SUSTAINED RELEASE FORMULATIONS USEFUL IN THE TREATMENT OF

DISEASES

FIELD OF THE INVENTION

The present invention concerns sustained release formulations and related kits. The present invention further relates to such sustained release formulations for use in the treatment of diseases such as osteoarticular diseases.

BACKGROUND OF THE INVENTION

Osteoarthritis, the most common form of arthritis, is a disease characterized by slow degenerative processes in the articular cartilage, subchondral bone associated with marginal osteophyte formation and low grade inflammation. Most cases of osteoarthritis are characterized by unknown causes and are referred as primary osteoarthritis. When the cause of the osteoarthritis is known, the condition is referred as secondary osteoarthritis. Secondary osteoarthritis is caused by another disease or environmental condition. Conditions that can lead to secondary osteoarthritis include repeated trauma or surgery to the joint structures, abnormal joints at birth (congenital abnormalities), gout, diabetes and other hormonal disorders. Other forms of arthritis are systemic illnesses such as rheumatoid arthritis and systemic lupus erythematosus (SLE).

Osteoarthritis involves mainly the hips, knees, spine and the inter-phalangeal joints. The most common symptom of osteoarthritis is pain in the affected joint(s) after repeated use. Pain and stiffness of the joints can also occur after a long period of inactivity. In severe osteoarthritis, complete loss of cartilage cushion causes friction between bones, causing pain at rest or pain with limited motion.

Currently, available pharmacological therapies target palliation of pain and include analgesic (i.e. non-steroidal anti-inflammatory drugs (NSAIDS), tramadol or opioids). However, the clinical presentation of osteoarthritis is usually monoarticular with fluctuations in intensity and localization over time. Therefore, local drug delivery is of great interest in order to avoid systemic side-effects. Several compounds have been used intra-articularly such as glucocorticoids and hyaluronic acid. Glucocorticoids are used for intra-articular treatment of acute arthritic flares: the short-term benefit of intra-articular corticosteroids in mono-arthritis flare is well established, but its longer term benefits have not been confirmed yet. Glucocorticoids are thought to be responsible for accelerating cartilage damage by inducing osteonecrosis or increasing the risk of septic arthritis (Bellamy et al., 2006, Cochrane Database Syst. Rev, Apr 19;(2):CD005328). Due to their toxicity, injectable corticosteroids can only be administered a few times, which leads, in absence of alternative, to early surgical intervention. Intra-articular injection of hyaluronic acid of high molecular weight is effective for restoring the mechanical integrity of the joint. Nevertheless, due to a high enzymatic activity in inflammatory joints, its half-life after injection is only about 6 to 8 hours. However, treatments using chemically cross-linked hyaluronic acid have suggested an increase in the half-life and the residence time of the reticulated hyaluronic acid in the joint.

In addition to the alteration of the joint due to the degeneration of endogenous glycosaminoglycans, it is known that pro-inflammatory cytokines play a fundamental role in the pathogenesis of synovial inflammation, and that the levels of TNF-a, IL-Ι β or IL-6 are increased in inflamed joints. Several novel therapies, that have recently been developed or are currently undergoing clinical trials for inflammatory rheumatic diseases, target several of these cytokines, such as TNF-a (etanercept, adalimumab and infliximab), IL-Ι β (anakinra) and IL-6 (MRA). However, although there is evidence that anti-cytokine therapies might be effective for arthritic patients, it is likely that targeting a single cytokine might benefit only specific patient subgroups. The most important TNF- α inhibitors have been shown to be effective on mono-arthritis when administered systemically. However, due to the potential severity of their side-effects (infections, neoplasia...) when administered systemically, their use is limited. In addition, the cost of injectable TNF-a inhibitors is high and their use is thereby limited to patients suffering to adverse reactions to steroids (Bliddal et al., 2006, Scand J Rheumatol. Sep-Oct;35(5): 341-5).

Recent developments in molecular biology have allowed discovering of novel mechanisms of action for known therapeutic classes such as alpha-2 adrenergic receptor agonists. Clonidine is a centrally acting alpha-2 adrenergic receptor agonist that stimulates the alpha-2 adrenoreceptors in the brain stem. It is generally used as an anti-hypertensive agent. Clonidine is also commonly referred to as 2,6-dichloro-N-2-imidazolidinyldenebenzenamine (C ₉ H ₉ CI ₂ N ₃ ). Recently, antiinflammatory properties have been demonstrated for this molecule and new applications such as the treatment of arthritic diseases have been considered. For instance, WO 2010/031819 describes a pharmaceutical formulation comprising an alpha-2 adrenergic receptor agonist for preventing or treating inflammatory pain and diseases in mucosa of oral cavity, pharynx and larynx.

WO 2009/129460 further describes the administration of an effective amount of clonidine, provided within biodegradable polymers, at or near a target site in order to relieve pain caused by diverse sources, including but not limited to spinal disc herniation (i.e. sciatica), spondilothesis, stenosis, discogenic back pain and joint pain, as well as pain that is incidental to surgery.

A formulation of clonidine with hyaluronan has been described. For example, WO 2009/101194 describes a pharmaceutical formulation comprising optionally a pharmaceutical carrier or a diluent, a glycosaminoglycan and an alpha-2 adrenergic receptor agonist, for use in the treatment and/or the prevention of acute or chronic osteoarticular diseases and/or symptoms by intra-articular injection. However, as indicated the half-life in the joint of the high molecular weight hyaluronic acid is only about 6 to 8 hours. Therefore, it is necessary to repeat injections once a week for 3-5 weeks resulting in discomfort for the patient.

In view of the above, a need exists for further and/or improved formulations. In particular, there remains a need for a formulation which allows prolonged drug release, particularly but without limitation for use in the treatment of osteoarticular diseases. It is an object of the present invention to provide formulations with sustained drug release.

SUMMARY OF THE INVENTION

The present inventors have found through extensive testing a formulation addressing one or more of the above-mentioned problems of the prior art.

Hence, a first aspect relates to a gel-forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients. Formulations applying the principles of the invention advantageously allow to prolong the release and hence the effect of the one or more pharmaceutical active ingredients, such as of an alpha-2 adrenergic receptor agonist and/or a corticosteroid, for instance in a joint. Furthermore, the present gel-forming formulation is a thermodynamically stable one phase formulation. Because of the regular structure, the present formulation provides a highly reproducible sustained release system in contrast to solutions involving biopolymers, polymers or formulations based on liposomes.

Surprisingly, the inventors also found that the present formulations protect their enclosed active ingredients or other components from the physical environment, thereby improving the stability of the active ingredients or other components in vivo. For example, the present formulations provide a protection of their enclosed active ingredients or other components against enzymatic degradation.

In addition, the inventors found that the present formulations advantageously allow to improve the pharmacological effect of the pharmaceutical active ingredients. For example, the present formulations allow to improve the articular function by an extended lubricating action on the joint and are thus effective for restoring the mechanical integrity of the joint.

Also disclosed is a method for producing said formulation comprising combining (e.g., admixing) the glycosaminoglycan, the sustained release agent and the one or more pharmaceutical active ingredients.

The formulation may additionally comprise one or more pharmaceutically acceptable excipients (e.g., solvents, carriers, diluents, etc.), particularly excipients compatible with the intended mode of administration of the formulation, such as in particular parenteral and preferably intra-osseous or intra-articular administration of the formulation.

Any formulations disclosed herein that are configured for use in medicine, whether or not comprising one or more pharmaceutically acceptable excipients in addition to the other herein recited elements, may be denoted by as pharmaceutical formulations or pharmaceutical compositions.

Another aspect provides a kit of parts comprising the above gel-forming formulation. Hence, disclosed is a kit of parts comprising a gel- forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids. Disclosed is as well a method for producing said kit of parts comprising including the formulation in a kit of parts.

Further provided is a kit of parts comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids, wherein the glycosaminoglycan, the sustained release agent and the one or more pharmaceutical active ingredients are configured to allow for producing (forming or obtaining) a gel- forming formulation as defined herein. Disclosed is as well a method for producing said kit of parts comprising including a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids, in a kit of parts.

A further aspect provides a medical device comprising any one of the gel-forming formulations or kits of parts as taught herein. In particular, a medical device is disclosed comprising a gel-forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients. Such medical device advantageously allows for parenteral administration, such as intra-osseous or intra-articular administration, of the formulation or kit of parts to a subject in need thereof.

An aspect provides any one of the formulations or kits of parts as taught herein for use as a medicament, preferably for use in the treatment (including throughout the present specification therapeutic and/or preventative measures) of osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably but without limitation osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis.

Hence, preferably disclosed is a gel-forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids, for use in the treatment of acute or chronic osteoarticular diseases such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis. The use of formulations of the present invention in the treatment of osteoarticular diseases is advantageous inter alia because these formulations allow efficient treatment during longer periods due to the extended release of the active ingredients. A further advantage of the present formulations is that they can allow simultaneous delivery of several active ingredients over a prolonged period of time. In addition, the inventors advantageously found that the present formulations allow for improving the articular function by their extended lubricating action on the joint. Hence, the present formulations advantageously provide increased patient compliance.

Also provided is use of any one of the formulations or kits of parts as taught herein for the manufacture of a medicament for the treatment of osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, monoarthritis and poly-arthritis. Thus, particularly intended is use of a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids, for the manufacture of a medicament for the treatment of acute or chronic osteoarticular diseases such as preferably osteoarthritis, rheumatoid arthritis, mono- arthritis and poly-arthritis.

Such treatment may typically involve parenteral administration, more preferably intra-osseous or intra-articular administration (injection) of the formulation.

Further provided is a method for treating osteoarticular diseases, including acute or chronic osteoarticular diseases, in a subject in need of such treatment, comprising administering to said subject any one of the formulations or kits of parts as taught herein. Particularly intended is a method for treating acute or chronic osteoarticular diseases in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of a formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids.

Such method of treatment may typically involve parenteral administration, more preferably intra- osseous or intra-articular administration (injection) of the formulation.

The formulations of the present invention are gel-forming formulations. The terms "gel- forming", "one phase" or "monophasic" can be used interchangeably herein. The recitation "gel-forming formulation" as intended throughout this specification refers to the capacity of the formulation to form a solid, jelly-like material (gel) for instance with a pseudoplastic behaviour. In particular, a gel- forming formulation forms a gel when combined with or exposed to materials and/or conditions conducive to gel formation, for example but without limitation, when dissolved or dispersed in a suitable liquid phase, such as in an aqueous solution or dispersion.

In preferred embodiments, the formulations as taught herein are not liposome-based, i.e., the glycosaminoglycan is substantially not, or is not, encapsulated within lipid bilayer liposomes or microspheres. Such non-liposome-based formulations are thus not subjected to a procedure or treatment intended to stimulate the formation of liposomes or microspheres such as typically sonication. Preferably the present formulations may be solutions, which is particularly advantageous for parenteral and more preferably intra-osseous or intra-articular administration of the formulations.

Preferably the present formulations may be configured for parenteral administration, such as parenteral injection. More preferably, the present formulations may be configured for intra-articular administration, such as intra-articular injection. Further preferred, the present formulations may be configured for intra-osseous administration, such as intra-osseous injection.

Without being bound to theory, the formulations as intended herein may consist of a solution or lamellar phase outside the body. Hence, the present formulations comprising a combination of a monoglyceride such as glycerol monooleate with a glycosaminoglycan are characterized by a low viscosity, whereby they can be easily administered such as for example injected in the joint. The formulations of the present invention can be converted into the cubic state as soon as the ambient water increases after parenteral administration such as for example after intra-osseous or intraarticular injection. Thereby, the viscosity of the present formulations may increase in situ. The formulations of the invention thus advantageously reach a suitable viscosity in vivo, leading to optimal release of the glucosamine and other advantages as described herein.

As noted, the gel-forming formulations taught herein comprise a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients. The sustained release agent as intended herein can be preferably a gel-forming agent.

The term "gel-forming agent" as intended throughout this specification encompasses agents capable of forming, a solid, jelly-like material (gel). In particular, gel-forming agents form a gel when combined with or exposed to materials and/or conditions conducive to gel formation, for example but without limitation, when dissolved or dispersed in a suitable liquid phase, such as in an aqueous solution or dispersion.

The sustained release agent and particularly the gel- forming agent as intended herein can preferably be a glyceride. The term "glyceride", as used herein, refers to an ester formed from glycerol and one or more same or distinct fatty acid(s). The terms "glyceride" and "acylglycerol" can be used interchangeably. The term glyceride encompasses monoglycerides (monoacylglycerol), diglycerides and triglycerides depending on whether one, two, or three fatty acids are esterified with glycerol. The sustained release agent and particularly the gel-forming agent as intended herein can further be a glycerate such as for instance but without limitation oleyl glycerate or phytanyl glycerate.

Preferably, the glyceride is a monoglyceride. A monoglyceride can be a 1 -monoacylglycerol or a 2- monoacylglycerol depending on the position of the ester bond on the glycerol moiety. Non- limiting examples of monoglycerides are for instance glycerol mono(o)leate (GMO), glycerol monolinoleate, glycerol monolinolenate, glycerol monopalmitate, glycerol monostearate or glycerol monolaurate.

The glyceride may be preferably an ester of glycerol and oleic acid. The term "oleic acid" refers to a monounsaturated omega-9 fatty acid, more particularly (9Z)-Octadec-9-enoic acid also known as cis-9-Octadecenoic acid or 18:1 cis-9. Particularly preferably, the glyceride is a monoglyceride with oleic acid, i.e., glycerol monooleate, also commonly denoted as glycerol monoleate, mono(o)lein, glyceryl monooleate, glyceryl oleate, (Z)-l-oleoyl-sn-glycerol, or 1,2,3-propanetriol 9-octadecenoic acid.

