U.S. patent application number 11/634383 was filed with the patent office on 2007-06-21 for use of anabolic agents, anti-catabolic agents, antioxidant agents, and analgesics for protection, treatment and repair of connective tissues in humans and animals.
This patent application is currently assigned to NUTRAMAX LABORATORIES, INC.. Invention is credited to Carmelita Frondoza, Todd R. Henderson.
Application Number | 20070141181 11/634383 |
Document ID | / |
Family ID | 39492846 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141181 |
Kind Code |
A1 |
Henderson; Todd R. ; et
al. |
June 21, 2007 |
Use of anabolic agents, anti-catabolic agents, antioxidant agents,
and analgesics for protection, treatment and repair of connective
tissues in humans and animals
Abstract
The present invention relates to compositions for the modulation
of inflammation in connective tissues in humans and animals and the
modulation of markers of such inflammation, including COX-2,
TNF-.alpha., IL-1.beta., iNOS, p38, and chemokines, comprising any
or all of anabolic, anti-catabolic, anti-oxidant and analgesic
agents, including aminosugars, S-adenosylmethionine, arachadonic
acid, GAGs, including pentosan, collagen type II, tetracyclines or
tetracycline-like compounds, diacerin, super oxide dismutase,
L-ergothioneine, methylsulfanylmethane, one or more avocado/soybean
unsaponifiables, and an analgesic, e.g., acetaminophen, and to
methods of treating humans and animals by administration of these
novel compositions to humans and animals in need thereof.
Inventors: |
Henderson; Todd R.;
(Jarrettsville, MD) ; Frondoza; Carmelita;
(Woodstock, MD) |
Correspondence
Address: |
COVINGTON & BURLING, LLP;ATTN: PATENT DOCKETING
1201 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20004-2401
US
|
Assignee: |
NUTRAMAX LABORATORIES, INC.
Edgewood
MD
|
Family ID: |
39492846 |
Appl. No.: |
11/634383 |
Filed: |
December 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824498 |
Apr 15, 2004 |
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11634383 |
Dec 6, 2006 |
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10192318 |
Jul 11, 2002 |
6797289 |
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10824498 |
Apr 15, 2004 |
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09274881 |
Mar 23, 1999 |
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10192318 |
Jul 11, 2002 |
|
|
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09249335 |
Feb 12, 1999 |
6451771 |
|
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09274881 |
Mar 23, 1999 |
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60074594 |
Feb 13, 1998 |
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60088205 |
Jun 5, 1998 |
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Current U.S.
Class: |
424/735 ;
424/757; 514/171; 514/54; 514/62 |
Current CPC
Class: |
A61P 19/00 20180101;
A61K 36/48 20130101; A61P 29/00 20180101; A61P 19/04 20180101; A61P
43/00 20180101; A61K 36/54 20130101; A61K 31/7008 20130101; A61K
31/737 20130101; A61K 31/56 20130101; A61K 31/56 20130101; A61K
2300/00 20130101; A61K 31/7008 20130101; A61K 2300/00 20130101;
A61K 31/737 20130101; A61K 2300/00 20130101; A61K 36/48 20130101;
A61K 2300/00 20130101; A61K 36/54 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/735 ;
514/171; 514/062; 514/054; 424/757 |
International
Class: |
A61K 36/736 20060101
A61K036/736; A61K 31/56 20060101 A61K031/56; A61K 31/7008 20060101
A61K031/7008; A61K 31/737 20060101 A61K031/737; A61K 36/48 20060101
A61K036/48 |
Claims
1. A formulation for modulating inflammation associated with damage
to connective tissue in humans and animals, comprising: one or more
avocado/soybean unsaponifiables; an aminosugar; and a
glycosaminoglycan component, wherein the amounts of the one or more
avocado/soybean unsaponifiables, glucosamine, and glycosaminoglycan
component in the formulation are effective in combination to at
least one of: substantially inhibit or downregulate gene expression
of at least one of COX-2, TNF-.alpha., IL-1.beta., iNOS, p38, and
chemokines; and reduce PGE-2 levels.
2. The formulation of claim 1, wherein the amounts of the one or
more avocado/soybean unsaponifiables, aminosugar, and
glycosaminoglycan in the formulation are effective in combination
to substantially inhibit gene expression of at least one of COX-2,
TNF-.alpha., iNOS, p38, IL-1.beta., and chemokines in tissue
cells.
3. The formulation of claim 2, wherein the tissues cells are
selected from the group consisting of chondrocytes, macrophage
monocytes, and fibroblasts.
4. The formulation of claim 1, further comprising
methylsulfanylmethane (MSM).
5. The formulation of claim 1, wherein the one or more
avocado/soybean unsaponifiables comprises one or more
phytosterols.
6. The formulation of claim 5, wherein the phytosterols are
selected from the group consisting of campesterol, stigmasterol,
dihydro-brassisterol, and Beta-sitosterol.
7. The formulation of claim 1, wherein the formulation is in the
form of a powder, and wherein the powder is created by combining
the one or more avocado/soybean unsaponifiables with one or more
excipients or carriers.
8. The formulation of claim 1, wherein the aminosugar is natural,
synthetic or semi-synthetic.
9. The formulation of claim 1, wherein the one or more
avocado/soybean unsaponifiables is natural, synthetic or
semi-synthetic.
10. The formulation of claim 1, wherein the aminosugar has been
chemically modified by one or more of esterification, sulfation,
polysulfation, acetylation and methylation.
11. The formulation of claim 1, wherein the aminosugar is selected
from the group consisting of glucosamine, glucosamine salts, and
mixtures thereof.
12. The formulation of claim 1, wherein the aminosugar is selected
from the group consisting of glucosamine hydrochloride, glucosamine
sulfate, glucosamine phosphate, mannosamine and salts of
N-acetylglucosamine.
13. The formulation of claim 1, wherein the glycosaminoglycan
component comprises a natural, synthetic or semi-synthetic
glycosaminoglycan, a glycosaminoglycan-like compound, a
glycosaminoglycan precursor or fragments of a
glycosaminoglycan.
14. The formulation of claim 1, wherein the glycosaminoglycan
component has been chemically modified by one or more of
esterification, sulfation, polysulfation, acetylation and
methylation.
15. The formulation of claim 1, wherein the glycosaminoglycan
component is selected from the group consisting of chondroitin,
chondroitin salts, hyaluronic acid, pentosan polysulfate and
mixtures thereof.
16. The formulation of claim 1, wherein the glycosaminoglycan
component is chondroitin sulfate.
17. The formulation of claim 1, wherein a dose of the one or more
avocado/soybean unsaponifiables ranges from about 5 milligrams to
about 5 grams.
18. The formulation of claim 1, wherein a dose for a small animal
of the one or more avocado/soybean unsaponifiables ranges from
about 5 milligrams to about 1000 milligrams.
19. The formulation of claim 1, wherein a dose for a human of the
one or more avocado/soybean unsaponifiables ranges from about 25
milligrams to about 1500 milligrams.
20. The formulation of claim 1, wherein a dose for a large animal
of the one or more avocado/soybean unsaponifiables ranges from
about 100 milligrams to about 5 grams.
21. The formulation of claim 1, wherein a dose of the one or more
avocado/soybean unsaponifiables ranges from about 0.5 mg/kg to
about 25 mg/kg.
22. The formulation of claim 1, wherein a dose of the one or more
avocado/soybean unsaponifiables for a small animal ranges from
about 0.5 mg/kg to about 25 mg/kg.
23. The formulation of claim 1, wherein a dose of the one or more
avocado/soybean unsaponifiables for a human ranges from about 0.5
mg/kg to about 25 mg/kg.
24. The formulation of claim 1, wherein a dose of the one or more
avocado/soybean unsaponifiables for a large animal ranges from
about 0.5 mg/kg to about 25 mg/kg.
25. The formulation of claim 1, wherein a dose of the aminosugar
ranges from about 25 milligrams to about 12 grams.
26. The formulation of claim 1, wherein a dose for a small animal
of the aminosugar ranges from about 25 milligrams to about 3
grams.
27. The formulation of claim 1, wherein a dose for a human of the
aminosugar ranges from about 100 milligrams to about 4 grams.
28. The formulation of claim 1, wherein a dose for a large animal
of the aminosugar ranges from about 300 milligrams to about 15
grams.
29. The formulation of claim 1, wherein a dose of the aminosugar
ranges from about 3 mg/kg to about 125 mg/kg.
30. The formulation of claim 1, wherein a dose of the aminosugar
for a small animal ranges from about 3 mg/kg to about 125
mg/kg.
31. The formulation of claim 1, wherein a dose of the aminosugar
for a human ranges from about 3 mg/kg to about 125 mg/kg.
32. The formulation of claim 1, wherein a dose of the aminosugar
for a large animal ranges from about 3 mg/kg to about 125
mg/kg.
33. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component ranges from about 15 milligrams to
about 12 grams.
34. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a small animal ranges from about 15
milligrams to about 2 grams.
35. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a human ranges from about 75
milligrams to about 4 grams.
36. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a large animal ranges from about
300 milligrams to about 12 grams.
37. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component ranges from about 1 mg/kg to about 75
mg/kg.
38. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a small animal ranges from about 1
mg/kg to about 75 mg/kg.
39. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a human ranges from about 1 mg/kg
to about 75 mg/kg.
40. The formulation of claim 1, wherein a dose of the
glycosaminoglycan component for a large animal ranges from about 1
mg/kg to about 75 mg/kg.
41. The formulation of claim 1, wherein the formulation is prepared
for intramuscular, intravenous, oral, subcutaneous, rectal,
topical, transcutaneous, intranasal, intraarticular, sublingual and
intraperitoneal administration.
42. The formulation of claim 1, wherein the formulation is prepared
for administration in the form of one or more pills, one or more
tablets, one or more capsules, one or more creams, powders, liquid
form, aerosol form, extended release form, or in the form of an
injectable.
43. The formulation of claim 1, wherein the formulation is prepared
for administration in the form of one or more liquid-filled
capsules, one or more softgel capsules, a paste, chewable tablets,
one or more scoops of powder, one or more sachets, or one or more
treats.
44. A method of preventing, treating, managing, repairing or
modulating inflammation associated with damage to connective tissue
in humans and animals comprising administering the formulation of
any of claims 1 to 43 to a human or animal in need thereof.
45. A method of preventing, treating, managing, repairing or
modulating inflammation associated with damage to connective tissue
in humans and animals comprising administering the formulation of
claim 1 to a human or animal in need thereof, wherein the
formulation is administered daily to reach a steady state
concentration in a body fluid of the human or animal that bathes a
target cell type that affects at least one of COX-2, TNF-.alpha.,
IL-1.beta., iNOS, p38, and chemokines, and wherein the dosage is
subsequently reduced in at least one of frequency and amount to
maintain a desired response in the human or animal.
46. The formulation of claim 1, wherein the one or more
avocado/soybean unsaponifiables are standardized to about 30%
sterol.
47. The formulation of claim 1, wherein the one or more
avocado/soybean unsaponifiables are standardized to about 30%
sterol in approximately a 2-1 ratio of soybean unsaponifiables to
avocado unsaponifiables.
48. The formulation of claim 1, wherein the one or more
avocado/soybean unsaponifiables are a solid at room temperature and
a liquid at human body temperature.
49. The formulation of claim 1, wherein the amounts of the one or
more avocado/soybean unsaponifiables, glucosamine, and
glycosaminoglycan component in the formulation are effective in
combination to substantially inhibit or downregulate gene
expression of one or more chemokines, wherein the one or more
chemokines are selected from the group consisting of IL-8 and
MCP.
50. The formulation of claim 1, wherein the amounts of the one or
more avocado/soybean unsaponifiables, glucosamine, and
glycosaminoglycan component in the formulation are effective in
combination to reduce a level of PGE-2.
51. The formulation of claim 1, wherein the amount of the one or
more avocado/soybean unsaponifiables is selected to achieve a level
of about 8 to about 25 .mu.g/mL of body fluid that bathes a target
cell type that affects at least one of COX-2, TNF-.alpha.,
IL-1.beta., iNOS, p38, and chemokines.
52. The formulation of claim 1, wherein the aminosugar comprises
glucosamine in a concentration effective to achieve a dosage of
about 10 .mu.g/mL in the target human or animal, and wherein the
glycosaminoglycan component comprises chondroitin sulfate in a
concentration effective to achieve a dosage of about 20 .mu.g/mL in
the target human or animal.
53. A method of modulating markers of inflammation of connective
tissue in a target human or animal, comprising: administering a
composition comprising one or more avocado/soybean unsaponifiables,
glucosamine, and chondroitin sulfate to a human or animal, wherein
the composition is administered to the target human or animal to
modulate inflammation in an amount effective to moderate gene
expression of at least one of COX-2, TNF-.alpha., IL-1.beta., iNOS,
p38, and chemokines to approximately normal values.
