US 2010O285159A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0285159 A1 HENDERSON et al. (43) Pub. Date: Nov. 11, 2010

(54) COMBINATION OF UNSAPONIFIABLE (22) Filed: May 14, 2010 LPDS COMBINED WITH POLYPHENOLS AND/OR CATECHNS FOR THE Related U.S. Application Data PROTECTION, TREATMENT AND REPAIR (60) Division of application No. 1 1/889,060, filed on Aug. OF CARTILAGE IN JOINTS OF HUMANS 8, 2007, now abandoned, which is a continuation of AND ANIMALS application No. 11/192,362, filed on Jul. 29, 2005, now abandoned. (75) Inventors: Todd R. HENDERSON, (60) Provisional application No. 60/592,322, filed on Jul. Jarrettsville, MD (US); Louis 30, 2004. LIPPIELLO, Forest Hill, MD Publication Classification (US); Charles FILBURN, Forest Hill, MD (US); David GRIFFIN, (51) Int. Cl. Belair, MD (US) A6IR 36/48 (2006.01) A63L/353 (2006.01) A6IP 9/04 (2006.01) Correspondence Address: A6IP 29/00 (2006.01) ARENT FOX LLP (52) U.S. Cl...... 424/757: 514/456 1050 CONNECTICUT AVENUE, N.W., SUITE 400 (57) ABSTRACT WASHINGTON, DC 20036 (US) The present invention relates to a composition and a kit for the protection, treatment and repair of cartilage in humans and (73) Assignee: animal joints. The composition or kit contains a combination NUTRAMAX LABORATORIES, of unsaponifiable lipids together with one or more of INC. Edgewood, MD (US) polyphenols and/or catechins. Preferably, the composition or kit contains avocado:Soybean unsaponifiables (ASU) and (21) Appl. No.: 12/780,642 green tea. US 2010/0285159 A1 Nov. 11, 2010

COMBINATION OF UNSAPONIFLABLE inhibits IL-1 induced expression of nitric oxide synthase and LPDS COMBINED WITH POLYPHENOLS nitric oxide production and Suppresses activation of nuclear AND/OR CATECHNS FOR THE factor-kB, a key step in initiation of the cytokine effects (5). PROTECTION, TREATMENT AND REPAIR Furthermore, the catechins in green tea were recently shown OF CARTILAGE IN JOINTS OF HUMANS to potently inhibit aggrecanase activities known to be AND ANIMALS involved in the early stages of destruction of cartilage pro teoglycans (6). Thus, the components of green tea have the RELATED APPLICATIONS potential to ameliorate the cause and the symptoms of DJD 0001. This application claims the benefit, pursuant to 35 through multiple mechanisms. The green tea may be admin U.S.C. S 119, of U.S. Provisional Patent Application No. istered as an extract or standardized to polyphenols or cat 60/592,322, filed Jul. 30, 2004, the entirety of which is incor echins. porated herein by reference. Unsaponifiable Lipids FIELD OF THE INVENTION 0005 Vegetable oils, including coconut, peanut and saf 0002 The present invention relates to a composition for flower oil to name a few, are possible components of the the protection, treatment and repair of cartilage in humans human diet that contain an important class of compounds and animal joints. termed unsaponifiable lipids. One rich source of unsaponifi able lipids are avocados and Soybean oil, with avocado having BACKGROUND OF THE INVENTION approximately four times more lipids than those found in other commonly eaten fruits. Many of the health benefits of 0003) Degenerative joint disease (DJD) results from the Soy, widely used as a staple food in Asian countries, may be cumulative effects of an imbalance between synthesis and due to this unsaponifiable fraction which also includes isofla degradation of components of cartilage extracellular matrix. vones. Unsaponifiable lipids are defined as the material which This imbalance is associated with disregulated activity of does not react with basic agents to form soaps. Included in inflammatory cytokines and production of prostaglandins this class are phytosterols, fat soluble vitamins A, D, E and K. that mediate pain. Progression of the disorder occurs when bioflavinoids, phytoestrogens and Small organic molecules this imbalance persists. Agents that act to increase synthesis called terpenes. The major components by weight of the of cartilage