U.S. patent application number 14/136916 was filed with the patent office on 2014-04-17 for osteoarthritis diet formulations.
This patent application is currently assigned to NESTEC S.A.. The applicant listed for this patent is Nestec S.A.. Invention is credited to Steven S. HANNAH, Mark K. WALDRON.
Application Number | 20140107063 14/136916 |
Document ID | / |
Family ID | 34981795 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140107063 |
Kind Code |
A1 |
WALDRON; Mark K. ; et
al. |
April 17, 2014 |
OSTEOARTHRITIS DIET FORMULATIONS
Abstract
Dietary formulations and methods for treating, preventing or
delay onset of osteoarthritis in canines and other animals are
disclosed. The formulations are enriched in n-3 fatty acids and
limited in n-6 fatty acids.
Inventors: |
WALDRON; Mark K.; (Sunset
Hill, MO) ; HANNAH; Steven S.; (Chesterfield,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nestec S.A. |
Vevey |
|
CH |
|
|
Assignee: |
; NESTEC S.A.
Vevey
CH
|
Family ID: |
34981795 |
Appl. No.: |
14/136916 |
Filed: |
December 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11173360 |
Jul 1, 2005 |
8637495 |
|
|
14136916 |
|
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|
60584703 |
Jul 1, 2004 |
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Current U.S.
Class: |
514/54 ;
514/560 |
Current CPC
Class: |
A61P 19/02 20180101;
A23K 50/45 20160501; A61K 31/202 20130101; A23K 50/40 20160501;
A61P 3/04 20180101; A61P 43/00 20180101; A23K 20/163 20160501; A61P
39/06 20180101; A61K 31/225 20130101; A23K 20/158 20160501; A61P
19/10 20180101; A61P 29/00 20180101; A61K 45/06 20130101; A23K
50/42 20160501 |
Class at
Publication: |
514/54 ;
514/560 |
International
Class: |
A61K 31/202 20060101
A61K031/202; A23K 1/18 20060101 A23K001/18; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating, preventing or delaying onset of arthritis
in a mammal, comprising: (a) identifying a mammal susceptible to or
suffering from arthritis; and (b) administering to the mammal a
dietary formulation comprising long chain n-3 fatty acids in an
amount of at least about 0.1% to 1.5% by weight of the dietary
formulation, thereby treating, preventing or delaying the onset of
arthritis in the mammal.
2. The method of claim 1, wherein the arthritis is osteoarthritis
or rheumatoid arthritis.
3. The method of claim 1, wherein the mammal is a dog or cat.
4. The method of claim 1, wherein the dietary formulation comprises
at least one of .alpha.-linolenic acid, eicosapentaenoic acid,
docosapentaenoic acid or docosahexaenoic acid.
5. The method of claim 1, wherein the dietary formulation comprises
n-6 fatty acids in an amount less than about 3% of the dietary
formulation.
6. The method of claim 5, wherein the dietary formulation comprises
arachidonic acid in an amount less than about 0.125% of the dietary
formulation.
7. The method of claim 5, wherein the dietary formulation comprises
linoleic acid in an amount less than about 2% of the dietary
formulation.
8. The method of claim 1, wherein the dietary formulation comprises
n-3 and n-6 fatty acids in a ratio of at least about 1:2.
9. The method of claim 1, wherein the dietary formulation further
comprises at least one of glucosamine or chondroitin.
10. The method of claim 1, wherein the dietary formulation further
comprises at least one antioxidant.
11. The method of claim 1, wherein the dietary formulation further
comprises at least one nonsteroidal anti-inflammatory drug.
12. The method of claim 1, wherein the dietary formulation is in
the form of a dietary supplement.
13. The method of claim 3, wherein the formulation is in the form
of a pet food or pet treat for the dog or cat.
14. The method of claim 1, wherein the dietary formulation further
comprises ingredients to promote weight loss in the mammal.
15. The method of claim 1, further comprising subjecting the mammal
to caloric restriction, or controlled exercise, or a combination
thereof, to promote weight loss.
16. A method of decreasing production of at least one matrix
metalloproteinase in synovial fluid of a mammal comprising: (a)
identifying a mammal in need of decreased production of at least
one matrix metalloproteinase in its synovial fluid; and (b)
administering to the mammal a dietary formulation comprising long
chain n-3 fatty acids in an amount of at least about 0.1% to 1.5%
by weight of the formulation, thereby decreasing the production of
the at least one matrix metalloproteinase in the synovial fluid of
the mammal.
17. The method of claim 18, wherein the matrix metalloproteinase is
MMP-2 or MMP-9.
18. A method of decreasing production of inflammatory cytokines in
a mammal comprising: (a) identifying a mammal in need of decreased
production of inflammatory cytokines; and (b) administering to the
mammal a dietary formulation comprising long chain n-3 fatty acids
in an amount of at least about 0.1% to 1.5% by weight of the
formulation, thereby decreasing the production of the inflammatory
cytokines in the mammal.
19. The method of claim 20, wherein the inflammatory cytokine is
interleukin-1, interleukin-6 or tissue necrosis factor-.alpha..
20. A method of reducing arachidonic acid in the membranes of a
mammal in vivo, comprising (a) identifying a mammal in need of
reduced arachidonic acid in its membranes in vivo; and (b)
administering to the mammal a dietary formulation comprising long
chain n-3 fatty acids in an amount of at least about 0.1% to 1.5%
by weight of the formulation, thereby reducing the arachidonic acid
in the membranes of the mammal in vivo.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Divisional of U.S. application Ser. No. 11/173,360, filed
Jul. 1, 2005, which claims benefit of U.S. Provisional Application
No. 60/584,703, filed Jul. 1, 2004, the entire contents of both of
which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods of treating canines and
other animals with osteoarthritis, and specially-formulated foods,
supplements and pharmaceuticals to treat, prevent or delay onset of
osteoarthritis in canines and other animals, and to maintain
healthy joints in animals.
BACKGROUND OF THE INVENTION
[0003] Osteoarthritis (OA), also called degenerative joint disease,
is the most prevalent joint disorder in humans and animals (Romich,
J. A. (1994) Top. Vet. Med. 5:16-23; Brooks, P. (2003) Bull. World
Health Org. 81:689-690). As many as 20% of adult dogs are affected
with OA, and suffer pain and disability as a result (Roush et al.
