Note: Descriptions are shown in the official language in which they were submitted.
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A FORMULATIONS COMPRISING OMEGA 3 FATTY ACIDS AND AN ANTI OBESITY AGENT FOR
THE
REDUCTION OF BODY WEIGHT IN CARDIOVASCULAR DISEASE PATIENTS (CVD) AND
DIABETICS
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of the priority date of U.S.
Provisional
Application 61/457,269, filed on February 16, 2011, the contents of which is
incorporated
herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to combinations of one or more anti-obesity
drugs with
mixtures of omega-3 fatty acid compositions designed to mediate omega-3
deficiencies in
individuals in need thereof; and to methods of administering such
combinations, and to
unit dosages of such combinations for the reduction of body weight in
cardiovascular
disease patients (CVD) and diabetics. The invention particularly relates to
compositions
wherein the omega-3 formulation is designed to mediate omega-3 deficiencies in
individuals in need thereof; particularly to compositions containing specific
ratios of highly
purified long chain fatty acid compositions which are effective in elevating
omega-3 levels
to a point at which the risk factors for cardiovascular disease are mitigated,
and most
particularly to a composition having an EPA:DHA ratio and level of omega-3
purity which
enable them to be effective in providing a sustained vasodilatory effect,
defined as a
vasodilatory effect lasting at least 6 hours.
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BACKGROUND OF THE INVENTION
[0002] In accordance with the findings of the U.S. 2005 Dietary
Guidelines
Advisory Committee, 70% of Americans are omega-3 fatty acid deficient. Further
studies
indicate that over 84% of CVD patients are deficient in omega-3 fatty acids,
specifically
Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosapentaenoic
acid
(DPA).
[0003] Cardiovascular disease (CVD) represents the primary cause of
mortality for
men and women in developed countries globally. These premature deaths come at
great
cost to both the individuals and their families, as well as representing a
huge burden to the
health care system of the country. The risk factors for cardiovascular disease
are well-
recognized and include: higher than average serum cholesterol, elevated levels
of LDL; a
low level of HDL in proportion to the LDL level; higher than average serum
triglycerides;
and higher levels of lipid oxidation products and inflammatory processes
creating plaques
and streaks which cause blockages of coronary arteries. An additional risk
factor for
cardiovascular disease and stroke is high blood pressure. Reduction in these
risk factors is
effective to reduce the prevalence of CVD and its many costs.
[0004] Although in some cases, genetic predisposition contributes to
CVD incidence, poor diet and sedentary lifestyle are major factors that
contribute
to increased risk for the development, and progression of CVD. Because of
this,
clinical management of CVD often includes an attempt to modify a patient's
lifestyle
to increase exercise, and incorporate a balanced diet, rich in omega-3 fatty
acids.
Due to non-compliance, and often an inability of a patient to adhere to
lifestyle
modifications, optimal patient care is not achieved through these efforts
alone, and
other therapeutic interventions or strategies must be considered.
[0005] Treatment options may include lipid-regulating medications, such
as
statins, or fibrates that act to lower low density lipoprotein (LDL)
cholesterol and/or
triglycerides (TG), metabolic components that are thought to contribute to
atherosclerotic
plaque buildup, and increase the risk for heart attack or stroke. However,
many of
these treatment options come with unwanted side effects that could add
additional
health risks, or cause physical discomfort.
[0006] A condition which often exists at the same time as omega-3
deficiency in a
patient population displaying risk factors for CVD is obesity. Obesity has
reached
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epidemic proportions in many of the world's industrialized nations, and a
majority of the
patient population fails to respond to single-agent therapy. Therefore, there
is a recognized
need for combination therapies, particularly in an obese patient population
suffering from
obesity, type II diabetes, hypertension, metabolic syndrome, as well as risk
factors for
cardiovascular disease.
[0007] Omega-3 fatty acids are natural polyunsaturated fats found in sea
foods like
fish and which are presently also available as dietary supplements. They
contain more than
one double bond in the aliphatic chain. They are named according to the number
(>1),
position and configuration of double bounds. The three major types of omega-3
fatty acids
are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and
docosahexaenoic acid
(DHA). These omega-3 polyunsaturated fatty acids have been shown to protect
against
several types of cardiovascular diseases such as myocardial infarction,
arrhythmia,
atherosclerosis, and hypertension (Abeywardena and Head, 2001; Kris-Etherton
et al.,
2003). It is widely accepted that (EPA) (C20:5n-3) and (DHA) (C22:6n-3) are
the major
biological active polyunsaturated fatty acids contributing to the prevention
of a variety of
cardiovascular disorders by improving endothelium-dependent vasodilatation and
preventing activation of platelets. Fish oil, EPA and DHA have been shown to
induce
relaxation and inhibit contraction by mechanisms which are endothelium-
dependent
(Shimokawa et al., 1987; Yanagisawa and Lefer, 1987). High contents of omega-3
polyunsaturated fatty acids, especially EPA, inhibited platelet aggregation
and increased
bleeding time, presumably due to a reduced generation of thromboxane A2.
Previous
studies have also shown that dietary supplementation with cod-liver oil
purified omega-3
fatty acids potentiated endothelium-dependent relaxations in isolated porcine
coronary
arteries (Shimokawa et al., 1988).
[0008] If a combination therapy comprising a novel omega-3 formulation in
combination with one or more anti-obesity agents could be provided which
alleviated
symptoms of obesity while simultaneously enhancing the patient's lipid profile
and
mitigating the various risk factors for cardiovascular disease, a long felt
need would be
realized.
SUMMARY OF THE INVENTION
[0009] The prior art fails to disclose a pharmaceutical formulation as
set forth in
the instantly disclosed invention, containing about 90% or greater omega 3
fatty acids by
weight having a combination of Eicosapentaenoic acid (EPA) and Docosahexaenoic
acid
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(DHA) in a weight ratio of EPA: DHA of from 5.7 to 6.3, wherein the sum of the
EPA,
DHA and DPA is about 82% by weight of the total formulation and about 92% of
the total
omega 3 fatty acid content of the composition. EPA + DHA are about 80% of the
total
formulation and about 89% of the total omega 3 fatty acid content of the
composition. The
prior art further fails to disclose an omega 3 composition as described above
in
combination with an anti-obesity agent.
[0010] It is noteworthy that tailoring the ratios, content and purity of
omega fatty
acid formulations provides the skilled artisan with a significant set of
specific parameters,
whereby formulations having a desired utility or pharmacological action may be
derived.
[0011] The present inventors have discovered that the ability of omega-3
fatty acid
preparations to cause endothelium-dependent relaxations depends on their
relative content
of EPA and DHA, as well as the purity of the overall formulation and the
presence of
additional key omega-3 fatty acids, particularly DPA.
[0012] Indeed, formulations in accordance with the present invention
having an
EPA:DHA ratio of about 6:1 induced significantly greater relaxations than an
EPA:DHA
1:1 preparation despite their similar content of omega-3 fatty acids. These
findings also
suggest that EPA is likely to be a more potent endothelium-dependent
vasorelaxant agonist
than DHA. The fact that the two major omega-3 fatty acids do not have similar
biological
activity to cause endothelium-dependent relaxation is important since the
leading
commercial omega-3 preparation (Lovaza ,GSK, Middlesex, UK) has a ratio of
EPA:DHA 1.2:1. Thus, the optimization of the ratio of EPA:DHA in omega-3
preparations
may provide new products with an enhanced vascular protective potential.
[0013] The present invention provides a novel composition, which may be
incorporated into an orally administered formulation for the reduction of risk
factors
associated with CVD, and a novel treatment method. A composition of the
formulation of
the invention may be used orally to treat and/or prevent risk factors of CVD
and stroke,
including reduction of high blood pressure and improving overall lipid
profiles, e.g. low
density lipoprotein (LDL), high density lipoprotein (HDL) and triglycerides.
While not
wishing to be bound by theory, the inventors believe that the compositions
work by acting
at different sites and aspects of cardiovascular disease. The compositions of
the present
invention are preferably presented for administration to humans and animals in
unit dosage
forms, such as tablets, capsules, pills, powders, granules, and oral solutions
or suspensions
and the like, containing suitable quantities of an active ingredient.
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[0014] The present invention also provides methods of treatment, for
example
administering to a patient having an omega-3 fatty acid deficiency, that may
be evidencing
one or more risk factors for CVD, a therapeutically effective amount of a
formulation in
accordance with the invention to achieve a therapeutic level of omega-3;
whereby
mitigation of said one or more risk factors for CVD is achieved. In
embodiments, the
invention is also a method for providing a sustained vasodilatory effect in a
patient by
administering a therapeutically effective amount of a formulation in
accordance with the
invention, whereby an indomethacin-independent sustained vasodilatory effect
is achieved.