components and Suppress the activity of specific unsaponifiable lipid fraction are a group of compounds called cytokines, particularly of interleukin-1-beta (IL-1-B) and phytosterols which differ slightly in structure and include tumor necrosis factor alpha (TNF-a), have the potential to beta Sitosterol, campesterol and stigmasterol. Of the many relieve symptoms of DID and stop its progression. Both of beneficial actions attributed to phytosterols, their ability to these cytokines act through mechanisms that involve genera reduce pain, Swelling, and tissue injury in joints is proposed. tion of reactive oxygen species (ROS) and reactive nitrogen With aging and stress, increased amounts of phytosterols are species (RNS) that lead to suppression of synthesis of carti thought to be necessary to maintain nomial biological func lage matrix proteoglycans along with increased production of prostaglandins, nitric oxide, and activation of metalloprotein tions (1.2). ases that degrade cartilage proteins. A combination of agents Biological Activity that is capable of interacting with these mechanisms in dif ferent ways and to stimulate the anabolic response needed in 0006 Phytosterols have potent biological activities, some chondrocytes for maintenance and/or restoration of matrix of which reduce the joint pain and Swelling characteristic of components could be effective in the protection, treatment inflammatory joint disorders as well as symptoms character and repair of connective tissue. istic of trauma or aging related joint disorders. In addition, these agents beneficially alter the bodies immune response in Polyphenols and Catechins a fashion which helps to minimize joint tissue destruction attributed to an allergic tissue reaction. Studies indicate phy 0004 Various polyphenols can be sourced from berries tosterols are active in immune modulation (3) and have anti and other fruits. Catechins are found primarily in teas. The inflammatory and antipyretic activity (4). Most of these preferred source of polyphenols and catechins are sourced activities are a result of the effect of phytosterols inhibiting from green tea. Green tea contains a mixture catechins, the production by inflammatory cells of chemical agents (cy including epicatechin (EC), epigallocatechin (EGC), epicat tokines) which cause tissue damage and stimulation of ana echin gallate (ECG) and epigallocatechin gallate (EGCG). bolic activity of chondrocytes. Avocado/Soybean oil unsa These catechins have potent antioxidant activity, acting as ponifiables (ASU) is a preferred source of unsaponifiable scavengers of the free radicals (ROS and RNS) involved in damage to cells. They also act by chelating metals that cata lipids for the protection, treatment and repair of connective lyze production of ROS (1). This antioxidant activity may tissue conditions. interfere with the damaging effects of agents, e.g. fibronectin In Vitro Tests fragments (Fn-f) and cytokines, that can cause DJD. Antioxi dants block the effects of Fn-f, which include increased 0007 Prior researchers have conducted in vitro tests on expression and activity of both cytokines IL-1 and TNF-a ASU. The in vitro model previously used to test ASU is based (2.3). In addition, recent studies have shown that green tea on monitoring reduction of interleukin-1 (IL-1)-induced met polyphenols significantly reduce the incidence of collagen alloprotease activity, nitric oxide or prostaglandin synthesis induced arthritis in mice that was associated with reduced (all agents associated with cartilage degradation, tissue expression of TNF-a and cyclooxygenase 2, a TNF-a regu inflammation, or pain) (5-9). ASU was shown to inhibit the lated enzyme that catalyzes the production of prostaglandin action of IL-1 at doses of 1 to 10 ug/ml. A combination of 2:1 E2 (4). Other studies have shown that the EGCG in green tea soy:avocado was found to be even more effective than either US 2010/0285159 A1 Nov. 11, 2010