(2002) Vet. Med. 97:108-112). OA can be defined as a disorder of
movable joints, with associated deterioration of articular
cartilage; osteophyte formation and bone remodeling; and changes in
periarticular tissues. Although the condition is classified as a
noninflammatory arthropathy, a low-grade, nonpurulent inflammation
is common and several inflammatory components have been strongly
associated with OA (Johnston et al. (1997) Vet. Clin. N Am. Sm.
Anim. Pract. 27:699-723; Amin et al. (1997) J. Clin. Invest.
99:1231-1237; Brooks et al. (2003) Bull. World Health Org.
81:689-690; Haynes et al. (2002) Clin. Immunol. 105:315-325). On a
cellular and biochemical level, OA is associated with increases in
degradative enzymes (especially the matrix metalloproteinases)
released from chondrocytes in response to inflammatory cytokines.
Inflammatory cytokines, such as interleukin-1.beta. (IL-1),
interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF.alpha.),
as well as other inflammatory mediators, are increased in the
synovial fluid of patients with OA.
[0004] Matrix metalloproteinases (MMPs), which are variably
produced by chondrocytes, leukocytes and fibroblasts, include
collagenases, stromelysins, gelatinases, elastase and others. All
these enzymes break down cartilage matrix in some manner, and play
an important role in physiologic remodeling of cartilage and other
connective tissues. In OA, MMPs degrade glycosaminoglycans,
including matrix glycoproteins, and collagen. They also reduce
hyaluronic acid concentrations in the synovial fluid, leading to
less viscous synovial fluid and impairing joint lubrication. Under
normal conditions, the degradative processes of MMPs are
appropriately balanced through the inhibitory function of tissue
inhibitors of metalloproteinases (TIMPs). However, in OA, this
balance is disrupted, with a disproportionate increase in MMPs. In
addition, inflammatory cytokines, especially IL-1 and TNF.alpha.,
stimulate the activation and release of MMPs.
[0005] Numerous studies, in dogs and other species, have documented
increases in active MMPs, reductions in TIMP, or both, in OA. For
example, it has been demonstrated that the degree of cartilage
degradation in knee OA, as determined by arthroscopy, was strongly
related to the activities of MMP-2 and MMP-13, as well as to the
reduced inhibitory effect of TIMP-2 on MMP-2. Synovial fluid from
dogs with naturally occurring OA has been shown to have higher
MMP-2 activity, and dramatic increases in MMP-9 activity, compared
to healthy controls (Volk S. W. et al. (2003) Am. J. Vet. Res.
64(10):1225-1233). MMP-9 has been correlated with rapidly
destructive OA in the hip joint of women undergoing total hip
replacement. Similarly, MMP-3 and MMP-9 were shown to be increased
in blood, and MMP-1, MMP-3, MMP-9 and TIMP-1 all were shown to be
increased in tissue samples from patients with this severe form of
OA.
[0006] Given their important role in OA, it has been suggested that
MMPs could serve not only as a therapeutic target for agents aimed
at ameliorating cartilage destruction, but also may serve as useful
markers for diagnosing and monitoring the progression of OA. An
increase in MMP activity is stimulated by prostaglandins, including
prostaglandin E.sub.2 (PGE.sub.2), which may be inhibited by
non-steroidal anti-inflammatory drugs or other compounds that
decrease PGE.sub.2 production.
[0007] The cytokines believed to be of greatest importance in OA
include IL-1, IL-6 and TNF.alpha.. The cytokines and other
inflammatory mediators in OA come from macrophages, lymphocytes,
fibroblasts, synoviocytes and chondrocytes. Elevated concentrations
of IL-1 and TNF.alpha. cause synovial inflammation as well as
degradation of cartilage and proteoglycans through activation of
MMPs. IL-1 stimulates the release of PGE.sub.2 from fibroblasts,
which subsequently stimulate pain receptors. In addition, these
cytokines stimulate the production of inflammatory free radicals,
especially nitric oxide (NO).
[0008] The activity of IL-6 in synovial fluid is greatly increased
in both dogs and humans suffering from OA. IL-6 can promote
anabolic activity in OA through inhibition of MMP activation and
promotion of matrix synthesis. On the other hand, IL-6 can
stimulate MMP-2, MMP-9 and MMP-13. Thus, this pleiotropic cytokine
helps reduce proteoglycan loss in the acute phase of OA, but
enhances osteophyte formation in chronic phases. Several studies
using IL-6.sup.-/- knock-out mice models have shown that IL-6 is
critical to the development of arthritic lesions.
[0009] Other inflammatory agents involved in the pathogenesis of OA
include the eicosanoids PGE.sub.2, thromboxane A.sub.2 (TXA.sub.2)
and leukotriene B.sub.4 (LTB.sub.4), produced from arachidonic acid
via cyclooxygenase-2 (COX-2) or 5-lipooxygenase (LOX) enzymes. The
activity of these enzymes, and resulting eicosanoids, are increased
in OA: osteoarthritic cartilage spontaneously releases 50 times
more PGE.sub.2 compared to normal cartilage. LTB.sub.4 promotes the
synthesis and release of IL-1 and TNF.alpha.. Further, LTB.sub.4 is
a potent chemotactic agent and can increase neutrophil-induced
damage to local tissues. TXA.sub.2 stimulates monocytes to release
TNF.alpha. and IL-1, which subsequently promote MMP production and
joint destruction. PGE.sub.2 promotes local inflammation and pain.
It can promote osteoclastic bone resorption, increased destruction
of Type II collagen and loss of proteoglycans. PGE.sub.2 stimulates
IL-6 release from fibroblasts, and it also sensitizes chondrocytes
to the effects of the free radical NO Inhibition of the COX-2
enzyme results in a decrease in PGE.sub.2, as well as a reduction
in IL-6.
[0010] There is no known cure for OA, so treatment is focused on
controlling pain, improving joint function and slowing the
degenerative process within the joint. Therapy usually involves
weight management, controlled exercise, and anti-inflammatory and
analgesic medications. It may also include nutritional supplements
to help reduce inflammatory mediators, promote chondrocyte health
and repair, and reduce oxidative damage.
[0011] Inhibition of the COX-2 enzyme responsible for PGE.sub.2
production is one means of providing relief for OA patients.
