USOO6492429B1 (12) United States Patent (10) Patent No.: US 6,492,429 B1 Graus et al. (45) Date of Patent: Dec. 10, 2002

(54) COMPOSITION FOR THE TREATMENT OF 5,401.777 A 3/1995 Ammon et al. OSTEOARTHRITIS 5,494.668 A 2/1996 Patwardhan 5,629,351 A 5/1997 Taneja et al. (75) Inventors: Ivo Maria Franciscus Graus, Wg Ede 5,872,124. A * 2/1999 Koprowski et al...... 514/261 (NL); Hobbe Friso Smit, As Utrecht 5,888,514 A * 3/1999 Weisman ...... 424/195.1 (NL) FOREIGN PATENT DOCUMENTS (73) Assignee: N.V. Nutricia, Zoetermeer (NL) WO 95 22323 8/1995 WO 97 O7796 3/1997 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Balch et al. Prescription for Nutritional Healing; Avery Publishing 2nd Ed. pp. 138–144, Oct. 1997.* (21) Appl. No.: 09/662,123 Lafeber et al., “Apocynin, a plant-derived, cartilage-Saving drug, might be useful in the treatment of rheumatoid (22) Filed: Sep. 14, 2000 arthritis", Rheumatology, (1999), pp. 1088–1093, vol. 38, O O British Society for Rheumatology. Related U.S. Application Data * cited by examiner (63) Continuation of application No. 09/613,562, filed on Jul. 10, 2000. Primary Examiner-Christopher R. Tate 7 ASSistant Examiner Patricia A Patten (51) Int. Cl." ...... A01N 35/00; AO1N 65/00 (74) Attorney, Agent, or Firm-Browdy and Neimark, (52) U.S. Cl...... 514/688; 424/725 PL.L.C. (58) Field of Search ...... 424/195.1, 196.1, 424/523, 547; 514/54, 62, 365, 783, 688; (57) ABSTRACT 260/468, 294.8, 470 Osteoarthritis is treated by a composition containing both (56) References Cited apocynin and an inhibitor of inducible nitric oxide synthase Such as curcumin. Further components Such as boswellic U.S. PATENT DOCUMENTS acids, glucosamine, acetylcysteine and boron further

3,950,387 A * 4/1976 Joullie et al...... is enhance the beneficial effect of apocynin and curcumin. 4,315,028 A * 2/1982 Scheinberg ...... 424/290 5,120,538 A 6/1992 Oei ...... 424/195.1 8 Claims, No Drawings US 6,492.429 B1 1 2 COMPOSITION FOR THE TREATMENT OF (4-hydroxy-3-methoxy-acetophenone, acetovanillone) for OSTEOARTHRITIS the treatment of rheumatoid arthritis. WO95/22323 describes the use of analogues of apocynin, CROSS REFERENCE TO RELATED in particular the 4-hydroxyethoxy analogue, for controlling APPLICATION free radical generation by inflammatory cells in e.g. inflam The present application is a continuing application from matory bowel disease, rheumatoid arthritis and Several other copending application Ser. No. 09/613,562, filed Jul. 10, inflammatory conditions. 2OOO. U.S. Pat. No. 5,629,351 discloses a fraction of boswellic FIELD OF THE INVENTION acids isolated form BOSwellia Serrata, including a hitherto unknown 2-hydroxybos wellic acid, having anti The invention is concerned with compositions for the inflammatory, anti-arthritic and anti-ulcerogenic activities. treatment of Osteoarthritis and related diseases and with WO 97/07796 claims the use of boswellic acid for the methods for treating osteoarthritis. treatment of diseases, Such as lung emphysema, cystic BACKGROUND OF THE INVENTION fibrosis, rheumatoid arthritis etc., which are induced by 15 leucocytic elastase or plasmin activity. Osteoarthritis (OA) is a degenerative joint disease, which develops by wear and tear of the joints during aging. OA U.S. Pat. No. 5,401,777 relates to the use of curcumin for mostly affects the weight-bearing joints Such as Spine, knees treating conditions associated with formation of leucotrienes and hips, but thumb and finger joints may also be affected. from arachidonic acid, Such as inflammatory bowel diseases, Repetitive mechanical injury of the cartilage eventually chronic hepatitis, bronchial asthma and psoriasis. results in loss of cartilage and damage to joint Surfaces and U.S. Pat. No. 5,494,668 describes a product to be used for adjacent bone. As a result of the tissue destruction, inflam the treatment of OA and other musculoskeletal diseases, matory cells invade the joint and the Synovial membrane containing combined extracts from Withania SOmnifera which is manifested by pain, Swelling and Stiffness of the (Ashwaganda) roots, Boswellia Serrata (Sallaiguggul) gum affected joints. The repetitive mechanical injury leads to 25 exudate (boSwellic acids), Curcuma longa (turmeric) rhi pathological changes that result in loSS of proteoglycans and Zomes (curcumin) and Zingiber officinale (ginger). collagen from the cartilage matrix, which in turn leads to SUMMARY OF THE INVENTION Surface erosion and decreased loading capacity, In OA, chondro-cytes show a reduced potential to Synthesize matrix It was found that osteoarthritis can be effectively treated constituents (proteoglycans and collagen fibers) and the by administration of