PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT AND/OR THE PREVENTION OF OSTEOARTICULAR DISEASES

Abstract

An intra-articular pharmaceutical composition is used for the treatment and/or the prevention of acute or chronic osteoarticular diseases and acute or chronic osteoarticular symptoms especially osteoarthritis. The composition includes a possibly adequate pharmaceutical carrier or diluent, a glycosaminoglycan, a compound activating the alpha 2 adrenergic receptor, an anti-inflammatory agent and stem cells.

Claims

1.-31. (canceled)

32. A method for treating an osteoarticular disease in a mammalian subject, the method comprising intra-articularly injecting in a joint of the subject in need thereof a pharmaceutical composition comprising: an effective amount of one or more glycosaminoglycan(s); and an effective amount of one or more compound(s) activating the alpha 2 adrenergic receptor; wherein the pharmaceutical composition reduces joint degeneration by providing a synergic decrease in articular accumulation of liquid and inflammation, a synergic reduction of joint pain or synergic improvement of joint mobility and function for two weeks or more.

33. The method of claim 32, comprising administering the pharmaceutical composition by intra-articular injection to the patient once a week.

34. The method of claim 32, comprising administering the pharmaceutical composition by intra-articular injection to the patient twice a week.

35. The method of claim 32, comprising administering the pharmaceutical composition by intra-articular injection to the patient once in two weeks or less.

36. The method of claim 32, wherein the pharmaceutical composition comprises from about 0.1 to about 100 mg of the glycosaminoglycan per kg of body weight of the subject and from about 0.1 g to about 1 mg of the compound activating the alpha 2 adrenergic receptor.

37. The method of claim 32, wherein the intra-articular injection of the pharmaceutical composition reduces nociceptive pain associated with the osteoarticular disease.

38. The method of claim 32, wherein the compound activating the alpha 2 adrenergic receptor is an agonist of the alpha 2 adrenergic receptor.

39. The method of claim 32, wherein the osteoarticular disease is selected from the group consisting of osteoarthritis, degenerative arthritis, gonarthrosis, and coxarthrosis.

40. The method of claim 32, wherein the pharmaceutical composition further comprises human adult mesenchymal stem cells or non-human embryonic mesenchymal stem cells.

41. The method of claim 32, wherein the pharmaceutical composition further comprises a pharmaceutical carrier or diluent.

Description

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0049] FIG. 1 shows the VAS scale assessing pain from 0 no pain at all to 100 maximum pain after 3 intraarticular administration of clonidine 150 g and hyaluronic acid at day 0, day 7 and day 14 in 2 patients suffering from gonarthritis.

[0050] FIG. 2 shows the WOMAC score assessing pain and function of the knee after 3 intra-articular administration of clonidine 150 g and hyaluronic acid at day 0, day 7 and day 14 in 2 patients suffering from gonarthritis (worse situation=96).

[0051] FIG. 3 shows the Lequesne score assessing algofunctional score of the knee after 3 intra-articular administration of clonidine 150 g and hyaluronic acid at day 0, day 7 and day 14 in 2 patients suffering from gonarthrosis (worse

[0052] FIG. 4 shows the VAS scale assessing pain from 0 no pain at all to 100 maximum pain after one intra-articular administration of clonidine 150 g and hyaluronic acid at day 0 in 1 patient suffering from coxarthrosis.

[0053] FIG. 5 shows the WOMAC score assessing pain and function of the hip after one intraarticular administration of clonidine 150 g and hyaluronic acid at day 0 in 1 patient suffering from coxarthrosis.

[0054] FIG. 6 shows the Lequesne score assessing pain and function of the hip after one intra-articular administration of clonidine 150 g and hyaluronic acid at day 0 in 1 patient suffering from coxarthrosis.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The term intra-articular as used herein refers to a percutaneous injection of a joint with the pharmaceutical composition of the invention.

[0056] The term local administration as used herein refers to a percutaneous injection in or in the vicinity of an inflamed joint. Local administration injection thus concerns the epidermis, the dermis, the muscle, or any deep organ.

[0057] The main advantages to local administration are to selectively restrict the analgesic and other effects to the injured areas. Furthermore, the local administration allows for high local concentration levels with little or no systemic release. The local administration and the intra-articular injection of hyaluronic acid is a recognised treatment of the above mentioned conditions.