Another means of reducing PGE.sub.2 production is through the use
of dietary long chain omega-3 (n-3) polyunsaturated fatty acids
(PUFA), which compete with arachidonic acid as substrates for the
COX and LOX enzymes. Dietary long chain n-3 PUFA also suppress the
pro-inflammatory mediators IL-1, IL-2 and TNF in cartilage tissue
(Curtis, C. L. et al. (2000) J. Biol. Chem. 275(2):721-724).
[0012] Polyunsaturated fatty acids in both the n-6 and n-3 families
can have immunomodulatory effects. The primary n-6 fatty acid in
canine cell membranes is arachidonic acid (AA; 20:4n-6), which
serves as the precursor for the production of PGE.sub.2, TXA.sub.2
and LTB.sub.4, potent inflammatory mediators in OA.
[0013] Polyunsaturated fatty acids of the omega-3 (n-3) or omega-6
(n-6 type) are not synthesized de novo in animal tissue and are
required for normal cellular function. Thus, they are considered
essential. The essential polyunsaturated fatty acids are linoleic
acid (LA: 18:2n-6) and .alpha.-linolenic acid (ALA; 18:3n-3). When
an animal is fed with a source of n-3 or n-6 polyunsaturated fatty
acids, including 18:2n-6, 18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-6,
there is a corresponding enrichment of n-3 and n-6 highly
unsaturated fatty acids (HUFAs), specifically 20:4n-6, 20:5n-3,
22:5n-3, 22:6n-3, into the circulation and in tissue enrichment.
Because the precursors of the n-3 and n-6 HUFAs can only be
obtained from dietary sources, their relative abundance in tissues
is limited by the availability of these precursors in the diet.
[0014] If the diet is enriched with long chain n-3 PUFA,
specifically eicosapentaenoic acid (EPA; 20:5n-3) and
docosahexaenoic acid (DHA; 22:6n-3), part of the AA in cell
membranes will be replaced by these long chain n-3 fatty acids. EPA
can serve as alternate substrate for the COX-2 and 5-LOX enzymes,
resulting in a different and less inflammatory set of compounds,
e.g., PGE.sub.3, TXA.sub.3 and LTB.sub.5 instead of PGE.sub.2,
TXA.sub.2 and LTB.sub.4.
[0015] The majority of clinical studies evaluating long chain n-3
PUFA in arthritis have been in human patients with rheumatoid
arthritis. Most of those studies showed positive benefits from
long-chain n-3 PUFA supplementation. Patients were able to reduce
or discontinue the use of non-steroidal anti-inflammatory drugs
(NSAIDs) without experiencing pain or joint stiffness. The
beneficial response appeared to be directly linked to the dosage
and duration of time receiving the long chain n-3 PUFA supplements.
Similar effects have been shown in dogs with OA. Twenty-two dogs
with OA of the hip were given a fatty acid supplement marketed for
dogs with inflammatory skin conditions (DVM Derm Caps, DVM
Pharmaceuticals, Miami, Fla.) (Miller et al. (1992) Canine Pract.
17:6-8). When dosed according to the manufacturer's recommendation,
13 of 22 dogs showed noticeable improvement in their arthritic
symptoms within two weeks (Miller et al., 1992, supra).
[0016] Glucosamine, an amino-sugar, is the principal component of
the O-linked and N-linked glycosaminoglycans (GAGs) that form the
matrix in connective tissues. Hyaluronan and keratan sulfate are
composed, in part, of repeating units of acetyl glucosamine. A
decrease in glucosamine synthesis by chondrocytes has been
implicated in the decline in matrix GAGs found in OA. Oral
supplementation with glucosamine in the management of OA has been
evaluated. Essentially all trials evaluating glucosamine have been
done with a purified salt, such as glucosamine sulfate or
glucosamine hydrochloride. The applicability of these data to
glucosamine from natural sources (animal or poultry cartilage) has
not been described.
[0017] More than 50% of orally administered glucosamine is
non-ionized at the physiologic pH of the small intestine and, as a
small molecule, is readily absorbed. Most orally administered
glucosamine is oxidized, with 70% of the associated radiolabel
detected in exhaled CO.sub.2. However, approximately 10% is
retained in tissue. Glucosamine has a stimulatory effect on
chondrocytes, and is incorporated into the proteoglycans and
collagen of extracellular matrix.
[0018] Several short and long-term, double-blinded, randomized
trials evaluating glucosamine supplementation in human patients
with OA of the knee were recently reviewed via meta-analysis. These
studies documented significant improvement in clinical signs of OA
with 1500 mg glucosamine per day. Two studies followed patients for
three years and documented that oral glucosamine efficiently
inhibited the long-term progression of OA. Similar studies on
glucosamine alone in dogs are lacking. However, several in vitro
and in vivo canine studies showed a benefit to a combination of
glucosamine and chondroitin sulfate.
[0019] Oxidative stress plays an important role in both
inflammation and tissue destruction in arthritis. Arthritic
patients have reduced concentrations of serum vitamins A, E and C
and other antioxidants, as well as increased markers of oxidative
damage. These anomalies could be reversed with antioxidant
supplementation. Several studies support the benefit of
supplemental antioxidants for controlling the oxidative damage in
OA.
[0020] In addition to nutrient modifications that may help address
changes associated with OA directly, dogs need appropriately
balanced nutrition to support normal maintenance and regeneration.
Dietary deficiencies have been reported for antioxidant nutrients,
B-vitamins, zinc, calcium, magnesium and selenium. Each of these
nutrients plays a role in the normal maintenance of cartilage and
other tissues. Therefore, it is important that dogs with OA receive
diets that provide complete and balanced nutrition.
[0021] In addition to providing a source of amino acids for
proteoglycan and collagen synthesis, dietary proteins are important
for their role in helping to maintain an optimum body condition.
Protein has several physiologic effects that may be beneficial for
weight control: protein stimulates metabolism and protein turnover,
induces thermogenesis and promotes satiety. During weight loss and
subsequent weight maintenance, increased protein intake promotes
loss of body fat with retention of lean body mass. These features
of protein may be beneficial to help address excess body weight in
dogs with OA.
[0022] Standard medical care for arthritic dogs includes weight
management, controlled exercise, and anti-inflammatory and
analgesic medications. There is a need in the art for additional
methods of therapy for canines and other animals with
osteoarthritis, as well as therapies for humans to reduce the
effects of osteoarthritis.
