US20070141181A1 - Use of anabolic agents, anti-catabolic agents, antioxidant agents, and analgesics for protection, treatment and repair of connective tissues in humans and animals - Google Patents

Use of anabolic agents, anti-catabolic agents, antioxidant agents, and analgesics for protection, treatment and repair of connective tissues in humans and animals Download PDF

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US20070141181A1
US20070141181A1 US11/634,383 US63438306A US2007141181A1 US 20070141181 A1 US20070141181 A1 US 20070141181A1 US 63438306 A US63438306 A US 63438306A US 2007141181 A1 US2007141181 A1 US 2007141181A1
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avocado
dose
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Todd Henderson
Carmelita Frondoza
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Nutramax Laboratories Inc
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Priority claimed from US09/249,335 external-priority patent/US6451771B1/en
Priority claimed from US10/192,318 external-priority patent/US6797289B2/en
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Priority to US11/634,383 priority Critical patent/US20070141181A1/en
Assigned to NUTRAMAX LABORATORIES, INC. reassignment NUTRAMAX LABORATORIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRONDOZA, CARMELITA, HENDERSON, TODD R.
Publication of US20070141181A1 publication Critical patent/US20070141181A1/en
Priority to ES07862507.6T priority patent/ES2535213T3/es
Priority to PCT/US2007/024853 priority patent/WO2008070086A2/en
Priority to PT78625076T priority patent/PT2101799E/pt
Priority to EP07862507.6A priority patent/EP2101799B1/de
Priority to CA2672108A priority patent/CA2672108C/en
Priority to PL07862507T priority patent/PL2101799T3/pl
Priority to JP2009540264A priority patent/JP5606071B2/ja
Priority to AU2007328265A priority patent/AU2007328265B2/en
Priority to HK10102909.4A priority patent/HK1136202B/en
Priority to NZ577602A priority patent/NZ577602A/xx
Priority to DK07862507.6T priority patent/DK2101799T3/en
Priority to US12/538,000 priority patent/US8568803B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compositions for the protection, treatment, repair, and modulation of inflammation of connective tissues in humans and other animals.
  • the tissues of mammals 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.
  • inflammation occupies a key position in this balance.
  • inflammation initiates the biochemical processes that result in tissue repair.
  • 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.
  • pharmacies are full of “anti-inflammatory drugs” (such as corticosteroids and the non-steroidal anti-inflammatory drugs, such as aspirin).
  • 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.
  • eicosanoid group 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.
  • FAs essential fatty acids
  • AA arachadonic acid
  • EPA eicosapentanoic acid
  • Eicosanoids produced from arachidonic acid include the 2-series of prostaglandins and the 4-series of leukotrienes, which are generally proinflammatory.
  • 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.
  • the eicosanoids are short-lived and locally active. They are responsible for the initial events of inflammation, including vasodilation, increased vascular permeability, and chemotaxis. Moreover, the eicosanoids are instrumental in the early steps of the healing process. For example, the eicosanoids trigger the release of cytokines such as TGF-B, which in turn stimulates the migration and proliferation of connective tissue cells, and the deposition of extracellular matrix. Specific constitutive eicosanoids also have protective effects in the gastrointestinal mucosa and kidney, because they maintain glycosaminoglycan synthesis and normal perfusion of these organs.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • 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.
  • 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.
  • the body also contains anabolic compounds that stimulate tissue growth.
  • Glucosamine is an amino sugar naturally formed in the body from glucose. When supplied exogenously, glucosamine stimulates connective tissue cell synthesis, and thereby increases the amounts of normal extracellular matrix. Glucosamine is also the building block for glycosaminoglycans in cartilage and other connective tissues. Supplying additional glucosamine thus supplies the body with extra raw materials for matrix synthesis in connective tissues.
  • Other examples of anabolic compounds in the body include somatotropin, which stimulates protein synthesis, and the somatomedins or insulin-like growth factors, which stimulate the proliferation of chondrocytes and fibroblasts and enhance matrix synthesis.
  • a given compound may have different effects in different tissues. For example, somatotropin increases protein synthesis (anabolism), but also speeds fat breakdown (catabolism).
  • 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.
  • 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.
  • 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.
  • the present invention provides novel compositions and methods of treating repairing, and preventing damage to connective tissues in humans and animals using such compositions. Therefore, it is an object of the invention to provide novel compositions of any or all of anabolic, anti-catabolic, anti-oxidant and/or analgesic agents for the protection, treatment and repair of connective tissues in humans and animals.
  • compositions comprising any or all of anabolic, anti-catabolic, anti-oxidant and/or analgesic agents selected from the group consisting of aminosugar, S-adenosylmethionine (SAMe), arachadonic acid (AA), GAG, pentosan sulfate, collagen type II, tetracyclines, diacerin, super oxide dismutase (SOD), L-ergothioneine, one or more avocado/soybean unsaponifiables (ASUs) and analgesics, such as acetaminophen.
  • anabolic, anti-catabolic, anti-oxidant and/or analgesic agents selected from the group consisting of aminosugar, S-adenosylmethionine (SAMe), arachadonic acid (AA), GAG, pentosan sulfate, collagen type II, tetracyclines, diacerin, super oxide dismutase (
  • compositions to repair, treat, and prevent damage to connective tissue in humans and animals that contain one or more of the elements selected from the group consisting of aminosugar, SAMe, arachodonic acid, GAG, pentosan sulfate, collagen type II, tetracyclines, diacerin, SOD, L-ergothioneine, one or more ASUs and analgesics, e.g, acetaminophen.
  • FIG. 1 provides a detailed description of the biosynthetic pathway for the creation of GAGs such as chondroitin sulfate.
  • FIG. 2 is the molecular structure of SAMe and its immediate precursor.
  • FIG. 3 provides a simplified diagram of the function of SOD.
  • FIG. 4 provides some examples of unsaponifiable lipids.
  • FIG. 5 is the molecular structure of acetaminophen.
  • FIGS. 6A and 6B show images of extracellular matrix (ECM) and chondrocytes on microcarriers as observed at two different time periods in Example 7.
  • ECM extracellular matrix
  • FIG. 7 is an image of chondrocytes stained for type II collagen as observed in Example 7.
