AU2006202129B2 - Nutraceuticals for the treatment, protection and restoration of connective tissues (D2) - Google Patents

Description

AUSTRALIA Patents Act 1990 INSTITUTE OF NUTRACEUTICAL RESEARCH PTY LTD COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Nutraceuticals for the treatment, protection and restoration of connective tissues The following statement is a full description of this invention including the best method of performing it known to us:- NTACBUTIALS FOR THE TBATm.POBCIN N B-EORATION OF ONCIBISB 5 The invention relates to a method for Isolating from connective tissue a variety of glycosaminoglycan (GAG)-peptide complete and polypeptides which am substantially free of contaminntng DNA and other molecules such as vUses which may be associated with the DNA in the cell. The invention also relates to uses of GAG-peptide complexes and polypeptides substantially free of DNA either directly, or 10 afer further processing, for the treatment, protection and restoration of connective tissues in inflammatory and degenerative disorders such as rheumatoid arthritis and osteoarthritis in any of their multiple forms or other degenerative conditions in mamans without potential contamination with viruses and other pathogens which are known to be localized within cells and bound to some of their intracellular components. 15 The invention is also directed to the use of a mixture of the Calcium salt of a GAG-peptide complexes) and polypeptides, preferably substantially free of DNA, which has been found to enhance connective tissue cell anabolism and macromolecular biosynthesis as well as exhibit immuno-suppressive and anti-inflammnatory activities in an animal model. By improving connective tissue cell proliferation, matrix gene 20 expression and inflammation by the above mentioned preparations, even in the presence proihflammatory mediators, such as Interleuki-1 which are known to suppress cellular biosynthesis of matrix components and promote their degradation, these naturally derived GAG-peptide complexes and polypeptides can slow or halt the progression of tissue destruction and inflammation which are characteristic 25 pathological features of arthritis and related degenerative diseases. Background Art Musculoskeletal disorders such as rheumatoid arthritis (RA), osteoafthritis (OA), disc degeneration (DD) and osteoporosis (OP) are the major cause of morbidity throughout the world. These diseases have a substantial influence on health and quality 30 of life and inflict an enormous cost on health systems (Scott JC, Hochberg MC. Arthritic and other musculoskeletal diseases, In: Chronic Disease Epidemiology, Brownson RC, Remington PL and Davis JL, eds. Washington, DC. American Public Realth Association, 1993). It has been estimated that musculoskeletal diseases cost the Australian community $1.3 billion annually in direct costs and $4.2 billion in indirect 35 cost. This represents 1.3% of GNP (Artiitis Foundation of Australia, Access Economics Pty Ltd report, 2001). Population based studies conducted in other 2 deveopd axmtflea show similar incidence and health burdens to &a of An&ia with Or eraple mom than 23 miln Aecan se medical at for afrthic, disorders with as with their managementt Meeding 70 biWon dollars animally (IRnohljn HS, Elkin RB, Paget SA, MAcssam 8 oW etcia 5 analyses in rheumaoid atitis ad oeoar W, Arthritis C Res 1997;10:41341) Moreover, the pralence of OA, DD and OP is rising p W y as the lfe span of the Peoples of developed nations increases. Indeed it has bn eimaed tt 20% of the POPUlation in developed counties wil be acvely seeking media treatments or Professional health support fbr 'musculoskeleta disabilities byte year 2020 (Croft p. 10 The ocuence of osteartt outside Bumpe. Ann Rheum Dis 1996;55:661-4). The aetiology of musIokee Conditions such as OA is multifactorial and alhough ageing is the mos tongly associated risk factor mechanical, hormonal and genetic factors al contribute to vOryig degrees- OA emerges as a clinical syndrome when these etiological determinants rowt in ficient joint damage to cause 15 impairment of rade0b01 and t appearace of symptoms. This clinical syndrome is m1nanifest radiology by joint space narrowing due to loss of articular ftgag (AC) and extensIve re-modelling of subchondral bone with pm-ifeati-t at the joint mA)ins (ostePhYtosis). Is the late sages of OA. joints are charaoterised pathologically by 20ensive AC fibrillation; loss of staining for proteoglycans (PGs) and eburnation of 20 bone at Sites of high contact stress. The subchondral bone beneath those region$ of OA where cartilage is fibrillated or eroded is generally sclerotic and conssts of immature woven bone (Felson DT. Osteoartbritis. Rheum Dis Clin Nth Amer 1990;16:499,512) The PGs of AC consist of a protein core to which several hundred GA- chains are covalently attached. The major GAG substituents of the P~s of AC are the 25 Chondroitin sulfates (CbS) whose isomeric structures and their distibuon are te known (Poole AR. Changes in Collagen and proteogyc of artheiar cartilage i, arthritis, Rheumtology, 1986, 10; 316-371). Thus in adult cartilage Ch.6-S is more abundant than the confesponding 4-sulfated isomer (Ch-4-S) which predominates in AC of verny Young animals. Within 1 the Oxbuacllular matrix of AC the hydraed PO 30compexes arm entrapped in the form of nmaro04o0ewlar AW0reat by a three dimensional aeitwork of Type th cllagen frea. S u unique structural orga satd of PC*, waer and a fibrous collagen network which is anchored in the subohondral bone Plate confers to AC the biomechanical properties of resilience necessary for normal biomechai function (Poole A 9 Canges in colgen and proteognycan of cular 35 Cartlage in arthritis. Rheumatology 1986, 10;-316-371).

S Products of cartilage breaklown in OA and RA joints have been Wn to be antigenic[Glaut TT, PMJOp C, C-Szab6 G, Buzas B Ragasa D, Mikcz . Mapping of artbrgM ptp swof cartilage Inggrensn protogycannd arthtis. Sand I Eheinmtal 1995;24:43-9, Rowley M Tait B, Mackay f 5 Cuningam T, Phillips B. CoUa antbodie in rhumatold arthritis: Significant of antibodies to denatured colagen and their association wih HLA-DR4. Arts Rheum 198629:174-84, Seibel M Jelsma R, Saed-Nejad V, Radliffe A. Variability in the immunochemical quanocation of keratan sulfate in 1Hman and bovine cartilap proteoglyoan, Biochem Soc Trans 1990;18(5):969-70] and when released into 10 synovial fluid (SF) may provoke a synovial inflammation. TIS ynoyitis, once established, can alter the metabolism of resident synoviocytes, the major cellular source of synovlal hyaluronan (HA) in joints. Inflammatory mediators released from local macrophage and infiltrating leukooytes can also promote increased vascular permeability and the dilution of SF by plasma fluid, thereby decreasing local HA 15 concentration (Mller..Ladner U. Gay RFu Gay S. Strucure and fiction of synoviocytes. .In Arthritis and Allied Conditions, Koopran W, ed. Baltimore. Williams and Wilkins, 1997, 243). This dilution of HA coupled with a reduction in its molecular size due to abnormal synthesis by synoviocytes results in a substaa decrease in the rheological properties of SF and consequently its ability to lubricate and 20 protect AC (Balazs EA. The physical properties of synovial fluid and the special role of hyaluronic acid. In: Disorders of the Knee, Helfet AJ, ed. Philadelphia. JP Lippincott, 1982, 61-74). Macrophage of the synovium, together with the leukocytes which enter the synovial cavity due to the local inflammation 7 are also an abundant source of cytokines (eg interleukin-1 (IL-i)], procoagulant factors, proteinases and oxygen 25 derived free radicals including nitric oxide medical (NO) (Pelletier Y-P, DiBattista A, Roughley P, McCollum R, Martel-Pelletier I Cytokines and inflammation in cartilage degradation. Rheumn Dis Clin N Am 1993;19:545-68, Dean DD. Proteinase-mediated cartilage degradation in osteoarthritis. Sam Arthritis Rheum 1991;20:2-11). While much of the excess proteolytic activity released into synovial fluid is abrogated by the 30 cndogenous inhibitors present, cytokines and free radicals can freely diffuse into cartilage and down-regulate PG and collagen synthesis by chondrocytes. These Pronflamrnatoiry mediators can also initiate the Production of catabolic proteinases, cytokines and f&e radicals such as NO - by the cartilage cells which via autocrine and paracrine pathways contribute to further AC matrix destruction (Evans CB, Watldns 35 SC, Stefanovic-Raio M. Nitric oxide and cartilage metabolism. Methods Enzymol 1996;269:75-88), 4 it is Cle" fium the abova tat In atbritic disease ouch as OA all tiues of the itam acted their ecessive breakdown and the conoinmitan elictton of an inflammtory reaction can lead not only to the progression of the disease state but also the initiation of symptoms tho most common being pain and impairment of joint 5 fimction. Phamacological management of rheumaic disorders and back pain of disoal origin, has up until quite recently, targeted the symptoms of these diseases rather tan the undedying pathologies which are the cause of the symptoms. Analgesics, steroidal and non-steroidal anti-inflammatory drugs (NAJDs) have over the last 50 years, 10 reprsented the mainstay of pharmacological treatment for the tieunatlo dismses. However, the deleterious side effects associated with the use of many of these synthetic drugs (Lichtenstein DR, Syngal S, Wolfe MM. Nonstemijdal antiinflammatoxy drugs and the gastrointestinal tract. The double-edged sword. Arthritis Rheum 1995;38:5-18, Davies MN, Wallace JL. Nonsteuidal anti-inflammatory drug-induced gastrointestinal 15 toxicity. New insights into an old problem. I Gastroenterol 1997;32:127-33, Manoukian AV, Carson JL. Nonstemridal anti-inflammatory drug-induced hepatio disorders. Jacidence and prevention. Drug Safbty 1996;15:64-71, Huskisson EC, Berry H, Gisen P, Jubb RW, Whitehead Y. Effects of antlinflammtoxy drugs on the progression of osteoarthritis of the knee. J Rheumatol 1995;22:1941-6) has prompted the evaluation 20 and development of alternative treatments, particularly remedies fi-om edible plant and animal sources which, by their very nature, are expected to be free of adverse side effects. The most widely used products in this regard are glucosamine and chondroitin sulphate (ChS) which are both constituents of cartilage PGs. Although it should be 25 noted that the glucosamine used commercially is generally isolated from the chitosan present in the exoskeleton of crustacea. Controlled clinical studies conducted with glucosamine and chondroltin sulfate, alone or in combination, have indicated that they can provide relief of symptoms In OA (McAliudon TB, LaValley MP, Gulin JP, Felson DT, Glucosanine and Chondroitin for the treatment of Osteoarthritis: a systematic 30 quality assessment arid meta-analysis. JAMA, 2000; 263: 1469-1475). These agents have been categorised as slow acting disease modifying anti-osteoatiaitis nutraceuticals, We are also aware of a number of patent disclosures describing the use of these agents alone and in combination with various other medicants for the treatment of OA and other musculoskeletal disorders (US Patent5,364,845, Nov 15 1994; US 35 Patent 6,136,795, Oct 24 2000; US Patent 6,162,787, December 19, 2000, US Patent 5 6,271,213, Aug 7, 2001, US Patent 6,432,929, August 13 2002 and nef es cited Since the ChSs a obtained from natural somrs ey can be sold diretly to the public as food addves or supplements and ae not presently require to comply with 5 h norus quaty control criteda used for syntheticalHy pharmaceuticals as required by government agencies such as the FDA. Commerdally available chondritin sle a nonnaly Man tuWed fom bovine tissues such as lumg and trachea by hydrolysis of the GAG protein com linkage of the cadla PCs using either chemical or enzymatic procedures (US Patent , 1,950,100 Mar 1932, 10 AustrUlian Patents AU-A1-66307/80 Jan 1981, AU-A-70540187 Deo 1987, US Patent 6,162,787, IDecember 19, 2000 and references cited therein). The negatively charged water soluble ChS may be separated and puified from the proteins and peptides also generated by the hydrolysis of cartilage by multiple precipitations with acetone aliphatic alcohols or the formation of water insoluble complexes with quaternary 15 ammonium salts such as cetyl pyridinium chloride (CPC) (US Patent, 1,950,100 Mar 1932, Australian Patents At-Al-66307/80 Jan 1981, ATJ-A-70540187 Dec 1987). However, none of these methods readily remove the contaminating nucleio acids (DNA and RNA) and other intracellular components also released during the chemical or enzymatic disruption of cartilage since these macromolecules are also anionic and 20 would co-precipitate with the anionically charged ChS. Contaminating nucleic acids could be selectively removed from the ChS by digestion with enzymes which degade these contaminating molecules (e& ribonucleases such as Benzonase (Mercke)), however this is an expensive procedure and the enzymes used would still have to be removed from the ChS preparation at some 25 stage. Choatropic solvents such as guanidine hydrochloride and salt solutions of high ionic strength, such as potassium chloride have also been previously used to extract native proteoglycans frotn cartilaginous tissues but at low temperatures (below 4 degrees C) and with the addition of protease inhibitors to prevent degrdation of the required macromolecues by endogenous enzymes (Hascall VC and Sjdera SW, 30 Pmtein-polysacclaride complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. J.Biological Chem 1969,244;2384-2396; Oegema T, Hascall V, Dziewiatkowski D, Isolation and characterisation of proteoglycns from the rat chondrosarcoma. J. Biol. Chem. 1975, 250: 6151-6159; Inerot S and Heinegard D, Bovine tracheal cartilage proteoglycans. Variations in 35 structure and composition with age. Collagen and Related Research, 1983, 3: 245 262). However, in order to release the GAG-peptides or ChS chains from these so C isolated proteoglycan complexes it is necessary to subject them to proteolytio digestion by the addition of exogenous enzymes such as papain to degade the he. ptin core( US Patent 6,162,787, December 19, 2000, Inerot S and Hainegard D, Bovine tracheal cartilage proteoglycans. Variations in structure and composition with age. Colagen 5 and Related Research, 1983, 3: 245-262, and rebrences cited therein ). Furthermore, use of these high ionic strength or chatropic conditions to tract cartilage also disrupts cell membranes and thus release intra-cellular componens, such as nucleic acids into the aqueous medium along with the PCs. Uncharacterised GAG-peptides have also been prepared from bovine nasal 10 cartilage but not bovine tracheal cartilage using sodium acetate or water at pH 4.5 (Nakano T, Nakano 'K, Sim IS, Extraction of glycosnminglycan peptide fom bovine nasal cartilage with 0.1 M sodium acetate, J Agriculture and Food Chemisty, 1998, 46; 772-778, Nakano T, Iawa N, Ozimek L, An economical method for the estraction of chondroitin sulfate-peptide from bovine nasal cartilage. Can Agric Engineering, 2000, 15 42; 205 -208). However, neither the presence or absence of DNA in these GAG-peptide preparations nor their respective pharmacological activities nor direct use for the treatment of musculoskeletal conditions described could be infected from the information recorded in these publications. Again, in the Nakano et at disclosures subsequent proteolytic or chemical hydrolysis of the products isolated by this method 20 of extraction was necessary to obtain the CMS ultimately required by the authors. In this regard it is important to note that most of the proteolytic enzymes used to exhaustively digest connective tissues to manufacture products of commercial interest, such as the ChSs are derived from bacterial or plant sources because of their broad range of substrate specificity and widespread availability. The amino acid sequences 25 which are recognised and cleaved by these enzymes, as well as the amino acid sequences of the polypeptide fragments generated by their proteolytic actions, are therefore different to the sites of cleavage and polypeptide sequences produced by the endogenous proteinases of mammalian connective tissues. For example, studies with the mammalian class of cysteine proteinases, the Cathepsins, have shown that their 30 preferred substrate binding and catalytic cleavage sites are different from that of the plant derived cysteine proteinase, papain (Barrett Aj, Buttle DJ, Mason Rw, Lysosomal cysteine proteinases, ISI Atlas of Science, 1988: 256 - 260). In addition, while digestion of purified preparations of PGs with papain released single ChS chains with about 10 atnino acid stubs still glycosidically attached the corresponding digestions of 35 cartilage PGs with the cathepsins, D or B or 0 produced clusters containing 2 or more ChS chains and longer amino acid stubs with amino acid sequences different to those 7 geneated by the papain digested PGs (Roughley PI atxiBarrett AJ, The degradation of cartilage proteoglycans by tissue prtens, Biohe J,1977,167: 629-637). Certain patent disclosures cite methods of preparation and use of hydrolysates of cartilage for the treatment of musculoskeletal disorders and joint cartilage defects but 8 these inventions am limited to the use of the peptides produced from type I and type H collagens for such treatments and make no refren to pharmacological activities of any GAG-peptide complexes when used alone or in combination with these collagen derived polypeptides. Furthermore none of these previous disclosums recognises the absence or presence of intra-cellular contaminants such as nucleic acids in their 10 preparations (US Patent 3.966,908, June 29, 1976, US Patent 4,804,745 Feb 1989, US Patent 5,399.347 Mar 1995, US Patent 5,364,845 Dec 1996, US Patent 6,025,327 Feb 2000, US Patent 6,372,794 April 2002). While the consequences of long term human consumption of bovine or other animl nucleic acids in commercial ChS preparations sold as nutraceuticals or food 15 supplements is presently unknown, it should be noted that these ntraellular anionic macromolecules are strongly bound to or frm- complexes with retroviruses and heat/protease resistant prion proteins which have been implicated in the spread of transmissible spongiform encephalopathies such as Creutzfeld-Jakob disease, kunu, Gerstmann-Straussler-Scheiner syndrome in humans, scrapie in sheep and goats, and 20 bovine spongioform encaphalopathies In cattle. (Welssmann C et A Transmission of prions, www.pnas.orglcgi/doi/1073/pnas. 172403799; Narang H, A critical review of the nature of the spongiform encephalopathy agent: protein theory versus virus theory, Exp Biol. Med, 2002,227: 4-19; C, et al, The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein, J. Mol. Biol., 2001, 307: 1011 25 1021; Nandi PK and Sizaret P-Y, Murine recombinent prion protein induces ordered aggregation of nucleic acids to condenced globular structures, Arch Virol, 2001, 146: 327-45; Cominicini S, et a, Genomic organisation, comparative .analysis and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND), Mamm Genome 2001, 9: 729-33, ). These intracelhdar entities could therefore be consumed by the 30 subject in appreciable amounts when they comply with the manufactures recommended dosage of one or more grams of ChS daily fbr the suppression of the symptoms arising from osteoartlritis and related conditions. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a 35 context for the present invention. It is not to be tak as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the 8 field Mleant to the present invention as it exdsted in Australia before the priority date of each claim of this application. DISCLOSURE OF NETO 5 In a first aspect the invention provides a method for the preparation of a connective tissue-derived GAG-peptide complex and polypeptide said method comprising the steps ofe subjecting particles of connective tissue to enzyme mediated autolysis by contacting with an autolysis medium containing a monovalent or divalent salt at an 10 effective pH and temperature sach that at least one GAG-peptide complex and at least one polypeptide are released substantially free of DNA, by the proteolytic actions of an endogenous enzyme(s) frum within the tissue particles into the autolysis medium, leaving residual tissue particles; and recovering the GAG-peptido complex(es) and polypeptide(s) from the autolysis medium. 15 Connective tissues are rich .In collagens, non-collagenous proteins and proteoglycans. Connective tissue proteoglycans are macromolecular complexes consisting of chains made of sulfated polysaccharides (about 95%) covalently linked to a protein core (about 5%). Glycosaminoglycan (GAG) refers to the polysaccharide chains of proteoglycans, 20 which are composed of repeating disaccharide units containing a derivative of an amino sugar (either glucosamine or galactosamine) glycosidically linked to glucuronic or iduronic acid. The most common derivatives being O-sulfated esters substituted in the 4 or 6 'positions of the N-acetylated glucosamine or galactosamine rings. Examples of GAGs include hyaluronio acid (hyaluronan) (which is non 25 sulfated), chondroitin sulfate, keratan sulfate and heparan sulfate. Cleavage of a proteoglycan structure produces complexes comprising one or more polyaccharide chains attached to a polypeptide fragment derived from the~ proteoglycan protein core. These complexes are referred to herein as "GAG-peptide complexes". 30 The term "polypeptide" includes peptides comprising two or more amino acids, but typically having 10 or more amino acids. Polypeptides can typically be derived by chemical or enzymatic cleavage of proteins, wherein proteins include glycoproteins and non-glycosylated proteins. The term polypeptides includes matrix derived potypeptides which may be obtained from cleavage of non-glycosylated or gicosylated proteins. 35 The properties of a polypeptide are determined by the type and sequence of its constituent amino acids.