agent alone (5.8). It was also noticed that this mixture was the and functional index. All indices showed improvement at main combination that was effective in decreasing production p-0.01 or better (17). In a double-blind study, individuals of collagenolytic activity (9). Fibroblasts also appear to be with knee OA (femoro-tibial) were dosed with 300 or 600 mg responsive to ASU. Metalloprotease activity (MMP-2 and of an avocado:Soybean unsaponifiable for three months. Indi MMP-3) was inhibited at low doses of ASU and at higher ces measured included NSAID and analgesic intake between doses the tissue-inhibitors of metalloproteases was increased day 30 and day 90. All indices improved with treatment at (7). The above mentioned assays could be related to the p-0.01 or better with NSAID intake decreasing by more than beneficial physiological (symptomatic relief) effects. Other 50% in 71% of the individuals compared to 36% in individu indices tested relate to a possible increase capacity for repair als receiving placebo. Lequesne's index dropped by 3.9 and and regeneration of articular cartilage. For example, the anti 2.9 points in ASU 300 and 600 mg respectively against 1.6 in catabolic activity of ASU was shown to be paired with a direct placebo. Similar results were observed in individuals given effect of ASU on stimulating collagen and proteoglycan pro 300 or 600 mg (18). Eighty-five out of 164 individuals with duction, possibly by increasing transforming growth factor painful primary OA of the knee or hip were dosed for 6 beta 1 and 2 synthesis (6). Research in our laboratory also has months with 300 mg unsaponifiables after a 15 day washout shown that ASU is effective in improving cartilage synthesis period for NSAIDs. Efficacy was determined by Lequesne's in-vitro. functional index, visual analog pain scale, intake of NSAIDs and overall disability score. Pain decreased from a score of In Vivo Tests in Animals 56.1 to 35.3 in the ASU group and from 56.1 to 45.7 in the placebo group (p<0.003). Values for NSAID intake showed 0008 Prior researchers have conducted in vivo animal consumption lower in ASU group (48% versus 63%). tests on ASU. An increase in collagen synthesis was observed Lequesne's test score decreased from 9.7 to 6.8 in the ASU in a carrageenan induced granuloma in the "Hairless' rat group and from 9.4 to 8.9 in the placebo group. The overall following 15 days of percutaneously applied ASU (10). Mice success rate was 39% in the ASU group and 18% in the that had subcutaneous implantation of rat articular cartilage placebo group. Improvement was more marked in individuals wrapped in cotton were treated orally for 2 weeks with the with hip OA and a residual effect was observed at month eight unsaponifiable fraction of either ASU, avocado alone or soy (19). bean alone daily for 2 weeks (13 mg/kg avocado or 26 mg/kg 0012. One hundred sixty three individuals in this pilot 2 soya or both in a ratio of 1:2 at a dose of 39 mg/kg). Param year study evaluating structural changes in the hip joint. Indi eters measured included proteoglycans and hydroxyproline viduals with painful primary OA of hip and joint space>or to content of the cartilage. Results indicate that unsaponifiables 1 mm (Kellgren grade 1 to 3) with at least a 6 month history of both avocado and Soybean reduced the degeneration of of pain and AFI index>or to 4. Primary assessment was proteoglycans and hydroxyproline content of the implanted decrease in joint space width performed in standing position. cartilage that was induced by the granulomatissue. A greater Results indicate a failure to demonstrate significant reduction effect was seen using a combination of avocado and Soybean, in progression of joint space loss compared to placebo. ASU an effect which was dose dependent (11). did reduce progression of joint space loss (20). 