Note: Descriptions are shown in the official language in which they were submitted.
CA 02710054 2011-12-23
OIL EXTRACT FROM A BIVALVE MOLLUSC
THAT IS RICH IN N-ACYLETHANOLAMIDES
TECHNICAL FIELD
The invention relates to an extract. More specifically, the invention relates
to oil extracts
from marine based raw materials rich in fatty acid amide compounds including N-
acylethanolamide (NAE) compounds such as N-palmitoylethanolamide (PEA) and N-
arachidonoylethanolamide (anandamide, AEA).
BACKGROUND ART
N-palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to
the family
known as N-acylethanolamines (NAE's; or N-acylethanolamides), including
'cannabis-like'
compounds such as N-arachidonoylethanolamide (anandamide, AEA). PEA and AEA,
have
the potential to treat a range of human/ animal conditions involving abnormal
inflammatory
and/or immune responses and associated pain (see Lambert et al., 2002, for a
review).
History and Scientific Literature Overview of PEA and other NAE's
a) Origin
PEA is a naturally occurring lipid found in many different cells of animal,
marine and plant
origin (see Lambert et al., 2002, for a review). In living organisms, PEA
synthesis is rapidly
induced in response to cellular stressors, such as tissue damage or
pathological insults,
which are often accompanied by inflammation and pain (Darmani, et al., 2005).
While the
biological functions of PEA in humans are not completely understood, it has
been
hypothesized that PEA constitutes one of several natural anti-inflammatory and
analgesic
chemicals (Darmani, et al., 2005).
The crude lipid extracted from the freeze-dried flesh of Perna canaliculus
using chloroform
and methanol consists of a complex mixture of triglycerides, sterol esters,
sterols, polar
lipids, free fatty acids and their derivatives, such as NAEs (Sepe et al.,
1998; Murphy et al.,
1
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
2002; Murphy et al., 2003). PEA and N-stearoylethanolamide are the most
abundant NAEs
in blue mussel lipid extracts along with much smaller amounts of N-m.yristoyl
(C14:0)-, N-
oleoyl (C18:1)-, N-linoleoyl (C18:2)-, and N-arachidonoyl (C20:4; anandamide/
AEA)-
ethanolamides. The nature and number of NAE's present in .a crude or purified
extract may
have therapeutic implications since the prior art has revealed the existence
of complex
synergistic interactions between NAE's in whole animals and animal tissues.
For example,
data from animal studies indicates that some actions of PEA may be mediated
via AEA and
possibly other NAEs. Furthermore, evidence of synergistic effects between NAEs
have
been reported (see below for details).
b) Chemistry
PEA is a saturated 16 carbon fatty acid ethanolamide (C16:0), with the
structure:
Ho
'~~ NH
C443
AEA is a polyunsaturated 20 carbon fatty acid ethanolamide (C20:4) with the
structure:
ki
0
C) Historical Perspective
Interest in the anti-inflammatory properties of PEA was first noted in the
early 1950's by
Coburn et al. (1954) who found that feeding guinea pigs a diet high in egg
yolk protected
them from experimental allergy. Subsequent studies isolated and purified PEA
from egg
yolk and identified the anti-inflammatory properties in animals (see Lambert
et al., 2002, for
a review). Research interest in NAEs was revived in the 1990's following the
discovery of
another endogenous NAE, AEA, with similar properties to the active component
of cannabis.
The cloning of the cannabinoid receptors (designated CBS and CB2) and the
generation of
selective CB receptor ligands provided the tools to further accelerate
research activity
(Devane et al., 1992). Since then a substantial body of evidence gathered from
animal and
2
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
human studies has shown that PEA has anti-inflammatory and analgesic
properties when
given via different routes of administration.
d) Pre-Clinical Studies
A number of studies have shown that synthetic PEA has anti-inflammatory and
analgesic
properties in a range of animal models. Typically, an inflammatory substance,
such as
carrageenan, collagen or phorbol ester, is injected beneath the skin and the
resulting
pathological and behavioural changes are measured within hours (acute model)
or several
days (chronic model). To date, the vast majority of these studies have been
conducted in
acute animal models of inflammation (e.g., Aloe et al., 1993; Mazzari et al.,
1996; Conti et
al., 2002; Costa et al., 2002).
e) Clinical Trials
In comparison to animal studies, there have only been a small number of human
clinical
trials to investigate the anti-inflammatory effects of PEA. During the early
1970's several
trials were conducted in Czechoslovakia using an oral formulation given the
trade name
lmpulsinT"" (N-2-Hydroxyethyl palmitamide, SPOFA United Pharmaceutical.
Works). The
first set of trials evaluated the efficacy of PEA (3 times/ day/ 12 days) to
reduce the
incidence and severity of respiratory tract infections in 1345 adult
volunteers (either young
male soldiers or employees of the Skoda Car Co.; Masek et al., 1974). Results
indicated
that Impulsin' helped to prevent viral infections when given before an
infectious episode,
but did not reduce the duration of infectious symptoms. A further series of
similar trials
conducted between 1973 and 1975, on a total of 1864 young male soldiers (same
dose
regime), confirmed that prophylactic ImpulsinTM significantly. reduced the
incidence of acute
respiratory infections in this population (Kahlich et al., 1979). The
incidence of unwanted
effects during the 12 week trial was particularly low (just a few percent;
Kahlich et al., 1979).
The apparent success of these trials led to the use of Impulsin' in the former
Czechoslovakia for acute respiratory disease. After several years on the
market, the drug
was withdrawn for unknown reasons which do not appear to be related to
toxicity (see Lo
Verme et al., 2005b, for a review). Two subsequent clinical trials have been
initiated to
investigate the efficacy of PEA for chronic back pain (lumbosciatalgia) and
multiple sclerosis
(see Lambert et al., 2002, for a review).
A cream containing "structured natural lipids" with PEA for topical
administration has also
been developed (PhysiogelTM A.I., Stiefel Laboratories) and evaluated in two
small-scale
3
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
clinical trials. The first was an observational study in which 19 adult
patients diagnosed with
anal eczema were instructed to apply the cream to the affected area for
between 6 and 63
days (Rohde & Ghyczy, 2003). After 4 weeks of the trial, 68% of patients
reported a
reduction in pain, burning sensation and itch, with 21% recording a worsening
of symptoms.