SUMMARY OF THE INVENTION
[0023] The invention provides dietary formulations and methods for
treating a canines and other animals with osteoarthritis. One
aspect of the invention features a dietary formulation comprising
long chain n-3 fatty acids, such as .alpha.-linolenic acid (ALA),
eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) or
docosahexaenoic acid (DHA), in an amount of at least about 0.1-1.5%
by weight of the formulation. In certain embodiments, the long
chain n-3 fatty acids are present in an amount of at least about
0.2% to 0.6% of the dietary formulation, or at least about 0.3% to
0.4% of the dietary formulation. In certain embodiments the dietary
formulation comprises about 0.2-0.6% EPA or DHA.
[0024] In some embodiments, the dietary formulation contains n-6
fatty acids in an amount less than about 3% of the dietary
formulation. In certain embodiments, the formulation contains less
than about 0.125% arachidonic acid. In other embodiments, the
formulation contains less than about 1-2% linoleic acid.
[0025] In some embodiments, the dietary formulation contains n-3
and n-6 fatty acids in a ratio of at least about 1:2. In certain
embodiments, the ratio of n-3 to n-6 fatty acids is at least about
1:1 or at least about 2:1.
[0026] The dietary formulation may comprise additional ingredients,
independently selected from glucosamine, chondroitin, antioxidants
and nonsteroidal anti-inflammatory drugs.
[0027] In certain embodiments, the dietary formulation is a pet
food or pet treat product for a dog or cat. Such pet food products
may be dry (kibble), semi-moist or moist (canned) food products. In
other embodiments, the dietary formulation may be a nutritional
supplement.
[0028] Another aspect of the invention features a method of
treating, preventing or delaying onset of arthritis in a mammal,
comprising administering to the mammal a dietary formulation of the
type described above. In various embodiments, the arthritis is
osteoarthritis or rheumatoid arthritis. In certain embodiments of
the method, the mammal is a companion animal such as a dog or cat.
In other embodiments, the mammal may be a human.
[0029] In certain embodiments, the method utilizes a dietary
formulation further comprises ingredients to promote weight loss in
the mammal. The method may also further comprise subjecting the
mammal to caloric restriction to promote weight loss, or providing
controlled exercise to the mammal.
[0030] Another aspect of the invention features a method of
decreasing production of at least one matrix metalloproteinase in
synovial fluid of a mammal comprising administering to the mammal a
dietary formulation comprising long chain n-3 fatty acids in an
amount of at least about 0.1% to 1.5% by weight of the formulation,
as described herein. In certain embodiments, the matrix
metalloproteinase is MMP-2 or MMP-9.
[0031] Another aspect of the invention features a method of
decreasing production of inflammatory cytokines in a mammal
comprising administering to the mammal a dietary formulation
comprising long chain n-3 fatty acids in an amount of at least
about 0.1% to 1.5% by weight of the formulation, as described
herein. In certain embodiments, the inflammatory cytokine is
interleukin-1, interleukin-6 or tissue necrosis factor-.alpha..
[0032] Another aspect of the invention features a method of
reducing arachidonic acid in the membranes of a mammal in vivo,
comprising administering to the mammal a dietary formulation
comprising long chain n-3 fatty acids in an amount of at least
about 0.1% to 1.5% by weight of the dietary formulation, as
described herein.
[0033] Other features and advantages of the present invention will
become apparent from the following description and appended claims,
taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a histogram showing concentration of plasma
arachidonic acid (mg/dl) in animals provided n-3 fatty acid
enriched diets (TRT) or control diets (CTL) before and following
corrective surgery to repair ruptured cruciate ligaments. *
p<0.05. Open bars (TRT); stippled bars (CTL).
[0035] FIG. 2 is a histogram showing concentration of plasma
eicosopentaenoic acid (mg/dl) in animals provided n-3 fatty acid
enriched diets (TRT) or control diets (CTL) before and following
corrective surgery to repair ruptured cruciate ligaments. *
p<0.05 on all days except day -7. Open bars (TRT); stippled bars
(CTL).
[0036] FIG. 3 is a histogram showing concentration of plasma
docosahexaenoic acid (mg/dl) in animals provided n-3 fatty acid
enriched diets (TRT) or control diets (CTL) before and following
corrective surgery to repair ruptured cruciate ligaments. *
p<0.05 on all days except day -7. Open bars (TRT); stippled bars
(CTL).
[0037] FIG. 4 is a histogram showing amounts of synovial fluid n-6
fatty acids (AA--arachidonic acid; LA--linolenic acid) (g/100 g
sample) in animals provided n-3 fatty acid enriched diets (TRT) or
control diets (CTL) before and following corrective surgery to
repair ruptured cruciate ligaments. Samples were taken on day 28
following surgery. * p<0.005. Open bars (TRT); stippled bars
(CTL).
[0038] FIG. 5 is a histogram showing amounts of synovial fluid n-3
fatty acids (EPA- eicosopentaenoic acid; DHA--docosahexaenoic acid)
(g/100 g sample) in animals provided n-3 fatty acid enriched diets
(TRT) or control diets (CTL) before and following corrective
surgery to repair ruptured cruciate ligaments. Samples were taken
on day 28 following surgery. * p<0.005. Open bars (TRT);
stippled bars (CTL).
[0039] FIG. 6 is a histogram showing the percent change in levels
of plasma bicyclo PGE.sub.2 in animals provided n-3 fatty acid
enriched diets (TRT) or control diets (CTL) before and following
corrective surgery to repair ruptured cruciate ligaments. Percent
change was measured as a change from initiation to the end of the
study. * p<0.05. Hatched bars (TRT); stippled bars (CTL).
[0040] FIG. 7 is a histogram showing concentration of pro-MMP-2 and
active MMP-2 (ng/10 .mu.l) in the synovial fluid of non-surgical
joints of animals provided n-3 fatty acid enriched diets (TRT) or
control diets (CTL) before and following corrective surgery to
repair ruptured cruciate ligaments. * p<0.05 due to diet. Open
bars (TRT); stippled bars (CTL).