0009. A third more pertinent study involved oral adminis tration of ASU (900 mg/weekday) to meniscectomized sheep Rationale for 3 and 6 months (12). In this model a “subtle but statisti cally significant protective effect on articular cartilage' was 0013 We believe that polyphenols and catechins, espe noted from computerized image analysis of histological cially from green tea, together with unsaponifiable lipids, stained cartilage. especially from ASU, can have beneficial effects on the bio 0010 Many plants contain similar type agents generally chemical processes that underlie development and/or pro classified as unsaponifiable lipids. Potent anti-inflammatory gression of DJD. In our invention, by “green tea” we mean activity has also been associated with these sources. For green tea available as a tea, as well as green tea available as an example, Park et al (13) found anti-inflammatory activity in extract, such as in a powderform. The anti-oxidant activity of an ethanolic extract of cactus which was identified as beta components of green tea have the capacity to attack the free sitosterol. Using the carrageenan paw oedema model in rats, radicals known to be involved in disregulated cytokine activ beta sitosterol was found to be one of the potent anti-inflam ity in osteoarthritis. The additional capacity to inhibit specific matory agents in extracts of Verbena officinalis (14). The proteases involved in cartilage degradation provides an addi isolated Sterols Stigmasterol and beta Sitosterol were potent tional mechanism for combating DJD. The anti-inflammatory anti-inflammatory agents when administered topically and in activity of avocado and soybean unsaponifiables can also a chronic inflammation model in the mouse (15). A sterol attack cytokine signaling, but apparently by less understood fraction containing 7.6% campesterol. 28.4% stigmasterol mechanisms. They also provide directanabolic activity that is and 61.1% beta sitosterol demonstrated anti-inflammatory needed to restore the balance between anabolic and catabolic activity in the carrageenan paw oedema model in mice after activity. A combination of unsaponifiable lipids with one or oral administration of 30 and 60 mg/kg (16). more of polyphenols or catechins should work well together. Thus the present invention consists of a combination of these In Vivo Tests in Humans agents over a range of doses. 0.011 Prior researchers have conducted in vivo tests in Dosage Ranges and Preferred Sources humans on ASU. Individuals with primary femorotibial or hip OA of at least six months duration were dosed with 300 mg of 0014 Polyphenols/catechins: about 3 mg to about 10 a 2:1 avocado:Soybean unsaponifiable preparation for 3 grams, with the preferred source of polyphenols being green months. Indices measured included NSAID intake, tea. The green tea may be combined with phosphatidylcho Lequesne's index and physician visual analog scale for pain line to improve absorption. US 2010/0285159 A1 Nov. 11, 2010

0015 Unsaponifiable lipids: about 5 mg to about 12 Production of Nitric Oxide in Human Chondrocytes. grams, with the preferred source being ASU. Arthritis & Rheumatism 45:2079-2086, 2002. 0027 6. Vankemmelbeke MN, Jones, GC, Fowles C, Ilic EXAMPLES MZ, Handley C J, Day A. J. Knight CG, Mort J S and 0016. The following examples are merely illustrative of Buttle, DJ. Selective inhibition of ADAMTS-1-4, and -5 the present invention and are not to be considered as limiting by Catechin Gallate Esters. European Journal Biochemis the invention, which is properly delineated in the following try 270: 2394-2403, 203. claims. Moreover, it should be noted that the use of the present or future tense in these examples is reflective of the References for ASU fact that the examples are prophetic. MPEP608.01(p). 0028. The following references regarding ASU are herein incorporated by reference in their entirety into this specifica Example 1 tion. 0017. A 70 kg 60 year old man has knee joint pain and (0029. 