[0058] The local administration and the intra-articular injection of alpha 2 adrenergic agonists are easily realizable and provide long lasting effect in combination with proteoglycans. In consequence, the problems related to placement of an invasive drug delivery system and the problems of bothersome side-effects, due to systemic release, can be strongly minimized. Health-related quality of life, patient satisfaction and economic assessment might be improved with such a treatment, especially in chronic conditions.

[0059] Preferably, the administrated compound is selected from the group consisting of a glycosaminoglycan, for example hyaluronic acid or a salt thereof or an ester of hyaluronic acid with an alcohol of the aliphatic, heterocyclic or cycloaliphatic series, or a sulphated form of hyaluronic acid or combination of agents containing hyaluronic acid. Suitable dosages of a proteoglycan-based hydrogel or a glycosaminoglycan or a derivative thereof will typically be from about 0.1 mg to about 100 mg/kg body weight per day or from about 0.5 mg to about 10 mg/kg body weight per day more preferably from about 2 mg to about 8 mg by body weight per day and suitable dosage amounts for alpha 2 adrenergic receptor agonists.

[0060] In a preferred embodiment, the alpha 2 adrenergic receptor agonist may be clonidine, p-aminoclonidine, tiamenidine, 5-bromo-6-(2 imidazolidine-2-ylamino) quinoxaline, dexmedetomidine, detomidine, medetomidine, oxymetazonline, tizanidine, mivazerol, lofexidine, xylazine, guanfacine, guanclofine, guanoxabenz, or a derivative or structural analogue thereof, alpha-methyinorepherine, azepexole, indoramin, 6-allyl-2-amino-5,6,7,8-tetrahydro4H-thiazolo [4,5-d] azepine diHCl or a compound selected from the table 1 and analogs thereof.

TABLE-US-00001 TABLE 1 Classification of the alpha-2-adrenergic receptor agonists guanidines agmatine betanidine biguanides cimetidine creatine gabexate guanethidine guanfacine guanidine impromidine iodo-3 benzylguanidine methylguanidine mitoguazone nitrosoguanidines pinacidil robenidine suIfaguanidine zanamivir imidazoles 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one urocanic acid amino-imidazole carboxamide antazoline biotine bis (4-methyl-l-homo piperazinylthiocarbonyl)disulfide carbimazole cimetidine clotrimazole creatinine dacarbazine dexmedetomidine econazole enoximone ethymizol etomidate fadrozole fluspirilene histamine histidinol idazoxan imidazolidines imidazolines clonidine tolazoline impromidine levamisole losartane medetomidine miconazole naphazoline niridazole nitroimidazoles ondansetron oxymetazoline phentolamine tetramisole thiamazole trimetaphan Derivatives of clonidine 2,6-dimethylclonidine 4-azidoclonidine 4-carboxyclonidine-methyl 3,5-dichlorotyrosine 4-hydroxyclonidine 4-iodoclonidine alinidine apraclonidine chlorethylclonidine clonidine 4-isothiocyanate clonidine 4-methylisothiocyanate clonidine receptor clonidine-displacing substance hydroxyphenacetyl aminoclonidine N,N-dimethylclonidine

[0061] The active compounds used in accordance with the invention are known. Pharmaceutical preparations containing hyaluronic acid are commercially available as are clonidine and other alpha 2 adrenergic receptor agonists. The compounds can be manufactured in a known manner essentially in accordance with the processes described in the prior art.

[0062] The term clonidine as used, herein refers to N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine and includes the pharmaceutically acceptable salts thereof, e.g., salts with inorganic acids, such as hydrohalic acids, or with organic acids, for example lower aliphatic monocarboxylic or dicarboxylic acids such as acetic acid, fumaric acid or tartaric acid or aromatic carboxylic acids such as salicylic acid are also suitable.

[0063] Clonidine is employed in a therapeutically effective amount. The present invention also encompasses the use of alpha 2 adrenergic receptor agonist for the manufacture of injectable formulations for a delivery to a joint or a close region thereof, a therapeutic dose of said agonist by intra-articular injection, wherein said solution comprises from about 3 g to about 1500 g of said alpha-2-adrenergic receptor agonist. Preferably, said formulation comprises from about 30 g to about 500 g of said alpha-2-adrenergic receptor agonist. More preferably said solution comprises from 50 g to 350 g of said alpha-2 adrenergic receptor agonist. In a preferred embodiment, the alpha 2 adrenergic receptor agonist is clonidine. The actual concentration of clonidine may vary, depending on the nature and degree of the pain syndromes being treated and whether the drug is being administered for therapeutic or prophylactic purposes.