[0015] By providing a method of treatment for mediating omega-3
deficiencies, use
of the instant invention to improve the health of the heart and to reduce risk
factors
associated with cardiovascular disease by delivering to an individual the
composition of
the invention is realized. Delivery of the composition of the invention, e.g.,
by oral
administration, has been shown to be useful for preventing oxidation of low
density
lipoprotein (LDL), increasing high density lipoprotein (HDL), and for reducing
total
cholesterol. Delivery of the composition of the invention is also useful for
reducing
triglycerides and reducing homocysteine. Desirably, the compositions of the
invention are
formulated such that an effective amount is delivered by multiple tablets (or
other suitable
formulation) a day. Suitably, these doses may be taken with meals, mixed into
food, or
taken on an empty stomach. Generally improvement is observed after two to
eight weeks
of daily use. Optionally, the compositions of the invention may be delivered
daily in a
suitable form (e.g., a chewable treat or bar). Other suitable dosage regimens
may be readily
developed by one of skill in the art. Such dosage regimens are not a
limitation of the
invention. The compositions of the present invention, in addition to their use
in treating
CVD in humans, may also be useful in treating non-human animals, particularly
mammals.
For example, these dietary supplements may be useful for companion animals
such as dogs
and cats, for cattle, horses, and pigs, among other animals.
[0016] A composition and method are taught which are useful in the
treatment of
obesity in cardiovascular patients, those having impaired glucose tolerance,
impaired
fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension,
hyperuricacidemia, gout, coronary artery disease, myocardial infarction,
metabolic
syndrome, hyperlipidemia, coronary heart disease, heart arrhythmias,
cerebrovascular
disease, stroke, peripheral vessel disease, sleep apnea and diabetics who are
at risk of
cardiovascular, cardiac and vascular events. The anti-obesity agents that are
useful in
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treating obesity are agents that affect energy expenditure, glycolysis,
gluconeogenesis,
glucogenolysis, lipolysis, fat absorption, fat storage, fat excretion, hunger
and/or satiety
and/or craving mechanisms, appetite/motivation, food intake, or G-I motility.
[0017] A primary objective of the instant invention is to teach
combinations of one
or more anti-obesity drugs with mixtures of an omega-3 fatty acid formulation
containing a
minimum of 90% omega 3 fatty acids by weight comprised of a combination of
Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) in a weight ratio
of EPA:
DHA of from 5.7 to 6.3, wherein the sum of the EPA, DHA and DPA comprise about
82%
by weight of the total formulation and about 92% of the total omega 3 fatty
acid content of
the composition. EPA + DHA are about 80% of the total formulation and about
89% of
the total omega 3 fatty acid content of the composition. The fatty acids of
the present
invention are understood to include biologically active glyceride forms, e.g.
triglycerides,
biologically active ester forms, e.g. ethyl ester forms, and biologically
active phospholipid
forms, their derivatives, conjugates, precursors, and pharmaceutically
acceptable salts and
mixtures thereof It is understood that the combination of omega-3 formulation
and anti-
obesity drug may be provided as a single unit dosage form, or as separate and
distinct unit
dosage forms.
[0018] A further objective of the instant invention is to teach methods
of
administering such combinations, and unit dosages of such combinations for the
reduction
of body weight in cardiovascular disease patients (CVD) and diabetics.
[0019] It is a further objective of the instant invention to provide a
method and
system for its practice to mediate omega-3 deficiency in patients having a
need therefore,
while assisting such patients in the loss of excess body weight.
[0020] It is yet an additional objective of the instant invention to
provide a novel
combination therapy for assisting such patients in the loss of excess body
weight while
providing a novel omega-3 containing formulation capable of providing a
sustained
vasodilatory effect.
[0021] These and other advantages of this invention will become apparent
from the
following description taken in conjunction with any accompanying drawings
wherein are
set forth, by way of illustration and example, certain embodiments of this
invention. Any
drawings contained herein constitute a part of this specification and include
exemplary
embodiments of the present invention and illustrate various objects and
features thereof.
All examples are illustrative and non-limiting in view of the disclosure.
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BRIEF DESCRIPTION OF THE FIGURES
[0022] Figure 1 illustrates the study design for the VASCAZENTM open
label
study;
[0023] Figure 2 is a plot of improved whole blood EPA + DHA + DPA levels
baseline to week 6;
[0024] Figure 3 illustrates the normal distribution curves for Groups A-C
during
the Open Label Study;
[0025] Figure 4 illustrates the effect of differing EPA:DHA ratios on the
relaxation
of coronary artery rings with and without the presence of the endothelium;
[0026] Figure 5 discloses the relaxation effect of an EPA:DHA 6:1 control
versus
the effect of eNOS and EDHF inhibitors;
[0027] Figure 6 discloses how the presence of Src kinase and P13-kinase
impacts
the relaxation effect of an EPA:DHA 6:1 product;
[0028] Figure 7 illustrates the shift in relaxation effect of an EPA:DHA
6:1 product
by membrane permeant analogues;
[0029] Figure 8A illustrates the effect EPA:DHA 6:1 has on both Akt and
eNOS
phosphorylation;
[0030] Figure 8B illustrates Western Blot Data Showing Sustained eNOS
Activation of Vascazen at 6 hours at a Concentration of 0.4 % and 40 1.tg of
Protein;
[0031] Figure 9 demonstrates the relation of purity to the sum of EPA +
DHA
relative to total Omega-3 ratios on the relaxation of coronary artery rings in
the presence or
absence of endothelium;
[0032] Figure 10 illustrates that the relaxation effect of the subject
EPA:DHA 6:1
formulation is insensitive to the presence of indomethacin;
[0033] Figure 11A and Figure 11B illustrate the indomethacin sensitivity
of the
relaxation effect of the subject EPA:DHA 6:1 formulation relative to several
over the
counter Omega-3 products;
[0034] Figure 12 illustrates the indomethacin sensitivity of the
relaxation effect of
the EPA:DHA 6:1 formulation relative to a formulation of like ratio containing
certain
additives;
[0035] Figure 13 illustrates the comparative vasorelaxing effect of
EPA:DHA 6:1
according to the present invention as compared to EPA:DHA 1:1, EPA alone and
DHA
alone;
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[0036] Figure 14 illustrates the mechanism by which EPA:DHA 6:1
stimulates the
endothelial formation of NO via the redox-sensitive activation of the
Phosphoinositide 3-
Kinase (PI3-Kinase)/ Protein Kinase (Akt) pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention provides a combination therapy and a method
for its
use in the treatment of obesity in cardiovascular patients, and those
experiencing impaired
glucose tolerance, impaired fasting glucose, insulin resistance syndrome,
dyslipidemia,
hypertension, hyperuricacidemia, gout, coronary artery disease, myocardial
infarction,
metabolic syndrome, hyperlipidemia, coronary heart disease, heart arrhythmias,
cerebrovascular disease, stroke, peripheral vessel disease, sleep apnea and
diabetics who
are at risk of cardiovascular, cardiac and vascular events. The anti obesity
agents that are
useful in treating obesity are selected from those which affect at least one
mechanism of
action selected from the group consisting of energy expenditure, glycolysis,
gluconeogenesis, glucogenolysis, lipolysis, fat absorption, fat storage, fat
excretion,
hunger, satiety, craving mechanisms, appetite, food intake, gastrointestinal
motility, and
combinations thereof
[0038] In a particular embodiment, the method of treatment is directed
toward anti-
obesity agents which may be selected from a group including active compounds
(antagonists or inverse agonists) against the receptor product of the
cannabinoid 1 (CB1)
gene; cathespsin K inhibitors; PYY; PYY3-36; a PYY agonist; 5HT transporter
inhibitor;
NE transporter inhibitor; ghrelin antagonist; H3 antagonist/inverse agonist;
MCH1R
antagonist; MCH2R agonist/antagonist; MC3R agonist; NPY1 antagonist; NPY4
agonist;
NPY5 antagonist; leptin; leptin agonist/modulator; leptin derivatives; opioid
antagonist;
orexin antagonist; BRS3 agonist; 11.beta. HSD-1 inhibitor; CCK-A agonist;
CNTF; CNTF
agonist/modulator; CNTF derivative; Cox-2 inhibitor; OHS agonist; 5HT2C
agonist; CB1
antagonists; neuropeptide Y5, appetite suppressants; lipase inhibitors; 5HT6
antagonist;
monoamine reuptake inhibitor; UCP-1, 2, and 3 activator; 133 agonist; thyroid
hormone
.beta. agonist; PDE inhibitor; FAS inhibitor; DGAT1 inhibitor; DGAT2
inhibitor; ACC2
inhibitor; glucocorticoid antagonist; acyl-estrogens; lipase inhibitor; fatty
acid transporter
inhibitor; dicarboxylate transporter inhibitor; glucose transporter inhibitor;
serotonin
reuptake inhibitors; aminorex; amphechloral; amphetamine; axokine;
benzphetamine;
chlorphentermine; clobenzorex; cloforex; clominorex; clortermine;
cyclexedrine;
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dextroamphetamine; diphemethoxidine, N-ethylamphetamine; fenbutrazate;
fenisorex;
fenproporex; fludorex; fluminorex; furfurylmethylamphetamine; levamfetamine;
levophacetoperane; mefenorex; metamfepramone; methamphetamine; nalmefene;
norpseudoephedrine; pentorex; phendimetrazine; phenmetrazine; phytopharm
compound
57; picilorex; topiramate; zonisamide; IGF-IR antagonists; MetAP2 modulators;
Alpha-
Arrestin ARRDC3 Modulators; Single Minded 1 (SIM1) modulator; Methionine
Aminopeptidase 2 (MetAP2); Sirtuin 1(SIRT1) modulators; or any combinations
thereof
[0039] The ability of endocannabinoid receptor blockers to attenuate
weight loss
has led scientists to explore their utility as anti-obesity drug candidates. A
concerted effort
to develop drugs that target this receptor complex by pharmaceutical companies
have
resulted in multiple anti-obesity drug candidates. These include Sanofi-
Aventis'
ACOMPLIA (RIMONIBANT) chemical formula 5-(4-Chloropheny1)-1-(2,4-dichloro-
pheny1)-4-methyl-N-(piperidin-1-y1)-1H-pyrazole-3-carboxamide, Merck's
TARANABAN
T chemical formula N-[(lS,2S)-3-(4-Chloropheny1)-2- , and Pfizer's CP-945,598,
chemical formula C25H25C12N70.1-1C1 , all CB1R blockers that reached, and
failed phase III
clinical trails. These trials failed in the late stages of development, due to
psychiatric
adverse reactions, including severe depression and anxiety.