  • FIG. 8 shows two charts illustrating PGE-2 response to IL-1 ⁇ activation as measured in Example 7.
  • FIG. 9 shows a chart illustrating the modulation of PGE-2 response to avocado/soybean unsaponifiables (ASUs), chondroitin sulfate, and glucosamine in accordance with Example 7.
  • ASUs avocado/soybean unsaponifiables
  • chondroitin sulfate chondroitin sulfate
  • glucosamine glucosamine
  • FIG. 10 shows a chart illustrating TNF- ⁇ expression in THP-1 cells in accordance with Example 8.
  • FIG. 11 is a chart illustrating IL-1 ⁇ in accordance with Example 8.
  • FIG. 12 is a chart illustrating iNOS expression in THP-1 cells as measured in Example 8.
  • FIG. 13 is a chart illustrating p38 expression in THP-1 cells as measured in Example 8.
  • FIGS. 14A and 14B illustrate the inhibition of COX-2 in activated chondrocytes, as measured in Example 9.
  • FIG. 15 is a chart illustrating secreted PGE-2 levels as measured in Example 9.
  • FIG. 16 is a chart illustrating interleukin-8 (IL-8) expression in human chondrocytes as measured in Example 11.
  • IL-8 interleukin-8
  • FIG. 17 is a chart illustrating monocyte chemotactic protein (MCP) expression in human chondrocytes as measured in Example 11.
  • MCP monocyte chemotactic protein
  • compositions of the present invention used to treat, repair, and prevent damage to connective tissue, include combinations of anabolic, anti-catabolic, and/or anti-oxidant agents.
  • Ingredients of preferred embodiments include compositions selected from the group consisting of aminosugars, SAMe, AA, GAGs, including pentosan, collagen type II, tetracyclines, diacerin, SOD, L-ergothioneine, methylsulfanylmethane (MSM), and one or more ASUs.
  • the combinations of the present invention also include one or more analgesics, such as acetaminophen.
  • the present invention covers methods of administering these novel compositions to humans and animals in need thereof.
  • 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.
  • GAGs glycosaminoglycans
  • the aminosugar component of the compositions of the present invention may comprise natural, synthetic or semi-synthetic aminosugars including but not limited to salts of glucosamine including glucosamine hydrochloride and glucosamine sulfate, glucosamine phosphate, and N-acetylglucosamine and salts and/or mixtures thereof.
  • 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.
  • aminosugar can extend to any composition of matter that is insubstantially different from the aminosugar as above-described.
  • 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.
  • 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.
  • GAGs are a preferred component of the compositions of the present invention.
  • mono-sulfated and polysulfated (or oversulfated) GAGs are preferred GAG components of the compositions of the present invention.
  • GAGs also is intended to encompass alternative nomenclature for the same group of above-described compounds—e.g., mucopolysaccharides, proteoglycans, and heparanoids.
  • 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.
  • 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.
  • chondroitin sulfate must have at least two, and potentially many, of these repeating units of glucuronic acid and sulfated galactosamine.
  • FIG. 1 provides a detailed description of the biosynthetic pathway for the creation of GAGs, such as chondroitin sulfate.
  • the present invention may include fragments of GAGs, such as fragments of chondroitin sulfate.
  • 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.
  • 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.
  • a molecule composed of a glucuronic acid and sulfated galactosamine would constitute a fragment of chondroitin sulfate.
  • glycosaminoglycans may be used in this invention, for example, hyaluronic acid.
  • 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.
  • 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.
  • 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.
  • 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.
  • PPS pentosan polysulfate
  • salts thereof such as calcium-derived PPS and sodium PPS.
  • a preferred GAG-like compound that may be used in the compositions of the present invention is PPS.
  • 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.
  • GAG-like compounds 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.
  • Pentosan's mechanism of action can be summarized as follows:
  • glycosaminoglycans or glycosaminoglycan-like compounds such as polysulfated glycosaminoglycans, may be used in this invention.
  • Diacerein a recently recognized organic compound found in plants of the genus Cassia has anti-inflammatory effects through inhibition of interleukin-1B consequently collagenase production in articular cartilage is reduced. It reduces the fibrinolytic activity of synovial fibroblasts as well. It also dose-dependently inhibits chemotaxis (attraction of white blood cells) and superoxide anion production (this is one of the “toxic oxygen species” or “free radicals”). These harmful compounds occur spontaneously in the body, especially during destructive inflammation. Diacerein has analgesic and antipyretic activities. It reduces the breakdown of chondroitin-4-sulfate resulting in an increase in the ratio of chondroitin-4-sulfate to chondroitin-6-sulfate. (This ratio is pathologically decreased in degenerating cartilage.) It mildly increases prostaglandin synthesis, which allows it to have protective effects on the gastric mucosa.
  • SAMe S-adenosylmethionine
  • 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.
  • 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.
  • Superoxide dismutase is an enzyme present naturally in the tissues of animals and plants, which has recently been investigated as an agent in the management of inflammation. It acts by intercepting toxic oxygen radicals in the intracellular space during destructive inflammatory processes. It does not inhibit prostaglandin biosynthesis, but stops the overproduction of prostaglandins resulting from destructive inflammation. Some of its effects include inhibition of edema formation and inhibition of acute signs of inflammation and the secondary articular changes (stiffness and calcification) in adjuvant-induced arthritis. Having no analgesic effects, it does not contribute to the overuse of the affected joints that eventually leads to more damage of the articular cartilage, as NSAIDs can. Also, it has no adverse effects on the cardiovascular, central nervous or endocrine systems.
  • FIG. 3 provides a simplified diagram of the function of SOD.
  • L-ergothioneine is an intracellular antioxidant naturally occurring in plants and animals, but not synthesized in human bodies: it comes only from dietary sources.
  • the antioxidant properties of L-ergothionein appear to be related to its ability to scavenge reactive oxygen species (free radicals), chelate various metallic cations, activate antioxidant enzymes such as glutathione peroxidase (SeGPx) and manganese superoxide dismutase (Mn SOD) and to inhibit superoxide-generating enzymes such as NADPH-Cytochrome C reductase, and to affect the oxidation of various hemoproteins such as hemoglobin and myoglobin. Because all body tissues depend on these two oxygen carrier molecules, this characteristic is extremely beneficial. [Brummel, M.
