrated forms.
[000108] In some embodiments, the composition may further comprise vitamins
and minerals including, but not limited to, calcium phosphate or acetate,
tribasic;
22
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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. Suitable
dosages for vitamins and minerals may be obtained, for example, by consulting
the
U.S. RDA guidelines. In still other embodiments, the particles comprise an
amino
acid supplement formula in which at least one amino acid is included (e.g., 1-
carnitine or tryptophan).
[000109] In further embodiments, 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
aldehyde, C16 aldehyde), ethyl vanillin, geraniol (3,7-dimethylocta-2,6-dien-1-
ol),
geranyl acetate (geraniol acetate), limonene (d, I, and dl), linalool (3,7-
dimethylocta-
1,6-dien-3-ol), linalyl acetate (bergamol), methyl anthranilate (methyl-2-
aminobenzoate), piperonal (3,4-methylenedioxy benzaldehyde, heliotropin),
vanillin,
alfalfa (Medicago sativa L.), allspice (Pimenta officinalis), ambrette seed
(Hibiscus
abelmoschus), angelic (Angelica archangelica), Angostura (Galipea
officinalis), anise
(Pimpinella anisum), star anise (lllicium verum), balm (Melissa officinalis),
basil
(Ocimum basilicum), bay (Laurus nobilis), calendula (Calendula officinalis),
chamomile (Anthemis nobilis), capsicum (Capsicum frutescens), caraway (Carum
carvi), cardamom (Elettaria cardamomum), cassia (Cinnamomum cassia), cayenne
pepper (Capsicum frutescens), Celery seed (Apium graveolens), chervil
(Anthriscus
cerefolium), chives (Allium schoenoprasum), coriander (Coriandrum sativum),
cumin
(Cuminum cyminum), elder flowers (Sambucus canadensis), fennel (Foeniculum
vulgare), fenugreek (Trigonella foenum graecum), ginger (Zingiber officinale),
horehound (Marrubium vulgare), horseradish (Armoracia lapathifolia), hyssop
(Hyssopus officinalis), lavender (Lavandula officinalis), mace (Myristica
fragrans),
23
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
marjoram (Majorana hortensis), mustard (Brassica nigra, Brassica juncea,
Brassica
hirta), nutmeg (Myristica fragrans), paprika (Capsicum annuum), black pepper
(Piper
nigrum), peppermint (Mentha piperita), poppy seed (Papayer somniferum),
rosemary
(Rosmarinus officinalis), saffron (Crocus sativus), sage (Salvia officinalis),
savory
(Satureia hortensis, Satureia montana), sesame (Sesamum indicum), spearmint
(Mentha spicata), tarragon (Artemisia dracunculus), thyme (Thymus vulgaris,
Thymus serpyllum), turmeric (Curcuma longa), vanilla (Vanilla planifolia),
zedoary
(Curcuma zedoaria), sucrose, glucose, saccharin, sorbitol, mannitol,
aspartame.
Other suitable flavoring are disclosed in such references as Remington's
Pharmaceutical Sciences, 18th Edition, Mack Publishing, p. 1288-1300 (1990),
and
Furia and Pellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical
Rubber Company, Cleveland, Ohio, (1971), known to those skilled in the art.
[000110] In other embodiments, the 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).
[000111] In still further embodiments, the 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). 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.
24
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
EXAMPLES
[000112] The present invention is further described in the following non-
limiting
Examples.
EXAMPLE 1
[000113] Antarctic krill (Euphausia superba) was captured and brought on board
alive, before it was processed into krill meal, an oil (asta oil), and
stickwater. During
the krill meal processing a neutral oil (asta oil) is recovered.
EXAMPLE 2
[000114] 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.
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 1
l== ti. acid (listrt ti'i n ht l;ril :ncal 4811Ã ; .1
.inrl lne ei0aa I r. t 1 kri1.1 s:01.
Dal l:y .. w id
;; le KtriI meal 8t()1[ .t
C4:0 (k,
C'l - Ã1.
C8
C'12.'' (t=
C~14: 7.8 (04
C14: ` 0.
C15: (I
(`l0:00 15,8 14.
C15:: 5. _ 4.2
C:18:E: 0. r 0.7
('18;1 13,4 1 1.S
('18:21= 1. 1.2
CÃ18:38 R4 Ã..4
0,6
~..:_ ';,'1\r? <II, <(1.
<f.1.
( 1 1~.( Ã1.2 0.2
('1 1'. <0.1 <ÃI,1
C'0:4\ ? ?
(`20:5'~."sr8155i 11,5 1ÃL4
(-22''.0 <0.1 <(r."[
(1_: 1 i.5 Ã1.4
('.: 0 - =, <I , l < i. I
(.;.' V", ) <!i,I
Of)
2N3(1)1.1.50 5.4 4.8
('24:1 ).01
24,6 21.9
td[ ,;o-En ::~~ it i'rd 19.(d 17.0
21.0 16.4
Total 65.5 58,2
1
Omegn-3 1 .2 17.0
()il'3e=.3-6- 1.3
26
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 2
t~rrt)q{ -r'I, ft i { ~~~, UfPh~ l.ri` rriettt
T Y~II~~ r`t~rl > T
ri1tr'e.t'.' I'l dl flit II
lThtt (F'k11 2"1
-,4- 85
V''4 ". _~=f is l':; - li 3'
1 1",l 1:CIrl 1i. .
ni,b.r 4,k
9 ltg N.,l6)tJ _; } :~ It16+ N; I U) '..
2 1IS irtg';Trotg 46tE
Table 3
T
-tJi;~It13 ;E I;ri I
Krill rrtc<t rc tl l ':1' tc tct1 K=)
c' L: i c?s1cC
+ 31
.
1 f. 7, % 14.1
t1.1 %.tt 12.r
11t 1.1
Iiri -1 4.2
PI 1.1 11.0
PS 1.4
1'C 28,6 211.2 5.3
1.1'1' ? 0 2.6 6.2
Total aolar lipids 'i?,6? 4+).3 36.
Tot :1L.Lit it lpi:ils 5-.2 59.5 ti;.
27
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
EXAMPLE 3
[000115] 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 CO2 only at 300 bars, 600 C and for 30
minutes. In
stage 2, the pressure was increased to 400 bar and 20% ethanol was added (vlv)
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. In addition the 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 (PI) and phosphatidylserine (PS) (Table 10) are shown.
Table
11 shows the level of astaxanthin and cholesterol for the different fractions.
28
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 4
lift, l 1. V L41t1"e C%iilil 1 3s t'Ãf~ f?11. )P iili
ai7l.fi FaCi ; 1,s i
F 1:; .tertl 1.. _I Polar
File KO K)) N KO
(0) Cool
('Ãi:)1 "t11
C:" ('.O0 fil)
( I WO (,,00
C1 To (+.47 C,.fi4 (1.24
C'14:0 22.08 3.28 12.48
C14:1 (?.33 1101 0.17
C'15:! f .$ J (.52
C 16:0 223.25
016:1 (.1)7 01. () 1 8.44
C18:0 1.72 1.03 1.42
018:1 31.29 13.57 18.:)2
C 1' :2X6 110 106 1.31
("115:3\o 03) v.'1 (i.i)(
C 18:3N3 0.69 1.02 1.32
C 18:4N 3 0. 35 1.81 310
2":' 0.06
(' :1 1.87 s`. r) 1," ,
C MV ,.2.1 9.73 I..14
C20:4N6 6.01' ,IØ) 0.49
( }9 3 3 111)9 (I, )(?
