U.S. patent application number 09/249335 was filed with the patent office on 2002-08-29 for useof anabolic agents anti-catabolic agents and antioxidant agents for protection treatment and repair of connective tissues in humans and animals.
Invention is credited to CORSON, BARBARA E.RN. DVM, HAMMAD, TAREK, HENDERSON, TODD R. DVM, LIPPIELLO, LOUIS, SOLIMAN, MEDHAT.
Application Number | 20020119950 09/249335 |
Document ID | / |
Family ID | 22943035 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119950 |
Kind Code |
A1 |
HENDERSON, TODD R. DVM ; et
al. |
August 29, 2002 |
USEOF ANABOLIC AGENTS ANTI-CATABOLIC AGENTS AND ANTIOXIDANT AGENTS
FOR PROTECTION TREATMENT AND REPAIR OF CONNECTIVE TISSUES IN HUMANS
AND ANIMALS
Abstract
The present invention relates to compositions for the
protection, treatment and repair of connective tissues in humans
and animals comprising any or all of anabolic, anti-catabolic, and
anti-oxidant agents, including aminosugars, S-adenosylmethionine,
arachadonic acid, GAGs, pentosan, collagen type II, tetracyclines
or tetracycline-like compounds, diacerin, super oxide dismutase,
and L-ergothionine 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. DVM;
(JARRETSVILLE, MD) ; CORSON, BARBARA E.RN. DVM;
(FAWN GROVE, PA) ; HAMMAD, TAREK; (BALTIMORE,
MD) ; SOLIMAN, MEDHAT; (MINYA, EG) ;
LIPPIELLO, LOUIS; (SCOTTSDALE, AZ) |
Correspondence
Address: |
COVINGTON & BURLING
ATTN: PATENT DOCKETING
1201 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20004-2401
US
|
Family ID: |
22943035 |
Appl. No.: |
09/249335 |
Filed: |
February 12, 1999 |
Current U.S.
Class: |
514/62 ; 514/152;
514/154; 514/392; 514/560 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 45/06 20130101 |
Class at
Publication: |
514/62 ; 514/560;
514/152; 514/154; 514/392 |
International
Class: |
A61K 038/00; A61K
031/70; A61K 031/65; A61K 031/415; A61K 031/20 |
Claims
We claim:
1. A composition for the treatment, repair or prevention of damage
to connective tissue comprising an aminosugar and one or more
compounds selected from the group consisting of L-ergothionine,
diacerin, arachadonic acid, and tetracycline-like compounds.
2. A composition for the treatment, repair or prevention of damage
to connective tissue comprising a GAG and one or more compounds
selected from the group consisting of L-ergothionine, diacerin,
arachadonic acid, and tetracycline-like compounds.
3. A composition for the treatment, repair or prevention of damage
to connective tissue comprising SAMe and one or more compounds
selected from the group consisting of SOD, L-ergothionine, collagen
type II, diacerin, arachadonic acid, and tetracycline-like
compounds.
4. A composition for the treatment, repair or prevention of damage
to connective tissue comprising pentosan and one or more compounds
selected from the group consisting of SOD, L-ergothionine, collagen
type II, diacerin, arachadonic acid, and tetracycline-like
compounds.
5. A composition for the treatment, repair or prevention of damage
to connective tissue comprising a GAG-like compound and one or more
compounds selected from the group consisting of SOD,
L-ergothionine, collagen type II, diacerin, arachadonic acid, and
tetracycline-like compounds.
6. A composition for the treatment, repair or prevention of damage
to connective tissue comprising SOD and one or more compounds
selected from the group consisting of L-ergothionine, collagen type
II, diacerin, arachadonic acid, and tetracycline-like
compounds.
7. A composition for the treatment, repair or prevention of damage
to connective tissue comprising L-ergothionine and one or more
compounds selected from the group consisting of collagen type II,
diacerin, arachadonic acid, and tetracycline-like compounds.
8. A composition for the treatment, repair or prevention of damage
to connective tissue comprising collagen type II and one or more
compounds selected from the group consisting of diacerin,
arachadonic acid, and tetracycline-like compounds.