9 The pferred monovalent salt is selected from hydrogen, sodium, potassium, ammonium and mixtures theof while the divalent salt is preferably selected from Caium, magnim, copper, zino and mixtures thereof The more preferred salts a calcium and magnesium. SThe pH may be in the range of from 2.5 to 8.5, preferably 3.5 to 8.0, more preferably 4 to 7 and most preferably 4.5 to 7. The temperature may be in the range of fom 20 to 45'C, preferably 2 5 t 4 5 C, more preferably 32 to 45*C most preferably 3TC. . The invention also provides GAG-peptide complexes and polypeptides 10 substantially free of DNA prepared by the method described above. The invention still further provides monovalent or divalent salts of OAG-peptide complexes substantially fee of DNA and polypeptides substantially free ofDNA. The connective tissue suitable for the method of the present invention includes skin, bone, tendon, ligament and cartilage. The source of connective tissue may be 15 from a wide variety of species including bovine, ovine, porcine, equine, avian, cervine and piscine species. Cartilage is preferred and may be hyaline, elastic or fibro type cartilage. Preferred animal species and anatomical locations of cartilage are bovine, vine, porcine, cervine or equine, tracheal , articular, auricular or nasal cartilage, chicken sternal or rib cartilage, shark skleetal cartilage and growing deer -antler 20 cartilage. Tissues from young animals are preferable to those obtained frommore mature, which in the case of the bovine species Is greater than 3 years, for example. The connective tissue may be treated and washed as required by methods known in the art to remove any adhering soft tissues and reduced to the required particle size by means including, but cot limited to, mincing, dicing, grinding and the like. 25 Appropriate particle size may be selected on the basis of commercial convenience. Preferred particle sizes are in the order of up to 5mm, preferably 1-3mm. A person skilled in the art will appreciate that the rate of autolysis may vary with many factors including pH, temperature, concentration, tissue type, tissue particle size and time of incubation. In a preferred embodiment, cartilage particles of size 1 30 3mm are subject-to autolysis in an aqueous medium at a pH of 4-5 and temperature of 32-42'C for up to 36 hours, preferably 16-24 hours. The person skilled in the art will also appreciate that there are a number of methods for assaying for DNA content and that sample to sample variations, independent of method used, can be expected. The present inventors have used a DNA 35 fluorescence dye binding assay and a direct spectroscopic method to estimate DNA levels in these preparations. The spectroscopic method Is that normally utilized by 10 Investigators to quantitate the levels of DNA in water soluble preparations which have a low protein Content, while the fluorescence dye binding assay is employed to determine the amounts ofDNA-in samples which may contain proteins and other tissue derived molecules (Davis LG, Kuehl WM, Battey 3F, BasiO methods in Molecular S Biology, 2nd Edition, Appleton and Lange, Norfolk, Connecticut, 1994). The term "substantially free of DNA" is to be understood In the context of the present invention to indicate a substantial reduction of DNA relative to the known DNA content of raw matedal (tissue particles prior to autolysis) or to commercial chondroitin sulfate preparations manufactured by alternative methods to those 10 described here. For example, the ratio of suited glycosaminoglycans (S-GAG) to DNA in three commercially available chondroitin sulfate preparations was found by the present inventors to avenge about 300:1 (Table 3). By contrast, GAG-peptides and Polypeptides released into an autolysis medium in accordance with the present invention provide ratios of S-GAG to DNA in the autolysis medium of greater than 15 1000 to 1 and the DNA being undetectable by the DNA Hoechst 33258 fluorescence dye binding assay or spectroscopically- A person skilled in the art would apprciate that ratigs of greater than 1000:1 would fall within the scope of the term "substantially free of DNA". By contrast , the ratio of S-GAG to DNA in commercially available ChSA for human consumption with ratios around 300:1 ablee 3) are considered by 20 the inventors not to be substantially free of DNA. In the context of the present invention, DNA content is used as a marker for intra-cellular macromolecular components. For example, the substantial absence of DNA Indicates a substantial absence of intra-cellular components in general such as nucleio acids and any associated molecules. 25 Methods Of recovering the GAG-peptide complexes and polypeptides from the antolysis media are well known in the art. For example, the residual tissue particles are removed by filtration from the autolysis media and the GAG-peptide complexes and polypeptides isolated from the media. Suitable isolation methods include neutralisation of the supernatant followed by: 30 freeze drying; or precipitation with acetone or aliphatic alcohols; or by the fbrmation of water insoluble complexes with quaternary ammonium salts such as cetyl, pyridinium, chloride: or by separation using size exclusion or ion-exochange or other forms of column 35 chromatography or membrane filtration technology.

11 GAG-pep&d complexes and polypeptddes may be further purified and/or separated using conventional procedure. ChSs as their univalent ( eg H, Na, K, ammonium) or divalent (eg Ca, M& ZN Cu ) salts obtained by mild chemical or eymatic hydrolysis of the GAG-peptide complexes prepared by the methods de~iied herein are also substantially free of DNA, the ratios of S-GAG to DNA being greater than 1000: 1. Collagen peptides may be isolated by methods known in the art (US Patent 4804,745 Peb 1989, US Patent 5,399.347 Mar 1995i US Patent 5,364,845 Dec 1996, US Patent 6,025,327 Feb 2000). In a second aspect the present invention provides a Chondroltin sulfate salt 10 substantially fre of DNA, derived ftom a GAG peptide complex, the GAG peptide complex being obtainable by the method described above. The present invention also provides a monovalent or divalent salt of ohondroitin sulfate substantially free of DNA wherein the monovalent sAt is selected from the group consisting of hydrogen, sodium, potassium, ammonium and mixtures thereof 5 and the divalent salt is selected from the group consisting of calcium, magnesium, copper, zinc and mixtures thereof GAG-peptide complexes substantially free of DNA, chondroitin sulfate salts substantially free of DNA and mixtures of GAG-peptide complexes with polypeptides substantially free of DNA may be used for the treatment, protection and restoration of 20 connective tissues in ina mmatory and degenerative tissue disorders and as anti angiogenic agents for the treatment of cancers or antilipideamiproftbrinolytic agents for improving blood flow in dyafhnction blood vessels. In this regard the inventors were surprised to discover that the pharmacological activities exhibited by the GAG-peptide complexes substantially free of DNA and 25 GAG-peptide complex/polypeptide mixtures substantialy free of DNA were superior to the corresponding activities of the commercially available ChS when their pharmacological -activities were compared on an equivalent weight basis. Accordingly, in a third aspect, the present invention provides use of at least one GAG-peptide complex substantially free of DNA, and/or chondroltin sulfate salt 30 substantially free of DNA; at least one GAG-peptide complex substantially free of DNA and at least one polypeptide substantially free of DNA, for the manufacture of a medicament for the treatment of inflammatory and degenerative tissue disorders such as rheumatoid arthritis and osteoarthritis in any of their multiple fonn, atherosoerosis and myoardial ischemia, degenerative and 3 - diabetic arteriopate, thrombosis and embolisms, hyperlipideamis and for the treatment of cancers, 12 7he present invention also provides a method for the treatment of n mmtr and degenerative tissue disorders such as rhematoid arthritis and osteoarthritis in any of thei multiple forms, atherosclerosis and myocardial ischemia, degenerative and diabetic arteiopathies, thrombosis and embolisms, hyperlipideamis and for the 5 treatment of cancers composing the administration to a subject in need of such tenantnt an effective amount of at least one GAG-peptide complex substantially free of DNA; and/or chondroitin sulfate salt substantially free of DNA; at least one GAG. peptide complex substantially free of DNA and at least one polypeptide substantially free ofDNA. S no chondroitin sulftes may be present as univalent (preferably Na, K, anMonium) or divalent (preferably Ca, Mg, Zn, Cu, more prefrably Ca, Mg) sat. In a fourth aspect the present invention provides a pharmaceutical composition comprising at least one GAG-peptide complex substantially free of DNA; and/or chondroitin sufte salt substantially free of ]DNA; at least one GAG-peptide complex 15 substantially free of DNA and at least one polypeptide substantially free of DNA, together with a pharmaceutically acceptable carrier. The chondroitin sulfates may be present as univalent (preferably Na, K, ammonium) or divalent (preferably Ca, Mg, Zn, .Cu, more preferably Ca, Mg salts). The pharmaceutical compositions may include excipients known -to be used in combination with chondroitin sulfate such as 20 glucosamaine. In a prefered aspect the present invention provides use of a calcium salt of at least one GAG-peptide complex substantially free of DNA and/or a calcium salt of chondroitin sulfate substantially free of DNA for the manufacture of a medicament for the treatment of inflammatory and degenerative tissue disorders such as rheumatoid 25 arthritis and osteoarthritis in any of their multiple fbrms, atherosclerosis and myocardial ischemia, degenerative and diabetic arteriopathies, thrombosis and embolisms, hyperlipideamis and for the treatment of cancers. The present invention also provides a method of treatment of inflammatoy and degenerative tissue disorders such as rheumatoid arthritis and osteoartbritis in any of so their multiple tbrms, atherosclerosis and myocardial ischemia, degenerative and diabetic arteriopathies, thrombosis and embolisms, hyperlipideamis and for the treatment of cancers comprising the administration to a subject in need of such treatment an effective amount of a calcium salt of at least one GAG-peptide complex substantial free of DNA and/or a calcium salt of chondroitin sulfate substantialy fee 35 of DNA.