C214\3 (;.'4 11.51 0.33
( 5\3 (ERA) 733 ? 112
( :0 I,.01 ..06 11,95
( 1 11,04 .74 11,1"
( K'4\ (,00 Jill
( ''11 (:,II)' I) 3
t 2:,\ (:.n .J7
C .63 fI1 '.; 3.51 12.51 K.`.7
(124:0 5 0.91
('24:1 0.11
1' 1)11 .111 i) ...[1') 11111. 9
521,)) 31.')) 38.)1
A i r.; I.uatbdi +3.22 16.43 29.61
14.77 49.55 32.37
1.11:1 11)1-.9) 1010.01, 1(1(,,1))
C1nie a 3 127.11 46.58 30. 02
Orne -6 1(7 2.98 2.35
29
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 5
i1 , i; i1 ,i ck,,_,; n Mfr :,1'1;)te 11 (c1 lhkk~ lie d fi 1: Ãi , {o,;, f t
"~c 1.
l ;, I P rE 1spP lipid
Irv' \c'''d Polar NcIpli ,_
I'll 1<)) KO K.)
C4:1) r )l)l :. i t).'1=)C'
C8:i) (11)0 :.0') 0,71U
C. 1(1:)) (ill tl.
(.14:1) 0,01
1:1.4:1 4 ; ,t)1 1.I11
(J;)) E!,i7i7
t 16:1) 4,71
35.78 32.81
( 16:1 . C,1c `7,1 ' fr,iS'
(:1#:i;.i 3 1 1.1% 1.55
('115:14() 15.58 13.54
t"lk. i~i) 418 2.16 [.9t)
(1% 0\n (. a6 '.22 3,19
1 , 31;3 l,(72 ',i)5 1.4%
t 18:41 \' 1012 115
t'2011 `y iJ12 N
(=20 'In;' (1.19
( r.:, E),()() . 14 EI.t)
t" (1:4 r :i2 Ã1.64
('20:41'\.; t7 0145 1.42
C 20:5N' (I'.l1)\) <' 25.:4; 25.47
C'22:Ã0 14 ~~.06 ){'32:1 {) '.!)} 4.94
(2 :21\6 3.2 `.71 (), 85
t 240 1 11;44 "".00 ().03
C 1 :5186 0.11 ,jj tl)(i
C 2 `:S1\2 .()0 I.!) ! 0.63
022:6N3 (DFH_ I 10,93 10.3!:1 1_.34
('24:0 1.7 3') 1 r
( 24:1 C 28 i
Tot:r, 1011:00 11 .00 1 r kE i, ,ltj
K I 15.74 37.32 34.81
c r1 42.14 1:1.15 16.84
P 'C11 11 n i' 42.12 43.53 48.34
?I:, 1fi~ HI 1f'l.'ft 1(3(,181
{ 44.56
Or ,g E-ti 5.1)1 3.90 3,7%
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 6
1=;i1t :ICit ci ripc 4i ici ,'d'J[e 1c4 ! rie..ttr:i lipi_d I ctr.,rt f
t l ltttztlil.lipit
e 1~. 1 Polar
KO KO Fi )
(i C(J !i1 Ã,1r)(7 (arts
( 13:O 111 Ã.1,;1(.1 (!Iq)
(14:4 3'+ 11.31 li-
( 14:1 3 4.29 ( i
(' 15:))
(1,49 64.11.
12,42 5. 112)6
( 111:!1 ,,54 3.?' l r
( 1H. A 2-17
1.
( 1``:k~~r 2U (1..,.. t!._.
lti, .'J 0,59 4,0_. I) (!,?
f'1 ;3 ).(13 1,27 [j.(15
( 17,(17 (j.(14 (),(!C
1.63 1.41 129
f I':2 16 0.04 0.04 0,(15
C-)0:3
N'S I,, l h (04 0.01
C2 1)4" Ii.(((I 0'(10 (),(1),
( 2tl:'1_ tt<) 0.(1)3 (`).(:i1
0.41 1),2.3
52)11 9.26 9.61)
1.;22: 1).(12) 0.013 0.0
( 22:. 1i. 1i ti.60 0,53
0.(:1(3 0,00 t? 00
(22:47(1 ((.(1(7 0.04
('22;15,0 '(.01 (U H)
tl.(i(i
C' 7:5 0.17 027 0.22
A) 2.74 17.22 4.64
C`24:0 0.15 1-1.01) 1.17
('24:1 (1.(? (1,21 1 ;
11)t1a l) (Iti.(Ii)
100.00
46,45 1 fi 001 45,10
r, ti;>ttil[' 41.7 4UI. :3 37.91
1 11,810 1 ',5,9 16.99
10O (II_i 1 _ c ii i.(I )
( J. ( 14.nri
Of I's 2.11 3,54 2.14
31
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 7
Fiat"{ acid , comp shoo Abe igly01761 and We
[Oat :t r S (`Vi'a c~.u: j).
1)it11~3'Ueride 1 ~re 14 iii', ,ic.rs
Fw r :i 11~,71:t1t -
He 151a.uteal KO i Polcaa= KÃ) KC 1 outral KC) Polar KO KC)
C0') O,( AN 0.00 sou 000 000
011,:)) 1).0() i),(a) (t, ;;() i) (1i Wit)
(41) O,(1(. 1l,OO 6,iii) (1,0'00 ),0O (i,0 )
(110:( ((3)1;; 0,00 ),(1O 0,10) il"( 9 (r,0O
(14:0 13.55 14.35 12.>2 5.86 .19 5.45
(14:1 (1.18 ti.0(! l),1" 0 :5 1)119 (;x.(15
('l ) {).. % 4,08 o,66 0.46 1.60 Ci.45
CI o) 35.24 25.51 25,310 2117 21,12
( ' 16: l 3. 4 ; 6.80 o119 3.27 1 08 4,91
(181) IN 313 1.59 1.1,3 143 (1,99
C1';:1 23.9.7 19.85 23,52 14.50 1477 17.41
C'18:;''1.7 1).21 1.9u 1.69 0"97 (.546
( 15:".. ().17 0"00 0.01 0.114 0 00 0.32
CI s: ',,l (09 11.4!(1 (.19 (1,85 11.(71) 134
C 11+:4' 3 112 0,00 2.75 1.30 0.00 _.72
C200 ON coo ON (910 (3,(1O
C20:1 1,051 1)410 1"01 148 (101) 0.57
C'0:'\' Opt 001) ()Ã0 rrOO 11.00 1100
4:203\6 0.13 1131() '3,041 93(1 0 (141 0,05
C'20:47(3 0.45 11.00 0,54 41,7(1 1.43
('20_,3\3 t) (4; 6).0() I),Ã)(1 (I, O 0,00 11,00
("20:47(3 0,35 11,41(( (),43 0.1.3'x) 0'00 0.43
('7037(1 i6l01'i 14.03 191 15Ã1() 24.31 2157 2536
(12P 0,15 (ME wk! 0.00 WE, 0.05
023:1 0.4 0,041 0.57 (1,80 0,69 0.37
( 008 0.00 0,50 006 11 0)) 0.54
(12'':47(3 0.1 0,00 0.00 0. 0 0.00 0.00
(21:57(6 f0'..: 040 0.00 (1010 0.00 10.00
(22:37(3 0- 1 1100 0.127 0.34 0.00 032
(122:9\3 41111:'1) 434 9.04 7.53 14.31 1133 1315
(24:0 1.54 11(1(1 0"42 0.49 q ml 039
(124.1 O j,,0." 11"00 J,((1 0.30 0,00 0,00
1()(.(4 (Ã11),(1(1 10,00 WIN) 100,06 1(0.0()
"'alk agi
ld 411.4() 513U 41.1() 36,24 4039 2545
~C" ru 1 n5 1 ,r.000 34.84 '6.6 4 25,66 1109 15.54 2134
F ;lyunslnuated1 24.77 1156 3314 44.67 40.87 48.2'
Total 1,1+0.00 100 : 100.01 100,00 100100 1(1 ;,00
C)nwga-3 21.95 15,55 30,18 41.51 39.0 44.13
Ori ega: 2.112 121$}1 30 IN 0,97 40)
32
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 8
[:F':1~" ,IC d c'l t21~s1(i: [ of th ttiglycet=ide .lad I =so-
ph{Yf111:11dylc1Ãol ne
fractions ('6 I w'~w't1,
TIIUl cerces 1 ial ('C.
