Europaisches Patentamt J European Patent Office (ji) Publication number: 0 450 398 A2 Office europeen des brevets EUROPEAN PATENT APPLICATION A61 K 31/725 © Application number: 91104299.2 © int. CIA C08B 37/08, © Date of filing: 20.03.91 © Priority: 05.04.90 IT 1994890 © Applicant: BIOINDUSTRIA FARMACEUTICI S.p.A. © Date of publication of application: Via De Ambrosiis, 2-4-6 09.10.91 Bulletin 91/41 1-15067 Novi Ligure Alessandria(IT) © Designated Contracting States: @ Inventor: Federici, Giorgio AT BE CH DE DK ES FR GB GR IT LI LU NL SE Via De Ambrosiis 2-4-6 1-15067 Novi Ligure, (Alessandria)(IT) Inventor: Bertolotto, Rosa Maria Via De Ambrosiis 2-4-6 1-15067 Novi Ligure, (Alessandria)(IT) © Representative: Minoja, Fabrizio Studio Consulenza Brevettuale Via Rossini, 8 1-20122 Milano(IT) © Chondroitin sulfate complexes having anti-inflammatory activity, processes for the preparation thereof and pharmaceutical compositions containing them. © Copper-chondroitin sulfate complexes having anti-inflammatory, antiphlogistic and antioxidant ac- tivities, the process for the preparation thereof as well as pharmaceutical compositions containing them. 00 O) CO in a. Hi Rank Xerox (UK) Business Services EP 0 450 398 A2 The present invention relates to a copper-chon- tained from sodium chondroitin sulfate or chon- droitinsulfuric acid complex, to a process for the droitinsulfuric acid and a cupric salt such as preparation thereof and to pharmaceutical composi- CuCI2.2H2O or CuSC>4..5H2O and they can contain tions containing it. from about 0.5% to about 10% Cu by weight. The The use of copper salts or complexes in the 5 starting chondroitin sulfate can have either low treatment of patients suffering from acute or chron- (1,000-3,000), medium (2,000-12,000) or high mo- ic arthritis dates back to the 40's and 50's, when it lecular weight. was an alternative to the use of gold salts, that are The reaction of chondroitin sulfate with the often poorly tolerated. However, the use of copper cupric salt is carried out in an aqueous solution and salts and complexes, particularly cuprolene, w the complex is precipitated by adding organic sol- dicuprene and a copper-salicylic complex, was vents, preferably C1-C4. alcohols. abandoned due to two basic reasons : The pharmacological properties of the com- - discovery of hydrocortisone; plexes of the invention have been studied by - lack of knowledge of the importance of cop- means of the traditional experimental patterns for per in biological systems. 75 articular inflammation, such as the cotton pellet- More recently, intra-articular administration of induced granuloma and antigenic monoarticular ar- orgotein, also named superoxide dismutase, has thritis, induced by ovoalbumin emulsified with been proposed. Superoxide dismutase is a copper- Freund's adjuvant containing tuberculosis containing enzyme which can prevent superoxides, mycobacteria. Moreover, superoxide dismutase mi- responsible for hyaluronic acid degradation in the 20 metic activity was evaluated by measuring the in- synovial fluid, from being accumulated. Unfortu- hibition of cytochrome C reduction by the superox- nately, the efficacy of orgotein in the treatment of ide ion in the xanthine-xanthine oxidase enzyme arthritis or infective diseases is severely restricted, reaction. since this substance has a 6 minute half-life. In all the considered tests, the compounds of Complexes of copper with amino acids 25 the invention proved to be remarkably active, there- (tryptophan, Iysine), with non steroid anti-inflam- fore they could be used in the therapeutical treat- matory drugs (indomethacin, ketoprofen, acetylsali- ment of inflammatory diseases of various origin, cylic acid) or with fatty acids (oleic, lauric and such as degenerative arthropathies, rheumatoid ar- caprylic acids) have been proposed, but the devel- thritis, hip arthrosis, spondylarthrosis. For the en- opment thereof did not surpass the experimental 30 visaged therapeutic uses, the complexes of the and pre-clinical phases (Fourth International Con- invention will be administered in suitable pharma- ference on Superoxide and Superoxide Dismutase ceutical formulations for the oral or parenteral ad- - N A Roberts, P A Robinson). ministrations (i.m., i.v. or intra-articular), such as Chondroitinsulfuric acid, or chondroitin sulfate, capsules, tablets, syrups, vials and the like. belongs to the class of glycosaminoglycans, which 35 Daily dosages can vary within wide limits, are biological polymers abundant in soft connective thanks to the low toxicity of the complexes (i.p. and skeletal tissues, and it has been used for a LD50 in the mouse: 675 mg/kg): generally they will long time as an hypolipemizing agent. European range from 0.3 mg/kg/day to 3 mg/kg/day. Patent N. 66,283 discloses "eustatic" compositions The following examples further illustrate the containing one non-toxic metal ion and a 40 invention. glycosaminoglycan selected from hyaluronic acid or chondroitin sulfate, useful as cicatrizants and EXAMPLE 1 generally to improve cell and tissue trophism. How- ever, anti-inflammatory and anti-arthritis activities 20 g of sodium chondroitin sulfate (MW are not mentioned. 