54. A formulation for modulating inflammation associated with
damage to connective tissue in humans and animals, comprising: one
or more avocado/soybean unsaponifiables; an aminosugar component
comprising comprising glucosamine, glucosamine salts, or mixtures
thereof, and a glycosaminoglycan component comprising chondroitin,
chondroitin salts, or mixtures thereof, wherein the amounts of the
one or more avocado/soybean unsaponifiables, aminosugar component,
and glycosaminoglycan component in the formulation are effective in
combination to substantially inhibit or downregulate gene
expression of COX-2, TNF-.alpha., IL-1.beta., iNOS, p38, and
chemokines, and to reduce PGE-2 levels.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 10/824,498, filed Apr. 15, 2004, which
is a continuation of U.S. Ser. No. 10/192,318, filed Jul. 11, 2002,
which is a continuation of U.S. Ser. No. 09/274,881, filed Mar. 23,
1999, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/088,205, filed Jun. 5, 1998, entitled "A
COMPOSITION OF ACETAMINOPHEN, AN AMINOSUGAR AND A
GLYCOSAMINOGLYCAN," and which further is a continuation-in-part
application of U.S. Ser. No. 09/249,335, filed Feb. 12, 1999, which
claims the benefit of U.S. Provisional Application Ser. No.
60/074,594, filed Feb. 13, 1998, entitled "THE USE OF ANABOLIC
AGENTS, ANTI-CATABOLIC AGENTS, ANTIOXIDANT AGENTS, AND ANALGESICS
FOR PROTECTION, TREATMENT AND REPAIR OF CONNECTIVE TISSUES IN
HUMANS AND ANIMALS." The disclosures of the above-referenced
applications are hereby incorporated by reference herein in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions for the
protection, treatment, repair, and modulation of inflammation of
connective tissues in humans and other animals.
BACKGROUND OF THE INVENTION
[0003] The tissues of mammals, including humans, are in a constant
state of flux between the anabolic processes that build up tissues,
and the catabolic processes which degrade tissues. The state of
health exists when there is a balance between these two processes,
and derangements of the balance produce disease. This holds true
for all tissues of the body. Connective tissues are of particular
importance for several reasons. First, they support the "functional
cells" of the body, i.e., epithelial, muscle and neural cells.
Second, they play critical roles in intercellular communication,
which is essential for multicellular life.
[0004] The inflammatory process occupies a key position in this
balance. When injury to tissues occurs, inflammation initiates the
biochemical processes that result in tissue repair. Because
inflammation results in the symptoms of pain, inflammation, and
swelling of the tissues involved, it is often-regarded by both
patients and physicians as an abnormal and undesirable state, which
should be treated and relieved as soon and as completely as
possible. As a result, pharmacies are full of "anti-inflammatory
drugs" (such as corticosteroids and the non-steroidal
anti-inflammatory drugs, such as aspirin). Under certain
circumstances, inflammation can indeed be destructive; however, it
is important to remember that inflammation is closely linked with
tissue healing. Indeed, inflammation is not easily categorized as
strictly anabolic or catabolic--it may have either effect. Its
purpose in the body is to remove, dilute or wall-off the injurious
agent(s). It also sets into motion the biochemical processes that
repair and reconstruct the damaged tissue. Because it is essential
to healing, and because it can also cause tissue destruction,
inflammation and its mediators are important factors in the
anabolic and catabolic balance.
[0005] One very important class of inflammatory mediators is the
eicosanoid group. The eicosanoids are synthesized in the body from
essential fatty acids ("FAs"). Through a series of biochemical
reactions, the precursor fatty acids are modified to produce
intermediate metabolites, arachadonic acid ("AA"), an omega-6 FA;
and eicosapentanoic acid ("EPA"), an omega-3 FA. Eicosanoids
produced from arachidonic acid include the 2-series of
prostaglandins and the 4-series of leukotrienes, which are
generally proinflammatory. The eicosanoids derived from EPA, such
as the 3 series prostaglandins and hydroxyeicosapentaenoic acid
("HEPE"), are less inflammatory than those derived from AA. In
addition, such eicosanoids may even have anti-inflammatory
effects.
[0006] As a class, the eicosanoids are short-lived and locally
active. They are responsible for the initial events of
inflammation, including vasodilation, increased vascular
permeability, and chemotaxis. Moreover, the eicosanoids are
instrumental in the early steps of the healing process. For
example, the eicosanoids trigger the release of cytokines such as
TGF-B, which in turn stimulates the migration and proliferation of
connective tissue cells, and the deposition of extracellular
matrix. Specific constitutive eicosanoids also have protective
effects in the gastrointestinal mucosa and kidney, because they
maintain glycosaminoglycan synthesis and normal perfusion of these
organs.
[0007] Because of anabolic processes such as these, and because of
the influence of natural anti-catabolic and anti-oxidant agents in
the body, the outcome of the majority of cases of inflammation is
resolution of the injury and healing of the damaged tissues. Only
in pathologic situations does inflammation itself become a
contributor to disease.
[0008] Research on the therapeutic use of eicosanoid precursor FAs
(including cis-linoleic and alpha-linolenic acids, the so-called
omega-3 and omega-6 fatty acids) has been primarily directed
towards their use as competitive inhibitors of the synthesis of
eicosanoids, and therefore, their anti-inflammatory effects. Except
in cases of severe or absolute dietary deficiency, little attention
has been given to the beneficial, anabolic effects that the
eicosanoids have in connective tissues. However, naturally
occurring "subclinical" deficiencies of eicosanoids probably
contribute significantly to disease, and are under diagnosed. For
example, the enzyme delta-6-desaturase is responsible for the
committed step in the synthesis of AA. Activity of this enzyme,
(delta-6-desaturase) decreases with age. This is likely to prove a
significant factor in the increased incidence of connective tissue
dysfunction in older population segments since a deficiency of AA
would decrease anabolic processes and allow catabolic events to
dominate.
[0009] Given the importance of inflammation in the healing of
tissues, and the protective role that some eicosanoids play, it is
not surprising that pharmaceuticals that decrease inflammation by
blocking eicosanoid production should also have negative effects on
healing and anabolic processes. It has long been known that
corticosteroid drugs, which are strongly anti-inflammatory, also
delay healing and decrease the production of extracellular matrix
components. This is because cortisol and related compounds
stabilize cell membranes and therefore inhibit the release of
phospholipase A2, the precursor of AA. Recently attention has
turned to the non-steroidal anti-inflammatory drugs ("NSAIDs").
Numerous studies have shown that NSAIDs, like corticosteroids, can
decrease the synthesis of matrix components by connective tissue
cells, because they inhibit prostaglandin endoperoxide synthase,
and thus block the cyclooxygenase pathway.
[0010] Since the inflammatory process is the sine qua non of tissue
healing, and since the eicosanoids are the mediators of the
inflammatory process, the use of AA (and other eicosanoid
compounds) is a novel approach to therapy of injured tissues.
Kirkpatrick et al. investigated the use of prostanoid precursors on
chick embryonic cartilage in organ culture and found no significant
effects. [Kirkpatrick, C. J., "Effects of Prostanoid Precursors and
Indomethacin on Chick Embryonic Cartilage Growth in Organ Culture,"
Expl. Cell Biol., 51:192-200 (1993)]. The experimental model in
this work may have contributed to the absence of significant
effects, because avian cartilage and embryonic cartilage differ
significantly from mammalian, postnatal cartilage. For example,
embryonic cartilage of any species is hypermetabolic and anabolic
to begin with because it is in a period of exponential growth. Kent
et al. examined the effects of AA in lapine cartilage and found a
positive effect, although previous and subsequent research failed
to confirm this. [Kent, L. et al., "Differential Response of
Articular Chondrocyte Populations to Thromboxane B2 and Analogs of
Prostaglandin Cyclic Endoperoxidases," Prostaglandins. 19:391-406
(1980)]. Kirkpatrick and Gardner found that AA and various
metabolites of AA had insignificant or inhibitory effects on
biosynthesis. [Kirkpatrick C. J. and Gardner, D. L., "Influence of
PGAI on Cartilage Growth," Experientia, 33(4):504 (1976)].
Lippiello, et al. found, however, that AA and other omega-6 fatty
acids had beneficial effects on chondrocyte metabolism in cell
culture. [Lippiello, L., Ward, M., "Modification of articular
cartilage chondrocyte metabolism by in vitro enrichment with fatty
acids (abstract)," Trans. Orthop. Res. Soc. 13:162 (1988);
Lippiello, L., "Prostaglandins and articular cartilage; does
Prostaglandin perturbation perpetuate cartilage destruction?" Semin
Arthritis Rheum 11:87 (1981).] These variable results are not
unexpected, since the balance between anabolic and catabolic
processes in the body is delicate and easily perturbed. Phan et
al., suggest that products of AA via the cyclooxygenase pathway are
anti-fibrogenic while AA products via the lipoxygenase pathway are
pro-fibrogenic. This phenomenon demonstrates the complexity of the
eicosanoids' interactions.
[0011] Catabolic events are typically mediated in the body by
enzymes that break apart body constituents. Catabolism is essential
for health and deficiency of necessary enzymes results in disease,
such as the so-called storage diseases like mucopolysaccharhidosis.
Excessive catabolism may also result in the breakdown of tissues
and lead to disease, as in degenerative diseases like
osteoarthritis or autoimmune diseases like multiple sclerosis.
Various anti-catabolic substances in the body help contain and
balance catabolism. For example, chondroitin sulfate counteracts
metalloproteinases that catabolize collagen and proteoglycans in
the cartilage matrix. Similarly, alpha-one anti-trypsin inhibits
the effects of elastase, which contributes to alveolar breakdown in
emphysema.
[0012] Oxidative damage also has an impact on the balance of
anabolism and catabolism in the body. This damage is the result of
the effects of free radicals, substances that have an unpaired
electron. Free radicals form constantly in the body as the result
of normal reactions like the production of ATP. They also form
during the inflammatory process. Free radicals cause cellular
damage because they are highly chemically reactive. Because they
have only a single electron, (a condition that nature abhors as it
does a vacuum), these substances "steal" electrons from molecules
in their vicinity. The molecules making up cell structures, such as
the cell membrane or DNA are thereby rendered electron-deficient.
The deficiency of electrons in turn makes the cell structure
unstable and cell dysfunction occurs, including manufacture of
abnormal proteins, cell rupture, and cell death. Oxidative damage
is implicated in many catabolic events in the body, including the
aging process. Anti-oxidants, such as vitamin C, vitamin E,
superoxide dismutase (SOD), selenium, and glutathione are
substances that scavenge free radicals before oxidative damage
occurs. In the sense that they prevent cell damage, anti-oxidants
are a specific type of anti-catabolic agent.
[0013] The body also contains anabolic compounds that stimulate
tissue growth. Glucosamine is an amino sugar naturally formed in
the body from glucose. When supplied exogenously, glucosamine
stimulates connective tissue cell synthesis, and thereby increases
the amounts of normal extracellular matrix. Glucosamine is also the
building block for glycosaminoglycans in cartilage and other
connective tissues. Supplying additional glucosamine thus supplies
the body with extra raw materials for matrix synthesis in
connective tissues. Other examples of anabolic compounds in the
body include somatotropin, which stimulates protein synthesis, and
the somatomedins or insulin-like growth factors, which stimulate
the proliferation of chondrocytes and fibroblasts and enhance
matrix synthesis.
[0014] The actions and interactions of these compounds are complex.
A given compound may have different effects in different tissues.
For example, somatotropin increases protein synthesis (anabolism),
but also speeds fat breakdown (catabolism). The effects that a
particular compound or combination of compounds will have depend on
many factors, including route of administration, dosage, and
duration of therapy.
[0015] Previous researchers have investigated the use of individual
compounds for their anabolic, anti-oxidant or anti-catabolic
effects. Glucosamine has been found in cell culture to stimulate
connective tissue cells to produce the components of the matrix:
collagen and glycosaminoglycans (GAGs). [Jimenez, S., "The Effects
of Glucosamine sulfate on Chondrocyte Gene Expression," Eular
Symposium, Madrid October 1996 Proceedings, page 8-10].
S-adenosylmethionine is known to participate in several synthesis
reactions, including the sulfation of GAGs. [Champe, P.
Biochemistry, 2.sup.nd edition, J. B. Lippincott Co, Philadelphia,
1994, pp. 248, 250, 265]. Arachadonic acid has been found to
stimulate corneal healing. [Nakamura, M., "Arachidonic Acid
Stimulates Corneal Epithelial Migration", J. Ocul. Pharmacol.,
Summer:10(2): 453-9 (1994)]. These compounds therefore have
anabolic effects.
[0016] Chondroitin sulfate has been shown to inhibit degradative
enzymes, including the metalloproteinases that destroy cartilage
matrix. [Bartolucci, C., "Chondroprotective action of chondroitin
sulfate," Int. J. Tiss. Reac., XIII(6):311-317 (1991)]. Studies
with pentosan sulfate have shown that it prevents
complement-mediated damage in a rabbit myocardial cells. [Kilgore,
K., "The Semisynthetic Polysaccharide Pentosan Polysulfate Prevents
Complement-Mediated Myocardial Injury in the Rabbit Perfused
Heart," J. Pharmocol. Exp. Ther., 285(3):987-94 (1998)]. Oral
administration of collagen type II has been shown to decrease the
deleterious immune response that destroys joint tissue in
rheumatoid arthritis. Tetracycline analogues are potent inhibitors
of matrix metalloproteinases. [Ryan, M., "Potential of
Tetracyclines to Modify Cartilage Breakdown in Osteoarthritis."