[0040] During phase III clinical evaluation of these drugs, it was
discovered that
patients with a predisposition to depression, anxiety, or other mood
disorders, were most
susceptible to adverse events with RIMONIBANT treatment, suggesting that
blockade of
the CB1R complex and natural endocannabinoid regulation exacerbates pre-
existing
conditions. Patients that did not present with psychological health issues
were less
susceptible to adverse events of this nature.
[0041] During their analyses with respect to combining CB1 receptor
blockers with
the novel omega-3 formulation of the present invention, the inventors were
sensitive to the
fact that the presence of anxiety and major depressive disorders were often
linked to
omega-3 fatty acid nutritional deficiency. They noted that correcting this
deficiency with
high purity prescription omega-3 fatty acids often reduces or alleviates these
disorders.
[0042] Endocannabinoids, produced by the body, serve to regulate mood by
binding to the CB1R, Gi/Go receptor complex. Drugs, like RIMONIBANT and others
that
block the interaction of endocannabinoids to their receptors, interrupt this
regulatory
mechanism, putting patients at risk for unstable mood, and depressive
disorders.
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[0043] The cannabinoid-1 receptor (CB 1R), G(i/o) receptor complex is
regulated
by omega-3 and chronic omega-3 deficiency affects presynaptic neuronal
functions
through modulation of the CB1R, G(i/o) receptor complex directly. With omega-3
deficiency, strong interaction that is normally observed in this receptor
complex is
weakened. Ultimately, G(i/o) becomes uncoupled from the receptor complex,
limiting
endocannabinoid signaling capacity through CB1R and resulting in unstable
mood. While
not wishing to be bound to any particular theory or mechanism of action, the
present
inventors propose that by correcting omega-3 deficiency, the CB1R receptor
complex
would become stabilized, in turn, improving endocannabinoid mood-stabilizing
effects.
Therefore, patients predisposed to depressive episodes in the clinical studies
of
RIMONIBANT, or similar CB1R-targeting therapies could benefit from omega-3 pre-
, and
ongoing treatment, in combination with RIMONIBANT, or similar drugs, as an
adjunct
therapeutic approach. These patients would be better candidates for the safe
administration
of RIMONIBANT, or related drugs.
[0044] With respect to the omega-3 component of the combination therapy
and
method of its use, the present invention provides a long chain fatty acid
composition that
includes a formulation containing a minimum of about 90% omega 3 fatty acids
by weight
having a combination of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid
(DHA)
in a weight ratio of EPA: DHA of from 5.7 to 6.3, wherein the sum of the EPA,
DHA and
DPA is about 82% by weight of the total formulation and about 92% of the total
omega 3
fatty acid content of the composition. EPA + DHA are about 80% of the total
formulation
and about 89% of the total omega 3 fatty acid content of the composition. The
fatty acids
of the present invention are understood to include biologically active
glyceride forms, e.g.
triglycerides, biologically active ester forms, e.g. ethyl ester forms, and
biologically active
phospholipid forms, their derivatives, conjugates, precursors, and
pharmaceutically
acceptable salts and mixtures thereof.
[0045] The pharmaceutical formulation of the instant invention is
contemplated as
being administered in amounts providing a daily dosage of 1 to 4 gm of said
formulation.
The pharmaceutical formulation at such dosage level being effective for the
treatment or
prophylaxis of risk factors of cardiovascular disease and the protection
against sudden
death in patients with CVD.
[0046] Pharmaceutical formulations of the instant invention may be
provided
wherein a unit form is a gel or liquid capsule.
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[0047] An exemplary unit dosage form includes from about 645 to about 715
mg/gm EPA, for example about 680 mg/gm EPA and from about 105 to 115 mg/gm,
for
example about 110 mg/gm DHA. The unit dosage can include from about 22 to
about 28
mg/gm DPA for example about 25 mg/gm DPA. Unit doses may additionally include
a
stabilizer, e.g. tocopherol in amounts up to about 0.5 %, for example about
0.15% to about
0.25% or about 0.2% by weight. The effective unit dosage is generally 3 gm to
4 gm of the
pharmaceutical formulation which are provided daily to CVD patients in one or
more unit
doses, for example about 3 - 4 one gram capsules per day. As set forth below,
one or more
optional ingredients can be included in the formulations. Such ingredients may
be
separately added or may be components of the source from which the omega 3
fatty acids in
the formulation are derived.
[0048] In some embodiments, the formulation may further contain about 30
mg/gm
of arachidonic acid (AA). In some embodiments, the formulation may further
contain up to
about 5%, for example about 3% or about 30 mg/gm of arachidonic acid (AA). It
has been
discovered that aspirin-acetylated COX-2 is also able to convert Omega-6 AA
through
lipoxygenases (LOX) to lipoxins (LXs), which are potent anti-inflammatory
mediators
(Nature Chemical Biology, Vol. 6, June 2010, Pp 401-402).
[0049] Some embodiments of the formulation contains >2%, for example >3%,
of
18 carbon Omega-3 fatty acids, either individually or in total. Exemplary 18
carbon atom
omega-3 fatty acids include alpha-linolenic acid (ALA) and Stearidonic acid
(SDA), either
alone or in combination. Studies have shown that the presence of 18 carbon
Omega-3s,
such as ALA elicit anti-inflammatory effects (Zhao et al, Am J Clin Nutr 2007;
85:385-
91).The composition is formulated with a specific amount of DHA consisting of
about 400
mg per daily dose.
[0050] The composition can contain additional fatty acids in lesser
amounts, usually
less than about 1% of each that is present. Exemplary embodiments contain
about 0.3-
0.7%, or about 5% of any of the additional fatty acids, These additional fatty
acids can
include, for example, omega -6 fatty acids such as Dihomo-gamma-linolenic acid
(DGLA;
20:3n6), Docosapentaenoic acid (Osbond acid; 22:5n6); omega-9 fatty acids such
as Oleic
acid (18:1n9) and others such as 7,10,13,15-hexadecatetraenoic acid and
(16:4n1),
9,12,15,17-octadecatetraenoic acid (18:4n1). Other fatty acids may be present
in higher
quantities. For example, Eicosatetraenoic acid (ETA; 20:4n3) may be present in
amounts
up to about 2%, for example about 1.5%, and Heneicosapentaenoic acid (HPA;
21:5n3)
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may be present in amounts up to about 3%, for example at about 2.3%. These
additional
fatty acids may be added separately or may be present in formulations obtained
from
particular sources using particular methods. Other additional components and
fatty acids
may also be present in small amounts, for example 0-0.25% of the formulation.
[0051] The composition is formulated with a DHA content to provide about
400
mg per daily dose.
[0052] Daily administration of the formulation can reduce the level of
triglycerides
(TG) and increases high density phospholipids (HDL) levels in CVD patients.
