AU7822798A - Aminosugar, glycosaminoglycan, and s-adenosylmethionine composition for the treatment and repair of connective tissue - Google Patents

Description

WO 99/62524 PCTIUS98/11748 5 AMINOSUGAR, GLYCOSAMINOGLYCAN, AND S-ADENOSYLMETHIONINE COMPOSITION FOR THE TREATMENT AND REPAIR OF CONNECTIVE TISSUE 10 BACKGROUND OF THE INVENTION Cross-Reference to Related Application The present application is a continuation-in-part of co-pending United States 15 Patent Application Serial No. 08/779,996, filed December 23, 1996, the disclosure of which is incorporated by reference herein in its entirety. 1. Field of the Invention The present invention relates to compositions for the repair and reduction of inflammation of connective tissue in humans and animals and, in particular, to 20 compositions capable of promoting anti-inflammation, chondroprotection, chondromodulation, chondrostabilization, chondrometabolization and the repair and replacement of human and animal connective tissue. 2. Background of the Invention The connective tissues of humans and animals are constantly subjected to stresses 25 and strains from mechanical forces and from diseases that can result in afflictions, such as arthritis, joint inflammation and stiffness. Indeed, connective tissue afflictions are quite common, presently affecting millions of Americans. Further, such afflictions can be not only painful but, in their extreme, debilitating. The treatment of connective tissue afflictions can be quite problematic. A simple 30 decrease in the stress to which the connective tissue is subjected is often not an option, - 1 - WO 99/62524 PCT/US98/11748 especially in the case of athletes and animals such as race horses. Consequently, treatment is often directed at controlling the symptoms of the afflictions and not their causes, regardless of the stage of the degenerative process. Presently, steroids, such as corticosteroids and NSAIDs, are widely used for the 5 treatment of these ailments. [Vidal, et al., Pharmocol. Res. Commun., 10:557-569 (1978)]. However, drugs such as these, which inhibit the body's own natural healing processes, may lead to further deterioration of the connective tissue. Connective tissue, for example articular cartilage, is naturally equipped to repair itself by manufacturing and remodeling prodigious amounts of collagen (a chief 10 component of connective tissue such as cartilage) and proteoglycans (PGs) (the other major component of connective tissue such as cartilage). This ongoing process is placed under stress when an injury occurs. In such cases, the production of connective tissue matrix (collagen and PGs) can double or triple over normal levels, thereby increasing the demand for the building blocks of both collagens and proteoglycans. 15 The building blocks for collagen are amino acids, especially proline, glycine and lysine. PGs are large and complex macromolecules comprised mainly of long chains of modified sugars called glycosaminoglycans (GAGs) or mucopolysaccharides. The terms GAGs and mucopolysaccharides are understood in the art to be interchangeable. PGs provide the framework for collagen formation and also hold water to give flexibility, 20 resiliency and resistance to compression. Like almost every biosynthetic pathway in the body, the pathways by which both collagen and GAG form single molecule precursors are quite long. As is also characteristic of other biosynthetic pathways, the pathways by which collagen and GAGs are produced include what is called a rate-limiting step -- that is, one highly regulated 25 control point beyond which there is a commitment to finish. The presence of such rate - 2 - WO 99/62524 PCTIUS98/11748 limiting steps permits complicated biosynthetic processes to be more easily and efficiently controlled by permitting the organism to focus on one point. For example, if conditions demand production and all the requisite raw materials are in place, then stimulation of the rate-limiting step will cause the end product to be produced. To stop or 5 slow production, the organism needs simply to regulate the rate-limiting step. In the production of PGs, the rate-limiting step is the conversion of glucose to glucosamine for the production of GAGs. Glucosamine, an aminosugar, is the key precursor to all the various modified sugars found in GAGs, including glucosamine sulfate, galactosamine, N-acetylglucosamine, etc. Glucosamine also makes up to 50% of 10 hyaluronic acid -- the backbone of PGs -- on which other GAGs, like chondroitin sulfate are added. The GAGs are then used to build PGs and, eventually, connective tissue. Once glucosamine is formed, there is no turning away from the synthesis of GAG polymers. Glucosamine has been shown to be rapidly absorbed into humans and animals is after oral administration. A significant portion of the ingested glucosamine localizes to cartilage and joint tissues, where it remains for long periods. This indicates that oral administration of glucosamine reaches connective tissues, where glucosamine is incorporated into newly-synthesized connective tissue. Glycosaminoglycans and collagen are the chief structural elements of all 20 connective tissues. Their synthesis is essential for proper maintenance and repair of connective tissues. In vitro, the introduction of glucosamine has been demonstrated to increase the synthesis of collagen and glycosaminoglycans in fibroblasts, which is the first step in repair of connective tissues. In vivo, topical application of glucosamine has enhanced wound healing. Glucosamine has also exhibited reproducible improvement in 25 symptoms and cartilage integrity in humans with osteoarthritis. [L. Bucci, Nutritional - 3 - WO 99/62524 PCTIUS98/11748 Supplement Advisor, (July 1992)]. The pathway for the production of proteoglycans may be briefly described as follows. Glucosamine is the main building block of connective tissue and may be provided either through the enzymatic conversion of glucose or through diet or external 5 administration (see FIG. 1). Glucosamine may be converted into the other main component of connective tissue, namely PGs, upon incorporation of glucosamine into GAGs (see FIG. 2). More specifically, GAGs are large complexes of polysaccharide chains associated with a small amount of protein. These compounds have the ability to bind large amounts 10 of water, thereby producing a gel-like matrix that forms the body's ground substance. GAGs stabilize and support cellular and fibrous components of tissue while maintaining the water and salt balance of the body. The combination of insoluble protein and the ground substance forms connective tissue. For example, cartilage is rich in ground substance while tendon is composed primarily of fibers. 