13 The.inventors have found that the ueof a mixte a calcium salt of a GAG peptide complex and a calcium salt of a polypeptide generated by the method described above enhances connective tissue cell anabolism and cell biosynthesis of macromolecular components such as PGs and HA, CVee when the cells are exposed to 5 suppressive proinflammatory cytokines and also together exhibit immunosuppressive and anti-inflmmatory activities when administered to a* animal model of arthritis. Accordingly , in a fifth aspect the present invention provides use of a calcium salt of at least one GAG-peptide complex and a calcium salt of at least one polypeptide substantially free of DNA for the manfcture of a medicament for the treatment of 10 inflammatory and degenerative tissue disorders such as rheumatoid arthritis and osteoarthritis in any of their multiple forms, atherosclerosis and myocardIal ischemia, degenerative and diabetic arteriopathies, thrombosis and embolisms, hyperlipideamis and for the treatment of cancers. The present invention also provides a method of treatment of inflammatory and i5 degenerative tissue disorders such as rheumatoid arthritis and osteoartritis in any of their multiple forms, atherosclerosis and myocardial ischemia, degenerative and diabetic arteriopaties, thrombosis and embolisms, hyperlipideamis and for the treatment of cancers comprising the administration to a subject in need of such treatment a calcium salt of at least one GAG-peptide complex and a calcium salt of at 20 least one polypeptide substantially free of DNA. Preferably the calcium salt of the GAG-peptide complex is substantially free of DNA. The calcium salts of the GAG peptide complexes and polypeptides may be co-isolated from connective tissue, preferably tracheal cartilage, more preferably bovine tracheal cartilage by the method described above. 25 In a sixth aspect, the present invention provides a pharmaceutical composition comprising a calcium salt of at least one GAG-peptide complex and a calcium salt of at least one polypeptide substantially free of DNA together with a pharmaceutically acceptable carrier. Preferably the calcium salt of the GAG-peptide complex is substantially free of DNA. 30 In a seventh aspect, the present invention provides use of at least one polypeptide substantially free of DNA fbr the manufacture of a medicament for the treatment of inflammatory and degenerative tissue disorders such as rheumatoid arthritis and osteoarthritis in any of their multiple forms. The present invention also provides a method of treatment of inflammatory and 35 degenerative tissue disorders such as rheumatoid arthritis and osteoarthritis in any of 14 their multiple forms comprising the admisetrution to a subject in need of such treatment an effective amount of at lest one polypeptide substantially free of DNA. In an eighth aspect the present invention provides a pharmaceutical composition comprising at least one polypeptide substantially free of DNA together with a 5pharmaeuic1y acceptable carrier. The method of the first aspect of the present invention is essentially nondisruptive to the connective tissue used. Thus the residual tissue particles removed from the autolysis medium after release of the GAG-peptide complexes and polypeptides into the medium can be used as a source of collagen derivatives. In this 10 regard it should be noted that traditional methods for preparing ChS from connective tissues such as cartilage use exhaustive proteolytic or chemical digestion to destroy the proteinaceous components of the matrix to allow the ChS to be released into aqueous solution and isolated. The residual tissue particles obtained by the present method can be hydrolysed proteolytically or chemically and subsequently purified to obtain 15 collagen peptides of variable molecular size to be used for the treatment, protection and restoration of connective tissues in inflammatory and degenerative disorders such as rheumatoid arthritis and osteoarthdtis in any of their multiple forms or for the enhancement of wound healing or for the preparation of artificial biomatrixes for cell culture, cell transplantation, or delivery of bioactive COmpounds including drugs and 20 growth factors into a host tissue. In a ninth aspect, the present invention provides a method according to the first aspect of the invention further including the step of hydrolysing the residual tissue particles to obtain collagen peptides. 25 above. The invention also provides collagen peptides prepared by the method described Throughout this specification, unless the context requires otherwise, the word "Comprise" or variatioUs such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step,.or group of 30 elements, integers or steps. In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and drawings.

15 FIGURB 1 xiaeties of Glycosamiycan-peptide (GAG-peptide) Released from 1 mm and 3 mm S Bovine Trachea Cartilage Powders with 100mMwith autolysis bufer, 1O0uM cum acetate, pH 4.5 at 37 degrees C. FIGUR2 Photcrogim hs of histological sections of 3mm bovine tracheal cartilage powders 10 particles before and after subjecting them to the autolysis procedure using 100mM calcium acetate buffer, pH 4,5 at 37 degrees C. Panels A and C are sections of cartilage before antolytic processing and B and ID after. Panels A and B show the results of staining with Toluidine Blue (TB), a dye which binds to glycosaminoglycans (GAGs) while C and D show sections stained with Masson Trichrome (Masson TC), a 15 dye known to stain native collagen fibres magenta colour. Note the loss of staining for GAGS in section 3 after autolysis but a slightly increased intensity of staining for collagen following the removal of the majority of GAGs as shown in D. Cell nuclei, identified in these sections by the green counter-stains are clearly unchanged by the antolytio process. All sections are shown at magnification X 400. 20 FIGURE 3 Kinetics of release of glycosatinoglycans-peptides (GAGa) from bovine tracheal cartilage in the presence of 100mM calcium acetate at 37 degrees C at various pHs. 25 IGURB4 Kinetics of release of glycosaminoglycans-peptides (GAGs) from bovine tracheal cartilage in the presence of 100mM calcium acetate at pH 4.5 b at various temperatures. 30 FIGURE 5 Release of glycosaminoglycan-peptides (GAGs) kom bovine tracheal cartilage in 100mM calcium acetate buffer at pH 4,5 C at 37 degrees C in the absence and presence of proteolytic inhibitors. The cysteine protease inhibitor, N-Ethylmalelmide was the most eftbctive inhibitor reducing release by approximately 50% that of the control 35 incubations which codained no inhibbors.

16 FIGUE 6 Superdex-200 Gel Filtration of Calcium Peptacan (CaP) showing s"fated GAG (As35) and protein(Asa profiles uing assays described in the tex. 5 FIGURE Superdex-200 GeW Filtration profiles of Calcium Peptacan (CaP) and Papain Digested CaP showing decrease in molecular size to that of chondroitin sulfate. FIGURE 8 10 Superdex-200 Gel Filtration chromatogras of Peptacans (CaP and 20P) and ChSAs (Sigma and Bioiberica). TGURE 9 Standard Curve of distribution coefficient (Kav) versus molecular mass of ChS 15 standards determined on Sephadex-G 200 using the Method of Melrose and Ghosh (1993) as described inthe text. FIGURE 10 Composite Agarose Polyacrylamide Gel (CAPAGE) Analysis of chondroitin sulfate 20 (ChS) and Calcium Peptacans (CaP). Lane 1: ChS (Sigma), lane 2: CaP, lane 3: purified CaP, lane 4: papain digested Cap. FIGURE 11 Analysis of proteins and peptides in Peptacans Using SDS-Polyacrylamide gel 25 electrophoresis. Lane 1= protein markers with molecular weights shown, lane 2= CaP, lane 3 = purified CaP, lane 4= chondroitiaseABC digested and purified CaP. FIGURE 12 Determination of DNA contamination in ChSA and Peptacans from their UV Spectra. go The presence of DNA is indicated by absorption at A 26 and protein at A no. FIGURE 13 Binding and release of Chondroitin Sulfate A (ChSA) and Calcium Peptacan (Ca?) from immobilized lysozyme, hyaluronadase, and bun granulocyte elastase using tire 35 BIAcore2000 technique. The extent of binding affnity of drgs to immobilized enzymes was recorded as resonance units (RU).

17 FIGURE 14 Short-Term (2 days) Conctron-Dependent Effects of Chondrotin Sulfte A (ChSA) and Calcium Peptwan (CaP) oa the corporation of 3 H-methyl Thymidine s into DNA of Ovine Chondwcytes in Monolayer Cultures. FIGURE 15 Loig-Tern (7 days) Concenttion-Dependent Effects of Chondroitin Sulfhte (ChSA) and Calcium Peptaoan (CaP) on the Incorporation of 'H-methyl Thymidine into DNA 10 of Ovine Articular Chondrocytes in Monolayer Cultures. FIGURE 16 Concentlation-Depende Effects of Chondroitin Sulfate A (ChSA) and Calcium leptacan (CaP) OnProteogycan (PG) Synthesis by Ovine Chondrocytes. .15 FIGURE 17 Effects Of Chondroitin Sulfate A (ChSA) and Calcium Peptacan (CaP) on Proteoglycan (PG) Synthesis by Ovine Chondrocytes in the Absence and Presence of Interleukin 1beta (IL-ibeta). 20 .FIGURE 18 Concentration Effects of ChSA and Peptacans on 3S Incorporation into PGs of the Extracellular Matrix of Fibrochondrocytes isolated from the Inner Region of Ovine Menisci. 25 FIGURE 19 Concentration Effects of ChSA and Peptacans on 35 S-PGs released into condition medium from the Extracellular Matrix of Fibrochondrocytes isolated from the inner Region of Ovine Menisci. 30 FIGURE 20 Relative Effects of ChSA and Peptacans (2.0 mg/I) on 35 S-incorporatlon into PDs of .. the Extracellular Matrix of Fibrochondrocytes of isolated from the inner Region of Ovine Menisci. 35.

18 FIGURB 21 Relative Efft of ChSA and Peptacans (2.0 mg/ml) on 3S-PGs released into conditioned mediu from the Extraceflular Matrix of Fibrochondrocytes isolated from 6 the inner Region of Ovine Menisci FIGURE 22 Concentration Effects of ChSA and Peptacans on "S-incorporation into PGs of the xtracellular matrix of Pibrochondrocytes isolated from the outer Region of Ovine 10 Menisci. FIGURE 23 Concentration Effects of ChSA and Peptacans on "S-PGs Released into Conditioned Media from Fibrochondrocytes isolated from the Outer Region of Ovine Menisci. 15 FIGURE 24 Relative Effects of ChSA and Peptacans (2.0 mg/mil) on US-incorporation into PGs of the extracellular matrix of Fibrochondmoytes isolated from the outer Region of Ovine Menisci. 20 IGURB 25 Relative Effects of ChSA and Peptacans (2.0 mg/mli) on 3S-PGs Released into Conditioned Media f-om Fibrochondrocytes isolated from the outer Region of Ovine Menisci. 25 FIGURE 26 Concentration Effects of Chondroitin Sulfate A (ChSA) and Calcium Peptacan (CaP) on ]DNA Synthesis by cultures of Human Synovial Fibroblasts (HSP) from Joints of Osteoarthritic Patients. Concentrations of CaP between 62 and 250 micrograms/mL 30 produced significant stimulation of synthesis (p <0.05). FIGU 27 Effects of ChSA, CaP and H2OP (2.0 mg/ml) on HA Synthesis by Human Synovial Fibroblasts (HSF) as shown by the radioactivity profiles obtained on Superose 6 Gel 35 Filtration chromatography.

19 MGUB 28 Relative Effects of Chondroitin Sulfate A (ChSA) and Peptacans (2.0'mg/ml) on Hyaluronan (HA) Synthesis by Human Synovial Fibroblasts (HSF) Derived from Joints 6 of Osteoarthritie Patients. FIGURE 29 Relative effects of Chondroitin Sulfate A (ChSA) and Peptacans and highly purifed Peptacans (2.0 mgfml) on Hyaluronan (HA) Synthesis by Human Synovial Fibroblasts 10 (HSP) Derived from Joints of Osteoarthritic Patients. Note the diminished activity of pCaP relative to Ca. FIGURE 30 Superose-6 Gel Filtration chromatograms of media removed from cultures of synovial 15 fibroblasts obtained f-omjoints of arthritic rabbits of non-drug treated controls (broken lines) and 4 animals treated daily with 300mg/kg of CaP for 14 days(solid lines) showing increased synthesis of radioactively labeled HA. FIGURE 31 20 Mean (tSEM) group effects of ChS and CaP treatments on the ex-vivo synthesis, of radioactively labelled HA by synovial fibroblasts from rabbit joints. Only cells from the CaP treated groups at 300mg/kg showed a significant increase in synthesis relative to the non-drug treated control group (p< 0.05). 25 FIGURE 32 Photomicrograph of histological sections of patella cartilage (AC) and adjacent synovium from joints of the rabbit PC arthritis model showing the extensive synovial iaflaumation and pannus formation accompanied by antibody staining for inducible nitric oxide synthase (iNOS) in chondrocytes. Panels A and B; sections from saline 30 injiected joints but processed and stained with the INOS primary and secondary antibodies as described in the text. Note the strong non-specifio brown staining of synovial tissue but not chondrocytes (which appear blue f-om the counter-stain) or the cartilage matrix in these control sections. A-100X, B-400X magnification. Panels C and D are sections from PC injected non-drug treated joints. C-100X, D=400X 85 magnfication. Intense staining for INOS in ohondrocytes is evident in these sections, Panel E is a section f-om PC injected joint of a ChS treated animal magnification X400, 20 Staining for expression of iNOS by chondrocytes is less than in the nondrg treated control but is still apparent Panel F is a rePresentative section fmm a PC injected joint of a CaP treated animal (300mg/kg) where staining for INOS in chondrocytes is reduced relative to control and ChS treated groups. Magnification X400. 5 FIGURE 33 Photomicrograph of histological sections of patella cartilage and adjacent synovium from joints of the rabbit PC arthritis model showing the extensive synovial Infammation and pannus formation accompanied by brown antibody staining In 10 chondrocytes for the nitric oxide sytbase oxydation product, nfmtyrosine. The section shown in Panel A is from PC injected non-drug treated rabbit joints process and stained with the primary and secondary bodies for nitrotyrosine as described in the text. Note as in FIGUB 32 the strong brown non-specific staining of synovial tissue but specific shining for the antigen in chondrocytes, particularly in the cartilage superi~ial zone. 15 Magnification X400. Panel B shows a representative section ftom a PC injected joint of a ChS treated animal where staining is reduced in intensity but not absent. Magnification X400. Panel C is a section kom a PC injected joint Of an animal given CaP ( 3 00mg/kg). Magnification X200. Staining for nitrotyrosine in chondrocytes and in the superficial cartilage zone was slight and less than in the non-drug treated control. 20 Panel C is a negative control section from a PC injected joint which confirms the non specific binding of the nitrotyrosine antibody to synovial tissues but not cartilage or chondrocytes. FIGURE 34 25 Histogratos showing the mean values (+ SEM) for neutrophils in the blood of animals from the ChS and CaP treated rabbit groups. Note the suppression of neutrophil levels in blood from the high dose CaP treated group. FIGU RE35 30 Histograms showing the mean values (±SEM) for mononuclear cells in the blood of animals fom the ChS and Ca? treated rabbit groups. Note the reduction of monouolear cells levels in the 300 and 2 0 0ng/kg Cap treated groups.