[Curr. Opin. Rheumatol., 8(3): 238-47 (1996)]. Diacerein modifies
the inflammatory process by inhibiting interleukin-1 activity, and
also by direct effects on lymphocytes and neutrophils. [Beccerica,
E., "Diacetylrhein and rhein: in vivo and in vitro effect on
lymphocyte membrane fluidity," Pharmocol. Res., 22(3):277-85
(1990); Mian, M., "Experimental Studies on Diacerhein: Effects on
the Phagocytosis of Neutrophil Cells from Subcutaneous
Carregeenan-Induced Exudate," Drugs Exp. Clin. Res., 13(11):695-8
(1987); Spencer, C., "Diacerein", Drugs, 53(1):98-106 (1997)].
These compounds can be classed as anti-catabolic agents.
[0017] L-ergothioneine scavenges hydroxyl radicals and may inhibit
singlet oxygen formation, [Han J S. "Effects of Various Chemical
Compounds on Spontaneous and Hydrogen Peroxide Induced Reversion in
Strain TA104 of Salmonella typhimurium," Mutant Res., 266(2):77-84
(1992)], while superoxide dismutase scavenges superoxide radicals
[Mathews C., Biochemistry 2.sup.nd ed., Benjamin/Cummings Pub. Co.,
Menlo Park Calif., 1996, page 551]. These compounds can be
classified as anti-oxidants.
[0018] Although these compounds have been investigated
individually, to our knowledge no one other than the present
inventors has examined the effects of certain combinations of any
or all of anabolic, anti-catabolic and anti-oxidant agents to
maintain health and to promote healing. According to the present
invention, combinations of these agents can be used to maximize
appropriate anabolic effects (healing) and decrease undesirable
catabolic effects (degradation) and oxidative damage, while at the
same time, causing minimal or no adverse reactions. Therefore, it
can be seen that there exists a need to provide compositions that
will make use of the beneficial effects of combinations of anabolic
agents, anti-catabolic agents, anti-oxidant and/or analgesic agents
for the maintenance and repair of connective tissues in humans and
animals.
SUMMARY OF THE INVENTION
[0019] The present invention provides novel compositions and
methods of treating repairing, and preventing damage to connective
tissues in humans and animals using such compositions. Therefore,
it is an object of the invention to provide novel compositions of
any or all of anabolic, anti-catabolic, anti-oxidant and/or
analgesic agents for the protection, treatment and repair of
connective tissues in humans and animals.
[0020] It is another object of the present invention to provide
methods of treating and repairing connective tissue in humans and
animals with compositions containing any or all of anabolic,
anti-catabolic, anti-oxidant and/or analgesic agents.
[0021] It is still another object of the present invention to
provide compositions comprising any or all of anabolic,
anti-catabolic, anti-oxidant and/or analgesic agents selected from
the group consisting of aminosugar, S-adenosylmethionine (SAMe),
arachadonic acid (AA), GAG, pentosan sulfate, collagen type II,
tetracyclines, diacerin, super oxide dismutase (SOD),
L-ergothioneine, one or more avocado/soybean unsaponifiables (ASUs)
and analgesics, such as acetaminophen.
[0022] It is a further object of the present invention to provide
compositions to repair, treat, and prevent damage to connective
tissue in humans and animals that contain one or more of the
elements selected from the group consisting of aminosugar, SAMe,
arachodonic acid, GAG, pentosan sulfate, collagen type II,
tetracyclines, diacerin, SOD, L-ergothioneine, one or more ASUs and
analgesics, e.g, acetaminophen.
[0023] It is a further object of the present invention to provide
compositions to modulate inflammation associated with damage to
connective tissue in humans and animals.
[0024] These and other objects of the present invention are
apparent from the detailed description and claims below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 provides a detailed description of the biosynthetic
pathway for the creation of GAGs such as chondroitin sulfate.
[0026] FIG. 2 is the molecular structure of SAMe and its immediate
precursor.
[0027] FIG. 3 provides a simplified diagram of the function of
SOD.
[0028] FIG. 4 provides some examples of unsaponifiable lipids.
[0029] FIG. 5 is the molecular structure of acetaminophen.
[0030] FIGS. 6A and 6B show images of extracellular matrix (ECM)
and chondrocytes on microcarriers as observed at two different time
periods in Example 7.
[0031] FIG. 7 is an image of chondrocytes stained for type II
collagen as observed in Example 7.
[0032] FIG. 8 shows two charts illustrating PGE-2 response to
IL-1.beta. activation as measured in Example 7.
[0033] FIG. 9 shows a chart illustrating the modulation of PGE-2
response to avocado/soybean unsaponifiables (ASUs), chondroitin
sulfate, and glucosamine in accordance with Example 7.
[0034] FIG. 10 shows a chart illustrating TNF-.alpha. expression in
THP-1 cells in accordance with Example 8.
[0035] FIG. 11 is a chart illustrating IL-1.beta. in accordance
with Example 8.
[0036] FIG. 12 is a chart illustrating iNOS expression in THP-1
cells as measured in Example 8.
[0037] FIG. 13 is a chart illustrating p38 expression in THP-1
cells as measured in Example 8.
[0038] FIGS. 14A and 14B illustrate the inhibition of COX-2 in
activated chondrocytes, as measured in Example 9.
[0039] FIG. 15 is a chart illustrating secreted PGE-2 levels as
measured in Example 9.
[0040] FIG. 16 is a chart illustrating interleukin-8 (IL-8)
expression in human chondrocytes as measured in Example 11.
[0041] FIG. 17 is a chart illustrating monocyte chemotactic protein
(MCP) expression in human chondrocytes as measured in Example
11.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The compositions of the present invention, used to treat,
repair, and prevent damage to connective tissue, include
combinations of anabolic, anti-catabolic, and/or anti-oxidant
agents. Ingredients of preferred embodiments include compositions
selected from the group consisting of aminosugars, SAMe, AA, GAGs,
including pentosan, collagen type II, tetracyclines, diacerin, SOD,
L-ergothioneine, methylsulfanylmethane (MSM), and one or more ASUs.
Optionally, the combinations of the present invention also include
one or more analgesics, such as acetaminophen. In addition, the
present invention covers methods of administering these novel
compositions to humans and animals in need thereof.
[0043] Glucosamine--an example of an aminosugar--is naturally
formed in the body from glucose. When supplied exogenously,
glucosamine stimulates connective tissue cell synthesis, increasing
the amounts of normal extracellular matrix. Glucosamine is also the
building block for glycosaminoglycans ("GAGs") in cartilage and
other connective tissues, thus, supplying additional glucosamine
supplies the body with extra raw materials for matrix synthesis in
connective tissues. The aminosugar component of the compositions of
the present invention may comprise natural, synthetic or
semi-synthetic aminosugars including but not limited to salts of
glucosamine including glucosamine hydrochloride and glucosamine
sulfate, glucosamine phosphate, and N-acetylglucosamine and salts
and/or mixtures thereof. In addition, the term aminosugar is also
used herein to encompass aminosugars that may have been chemically
modified yet retain their function. Such chemical modifications
include but are not limited to esterification, sulfation,
polysulfation, acetylation, and methylation. Moreover, it is
contemplated that the term aminosugar can extend to any composition
of matter that is insubstantially different from the aminosugar as
above-described.
[0044] The GAG component of the compositions of the present
invention may comprise natural, synthetic or semisynthetic GAGs,
GAG-like compounds, or GAG precursors, including but not limited to
chondroitin, hyaluronic acid, glucuronic acid, iduronic acid,
keratan sulfate, heparan sulfate, dermatin sulfate, and fragments,
salts, and mixtures thereof. In addition, the term GAG as used
herein further encompasses GAGs that have been chemically altered
yet retain their function. Such modifications include but are not
limited to esterification, sulfation, polysulfation, and
methylation. In fact, sulfated GAGs are a preferred component of
the compositions of the present invention. Hence, mono-sulfated and
polysulfated (or oversulfated) GAGs are preferred GAG components of
the compositions of the present invention. The term GAGs also is
intended to encompass alternative nomenclature for the same group
of above-described compounds--e.g., mucopolysaccharides,
proteoglycans, and heparanoids. In addition, the GAG or GAG-like
component of the compositions of the present invention may be
derived from plant or animal sources, including but not limited to
beechwood tree, to forms of animal cartilage including shark
cartilage, bovine trachea, whale septum, and porcine nostrils, and
to invertebrates such as Perna canaliculus and sea cucumber.
[0045] Chondroitin sulfate is a preferred GAG. Chondroitin sulfate
is the most abundant glycosaminoglycan in articular cartilage and
is also present in many other connective tissues in the body.
Additionally, chondroitin sulfate competitively inhibits
degradative enzymes that degrade connective tissues under
conditions of abnormal, excessive inflammation. Chondroitin sulfate
is a polymer composed of repeating units of glucuronic acid and
sulfated galactosamine. [Lester M. Morrison, M. D. and O. Arne
Schjeide, Ph.D., Coronary Heart Disease and the Mucopolysaccharides
(Glycosaminoglycans) 12 (1974); Philip C. Champe and Richard A.
Harvey, Lippincott's Illustrated Reviews: Biochemistry, 148-50
(2.sup.nd ed. 1994)]. One of ordinary skill in the art understands
that chondroitin sulfate must have at least two, and potentially
many, of these repeating units of glucuronic acid and sulfated
galactosamine.
[0046] FIG. 1 provides a detailed description of the biosynthetic
pathway for the creation of GAGs, such as chondroitin sulfate. In
addition, the present invention may include fragments of GAGs, such
as fragments of chondroitin sulfate. One of ordinary skill in the
art at the time the invention understands that "fragments of
glycosaminoglycans" are groups of saccharides that constitute less
than two repeating units of the glycosaminoglycan. Hence, it is
understood that fragments of these substances would be composed of
groups of saccharides that constitute fewer than two of the
repeating units of the respective polymer.
[0047] For example, one of ordinary skill in the art understands
that fragments of chondroitin sulfate are molecules composed of the
saccharides that comprise the repeating units of chondroitin
sulfate, but that are present in groups of less than the two
repeating units described above. Thus, a molecule composed of a
glucuronic acid and sulfated galactosamine would constitute a
fragment of chondroitin sulfate. Indeed, there are eight different
disaccharide structures that may constitute fragments of
chondroitin sulfate. [Timothy. E. Hardingham and Amanda J. Fosang,
Proteoglycans: Many Forms and Many Functions, FASEB J., 6:861-862
(1992)].
[0048] Other naturally occurring glycosaminoglycans may be used in
this invention, for example, hyaluronic acid. Also, fragments of
the glycosaminoglycans may also be utilized. A person of ordinary
skill in the art understands the terms "fragments of chondroitin,"
"fragments of chondroitin sulfate," "fragments of chondroitin
salts," "fragments of glycosaminoglycan" and "chondroitin sulfate
fragments," and further understands them to mean groups of
saccharides (or salts thereof) that constitute less than two
repeating units of the glycosaminoglycan.
[0049] One of skill would expect that fragments of chondroitin
sulfate, for example, would have the same utility as chondroitin
sulfate itself. Chondroitin sulfate is broken down into smaller
units within the body, and that it is reformulated in the
production of cartilage and other connective tissue. Therefore, it
is understood that the body utilizes fragments of chondroitin
sulfate in the same manner as it utilizes chondroitin sulfate
itself. The same is true with respect to "fragments of
chondroitin," "fragments of chondroitin salts," and "fragments of
glycosaminoglycan." Each of chondroitin, chondroitin salts and
other glycosaminoglycans, if ingested, is broken down by the body
and reformulated in the production of cartilage and other
connective tissue. Therefore, the body utilizes fragments of
chondroitin in the same manner as it utilizes chondroitin itself,
utilizes fragments of chondroitin salts in the same manner as it
utilizes chondroitin salts, and utilizes fragments of
glycosaminoglycans in the same manner as it utilizes
glycosaminoglycans.
[0050] Moreover, it is intended that the term GAG can extend to any
composition of matter that is insubstantially different from the
GAGs as above-described. An example of such a GAG-like compound
that is within the scope of the present invention is pentosan
polysulfate (PPS) as well as salts thereof such as calcium-derived
PPS and sodium PPS. Accordingly, a preferred GAG-like compound that
may be used in the compositions of the present invention is
PPS.
[0051] PPS is a semi-synthetic polysulfated xylan that is a
sulfated form of a compound extracted from beechwood hemicellulose
consisting of repeating units of (1-4) linked
.beta.-D-xylano-pyranoses. More specifically, PPS is produced by
extracting these hemicellulose compounds via a series of chemical
reactions from the wood, and then adding numerous sulfate groups to
the purified polysaccharide chains. This process results in low
molecular weight linear polysaccharide chains that carry numerous
negatively charged sulfate groups. PPS is a semi-synthetic
heparinoid that is considered an oversulfated form of a GAG.