15 GAGs are long chains composed of repeating disaccharide units of monosaccharides (aminosugar-acidic sugar repeating units). The aminosugar is typically glucosamine or galactosamine. The aminosugar may also be sulfated. The acidic sugar may be D-glucuronic acid or L-iduronic acid. GAGs, with the exception of hyaluronic acid, are covalently bound to a protein, forming proteoglycan monomers. These PGs 20 consist of a core protein to which linear carbohydrate chains formed of monosaccharides are attached. In cartilage proteoglycan, the species of GAGs include chondroitin sulfate and keratin sulfate. The proteoglycan monomers then associate with a molecule of hyaluronic acid to form PG aggregates. The association of the core protein to hyaluronic acid is stabilized by link proteins. 25 The polysaccharide chains are elongated by the sequential addition of acidic - 4 - WO 99/62524 PCT/US98/11748 sugars and aminosugars, and the addition is catalyzed by a family of transferases. Aminosugars, such as glucosamine, are synthesized through a series of enzymatic reactions that convert glucose to glucosamine, or alternatively may be provided through the diet. The glucosamine is then incorporated into the GAGs as described above. Acidic 5 sugars may be provided through the diet, may be obtained through degradation of GAGs by degradative enzymes, or produced through the uronic acid pathway. Since repeating disaccharide units contain one aminosugar (such as glucosamine), it is clear that the presence of an aminosugar in the production of connective tissue is important. Glucosamine is, by far, the more important ingredient in the production of 10 connective tissue since it is the essential building block of GAGs. See FIG 1. All GAGs contain hexosamine or uronic acid derivative products of the glucose pathway and from exogenous glucosamine, for example: Hyaluronic acid Glucosamine + Glucuronic Acid Keretan-Sulfate Glucosamine + Galactose 15 Chondroitin Sulfate Glucuronic Acid + Galactosamine Heparin Sulfate Glucosamine + Glucuronic or Iduronic Acid Heparan Sulfate Glucosamine + Glucuronic or Iduronic Acid Dermatin Sulfate Iduronic Acid + Galactosamine Chondroitin sulfate is a GAG that provides a further substrate for the synthesis of 20 the proteoglycans. The provision of the chondroitin in its salt (sulfate) form facilitates its delivery and uptake by the humans and animals in the production of connective tissue. In addition, the sulfate portion of chondroitin sulfate is available for use in catalyzing the conversion of glucosamine to GAGs. Fragments of GAGs, including chondroitin sulfate, may also be used to provide a substrate for synthesis of proteoglycans since the assembly 25 of PG occurs in the extracellular space. In addition, chondroitin sulfate has been shown to have cardiovascular health benefits. [Morrison et al., Coronary Heart Disease and the Mucopolysaccharides - 5 - WO 99/62524 PCTIUS98/11748 (Glycosaminoglycans), pp. 109-127 (1973)]. Thus, the preferred form of glycosaminoglycan included in the compositions of the present invention is chondroitin sulfate or fragments thereof. Chondroitin may be more efficacious than glucosamine for injury rehabilitation. 5 [Christensen, Chiropractic Products, pp. 100-102 (April 1993)]. An evaluation of glucosamine versus chondroitin for treatment of osteoarthritis has been conducted and concludes, contrary to Christensen, that glucosamine is preferred. [Murray, MPI's Dynamic Chiropractic, pp. 8-10 (September 12, 1993)]. Neither reference teaches or suggests combining the materials. Bucci (Townsend Letter for Doctors, pp. 52-54, 10 January 1994), discloses the combination of glucosamine and chondroitin for treatment of osteoarthritis. Bucci has acknowledged that this combination was personally disclosed to him by one of the present inventors. Chondroitin sulfate also acts to inhibit the degradative enzymes that break down connective tissue. In so doing, chondroitin sulfate promotes the maintenance of healthy 15 connective tissues. When combined with glucosamine, which functions primarily as a building block for the synthesis of connective tissue, chondroitin sulfate works in concert with the glucosamine but may work in a different fashion. The ability of chondroitin sulfate to block degradation is one of its important functions. S-Adenosylmethionine (SAM) is a significant physiologic compound which is 20 present throughout body tissue and takes part in a number of biologic reactions as a methyl group donor or as an enzymatic activator during the synthesis and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and proteins. SAM may be second only to adenosine triphosphate (ATP) in the variety of reactions in which it is a cofactor. SAM is metabolized via three metabolic pathways of transmethylation, 25 transsulfuration, and aminopropylation. [Stramentinoli, Am. J. Med., 83(5A):35-42 - 6 - WO 99/62524 PCTIUS98/11748 (1987)]. In higher organisms, SAM plays a significant role in transmethylation processes with more than 40 anabolic or catabolic reactions involving the transfer of the methyl group of SAM to substrates such as nucleic acids, proteins, and lipids, among others. Also, the release of the methyl group from SAM is the start of a "transsulfuration" 5 pathway that produces all endogenous sulfur compounds. After donating its methyl group, SAM is converted into S-adenosylhomocysteine, which in turn is hydrolyzed to adenosine and homocysteine. The amino acid cysteine may then be produced from the homocysteine. The cysteine thus produced may exert a reducing effect by itself or as an active part of glutathione, which is a main cell anti-oxidant. [Stramentinoli, cited above]. 10 SAM has been used to treat various disorders. In various forms of liver disease, SAM acts as an anticholestatic agent. [Adachi et al., Japan Arch. Inter. Med., 33:185-192 (1986)]. SAM has also been administered as an antidepressant for use in the management of psychiatric disorders [Caruso et al., Lancet, 1: 904 (1984)], and as an anti inflammatory compound in the management of osteoarthritis [Domljan et al., Int. J. Clin. 15 Pharm. Toxicol., 27(7):329-333 (1989)]. Low levels of SAM are believed to play a role in reducing the risk of certain cancers. [Feo et al., Carcinogenesis, 6:1713-20 (1985)]. In addition, the administration of SAM has also been associated with a fall in the amount of early reversible nodules and the prevention of the development of late pre-neoplastic lesions and hepatocellular 20 carcinomas. [Garcea et al., Carcinogenesis, 8:653-58 (1987)]. Unfortunately, SAM per se is unstable due to its high reactivity. The relatively recent synthesis of stable salts, however, has made SAM available for research and therapeutic use. [See, e.g., U.S. Patent Nos. 4,990,606 and 5,102,791]. SAM has been used outside of the United States in a number of clinical trials 25 concerning the treatment of osteoarthritis. While used in these trials primarily as an -7 - WO 99/62524 PCT/US98/11748 analgesic and replacement for NSAID therapy, SAM is a precursor of polyamines. In addition to their analgesic and anti-inflammatory properties, and their ability to scavenge free radicals, polyamines may stabilize the polyanionic macromolecules of proteoglycans. [Schumacher, Am. J. Med., 83(5A):2 (1987)]. 