21 F C OUT TE In order to avoid repetition and improve the clarity of this description of the 5 products isolated from cartilages by the methods of the invention they have been abbreviated and given the collective title of PEPTACANS (P). The different Peptacans isolated by the methods herin are identified by the addition of prefixes, Thus: Calcium Peptacan (CaP) - is the aqueous soluble product obtained by subjecting bovine tracheal cartilage powder to autolysis at 370C with 0.1 M calcium acetate at pH I0 4.5. Sodiuro Peptacan (NaP) - is the aqueous soluble product obtained by subjecting bovine tracheal cartilage powder to autolysis at 37)C with 0.1 M sodium acetate at pH 4.5. Water Peptacan (20P) - is the aqueous soluble product obtained by subjecting 15 bovine tracheal cartilage powder to autolysis at 37 0 C with dilute acetic acid pH at 4.5. dCaP - is CaP dialysed against H20 dNaP -is NaP dialysed against HO dB20P -is H1 2 0P dialysed against f20 pCaP -is CaP purified by gel filtration or ion exchange chromatography 20 pNaP - is NaP purified by gel filtration or ion exchange chromatography pH2OP - is H2OP purified by gel filtration or ion exchange chromatography Experimental Protocols 25 Bovine, ovine, cervine or porcine tracheal cartilage or nasal cartilage, chicken sternal cartilage, or skeletal shark cartilage or deer antler cartilage were freed of adhering soft tissues mechanically or as described previously US Patent 5,399.347 Mar 1995, US Patent 5,364,845 Dec 1996, US Patent 6,025,327 Feb 2000). These cleaned hyaline cartilages were rinsed with water, minced into 1 mm or 3 tum sizes, freeze 30 dried and stored at -20 *C. Bovine tracheal chondroitin sulfte A (ChSA) was Purchased from Sigma Chemical Co, USA or was obtained as a gift from Biolberica, Barcelona, Spain (batch 1/0015, batch 05/2001, batch 18/11/99). All other chemicals were of analytical grade and were purchased from local suppliers.

22 WOWs of Glcsaioaya Pentde GGetdlCpee andO- 3 Iolyentde Rom the CAtiAMnm31_a Studies on the kinetics of release of the GAG-peptides and polypeptides from the cartilage powders using the different buffers (eg sodium or calcium acetate or dilute 5 acetic acid to give the vadous Peptacan products listed above in abbreviations was undertaken under a variety of conditions. The objective of these experiments was to determine the effects of 0) particle size - 3mn5mm, (i) different pHs eg. pH range 3.5-7.0, (tit) different teperatures, 4*C, 25*C and 37 0 C, and (Qv) animal species and tissue locations on the rate of autolysis and product release Iuto the aqueous phase. All 10 the experiments were performed, with stirring and release of sulphated GAGs and polypeptides monitored over 24 hours. Studies on the kinetics of release of the GAG-. peptide complexes from the cartilages showed that more than 80% of the GAG content could be mobilised into the aqueous medium after 24 hours. Studies also showed that the rate of release was dependent on the cartilage particle size, the'smaller preparations 15 undergoing more rapid release. However, by 24 hours the yields obtained were the same. The pH and temperature were found to be important determinants of the rate of release which indicated that the release process was mediated by endogenous enzymes present within the- solid tissues. This proposed mechanism was confirmed by undertakiug autolysis experiments In the absence and presence of specific enzyme 20 inhibitors. Since it was found that the addition of N-ethylmaleimide produced the most significant inhibition of GAG-peptide and polypeptides release into the aqueous medium we consider that the cysteine class of proteinases, such as the Cathepsins, were the major, but not exclusive, contributors to the autolytic process. In all experiments the aqueous phase was separated from the cartilage powders 26 by filtration and-the filtrate centrifuged to remove fine particles and thou neutralized to pH 7.0 by addition of an alkaline solution containing the desired cation. These Peptacan solutions after chemical analysis were either feeze dried and used directly for pharmacological studies or purified and/or converted to chondroitin suathes. The freeze dried Peptacans were also used as stock material for the preparation of dialysed 30 and fractionated preparations as described below. Alternatively the Peptacans could be isolated from the aqueous solutions obtained from the cartilage digests by precipitation with excess quantities of acetone, ethanol or methanol, usually by adding 3-5X the volume of the aqueous extracts. The precipitates so obtained would be washed with absolute ethanol and dried under vacuum then stored in a vacuum dessicator. 35 23 Bistoloaical examination of bovine tracheal cartilage samies before and ater calcium Minced 3 mm cartilage samples before and after treatment with 0.dM calcium acetate, pH 4.5 at 37 0 C for 24 hours were fixed in 10% (v/v) neutral bufered formalin 5 for 48 hours. Specimens were then washed, dehydrated in increasing alcohol concentrations (70 - 100% v/v), and double-embedded in methyl beaoate / celloidin then paraffin wax. Sections (4um) were out on a rotary microtome and adhered to Superfrost Plus (Menzel Glaser, USA) glass slides. Histochemical staining with Toluldhie blue (TB), a dye which binds to sulfated GAGs and Masson trichrom(C), a 10 dye that binds to native collagen, wer perfonned in batches under controlled conditions as described previously ( Hwa S-Y, Burkbadt ), Uttle C, Ghosh P, The effects of Diacerein on cartilage and subchondral bone in an ovine model of osteoarthritis, J Rheumatology, 2001, 28: 825-834). Briefly, sections were de paraffnised, and equilibrated in 70% (v/v) alcohol for 15 minutes, then stained in 15 0.04% (w/v) Dye / 0.IM sodium acetate bufler (pH 4.0) for 10 minutes. They were then counter-stained in 0.1% (w/v) fast green for 2 minutes, dehydrated in isopropanol followed by xylene and cover-slips applied. Purification f Glycosaninoglycan Penide Samoles by Gel Filtration 20 The freeze-dried Peptacans were dissolved in F6O to afford concentrations of 4.0 mg/mil of clear solutions. 1.0 ml of above Peptacan solutions was injected into a pre-equilibrated HiLoad 16/60 Superdex 200 column and eluted with 0,5 M NaCl at the flow rate of 1.0 ml/min. Fractions (2.0 mi) were collected in 5-ml plastic tubes. GAG peptide and protein content of each fraction was -determined using sulphated 25 glycosaminoglycan (S-GAG) (Farundale RW, Buttle DS and Barrett AL Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta: 883, 173-177) and BCA assay (Smith PK, Krobn R, Hermanson GT, Mallia AY, Gartner FM, Provenzano MD, Fujimoto EK, Goeke NM, Olson BY and Kienk DC. Anal. Biochem. 150, 76 - 85, 30 1985) respectively. The fractions which were S-GAG-positive were pooled and freeze dried. These were designated as pCaP or pNaP or pHOP. 8ialfted Glyosaminoglycan (8-GAG) DMMB AgAY The total S-GAG content of Peptacans was determined by reaction using the 35 metachronmtic dye 1,9-dimethylmethylene blue (DMMB) (Furadale RW, Buttle DJ and Barrett Al. Improved quantitation and discrimination of sulfated glycosaminoglycans 24 by Use of dimethylmiethylene blue. Biochim Biophys. Acta 883, 173-177). A standard cuve was prepared using a commerially available chondmitin sulfte A (ChSA) derived from bovine tracheal cartilage (ICN, USA) in 96-wa microtitre plates. ChSA standard and Peptacan samples were diluted in 0.2% sodium formate before DMMB S reagnt was added and the absorbence at 535 nm read immediately. Somax software was used to construct a standard curve and calculate the concentration of S-GAG Ia Peptacans. Analysis Of l Using igh Perfrmance Liqnid Chromatomauhv(HPIO 10 The ratio of lhondroitin4-sufte (Ch4S) and chondroitin-6sulfat (Ch6S) isomers in Peptacss were determined -using high performance liquid chmrmatography (HPLC) basically as described by Lee and Tieckelman (Lee GJL and Tieckelman H. The application of high perform ,ace liquid chromatography in enzymatic assays of chondroitin sulphate isomers in normal urine. I Chomatography. 222- 23-31, 1981). 15 Freeze dried CaP was dissolved in H0 and then digested by incubation with 0.125 units of chondroitinase ABC (SKK, Tokyo japan) at 37 0 C overnight. The unsaturated disaccharides were separated in a high performance amino column using 0.2 M ammonium acetate, pH 5.5 as mobile phase and photometrically detected at 232 nm. Peaks were identified by comparison with those of chondroitin sulphate A (Sigma 20 Chemical Co, USA). The area of each peak was measured using NM Image 1.61.1 software. Dcolagen or colleen etide content in preparations by assay for hydroxprline prt 25 The collagen content of cartilage powder, freeze dried Peptacans or cartilage residue after extraction was estinated by measuring the concentration of the amino acid hydroxyproline which is unique to this protein. Each freeze dried Peptacan sample was directly dissolved in HO (10 mg/ml) and then hydrolysed in 5 N HCl at 110 *Cfor 24 h. The unprocessed cartilage powders or residues were papain digested for 24 h first -0 and then centrifuged and the supernatant collected, which was then subjected to S1 HC hydrolysis as described above. The hydrolysed sample solution was neutralised to pH 7 before dilution and analysis. The hydroxyproline concentration in these solutions was determined using the method of Stegman and Stalder (Stegman H and Stalder K. Determination of Hydrmxyproline, CliU. Chim. Acta 18; 267-273, 1967) by using a L 35 hydroxyproline standard and measuring the absorbance at 562 m after the addition of chloramine T and p-dimethyaminobenzaldehyde to develop the chromophore. The 25 hydroxyproline concentration was multiplied by 7.4 to give an estimate of the collagen content. Detemination of Protein content of preparations by the icinchoninic Add (BCA) 5 asrsY The total protein content of cartilage powder, freeze dried Peptacans or cartilage residue after extraction was determined using BCA assay (Smith PK, Krohn RI, Hermanson GT, Mallia AX, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ and Kienk DC. Anal. Biochem. 150, 76 - 85, 1985). Cartilage powder and 10 cartilage residue were papain-digested for 16 h and centrifuged to provide clear supernatants. Each freeze dried Peptacan was directly dissolved in H0 to provide a 2.0 mg/ml solution. 20 pl of each sample solution was added to a well of 96-well plates. Just prior to assay, 50 parts of reagent 1 (0.4% NaOH; 1,7% Na2COs; 0.95% NaHCO 3 ; 1.0% bicinchoninic acid; 0,16% Na4artrate) was mixed with reagent 2 (4% 15 CnSO 4 .5H 2 0). 200 pl of this working reagent was added to the sample solution. After incubation at 37 "C for 60 min the absorbance Am 2 was read using a Thermonax microplate reader. Bovine serum albumin (BSA) or highly purified gelatine (Sigma Chemical Co) at 0 -10 gg/well were used to construct a standard curve. 20 Composite Agarose Polyacrylamide Gel Electrophoresis (CAPAGB) Each of ChSA and Peptacan samples (CaP, pCaP or papain digested CaP) were dissolved in H 2 0 at the concentrations of 1.0 - 3.0 mg/ml and then mixed 1:1 with CAPAGE sample loading buffer (20 mM Tris-acetate, pH6.3, 1 mM Na 2

SO

4 , 60%/o sucrose and 0.01% bromophenol blue). 20 pl of each sample equivalent to 10 tg of 25 GAG was loaded into wells of 2 mm thick CAPAGB gel (0.6% agarose, 1.2% acrylamide, 10 mM Tdis-acetate pH 6.3 and 0.25 mM sodium sulfate) and electrophoresed in the CAPAGE running buffer (10 mM Tris-acetate pH 6.3,' 0.25 mM Na 2

SO

4 ) at 150 V for 2 h. The gel was stained in a solution of 0.02% toluidine blue in 0.1 M acetic acid for 1 h, destained in 0.5 M acetic acid for 2 h and dried on an agarose 30 gel-bound film. The dried gel was rinsed with H2O and dried again. Determination of Aversze Molecular Size of Peptacans Using Gel Eiltration A HiLoad Superdex-200 prep grade prepacked column (16 mm x 60 mm) and 35 previously characterised ChS standards were used to determine the molecular size of the various Peptacans and their digested products. The column was equilibrated with 26 0.25 M NaCi for at. least 3 h prior to loading samples. Sephadex-G200 chromatographed bovine tracheal chondroitin sulfate fractions (CSI - CS7) pmpared previously were used as molecular size standards (Meirose I and Ghosh P, Determination of the average molecular size of glycosaminoglycans by fast protein 5 liquid chromatography. J Chromatography, 1993, 637; 91-95). 1.0 ml of standard (0.5 mg/ml) or Peptacans (CaP, %OP and pCaP) (1.0 mg/nil) were loaded into cbhuns and chromatographed at a flow rate of 1 mil/min using 0.25 MNaCl solution. Fractions (1.0 ml) were collected in 5-ml plastic tubes. GAG content of each fraction was determined with S-GAG assay. A standard curve was constructed using molecular mass versus 10 distribution coefficient (Kay) of CS1-CS7. Analysis of Prefeins in Peptacans by SDS-PAGB Blectrophoresis ChSA (Sigma) and CaP were dissolved in 140 and then mixed 1:1 with 2 x sample loading buffer (0.07 M TrisHC, 1.5% SDS, 20% glycerol, 0.2MDTT and 0.1% 15 )3PB) to achieve the final concentrations of4.0 -20 mg/m. The samples were boiled in a water bath for 5 min. 20 pl of above samples were loaded into the wells of 8 - 16 % pre-cast Tris-glycine gel (Norvex). SeeBlue pro-stained low molecular weight range protein markers (Norvex) were loaded into wells on the left-hand side of the gel and electrophoresis was performed at 125 V for 2 h. The gel was stained in Coomassic blue 20 R250 solution (40% ethanol, 10% acetic acid and 0.2% Coomassie R250) for 30 min and destained in a solution containing 10% ethanol and 7.5% acetic acid for 16 h. The gel was dried in a Bio-Rad Gelair drier. Determination of DNA in Peptacans and Commercial Chondroitin Sulfates Using UV 25 Spectroscopy Chondroitin sulfates (Bioiberica or Sigma) and Peptacans (CaP, Nap and H2OP) were prepared at 1.0 mg/ml in 140. 100 pl of each sample solution were loaded into a micro-cuvette and scanned spectrophotometrically over the wavelength range of 220 320 nm. The absorbence eurves, A, 60 and Ano were recorded for each sample in 30 triplicate. The absorption of light by concentrated aqueous solutions of these preparations at the wavelength of 260nM in their UV spectra provides a measure of the levels of DNA present; whereas absorption by these solutions at 280nM is taken as a measure of protein content. The ratio of the A m and A 2sg values is commonly used as an index of DNA purity, with ratios of more than 1.5 being considered to indicate 35 preparations high In DNA.