[0053] A highly potent omega-3 formulation in accordance with the present
invention is marketed by Pivotal Therapeutics, Inc., under the trade name
VASCAZENTM, to alleviate the cardiovascular risks associated with omega-3
deficiency. VASCAZENTM, has been formulated for the dietary management of
omega-3
deficiency in patients with CVD, providing EPA and DHA to levels not
attainable
through normal dietary modifications. More specifically, the VASCAZENTM
product
exemplifies the present invention in being composed of about 90% or more omega-
3 fatty
acids at a ratio of eicosapentaenoic acid (EPA) to docosahexaenoic acid (DHA)
within the range of 5.7:1 ¨ 6.3:1, respectively. The formulation contains
about
680mg/g of EPA, about 110mg/g of DHA, and about 25 mg/g of DPA per
capsule. Each capsule has a total weight of about 1000 mg. his generally
contemplated that a daily regimen of VASCAZENTM includes 4 tablets per day
given either in one dose or in separate doses throughout the day. With respect
to a
1000 mg fill, the formulation contains at least about 90% or more omega-3
fatty
acids, wherein about 80% is the sum of EPA+DHA, and about 82% the sum of
EPA + DPA + DHA. Embodiments can also contain about 30mg/g of
arachidonic acid, an omega-6 fatty acid, and/or >3% of 18 carbon Omega-3 fatty
acids.
[0054] The levels of low density lipids (LDL), HDL and TG are monitored.
[0055] According to a US study, and the Dietary Guidelines Committee, 70%
of Americans are omega-3 deficient due to lack of consumption of this
essential
nutrient in the typical "western diet", which includes an overabundance of pro-
inflammatory omega-6 fatty acid intake, by comparison. In patients with CVD,
this
dietary trend can be particularly dangerous. Coupled with other
cardiometabolic risk
factors, omega-3 deficiency further exacerbates the chronic progression of
this
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disease. A growing body of evidence has demonstrated the cardiovascular health
risks associated with chronic omega-3 deficiency. A dietary deficiency of EPA
acid
and DHA in particular, allows for downward pro-inflammatory pressures created
by the
metabolism of arachidonic acid (AA) that is typically very high in the diets
of most
Americans. Overall, omega-3 fatty acid deficiency contributes to a pro-
inflammatory
state, the consequences of which include negative effects on cardiovascular
health,
including increased risk for development of dyslipidemia (high cholesterol,
high
triglycerides), atherosclerotic plaque buildup, hypertension, and cardiac
arrhythmia.
[0056] Chronic omega-3 deficiency can subsequently lead to increased risk
for
suffering a fatal heart attack. Maintenance of blood levels of EPA, DHA and
DPA
above 6.1% of total blood fatty acids, compared to levels between 2.1% - 4.3%
is
associated with an 80.0% lower risk of sudden cardiac death. To counterbalance
the cardiovascular risks associated with an overabundance of AA, and the pro-
inflammatory influences upon this metabolic pathway, one would need to
increase
EPA and DHA consumption to levels that can not be attained through dietary
changes alone. Filling the "omega-3 nutritional void" thus requires
additional supplementation with a highly potent EPA and DHA formulation, which
provides high levels of EPA, as well as DHA, for full clinical benefit,
removing a key
risk factor in patients with CVD.
[0057] In an open label study to analyze the safety and efficacy of
VASCAZENTM, whole blood omega-3 fatty acid levels were examined in 143
patients, and the inventive formulation was administered to patients for two
or six-week
follow-ups, providing about 2800 mg/day EPA and about 480 mg/day DHA. The
primary outcome measure was the change in the sum of blood EPA+DHA+DPA.
levels (the Omega-ScoreTm), expressed as a percentage of total blood fatty
acid levels
over a two or six-week duration.
[0058] The normalized baseline Omega-ScoreTM was 3.4% (N=143). In the
two-week and six-week treated groups, the inventive formulation increased
Omega-
ScoreTM levels by 52.8% (N= 63, p = <0.0001) and 120.6% (N = 31, p = <0.0001)
respectively, compared to baseline levels measured in each group. After six
weeks of
intervention, maximal, and stable levels were maintained at an average score
of 7.5%.
The formulation in accordance with the present invention was generally well
tolerated,
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with only minor adverse events reported in a small proportion of study
participants. (See Table 4)
METHODOLOGY:
[0059] The 6-week open label study was conducted at a single site in
Canada.
Subjects were eligible for the study if they met all inclusion and exclusion
criteria set
out in the clinical study protocol. All eligible subjects provided informed
consent
prior study enrollment, and entered Group A (Figure 1.). Sixty three subjects
were
provided 4 capsules per day of VASCAZENTM (Group B), an oral dose of 2720mg
EPA and
440mg DHA per day. After two weeks of treatment, whole blood omega-3 blood
level
was assessed, and 31 subjects entered into Group C, for continued treatment.
Group C
subjects provided whole blood samples at weeks 4 and week 6, for follow-up
Omega-ScoreTM assessment.
[0060] The primary outcome measure was the change in Omega-ScoreTM values
expressed as a percentage of total blood fatty acid levels over a 2-week
period for Group
B, and 6-week period for Group C. The baseline Omega-ScoreTM value for Group A
was
calculated as the mean percentage at week 0, prior to VASCAZENTM intervention,
and
Groups B and C Omega-ScoreTM means were evaluated at the specified time points
accordingly.
[0061] The study included both men and women >15 years of age, in stable
medical condition. Exclusion criteria included the following: A history of
ventricular arrhythmia, known bleeding or clotting disorder, liver or kidney
disease, autoimmune disorder or suppressed immune systems, seizure disorder or
taking anticonvulsant medication; allergies to fish; or subjects with an
implantable
cardioverter defibrillator. Medical histories, and current medications were
also
documented.
[0062] Laboratory analysis of total blood fatty acids in whole blood was
conducted by a central laboratory, (University Health Network Laboratory,
Toronto,
Ontario), accredited by the College of American Pathologists' Laboratory
Accreditation Program. Analysis was carried out by derivatizing fatty acids
into
methyl esters followed by Gas Chromatography-Mass Spectrometry (GC-MS)
analysis
(Agilent Technologies 6890N series gas chromatograph, 5975C detector,
Mississauga, Ontario). Fatty acids were extracted from 200 !IL of whole blood
sample
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using a mixture of methanol and chloroform. Fatty acids were then methylated
with
10% (w/v) BC13 in methanol by incubation at 90 C for 25 mm to form fatty acid
methyl
esters (FAMEs). After cooling the FAMEs were extracted with water/hexane
mixture and 1
uL of n-hexane extract was injected for GC-MS analysis.
[0063] Sample size was justified accordingly. Assuming a mean baseline
level of blood Omega-ScoreTM levels of at least 3.0% and a standard deviation
in
change of blood Omega-ScoreTM levels of 1.8% in the study population, the
minimum
sample size of 63 study subjects would result in a minimum power of 90.2% to
detect an increase in blood Omega-ScoreTM levels following 2 weeks of study
intervention of at least 25.0% relative to baseline, at a significance level
of a = 0.05.
The minimum sample size of 30 subjects taking VASCAZENTM for six weeks would
result in a minimum power of 94.2% to detect an increase in blood Omega-
ScoreTM
levels following 6 weeks of study intervention of at least 40.0% relative to
baseline,
at a significance level of a = 0.05. The safety population was defined as a
patient
group that had a minimum of 2 weeks and maximum of 6 weeks VASCAZENTM, at a
dose of 4 capsules per day. Primary analyses of treatment efficacy was
performed on
the subset of enrolled study subjects for whom blood measurements were taken
at
baseline and after 2 weeks of study treatment. The change in blood Omega-
ScoreTM
levels over the 2-week period (expressed as a percentage change from baseline)
was
computed for each study subject. The distribution of changes in blood Omega-
ScoreTM levels over 2 weeks were tested for normality using the Pearson-
D'Agostino test. A paired t-test was conducted in order to test the change in
blood
Omega-ScoreTM levels over the 2-week period.
[0064] Secondary analyses of treatment efficacy was performed on the
subset of enrolled study subjects for whom blood Omega-ScoreTM levels were
taken at
baseline and at time points of 2 weeks, 4 weeks and 6 weeks following
baseline. An
analysis of variance (ANOVA), utilizing subjects as blocks, was conducted to
test the
change in blood OmegaScoreTM levels between any pair of time points over the 6-
week period; multiple comparisons were conducted at a family-wide significance
level of a = 0.05 in order to determine which pairs of time points (if any)
differ
significantly in terms of mean blood EPA + DHA + DPA levels. A linear contrast
was
carried out in order to test the hypothesis that mean blood EPA + DHA + DPA
levels increase linearly within this subset of study subjects over the 6-week
period.