  • Collagen Type II also has beneficial effects that help maintain the normal balance between anabolism and catabolism.
  • 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).
  • GALT gut-associated lymphoid tissues
  • tetracycline 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.
  • 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.
  • doxycycline has been shown to inhibit collagenase activity in articular cartilage.
  • Nonsaponifiable of avocado (genus Persea , especially P. americana ) and the soybean ( Glycine max ) have also been studied for their beneficial effects on connective tissues.
  • These nonsaponifiable compounds are that part of the plant lipids that do not undergo saponification, i.e., they do not react with alkali to form a soap.
  • any particular avocado extract may contain any number.
  • Examples include fat soluble vitamins (A, D, E, and K), steroids such as phytoestrogens, sterols (bioflavonoids) and volatile essentials oils (terpenes such as menthol, camphor, lycopene, gibberellic acid, limonene, cinnamaldehyde, carotenoids, and ubiquinone, also known as coenzyme Q.) [Mathews, C. K. & van Holde, K. E. Biochemistry, 2.sup.nd ed., The Benjamin/Cummings Pub. Co., Inc., 1996, p. 691.]
  • ASU avocado/soybean unsaponifiables
  • Piascledine has been used to treat osteoarthritis and other forms of arthritis
  • Thiiers, M. H. “Unsaponifiable constituents of avocado and soya oils. Treatment of certain forms of arthralgia,” J. Med. Lyon 53(222):195-8 (February 1972) (article in French)]
  • soft-tissue inflammatory conditions [Trevoux, R., “Unsaponifiable fractions of the avocado and soybean in gynecology,” J. Bynecol. Obstet. Biol. Reprod. 6(1):99-105 (January 1977) (article in French); Lamaud, M.
  • ASU blocks the cascade that leads to metalloprotease activation [Boumediene K., et al., “Avocado/soya unsaponifiables enhance the expression of transforming growth factor beta 1 and beta 2 in cultured articular chondrocytes,” Arthritis Rheum. 42(1): 148-56 (January 1999)].
  • ASU mixtures also reduce the spontaneous production of stromelysins, IL-6, interleukin-8 (IL-8) and prostaglandin E2 by chondrocytes. Additionally, ASUs decrease the effects of IL-1, and thereby reduce chondrocyte and synoviocyte production of collagenase. [Henrotin, Y.
  • TGF beta 1 and 2 are members of a family of homologous polypeptidecytokines. These locally-acting hormones can have paracrine or autocrine effects and are made by a variety of cell types, including lymphocytes, endothelial cells and macrophages. TGF beta has varied effects in different tissues; it generally inhibits epithelial cell metabolism. In connective tissues, however, it has been shown to be an indirect mitogen for fibroblasts and other cells of mesenchymal origin. It also can stimulate cellular production of fibronectin and collagen, and decrease protease activity, resulting in a net increase in matrix production. [Cotran, R. F., Kumar, V. and Robbins, S. L., Eds., Pathologic Basis of Disease, 5.sup.th ed., Saunders, 1994, pp. 40-42.]
  • Stromelysins are a subtype of proteinases that act on a variety of extracellular matrix components, including proteoglycans, laminin, fibronectin, and collagen. Stromelysins are produced by fibroblasts, synoviocytes, and macrophages, among other cell types, under the influence of cytokines such interleukin-1 and tumor necrosis factor alpha Interleukins and prostaglandins are among the many mediators of inflammation. Reductions in levels of all of these compounds result in a decrease in pain and swelling, which are hallmarks of inflammation.
  • ASU mixtures are necessary for growth, and augment the anabolic effects of TGF-beta. Because they stimulate TGF beta and also decrease degradative enzymes, as explained above, ASU mixtures can be said to have both anabolic and anti-catabolic effects. Although some of the effects of ASUs overlap the effects of other compounds in the present invention, ASUs contribute unique properties to the group of compounds and provide very beneficial effects when used in combination with those other compounds. For example, while glucosamine and ASUs both stimulate anabolic processes in connective tissue cells, these compounds have different cellular mechanisms of action. Glucosamine acts in part through protein kinase C, while the effect of ASUs, as stated above, is through transforming growth factor.
  • chondroitin and ASUs have inhibitory effects of IL-1. ASUs, however, inhibit the plasmin cascade, while chondroitin decreases activation of the complement cascade.
  • Osteoarthritis is a complex disease involving interplay of many cytokines at the cellular level. Because the different compounds of the present invention act on different cytokines, they will have synergistic effects when used in appropriate combinations.
  • ASUs have been shown effective in reducing symptoms of osteoarthritis [Maheu, E., et al., “Symptomatic efficacy of avocado/soybean unsaponifiables in the treatment of osteoarthritis,” Arthritis Rheum. 41(1): 81-91 (January 1998); Blotman, F., et al., “Efficacy and safety of avocado/soybean unsaponifiables in the treatment of symptomatic osteoarthritis,” Rev. Rheum. Engl. Ed. 64(12): 825-34 (December 1997)].
  • side effects in the intervention groups were similar to those seen in the placebo groups, indicating that ASUs are safe and well tolerated substances.
  • ASUs as used in this invention can include any or all unsaponifiable lipids and/or combinations thereof.
  • ASUs examples include but are not limited to: limonene, beta carotene, phyloquinone, and giberellic acid.
  • ASUs can include any of a number of classes of compounds including but not limited to fat soluble vitamins, steroids, sterols and volatile essentials oils, or any combinations thereof.
  • the avocado/soybean unsaponifiables (ASU) can include one or more phytosterols, such as campesterol, stigmasterol, dihydro-brassisterol, and Beta-sitosterol.
  • the invention includes, moreover, compositions which contain one avocado/soybean extract or mixtures or combinations of such extracts (more than one ASU). There are many such combinations and all are intended to be included within the present invention.
  • Various formulations of the present invention may include one or more avocado/soybean unsaponifiables in a variety of forms or amounts.
  • one or more avocado/soybean unsaponifiables may be included in a composition so that they are standardized to about 30% sterol.
  • avocado/soybean unsaponifiables may be included in a composition in approximately a 2-1 ratio of soybean unsaponifiables to avocado unsaponifiables.