27 Determination of DNA in Peptavans and Commercial Chondroitin Sulfates Usin the Hoedhst 33258 dye binding assay Chondroitin sulfates (Bioiberica or Sigma) and Peptacans (CaP, NaP and U2OP) were prepared at 1.0.mg/ml in H20 and the DNA content was determined in duplicate 5 by using a fluorometric assay in which the Hoechst 33258 dye on binding to DNA shows a change in fluorescence (Kim YJ, Sah RLY, Doong J-YH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem. 1988;174:168-176). For the cartilage preparations prior digestion with papain was employed as described by Kim et a (1988) in the in the above publication. Briefly, 10 DNA was determine by adding 100 pl/well of Hoechst 33258 dye solution to each well of a 96-well plate followed by adding 100 pi of CbS or Peptacan or papain digested samples. The plate was gently agitated for 5 min before measuring the fluorescence using excitation and emission wavelengths of 350 nm and 450 nm respectively and slit widths of 10 nm and 15 nm. Calf thymus DNA (Sigma Chemical CoXO-25 ptg/ml) 15 with a uv spectral ratio of A26o to A 2 so of 1.85 was used to construct a standard curve and the DNA content of the preparations determined relative to this standard. DNA results were expressed as a % of the dry weight of the samples. Determination of Binding Affiuity of CaP and ChSA to Lysozye. Elastase and 20 llyaluronidase Using BlAcore 2000 The molecular interactions between CaP or ChSA and lysozyme, elastase or hyaluronidase were investigated using a surface plasmon resonance (SPR) biosensor device - BlAcore 2000 system (Pharmacia Blosensor AJB). Lysozyme (chicken egg white, CalBiochem), elastase (human neutrophil, ICN) or hyaluronidase (bovine testes 25 type IV, Sigma) was dissolved in 100 ig/l in 10 mM sodium acetate, pH 6.0 and immobilized on a CM5 sensor-chip by using amine coupling procedure. CaP or ChSA was firstly dissolved in H20 and then diluted in standard BBS running buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) to 2.5 Ig/ml. The binding of CaP or ChSA to immobilized enzymes was determined in four flow 30 cells separately including one of the flow cells (minus ligand) used to record background sensorgrams. 200 pl of CaP or ChSA solution were injected individually on to the immobilized chip surface and the molecular interactions were monitored at the flow rate of 50 pg/ml at 25 "C. The sensorgrams were recorded and evaluated using the BIAevcaluation 3.1 software provided with the system. 35 28 Inhibition ofHuman Neutrophil Elastase (M ) by eptacans and Clandroitin Sulfate A Stock solutions of chondroitin sulfate A (ChSA) and Peptacana were prepared at 2,5 mg/mI in HNE assay buffer (50 mM sodium phosphate, pH 7.4, 0.1% BSA, 0.025% 5 Triton X100) and serially diluted to give solutions with concentrations within the range 4.9 - 1250 pg/ml. Triplicate diluted samples (1OOpI) were transferred to wells of a microtitre plate to which was added 50 pl of 2.0 pg/ml human neutrophil elastase (HNE) in the HE assay buffer and the plate incubated at 37 *C for 10 in. Substrate solution (100pl of 0.5 mM SAAVNA in 12.5% DMSO and 87.5% assay buffer) were 10 added to each well and the absorbence was read at 405 nm 4 times at 30 minute intervals. The elastase inhibition rates were expressed as a percentage of the control rate and a plot of % residual HNE activity versus inhibitor concentrations In the inhibition assay was prepared. 15 Ovine Chondrocyte Cultures Freshly prepared or cryopreserved primary ovine articular ohondrocytes were removed from liquid nitrogen storage and were immediately thawed at 37 "C in a water bath and rinsed twice in culture medium containing 50 pig/ml gentamicin. The cells were seeded at the density of 2 x 10s cells /well in 6-well plates and grown in an 20 atmosphere of 5% CO 2 in air at 37 *C for 4 days. Medium was changed every 2 days. Effects of Peptacans and Chondroitin Sulfate A on DNA Synthesis by Ovine Chondrocytes Using 3 H-methyl Thymidine Incorporation Assay DNA synthesis in ovine chondrocytes stimulated with chondroitin sulfate A 25 (ChSA) or Peptacans (CaP, NaP or H2OP) was determined in duplicate by the incorporation of 3H-methyl thymidine into this nucleic acid. The cell cultures were maintained in monolayer culture in 2.0 ml of DMEMIFI2 medium containing 10% FCS and 50 pg/ml gentamicin for 3 days before adding the samples. ChSA or Peptacans were added at the concentrations of 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 30 250, 500 and 1000 pg/ml in the above medium and incubated in an atmosphere of 5%

CO

2 in air at 37 *C for 24 h (short-term) or 6 days (long-term) followed by incubation with 0.25 iCi/well 3 H-methyl thymidine for a further 24 h. Cells were then harvested and radioactivity in DNA was determined using a Obeta-counter. Data were expressed as mean values ± SEM. 35 29 Effects of Peptacans and Chondroitin Sulfate A on DNA Synthesis Using a flom etric Assay DNA synthesis in human synovial fibroblasts (HSP) was determined in duplicate by using fluorometric assay with Hoechst 33258 dye. The HSF, from joints of 5 osteoarthritic patients, was cultured in monolayer in 6-well plates for 24 b. Then the serial concentrations of ChSA or Peptacans were added at 62.5, 125, 250 500, 1000 and 2000 ig/ml in DMM medium containing 10% FCS and 50 pg/ml gentamicin. The cultures were maintained in an atmosphere of 5% Co2 in air at 37 "C for a further 24 h. The cells were detached by trypain/EDTA (0.2%/0. 1%) treatment and then papain 10 digested. 100 pl/well of Hoechst 33258 dye solution was added to a 96-well plate followed by adding 100 pl of papain digested samples. The plate was gently agitated for 5 min before measuring the fluorescence using excitation and emission wavelengths of 350 nm and 450 nm respectively and slit widths of 10 nn and 15 nm. Calf thymus DNA (0-25 pg/ml) was used as a standard. 15 Effects of Petacans and Chondroitin Sulfate A (ChSA on ProteogJycan (PG) Synthesis by Ovine Chondrocytes using a "SO4 - Incorporation Assay The primary ovine articular chondrocytes prepared as described above were seeded at a density of 2 x 10 5 cells/well in 6-well plates and grown for 4 days before 20 addition of 2.0 ml of different concentrations ChSA or Peptacans (CaP, NaP or H 2 OP) in DMEMF12 culture medium containing 10% FCS and 50 g/Il gentamicin. Compounds were added in the presence or absence of 10 ug/ml recombinant human IL 1. The cultures were grown for finther 3 days. Then 40 pCi /well 35

SO

4 were added 24 h prior to the termination of cultures. After a further 24 h incubation at 37 *C, 25 conditioned rmedia were collected in microfuge tubes. The cells were rinsed with cold PBS twice and then trypsinized with 0.2% trypsin/0.1% EDTA solution. The cell suspension was collected and centifuged at. 1310 x g for 5 min. Papain digestion to release 5,04. labelled GAGs was performed by adding 0.5 ml of papain digestion buffer (2 pl/ml papain suspension, 5 mM cysteine in PBS pH 30 6.2) into 0.5 ml of conditioned media, while the cell pellets and their extacellular matrix were directly resuspended in 0.5 ml of papain digestion buffer and incubated at 60 *C overnight. Newly synthesised "Sulfated PGs (asS-GAGs) were separated from free 3sS0 4 by the following procedure: To 0.4 ml of each papain digested sample, 0.2 ml of 0.2 M Na2SO 4 plus 50 mg/ml CSA (1:1) was added and vortexed. Then 0.1 ml of 35 0.4 M BaC was added to each tube and vortexed. The mixture was centrifuged at 1310 x g for 10 min. 0.4 ml ofthe supernatant was transferred into another tube. 0.1 ml 30. of 50 mg/ml CSA plus 0.4 M BaC 2 (2:5) mix was added. Then 0.1 ml of 0.2 M NaSO4 was added and ce gd at 1310 x g for 10 min The last tep was repeated once. 0.1 ml of the supematant was transferred into a pony vial followed by adding 3 ml / vial Eco-lite scintillation fluid and thoroughly mixed. Samples were then counted 5 in an automated B-counter (Canberra Packard 1500 Liquid Saintillaon Analyzer). Ovine Fibrochondroeyte Cultures The skin and overlying musculature of ovine stifle joints were moved and the external joint capsule sprayed with 70% ethanol. The joints were then opened in a 10 lamina flow hood. The menisci were removed, rinsed with sterile PBS and cut into two parts - the inner and outer regions. The inner and outer meniscus tissues were individually dissected into small pieces and digested separately with 0.2% pronase in culture medium (DMEM containing 10% FCS and 50 pg/mI gentamicin) for 2, h followed by 0.04% collagenase digestion overnight. 15 The digests were filtered 3 times through 70 sm mesh and rinsed with DMBM culture medium containing 50 pg/mi of gentamicin. The cells were seeded into 75 cm flasks and incubated in an atmosphere of 5% CO 2 in air at 37 *C for 7 days to reach confluency. The cells were rinsed twice with sterilised PBS, trypsinized with 0.2% trypsin/0.1% EDTA solution and rinsed twice with culture medium. The cells were 20 subltured at the density of 2 x 10cells/well in 6-well plates and grown for 2 days in an atmosphere of 5% CO2 in air at 37 "C. The Effects ofPentacans and Chondroitin Sulfate A on Proteoglycan (PG) Synthesis by Fibrochondrocytes using the "SO -Incorporation Assay 25 The first passage fibrochondrocytes from either inner or outer region of ovine meniscus were seeded at the density of 2 x 105 cells /well in 6-well plates and grown for 2 days before 2.0 ml of different concentrations of chondroitin sulfate A (ChSA) or Peptacans (CaP, NaP or H2OP) in DMEM/F12 was added. The cultures were grown for a further 3 days then 40 ICi/well "SO 4 was added 24 h prior to the termination of 30 cultures. After 24 h incubation at 37*C, conditioned media were collected in microfuge tubes. The cells were rinsed with cold PBS twice and then trypsinized with 0.2% trypsin/0.1%EDTA solution. The cell suspension was collected and centrifuged at 1310 x g for 5 min. Papain digestion was performed by adding 0.5 ml of papain digestion buffer (2 35 pl/ml papain, 5 mM cysteine in PBS pH 6.2) into 0.5 ml of conditioned media, while the cell pellets were directly resuspended in 0.5 ml of papain digestion buffer and Si incubated at 60 *C overnight. Newly synthesised "Sulfted PGs (as"S-GAGs) wer separate from free "804 by the following procedure: To 0.4 ml of each papain digested ample, 0.2 ml of 0.2 M Na 2

SO

4 plus 50 mg/ml CSA (1:1) was added and vortexed. Then 0.1 ml of 0.4 M BaCh was added to each tube and vortexed. The 5 mixu was cent ed at 1310 x g for 10 min. 0.4 ml of the supernatant was transferred into another tube. 0.1 m1 of 50 mg/ml CSA plus 0.4 M BaCh (2:5) mix was added. Then 0.1 ml of 0.2 M NaaSO4 was added and centrifged at 1310 x g for 10 min. The last step was repeated once. 0.1 ml of the supernatant was transferred into a pony vial followed by adding 3 ml/vial Eco-lite scWttlation fluid and thoroughly 10 mixed. Samples were then counted in an automated B-counter (Canberra Packard 1500 Liquid Scintillation Analyzer). Human Synovial Fibroblast Cultures Cryopreserved human synovial fibroblast (HS) derived from joints of patients 15 with osteoarthritis were thawed quickly at 37 C in a water bath. The entire cell suspension in cryo-vials was transferred to a 15 ml sterile centrifuge tube using a transfer pipette in a lamina flow hood. The tubes were centrifuged at room temperature, at 8Og for 10 min. The supernatant was discarded and the cell pellet completely resuspended in 10 ml DME3M medium containing 10% FCS and 50 pg/mil gentamicia 20 by aspirating with a sterile transfer pipette. The tubes were re-centrifuged at 800g for .10 min at room temperature. The supernatant was again discarded and the cell pellet completely resuspended in 10 ml DMEM complete medium. The cell suspension was seeded in appropriate numbers of labelled flasks followed by the addition of 15 ml of DMEM complete medium. The cells were incubated in an atmosphere of 5% CO 2 in 25 air at 37 *C until confluency. Culture medium was changed .every 2 days. To subculture the cells, the flasks were removed from the 37"C incubator and the cells were rinsed twice at room temperature with PBS. Trypsin/EDTA (0.2/0.1%) solution was added and swirled over the cell layer. The flasks were recapped and maintained at 37 *C for 3 - 5 min. The cells were checked under a microscope for detachment. 30 DMEM complete medium (2 - 3 mil per flask) was added and washed over the surface of flasks to collect the released cells. The cells were transferred to centrifuge tubes and centrifhged at 800g for 10 miu at room temperature. The cells were washed twice and then resuspended in 10 ml DMEM complete medium. The cells were then counted using a haemocytometer. The cells were seeded at the density of 1.5 x 10' cells /2 35 ml/well in 6-well plates and grown for 24 h in an atmosphere of 5% CO2 in air at 37 *c.

