Oral administration compositions and methods of administration comprising avocado / soybean unsaponifiables and lipolic acids

Abstract

An orally administered composition for treating or reducing damage to connective tissue or for treating or reducing inflammatory symptoms associated with damage to connective tissue includes a synergistic combination of (i) avocado / soybean unsaponifiables; and (ii) a lipoic acid or a salt or derivative thereof. A method for treating or reducing damage to connective tissue, for treating or reducing inflammatory symptoms associated with damage to connective tissue, or for reducing the level of one or more inflammatory mediators in connective tissue includes administering the orally administered composition to an avian or mammalian subject.

Description

[0001] This application is a divisional application of the application filed on May 16, 2012, with application number 201280035766.2 and entitled "Oral administration composition and method of administration containing avocado / soy unsaponifiables and fatty acids". Technical Field

[0002] This invention provides a method comprising administering (i) avocado / soybean unsaponifiables (ASU) and (ii) lipoic acid (LA) or derivatives thereof to mammalian or avian subjects. This invention also provides an oral administration composition comprising avocado / soybean unsaponifiables and lipoic acid or derivatives thereof. Background Technology

[0003] Connective tissue is the structural framework connecting cartilage, bone, synovium, ligaments, menisci, and tendons in a joint. Components of connective tissue are produced by resident cells and then secreted to form the tissue's extracellular matrix (ECM). Besides serving as a structural framework, the ECM plays a crucial role in cell communication and function. In articular cartilage, chondrocytes are arranged in a distinctive pattern within a type II collagen ECM framework. Bone-forming osteoblasts and osteocytes, as well as bone-resorbing osteoclasts, are organized within a mineralized type I collagen ECM. A small number of fibroblast-like and macrophage-like cells in the synovium are also held in place by the ECM. Similarly, tendon cells and ligament cells assemble within the ECM. The synthesis and breakdown of connective tissue ECM are controlled by a network of regulatory molecules, also produced by resident tissue cells. This network includes growth factors and various molecules known as pro-inflammatory mediators. These include cytokines, chemokines, prostaglandins, and nitric oxide. These molecules exhibit a wide range of biological activities. They can induce cell proliferation or cell death. These substances can also induce anabolic pathways that generate ECM or induce catabolic enzymes that break down ECM. Under physiological conditions, such as those involving cell survival or death, the generation or breakdown of connective tissue ECM is strictly controlled to maintain homeostasis. The generation and function of regulatory molecules are regulated by many factors, including mechanical forces, physiological factors such as temperature and pH, chemicals, microorganisms and their products. Under certain conditions, these factors can lead to the generation of excessive and untimely regulatory molecules, causing irreparable tissue damage, loss of function, and death.

[0004] Inflammation and pro-inflammatory mediators

[0005] Tissues respond to mechanical, physical, and chemical damage and infection through inflammatory responses. Inflammation is known to lead to recovery, cure, defense against infection, and often sustain life. The inflammatory response in humans and animals consists of two phases. The initial phase is characterized by the local synthesis of pro-inflammatory mediators such as prostaglandins and leukotrienes. These are derived from arachidonic acid via the action of cyclooxygenases and lipoxygenases. These pro-inflammatory mediators increase local blood flow and enhance cell permeability to allow leukocyte recruitment and accumulation. Other pro-inflammatory mediators subsequently generated include cytokines (IL-1β, TNF-α), chemokines (IL-8), and nitric oxide. In the second phase, the resolution phase, the prostaglandins generated in the initial phase activate an enzymatic pathway, through which arachidonic acid is converted into chemical mediators with anti-inflammatory properties. Prostaglandin E2 (PGE2) has been reported to activate the expression of 15-lipoxygenase, which converts arachidonic acid into anti-inflammatory lipoxygenases. Therefore, the resolution of inflammation is driven by a pro-inflammatory response. These studies suggest that the initiation, progression, and termination of the inflammatory process are tightly controlled. Prolonged, exaggerated inflammation is associated with many conditions, including osteoarthritis (OA), rheumatoid arthritis (RA), Alzheimer's disease, and cardiovascular disease.