5 SAM may also function as a source of endogenous sulfur, which will increase sulfation of GAGs to be incorporated in proteoglycans. The inclusion of SAM is particularly beneficial in instances of subclinical deficiencies of SAM, occurring especially in elderly populations with higher risk of osteoarthritis [Frezza et al., Gastroenterol., 99:211-215 (1990)]. The supplementation of SAM may aid in instances 10 of SAM deficiency where the ability of the body to sulfate GAGs may be compromised. In addition, a number of metabolites of SAM aid in the repair of connective tissue, including glutathione, polyamines, methylthioadenosine, and adenosine. Glutathione works as a scavenger of oxygen-related products [Shumacher, Am. J. Med., _8(Supp 5a):1-4 (1987); Matthew & Lewis, Pharmacol. (Life Sci. Adv.), 9:145-152 (1990); Szabo is et al., Science, 214:200-202 (1981)] and thus has an anti-inflammatory effect. Polyamines, including spermine, spermidine, and putrescine, stabilize polyanionic macromolecules of proteoglycans [Schumacher, cited above; Conroy et al., Biochem. J., 162:347-350 (1977)] and thus protect proteolytic and glycolytic enzymes. These polyamines also have an anti-inflammatory effect [Bird et al., Agents Actions, 13:342 20 347 (1983); Oyangui, Agents Actions, 14:228-237 (1984)], probably as a scavenger of oxygen-related products [Kafy et al., Agents Actions, 18:555-559 (1986); Matthews & Lewis, cited above], and have an analgesic effect [Bird et al., cited above; Oyangui, cited above]. The SAM metabolite methylthioadenosine has a pronounced anti-inflammatory effect [Matthews & Lewis, 1990] while adenosine has a more modest anti-inflammatory 25 effect [Matthews & Lewis, 1990]. -8- WO 99/62524 PCTIUS98/11748 Studies have shown that some forms of exogenous SAM are stable in digestive juices when given orally. [Stramentinoli et al., cited above; Vendemiale et al., Scand. J. Gastroenterol., 24:407-415 (1989)]. The metabolism of exogenous SAM appears to follow known pathways of endogenous SAM metabolism. [Kaye et al., Drugs, 40(Suppl. 5 3):124-138 (1990)]. In humans, oral SAM was tolerated to the same extent as placebo with very mild nonspecific side effects. [Schumacher, cited above; Frezza et al., cited above]. Manganese plays a role in the synthesis of GAGs, collagen and glycoproteins which are important constituents of cartilage and bone. Manganese is important for 10 enzyme activity of glycosyltransferases. This family of enzymes is responsible for linking sugars together into glycosaminoglycans, adding sugars to other glycoproteins, adding sulfate to aminosugars, converting sugars into other modified sugars, and adding sugars to lipids. The enzymatic functions of glycosyltransferases are important in glycosaminoglycan synthesis (hyaluronic acid, chondroitin sulfate, keratan sulfate, 15 heparin sulfate and dermatin sulfate, etc.), collagen synthesis, and in the functions of many other glycoproteins and glycolipids. Manganese also plays a role in the synthesis of glycosaminoglycans and glycoproteins, which are important constituents of cartilage and bone. Many reproductive problems in horses and skeletal abnormalities in foals have been ascribed to manganese 20 deficiency. [Current Therapy in Equine Medicine, 2:402-403 (1987)]. Manganese deficiency leads to abnormal bone growth, swollen and enlarged joints, and slipped tendons in humans and animals. In humans, manganese deficiencies are also associated with bone loss and arthritis. Levels of all glycosaminoglycans are decreased in connective tissues during manganese deficiencies, with chondroitin sulfates 25 being most depleted. Manganese-deficient organisms quickly normalize - 9 - WO 99/62524 PCT/US98/11748 glycosaminoglycans and collagen synthesis when manganese is replenished. Approximately 40% of dietary manganese is absorbed by the body tissue. Storage of manganese in the body is minimal -- a mere 12 to 20 mg is present in the body at any one time. Large amounts of calcium and phosphorus in the intestine are also known to 5 interfere with manganese absorption. The richest dietary sources are the foods least consumed by the general public, such as whole grain cereals and breads, dried peas, beans and nuts. The ascorbate form of manganese is preferred due to the high bioavailability and the need for vitamin C (ascorbic acid) for collagen production. Vitamin C also enhances manganese uptake by the body. 10 Other optional ingredients in the compositions of the present invention are methyl donors or methyl donor cofactors, such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine. These ingredients augment the function of SAM in that they are cofactors in methylation. In addition, these compounds are likely to be lacking in patients suffering from connective tissue disorders. For example, it is 15 estimated that 12% of the elderly population in the United States suffers from a vitamin B 12 deficiency, a group more likely to suffer from connective tissue disorders. An adequate amount of vitamin B12, for example, has an important environmental influence on the accumulation of homocysteine that results from the metabolism of SAM. In other words, methyl donors or methyl donor cofactors, such as 20 vitamin B12 and the others listed in the preceding paragraph, can reduce levels of homocysteine when administered either alone or in combination. Vitamin B 12 is generally known to function as a coenzyme in biochemical reactions such as the synthesis of proprionic acid and of methionine. Recent evidence suggests that the elevated levels of plasma homocysteine increase the risk of occlusive 25 vascular disease. Adequate amounts of vitamin B12 are considered the most important - 10 - WO 99/62524 PCT/US98/11748 environmental influence on the accumulation of unnecessary homocysteine. [Joosten et al., Am. J. Clin. Nutr., 58(4): 468-76 (1993)]. In addition, it is also understood that vitamin B12 may play a role in the methylation of selenium. [Chen and Whanger, Tox. and Appl. Pharm., 118:65-72 (1993)]. Specifically, increased levels of vitamin B12 5 significantly contribute to selenium methylation and might decrease overall selenium toxicity by preventing its accumulation in tissues. [Chen and Whanger, cited above]. 3. Description of Background Art Several disclosures suggest provide exogenous quantities of glucosamine in order to bypass the rate-limiting step of the conversion of glucose to glucosamine in those 10 pathways that produce PGs. For example, the intravenous administration of glucosamine (a precursor of the GAGs) and derivatives thereof has been disclosed in United States Patent No. 3,232,836, issued to Carlozzi et al., for assisting in the healing of wounds on the surface of the body. In United States Patent No. 3,682,076, issued to Rovati, the use of glucosamine and salts thereof is disclosed for the treatment of arthritic conditions. 