9. A composition for the treatment, repair or prevention of damage
to connective tissue comprising diacerin and one or more compounds
selected from the group consisting of arachadonic acid, and
tetracycline-like compounds.
10. A composition for the treatment, repair or prevention of damage
to connective tissue comprising arachadonic acid and
tetracycline-like compounds.
11. A method of preventing, treating or repairing damage to
connective tissue in humans and animals comprising administering
the compositions of any one of claims 1-10 to a human or animal in
need thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions for the
protection, treatment and repair of connective tissues in humans
and animals.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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-.beta., 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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
PGA1 on Cartilage Growth," Experientia, 33(4):504 (1976)]. 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.
[0010] 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.
[0011] 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.
[0012] 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 extracelluiar 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] L-ergothionine scavenges hydroxyl radicals and may inhibit
singlet oxygen formation, [Han JS. "Effects of Various Chemical
Compounds on Spontaneous and Hydrogen Peroxide Induced Reversion in
Strain TA104 of Salmonella typhimuriu." 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.
[0017] 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 and anti-oxidant agents for the
maintenance and repair of connective tissues in humans and
animals.
SUMMARY OF THE INVENTION
[0018] 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, and anti-oxidant agents for
the protection, treatment and repair of connective tissues in
humans and animals.
[0019] 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, and anti-oxidant agents.
[0020] It is still another object of the present invention to
provide compositions any or all of anabolic, anti-catabolic, and
anti-oxidant 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), and L-ergothionine.
[0021] 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,
arachidonic acid, GAG, pentosan sulfate, collagen type II,
tetracyclines, diacerin, SOD, and L-ergothionine.
[0022] These and other objects of the present invention are
apparent from the detailed descripton and claims below.
[0023] Priority Claim:
[0024] In connection with this application, priority is claimed to
the following provisional application: THE USE OF ANABOLIC AGENTS,
ANTI-CATABOLIC AGENTS AND ANTIOXIDANT AGENTS FOR PROTECTION,
TREATMENT AND REPAIR OF CONNECTIVE TISSUES IN HUMANS AND ANIMALS,
U.S. Ser. No. 60/074,594, filed Feb. 13, 1998.
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.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The compositions of the present invention, used to treat,
repair, and prevent damage to connective tissue, consist of
anabolic, anti-catabolic, and anti-oxidant agents selected from the
group consisting of glucosamine, SAMe, AA, chondroitin sulfate,
pentosan sulfate, collagen type II, tetracyclines, diacerin, SOD,
and L-ergothionine. In addition, the present invention covers
methods of administering these novel compositions to humans and
animals in need thereof.
[0029] 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, 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.
[0030] 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.
[0031] 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.
[0032] 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. 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)].
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] Pentosan's mechanism of action can be summarized as
follows:
[0039] 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).
[0040] 2. Inhibition of chemotaxis of granulocytes, which are white
blood cells that contribute to inflammation.
[0041] 3. Stimulatory effect on proteoglycan synthesis.
[0042] 4. Stimulatory effects on hyaluronic acid synthesis by
synovial fibroblasts.
[0043] 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.
[0044] Other synthetic or semi-synthetic glycosaminoglycans or
glycosaminoglycan-like compounds, such as polysulfated
glycosaminoglycans, may be used in this invention.
[0045] Diacerein, a recently recognized organic compound found in
plants of the genus Cassia has anti-inflammatory effects through
inhibition of interleukin-1.beta.; 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 turnover of chondroitin-4-sulfate resulting in a
decrease in the ratio of chondroitin-6-sulfate to
chondroitin-4-sulfate. (This ratio is pathologically increased in
arthritic joints.) It mildly increases prostaglandin synthesis,
which allows it to have protective effects on the gastric
mucosa.
[0046] S-adenosylmethionine (SAMe) is an important endogenous
compound, resent throughout the body, and taking part in a great
number of biologic reactions such s 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.
[0047] 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. [Champ 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); Carney, M., "Neuropharmacology of S-Adenosyl
Methionine," Clin. Neuropharmacol., 9(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. [Champ
and Harvey, Biochemistry, 1994].