[0064] Due to their anti-inflammatory properties, inhibition of pro-inflammatory cytokines TNF-alpha and IL1-beta and the increase of anti-inflammatory cytokines, such as TGF beta, alpha 2 receptor agonists in injectable administration will have applications in osteoarticular inflammatory conditions and in diseases where there are osteoarticular inflammatory conditions, such as those above mentioned.

[0065] According to the method and the formulation of the invention, an injection of the combination of hyaluronic acid and an alpha 2 adrenergic receptor agonist form induces a long lasting pain relief, both in human case and in an animal model of osteoarticular pain. The method is safe, devoid of major drug's side effects and allows for acute as well as chronic treatment and or prophylaxis without the use of too invasive technique.

[0066] Pharmaceutical carriers or adjuvant such as preservatives may be incorporated in an amount effective for inhibiting growth of microbes, such as bacteria, yeast and molds, in the composition. Any conventional preservative against microbial contamination of the product can be employed so long as it is pharmaceutically acceptable and is unreactive with clonidine. Preferred preservatives are antimicrobial aromatic alcohols, such as benzyl alcohol, phenoxyethanol, phenethyl alcohol, and the like, and esters of parahydroxybenzoic acid commonly referred to as paraben compounds, such as methyl, ethyl, propyl, and butyl esters of parahydroxybenzoic acid and the like and mixtures thereof, but are not limited thereto. Particularly preferred are benzyl alcohol and phenoxyethanol.

[0067] Optionally, anaesthetic agent, such as lidocaine and the like, can be included. For administration according to the invention the active quantities of the compounds that alleviate neuropathic pain can be contained together with customary pharmaceutical excipients and/or additives in solid or liquid pharmaceutical formulations.

[0068] Liquid, preparations such as solutions, suspensions or emulsions of the active ingredients, prepared by dissolving or dispersing the compounds, can contain the usual diluents such as water, oil and/or suspending aids such as polyethylene glycols and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension and such like. Further additives such as preservatives, flavouring agents and such like may also be added.

EXAMPLES

Example 1

[0069] Four patients suffering from gonarthritis with knee effusion were administered one intraarticular injection of about 20 mg of hyaluronic acid and about 150 g of clonidine in the injured knee. A second and a third intra-articular injections were realized 7 and 14 days later. Before each injection, the intra-articular liquid was removed and measured. Pain and mobility were evaluated at baseline (day 0), 48-72 hours after the intra-articular injections, before the next injection (day 7 and day 14) and at day 21. The evaluations consisted in a 0-100 mm visual analogue scale filled in by the patient recording pain intensity (0=no pain, 100=intolerable pain), the WOMAC score (worse possible score=96) recording pain and mobility and an algofunctional scale (Lequesne Index, worse score=24).

[0070] As shown in FIGS. 1, 2 and 3, the intra-articular administration of the combination induced unexpectedly an relief in pain (decrease in VAS from 86.5 mm to 14.5 mm) in 48 hours and the 2 re-administrations induced further improvements (VAS score=11 at day 9, 12 at day 16). This unexpected effect by comparison to the effect of either product was maintained for at least 1 week after the 3.sup.rd injection as VAS score was 13 at day 21. Same results were obtained using the WOMAC score which additionally evaluated mobility of the injured knee. The WOMAC score decreased from 61 at baseline to 44, 41 and 37 at days 7, 14 and 21. Likewise the algofunctional score evaluated using the Lequesne scale showed a dramatic improvement with scores decreasing from 20 at baseline to 15, 13, and 12 respectively at day 7, 14 and 21. These results were totally unexpected as hyaluronic acid has not been shown to decrease pain score by more than 15 mm in meta-analyses that reassessed its efficacy and the effects of hyaluronic acid do not occur before 3 to 4 weeks. No data are available for clonidine in osteoarthritis.

