FIELD OF THE INVENTION

Oral compositions for the treatment of damaged connective tissue in joints with improved and efficient supply of exogenous precursors of chondroitin and hyaluronan, the key biosynthetic building-block for the repair of joint cartilages and synovial fluids. BACKGROUND

Proteoglycans (PG) are complex macromolecutes comprised mainly of long chains of modified sugars called glycosaminoglycans (GAG) which attract and retain water within the cartilage formation, providing unique mechanical properties for flexibility, resiliency, and resistance to and recovery under compressive forces.

GAG have mainly a linear structure repetition of a disaccharide units with five main kinds of GAG found in tissues and fluids of vertebrates: chondroitin sulfates, keratin sulfates, dermatan sulfates, heparin sulfates, and hyaluronan.

Hyaluronan is a non-sulphated GAG with polymeric structure of N-acetyl-D- glucosamine and D-glucuronic acid, being a key element in synovial fluid and articular cartilage. Chondroitin sulfate is a sulphated GAG with a polymeric structure of disaccharides of N-acetyl-galactosamine and D-glucuronic acid. Chondroitin sulfate, as

CS4 and CS6 is found in articular cartilage matrix and is a key factor in PG formation.

Synovial fluid is normally highly viscous due to its hyaluronan content and plays a crucial role in maintaining healthy cartilage and protecting the joint surface. Lack of hyaluronan leads therefore to joint disarrays. Synovial fluid is essentially an ultrafiltrate of plasma with the exception of the hyaluronan secreted by the synovium.

Connective tissues of the articular cartilage are naturally equipped to attempt to repair itself by manufacturing large amounts of collagen and PG. The process is placed under stress by injuries or chronic misuse. In such cases, the production of connective tissue matrix (collagen and PG) can double or triple over normal levels, thereby increasing the demand for the building blocks of both collagens and PG.

Under normal conditions, the body synthesizes sufficient amounts of base components to maintain and grow healthy articular cartilage, while limiting the production and release of destructive proteinases, inflammatory mediators and catabolic enzymes. There have been countless therapeutic approaches for management of joint disease, providing nutritional supplementation of metabolic precursors to the diet to aid in the biosynthesis of PG, GAG, hyaluronan, and collagen (see, U.S. Pat. Nos. 5,364,845 and

5,587,363). Numerous other disclosures also suggest the introduction of nutritional supplements comprising glucosamine salt, chondroitin sulfate, collagen and hyaluronan and others as therapy for the treatment of connective tissues, arthritic conditions, e.g.

U.S. Pat. Nos. 3,683,076; 3,697,652; 5,364,845, 5,587,363; 5,916,565; 5,922,692;

6,271,213; 6,492,349; 6,583,123; and 6,645,948.

Pharmacokinetic studies on humans and experimental animals revealed that chondroitin sulfate is absorbed orally with a relatively straight 1° order kinetics for single doses up to 3,000 mg.

However, the studies from Ronca et al. (Arzneimittelforschung. 1990; 40:319-23; ibid 1995 ;45:918-25) evidenced that chondroitin sulfate is not rapidly absorbed in the gastro-intestinal tract, although labelled GAG was found in synovium and cartilage. The actual bioavailability of chondroitin sulfate seem to fall in the range from 15% to

24% of the orally administered dose, which is a limiting factor in a nutritional supplement/pharmaceutical composition aiming to supply the building blocks for GAG and PG biosynthesis.

Paradoxically, a health intestinal flora may interfere with absorption and utilization of supplemented chondroitin. Kin D-H et al., J. Microbiol. Biotechnol. (2009), 19(2), published online 13-01-09 have measured the inhibitory effect of several lactic acid bacteria isolated from intestinal microflora and commercial probiotics against the GAG degradation by intestinal bacteria. Bifidobacterium longum HYl 30101 and Lactobacillus plantarum AK8-4 exhibited the most potent inhibition. This is another evidence that an effective therapeutic amount of chondroitin sulfate may be hampered by its scarce digestion pattern in gut either due to a low chodroitin lyasing microflora or to a high content of lactic microflora having an anti-chondroitinase action, or both.

Therefore, it remains a need in the art to enhance absorption the building blocks of connective and synovial tissues so to achieve a higher effectiveness in the treatment thereof. The present invention aims to meet these needs, at least in part. SUMMARY

The present invention provides pharmaceutical, nutritional or veterinary compositions for treating connective tissue and joint damages in man and in animals which comprise: i) chondroitin sulphate, ii) a stabilized, enteric-coated chondroitin-lyase or a microbial strain producing thereof, and, optionally, iii) a source of glucosamine.