[0052] There are several forms of PPS that display the
above-described activities. Sodium PPS and a calcium-derived PPS
(called CAPPS) may both be used to accomplish the functions of PPS.
Each of these forms of PPS exhibit GAG-like activity, and will
hereinafter be referred to as GAG-like compounds.
[0053] Pentosan's mechanism of action can be summarized as
follows:
[0054] 1. Anti-inflammatory activities through stabilization and
improvement of micro-circulation in the inflamed tissues and
through anti-Complement effects (decreases the release of the
humoral mediators of inflammation called the Complement
cascade).
[0055] 2. Inhibition of chemotaxis of granulocytes, which are white
blood cells that contribute to inflammation.
[0056] 3. Stimulatory effect on proteoglycan synthesis.
[0057] 4. Stimulatory effects on hyaluronic acid synthesis by
synovial fibroblasts.
[0058] 5. Potent inhibition of catabolic enzymes including, human
granulocyte elastase (noncompetitive inhibition), hyaluronidase
(competitive inhibition), chondroitin-4-sulfatase and
N-acetyl-glucosaminidase at concentrations much more lower than
that of NSAIDs.
[0059] Other synthetic or semi-synthetic glycosaminoglycans or
glycosaminoglycan-like compounds, such as polysulfated
glycosaminoglycans, may be used in this invention.
[0060] Diacerein, a recently recognized organic compound found in
plants of the genus Cassia has anti-inflammatory effects through
inhibition of interleukin-1B consequently collagenase production in
articular cartilage is reduced. It reduces the fibrinolytic
activity of synovial fibroblasts as well. It also dose-dependently
inhibits chemotaxis (attraction of white blood cells) and
superoxide anion production (this is one of the "toxic oxygen
species" or "free radicals"). These harmful compounds occur
spontaneously in the body, especially during destructive
inflammation. Diacerein has analgesic and antipyretic activities.
It reduces the breakdown of chondroitin-4-sulfate resulting in an
increase in the ratio of chondroitin-4-sulfate to
chondroitin-6-sulfate. (This ratio is pathologically decreased in
degenerating cartilage.) It mildly increases prostaglandin
synthesis, which allows it to have protective effects on the
gastric mucosa.
[0061] S-adenosylmethionine (SAMe) is an important endogenous
compound present throughout the body, and taking part in a great
number of biologic reactions such as transsulfation reactions. In
this role it is an important reactant in the synthesis of many
structural components of connective tissues, including proteins and
proteoglycans. Thus, SAMe has significant anabolic effects which
would enhance the actions of other anabolic agents. SAMe also has
anti-inflammatory effects by virtue of its antioxidant action.
[0062] SAMe is compound synthesized in the body from adenosine
triphosphate ("ATP") and methionine (FIG. 2). It is present in many
tissues, including the central nervous system. The primary CNS
function of SAMe is to donate methyl groups in the reactions
synthesizing various crucial compounds, including neurotransmitters
and phospholipids. For example, SAMe facilitates the conversion of
phosphatidylethanolamine to phosphatidylcholine, which forms part
of the inner, lipid layer of the plasma membrane. In so doing, SAMe
increases membrane fluidity and enhances effectiveness of
receptor/ligand binding. [Champe and Harvey, Biochemistry, 1994;
Stramentinoli, G., "Pharmacologic Aspects of S-Adenosylmethionine,"
American J. Med., 83(5A):35 (1987); Baldessarini, F.,
"Neuropharmacology of S-Adenosyl Methionine," American J. Med.,
83(5A):95 (1987); Camey, M., "Neuropharmacology of S-Adenosyl
Methionine," Clin. Neuropharmacol., 2(3):235 (1986); Janicak, P.,
"S-Adenosylmethionine in Depression," Alabama J. Med. Sci.
25(3):306 (1988)]. These functions may also pertain to other methyl
donors such as betaine (trimethylglycine),
5-methyltetrahydrofolate, folic acid, and dimethylglycine. [Champe
and Harvey, Biochemistry, 1994].
[0063] Superoxide dismutase is an enzyme present naturally in the
tissues of animals and plants, which has recently been investigated
as an agent in the management of inflammation. It acts by
intercepting toxic oxygen radicals in the intracellular space
during destructive inflammatory processes. It does not inhibit
prostaglandin biosynthesis, but stops the overproduction of
prostaglandins resulting from destructive inflammation. Some of its
effects include inhibition of edema formation and inhibition of
acute signs of inflammation and the secondary articular changes
(stiffness and calcification) in adjuvant-induced arthritis. Having
no analgesic effects, it does not contribute to the overuse of the
affected joints that eventually leads to more damage of the
articular cartilage, as NSAIDs can. Also, it has no adverse effects
on the cardiovascular, central nervous or endocrine systems. FIG. 3
provides a simplified diagram of the function of SOD.
[0064] L-ergothioneine is an intracellular antioxidant naturally
occurring in plants and animals, but not synthesized in human
bodies: it comes only from dietary sources. The antioxidant
properties of L-ergothionein appear to be related to its ability to
scavenge reactive oxygen species (free radicals), chelate various
metallic cations, activate antioxidant enzymes such as glutathione
peroxidase (SeGPx) and manganese superoxide dismutase (Mn SOD) and
to inhibit superoxide-generating enzymes such as NADPH-Cytochrome C
reductase, and to affect the oxidation of various hemoproteins such
as hemoglobin and myoglobin. Because all body tissues depend on
these two oxygen carrier molecules, this characteristic is
extremely beneficial. [Brummel, M. C., "In Search of a
Physiological Function for L-ergothioneine," Med. Hypotheses,
18(4):351-70 (December 1985); Brummel, M. C., "In Search of a
Physiological Function for L-ergothioneine,--II," Med. Hypotheses,
30(1):39-48 (September 1989); Han, J. S., "Effects of Various
Chemical Compounds on Spontaneous and Hydrogen Peroxide-Induced
Reversion in Strain TA104 of Salmonella typhimurium," Mutat. Res.,
266(2):77-84 (April 1992); Arduini, A., "Possible Mechanism of
Inhibition of Nitrite-Induced Oxidation of Oxyhemoglobin by
Ergothioneine and Uric Acid," Arch. Biochem. Biophys.,
294(2):398-402 (May 1992)].
[0065] Collagen Type II also has beneficial effects that help
maintain the normal balance between anabolism and catabolism.
Specifically, connective tissue diseases may result from autoimmune
processes, in which the immune system attacks and catabolizes the
individual's own connective tissues as if it were a "foreign
invader." Oral administration of collagen Type II can desensitize
the immune system, preventing further attack and normalizing immune
responses in these individuals. This decreases catabolic processes
in the connective tissues and maximize anabolism. Ingestion of
collagen type II presents this molecule to the immune cells in the
gut-associated lymphoid tissues (GALT, a.k.a., Peyer's patches).
Interactions between the collagen molecule and specific cells
within the GALT activates mobile immune cells called T suppressor
cells. These cells, in turn, moderate the destructive immune
reaction against the individual's own collagen type II (in
connective tissues).
[0066] Compounds in the tetracycline family include tetracycline,
doxycycline, tetracycline analogs, and "tetracycline-like"
compounds, and have been used therapeutically for their
anti-microbial effects. Current research has focused on
"tetracycline-like" compounds which possess insignificant
antimicrobial effects, but with anti-catabolic effects.
Specifically, "tetracycline-like" compounds are polycyclic
compounds that inhibit tissue metalloproteinases which degrade
extracellular matrix components including collagen and
proteoglycans yet have insubstantial anti-microbial effects. This
function of these compounds, as well as other compounds in the
tetracycline family, may be related to the ability of these
compounds to chelate calcium and zinc ions. For example,
doxycycline has been shown to inhibit collagenase activity in
articular cartilage.
[0067] Certain lipid extracts, termed nonsaponifiable, of avocado
(genus Persea, especially P. americana) and the soybean (Glycine
max) have also been studied for their beneficial effects on
connective tissues. These nonsaponifiable compounds are that part
of the plant lipids that do not undergo saponification, i.e., they
do not react with alkali to form a soap. There are many such
compounds, and any particular avocado extract may contain any
number. Examples include fat soluble vitamins (A, D, E, and K),
steroids such as phytoestrogens, sterols (bioflavonoids) and
volatile essentials oils (terpenes such as menthol, camphor,
lycopene, gibberellic acid, limonene, cinnamaldehyde, carotenoids,
and ubiquinone, also known as coenzyme Q.) [Mathews, C. K. &
van Holde, K. E. Biochemistry, 2.sup.nd ed., The Benjamin/Cummings
Pub. Co., Inc., 1996, p. 691.]
[0068] The avocado/soybean unsaponifiables (ASU) have been used in
Europe under the trade name Piascledine and have been used to treat
osteoarthritis and other forms of arthritis [Thiers, M. H.,
"Unsaponifiable constituents of avocado and soya oils. Treatment of
certain forms of arthralgia," J. Med. Lyon 53(222):195-8 (February
1972) (article in French)], as well as soft-tissue inflammatory
conditions [Trevoux, R., "Unsaponifiable fractions of the avocado
and soybean in gynecology," J. Bynecol. Obstet. Biol. Reprod.
6(1):99-105 (January 1977) (article in French); Lamaud, M. E., et
al., "Biochemical modifications of connective tissue induced by the
non-saponifiables of avocado and soybean oils administered
percutaneously in the `hairless` rat," Pathol. Biol. 26(5):269-74
(May-June 1978) (article in French)]. The mechanism of action of
this compound is to stimulate chondrocyte expression of TGF
(transforming growth factor) beta 1, TGF beta 2 and plasminogen
activator inhibitor 1 ("PAI-1"). By increasing PAI-1, ASU blocks
the cascade that leads to metalloprotease activation [Boumediene
K., et al., "Avocado/soya unsaponifiables enhance the expression of
transforming growth factor beta 1 and beta 2 in cultured articular
chondrocytes," Arthritis Rheum. 42(1): 148-56 (January 1999)]. ASU
mixtures also reduce the spontaneous production of stromelysins,
IL-6, interleukin-8 (IL-8) and prostaglandin E2 by chondrocytes.
Additionally, ASUs decrease the effects of IL-1, and thereby reduce
chondrocyte and synoviocyte production of collagenase. [Henrotin,
Y. E., et al., "Effects of three avocado/soybean unsaponifiable
mixtures on metalloproteinases, cytokines and prostaglandin E2
production by human articular chondrocytes," Clin. Rheumatol.
17(1): 31-9 (1998).]
[0069] TGF beta 1 and 2 are members of a family of homologous
polypeptidecytokines. These locally-acting hormones can have
paracrine or autocrine effects and are made by a variety of cell
types, including lymphocytes, endothelial cells and macrophages.
TGF beta has varied effects in different tissues; it generally
inhibits epithelial cell metabolism. In connective tissues,
however, it has been shown to be an indirect mitogen for
fibroblasts and other cells of mesenchymal origin. It also can
stimulate cellular production of fibronectin and collagen, and
decrease protease activity, resulting in a net increase in matrix
production. [Cotran, R. F., Kumar, V. and Robbins, S. L., Eds.,
Pathologic Basis of Disease, 5.sup.th ed., Saunders, 1994, pp.
40-42.]
[0070] Stromelysins are a subtype of proteinases that act on a
variety of extracellular matrix components, including
proteoglycans, laminin, fibronectin, and collagen. Stromelysins are
produced by fibroblasts, synoviocytes, and macrophages, among other
cell types, under the influence of cytokines such interleukin-1 and
tumor necrosis factor alpha Interleukins and prostaglandins are
among the many mediators of inflammation. Reductions in levels of
all of these compounds result in a decrease in pain and swelling,
which are hallmarks of inflammation.
[0071] The fat-soluble vitamins present in ASU mixtures are
necessary for growth, and augment the anabolic effects of TGF-beta.
Because they stimulate TGF beta and also decrease degradative
enzymes, as explained above, ASU mixtures can be said to have both
anabolic and anti-catabolic effects. Although some of the effects
of ASUs overlap the effects of other compounds in the present
invention, ASUs contribute unique properties to the group of
compounds and provide very beneficial effects when used in
combination with those other compounds. For example, while
glucosamine and ASUs both stimulate anabolic processes in
connective tissue cells, these compounds have different cellular
mechanisms of action. Glucosamine acts in part through protein
kinase C, while the effect of ASUs, as stated above, is through
transforming growth factor. Similarly, chondroitin and ASUs have
inhibitory effects of IL-1. ASUs, however, inhibit the plasmin
cascade, while chondroitin decreases activation of the complement
cascade. Osteoarthritis is a complex disease involving interplay of
many cytokines at the cellular level. Because the different
compounds of the present invention act on different cytokines, they
will have synergistic effects when used in appropriate
combinations.
[0072] In placebo controlled, double blind trials, ASUs have been
shown effective in reducing symptoms of osteoarthritis [Maheu, E.,
et al., "Symptomatic efficacy of avocado/soybean unsaponifiables in
the treatment of osteoarthritis," Arthritis Rheum. 41(1): 81-91
(January 1998); Blotman, F., et al., "Efficacy and safety of
avocado/soybean unsaponifiables in the treatment of symptomatic
osteoarthritis," Rev. Rheum. Engl. Ed. 64(12): 825-34 (December
1997)]. In these studies, side effects in the intervention groups
were similar to those seen in the placebo groups, indicating that
ASUs are safe and well tolerated substances. ASUs as used in this
invention can include any or all unsaponifiable lipids and/or
combinations thereof.