The inventors have found that the monoglyceride and preferably glycerol monoleate allows gel formation of the formulation upon contact of said formulation with an aqueous liquid such as physiological or bodily fluids, providing for advantages as discussed herein. Hence, the present formulation can be preferably configured such that the monoglyceride performs or acts as a gel- forming agent in the formulation (i.e., the monoglyceride is provided as a gel-forming agent). In other words, the present formulations can be preferably configured such that the monoglyceride allows the formulations to form a gel once administered parenterally, preferably intra-osseously or intra-articularly.

In some embodiments, the formulation can comprise the sustained release agent in a concentration ranging between 5 and 85% by weight (w/w). For example, the formulation can comprise the sustained release agent in a concentration ranging between 35 and 75% by weight (w/w), for example between 40 and 70% by weight (w/w), for example between 45 and 65% by weight (w/w), for example between 50 and 60% by weight (w/w). Preferably, the formulation can comprise the sustained release agent in a concentration ranging between 45 and 65% by weight (w/w).

Advantageously, the combination of a sustained release agent, and more preferably a sustained release agent as discussed above, with a glycosaminoglycan and one or more pharmaceutical active ingredients such as for instance but without limitation one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids, can enhance and improve the sustained release of any one of and preferably all of the one or more pharmaceutical active ingredients by amplification of the gel-forming process in situ. Indeed, the inventors have found that a sustained release agent preferably intended herein, such a monoglyceride, allows or enhances gel-formation of the formulation upon contact of the formulation with an aqueous liquid such as physiological or bodily fluids, for example synovial fluid. As an additional advantage, in situations where the glycosaminoglycan can also provide therapeutic benefit (e.g., in osteoarticular diseases), the present formulation can also be advantageous because it can enhance and improve the sustained release of the glycosaminoglycan.

Formulations as intended herein may preferably comprise glycerol monooleate as a sustained release agents and preferably gel- forming agent. In some embodiments, the present formulations can comprise glycerol monooleate in a concentration ranging between 5 and 85% by weight (w/w).

For example, the present formulations can comprise glycerol monooleate in a concentration ranging between 35 and 75% by weight (w/w), for example between 40 and 70% by weight (w/w), for example between 45 and 65% by weight (w/w), for example between 50 and 60% by weight (w/w). Preferably, the present formulations can comprise glycerol monooleate in a concentration ranging between 45 and 65% by weight (w/w), and more preferably in a concentration of about 55% by weight (w/w).

The inventors have thus found that the formulations of the invention allow advantageously prolonged and potentially at least partly concomitant release of the active ingredients, namely the one or more pharmaceutical active ingredients such as the one or more alpha-2 adrenergic receptor agonist and/or one or more corticosteroids, and where applicable potentially also of the glycosaminoglycan. Indeed, the formulations of the invention ensure an immediate release of the active ingredients within the first hours after injection followed by a sustained release of any one of and preferably more of the active ingredients over an extended period of time.

The formulations embodying the principles of the invention comprising the monoglyceride such as preferably glycerol monooleate and the glycosaminoglycan such as preferably sodium hyaluronate advantageously provide a highly reproducible sustained release system. Prior art formulations comprising (bio)polymers such as for instance suspensions, emulsions and vesicles are thermodynamically unstable and typically consist of at least two phases. The present formulations can differ from these in that the present formulations are thermodynamically stable one phase formulations providing controlled release of the glycosaminoglycan over an extended period of time.

Further, the formulations embodying the principles of the present invention comprising a sustained release agent such as a gel forming agent allow to protect their enclosed active ingredients or other components from the physical environment, thereby improving the stability of their active ingredients or other components in vivo. For example, the present formulations comprising a sustained release agent such as a gel forming agent can provide a protection of their enclosed active ingredients or other components against enzymatic degradation.

The formulations according to the present invention comprising a sustained release agent advantageously have mechanical and/or rheological properties close to healthy synovial fluid. The present formulations comprising a sustained release agent such as a gel-forming agent therefore allow to improve the articular function by their lubricating action on the joint. The present formulations further allow to effectively restore the mechanical integrity of the joint.

Expanding on these findings, the present inventors have realised the use of a glycosaminoglycan and a gel-forming agent as a sustained release pharmaceutical excipient or as a protectant against a glycosaminoglycanase, preferably selected from hyaluronidase, chondroitin B lyase, proteoglycanase, keratanase, chitosanase and chitinase.

Accordingly, another aspect relates to the use of a glycosaminoglycan and a gel-forming agent as a sustained release pharmaceutical excipient or as a protectant against a glycosaminoglycanase. Such use of the present formulations or kits of parts advantageously increases the stability of the pharmaceutical active ingredients or other components and hence, prolongs the presence of the pharmaceutical active ingredients or other components in situ. These aspects use a glycosaminoglycan and a gel-forming agent, which components may thus be suitably denoted as being in combination.

Also disclosed are thus a glycosaminoglycan and gel-forming agent as taught herein for use as a sustained release pharmaceutical excipient, preferably for use as a sustained release pharmaceutical excipient in any one of the diseases as taught herein, and more preferably for use as a sustained release pharmaceutical excipient in the treatment of osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, monoarthritis and poly-arthritis. Also provided is use of a glycosaminoglycan and gel-forming agent as taught herein for the manufacture of a sustained release pharmaceutical excipient, preferably for the manufacture of a sustained release pharmaceutical excipient for the treatment of any one of the diseases as taught herein, and more preferably for the manufacture of a sustained release pharmaceutical excipient for the treatment of osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis. Further provided is a method for treating any one of the diseases as taught herein and preferably osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis, in a subject in need of such treatment, comprising administering to said subject a glycosammoglycan and gel-forming agent as a sustained release pharmaceutical excipient. Particularly intended is a method for treating any one of the diseases as taught herein and preferably osteoarticular diseases in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of a glycosammoglycan and gel-forming agent as a sustained release pharmaceutical excipient. When used as a sustained release pharmaceutical excipient, the glycosammoglycan and gel-forming agent as taught herein can be administered in combination with one or more pharmaceutical active ingredients, preferably one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids.

Hence, particularly disclosed is a glycosammoglycan and gel- forming agent as taught herein for use as a sustained release pharmaceutical excipient, preferably for use as a sustained release pharmaceutical excipient in any one of the diseases as taught herein, and more preferably for use as a sustained release pharmaceutical excipient in the treatment of osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis, wherein the glycosammoglycan and sustained release agent is to be administered in vivo together with one or more pharmaceutical active ingredients, preferably one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids. Further provided is a method for treating any one of the diseases as taught herein and preferably osteoarticular diseases, including acute or chronic osteoarticular diseases, such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis, in a subject in need of such treatment, comprising administering to said subject a glycosammoglycan and gel-forming agent as a sustained release pharmaceutical excipient together with one or more pharmaceutical active ingredients, preferably one or more alpha-2 adrenergic receptor agonists and/or one or more corticosteroids.

Another aspect provides use of a glycosammoglycan and gel-forming agent as taught herein as a protectant against a glycosammoglycanase. Also provided is the use of a glycosammoglycan and gel-forming agent as disclosed herein for protecting a glycosammoglycan, such as particularly the glycosammoglycan contained in the combination, against a glycosammoglycanase. A related aspect provides a glycosammoglycan and gel-forming agent as disclosed herein for use as a protectant against a glycosammoglycanase. Further provided is a glycosammoglycan and gel- forming agent as disclosed herein for use as a protectant of a glycosammoglycan against a glycosammoglycanase. It shall be understood that in any one of these above aspects, the glycosammoglycan and gel-forming agent may advantageously but without limitation act as a protectant against a glycosammoglycanase particularly for the glycosammoglycan contained in the combination. In certain indications such as in osteoarticular diseases, particularly where the glycosaminoglycan and gel-forming agent are administered intra-osseously or intra-articularly, the glycosaminoglycan and gel- forming agent can potentially provide additional advantages as described herein.

Hence, also provided is the use of a glycosaminoglycan and a gel-forming agent, such as particularly in combination, as a visco-elastic agent or as a lubricant.

It shall be understood that specific or preferred features of the present formulations, kits, methods and uses, such as but without limitation, the nature and quantities of the glycosaminoglycan and gel- forming agent, as described elsewhere in this application, also apply to the aforementioned uses of the glycosaminoglycan and the gel-forming agent as a pharmaceutical excipient; as a visco- elastic agent; as a lubricant; or as a protectant against a glycosaminoglycanase.

In any one of these above aspects, said glycosaminoglycanase may be preferably selected from a group consisting of a hyaluronidase, chondroitin B lyase, proteoglycanase, keratanase, chitosanase and chitinase. In a non-limiting example where the formulation or the kit of parts embodying the principles of the invention comprise hyaluronic acid, the formulation or the kit of parts may provide for protection of the hyaluronic acid from hyaluronidase.

As noted, the gel-forming formulations taught herein comprise a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients. Particularly herein, the glycosaminoglycan may be selected from the group consisting of hyaluronic acid and derivatives thereof, a proteoglycan and derivatives thereof, a chondroitin sulfate, a keratan sulfate, a chitosan and derivatives thereof, a chitin and derivatives thereof. The present formulations may comprise one or more glycosaminoglycans. The present formulations may thus comprise one glycosaminoglycan or a mixture of glycosaminoglycans selected from the group consisting of hyaluronic acid and derivatives thereof, a proteoglycan and derivatives thereof, a chondroitin sulfate, a keratan sulfate, a chitosan and derivatives thereof, a chitin and derivatives thereof.

The term "hyaluronic acid" or "HA" may be used interchangeably with "hyaluronan" or "hyaluronate". The term "hyaluronic acid" refers to an anionic, non-sulfated polymer of disaccharides composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating β-1,4 and β-1,3 glycosidic bonds. Hyaluronic acid derivatives include but are not limited to salts of hyaluronate such as sodium hyaluronate 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.

The term "proteoglycan" refers to proteins with one or more covalently attached glycosaminoglycan (GAG) chain(s). The glycosaminoglycan can be a proteoglycan selected from decorin, biglycan, testican, fibromodulin, lumican, versican, perlecan, neurocan or aggrecan. The term "chondroitin sulfate" refers to a polymer of disaccharides composed of N- acetylgalactosamine and glucuronic acid, each of which can be sulfated in variable positions and quantities. The chondroitic sulfate can be selected from chondroitin-4-sulfate, chondroitin-6-sulfate, chondroitin-2,6-sulfate, chondroitin-4,6-sulfate.

The term "keratan sulfate" may be used interchangeably with "keratosulfate" and refers to a polymer of repeating disaccharides -3Gai i-4GlcNAc i- which can be sulfated at carbon position 6 (C6) of either or both the Gal or GlcNAc monosaccharides.

The term "chitosan" refers to a linear polymer composed of randomly distributed -(l-4)-linked D- glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).

The term "chitin" refers to a polymer composed of -(l,4)-linked N-acetylglucosamine.

The glycosaminoglycans used in accordance with the invention are known and commercially available.

Without limitation, the present formulations can comprise the glycosammoglycan in a concentration ranging between 0.1 and 15% by weight (w/w). For example, the formulations can comprise the glycosammoglycan in a concentration ranging between 0.5 and 10% by weight (w/w), for example between 0.6 and 7.5% by weight (w/w) for example between 0.7 and 5.0% by weight (w/w), for example between 0.7 and 2.5% by weight (w/w), for example between 0.7 and 2.0% by weight (w/w) for example, the formulations can comprise the glycosammoglycan in a concentration ranging between 0.7 and 1.0% by weight (w/w). Preferably, the formulations can comprise the glycosammoglycan in a concentration ranging between 0.7 and 1.5% by weight (w/w). More preferably, the formulations can comprise the glycosammoglycan in a concentration of 0.75% by weight (w/w). Where the glycosammoglycan displays therapeutic benefit of its own, such as but without limitation in osteoarticular diseases, it may be included in a therapeutically effective amount, such as the exemplary amounts recited in this paragraph.

In the formulations of the invention, the glycosammoglycan and the alpha-2 adrenergic receptor agonist are not covalently linked or are covalently linked. In the formulations of the invention, the glycosammoglycan and the corticosteroid are not covalently linked or are covalently linked.

In preferred embodiments, the glycosammoglycan is hyaluronic acid or a derivative thereof. Without limitation, suitable derivatives may be salts of hyaluronic acid, such as preferably sodium hyaluronate. The hyaluronic acid or derivative thereof can have a low (< 900 kDa) or high (> 900 kDa) molecular mass. Particularly preferred glycosaminoglycans are hyaluronic acid or derivatives thereof with high (> 900 kDa) molecular mass. The glycosammoglycan can be for instance but is not limited to sodium hyaluronate characterized by a high molecular weight of about 1.9 MDa. Without limitation, the present formulations can comprise the hyaluronic acid or derivative thereof in a concentration ranging between 0.1 and 15% by weight (w/w). For example, the formulations can comprise the hyaluronic acid or derivative thereof in a concentration ranging between 0.5 and 10%) by weight (w/w), for example between 0.6 and 7.5% by weight (w/w), for example between 0.7 and 5.0% by weight (w/w), for example between 0.7 and 2.5% by weight (w/w), for example between 0.7 and 2.0%> by weight (w/w), for example, the formulations can comprise the hyaluronic acid or derivative thereof in a concentration ranging between 0.7 and 1.0% by weight (w/w). Preferably, the formulations can comprise the hyaluronic acid or derivative thereof in a concentration ranging between 0.7 and 1.5% by weight (w/w).