Ir.t~, cl cpttu.e Neptu-,re
L_Ie N :ut_y l K0 Polar KCt KC) N= urral K0 Polar KCf K(
('4:.r (1"(31) 1,(101 ( (r 1i (,tit;i
(1(:(J (ir:jti '1,(111 (1.1!(1 (_,s itl (I, fi
(: :11 (1,,jo 11,0Ã1 (I (7Q (!.(3f1 (I.
(1:i() "1ÃT thlj(? (i,Ãli) (t,
(:12x;1 (f,III I (_ II,Iu1 (1.(11 l Il
('14:f 1 2 1.06 ' S 3 19. ",8 4.27 1.13
('14:1 0.36 0 1.; (1.'" u.titl 6.08
('1`.!1 its 2.64 (1,' 1 l"[i(j (,-?2 11.45
('111:!;! 23 7 4,93 41,(10 44,14 30,15 6
('16:1 17,69 (1.58 tl. 4 (10(.1 1.84 2,. 14
C'lk:0l 1,5' .12 1.;'' ( 1,76 (. ;) 1.32
(218:1 27,93 34,39 27.,2 (1,65 14.24 11.29
C 18;2N, I,t?-w 2,01:5 1,92 (1.1(01
{'19:11'`. Ã1, 2i) (t1 CI. j`; 11,1)0, (,ÃX, (1,'Ã,
{ 1 i= (1,51 1"(1Ã1 (1, 'i' ,95 0.+67 975
(218:40 I. 99 11.011 4,93 01,00 1.11. 2.46
( 21 ;01 0.0 6 1Ã1 (i,:` 8 1(1(1' 01011 (1,
C'20:1 1,67 G)00 1.76 0.00 0.52 Ãt 90
C20:21\I' (Isl4 [0 141 (1, (I, [)(I 0.00 (t,;d(1
C 2(I;z1\1 I,E'l 11.(1Ã1 1I 11,ti0 (I.Ã1() (1,54
( 2(i:4164 11,1U' '1 t)Ã1 11,1)(1 01.41, (l,i iF
C 20:3N3 41,:35 (},Gift 0,01' 0.!10 (r,i 0
00:4164 (1.11 0,01Ã1 C, 0, ('J.31 0.55
C-2 r': SN I I:LP. II 7,t) 44 0, 00 18.S9 2
R. 4
(922:t) (3,.`,12 0.00 ('' (} 00 (1.0 (1,.^10
(2 2:1 0.3 i 0.010 9,41 Ã'1.00 1.46 01,91
C 1~'t? (3'.11.1 0.1,01(( J _ rP,(t(1 r."1,P;t(1 (1..'1
022:41 3 1.Ã:f0
C22: `M f l jl,l }Ea (. - .1,1X, (3,Ã,f1I ( ` j
(.2,:61~,3 u, 11) (1.(IEI (! 0. HO 0,41 (t 1
C"- 2: 6N' (DHA;= (1.(7 ,,97 1.41 24.26 7.79 13.82
1,26 1,78 6.1 0 00 0.32 1. i
(.'14:1 (11)01 1).9(01 ( Y i (II' 0,(10 ~1
10Ã '.x) 711 1 1 00 ;11(,.(71) 100,40
w:11 48,64 3ti,1_ 5, IS 14 5 (f.83 35.)1
\1 , Io I ~ C1 4,911 46.8) 31 152 f; 1.8,1-1 14,14
t'c, :tt :t 7,45 -:9 1 ry 2 22.0, 31,02 50,35
10(1,01) II ~11(1(1.1,(1 ;;,:,1(1.(11,1 1011,Ãi(;1
{7tt=e :t 5.51 1 14 It 32,20 7'9,87 47.69
{)tie4a-6 L94 2,05 39 0.010 2.Ã5 2.56
33
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 9
Patty acid cortpesition cif tlic l lx 11 ratio: 115111 1c
and tit I hosp1icti tt s 'inc fractions t f 1 u i).
PC' 2S
7'iM~ Acid
e Ycutrtrl KC) Po_ar KO KO Acutiat Kt) P Ã .r KO
KO
'4:0 J'.+:) t~, U.iFYi r:n n ' Ã L.iICI
(1f :1' C t' (1.111 (;,(}(}
(:1:t! UO aril}
C' 11):f) El.Ãi) G.( i(1
(' 12:0 ~ 1) P ' 1)!) J.' cj)l 141} ,,.75 _.i 2.+1
(.14:) D)7 t o.02 'i, C l (1,20 0,1)51
'.34 1: 1111) ~,))) (1.11 (..(7(1
(16:0 1.1;_05 2'3.I1 3:1,44 43.61 19.49
('1 :t )1, (1.17 9,96 3.47 _.79
C )):)' 1.3 1 I.33 2.118 3,34 2.24
C19:1 34.34 13. 11.16 400 7.37 11.87
C'1246 16.55 2.27 1.90 0,011 11.,;,11 (;,Op
t lCC:3 lh 1.44 0,25 51.21) (.,5151
t 153 '? 2.49 1.1> 1.14 ; jII ti,; t) (=,rjO
C1%:4N3 2.38 ).41 00 j 1.i{)
1111,)
C'20:0 2.79 U.03 0'05
0.20:1 2.42 0.82 1.74 0 ,10 51.51!1 Ã).0(1
C 20: 246 2.511 (1 , 5 (1,06 1) Y' 0'1"1(j i 1'iCl
('20:4246 1.85 (1,6: 0.56_. 0.00
i1(? 1151
C20:3243 3.94 Q. 7 0.06 0.00 010 t:.33
C 2,:4243 4.32 i1 1 4( 0.00 n at; C=.(aU
(2++. ; 1118 21.145 30,09 215.x4 15.81 16.35
L2 :(i 1)t! 0.05 E.1=1^ '} fit I 1.1() 0.00
C22:1 2.7 7 ti 1.87 '.01 (090 0.60
022:226 1,11)1) 0. 0.97 1'1 0. G (1,61,
C .12:4M :;ill) 0., _ 0,02
U. (l t;.i#}
('2:5Jti .4') 0i 0=:10 11.0))
( L:5N3 1.46 ON ! (Iii)' Ci.)iO
076312)1,1) 1,1111 1(I 53 1? "3 16,99 44.63
C24:0' '.34 C1 11.18 t
('24:1 ';,1.11) (I,'1 (1õ3, if!