[0006] In joint tissues, chondrocytes, synovial cells, osteoblasts, osteoclasts, ligament cells, and tendon cells produce a large number of pro-inflammatory mediators. One of these is prostaglandin E2 (PGE2), which is known to play a regulatory role by inducing the production of other mediators, including cytokines, nitric oxide, and connective tissue degrading metalloproteinases (MMPs). Due to its ability to induce MMPs, PGE2 contributes to the degradation of cartilage ECM. Furthermore, PGE2 promotes bone resorption and osteophyte formation. PGE2 sensitizes nociceptors on peripheral nerve endings, thus contributing to the development of inflammatory pain. PGE2 levels are locally regulated by inducible cyclooxygenase-2 (COX-2), a nitric oxide synthase in chondrocytes that inhibits the degradation of cartilage and proteoglycans. Under pathological conditions such as osteoarthritis, COX-2 expression is upregulated, accompanied by increased PGE2 production.

[0007] The roles of other tissues in the inflammatory process have also been well-established. Synovial inflammation is now recognized as playing a crucial role in cartilage degeneration in osteoarthritis, particularly in the early stages of the disease. Synovitis is characterized by the activation of resident macrophage-like and fibroblast-like cells in the synovium, leading to the overproduction of pro-inflammatory mediators (including TNF-α, IL-1β, and PGE2). Recent evidence suggests that synovial macrophages are the primary source of cytokines in the earliest stages of osteoarthritis and are significant contributors to cartilage damage throughout the disease process. Cytokines also induce the production of PGE2 and active metalloproteinases (MMPs). It is now generally accepted that these mediators control the balance between ECM destruction and repair, making these molecules preferred targets for therapeutic interventions. Other tissues in the joint, such as subchondral bone, also produce pro-inflammatory mediators that regulate joint health.

[0008] In addition to pro-inflammatory mediators such as cytokines and prostaglandins, reactive oxygen species (ROS) are also involved in joint degeneration observed in osteoarthritis. ROS-induced oxidative stress (such as nitric oxide and hydrogen peroxide) has been shown to cause chondrocyte apoptosis and chondrocyte emulsion (ECM) degradation. Furthermore, ROS-activated signaling pathways have been reported to lead to increased production of pro-inflammatory mediators, including cytokines and prostaglandins. In vitro studies have confirmed the association between the pathways involved in ROS production and pro-inflammatory mediators. These studies support the idea that agents that simultaneously inhibit oxidative stress and inflammatory pathways will be particularly useful in the regulation of inflammation.

[0009] Treatment of inflammation in joint tissues with medication

[0010] The widespread use of selective COX-2 inhibitors and various non-selective nonsteroidal anti-inflammatory drugs (NSAIDs) in the treatment of this condition reflects the central role of COX-2 and PGE2 in the pathophysiology of osteoarthritis. However, long-term administration of these drugs has adverse side effects, including gastrointestinal disturbances and disruption of chondroitin proteoglycan metabolism. Studies in human and animal models have demonstrated that COX inhibitors impair bone healing and repair. Therefore, alternative therapies for managing inflammation are needed that do not focus on using NSAIDs to inhibit the production of PGE2 and other pro-inflammatory mediators.

[0011] Treating joint tissue inflammation with health supplements

[0012] Avocado / soy unsaponifiables (ASU)

[0013] Numerous studies have demonstrated the benefits of avocado / soy unsaponifiables (ASU) in promoting joint health and managing osteoarthritis. Clinical studies have reported the beneficial effects of ASU in human and equine osteoarthritis patients, as well as in experimental OA animal models. In vitro studies using bovine and human joint tissue cells have elucidated the mechanism of ASU's beneficial effects on osteoarthritis. These studies show that ASU inhibits the expression and production of cytokines, chemokines, PGE2, nitric oxide, and MMPs. ASU also exerts an anabolic effect on cartilage metabolism by increasing the synthesis of cartilage matrix components while inhibiting their degradation.