1. Ling W H, Jones P J H: Minireview. Dietary radiologically diagnosed degenerative joint disorder. Intake Phytosterols: A Review of Metabolism, Benefits and Side on a daily basis of a Supplement that contains 100 mg green Effects. Life Sciences 57: 195-206, 1995 tea extract and 300 mg avocado-Soybean unsaponifiables 0030) 2. DeJong A, PlatJ, Mensink RP: Metabolic Effects reduces these symptoms. of Plant Sterols and Stanols (Review). J Nutri Biochem 14: 362-369, 2003 Example 2 0031. 3. Bouic PJ: The Role of Phytosterols and Phy 0018. A 20 kg 6 year old Labrador retriever has degenera tosterolins in Immune Modulation: A Review of the Past 10 tive joint disorder localized to the hip and has difficulty rising Years. Curr Opin Clin Nutr Metab Care 4: 471-475, 2001 and ascending stairs. Intake on a daily basis of a Supplement 0032 4. Gupta MB, Nath R, Srivastava N. Shanker K, containing 20 mg green tea extract and 30 mg avocado-Soy Kishor K. Bhargava K P: Antiinflammatory and Anti bean unsaponifiables reduces these symptoms. pyretic Activities of Beta Sitosterol. Planta Med 39: 157 163, 1980 Example 3 0033 5. HenrotinYE, Labasse AH, Jaspar JM, DeGroote D D, Zheng S X, Guillou G. B. Reginster J Y. Effects of 0019. A 5 kg cat is diagnosed with spinal arthritis and Three Avocado/Soybean Unsaponifiable Mixtures on Met cannot jump on the windowsill. The cat is administered 3 mg alloproteinasses, Cytokines and PGE2 Production by of polyphenols and catechins and 5 mg avocado Soybean Human Articular Chondrocytes. Clin Rheumatol 17: unsoponifiables. The cat improves dramatically and is now 31-39, 1998 capable of jumping on the windowsill. 0034 6. Boumediene K, Felisaz, N, Bogdanowiez. P. Cal Example 4 era P. Guillou G. B. Pujol J P: Avocado/Soya Unsaponifi ables Enhance the Expression of Transforming Growth 0020. A 1000 kg horse is diagnosed with bone spavin and Factor Beta 1 and Beta 2 in Cultured Articular Chondro treated with 3 grams of epigallocatechin gallate and 4 grams cyctes. Arthritis Rheum 42: 148-156, 1999 of ASU. The horse responds dramatically. 0035 7. Kut-Lasserre C, Miller C C, Ejeil A. L. Gogly B, Dridi M, Piccardi N. Guillou B, Pellat B, and Godeau. References for Green Tea Effect of Avocado and Soybean Unsaponifiables on Gelati 0021. The following references regarding green tea are nase A (MMP-2), Stromelysin 1 (MMP-3) and Tissue herein incorporated by reference in their entirety into this Inhibitors of Matrix Metalloproteinase (TIMP-1 and specification. TIMP-2) Secretion by Human Fibroblasts in Culture. 3 0022. 1. Higdon J V and Frei B Tea Catechins and Periodontal 72: 1685-1694, 2001 Polyphenols: Health Effects, Metabolism, and Antioxidant 0036 8. Henrotin Y E. Sanchez C, Deberg M A. et al: Functions. Critical Reviews in Food Science and Nutrition Avocado/Soybean Unsaponifiables Increase Aggrecan 4389-143, 2003. Synthesis and Reduce Catabolic and Proinflammatory 0023 2. Homandberg GA and Wen FH Fibronectin Frag Mediator Production by Human Osteoarthritic Chondro ment Mediated Cartilage Chondrolysis. I. Suppression by cytes. 3 Rheumatol 30: 1825-1834, 2003 Anti-oxidants. Biochimica Biophysica Acta 0037 9. Mauvi el A, Loyau G. Pujol J P: Effect of Unsa 1317:134-142, 1996. ponifiable Extracts of Avocado and Soybean (Piascledine) 0024 3. Homandberg GA, Hui F, Wen C, Purple C, Bew on the Collagenolytic Action of Cultures of Human Rheu sey K, Koepp H, Huch K and Harris A. Fibronectin-Frag matoid Synoviocytes and Rabbit Articular Chondrocytes ment-Induced Cartilage Chondrolysis is Associated with Treated with Interleukin-1. Rev. Rhum Mal Osteoartic 58: Release of Catabolic Cytokines. Biochemical Journal 321: 241-245, 1991 751-757, 1997. 0038 10. Lamaud M. E. Miskulin M, Robert A M, Wepi 0025 4. Haqqi T, Anthony D D, Gupta S. Ahmad N. Lee M erre 3: Biochemical Modifications of Connective Tissue S. Kumar G K and Mukhtar H. Prevention of Collagen Induced by the Non-Saponifiables of Avocado and Soya induced Arthritis in Mice by a Polyphenolic Fraction From bean Oils Administered Percutaneously in the Hairless Green Tea. Proceedings National Academy Science USA Rat. Pathol Biol 26: 269-274, 1978 96:4524-4529, 1999. 0039 11. Khayyal M T. El-Ghazaly MA: The Possible 0026. 5. Singh R, Ahmed S, Islam N, Goldberg VM and “Chondroprotective” Effect of the Unsaponifiable Con Haqqi T M. Epigallocatechin-3-Gallate Inhibits Interleu stituents of Avocado and Soya. In Vivo. Drugs Exptl Clin kin-1B-Induced Expression of Nitric Oxide Synthase and Res 24: 41-50, 1998 US 2010/0285159 A1 Nov. 11, 2010