,;11 1115;,)(1 ii?0 0511115 1... 1) 11151.00 laÃ,t)n
San07eii 25.19 .136.46 34.16 43.95 56.47 24.111
LI I tinteft 42.50 14,67 14.29 9.95 10.84 14.05
d 32.25 48,87 51.53 46.119 32,1+9 (11,31
1.`.'. 101, 00 1:10.26 100.00 i('1 .00 l+I . =ti 11)13, ))
(}rrie 9.- 15.6`) 44,73 47.7 46.0') 32.69 61..31
C)m ;i 6 1(31 4.(3 3.81 111::10 0,00
('4:0 11,(50 1) )1) }.1)0 f),[i1) (i j)(
0,01) 1) 000 111111 0.00
1'8;1:1 (),1111 (1,1:10 (1.()0 (,tnt, (ii),)
CIO:0 1;,111) 0,tI6 1100 1.)111 U1)11
( i 7:(1 flu,) (i C(j 11,11(1 1)16 (~(j)
0140 5.92 72 14.42 4.60 0.33
014:1 3.: ` (1.6$ 01.0) (1.00 1.000 0.1(1
('15:0 3.81, 1.95 3.18 1.0 1.30 (1.23
('116:(1 3 2 31;.566 31.39 35.91 31.21 16.38
0'16:1 19.05 224 1.16 (1.(151 151 0.75
('18:0 (.72 2.83 5.511 12.72 16.7[) 1.94
0'18:1 18.1.5 24,77 14,23 36.96 191;)1 18.4i
34
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 10
(iii] = 0 .it,
I'1 PF
Dm- Acid
File \ In iI K() .04 i K)) )) NOW K() ('u], r K{) K()
( ]8:2N( (1?0 2.67 C1(1'} 11.11O ,2. 62 (I,$5
C'101;31;6 (l,{ Q 11.011 off: 11,110 1.1.1111 1)õ`.tV
t']1(:3113 11:.11 1itK 1 "`' 11.111 (1.111) 0.13
(16:4h3 O;:0 0,1)11 (I (1,110 (i,In) (1.; [)
1.2(1:1) 11 711 17.00 111 (1.00 1) 111) 11,1)1)
C20:1 ii{? 11,17(1 (()j I'),;
('20:2176 1 0 (If)1l (1:21)
C 211:3N(? Gii uO (HI) (1,11)1 1.5
C 20:416, 0.0) n 0.00 0.00 C1I:O
C 20:3N1 11,:10 0.00 0. 0.110 0.0) 0.1210
1'20:41'?. I)(li) h('l (1.11(1 (1,111) OD)
L (?; N, (EL'1) Ã ..,') 1160 20.45 11.01' 10.76 21.26
c n, i )`}1. 0.00 1 -1 0,110 (1)0 (1-1-110
('22:] 11,711 11th) III O.I)0 (1.(10 (1,(111
C22:2N6 0,11-110 11,1)1( (I, (1,10 (110(7 11,"'0
C2 2:4!76 11: 41 Omit (..i: O,l)(i 1!,(117 {l, i't{j
.
C ~.5jtifi (1:(111. 0,110 [I 111)1)
( ':tiN^ 0,(li 01111 (. Ii,i")0 (!.1#r) (:
( 201ti3 (I}]1<4'. ():"ii) 179 ]3.32 11.1.1)1 1139 3A 116
C200 000 0.011 01. (1.11(1 0.00 0.:,O
0 010 11.(1(1 11,1)0 0.011 (!.'V)
11111,00 17O.(n) 1(1(1.1.+[)
41.266 } 63.114 53.81 21.2$
Lles1 39 CA 27A7 1111 311.96 21.42 19.311
Y !s 31A7 7 11.110 2177 59.42
l'cit: 1111) II 1;11,1)1) lUh),l;t; 100.110 ])t)).u,)
Orr.eg 7.11 ".17 (1.1711 2115 5743
Ull c ,a (; (1,11(1 '?.62 ion
Table 11
C mp mlttw ll(1 J3tt0 111(1' the I and INH 011 m 4w )ti fl
~`h:;3ll1f ( a7)([ "1~(1 Neill oil.
Nepni11e LtllalA
C'ns,; c'unds KU extl bet, rl KU P 11 i K() tiieuti- ll KU
i=,1 n in] 1m; ehlo" 472 m r k ,-r 117 ~2 I":_r i1k1õ 1(122 I;,_ Irk lu_ 1,v,
.6 1 1': 1& 1111 L 1 11 I ].11 el 1 ). 77 <1 nu k <1 Isla A,
- U 'MA g 1- - I ' l l ; . ' 1. 17 u)
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
EXAMPLE 4
[000116] Neutral lipids were extracted from krill meal (138 kg) using SFE with
neat C02 (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 (S1 - 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. Next, the polar lipids were extracted
using
C02 at 500 bar, 20% ethanol and at a temperature of 75 C. Using a solvent
ratio of
32 (kg/kg) and collecting an extract of 18.2 kg using a separator at 60 bars
and
35 C. The polar lipids were collected and analyzed (see Table 13). Next, the
polar
lipids were mixed in a 50/50 ratio with the neutral lipids collected from the
first
separator before finally the ethanol was removed carefully by evaporation. The
product obtained was red and transparent. If the ethanol is removed before the
mixing if the fractions a transparent product is not obtained. The composition
of the
50/50 red and transparent product can be found in Table 14.
36
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 12
I n l 1 it rut f[ ,,tract c 1 Cc*cd in 51
I i:tv <<c:id i ni` Amount
l:r `a'l00 1.8.4
It':{t l!n, 2 ,2
It=:1 it- I! )
(11-7"1 41l 1 H 25,0
+ r,t [ + ;_t l
1t r. Tin _3
[i
18,:2 t [1110 1,3
1 8 : : [t r II i - ;.
t R:= fl-s
2(}:7 rt-? 4,1
22:6 rt-4 l`7
nII).: I ,t fllc cxtr - 4ol.l's 1cd _tt Si
1
(.ipit [_1,.111; 1 is itnt
ri 1 ,`cer~itl )_ 84
l E>i 11),,3; 1?.,7
F r, t 1.-11 ,' mils ~' 1r4i% 1,.4
t, l~:irl<7-gt ait;i11hU o Ilic c [-i~1 in SI,
tst t i?unir 1i[td1. t` I: I!'1'
t'r-~ 4-3
t . 4 462
Trip i J i', Fnmin t-_i,r ti 106 1
Trimetiy1a liinccxide mg N.'1('1) p 2
37
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 13
Unit ~.rri-r r~
t4:0 4"l tii! 1.3
t6:ti E..1C (< 13.S
1 :iJ g,'1J0a 0.6
10:1 n-7 at 1(,_I() 4:^.
18 1601 + (n- 7"1 +
i=1' 1 1(,( 0-
,W (tk-9 + i tl- 11 -1,00c 0.6
tIl `) tl } 'li;l(.i e ?.1
2':1 C11 11 i +
16:-- ii-4)
16:4 (n-11 1ri0
4F c!_i.1
iK2, 11-6 0C) 0i.8
IM 11-3, 0.6
18:4n-3 glO',g Iii
Ct: li ltit w 14.7
,2:6 11-4 t l llij ~ j,j
chs's
Llpi_1 _ rrs
c;t,,=. 4 1cit'1g
iul::l~ruj <10,5
1.6
1tics~ ~'1 I~1-u~ iits +linr 4.4
t 1 L u '\ riOi l tr
,ir eif I l i r i . , lag 1x lti'`,g 422
rinietl 'In in,cixirie ;rigN 1(.1:1 23
38
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 14
Fatty õcid compcositionof the f'mal blended produce
obtained in Exam ale 4 in S l .
Fatty ii.cd W lit ?tti<rt.i t
14; 9,7
l6',,' 1.
lw:. rr-9+lrt tr ~;i 1 16.0
20:(.11-9) + f,t 7 1
16:? ti-4)
16:4 r rt- l i 1 i <f r.'1
18:2ri-; In
1 h: rt t1.K
18:4 ft- 1 2.1.
2 0:5 rt :T 10 r
2.2:6 ra-4 1 i 4.>
Lipid class c o1 Ieosi`ion of the 11:1.1; blended product
obtained i;r J x 1<rl?=c: 4.
Liaid iJEtit tr ount
I r; 1 r L' rft' 1 ' z A
1.'t
i .'Ills ' ' . g 0. `}
1 .' I, sl:'Yc; l (1.0
C h o ] yl'i ester I'' JO
1'1 1 {, Ir'} r r ~.arii r I <1
11 l1c - 1 1 _~~ 1 r 42
1 -hF p[ iÃI,1,h:Oine g I''
NJ 1, t
E[irr {iie:rr~Ie4.
Co' li . i l
Asta:e 1i
Trin ete l 10 a 1)
Trip e:th l ;ii,:. ,ccxi i 1t:1 I 8)
39
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
EXAMPLE 5
[000117] 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.
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 15
Fatty "1i:-iu co111p0s1t:o_'1 of the final :)iC11uCCi product
oht1 r. '.1117 3"1. l = 5.