The cream was reportedly well tolerated by 95% patients. In the second trial,
21 adult
patients with end-stage kidney failure and suffering from uremic itch, applied
the cream
twice daily for three weeks (Szepietowski et al., 2005). At the conclusion of
the 3 week test
period there was a statistically significant reduction in itch (completely
absent in just under
40% patients) and 81 % of patients reported an elimination of dryness in the
affected area
(xerosis). The cream was well tolerated by all patients with no adverse
effects reported.
These results provide an encouraging sign that a PEA-containing cream may
provide an
alternative therapeutic option in the treatment of inflammatory skin
conditions.
f) Mode of Action
The precise mechanism(s) by which PEA exerts its anti-inflammatory and
analgesic effects
are not completely understood. It is generally accepted that PEAs effects are
not mediated
via classical cannabinoid receptors, a finding that may account for PEAs lack
of
psychotropic effects. In contrast, the majority of AEAs actions appear to be
mediated via
CB1 and/or CB2 receptors in the brain and periphery. One problem with the use
of AEA is its
psychotropic side effects, which are thought to be mediated by CB1 receptors.
However,
there is evidence that such unwanted effects may be reduced or eliminated by
co-
administering low (sub-therapeutic) doses of AEA and another NAE (Calignano et
al., 1998;
Di Marzo et al., 2001; De Petrocellis et al., 2001; Lo Verme et al., 2005b).
For example,
sub-analgesic doses of PEA or AEA provide analgesia when given in combination
at the
same low doses (Calignano et al., 2001).
At the molecular and behavioural levels, PEA interacts with a number of
important targets in
the body. Evidence gathered from in vitro and in vivo studies indicates that
PEA reduces
oedma, mast cell proliferation, neutrophil infiltration and a number of
endogenous mediators
of inflammation including: mast cell degranulation (preventing release of
histamine and
serotonin), cyclo-oxygenase-2 (COX-2) activity, endothelial nitric oxide
synthase activity,
nitric oxide production from macrophages and lipid peroxidation during acute
hypoxia
(Gulaya et al., 1998). In addition, PEA reduces hyperalgesia in animal models
of
inflammatory pain (Jaggar et al., 1998; Farquhar & Smith, 2001; Conti et al.,
2002).
4
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
Recent data suggests that peroxisome proliferator-activated receptor alpha
(PPAR-a) is of
key importance to the anti-inflammatory actions of PEA (Lo Verme et al.,
2005a).
Standard Therapeutic Approaches to the Control of Inflammatory Conditions
A. Diseases Involving Chronic Inflammation
Inflammation is part of the body's normal response to injury and infection and
is
characterised by the classic signs of localised redness, swelling, heat and
pain. A normal
inflammatory response is an acute process that resolves following removal of
the stimulus
and the initiation of repair and tissue healing. In some circumstances, acute
inflammation
can progress to chronic inflammation, which is a key component of a large
number of
pathologies affecting bone and joint, respiratory, skin, gastrointestinal
tract, cardiovascular
and neural systems. Importantly, the processes underlying acute and chronic
inflammation
are to a large distinct.
The popularity of non-steroidal anti-inflammatory drugs (NSAIDs) for the
treatment of
inflammation and pain is largely due to their more favourable risk profiles
compared to other
anti-inflammatory drugs. However, there are several major limitations to long
term NSAID
use including: their propensity to cause gastric damage (peptic ulcers and
gastrointestinal
bleeding), renal impairment and an increased risk of bleeding. Furthermore,
since NSAIDs
do not alter disease progression, sufferers may require an additional class of
drugs, the
Disease Modifying Anti-inflammatory Drugs (DMARDs; e.g., methotrexate,
etanercept,
corticosteroids). Unfortunately, most of these agents have narrow margins of
safety and are
characterised by frequent unwanted effects that negatively impact upon quality
of life.
B. Atopic dermatitis/ Eczema
Atopic dermatitis/ eczema (AD) is a chronic skin condition classically
presenting as
reddening, itch and the formation of vesicles which may lead to weeping and
crusting. AD
affects at least 15% of the developed world and is often associated with other
forms of
allergy, such as asthma and hay fever (Lee, Y-A., et al., 2000).
Topical corticosteroids are the gold standard of drug treatment for AD.
However, one of the
main drawbacks to long-term topical corticosteroid use is the risk of adverse
effects such as
skin shrinkage, oral and allergic contact dermatitis, acne, decreased skin
pigmentation and
excessive hair growth within the treatment area. Although a number of non-
steroidal
products are available (e.g., pimecrolimus, tacrolimus, antibiotics,
cyclosporine,
5
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
methotrexate, etc), these are often less efficacious and none are without
adverse effects
that are occasionally serious (see Abramovits, 2005, for a review). For
example,
pimecrolimus and tacrolimus recently received FDA Black Box warnings regarding
a
possible cancer risk. Clearly, there is a need for effective anti-inflammatory
products with
fewer adverse effects.
Anti-inflammatory effects of Perna Canaliculus
"Stabilized" lipid extracts of the New Zealand Green-lipped mussel (Perna
canaliculus,
NZGLM) have demonstrated beneficial effects in several different animal models
of
inflammation as well as chronic inflammatory conditions in humans (Whitehouse
et al.,
1997; Whitehouse et al., 1999; Shiels & Whitehouse, 2000; Tenikoff et al.,
2005; Gibson &
Gibson, 1998; Emelyanov et al., 2002; Cho et al., 2003; Gruenwald, et al.,
2004).
Stabilization typically involves the addition of an organic acid, such as
tartaric acid, to reduce
oxidation of PUFA prior to processing (W085/05033; NZ211928). However not all
human
studies in which stabilized mussel lipid extracts were used to treat
inflammation have
reported positive outcomes (Lau et al., 2004; see Cobb and Ernst, 2006, for a
review) or
have been able to demonstrate a reduction in blood markers of inflammation in
healthy
volunteers (Murphy et al., 2006).