[0014] Early studies using human osteoarthritis chondrocyte cultures found that ASU significantly reduced the stimulatory effect of IL-1β on PGE2 production. Of the two cyclooxygenase isoforms involved in prostaglandin synthesis, COX-2 is highly inducible in response to cytokine exposure. High levels of COX-2 expression have been confirmed in human synovial tissue. Several studies in experimental animals and humans have shown that PGE2 synthesis and COX-2 expression are upregulated in the synovium of osteoarthritis (OA). Elevated PGE2 levels have been detected in synovial tissue and synovial fibroblasts of OA. Experimental evidence suggests that synovial tissue is a major source of arachidonic acid found in the synovial fluid of osteoarthritis. Cytokines IL-1β and TNF-α increase PGE2 production in synovial cells. The reported reduction in PGE2 synthesis by ASU appears to be related to decreased COX-2 gene expression.

[0015] Fatty acids (LA)

[0016] Fatty acids (LAs), also known as 1,2-dithiopentane-3-valeric acid, 1,2-dithiopentane-3-valeric acid, or 6,8-lipoic acid, are potent, naturally occurring low-molecular-weight antioxidants. Fatty acids are synthesized enzymatically from octanoic acid in mitochondria. They are a key cofactor in mitochondrial decarboxylation and are essential for sufficient ATP production. Fatty acids exist in enantiomeric forms, R-fatty acids (R-LA) and S-fatty acids (S-LA). In biological systems, only R-LA binds to lysine residues in amide bonds. The oxidized (LA) and reduced (DHLA) forms represent potent redox pairs. The biological effects of LA include scavenging reactive oxygen species (ROS); regenerating endogenous antioxidants such as glutathione and vitamin E; chelating metal ions; and repairing oxidative damage in macromolecules. Both LA and DHLA scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) and have the ability to inhibit protein carbonyl formation. LA and DHLA can regenerate other endogenous antioxidants such as vitamin C, vitamin E, and glutathione, thus protecting cells against oxidative stress. Recent evidence suggests that LA acts not only as a true oxidant scavenger but also as an activator of cellular stress response pathways.

[0017] Studies have shown that orally administered LA produces key biological activity in the defense against oxidative stress-related damage. Increasing evidence suggests that orally administered LA is bioavailable, safe at appropriate doses, and produces several metabolic and clinical effects. The reported clinical benefits of alpha-lipoic acid (LA) involve the following conditions: diabetic polyneuropathy (Ametov et al., Thesensory symptoms of diabetic polyneuropathy are improved with alpha-lipoic acid: the SYDNEY trial. Diabetes Care. 2003, 26:770-776); conditions affecting the vascular system, such as hypertension; inflammatory diseases, such as coronary atherosclerosis; and cognitive-neurological disorders, such as Alzheimer's disease (Hager et al., Alpha-lipoic acid as a new treatment option for Azheimer typedementia, Archives of Gerontology and Geriatrics, 2001, 32:275-282 and Hager et al., Alpha-lipoic acid as a new treatment option for Alzheimer's disease--a 48-month follow-up analysis J Neural Transm. Suppl. 2007, 72:189-93. However, little is known about the role of LA in joint inflammation. LA has diverse roles at the cellular level and its mechanisms of action involve a variety of biological activities, such as antioxidation, anti-inflammation, anti-chelation, and enhancement of kinases and phosphatases.

[0018] Derivatives of fatty acids have been described in the art. Some derivatives of fatty acids offer improved biological activity, improved pharmacokinetic properties such as longer half-life, improved bioavailability, and reduced drug interaction profiles. Derivatives of alpha-lipoic acids have been described in the following publications, which are hereby incorporated by reference: Gruzman et al., Synthesis and characterization of new and potent alpha-lipoic acid derivatives. Bioorganic & Medicinal Chemistry, 2004, 12: 1183-1190; Melagraki et al., Synthesis and evaluation of the antioxidant and anti-inflammatory activity of novel coumarin-3-aminoamides and their alpha-lipoic acid adducts. European Journal of Medicinal Chemistry, 2009, 44: 3020-3026; Gurkan et al., Syntheses of novel indole lipoic acid derivatives and their antioxidant effects on lipid peroxidation. Archiv der Pharmazie, 2005, 338: 67-73; Ortial et al., Fluorinated amphiphilic amino acid derivatives as antioxidant carriers: a new class of protective agents. J Med Chem 2006; 12-2820; and Koufaki et al., Sign and synthesis of antioxidant alpha-lipoic aicdhybrids. Methods Mol Biol, 2010, 594: 297-309. Invention Summary

[0019] This invention provides an oral administration composition comprising: (i) avocado / soy unsaponifiables (ASU) and (ii) fatty acids or derivatives thereof. The composition may also be used in other forms of administration, such as topical, rectal, and sublingual.