The present invention also relates to such compositions and the oral use thereof to efficiently treat the joint and connective tissue damages by enhancing the bioavailability of the glycosoaminoglycans (GAG) and proteglycanes (PG) precursors. DETAILED DESCRIPTION In accordance with the invention, a composition comprising chondroitin sulphate and a stabilized, enteric-coated chondroitin-lyase or a microbial strain producing thereof and, optionally, a glucosamine source is provided.

Any species or combination of species of chondroitin sulfate may be used in practicing the invention, i.e. chondroitin 4-sulfate, chondroitin 6-sulfate, chondroitin 2,6-sulfate, chondroitin 4,6-sulfate, and chondroitin sulfate chain moiety in chondroitin sulfate proteoglycan. Both ichthyic and mammalian collagen chondroitins are suitable.

Preferred chondroitin sulfate are chondroitin 4-sulfate, alias chondroitin A or ChS-A; and chondroitin-6-sulfate, alias chondroitin C or ChS-C; and mixture thereof.

In a preferred embodiment, present composition shall comprise chondroitin sulphate in an amount from 150 mg to about 2500 mg, preferably 200 to 800 mg per unit dose.

The expressions "chondroitin-lyase or a microbial strain producing thereof as used herein means a chondroitin-lyase enzyme, purified, crude or lysate from a fermentation of microbial strain producing chondroitin-lyase, or a viable cell population thereof.

The term "chondroitin-lyase" as used herein means the hydrolytic enzymes acting on chondroitin with the catalytic cleavage of N-acetylhexosaminide links in said substrates. It comprises classic chondroitinase such as chondroitinase ABC, chondroitinase ABCI, chondroitinase ABCII, chondroitinase AC, chondroitinase B; or enzymes with chondroitinase-like activity such as hyaluronidase, collectively referred hereinafter as "chondroitinase". Chondroitinase may be obtained from a microorganism that naturally expresses a chondroitinase; e.g., but not limited to, Proteus vulgari;, or from the expression of a recombinant protein in a host cell a prokaryotic cell (such as E. colϊ) or an eukaryotic cell (such as yeast, a mammalian cell or an insect cell). Both native and recombinant chondroitinase are suitable for the present purpose.

In an embodiment, a suitable chondroitinase are selected from chondroitinase ABCx of different sources, for example from Proteus vulgaris e.g. as supplied by United States Biological (Swampscott; MA, USA); native chondroitinase ABCI e.g. as supplied by Sikagaku Biobusiness Corp. (Tokyo, Japan); recombinant chondroitinase ABC e.g. as supplied by Glyko Biomedical (Novato, CA, USA); and recombinant chondroitinase ABCI as supplied by Acorda Therapeutics Inc. (Hawthorne, NY, USA). Another suitable chondroitin-lyase is chondroitinase AC, e.g. native AC as supplied by United States Biological; and AC II Arthro (from Arthrobacter aurescens) by Sikagaku.

The expression "chondroitin producing microbial strains" as used herein comprise non-pathogenic or inactivated, native or recombinant form of E. coli, Arthrobacter aurescens, Flavobacterium heparinum, Bacteroides stercoris (e.g. HJ- 15), P. vulgaris or other human intestinal anaerobic bacteria producing chondroitinases.

In another embodiment, a suitable chondroitin-lyase is hyaluronidase (CAS#: 37326- 33-3; EC#: 3.2.1.35), which is actually also known as chondroitinase or chondroitinase I since it hydrolysis both hyaluronan and chondroitin sulfate A and C, primarily to tetrasaccharide residues.

Preferred source of hyaluronidase is testicular, i.e. from bovine or ovine tests, e.g. as supplied by Reliable Biopharmaceutical Corp. (St. Louis, MO, USA), Seravac Biotech PTY (Cape Town, South Africa), Worthington Biochem Corp. (Lakewood, NJ, USA), Diosynth BV (Apeldoorn, The Netherlands), Sigma-Aldrich (St. Louis, MO, USA), Ambipro Inc. (San Diego, CA , USA), or recombinant hyaluronidase, e.g. Hylenex™ supplied by Baxter (Deerfield, IL; USA).