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PCT/US2012/025014
RESULTS:
[0065] Baseline characteristics of each study group are outlined in Table
1.
Across all groups, age demographics were comparable, with the majority of
study
participants being middle-aged. Within group A, the mean age of the total
group
(N=143), consisting of mostly males (74.1%), was 50.9 years, and similar age
distributions were observed between men (52.1), and women (46.9). Group B
(N=63,
74.2% men), the two-week treatment group, had a mean age of 53.7, with
comparable
mean ages between men (55.8) and women (47.9). Finally, study subjects within
group C (N=31, 87% men) had a mean age of 55.0 years (men, 54.0; women, 61.5).
Baseline OmegaScoreTM values were measured and all three groups, including men
and
women were found to have comparable, omega-3 deficient (defined as less than
6.1% Omega-Score)(N Engl J Med, Vol. 346, No. 15, April 11, 2002, Pp. 1113-
1118), scores between 3.3% and 3.8%.
Table 1- Baseline Characteristics*:
Grou = A
Characteristic Men (N=106) Women (N=37) Total (N=143)
Age, mean (SD) (years) 52.11.3.6 46.9 15.0 50.9 14.6
*Omega-Scorelm(%)
Mean 3.4 1.4 3.5 1.2 3.4 1.3
95%0 3.2 to 3.7 ( 1.1 to 1.6) 3.2 to 3.7 ( 1.0 to 1.4)
3.2 to 3.6 ( 1.1 to 1.6)
Grou B
Characteristic Men (N=47) Women (N=16) Total (N=63)
Age, mean (SD) (years) 55.8 10.9 47.9 16.7 53.7 13.1
*Omega-Scorelm(%)
Mean 3.8 1.4 3.3 1.3 3.6 1.3
95%C1 3.4 to 4.1 ( 1.0 to 1.8) 2.9 to 3.7 (4.9 to 1.7)
3.2 to 3.9 ( 1.0 to 1.7)
Group C
Characteristic Men (N=27) Women (N=4) Total (N=31)
Age, mean (SD) (years) 54.0 8.7 61.5 11.0 55.0 9.2
*Omega-Scorelm(%)
Mean 3.7 1.2 N/A 3.4 1.2
95%0 3.3 to 4.0 ( 0.8 to 1.5) N/A 3.1 to 3.7 (
0.8 to 1.5)
Omega-ScoreTm calculated as the mean +/- SD (where N = number of subjects)
from a normal distribution of raw data.
Group C (women) did not have sufficient numbers to fit a normal distribution
curve. The mean baseline score of the raw
data for this group was 2.98%.
Results of the primary outcome measure are illustrated in Figure 2 and Table
2, and
calculated/fit to a normal distribution in Figure 3, Table 3. Baseline levels
of whole blood
omega-3 blood levels revealed an omega-3 deficiency (group A) in a large study
group
(N=143). Within this group, subjects had a mean score of 4.4%, or 3.4% (normal
distribution curve fit), representing 84.5% of individuals with scores below a
6.1% score
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cutoff, cardiovascular disease risk quartile. Study participants that received
VASCAZENTM
intervention for 2 weeks (group B) had a significant (P<0.0001) improvement in
their
scores (Figure 2, Table 2), with mean values improving from 3.6% to 5.5%
(Figure 3,
Table 3), a 52.8% score increase. Over two weeks of intervention, study
participants
considered "at risk" were reduced from 80.6% to 46.8% (Table 3). Over the
course of 6
weeks VASCAZENTM intervention, group C subjects had significant mean score
improvements (P<0.0001)(Figure 2, Table 2), with mean values improving from
3.4% to 7.5% between baseline and week 6 (Figure 3, Table 3), and representing
a
120.6% increase in whole blood levels of EPA+DHA+DPA Omega-ScoreTM values.
After 6 weeks of VASCAZENTM intervention, 13.2% of participants remained at
higher
risk (<6.1% Omega-ScoreTI"), Table 3.
TABLE 2
Omega-ScoreTm Mean SD (%)
Group A (% change from baseline)
(N=143)
Baseline 4.4 1.7
Group B
(N=63)
Baseline 4.7 1.9
Week 2 6.7 1.9(52.8%)
Group C
(N=31)
Baseline 4.3 I.5
Week 2 6.4 2.1 (48.8%)
Week 4 8.6 2.4 (100.0%)
Week 6 8.2 2.0 (90.7%)
Table 2. Primary Outcome Measure: Change in the sum of blood EPA+DHA+DPA
levels expressed as a percentage
of total blood fatty acid levels over a two or six-week intervention
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TABLE 3
Omega-Scorinvi (%) % of Patients At Risk (<6.1%
Omega-
Group A Mean SD (% change 95%C1 Scorem)
(N=143) from baseline
Baseline 3.4 1.3 3.1 to 3.7 84.5%
( 0.9 to 1.4)
Group B
(N=63)
Baseline 3.6 1.3 3.2 to 3.9 80.6%
( 1.0 to 1.7)
Week 2 5.5 1.6 5.1 to 6.0 46.8%
(52.8%) ( 1.2 to 2.1) (-33.8%)
Group C
(N=31)
Baseline 3.4 1.3 3.1 to 3.7 84.5%
( 0.9 to 1.4)
Week 2 5.7 1.9 5.4 to 6.3 43.2%
(67.6%) ( 1.4 to 2.3) (-41.3%)
Week 4 7.9 2.4 6.6 to 9.1 15.0%
(132.4%) ( 1.2 to 3.7) (-69.5%)
Week 6 7.5 1.2 7.0 to 8.0 13.2%
120.6%) ( 0.7 to 1.7 -71.3%
Table 3. Primary Outcome Measure: Change in the sum of blood EPA+DHA+DPA
levels expressed as a
percentage of total blood fatty acid levels over a two or six-week
intervention, and represented as a normal
distribution.
[0066] Patients with >6.1% (ideal) scores had an 80% less chance of death
from
sudden cardiac arrest, compared to individuals in the 2.1%-4.3% risk quartile
(score)
range. In this study, the mean baseline value of the study population
indicated that
84.5% of study participants, many of which with cardiovascular health issues,
on
statin, and/or blood pressure medication, had scores less than 6.1%, leaving
themselves
at greater risk for adverse events, especially in patients with known
dyslipidemia,
type 2 diabetes, and/or hypertension. After six weeks of VASCAZENTM
intervention,
71.3% of group C participants with previous baseline scores less than 6.1%
were able
to increase their score to a level above this threshold.
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TABLE 4
Adverse Event 2-6 Weeks Treatment Severity Relationship to
Description (N=63) Study Treatment
Reflux/ Aftertaste 2 Mild Definite
Minor Leg Bruising 1 Mild Unrelated*
*Minor bruising appeared after two weeks of treatment and disappeared within 3
days. The subject continued taking
VASCAZENTm for additional four week without any adverse event.
[0067] Group B study participant scores significantly increased
(P<0.0001) by
52.8% from 3.6% to 5.5%. With prolonged VASCAZENTM intervention, group C
individuals had significant score improvement over the course of 6 weeks
(P<0.0001,
ANOVA), with similar improvements as the group B individuals within two weeks.
After 4
weeks, VASCAZENTM significantly (P<0.0001) increased mean scores from 3.4% to
7.9%, representing a 132.4% improvement, bringing the mean score of the total
population
to well within the >6.1% low risk quartile. Indeed, only 15% of study
participants
remained below this benchmark level after 4 weeks, a level that is sustained
in the study
group through 6 weeks of VASCAZENTM intervention. VASCAZENTM was generally
well
tolerated with a low incidence, of minor adverse events that are typical for
omega-3
polyunsaturated fatty acid ethyl esters. This study has highlighted the
prevalence of
chronic omega-3 deficiency in the majority of people (84%), both men and
women.
[0068] The consequences of omega-3 deficiency in patients with CVD are
well
documented, with numerous studies linking EPA and DHA deficiency. Many
studies and current therapeutic approaches have categorized omega-3 as a
therapeutic agent for the treatment of symptoms that accompany CVD.
Unfortunately
the common thread of thought around omega-3 fatty acid therapy does not lead
to
optimal results. EPA and DHA should not be considered therapeutic agents,
rather, they should be considered essential nutrients, which should ideally be
consumed regularly as part of a healthy balanced diet. Omega-3 deficiency in
patients with CVD adds unnecessary risks, which can be avoided with suitable
omega-3 supplementation. The present invention as exemplified by VASCAZENTM
intervention provides essential balanced levels of EPA and DHA that are
difficult for
many CVD patients to incorporate into their daily diet through food alone. In
the
typical western diet, the average American consumes 15 times less omega-3
fatty acids
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from fish than what is required to attain and maintain clinically beneficial
levels of
EPA and DHA. In order to consume enough of this essential nutrient to provide
the
daily dose that the present invention can provide, one would have to eat fish
every
single day, for more than one meal per day. This is unrealistic for most
people.