15 Finally, the use of glucosamine salts has also been disclosed for the treatment of inflammatory diseases of the gastrointestinal tract in United States Patent No. 4,006,224 issued to Prudden. In vitro, glucosamine increases synthesis of collagen and glycosaminoglycans, the first step in repair of connective tissues, in fibroblasts. In vivo, topical application of glucosamine has enhanced wound healing. 20 Several disclosures also suggest going one step further in bypassing the glucose to-glucosamine rate-limiting step, by providing exogenous quantities of various of the modified sugars found in the GAGs for producing proteoglycans. For example, in United States Patent No. 3,6797,652 issued to Rovati et al., the use of N-acetylglucosamine is disclosed for treating degenerative afflictions of the joints. 25 In still other disclosures of which we are aware, it has been taught to go still one - 11 - WO 99/62524 PCT/US98/11748 step further in bypassing the glucose-to-glucosamine rate-limiting step by providing exogenous quantities of the GAGs themselves (with and without various of the modified sugars). For example, in United States Patent No. 3,371,012 issued to Furuhashi, a preservative is disclosed for eye graft material that includes galactose, N 5 acetylglucosamine (a modified sugar found in the GAGs) and chondroitin sulfate (a GAG). Additionally, United States Patent No. 4,486,416 issued to Soll et al., discloses a method of protecting comeal endothelial cells exposed to the trauma of intraocular lens implantation surgery by administering a prophylactically effective amount of chondroitin sulfate. Also, United States Patent No. 5,141,928 issued to Goldman discloses the 10 prevention and treatment of eye injuries using glycosaminoglycan polysulfates. United States Patent No. 4,983,580 issued to Gibson, discloses methods for enhancing the healing of comeal incisions. These methods include the application of a corneal motor composition of fibronectin, chondroitin sulfate and collagen to the incision. In United States Patent No. 4,801,619 issued to Lindblad, the intraarticular 15 administration of hyaluronic acid is disclosed for the treatment of progressive cartilage degeneration caused by proteoglycan degradation. The use of a SAM and selenium composition as a nutritional supplement is disclosed in United States Patent Application Ser. No. 08/725,194 filed by one of the present inventors and is herein incorporated by reference. In addition, one of the 20 inventors of the present invention has taught, in United States Patent No. 5,587,363 the combination of an aminosugar, such as glucosamine, and a glycosaminoglycan, such as chondroitin, for treatment of degenerative joint diseases. One of the present inventors has further taught the optional inclusion of manganese in a composition of an aminosugar and a glycosaminoglycan in United States Patent No. 5,364,845. 25 Accordingly, it can be seen that there remains a need for compositions which - 12 - WO 99/62524 PCT/US98/11748 include analgesic, anti-inflammatory, and antidepressant components, as well as components that provide the building blocks for the production of connective tissue in humans and that also protect against the degradation of that tissue. 5 SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a composition for the protection and repair and for reducing the inflammation of connective tissue in humans and animals. It is a further object of the present invention to provide compositions which 10 contain S-Adenosylmethionine and an aminosugar or salts thereof, such as glucosamine, for facilitating the repair and reducing the inflammation of connective tissue in humans and animals. It is another object of the present invention to provide compositions which contain S-Adenosylmethionine and GAGs, such as chondroitin salts and fragments thereof, for 15 facilitating the repair and for reducing the inflammation of connective tissue in humans and animals. It is yet a further object of the present invention to provide compositions which contain S-Adenosylmethionine, an aminosugar or salts thereof, and GAGs or fragments thereof for facilitating the repair and for reducing the inflammation of connective tissue in 20 humans and animals. It is another object to optionally provide manganese to any of these compositions for humans and animals. It is still a further object to optionally provide methyl donors or methyl donor cofactors, such as vitamins B 12 and B6, folic acid, dimethylglycine, and trimethylglycine, 25 to the compositions of the present invention for humans and animals if desirable. - 13 - WO 99/62524 PCT/US98/11748 It is a further object of the present invention to provide methods of administering these compositions. These and other objects of the present invention will become readily apparent from a reading of the following detailed description and examples. 5 BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a sequence for the biosynthesis of hexosamines. FIG. 2 is a schematic flowchart illustrating the biological pathway by which the composition of the present invention aids in protection and repair of connective tissue. 10 FIG. 3 is an enlarged portion of the flowchart of FIG. 2. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, a composition selected from the group consisting of SAM and an aminosugar or salts thereof (e.g., glucosamine); SAM and 15 GAGs (e.g., chondroitin salts) or fragments thereof; and SAM, an aminosugar (or salts thereof), and GAGs (or fragments thereof) is provided to humans and animals for stimulating both collagen and PG synthesis and for reducing inflammation. Manganese, preferably manganese salts, may optionally be included to any of these compositions. In addition, other optional ingredients include methyl donors or methyl donor cofactors, 20 such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine. These compositions may act to accomplish several functions, including bypassing the glucose to glucosamine rate-limiting step in the natural production of proteoglycans in humans and animals, and producing additional quantities of collagen and proteoglycans for use in the repair of damaged connective tissue. In addition, inflammation of connective tissue may 25 be reduced by the compositions of the invention. The compositions of the present - 14 - WO 99/62524 PCTIUS98/11748 invention may achieve these functions directly or through indirect pathways -- i.e., through their effect on other components in the living system which in turn can increase connective tissue synthesis or reduce inflammation. In one embodiment, a composition of the present invention include S 5 Adenosylmethionine (SAM) and an aminosugar, such as glucosamine, preferably