Example 2

[0071] To two patients suffering from coxarthritis, was administered a single intra-articular injection of about 20 mg of hyaluronic acid and about 150 g of clonidine in the injured hip. Pain and mobility were evaluated at baseline (day 0), and at day 7, day 14, day 21 and day 28. The evaluations consisted in a 0-100 mm visual analog scale filled in by the patient recording pain intensity (0=no pain, 100=intolerable pain), the WOMAC score (worse possible score=96) recording pain and mobility and an algofunctional scale (Lequesne, worse score=24).

[0072] In patients with coxarthritis who were treated with this combination, the effect of a single intra-articular injection of about 20 mg of hyaluronic acid and about 150 g of clonidine induced an immediate, marked, and prolonged effect as shown in FIGS. 4, 5, and 6. VAS decreased from 52 mm at baseline to 10, 11, 42 and 51 at days 7, 14, 21 and 28. Likewise, the WOMAC score decreased from 69 at baseline to 27, 28, 43 and 72 at days 7, 14, 21 and 28 respectively and the algofunctional score from 13 to 8 and 8 at days 7 and 14 and returned to pre injection values at days 21 and 28: (13 and 14 respectively).

[0073] These results were unexpected as there is no reports of such improvement in the 3 weeks following the administration of hyaluronic acid. No data are available with clonidine. The prolonged antalgic effect was also totally unexpected.

[0074] In summary, patients treated by simultaneous intra-articular injection of clonidine and of hyaluronic, showed a dramatic improvement of pain score (80%) and of joint functional score (60%); improvements which were maintained for 2-3 weeks.

[0075] Similar favourable evolution was observed for swelling, which appeared to be significantly reduced. In this feasibility study, no major side-effects have been reported. Interestingly, only partial treatment escape was observed.

Example 3

Clinical Experience with Intra-articular Products

[0076] The most comprehensive clinical data have been reported for intra-articular (i) corticosteroids, (ii) hyaluronic acid and (iii) TNFCC inhibitors.

[0077] In a recent paper, Uthman et al. (Postgrad Med J, 2003 August; 79(934):449-53) reviewed all published data with intra-articular products. Besides corticosteroids and hyaluronic acid, only anecdotic reports have been published on substances or approaches as diverse as orgotein, radiation synovectomy, dextrose prolotherapy, silicone, saline lavage, saline injection without lavage, analgesic agents, glucosamine, somatostatin, sodium pentosan polysulfate, chloroquine, mucopolysaccharide polysulfuric acid ester, lactic acid solution, and thiotepa cytostatics, without conclusive evidence of efficacy.

Example 4

Comparison of Clonidine/Hyaluronic Acid with Intra-articular Anti-Inflammatory Treatments

[0078] A (historical/published data) comparison of standard mono-arthritis treatments with the combination clonidine-hyaluronic acid tends to favour this combination for the efficacy on mono-arthritis symptoms.

[0079] The efficacy is measured by a reduction in score value (which demonstrates an improvement), such as pain scores or functional scores.

TABLE-US-00002 Improve- Reduction ment in Reduction Side- in Pain Function in Swelling Effects** A.sub.2A/HA 85% 55% +++ None Corticosteroids* 30% 30% +++ 5% Hyaluronic acid* 10% 15% n.r. None TNF inhibitors* 30% n.r. ++ None n.r. = not reported, n.m. = not measured; *average data from available meta-analyses and/or published studies (Bliddal et al, 2006, Bliddal et al 2006, Fernandez Lopez and Ruano-Ravina, 2006, Arrich et al., 2005; Bellamy et al. 2006; Bellamy et al. 2006); **major side effects only

Example 5

Comparison of A2A/HA With Intra-Articular HAs

[0080]

TABLE-US-00003 Reduction in Pain Improve- Reduction (max effect ment in Reduction in Pain reported) Function in Swelling A.sub.2A/HA 85% 55% +++ Hyaluronic Acid Arich et al., 2005 0% 17% 11% n.r. Wang et al., 2004 8% 24% n.r. n.r. Kolarz et al., 2003 55% 23% n.r. Lo et al., 2004 7% 47% n.r. n.r.

[0081] The maximal hyaluronic acid, effects reported in literature is a 55% improvement in pain and a 23% in function (Kolarz et al. 2003), but these effects seem, to have never been replicated (Arich et al., 2005; Wang et al., 2004; Lo et al., 2003).