a Suitable amount of apocynin or its expression of proteolytic enzymes (called matrix metallo functional and structural analogues, preferably in the form of proteases, MNMPs) contributes to cartilage destruction and an extract from a Picrorhiza plant, and preferably together release of proteoglycan fragments in the Synovial fluid. The with further components enhancing the beneficial effect of inflammatory responses to the mechanical insults further the apocynin. Thus, the invention in a first aspect provides contribute to cartilage destruction. The inflammatory media 35 compositions for the treatment of Osteoarthritis, containing tor interleukin I (IL-1) is considered as the principal media an effective amount of apocynin or its analogues. tor of cartilage destruction. It induces a number of changes In a Second aspect, the invention provides methods for in chondrocytes, including the concurrent generation of treating Osteoarthritis comprising administering to a perSon Significant amounts of NO (nitrogen oxide) and Superoxide in need of Said treatment an effective amount of apocynin or radicals by inducible NO synthase (iNOS) and NADPH 40 its analogues. In a third aspect, the invention provides oxidase, respectively. NO reacts with Superoxide to form compositions comprising apocynin or analogues and meth peroxynitrite, which is largely responsible for the decrease ods for the treatment of arthritis in general as caused by in proteoglycan Synthesis induced by IL-1. The role of wear-and-tear processes, due to labour, ageing or obesity, for Superoxide was further demonstrated by the ability of Super the treatment of developed arthritis and for the-treatment of oxide dismutase to reverse the decrease in proteoglycan 45 the first Symptoms of joint disorders in patients not having Synthesis. The prevention of peroxynitrite formation via a familial history of rheumatism. Selective inhibition of iNOS and thus NO formation in vivo In a fourth aspect the invention provides compositions resulted in a marked decrease of MMPs. In addition, that are characterized by the Simultaneous presence of intra-articular treatment of OA patients with Superoxide apocynin or analogues and other components, in particular dismutase reduces Symptoms of OA for prolonged periods. 50 an inhibitor of inducible nitric oxide synthase and methods These Studies demonstrate that induction of catabolic for treatment of arthritis in general caused by wear-and-tear, enzymes (MMPs) and cartilage destruction is mediated via autoimmune responses in the joints, goute, or other causes of the formation of NO and Oradicals by chondrocytes or cartilage degradation. inflammatory cells. While the primary cause of OA is DETAILED DESCRIPTION OF THE mechanical damage, rheumatoid arthritis (RA) is mainly the 55 result of an autoimmune response that leads to chronic INVENTION inflammation in the joint. Contrary to OA, typical manifes According to the invention apocynin is a highly useful tations of RA are an increase of parameters that are associ component for treating osteoarthritis and related conditions. ated with inflammation, Such as haematocrite and white In particular, apocynin was found to be effective in retarding blood cell count and pannus formation or hyperplasia of the 60 progression of Osteoarthritis. It was found furthermore, that joint capsule that leads to deformed joints. This in turn leads the inflammatory processes in arthritis can be controlled and to morning Stiffness. Although the (primary) aetiology of RA the accompanying Symptoms of Stiffness and pain can be and OA are different, the pathological processes at a later alleviated by combined administration of an inhibitor of Stage result in Some manifestations that are similar Such as NADPH oxidase, such as apocynin, and an inhibitor of joint pain, cartilage degradation and general joint disability. 65 inducible nitric oxide synthase (iNOS), such as curcumin. Lafeber et al. (Rheumatology, 1999 (38) 1088–1093) Consequently, the composition of the invention contains describe in vitro Studies on the utility of apocynin at least 50 lug, preferably at least 100 tug, up to 1000 mg of US 6,492.429 B1 3 4 apocynin on the basis of daily intake. The preferred daily CO-, p-coumaroyl), preferably a 3,4-di-hydroxycinnamoyl intake is between 1 and 100 mg. Apocynin (acetovanillone) (caffeoyl) or 4-hydroxy-3-methoxycinnamoyl (feruloyl) is a known compound (4-hydroxy-3-methoxy group, and/or a dihydroxyphenyl group, preferably a dihy acetophenone); most preferred is a dosage of at least 15 droxybenzopyran group, were found to be useful in this mg/day. According to the invention, analogues of apocynin respect. Examples