  • one or more avocado/soybean unsaponifiables may be a solid at room temperature and a liquid at human body temperature.
  • one or more avocado/soybean unsaponifiables may be combined with one or more excipients or carriers to create a powder.
  • the compounds of the present invention have several advantages over existing therapies for connective tissue disorders, such as excellent safety profiles. This is in part related to the fact that these compounds occur normally in the body and in various foods. Another characteristic shared by the compounds is tendency for a slow onset of action. Pharmaceuticals, such as NSAIDs, tend to cause sudden changes in the symptoms of disease. The endogenous compounds in the present invention work more slowly, by normalizing structures and functions within the body. While this action is beneficial, it does mean that symptoms will typically not be relieved immediately. For this reason, an analgesic is included as an optional component of the compositions of the present invention.
  • the analgesic is to be chosen from the group of analgesic compounds that have been shown to have minimal side effects at therapeutic doses, and also to have minimal negative effects on connective tissue synthesis, as corticosteroid drugs and many NSAIDs have been shown to have.
  • the analgesic that may be included in the composition of the present invention therefore is a nonsteroidal analgesic that does not have anti-inflammatory effects.
  • the analgesic is a nonsteroidal drug that is not an NSAID.
  • Examples of the analgesics of the present invention include acetaminophen and tramadol. Except as discussed below, the preferred analgesic of the present invention is acetaminophen.
  • Acetaminophen is an analine derivative analgesic and antipyretic compound working centrally through reversible inhibition of the enzyme cyclo-oxygenase in the central nervous system. Acetaminophen also blocks the peripheral pain impulse generation in nerve endings throughout the body. It has been used extensively for symptomatic pain relief. The relief of pain is beneficial for more than the obvious humane reasons. Since there are also important links between the emotional centers of the brain and the immune system, the relief of pain, and the resulting elevation in mood, has beneficial effects on inflammation and the many other processes that are modulated by the immune system. Although it blocks cyclooxygenase activity, acetaminophen has very little anti-inflammatory activity.
  • acetaminophen does not inhibit connective tissue anabolism, as NSAIDs and corticosteroids do, and because it has minimal side effects at therapeutic doses, it is an ideal analgesic agent in the present invention.
  • Another advantage to including a safe analgesic in the present invention is that it will increase the likelihood that patient compliance would be high, i.e., that patients would continue taking the preparations long enough for disease-modifying effects to occur.
  • Studies of isolated chondroprotective agents often have a high rate of drop-out in the early weeks of therapy due to patient perception that the agent is not working. With the addition of an analgesic, patients would be more inclined to continue therapy.
  • Cats are sensitive to acetaminophen because they do not metabolize it effectively (poor hepatic conjugation with glucuronic acid and subsequent depletion of glutathione occurs) [Goodman, A. and Goodman, L., The Pharmacological Basis of Therapeutics, 7.sup.th ed., MacMillan Publishing Co., 1985, pp. 692-95; Ahrens, F., Pharmacology, Williams & Wilkins, 1996, pp. 174-75]. Accordingly, acetaminophen is not recommended for use in cats.
  • Methylsulfonylmethane is an organic sulfur compound belonging to a class of chemicals known as sulfones. It occurs naturally in some primitive plants and is present in small amounts in many foods and beverages. researchers have suggested that MSM has anti-inflammatory effects.
  • 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.
  • 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.
  • each compound has a number of functions, they can be roughly grouped as: (1) anabolic agents, including glucosamine, SAMe, AA, and ASUs, which promote growth processes in the body; (2) anti-catabolic agents, such as chondroitin sulfate, pentosan sulfate, collagen type II, tetracyclines, diacerin and ASUs, which inhibit destructive or catabolic processes; and (3) antioxidants, such as SOD, and L-ergothioneine which prevent tissue damage by scavenging toxic oxygen species (free radicals).
  • anabolic agents including glucosamine, SAMe, AA, and ASUs, which promote growth processes in the body
  • anti-catabolic agents such as chondroitin sulfate, pentosan sulfate, collagen type II, tetracyclines, diacerin and ASUs, which inhibit destructive or catabolic processes
  • antioxidants such as SOD, and L-ergothioneine which prevent
  • an analgesic could optionally be added to any of the individual compounds recited above or to a combination of them to provide relief from pain.
  • Acetaminophen is the analgesic of choice because it does not have powerful anti-inflammatory effects and therefore does not interfere with healing of connective tissue. It also has minimal side effects at therapeutic doses, unlike NSAIDs which may cause gastrointestinal ulceration or poor renal perfusion even at therapeutic doses.
  • the present invention consists of various combinations of two or more of the following agents: AA, glucosamine, chondroitin sulfate, pentosan, diacerin, S-adenosylmethionine, superoxide dismutase, L-ergothionein, collagen type II, tetracycline-like compounds, one or more ASUs and, optionally, one or more analgesics, e.g., acetaminophen.
  • agents AA, glucosamine, chondroitin sulfate, pentosan, diacerin, S-adenosylmethionine, superoxide dismutase, L-ergothionein, collagen type II, tetracycline-like compounds, one or more ASUs and, optionally, one or more analgesics, e.g., acetaminophen.
  • Examples include, but are not limited to such combinations as: two anabolic agents (e.g., AA and glucosamine); an anabolic agent and an anti-catabolic agent (e.g., AA and pentosan); an anti-catabolic and an antioxidant (e.g., tetracyclicline and superoxide dismutase); or combinations of more than two agents (e.g., glucosamine, SAMe and AA) or SAMe, ASUs, acetaminophen and diacerin.
  • Examples of specific compounds that may be present in ASU extracts include but are not limited to: limonene, beta carotene, ubiquinone, and undecaprenol phosphate.
  • 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:
  • One or more avocado/soybean unsaponifiables are one or more avocado/soybean unsaponifiables.
  • Analgesic e.g., acetaminophen
  • the first X in the first row means a combination of glucosamine and L-ergothioneine or glucosamine and diacerin.
  • the compositions of the present invention additionally comprise any aggregation or addition of the combinations marked by X in any given row or column.