[0020] The present invention also provides a method for preventing, treating, protecting, repairing or reducing connective tissue damage or alleviating symptoms associated with connective tissue damage in avian or mammalian subjects, the method comprising administering to the subject (i) an avocado / soy unsaponifiable and (ii) fatty acids or derivatives thereof.

[0021] The present invention further provides a method for reducing the level of one or more inflammatory mediators in connective tissue, the method comprising administering (i) avocado / soy unsaponifiables and (ii) fatty acids or derivatives thereof to a mammalian subject.

[0022] Other novel features and advantages of this invention will become apparent when those skilled in the art examine the invention or learn of it through practice. Attached Figure Description

[0023] Figure 1 The effects of avocado / soybean unsaponifiables and fatty acids on PGE2 production in LPS-activated horse chondrocyte cultures were illustrated using graphs.

[0024] Figure 2 The effects of avocado / soybean unsaponifiables and fatty acids on PGE2 production in H2O2-activated horse chondrocyte cultures were illustrated using graphs. Invention Details

[0025] This invention provides a method comprising administering (i) avocado / soy unsaponifiables (ASU) and (ii) fatty acids or derivatives thereof to a mammalian subject. Avocado / soy unsaponifiables (ASU) and fatty acids or derivatives thereof may be administered together in a composition or dosage form, or they may be administered separately. In a preferred embodiment, avocado / soy unsaponifiables (ASU) and fatty acids or derivatives thereof are administered together in a composition or dosage form, or separately, within a period of overlapping therapeutic efficacy, preferably within 1 hour, more preferably within 30 minutes, and most preferably within 5 minutes.

[0026] The term "mammal subject" refers to any mammal, including but not limited to humans, dogs, cats, horses, cows, and camels. The term "avian subject" refers to birds.

[0027] The term "avocado / soybean unsaponifiables (ASU)" refers to a mixture of avocado unsaponifiables and soybean unsaponifiables. "Unsaponifiables" are compounds that do not react with alkali to form soap. The term "avocado unsaponifiables" refers to a compound extract obtained from any part of the avocado (Persea genus). The avocado can be any species, such as, but not limited to, Persea americana and Persea schiedeana. The term "soybean unsaponifiables" refers to a compound extract obtained from any part of soybean (Glycine max sorghum). The soybean can be any species, such as, but not limited to, Glycine willd sorghum.

[0028] Avocado / soybean unsaponifiables are well known in the art and described in numerous patents and publications, including but not limited to: U.S. Patent Nos. 6,797,289, 7,449,487, and 6,759,543; U.S. Patent Application Publication Nos. 20080176935 and 20090087503; Their, "Unsaponifiable constituents of avocado and soybean oils. Treatment of certain forms of arthralgia," J. Med. Lyon 53(222):195-8 (February 1972); Trevoux, "Unsaponifiable fractions of the avocado and soybeanin gynecology," J. Gynecol. Obstet. Biol. Reprod. 6(1):99-105 (January 1977); Lamaud et al., "Biochemical modifications of connective tissue induced by the non-saponifiables of avocado and soy-bean oils administered percutaneously in the hairless rat," Pathol. Biol. 26(5):269-74 (May-June 1978); Boumediene et al., "Avocado / soya unsaponifiables enhance the expression of transforminggrowthfactor beta 1and beta 2 in cultured articular chondrocytes, "ArthritisRheum. 42(1):148-56 (January 1999); Henrotin et al., "Effects of three avocado / soybeanunsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes," Clin. Rheumatol.17(1):31-9 (1998); Maheu et al., "Symptomatic efficacy of avocado / soybean unsaponifiables in the treatment of osteoarthritis," Arthritis Rheum. 41(1):81-91 (January 1998); and Blotman 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), each of these patents and publications is incorporated herein by reference in its entirety. Furthermore, avocado / soybean unsaponifiables in combination with another ingredient (glucosamine) are currently marketed in the United States under the trademark AVOCA. Available on the market. Avocado / soy unsaponifiables are also marketed under a trademark in Europe. Going public.