As noted, the gel- forming formulations taught herein comprise a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients. The applicability of the present invention is not limited to any pharmaceutical active ingredient or class of pharmaceutical active ingredients. The pharmaceutical active ingredient may be pharmacologically or chemotherapeutically active itself, or may be converted into a pharmacologically or chemotherapeutically active species by a chemical or enzymatic process in the body, i.e., the pharmaceutical active ingredient may be a prodrug. Moreover, due to its ability to sustain the release of the one or more incorporated pharmaceutical active ingredients in presence of aqueous fluids, the developed delivery system may be administered parenterally, such as intra-osseously, intra-articularly, subcutaneously, intramuscularly and/or in any biological cavity.

The present formulations may also be particularly useful for poorly-stable pharmaceutical active ingredients, used in chronic diseases or in any osteoarticular pathology.

Illustrative non-limiting examples of poorly-stable pharmaceutical active ingredients include peptides and proteins, peptide-like active ingredients, antibodies and vaccines, sRNA, DNA, hormones (including glucocorticoids), immunoglobulins, insulin, thyroid hormones, estrogens, androgens, testosterone, somatorelin, growth hormone, somatostatin, desmopressin, terlipressin, monoclonal antibodies, recombinant antibodies, gonadorelin analogues, gonadorelin antagonists, etc.

Advantageously, the present invention can be used for pharmaceutical active ingredients characterized by anticancer, antiviral, antimicrobial, anthelmintic, antimycotic activities such as beta-lactam antibiotics, macrolides, tetracyclines, aminoglycosides, quinolones, azolic derivates, AIDS antiviral agents, hepatite B and C drugs, herpetic drugs, polyen antimycotic drugs, echinocandins, antiprotozoaire drugs, alkylating agents, antimetabolites, antitumoral antibiotics, topo-isomerase inhibitors, microtubules inhibitors, tyrosin kinase inhibitors. Chronic diseases may also be treated with the present formulations. The pharmaceutical active ingredients which may be administered for this purpose include one or more of antithrombic and antihemorrhagic drugs, therapeutic agents for cardiovascular diseases when chronic administration is needed, antipsychotics, antidepressants, stimulating agents, antiparkinsonians, antiepileptics, and active compounds used in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.

In addition, antalgic drugs for chronic pain such as NSAI drugs, foot drop drugs, active agents for osteoporosis and Paget diseases may also be administered.

The term "pharmaceutical active ingredient" or "pharmaceutical drug" also encompasses any pharmacologically active salts, esters, N-oxides or prodrugs of the title compound or substance.

Moreover, combination of two or more pharmaceutical active ingredients or doses combinations may be included as the drug component. In this case, the release of each active ingredient may be identical or different such as for instance in case of a combination of two active ingredients in which the first one is presented as an immediately release form and the second one as a controlled release. Similarly, a combination of immediate release and controlled release form may also be obtained for the same active ingredient, in order to provide a rapid and sustained effect.

In preferred embodiments, the pharmaceutical active ingredient may be an alpha-2 adrenergic receptor agonist.

Particularly herein, the alpha-2 adrenergic receptor agonist may be selected from the group consisting of clonidine and derivatives thereof, including 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, Ν,Ν'- dimethylclonidine, p-aminoclonidine, and tiamenidine; imidazolidines, including imidazolines, impromidine, detomidine, medetomidine, dexmedetomidine, levamisole, losartane, lofexidine, miconazole, naphazoline, niridazole, nitroimidazoles, ondansetron, oxymetazoline, phentolamine, tetramisole, thiamazole, tizanidine, tolazoline, trimetaphan; imidazoles, including 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, idazoxan, mivazerol; guanidines, including agmatine, betanidine, biguanides, cimetidine, creatine, gabexate, guanethidine, guanethidine sulfate, guanclofine, guanfacine, guanidine, guanoxabenz, impromidine, iodo-3 benzylguanidine, methylguanidine, mitoguazone, nitrosoguanidines, pinacidil, robenidine, sulfaguanidine, zanamivir; alpha- methyinorepherine, azepexole, 5-bromo-6-(2 imidazolidine-2-ylamino) quinoxalin, formoterol fumarate, indoramin, 6-allyl-2-amino-5,6,7,8-tetrahydro4H-thiazolo [4,5-d]azepine diHCl, nicergoline, rilmenidine, and xylazine.

Further, the alpha-2 adrenergic receptor agonist as intended herein may be clonidine, p- aminoclonidine, tiamenidine, 5-bromo-6- (2 imidazolidine-2- ylamino) quinoxaline, dexmedetomidine, detomidine, medetomidine, oxymetazonline, tizanidine, mivazerol, lofexidine, formoterol fumarate, nicergoline, rilmenidine, 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.

By means of further guidance, Table 1 provides a view of an applicable classification of alpha-2- adrenergic receptor agonists.

Table 1 : Classification of the alpha-2-adrenergic receptor agonists

Guanidines

agmatine

betanidine

biguanides

cimetidine

creatine

gabexate

guanethidine

guanethidine sulfate

guanfacine

guanidine

impromidine

iodo-3 benzylguanidine

methylguanidine

mitoguazone

nitrosoguanidines

pinacidil

robenidine

sulfaguanidine

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

chlorethy 1 clonidine

clonidine 4-isothiocyanate

clonidine 4-methylisothiocyanate

clonidine receptor

clonidine-displacing substance

hydroxyphenacetyl aminoclonidine

N, N ' -dimethylclonidine

The alpha-2 adrenergic receptor agonists used in accordance with some embodiments of the invention are known. Pharmaceutical preparations of alpha-2 adrenergic receptor agonists are commercially available. The alpha-2 adrenergic receptor agonists can be manufactured in a known manner essentially in accordance with processes described in the prior art. The alpha-2 adrenergic receptor agonist including salts thereof can be employed in a therapeutically effective amount. Hence, the formulations can comprise the alpha-2 adrenergic receptor agonist in a concentration ranging between 0.0150 and 6.75% by weight (w/w). For example, the formulations can comprise the alpha-2 adrenergic receptor agonist in a concentration ranging between 0. 0150 and 4.50% by weight (w/w), for example between 0.0150 and 2.25% by weight (w/w), for example between 0.0150 and 0.675%> by weight (w/w), for example between 0.0150 and 0.45% by weight (w/w), for example, the formulations can comprise the alpha-2 adrenergic receptor agonist in a concentration ranging between 0.0150 and 0.225%) by weight (w/w). Preferably, the formulations can comprise the alpha-2 adrenergic receptor agonist in a concentration ranging between 0.0150 and 0.0675%) by weight (w/w).

Preferably, the alpha-2 adrenergic receptor agonist is clonidine. The term "clonidine", as used herein, refers to N-(2,6-dichlorophenyl)-4,5-dihydro-lH-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.

Particularly preferred are formulations comprising clonidine in a therapeutically effective amount. Hence, the formulations can comprise clonidine in a concentration ranging between 0.0150 and 6.75%) by weight (w/w). For example, the formulations can comprise clonidine in a concentration ranging between 0.0150 and 4.50% by weight (w/w), for example between 0.0150 and 2.25% by weight (w/w), for example between 0.0150 and 0.675% by weight (w/w), for example between 0.0150 and 0.45% by weight (w/w), for example, the formulations can comprise clonidine in a concentration ranging between 0.0150 and 0.225%) by weight (w/w). Preferably, the formulations can comprise clonidine in a concentration ranging between 0.0150 and 0.0675%) by weight (w/w). In particularly preferred formulations in accordance with the invention the glycosaminoglycan is sodium hyaluronan, the sustained release agent is glycerol monooleate and the pharmaceutical drug is clonidine. For example and preferably such formulations can comprise sodium hyaluronan in a concentration ranging between 0.1 and 15%) by weight (w/w), glycerol monooleate in a concentration ranging between 5 and 85% by weight (w/w) and clonidine in a concentration ranging between 0.0150 and 6.75%) by weight (w/w). Preferably, the present formulations may comprise sodium hyaluronan in a concentration ranging between 0.7 and 1.5% by weight (w/w), glycerol monooleate in a concentration ranging between 45 and 65% by weight (w/w) and clonidine in a concentration ranging between 0.0150 and 0.0675%) by weight (w/w). As mentioned above, the present formulations may be particularly useful for pharmaceutical active ingredients including hormones, such as inter alia glucocorticoids. In certain embodiments, the pharmaceutical active ingredient may thus be a steroid hormone, preferably a corticosteroid, such as a glucocorticoid or a mineralocorticoid. Preferably, the pharmaceutical active ingredient may be a glucocorticoid. Corticosteroids, such as in particular glucocorticoids, are currently used for treatment of acute arthritic flares such as mono-arthritis flares, in particular by intra-articular administration. The sustained-release formulation described herein can provide a new suitable alternative to the current ways of administration of corticosteroids.

Hence, in preferred embodiments, the pharmaceutical active ingredient may be a corticosteroid, preferably a glucocorticoid.

A corticosteroid, such as a glucocorticoid, as intended herein may be, without limitation, of hydrocortisone type (e.g., hydrocortisone (i.e., Cortisol), hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, or prednisone); or an acetonide (e.g., triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, or halcinonide); or of betamethasone type (e.g., betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, or fluocortolone); or of ester type (e.g., hydrocortisone- 17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17- butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, fluprednidene acetate, hydrocortisone- 17-butyrate, 17-aceponate, 17-buteprate, or prednicarbate).

Particularly herein, the corticosteroid, such as a glucocorticoid, may be selected from the group consisting of betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone- 17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone- 17-butyrate, clobetasol-17- propionate, fluocortolone caproate, fluocortolone pivalate, fluprednidene acetate, hydrocortisone- 17-butyrate, 17-aceponate, 17-buteprate, prednicarbate, and analogs thereof.

The corticosteroids, such as glucocorticoids, used in accordance with certain embodiments of the invention are known. Pharmaceutical grade preparations of corticosteroids, such as glucocorticoids, are commercially available. The corticosteroids, such as glucocorticoids, can be manufactured in known manners essentially in accordance with processes described in the prior art. The corticosteroids, such as glucocorticoids, including salts thereof can be employed in a therapeutically effective amount. Hence, the formulations can comprise the corticosteroid, such as glucocorticoid, in a concentration ranging between 0.100 and 10.00% by weight (w/w). For example, the formulations can comprise the corticosteroid, such as glucocorticoid, in a concentration ranging between 0.200 and 5.00% by weight (w/w), for example between 0.250 and 2.50% by weight (w/w), for example, the formulations can comprise the corticosteroid, such as glucocorticoid, in a concentration ranging between 0.250 and 1.00% by weight (w/w). Preferably, the formulations can comprise the corticosteroid, such as glucocorticoid, in a concentration ranging between 0.250 and 0.750%> by weight (w/w).

Preferably, the corticosteroid is betamethasone. The term "betamethasone", as used herein, refers to a glycocorticoid steroid and includes the pharmaceutically acceptable salts or esters thereof.

Preferably, the pharmaceutically active ingredient may be selected from the group consisting of betamethasone, betamethasone sodium phosphate, betamethasone valerate, and betamethasone dipropionate.

Betamethasone is available in a number of compound forms: betamethasone dipropionate for example branded as Diprosone, Diprolene, Celestamine, and others; betamethasone sodium phosphate for example branded as Bentelan; and betamethasone valerate for example branded as Betnovate, Celestone, Fucibet, and others. In the United States and Canada, betamethasone may be mixed with clotrimazole and may be sold as Lotrisone and Lotriderm.

Particularly preferred are formulations comprising betamethasone in a therapeutically effective amount. Hence, the formulations can comprise betamethasone in a concentration ranging between 0.100 and 10.00%) by weight (w/w). For example, the formulations can comprise betamethasone in a concentration ranging between 0.200 and 5.00%> by weight (w/w), for example between 0.250 and 2.50%) by weight (w/w), for example, the formulations can comprise betamethasone in a concentration ranging between 0.250 and 1.00%> by weight (w/w). Preferably, the formulations can comprise betamethasone in a concentration ranging between 0.250 and 0.750%o by weight (w/w).

In particularly preferred formulations in accordance with certain embodiments of the invention, the glycosaminoglycan is hyaluronic acid or a derivative thereof, more preferably sodium hyaluronan, the sustained release agent is a glyceride, preferably a monoglyceride, more preferably glycerol monooleate, and the pharmaceutical active ingredient is a steroid hormone, preferably a corticosteroid, more preferably a glucocorticoid.

In further particularly preferred formulations in accordance with certain embodiments of the invention, the glycosaminoglycan is hyaluronic acid or a derivative thereof, more preferably sodium hyaluronan, the sustained release agent is a glyceride, preferably a monoglyceride, more preferably glycerol monooleate, and the pharmaceutical active ingredient is betamethasone, such as, e.g., betamethasone, betamethasone sodium phosphate, betamethasone valerate, or betamethasone dipropionate, preferably betamethasone dipropionate.

In particularly preferred formulations in accordance with certain embodiments of the invention, the glycosaminoglycan is sodium hyaluronan, the sustained release agent is glycerol monooleate and the pharmaceutical active ingredient is betamethasone. For example and preferably such formulations can comprise sodium hyaluronan in a concentration ranging between 0.1 and 15% by weight (w/w), glycerol monooleate in a concentration ranging between 5 and 85% by weight (w/w) and betamethasone in a concentration ranging between 0.100 and 10.00%) by weight (w/w). Preferably, the present formulations may comprise sodium hyaluronan in a concentration ranging between 0.7 and 1.5% by weight (w/w), glycerol monooleate in a concentration ranging between 45 and 65%) by weight (w/w) and betamethasone in a concentration ranging between 0.250 and 0.750% by weight (w/w).