The aforesaid chondroitin-lyases can be used in semi-purified or purified form, e.g. as in WO2005112986, or in crude form, i.e. obtained as cellular lysate of chondroitin-lyase producing microbial strains; or a crude testicular extract, e.g. bovine "orchic extracts". The amount of chondroitin-lyase or a microbial strain producing thereof will depend on the amount of chondroitin sulphate supplied per single administration, for it must be in condition to hydrolize the chondroitin in the lower gastrointestinal tract in a period from 1 to 6 hours. Since 1 units of enzyme liberates 1 μmole of di- or tetrasaccharides, the composition will comprise a chondroitin-lyase or a microbial strain producing thereof in an amount from 1 to about 10,0000 units, preferably from 10 to 100 units per unit dose for an weighted amount from 180 to 1800 mg of chondroitin sulphate. Taking into account the possible progressive decay of the enzyme strength at room temperature, an over-dosage from 2 to 10 times chondroitin-lyase content calculated accordingly shall be provided.

Chondroitin-lyase or a microbial strain producing thereof are herewith enteric-coated to protect their structure from degradation by the gastric acid. Instead, chondroitin sulphate and glucosamine source(s) are preferentially not gastroprotected, since the pre- digestion facilitates their absorption. The coating can be conceived in microsphere or microgranules dispersed in tablets, capsules, powder and so on; or as multilayer tablets, granulates etc. having the chondroitin-lyase or a microbial strain producing thereof in enteric-coated form with the remaining components present as a ready-soluble phase.

The enteric coating of chondroitin-lyase or a microbial strain producing thereof can be carried out according to conventional techniques, e.g. delivering chondroitin-lyase or a microbial strain producing thereof into microspheres or microtablets.

Various microcapsulation techniques with different outcomes in final particle size and composition can be applied, e.g. air suspension, phase-separation, airspraying, orifice/centrifugal method, supercritical -fluid technic, pancoating, solvent-evaporation, spray drying and coagulation, surface polymerization, melt cooling, and multiple emulsion solvent evaporation. In the former case, polyvinylalcohol (PVA) is commonly used but polyvinylpirrolidone, alginates, methylcellulose, gelatine can be used also as an emulsifier. Organic solvents can be removed under normal or reduced pressure.

The enteric-coated chondroitin-lyase or a microbial strain producing thereof can be produced by using a variety of materials including shellac, hydroxypropyl- methylcellulosephthalate (HPMCP, Pharmacoat 606, Pharmacoat 645), polyvinylacetate phthalate, cellulose acetate phthalate, gelatin, starch, albumin, collagen, fibrinogen, polyamino acid, polylactides (PLA), polyglycolides (PGA), poly D-hydroxybutyric acid (PHB), polycaprolactone, polyanhydrides, polyorthoesters, methacrylic acid copolymer Eudragit™ E-IOO, Eudragit™ L3D (Rohm & Hass, Germany), corn protein extract (Zein-DP), sodium alginate, alginic acid, shellac, carboxyvinylpolymer (Carbomer™), hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, hydroxyproprylmethyl acetate succinate, carboxymethylcellulose, cellulose acetate phthalate, hydroxypropylcellulose, ethylcellulose, methylcellulose, polyvinylacetate phthalate, soy protein, wheat protein, processed chitin or chitinic acids, carrageenan, pectin, guar gum, locust bean gum, xanthan gum, gellan gum, arabic gum, kollicoat™ MAE 30 DP (BASF company), medium chain triglycerides, hydrogenated palm oil, and the like.

In a preferred embodiment, enteric coating is carried out with shellac or analogue thereof usually in the range from about 1 to about 10% by weight Customary auxiliary agent in the coating process include anti-adhesives such as talc, lactose, plasticizers such as PVP, glycerol, fatty acids; and optionally dyes or pigments.

Another example of enteric coating are disclosed in U.S. Pat. N.os 4,079,125 and 5,750,104 wherein a chondroitin-lyase or a microbial strain producing thereof shall substitute the original digestive enzymes. Such compositions comprise the enzymatic source in a binder system consisting of polyvinylpyrrolidone (PVP), microcrystalline cellulose (Avicel™), cellulose acetate phthalate, methylcellulose and alginic acid, starch or modified starches, with disintegrant such as citric acid, alkaline carbonates.

In a preferred embodiment, the coated microgranules or microtablets comprising chondroitin-lyase or a microbial strain producing thereof are placed into soft or hard- shell capsules that contain chondroitin sulphate and other ingredients in free form.

Noteworthy, any enteric resistant compatible polymer or lipid complying with the regulations concerning nutritional and pharmaceutical use which allows the enzyme to be released in the intestine can be applied. For example, the enteric protection may be carried out by a non-porous, physiologically acceptable, gastric acid-resistant coating, e.g. insoluble in the pH range of 1.5 to 5 but soluble in the pH range of 5.5 to 9. In another embodiment, the form is a multilayer tablet in which chondroitin-lyase or a microbial strain thereof is inserted in an enteric-coated layer whilst chondroitin sulphate and other ingredients are located in the fast-releasing layer (e.g. Multi-layer™).