32 Effects of Peptacans and Chondroitin Sulfate A on Hvaluronan(HA) Synthesis by Human Synovial Fibroblasts (HS) Using a _T-glucosamine hLE ai Say The HSF cells were seeded at 1.5 x 101 cells/well in the 6-well plates and allowed to attach for 24 h before addition of compounds. Chondroitin sulfate A 5 (ChSA) and Peptacans (CaP and H2OP) solutions were prepared in DMEM culture medium containing 10% FCS and 50 pg/mI gentamicin at double the concentration required in the cultures. The highest concentration prepared was 4.0 mg/mil which was sterilised through a 0.22 pm filter and then serially diluted to give final concentrations of 2000, 1000, 500, 250, 125 pg/ml, 62.5 pg/ml of compounds. One ml of each drug 10 solution was added to each well of 6-well plate. Stock 3 H-glucosamine was diluted in culture medium to give a 1 pCi/ml solution which was immediately added to each well (1 pCi/well). The plates were incubated for a further 24 h. At culture termination, media was collected into 5 ml capped tubes and stored at -20" C for 3 H-HA analysis. 15 Isolation and Quantitatien of !H-Hyaluronan ( 3 H-HA) in Cultums Using Superose 6 Gel Filtration Chromatography Two aliquots of 0.5 ml from each media sample were labelled A and B. 20 p 1 of 1 M acetic acid, pH 6.0 was added to all aliquots. 50 pl of reaction buffer (20 mM Na acetate and 0.15 M NaCl, pH 6.0) was added to aliquot A and 50 pl of 5 TRU 20 Streptomyces hyaluronidase in reaction buffer was added to aliquote B. All samples were incubated at 60 0C for 3 h followed by boiling for 5 min to inactivate the added hyaluronidase. The samples were store at -20 "C prior to gel filtration, A gel filtration column prepacked with Superose 6 was used to isolate and identify 3 H-HA in culture media. Media samples were routinely centrifuged at high 25 speed on a bench Microfuge for 10 min immediately before loading to the column. Samples (200 pl of each) were injected into the column through sample loop and the column was eluted with PBS buffer (0.15 M NaCl, 0.05 M Na 2

PO

4 , pH 7.2) at the flow rate of 0.2 ml/I min. The column eluent was collected at 0.5 ml/fraction for total of 46 fractions and radioactivity was determined using a B-scintillation counter. 30 Studies on the effects of oral administraion of Chondroitin Sulfate or Calcium Peptacan on joint inflammatory mediators, white blood cell numbers and ex-vivo synovial fibroblast biosynthesis of Hyaluronan in a rabbit model of arthdtis. The rationale, methodology and pathological outcomes obtained for the rabbit 35 model of arthritis used in the present experiments has been described in detail elsewhere (Page-Thomas DP: Aspects of synovial degradation. Bayer-Symposium, 33 Experimental models of chronic inflammatory disease, 353-365 (1977) Springer Verlag Berlin; Cambray et al., The effects of dexamethasone in vitro on the production of collagenase and inhibitor by synovial and cartilage explants from joints of rabbits with a proliferative arthritis, Rheumatol Int, 1981, 1: 69-72; Smith et al., The effects of 5 orally administered calcium pentosan polysulfate on inflammation and cartilage degradation produced in rabbit joints by intrmarticular injection of a hyaluronate polylysine complex. Arthritis and Rheumatism, 1994, 37: 125-136). In the present studies, male New Zealand White rabbits of approximately 4 months of age were used and acclimatised to holding cages for 7 days. Animals were then divided into 5 10 experimental groups according to the criteria provide in the following protocol. Experimental Protocol: Group A (n - 4) Non-drug-treated, Polycation(polylysine)-Hyaluronan complex (PC)-injected intra-articularly into one rabbit knee joint and an equal volume of 15 isotonic saline injected in the contralateral knee joint on day 7. Group B (n - 4) PC-injected intra-articularly into one knee joint and isotonic saline injected into the contralateral knee joint but animals administered 300mg/kg (body weight) chondroitin sulfate (Bioiberica batch # 18/11/99) orally daily for 14 days beginning 7 days before the PC injection. 20 Group C (n = 4) PC-injected intra-articularly in one knee joint and isotonic saline injected in the contralateral knee joint, administered 30 0mg/kg (body weight) CaP orally daily for 14 days beginning 7 days before PC injection. Group D (n = 4) PC-injected intra-articularly in one knee joint and isotonic saline injected in the contralateral knee joint but animals administered 200mg/kg (body 25 weight) CaP orally daily for 14 days beginning 7 days before PC injection. Group E (n = 4) PC-injected intra-articularly in one knee joint and isotonic saline injected in the contralateral knee joint but animals administered with 1I00mg /kg (body weight) CaP orally daily for 14 days beginning 7 days before PC injection. After animals in groups B to B had received drug treatment for 7 days they were 30 anaesthetised with halothane/nitrous oxide/oxygen (2:1:2; flow rates for 02 and N20= 1 and 2 litres/minute respectively). Each animal was under anaesthetic for at least 5 minutes before intra-articular injection and for a total of 10 minutes thereafter. Animals were given a single intra-articular injection of a preformed insoluble complex (PC) of poly-D-lysine (7.5 mg) and hyaluronan (HA) (7.5 mg) in 1 mL sterile, pyrogen 35 free isotonic saline in one knee. This preparation was prepared immediately prior to injection by quickly mixing 0.5mL sterile 15mg HA/mL with 0.5mL sterile poly-D- 34 lysine/mL in a 2mL sterile syringe. The contralateral knee joint of each animal was injected with I mL sterile isotonic saline ouly to serve as an internal control. Animals in groups B to B continued to receive daily oral drug preparations for further 7 days post PC injection. S Seven days post arthritis induction, rabbits were anaesthetised with halothane/nitrous oxide/oxygen as before and blood (20 - 40 mL) was obtained directly from the heart using a sterile 14 gauge catheter and a 50 mL syringe. 10 mL of this blood was deposited into sterile glass EDTA (K3) vacutainer tubes with a final dilution of 0.7 % EDTA. A further 9mL was added to ImL 5%(w/v) trisodium citrate and the 10 remainder was added to serum tubes. Within 2 hours of blood collection the separation of mononuclear cells and neutrophils was performed using the centrifugation procedure as described below. All blood samples were kept at room temperature at all times. Separation and determination of blood white cells numbers 15 The EDTA-containing blood collected as described above was used to prepare white cell fractions. 20 mL of Percoll (AMRAD Pharmacia, Boronia, Melbourne Australia) at a density rage between 1.070-1.072 g/mL, was prepared in sterile 40 mL Sorvall centrifuge tubes. This optimum density was achieved by first adding 9 parts of neat Percoll to 1 part of sterile 1.5M NaCl. This stock solution was then diluted using 20 0. 15M NaCi until the desired density was obtained. Centrifugation of Percoll and blood The Percoll, at an optimum density range of 1.070-1.072 g/mL, was centrifuged at 10,000 x g for 20 minutes at room temperature, using a fixed angle rotor head in a 25 Sorvall RC-5 centrifuge to form a. gel which provides better separation of the white cells. 4 mL of the collected blood was then layered on top of the preformed density gradient and centrifuged at 1000 x g for 20 minutes at room temperature using a swinging bucket rotor in a Beckman TJ-6 centrifuge. From each of the density vials 1 7 (AMRAD, Pharmacia) 40 sL was added to 3720 pL of sterile 0.15M NaCI to give a 30 total volume of 4 mL. Each AMRAD vial contained coloured density marker beads of a given density. The density range of the 7 vials was 1.017-1.102 g/mL. The 4 ML density bead solution was then layered on top of a 20 mL preformed Percoll density gradient (1.07-1.072 g/ml), and centrifuged at 1000 x g for 20 minutes at room temperature with the sample tubes prepared above, using a swinging bucket rotor in a 35 Beckman TJ-6 centrifuge. This density marker tube served to calibrate the gradients 35 formed in the sample tubes. The following density ranges were used for the rabbit's blood (these differ fromn the manufacturer's instruction for human blood): 1. Mononuclear cells: 1.04-1.06 g/mL 2. Neutrophils: 1.06- 1.08 g/mL 5 3. Brythrocytes: 1,08-1.09 g/mL After centrifugation of each sample tube, mononuclear cells and neutrophils were separated by aspiration at their designated density range and placed into separate centrifuge tubes. These cells were washed twice with 3 volumes of 0.15M NaC1 at 10 1000 x g for 10 minutes at room temperature using a swinging bucket rotor in a Beckman TJ-6 centrifuge. The cell pellets of the mononuclear cells and neutrophils were resuspended with 500 pL PBS. From each cell suspension 50 pL was added to 50 pL of trypan blue solution and the cell numbers quantitated using a haemnocytometer. From the mononuclear cells another 50 sL aliquot was used to form differential 15 slides. This procedure allowed the percentage of neutrophils, lymphocytes and monocytes to be determined. Smears were formed using a Cytospin centrifuge. Dissection and collection of synovial membrane from joints for cell culture Joints were sprayed liberally with 70% (v/v) ethanol and placed in a clean 20 plastic bag at 4'C for transport to the sterile environment of a laminar flow cabinet. Using rat-toothed forceps and a no. 22 scalpel blade, the tendons and excess muscle tissue were removed from the joints without opening the capsule. Using small rat-toothed forceps and no.11 scalpel blade the joint capsule was opened aseptically using a posterior approach, severing the posterior insertions of the 25 cruciate ligaments to allow dis-articulation. The patella and the attached suprapatella mound of synovium was removed whole using a no. 11 blade. This tissue was then placed in a labelled specimen container filled with 10%(v/v) neutral buffered formaldehyde for histological processing. The remaining synovia was then barvested by excising pieces of the joint lining with the small rat-toothed forceps and discection 30 with a no.11 blade, avoiding inclusion of excessive subsynovial tissue. Collected pieces were then floated into a labelled petridish containing lOmL sterile PBS. Synovial fibroblast isolation The synovia in the petridish was diced finely then transferred to a 15 miL sterile 35 centrifuge tube, using a sterile plastic transfer pipette with the fine end cut off (using a sterile no. 22 blade). The tubes were centrifuged at 1500 - 2000 rpm at 20*C for 36 10 minutes and the cell pellet resuspended in 5 mL sterile T&E [0.2% (w/v) trypsin/ 0.1% (w/v) EDTA in PBS]. The tubes were placed at 4 0 C overnight (cold trypsinisation) then were incubated at 37*C for one hour. After incubation 5 mL DMFM100% FCS was added (to inactivate the trypsin) and then the tubes were 5 recentrifuged at 20*C, 2000 rpm for 10 minutes and trypsin/PCS solution between the pellet and floating fat layer was removed. Sterile DMBM/10% FCS was added to a total volume of 10 mL and pellet was resuspended. The tubes were recentrifuged at 20 0 C, 2000 rpm for 10 minutes. The DMEMIO 0 / FCS solution was removed and the pellet was resuspended in 2 mg/mL collagenase in DMEM/10%FCS. The tubes were placed 10 in the incubator at 37 0 C for 3 hours then the collagenase solution, including fat layer, was removed to waste. Sediment pellet was resuspended in 10mL DMBMI1O%FCS using a sterile plastic transfer pipette. Two x 5mL aliquots of the cell suspension were placed into 2 x 25 cm 2 tissue culture flasks and incubated at 37 0 C. Media routinely was changed every two or three days. When one of the flasks was ~90% confluent it 15 was used to determine HA synthesis. When the other flask was confluent, the cells were subcultured. In vitro HA biosynthesis by synovial fibroblast Synovial fibroblasts as prepared above were plated into 6 well plates at 200,000 cells 20 per well and allowed to attach for 24 hours. To each well was added 2mL DMEM+10%(v/v) FBS followed by 3H-glucosamine (stock is lsCi per microlitre) and plates incubated for 24 hours. After which, flasks were removed from the incubator and chilled at 4 0 C and media was removed to labelled 15mL centrifuge tubes. As media was removed, each flask was twice gently washed with 0.5mL nonsterile 25 PBS twice, which was added to the same labelled l5mL centrifuge tube. After the PBS rinses, ImL T+E was added directly to the cell layer in each well. The plate was incubated at 37 0 C for 10 minutes. The released cell suspensions were transferred to labelled SmL tubes, rinsing the flasks twice with 0.5mL papain buffer and adding the rinses to the same tubes. 30 Determination of radiolabelled HA synthesised by cells Two x 0.5niL aliquots of each media sample were labelled A and B. To all aliquots were added 20pL 1M acetic acid (to adjust pH to approx. 4.0). To each sample labelled A was added 50pL 20mM sodium acetate/0.15MNaCl pH 6.0. To each sample 35 labelled B was added 50pL 20mM sodium acetate/0.15M NaCl pH 6.0 containing 5 TRU Streptomyces hyaluronidase.( Sigma Chemical Co). All samples were incubated 37 at 60*C for 3 hours the boiled for 5 minutes to inactivate the Streptomyces hyaluronidase. Samples were then be stored at -20*C prior to Superose 6 gel chromatography and determination of incorporated radioactivity into HA as already described above for the synthesis of HA by human synovial fibroblasts. 5 Immunohistology of Synovial Tissue The patellae and suprapatella mound of synovium was removed whole from joints using the dissection procedure described above and the patellae and adhering synovium (and any pannus) sliced in half and each half placed in a specimen container 10 with neutral buffered formalin. Processing and preparation of stained and unstained tissue sections were performed as already described above Immunolocaisation of Inducible Nitric Oxide Synthase (INOS) Four-micron patellaelsynovium sections were deparaffinised to water. 15 Endogenous peroxidase was blocked with 3% H202 for 5 minutes. A high temperature heat retrieval was used to unmask antigenic sites using 10mM citrate buffer; pH 6.0. Non-specific binding was blocked with a serum-free protein block for 10 minutes. Sections were then incubated with the primary antibody polyclonal antiserum for iNOS (Cayman Chemicals Inc) 1:1000 overnight at 40C. The negative control reagent used 20 was a protein concentration matched non-immune rabbit serum. The detection system utilised a commercial avidin-biotin with horseradish peroxidase as an enzyme label (Dako Biologics Pty Ltd). The secondary antibody cocktail of biotinylated goat anti rabbit and biotinylated goat anti-mouse (Dako Biologics Pty Ltd) was applied for 15 minutes at room temperature. Slides were then incubated for 15 minutes in a 25 streptavidin conjugated to peroxidase (Dako Biologics Pty Ltd). Peroxidase activity was detected using 3,3'-diaminobenzidine (DAB). Sections were counterstained with Mayer's haematoxylin for 1 minute, dehydrated, cleared in xylene and mounted in Buckitt.. 30 Jmmunolocaisation of Nitro-Tyrosine The method used was identical to that described by Kobayashi et al (2001) for rabbit cartilages in another arthritis model( Kobayashi K, et al., Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy. J Orthopaedic Res, 2001,19: 802-808) 35 9 38 Statitica methods Differences between PC and saline injected joint data were assessed using paired t-tests. Non-treated, drug treated and non-drug treated PC or saline joint data were compared on each day using unpaired t-tests. UHstological scoring (nonparametric 6 data) was analysed using Wilcoxon signed rank tests. Analyses were performed using Microsoft Excel 98 or Statview 5.0 on an Apple Macintosh PowerPC. P values less than 0.05 were considered to be significant. RESULTS AND DISCUSSION 10 The present inventors have made the unexpected and surprising discovery that subjecting cartilage particles to autolysis in aqueous buffers maintained within the pH range of 4.0-7.0, particularly 4.5, at 37'C for periods up to 36 hours, particularly 16 or 24 hours specifically released GAG-peptide complexes and matrix derived polypeptides into solution while leaving the tissue cells and their intracellular 15 macromolecular components essentially in tact. In this context we used DNA as a marker for intracellular macromolecular components and demonstrated that the autolysis medium obtained by the present invention was substantially free of DNA. . Moreover, these released GAG-peptide complexes, defined arbitrarily as Peptacans when used alone or in combination with other co-released polypeptides are 20 pharmacologically more active than commercially available ChS. The release of these cartilage products was found to be more rapid with smaller particles than larger ones (FIGURE 1). However, when either cartilage was incubated at 37'C over a 24 hour period 75-83% of the total tissue sulfated GAGs was released into solution as shown by analysis for this component irrespective of the particle size 25 (FIGURE 1). The efficiency and selectivity of this method was also confirmed by histochemical analysis of the cartilage particles before and after subjecting them to the inventive method. FIGURE 2 shows photomicrographs of histological sections of 3mm bovine tracheal cartilage powders particles before and after subjecting them to the 30 autolysis procedure using 100mM calcium acetate buffer, pH 4,5 at 37 degrees C. Panels A and C are sections of cartilage before autolytic processing and B and D after. Panels A and B show the results of staining with Toluidine Blue (TB), a dye which binds to glycosaminoglycans (GAGs) while C and D show sections stained with Masson Trichrome (Masson TC), a dye known to stain native collagen fibres magenta 35 colour. Note the loss of staining for GAGs in section B after autolysis but a slightly increased intensity of staining for collagen following the removal of the majority of 39 GAGs as shown in D. Cell nuclei, identified in these sections by the green counter stains are clearly unchanged by the autolytic process. All sections are shown at magnification X 400. As is evident from FIGURE 2 the level of staining for the presence of sulfated 6 GAGs in cartilage particles using the cationic stain for these molecules, Toluidine Blue, before processing was extensive but after incubation was diminished. On the other hand, Masson Trichrome staining of native collagen fibres was essentially unchanged by the autolytic process ( FIGURE 2). Significantly, the cells and importantly their nuclei which were clearly visible using both staining procedures of the residual 10 cartilage, remaining after removing the supernatant and washing with the incubation buffer, were observed to be largely undisturbed (FIGURE 2). o The autolytic process was affected by both pH and temperature . As is evident from FIGURE 3 the most rapid release of peptacans from bovine tracheal cartilage occurred within the pH range 4.0 - 7.0. The normal mammalian blood temperature of 15 37 degrees C was found to be more effective than lower temperatures, most notably 4*C in facilitating peptacan release while intermediate levels of release occurred at 25 degrees C (FIGURE 4). The influence on this release process of both temperature and pH was consistent with the notion that autolysis was proceeding via the cleavage of matrix proteoglycans and other structural proteins by endogenous enzymes whose 20 catalytic activities were optimum within the pH range 4.0-7.0 at 37 degrees C. This explanation was confirmed by the observation that the release process was substantially slowed by maintaining the cartilages particles in the autolysis buffers at 4 0 C as well as. the observation that the rate of release was markedly reduced by including specific inhibitors of proteinases in the incubations in buffers at 37 degrees C (FIGURE 5). As 25 is evident from FIGURE 5, approximately 50% inhibition of the autolytic process was achieved by the addition of the cysteine proteinase inhibitor , N-ethylmaleimide to the buffer solution. Since the major cysteine proteinases of cartilage are the Cathepains (Dingle .T, The secretion of enzymes into the pericellular enviroment. Phil Trans Royal Soc (London), 1975, 271: 315-324; Barrett AJ and McDonald yX, Mammalian 30 Proteases, Academic Press, London, New York, 1980, pp338-350; Barrett AJ et at., Lysosomal cysteine proteinases, ISC Atlas Sci Biochemistry, 1988,1: 256-260, Muller Ladner U, Gay RE, Gay S, Cysteine proteinases in arthritis and inflammation, Perspectives in Drug Discovery and Design, 1996, 6:87-98) which have pH optima between 3.5 - 6.0, it is most likely that these enzymes are largely responsible fbr the 35 autolytic degradation of matrix components in this invention. However, as matrix component degradation was not completely abrogated by N-ethylmnaleimide other WO 03/062279 A 40 classes of proteinases are clearly involved. Since the shrine proteinase inhibitor, Bennmidine had no observable effect on release, this class of proteinases would seem to excluded. However, the matrix metalloproteinases (Birkedal Hansen I, et al., Matrix wetalloproteinases: a review. Critical Rev Oral Bio Med 1993, 197: 197-250) and 5 aggrecanases (Ilic MZC, Handley CJ, Robinson HC, Mok MT, Mechanism of catabolism of aggrecan by articular cartilage, Arch Biochem Biophys, 1992, 294:115 122) which have previously been shown to degrade cartilage matrix components at neutral pH are potential candidates. As already discussed herein the matrix degradation products generated by these 10 endogenous enzymes are structurally different from those produced by digesting connective tissue with papain or proteinases of other origins. Since the pharmacological activity of drugs and biopharmaceuticals is critically dependent on their composition and structure , we consider that the peptacans and the other autolytic products generated by the present inventive method cannot be reproduced by simply 15 digesting cartilages with exogenous enzymes such as papain, chymopapain, bromolein, pepsin, trypsin, chymotrypsin, elastase or-other enzymes which are not specifically derived from the tissues themselves. The efficiency of the autolytic process in the present invention was also influenced by the animal species and anatomical location from where the cartilage 20 was derived. As is evident from TABLE 1 below, bovine tracheal and nasal cartilages afforded the highest yields of peptacans when incubated at 37*C over 24 hours, However, deer antler cartilages and avian (chicken ) sterna provided more than 60% yield of peptacans under the same conditions. Surprisingly, commercially available samples of shark cartilage, of unknown location or method of preparation afforded very 25 low yields of peptacans. Nevertheless, it is clear from these examples that the method of the invention is applicable to a wide range of connective tissues but the efficiency of release of peptacans into the aqueous medium is related to their ultrastructural assembly and means of preparation prior to autolytic treatment.