In preferred embodiments, the pharmaceutical active ingredients may be i) an alpha-2 adrenergic receptor agonist as defined herein, preferably clonidine or a derivative thereof; and ii) a steroid hormone as defined herein, preferably a corticosteroid, more preferably a glucocorticoid, even more preferably betamethasone.

Hence, in particularly preferred formulations in accordance with certain embodiments of the invention, the glycosaminoglycan is hyaluronic acid or a derivative thereof, more preferably sodium hyaluronan, the sustained release agent is a glyceride, preferably a monoglyceride, more preferably glycerol monooleate, and the pharmaceutical active ingredients are i) an alpha-2 adrenergic receptor agonist, preferably clonidine or a derivative thereof; and ii) a steroid hormone, preferably a corticosteroid, more preferably a glucocorticoid, even more preferably betamethasone.

In particularly preferred formulations in accordance with the invention, the glycosaminoglycan is sodium hyaluronan, the sustained release agent is glycerol monooleate and the pharmaceutical drugs are clonidine and betamethasone.

In certain embodiments, the gel- forming formulations may further comprise one or more excipients selected from a solvent, an oil and an antioxidant.

As noted, in some embodiments the present formulations may further comprise one or more solvents. Preferably, such solvents may be polar solvents, more preferably protic solvents. The term "protic solvent" generally refers to a solvent which has a dissociable hydrogen, for instance, a solvent that has a hydrogen atom bound to an oxygen such as in a hydroxyl group or to a nitrogen such as in an amine group. Particularly, the one or more solvents may be selected from water, ethanol, glycerol, ethylene glycol and/or propylene glycol. Preferably, the one or more solvents may be selected from water, ethanol or propylene glycol. In a preferred embodiment, the formulation may comprise water, ethanol and propylene glycol. The one or more solvents may allow the optimization of the viscosity of the present formulations. Hence, the one or more solvents may further increase the injectability of the present formulations. In addition, the one or more solvents may allow the sterilization of the lipidic phase of the formulations by filtration in an aseptic environment.

In certain embodiments, such formulations may comprise ethanol in a concentration ranging between 0 and 95% by weight (w/w). For example, the formulation can comprise ethanol in a concentration ranging between 0 and 20% by weight (w/w), for example, between 5%> and 15%> by weight (w/w). Preferably, the formulation can comprise ethanol in a concentration ranging between 5 and 10%> by weight (w/w).

In certain embodiments, the formulations may comprise propylene glycol in a concentration ranging between 0 and 95% by weight (w/w). For example, the formulation can comprise propylene glycol in a concentration ranging between 0 and 40% by weight (w/w), for example, between 5 and 20% by weight (w/w), for example, between 10 and 17.5 %> by weight (w/w). Preferably, the formulation can comprise propylene glycol in a concentration ranging between 5 to 15%) by weight (w/w).

In certain embodiments, the formulations may comprise water in a concentration ranging between 0 and 50%) by weight (w/w). Preferably, the present formulation can comprise water in a concentration ranging between 5 to 25% by weight (w/w).

In a particularly preferred embodiment, the present formulation can comprise ethanol, propylene glycol and water as solvents, more preferably may comprise ethanol in a concentration of 10%> by weight (w/w), propylene glycol in a concentration of 15%> by weight (w/w) and water in a concentration of 15%> by weight (w/w).

As noted, in some embodiments the present formulations may further comprise one or more oils. The oil may be a vegetable oil, a mineral oil or an organic oil. The term "vegetable oil" as used herein may encompass any lipid material derived from plants. Suitable non-limiting examples of vegetable oils which can be added to the present formulation are for instance soybean oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil (ground nut oil), rapeseed oil, safflower oil, sesame oil, sunflower oil or nut oil. . The term "mineral oil" as used herein may encompass any lipid material derived from a mineral (non- vegetable) source, particularly a distillate of petroleum. A suitable non-limiting example of a mineral oil which can be added to the present formulation is for instance paraffin oil. The term "organic oil" as used herein may encompass any lipid material produced by plants, animals, or other organisms through natural metabolic processes. The oil may allow to induce a decrease in the water uptake of the formulations. In addition and possibly related to the aforementioned, the oil may further prolong the sustained release of the active ingredients from the formulations.

Preferably, the formulations may further comprise one or more vegetable oils. In certain embodiments, the formulations can comprise the vegetable oil in a concentration ranging between 0 and 95% by weight (w/w). For example, the present formulations can comprise the vegetable oil in a concentration ranging between 0 and 10% by weight (w/w), for example, the present formulations can comprise the vegetable oil in a concentration ranging between 2.5 and 7.5% by weight (w/w). Preferably, the present formulations can comprise the vegetable oil in a concentration ranging between 2 and 5% by weight (w/w).

Preferably, the present formulations as described herein may comprise soybean oil as a vegetable oil. The present formulations may preferably comprise soybean oil in a concentration ranging between 0 and 95% by weight (w/w). For example, the present formulation can comprise soybean oil in a concentration ranging between 0 and 10% by weight (w/w), for example, the present formulation can comprise soybean oil in a concentration ranging between 2.5 and 7.5% by weight (w/w). Preferably, the present formulation can comprise soybean oil in a concentration ranging between 0 and 5% by weight (w/w).

As noted, in some embodiments the present formulations may further comprise one or more antioxidants. The term "antioxidant" as used herein may encompass any molecule capable of inhibiting the oxidation of other molecules thereby preventing the production of free radicals. Suitable non-limiting examples of antioxidants which can be added to the present formulation are for instance ascorbic acid (vitamin C), polyphenols, sulphites, sodium metabisulphite and tocopherols. Preferably, antioxidants included in such formulations may be those approved for parenteral administration such as for intra-osseous or intra-articular administration.

The present formulations can comprise an antioxidant or a combination of antioxidative agents in a concentration ranging between 0 and 3% by weight (w/w). For example, the present formulations can comprise the antioxidant in a concentration ranging between 0.01 and 1% by weight (w/w), for example, the formulation can comprise the antioxidant in a concentration ranging between 0.1 and 0.6% by weight (w/w). Preferably, the present formulations can comprise the antioxidant in a concentration ranging between 0.1 and 0.3% by weight (w/w). Further preferably, the present formulations can comprise the antioxidant in a concentration ranging between 0.01 and 0.6% by weight (w/w). Particularly preferably, the present formulations can comprise the antioxidant in a concentration ranging between 0.01 and 0.06% by weight (w/w), such as, e.g., 0.02% w/w, 0.03% w/w, 0.04% w/w, or 0.05% w/w. The antioxidant(s) may prevent both the oxidation of the lipidic and hydrophilic compounds present in the formulations.

Preferably, the present formulations may comprise alpha-tocopherol acetate as an antioxidant. The present formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0 and 3% by weight (w/w). For example, the present formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.01 and 1% by weight (w/w), for example, the present formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.1 and 0.6% by weight (w/w). Preferably, the present formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.1 and 0.3%> by weight (w/w). Further preferably, the present formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.01 and 0.6% by weight (w/w). Particularly preferably, the present formulations can comprise alpha- tocopherol acetate in a concentration ranging between 0.01 and 0.06%> by weight (w/w), such as, e.g., 0.02% w/w, 0.03% w/w, 0.04% w/w, or 0.05% w/w.

In certain embodiments, the formulations may have a pH ranging from 6.0 to 7.0. Such a pH advantageously improves or maintains the rheological properties of the present formulations during storage in particular at temperatures equal to or above room temperature. Such a pH advantageously improves or maintains the stability of the glycosaminoglycan such as sodium hyaluronate in the present formulations during storage in particular at temperatures equal to or above room temperature. In preferred embodiments, the formulations may have a pH ranging from 6.2 to 6.8. For instance, the present formulations may have a pH of 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8. Preferably, the present formulations have a pH of 6.5. As described in the example section, adjusting the pH of formulations illustrating the present invention to the aforementioned values, such as in particular to 6.5, advantageously maintained the rheological properties of the formulation at least after 1 month of storage, for example after 3 months, for example after 6 months, for example after 12 months (1 year) of storage at a temperature above room temperature such as at 25 °C or 30°C.

The pH of the formulation may be adjusted to the desired pH during the preparation of the formulation. In particular, the pH of the formulation may be adjusted after dissolution of its constituting components. The pH of the present formulations may be adjusted to the desired pH as known in the art. Preferably, the pH of the present formulations is adjusted with a base, such as preferably with an alkali hydroxide, more preferably with NaOH.

The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of appended claims is hereby specifically incorporated in this specification. BRIEF DESCRIPTION OF FIGURES

Figure 1 represents a graph illustrating the dissolution profiles of clonidine loaded in a cubic phase structure of GMO (CUBIC REF), in a mixture of GMO with solvent (CGS REF) and in a formulation illustrating the invention (F2, see Tables 14 and 15).

Figure 2 represents a graph illustrating the dissolution profiles of clonidine loaded in a cubic phase structure of GMO (CUBIC REF) and in two formulations illustrating the invention with different concentrations of clonidine (F2 and F6, see Tables 14 and 15).

Figure 3 represents a graph illustrating the dissolution profiles of clonidine loaded in a cubic phase structure of GMO (CUBIC REF), in a mixture of GMO with solvent (CGS REF) and in four formulations illustrating the invention with different concentrations of sodium hyaluronate (Fl to F4, see Table 14 and 15).

Figure 4 represents a graph illustrating the dissolution profiles of clonidine loaded in two formulations illustrating the invention with different concentrations of propylene glycol (F2 and F7, see Table 14).

Figure 5 represents a graph illustrating the dissolution profiles of clonidine loaded in two formulations exemplifying the invention with different concentrations of sodium oleate (F2 and F8, see Table 14).

Figure 6 represents a graph illustrating the dissolution profiles of clonidine loaded in three formulations illustrating the invention with different concentrations of soybean oil and propylene glycol (F2, F9 and F10, see Table 14).

Figure 7 represents a graph illustrating the water uptake of different formulations exemplifying the invention in function of time.

Figure 8 represents a graph illustrating the rheological properties of a formulation illustrating the invention (26K10/2 = Fl 1) and a commercial product (Structovial ^® ). Elastic modulus (G') and loss modulus (G") are represented by the dark and light curves, respectively.

Figure 9 represents a graph illustrating the chromatograms obtained by gel permeation chromatography (GPC) analysis showing the fast degradation of a solution of 1 mg/ml of sodium hyaluronate at pH 7 and 37°C in presence of 2 Ul/ml of ovine hyaluronidase.

Figure 10 represents a graph illustrating the degradation of a solution of 1 mg/ml of sodium hyaluronate, a commercial comparator (Structovial ^® ) and a formulation illustrating the invention (26K10/2 = Fl 1) in the presence of 2 Ul/ml of ovine hyaluronidase at pH 7 and 37°C. The control comprises 1 mg/ml sodium hyaluronate, without hyaluronidase in the medium. Figure 11 represents a graph illustrating the chromatograms obtained by gel permeation chromatography (GPC) analysis showing the protection of sodium hyaluronate against degradation by 2 Ul/ml of ovine hyaluronidase at pH 7 and 37°C of a solution of 1 mg/ml of sodium hyaluronate and a formulation illustrating the invention.

Figure 12 represents an agarose gel illustrating the release of hyaluronic acid from (1) a hyaluronic acid standard, (2) 0.5 mg/ml hyaluronic acid after incubation in cell culture medium with serum at day 0, (3) 0.5 mg/ml hyaluronic acid after incubation in cell culture medium with serum at day 21, and (4) a comparative formulation without clonidine (Fl l of Table 14 without clonidine) at day 0, (5) at day 7, (6) at day 14 and (7) at day 21, and (8) a formulation illustrating the invention (Fl l, Table 14) at day 0, (9) at day 14 and (10) at day 21.

Figure 13 represents a graph illustrating the release of hyaluronic acid (as a percentage) in function of time from a formulation illustrating the invention (Fl l, Table 14) and a comparative formulation (Fl l of Table 14 without clonidine) (n=\).

Figure 14 represents a graph illustrating the cytokine secretion in the supernatant of stimulated peripheral blood mononuclear cells (PBMCs) left untreated (LPS) or treated with dexamethasone (DEX) or clonidine (Clo).

Figures 15A and 15B represent graphs illustrating the secretion of tumor necrosis factor alpha (TNFa) and interleukine-6 (IL6) respectively, in the supernatant of stimulated peripheral blood mononuclear cells (PBMCs) left untreated (LPS) or treated with triamcinolone (Triam) or clonidine (Clo) and different concentrations of hyaluronic acid (HA).

Figure 16 represents a graph illustrating the rheological properties of a formulation illustrating the invention (26K10/2 = Fl 1) after 6 months of storage at 30°C (n=3, Mean ± SD)

Figure 17 represents a graph illustrating the rheological properties of a formulation illustrating the invention (26K10/2 = Fl 1 with pH adjusted to 6.5) after 6 months of storage at 30°C (n=3, Mean ± SD)

Figure 18 represents a graph illustrating in vitro dissolution profiles of betamethasone from a formulation illustrating the present invention in saline phosphate buffer (pH 7.40) with 0.1% Tween 80 at 50 rpm and 37°C (n=6, Mean ± SD)

Figure 19 represents a graph illustrating in vitro dissolution profiles of clonidine and betamethasone from a formulation illustrating the present invention in saline phosphate buffer (pH 7.40) with 0.1% Tween 80 at 50 rpm and 37°C (n=6, Mean ± SD) DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of and "consisting essentially of.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/-10% or less, preferably +1-5% or less, more preferably +1-1% or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

Whereas the term "one or more", such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

All documents cited in the present specification are hereby incorporated by reference in their entirety.

Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.

The present inventors found through extensive testing formulations with prolonged release of a desired active ingredient. Hence, the invention broadly relates to a gel-forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients, as well as to related kits, uses and methods.