1 l'1 :' 1 1 [:11i* Amount
14: ?,'1(1+) t?.2
1 r.,r - '1(111 '.7
11r.'( g 10 1.0
i tr:1 11-7
16(' 4.9
11a1'r,s +(11-7!+(.1_)) g.100 14.9
l-a"I+tr1-li p IHI g 1.1
lt[A11+it,9 +ir1_
10:2 (1t-41 9,11 t1
16:4rrr Li 100 1 r1- 10( 1.2
18:1 n-: 1rit t
111:4
0:5 n n 1 iti 7 `. 1 l ).
6
.2:6 i-4 4.;
i i :1~,. 'rrr1,-5rtrC~1. > l 1 l i e Gr- [ I 1-'tftl + 1 riii~- a
1.;111: Unit Ar'1Onn`
T'r i 1 1, [' O g 41
I.
1=:~ r 1C 1.-
t 0A
Phoph 1 , c J +_ 1111ine ! { .0
51
1 Ali ~nl rilr1~, LIiclU c- "t`- . t),5
1c,r:r 5 :.4
1.. 43 ,1.
,rrC an I~ Ilit Ii i i bl,~ i.ikl 1' .iIacr'A
t('Yfl_L '.1 ill E in.7-1G'
C'om ounc' T.-nIt amount
Free stlxr lt11i11 12
1~'i esters
Biel 1,, 1;111 ill 9 1`1(111 m 19r"ik'.i:1(7rtg 1.:J
41
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
EXAMPLE 6
[000118] Krill lipids were extracted from krill meal (a food grade powder)
using
supercritical fluid extraction with co-solvent. Initially, 300 bar pressure,
333 K and
5% ethanol (ethanol: C02, 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.
Table 16 !a I r:,I, r 'kiÃIttC
C'2:
: ( 2 7 H 1
(. i
. if'r.r 135.7
Per, I I C
EXAMPLE 7
[000119] Krill oil was prepared according to the method described in Example 6
by extracting from the same krill meal. The oil was subjected to 31P 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 dal-10% Na
cholate, 1% EDTA, pH 7.0 in H2O+D20, 0.3 g L-1 PMG internal standard) was
added. Next, the tube was placed in an oven at 60 C and periodically
shaken/sonicated until completely dispersed. The solution was then transferred
to a
ml NMR tube for analysis. Phosphorus NMR spectra were recorded on the two-
42
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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.
[000120] 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. Example spectra are
presented
in FIG. 1. Peak area integration gave relative molar amounts of each lipid
class.
Weight percent values were calculated using molecular masses calculated from a
krill sample fatty acid profile (average chain length=18.6). Total PL levels
were
calculated from the PMG internal standard peak. The quantification of the
phospholipids are shown in Table 17 for both the raw material, the final
product and
for a commercially available krill oil (Neptune Krill Oil). 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.
43
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 17
1 1.osp iolipid pro Eller
T`; -'- 'il
!: N'KC3 K :11 Oil cil C ~.i tc,l in Px, n:pke 7
PC' 6C0 0,,. 6 75.3
AAPl'. 1,O 13.0:
1I PC 1. 1.3 0.4
PS
2LPC' -.4 1 3.8 2.9
L111't: 1.2
r.
1?õ (i.;.) 3.4 1.4
AMT.' -S
Sv'1
(.7Ã~C... 1.3
1.?1S"
NA";I ', 3.=
(`'1, 53
2.1
991.,
lipid vurl is
44
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[000121] Analysis has been carried out on the fatty acid and ether/alcohol
profiles of the AAPC. The results are presented in Table 18.
Table 18
A..PC' I.cnh 1
A,\P( f t i' 9C1i1 }Tl~C7~It1T1
L fll ?C'S1tion &.Jeo hool "%;x
-3)- 16:1.1 41',()
ti.1 1-9! - 4,6% 16:1 14.1
14:1)
`.4P41 18:1) #?.h
18:2 5.1
(1.9".1, 17:1) 4.4
, % 15:4.)-i 2.1
1.>:f) 1.7
3 h 211:1 .=1
no 15:0-,"s
i f"U 18:13-i i.
41%%
24:1 .
[000122] The rest of alcohols (i17:0, etc.), were less than 0.3% each. Only
part
of 20:1 was confirmed by GC-MS. The alcohol moieties composition of Krill AAPC
was determined (identification was performed in the form of 1-alkyl-2,3-diTMS
glycerols on GC-MS, % of total fatty alcohols were obtained by GC with FID).
Ten
other fatty acids were all below 0.3% by mass.
EXAMPLE 8
[000123] 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). Four groups of rats (n=6 per group) were
fed
ad lib either a control diet (CTRL) or a diet supplemented with a source of
omega-3
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
fatty acids (FO, NKO, Superba). All diets supplied same amount of dietary
fatty
acids, oleic acid, linoleic acid and linolenic acid. Omega-3 diets (FO, NKO
and
SuperbaTM) were additionally balanced for EPA and DHA content. 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 characterized as being pre-diabetic. Rats were
fed the
test diets for 4 wk after which they were sacrificed and blood and tissue
samples
were collected. This example shows that supplementation of the Zucker rat with
krill
oil prepared as in Example 7 results in an improvement of metabolic parameters
characteristic of the obesity induced type two diabetic condition. 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. Specifically, 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).
Furthermore,
the efficacy of transfer of DHA from the diet to the brain tissue was greatest
with the
krill oil prepared as in Example 6 (FIG. 10).
EXAMPLE 9
[000124] The purpose of this experiment was to investigate the effect of
dietary
krill oil on metabolic parameters in high-fat fed mice and to compare the
effect of
dietary krill oil with that of fish oil containing the same amount of omega-3
fatty acids.
Four groups of C57BL/6 mice (n=10 per group) were fed 1) chow (N), 2) high fat
diet
comprising 21% butter fat and 0.15% cholesterol (HF), 3) high fat diet+krill
oil
(HFKO) or 4) high fat diet+fish oil (HFFO). Treatment 3 contained 2.25% (w/w)
krill
oil as prepared in example 5 (except that the astaxanthin content was 500 ppm)
which were equivalent to 0.36% omega-3 fatty acids. 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.
N=normal chow diet (n=10); HF=high-fat diet (n=10); HFFO=high-fat diet
supplemented with fish oil (n=9); HFKO=high-fat diet supplemented with krill
oil
(n=8). The data are presented in FIGS. 12-19.
[000125] This example shows that supplementation of high-fat fed mice with
krill
oil results in an amelioration of diet-induced hyperinsulinemia, insulin
resistance,
46
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
increase in muscle lipid content (measured as a change in muscle mass), serum
adiponectin reduction and hepatic steatosis. These potentially beneficial
atheroprotective effects were similar or greater than those achieved with a
supplement containing a comparable level of omega-3 fatty acids (see FIGS. 12-
19).
EXAMPLE 10
[000126] The effects of different omega-3 fatty acid sources on metabolic
parameters in the Zucker rat were also investigated. 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 krill oil (KO). The KO was prepared by extracting
the
triacylglycerides and the phospholipids from the krill meal using
supercritical CO2
with ethanol so that the final oil consisted of at 50% phospholipids, 30%
omega-3
fatty acids and around 1300 ppm astaxanthin. Three groups of rats (n = 6 per
group)
were fed ad lib either a control diet (CTRL) or a diet supplemented with a
source of
omega-3 fatty acids (FO, KO). All diets supplied same amount of dietary fatty
acids,
oleic acid, linoleic acid and linolenic acid. Omega-3 diets were additionally
balanced
for EPA and DHA content (see Table 19).
Table 19. Fatty acid content of feeds used.
t+Ei i3 k0116 n( u3 t(tt t t- A ttit '`4 \ t P, A t, \
0. 82 "SLR "S04 " !. t 4. 4 I
[000127] 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.
47
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 20. Fatty acid composition of the VAT, SAT, Liver and Heart.