Mussel Patents
Mussel patents in the art are related to various aspects of mussel extract
formulation, for
example:
NZ211928 describes a formulation for stabilising green shell mussel extract by
adding an
organic acid (acetic acid, citric acid, tartaric acid, lactic acid) and/or a
metal salt to the
mussel flesh suspended in saline once harvested.
NZ270754 describes a combination of finely ground mussel extract suspended
within fish
oil.
NZ314867 describes a protein extract from green shell mussel combined with
glycosamino-
glycans.
NZ514389 describes delivery of a green shell mussel extract to a pet in a pet
food at a rate
of 0.18 to.114 mg/kg of animal body weight per day.
6
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
Other patents describe extraction processes to enrich the extract in selected
components,
for example:
NZ329018 describes a process for extracting glycogen from mussels by treating
with
proteolytic enzyme in water, separating the solid residue and recovering the
glycogen from
the aqueous solution.
NZ510407 describes an extract of green shell mussel containing carbohydrate
and lipids
and with the protein fraction removed.
NZ328489 describes a protein extract from green shell mussel as well as a
method of
producing the extract by stirring the flesh for 45 minutes in phenol solution
then centrifuging
/ aspirating the upper layer and precipitating out the protein containing
product using
ethanol.
None of the above patents refer to producing an enriched NAE compound extract
nor
recognize the usefulness of these compounds in various treatments.
Commercial Mussel Products
Two widely available commercial preparations of the NZGLM are SeatoneT"' and
LyprinoIT".
Seatone consists of a stabilised freeze-dried powder obtained from whole
mussel flesh,
whereas LyprinolTm is an oil extracted from stabilized freeze-dried flesh (via
supercritical
CO2 fluid) with the addition of olive oil and vitamin E and formulated into
capsules
(Pharmalink International Ltd., Cayman Islands). LyprinolT"' contains five
major classes of
lipid: free fatty acids, triglycerides, sterol esters, sterols and
phospholipids (Sinclair et al.,
2000; Wolyniak et al., 2005). The most abundant free fatty acids in
LyprinolT"" are: palmitic
(C16:0), linoleic (C18:2n-6), EPA (C20:5n-3), DHA (C22:6n-3), palmitoleic
(C16:1 n-7),
C16:ln-9,7,5 and myristic (C14:0) (Sinclair et al., 2000; Wolyniak et al.,
2005). Although a
large number of other fatty acids have also been identified (some 91 in all),
individually
these are present in only small quantities (below 5% w/w total lipid; Wolyniak
et al., 2005).
The omega-3 PUFA's comprise 40% of the total fatty acids and EPA and DHA are
the most
abundant (Wolyniak et al., 2005). There is evidence to suggest that most of
the anti-
inflammatory activity of LyprinolTM resides in the fatty acid fraction
(Whitehouse et al., 1999;
Treschow et al., 2007).
Following a course of LyprinolTM human subjects had reduced levels of several
pro-
inflammatory compounds including thromboxane B2, prostaglandin E2 and
interleukin-1R,
7
CA 02710054 2011-12-23
similar to those observed following low-dose omega-3 polyunsaturated fatty
acid
supplements (Sinclair et al., 2000). This suggests that a significant
component of
Lyprinol'sTM activity may be due to its omega-3 content, a possibility that is
consistent with
recent in vitro evidence (McPhee et al., 2007; Treschow et al., 2007). The
hypothesized
mode of action of LyprinolTM is via inhibition of both the 5-lipoxygenase and
COX pathways.
Given the above evidence outlining the therapeutic benefits of PEA and other
NAE
compounds, it should be appreciated that a product rich in these compounds may
be
advantageous.
It is an object of the present invention to address the foregoing problems or
at least to
provide the public with a useful choice.
No admission is made that any reference constitutes prior art. The discussion
of the
references states what their authors assert, and the applicants reserve the
right to challenge
the accuracy and pertinency of the cited documents. It will be clearly
understood that,
although a number of prior art publications are referred to herein; this
reference does not
constitute an admission that any of these documents form part of the common
general
knowledge in the art, in New Zealand or in any other country.
It is acknowledged that the term 'comprise' may, under varying jurisdictions,
be attributed
with either an exclusive or an inclusive meaning. For the purpose of this
specification, and
unless otherwise noted, the term 'comprise' shall have an inclusive meaning -
i.e. that it will
be taken to mean an inclusion of not only the listed components it directly
references, but
also other non-specified components or elements. This rationale will also be
used when the
term 'comprised' or'comprising' is used in relation to one or more steps in a
method or
process.
Further aspects and advantages of the present invention will become apparent
from the
ensuing description which is given by way of example only.
DISCLOSURE OF INVENTION
For the purposes of this specification, the term 'marine based' refers to
shellfish
organisms living in or around the seawater or freshwater.
8
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
The term 'N-acylethanolamine' (NAE) is used but includes other names common to
the art for this group of compounds including 'N-acylethanolamides' or fatty
acid
ethanolamides.
According to a first aspect of the present invention, there is provided a
composition
containing an oil extract from a marine based organism that is rich in fatty
acid amide
compounds.
According to a further aspect of the present invention, there is provided a
composition containing an oil extract from a marine based organism that is
rich in N-
acylethanolamine (NAE) compounds.
Preferably, the extract is rich in N-palmitoylethanolamide (PEA). It should be
appreciated by those skilled in the art that PEA also has a number of
different names
including but not limited to palmidrol, and N-(2-hydroxyethyl) -
hexadecanamide.
According to a further aspect of the present invention, there is provided a
composition containing an oil extract from a marine based organism containing
at
least 0.10 pg/g of PEA as measured in the wet weight of the marine organism
flesh
prior to extraction.
According to a further aspect of the present invention, there is provided a
composition containing an oil extract from a marine based organism that is
rich in
NAE compounds including at least 0.10 pg/g of PEA as measured in the wet
weight
of the marine organism flesh prior to extraction.