The term "sustained release" broadly refers to the release of a compound from a formulation over an extended, prolonged or increased period of time compared with the release of said compound from a formulation know in the prior art. As used herein, the sustained release refers to the prolonged release of the active ingredients from the present formulations. In particular, the sustained release refers to the extended release of one or more pharmaceutical active ingredients such as one or more alpha-2 adrenergic receptor agonists and/or optionally a glycosaminoglycan from the present formulations. For instance, it is know that the half-life of high molecular weight hyaluronic acid in the joint is about 6 to 8 hours. The sustained release as used herein refers to the extended release of one or more pharmaceutical active ingredients such as one or more alpha-2 adrenergic receptor agonists and/or optionally a glycosaminoglycan such as hyaluronic acid from the present formulations, for example release during one or more days, such as during 2 days, 3 days, 4 days, 5 days, 6 days, or during one or more weeks such as during 1.5 week, 2 weeks, 3 weeks, or during one or more months. These terms may thus also specifically encompass extended release, delayed release or controlled release.

In the present formulations, the one or more pharmaceutical active ingredients can be conveniently considered to represent a therapeutically active principle, i.e., an active ingredient. However, in certain indications where the glycosaminoglycan can have some therapeutic benefit (e.g., in osteoarticular diseases), the glycosaminoglycan can also be conveniently considered to represent a therapeutically active principle.

As used herein, the term "active ingredient" broadly refers to a compound, substance or component which, when provided in an effective amount, achieves a desired therapeutic and/or prophylactic outcome(s). Typically, an active ingredient may achieve such outcome(s) through interacting with and/or modulating living cells or organisms.

An aspect relates to the use of a glycosaminoglycan and a sustained release agent as a pharmaceutical excipient; as a visco-elastic agent; as a lubricant; or as a protectant against a glycosaminoglycanase, preferably selected from hyaluronidase, chondroitin B lyase, proteoglycanase, keratanase, chitosanase and chitinase.

The glycosaminoglycan and sustained release agent are suitable for use as a protectant against a glycosaminoglycanase. The term "protectant" as used herein refers to the ability of the glycosaminoglycan and sustained release agent to provide or afford protection of one or more compounds of a formulation against degradation or inactivation. The terms "sustained protection", "improved protection" or "prolonged protection" broadly refer to the protection of a compound of a formulation against degradation or inactivation over an extended, prolonged or increased period of time compared with the protection of said compound in a reference formulation such as a formulation know in the prior art.

The glycosaminoglycan and sustained release agent are further suitable for use as a visco-elastic agent. The term "visco-elastic agent" as used herein refers to the ability of the glycosaminoglycan and sustained release agent to provide visco- elasticity to a formulation. The term "visco-elasticity" generally refers to the property of a material to exhibit both viscous and elastic characteristics, i.e. time dependent strain when undergoing deformation.

The glycosaminoglycan and sustained release agent are further suitable for use as a lubricant. The term "lubricant" as used herein refers to the ability of the glycosaminoglycan and sustained release agent to reduce friction between moving surfaces. For instance, in certain indications such as in osteoarticular diseases where the glycosaminoglycan and sustained release agent are administered intra-articularly, the glycosaminoglycan and sustained release agent can be conveniently considered to reduce friction between (i.e., lubricate) the joint surfaces.

The formulation of the invention can be a pharmaceutical formulation. Accordingly, the invention encompasses pharmaceutical formulations as taught herein. A pharmaceutical formulation generally comprises the active ingredient(s) and one or more pharmaceutically acceptable excipients. Suitable pharmaceutical excipients depend on the dosage form and identities of the active ingredients and can be selected by the skilled person (e.g. by reference to the Handbook of Pharmaceutical Excipients 6 ^th Edition 2009, eds. Rowe et al.).

Suitable pharmaceutical excipients depend on the dosage form and identities of the active ingredients and can be selected by the skilled person (e.g. by reference to the Handbook of Pharmaceutical Excipients 6 ^th Edition 2009, eds. Rowe et al.). As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the cells.

The precise nature of the carrier or other material will depend on the route of administration. For example, the composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen- free and has suitable pH, isotonicity and stability.

The formulations may comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, preservatives, complexing agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium phosphate, sodium hydroxide, hydrogen chloride, benzyl alcohol, parabens, EDTA, sodium oleate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. Preferably, the pH value of the formulation is in the physiological pH range, such as particularly the pH of the formulation is between about 5 and about 9.5, more preferably between about 6 and about 8.5, even more preferably between about 7 and about 7.5. The preparation of such pharmaceutical formulations is within the ordinary skill of a person skilled in the art.

Another aspect of the invention provides a kit of parts comprising the formulations as defined herein. A further aspect provides a medical device comprising any one of the formulations or kits of parts as taught herein.

Also disclosed is an arrangement or kit of parts comprising a surgical instrument or medical device for administration of any one of the formulations or kits of parts as taught herein to a subject, such as for example systemically or locally, for example at a site of osteoarticuar disease, for example, by injection, and further comprising the formulation or kit of parts as taught herein.

For example but without limitation, such arrangement or kit of parts may comprise a vial with any one of the formulations as taught herein, a medical device for delivering the formulation to an inflamed joint of a mammal subject and having reservoir means for storing the formulation, piston means movable along the longitudinal axis of the reservoir for dispensing the formulation, and a hollow needle mounted on said reservoir means for delivering the formulation to an inflamed and/or damaged joint of the mammal subject. Hence, particularly disclosed are kits of parts comprising a vial with a gel-forming formulation comprising a glycosaminoglycan, a sustained release agent and one or more pharmaceutical active ingredients such as one or more alpha-2 adrenergic receptor agonists; a device for delivering the formulation to an inflamed and/or damaged joint of a mammal subject and having reservoir means for storing the formulation, piston means movable along the longitudinal axis of the reservoir for dispensing the formulation, - and an hollow needle mounted on said reservoir means for delivering the formulation to an inflamed and/or damaged joint of the mammal subject.

The formulations or kit of parts as taught herein may be configured for local administration. The present formulations or kits of parts may be configured for parenteral administration i.e., including one or more of intra-osseous, intra-articular, intramuscular, subcutaneous, intravenous and intrasternal administration.

Preferably, the formulations or kit of parts as taught herein are configured for intra-osseous administration. Intra-osseous administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the marrow of a bone.

Particularly preferred, the formulations or kit of parts as taught herein are configured for intraarticular administration. Intra-articular administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the synovial capsule of an articulating joint.

The formulations or kit of parts as taught herein display excellent characteristics such as gel- formation upon administration and sustained release of the active ingredient, which make said formulations or kit of parts suited for prophylactic or therapeutic use.

Accordingly, the formulations or kit of parts as taught herein may be used for the treatment and/or prevention of any disease wherein a subject would benefit from the sustained release of one or more pharmaceutical active ingredients (and potentially one or more glycosaminoglycans) after the administration of the one or more pharmaceutical active ingredients (and potentially one or more glycosaminoglycans) to the subject. In particular, the formulations or kits of parts as taught herein may be used for the treatment and/or prevention of one or more diseases selected from osteoarticular diseases, chronic diseases, cancer, viral infections, bacterial infections, parasitic diseases, fungal diseases, vascular diseases, cardiac diseases, cardiovascular diseases, mental disorders, depression, Parkinson, epilepsy and Alzheimer's disease, etc.

Preferably, the formulations or kit of parts as taught herein may be used for the treatment and/or prevention of osteoarticular diseases or chronic diseases.

The chronic diseases as intended herein encompass but are not limited to chronic pain, osteoporosis and Paget' s disease of bone.

More preferably, the formulations or kit of parts as taught herein may be used for the treatment and/or prevention of osteoarticular diseases.

The osteoarticular diseases as intended herein encompass bone diseases, articular diseases or combinations thereof.

The osteoarticular diseases as intended herein encompass osteoporosis including focal osteoporosis, multifocal osteoporosis, primary osteoporosis or secondary osteoporosis; fracture; non-union fracture; delayed union fracture; malunion fracture; pseudarthrosis; osteonecrosis; bone cyst; bone defect; osteoarthritis; degenerative arthritis; gonarthrosis; coxarthrosis; rheumatoid arthritis; spondyloarthropathies, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthropathy, undifferentiated spondylarthritis, reactive arthritis; systemic lupus erythematosus and related syndromes; scleroderma and related disorders; Sjogren's Syndrome; systemic vasculitis, including Giant cell arteritis (Horton's disease), Takayasu's arteritis, polymyalgia rheumatica, ANCA-associated vasculitis (including Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss Syndrome), Behcet's Syndrome; other polyarteritis and related disorders including polyarteritis nodosa, Cogan's Syndrome, and Buerger's disease; arthritis accompanying other systemic inflammatory diseases, including amyloidosis and sarcoidosis; crystal arthropathies, including gout, calcium pyrophosphate dihydrate disease, disorders or syndromes associated with articular deposition of calcium phosphate or calcium oxalate crystals; traumatic arthritis, focal cartilage and/or joint defect, focal degenerative arthritis; chondrocalcinosis; neuropathic arthropathy; Felty's Syndrome; Reiter's Syndrome; Lyme disease and rheumatic fever.

Preferably, the osteoarticular disease as intended herein may be selected from the group consisting of osteoarthritis, degenerative arthritis, gonarthrosis, coxarthrosis, and other inflammatory general conditions or symptoms in which joints are involved, such as systemic lupus erythematosus, spondyloarthropathies, polymyalgia rheumatica, ankylosing spondylitis, Reiter' s Syndrome, psoriatic arthropathy, enteropathic arthritis (related to inflammatory bowel disease such as haemorrhagic colitis and Crohn's disease), rheumatoid arthritis, neuropathic arthropathy, acute rheumatic fever, gout, chondrocalcinosis, calcium hydroxyapatite crystal deposition disease, and Lyme disease.

Particularly preferred, the osteoarticular disease as intended herein may be selected from the group consisting of osteoarthritis, rheumatoid arthritis, mono- arthritis and poly-arthritis. The term "monoarthritis" generally refers to inflammation of one joint at a time. The term "poly-arthritis" generally refers to any type of arthritis which involves at least five joints simultaneously.

Osteoarticular diseases treatable with the formulations of the present invention include chronic and acute osteoarticular diseases. The term "chronic", as used herein, refers to long-lasting pain. The term "acute", as used herein, refers to (often severe) pain for a short period of time. The treatment may also be used to prevent or delay the osteoarticular disease.

Further disclosed herein are the gel-forming formulations or kits of parts as taught herein, preferably for use as a medicament and more preferably for use in the treatment and/or prevention of osteoarticular diseases, wherein the gel-forming formulations or the kits of parts are to be administered parenterally. Preferably, the gel-forming formulations or the kits of parts are to be administered intra-osseously or intra-articularly. Accordingly, the present gel-forming formulations or kits of parts may be administered by parenteral, more preferably intra-osseous or intra-articular route.

Also disclosed herein are the gel-forming formulations or kits of parts for use as taught herein, preferably for use as a medicament and more preferably for use in the treatment and/or prevention of osteoarticular diseases, wherein the gel-forming formulations or the kits of parts may be administered in the form of an injection. Preferably, the gel-forming formulation or the kits of parts are to be administered in the form of a parenteral injection, more preferably intra-osseous or intra- articular injection. The present formulations and kits of parts advantageously allow local and hence effective treatment of the osteoarticular diseases.

Further disclosed herein are the gel-forming formulations or kits of parts for use as taught herein for use as a medicament and more preferably for use in the treatment and/or prevention of osteoarticular diseases, wherein the gel-forming formulations or the kits of parts may be administered twice a month, once a month, or once in two or more months. Accordingly, disclosed are also the formulations or kits of parts as taught herein, preferably for use as a medicament and more preferably for use in the treatment and/or prevention of osteoarticular diseases, wherein the gel- forming formulations or kits of parts may be parenterally injected twice a month, once a month, or once in two or more months. Further disclosed are also the formulations or kits of parts as taught herein, preferably for use as a medicament and more preferably for use in the treatment and/or prevention of osteoarticular diseases, wherein the gel- forming formulations or kits of parts may be intra-osseously or intra-articularly injected twice a month, once a month, or once in two or more months. Advantageously, the present formulations allow a reduction of the administration frequency and increase the compliancy of the patients by decreasing the pain produced by repeated injections. Furthermore, the present formulations allow a prolonged therapeutic effect in the treatment of osteoarticular diseases.

Suitable dosage forms include solutions for intra-osseous or intra-articular injection.

The term "prophylactically effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the present formulations or pharmaceutical formulations.

In the context of the present invention a "therapeutically effective dose" means an amount of an active ingredient or formulation that when administered brings about a positive therapeutic response with respect to treatment of a patient with osteoarticular disease.

Appropriate therapeutically effective doses of a pharmaceutical active ingredient in a formulation or kit of parts as taught herein can be determined by a qualified physician with due regard to the nature of the glycosaminoglycan, the disease condition and severity, and the age, size and condition of the patient. For example, appropriate therapeutically effective doses of an alpha-2 adrenergic receptor agonist in a formulation or pharmaceutical formulation of the invention can be determined by a qualified physician with due regard to the nature of the alpha-2 adrenergic receptor agonist, the disease condition and severity, and the age, size and condition of the patient. For example, the dose to be administered may range from about 0.30 to 1.5 mg of the alpha-2 adrenergic receptor agonist such as clonidine per injection, for example, from about 0.35 to 1.40 mg of the alpha-2 adrenergic receptor agonist such as clonidine per injection. Preferably, the dose to be administered ranges from about 0.450 to 1.35 mg of the alpha-2 adrenergic receptor agonist such as clonidine per injection.