[0048] Superoxide dismutase is an enzyme present naturally in the
tissues of animals, 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.
[0049] L-ergothionine 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 (Sept. 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)].
[0050] 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).
[0051] 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.
[0052] Although the effects of these compounds have been
investigated in isolation, 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.
[0053] 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, and AA, which promote growth processes in the body; (2)
anti-catabolic agents, such as chondroitin sulfate, pentosan
sulfate, collagen type II, tetracyclines and diacerin, which
inhibit destructive or catabolic processes; and (3) antioxidants,
such as SOD, and L-ergothionine which prevent tissue damage by
scavenging toxic oxygen species (free radicals). Naturally, some
compounds 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. 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, and tetracycline-like compounds.
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., glucosamine and collagen type II);
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).
[0054] 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:
[0055] Glucosamine
[0056] Chondroitin
[0057] SAMe
[0058] Pentosan
[0059] Superoxide Dismutase (SOD)
[0060] L-Ergothionine
[0061] Collagen Type II
[0062] Diacerin
[0063] Arachadonic Acid
[0064] Tetracycline like compounds
[0065] 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-ergothionine. 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-ergothionine plus
diacerin, or glucosamine plus diacerin plus tetracycline-like
compounds or glucosamine plus L-ergothionine 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-ergothionine, and so on.
1 Superoxide Tetracycline Dismutase L- Collagen Arachadonic like
(SOD) Ergothionine Type II Diacerin Acid compounds Glucosamine X X
X X Chondroitin X X X X SAMe X X X X X X Pentosan X X X X X X
Superoxide X X X X X Dismutase (SOD) L- X X X X Ergothionine
Collagen X X X Type II Diacerin X X Arachadonic X Acid
[0066] The present inventors have investigated certain combinations
of the above agents and have documented a novel response in several
combinations. 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.
[0067] 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. Also, any salt of any of the
present compounds may be used to aid in absorption, e.g,
glucosamine HCl, glucosamine sulfate, sodium chondroitin sulfate,
etc. In addition, the composition can be given in all common dosage
forms including extended release dosage forms, e.g., pills,
tablets, capsules, etc.
[0068] 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 dosage ranges for
the various components of the presently claimed compositions are as
follows:
2 Compound Daily Dose Glucosamine Total dose range: 25 mg to 12g
small animal: 25 mg-3 g. human: 100 mg-4 g large animal: 300 mg.-12
g Chondroitin sulfate Total dose range: 15 mg-12 g small animal: 15
mg-2 g human: 75 mg-4 g large animal: 300 mg-12 g 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 dismutase Total dose range: 3 mg to 6 g (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-5 g human: 10 mg-8 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
[0069] 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.
EXAMPLE 1
[0070] 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- l 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
[0071] Procedure:
[0072] 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.
[0073] 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
20-40 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.
[0074] GAG Synthesis:
[0075] 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.
[0076] Counting of 96 Well Plates:
[0077] 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 n sulfate program was used with 1 minute counting
time. The data was expressed as CPM/100,000 cells.
3 Indv. Agents: Agents Evaluation 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
10 Paleos 86 209 62 -147 1% Sam 123 1 Paleos 74 197 80 -117 3% Sam
42 1 Paleos 74 116 100 -16 3% Sam 42 10 Paleos 86 128 83 -45 3% Sam
42 Collagen 118 160 90 -70 3% Sam 42 AA 70 112 104 -8 AA 70 10
Pentos 76 146 106 -40 Collagen 70 10 Paleos 86 156 82 -74 Collagen
118 10 Pentos 76 194 65 -129 Collagen 118 10 Paleos 86 204 77 -127
ChSO4 = Chondroitin AA = Arachadonic Acid SAMe =
S-adenosylmethionine Paleos = SOD Collagen = Collagen Pentos =
Pentosan H = High concentration L = Low concentration
[0078] 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
[0079] A 4 year 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
[0080] A 6 yr 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.
[0081] 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 have
a 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.
[0082] Many modifications may be made without departing from the
basic spirit of the present invention. 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. Hence, the attached claims are
intended to cover the invention embodied in the claims and
substantial equivalents thereto.
* * * * *