45 30,000±5,000) are dissolved in distilled water (400 Finally, interactions between Cu ion and ml) to a 5% concentration. The resulting solution is chondroitin sulfate have already been studied from added with 2.5 g of CuCI2.2H2O (equivalent to the chemicophysical point of view (e.g. in Inorg. 0.636 g Cu++) dissolved in 20 ml of water. The Chim. Acta, 1983, 78(3)-1 29-34), but not from the reaction mixture is stirred overnight, then treated biological and pharmacological one. 50 with 95° ethanol (3 volumes) to obtain a precipitate Now it has been found that copper-chondroitin- which is pump filtered and washed with absolute sulfuric acid complexes have excellent pharmaco- ethanol. 20.7 g of a Cu++ -chondroitinsulfuric acid logical properties, particularly anti-inflammatory, complex are obtained, containing 3% copper and anti-phlogistic, antioxidant and antiradical activities. 85% mucopolysaccharides. The product is in the Said copper-chondroitinsulfuric acid complexes can 55 form of a soft light blue powder, which is soluble in inhibit elastase, cathepsin, galactosidase and water, insoluble in organic solvents (ethanol, ac- glucuronidase at micromolar concentrations. etone, methanol). pH of a 1% aqueous solution is The complexes of the invention can be ob- from 5 to 6.5. EP 0 450 398 A2 times By electrophoresis for 10 min at 450 Volts on overnight, then centrifuged and washed 3 Cellogel strips in 1M carbonate buffer, pH 11, an with absolute ethanol, once with acetone, pump 10.3 homogeneous band is evidenced which migrates to filtered and dried under vacuum in a dryer. g the anode. The band contains copper, which is of the complex are obtained, in the form of a quantitatively titrated by atomic absorption, and 5 hygroscopic light blue powder, having the following chondroitinsulfuric acid which is revealed by the characteristics : copper : 0.84%; glucuronic acid : sulfur light blue coloration obtained in the presence of a 25%; hexosamine : 26%; nitrogen : 3%; : toluidine blue solution. 6%; proteins : 2%; chlorides : 2%. Studies on the Cu-chondroitinsulfuric acid bond were carried out subjecting a 2% aqueous solution 10 EXAMPLE 5 of the complex to ultrafiltration through membranes of various cut-off (2,000-5,000-20,000-30,000) to A solution of 50 g of sodium chondroitin sulfate confirm the stability of the complex. (MW 30,000±5,000) in 750 ml of water is added with 20 g of CuSO4.5H2O (equivalent to 5 g of EXAMPLE 2 75 Cu++). The mixture is stirred overnight, then treat- ed with 95° ethanol (3 volumes) to obtain a and washed with 60 g of sodium chondroitin sulfate (MW precipitate which is centrifuged in 30,000±5,000) are dissolved in water (1,000 ml) to absolute ethanol, to yield 56.2 g of a complex the follow- a 6% w/v concentration and transformed into the the form of a light blue powder, having acidic form by passing them through a resin col- 20 ing characteristics: soluble in water, pH = 4.5; cop- chlorides umn Amberlite IR/120 (H+). The obtained acid solu- per : 7.37%; mucopolysaccharides : 83%; sodium tion (pH 1.2) is slowly added with 10 ml of a 20% : 2%; sulfur : 5.8%; nitrogen : 2.9%; : solution of CuSO4..5H2O (equivalent to 0.508 g of 6.20%. Cu++), under strong stirring. Stirring is continued for 4 hours, pH is adjusted to 6 with 1N NaOH, 25 EXAMPLE 6 then the reaction mixture is treated with 95 ethanol to obtain a precipitate which is pump fil- A solution of 50 g of sodium chondroitin sulfate is added tered and dried under vacuum in a dryer, to+give (MW 30,000±5,000) in 500 ml of water CuSO4.5H2O to 7.63 of 60.5 g of the complex, containing 0.75% Cu and with 30 g of (equivalent g + is 85% mucopolysaccharides. The product is in the 30 Cu +) dissolved in 200 ml of water. The mixture 95° ethanol form of a light blue soft powder which is soluble in stirred overnight, then treated with (3 is water, pH= 6. Chlorides : 2%; nitrogen : 2.5%; volumes) to obtain a precipitate which centrifug- 57.7 sulfur : 5.9%; proteins : 1.3%; humidity : 5.3%. ed and washed with absolute ethanol, to yield g of a complex containing 8.7% copper. EXAMPLE 3 35 EXAMPLE 7 A solution of 10 g of depolymerized sodium chondroitin sulfate (MW 3,000±1,000) in 150 ml of 20 g of sodium chondroitin sulfate (injectable distilled water is added with 40 ml of a CUSO4..5H2O solu- grade, MW 30,000±5,000) are dissolved in obtained clear tion (equivalent to 84.7 mg of Cu++). The mixture is 40 water to a 5% concentration.