[0029] Dosage calculations can be determined by those skilled in the art by assessing body weight, surface area, and species differences. The conventional daily dose of avocado / soybean unsaponifiables (ASU) is from about 1 mg / kg to about 12 mg / kg, preferably from about 2 mg / kg to about 5 mg / kg, and more preferably from about 3 mg / kg to about 4 mg / kg. In some embodiments, the conventional daily dose is at least 5 mg for small animals and up to 12 g for large animals. The daily dose refers to the total dose administered over a 24-hour period.

[0030] In some embodiments, the avocado / soy unsaponifiables are applied daily. In other embodiments, the avocado / soy unsaponifiables are applied at a lower frequency, such as every other day, once a week, or once a month. In some embodiments, the avocado / soy unsaponifiables are applied at a daily dose of about 1 mg / kg to about 12 mg / kg, preferably about 2 mg / kg to about 5 mg / kg, most preferably about 3 mg / kg to about 4 mg / kg, for three days to one month, preferably about one week, after which the daily dose is reduced to about 25% to about 90% of the initial dose, preferably about 50% to about 80% of the initial dose, and most preferably about 60% to about 75% of the initial dose. Dosage calculations can be determined by those skilled in the art by assessing body weight, surface area, and species differences.

[0031] Avocado / soy unsaponifiables can be applied once a week to five times a day, preferably once every two days to three times a day, and more preferably once or twice a day. In a preferred embodiment, avocado / soy unsaponifiables are applied once a day. Avocado / soy unsaponifiables can be taken with or without food.

[0032] The term "lipidic acid or its derivative" refers to compounds and their salts or derivatives having the following structures:

[0033]

[0034] Lipoic acids are also known as α-lipoic acids; lipoic acid; 6,8-dithiooctanoic acid; and 1,2-dithiopentane-3-valeric acid. Derivatives of lipoic acids include, but are not limited to, esters and amides of lipoic acids, conjugates of lipoic acids, and lipoic acid analogs. Esters and amides of lipoic acids include, but are not limited to, 3-(5-[1,2]-dithiocyclopentane-3-yl-valeric acid 3-(5-[1,2]-dithiocyclopentane-3-yl-valeric acid 3-(5-[1,2]-dithiocyclopentane-3-yl-valeric acid 3-(5-[1,2]-dithiocyclopentane-3-yl-valeric acid 3-(5-[1,2]-dithiocyclopentane-3-yl-valeric acid 3-propyl-amide). Fatty acid conjugates include, but are not limited to, coumarin-fatty acid conjugates; indole-α-fatty acid conjugates such as 5-[1,2]-dithiocyclopentan-3-yl-valeric acid [1-(4-fluoro-benzyl)-1H-indole-5-yl]-amide; and amphiphilic fatty acid derivatives such as N-lactosyl-N ε -(5-[1,2]-dithiocyclopentan-3-yl-valeryl)-L-lysine-1H,1H,2H-perfluorooctylamide (N-lactobionyl-N) ε-(5-[1,2]-dithiolan-3-yl-penoyl)-L-lysinyl-1H,1H,,2H-perfluorooctylamide). Fatty acid analogs include, but are not limited to, 1,2-diselenolane-3-pentanoic acid and 1,2-dithiopentane derivatives of fatty acids containing a catechol moiety linked via a heteroaromatic ring. Derivatives of alpha-lipoic acids have been described in the following publications, which are hereby incorporated by reference: Gruzman et al., Synthesis and characterization of new and potent alpha-lipoic acid derivatives. Bioorganic & Medicinal Chemistry, 2004, 12: 1183-1190; Melagraki et al., Synthesis and evaluation of the antioxidant and anti-inflammatory activity of novel coumarin-3-aminoamides and their alpha-lipoic acid adducts. European Journal of Medicinal Chemistry, 2009, 44: 3020-3026; Gurkan et al., Syntheses of novel indole lipoic acid derivatives and their antioxidant effects on lipid peroxidation. Archiv der Pharmazie, 2005, 338: 67-73; Ortial et al., Fluorinated amphiphilic amino acid derivatives as antioxidant carriers: a new class of protective agents. J Med Chem2006; 12-2820; Koufaki et al., Sign and synthesis of antioxidant alpha-lipoic acid hybrids.Methods Mol Biol, 2010, 594:297-309; Sen et al., A positively charged alpha-lipoic acid analogue with increased cellular uptake and more potent immunomodulatory activity. Biochem Biophys Res Commun, 1998, 247:223-228; Harnett et al., Novel lipoic acid analogues that inhibit nitric oxide synthase. Bioorg Med Chem Lett, 2002, 12:1439-1442; and Acker, Syntheses of Reduced Lipoic Acid and Analogs of Lipoic Acid, Journal of Organic Chemistry, 1963, 28:2533-2536. Lipoic acids and their derivatives may include racemic compounds, enantiomers, or mixtures thereof.