Similarly, appropriate therapeutically effective doses of a glycosaminoglycan in a formulation or kit of parts as taught herein can be determined by a qualified physician with due regard to the nature of the glycosaminoglycan, the disease condition and severity, and the age, size and condition of the patient. Without limitation, a typical dose to be administered may range from about 5 mg to 100 mg of the glycosaminoglycan per injection. For example, the dose to be administered may range from about 7.5 mg to 60 mg of the glycosaminoglycan such as hyaluronic acid per injection, for example, from about 15 mg to 50 mg of the glycosaminoglycan per injection. Preferably, the dose to be administered ranges from about 7.5 mg to 30 mg of the glycosaminoglycan per injection.

It is recognized that the treatments of the invention may comprise administration of a single therapeutically effective dose or administration of multiple therapeutically effective doses of formulations or pharmaceutical formulations.

Except when noted, "subject" or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred patients are human subjects.

As used herein, a phrase such as "a subject in need of treatment" includes subjects that would benefit from treatment of a given condition, particularly of osteoarticular disease. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in whom said condition is to be prevented.

The terms "treat" or "treatment" encompass both the therapeutic treatment of an already developed disease or condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent the chances of progression of the disease or condition. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. "Treatment" can also mean prolonging survival as compared with expected survival if not receiving treatment.

The above aspects and embodiments are further supported by the following non- limiting examples.

EXAMPLES

Comparative Example 1 : Preparation of a glycerol monooleate (GMO) based formulation comprising clonidine

A GMO based formulation comprising 0.450 mg of clonidine was prepared as follows. Basically, under aseptic conditions, l . lg of glycerol monooleate (GMO) was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 0.3 g of an aqueous solution of clonidine (clonidine HC1; 1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring and stirred until complete dissolution.

Comparative Example 2: Preparation of a glycerol monooleate (GMO) based formulation comprising clonidine and purified Soybean oil

A GMO based formulation comprising 0.450 mg of clonidine was prepared as follows. Basically, under aseptic conditions, l .lg of glycerol monooleate (GMO) was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 0.3 g of an aqueous solution of clonidine (clonidine HC1; 1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring and stirred until complete dissolution.

Comparative Example 3: Preparation of a glycerol monooleate (GMO) based formulation comprising sodium hyaluronate

A GMO based formulation comprising 15 mg of sodium hyaluronate was prepared as follows. Basically, under aseptic conditions, l . lg of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of sterile and apyrogenic water was added under magnetic stirring till sodium hyaluronate was completely dissolved.

Comparative Example 4: Preparation of a glycerol monooleate (GMO) based formulation comprising sodium hyaluronate and purified Soybean oil A GMO based formulation comprising 15 mg of sodium hyaluronate was prepared as follows. Basically, under aseptic conditions, l . lg of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of sterile and apyrogenic water was added under stirring till sodium hyaluronate was completely dissolved.

Example 1: Preparation of a GMO based gel-forming formulation comprising sodium hyaluronate and clonidine

A. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate and 0.450 mg of clonidine

A formulation as described in Table 2 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 2: Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate and 1.350 mg of clonidine

A formulation as described in Table 3 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μηι filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 3 : Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate and 0.450 mg of clonidine

A formulation as described in Table 4 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 4: Composition of a formulation according to an embodiment invention

D. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate and 1.35 mg of clonidine

A formulation as described in Table 5 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol and 0.4 g of PG at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 5: Composition of a formulation according to an embodiment of the invention

Example 2: Preparation of a GMO based gel-forming formulation comprising sodium hyaluronate, clonidine and purified Soybean oil

A. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate; 0.450 mg of clonidine and purified Soybean oil

A formulation as described in Table 6 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved. Table 6: Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate; 1.350 mg of clonidine and purified Soybean oil

A formulation as described in Table 7 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22μιη filter was added under stirring (e.g. magnetic stirring) till sodium hyaluronate was completely dissolved.

Table 7: Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate; 0.450 mg of clonidine and purified Soybean oil

A formulation as described in Table 8 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil at 45°C. The obtained solution was filtrated through a 0,22 μηι filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 8: Composition of a formulation according to an embodiment of the invention

D. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate; 1.350 mg of clonidine and purified Soybean oil

A formulation as described in Table 9 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring with 0.2 g of ethanol, 0.3 g of PG and 0.1 g of purified soybean oil at 45°C. The obtained solution was filtrated through 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 9: Composition of a formulation according to an embodiment of the

Example 3: Preparation of a GMO based gel-forming formulation comprising sodium hyaluronate, clonidine, purified Soybean oil and alpha-tocopherol acetate

A. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate; 0.450 mg of clonidine; purified Soybean oil and alpha-tocopherol acetate

A formulation as described in Table 10 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG, O.lg of purified soybean oil and 600 μg of alpha- tocopherol acetate at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 10: Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel-forming formulation comprising 15 mg of sodium hyaluronate; 1.350 mg of clonidine; purified Soybean oil and alpha-tocopherol acetate

A formulation as described in Table 11 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of alpha- tocopherol acetate at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved. Table 11 : Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate; 0.450 mg of clonidine; purified Soybean oil and alpha-tocopherol acetate

A formulation as described in Table 12 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of alpha- tocopherol acetate at 45°C. The obtained solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 12: Composition of a formulation according to an embodiment of the invention

D. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate; 1.350 mg of clonidine; purified Soybean oil and alpha-tocopherol acetate

A formulation as described in Table 13 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under stirring (e.g. magnetic stirring) with 0.2 g of ethanol, 0.3 g of PG, 0,lg of purified soybean oil and 600μg of alpha- tocopherol acetate at 45°C. The obtained solution was filtrated through a 0.22 μηι filter in order to ensure the sterility of the GMO phase. To the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with an Ultra-Turrax device at 24000 rpm. To this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under stirring till sodium hyaluronate was completely dissolved.

Table 13: Composition of a formulation according to an embodiment of the invention

Example 4: Dissolution profiles of clonidine from GMO based gel-forming formulations and water uptake profiles of said GMO based gel formulations

A. Formulations

Different formulations according to the invention as described in Table 14 were prepared as described in Examples 1 to 3.

Table 14: Composition of formulations Fl to Fl 1 as examples of the invention

Abbreviations: CLO: clonidine; HA: sodium hyaluronate; PG: propylene glycol; GMO: glycerol monooleate Two comparative formulations as described in Table 15, CGS REF and BK CLO, comprising either hyaluronic acid or clonidine were prepared as described in Comparative Example 1 and Comparative example 3, respectively. The control formulation CUBIC REF was prepared analogously. This control formulation is an uninjectable high viscosity cubic phase structure gel of GMO comprising clonidine.

Table 15: Composition of comparative and control formulations

Oleate a- Soybean

[CLO] [HA] Ethanol PG GMO Water

Formulations Na Tocopherol oil (%)

(mg/g) (%) (%) (%) (%)

(%) (%)

CGS REF 225 - 10 20 55 15 - - -

BK CLO - 7.5 10 20 55 15 - - -

CUBIC REF ^{" "} 225 ^" - - - ^{" "} 75 ^" - - -

Abbreviations: CLO: clonidine; HA: sodium hyaluronate; PG: propylene glycol; GMO: glycerol monooleate

B. Dissolution profiles

In order to test the release of clonidine from different formulations, dissolution tests were perfomed. A Disteck 2100C USP 32 dissolution apparatus (Distek Inc. North Brunswick, NJ, USA), Type II (paddle method) was used for the dissolution tests. This dissolution apparatus was equipped with small volume bath vessels (200 ml) characterized by a flat bottom. Topical drug dissolution cells (Distek ^® 2100C Series, Etten-Leur, Holland) were used as sinker devices in order to immerse the samples.

The dissolution tests were performed in triplicate on lg of the formulation. Release testing was carried out in 50 ml of saline phosphate buffer solutions (1.06mM Potassium phosphate monobasic / 2.97mM Sodium phosphate dibasic / 155.17mM Sodium Chloride) at pH 7.4 ± 0,05 (Invitrogen ^® , Merelbeke, Belgium). The temperature of the dissolution media was maintained at 37 ± 0.1 °C. The rotational speed employed was 50 rpm. Sodium azide (0.02% w/w) was added to the dissolution medium in order to prevent bacterial contamination.

Aliquots (250 μΐ) of the dissolution medium were withdrawn at predetermined time intervals: 6, 12, 24, 48, 72, 96, 144, 240 hours and replaced by fresh dissolution medium, maintained at room temperature. A suitable extraction method of HA was developed by using Amicon ^® centrifugal filter unit, which is an ultrafiltration device with a nominal molecular mass limit of 30 kDa. Centrifugation of the samples through this filter at 14 OOOg during 30 min allowed the separation of HA from the filtrate. Then, a methanolic solution of sodium octane sulfonate (2.2g/liter) was added to the filtrate. As recommended in the FDA's Guidances for Industry, the similarity f ₂ factor was used in order to determine the similarity of dissolution profiles (Shah V.P. and al., 1998). The compared dissolution profiles were obtained under the same conditions and their dissolution time points were similar.

The apparent drug diffusion coefficient (D) within the formulations was determined by a mathematical model described by Siepmann and co-workers (Siepmann J. and Siepmann F, 2008). This model allows the quantification of the effect of the composition of the formulation on D within the gel. The higher the value of D, the higher the release rate of the drug or active ingredient from the gel.

The apparent drug diffusion coefficient within the formulations was determined by fitting an analytical solution of Fick's second law of diffusion to experimentally determined drug release kinetics from gels, in which the drug was molecularly dispersed. As only one edge was exposed to the release medium, the mathematical analysis could be restricted to one dimension. Hence, the release kinetics could be described by Fick's second law of diffusion in a plane sheet as shown in Formula (I), dt dx ² (I)

wherein c denotes the concentration of the drug within the formulation, being a function of time and position x.

The mathematical equation of Formula (II) was used to describe the release of the active ingredients from the investigated formulations (only one circular surface being exposed to the release medium),

(Π)

wherein M _t and M _∞ represent the absolute cumulative amounts of active ingredient released at time t and infinite time, respectively; L denotes the height of the gel.

The dissolution profile of clonidine from a cubic phase structure gel of GMO (CUBIC REF in Figure 1) was performed as a reference because this known formulation presented high viscosity and the cubic phase is the most suitable crystalline phase structure for sustained delivery systems. Nevertheless, the CUBIC REF preparation is uninjectable. In consequence of uninjectability of this kind of preparations, the addition of solvents was considered (CGS REF in Figure 1). Nevertheless, the CGS REF formulation provided a very fast release of clonidine in about 2 hours as shown in Figure 1.

The addition of a viscoelastic scaffold, such as sodium hyaluronic acid, to this formulation containing GMO and solvent was then considered. Unexpectedly, formulations containing only small amounts of HA (F2 in Figure 1) presented a similar sustained-release profile of clonidine to the reference cubic phase preparation. The formulation F2 surprisingly showed relatively low viscosity in comparison to cubic reference formulations. This observation showed the important role played by the incorporation of HA in the formulation. Moreover, the dissolution studies as shown in Figure 1 show that the clonidine release from the formulation F2 is slightly slower even that not significantly in comparison to a cubic phase preparation (CUBIC REF) composed of GMO (75% w/w) and water (25% w/w).

Therefore, formulation F2 was considered as a reference gel-forming formulation for further testing. The influence of the addition and/or variation of the formulation with different compounds such as clonidine, sodium hyaluronate, propylene glycol, sodium oleate and purified oil, was evaluated.

The release profile of clonidine seemed to not be influenced by the concentration of clonidine in the formulation (Figure 2). A formulation comprising 0.0225%) of clonidine (F2, Figure 2) had a similar release profile compared with a formulation comprising 0.0675%) of clonidine (F6, Figure 2). Indeed, f2 ^' factor values of a formulation comprising 0.0225%o of clonidine (F2) and a formulation comprising 0.0675%o of clonidine (F6) showed no significant difference with both references (Table 16). Moreover, there is no significant difference of D values of a formulation comprising 0.0225%o of clonidine (F2) and a formulation comprising 0.0675%o of clonidine (F6) (Table 16).

A formulation without sodium hyaluronate (CGS REF in Figures 1 and 3) showed a significant effect on the release profile of clonidine. This observation showed the important role played by the incorporation of sodium hyaluronate in the formulation.

The concentration of sodium hyaluronate, higher than 7.5 mg sodium hyaluronate/g of formulation, seemed to have no effect on the release profile of clonidine (Figure 3). Indeed,^ factor values of a formulation comprising 7.5 mg sodium hyaluronate/g of formulation (F2), 10 mg sodium hyaluronate/g of formulation (F3) and 15 mg sodium hyaluronate/g of formulation (F4) showed no significant difference with both references (Table 16). A concentration of sodium hyaluronate of 7.5mg/g of formulation, 10 mg/g of formulation and 15 mg/g of formulation allowed a similar sustained release of clonidine compared to the cubic phase preparation (Figure 3). Moreover, there is no significant difference of D values of a formulation comprising a concentration of sodium hyaluronate of 7.5 mg/g of formulation, 10 mg/g of formulation and 15 mg/g of formulation (Table 16).

On other hand, when the concentration of sodium hyaluronate was lower than 7.5mg/g of formulation (Fl comprising 5 mg sodium hyaluronate/g of formulation), a significant effect on the release profile of clonidine was observed (Figure 3). Indeed, f2 factor values of a formulation comprising a concentration of sodium hyaluronate of 5 mg/g of formulation (Fl) on the one hand and 7.5 mg/g of formulation (F2), 10 mg/g of formulation (F3) or 15 mg/g of formulation (F4) on the other hand showed significant difference with both references (Table 16). As shown in Figure 3, the release of the alpha-2 adrenergic receptor agonist was increased from the formulation comprising 5 mg sodium hyaluronate/g of formulation. The high D value confirmed this statement (Table 16).