[0082] These maximum hyaluronic acid effects are significantly below the effects reported with the combination clonidine-hyaluronic acid (on pain 55% vs 85% as well, as function 23% vs 55%, respectively). There is no reported benefit on swelling with hyaluronic acid, a major pain component in osteoarthritis and other inflammatory joint disorders, while there is a strong reduction in swelling with the combination clonidine and hyaluronic acid.

[0083] The effect of clonidine has never been studied in chronic knee pain before.

[0084] The effects of clonidine in acute pain studies have been show not to exceed 72 hours (Gentili et al, 2001), with even a minimum effect duration of 4 hours (Alagol et al, 2005), while the combination clonidine-hyaluronic acid has been shown to have an effects of up to 2 weeks.

Example 6

[0085] The effects of clonidine above were

[0086] The inhibition of IL-1beta release by alpha-2-adrenergic agonists, induced by LPS stimulation of TH1 cells, is measured under control conditions (dexamethasone).

[0087] The results of the study are: [0088] Dexamethasone 10.sup.7 M inhibits 65% of IL-1 beta secretion by LPS-stimulated TH1 cells [0089] Mivazerol and clonidine, at 10.sup.7 M and 10.sup.5 M, display 80-90% of DEX (10.sup.7 M) efficacy [0090] Guanfacine and moxonidine are at 40 to 60% of DEX effect, while others have only marginal effects [0091] Guanabenz, Brimonidine and Methyldopa have no effects

Example 7

[0092] The effects of the combination clonidine and hyaluronic acid were tested in a model of Peripheral Blood Mononuclear Cells. In brief, PBMCs (Peripheral blood mononuclear cells) from heparinized venous blood of healthy volunteers were isolated by Ficoll-gradient centrifugation. MNC (Mononuclear cells) were washed three times in PBS and resuspended in RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, and 10% heat-inactivated FBS. Cells were seeded at 100.000 cells in 96-well plate in a total volume of 200 L/well.

[0093] PBMC, at 100.000 cells/200 L, were plated in 96-well microtiter plates and stimulated or not 10 g/mL phytohemagglutinin (PHA). Effects of hyaluronic acid HA (200 g/mL) and p-aminoclonidine A2A (5 M) were tested on PBMC.

[0094] Proliferation Assay. The culture was incubated with 1 Ci/mL 3H-thymidine for 24 h before end of culture to measure the proliferation. Then, cells were washed twice with ice-cold PBS and twice with ice-cold 5% trichloroacetic acid (TCA). Finally, cells were lysed with 0. IN NaOH-0.1% Triton-X100. Supernatants were collected and analyzed on a beta-counter in presence of scintillation liquid. Results were done in cpm (count per minute=disintegration number of radioactive element observed in series of successive counts of one minute). IL-1 detection (By ELISA). Levels of IL-1 from PBMC or culture supernatants were measured by Quantikine ELISA kit (R&D Systems Inc, Minneapolis, USA). The minimum detectable concentration of IL-1 was estimated at 1.0 pg/mL.

[0095] The release of IL-1beta by PBMC and the proliferation of PBMC, induced by PHA stimulation, were set as controls, and compared with the measures for PBMC treated with HA and/or A2A.

[0096] The inhibition of PBMC release of IL-1beta and the inhibition of PBMC proliferation, induced by LPS stimulation of TH1 cells, were also measured and provided similar trends as for PHA stimulated cells.

Effects of HA Alone

[0097]

TABLE-US-00004 IL-1 detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND* 769 PBMC + PHA 2185 16069 PBMC + HA 2095 364 PBMC + PHA + HA 6394 9087 IL-1 detection Proliferation Conditions tested (%) (%) PBMC ND* 5 PBMC + PHA 100 100 PBMC + HA 96 2.2 PBMC + PHA + HA 293 57

[0098] IL-1beta secretion induced by PHA stimulation shows a significant increase over basal condition (where IL-1beta levels were undetectable) and was set at 100. Proliferation of PMBC cells also significantly increased, by a factor of about 20. Surprisingly, a similar cytokine increaseover basal conditionwas observed with addition of HA alone, while PBMC proliferation was not affected. When stimulated by PHA, the addition of HA synergistically increases IL-1beta secretion, 3 times over the stimulated conditions, but was able to reduce PBMC proliferation by a factor 2.