in a salt form. In another embodiment of the present invention, the composition includes SAM and a glycosaminoglycan, such as chondroitin (preferably in a salt form such as chondroitin sulfate). In another embodiment, the composition of the present invention includes SAM, an aminosugar, such as glucosamine, preferably in a salt form, and a 10 glycosaminoglycan, such as chondroitin (preferably in a salt form, such as chondroitin sulfate). Alternatively, fragments of a glycosaminoglycan may be used in a composition of the invention in addition to or in substitution for the glycosaminoglycan. Each of these compositions may optionally include manganese. A preferred form of manganese in such compositions is a manganese salt, such as manganese ascorbate, because the ascorbate is a is soluble form of manganese which further provides ascorbic acid, a substance needed for collagen synthesis. Other manganese salts such, as for example, sulfate or gluconate, may be used however. Each of these compositions may optionally contain one or more methyl donors or methyl donor cofactors selected from the group consisting of vitamins B 12 and B6, folic acid, dimethylglycine, and trimethylglycine. 20 Referring to FIGS. 2 and 3, the biosynthetic pathway for the production of connective tissue, which is affected by the method of the present invention by virtue of the components of the composition of the present invention which aid in connective tissue repair, functions as described in the above background section of this application. In a preferred embodiment, the aminosugar glucosamine is the base of the 25 composition, providing the primary substrate for both collagen and proteoglycan - 15 - WO 99/62524 PCT/US98/11748 synthesis. Glucosamine is the preferred substrate for proteoglycan synthesis, including chondroitin sulfates and hyaluronic acid. The glucosamine preferably is in a salt form so as to facilitate its delivery and uptake by humans and animals. The preferred salt forms are glucosamine hydrochloride, glucosamine sulfate and N-acetylglucosamine. 5 Administration of a preferred embodiment of the composition of the present invention provides the human or animal organism with exogenous quantities of SAM, an aminosugar or salts thereof, and a glycosaminoglycan or fragments thereof. If desired, the composition also provides the human or animal organism with exogenous quantities of manganese cofactors. Also if desired, the compositions of the present invention may 10 include methyl donors or methyl donor cofactors, such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine. The exogenous glucosamine provided by the composition of present invention is converted to proteoglycans as is seen in FIG. 2 and as described above. In the former case, the glucosamine may be converted with the aid of manganese 15 directly into GAG, including hyaluronic acid (which is 50% glucosamine and which forms the backbone of the proteoglycans). This core protein is then linked to the hyaluronic acid via the link protein, as is seen in FIG. 3. In the latter case, the free amino acids are, with the aid of manganese and zinc cofactors (and ascorbic acid or vitamin C), converted to procollagen. The procollagen is 20 then converted into collagen with the aid of copper or iron cofactors and vitamin C (ascorbic acid) and sulfate chelates. Thus, preferred compositions of the present invention containing SAM and glucosamine advantageously stimulate the synthesis of collagen and glycosaminoglycans or mucopolysaccharides (GAGs), including hyaluronic acid, the backbone of 25 proteoglycans (PG's), thereby providing a natural tissue repair function. These - 16 - WO 99/62524 PCT/US98/11748 compositions provide the connective tissue repair function of glucosamine, the increased sulfation of GAGs by SAM, the stabilization by SAM metabolites of the polyanionic macromolecules of proteoglycans, and the additional analgesic, anti-inflammatory, and anti-depressant effects of SAM. The optional addition of manganese provides a further 5 benefit if a deficiency of the mineral exists or if it is otherwise desired. The optional inclusion of methyl donors or methyl donor cofactors, such as vitamins B 12 and B6, folic acid, dimethylglycine, and trimethylglycine, helps to promote methylation and thereby convert homocysteine to methionine. Another preferred composition of the invention comprises SAM and chondroitin 10 salts (such as chondroitin sulfate). SAM operates in this composition, in conjunction with endogenous glucosamine, as described above. Chondroitin salts operate with SAM and endogenous glucosamine by inhibiting the synovial degradative enzymes. Chondroitin salts (such as chondroitin sulfate) also directly contribute to the pool of GAGs of cartilaginous tissue. Manganese salts may also be included in this composition in those 15 cases where a deficiency of manganese exists. Methyl donors or methyl donor cofactors, such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine, may optionally be included in these compositions to help promote methylation and thereby convert homocysteine to methionine. Another preferred embodiment of the composition of the present invention 20 contains SAM, glucosamine, and chondroitin salts (such as chondroitin sulfate) and mixtures and fragments thereof, and also advantageously stimulates the synthesis of collagen and glycosaminoglycans or mucopolysaccharides (GAGs), including hyaluronic acid, thereby providing a natural tissue repair function. This composition provides the superior connective tissue repair function of glucosamine, the above-described benefits of 25 SAM, and the above-described benefits from chondroitin salts (including chondroitin - 17 - WO 99/62524 PCTIUS98/11748 sulfate) and fragments of chondroitin salts. Chondroitin salts (including chondroitin sulfate) also operate with SAM and glucosamine by inhibiting the synovial degradative enzymes. Chondroitin salts (including chondroitin sulfate) also directly contribute to the pool of GAGs of cartilaginous tissue. Manganese provides a further benefit if a 5 deficiency of the mineral exists. As with the compositions described above, methyl donors or methyl donor cofactors, such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine, may optionally be included in these compositions to help promote methylation and thereby convert homocysteine to methionine. Tissue repair can thus be accomplished, in the context of the treatment and repair of connective 10 tissue and the treatment of arthritic conditions, in almost all areas of the body both human and animal. In the present method for the treatment and repair and for reducing the inflammation of connective tissue in humans and animals, preferred compositions comprising amounts of SAM in combination with glucosamine including salts thereof in 15 combination with chondroitin salts (including chondroitin sulfate) or fragments thereof, or