  • the compositions disclosed in the first row include combinations of glucosamine plus L-ergothioneine plus diacerin, or glucosamine plus diacerin plus tetracycline-like compounds or glucosamine plus L-ergothioneine plus diacerin plus AA plus tetracycline-like compounds, and so on.
  • compositions disclosed in the column designated “Collagen Type II” would include combinations of collagen Type II plus SAMe plus pentosan, or collagen Type II plus SAMe plus pentosan plus superoxide dismutase plus L-ergothioneine, and so on.
  • compositions disclosed in the column designated “ASU” would include combinations of one or more ASUs plus glucosamine, or one or more ASUs plus SAMe plus pentosan, or one or more ASUs plus collagen Type II plus SAMe plus pentosan plus superoxide dismutase plus L-ergothioneine, and so on.
  • 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.
  • any salt of any of the present compounds may be used to aid in absorption, e.g., glucosamine HCl, glucosamine sulfate, glucosamine phosphate, sodium chondroitin sulfate, calcium chondroitin sulfate, potassium chondroitin sulfate, etc.
  • the compositions of the present invention can be given in all common dosage forms including extended release dosage forms, pills, tablets (such as chewable tablets), capsules (such as hard gelatin capsules, liquid-filled capsules, softgel capsules, etc.), creams, pastes, powders (such as scoops of powder), liquids, aerosols, extended release forms, injectables, etc.
  • the compositions of the present invention may also be given in dosage forms such as sachets and treats.
  • compositions of the present invention will vary depending upon the needs of the human or animal to which the compositions are administered.
  • the frequency of dosage may also vary depending on the needs of the human or animal to which the compositions are administered.
  • a combination of ASU, chondroitin sulfate (CS), and glucosamine (Gluc) may be administered to an animal such as a dog or horse.
  • the composition may be administered to the animal daily (or every other day) at a specific dose (or varying dose) for an initial period, such as 2-4 weeks or 4-6 weeks.
  • Individual dose regimens may vary by target subject.
  • the dosage regimen for the initial period may be designed so that the active components achieve a steady state in the body fluids that bathe inflamed tissue in the animal.
  • daily dosages of the composition in powdered form may be administered to horses in the following amounts: horses under 600 pounds may be administered one scoop, horses between 600 and 1200 pounds may be administered two scoops, and horses over 1200 pounds may be administered 3 scoops.
  • horses under 600 pounds may be administered one scoop
  • horses between 600 and 1200 pounds may be administered two scoops
  • horses over 1200 pounds may be administered 3 scoops.
  • a long term administration for horses may be 1 ⁇ 2 scoop, 1 scoop, and 1-2 scoops daily for horses weighing up to 600 pounds, 600-1200 pounds, and over 1200 pounds, respectively.
  • dosage Prior to and during strenuous exercise or an event that may affect inflammation of connective tissue in the animal, dosage may be increased in amount and/or frequency, e.g., to the initial administration level, for extra support. Also, if the animal's comfort level appears to decrease, the dosage and frequency may be increased, e.g., to the initial administration levels, for an additional period, such as 2-4 weeks, before reducing level and frequency again.
  • administration may be further reduced in frequency and/or amount, e.g., to half dosage and half frequency.
  • a subject's dosage and frequency may be increased at any time as needed, e.g., on weekends or other times when the subject is more active, in order to provide extra support to the connective tissues as needed.
  • ASU ASU
  • CS Gluc
  • Gluc Gluc
  • the dosage ranges for the various components of the presently claimed compositions are as follows: Compound Daily Dose Glucosamine Total dose range: 25 mg to 15 g Or: 3-125 mg/kg for small or large animals or humans Small animal: 25 mg-3 g; or 3-125 mg/kg Human: 100 mg-4 g; or 3-125 mg/kg Large animal: 300 mg-15 g; or 3-125 mg/kg Chondroitin Total dose range: 15 mg-12 g sulfate Or: 1-75 mg/kg for small or large animals or humans Small animal: 15 mg-2 g; or 1-75 mg/kg Human: 75 mg-4 g; or 1-75 mg/kg Large animal: 300 mg-12 g; or 1-75 mg/kg SAMe Total dose range: 10 mg-8 g Small animal: 10 mg-1 g Human: 75 mg-3 g Large animal: 400 mg-8 g Pentosan Total dose range: 3 mg to 3 g Small animal: 3 mg-1 g Human: 50 mg-2 g Large animal: 100 mg-3 g Superoxide Total dose range: 3
  • 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.
  • physiological concentration ranges as determined by the best data available to the inventors, were used.
  • 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.
  • the cell digest was repeatedly aspirated to increase release of cells.
  • the cell digest was then placed into a 50 mL sterile centrifuge tube and centrifuged in the Beckman at 1000 RPM for 10 minutes.
  • the medium was discarded by pipette and fresh DMEM/F-12 containing 1% FCS added. Depending on the size of the pellet, about 2040 mL medium was added. Cell counts were determined by haemocytometer and the digest made up to a concentration of 100,000 cells/0.2 mL.
  • 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.
  • 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.
  • the cell layer for both the synthesis experiments were heated at 50 degrees after adding 100 ul 1 N NaOH for a period of 2 hours. 200 ul scintillant was added and the plates were placed in the counter. The data was expressed as CPM/100,000 cells. Indv.
  • a 4 year old child has juvenile rheumatoid arthritis in which the immune system inappropriately targets endogenous connective tissues with antibodies against native collagen type II.
  • the resulting inflammation and degradation of cartilage causes pain and dysfunction in the synovial joints.
  • Present treatments include corticosteroids which non-selectively suppress the immune system, thus leaving the body vulnerable to infectious disease, or methotrexate, which inhibits DNA synthesis, repair, and cellular replication, thus affecting not only the immune system but also intestinal mucosa, and the bone marrow.
  • This child is given 2 mg of collagen type II daily, and SOD 10 mg daily.
  • the collagen decreases the inappropriate immune attack, and the SOD inactivates destructive free radicals that damage cells. By preventing cellular damage, the SOD helps maximize the normal function of joint tissue cells. This combination has no harmful side effects at therapeutic doses and is a beneficial addition to existing therapies for rheumatoid arthritis.
  • a 6 year old thoroughbred race horse has neutrophilic inflammation of the carpus.
  • 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.
  • 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.