[0035] The usual daily dose of fatty acids or fatty acid derivatives can range from about 1 mg / day to about 15,000 mg / day. The usual daily dose of fatty acids or fatty acid derivatives can range from about 0.25 mg / kg / day to about 50 mg / kg / day. The daily dose of a fatty acid refers to the total amount of fatty acid administered over a 24-hour period. The daily dose can be provided by one or more administrations. For example, the daily dose can be administered once daily, twice daily, or three or more times daily. In a preferred embodiment, the daily dose is administered one to five times over a 24-hour period, preferably one to three times, more preferably one or two times. The daily dose can be varied depending on the subject type. For example, for humans, the daily dose is preferably about 25 to about 2,000 mg / day, more preferably about 50 to about 1,500 mg / day, and most preferably about 100 to about 1,200 mg / day, or preferably about 0.5 to about 50 mg / kg / day, more preferably about 0.6 to about 40 mg / kg / day, and most preferably about 1.25 to about 30 mg / kg / day. For dogs, the daily dose is preferably about 1 to about 1,500 mg / day, more preferably about 10 to about 750 mg / day, and most preferably about 20 to about 600 mg / day, or preferably about 1 to about 100 mg / kg / day, more preferably about 10 to about 60 mg / kg / day, and most preferably about 20 mg / kg / day. For cats, the daily dose is preferably about 1 to about 100 mg / day, more preferably about 3 to about 50 mg / day, and most preferably about 3 to about 15 mg / day, or preferably about 0.5 to about 25 mg / kg / day, more preferably about 1 to about 20 mg / kg / day, and most preferably about 3 to 15 mg / kg / day. For horses, the daily dose is preferably about 125 to about 15,000 mg / day, more preferably about 500 to about 12,500 mg / day, and most preferably about 200 to about 10,000 mg / day, or preferably about 0.5 to about 100 mg / kg / day, more preferably about 1 to about 50 mg / kg / day, and most preferably about 1 to 25 mg / kg / day.

[0036] In some preferred embodiments, a fatty acid or its derivatives, analogs, racemates, or enantiomers, or mixtures thereof, is administered for three days to one month, preferably about one week, after which the daily dose is reduced to 75% of the initial dose. Dosage calculations can be determined by those skilled in the art by assessing body weight, surface area, and species differences.

[0037] Fatty acids or their derivatives may be applied once a week to five times a day, preferably once every two days to three times a day, and more preferably once or twice a day. In a preferred embodiment, fatty acids or their derivatives are applied once a day. Fatty acids or their derivatives may be taken with or without food.

[0038] In some embodiments, the combination of (i) avocado / soy unsaponifiables and (ii) fatty acids or their derivatives exhibits a synergistic effect. A synergistic effect means that the combination of two or more components provides a result greater than the sum of the effects produced by each agent alone. In preferred embodiments, the result is statistically significant and greater than an additive effect. In some embodiments, the combination of avocado / soy unsaponifiables and fatty acids or their derivatives has a statistically significant and better effect than each component alone. In preferred embodiments, the combination of avocado / soy unsaponifiables and fatty acids or their derivatives exhibits a synergistic effect in one or more of the following ways: preventing, treating, repairing, or reducing connective tissue damage; alleviating symptoms associated with connective tissue damage in avian or mammalian subjects; and reducing the levels of one or more inflammatory mediators in connective tissue.