[0069] The present study has demonstrated that maintenance of EPA+DHA+DPA
to levels >6.1% can be achieved with the present invention within 4 weeks of
intervention,
and that over 85% of patients can achieve these levels at a dose of 4 capsules
per day,
supplying about 2720mg EPA and 440mg DHA. These findings support the use of
omega-
3 fatty acid supplements according to the present invention for the
maintenance of
routinely measured (via Omega-ScoreTM assessment), clinically beneficial
EPA+DHA+DPA blood levels in patients with CVD.
SUSTAINED VASODILATORY EFFECT:
[0070] In addition to the benefits outlined above with respect to omega-3
supplementation for an omega-3 deficient patient population, formulations
according to
the invention have been shown to provide a sustainable eNOS vasodilatory
effect, defined
as a vasodilatory effect persisting for 6 hours or more, which has heretofore
not been
achievable with either prescription or OTC grade omega-3 supplements.
[0071] To understand this vasodilatory effect in the context of treatment
and
prevention of cardiovascular disease, it is first necessary to understand the
mechanism of
vasodilation via the endothelium lining of blood vessels.
[0072] The following list of Abbreviations will be relied upon for the
following
discussion.
ABBREVIATION LIST
Abbreviation Signification
[Cal i Intracellular free calcium concentration
APA Apamin
CaM Calmodulin
CaMK-2 Calmodulin kinase-2
cAMP Cyclic adenosine 3 ':5' monophosphate
cGIVIP Cyclic guanosine 3 ': 5' monophosphate
EDHF Endothelium-derived hyperpolarizing factor
eNOS Endothelial NO synthase
ET-1 Endothelin-1
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H202 Hydrogen peroxide
IKCa Calcium-dependent Intermediate conductance
Potassium Channels
Indo Indomethacin
L-NA N-co-nitro-L-arginine
MnTMPyP Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin
NO Nitric oxide
02 - Superoxide anion
PEG-Catalase Polyethylene glycol-catalase
PGI2 Prostacyclin 12
P13-K Phosphoinositide-3 kinase
PKC Protein kinase C
PP2 4-amino-5-(4-chloropheny1)-7-(t-butyl) pyrazolo [3,4]
pyrimidine
ROS Reactive oxygen species (Reactive Oxygen Species)
sGC Soluble guanylyl cyclase
SKCa Ca2+ -dependent small conductance potassium channels
SOD Conductance Superoxide dismutase
TRAM34 1-[(2-Chlorophenyl) diphenylmethyl]- 1H-pyrazole
TX A2 Thromboxane A2
U46619 9, 11-dideoxy-9-prostaglandin F2 methanoepoxy
[0073] The endothelium consists of a single endothelial cell layer lining
the luminal
surface of all blood vessels. Endothelial cells play an important function in
the regulation
of vascular homeostasis. They regulate the contact of blood with the
underlying
thrombogenic arterial wall. They respond to numerous physiological stimuli
such as
circulating hormones and shear stress by releasing several short-lived potent
endothelium¨
derived vasoactive factors such as nitric oxide (NO) and endothelium- derived
hyperpolarizing factor (EDHF), these two factors playing a major role in the
control of
vascular tone (Busse et al., 2002; Michel and Feron, 1997). In addition,
endothelial cells
can also generate prostacyclin (PGI2), a prostanoid causing relaxation of some
blood
vessels.
ENDOTHELIUM¨DERIVED NITRIC OXIDE (NO):
[0074] NO is produced by endothelial nitric oxide synthase (eNOS) from L-
arginine, NO plays critical roles in normal vascular biology and
pathophysiology. NO
induces relaxation of the vascular smooth muscle by activating soluble
guanylyl cyclase
resulting in the formation of cyclic guanosine 3'-5'monophosphate (cGMP). In
addition to
the regulation of vascular tone and inhibition of platelet aggregation, NO
also inhibits
many key steps involved in atherogenesis including vascular smooth muscle cell
proliferation, monocyte adhesion (Dimmeler et al., 1997; Hermann et al., 1997;
Tsao et al.,
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1996) and cell death. eNOS can be activated by receptor-dependent and
¨independent
agonists as a consequence of an increase in the intracellular concentration of
free Ca
([Ca2]0 and the association of a Ca2+/ calmodulin (CaM) complex with eNOS
leading to
its activation (Fleming et al., 2001). Indeed both the agonist-induced NO
formation and
subsequent vasorelaxation are abolished by the removal of Ca2+ from the
extracellular
space as well as by CaM antagonists. eNOS is also regulated in endothelial
cells at a post-
translational level primarily through protein/protein interactions and
multisite
phosphorylation at Ser116, Thr497, Ser635, and Ser1179 (residue numbers are
for the
bovine sequence, equivalent to Ser114, Thr495, Ser633, and Ser1177 in the
human
sequence (Bauer et al., 2003; Boo et al., 2002; Dimmeler et al., 1997).
Indeed, eNOS has
been shown to be regulated by the interaction with positive and negative
protein
modulators such as caveolin (Cav-1) and heat shock protein 90 (Garcia-Cardena
et al.,
1998; Ju et al., 1997; Pritchard et al., 2001). In the basal state, the
majority of eNOS
appears to be bound to caveolin-1 with its enzymatic activity being repressed
in the
caveolae (Michel et al., 1997). This tonic inhibition of eNOS can be released
by displacing
caveolin-1 with Ca2+/CaM in response to Ca2+ mobilizing agonists (Ju et al.,
1997). In
addition to these modulators, phosphorylation of eNOS at key regulatory sites
plays an
important a role in the regulation of enzyme activity in response to several
physiological
stimuli (Ju et al., 1997). It has been shown that phosphorylation of eNOS at
Ser1179 is
associated with increased enzyme activity (Gallis et al., 1999; McCabe et al.,
2000).
Phosphorylation of eN0S-Ser1179 is regulated by P13-kinase-dependent
mechanisms
(Gallis et al., 1999). Akt, one of the major regulatory targets of P13-kinase,
has been
shown to directly phosphorylate eNOS at Ser1179 and activate the enzyme in
response to
vascular endothelial growth factor (VEGF), sphingosine-l-phosphate, and
estrogens
(Dimmeler et al., 1997; Fulton et al., 1999). However, eN0S-Ser1179 can also
be
phosphorylated by AMP-activated protein kinase (Busse et al., 2002), protein
kinase A
(PKA), and protein kinase G (PKG). Exactly which protein kinase(s)
phosphorylates
eN0S-Ser1179 in intact cells appears to be dependent on the type of
endothelial cells and
stimuli. For example, shear stress phosphorylates eNOS Ser1179 by a P13-kinase-
and
PKA-dependent manner without involving Akt whereas EGF phosphorylates eNOS
Ser1179 by a P13-kinase- and Akt-dependent manner (Boo et al., 2002). In
addition, the
ischemia-reperfusion injury activates the PKA pathway leading to the
phosphorylation of
eNOS at Ser1179 and Ser635 (Li et al., 2010). In addition, the level of eNOS
expression
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can be modulated by several factors including shear stress (Butt et al.,
2000), hypoxia, low-
density lipoproteins (LDL) (Chen et al., 2008; Chen et al., 1999) and oxidized
fatty acids
(Corson et al., 1996).
ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR (EDHF):
[0075] Endothelium-dependent vasorelaxation has also been observed in
some
blood vessels following inhibition of NO and PGI2 synthesis and has been
attributed to
endothelium-derived hyperpolarizing factor (EDHF). EDHF relaxes blood vessels
through
hyperpolarization of the vascular smooth muscle. This effect will close
voltage-operated
Ca2+ channels resulting in reduction of the intracellular free Ca2+ level and
subsequent
relaxation of the vascular smooth muscle. Potassium (K+) channels underlie the
hyperpolarization induced by EDHF and involve intermediate conductance Ca2+-
activated
K+ (IKCa) channels and small conductance Ca2+ -activated K+ (SKCa channels).
In several
disease conditions including the presence of cardiovascular risk factors, the
endothelium
undergoes functional and structural alterations and it loses its protective
role, and becomes
proatherosclerotic (Vanhoutte, 1989).The loss of the normal endothelial
function is
referred to as endothelial dysfunction, which is characterized by impaired NO
bioavailability subsequent to a reduced generation of NO by eNOS and/or an
increased
breakdown of NO by reactive oxygen species (ROS) and, in particular,
superoxide anions
(Vanhoutte, 1989).
[0076] Previous studies by the present inventors have indicated that
natural
products such as Concord grape juice (Anselm et al., 2007) and red wine
polyphenols
(Ndiaye et al., 2005) activate the endothelial formation of NO by causing the
redox-
sensitiveSer/PI3-kinase /Akt pathway-dependent phosphorylation of eNOS at
Ser1177.