41 1 II %0 00 cn0 C;c 5 56C C4) Zoo o ;C \0 m4 ____0 *1lie.

42 When the supernatants derived from the tracheal cartilage incubated with 0.1M Calcium Acetate were applied to a Superdex gel filtration column and fractions monitored for sulfated-GAGs and protein two resolved peaks corresponding to the GAG-peptide and polypeptides were obtained (FIGUBB 6). Evidence that the GAG 5 peptide was not simply ChS but contained more than one sulfated GAG chain was provided by its digestion with the proteolytic enzyme papain which shifted the DMMB positive peak to the same column fractions obtained when pure ChS was chromatographed under the same conditions (FIGURE 7). When the supernatants obtained from incubation of 0.1M Calcium acetate pH 4.5 (CaP) and dilute acetic acid 10 pH 4.5 (H2OP) were chromatographed under the same conditions on the Superdex-200 column it was observed that the GAG-peptide obtained with the Calcium Acetate buffer was of smaller molecular size than that released by the acetic acid digestions. Both were, however larger than two commercially available ChSs (FIGURE 8). The weight average molecular masses of the CaP, H20P, purified CaP (pCaP) and the two 15 commercially available ChSs were then assessed using a Sephadex-G 200 high resolution gel exclusion column and seven ChS standards prepared and characterised previously (Melrose J and Ghosh P, Determination of the average molecular size of glycosaminoglycans by fast protein liquid chromatography. I Chromatography, 1993, 637; 91-95). The correlation between MW and Kay obtained using these ChS 20 standards and the Sephadex column is shown in FIGURE 9 and the results obtained for the unknown preparations is shown in TABLE 2 below. These experiments confirmed that the GAG-peptide in CaP contained 2 ChS chains while that in H2OP consisted of 3 ChS chains. 25 TABLE 2 Average Molecular Size (KDa) of commercial chondroltin sulfates and Peptacans as determined by Gel Chromatography and standards ChSA (Sigma) 17.3 ChSA (Bioiberica) 20.0 CaP 33.7 pCaP 31.1 H20P 46.1 MgP 32.3 ZnP 50.4 30 CAPAGE of CaP, pCaP and papain digested CaP indicated that the only sulfated GAG present was ChS (FIGURE 10). However, SDS-PAGE showed that CaP also 43 contained a number of peptides with MWs of 21 KDa or less which were not present in the purified CaP (FIGURE 11). Figure 11 shows analysis of proteins and peptides in Peptacans Using SDS-Polyacrylamide gel electrophorsis. Lane 1= protein markers with molecular weights shown, lane 2= CaP, lane 3 = purified CaP, lane 4 = chondroitinase 5 ABC digested and purified CaP. Analysis for total protein and hydroxy proline content as a marker of collagen of these samples suggested that the dominant polypeptides released were largely derived f-om cleavage of collagen chains, 10 TABLE 3 Analysis of Peptacans and Commercial Chondroitin Sulfates For Sulfated Glycosaminoglycans, Protein and DNA content Samples S-GAG(DMMI) Protein (BCA) DNA(Hoechst) ChSA (Sigma) 98.5 0.16' 0.36 ChSA 97.4 1.25" 0.32 (Bb 1/0015,05/2001) ChSA (Bb 18/11/99) 98.1 0.63a 0.35 ChSA (NaP) 97.8 0.74a UN ChSA (CaP) 96.5 1.40a UN CaP 39.8 35.12" UN pCaP (BtOH ppt) 49.0 40.5 1 ' UN -H2OP 65.6 4 4

.

4 8 ' UN NaP 53.3 3 2

.

9 3 ' UN pH20P (column) 85.0 9

.

60 b UN pCaP (column) 44.8 6 5 2 UN MgP 32.5 3 6

.

59 ' UN ZnP 18.6 38

.