amounts of SAM and chondroitin salts (including chondroitin sulfate) or fragments thereof in combination with glucosamine including salts thereof, may be administered to humans and animals thereof for stimulating both collagen and proteoglycan synthesis. An additional preferred composition comprising amounts of SAM and chondroitin salts 20 (including chondroitin sulfate) or fragments thereof may be administered to humans and animals for stimulating proteoglycan synthesis and reducing inflammation. Manganese salts may also be optionally included in each composition in cases where a deficiency of manganese exists. Methyl donors or methyl donor cofactors, such as vitamins B12 and B6, folic acid, dimethylglycine, and trimethylglycine may optionally be included to these 25 compositions as well. - 18 - WO 99/62524 PCTIUS98/11748 The compositions of the present invention are administered to promote tissue repair, including cartilage repair, and the treatment of arthritic conditions as well as connective tissue damage in humans and animals. The anti-depressant effect of SAM may help to alleviate the burden of sickness for some patients, thus enhancing their 5 quality of life. This effect, as well as the analgesic and anti-inflammatory effects of SAM which will help alleviate the pain associated with arthritic conditions, may help remove impediments to physical activity. Increased levels of physical activity, in turn, can supply the loading and unloading forces necessary for the regeneration of articular cartilage. Supplementation with glucosamine, with its chondroprotective role, thus helps to ensure 10 that the raw materials are available to support the increased regeneration of cartilage. The compositions of the present invention are also understood to play a role in chondromodulation, chondrostabilization, and chondrometabolizaton. The dosage of SAM in the nutritional supplements of the present invention ranges from about 5 mg to about 5,000 mg in humans and small animals, and from about 2 mg to 15 about 20,000 mg in large animals (e.g., equine). The dosage of glucosamine in the nutritional supplements of the present invention ranges from about 50 mg to about 5,000 mg in humans and small animals, and from about 250 mg to about 40,000 mg in large animals (e.g., equine). The dosage of chondroitin salts in the nutritional supplements of the present invention ranges from about 15 mg to about 5,000 mg in humans and small 20 animals, and from about 100 mg to about 30,000 mg in large animals. When included in the compositions of the present invention, manganese may optionally be present in the range of about 2 to about 75 mg in humans and small animals, and from about 10 mg to about 500 mg in large animals. The ascorbate component of the manganese ascorbate may range from about 10 mg to about 500 mg in humans and small animals, and from 25 about 50 mg to about 2,500 mg in large animals. When included in the compositions of - 19 - WO 99/62524 PCT/US98/11748 the present invention, the methyl donors or methyl donor cofactors, such as vitamins B 12 and B6, folic acid, dimethylglycine, and trimethylglycine may be present in the range of about 0.1 mg to about 10 mg in humans and small animals, and from about 1 mg to about 100 mg in large animals. 5 As a preferred embodiment, a dosage of the nutritional supplement composition of the present invention may consist of one or more capsules or tablets for human oral consumption. In such an embodiment, the preferred weight of the dosage is between about 5 mg to about 5,000 mg, and preferably about 2,500 mg. The dosage may be administered in a single daily dosage form in which all components are present, e.g., a 10 capsule or tablet of preferably 2,500 mg. The dosage may also be administered in more than one dosage form in which each dosage form contains at least one component. When a single dosage is administered in more than one dosage form, the multiple dosage forms may be co-administered as a single dosage. Thus, for example, a single dosage may be comprised of a SAM dosage form co-administered with a glucosamine and chondroitin 15 salts dosage form. Alternatively, the nutritional supplement compositions of the present invention may be administered more than once daily. Hence, for example, the nutritional supplement compositions of the present invention may be in the form of an oral dosage form of 1250 mg administered twice daily or 833 mg administered three times daily. The 20 number of daily administrations will depend upon the needs of the human or animal recipient. Different connective tissue disorders and injuries require different amounts of the compositions of the present invention. In that regard, several dosages may be administered depending on the particular needs of the human or animal. Alternatively, and of particular use in large animals, the compositions of the 25 present invention may for example be administered in scoops. Such administration may - 20 - WO 99/62524 PCT/US98/11748 take the form, for example, of a level scoopful containing about 1,800 mg glucosamine, about 600 mg chondroitin salts, about 16 mg of manganese (when included in the form of manganese ascorbate), and about 104 mg of ascorbate (when included in the form of manganese ascorbate). 5 These preparations may be made by conventional methods. For example, to prepare the compositions of the invention, the above-described ingredients are combined as the active ingredient in intimate admixture with a suitable carrier according to conventional compounding techniques. This carrier may take a wide variety of forms depending upon the form of preparation desired for administration, e.g., oral, sublingual, 10 nasal, guttural, rectal, transdermal or parenteral. In preparing the compositions in oral dosage form, any usual pharmaceutical medium may be employed. For oral liquid preparations (e.g., suspensions, elixirs, and solutions), media containing for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. Carriers such as starches, sugars, 15 diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to prepare oral solids (e.g., powders, capsules, pills, caplets, tablets, microencapsulated granules, microtablets, coated granules and lozenges). Capsules or tablets are a preferred oral dosage form. Controlled release forms may also be used. Because of their ease in administration, lozenges, tablets, pills, caplets, and capsules 20 represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. The compositions of the present invention may be in the form of one or more of these oral dosage forms -- i.e., a single dosage may be in multiple forms. - 21 - WO 99/62524 PCTIUS98/11748 For parenteral products, the carrier will usually comprise sterile water, although other ingredients may be included, e.g., to aid solubility or for preservation purposes. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be employed. 