[0077] Fish oil omega-3 is a rich source of EPA and DHA. Omega-3 fatty
acids
have been shown to cause endothelium-dependent vasorelaxation in vitro in rat
aortic rings
and coronary artery rings by stimulating the endothelial formation of NO
(Engler et al.,
2000; Omura et al., 2001). However, the signal transduction pathway leading to
eNOS
activation remains poorly studied. Moreover, little information is currently
available
regarding the optimal ratio of EPA: DHA for the activation of eNOS. Therefore,
the
following experiments were carried out to characterize the fish oil-induced
activation of
eNOS in isolated blood vessels and cultured endothelial cells.
[0078] The initial experiment was designed to determine the ability of
omega-3
fatty acids (EPA, DHA and different ratios of EPA: DHA) to cause endothelium-
dependent
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relaxations in rings of porcine coronary arteries, thereby enabling the
characterization of
the role of NO and EDHF in endothelium-dependent relaxation and identification
of the
signal transduction pathway involved.
[0079] Additional experiments were designed to determine the ability of
omega-3
fatty acids (EPA, DHA and different ratios of EPA: DHA) to cause activation of
eNOS in
cultured endothelial cells and to determine the underlying signal transduction
pathway.
[0080] In order to make the above determinations we designed an
experiment to
codify vascular reactivity. Initially, the left circumflex coronary artery
harvested from
fresh pig hearts is cleaned of its fat and adherent tissue and cut into rings
2 to 3 mm in
length. Rings without endothelium were obtained mechanically by rubbing with a
pair of
pliers inserted into the vessel lumen. Rings with or without endothelium were
suspended in
organ baths containing Krebs bicarbonate solution (composition in mM: NaC1
118.0, KC1
4.7, CaC12 2.5, MgSO4 1.2, NaHCO3 23.0; KH2PO4 1.2 and glucose 11.0, pH 7.4,
37 C)
oxygenated with a mixture of 95% 02 and 5% CO2. After equilibrating rings for
90 mM at
a basal tension of 5 g, rings were contracted with KC1 (80 mM) to verify the
responsiveness of the vascular smooth muscle. After a 30 min washing period,
the integrity
of the endothelium was verified. Rings were contracted with U46619 (1-60 nM,
an
analogue of thromboxane A2) to 80% of the maximal contraction, and at the
plateau of the
contraction, bradykinin (0.3 M) was added to check the presence of a
functional
endothelium. After repeated washings and return to baseline, rings were
contracted again
with U46619 before applying an increasing range of omega-3 fatty acids (0.001%
to 0.4%
v /v) to test their ability to induce relaxation of coronary artery rings.
During the
stabilization phase (30 min before contraction with U46619) different
pharmacological
tools were added to the Krebs bicarbonate solution to characterize the
signaling pathway
leading to endothelium-dependent relaxations:
a. Indomethacin (10 M), an inhibitor of cyclooxygenases (COX) to prevent the
formation
of vasoactive prostanoids, particularly prostacyclin,
b. No)-nitro-L-arginine (L-NA, 300 M), a competitive inhibitor of NO synthase
(NOS) to
overcome the NO component, and
c. TRAM 34 (100 nM) and apamin (100 nM) inhibitors of Ca2+-activated potassium
channels (IKCa and SKCa) respectively, to overcome the EDHF component.
[0081] Pig coronary artery endothelial cells were harvested, cleaned with
phosphate buffered saline solution (PBS) without calcium to remove any
residual blood.
24
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WO 2012/112520 PCT/US2012/025014
Endothelial cells were isolated by collagenase (type I, Worthington, 1 mg/ml,
14 min at
37 C) and cultured in medium MCDB131 (Invitrogen) supplemented with 15% v/v
fetal
calf serum, 2 mM glutamine, 100 U/mL penicillin, 100 U/mL streptomycin and 250
mg/ml
fungizone (Sigma, St Louis, MO) at 37 C in 5% CO2. All experiments were
performed
with confluent endothelial cells used at first passage. Endothelial cells were
exposed to
MCDB131 with 0.1% v/v fetal calf serum 5 h before treatment with different
substances.
[0082] After treatment, endothelial cells were rinsed twice with PBS and
lysed with
extraction buffer (composition in mM: Tris / HC1 20, pH 7.5 (QBiogene), NaC1
150,
Na3VO4 1, Na4P207 10, NaF 20, okadaic acid 0.01 (Sigma), protease inhibitors
(Complete
Roche) and 1% Triton X-100). 25 lig of total proteins were separated on SDS-
polyacrylamide (Sigma 8%) at 100 V for 2 h. Separated proteins were
transferred onto a
polyvinylidene fluoride membrane (Amersham) by electrophoresis at 100 V for 2
h. The
membranes were blocked with blocking buffer containing 3% bovine serum albumin
in
TBS-T (Tris-buffered saline solution, Biorad, containing 0.1% Tween 20, Sigma)
for 1 h.
For detection of proteins, membranes were incubated in TBS-T containing the
respective
primary antibodies (p-eNOS Ser 1177, p-eNOS Thr 495 and p-Akt Ser 473
(dilution
1:1000), 13-tubulin (dilution 1:5000, Cell Signaling Technology) overnight at
4 C. After a
washout period, the membranes were incubated with secondary antibodies (anti-
rabbit for
p-eNOS, p-Akt, and anti-mouse for 13 tubulin) coupled to horseradish
peroxidase (Cell
Signaling Technology, dilution 1:5000) at room temperature for 1 h. Stained
protein
markers (Invitrogen) were used for the determination of the molecular weight
of separated
proteins. Immunoreactive bands were detected using chemiluminescence
(Amersham).
[0083] All results were presented as mean standard error of mean (SEM).
n
indicates the number of different coronary arteries studied. Statistical
analysis was
performed using Student t test or analysis of variance (ANOVA) test followed
by
Bonferoni post-hoc test. A P value of <0.05 is considered statistically
significant.
RESULTS:
[0084] The omega-3 fatty acid preparation EPA:DHA 1:1 induced
concentration-
dependent relaxations of coronary artery rings with endothelium whereas only
small
relaxations were obtained in those without endothelium contracted with U46619
(Figure
4). The relaxations to EPA:DHA 1:1 was observed at volumes greater than 0.01 %
v/v and
they reached about 75% at 0.4 % v/v (Figure 4). In addition, the omega-3 fatty
acid
CA 02827577 2013-08-16
WO 2012/112520 PCT/US2012/025014
preparation EPA: DHA 6:1 also induced endothelium-dependent relaxations which
were
more potent than those induced by EPA: DHA 1:1 (Figure 4). Relaxations to
EPA:DHA
6:1 started at 0.01 % v/v and they reached about 98% at 0.4% v/v (Figure 4).
These
findings indicate that the omega-3 fatty acid preparation EPA:DHA 6:1 is more
effective to
induce endothelium-dependent relaxations of coronary artery rings than the
EPA:DHA 1:1
preparation. Thereafter, all subsequent experiments were performed with the
omega-3 fatty
acid preparation EPA: DHA 6:1.
[0085] It was determined that the omega-3 fatty acid preparation EPA:DHA
6:1
induces endothelium-dependent relaxations involving both NO and EDHF.
[0086] Previous studies have indicated that EPA and DHA induce relaxation
of
coronary artery rings by a mechanism mainly endothelium-dependent and
sensitive to
inhibitors of the formation of NO and EDHF (Omura et al., 2001). Therefore, a
study to
determine whether the endothelium-dependent relaxations induced by omega-3
fatty acid
formulations having an EPA:DHA ratio of about 6:1 according to the present
invention
(referred to as EPA:DHA 6:1 herein) involve NO and EDHF was undertaken. The
endothelium-dependent relaxation to EPA:DHA 6:1 was not significantly affected
by
inhibitors of the EDHF component, TRAM 34 and apamin (inhibitors of Ca2+-
dependent
potassium channels of intermediate and low conductance IKCa and SKCa,
respectively,
Figure 5). In contrast, relaxations were partially inhibited, but in a
statistically significant
amount, by L-NA (a competitive inhibitor of eNOS), indicating the involvement
of NO
(Figure 5). In addition, the combination of L-NA plus TRAM 34 and apamin
abolished the
endothelium-dependent relaxation to EPA:DHA 6:1 (Figure 5). Altogether, these
findings
indicate that EPA:DHA 6:1 induces endothelium-dependent relaxations which are
mediated predominantly by NO and also, to a lesser extent, by EDHF.