- --- - -------
K
[000128] This example shows that supplementation of the Zucker rat with krill
oil
prepared as described above resulted in a reduction in the levels of
anandamide
(AEA) and 2-arachidonoyl glycerol (2-AG) in visceral adipose tissue (FIG. 20A-
B). In
subcutaneous fat, the level of 2-AG were reduced compared to fish oil and
control
(FIG. 21A-B). In liver and heart (FIGS. 22A-B and 23A-B, respectively) the
level of
AEA was most efficiently reduced with krill oil.
[000129] Furthermore, the triacylglycerol content in tissues was measured as
well. FIGS. 24 and 25 show the TAG deposition in the liver and heart,
respectively.
In both tissues, 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. Clearly,
krill oil is
48
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
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.
EXAMPLE 11
[000130] In this example, the effects on lipid metabolism, ectopic fat
deposition,
and susceptibility to inflammation in Zucker fa/fa rats were studied.
Relatively low
doses of dietary (n-3) LCPUFA were administered as FO or KO. Fatty acid
profiles
and endocannabinoid concentrations were determined in different tissues to
examine
the possible impact of (n-3) LCPUFA on the dysregulated endocannabinoid system
of Zucker rats, which were fed a diet containing 0.8% of energy (n-3) LCPUFA,
a
level lower than that typically used in rodent studies, to allow a more
meaningful
comparison with human studies.
[000131] Eighteen male Zucker rats (Harlan) 4 wk of age were divided into 3
groups and fed for 4 wk a control diet (C) or diets supplemented with either
FO (GC
Rieber Oils) or KO (Superba, Aker BioMarine). The diets were based on the AIN-
93G formulation, with substitution of soybean oil with a blend of oils
(rapeseed oil,
sunflower oil, coconut oil, and linseed oil). This allowed the 3 diets to be
similar for
total fatty acids and for oleic, linoleic (LA), and a-Iinolenic (ALA) acids.
FO and KO
diets were further balanced for EPA and DHA content (see Table 21). The 3
diets
were prepared by Altromin GmbH & Co. KG and stored in vacuum bags to reduce
(n-3) LCPUFA oxidation. The amount of 0.5 g EPA + DHA/100 g of diet,
equivalent
to 0.8% of energy in the rat diet, was chosen to provide a level of (n-3)
LCPUFA
intake achievable in humans and corresponds to 1.8 g/d in an 8.4-MJ/d diet in
humans. All experiments were performed according to the guidelines and
protocols
approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116)
and
by the Animal Research Ethics Committee of the University of Cagliari, Italy.
49
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 21. Dietary Fatty Acid Composition.
931
[000132] 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.
[000133] Cells were centrifuged at 300 x g; 10 min 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 105
cells cm2
and incubated for 2 h at 37 C and 5% CO2 atm. After removing nonadherent
cells,
macrophages were cultured in DMEM with 10% fetal calf serum in the presence of
lipopolysaccharide (LPS) from Escherichia coli 026:66 (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
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
time indicated, supernatants and cells were separated and stored at 280 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.
[000134] Serum C-reactive protein (Chemicon International), tumor necrosis
factor-a (TNFa), interleukin (IL)-10, and tumor growth factor-b (TGFb)
(Biosource)
were determined by a sandwich ELISA. Moreover, to evaluate macrophage
susceptibility to inflammatory ligands, the secretion of TNFa, IL-6 (Bender
MedSystem), IL-1b, IL-10, and TGFb were assessed in culture supernatants from
peritoneal macrophages activated with LPS. Media were assayed at 800 x g; 10
min
to remove debris. Supernatants were frozen at 280 C until assayed with a
sandwich
ELISA (Biosource).
[000135] Total lipids were extracted from tissues using chloroform: methanol
2:1
(v:v). Separation of total lipids into TAG and PL was performed as previously
reported. Aliquots were mildly saponified as previously described to obtain
FFA for
HPLC analysis. Separation of fatty acids was conducted with a Hewlett-Packard
1100 HPLC system (Hewlett-Packard) equipped with a diode array detector as
previously reported. Because 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. H2 served as carrier gas (1 mL/min) and the
flame
ionization detector gases were H2 (30 mL/min), N2 (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.
[000136] AEA and 2-AG were measured. MAGL and FAAH activities were
determined in the heart, liver, VAT, and SAT from C and FO- and KO-
supplemented
rats. In particular, 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-[3H]-
glycerol (40
Ci/mmol, ARC) properly diluted with 2-AG (Cayman Chemicals). After incubation,
the
amount of [3H]-glycerol produced was measured by scintillation counting of the
51
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
aqueous phase after extraction of the incubation mixture with 2 volumes of
CHC13:MeOH 1:1 (v:v). AEA hydrolysis (by FAAH) was measured by incubating the
10,000 X g membrane fraction of tissues (70 mg per sample) in Tris-HCI 50
mmol/L,
at pH 9.0-10.00 at 37 C for 30 min, with synthetic N-arachidonoyl-[14C]-
ethanolamine
(110 mCi/mmol, ARC) properly diluted with AEA (Tocris Bioscience). After
incubation, the amount of [14C]-ethanolamine produced was measured by
scintillation
counting of the aqueous phase after extraction of the incubation mixture with
2
volumes of CHCI3:MeOH 1:1 (by vol.). Values in the text are means SD.
[000137] The one-way ANOVA and the Bonferroni test for post hoc analyses
were applied to evaluate statistical differences among groups. Where variances
were
unequal, Kruskal-Wallis non-parametric 1-way ANOVA was used.
[000138] Growth and food intake did not differ among the 3 groups and none of
the rats exhibited adverse effects (data not shown). At the end of the 4-wk
treatment,
the body weight of the rats was 400 35 g.
[000139] In KO-supplemented rats, and to a lesser extent in the FO group, the
liver TAG concentration was significantly lower than in C (FIG. 29A). The
heart TAG
concentration was significantly lower than C only in KO-supplemented rats
(FIG.
29B).
[000140] Rats supplemented with FO or KO had 75% lower plasma LDL
cholesterol concentrations than C, whereas HDL cholesterol did not differ
among the
groups (FIG. 32A). Conversely, triglyceridemia was -30% higher than C in both
(n-3)
LCPUFA-supplemented groups (FIG. 32B).
[000141] Plasma proinflammatory (TNFa, IL-6, IL-1b) and anti-inflammatory
cytokines (IL-10 and TGFb) and C-reactive protein did not differ among the
experimental groups (see Table 22).
52
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 22. Plasma levels of inflammatory markers in rats fed Control (C), Fish
Oil
(FO), or Krill Oil (KO) diets.
17.2
41 f I
[000142] In macrophages incubated for 24 h in the presence of LPS, TNFa
secretion was significantly lower in FO and KO rats compared with C (see Table
23).
Plasma IL-1b, IL-6, and IL-10 concentrations did not differ among dietary
groups
following LPS stimulation.
Table 23. TNF-alpha release in LPS-treated peritoneal macrophages from Obese
Zucker rats fed C, FO, or KO diets for four weeks.
[000143] The VAT AEA concentration was lower in the FO and KO groups than
in C (Fig. 30A), whereas 2-AG was significantly lower than C only in the KO-
supplemented rats (Fig. 3013). Endocannabinoid concentrations in SAT did not
differ
among the 3 groups.
[000144] Liver and heart endocannabinoids were similarly affected in the KO-
supplemented rats (Fig. 31A-D). AEA concentrations were -25% of, and 2-AG
concentrations -200% of, those of C in both tissues. In FO-fed rats, liver but
not
heart AEA concentrations were less than in C.
53
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[000145] Activities of enzymes involved in endocannabinoid degradation.
Changes in FAAH and MAGL activity have been observed in adipose tissues and
liver of obese individuals and rodents. In this study, heart, liver, VAT, and
SAT FAAH
activities did not differ among the experimental groups (see Table 24).
Conversely,
MAGL activity was significantly lower in the VAT of the FO and KO groups, and
in
the heart tissue of the KO group, compared with C (see Table 24). Liver MAGL
activity tended to be lower in both groups compared with C (P = 0.1).
