FORMULATIONS USEFUL IN THE TREATMENT OF OSTEOARTICULAR DISEASES FIELD OF THE INVENTION

The present invention relates to sustained release formulations and related kits. The present invention further relates to sustained release formulations for use in the treatment of 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, the half-life of the glycosaminoglycan such as hyaluronic acid after injection into the joint is only about 6 to 8 hours.

Compositions for the sustained delivery of glycosaminoglycans have been described. For instance, mono-injections of hyaluronic acid stabilized by chemical modification are commercially available and have been suggested to have an increased residence time in the joint. Furthermore, WO 2006/071694 discloses a visco-supplement composition comprising a biodegradable polymer, a solvent, a visco-supplement such as particularly hyaluronic acid or a salt thereof, and a surfactant and further provides a method of ameliorating the symptoms of osteoarthritis in a patient, comprising injecting the composition into an afflicted joint of the patient. In addition, WO 03/000190 describes a composition useful for the treatment of arthritic joints comprising at least one glycosaminoglycan, at least part of which are encapsulated in at least one liposome.

However, there exists a need for the development of further and/or improved formulations. In particular, there exists a need for a formulation with prolonged drug release for use in the treatment of osteoarticular diseases.

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 and a monoglyceride. A formulation applying the principles of the invention displays advantageously enhanced gel-formation upon contact of the formulation with an aqueous liquid such as physiological or bodily fluids, for example synovial fluid. Such a formulation further allows to prolong the release of the glycosaminoglycan for example in a joint, advantageously over a few weeks. 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 contrary to solutions involving biopolymers, polymers or formulations based on liposomes.

Surprisingly, the inventors also found that the present formulation protects the glycosaminoglycan from the physical environment, thereby improving its stability in vivo. The inventors indeed found that the present formulation provides a protection of the glycosaminoglycan against enzymatic degradation. Also disclosed is a method for producing the present formulation comprising combining the glycosaminoglycan and the monoglyceride.

The formulation may additionally comprise one or more pharmaceutically acceptable excipients (e.g., solvents, carriers, diluents, etc.), preferably 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 a glycosaminoglycan and a monoglyceride, wherein the glycosaminoglycan and the monoglyceride are configured to allow for producing (forming or obtaining) any one of the formulations as taught herein. Disclosed is as well a method for producing said kit of parts comprising including the glycosaminoglycan and the monoglyceride in a kit of parts.

Further provided is a kit of parts comprising the above formulation. Hence, disclosed is a kit of parts comprising a gel-forming formulation comprising a glycosaminoglycan and a monoglyceride. Disclosed is as well a method for producing said kit of parts comprising including the gel-forming formulation in a kit of parts.

A further aspect provides a medical device comprising any one of the gel-forming formulations or the kits of parts as taught herein. Hence, particularly disclosed is a medical device comprising a gel-forming formulation comprising a glycosaminoglycan and a monoglyceride. 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.

As mentioned above, in the present formulation the glycosaminoglycan is protected from its environment, and particularly from glycosaminoglycan-degrading enzymes, thereby improving the stability of the glycosaminoglycan in vivo when the formulation is administered to a subject, compared to glycosaminoglycan administered not in combination with a monoglyceride.

Another aspect thus provides use of any one of the gel-forming formulations or kits of parts as disclosed herein as a protectant against a glycosaminoglycanase. Particularly disclosed is thus the use of a gel-forming formulation comprising a glycosaminoglycan and a monoglyceride as a protectant against a glycosaminoglycanase. Also provided is the use of any one of the gel-forming formulations or kits of parts as disclosed herein for protecting a glycosaminoglycan, such as particularly the glycosaminoglycan contained in the formulations, against a glycosaminoglycanase. A related aspect provides any one of the gel-forming formulations or kits of parts as disclosed herein for use as a protectant against a glycosaminoglycanase. Further provided is any one of the gel-forming formulations or kits of parts as disclosed herein for use as a protectant of a glycosaminoglycan against a glycosaminoglycanase. Another related aspect provides a method for protecting a glycosaminoglycan against a glycosaminoglycanase comprising formulating the glycosaminoglycan in a gel-forming formulation comprising the glycosaminoglycan and a monoglyceride. Also provided is the use of a monoglyceride for protecting a glycosaminoglycan against a glycosaminoglycanase, wherein the glycosaminoglycan and the monoglyceride are formulated in any one of the gel- forming formulations or kits of parts as described herein. A related aspect provides a monoglyceride for use as a protectant of a glycosaminoglycan against a glycosaminoglycanase, wherein the glycosaminoglycan and the monoglyceride are formulated in any one of the gel-forming formulations or kits of parts as disclosed herein. It shall be understood that in any one of these above aspects, the formulation or the monoglyceride may advantageously but without limitation act as a protectant against a glycosaminoglycanase particularly for the glycosaminoglycan contained in the formulations. 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.