[0041] FIG. 8 is a histogram showing concentration of pro-MMP-9 and
active MMP-9 (ng/10 .mu.l) in synovial fluid of non-surgical joints
of animals provided n-3 fatty acid enriched diets (TRT) or control
diets (CTL) before and following corrective surgery to repair
ruptured cruciate ligaments. * p<0.05 due to diet. Open bars
(TRT); stippled bars (CTL).
[0042] FIG. 9 is a graph showing a time course of concentration of
TIMP-2 in synovial fluid (ng/10 .mu.l) of animals provided n-3
fatty acid enriched diets (TRT -T-) or control diets (CTL
-.gamma.-) before and following corrective surgery to repair
ruptured cruciate ligaments. * p<0.05. Open bars (TRT); stippled
bars (CTL).
[0043] FIG. 10 is a diagram illustrating mechanisms by which n-3
fatty acids may affect joint destruction.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0044] The reference works, patents, patent applications, and
scientific literature that are referred to herein are hereby
incorporated by reference in their entirety to the same extent as
if each was specifically and individually indicated to be
incorporated by reference. Any conflict between any reference cited
herein and the specific teachings of this specification shall be
resolved in favor of the latter.
[0045] Various definitions are made throughout this document. Most
words have the meaning that would be attributed to those words by
one skilled in the art. Words specifically defined either below or
elsewhere in this document have the meaning provided in the context
of the present invention as a whole and as are typically understood
by those skilled in the art. Any conflict between an art-understood
definition of a word or phrase and a definition of the word or
phrase as specifically taught in this specification shall be
resolved in favor of the latter. Headings used herein are for
convenience and are not to be construed as limiting.
[0046] The present invention relates to any animal, preferably a
mammal; particularly cats and dogs. In some embodiments, the
methods and dietary formulations set forth herein are applicable to
humans, as will be appreciated by the person of skill in the
art.
[0047] As used herein, "treating, preventing or delaying onset" in
connection with inflammatory conditions such as arthritis, refers
to partially or fully ameliorating the condition or one or more
symptoms associated with the condition, completely inhibiting the
occurrence of the condition, or retarding the presentation or
development of the condition.
[0048] The invention provides dietary formulations for canines and
other animals rich in n-3 fatty acids. This class of fatty acids,
also referred to as omega 3 fatty acids, typically contain 12-26
carbon atoms containing one or more carbon-carbon double bonds.
Preferred for use in the present invention are long chain (18 or
more carbon atoms) polyunsaturated n-3 fatty acids (LPUFAs).
Examples of such n-3 fatty acids include but are not limited to the
essential n-3 fatty acid, .alpha.-linolenic acid (LNA or ALA), and
other n-3 fatty acids, such as eicosapentaenoic acid (EPA),
docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Dietary
sources of n-3 fatty acids include, but are not limited to flax
seed, flax oil, walnuts, cold-pressed canola oil, wheat germ, dark
green, leafy vegetables, and oily cold-water fish.
[0049] As discussed in greater detail below, the formulations
preferably contain an amount of long chain n-3 fatty acids that is
greater than about half the amount of any long chain n-6 fatty
acids present in the formulation. Examples of n-6 fatty acids
include the essential n-6 fatty acid linoleic acid (LA) and other
n-6 fatty acids, such as arachidonic acid (AA) and .gamma.-linoleic
acid (GLA). Dietary sources of n-6 fatty acids include, but are not
limited to soy oil, sunflower seeds, safflower seeds, pumpkin
seeds, sesame seeds, tahini, corn oil, peanuts and most nuts. In
general, the amounts of n-6 fatty acids are limited. For example,
the amount of arachidonic acid in the formulation is less than
about 0.125% by weight. Further, the linoleic acid content of the
formulation is in the range of about 1-2% by weight.
[0050] The formulations of the invention contain an effective
amounts of long chain n-3 fatty acids. As used herein "effective
amount" refers to an amount of long chain n-3 fatty acids that
ameliorates at least one sign or symptom of osteoarthritis,
including, but not limited to pain, lameness, cartilage loss, joint
swelling, crepitus, difficulty in posturing to defecate or urinate,
stiffness, gait abnormality, joint laxity, joint effusion,
increased synovial fluid volume, and the like.
[0051] In some embodiments, the formulation contains long chain n-3
fatty acids in an amount to deliver about at least about 20 mg/kg
bodyweight/day to the animal. In other embodiments, the formulation
contains long chain n-3 fatty acids in an amount to deliver at
least about 30 mg/kg/day to the animal. In other embodiments, the
formulation contains long chain n-3 fatty acids in an amount to
deliver at least about 40 mg/kg/day to the animal. In other
embodiments, the formulation contains long chain n-3 fatty acids in
an amount to deliver at least about 50 mg/kg/day to the animal. In
other embodiments, the formulation contains long chain n-3 fatty
acids in an amount to deliver at least about 60 mg/kg/day to the
animal. In other embodiments, the formulation contains long chain
n-3 fatty acids in an amount to deliver at least about 70 mg/kg/day
to the animal. In other embodiments, the formulation contains long
chain n-3 fatty acids in an amount to deliver at least about 80
mg/kg/day to the animal. In other embodiments, the formulation
contains long chain n-3 fatty acids in an amount to deliver at
least about 90 mg/kg/day to the animal. In other embodiments, the
formulation contains long chain n-3 fatty acids in an amount to
deliver at least about 100 mg/kg/day to the animal. In other
embodiments, the formulation contains long chain n-3 fatty acids in
an amount to deliver at least about 110 mg/kg/day to the animal In
other embodiments, the formulation contains long chain n-3 fatty
acids in an amount to deliver at least about 120 mg/kg/day to the
animal. In some embodiments of the invention the long chain n-3
fatty acid is one or more n-3 fatty acids selected from
.alpha.-linolenic acid (ALA), eicosapentaenoic acid (EPA),
docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).
[0052] The amount of long chain n-3 fatty acids as a percentage of
the dietary formula is in the range of about 0.1-1.5% of the
dietary formulation on a dry matter basis, though a greater
percentage can be supplied. In various embodiments, the amount is
about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%,
1.2%, 1.3%, 1.4% or 1.5% of the dietary formulation on a dry matter
basis. In some embodiments of the invention, the long chain n-3
fatty acid is eicosapentaenoic acid (EPA). In some embodiments, the
long chain n-3 fatty acid is docosapentaenoic acid (DPA). In other
embodiments, the long chain n-3 fatty acid is docosahexaenoic acid
(DHA). In still other embodiments, the long chain n-3 fatty acid is
.alpha.-linolenic acid (ALA). In other embodiments, the dietary
formulation contains a mixture of two or more of these n-3 fatty
acids.