5 Having discussed the composition of the present invention, it will be more clearly perceived and better understood from the following specific examples which are intended to provide examples of the preferred embodiments and do not limit the present invention. EXAMPLE 1 10 The composition of the present invention is made in one or more capsules for oral administration in humans and small animals. In a preferred embodiment, each dosage contains: Human & Small Animal Range/Dose 15 SAM 5-5,000 mg Glucosamine 50-5,000 mg Chondroitin Sulfate 15-5,000 mg 20 EXAMPLE 2 For those situations in which manganese supplementation is desired, a manganese salt is added to the composition of Example 1 so that each dosage contains: Human & Small Animal Range/Dose 25 SAM 5-5,000 mg Glucosamine 50-5,000 mg Chondroitin Sulfate 15-5,000 mg Manganese (as Ascorbate) 2-75 mg Ascorbate (as Manganese 30 Ascorbate) 10-500 mg - 22 - WO 99/62524 PCTIUS98/11748 EXAMPLE 3 For larger animals, such as horses, the composition of Example 1 is administered as filled scoops. Large Animal (Equine) Range/Dose 5 SAM 2-20,000 mg Glucosamine 250-40,000 mg Chondroitin Sulfate 100-30,000 mg 10 EXAMPLE 4 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 3 so that each dosage contains: Large Animal (Equine) Range/Dose 15 SAM 2-20,000 mg Glucosamine 250-40,000 mg Chondroitin Sulfate 100-30,000 mg Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese 20 Ascorbate) 50-2,500 mg EXAMPLE 5 For a further preferred composition, each dosage contains: Human & Small Animal Range/Dose 25 SAM 5-5,000 mg Glucosamine 50-5,000 mg 30 EXAMPLE 6 For those situations in which manganese supplementation is desired, a manganese salt is added to the composition of Example 5 so that each dosage contains: Human & Small Animal Range/Dose - 23 - WO 99/62524 PCT/US98/11748 SAM 5-5,000 mg Glucosamine 50-5,000 mg Manganese (as Ascorbate) 2-75 mg Ascorbate (as Manganese 5 Ascorbate) 10-500 mg EXAMPLE 7 For larger animals, such as horses, the composition of Example 5 is administered as filled scoops. 10 Large Animal (Equine) Range/Dose SAM 2-20,000 mg Glucosamine 250-40,000 mg 15 EXAMPLE 8 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 7 so that each dosage contains: Large Animal (Equine) Range/Dose 20 SAM 2-20,000 mg Glucosamine 250-40,000 mg Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese Ascorbate) 50-2,500 mg 25 EXAMPLE 9 For a further preferred composition, each dosage contains: Human & Small Animal Range/Dose 30 SAM 5-5,000 mg Chondroitin Sulfate 15-5,000 mg - 24 - WO 99/62524 PCTIUS98/11748 EXAMPLE 10 For those situations in which manganese supplementation is desired, a manganese salt is added to the composition of Example 9 so that each dosage contains: 5 Human & Small Animal Range/Dose SAM 5-5,000 mg Chondroitin Sulfate 15-5,000 mg Manganese (as Ascorbate) 2-75 mg 10 Ascorbate (as Manganese Ascorbate) 10-500 mg EXAMPLE 11 For larger animals, such as horses, the composition of Example 10 is administered 15 as filled scoops. Large Animal (Equine) Range/Dose SAM 2-20,000 mg Chondroitin Sulfate 100-30,000 mg 20 EXAMPLE 12 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 11 so that each dosage contains: 25 Large Animal (Equine) Range/Dose SAM 2-20,000 mg Chondroitin Sulfate 100-30,000 mg Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese 30 Ascorbate) 50-2,500 mg EXAMPLE 13 For those situations in which methyl donors or methyl donor cofactors are desired, - 25 - WO 99/62524 PCT/US98/11748 such compounds may be added to the composition of Example 1 so that each dosage contains: Human & Small Animal Range/Dose 5 SAM 5-5,000 mg Glucosamine 50-5,000 mg Chondroitin Sulfate 15-5,000 mg vitamin B 12 0.1-10 mg 10 EXAMPLE 14 For those situations in which manganese supplementation is desired, a manganese salt is added to the composition of Example 13 so that each dosage contains: Human & Small Animal Range/Dose 15 SAM 5-5,000 mg Glucosamine 50-5,000 mg Chondroitin Sulfate 15-5,000 mg Manganese (as Ascorbate) 2-75 mg 20 Ascorbate (as Manganese Ascorbate) 10-500 mg vitamin B 12 0.1-10 mg EXAMPLE 15 25 For larger animals, such as horses, the composition of Example 13 is administered as filled scoops. Large Animal (Equine) Range/Dose SAM 2-20,000 mg 30 Glucosamine 250-40,000 mg Chondroitin Sulfate 100-30,000 mg vitamin B 12 1-100 mg EXAMPLE 16 35 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 15 so that each dosage contains: - 26 - WO 99/62524 PCTIUS98/11748 Large Animal (Equine) Range/Dose SAM 2-20,000 mg Glucosamine 250-40,000 mg Chondroitin Sulfate 100-30,000 mg 5 Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese Ascorbate) 50-2,500 mg vitamin B 12 1-100 mg 10 EXAMPLE 17 For a further preferred composition, each dosage contains: Human & Small Animal Range/Dose SAM 5-5,000 mg 15 Glucosamine 50-5,000 mg vitamin B 12 0.1-10 mg EXAMPLE 18 For those situations in which manganese supplementation is desired, a manganese 20 salt is added to the composition of Example 17 so that each dosage contains: Human & Small Animal Range/Dose SAM 5-5,000 mg Glucosamine 50-5,000 mg 25 Manganese (as Ascorbate) 2-75 mg Ascorbate (as Manganese Ascorbate) 10-500 mg vitamin B 12 0.1-10 mg 30 EXAMPLE 19 For larger animals, such as horses, the composition of Example 17 is administered as filled scoops. Large Animal (Equine) Range/Dose 35 SAM 2-20,000 mg Glucosamine 250-40,000 mg vitamin B12 1-100 mg - 27 - WO 99/62524 PCT/US98/11748 EXAMPLE 20 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 19 so that each dosage contains: 5 Large Animal (Equine) Range/Dose SAM 2-20,000 mg Glucosamine 250-40,000 mg Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese 10 Ascorbate) 50-2,500 mg vitamin B 12 1-100mg EXAMPLE 21 For a further preferred composition, each dosage contains: 15 Human & Small Animal Range/Dose SAM 5-5,000 mg Chondroitin Sulfate 15-5,000 mg vitamin B 12 0.1-10 mg 20 EXAMPLE 22 For those situations in which manganese supplementation is desired, a manganese salt is added to the composition of Example 21 so that each dosage contains: Human & Small Animal Range/Dose 25 SAM 5-5,000 mg Chondroitin Sulfate 15-5,000 mg Manganese (as Ascorbate) 2-75 mg Ascorbate (as Manganese 30 Ascorbate) 10-500 mg vitamin B 12 0.1-10 mg - 28 - WO 99/62524 PCT/US98/11748 EXAMPLE 23 For larger animals, such as horses, the composition of Example 21 is administered as filled scoops. Large Animal (Equine) Range/Dose 5 SAM 2-20,000 mg Chondroitin Sulfate 100-30,000 mg vitamin B12 1-100 mg 10 EXAMPLE 24 For those situations in which manganese supplementation is desired, manganese salts may be added to the composition of Example 23 so that each dosage contains: Large Animal (Equine) Range/Dose SAM 2-20,000 mg 15 Chondroitin Sulfate 100-30,000 mg Manganese (as Ascorbate) 10-500 mg Ascorbate (as Manganese Ascorbate) 50-2,500 mg vitamin B 12 1-100 mg 20 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. - 29 -