Table 24. FAAH and MAGL activities in the heart, liver, VAT, and SAT of Obese
Zucker rats fed C, FO, or KO diets for four weeks.
[000146] Plasma EPA and DHA concentrations were higher and that of ARA
was lower in the FO and KO groups compared with C (see Table 25).
Interestingly,
the levels of ALA and LA (only in the KO group) were higher than in C despite
the
similar levels of these fatty acids in the diets (see Table 21). Peritoneal
macrophages
from FO- and KO-fed rats had significantly higher EPA and DHA and lower ARA
concentrations than those from the C group (see Table 26).
54
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 25. TNF-alpha release in LPS-treated peritoneal macrophages from rats
fed
Control (C), Fish Oil (FO), or Krill Oil (KO) diets.
{
Table 26. 20:5(n-3), 22:6(n-3), and 20:4(n-6) concentrations in peritoneal
macrophages from rats fed Control (C), Fish Oil (FO), or Krill Oil (KO) diets
(expressed as mol% of total fatty acids).
U 11
[000147] Dietary (n-3) LCPUFA influenced the TAG fraction of VAT and SAT
with greater incorporation of EPA, DPA, and DHA in both experimental groups
compared with C (see Table 27). The EPA and DHA levels in SAT were higher in
FO-than in KO-treated rats. ARA was significantly lower than in C only in SAT
of KO-
supplemented rats; there was no difference among the groups in VAT. As in
plasma,
ALA was higher than in C in both VAT and SAT of FO and KO groups. On the
contrary, LA was significantly higher only in VAT of (n-3) LCPUFA supplemented
rats
compared with C. In all 3 dietary groups, remarkable differences in fatty acid
profiles
of the PL fraction were observed, also between VAT and SAT (see Table 27). PL
ARA levels of the VAT, but not SAT, were significantly less in FO- and KO-
supplemented rats than in C. Levels of EPA, DPA, and DHA were higher in the (n-
3)
LCPUFA-supplemented rats compared with C in VAT PL, while only EPA changed
significantly in SAT PL.
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
L- I .
O
o
U-
0
LL
U
N+r
ryU)
O
U
CJ
O
O
U
O
Q
E
O
U
LL (D
O
t` Y
N -_
a) O
(~ 'C
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
4)
=L
L
O
O
LL
O
LL
wr
O
0
O
L
0)
C
4-
0
a
Q
E
O
U
LL
co
N
a)
(CS
H
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[000148] In 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. In liver PL, 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. In the
PL
fraction of the (n-3) LCPUFA-supplemented groups, concentrations of EPA, DPA,
and DHA were also greater than C, with higher levels in the KO group, whereas
ARA
was significantly less than in C only in the FO group (see Table 28).
EXAMPLE 12
[000149] 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.
Table 29. Lipid and protein sources for the diets.
Control Fish oil Krill oil
Lipid source 0 g soybean oil 29 g soybean oil; 25 g soybean oil;
11 g fish oil 15g krill oil
Protein source 140 g casein 140 g casein 140 g casein
58
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
[000150] Body weight was measured approximately two times per month and
were similar in all groups. At five months of age, 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.
[000151] To identify functional classes of genes changed by treatment
Parametric Analysis of Gene set Enrichment (PAGE) was performed. This
technique
allows for an unbiased and highly sensitive method of detecting classes of
genes
that are modulated by treatment. In addition, PAGE determines a z-score
indicating if
a gene class was activated (z-score >0) or repressed (z-score <0) by
treatment.
Genes were grouped into functional classes using the Gene Ontology (GO)
hierarchy, and GO terms that were annotated with at least 10 but not more than
1000 genes per term were considered. GO terms were considered to be
significantly
altered by treatment at p<0.001. A global comparison of the GO Biological
Processes modulated by diet was made, which showed that fish oil is less
bioactive
than krill oil. Moreover, in several cases (e.g., lipid biosynthesis and fatty
acid
metabolism), the overall effect of fish oil was in the opposite direction as
observed
with the KO diet.
[000152] The effect of diet on glucose metabolism was studied. PAGE analysis
revealed that the GO term "glucose metabolism" was decreased in the FO and KO
diets (p=0.004-6). The ability of n-3 PUFAs to decrease glucose metabolism
compared to the control diet with the same energy intake may be regulated at
the
early stages of glycolysis by decreasing glucose uptake though the liver
glucose
transporter (Glut2 / Slc2a2) and by decreased phosphorylation through the
liver
enzyme glucokinase (Gck). KO showed a trend for decreased Slc2a2 expression
(p=0.030; see FIG. 34). It is important to note that the majority of the
carbohydrate in
59
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
the diets used in this study comes from sucrose (composed of glucose and
fructose),
and interestingly, there appears to be a shift favoring fructose metabolism in
mice
fed diets containing KO. As shown in FIG. 34, expression of two genes involved
in
fructose metabolism tended to be greater in the KO group: Ketohexokinase (Khk)
converts fructose to fructose- l-phosphate, and aldolase B (Aldob) converts
fructose-
1-phosphate into compounds which can enter glycolysis or be used to synthesize
glycogen. Taken together, the decrease in glucose metabolism and shift towards
hepatic fructose metabolism suggest that krill oil supplementation acts to
preserve
glucose for tissues such as brain or muscle.
[000153] PAGE also revealed a trend for decreased gluconeogenesis in the KO
group, with a nearly significant decrease in this pathway (p=0.040). In
addition to the
genes annotated in this Gene Ontology term, there are several well-known genes
that regulate hepatic glucose production but are not annotated by the Gene
Ontology
consortium; interestingly, these genes showed a strong trend to be regulated
in the
KO group (see FIG. 35) providing further evidence for decreased hepatic
gluconeogenesis. Two of these genes (Ppargcla and Hnf4a) are master regulators
of metabolic gene transcription, and they have potent physiological effects on
hepatic gluconeogenesis/glucose production. These genes encode proteins that
regulate metabolism by binding to DNA and enhancing the expression of other
metabolic genes in many tissues. In the liver of humans with type 2 diabetes
and in
mouse models of diabetes, the expression of Ppargcla and Hnf4a are increased
which results increases the expression of genes that result in gluconeogenesis
(phosphoenolpyruvate carboxykinase 1; Pckl) and aberrant glucose export from
the
liver (glucose-6-phosphatase, G6pc). In the current study, Ppargcla expression
was
decreased in KO (p=0.014); and Hnf4a was significantly decreased in expression
by
the KO diet. There were also marked reductions by KO in the expression of two
targets of Ppargcl a/Hnf4a (see FIG. 35), with Pck1 being significantly
decreased by
KO and G6pc showing a trend for a decrease by KO (p=0.04). These data strongly
suggest that krill oil has the ability to suppress hepatic glucose production
which is
increased in type 2 diabetes. Suppression of gluconeogenesis and hepatic
glucose
output via modulation of Ppargcla activity has been proposed as a strong
therapeutic target for the treatment of diabetes, provided that the
intervention does
not oppose the beneficial effects of Ppargcla expression in other tissues. The
krill
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
supplements used in this study may functionally decrease hepatic glucose
production and increase glucose uptake in tissues other than liver.
[000154] The effect of diet on hepatic lipid metabolism was also studied. The
KO
diet resulted in a gene expression profile suggesting decreased hepatic lipid
accumulation. There is a significant modulation of the GO term "lipid
biosynthesis."
While decreased hepatic lipid synthesis in the KO group may simply be a
consequence of decreased substrate availability as a result of decreased
glucose
metabolism, this result may be of clinical significance as hepatic lipid
accumulation
(hepatic steatosis) is associated with insulin resistance and the metabolic
syndrome
in humans. Interestingly, fish oil did not significantly modulate lipid
biosynthesis in
this study. Thus, it is tempting to speculate that 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.