17 U 15 S-GAGs = sulfated glycosaminoglycans (method of assay used, see text) UN = undetectable, a = using BSA as standard, b =using Gelatin as standard Spectroscopic analysis of concentrated aqueous solutions (1.0mg/ml ) of the 20 Peptacans ( CaP, NaP, H2OP) and commercial ChSs (FIGURE 12) and ChSs derived from Peptacans revealed low absorption for protein at 280nM and DNA at 260nM for the Peptacans and the ChSs derived from them. In contrast high values for DNA were evident in the commercial ChS preparations as indicated by the ratios of A260/A280 in their uv spectra which were in the order of 1.85 or more (FIGURE 12). Using a 25 published fluorescent dye binding assay for DNA in the same samples and calf thymus DNA as a standard confirmed that the Peptacan preparations and the ChSs derived from 44 them were substantially free of DNA, whereas the pharmaceutical quality commercial ChS examined contained approximately 0.3% DNA (TABLE 3). Analysis of the relative binding of CaP and ChS to the enzymes, lysozyme, hyaluronidase and human neutrophil elastase which are mediators of matrix destruction in arthritis using the 5 BlAcore 2000 system showed that CaP interacted more strongly with lysozyme and elastase than ChS but with hyaluronidase there was little difference between the two preparations (FIGURE 13). The higher binding affinity of CaP for the proteolytic euzyme, elastase derived from human neutrophils was confirmed using a conventional fimutional assay and the synthetic elastase substrate, succinyl-alanine-alanine-valine 10 ndtroanilide (SAAVN). Using this assay system the concentration range of ChS which produced 50% inhibition of elastase (IC50) was found to be 4-5 micrograms/ml while the IC50 for the CaP preparation was between 1-3 micrograms/ml. In the short term (2 days) chondrocyte cultures CaP at concentrations of 250 micrograms/ml and above stimulated cell division as indicated by tritlated thymidine 15 incorporation into DNA while ChS had no effect (FIGURE 14). In the longer term chondrocyte cultures (7 days) CaP at 62.5 micrograms/nil stimulated DNA synthesis (FIGURE 15). These data clearly identify the anabolic effects of the CaP which would support the repair and regeneration of connective tissues. As already indicated PGs are essential components of the cartilage extracellular 20 matrix and are responsible for its unique biomechanical properties. Since these molecules are degraded and depleted from cartilage in arthritic joints, agents which can prevent this event or stimulate their biosynthesis by ohondrocytes and other cells would be beneficial to the recovery of joint function and a reduction in patient symptoms, In contrast to ChS, CaP exhibited a concentration dependent stimulation of 25 proteoglycan synthesis by chondrocytes with more than 150% deposition of PGs into the extracellular matrix at CaP concentrations of 1.0 and 2.0 mg/ml (FIGURE 16). At concentrations between 62.5 - 500 micrograms/ml ChS released slightly more POs into the media than CaP but this was reversed at 2.0mg/ml. (FIGURE 16). Significantly, when the effects of CaP and ChS on PG synthesis by chondrocytes was studied in 30 cultures to which the proinflammatory cytokine interleukin-1 (IL-1) had been added. CaP produced a 500% increase in synthesis at concentrations of 1.0mg/mi while ChS had no effect (FIGURB17). Interestingly this stimulatory effect by CaP on PG synthesis was greater than when cells were cultured in the absence of IL-1 (FIGURE 17). 35 The knee joint menisci are structurally complex fibrocartilages which perform an essential role for the weight bearing functions of diarthrodial joints. They are 45 crescent shaped cartilage in which the outer rim is under high tensile strsses and the inner region is subjected to more compressional loading. The fibroehoudrocytes from these two regions are therefore metabolically different and were for this reason studied separately. When fibrochondrocytes from the inner region of ovine menisci were 5 cultured with various concentrations of ChS and the Peptacans, only CaP stimulated PG synthesis (FIGURES 18 &19). At 2.0mg/ml a 200% stimulation of PG synthesis was obtained for CaP and a 130% increase for ChS but only for PGs released into the media (FIGURES 20 &21). CaP was observed to produce a similar PG stimulatory profile on fibrochondrocytes isolated from the outer region of the meniscus (FIGURES 22,23 & 10 24) but in these cultures ChS filled to elicit a significant increase in media PG levels (FIGURE 25). The major non-proteinaceous component of joint synovial fluid is hyaluronan (HA). As already indicated HA confers to synovial fluid its unique rheological properties which includes exceptionally efficient lubrication of articular cartilage and 15 peri-articular tissues. In rheumatoid and osteoaxthritic joints synovial fluid HA concentration and molecular weight are decreased (Dahl LB, Dahl IMS, Engstrom Laurent A, Granath K. Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies. Ann Rheum Dis 1985;44;817-22). Since the theological effects of HA are dependent on Its 20 molecular weight and concentration, a decline in either or both of these parameters decreases the ability of synovial fluid to efficiently lubricate and protect articulating surfaces. However, HA is also anti-inflammatory and exhibits a number of other important functions essential in joint physiology (Ghosh P and Guidolin D, Potential mechanism of action of intra-articular hyaluronan therapy in osteoaxthritis; Are the 25 effects molecular weight dependent?, Seinars in Arthritis and Rheumatism 2002, 32: 10-37. ). Diminished HA concentration and a reduction in its molecular weight could therefore have profound effects, not only on the rheological properties of synovial fluid, but synovial and inflammatory cell functions and their Macromolecular expression. Synovial fluid HA is almost exclusively synthesized by type B cells of the 30 synovial lining. These fibroblasts (synoviocytes) when Isolated from synovial joints and established in culture retain their phenotypic expression and elaborate HA Into culture media. Fibroblast cultures derived from rheumatoid or osteoarthritic joints show aberrant biosynthesis of HA reflecting the abnormal metabolic status of the joints f-om which they were derived (Smith MM, Ghosh P. The synthesis of hyaluronic acid by 35 human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment. Rheumatol Int 1987;7:113-22). Generally the amount of HA 46 synthesized is less than obtained from normal synoviocytes and its molecular weight is reduced. In the present experiments Peptacans and ChS were evaluated for their respective abilities to promote the biosynthesis of DNA and high molecular weight HA 5 by synovial fibroblasts obtained from joints of patients with OA. The results obtained are shown in FIGURES 26-29. As is evident from FIGURB 26 only CaP exhibited a significant effect on DNA synthesis at concentrations up to 250 micrograms/mL. However, HA synthesis by these cells was increased 230% at 250 micrograms/mL ( FIGURE 27) and 380% at ZOmg/mL (FGURE 28). The identity molecular weight of 10 the HA synthetised was confirmed by chromatography (FIGURE 27) and agarose gel electrophoresis (results not shown). The relative activities of ChS, CaP and H2OP when incubated with the cells at a concentration of 2.0mg/ml are shown in FIGURE 28. The reason for the significant differential effects between CaP and H2OP on HA synthesis by these cells is presently unresolved but was a surprising finding. 15 The relative activities of CaP, pCaP and pH2OP studied under the same conditions are included in FIGURE 29. This result shows the higher activity of CaP relative to the column purified (pCaP) preparation with regard to the stimulation of HA synthesis by human OA synovial fibroblasts. This result suggests that polypeptides in CaP may be acting synergistically with the calcium GAG-peptide complex. 20 The in vitro stimulation of the biosynthesis of HA by synovial fibroblasts derived from joints of humans with osteoarthritis by CaP was also demonstrated to occur in vivo using an animal model of arthritis. In these latter experiments ChS.at 300mg/kg or CaP at 100, 200, or 300mg/kg was administered orally to rabbits in which monoarticular proliferative arthritis had been induced by the intra-articular injection of 25 a PC-complex. The preparations were given daily for 7 days before arthritis was inducted and daily for 7 days thereafter. Animals were euthanased and blood and tissues collected for analysis on day 14. Synovial fibroblasts from the joints of these animals together with those from a non-drug treated arthritis control group were established in primary cell culture and the biosynthesis of radiolabelled HA determined 30 as described for the in vitro studies. The results of these experiments are shown in FIGURES 30 and 31. Compared to the non-drug treated control group synovial fibroblasts from all animals in the CaP treated group showed higher synthesis of HA (FIGURE 30). however, because of the inter-animal biological variation only the group receiving the dose of 300mg/kg were statistically significant (p < 0.05) relative to the 35 non-drug-treated group (FIGURE 31). If the comparison of HA stimulation by the treatments was based on their respective sulfated GAG content then CaP was more 47 than 3X more active than ChS. Promotion of HA synthesis by synovial cells in arthritic joints would contribute to improved joint lubrication and a reduction of cartilage degradation as has already been discussed in detail elsewhere in this disclosure. The extensive synovial inflammation provoked in the rabbit joints by the intra 5 articular injection of the arthritogen (PC complex) was very apparent from the histological sections of the synovial membranes which showed massive cellular infiltration, connective proliferation, hyperplasia and neovascularlization. In the non drug treated control group these inflammatory events were further characterised by the - presence -of an invasive pannus originating from the synovial tissues at the joint 10 margins. Beneath and adjacent to this pannus tissue destruction of cartilage and subchondral bone was evident (FIGURES 32 and 33). In the drug treated groups synovitis was not abrogated but the extent of pannus formation was reduced and the skturat tegrity-of-carti~lg- ely-preserved-Furtherm re, resion-bychondrocytes in the patella cartilage of inducible nitric oxide synthase (iNOS), the 15 intra-cellular enzyme responsible for the production of nitric oxide free radicals, was mitigated particularly in the high dose CaP treated animal (FIGURE 32). This observation was consistent with the level of immunohistochemical staining for nitrotyrosine, which is a characteristic product marker of the chemical action of nitric oxide radical on intra-cellular proteins ( Kobayashi K, et al., Chondrocyte apoptosis 20 and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy. J Orthopaedio Res, 2001,19: 802-808 ). As can be seen in Figure 33 staining for this product was substantially reduced in chondrocytes from carilages from joints of the CaP treated animals . This reduction in chondrocyte production of iNOS and thus nitric oxide free radicals by CaP treatment is considered to arise from its 25 ability to modulate the effects of pro-inflammatory cytokines (such as interleukin -1) as suggested by the in-vitro experiments. Interleukin -lis known to be a major initiators of nitric oxide and other free radical production, synovitis inflammation and tissue destruction within arthritic joints (Taskiran D, Stefanovic-Racio M, Georgescu H, Evans C. .Nitric oxide mediates suppression of cartilage proteoglycan synthesis by 30 interleukin-1. Biochem Biophys Res Commun 1994;200:142-8). However, CaP could also have a direct inhibitory effect on nitric oxide radical activity or its production by synovial cells and chondrocytes which in itself would be a useful means of suppressing inflammatinn and tissue destruction in arthritic joints . Such a strategy which has already been suggested by others using synthetic INOS inhibitors (US Patent 35 6,346,519, February 2002).

48 In addition to the beneficial effects mediated by CaP on the joint tissues of the rabbit arthritis model described above, it was also found to suppress the levels of both neutrophils and mononuclear cells circulating in the blood of the arthritic animals (FIGURES 34 and 35). Using the ChS treated animals as drug treated control group, it 6 was evident that this suppressive effect of CaP was dose dependent, the 300mg/kg dose being the more effective, particularly on circulating neutrophil nmmbers(FIGURE 34). While these blood borne white cells serve as an important vanguard in the immune defense against invading pathogens, once activated, as occurs in inflammatory states such as rheumatoid arthritis and osteoarthritis, emphysema, inflammatory bowel 10 disease, vasculitis and suchlike, they provide an ongoing source of destructive proteinases, oxygen derived free radicals and pro-inflammatory cytokines all of which contribute to the initiation and perpetuation of the disease process. Immunosuppression in these inflammatory conditions , has and continues to be, an important therapeutic objective for their treatment (St. Georgiev V, Immunomodulating drugs: major 15 advances in research and development. in Immunomodulating drugs, Editors : St, Georgiev V and Yamaguchi H, Ann NY Acad Science, 1993, 685, 1-10). Moreover, immunosuppressive therapy has been extensively used for the management of chronic rheumatoid arthritis for over 30 years (Weinblatt MB, Immunosuppressive therapy in rheumatoid arthritis, in: Therapeutic control of inflammatory disease, new approaches 20 to antirheumatic drugs, Editors: Otterness I, Lewis A, Capetola R, Advances in inflammation research , Vol 11, Raven Press, New York, 1986, pp265-276). Although the suppressive effects on white cells observed for high oral dose CaP in the rabbit model of mono-articular proliferative arthritis used here appear to be a novel finding for a nutraceutical preparation, it is known that oral administration of cartilage derived 25 antigens, such as type II collagen peptides can induce cell anergy and/or active cellular suppression of immune responses by promoting clonal selection and the secretion of anti-inflammatory cytokines, such as interleukins -4 and 10 and transforming growth factor- beta (TGF-beta). Furthermore, oral administration of these cartilage antigens have be used to treat inflammatory joint diseases such as rheumatoid arthritis( Kagnoff 30 MF, Oral tolerance: mechanisms and possible role in inflammatory joint diseases, Baillieres's Clinical Rheumatology, 1996, 10: 41-54; Weiner HL and Komagata Y,. Oral Tolerance and the treatment of rheumatoid arthritis. Sem Immunopatbol, 1998, 20: 289-308) 35 49 INDUSTRIAL APPLCABIUTY GAG-peptide complexes and polypeptides substantially free of DNA may be used, either directly or after further processing for the treatment, protection and restoration of connective tissues in inflammatory and degenerative tissue disorders such 5 as rheumatoid arthritis and osteoarthritis in any of their multiple forms, atherosclerosis and myocardial ischernia, degenerative and diabetic arteriopathies, thrombosis and embolisms, hyperlipideamis and as anti-angiogenic agents for tie treatment of cancers. Because the compounds are substantially free of DNA, they are also substantially free of other molecules such as viruses that may be associated with DNA 10 An avoidance of such associated molecules could avoid health risks. Bovine and other animal nucleic acids are strongly bound to or form complexes with retroviruses and heat/protease resistant prion proteins which have beep implicated in the spread of transmissible spongiform encephalopathies such as Creutzfeld-Jakob disease, kuru, Gerstmann-Straussler-Scheiner syndrome in humans, scrapie in sheep and goats, and 15 bovine spongioform encaphalopathies in cattle. Calcium salts of GAG-peptide complexes have been found to have particular pharmacological efficacy which is father enhanced in combination with matrix polypeptides. Ingestion of calcium salts would provide an added benefit in being a source of dietary calcium. 20 The method of the present invention is essentially non-disruptive to the connective tissue used. Thus the residual tissue particles remaining after the autolysis is completed and the medium containing the GAG-peptide complexes and polypeptides has been removed, can still be used as a source of collagen derivatives. In this regard it should be noted that traditional methods for preparing ChS from connective tissues 25 such as cartilage use exhaustive proteolytic or chemical digestion to destroy the proteinaceous components of the matrix to allow the ChS to be released into aqueous solutions and isolated. The residual cartilage particles obtained by the present method can be hydrolysed proteolytically or chemically to obtain collagen peptides of variable molecular size to be used for the treatment, protection and restoration of connective 30 tissues in inflammatory and degenerative disorders such as rheumatoid arthritis and osteoarthritis in any of their multiple forns or for the enhancement of wound healing or for the preparation of artificial biomatrices for cell culture, cell transplantation, or delivery of bioactive compounds including drugs and growth factors into a host tissue. It will be appreciated by persons skilled in the art that numerous variations 35 and/or modifications may be made to the invention as shown in the specific embodiments without departing krom the spirit or scope of the invention as broadly 50 described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (10)

1. A method for the preparation of a composition comprising residual cartilage tissue particles or collagen peptides or mixtures thereof the method comprising the 5 steps of: (a) subjecting particles of connective tissue to enzyme mediated autolysis by contacting with an autolysis medium containing a divalent salt at a pH of from about

2.5 to about 8.5 and at an effective temperature such that at least one GAG-peptide 10 complex and at least one polypeptide are released substantially free of DNA, by the proteolytic actions of an endogenous enzyme(s) from within the tissue particles, into the autolysis medium leaving at least one residual cartilage residue tissue particle; (b) recovering at least one residual cartilage tissue particle and/or 15 (c) hydrolysing the at least one residual cartilage tissue particle to obtain a collagen peptide. 2. The method according to claim I wherein the residual cartilage tissue particle is 20 proteolytically or chemically hydrolysed.

3. The method according to claim 1 or 2 wherein the method further comprises recovering a collagen peptide. 25

4. The method according to any one of claims 1-3 further comprising the step of recovering at least one GAG-peptide complex and at least one polypeptide.

5. A composition comprising a residual cartilage tissue particle prepared according to the method of any one of claims 1-4 30

6. A composition comprising a hydrolysed cartilage tissue particle prepared according to the methods of any one of claims 1-4

7. A composition comprising a collagen peptide prepared by the method according 35 to any one of claims 1-4. 52

8. Use of the residual cartilage tissue particle prepared according to the method of claim 1 as a source of a collagen peptide.

9. A method for the preparation of a composition comprising collagen peptides the 5 method comprising the steps of: (a) subjecting particles of connective tissue to enzyme mediated autolysis by contacting with an autolysis medium containing a divalent salt at a pH of from about 2.5 to about 8.5 and at an effective temperature such that at least one GAG-peptide 10 complex and at least one polypeptide are released substantially free of DNA, by the proteolytic actions of an endogenous enzyme(s) from. within the tissue particles, into the autolysis medium leaving at least one residual cartilage residue tissue particle; and (b) hydrolysing the at least one residual cartilage tissue particle to obtain a collagen 15 peptide.

10. Use of a collagen peptide prepared according to the method of any one of claims 1-4, or cy9 in the preparation of a medicament for treating or preventing arthritis or other degenerative disease. 20