In a preferred embodiment, the composition of invention comprises a glucosamine source as useful building-block in GAG, PG, synovial and collagen biosynthesis.

The term "glucosamine source" as used herein means glucosamine and derivatives and salt thereof, e.g. glucosamine sulfate, glucosamine hydrochloride, and N-acetyl D- glucosamine, which are readily available from the hydrolysis of chitin.

In a preferred embodiment, the composition comprise glucosamine as above defined in amount from 100 to about 3500 mg, preferably from 200 to 700 mg per unit dose.

Another suitable glucosamine source is hyaluronan, which is readily depolymerised by the chondroitin-lyase enzyme within the composition to provide glucosamine disaccharides. Any species or combination of hyaluronan may be used herein, e.g. in the acid form as well as salt thereof, preferably Na, K, ammonium, Ca, or Mg salts. In certain embodiments, hyaluronan or salt thereof has a molecular weight in the range of between 300,000 and 3,000,000; preferably between 600,000 and 1 ,800,000.

In a preferred embodiment present composition shall comprise hyaluronan in an amount from 1 to about 500 mg, preferably from 5 to 100 mg per unit dose.

In a preferred embodiment, the compositions further comprise a joint regenerator agent such as methylsulfonylmethane (MSM), e.g. at 100 to 3500 mg per unit dose.

The composition of invention can further comprise other auxiliary ingredient suitably employed in the supplemental treatment of connective disorders, including Zn, Mn, Fe, Cu and other essential multivalent cations; or antioxidants, particularly flavonoids such as apigenin, kaempferol quercetin, silybin, apigenin, hesperidin, luteolin, anthocyanins; saponins and sapogenis; tannins; S-adenosyl methionine (SAM or SAM-e); and a variety of other ingredients known to in the art.

The composition of invention may be medicinal, i.e. pharmaceutical and nutritional preparations obtainable in a variety of unit dosage forms such as tablets, capsules, coated pills, powders, powder packets, granules, wafers, and liquid preparations. The composition of invention may use solid, semi-solid, or liquid vehicle/carriers. The compositions of the invention can also comprise other therapeutic agents insofar as it is generally used as a therapeutic for joint disease (arthropathy). Examples of other such therapeutic agents include, but are not limited to corticosteroids, nonsteroidal antinflammatory drugs, antirheumatics, immunoregulators, immunosuppressors, articular function augmenters, and interleukin inhibitors.

The present invention also provides a method for treating joint and connective tissue damage including, but not limited to, arthritic disease, osteoarthritis, rheumatoid arthritis, osterochondrosis dessicans, cartilage damage, joint injury, joint inflammation, joint synovitis, degenerative joint disease (DJD), post surgical DJD, traumatic injury, fracture, tendon damage, ligament damage, skeletal damage, musculoskeletal damage, and fiber damage by administering a composition as disclosed herein. The invention is now elucidated by way of the following, non-restrictive examples.

EXAMPLES Example 1 — Capsules Ingredients Quantity

Chondroitin sulfate 1000 mg

Glucosamine sulfate 500 mg

Microgranules with hyaluronidase from bovine testes ⁽¹⁾ 20 mg

MSM (methylsulfonylmethane) 500 mg ( ^l) Type VI-S, (Sigma-Aldrich), 300-1500 units/mg of total solid, enteric-coated in Microcoat™ system. Other ingredients: gelatin (capsules). Example 2 - Capsules with SAM

Ingredients Quantity

Chondroitin sulfate 500 mg Glucosamine hydrochloride 350 mg

Microgranules with chondroitinase ABC from Proteus v. ⁽²⁾ 50 mg MSM (methylsulfonylmethane) 200 mg

S-adenosyl methionine (SAM) 100 mg

^ Cat.# C3667 (Sigma-Aldrich), 5-25 units/mg of total solid, enteric-coated in Microcoat™ system. Other ingredients: gelatin (capsules). Example 3 - Multi-layer tablets The composition is conceived with Multi-layer™ technique, i.e. layer #1 and #3 as fast release; layer #2 enteric-coated with shellac as described in US2005186278.