of Suitable phenolic compounds include may be used instead of or in addition to apocynin. Such curcuminoids, reSVeratrol, querce tin and other analogues are in particular those in which the 4-hydroxyl hydroxyflavones, catechins Such as epicatechin, catechin, group is etherified, especially with a hydroxylated alkyl gallocatechin, afzelechin, epigallocatechin gallate, epicat group, Such as 2-hydroxyethyl, 2,3-dihydroxypropyl or a echin gallate, compounds having activated phenolic groups Sugar moiety. The latter analogue in which the Sugar moiety as do occur in ginkgo biloba extracts, luteolin but also is B-D-glucose, is commonly known as androsin. This is the procyanidins, caffeic acid, chlorogenic acid, ferulic acid, usual form in which apocynin is present in fresh plants. carnosic acid and roSmarinic acid and their natural deriva Preferably apocynin and/or its analogues are present in an tives as occur in herb extracts appeared to be useful in this extract obtained from apocynin- or androsin-containing respect. The amount of iNOS inhibitor is preferably between plant or plant parts, Such as Picrorhiza Species, for example 15 200 lug and 2000 mg per day; most preferably between and P. kurrooa and P. Scrophulariiflora; the latter is also known 20 and 500 mg/day. as Neopicrorhiza Scrophulariiflora. Cucurbitacins are Especially Suitable are curcuminoids. These compounds known to be present in both Picrorhiza Species (Stuppner et are obtainable from plant parts, especially rhizomes of al., 1991, Wang et al., 1993). As some cucurbitacins are Zingiberaceae. A common prominent member of this family reported to induce immune responses and might potentially is Curduma longa. Curcumin (1,7-bis(4-hydroxy-3- lead to adverse health effects, a prepartion is preferably methoxyphenyl)hepta-1,6-dien-3,5-dione) is the most chosen in which cucurbitacins are largely absent, i.e. in prominent example of these active compounds. The recov which the cucurbitacin level is lower than 40%, preferably ery of curcumin from Curcuma rhizomes is well-known in lower than 20%, especially lower than 10% of the level of the art. It is especially preferred to use preparations that apocynin or its analogue. The total daily intake of cucurbi 25 comprise more than 85% curcumin, in particular more than tacins should preferably be below 10 mg, more preferably 92% curcumin in order to avoid undesired side effects. below 4 mg. Said combination of apocynin and iNOS inhibitors is The Picrorhiza extract can be obtained by extracting the advantageously combined with other components that can rhizomes (roots) of Picrorhiza species or by Subjecting an further enhance the effects Such as: extract to column chromatography. Alternatively, extracts boSWellic acids: these can be obtained by extracting with high amounts of phenolic compounds can be obtained Boswellia (Sabinsa) in particular Boswellia Serrata by by pretreating the plant material with mineral acid to convert applying methods known in the art. It is preferred to use glycosides to their respective aglycones (e.g. androsin to an acid fraction that comprises at least 15% and more apocynin). If desired, the material may then be defatted preferably more than 23% boswellic acids; between 10 Shortly to remove wax and other highly lipophilic matter. 35 and 1000 mg preferably 80-400 mg should be admin The material is extracted, for example with ethyl acetate istered per day; the boSwellic acids are usually assayed and/or ethanol. The organic Solvent is removed and an as 65% of the boswellia extract, based on acid titration; aqueous Solution is obtained. The pH of the extract is this level, however, corresponds to a true boSwellic acid increased to 10, e.g. with Sodium hydroxide, to deprotonate level of about 25%; phenolic compounds and to retain them in the aqueous 40 phase. The aqueous Solution is then washed e.g. with diethyl glucosamine can be administered as Sulfate or chloride ether to remove cucurbitacins. The aqueous phase is then Salt or other food-grade form in an amount between reacidified to neutralise phenolic compounds and again 750-2500 mg preferably between 1 and 2 g; extracted with e.g. diethyl ether. The organic phase is chondroitin can be administered in an amount of 0.6-2.4 collected and the solvent removed. 