As noted above, the composition is rich in a range of NAE compounds. By way of
illustration, the composition includes (but is not limited to) having
quantities of the
NAEs: N-stearoylethanolamide (18:0), 70 ng/ g; N-oleoylethanolamide (C18:1), 5
ng/
g; N-linoleoylethanolamide (C18:2), 5 ng/ g; and N-arachidonoylethanolamide
(C20:4, anandamide), 8 ng/ g in the wet flesh prior to extraction. This should
not be
seen as limiting as the composition may also contain other NAEs and may also
include NAEs in amounts varying above and below that disclosed.
Preferably, the marine based organism is a bivalve mollusc. More preferably,
the
organism is a mussel species. Most preferably, the organism is a mussel of the
species Perna or Mytilus (green or blue mussel respectively). This should not
be
9
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
seen as limiting as other species may also be included for example, clam and
oyster
species.
Preferably, the fatty acid amide compounds include NAE compounds.
Preferably, the NAE compounds include: N-myristoylethanolamide, C14:0; N-
palmitoylethanolamide (PEA), C16:0; N-stearoylethanolamide, C18:0; N-
oleoylethanolamide, C18:1; N-linoleoylethanolamide, C18:2; N-
arachidonoylethanolamide (anandamide, AEA), C20:4; N-eicosaenoylethanolamide,
C20: 1, and combinations thereof.
In preferred embodiments, the level of PEA in the oil extract is at least 0.10
pg/g as
measured. in the wet tissue of the marine. organism prior to any extraction.
More
preferably, the level is at least 0.50 pg/g. In selected embodiments the level
of PEA
may be greater than 3.0 pg/g.
Preferably, the level of AEA in the oil extract is at least 0.008 pg/g wet
tissue of the
marine organism prior to any extraction. More preferably, the level is greater
than
0.01 pg/g wet tissue of the marine organism prior to any extraction. In
selected
embodiments the level of AEA may be greater than 0.05 pg/g.
Preferably, the composition includes PEA in the oil extract at a level greater
than 1.0
pg/g as measured in the dried tissue of the marine organism prior to any
further
extraction.
Preferably, the composition includes AEA in the oil extract at a level greater
than
0.09 pg/g as measured in the dried tissue of the marine organism prior to any
further
extraction.
It should be appreciated that the above levels of at least PEA and AEA are
significantly higher in the oil extract of the present invention than that of
the prior art
which either does not teach of PEA levels at all or does not teach the levels
described. In the closest prior art, Sepe et al teaches of producing only
0.053
0.0039 pg/g of PEA and 0.0018 0.003 pg/g of AEA in wet tissue from
Mediterranean mussels.
It is emphasised that the above values refer to PEA and AEA concentration in
wet
flesh. It should be appreciated that methods which reduce the water content
(such
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
as drying) also concentrate the level of active compounds including PEA and
AEA.
The extract of the present invention is already substantially more
concentrated
before such water removal processes are completed.
According to a further aspect of the present invention, the fatty acid amide
levels in
the raw marine material substantially as described above may be enriched.
For the purposes of this specification, the term 'enriched' refers to an
increase in the
concentration/amount of fatty acid amide compounds in the marine material
before
any concentration occurs via a drying process.
In one preferred embodiment, enrichment occurs by harvesting, crushing and
then
holding the marine material in a crushed state at between.4 and 10 C for a
time
period of at least 24 hours. In one embodiment, the material may be held for
up to
144 hours.
The inventors have found that by completing the above enrichment step, the
concentration/amount of fatty acid amide compounds increases. This is thought
to
be due to biochemical reactions occurring post-mortem in the marine material.
Unexpectedly, this process resulted in a substantial enrichment of mussel meat
fatty
acid amide levels. Also unexpectedly, the inventors discovered that by using
the
parameters described, microbial contamination does not occur to an extent
detrimental to the extract being suitable for human consumption. As may be
appreciated, normal handling procedures for marine based materials requires
steps
to be taken as quickly as possible to prevent microbial growth such as
freezing or
drying. Allowing the material to be held for several days at 10 C goes against
current
practice and yet the level of microbial contamination was unexpectedly low for
this
time and temperature.
As noted above, the composition is rich in fatty acid amides including PEA and
AEA
but may also include other compounds (both fatty acid amides and other
compounds). In fact, it is the inventors understanding that it is highly
desirable to
have multiple NAE compounds present besides PEA as, based on prior art
experience with NAEs (published literature), synergies between NAEs are common
and therefore, the potency of a product containing multiple NAEs may be
substantially increased compared to that containing only one NAE.
11
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
In a further embodiment, the composition also includes at least one
polyunsaturated
fatty acid (PUFA) compound. More preferably, the PUFA compound or compounds
include omega-3 PUFAs. It may be appreciated that having an oil extract that
includes both fatty acid amide compounds and PUFA compounds is desirable. What
may also be appreciated is that as fatty acid amide compounds and PUFA
compounds have different chemical properties, obtaining an extract with
significant
levels of both types of compound is unexpected. It should however be
appreciated
that PUFA compounds are not essential to the composition of the present
invention
and the presence of PUFA compounds should not be seen as limiting.
Preferably, the oil extract includes at least one PUFA compound. More
preferably,
the PUFA or PUFAs are of the omega-3 class. Preferably, the PUFA or PUFAs are
selected from: 4,7,10,13,16,19-docosahexaenoic acid (DHA; 22:6n3),
5,8,11,14,17-
eicosatetraenoic acid (EPA, 20:5n3), 6,9,12,15-octadecatetraenoic acid (OTA,
18:4n3), 9,12,15-octadecatrienoic acid (ALA, 18:3n3), 7,10,13,16,19-
docosapentaenoic acid (DPA, 22:5n3), 11,14,17-eicosatrienoic acid (ETA,
20:3n3),
8,11,14,17-eicosatetraenoic acid (20:4n3) and combinations thereof. In
preferred
embodiments, the DHA content is greater than 3g/1 00g as measured in the
extract.
Preferably, the EPA content is greater than 5g/1 00g as measured in the
extract.
Preferably, the composition is formulated as an orally administered powder,
solution,
suspension, emulsion, oil, tablet or capsule. The composition may
alternatively be
formulated for topical administration, for example as a cream, lotion,
ointment or oil.
In a further embodiment, the composition is a solid or liquid food for
administration
as a `functional food'.