  • a 47 year-old woman has severe knee osteoarthritis.
  • she requires large doses of NSAIDs to control her symptoms.
  • her orthopedic surgeon has recommended taking glucosamine/chondroitin sulfate, she has been reluctant to do so because these compounds are extracted from animal tissues and the patient is a strict vegetarian. Instead she takes diacerein 25 mg and ASU 250 mg, and 500 mg of acetaminophen daily.
  • the diacerin inhibits chemotaxis and thereby reduces inflammation in the knee joint.
  • the ASU increases TGF beta 1 and 2, stimulating repair of damaged joint tissues.
  • the acetaminophen causes rapid analgesia, reducing the patient's symptoms without adversely affecting cartilage metabolism and without risk of gastrointestinal ulceration.
  • the patient decides to add a 15 minute walk to her daily schedule.
  • the controlled exercise further improves her physical and mental state.
  • a 5 year old Jersey dairy cow is diagnosed with severe osteoarthritis following an episode of fever and synovitis attributed to Lyme disease.
  • This animal is the source of the owner's family milk supply and the owner wishes to treat the lameness with compounds that are “natural,” i.e., compounds that normally occur in plants and animal bodies, rather than pursuing more traditional solutions such as 1) culling the animal 2) using non-steroidal anti-inflammatory drugs or 3) using steroids.
  • the animal is treated with ASU 900 mg, SAMe 600 mg and glucosamine 500 mg daily.
  • This approach is an improvement over existing options for several reasons. Because the compounds are natural components of plants and animal bodies with documented wide margins of safety, there is less concern over metabolites secreted in the milk. Because the compounds are available orally, and are active in small amounts, they are easy to administer to the animal in feed. The combined effect of the three compounds is to reduce inflammation, and pain, to support normal function, and to stimulate healing of connective tissues.
  • compositions in inhibiting or otherwise modulating the markers of inflammation and pain, such as COX-2.
  • various formulations were designed to reduce the markers of inflammation and pain, but not to eliminate those markers entirely due to the possibility of adverse side effects.
  • one goal achieved by some embodiments described in the following examples was the reduction of the inflammation and pain markers to approximately control levels, or slightly above or below control levels.
  • the ASU that was used i.e., ASU-NMX 1000TM, Nutramax Laboratories Inc., Edgewood, Md. USA
  • ASU-NMX 1000TM Nutramax Laboratories Inc., Edgewood, Md. USA
  • concentrations used in the studies were based on the minimum phytosterol content of the ASU composition.
  • the desired concentration of ASU for use in a specific experiment was first determined by incubating bovine chondrocytes (5 ⁇ 10 5 cells/well) for 72 hrs with: (i) control media alone or (ii) ASU at concentrations of 25, 8.3, 2.7, 0.9, and 0.3 ⁇ g/ml.
  • PGE-2 and nitrite concentrations were analyzed for secreted PGE-2 and nitrite concentrations. There was no significant effect on PGE-2 and nitrite levels at 0.3 or 0.9 ⁇ g/ml. At 2.7 ⁇ g/ml, there was a slight suppression of PGE-2 and nitrite levels. The highest levels of suppression were found to be between 8.3 and 25 ⁇ g/mL.
  • the ASU concentrations used in these Examples were selected based on the above data and on reported clinical dosage and previous published in vitro data for ASU.
  • Cyclooxygenase-2 (COX-2) is a protein that functions as an enzyme and specifically regulates the production of certain chemical messengers called prostaglandins (PGE-2). This PGE-2 molecule causes the pain and swelling of inflammation observed in arthritic conditions. When COX-2 activity is blocked, inflammation is reduced. COX-2 is active only at the site of inflammation.
  • PGE-2 Prostaglandin E2
  • PGE-2 is a chemical messenger that belongs to a group of hormone-like substances that participate in a wide range of body functions including inflammation. PGE-2 causes pain and swelling during inflammation.
  • MAP kinase is also known as mitogen-activated protein kinase 14.
  • MAP kinase p38 is involved in a signaling system that controls cellular responses to cytokines, stress and bacterial products like lipopolysaccharides (LPS).
  • LPS lipopolysaccharides
  • MAPK serine/threonine is a specific protein kinase that responds to extracellular stimuli and regulates various cellular activities including gene expression, proliferation, differentiation and function. It is involved in cell signaling and communication such as signaling pathways responsive to stimuli exemplified by physical stress and cytokines.
  • Cytokines are diverse proteins involved in cellular signaling and communication like hormones and neurotransmitters. They are critical to the functioning of both innate and adaptive immune response and play a major role in a variety of immunological, inflammatory and infectious diseases.
  • Tumor necrosis factor alpha belongs to a superfamily of proteins called cytokines which induce death (necrosis) of tumor cells and possess a wide range of pro-inflammatory activity. TNF- ⁇ is multifunctional, and inhibiting its activity is beneficial in reducing the inflammation in inflammatory diseases including arthritis.
  • Interleukin-1 beta is a protein belonging to the cytokine family produced by various cells, including chondrocytes, macrophages, and fibroblasts. It is a major regulator of inflammation. IL-1 ⁇ raises body temperature and the production of other chemical mediators involved in inflammation and innate immunity.
  • iNOS Inducible nitric oxide synthase
  • iNOS is a soluble enzyme that controls the production of nitric oxide (NO) following exposure to cytokines and other stimulators.
  • iNOS is important in inflammation and in defense against infection.
  • Chemokines are proteins that are produced by a variety of cells which have the ability to attract different cells to the site of inflammation and or injury and to help localize these cell in situ.
  • Two examples of chemokines are interleukin-8 (IL-8) and monocyte chemotactic protein (MCP).
  • Example 7 The study of Example 7 was designed to evaluate whether chondrocytes propagated in microcarrier spinner culture can be activated by interleukin-1 ⁇ (IL-1 ⁇ ) to produce prostaglandin E-2 (PGE-2); and whether this activation can be blocked by natural products known individually to have anti-inflammatory activity: Avocado Soybean Unsaponifiables (ASU), glucosamine (Glu), and chondroitin sulfate (CS).