Effects of A2A Alone

[0099]

TABLE-US-00005 IL-1 detection Proliferation Conditions tested (pg/mL) (cpm) PBMC ND* 769 PBMC + A2A ND* 786 PBMC + PHA 2185 16069 PBMC + PHA + A2A ND* 16212 IL-1 detection Proliferation Conditions tested (%) (%) PBMC ND* 5 PBMC + A2A ND* 4.9 PBMC + PHA 100 100 PBMC + PHA + A2A ND* 101

[0100] A2A has no effect on basalunstimulated-conditions. Interestingly, when stimulated by PHA, the addition of A2A totally suppresses the increase in IL-1beta secretion due to PHA stimulation, to undetectable levels, but was not able to inhibit PBMC proliferation.

Effects of Combination of A2A & HA

[0101]

TABLE-US-00006 IL-1 detection Proliferation Conditions tested (pg/mL) (cpm) PBMC + PHA 2185 16069 PBMC + PHA + HA 6394 9087 PBMC + PHA + HA + A2A 3053 5445 IL-1 detection Proliferation Conditions tested (%) (%) PBMC + PHA 100 100 PBMC + PHA + HA 292 56 PBMC + PHA + HA + A2A 139 34

[0102] Interestingly, the combination HA/A2A demonstrates a potent anti-inflammatory effect over the PHA/HA condition. Indeed, the addition of A2A was able to significantly revertby over 50%the increase in IL-1beta secretion due to PHA/HA stimulation, bringing its level to the one of the PHA alone condition. Surprisingly, A2A was further able to inhibit PBMC proliferation, by another 35%.

[0103] During inflammation, lower molecular weight fragments of hyaluronan are known to be inflammatory and immune-stimulatory agents by inducing the secretion of cytokines such as IL-6 and monocyte chemoattractant protein (MCP-1). HA has also the property to enhance the adhesion of lymphocytes and monocytes to the extracellular matrix (Yamawaki et al., 2009). These experiments show that hyaluronic acid has a potent stimulatory effect on cytokine (e.g., IL-1) production without PBMC proliferation.

[0104] It has been demonstrated that clonidine alters the Th1/Th2 cytokine production (Xu et al., 2007; Cook-Mills et al., 1998). These experiments show that the addition of alpha 2 adrenoreceptor agonists totally suppresses the increase in cytokine (e.g., IL-1beta) secretion due to PHA stimulation, but is not able to inhibit PBMC proliferation.

[0105] Surprisingly, the combination HA/A2A demonstrates a potent anti-inflammatory effect both on cytokine production and inflammatory cell proliferation. Indeed, the addition of A2A to HA was able to significantly inhibit the increase in IL-1beta secretion and was farther able to inhibit PBMC proliferation.

REFERENCES

[0106] Jasmin Arrich, et al. (Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee: systematic review and meta-analysis. CMAJ 2005; 172 (8)2005) [0107] Bellamy et al. (Cochrane Database Syst. Rev., 2006 19, (2) CD005321) [0108] Barron, M, G. (J. Am. Osteopath., 107, ES 21-27) [0109] Zouher et al. (Paediath. Anaesth. 2005, 15 (11) p. 964-970). [0110] Luiz-Cleber P. et al. (Anaesth. Analog 2005, 101 (3) p. 807-811) [0111] Murphy D R (Med. Health R. I., 2000, 83 (4) p. 104-107) [0112] Armand, S et al. (Br. J. Anaesth. 1998, 81 (2) p. 126-134) [0113] Gentili M et al. (Pain 1996, 64, p. 593-596) Reuben S. et al. (Anaesthesial, 1999, 91, p. 654-658) [0114] Uthman I, et al. (Intra-articular therapy in osteoarthritis Postgrad Med J. 2003 August; 79 (934): 449-53 2003) Kolarz Genoteal (seminars in Arthritis and Rheumatism vol 32, no 5, 2003) Rubin B. (the Journal of the American Osteopathic Association 2005, vol 105, no 9, suppl. 4, p. 523-528) [0115] Wang Chen-Ti et al. (The journal of bone and joint surgery American volume, 2004, vol. 86-A, no 3 p. 538-549) [0116] Lo Grace et al. (The journal of the American medical association, vol 900 no 23, p. 3115-3121). [0117] Cook-Mills et al., Immunology. 85 544-549 (1995). [0118] Yamawaki H et al., Glycobiology, 19, 83-92 (2009).