Ingredients Quantity Shark Cartilage (chondroitin sulphate content ~ 15% w/w) 2000 mg Chondroitinase AC from Flavobacterium heparinum in shellac 200 mg Glucosamine hydrochloride plus customary excipients 500 mg

⁽²⁾ Cat.# C2780 (Sigma- Aldrich), 0.1 units/mg of total solid, coated according to US

2005186278. Example 4 - Multi-component tablets

Ingredients Quantity

Chondroitin sulfate 200 mg

Shark cartilage 350 mg

Bovine testis (orchid) extract q.b. Glucosamine sulfate 250 mg

MSM (methylsulfonylmethane) 125 mg

Other ingredients of the multi vitamin/mineral/phyto complex: thiamine HCl 1 mg; riboflavin 1 mg, niacinamide 1 mg, pyridoxine HCl 1 mg, ascorbic acid 1 mg, cyanocobalamin 10 meg, Zn methionine 1 mg, Se (chelate) 2 meg, copper (chelate) 100 meg, Cr (chelate) 2 meg, tocopheryl acetate 1 IU, vitamin A acetate 100 IU, 1-histidine

5 mg, boron 0.5 mg, alfalfa powder 12 mg, yucca extract (29% saponins) 0.5 mg, devils claw (powder) 0.5 mg, rutin (20 mg), cetyl palmitate 0.5 mg. Further ingredients: rice powder, stearin, Mg stearate, silica, pigments and excipients for tablets.

Example 5 - Modified CondrosulF ^M 800 A sachets of CondrosulF ^M 800 (IBSA, Pambio-Noranco, Switzerland) is opened and mixed with the appropriate chondroitin-lyase and repackaged in sachets. Ingredients Quantity

Chondroitin sulphate sodium salt 800 mg

Microgranules with hyaluronidase from bovine testes ⁽¹⁾ 15 mg Other ingredients: saccharin, E 320 (BHT), excipients for granulations. Example 6 - Jerry cans for liquid supplementation to horses Ingredients Quantity (in a 40 ml dose)

Chondroitin sulfate 250 mg

Glucosamine hydrochloride 3750 mg Vitamin C Mn ascorbate (Manganese ascorbate) 15 mg

Microgranules chondroitinase ABC from Proteus v. ⁽²⁾ 15 mg

MSM (methylsulfonylmethane) 2500 mg

The aforesaid ingredients are suspended in purifies water and canned in 2 1 PE-bottles. Example 7 - Chewable tablets for pets (dogs, cats) Ingredients Quantity

Glucosamine hydrochloride 500 mg

.Chondroitin Sulfate 400 mg

Vitamin C Mn ascorbate (Manganese ascorbate) 30 mg

Microgranules with hyaluronidase from bovine testes ^ 20 mg MSM (methylsulfonylmethane) 200 mg

Other Ingredients: brewers yeast, bacon flavour, desiccated liver, Mg stearate. Applicative Example — Pharmacokinetic control

The oral absorption and the bioavailability of chondroitin sulfate in the composition of invention can be assessed according to the method of Volpi N., University of Modena and Reggio Emilia, Italy (Osteoarthritis Cartilage, 2003; 11(6) :433-41).

In brief, venous blood samples (15 ml) is taken from a vein of the forearm using an indwelling catheter. Blood samples are collected in tubes with citrate as anticoagulant at predose and at given intervals after supplementation. The samples are stored at 4 ⁰ C,

_^ centrifuged within 1 h for 10 min and immediately divided into 2x2.5 ml aliquots, transferred into labelled test tubes and stored frozen at -20 ⁰ C until analysis.

Extraction from plasma can be performed according to Volpi N. Osteoarthritis

Cartilage 2002; 10:768-77 and the amount of chndroitin sulphate absorbed by the oral route can characterized and quantified by agarose-gel electrophoretic technique, densitometric scanning. The incremental composition of the constituent disaccharides is shown in Table I. Table I - Tentatively estimative of incremental absorption and composition of unsaturated disaccharides of formula (I) from chondroitin sulphate administered with (A) with, or without (B) an exogenous source of chondroitin-lyase enzyme.

R ² R ⁴ R ⁶ Δ A-B

Δ Di-Os H H H + 22%

Δ Di-6s H H SO ₃ ^" + 17%

Δ Di-4s H SO ₃ ^" H + 38%

Δ Di-2,6dis SOf H SO ₃ ^" + 15%

Δ Di-4,6dis H SOf SO ₃ ^" -

Δ Di-2,4dis SO ₃ ^" SO ₃ - H + 94%

Δ Di-2,4,6tris SO ₃ ^" SO ₃ ^" SO ₃ ^" -

MW _n 25x1 O ³

MW _W 67x1 O ³ MW _n : number average molecular weight; MW _W = weight average molecular weight.