[0039] This invention provides a method for preventing, treating, repairing, reducing damage to, or controlling inflammation of connective tissue, protecting cartilage, or alleviating symptoms associated with connective tissue injury in avian or mammalian subjects, the method comprising administering to the subject: (i) an avocado / soy unsaponifiable and (ii) fatty acids or derivatives thereof. The term “connective tissue” includes, but is not limited to, cartilage, bone, synovium, ligaments, menisci, and tendons. In some embodiments, administration of an avocado / soy unsaponifiable and (ii) fatty acids or derivatives thereof may prevent, treat, repair, or reduce connective tissue injury. Connective tissue injury may be the result of physical injury or may represent “wear and tear” caused by continuous use, weight, and age (e.g., osteoarthritis). Connective tissue injury may also be caused by diseases such as rheumatoid arthritis, synovial disorders, infection-related rheumatic diseases, and inflammatory connective tissue disorders. In some embodiments, administration of an avocado / soy unsaponifiable and (ii) fatty acids or derivatives thereof may alleviate symptoms associated with connective tissue injury in avian or mammalian subjects. Symptoms associated with connective tissue damage include, but are not limited to: pain, discomfort, pressure, inflammation, stiffness, and / or swelling.

[0040] The present invention also provides a method for reducing the level of one or more inflammatory mediators in connective tissue, the method comprising administering to an avian or mammalian subject: (i) an avocado / soy unsaponifiable and (ii) fatty acids or derivatives thereof. Inflammatory mediators include, but are not limited to, prostaglandins such as prostaglandin E2 (PGE2); cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α); chemokines; leukotrienes; nitric oxide; and reactive oxygen species.

[0041] The application of avocado / soy unsaponifiables and fatty acids or their derivatives can also be used to treat, prevent, and reduce damage to or alleviate symptoms associated with diseases affecting the cardiovascular, nervous, musculoskeletal, and gastrointestinal systems. The present invention also provides oral administration compositions comprising: (i) avocado / soy unsaponifiables and (ii) fatty acids or their derivatives. Oral administration compositions are any dosage form known in the art that can be administered orally, such as, but not limited to: capsules; tablets; powders dispersible in beverages; liquids, such as solutions, suspensions, or emulsions; soft gels / chewable capsules; chewing sticks or other convenient dosage forms, such as oral liquids in capsules.

[0042] Orally administered compositions may contain one or more inactive pharmaceutical ingredients (also commonly referred to herein as "excipients"). Inactive ingredients are used, for example, to dissolve, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, and alter the shape of the active ingredient, to form a suitable and effective formulation that is safe, convenient, and otherwise acceptable in use. Excipients are preferably pharmaceutically acceptable. Examples of pharmaceutically acceptable excipients include lubricants, buffers, stabilizers, foaming agents, pigments, colorants, flavoring agents, fillers, bulking agents, aromatizers, release modifiers, adjuvants, plasticizers, flow promoters, release agents, polyols, granulating agents, diluents, binders, buffers, adsorbents, flow aids, adhesives, anti-adhesives, acidifiers, softeners, resins, modifiers, solvents, surfactants, emulsifiers, elastomers, and mixtures thereof.

[0043] The oral administration composition may further comprise one or more active ingredients. For example, the composition may further comprise one or more pharmaceutical or nutritional supplements. In some embodiments, the composition may further comprise compounds beneficial to connective tissue. Examples include, but are not limited to, glycosaminoglycans such as chondroitin; amino sugars such as glucosamine, methanesulfonylmethane (MSM), green tea extract, frankincense extract, scutellaria baicalensis extract, gum arabic extract, turmeric extract, curcumin, cetylmyristate complex (CMO), and eggshell membrane.

[0044] All references cited in this article are incorporated in their entirety through citation. Example

[0045] Example 1 Effects of avocado / soybean unsaponifiables (ASU) and fatty acids (LA) on prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated horse chondrocyte cultures.