[000155] Although it has been reported that n-3 PUFA supplementation
increases the expression of genes involved in fatty acid oxidation, pathway
analysis
of the gene expression data in this study showed that the GO term "fatty acid
metabolism" was significantly depressed by KO, with no effect of FO on this
pathway
(see FIG. 39). This effect is underscored by the decrease in the expression of
key
genes involved in mitochondria) fatty acid oxidation (see FIG. 36) including
the liver
isoform of the rate-limiting enzyme carnitine palmitoyl transferase 1 and
three
enzymes involved in mitochondrial fatty acid beta oxidation (Acads, Acadm, and
Acadl). The reason for the discrepancy between this example and previous
reports
showing that n-3 PUFAs increase fatty acid oxidation is not clear. However,
others
have shown that expression of the Cptla gene is regulated by Ppargcla, and the
data from this study are in agreement with that finding. Thus, there appears
to be
strong transcriptional evidence that fatty acid metabolism is decreased in
response
to krill oil supplementation.
[000156] PAGE analysis also revealed that 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.
Specifically, 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
61
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
a protein that catalyzes the fifth condensation reaction in cholesterol
synthesis (FIG.
37). Others have suggested that the hyperlipidemic effects of dietary
saturated fats
are mediated through increased activity of PPARgamma coactivator, l beta
(Ppargcl b) and sterol regulatory element binding factor 2 (Srebf2). As shown
in FIG.
37, we observed a significant downregulation of both of these genes by KO.
Thus,
previous studies and the current study provide evidence that the liver is
sensitive to
the saturation of dietary fatty acids, and that Ppargcl b and Srepf2 activity
may be
the important regulators of cholesterol synthesis in response to dietary fatty
acid
saturation.
[000157] The effect of diet on mitochondrial respiratory activity was also
studied,
as well as its implications for reduced oxidative damage. PAGE revealed that
the
KO diet resulted in a significant activation of the GO term "mitochondrial
respiratory
chain"; this was largely caused by an increased expression of genes encoding
subunits of Complex I (NADH dehydrogenase). KO was also associated with a
significant decrease in the expression of superoxide dismutase 2 (Sod2, FIG.
34), a
critical enzyme involved in the detoxification of reactive oxygen species in
mitochondria. Biochemical assays of oxidative damage (lipid peroxidation,
nucleic
acid oxidation) in banked tissues would reveal if the transcriptional effects
are seen
at the biochemical level. It seems plausible, therefore, that the increased
activity of
the mitochondrial respiratory chain in KO mice is related to increased
mitochondrial
proton leak which would result in decreased oxidative damage.
[000158] The effect of diet on inflammatory pathways was also studied,
although
no striking effects were observed. PAGE analysis revealed that there was a
trend
for KO modulation of several pathways involved in inflammation, most notably
an
increase in the activity of "negative regulation of lymphocyte proliferation"
(p<0.05),
an anti-inflammatory action of KO may be more pronounced in adipose tissue or
brain.
[000159] As a result of these studies, a proposed mechanism of action for
krill
oil-regulation of metabolism has been developed. The data above clearly
support a
role for krill oil-supplementation to have beneficial effects on hepatic
glucose and
lipid metabolism. Two key regulators of these metabolic pathways in liver are
sterol
regulatory element binding transcription factor 1 (Srebfl) and the
carbohydrate
recognition element binding protein (Mlxipl). Expression and activity of the
genes
encoding these transcriptional cofactors is increased by insulin (Srebfl) and
glucose
62
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
(MlxipI) which results in a stimulation of glycolysis and hepatic lipogenesis
leading to
lipid accumulation and insulin resistance in the liver. Conversely, inhibition
or
deficiency of these proteins ameliorates metabolic abnormalities in mouse
models of
the metabolic syndrome. The KO diet robustly decreased the expression of these
two genes (see FIG. 38), though FO had no effect. Post-translational
regulation of
these transcriptional cofactors by PUFAs appears to be specific to
particularly fatty
acids, although the current study suggests that the genes Srebfl and MlxipI
are also
regulated at the transcriptional level by certain PUFAs.
[000160] In addition to the decreased expression of these transcriptional
cofactors, four genes known to be targets of these regulatory genes were
decreased
in expression by a krill oil-supplemented diets (see FIG. 38). These genes
include:
liver pyruvate kinase (PkIr), a regulatory enzyme in hepatic glycolysis; ATP
citrate
lyase (Acly) which catalyzes the conversion of citrate to acetyl CoA which can
be
used for synthesis of fatty acids; fatty acid synthase (Fasn) and acetyl CoA
carboxylase (Acaca) which catalyze two of the initial steps of fatty acid
synthesis.
Because PAGE revealed that hepatic glucose and fatty acid metabolism are
suppressed by krill oil-supplementation, the data altogether confirm that
Srebfl and
MIxipI are master regulators of hepatic metabolism, and their transcription
and
activity are modulated by krill oil-supplementation.
EXAMPLE 13
[000161] This example, which was conducted using the study parameters set
forth above in Example 12, addresses the effect of fish oil and krill oil on
hepatic
gene expression in mice. Fish oil was much less potent in changing the gene
expression, compared to krill oil. Table 29 sets forth the pathways that are
differently
affected by krill oil and fish oil.
63
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
Table 29.
Pathway GO ID Genes FC CR FC FO FC KO
fatty acid GO:0006631 149 -6.17 1.94 -3.59
metabolic process _ _
monocarboxylic GO:0032787 202 -5.23 1.42 -4.08
acid metabolic
process
cellular lipid GO:0044255 485 -5.64 1.41 -5.02
metabolic process
lipid metabolic GO:0006629 568 1-5.49 1.50 -5.18
process
peroxisome GO:0005777 90 -7.23 3.59 -1.60
fatty acid GO:0019395 20 -5.49 1.95 -2.16
oxidation
fatty acid GO:0009062 18 -5.41 2.34 -2.52
catabolic process
GO ID = The list of genes within a GO pathway can be obtained at the website
for
the Gene Ontology Consortium.
CR=calorie restriction diet.
[000162] The difference between krill oil and fish oil on the regulation of
genes
within lipid metabolism can be ascribed to their influence on key regulators
of those
genes. The most important transcription factors regulating these genes include
PPAR alpha, PPAR delta, SREBP-1c, SREBP-2, ChREBP/Mlxipl, HNF-4 alpha,
PGC-1 alpha, and PGC-1 beta.
[000163] Krill oil led to a downregulation of all of these transcription
factors,
whereas fish oil had no significant effect. Also, the same pattern was seen on
the
expression of target genes for each of the transcription factors.
[000164] Mitochondrial genes are in general down-regulated after treatment
with
krill oil. However, treatment with krill oil leads to a significant
upregulation of certain
classes of mitochondrial genes. Those genes appear to be associated with the
electron transfer/respiratory chain, mitochondrial ribosomes, protein located
at
the inner mitochondrial membrane, and ATPases.
[000165] The majority of those genes are encoded in the nucleus. However,
some genes in the respiratory chain are encoded in the mitochondria. The
underlying mechanism behind this effect of krill oil is not known. However, it
might
64
CA 02763647 2011-11-25
WO 2010/136900 PCT/IB2010/001478
indicate that the energy production in the liver is in some way affected. This
effect is
not seen after treatment with fish oil.
[000166] It will, of course, be appreciated that the above description has
been
given by way of example only and that modifications in detail may be made
within
the scope of the present invention.
[000167] Throughout this application, various patents and publications have
been cited. The disclosures of these patents and publications in their
entireties are
hereby incorporated by reference into this application, for example, in order
to more
fully describe the state of the art to which this invention pertains.
[000168] The invention is capable of considerable modification, alteration,
and
equivalents in form and function, as will occur to those ordinarily skilled in
the
pertinent arts having the benefit of this disclosure.
[000169] While the present invention has been described for what are presently
considered the preferred embodiments, the invention is not so limited. To the
contrary, the invention is intended to cover various modifications and
equivalent
arrangements included within the spirit and scope of the detailed description
provided above.