  • ASU avocado Soybean Unsaponifiables
  • Glu glucosamine
  • CS chondroitin sulfate
  • Canine chondrocytes (4 ⁇ 10 3 /cm 2 ) seeded in collagen microcarrier beads were propagated in spinner culture for 14 days. They were next incubated with: media alone or the combination of ASU (NMX-1000TM, 25 ⁇ g/mL), CS (TRH122®, 20 ⁇ g/mL) and Glu (FCHG49®, 10 ⁇ g/mL) for 24 hrs.
  • the combination of ASU, Glu, and CS was supplied by Nutramax Laboratories, Inc. Cultures were then incubated with media alone or activated with IL-1 ⁇ (10 ng/mL) at 37° C., 5% CO 2 for 24 hrs. The supernatant was assayed for PGE-2 content. Chondrocytes were analyzed by microscopy and immunofluorescence for type II collagen. Data was analyzed by ANOVA with the Tukey post-hoc test. Values of p ⁇ 0.05 were considered statistically significant.
  • FIGS. 6A and 6B show a phase contrast photomicropgraph of chondrocytes growing on microcarriers indicating that the cells proliferate and produce extracellular matrix.
  • chondrocytes increased PGE-2 levels (19152 ⁇ 2721 pg/mL) when activated with IL-1 ⁇ .
  • ASU, CS, and Glu combination inhibited PGE-2 production (4020 ⁇ 468 pg/mL) by 79% when compared to IL-1 ⁇ activated control (p ⁇ 0.05).
  • microcarrier spinner culture system can be used to evaluate chondrocyte responses to pro-inflammatory stimuli and to identify agents that can modify these responses.
  • the dynamic condition in the microcarrier spinner bioreactor appears to recapitulate the biomechanical environment that chondrocytes encounter in the joint. Therefore, the microcarrier spinner culture system may represent a useful tool to evaluate the potential anti-inflammatory properties of natural products. Using this culture system, we observed that the combination of ASU, Glu, and CS effectively blocks activation of the inflammatory pathway.
  • Osteoarthritis is a degenerative joint disease characterized by erosion of articular cartilage and secondary inflammation of the synovial membrane.
  • the synovial membrane contains monocyte/macrophage-like cells that produce mediators critical to the pathogenesis of OA.
  • pro-inflammatory mediators include chemokines, cytokines, prostaglandins, and nitric oxide.
  • chemokines include chemokines, cytokines, prostaglandins, and nitric oxide.
  • chemokines include chemokines, cytokines, prostaglandins, and nitric oxide.
  • Recent clinical and in vitro studies have indicated that certain natural products such as Avocado Soybean Unsaponifiables (ASU), Glucosamine (Glu), and Chondroitin Sulfate (CS) each have anti-inflammatory properties.
  • ASU has been reported to reduce pain and functional disability in OA patients.
  • Example 8 seeks to determine whether the combination of Avocado Soy Unsaponifiables (ASU), glucosamine (Glu), and chondroitin sulfate (CS) was more effective in suppressing pro-inflammatory gene expression than ASU alone, or the combination of Glu and CS together.
  • ASU Avocado Soy Unsaponifiables
  • Glu glucosamine
  • CS chondroitin sulfate
  • THP-1 cells Human monocyte/macrophage surrogate THP-1 cells (5 ⁇ 10 5 cells) were incubated for 24 hrs at 37° C. and 5% CO2 with: (i) control media alone, (ii) ASU (8.3 ⁇ g/ml; NMX10000TM-ASU), (iii) Glu (15 mM; FCHG49®) and CS (20 ⁇ g/ml; TRH122®), or with (iv) a combination of ASU (8.3 ⁇ g/ml), Glu (15 mM), and CS (20 ⁇ g/ml). All test materials were supplied by Nutramax Laboratories, Inc., Edgewood, Md. The cells were then activated with 20 ng/ml LPS for 1 hour. Total RNA was extracted and subjected to RT-PCR analysis using primers specific to TNF- ⁇ , IL-1 ⁇ , iNOS, p38, and S14 as the housekeeping gene.
  • Results Pre-treatment with the combination of ASU, Glu, and CS profoundly suppressed the expression of TNF- ⁇ , IL-1 ⁇ , and iNOS by 50-80% in activated THP-1 cells.
  • the combination treatment reduced TNF- ⁇ and IL-1 ⁇ expression to levels similar to baseline non-activated controls and reduced iNOS expression to levels lower than baseline non-activated levels.
  • the inhibitory effect of the combined preparation on TNF- ⁇ , IL-1 ⁇ , and iNOS expression is more profound than ASU alone, or Glu and CS together.
  • the inhibition of cytokine and iNOS expression is associated with a profound suppression of p38 expression.
  • DISCUSSION/CONCLUSION This example demonstrates that the combination of ASU, Glu, and CS was more effective in suppressing pro-inflammatory gene expression than ASU alone, or Glu and CS together.
  • the suppression of TNF- ⁇ , IL-1 ⁇ , and iNOS was associated with down-regulation of p38, a key signal transduction mediator involved in joint inflammation.
  • Cyclooxygenase-2 (COX-2) is a critical enzyme involved in inflammation and plays a key role in the production of the pro-inflammatory mediator prostaglandin (PGE-2). Also known as prostaglandin G/H synthase, COX-2 catalyzes the stepwise conversion of arachidonic acid into two short-lived intermediates, prostaglandin G (PGG) and prostaglandin II (PGH). PGG isomerizes to different forms, including PGE-2.
  • Non-steroidal antiinflammatory drugs (NSAIDs) are used extensively to suppress inflammation and alleviate pain in osteoarthritis (OA) by inhibiting prostaglandin synthesis.
  • CAM Complementary and Alternative Medicines
  • Soeken K L. et al. Clin J. Pain. 20(1): 13-8, 2004 extracts from Avocado Soybean Unsaponifables (ASU).
  • ASU avocado Soybean Unsaponifables
  • the anti-inflammatory activity of ASU has been tested on tissue cell prototypes primarily derived from the immune-inflammatory system. Little is known about the effect of ASU on cartilage cells. As the only cellular component of cartilage, chondrocytes synthesize pro-inflammatory mediators such as PGE-2.
  • the study of Example 9 tested the hypothesis that ASU effectively inhibits COX-2 gene expression, thereby suppressing PGE-2 synthesis.