[0053] Average diets may contain a ratio of about 1:10 n-3:n-6
fatty acids. The present formulations limit n-6 fatty acids
(particularly AA and LA) to achieve ratios of n-3:n-6 that favor a
higher proportion of n-3 fatty acids than an average diet. In some
embodiments, the ratio of n-3:n-6 is greater than about 1:9, 1:8,
1:7 or 1:6. In other embodiments, the ratio of n-3:n-6 is greater
than about 1:5, 1.4 or 1:3. In another embodiment, the ratio of
n-3:n-6 is greater than about 1:2. In other embodiments, the ratio
of fatty acids is such that there is an equal or greater proportion
of n-3 fatty acids than n-6 fatty acids. For example, the ratio of
n-3:n-6 may be about 1:1 to about 15:1. In some embodiments, the
ratio is about 2:1 to about 3:1, but can be greater, e.g., 4:1,
5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1 or 14:1.
[0054] While not wishing to be bound by any particular theory of
operability, it is believed that the long chain n-3 fatty acids in
the formula, when consumed by the animal, replace arachidonic acid
in the membranes of the cells and will be used for the production
of anti-inflammatory eicosanoids, driving production of such
compounds as prostaglandin E.sub.3 (PGE.sub.3), thromboxane A.sub.3
(TXA.sub.3) and leukotriene B.sub.5 (LTB.sub.5), rather than
PGE.sub.2, TXA.sub.2 and LTB.sub.4. The reduction of PGE.sub.2
leads to a decrease in pro-MMP-2 and pro-MMP-9, thereby lessening
the proteolytic activation of these metalloproteinases, resulting
in reduction of inflammation and pain in the joints. In addition,
it is believed that the increase in long chain n-3 fatty acids
contributes to an increase in tissue inhibitor of
metalloproteinases-2 (TIMP-2). which blocks the activation of MMP-2
and MMP-9 by saturating binding sites of other MMPs (such as
membrane-type-1 matrix metalloproteinase) that are believed
necessary for the first activation step of MMP-2 and MMP-9. FIG. 10
diagrammatically illustrates some of these mechanisms.
[0055] Indeed, it has been demonstrated in accordance with the
present invention that a dietary formulation enriched in n-3 fatty
acids promotes an increase in TIMP-1 and a decrease in two matrix
metalloproteinases involved in the breakdown of gelatinase: MMP-2
(Gelatinase-A) and MMP-9 (Gelatinase B) (see Example 2, referring
to FIGS. 7, 8 and 9). The formulations of the invention are
expected to find practical utility in reducing levels of these and
other matrix metalloproteinases, including, but not limited to: (1)
collagenases such as interstitial collagenase (MMP-1), neutrophil
collagenase (MMP-8) and collagenase-3 (MMP-13); (2) stromelysins
such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and
matrilysin (MMP-7); and (3) membrane-type MMPs such as MMP-14 and
MT1-MMP.
[0056] The formula of the invention may also contain additional
factors to support joint health such as, but not limited to,
glucosamine and chondroitin sulfate.
[0057] In some embodiments glucosamine is provided in an amount of
500-1000 ppm of food. In other embodiments, glucosamine is provided
in an amount of at least about 1000-1500 ppm. In other embodiments,
glucosamine is provided in an amount of at least about 1500-2000
ppm or more, provided no untoward effect results from
administration of glucosamine.
[0058] In some embodiments chondroitin sulfate is provided in an
amount of to deliver about 100-300 mg/day. In other embodiments,
chondroitin sulfate is provided in an amount to deliver at least
about 300-500 mg/day. In other embodiments, chondroitin sulfate is
provided in an amount to deliver at least about 500-700 mg/day or
more, provided no untoward effect results from administration of
chondroitin sulfate.
[0059] The formulation may also include antioxidants, including,
but not limited to vitamin A, vitamin C, vitamin E, riboflavin,
selenium, and pyridoxine.
[0060] In some embodiments selenium is provided in an amount of
0.5-0.7 mg/kg of food. In other embodiments, selenium is provided
in an amount of at least about 0.7-0.9 mg/kg. In other embodiments,
selenium is provided in an amount of at least about 0.9-1.1 mg/kg
or more, provided no untoward effect results from administration of
selenium.
[0061] In some embodiments, vitamin A is provided in an amount of
at least about 20-30 IU/g of food. In other embodiments, vitamin A
is provided in an amount of at least about 30-40 IU/g of food. In
other embodiments, vitamin A is provided in an amount of at least
about 40-50 IU/g of food or more, provided no untoward effect
results from administration of vitamin A.
[0062] In some embodiments, vitamin E is provided in an amount of
at least about 0.5-1 IU/g of food. In other embodiments, vitamin E
is provided in an amount of at least about 1-1.5 IU/g of food. In
other embodiments, vitamin E is provided in an amount of at least
about 1.5-2.5 IU/g of food or more, provided no untoward effect
results from administration of vitamin E.
[0063] In some embodiments, vitamin C is provided in an amount of
at least about 50-150 ppm of food. In other embodiments, vitamin C
is provided in an amount of at least about 150-250 ppm. In other
embodiments, vitamin C is provided in an amount of at least about
250-350 ppm or more, provided no untoward effect results from
administration of vitamin C.
[0064] In some embodiments, riboflavin is provided in an amount of
at least about 5-15 mg/kg of food. In other embodiments, riboflavin
is provided in an amount of at least about 15-25 mg/kg. In other
embodiments, riboflavin is provided in an amount of at least about
25-35 mg/kg or more, provided no untoward effect results from
administration of riboflavin.
[0065] In some embodiments, pyridoxine is provided in an amount of
at least about 5-15 mg/kg of food. In other embodiments, pyridoxine
is provided in an amount of at least about 15-25 mg/kg. In other
embodiments, pyridoxine is provided in an amount of at least about
25-35 mg/kg or more, provided no untoward effect results from
administration of pyridoxine.