0040 12. Cake MA, Read R A, Guillou B. Ghosh P: 1. A method of treating or repairing connective tissue or Modification of Articular Cartilage and Subchondral Bone reducing inflammation in connective tissue of a human or Pathology in an Ovine Meniscectomy Model of Osteoar animal, comprising administering one or more polyphenols thritis by Avocado and Soya Unsaponifiables (ASU). or catechins and unsaponifiable lipids. Osteoarthritis & Cartilage 8: 404-411, 2000 2. The method of claim 1, wherein the unsaponifiable lipids 0041) 13. Park E. H. Kahng 31-1, Lee SH, Shin KH: An comprise avocado/soybean unsaponifiables. Antiinflammatory Principle from Cactus. Fitoterapia 72: 3. The method of claim 1, wherein the one or more 288-290, 2001 polyphenols or catechins are selected from the group consist 0042. 14. Deepak M, Honda SS: Antiinflammatory Activ ing of epicatechin (EC), epigallocatechin (EGC), ephicat ity and Chemical Composition of Extracts of Verbena Offi echin gallate (ECG), and epigallocatechin gallate (EGCG). cinalis. Phytother Res 14: 463-465, 2000 4. The method of claim 3, wherein the one or more 0043 15. Gomez MA, SaenzMT, Garcia MD, Fernandez polyphenols or catechins comprise epigallocatechin gallate MA: Study of the Topical Antiinflammatory Activity of (EGCG). Achillea Ageratum on Chronic and Acute Inflammation 5. The method of claim 1, wherein the one or more Models Z Naturforsch (C) 54.937-941, 1999. polyphenols and catechins are sourced from green tea or 0044 16. Navarro A. DeLasheras B. Villar A: Antiinflam green tea extract. matory and Immunomodulating Properties of a Sterol 6. The method of claim 1, further comprising administer Fraction from Sideritis Foetens Chem. Biol Pharm Bull ing phosphatidylcholine. 24:470-473, 2001 7. A method of regulating cytokine activity or cytokine 0045 17. Blotman F, Maheu E. Wulwik A, Caspard H. signaling in a human or animal, comprising administering Lopez A. Efficacy and Safety of Avocado/Soybean Unsa one or more polyphenols or catechins and unsaponifiable ponifiables in the Treatment of Symptomatic Osteoarthritis lipids. of the Knee and Hip. A Prospective. Multicenter. Three 8. The method of claim 7, wherein the unsaponifiable lipids Month Randomized, Double-Blind, Placebo-Controlled comprise avocado/soybean unsaponifiables. Trial. Rev. Rhum Engl Ed 64: 825-834, 1997 9. The method of claim 7, wherein the one or more 0046) 18. Appelboom T. Schuermans J. Verbruggen G. polyphenols or catechins are selected from the group consist Henrotin Y. Reginster JY: Symptoms Modifying Effect of ing of epicatechin (EC), epigallocatechin (EGC), ephicat Avocado/Soybean Unsaponifiables (ASU) in Knee echin gallate (ECG), and epigallocatechin gallate (EGCG). Osteoarthritis. Scand J Rheumatol 30: 242-247, 2001 10. The method of claim 7, wherein the one or more 0047. 19. Maheu E, Mazieres B, Valat J. P. Loyau G, polyphenols or catechins comprise epigallocatechin gallate LeLoet X, Bourgeois P. Grouin J M. Rozenberg S: Symp (EGCG). tomatic Efficacy of Avocado/Soybean Unsaponifiables in 11. The method of claim 7, wherein the one or more the Treatment of Osteoarthritis of the Knee and Hip. Arthri polyphenols and catechins are sourced from green tea or tis & Rheum 41:81-91, 1998 green tea extract. 0048, 20. Lequesne M, Maheu E. Cadet C, Dreiser R L: 12. The method of claim 7, further comprising administer Structural Effect of Avocado/Soybean Unsaponifiables on ing phosphatidylcholine. Joint Space Loss in Osteoarthritis of the Hip. Arthritis & Rheum 47: 50-58, 2002. ck ck ck ck ck