[0073] Examples of components of ASUs include but are not limited
to: limonene, beta carotene, phyloquinone, and giberellic acid. As
explained above, ASUs can include any of a number of classes of
compounds including but not limited to fat soluble vitamins,
steroids, sterols and volatile essentials oils, or any combinations
thereof. For instance, the avocado/soybean unsaponifiables (ASU)
can include one or more phytosterols, such as campesterol,
stigmasterol, dihydro-brassisterol, and Beta-sitosterol.
[0074] The invention includes, moreover, compositions which contain
one avocado/soybean extract or mixtures or combinations of such
extracts (more than one ASU). There are many such combinations and
all are intended to be included within the present invention.
[0075] Various formulations of the present invention may include
one or more avocado/soybean unsaponifiables in a variety of forms
or amounts. For instance, in some embodiments, one or more
avocado/soybean unsaponifiables may be included in a composition so
that they are standardized to about 30% sterol. In some
embodiments, avocado/soybean unsaponifiables may be included in a
composition in approximately a 2-1 ratio of soybean unsaponifiables
to avocado unsaponifiables. In some embodiments, one or more
avocado/soybean unsaponifiables may be a solid at room temperature
and a liquid at human body temperature. In some embodiments, one or
more avocado/soybean unsaponifiables may be combined with one or
more excipients or carriers to create a powder.
[0076] The compounds of the present invention have several
advantages over existing therapies for connective tissue disorders,
such as excellent safety profiles. This is in part related to the
fact that these compounds occur normally in the body and in various
foods. Another characteristic shared by the compounds is tendency
for a slow onset of action. Pharmaceuticals, such as NSAIDs, tend
to cause sudden changes in the symptoms of disease. The endogenous
compounds in the present invention work more slowly, by normalizing
structures and functions within the body. While this action is
beneficial, it does mean that symptoms will typically not be
relieved immediately. For this reason, an analgesic is included as
an optional component of the compositions of the present invention.
The analgesic is to be chosen from the group of analgesic compounds
that have been shown to have minimal side effects at therapeutic
doses, and also to have minimal negative effects on connective
tissue synthesis, as corticosteroid drugs and many NSAIDs have been
shown to have. The analgesic that may be included in the
composition of the present invention therefore is a nonsteroidal
analgesic that does not have anti-inflammatory effects. In other
words, the analgesic is a nonsteroidal drug that is not an NSAID.
Examples of the analgesics of the present invention include
acetaminophen and tramadol. Except as discussed below, the
preferred analgesic of the present invention is acetaminophen.
[0077] Acetaminophen is an analine derivative analgesic and
antipyretic compound working centrally through reversible
inhibition of the enzyme cyclo-oxygenase in the central nervous
system. Acetaminophen also blocks the peripheral pain impulse
generation in nerve endings throughout the body. It has been used
extensively for symptomatic pain relief. The relief of pain is
beneficial for more than the obvious humane reasons. Since there
are also important links between the emotional centers of the brain
and the immune system, the relief of pain, and the resulting
elevation in mood, has beneficial effects on inflammation and the
many other processes that are modulated by the immune system.
Although it blocks cyclooxygenase activity, acetaminophen has very
little anti-inflammatory activity. Therefore, acetaminophen does
not inhibit connective tissue anabolism, as NSAIDs and
corticosteroids do, and because it has minimal side effects at
therapeutic doses, it is an ideal analgesic agent in the present
invention. Another advantage to including a safe analgesic in the
present invention is that it will increase the likelihood that
patient compliance would be high, i.e., that patients would
continue taking the preparations long enough for disease-modifying
effects to occur. Studies of isolated chondroprotective agents
often have a high rate of drop-out in the early weeks of therapy
due to patient perception that the agent is not working. With the
addition of an analgesic, patients would be more inclined to
continue therapy.
[0078] Cats are sensitive to acetaminophen because they do not
metabolize it effectively (poor hepatic conjugation with glucuronic
acid and subsequent depletion of glutathione occurs) [Goodman, A.
and Goodman, L., The Pharmacological Basis of Therapeutics,
7.sup.th ed., MacMillan Publishing Co., 1985, pp. 692-95; Ahrens,
F., Pharmacology, Williams & Wilkins, 1996, pp. 174-75].
Accordingly, acetaminophen is not recommended for use in cats.
[0079] Methylsulfonylmethane (MSM, or dimethylsulfone) is an
organic sulfur compound belonging to a class of chemicals known as
sulfones. It occurs naturally in some primitive plants and is
present in small amounts in many foods and beverages. Researchers
have suggested that MSM has anti-inflammatory effects.
[0080] The present invention comprises novel combinations of
anabolic agents, anti-catabolic agents and antioxidant agents that
maximize beneficial, anabolic effects (healing) and minimize any
potential negative effects. In so doing, the present invention
provides novel combinations of these agents and anti-oxidant
agents, for the protection, treatment and repair of connective
tissues in humans and animals.
[0081] These compounds have a variety of beneficial effects on
animal and human connective tissues, and, because they function via
a variety of mechanisms, work well in combination with each other.
Although each compound has a number of functions, they can be
roughly grouped as: (1) anabolic agents, including glucosamine,
SAMe, AA, and ASUs, which promote growth processes in the body; (2)
anti-catabolic agents, such as chondroitin sulfate, pentosan
sulfate, collagen type II, tetracyclines, diacerin and ASUs, which
inhibit destructive or catabolic processes; and (3) antioxidants,
such as SOD, and L-ergothioneine which prevent tissue damage by
scavenging toxic oxygen species (free radicals). Naturally, some
compounds, such as ASUs, could be placed in more than one group, by
virtue of their overlapping functions. The present invention
establishes that combinations of these compounds would work well.
In addition, an analgesic could optionally be added to any of the
individual compounds recited above or to a combination of them to
provide relief from pain. Acetaminophen is the analgesic of choice
because it does not have powerful anti-inflammatory effects and
therefore does not interfere with healing of connective tissue. It
also has minimal side effects at therapeutic doses, unlike NSAIDs
which may cause gastrointestinal ulceration or poor renal perfusion
even at therapeutic doses. Thus, the present invention consists of
various combinations of two or more of the following agents: AA,
glucosamine, chondroitin sulfate, pentosan, diacerin,
S-adenosylmethionine, superoxide dismutase, L-ergothionein,
collagen type II, tetracycline-like compounds, one or more ASUs
and, optionally, one or more analgesics, e.g., acetaminophen.
Examples include, but are not limited to such combinations as: two
anabolic agents (e.g., AA and glucosamine); an anabolic agent and
an anti-catabolic agent (e.g., AA and pentosan); an anti-catabolic
and an antioxidant (e.g., tetracyclicline and superoxide
dismutase); or combinations of more than two agents (e.g.,
glucosamine, SAMe and AA) or SAMe, ASUs, acetaminophen and
diacerin. Examples of specific compounds that may be present in ASU
extracts include but are not limited to: limonene, beta carotene,
ubiquinone, and undecaprenol phosphate.
[0082] The following table shows possible combinations of pairs of
the compounds discussed above. The letter "X" marks novel
combinations of compounds that form the novel compositions of the
present invention. The invention also includes combinations of
three or more agents of the following compounds in the combinations
shown on the table:
[0083] Glucosamine
[0084] Chondroitin
[0085] SAMe
[0086] Pentosan
[0087] Superoxide Dismutase (SOD)
[0088] L-ergothioneine
[0089] Collagen Type II
[0090] Diacerin
[0091] Arachadonic Acid
[0092] Tetracycline like compounds
[0093] One or more avocado/soybean unsaponifiables.
[0094] Analgesic, e.g., acetaminophen
[0095] Methylsulfanylmethane (MSM)
[0096] As explained above, examples of desired combinations are
marked by X. For example, the first X in the first row means a
combination of glucosamine and L-ergothioneine or glucosamine and
diacerin. The compositions of the present invention additionally
comprise any aggregation or addition of the combinations marked by
X in any given row or column. For example, the compositions
disclosed in the first row include combinations of glucosamine plus
L-ergothioneine plus diacerin, or glucosamine plus diacerin plus
tetracycline-like compounds or glucosamine plus L-ergothioneine
plus diacerin plus AA plus tetracycline-like compounds, and so on.
Examples of compositions disclosed in the column designated
"Collagen Type II" would include combinations of collagen Type II
plus SAMe plus pentosan, or collagen Type II plus SAMe plus
pentosan plus superoxide dismutase plus L-ergothioneine, and so on.
Examples of compositions disclosed in the column designated "ASU"
would include combinations of one or more ASUs plus glucosamine, or
one or more ASUs plus SAMe plus pentosan, or one or more ASUs plus
collagen Type II plus SAMe plus pentosan plus superoxide dismutase
plus L-ergothioneine, and so on. Similarly, the table shows that an
analgesic, e.g., acetaminophen, can be combined with any other
compound listed in the table either singly or in any combination.
TABLE-US-00001 Superoxide Tetracycline Analgesic Dismutase L-
Collagen Arachadonic like e.g., (SOD) Ergothioneine Type II
Disceria Acid compounds ASU acetominophen Glucosamine X X X X X X
Chondroitin X X X X X X SAMe X X X X X X X X Pentosan X X X X X X X
X superoxide X X X X X X X dismutase (SOD) L-ergothioneine X X X X
X X Collagen X X X X X Type II Disceria X X X X Arachadonic X X X
Acid Tetracycline X X like compounds ASU X
[0097] Certain combinations of the above agents have been
investigated, and a novel response in several combinations was
documented. The effects of certain combinations of chondroitin
sulfate, glucosamine, SAMe, arachidonic acid, collagen, pentosan,
and superoxide dismutase were studied in cultures of adult bovine
cartilage cells in different experiments (see example 2). Certain
combinations had an inhibitory effect (hypometabolic) in this
particular study. Both stimulatory and inhibitory novel
interactions could be beneficial under various disease states. For
example, a hypermetabolic state is part of the pathogenesis of some
diseases. In such diseases, an inhibitory (hypometabolic) response
would be beneficial to the individual. Future studies are planned
to investigate the effects of a range of concentrations in the
agents studied under various experimental models. Note that both
increases and decreases in biosynthetic activity are novel
interactions and could be beneficial to organisms under selected
circumstances. For example, many researchers currently believe that
osteoarthritis has a hypermetabolic component, especially in the
early stages of pathogenesis. Researchers are divided as to whether
treatment should focus on agents that stimulate cartilage matrix
production, or agents that are inhibitory and therefore make the
cartilage environment more hypometabolic, which in turn could have
a stabilizing effect on the cartilage tissue.
[0098] The compositions of the present invention may be
administered via any route, including but not limited to
intramuscularly, intravenously, orally, subcutaneously, rectally,
topically, transcutaneously, intranasally, and intra-articularly,
sublingually, intraperitoneally.
[0099] Also, any salt of any of the present compounds may be used
to aid in absorption, e.g., glucosamine HCl, glucosamine sulfate,
glucosamine phosphate, sodium chondroitin sulfate, calcium
chondroitin sulfate, potassium chondroitin sulfate, etc. In
addition, the compositions of the present invention can be given in
all common dosage forms including extended release dosage forms,
pills, tablets (such as chewable tablets), capsules (such as hard
gelatin capsules, liquid-filled capsules, softgel capsules, etc.),
creams, pastes, powders (such as scoops of powder), liquids,
aerosols, extended release forms, injectables, etc. The
compositions of the present invention may also be given in dosage
forms such as sachets and treats.
[0100] The dosage ranges of the compositions of the present
invention will vary depending upon the needs of the human or animal
to which the compositions are administered. The frequency of dosage
may also vary depending on the needs of the human or animal to
which the compositions are administered.
[0101] For example, a combination of ASU, chondroitin sulfate (CS),
and glucosamine (Gluc) may be administered to an animal such as a
dog or horse. The composition may be administered to the animal
daily (or every other day) at a specific dose (or varying dose) for
an initial period, such as 2-4 weeks or 4-6 weeks. Individual dose
regimens may vary by target subject. The dosage regimen for the
initial period may be designed so that the active components
achieve a steady state in the body fluids that bathe inflamed
tissue in the animal. For example, during the initial period, daily
dosages of the composition in powdered form may be administered to
horses in the following amounts: horses under 600 pounds may be
administered one scoop, horses between 600 and 1200 pounds may be
administered two scoops, and horses over 1200 pounds may be
administered 3 scoops. Some or all of the initial period may pass
before a response to the composition is observed in the animal. For
this reason, various formulations may be considered relatively
slow-acting. Some subjects may respond sooner during the initial
period of administration. Once a clinical response is observed, the
amount and frequency of dosages may be reduced to a level that is
intended to keep the subject comfortable. For example, a long term
administration for horses may be 1/2 scoop, 1 scoop, and 1-2 scoops
daily for horses weighing up to 600 pounds, 600-1200 pounds, and
over 1200 pounds, respectively. Prior to and during strenuous
exercise or an event that may affect inflammation of connective
tissue in the animal, dosage may be increased in amount and/or
frequency, e.g., to the initial administration level, for extra
support. Also, if the animal's comfort level appears to decrease,
the dosage and frequency may be increased, e.g., to the initial
administration levels, for an additional period, such as 2-4 weeks,
before reducing level and frequency again. Once consistent
improvement at a particular dosage level has been observed,
administration may be further reduced in frequency and/or amount,
e.g., to half dosage and half frequency. A subject's dosage and
frequency may be increased at any time as needed, e.g., on weekends
or other times when the subject is more active, in order to provide
extra support to the connective tissues as needed.