The ratio of solvent seemed to have a significant effect on the release rate of clonidine (Figure 4). Indeed, f2 factor values of a formulation comprising 20% of propylene glycol (F2) and a formulation comprising 40%> of propylene glycol (F7) showed significant difference with both references (Table 16). Moreover, there is significant difference of D values (Table 16). As illustrated in Figure 4, the release of clonidine was increased from the formulation comprising 40%> of propylene glycol (F7). The high D value of the formulation comprising 40%> of propylene glycol (F7) confirmed this statement (Table 16).

The incorporation of sodium oleate seemed to have no significant effect on the release rate of the clonidine (Figure 5). Indeed,^ factor values of a formulation comprising no sodium oleate (F2) and a formulation comprising 0.3%> of sodium oleate (F8) showed no significant difference with both references (Table 16). Moreover, as illustrated in Table 16, there is no significant difference of D values of a formulation comprising no sodium oleate (F2) and a formulation comprising 0.3%> of sodium oleate (F8).

However, an improvement in the sustained release of clonidine was obtained with a formulation comprising 2.5% by weight of soybean oil (F9) or 5% by weight of soybean oil (F10) instead of propylene glycol (Figure 6). Indeed,^ factor values of a formulation comprising 2.5% by weight of soybean oil (F9) and a formulation comprising 5% by weight of soybean oil (F10) showed significant difference with both references (Table 16). The low D values of the formulation comprising 2.5% by weight of soybean oil (F9) and 5% by weight of soybean oil (F10) confirmed this statement (Table 16). This observation could possibly be explained by an increase of the hydrophobicity, and thus a decrease of the water uptake properties of the formulations as shown in Figure 7.

Table 16: Similarity factor and apparent drug diffusion coefficient D obtained with different formulations. A f ₂ factor value smaller than 50 (in bold) represents a significant difference between two dissolution profiles

*After 5 weeks

The release of clonidine from the investigated formulations seemed to be mainly controlled by a diffusion process during the entire release period which was about 1 or 2 week(s), as shown by the relative good agreement between the theory and the experiment (see R ² values close to 1 in Table 16).

C. Water uptake profiles

For each defined time point (2h, 4h, 6h, 8h, 16h, 24h, 48h and 1 week), 250 μΐ of each formulation (Fl, F2, F3, F4, F6, F7, F8, F9, F10 and Fl 1) were placed and weighted (Initial weight W;) in a 2ml Eppendorf ⁸ tube (n=3). Then, 1 ml of phosphate buffer pH 7.4 was added at the top of the sample and all were placed at 37°C. At fixed time points (2h, 4h, 6h, 8h, 16h, 24h, 48h and 1 week), phosphate buffer was removed and samples were blotted with tissue paper to take out the excess of water in order to be reweighted (Final weight W _f ).

The water uptake (W) was calculated by the equation of Formula (III):

(III)

As shown in Figure 7, the steady state values of the water uptake of forming gels were reached for all formulations 48h after immersion at 37°C. These values depended on the composition of the formulation. Indeed, the higher the concentration of the sodium hyaluronate (Fl, F2, F3 and F4 in Figure 7) or propylene glycol (PG) (F2 and F7 in Figure 7), the higher the water uptake of the gel- forming formulation (Figure 7). On the other hand, soybean oil induced a decrease in the water uptake of the formulations (F9, F10 and Fl l in Figure 7). Indeed, 5% by weight of soybean oil replacing PG considerably decreased the water uptake of the formulation. This could possibly be explained by an increase of the hydrophobicity of the formulation. Consequently, the formulation Fl l showed a low water uptake of the forming gel in function of time.

Example 5: Rheological properties of GMO based gel-forming formulations

In order to test the viscosity and elasticity of the formulations, the rheological properties of different formulations were analyzed. The analysis was performed using a TA-instrument rheometer ARES-G2 with a cone-plate geometry (diameter 25.0 mm, 0.0997 rad) at 25°C.

The instrument was used in the flow-sweep mode and shear stress was evaluated from 0,1 to 100 s ^"1 shear rate.

The instrument was also used in the oscillation- frequency mode where strain was set at 1% and angular frequency was set from 100 to 0.1 rad/s. This application allowed to study the evolution of the elastic modulus (G') and loss modulus (G") in function of the angular frequency applied.

The Power Law, as given in the equation of Formula (IV), is widely used as a model for non- Newtonian fluids,

= kf

(IV)

wherein τ is the shear stress (Pa), γ is the shear rate (s ¹ ) and k is the consistency coefficient (Pa.s ⁿ ).

This mathematical relationship allows the evaluation of shear-thinning and shear-thickening systems, depending on the power factor n (also called the flow behavior index). If n value is less than 1, the fluid can be considered as shear-thinning, and if it is greater than 1, the fluid can be considered as shear-thickening. A test of whether the power law applies and means of determining n are to plot the log shear stress vs log shear rate. If the plot is linear, the power law applies. The value of n, which is the reciprocal of the slope of the line, can be used as a measure of the degree of shear-thinning or shear-thickening (Kroschwitz, 1990). Indeed, the lower the flow behavior index n, the higher is the pseudoplasticity of the shear-thinning system. Table 17: Determination of the flow behavior and viscosity of different formulations (measures of shear stress were performed from 0.1 to 100 s ^"1 as shear rate)

It seems that there is no difference of viscosity and pseudoplasticity (no difference of flow behavior index ri) between the formulations containing similar quantity of sodium hyaluronate (F2, F5 and F6, Table 17). On the other hand, the higher the concentration of HA, the higher was the viscosity and pseudoplasticity of the formulations (Fl , F2, F3 and F4, Table 17).

Gel formulations characterized by pseudoplastic flow behavior means that their viscosity decreases with increasing rate of shear stress. This particularity of pseudoplastic fluid explained why the "syringeability" (Table 19) of the formulations was great in spite of the high initial viscosity. This observation is important regarding the ease of injection of the present formulations to the patients. For instance, by using hypodermic syringes, the shear rate values could reach about 10 000 s ^"1 .

The use of PG at 40% by weight (F7) seems to influence the viscosity but not the pseudoplasticity of the formulations (Table 17). The use of soybean oil in a concentration of 2.5% and 5% by weight (F9 and FIO, respectively) seems to influence the viscosity and also the pseudoplasticity of the formulations compared to a formulation without soybean oil (F2) (Table 17).

Structovial ^® (Croma-Pharma GmbH, Austria) is an example of a commercially available product currently used in the treatment of arthritis. Structovial ^® is an aqueous solution comprising 1% sodium hyaluronate.

As illustrated in Table 17, Structovial ^® showed lower viscosity values and lower pseudoplasticity (higher n value) compared with the formulations Fl-Fl 1 which illustrate the present invention. According to the results obtained for the dissolution studies, formulation FIO was selected as a preferred formulation. It was decided to add an antioxidant, alpha-tocopherol, in order to prevent any oxidation of lipidic compounds (formulation 26K10/2 or Fl l). This formulation was chosen for subsequent experiments and rheological properties were compared with the commercially available product, Structovial ^® (Figure 8).

As shown in Figure 8, in contrast with Structovial ^® , formulation Fl l did not present any cross-over which means that the structure of the formulation was conserved in the range of frequency studied. Moreover, the elastic function predominated which means that potential impacts could be strongly absorbed (Figure 8).

The frequency of crossover between the elastic modulus G' and the viscous modulus G" is equal to 0.41±0.12 Hz for an uncontaminated (non-osteoarthritic) synovial fluid of the knee (Mazzuco et al., J. Orthopedic Res., 1157-1163, 2002). This value of crossover frequency is confirmed by Fam et al., Biorheology, 44, 59-74, 2007.

Below 0.41 Hz: G">G', the synovial fluid has a predominantly viscous function which means that the joint is strongly lubricated when the patient is at rest.

Above 0.41 Hz: G'>G", the synovial fluid has a predominantly elastic function which means that impacts are strongly absorbed when the patient runs or jumps.

Table 18: Rheological properties of the selected formulation in comparison with the Structovial® and healthy synovial fluid

As shown in Table 18, in contrast to Structovial , formulation Fl 1 had rheological properties close to the healthy synovial fluid.

The syringeability of the formulations was tested using a Stable Micro Systems TA-XT.plus Texture Analyser (Stable Micro systems,UK) equipped with a 50 kg loading cell. Syringeability can be considered as the ability of a preparation to be successfully administered by syringe with an appropriate needle. The principle consists in applying a given displacement rate to the plunger of the syringe filled with the formulation and in measuring the resulting force (N).

The study was performed using a 5 ml syringe equipped with a 21GG needle.

The parameters of analysis were set as followed: Pre-test speed = 1.00 mm/s; Test speed = 1.00m m/s; Post-test speed = 10.0 mm/s; Distance = 15 mm; Trigger force = 0.04903N.

Table 19: Color, limpidity, pH and syringeability of the formulations

Color of Limpidity . . , , , , pH of Syringeabilit „ , , . Air bubbles

solutions of solutions solutions y (N)

Fl - Transparent 5.70 62.4 ± 1.6

F2 - Transparent 5.65 78.1 ± 1.8

F3 - Transparent + 5.65 89.7 ± 0.8

F4 - Transparent ++ 5.70 142.5 ± 3.2

F5 - Transparent 5.60 84.0 ± 0.2

F6 - Transparent 5.55 82.1 ± 0.6

F7 - Transparent 6.00 69.5 ± 1.0

F8 yellow Transparent 7.10 75.0 ± 0.5

F9 - Transparent 5.65 79.5 ± 1.2

F10 - Transparent 5.60 85.1 ± 2.6

Fl l Slighty Transparent 5.60 80.7 ± 0.6 yellow

Structovia - Transparent 7.4 5.3 ± 0.1 1

Syringeability of the formulations was directly dependent on the sodium hyaluronate concentration. The higher the concentration of sodium hyaluronate, the higher was the force required to apply a given displacement rate to the plunger of the syringe filled with the formulation. Indeed, the formulations comprising 5 mg sodium hyaluronate/g of formulation (Fl) and 15mg sodium hyaluronate/g of formulation (F4) showed a syringeability of about 62.4N ± 1.6N and 142.5N ± 3.2N, respectively (Table 19). For instance, the syringeability of formulation Fl l was significantly higher than the syringeability of the commercially available product Structovial ^® (Table 19). This could certainly be explained by the advantageous higher rheological properties of formulation Fl l (Tables 17 and 18). The H of the formulation seemed to be modified by addition of sodium oleate in the composition (F8). This neutral pH seemed to induce a color modification of the final formulation. Indeed, at pH 7 the formulation F8 was yellow.

Consequently, the present formulations show advantageous rheological properties and syringeability. These properties of the present formulations demonstrate the superiority of the present formulations in the treatment of osteoarticular diseases.

Example 6: Protection of a GMO based gel-forming formulation to degradation by hyaluronidase activity

In order to evaluate the rate of degradation of sodium hyaluronate in different formulations by the enzyme ovine hyaluronidase, first a suitable concentration of the enzyme for use in the test was determined. Three concentrations were tested in triplicate on a solution of 1 mg/ml of sodium hyaluronate: 0.4 Ul/ml, 2 Ul/ml and 10 Ul/ml. The study was performed at pH 7. The temperature of the solution was maintained at 37°C and the shaking speed employed was about 80 min ¹ . Sodium azide (0.02% w/w) was added in order to prevent bacterial contamination. The degradation of sodium hyaluronate was extremely fast at the three evaluated concentrations of enzyme (as shown in Figure 9 for the degradation with 2 Ul/ml of ovine hyaluronidase). As a consequence, the concentration of ovine hyaluronidase was set at 2 Ul/ml for the subsequent study.

The protection of sodium hyaluronate in different gel formulations was assessed in triplicate on lg of formulation immersed in 50 ml of phosphate buffer containing 2 Ul/ml of ovine hyaluronidase. The temperature of the dissolution media was maintained at 37°C and the shaking speed employed was about 80 min ^"1 . Sodium azide (0.02% w/w) was added in order to prevent bacterial contamination. Every three days, fresh solution of ovine hyaluronidase was added to the media. Finally, after two weeks, gels were withdrawn from the media, put in 7.5 ml of fresh phosphate buffer pH 7 and extraction of sodium hyaluronate was performed at 100°C during 20 minutes. The aqueous solutions were filtered through 0.22 μιη filters and analyzed by GPC.

This study was performed at pH 5, 6 and 7 in order to simulate the pH of an arthritic joint that is more acid than a healthy joint. Structovial ^® was chosen as a reference product.

As reported in Figures 10 and 11, the formulation Fl l (as given in Table 14 and prepared as in Example 3A) protected sodium hyaluronate inside the formulation against enzymatic degradation during at least 24 hours.

Consequently, the present formulations protect their enclosed active ingredients against enzymatic degradation during at least 24 hours. The protection of the active ingredients from the physical environment increases the stability of the present formulations in vivo. In this regard, the protection of the active ingredients of the present formulations demonstrates the superiority of the formulations of the invention in the treatment of osteoarticular diseases.

Example 7: Release profiles of clonidine and hyaluronic acid from GMO based gel-forming formulations

A. Release profiles of clonidine from a formulation according to an embodiment of the invention in vitro

The release profile of clonidine from a formulation according to an embodiment of the invention, formulation Fl l (composition as given in Table 14 and prepared as in Example 3 A), is compared with the release profile of clonidine from the comparative formulation CGS REF (composition of the formulation given in Table 15 and as prepared in Comparative Example 1) by in vitro assessment with HPLC.