According to a further aspect of the present invention there is provided a
formulation
for oral or topical administration containing a therapeutically effective
amount of an
oil extract from a marine based organism that is rich in fatty acid amide
compounds.
According to a further aspect of the present invention there is provided a
formulation
for oral or topical administration containing a therapeutically effective
amount of an
oil extract from a marine based organism that is rich in NAEs.
According to a further aspect of the present invention there is provided a
formulation
for oral or topical administration containing a therapeutically effective
amount of an
oil extract rich in PEA.
12
CA 02710054 2011-12-23
According to a further aspect of the present invention there is provided a
formulation
for oral or topical administration containing a therapeutically effective
amount of an
oil extract from a marine based organism containing at least 0.10 pg/g of PEA
as
measured in the wet weight of the marine organism flesh prior to extraction.
According to a further aspect of the present invention there is provided a
formulation
for oral or topical administration containing a therapeutically effective
amount of an
oil extract from a marine based organism that is rich in NAE compounds
including at
least 0.10 pg/g of PEA in the wet weight of the marine organism flesh prior to
extraction.
Preferably, the oil extract also includes at least 0.008 pg/g of AEA in the
wet weight
of the marine organism flesh prior to extraction.
Preferably, the formulation as described above also includes at least one
PUFA.
More preferably, the PUFA or PUFAs include omega-3 fatty acids.
In embodiments envisaged by the inventors, the formulation includes carrier
substances and may also include accepted food-grade antioxidants to assist in
long-
term stability of the extracted active compounds.
In one preferred embodiment, the formulation is a capsule for oral
administration wherein
the capsule is filled with oil extract sourced from mussel meat.
In an alternative preferred embodiment, the formulation is a cream or lotion
for topical
administration wherein the cream/ lotion includes an oil extract sourced from
mussel meat.
According to a further aspect of the present invention there is provided a
method of
treatment of inflammation and associated pain by oral or topical
administration of a
composition or formulation substantially as described above.
In one embodiment, the inflammation is of a chronic rather than acute nature.
By
way of example, disease conditions involving chronic inflammation include:
eczema/
atopic dermatitis, asthma, inflammatory bowel disease (including Crohn's
disease
13
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
and ulcerative colitis), rheumatoid and osteoarthritis, glomerulonephritis,
atherogenesis, Alzheimer's' disease and adult respiratory distress syndrome.
By
way of second example, disease conditions involving chronic pain include
neuropathic and arthritic pain.
In an alternative embodiment, the inflammation is of an acute nature and
includes
treatment of a soft tissue injury by topical application of a cream containing
a
composition or formulation of the present invention to the exterior of the
injury site.
According to a further aspect of the present invention there is provided a
method of
treatment of a disease associated with inflammation by oral or topical
administration
of a composition or formulation substantially as described above.
According to a further aspect of the present invention there is provided a
method of
ameliorating the development of inflammation by oral or topical administration
of a
composition or formulation substantially as.described above.
According to a further aspect of the present invention there is provided a
method of
treatment of a skin disease by topical administration of a composition or
formulation
substantially as described above.
According to a further aspect of the present invention there is provided a
method of
treatment of a skin disease by oral or topical administration of a composition
or
formulation substantially as described above.
According to a further aspect of the present invention there is provided a
method of
ameliorating the symptoms of a skin disease by oral or topical administration
of a
composition or formulation substantially as described above.
In preferred embodiments, the skin diseases to be treated include atopic
dermatitis/
eczema and contact dermatitis.
In preferred embodiments, the symptoms ameliorated include itch, dryness,
swelling
and reduced proliferation of human keratinocytes.
In one embodiment, the treatments substantially as described above may be
incorporated with at least one non-steroidal anti-inflammatory drug (NSAID).
It is
envisaged that the extract may enhance the activity of even low (sub-
therapeutic)
14
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
doses of NSAID thereby reducing the severity of side effects. In addition, the
compositions and formulations of the present invention may also act to enhance
the
NSAID activity therefore allowing for the advantage of either better efficacy
and/or
reduced dosage of NSAID required.
According to a yet further aspect of the present invention there is provided
the use of
an oil extract substantially as described above in the manufacture of a
formulation for
treatments as described above.
From the above description it should be appreciated that there is provided an
oil extract
containing substantially elevated levels of active compounds that according to
the art, are
beneficial for at least joint mobility. Associated oral and topical
formulations .are also
provided as well as treatments for various inflammation-related disorders and
the symptoms
thereof.
There is an urgent need for efficacious anti-inflammatory products that can be
used long-
term with fewer unwanted effects compared to current pharmaceuticals. The
extract of the
present invention may address this need. It is envisaged that daily
administration of a food
or formulation containing an NAE-enriched marine material extract represents
an ideal
treatment modality for the prevention or treatment of inflammatory symptoms.
BRIEF DESCRIPTION OF DRAWINGS
Further aspects of the present invention will become apparent from the
following description
which is given by way of example only and with reference to the accompanying
drawings in
which:
Figure 1 LC/MS chromatogram of a representative Perna canaliculus extract
showing the peak containing PEA (arrowhead, lower trace) at time =
10 minutes. The relative size of the peak demonstrates that PEA
/ makes up an appreciable portion of the total ion current (top trace)
suggesting a significant amount of PEA is present in the extract.
BEST MODES FOR CARRYING OUT THE INVENTION
Examples are now provided showing formulations produced and envisaged by the
inventors
relating to NAE enriched oil extracts.
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
EXAMPLE 1
An experiment was carried out to determine PEA levels in Blue mussels (Mytilus
edulis) and
Green-lipped mussels (Perna canaliculus) harvested commercially from New
Zealand
waters on a laboratory scale (Trial 1). A second experiment was conducted to
determine
PEA and AEA levels in Perna canaliculus on a simulated industrial scale (Trial
2).
Methods
PEA and AEA levels were measured by LC/MS in the crude lipid extracted from
the dried
meat of freshly harvested Blue and Green mussels and reported as pg/g in wet
flesh (Table
1 below; Giuffrida et al., 2000).
The presence of PEA in the lipid extract of P. canaliculus was confirmed by
LC/MS as
shown in Figure 1 where the arrowhead indicates the PEA peak.