[0102] The combination of ASU, CS and Gluc may also be used for
long-term treatments on normal subjects to help prevent and/or
modulate an inflammatory incident.
[0103] The dosage ranges for the various components of the
presently claimed compositions are as follows: TABLE-US-00002
Compound Daily Dose Glucosamine Total dose range: 25 mg to 15 g Or:
3-125 mg/kg for small or large animals or humans Small animal: 25
mg-3 g; or 3-125 mg/kg Human: 100 mg-4 g; or 3-125 mg/kg Large
animal: 300 mg-15 g; or 3-125 mg/kg Chondroitin Total dose range:
15 mg-12 g sulfate Or: 1-75 mg/kg for small or large animals or
humans Small animal: 15 mg-2 g; or 1-75 mg/kg Human: 75 mg-4 g; or
1-75 mg/kg Large animal: 300 mg-12 g; or 1-75 mg/kg SAMe Total dose
range: 10 mg-8 g Small animal: 10 mg-1 g Human: 75 mg-3 g Large
animal: 400 mg-8 g Pentosan Total dose range: 3 mg to 3 g Small
animal: 3 mg-1 g Human: 50 mg-2 g Large animal: 100 mg-3 g
Superoxide Total dose range: 3 mg to 6 g dismutase (each mg
containing >3000 McCord - Fridovich units Small animal: 3 mg-2 g
Human: 5 mg-3 g Large animal: 50 mg-6 g L-ergothioneine Total dose
range: 50 mg to 25 g Small animal: 50 mg-10 g Human: 50 mg-15 g
Large animal: 100 mg-25 g Collagen Type II Total dose range: 0.1 mg
to 10 g Small animal: 0.1 mg-10 g Human: 0.1 mg-7.5 g Large animal:
1.0 mg-10 g Diacerin Total dose range: 5 mg to 5 g Small animal: 5
mg-1 g Human: 20 mg-3 g Large animal: 50 mg-5 g Arachadonic acid
Total dose range: 10 mg to 12 g Small animal: 10 mg-3 g Human: 10
mg-5 g Large animal: 50 mg-12 g Tetracyclines Total dose range: 1.0
mg to 2 g Small animal: 1.0 mg-1 g Human: 2 mg-1.5 g Large animal:
50 mg-2 g Avocado/soybean Total dose range: 5 mg to 5 gram
unsaponifiables Or: 0.5-25 mg/kg for small or large animals or
humans Small animal: 5 mg to 1000 mg; 0.5-25 mg/kg Human: 25 mg to
1500 mg; or 0.5-25 mg/kg Large animal: 100 mg to 5 grams; or 0.5-25
mg/kg Analgesic, e.g., Total dose range: 4 mg to 10 grams
acetaminophen Small animal: (excluding cats): 4 mg to 1000 mg
Human: 100 mg to 4 grams Large animal: 100 mg to 10 grams
[0104] Doses are designed to cover the spectrum of body weights of
small animals to large animals, with humans in the middle. The
following examples are illustrative and do not in any way limit the
present invention. In particular, physiological concentration
ranges, as determined by the best data available to the inventors,
were used.
EXAMPLE 1
[0105] In our preliminary investigations, surgical instability was
induced in the stifle joint of New Zealand white rabbits by
modification of the Hulth technique. Post-operatively, animals were
exercised for 1 hour daily. Experimental dietary formulas were
evaluated for their cartilage stabilizing effect. The standard
Harland (Teklad) rabbit diet (control); a standard diet also
containing a 2% fungal oil containing 40% AA by weight (Arasco);
and a standard diet containing also arachidonic acid and
glucosamine/chondroitin were investigated. At 16 weeks, the medial
femoral condyles of all rabbits were removed and cartilage
degeneration quantitatively evaluated with a modified Mankin
histological-histochemica-1 grading system with safranin-O stained
slides. Cartilage from all joints with surgical instability
exhibited varying degrees of macroscopic degenerative lesions. Our
preliminary results indicated that adding arachidonic acid to
glucosamine/chondroitin sulfate has the potential to produce a
novel interaction in cartilage. This novel interaction has the
potential to have a cartilage modulating effect.
EXAMPLE 2
[0106] Procedure:
[0107] Articular cartilage was resected from human or animal joints
aseptically and placed into a large petri dish in a small amount of
DMEM/F-12 or F-12. The tissue was diced to 1-2 mm dimensions and
transferred to a small culture flask containing 20 mL DMEM or
F-12+400 u/mL collagenase. The flask was placed on the shaker and
incubated overnight.
[0108] The cell digest was repeatedly aspirated to increase release
of cells. The cell digest was then placed into a 50 mL sterile
centrifuge tube and centrifuged in the Beckman at 1000 RPM for 10
minutes. The medium was discarded by pipette and fresh DMEM/F-12
containing 1% FCS added. Depending on the size of the pellet, about
2040 mL medium was added. Cell counts were determined by
haemocytometer and the digest made up to a concentration of 100,000
cells/0.2 mL.
[0109] GAG Synthesis:
[0110] To conduct GAG synthesis, 0.2 mL was aliquoted into each
well of a 96 well plate using an 8 channel pipetter and the cells
allowed to attach for 24 hours. The media was removed and 0.3 mL of
fresh 1% FCS media added for 2-3 days. On the day of the
experiment, the media was removed and the experimental solutions
containing 35-sulfate isotope were added. The incubation was
continued for 4 hours. Termination: at the end of the incubation
period, the labeling media was removed, the cell layer was rinsed
repeatedly with cold 0.3 mL DMEM or F-12 (about 5 times.), and the
cell layer was frozen for counting.
[0111] Counting of 96 Well Plates:
[0112] The cell layer for both the synthesis experiments were
heated at 50 degrees after adding 100 ul 1 N NaOH for a period of 2
hours. 200 ul scintillant was added and the plates were placed in
the counter. The data was expressed as CPM/100,000 cells.
TABLE-US-00003 Indv. Agents: Agents Evaulation CPM/ Sum Combined
Difference Agent 100,000 cells (CPM) (CPM) (CPM) CHSO4-L 64 AA 70
134 18 -116 ChS04-H 50 AA 70 120 81 -39 Glu-H 117 AA 70 187 16 -177
1% Sam 123 10Paleos 86 209 62 -147 1% Sam 123 1Paleos 74 197 80
-117 3% Sam 42 1Paleos 74 116 100 -16 3% Sam 42 10Paleos 86 128 83
-45 3% Sam 42 Collagen 118 160 90 -70 3% Sam 42 AA 70 112 104 -8 AA
70 10Pentos 76 146 106 -40 Collagen 70 10Paleos 86 156 82 -74
Collagen 118 10Pentos 76 194 65 -129 Collagen 118 10 Paleos 86 204
77 -127 ChS0.sub.4 = Chondroitin AA = Arachadonic Acid SAMe =
S-adenosylmethionine Paleos = SOD Collagen = Collagen Pentos =
Pentosan H = High concentration L = Low concentration
[0113] In this model, at the concentrations studied, the
representative combinations had an inhibitory (hypometabolic)
effect in this particular study. This hypometabolic effect could be
beneficial under various disease states, indeed both stimulatory
and inhibitory novel interactions could be beneficial under various
disease states. For example, a hypermetabolic state is part of the
pathogenesis of some diseases. In such diseases, an inhibitory
(hypometabolic) response would be beneficial to the individual.
Future studies are planned to investigate the effects of a range of
concentrations in the agents studied under various experimental
models. Note that both increases and decreases in biosynthetic
activity are novel interactions and could be beneficial to
organisms under selected circumstances. For example, many
researchers currently believe that osteoarthritis has a
hypermetabolic component, especially in the early stages of
pathogenesis. Researchers are divided as to whether treatment
should focus on agents that stimulate cartilage matrix production,
or agents that are inhibitory and therefore make the cartilage
environment more hypometabolic, which in turn could have a
stabilizing effect on the cartilage tissue.
EXAMPLE 3
[0114] A 4 year old child has juvenile rheumatoid arthritis in
which the immune system inappropriately targets endogenous
connective tissues with antibodies against native collagen type II.
The resulting inflammation and degradation of cartilage causes pain
and dysfunction in the synovial joints. Present treatments include
corticosteroids which non-selectively suppress the immune system,
thus leaving the body vulnerable to infectious disease, or
methotrexate, which inhibits DNA synthesis, repair, and cellular
replication, thus affecting not only the immune system but also
intestinal mucosa, and the bone marrow. This child is given 2 mg of
collagen type II daily, and SOD 10 mg daily. The collagen decreases
the inappropriate immune attack, and the SOD inactivates
destructive free radicals that damage cells. By preventing cellular
damage, the SOD helps maximize the normal function of joint tissue
cells. This combination has no harmful side effects at therapeutic
doses and is a beneficial addition to existing therapies for
rheumatoid arthritis.
EXAMPLE 4
[0115] A 6 year old thoroughbred race horse has neutrophilic
inflammation of the carpus. In this condition, trauma to the
tissues of the joint injures cells and therefore results in
liberation of cytokines which attract large numbers of neutrophils
into the synovial space. This response is beneficial in cases of
sepsis, but in non-septic conditions the neutrophils provide no
useful service to the animal. Indeed, because neutrophils produce
various degradative compounds, including superoxide molecules,
their presence in the joint contributes to a vicious cycle of
inflammation, tissue damage, and increased inflammation. Currently
this condition is treated with nonsteroidal antiinflammatory drugs,
which suppress prostaglandin synthesis and therefore have many side
effects. This horse is given a mixture of diacerin 100 mg, pentosan
200 mg and SAMe, 1000 mg The diacerin and pentosan both inhibit
chemotaxis (the attraction of white blood cells into the affected
area) and thus reduce the numbers of neutrophils in the joint.
Additionally, pentosan stimulates the synthesis of synovial fluid
and thus supports normal function of the joint. Diacerin inhibits
superoxide production; since superoxide production is one of the
mechanisms through which neutrophils have their harmful effects,
this action of diacerin is obviously beneficial. SAMe supports the
structure and function of cell membranes, and therefore helps
repair injured joint tissue cells thus blocking the events that
start the harmful inflammation. This combination has no harmful
side effects at therapeutic doses and is a great improvement over
existing therapies.
EXAMPLE 5
[0116] A 47 year-old woman has severe knee osteoarthritis.
Currently she requires large doses of NSAIDs to control her
symptoms. Although her orthopedic surgeon has recommended taking
glucosamine/chondroitin sulfate, she has been reluctant to do so
because these compounds are extracted from animal tissues and the
patient is a strict vegetarian. Instead she takes diacerein 25 mg
and ASU 250 mg, and 500 mg of acetaminophen daily. The diacerin
inhibits chemotaxis and thereby reduces inflammation in the knee
joint. The ASU increases TGF beta 1 and 2, stimulating repair of
damaged joint tissues. The acetaminophen causes rapid analgesia,
reducing the patient's symptoms without adversely affecting
cartilage metabolism and without risk of gastrointestinal
ulceration. As a result of the reduction in pain, the patient
decides to add a 15 minute walk to her daily schedule. The
controlled exercise further improves her physical and mental
state.
EXAMPLE 6
[0117] A 5 year old Jersey dairy cow is diagnosed with severe
osteoarthritis following an episode of fever and synovitis
attributed to Lyme disease. This animal is the source of the
owner's family milk supply and the owner wishes to treat the
lameness with compounds that are "natural," i.e., compounds that
normally occur in plants and animal bodies, rather than pursuing
more traditional solutions such as 1) culling the animal 2) using
non-steroidal anti-inflammatory drugs or 3) using steroids. The
animal is treated with ASU 900 mg, SAMe 600 mg and glucosamine 500
mg daily. This approach is an improvement over existing options for
several reasons. Because the compounds are natural components of
plants and animal bodies with documented wide margins of safety,
there is less concern over metabolites secreted in the milk.
Because the compounds are available orally, and are active in small
amounts, they are easy to administer to the animal in feed. The
combined effect of the three compounds is to reduce inflammation,
and pain, to support normal function, and to stimulate healing of
connective tissues.
[0118] Various additional examples illustrate the effect of various
compositions in inhibiting or otherwise modulating the markers of
inflammation and pain, such as COX-2. In several of these examples,
various formulations were designed to reduce the markers of
inflammation and pain, but not to eliminate those markers entirely
due to the possibility of adverse side effects. Thus, one goal
achieved by some embodiments described in the following examples
was the reduction of the inflammation and pain markers to
approximately control levels, or slightly above or below control
levels.