Claims (23)

1. A composition for treatment and repair and for reducing the inflammation of connective tissue in humans and animals comprising an aminosugar selected from the 5 group consisting of glucosamine, glucosamine salts and mixtures thereof, in combination with S-Adenosylmethionine and a glycosaminoglycan selected from the group consisting of chondroitin, chondroitin salts, and fragments and mixtures thereof.

2. The composition of claim 1, wherein a dose of the aminosugar ranges 10 from about 50 mg to about 40,000 mg.

3. The composition of claim 2, wherein the dose of the aminosugar for humans and small animals ranges from about 50 mg to about 5,000 mg. 15

4. The composition of claim 2, wherein the dose of the aminosugar for large animals ranges from about 250 mg to about 40,000 mg.

5. The composition of claim 1, wherein a dose of the glycosaminoglycan ranges from about 15 mg to about 30,000 mg. 20

6. The composition of claim 5, wherein the dose of glycosaminoglycan for humans and small animals ranges from about 15 mg to about 5,000 mg.

7. The composition of claim 5, wherein the dose of glycosaminoglycan for 25 large animals ranges from about 100 mg to about 30,000 mg. - 30 - WO 99/62524 PCTIUS98/11748

8. The composition of claim 1, wherein a dose of the S-Adenosylmethionine ranges from about 2 mg to about 20,000 mg. 5

9. The composition of claim 8, wherein the dose of S-Adenosylmethionine for humans and small animals ranges from about 5 mg to about 5,000 mg.

10. The composition of claim 8, wherein the dose of S-Adenosylmethionine for large animals ranges from about 2 mg to about 20,000 mg. 10

11. A composition for treatment and repair and for reducing the inflammation of connective tissue in humans and animals comprising: an aminosugar selected from the group consisting of glucosamine, glucosamine salts and mixtures thereof, in combination with S-Adenosylmethionine and a glycosaminoglycan selected from the group consisting 15 of chondroitin, chondroitin salts, and mixtures or fragments thereof, wherein a dose of the aminosugar ranges from about 50 mg to about 40,000 mg, a dose of the glycosaminoglycan ranges from about 15 mg to about 30,000 mg, and a dose of the S Adenosylmethionine ranges from about 5 mg to about 40,000 mg. 20

12. The composition of claim 1, wherein the salt of glucosamine is selected from the group consisting of glucosamine hydrochloride, glucosamine sulfate, and N-acetylglucosamine.

13. A composition for treatment and repair and for reducing the inflammation 25 of connective tissue in humans and animals comprising: an aminosugar selected from the - 31 - WO 99/62524 PCTIUS98/11748 group consisting of glucosamine, glucosamine salts and mixtures thereof, in combination with S-Adenosylmethionine.

14. The composition of claim 13, wherein a dose of the aminosugar ranges 5 from about 50 mg to about 40,000 mg, and wherein a dose of the S-Adenosylmethionine ranges from about 2 mg to about 20,000 mg.

15. The composition of claim 13, wherein the dose of the aminosugar for humans and small animals ranges from about 50 mg to about 5,000 mg, and wherein the 10 dose of S-Adenosylmethionine for humans and small animals ranges from about 5 mg to about 5,000 mg.

16. The composition of claim 13, wherein the dose of the aminosugar for large animals ranges from about 250 mg to about 40,000 mg, and wherein the dose of S 15 Adenosylmethionine for large animals ranges from about 2 mg to about 20,000 mg.

17. A composition for treatment and repair and for reducing the inflammation of connective tissue in humans and animals comprising: a glycosaminoglycan selected from the group consisting of chondroitin, chondroitin salts, and mixtures or fragments 20 thereof in combination with S-Adenosylmethionine.

18. The composition of claim 17, wherein a dose of the glycosaminoglycan ranges from about 15 mg to about 30,000 mg, and wherein a dose of the S Adenosylmethionine ranges from about 2 mg to about 20,000 mg. 25 - 32 - WO 99/62524 PCT/US98/11748

19. The composition of claim 17, wherein the dose of the glycosaminoglycan for humans and small animals ranges from about 15 mg to about 5,000 mg, and wherein the dose of S-Adenosylmethionine for humans and small animals ranges from about 5 mg to about 5,000 mg. 5

20. The composition of claim 17, wherein the dose of the glycosaminoglycan for large animals ranges from about 100 mg to about 30,000 mg, and wherein the dose of S-Adenosylmethionine for large animals ranges from about 2 mg to about 20,000 mg. 10

21. The composition of any of claims 1 through 20 further comprising manganese or a salt thereof.

22. The composition of any of claims 1 through 21 further comprising a methyl donor or methyl donor cofactor selected from the group consisting of vitamin 15 B12, vitamin B6, folic acid, dimethylglycine, and trimethylglycine.

23. A method for the treatment and repair and for reducing the inflammation of connective tissue in humans and animals comprising the step of administering any of the compositions of claim 1 to claim 22 to a human and an animal. - 33 -