Results
TABLE 1 - NAE Concentrations in Three Different Mussel Species
Wet Mussel. Homogenate PEA AEA
(Ng/g)
M. galloprovincialis Sepe et al.' 0.021 - 0.053 0.0018 - 0.003
M. edulis Trial 11 0.15-0.39
P. canaliculus Trial 1 0.3-0.61
Trial 22 0.54-0.97 0.008 - 0.054
1. NAE levels were determined by back-calculation from lipid extract data.
2. NAE levels were estimated using a conversion factor based upon trial 1 data
(Table 1).
Conclusions - Trials I and 2
1) PEA levels in P. canaliculus were higher than both Mytilus species. Since
P.
canaliculus contains slightly more meat on an equal weight basis and therefore
a slightly
higher proportion of dry matter, this at least partly accounts for the higher
PEA levels
relative to Mytilus.
16
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
2) AEA was also present at higher levels in P. canaliculus compared to M.
galloprovincialis, although the absolute levels were low.
EXAMPLE 2
A method to enrich the endogenous levels of NAEs in the flesh of P.
canaliculus prior to
extraction is described. In this example, homogenised/ intact mussel meat from
commercially harvested mussels was incubated under various conditions, as
described
below, in a laboratory. Incubation times ranged from 0 to 144 hours at 4 or 10
deg C in
either aerobic or anaerobic conditions at atmospheric pressure. Tissue pH was
monitored
at intervals throughout the trial. At the end of the incubation period, the
tissue was frozen
and subsequently freeze-dried. The experimental conditions were tightly
controlled to
minimise potential confounding factors such as differences between batches of
mussels,
freezing or freeze-drying conditions, etc. NAE and omega-3 levels were
measured in the
freeze-dried mussel.
Results from the experiment showed that dried meat prepared from homogenised
mussels
incubated at 10 C under aerobic conditions for up to 144 hours had 5 to 10-
fold more PEA
and AEA relative to dried meat prepared from the same batch of mussels frozen
immediately after homogenisation.
The inventors also unexpectedly found that levels of the major PUFA omega-3s
(DHA, EPA
and ALA) in the same dried mussel samples were retained even after 144 hours
incubation.
In addition, microbial levels in the dried product were unexpectedly low given
the time and
temperatures involved.
EXAMPLE 3
As noted above in the description, it is understood that the invention oil
extract is rich in NAE
compounds including but not limited to PEA. By way of example, the invention
extract
contains PEA, AEA and/or PUFAs. A concentration profile within the lipid
extract is
anticipated to be: 3.0 - 57.0 pg/g PEA or more; 0.1-5.2 pg/g anandamide or
more and/or at
least 3g/ 100g DHA and at least 5g/ 100g EPA.
EXAMPLE 4
In one embodiment of the invention an oral formulation is described. By way of
example, a
17
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
typical oral formulation includes mussel lipid extract (oil) of the present
invention, with or
without carrier lipid and antioxidant(s), contained within a gel capsule. The
dosage
envisaged for an adult human is approximately 1-4 capsules taken 1-2 times per
day. It is
anticipated that this administration regime will provide relief of pain and
swelling due to
chronic inflammation, particularly involving joints (arthritis).
EXAMPLE 5
In a further embodiment, the invention oil extract is used in a topical
formulation. By way of
example a topical formulation may include a cream or lotion containing the
lipid extract (oil)
of the present invention, with or without carrier lipid and antioxidant(s).
Other substances
that may be used in the cream/ lotion, include, but are not limited to:
propylene glycol, water,
glycerin, glycerides, hydrogenated lecithin, betaine, hydroxyethylcellulose,
sodium carbomer,
squaline, xanthan gum.
The cream is applied topically to affected areas 1-3 times per day for the
relief of dry skin,
redness, swelling, itch and the reduction of dermal thickening.
EXAMPLE 6
In a further example, an oral or topical formulation, as described above, is
used for the
treatment of acute inflammation. In one example, the extract could be
administered within 4
hours of the onset of inflammation (e.g., soft tissue injury) in order to
reduce the severity of
the symptoms and possibly recovery time.
EXAMPLE 7
In a further example, the invention oil extract is utilised in a functional
food. In one such
embodiment, the food may be eaten in order to reduce the risk of developing an
inflammatory condition including those described above.
By way of example, the product may be the invention oil extract in a ready to
use state to be
added (e.g. mixed into) various food products either by the consumer or the
product
manufacturer.
In one example, the invention oil extract is atomized to produce microspheres
and added to
a range of food products by manufacturers.
18
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
EXAMPLE 8
The invention oil extract may be co-administered with an NSAID. For example, a
low dose
of NSAID and an oral dose of the invention extract are used to treat chronic
inflammation
(e.g. rheumatoid or osteoarthritis; adult dose: 1-4 capsules taken at the same
time and
frequency as the NSAID). The invention extract then potentiates the anti-
inflammatory
effect of a low dose of NSAID and thus reduce the risk of unwanted effects.
EXAMPLE 9
The invention extract was further investigated by comparison to a prior art
commercial
product (P. canaliculus product; described in US 6,083,536 and US 6,346,278).
The
invention extract is rich in fatty acid amides including NAE compounds such as
PEA and
AEA. The invention extract may also include PUFA compounds such as EPA and
DHA.
The relative amounts of each key component in the extract of the present
invention are
compared to the commercial P. canaliculus product (Table 2 below).
TABLE 2 - COMPARATIVE DATA
NAEs (pg/ g Omega 3 Fatty Acids (g/100g oil)2
Oil),
Test C18:3n-
Material C20:5n-3
Material Docosa- Eicosa- C1 8:4n-3 3 Alpha
PEA AEA hexanoic pentaenoic Stearidonic Linolenic
Acid (DHA) Acid (EPA) Acid (OTA) Acid
(ALA)
Invention 57.0 5.2 12.1 19.4 1.7 1.1
Extract
Commercial
P. 3.3 0.2 3.1 4.9 0.7 0.4
canaliculus
prod UCt3
' Ethanolamides were quantified by liquid chromatography and mass spectrometry
(LC/MS; triple
quadruple MS).