[0066] The formulation may be adjusted to a dietetic formulation to
allow animals to lose weight in addition to providing the
beneficial effects on osteoarthritis. Proper weight management of
animals can promote an enhanced palliative effect in addition to
the direct therapeutic effect on joints.
[0067] It may be desirable to adjust the formulation for specific
needs of the animal, taking into account parameters such as, but
not limited to, breed, age, size, weight, and general health
status, such as the degree or stage of osteoarthritis, rheumatoid
arthritis or other diseases causing an inflammatory response in the
animal. Methods for calculating the enrichment of n-3 and n-6 fatty
acids in membranes and tissues are known in the art and can be used
to adjust for levels of the n-3 and n-6 fatty acids in the diet.
For example, such calculations are described in Bauer, J. E. et al.
(2002) J. Nutr. 132:1642 S-1645S and PCT Publication No. WO
03/092405.
[0068] The dietary formulation of the invention may be in the form
of dry food, soft/moist food or canned food. The protein content of
dry food is generally in the range of about 15-30% by weight. The
overall fat content of dry food is generally in the range of about
5-20% by weight. The carbohydrate content of dry food is in the
range of about 30-60% by weight. The moisture content of dry food
is generally less than about 15% by weight. The content of protein,
carbohydrate and fat may be adjusted to suit the special needs of
different breeds of canines as would be well-known in the art.
[0069] The protein content of soft-moist food is generally in the
range of about 10-30% by weight. The overall fat content of
soft-moist food is generally in the range of about 2-15% by weight.
The carbohydrate content of soft-moist food is in the range of
about 20-40% by weight. The moisture content of dry food is
generally less than about 20-50% by weight. The content of protein,
carbohydrate and fat may be adjusted to suit the special needs of
different breeds of canines as would be well-known in the art.
[0070] The protein content of canned food is generally in the range
of about 5-20% by weight. The overall fat content of canned food is
generally in the range of about 1-20% by weight. The carbohydrate
content of canned food is in the range of about 15-40% by weight.
The moisture content of canned food is generally less than about
80% by weight. The content of protein, carbohydrate and fat may be
adjusted to suit the special needs of different breeds of canines
as would be well-known in the art.
[0071] The dietary formulation of the invention may be also be in
the form of a nutritional supplement which may be administered
admixed with food or water, or provided separately as a
pharmaceutical dosage form. Supplements and dosage forms include,
but are not limited to, tablets (including pills, chewable tablets,
quick dissolve tablets, multi-layer tablets, bi-layer tablets and
the like), powders, elixirs, liquids, solutions, suspensions,
emulsions, capsules, caplets, lozenges, chewable lozenges, beads,
powders, granules, particles, gels, pastes, dissolvable films,
microparticles, dispersible granules, health bars, animal treats,
and combinations thereof. The preparation of the above dosage forms
are well known to persons of ordinary skill in the art.
[0072] The invention also provides methods of treating
osteoarthritis in canines by feeding canines dietary formulations
of the invention to alleviate the symptoms of osteoarthritis. The
amount of the nutrients in the dietary formulations may be adjusted
according to the specialized needs of the breed of canine and the
degree or stage of osteoarthritis experienced by the animal A
veterinarian will be able to provide guidance as to dietary
formulations to be administered to the animal as well as adjusting
other parameters of the diet (e.g., to provide for weight
management) and may also provide guidance as to the type and
duration of other therapy (e.g., pain management, exercise, and the
like).
[0073] In general, canines in need of weight management as well as
treatment for osteoarthritis will be fed a diet with reduced
calories to promote weight loss while maintaining an increased
amount of long chain n-3 fatty acids to promote alleviation of at
least one symptom of osteoarthritis. The dietary formulation may
also include other ingredients as detailed above, such as
antioxidants, non-steroidal anti-inflammatory drugs, glucosamine,
and chondroitin sulfate.
[0074] The invention also provides methods of treating healthy
animals such that onset of OA may be delayed or prevented, and to
support joint health. Thus, the dietary formulations and/or
nutritional supplements of the invention may be provided to
animals, such as canines, as a prophylactic measure to prevent or
delay the onset of OA and to maintain healthy joints. The
formulations, supplements or pharmaceuticals contain long chain n-3
fatty acids in an amount of at least about 0.1-1.5% of formulation
on a dry matter basis, and the formulations may also include n-6
fatty acids in amounts described herein, as well as antioxidants,
glucosamine, chondroitin and/or NSAIDs. The amount of the nutrients
in the dietary formulations may be adjusted according to the
specialized needs of the breed of canine, for example, and the age
of the animal, in accordance with standard procedures.
[0075] The methods and dietary formulations provided herein to
treat and prevent osteoarthritis may also be used to treat or
prevent other inflammatory conditions. In particular, the
formulations and methods are expected to be advantageous for the
treatment of rheumatoid arthritis as well, in view of the
inflammatory nature of that condition.
[0076] The invention also provides a method of decreasing
arachidonic acid in the membranes of cells of mammals, particularly
canines comprising administering a dietary formulation of the
invention to the mammal. The amount of long chain n-3 fatty acid in
the dietary formulation will be sufficient to replace arachidonic
acid in the membranes of cells of the canines. In general, the
amount of long chain n-3 fatty acids is about 0.1-1.5% of the
dietary formulation on a dry matter basis.
[0077] The invention also provides method of reducing the effects
of osteoarthritis in a mammal comprising administering a dietary
formulation comprising an increased amount of long chain n-3 fatty
acid. In some embodiments, the long chain n-3 fatty acid is in an
amount of at least about 0.1-1.5% of the dietary formulation on a
dry matter basis. In some embodiments, the amount is about 0.3% of
the dietary formulation on a dry matter basis. In some embodiments,
the amount is about 0.4% of the dietary formulation on a dry matter
basis. In some embodiments, the amount is about 0.5% of the dietary
formulation on a dry matter basis. In some embodiments, the dietary
formulation is for mammals, particularly canines. In other
embodiments, the dietary formulation is for humans.
[0078] In some embodiments, the dietary formulation for humans is
in the form of a nutrition supplement, as is known in the art.