[0119] In several of the following Examples, the ASU that was used
(i.e., ASU-NMX 1000.TM., Nutramax Laboratories Inc., Edgewood, Md.
USA) was dissolved and diluted in 100% ethanol (Sigma-Aldrich) to
achieve the desired concentrations for use in the particular
experiment. The concentrations used in the studies were based on
the minimum phytosterol content of the ASU composition. The desired
concentration of ASU for use in a specific experiment was first
determined by incubating bovine chondrocytes (5.times.10.sup.5
cells/well) for 72 hrs with: (i) control media alone or (ii) ASU at
concentrations of 25, 8.3, 2.7, 0.9, and 0.3 .mu.g/ml. Cells were
activated with lipopolysaccharide (LPS, 20 ng/ml; Sigma-Aldrich)
for 24 hrs, and cellular supernatants were analyzed for secreted
PGE-2 and nitrite concentrations. There was no significant effect
on PGE-2 and nitrite levels at 0.3 or 0.9 .mu.g/ml. At 2.7
.mu.g/ml, there was a slight suppression of PGE-2 and nitrite
levels. The highest levels of suppression were found to be between
8.3 and 25 .mu.g/mL. The ASU concentrations used in these Examples
were selected based on the above data and on reported clinical
dosage and previous published in vitro data for ASU.
[0120] In Examples 7-11, the following terms and definitions will
be used:
[0121] Cyclooxygenase-2 (COX-2) is a protein that functions as an
enzyme and specifically regulates the production of certain
chemical messengers called prostaglandins (PGE-2). This PGE-2
molecule causes the pain and swelling of inflammation observed in
arthritic conditions. When COX-2 activity is blocked, inflammation
is reduced. COX-2 is active only at the site of inflammation.
[0122] Prostaglandin E2 (PGE-2) is a chemical messenger that
belongs to a group of hormone-like substances that participate in a
wide range of body functions including inflammation. PGE-2 causes
pain and swelling during inflammation.
[0123] p38: MAP kinase (MAPK) is also known as mitogen-activated
protein kinase 14. MAP kinase p38 is involved in a signaling system
that controls cellular responses to cytokines, stress and bacterial
products like lipopolysaccharides (LPS).
[0124] Mitogen-activated protein kinases (MAPK): MAPK
serine/threonine is a specific protein kinase that responds to
extracellular stimuli and regulates various cellular activities
including gene expression, proliferation, differentiation and
function. It is involved in cell signaling and communication such
as signaling pathways responsive to stimuli exemplified by physical
stress and cytokines.
[0125] Cytokines are diverse proteins involved in cellular
signaling and communication like hormones and neurotransmitters.
They are critical to the functioning of both innate and adaptive
immune response and play a major role in a variety of
immunological, inflammatory and infectious diseases.
[0126] Tumor necrosis factor alpha (TNF-.alpha.) belongs to a
superfamily of proteins called cytokines which induce death
(necrosis) of tumor cells and possess a wide range of
pro-inflammatory activity. TNF-.alpha. is multifunctional, and
inhibiting its activity is beneficial in reducing the inflammation
in inflammatory diseases including arthritis.
[0127] Interleukin-1 beta (IL-1.beta.) is a protein belonging to
the cytokine family produced by various cells, including
chondrocytes, macrophages, and fibroblasts. It is a major regulator
of inflammation. IL-1.beta. raises body temperature and the
production of other chemical mediators involved in inflammation and
innate immunity.
[0128] Inducible nitric oxide synthase (iNOS) is a soluble enzyme
that controls the production of nitric oxide (NO) following
exposure to cytokines and other stimulators. iNOS is important in
inflammation and in defense against infection.
[0129] Chemokines are proteins that are produced by a variety of
cells which have the ability to attract different cells to the site
of inflammation and or injury and to help localize these cell in
situ. Two examples of chemokines are interleukin-8 (IL-8) and
monocyte chemotactic protein (MCP).
EXAMPLE 7
Regulation of Prostaglandin E-2 Production in IL-1.beta. Activated
Chondrocytes Propagated on Microcarrier Spinner Culture
[0130] The study of Example 7 was designed to evaluate whether
chondrocytes propagated in microcarrier spinner culture can be
activated by interleukin-1.beta. (IL-1.beta.) to produce
prostaglandin E-2 (PGE-2); and whether this activation can be
blocked by natural products known individually to have
anti-inflammatory activity: Avocado Soybean Unsaponifiables (ASU),
glucosamine (Glu), and chondroitin sulfate (CS).
[0131] Method: Canine chondrocytes (4.times.10.sup.3/cm.sup.2)
seeded in collagen microcarrier beads were propagated in spinner
culture for 14 days. They were next incubated with: media alone or
the combination of ASU (NMX-1000.TM., 25 .mu.g/mL), CS
(TRH122.RTM., 20 .mu.g/mL) and Glu (FCHG49.RTM., 10 .mu.g/mL) for
24 hrs. The combination of ASU, Glu, and CS was supplied by
Nutramax Laboratories, Inc. Cultures were then incubated with media
alone or activated with IL-1.beta. (10 ng/mL) at 37.degree. C., 5%
CO.sub.2 for 24 hrs. The supernatant was assayed for PGE-2 content.
Chondrocytes were analyzed by microscopy and immunofluorescence for
type II collagen. Data was analyzed by ANOVA with the Tukey
post-hoc test. Values of p<0.05 were considered statistically
significant.
[0132] Results: By following this method, it was found that
chondrocytes attached, multiplied on microcarriers, and produced
extracellular matrix material, as illustrated in FIGS. 6A and 6B.
These figures show a phase contrast photomicropgraph of
chondrocytes growing on microcarriers indicating that the cells
proliferate and produce extracellular matrix.
[0133] As illustrated in FIG. 7 ("Light Microscope Image of
Immunostained Chondrocytes Confirming Their Continued Production of
Type II Collagen Which Is Characteristic of their Articular
Cartilage Phenotype"), the cultures formed aggregates and
immunostained for type II collagen, indicating continued production
of the protein.
[0134] As illustrated in FIG. 8 ("Production of PGE-2 by
Chondrocytes after IL-1.beta. indicating that the chondrocytes
continue to be responsive even at subsequent passage"), activation
of chondrocyte-seeded microcarriers at passage 3 and 4 showed
similar responsiveness to the cytokine with IL-1.beta., PGE-2
levels of 179% and 165% of non-activated controls,
respectively.
[0135] Pretreatment of chondrocyte-seeded microcarriers with the
combination of ASU, Glu, and CS significantly reduced PGE-2 levels
to about 60% below non-activated controls (p<0.05).
[0136] As illustrated in FIG. 9 ("PGE-2 Production by Chondrocytes
Is Inbited by the Combination of ASU-CS and Glu indicating that the
combination decreases the production of this pro-inflammatory
marker"), chondrocytes increased PGE-2 levels (19152.+-.2721 pg/mL)
when activated with IL-1.beta.. ASU, CS, and Glu combination
inhibited PGE-2 production (4020.+-.468 pg/mL) by 79% when compared
to IL-1.beta. activated control (p<0.05).
[0137] Conclusion: This evidence demonstrates that the microcarrier
spinner culture system can be used to evaluate chondrocyte
responses to pro-inflammatory stimuli and to identify agents that
can modify these responses. The dynamic condition in the
microcarrier spinner bioreactor appears to recapitulate the
biomechanical environment that chondrocytes encounter in the joint.
Therefore, the microcarrier spinner culture system may represent a
useful tool to evaluate the potential anti-inflammatory properties
of natural products. Using this culture system, we observed that
the combination of ASU, Glu, and CS effectively blocks activation
of the inflammatory pathway.
EXAMPLE 8
Suppression of TNF-.alpha., IL-1.beta., iNOS, and p38 Expression by
the Combination of Avocado Soy Unsaponifiables, Glucosamine, and
Chondroitin Sulfate in Human Macrophage-Like THP-1 Cells
[0138] Osteoarthritis (OA) is a degenerative joint disease
characterized by erosion of articular cartilage and secondary
inflammation of the synovial membrane. The synovial membrane
contains monocyte/macrophage-like cells that produce mediators
critical to the pathogenesis of OA. Such pro-inflammatory mediators
include chemokines, cytokines, prostaglandins, and nitric oxide.
Recent clinical and in vitro studies have indicated that certain
natural products such as Avocado Soybean Unsaponifiables (ASU),
Glucosamine (Glu), and Chondroitin Sulfate (CS) each have
anti-inflammatory properties. ASU has been reported to reduce pain
and functional disability in OA patients. Similarly, the
combination of Glu and CS was also shown to alleviate pain and
improve joint mobility in the subgroup of patients suffering with
moderate to severe OA. In the study of Example 8, we evaluated
whether the combination of ASU, Glu, and CS would have a more
profound effect in suppressing pro-inflammatory gene expression
than ASU alone, or the combination of Glu and CS together. These
treatments were tested in the well-documented surrogate
monocyte/macrophage THP-1 cell line.
[0139] The study of Example 8 seeks to determine whether the
combination of Avocado Soy Unsaponifiables (ASU), glucosamine
(Glu), and chondroitin sulfate (CS) was more effective in
suppressing pro-inflammatory gene expression than ASU alone, or the
combination of Glu and CS together.
[0140] Methods: Human monocyte/macrophage surrogate THP-1 cells
(5.times.10.sup.5 cells) were incubated for 24 hrs at 37.degree. C.
and 5% CO2 with: (i) control media alone, (ii) ASU (8.3 .mu.g/ml;
NMX10000.TM.-ASU), (iii) Glu (15 mM; FCHG49.RTM.) and CS (20
.mu.g/ml; TRH122.RTM.), or with (iv) a combination of ASU (8.3
.mu.g/ml), Glu (15 mM), and CS (20 .mu.g/ml). All test materials
were supplied by Nutramax Laboratories, Inc., Edgewood, Md. The
cells were then activated with 20 ng/ml LPS for 1 hour. Total RNA
was extracted and subjected to RT-PCR analysis using primers
specific to TNF-.alpha., IL-1.beta., iNOS, p38, and S14 as the
housekeeping gene.
[0141] Results: Pre-treatment with the combination of ASU, Glu, and
CS profoundly suppressed the expression of TNF-.alpha., IL-1.beta.,
and iNOS by 50-80% in activated THP-1 cells. The combination
treatment reduced TNF-.alpha. and IL-1.beta. expression to levels
similar to baseline non-activated controls and reduced iNOS
expression to levels lower than baseline non-activated levels. The
inhibitory effect of the combined preparation on TNF-.alpha.,
IL-1.beta., and iNOS expression is more profound than ASU alone, or
Glu and CS together. The inhibition of cytokine and iNOS expression
is associated with a profound suppression of p38 expression.
[0142] As illustrated in FIG. 10 ("Inhibition of TNF-.alpha.
Expression in Monocyte/macrophage-like THP-1 cells by the
Combination of ASU-CS-Glu indicating that the combination appears
to be better than individual components"), pre-incubation with the
combination of ASU, Glu, and CS suppressed TNF-.alpha. expression
by >75% in LPS-activated cells. The combination treatment
down-regulated TNF-.alpha. expression to levels similar to
non-activated control (C) levels. The inhibitory effect of the
combination was more profound than the individual agents alone.
[0143] As illustrated in FIG. 11 ("Inhibition of IL-1.beta.
Expression in Monocyte/macrophage-like THP-1 cells by the
Combination of ASU-CS-Glu indicating that the combination appears
to be better than individual components"), pre-incubation with the
combination of ASU, Glu, and CS suppressed IL-1.beta. expression by
50% in LPS-activated cells. The combination treatment was more
effective in suppressing IL-1.beta. expression than the individual
agents alone.
[0144] As illustrated in FIG. 12 ("Inhibition of iNOS Expression in
Monocyte/macrophage-like THP-1 cells by the Combination of
ASU-CS-Glu indicating that the combination appears to be better
than individual components"), pre-incubation with the combination
of ASU, Glu, and CS suppressed iNOS expression by 80% in
LPS-activated cells. The combination suppressed iNOS expression to
levels lower than non-activated control (C) levels. The combination
was more effective in suppressing iNOS expression than the
individual agents alone.
[0145] As illustrated in FIG. 13 ("Inhibition of p38 Expression in
Monocyte/macrophage-like THP-1 cells by the Combination of
ASU-CS-Glu indicating that the combination appears to be better
than individual components"), pre-incubation with the combination
of ASU, Glu, and CS suppressed p38 expression by 75% in
LPS-activated cells. The combination treatment down-regulated p38
expression to levels lower than non-activated control (C) levels.
The combination was more effective in suppressing p38 expression
than the individual agents alone.
[0146] DISCUSSION/CONCLUSION: This example demonstrates that the
combination of ASU, Glu, and CS was more effective in suppressing
pro-inflammatory gene expression than ASU alone, or Glu and CS
together. The suppression of TNF-.alpha., IL-1.beta., and iNOS was
associated with down-regulation of p38, a key signal transduction
mediator involved in joint inflammation. These findings suggest the
potential clinical utility of the combination treatment to
alleviate pain and inflammation in OA patients, particularly for
those who fail to respond to the single treatments alone.