2 Omega-3 Fatty acids were converted to their FAME and quantified by gas
chromatography
according to a validated method (AOAC # 963.22).
3 Each capsule contains: 50 mg P. canaliculus extract, 100 mg olive oil and
0.225 mg d-alpha-
tocopherol.
19
CA 02710054 2011-12-23
Data presented in Table 2 reveal that the extract of the present invention
contains
substantially higher levels of NAEs (17 fold higher PEA and 26 fold higher
AEA) and
Omega-3s (particularly the DHA and EPA) relative to the commercial P.
canaliculus product.
It should be appreciated that formulations using the composition of the
present invention
may simply be an undiluted extract or may be mixed with other ingredients,
therefore diluting
the components above, but to a different extent than in the commercial P.
canaliculus
product.
The scope of the claims should not be limited by the preferred embodiments set
forth in the
examples, but should be given the broadest interpretation consistent with the
claims.
REFERENCES
1. Calignano, A., La Rana, G., Giuffrida, A. & Piomelli, D. (1998) Control of
pain
initiated endogenous cannabinoids. Nature, 394: 277-281.
2. Calignano, A., La Rana, G., & Piomelli, D. (2001). Antinociceptive activity
of the
endogenous fatty acid amide, palmitoylethanolamide. European Journal of
Pharmacology, 419(2-3), 191-198.
3. Cho, S. H., Jung, Y. B., Seong, S. C., Park, H. B., Byun, K. Y., Lee, D.
C., et al.
(2003). Clinical efficacy and safety of Lyprinol, a patented extract from New
Zealand
green-lipped mussel (Perna Canaliculus) in patients with osteoarthritis of the
hip
and knee: a multicenter 2-month clinical trial. Allergie et Immunologie,
35(6), 212-
216.
4. Cobb, C. S., & Ernst, E. (2006). Systematic review of a marine
nutriceutical
supplement in clinical trials for arthritis: the effectiveness of the New
Zealand green-
lipped mussel Perna canaliculus. Clinical Rheumatology, 25(3), 275-284.
5. Coburn, A. F., C. E. Graham, and J. Haninger. 1954. The effect of egg yolk
in diets
on anaphylactic arthritis (passive Arthus phenomenon) in the guinea pig.
Journal of
Experimental Medicine, 100: 425-35.
6. Conti, S., Costa, B., Colleoni, M., Parolaro, D., & Giagnoni, G. (2002).
Anti-
inflammatory action of endocannabinoid palmitoylethanolamide and the synthetic
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
cannabinoid nabilone in a model of acute inflammation in the rat. British
Journal of
Pharmacology, 135(1), 181-187.
7. Darmani, N.A., Izzo, A.A., Degenhardt, B., Valenti, M., Scaglinone, G.,
Capasso, R.,
Sorrentini, I., Di Marzo, V. (2005) Involvement of the cannabimimetic
compound, N-
palmitoylethanolamine, in inflammatory and neuropathic conditions: review of
the
available pre-clinical data, and first human studies. Neuropharmacology,
48:1154-
1163.
8. De Petrocellis, L., Davis, J.B. & Di Marzo, V. (2001) Palmitoylethanolamide
enhances anandamide stimulation of human vanilloid VR1 receptors. FEBS
Letters, 506(3): 253-256.
9. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A.,
Griffin, .G.,
Gibson, D., Mandelbaum, A., Etinger, A., Machoulam, R. (1992) Isolation and
structure of a brain constituent that binds to the cannabinoid receptor.
Science,
258(5090): 1946-1949.
10. Di Marzo, V., Melck, D., Orlando, P., Bisogno, T., Zagoory, 0., Bifulco,
M., Vogel, Z.
& De Petrocellis, L. (2001). Palmitoylethanolamide inhibits the expression of
fatty
acid amide hyrolase and enhances the anti-proliferative effect of anandamide
in
human breast cancer cells. Biochemical Journal, 358(pt 1): 249-255.
11. Emelyanov, A., Fedoseev, G., Krasnoschekova, 0., Abulimity, A.,
Trendeleva, T., &
Barnes, P. J. (2002). Treatment of asthma with lipid extract of New Zealand
green-
lipped mussel: a randomised clinical trial. European Respiratory Journal,
20(3), 596-
600.
12. Farquhar-Smith, W. P., & Rice, A. S. (2001). Administration of
endocannabinoids
prevents a referred hyperalgesia associated with inflammation of the urinary
bladder. Anesthesiology, 94(3), 507-513.
13. Fowler, C.J. (2003) Plant-derived, synthetic and endogenous cannabinoids
as
neuroprotective agents non-psychoactive cannabinoids, `entourage' compounds
and inhibitors of N-acyl ethanolamine breakdown as therapeutic strategies to
avoid
psychotropic effects. Brain Research Reviews, 41: 26-43.
21
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
14. Gibson,. S. L. M., and R. G. Gibson (1998). The treatment of arthritis
with a lipid
extract of Perna canaliculus: a randomized trial. Complementary Therapies in
Medicine, 6: 122-26.
15. Gruenwald, J., Graubaum, H.-J., Hansen, K., & Grube, B. (2004). Efficacy
and
tolerability of a combination of Lyprinol and high concentrations of EPA and
DHA in
inflammatory rheumatoid disorders. Advances in Therapy, 21(3), 197-201
16. Gulaya, N.M., Kuzmenko, A.I., Margitich, V.M., Govseeva, N.M., Melnichuk,
S.D.,
Goridko, T.M. & Zhukov, A.D (1998). Long-chain N-acylethanolamines inhibit
lipid
peroxidation in rat liver mitochondria under acute hypoxic hypoxia. Chemistry
&
Physics of Lipids, 97(1): 49-54.
17. Jaggar, S. I., Hasnie, F. S., Sellaturay, S., & Rice, A. S. (1998). The
anti-
hyperalgesic actions of the cannabinoid anandamide and the putative CB2
receptor
agonist palmitoylethanolamide in visceral and somatic inflammatory pain. Pain,
76(1-2), 189-199.
18. Kahlich, R., Klima, J., Cihla, F., Frankova,.V., Masek, K., Rosicky, M.,
Matousek, F.