[0079] The invention further provides methods for decreasing
production of matrix metalloproteinase in canines and other mammals
by administering the dietary formulation of the invention. The
matrix metalloproteinases include, but are not limited to MMP-2 and
MMP-9. Generally, the amount of long chain n-3 fatty acids is in
the range of about 0.1 to 1.5% of the formulation on a dry matter
basis. The amount of arachidonic acid in the formulation is limited
in some embodiments to be less than 0.125%. In other embodiments,
the amount of linoleic acid is limited to no more than 1-2% of the
dietary formulation. The formulation may also be supplemented with
antioxidants, glucosamine and at least one NSAID.
[0080] The invention also provides a method of decreasing
production of inflammatory cytokines in canines and other mammals
by administering a dietary formulation of the invention. The
inflammatory cytokines include, but are not limited to IL-1, IL-6
and TNF.alpha.. Generally, the amount of long chain n-3 fatty acids
is in the range of about 0.1 to 1.5% of the formulation. The amount
of arachidonic acid in the formulation is limited in some
embodiments to be less than 0.125%. In other embodiments, the
amount of linoleic acid is limited to no more than 1-2% of the
dietary formulation. The formulation may also be supplemented with
antioxidants, glucosamine and at least one NSAID. The dietary
formulations may also be used to decrease production of matrix
metalloproteinase and/or decrease production of inflammatory
cytokines.
[0081] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
Example 1
Experimental Design
[0082] This example sets forth a protocol for determining the
effect of enriched n-3 fatty acid diets on biochemical parameters
associated with OA in canines A double blind, randomized, and
placebo controlled design was utilized.
[0083] Twenty four dogs with degenerative osteoarthritis resulting
in clinically confirmed acute anterior cruciate ligament (ACL)
injury (rupture of the anterior cruciate ligament) were used in the
study and randomly allocated to either the treatment diet or the
control diet (n=12). Dogs were stratified by degree of OA and
ligament injury. Dogs were given routine physical examinations by
the surgical clinicians and radiographs were taken prior to the
study.
[0084] The dogs were randomly divided into two groups of twelve
dogs each:
[0085] Group 1: n-3 LC PUFA supplement (3.5% added fish oil)
[0086] Group 2 (control): containing no long chain n-3 fatty acids,
but contained tallow as the added fat source.
[0087] Dogs were fed the supplements for a duration of 63 days (7
days before the corrective surgery and 56 days after the corrective
surgery).
[0088] Synovial fluid and serum for TIMP-2, PGE.sub.2, and MMP
analysis were obtained by needle and syringe via sterile
arthrocentesis and venipuncture, respectively, at the following
time points:day -7, day 0, day 7, day 14, day 28, and day 56.
Synovial fluid was obtained from the affected (ACL ruptured) and
contralateral joints of all dogs.
[0089] Gas Chromatography, utilizing an HP 5890 gas chromatograph,
was performed to evaluate serum fatty acids and to measure N-3 LC
PUFA in serum of the subjects
[0090] Analysis of the pro- and active form of MMP-2 and MMP-9 in
all serum and synovial samples was conducted by electrophoresis
using gelatin imbedded gels. Gel analyses used NOVEX Zymogram (San
Diego, Calif.) gels, buffers, stains, and equipment. Zymography was
run as directed by standardized NOVEX instructions. Gels were
scanned on a densitometer (Molecular Dynamics, Sunnyvale, Calif.).
Each sample band was compared and quantified against the standards
run on each gel.
[0091] Serum bicyclo-PGE.sub.2, the stable metabolite of PGE.sub.2,
analysis was conducted using commercial available enzyme linked
immunosorbant assay (ELISA) kits available from Caymen (Ann Arbor,
Mich.).
Example 2
Effect of Enriched n-3 Fatty Acid Diets on Biochemical Parameters
Associated with Canine Osteoarthritis
[0092] The following results were obtained utilizing the protocol
and evaluation procedures set forth in the previous example. First,
amounts of AA, EPA and DHA in plasma was measured. Referring now to
the figures, FIG. 1 shows that plasma AA was reduced in animals
provided n-3 fatty acid-enriched diets, as compared to animals fed
control diets. FIGS. 2 and 3 respectively show that plasma EPA and
DHA were increased in animals fed the n-3 fatty acid-enriched
diets, as compared to animals fed control diets. Additionally, as
illustrated in FIG. 6, plasma bicyclo PGE.sub.2 decreased by about
10% (from entry to completion of the protocol) in animals fed the
n-3 fatty acid enriched diet, but increased by more than 20% in
animals fed the control diet.
[0093] The fatty acid composition of synovial fluid was also
examined. As illustrated in FIGS. 4 and 5, respectively, animals
fed an n-3 fatty acid-enriched diet before and after surgery
exhibited a decrease in synovial fluid n-6 fatty acids and an
increase in synovial fluid n-3 fatty acids, as compared with
animals fed a control diet. Furthermore, as shown in FIGS. 7 and 8,
pro- and active MMP-2 and MMP-9 were decreased in the synovial
fluid of animals fed the n-3 fatty acid-enriched diets, as compared
with that of animals fed the control diet. As shown in FIG. 9,
TIMP-2 was increased in the synovial fluid of animals fed the n-3
fatty acid-enriched diets, as compared with that of animals fed the
control diet.
[0094] The results described hereinabove demonstrate that dietary
enrichment of n-3 fatty acids improves several physiological and
biochemical parameters associated with canine osteoarthritis.
Example 3
Exemplary Dietary Formulation
[0095] An exemplary, non-limiting dietary formulation of the
invention is as follows:
TABLE-US-00001 Moisture 9.1% Protein 27.8% Fat 12.9% Ash 8.02%
Carbohydrate (by subtraction) 42.20% Fatty Acid Composition:
Percent of fat 14:0 3.13 14:1 0.22 15:0 0.35 16:0 20.9 16:1 5.63
17:0 0.6 18:0 8.08 18:ln-9 29.8 18:2n-6 11.8 18:3n-6 0.18 20:0 0.2
18:3n-3 1.08 20:2n-6 0.25 20:3n-6 0.32 20:4n-6 0.66 20:5n-3 3.09
22:5n-3 0.67 22:6n-3 2.66 Unknowns 4.66 Percent of formulation EPA
0.39861% Arachidonic acid <0.125% Linoleic acid 1-2%
[0096] The present invention is not limited to the embodiments
described and exemplified above, but is capable of variation and
modification within the scope of the appended claims.
[0097] What is claimed:
* * * * *