B. Release profiles of hyaluronic acid from a formulation according to an embodiment of the invention in vitro

Release profile of HA from a formulation according to an embodiment of the invention, formulation Fl l (composition of the formulation as given in Table 14 and prepared as in Example 3A) is compared with the release profile of HA (i) from the comparative formulation CGS REF (composition of the formulation as given in Table 15 and as prepared in Comparative Example 2), (ii) from a formulation comprising HA and clonidine as known in the prior art and (iii) from a commercially available product (Structovial®). The release profile is studied in an inflammatory environment such as in the presence of stimulated peripheral blood monocytes (PBMC) or in other environments such as in the presence of degrading enzymes, for instance in the presence of hyaluronidases. The HA release profiles are assessed by agarose gel electrophoresis, by ELISA and by indirect HA quantification such as the measurement of the degradation time by degrading enzymes.

In an experiment, the hyaluronic acid release from a formulation illustrating the invention (formulation Fl l, Table 14), and from a comparative formulation (formulation Fl l without clonidine) was evaluated in complete cell culture medium containing 10% of serum. 500mg of formulations were incubated at 37°C during 7, 14 or 21 days in 0.5 ml of complete cell culture medium. After incubation, supernatant was retrieved and diluted in charging buffer containing Stain- All® (Sigma Aldrich) and charged on a 1% agarose gel. The gel migrated in TA buffer at 100V during 4 to 5 hours. After migration, the gel was stained in Stain- All® (Sigma Aldrich) overnight at room temperature under agitation followed by a destaining by natural light. HA was colored in blue and quality (molecular weight assessment) was evaluated by comparison to HA alone and to a HA standard ladder. Results indicated that the release of HA from the two formulations (Fl l and Fl l without clonidine) was qualitatively similar to non- formulated HA (Figure 12, lanes 2 and 3). The results further indicated that the release of HA from Fl l (Figure 12, lanes 8 to 10) was slower than from Fl l without clonidine (Figure 12, lanes 4 to 7), indicating a protective role of clonidine in the degradation of the formulation illustrating the invention.

In a second experiment, Enzyme Linked Immunosorbent Assay (ELISA) using an anti-HA antibody was used to evaluate the quantity of HA that was released from a formulation illustrating the invention (formulation Fl l, Table 14) and from a comparative formulation (formulation Fl l without clonidine).

As illustrated in Figure 13, the release of HA was progressive. Hyaluronic acid from Fl l without clonidine was released faster than from the formulation illustrating the invention, Fl l (Figure 13). After 21 days, only 38% of the HA was released from Fl 1 (Figure 13).

Example 8: Resistance of GMO based gel-forming formulations to degradation in situ

Similar in vitro experiments as described in Example 5 are performed but the results are focused on the remaining formulation and its resistance to degradation. The resistance to degradation is evaluated by measuring the residual HA inside the remaining formulation and compared with a formulation without clonidine in order to outline the possible protective role of clonidine. The resistance to degradation is tested in in vitro experiments and/or subcutaneous implantation and in diffusion in vivo experiments.

Example 9: Efficacy of GMO based gel-forming formulations in a rat model of osteoarthritis

A. Efficacy in vivo of a formulation according to an embodiment of the invention

The following formulations are tested for their efficacy in the treatment of osteoartitis by intraarticular injection in a rat model: (i) a formulation according to an embodiment of the invention, formulation Fl l as given in Table 14 and prepared as in Example 3 A, (ii) a comparative formulation CGS REF as given in Table 15 and as prepared in Comparative Example 1, (iii) a comparative formulation comprising HA and clonidine as known in the prior art and (iv) a commercially available product (Structovial®). The efficacy (protection, regeneration of cartilage) is studied by the investigation of the general behavior and clinical signs of the rat model, histology and (blood) biomarkers.

Efficacy studies of the formulations in a rat model of osteoarthritis may demonstrate the superiority of the formulations of the invention compared to the comparative formulations and to a commercially available product (Structovial®). B. Clonidine properties of a formulation according to an embodiment of the invention

The ancillary anti-inflammatory properties of clonidine were determined by the evaluation of cytokine release in the presence of stimulated peripheral blood mononuclear cells (PBMCs). When stimulated with Lipopolysaccharide (LPS), PBMCs generally secrete different pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFa) and interleukine-6 (IL6). PBMCs were co- treated with clonidine or a positive control, dexamethasone. Pro-inflammatory cytokine secretion was evaluated by ELISA using specific antibodies against TNFa and IL6.

PBMC were stimulated with LPS and treated immediately with clonidine or the positive control. After 48 hours of incubation, cells were discarded and the cell supernatant used in ELISA assays. The results indicated that clonidine inhibited significantly IL6 and TNFa secretion (Figure 14). However, clonidine inhibited IL6 secretion only at concentrations above 10 ^~4 M (Figure 14).

The anti-inflammatory properties of clonidine in combination with HA were further tested in PBMCs. PBMC were stimulated with LPS and treated immediately with clonidine or the positive control. After 48 hours of incubation, cells were discarded and the cell supernatant used in ELISA assays. The positive control in this experiment was triamcinolone, a glucocorticoid used in clinics with a similar action compared with dexamethasone.

The results indicated that clonidine inhibited significantly IL6 and TNFa secretion (Figures 15A and 15B). However, clonidine inhibited IL6 secretion only at concentration above 10 ^~4 M (Figure 15B). Moreover, the results indicated that HA did not have any anti- inflammatory properties when incubated with LPS-stimulated PBMC cells and that HA had no influence on the anti-inflammatory properties of clonidine (Figure 15A and 15B).

The efficacy studies of clonidine in vitro in stimulated peripheral blood monocytes may indicate decreased inflammation.

Conclusions

Collectively, the above examples show that formulations embodying the principles of the invention allow to prolong the release and effects of both an alpha-adrenergic receptor agonist such as clonidine and of a glycosaminoglycan such as hyaluronic acid in the joint over a few weeks. The formulations of the invention further allow to protect the glycosaminoglycan against enzymatic degradation. The formulations of the invention show rheological properties close to healthy synovial fluid. The present formulations provide by their lubricating action on the joint an improved and prolonged articular function. Example 10: Preparation of a GMO based gel formulation comprising sodium hyaluronate, clonidine, purified Soybean oil and acetate alpha-tocopherol wherein pH was adjusted to 6.5 with NaOH IN in order to improve the stability of sodium hyaluronate

A. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 0.450 mg of clonidine; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 20 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanol, 0.3 g of PG, O. lg of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 20: Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 1.350 mg of clonidine; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 21 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanol, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 21 : Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 0.450 mg of clonidine; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 22 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanol, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 22: Composition of a formulation according to an embodiment of the invention

D. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 1.350 mg of clonidine; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 23 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanol, 0.3 g of PG, 0,lg of purified soybean oil and 600μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with an Ultra-Turrax device at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 23: Composition of a formulation according to an embodiment of the invention

It is demonstrated by gel permeation chromatography (GPC) and rheological study that a hydrolysis of hyaluronic acid occurred inside the formulation depending on the pH of the formulation and the storage temperature. This is illustrated for instance for a storage temperature of 5°C, 25°C, and 30°C in Tables 24, 25, and 26 respectively. Figure 16 shows that the rheological properties of the developed formulation (Fl l) decreased considerably at least after 1 month of storage at 25°C and 30°C. Nevertheless, as illustrated in Tables 24, 25 and 26 and in Figure 17, this problem was solved by adjusting the pH of the formulation to pH between 6.0 and 7.0, such as preferably to pH between 6.2 and 6.8, such as particularly to pH of 6.5. Indeed, adjusting the pH of the formulation illustrating the present invention conserved the rheological properties of the formulation during storage, even at temperatures above room temperature such as at 30°C, as shown in Figure 17. Same was observed for the carrier without incorporated drug.

Other parameters studied such as the evolution of the pH of the formulations, the stability and dissolution profiles of clonidine and finally, the stability of GMO and soybean oil by measuring the concentration of free fatty acid did not provide significant modification after 6 months of storage at 5, 25 and 30°C regardless of the pH of the composition.

Table 24: Rheological properties of formulations at storage of 5°C ± 3°C

Table 25: Rheological properties of formulations at storage of 25°C ± 2°C/60% RH ± 5% RH

Table 26: Rheological properties of formulations at storage of 30°C ± 2°C/65% RH ± 5% RH

Example 11: Preparation of a GMO based gel formulation comprising sodium hyaluronate, betamethasone dipropionate, purified Soybean oil and acetate alpha-tocopherol wherein pH was adjusted to 6.5 with NaOH IN in order to improve the stability of sodium hyaluronate

Glucocorticoids are currently used for treatment of acute arthritic flares. The short-term benefit of intraarticular (IA) corticosteroids in mono-arthritis flare is well-established. Due to their sustained- release properties, the present formulations represent a suitable alternative for the administration of corticosteroids. A. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 5 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 27 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of an ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, O. lg of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of sterile and apyrogenic water was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 27: Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 10 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 28 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of an ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, O.lg of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of sterile and apyrogenic water was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added). Table 28: Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 5 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 29 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of sterile and apyrogenic water was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 29: Composition of a formulation according to an embodiment invention

D. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate;10 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 30 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of sterile and apyrogenic water was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 30: Composition of a formulation according to an embodiment of the invention

In-vitro dissolution profiles of betamethasone from a formulation illustrating the present invention in saline phosphate buffer (pH 7.40) with 0.1% Tween 80 at 50 rpm and 37°C is illustrated in Figure 18. Figure 18 shows substantially linear sustained release of betamethasone from the formulation over the duration of 20 days.

Example 12: Preparation of a GMO based gel formulation comprising sodium hyaluronate, clonidine, betamethasone dipropionate, purified Soybean oil and acetate alpha-tocopherol wherein pH was adjusted to 6.5 with NaOH IN in order to improve the stability of sodium hyaluronate

Glucocorticoids are currently used for treatment of acute arthritic flares. Indeed, the short-term benefit of intra-articular (IA) corticosteroids in mono-arthritis flare is well-established. Intraarticular use of alpha2-adrenergic receptor agonists such as clonidine are thought to produce analgesia mainly through an inhibition of the transmission of nociceptive stimulation. The present formulations represent a suitable alternative to the administration of both corticosteroids and clonidine. Indeed, the burst release of clonidine could produce analgesia a few times after injection and the sustained-release of corticosteroids could provide an extended period of activity.

A. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 0.450 mg of clonidine; 5 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 31 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, O.lg of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 31 : Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 1.350 mg of clonidine; 5 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 32 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 32: Composition of a formulation according to an embodiment of the invention

C. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 0.450 mg of clonidine; 5 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 33 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added). Table 33: Composition of a formulation according to an embodiment of the invention

D. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 1.350 mg of clonidine; 5 mg of betamethasone, purified Soybean oil and acetate alpha-tocopherol A formulation as described in Table 34 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 5 mg of betamethasone, 0.3 g of PG, 0,lg of purified soybean oil and 600μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with an Ultra-Turrax device at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 34: Composition of a formulation according to an embodiment invention

In-vitro dissolution profiles of clonidine and betamethasone from a formulation illustrating the present invention in saline phosphate buffer (pH 7.40) with 0.1% Tween 80 at 50 rpm and at 37°C is illustrated in Figure 19. Figure 19 shows substantially linear sustained release of betamethasone from the formulation over the duration of 20 days, and comparatively more burst like release of clonidine during this period.

E. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 0.450 mg of clonidine; 10 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 35 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, O.lg of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 35: Composition of a formulation according to an embodiment of the invention

F. Preparation of a GMO based gel formulation pH 6.5 comprising 15 mg of sodium hyaluronate; 1.350 mg of clonidine; 10 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 36 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μηι filter in order to ensure the sterility of the GMO phase. At the filtrate, 15 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 36: Composition of a formulation according to an embodiment of the invention

G. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate; 0.450 mg of clonidine; 10 mg of betamethasone; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 37 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with a high-speed homogenizer (e.g. Ultra-Turrax ^® ) at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (1.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added). Table 37: Composition of a formulation according to an embodiment of the invention

H. Preparation of a GMO based gel formulation pH 6.5 comprising 30 mg of sodium hyaluronate;

I.350 mg of clonidine; 10 mg of betamethasone, purified Soybean oil and acetate alpha-tocopherol A formulation as described in Table 38 was prepared as follows. Basically, under aseptic conditions, 1.1 g of GMO was gently melted at 45°C and then blended under magnetic stirring with 0.2 g of ethanolic solution of 10 mg of betamethasone, 0.3 g of PG, 0,lg of purified soybean oil and 600μg of acetate alpha-tocopherol at 45°C. Solution was filtrated through a 0.22 μιη filter in order to ensure the sterility of the GMO phase. At the filtrate, 30 mg of sodium hyaluronate was added and put in suspension during 3 minutes with an Ultra-Turrax device at 24000 rpm. At this suspension, 0.3 g of an aqueous solution of clonidine (4.5 mg/ml) passed through a 0.22 μιη filter was added under magnetic stirring till sodium hyaluronate was completely dissolved. Finally, pH of the formulation was adjusted to 6.5 with NaOH IN (about 3 μΐ of NaOH IN added).

Table 38: Composition of a formulation according to an embodiment invention

In-vitro dissolution profiles of clonidine and betamethasone from a formulation illustrating the present invention in saline phosphate buffer (pH 7.40) with 0.1% Tween 80 at 50 rpm and at 37°C is illustrated in Figure 19. Figure 19 shows substantially linear sustained release of betamethasone from the formulation over the duration of 20 days, and comparatively more burst like release of clonidine during this period.