[0046] Chondrocytes were pretreated for 24 hours with 8.3 μg / ml avocado / soybean unsaponifiables (ASU) and different concentrations (2.5, 1.25, and 0.625 μg / ml) of fatty acids (LA), followed by activation with lipopolysaccharide (LPS) (1 ng / ml). LPS is an endotoxin derived from bacterial cell walls and acts as a broad inflammatory stimulant to induce prostaglandin E2 (PGE2) production. After another 24 hours, the supernatant was collected and PGE2 levels were measured. Statistical significance between the activation control group and the pretreatment group was analyzed using Tukey post-hoc analysis (mean ± 1 SD, n = 3).

[0047] The combination of 8.3 μg / ml avocado / soybean unsaponifiables (ASU) and fatty acids (LA) at concentrations of 2.5, 1.25, and 0.625 μg / ml significantly reduced PGE2 levels compared to ASU alone (p < 0.001) or LA alone (p < 0.001). Results are presented graphically. Figure 1 middle.

[0048] Example 2 Effects of ASU and fatty acids (LA) on prostaglandin E2 (PGE2) production in hydrogen peroxide (H2O2) activated horse chondrocyte cultures.

[0049] Chondrocytes were pretreated for 24 hours with 8.3 μg / ml avocado / soy unsaponifiables (ASU) and different concentrations (2.5, 1.25, and 0.625 μg / ml) of fatty acids (LA), followed by activation with hydrogen peroxide (500 μM). Hydrogen peroxide is a strong oxidant used to induce prostaglandin E2 (PGE2) production. After another 24 hours, the supernatant was collected and PGE2 levels were measured. Statistical significance between the activation control and pretreatment groups was analyzed using Tukey post-hoc analysis (mean ± 1 SD, n = 3). The combination of 8.3 μg / ml avocado / soy unsaponifiables (ASU) and fatty acids (LA) at concentrations of 2.5 and 1.25 μg / ml significantly reduced PGE2 levels compared to ASU alone (p < 0.001) or LA alone (p < 0.05). Results are shown in figures. Figure 2 middle.

Claims

1. An oral administration composition for treating connective tissue damage in avian or mammalian subjects, comprising a composition of the following components: (i) avocado / soy unsaponifiables and (ii) fatty acids, In the composition, the concentration of avocado / soybean unsaponifiables is 8.3 μg / ml, and the concentration of fatty acids is 2.5 μg / ml or 1.25 μg / ml; The daily dose of avocado / soybean unsaponifiables ranges from 1 mg / kg to 12 mg / kg, and the daily dose of fatty acids ranges from 0.25 mg / kg to 50 mg / kg.

2. The use of (i) avocado / soybean unsaponifiables and (ii) fatty acids in the preparation of a medicament for treating connective tissue damage in avian or mammalian subjects, wherein the concentration of avocado / soybean unsaponifiables in the composition is 8.3 μg / ml and the concentration of fatty acids is 2.5 μg / ml or 1.25 μg / ml; The daily dose of avocado / soybean unsaponifiables ranges from 1 mg / kg to 12 mg / kg, and the daily dose of fatty acids ranges from 0.25 mg / kg to 50 mg / kg.

3. The use according to claim 2, wherein the connective tissue is selected from: cartilage, bone, synovium, ligament, meniscus, and tendon.

4. The use according to claim 2, wherein the mammalian subject is a human, horse, dog, cat, camel, or cow.

5. The use of a composition of (i) avocado / soy unsaponifiables and (ii) fatty acids in the preparation of a medicament for reducing the levels of one or more inflammatory mediators in the connective tissue of avian or mammalian subjects, wherein the concentration of the avocado / soy unsaponifiables in the composition is 8.3 μg / ml and the concentration of the fatty acids is 2.5 μg / ml or 1.25 μg / ml; The daily dose of avocado / soybean unsaponifiables ranges from 1 mg / kg to 12 mg / kg, and the daily dose of fatty acids ranges from 0.25 mg / kg to 50 mg / kg.

6. The use according to claim 5, wherein the one or more inflammatory mediators are selected from prostaglandin E2.

7. The use according to claim 5, wherein the poultry or mammal subject is a bird, human, horse, dog, cat, camel, or cow.

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