& Bruthans, J. (1979) Studies on prophylactic efficacy of N-2-Hydroxyethyl
palmitide
(Impulsin) in acute respiratory infections. Serologically controlled field
trials.
Journal of Hygiene, Epidemiology, Microbiology and Immunology, 23(1): 11-24.
19. Lambert, D.M., Vandevoorde, S., Jonsson, K-O. & Fowler, C.J. (2002) the
palm itoylethanolamide family: a new class of anti-inflammatory agents?
Current
Medicinal Chemistry, 9(6): 663-674.
20. Lau, C. S., Chiu, P. K. Y., Chu, E. M. Y., Cheng, I. Y. W., Tang, W. M.,
Man, R. Y.
K., et al. (2004). Treatment of knee osteoarthritis with Lyprinol, lipid
extract of the
green-lipped mussel - a double blind placebo-controlled study. Progress in
Nutrition,
6(1), 17-31.
21. Lee, Y. A., Wahn, U., Kehrt, R., Tarani, L., Businco, L., Gustafsson, D.,
et al.
(2000). A major susceptibility locus for atopic dermatitis maps to chromosome
3q21.
Nature Genetics, 26(4), 470-473.
22. Lo Verme, J., Fu, J., Astarita, G., La Ranna, G., Russo, R., Calignano, A.
&
Piomelli, D. (2005a) The nuclear receptor peroxisome proliferator-activated
22
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
receptor-alpha mediates the anti-inflammatory actions of
palmitoyleth.anolamide.
Molecular Pharmacology, 67: 15-19.
23. Lo Verme, J., La Ranna, G., Russo, R., Calignano, A. & Piomelli, D.
(2005b). The
search for the palmitoylethanolamide receptor. Life Sciences, 77: 1685-1698.
24. Masek, K., Perlik, F., Klima, J. & Kahlich, R. (1974) Prophylactic
efficacy of N-2-
Hydoxyethyl palmitamide (Impulsin) in acute respiratory tract infections.
European
Journal of Clinical Pharmacology, 7: 415-419.
25. McPhee, S., L. D. Hodges, P. F. A. Wright, P. M. Wynne, N. Kalafatis, D.
W.
Harney, and T. A. Macrides (2007). Anti-cyclooxygenase effects of lipid
extracts
from the New Zealand green-lipped mussel, Perna canaliculus. Comparative
Biochemistry & Physiology Part B, Biochemistry & Molecular Biology, 146: 346-
56.
26. Murphy, K.J. et at. (2002) Lipid, FA and sterol composition of New Zealand
green
lipped mussel (Perna canaliculus) and Tasmanian blue mussel (Mytilus edulis).
Lipids, 37(6): 587-595.
.27. Murphy, K.J. et at. (2003) Fatty acid and sterol composition of frozen
and freeze-
dried New Zealand green lipped mussel (Perna canaliculus) from three sites in
New
Zealand. Asia Pacific Journal of Clinical Nutrition, 12(1): 50-60.
28. Murphy, K. J., Galvin, K., Kiely, M., Morrissey, P. A., Mann, N. J., &
Sinclair, A. J.
(2006). Low dose supplementation with two different marine oils does not
reduce
pro-inflammatory eicosanoids and cytokines in vivo. Asia Pacific Journal of
Clinical
Nutrition, 15(3), 418-424.
29. Rohde, H. & Ghyczy, M. (2003). Behandlung des chronischen analekzems
miteiner
endocannabinoidhaltigen pflegecreme. Haut, 7 (Band XIV): 281-282.
30. Sepe, N., L. De Petrocellis, F. Montanaro, G. Cimino, and Di Marzo, V.
(1998)
Bioactive long chain N-acylethanolamines in five species of edible bivalve
molluscs:.
possible implications for mollusc physiology and sea food industry. Biochemica
et
Biophysica Acta, 1389:101-111.
23
CA 02710054 2010-06-18
WO 2008/075978 PCT/NZ2007/000370
31. Shiels, I. A., and M. W. Whitehouse (2000). Lyprinol: anti-inflammatory
and uterine-
relaxant activities in rats, with special reference to a model for
dysmenorrhoea.
Allergie et Immunologie, 32: 279-283.
32. Sinclair, A.J., Murphy, K.J. & Li, D. (2000). Marine Lipids: overview
"news insights
and lipid composition of Lyprinol. Allergie et Immunologie, 32(7): 261-71.
33. Szepietowski, J.C., Szepietowski, T. & Reich, A. (2005). Efficacy and
tolerance of
the cream containing structured physiological lipids with endocannabinoids in
the
treatment of uremic pruritis: a preliminary study. Acta Dermatovenerol Croat.,
13(2): 97-103.
34. Tenikoff, D., K. J. Murphy, M. Le, P. R. Howe, and G. S. Howarth (2005).
Lyprinol
(stabilised lipid extract of New Zealand green-lipped mussel): a potential
preventative treatment modality for inflammatory bowel disease. Journal of
Gastroenterology, 40: 361-5.
35. Treschow, A. P., L. D. Hodges, P. F. A. Wright, P. M. Wynne, N. Kalafatis,
and T. A.
Macrides (2007). Novel anti-inflammatory omega-3 PUFAs from the New Zealand
green-lipped mussel, Perna canaliculus. Comparative Biochemistry & Physiology
Part B, Biochemistry & Molecular Biology, 147: 645-56.
36. Whitehouse, M. W., T. A. Macrides, N. Kalafatis, W. H. Betts, D. R.
Haynes, and J.
Broadbent (1997). Anti-inflammatory activity of a lipid fraction (Lyprinol)
from the NZ
Green-lipped mussel. Inflammopharmacology, 5: 237-246.
37. Whitehouse, M.W., Roberts, M.S. & Brooks, P.M. (1999). Over the counter
(OTC)
oral remedies for arthritis and rheumatism: how effective are they?
Inflammopharmacology, 7(2): 89-105.
38. Wolyniak, C.J. et al. (2005) Gas Chromatography-chemical ionization-mass
spectrometric fatty acid analysis of a commercial supercritical carbon dioxide
lipid
extract from New Zealand Green-Lipped mussel (Perna canaliculus). Lipids, 40:
355-360.
24