45 g, e.g. as Sulfate but preferably in a form that is better Apocynin may also be obtained from other Sources, e.g. available, from the rhizome of Canadian hemp (Apocymum between 10 and 500 mg gingerols, preferably isolated cannabinum) or other Apocynum species (e.g. A. from ginger oil, androsaemifolium) or from the rhizomes of Iris Species, extract from (Urtica dioica especially from the leaves, provided that the extracts do not contain Substantial amounts 50 N-acetyl cysteine in an amount per daily dose of 50 mg of cardiac glycosides. and 1 g, preferably 0.1-0.8 g., optionally as equivalent Although the inventors do not wish to be bound by a cysteine or thioproline; specific theory, it is believed that the inhibitory effect of boron 1-20 mg preferably 2-10 mg, e.g. as inorganic Salt, apocynin on NADPH oxidase restores the human system's 55 manganese 0.5–20 mg, preferably 1-10 mg and as inor potential to prevent cartilage destruction that is associated ganic Salt Such as MnO, with osteoarthritis. Vitamins, minerals and trace elements Such as Vitamin B5, This effect of apocynin was found to be enhanced by B6, C, D, E, folic acid, Zinc and Selenium. compounds that are capable of inhibiting inducible NO The products according to the invention can be Supple synthase (iNOS) and thus to reduce the formation of per 60 ments Such as tablets, pills, concentrates, powders, bars or oxynitrite. These combined effects are believed to be impor cereals Sauces, emulsions, liquids, or other dietetic prepa tant in an effective control of the inflammation and its rations that can comprise the active components. The prepa consequences in OA and OA-related diseases. Thus, the rations are meant to be used for oral administration. composition according to invention preferably also contains EXAMPLE 1. a component that inhibits iNOS. Phenolic compounds, in 65 particular those which contain at least a The following components were mixed and tabletted to p-hydroxycinnamoyl group (HO-CH-CH=CH the following amounts per tablet: US 6,492.429 B1 6 EXAMPLE 3 The following active components were mixed into daily Component amount unit dosages: active ingredients Glucosamine sulfate (potassium) 331,6 mg Boswellia serrata extract (65% boswellic acids) 102,6 mg Component amount unit Picrorhiza kurrooa extract (10% apocynin) 33,3 mg Turmeric root extract (95% curcumin) 8,8 mg Glucosamine sulfate (potassium) 1500 mg N-Acetyl cysteine 99% 84.2 mg Chondroitin sulfate 1200 mg Boron citrate 5% 1O mg Picrorhiza kurrooa extract (10% apocynin) 20 mg D-C-Tocopherol succinate 1162 IU/g (vit. E) 15 mg Ginger oil 500 mg Ascorbic acid 95% (vit. C) 3,6 mg Ginkgo biloba extract 400 mg Pyridoxin.HCl, powder (vit. B6) 130 fig Pine bark extract 400 mg Vitamin D-3500 IU/mg 50 ug Green tea extract 400 mg Calcium D-pantothenate, USP 660 ug 15 Folic acid, USP, FCC 280 ug Zinc oxide, USP 10 mg We claim: Selenium yeast 0.2% Se OB 2.2 mg Manganese sulfate monohydrate 11 mg 1. A composition for treating Osteoarthritis comprising an excipients effective amount of apocynin or an analogue thereof effec povidone 28 mg tive for the treatment of osteoarthritis, from 200 lug to 2000 microcrystalline cellulose granulate 2OO mg mg of an inhibitor of inducible nitric oxide Synthase per dicalcium phosphate dihydrate 194 mg daily dose, and 10 mg to 1 g of at least one boSwellic acid croscarmellose Na, NF 55 mg per daily dose. silicon dioxide 6 mg stearic acid vegetable source 27 mg 2. The composition according to claim 1 wherein the vegetable stearate 9 mg 25 effective amount of apocynin or its analogues is between 1 white film coat solin 185 mg mg and 100 mg per daily dose. isopropyl alcohol USP 60 mg aqueos film coat base clear 79 mg 3. The composition according to claim 2 wherein the corn starch, bleached 18 mg weight ratio of apocynin or its analogue to curcubitacin in pharm. glazes DA45A, 4LB 16 mg the extract is at least 5. 4. The composition according to claim 1 wherein the Six tablets were administered per day for effective treat apocynin is part of an extract of a plant species of Picorhiza ment of arthritic conditions. or Neopicrohiza. 5. The composition according to claim 1 wherein the EXAMPLE 2 inhibitor of inducible nitric oxide Synthase comprises a 35 phenolic compound containing at least one member Selected The following active components were mixed into daily from the group consisting of 4-hydroxycinnamoyl groups dosages: and dihydroxyphenyl groups. 6. The composition according to claim 5 wherein the 40 inhibitor of inducible nitric oxide Synthase comprises cur Component amount unit cumin or an analogue thereof. 7. The composition according to claim 6 wherein Said Glucosamine sulfate (potassium) 15OO mg Chondroitin sulfate 1200 mg curcumin is part of an extract of a plant species of Curcuma. Picrorhiza kurrooa extract (10% apocynin) 2O mg 8. The composition according to claim 1 wherein Said Rosemary extract 250 mg 45 bOSwellic acids are part of an extract of a plant species of Resveratrol (grape skin extract) 500 ug Boswellia. Urtica dioica extract 750 mg