  • Articular chondrocytes were isolated from the metacarpal joints of mature Holsteins by collagenase digestion. Chondrocytes were plated (5 ⁇ 105/well) and maintained for 5-7 days prior to use. Chondrocytes were pre-incubated with: (i) ASU (25 ⁇ g/ml) for 72 ⁇ l s, or (ii) control media alone for 72 hrs.
  • chondrocytes were next re-incubated with control media alone or activated with 20 ng/ml of Lipopolysaccharide (LPS) at 37° C., 5% CO 2 , for: (a) 1 hour to determine COX-2 expression by RT-PCR analysis and (b) 24 hours to measure secreted PGE-2 levels by immunoassay.
  • LPS Lipopolysaccharide
  • Cells were lysed and total RNA was extracted with TRIzol® (Life TechnologiesTM. Equal amounts (1 ⁇ g) of total RNA were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Bovine primers specific for COX-2, and GAPDH as the housekeeping gene were used.
  • the gels containing ethidium bromide were electrophoresed to visualize the bands under UV light. Three to five separate runs were performed. Multiple comparisons by one-way ANOVA (Tukey post-hoc analysis) were performed using the SigmaStat statistical program where p ⁇ 0.05 was considered statistically significant.
  • cytokines TNF- ⁇ , and IL-1 ⁇ , and the enzyme cyclooxygenase-2 (COX-2), are known as the principal mediators in chronic inflammatory disorders.
  • COX-2 is the critical enzyme involved in inflammation by regulating the production of prostaglandin PGE-2.
  • Non-steroidal antiinflammatory drugs (NSAIDs) are used extensively to suppress inflammation and alleviate pain, particularly in osteoarthritis, by inhibiting cytokine and PG synthesis. More recently, alternative approaches to the management of pain and inflammation have provided encouraging results. Among these are extracts from Avocado Soybean Unsaponifiables (ASU). Clinical studies in humans suggest that ASU reduces pain associated with inflammation and reduces the extent of joint space narrowing.
  • Example 10 tested the hypothesis that ASU inhibits gene expression of COX-2, TNF- ⁇ and IL-1 ⁇ in chondrocytes and monocytes.
  • the surrogate monocyte-macrophage-like THP-1 cells were used.
  • Articular chondrocytes (5 ⁇ 10 5 /well) from the metacarpal joints of mature Holsteins and human THP-1 monocyte-like cells (5 ⁇ 10 5 /well) were pre-incubated with: (i) ASU (25 ⁇ g/mL) or (ii) control media alone for 72 and 24 hrs respectively. Cells were re-incubated with control media alone or 20 ng/ml of lipopolysaccharides (LPS) for: (a) 1 hr to determine gene expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis and (b) 24 hrs to measure secreted PGE-2 levels by immunoassay.
  • LPS lipopolysaccharides
  • Example 10 demonstrates for the first time that ASU dramatically suppresses the expression of TNF- ⁇ and IL-1 ⁇ in chondrocytes and monocytes, while confirming the reduction of COX-2 transcripts in chondrocytes. This observation supports the positive clinical findings that ASU ameliorates pain and inflammation. Our study supports the proposed utility of ASU in the management of painful conditions, exemplified by osteoarthritis.
  • Osteoarthritis is a degenerative joint disease characterized by erosion of articular cartilage and secondary inflammation of the synovial membrane. Cartilage erosion is induced by pro-inflammatory mediators produced by chondrocytes in cartilage and monocyte/macrophages localized in the synovial membrane.
  • Clinical studies have documented the benefit of using Avocado Soybean Unsaponifiables (ASU), glucosamine (Glu), and chondroitin sulfate (CS) in the management of OA.
  • ASU has been shown to reduce pain and minimize functional impairment in OA patients. See, e.g., Ernst E. Clin Rheum. 2003; 22(4-5): 285-8.
  • Equine chondrocytes were isolated from articular cartilage by collagenase digestion. Equine chondrocytes and human articular chondrocytes (ATCC) were plated at a density of 5 ⁇ 10 5 cells/well.
  • Example 11 The study of Example 11 demonstrated that the combination of ASU, Glu, and CS profoundly suppressed pro-inflammatory gene expression in chondrocytes.
  • the combination treatment was effective in reducing the expression of chemokines.
  • the suppression of pro-inflammatory mediators, such as chemokines, are critical in modulating the pro-inflammatory response in the osteoarthritic joint.
  • Our results reinforce the potential clinical utility of the combination of ASU, Glu, and CS in the management of OA, providing an alternative option to patients who fail to respond to the single agents alone.
  • glucosamine has stimulatory effects on chondrocyte metabolism which, by itself, aids in ameliorating diseases of cartilage degradation.
  • 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.
  • glucosamine with L-ergothioneine would be more beneficial than providing either alone.
  • the synergy that exists between certain compounds in the present invention also enables the use of lower doses of each compound. Although these compounds are quite safe, there may be a potential for side effects. For example, large doses of glucosamine sulfate or chondroitin sulfate can cause gastrointestinal disturbances in some individuals. In addition, these compounds are costly; for these reasons, the ability to minimize the dose and still achieve beneficial effects is desirable.

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DK07862507.6T DK2101799T3 (en) 2006-12-06 2007-12-05 Use of anabolic agents, anti-catabolic agents, antioxidant agents and analgesics to protect, treat and restore connective tissue in humans and animals
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AU2007328265A AU2007328265B2 (en) 2006-12-06 2007-12-05 A composition for treatment of connective tissues
PT78625076T PT2101799E (pt) 2006-12-06 2007-12-05 Utilização de agentes anabólicos, agentes anticatabólicos, agentes antioxidantes, e analgésicos para proteção, tratamento e reparação de tecidos conjuntivos em seres humanos e animais
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WO2008070086A3 (en) 2008-11-27
CA2672108A1 (en) 2008-06-12
AU2007328265A1 (en) 2008-06-12
US20100203177A1 (en) 2010-08-12
JP2010511708A (ja) 2010-04-15
HK1136202A1 (en) 2010-06-25
EP2101799A4 (de) 2010-11-17
AU2007328265B2 (en) 2014-02-27
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EP2101799A2 (de) 2009-09-23

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