[0087] Several studies have shown that relaxations mediated by NO in
response to
polyphenols derived from grapes involve the redox-sensitive Src/PI3-kinase/Akt
pathway
(Anselm et al., 2007; Ndiaye et al., 2005). Therefore, it was decided to
determine whether
this pathway is involved in NO-mediated relaxations to EPA:DHA 6:1. In order
to
selectively study the NO component, all experiments were conducted in the
presence of
inhibitors of the EDHF component (Apamin + TRAM 34) and the formation of
vasoactive
prostanoids (indomethacin). The relaxation induced by EPA:DHA 6:1 was
significantly
reduced by PP2 (an inhibitor of Src kinase, Figure 6) and wortmannin (an
inhibitor of PI3-
kinase, Figure 6). Furthermore, the relaxations to EPA:DHA 6:1 were shifted to
the right
26
CA 02827577 2013-08-16
WO 2012/112520 PCT/US2012/025014
by the membrane permeant analog of SOD, MnTMPyP and catalase (PEG-catalase)
and by
native SOD and catalase (Figure 7) in a statistically significant amount.
Altogether, these
findings suggest that Src kinase and the P13-kinase mediate the stimulatory
signal of EPA:
DHA 6:1 to eNOS via a redox-sensitive mechanism.
[0088] To obtain direct evidence that EPA:DHA 6:1 is able to
activate the PI3-
kinase/Akt pathway leading to eNOS activation, cultured coronary artery
endothelial cells
were exposed to EPA:DHA 6:1 up to 6 hours and the level of phosphorylated Akt
and
eNOS was determined using Western blot. The data indicate that EPA: DHA 6:1
increased
the level of phosphorylation of Akt and eNOS starting at 15 min and that this
effect
persists until 6 h (Figure 8A and Figure 8B). The level of total eNOS
expression remained
unaffected by the EPA: DHA 6:1 treatment (Figure 8A). In addition, the
stimulatory effect
of EPA:DHA 6:1 on phosphorylation of Akt and eNOS was inhibited by MnTMPyP,
PEG-
catalase and by native SOD and catalase (Figure 8A). Thus, these data provide
direct
evidence that EPA: DHA 6:1 activate eNOS via a redox-sensitive mechanism
Table 5 - Comparative Capsule Contents VASCAZENTM vs. German Omega-3 OTC
Brands
Product Weight/Capsule Omega- Vitamin E Vitamin E EPA DHA
EPA+DHA
(mg) 3 (mg)/Capsule (in (mg)/Capsule
(mg)/Capsule (in
(mg / %)/Capsule
%)/Capsule
%) per
Capsule
ABTEI 1767 390/ 15 0.85 230 160
22
22.1
TETESEPT 1350 350/ 15 1.1 180 120
22.2
25.9
DOPPELHERZ 1300 300/ 12 0.92 180 120
23.1
23.1
SCHAEBENS 1450 500/ 10 0.07 n/a (500 mg n/a
VEGETAL 34.5 linolenic
acid)
SCHAEBENS 900 195 / 10 1.1 117 78
21.7
FISH OIL 21.67
OPTISANA 708 130/ 6 0.85 80 50
18.4
(LIDL) 18.4
VASCAZENTM 1000 900/ 2 0.2 680 110
79
90%
Omega-3 in % signifies total omega-3 in % of total fatty acids as EE (ethyl
esters)
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[0089] Now referring to Figures 9-12, these figures help to illustrate
the importance
of both the purity of and the presence of additives in the formulation,
respectively in
providing a maximal relaxation response. For the purpose of this discussion,
omega-3
purity was defined as the percentage of the sum of EPA+DHA per capsule. The
use of
indomethacin as a determinant of the relaxation effect is based upon the
following
explanation. In some blood vessels vasorelaxing prostanoids such as
prostacyclin have
been identified as an endothelium-derived vasorelaxing factor. These
vasorelaxing
prostanoids are generated from the metabolism of arachidonic acid by
cyclooxygenase-1
(COX-1). Indomethacin is an inhibitor of COX-1 and thus will prevent the
formation of
vasorelaxing prostanoids. The magnitude of the endothelium-dependent
relaxation is
dependent on the purity of the formulation (Figure 9) and on the EPA:DHA ratio
(Figure
4). In addition, the EPA:DHA 6:1 formulation caused similar endothelium-
dependent
relaxation as the OTC Omega-3 product TETESEPTTm with an omega-3 purity (as
defined
above) of 22.2 % as compared to that of the EPA:DHA 6:1 formulation of 75.1 %
and was
much more effective than the other OTC Omega-3s tested (ABTEI LACHSOLTM 1300,
DOPPELHERZ , SCHAEBENSTM and OPTISANATm (Figure 11 A). The endothelium-
dependent relaxation induced by VASCAZENTM (as an example of EPA:DHA 6:1) is
not
affected by indomethacin at 10 p,M. In contrast, the relaxation induced by
TETESEPTTm
which was similar to that of EPA:DHA 6:1 was significantly reduced by
indomethacin
(Figure 11 A and B). Endothelium-dependent relaxations induced by SCHAEBENSTM
and
OPTISANATm were markedly reduced and those to ABTEITm and DOPPELHERZ were
slightly reduced (Figures 11 A and B). This data further indicate that the
indomethacin-
sensitive relaxation of the OTC Omega-3s cannot be attributed to EPA and DHA
nor to its
relative concentration ratio but most likely to the presence of additives such
as Vitamin E
(alpha-tocopherol), see Table 5. Indeed, the vitamin E content of EPA:DHA 6:1
is 0.2 %
whereas that of OTC Omega-3 formulations varies between 0.85 and 1.1 % (Table
5). The
importance of the vitamin E additive effect is further suggested by the fact
that
TETESEPTTm has a more than fivefold higher vitamin E content than that of the
EPA:DHA 6:1 formulation. Therefore, the selective inhibitory effect of
indomethacin
induced upon the TETESEPTTm but not upon the EPA:DHA 6:1 is most likely
explained
by the more than fivefold higher vitamin E content per capsule. Vitamin E has
been shown
to cause endothelium-dependent relaxation which is inhibited by indomethacin
(Wu et al.,
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CA 02827577 2013-08-16
WO 2012/112520 PCT/US2012/025014
J Nutr. 135: 1847-1853, 2005). Both omega-3 purity and additives, contribute
to the
endothelium-dependent relaxation observed with Omega-3 products. This is
further
illustrated by comparing the relaxation induced by the EPA:DHA 6:1 formulation
to that of
the METAGENICSTm EPA-DHA 6:1 formulation. Indeed, the latter is markedly
inhibited
by indomethacin as compared to the former (Figure 12). Thus, in the presence
of
indomethacin, the relaxation observed in the presence of Omega-3 products is
clearly
dependent on omega-3 purity. These experiments underscore the sustained
(greater than 6
hours) vasodilatory effect achieved due to the unique ratio and omega-3 purity
of the novel
EPA:DHA 6:1 product of the present invention. The combination of the 6:1 ratio
coupled
with the absence of exogenous impurities in the present invention lead to an
indomethacin
independent vasodilatory effect when compared to either EPA or DHA alone,
EPA:DHA
1:1 or to a 6:1 product which contains exogenous impurities (see Figures
4,9,11,12 and
13).
[0090] These findings indicate that omega-3 fatty acid preparations are
potent
endothelium-dependent vasodilators and that this effect is dependent on the
ratio and the
omega-3 purity of EPA and DHA within the capsule. They further suggest that
omega-3
fatty acids activate eNOS via a redox-sensitive P13-kinase/Akt pathway leading
to changes
in the phosphorylation level of eNOS as illustrated in Figure 14.
[0091] All patents and publications mentioned in this specification are
indicative of
the levels of those skilled in the art to which the invention pertains. All
patents and
publications are herein incorporated by reference to the same extent as if
each individual
publication was specifically and individually indicated to be incorporated by
reference.
[0092] It is to be understood that while a certain form of the invention
is illustrated,
it is not to be limited to the specific form or arrangement herein described
and shown. It
will be apparent to those skilled in the art that various changes may be made
without
departing from the scope of the invention and the invention is not to be
considered limited
to what is shown and described in the specification and any drawings/figures
included
herein.
[0093] One skilled in the art will readily appreciate that the present
invention is
well adapted to carry out the objectives and obtain the ends and advantages
mentioned, as
well as those inherent therein. The embodiments, methods, procedures and
techniques
described herein are presently representative of the preferred embodiments,
are intended to
be exemplary and are not intended as limitations on the scope. Changes therein
and other
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uses will occur to those skilled in the art which are encompassed within the
spirit of the
invention and are defined by the scope of the appended claims. Although the
invention has
been described in connection with specific preferred embodiments, it should be
understood
that the invention as claimed should not be unduly limited to such specific
embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which
are obvious to those skilled in the art are intended to be within the scope of
the following
claims.