EXAMPLE 9
Inhibition of Cyclooxygenase-2 Gene Expression and Prostaglandin E2
Production by Avocado Soybean Unsaponifiables (ASU) in
Chondrocytes
[0147] Introduction: Cyclooxygenase-2 (COX-2) is a critical enzyme
involved in inflammation and plays a key role in the production of
the pro-inflammatory mediator prostaglandin (PGE-2). Also known as
prostaglandin G/H synthase, COX-2 catalyzes the stepwise conversion
of arachidonic acid into two short-lived intermediates,
prostaglandin G (PGG) and prostaglandin II (PGH). PGG isomerizes to
different forms, including PGE-2. Non-steroidal antiinflammatory
drugs (NSAIDs) are used extensively to suppress inflammation and
alleviate pain in osteoarthritis (OA) by inhibiting prostaglandin
synthesis. More recently, Complementary and Alternative Medicines
(CAM), such as plant-derived products, have been documented to
exert potent anti-inflammatory activity. See, e.g., Soeken K L. et
al. Clin J. Pain. 20(1): 13-8, 2004. Among these are extracts from
Avocado Soybean Unsaponifables (ASU). The anti-inflammatory
activity of ASU has been tested on tissue cell prototypes primarily
derived from the immune-inflammatory system. Little is known about
the effect of ASU on cartilage cells. As the only cellular
component of cartilage, chondrocytes synthesize pro-inflammatory
mediators such as PGE-2. The study of Example 9 tested the
hypothesis that ASU effectively inhibits COX-2 gene expression,
thereby suppressing PGE-2 synthesis.
[0148] Materials and Methods: Articular chondrocytes were isolated
from the metacarpal joints of mature Holsteins by collagenase
digestion. Chondrocytes were plated (5.times.105/well) and
maintained for 5-7 days prior to use. Chondrocytes were
pre-incubated with: (i) ASU (25 .mu.g/ml) for 72 .mu.l s, or (ii)
control media alone for 72 hrs. The chondrocytes were next
re-incubated with control media alone or activated with 20 ng/ml of
Lipopolysaccharide (LPS) at 37.degree. C., 5% CO.sub.2, for: (a) 1
hour to determine COX-2 expression by RT-PCR analysis and (b) 24
hours to measure secreted PGE-2 levels by immunoassay. Cells were
lysed and total RNA was extracted with TRIzol.RTM. (Life
Technologies.TM.. Equal amounts (1 .mu.g) of total RNA were
subjected to reverse transcription-polymerase chain reaction
(RT-PCR). Bovine primers specific for COX-2, and GAPDH as the
housekeeping gene were used. The gels containing ethidium bromide
were electrophoresed to visualize the bands under UV light. Three
to five separate runs were performed. Multiple comparisons by
one-way ANOVA (Tukey post-hoc analysis) were performed using the
SigmaStat statistical program where p<0.05 was considered
statistically significant.
[0149] Results: Pre-incubation of chondrocytes with Avocado Soybean
Unsaponifiables reduced baseline expression of COX-2 in
non-activated bovine chondrocytes. As shown in FIGS. 14A and 14B
("Inhibition of COX-2 Expression in Activated Chondrocytes by
ASU"), pre-incubation of chondrocytes with ASU for 72 hrs, followed
by activation with LPS for 1 hour, profoundly blocked the
activation of COX-2 transcripts down to baseline levels.
[0150] In addition, as shown in FIG. 15 ("Inhibition of PGE-2
Production Activated Chondrocytes by ASU"), pre-incubation of
chondrocytes with ASU for 72 hours followed by activation with LPS
for 24 hours, significantly reduced the concentration of secreted
PGE-2 (P<0.01), as shown in FIG. 15. Multiple comparisons by
one-way ANOVA (Tukey post-hoc analysis) were performed using the
SigmaStat statistical program where p<0.05 was considered
statistically significant.
[0151] Discussion/Conclusion: The study of example 9 demonstrates
that ASU inhibits the activation of COX-2 expression in
chondrocytes. This inhibition results in decreased production of
the pro-inflammatory mediator PGE-2. Blockage of PGE-2 production
has been documented to relieve pain associated with inflammation.
Our findings support the proposed utility of ASU in the management
of painful conditions, exemplified by osteoarthritis.
EXAMPLE 10
Inhibition of Pro-Inflammatory Cytokine and COX-2 Expression in
Chondrocytes and Monocytes by Avocado Soybean Unsaponifiables
(ASU)
[0152] The cytokines TNF-.alpha., and IL-1.beta., and the enzyme
cyclooxygenase-2 (COX-2), are known as the principal mediators in
chronic inflammatory disorders. COX-2 is the critical enzyme
involved in inflammation by regulating the production of
prostaglandin PGE-2. Non-steroidal antiinflammatory drugs (NSAIDs)
are used extensively to suppress inflammation and alleviate pain,
particularly in osteoarthritis, by inhibiting cytokine and PG
synthesis. More recently, alternative approaches to the management
of pain and inflammation have provided encouraging results. Among
these are extracts from Avocado Soybean Unsaponifiables (ASU).
Clinical studies in humans suggest that ASU reduces pain associated
with inflammation and reduces the extent of joint space narrowing.
Little is known about the effect of ASU on cellular targets. The
study of Example 10 tested the hypothesis that ASU inhibits gene
expression of COX-2, TNF-.alpha. and IL-1.beta. in chondrocytes and
monocytes. The surrogate monocyte-macrophage-like THP-1 cells were
used.
[0153] Articular chondrocytes (5.times.10.sup.5/well) from the
metacarpal joints of mature Holsteins and human THP-1 monocyte-like
cells (5.times.10.sup.5/well) were pre-incubated with: (i) ASU (25
.mu.g/mL) or (ii) control media alone for 72 and 24 hrs
respectively. Cells were re-incubated with control media alone or
20 ng/ml of lipopolysaccharides (LPS) for: (a) 1 hr to determine
gene expression by reverse transcription-polymerase chain reaction
(RT-PCR) analysis and (b) 24 hrs to measure secreted PGE-2 levels
by immunoassay. Primers specific for bovine and human COX-2,
TNF-.alpha., IL-1.beta. and GAPDH as the housekeeping gene were
used. The gels containing ethidium bromide were electrophoresed to
visualize the DNA bands under UV light. Three to five separate runs
were performed. Multiple comparisons by one-way ANOVA (Tukey
post-hoc analysis) were performed using the SigmaStat statistical
program where p<0.05 was considered statistically significant.
ASU reduced baseline expression of COX-2, TNF-.alpha. and
IL-1.beta. in non-activated bovine chondrocytes.
[0154] Moreover, ASU blocked the activation of these mediators in
cells induced by LPS. Blockage of COX-2 expression led to
significant reduction of secreted PGE-2 by 93+/-1% (P<0.01).
Similarly, pre-incubation of THP-1 cells with ASU for 24 hrs
followed by activation with LPS for 1 hr profoundly blocked the
expression of TNF-.alpha. and IL-1.beta.transcripts compared to
control cells activated with LPS alone.
[0155] The study of Example 10 demonstrates for the first time that
ASU dramatically suppresses the expression of TNF-.alpha. and
IL-1.beta. in chondrocytes and monocytes, while confirming the
reduction of COX-2 transcripts in chondrocytes. This observation
supports the positive clinical findings that ASU ameliorates pain
and inflammation. Our study supports the proposed utility of ASU in
the management of painful conditions, exemplified by
osteoarthritis.
EXAMPLE 11
Pro-Inflammatory Gene Expression in Chondrocytes is Inhibited by
the Combination of Avocado Soybean Unsaponifiables, Glucosamine,
and Chondroitin Sulfate
[0156] Introduction: Osteoarthritis (OA) is a degenerative joint
disease characterized by erosion of articular cartilage and
secondary inflammation of the synovial membrane. Cartilage erosion
is induced by pro-inflammatory mediators produced by chondrocytes
in cartilage and monocyte/macrophages localized in the synovial
membrane. Clinical studies have documented the benefit of using
Avocado Soybean Unsaponifiables (ASU), glucosamine (Glu), and
chondroitin sulfate (CS) in the management of OA. ASU has been
shown to reduce pain and minimize functional impairment in OA
patients. See, e.g., Ernst E. Clin Rheum. 2003; 22(4-5): 285-8.
Similarly, the combination of Glu and CS significantly reduced
lameness and improved joint mobility in animals suffering from OA.
See, e.g., Hanson R R et al. Equine Practice. 1997; 19(9):16-22;
and Canapp S O et al. Amer. J. Vet. Res. 1999; 60(12): 1552-7. See
also, e.g., Henrotin Y E et al. Clin Rheum. 1998; 17(1): 31-9; and
Chan P S et al. Osteoarthritis Cart. 2005; 13(5): 387-94.
Expression of pro-inflammatory mediators is regulated through the
p38 MAPK signaling pathway. Our study evaluated whether the
combination of ASU, Glu, and CS profoundly down-regulated
pro-inflammatory gene expression through the p38 signaling pathway.
We evaluated the effect of these compounds in human and equine
chondrocytes.
[0157] Materials and Methods: Equine chondrocytes were isolated
from articular cartilage by collagenase digestion. Equine
chondrocytes and human articular chondrocytes (ATCC) were plated at
a density of 5.times.10.sup.5 cells/well. Cells were incubated at
37.degree. C., 5% CO.sub.2 for 24 hrs with control media alone, or
physiologic concentrations of: (i) ASU (8.3 .mu.g/ml;
NMX1000.TM.-ASU), (ii) Glu (11 .mu.g/ml; FCHG49.RTM.) and CS (20
.mu.g/ml; TRH122.RTM.), or (iii) a combination of ASU (8.3
.mu.g/ml), Glu (11 .mu.g/ml), and CS (20 .mu.g/ml). To induce
inflammation, cells were activated for 1 hr with lipopolysaccharide
(20 ng/ml, LPS) or interleukin-1-beta (10 ng/ml, IL-1.beta.). Total
RNA was isolated using TRIzol.RTM. (Life Technologies.TM.) and gene
expression was analyzed using RT-PCR.
[0158] Results: In activated equine chondrocytes, the combination
treatment suppressed COX-2 expression to levels similar to
non-activated control levels. In addition, the combination of ASU,
Glu, and CS suppressed chemokine expression in activated human
chondrocytes. See FIG. 16 ("Inhibition of Chemokine IL-8 Expression
in Activated Chondrocytes by the Combination of ASU-CS-Glu
indicating that the combination appears to be better than
individual components") and FIG. 17 ("Inhibition of Chemokine MCP
Expression in Activated Chondrocytes by the Combination of
ASU-CS-Glu indicating that the combination appears to be better
than individual components"). Interleukin-8 (IL-8) and MCP
expression was down-regulated to levels similar to non-activated
control levels.
[0159] Discussion/Conclusion: The study of Example 11 demonstrated
that the combination of ASU, Glu, and CS profoundly suppressed
pro-inflammatory gene expression in chondrocytes. The combination
treatment was effective in reducing the expression of chemokines.
The suppression of pro-inflammatory mediators, such as chemokines,
are critical in modulating the pro-inflammatory response in the
osteoarthritic joint. Our results reinforce the potential clinical
utility of the combination of ASU, Glu, and CS in the management of
OA, providing an alternative option to patients who fail to respond
to the single agents alone.
[0160] Based on the teaching of the present invention, one of skill
in the art would understand that combinations of the compounds
taught by the present invention would act synergistically. For
example, it is understood that glucosamine has stimulatory effects
on chondrocyte metabolism which, by itself, aids in ameliorating
diseases of cartilage degradation. However, an increase in cell
metabolism can also produce an increase in free-radical production,
as a natural by-product of oxidative phosphorylation. The increase
in free radical production would dilute the beneficial effects of
the glucosamine administration. By combining L-ergothioneine with
glucosamine, one would expect an increase in metabolism and a
reduction in free-radical damage, providing for a greater benefit
than if compounds leading to one of these effects were provided.
Therefore, one of skill in the art, based on the teaching of the
present invention, would understand that combining glucosamine with
L-ergothioneine would be more beneficial than providing either
alone. The synergy that exists between certain compounds in the
present invention also enables the use of lower doses of each
compound. Although these compounds are quite safe, there may be a
potential for side effects. For example, large doses of glucosamine
sulfate or chondroitin sulfate can cause gastrointestinal
disturbances in some individuals. In addition, these compounds are
costly; for these reasons, the ability to minimize the dose and
still achieve beneficial effects is desirable.
[0161] Many modifications may be made without departing from the
basic spirit of the present invention. While various embodiments of
the present invention have been described above, it should be
understood that they have been presented by way of example only,
and not limitation. Accordingly, it will be appreciated by those
skilled in the art that within the scope of the appended claims,
the invention may be practiced other than has been specifically
described herein. Thus, the breadth and scope of the present
invention should not be limited by any of the above-described
exemplary embodiments, but should be defined only in accordance
with the following claims and their equivalents.
* * * * *