A further aspect provides any one of the gel-forming 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, preferably by intra-osseous or intra-articular injection. Hence, particularly disclosed is a gel-forming formulation comprising a glycosaminoglycan and monoglyceride for use in the treatment of acute or chronic osteoarticular diseases such as preferably osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis, preferably by intra-osseous or intra-articular injection. The use of the formulations of the present invention in the treatment of osteoarticular diseases is advantageous because these formulations allow efficient treatment during longer periods due to the extended release of the glycosaminoglycan. In addition, the inventors advantageously found that the present formulations allow to improve the articular function by their extended lubricating action on the joint. The present formulations can also restore at least partly the mechanical integrity of the joint. Hence, the present formulations advantageously allow to obtain an improved therapeutic effect with fewer injections and thus provide increased patient compliance.

A related aspect thus provides the 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 but without limitation osteoarthritis, rheumatoid arthritis, mono-arthritis and poly-arthritis.

Thus, particularly disclosed is use of a glycosaminoglycan and a monoglyceride for the manufacture of a gel-forming formulation for the treatment of osteoarticular diseases, including 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 a therapeutically or prophylactically effective amount of any one of the formulations or kits of parts as taught herein. Particularly intended 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 a therapeutically or prophylactically effective amount of a gel- forming formulation comprising a glycosaminoglycan and a monoglyceride.

Such method of treatment may typically involve parenteral administration, more preferably intraosseous 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 and a monoglyceride. The term "monoglyceride", as used herein, refers to a glyceride consisting of one fatty acid chain covalently bonded to a glycerol molecule through an ester linkage. The terms "monoglyceride" and "monoacylglycerol", as used herein, can be used interchangeably. The 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 and glycerol monolaurate.

The inventors have found that the monoglyceride 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, without being bound to theory, in the formulations as taught herein the monoglyceride performs or acts as 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.

Without limitation, the present formulations can comprise the monoglyceride in a concentration ranging between 5 and 85% by weight (w/w). For example, the formulations can comprise the monoglyceride 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, ranging between 50 and 60% by weight (w/w). Preferably, the formulations as taught herein can comprise the monoglyceride in a concentration ranging between 45 and 65% by weight (w/w).

In preferred embodiments, the monoglyceride may be 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 preferred monoglyceride is 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.

Without limitation, such formulations can comprise glycerol monooleate in a concentration ranging between 5 and 85% by weight (w/w). For example, the formulations can comprise said 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 formulations can comprise said glycerol monooleate in a concentration ranging between 45 and 65% by weight (w/w).

The combination of a monoglyceride, more preferably glycerol monooleate, with a glycosaminoglycan can advantageously allow to increase the sustained release of the glycosaminoglycan by amplification of the gel-forming process in situ. Indeed, the inventors have found that the combination of a monoglyceride, more preferably glycerol monooleate, with a glycosaminoglycan allows or enhances gel-formation of the formulation upon contact of the formulation with an aqueous liquid such as physiological or bodily fluids.

The inventors have further found that the formulations embodying the principles of the invention comprising a combination of a monoglyceride, more preferably glycerol monooleate, with a glycosaminoglycan ensure a prolonged release of the glycosaminoglycan over an extended period of time.

Hence, also disclosed is the use of any one of the gel-forming formulations or kits of parts as disclosed herein as a sustained release formulation. Particularly disclosed is the use of a gel- forming formulation comprising a glycosaminoglycan and a monoglyceride as a sustained release formulation. Also provided is the use of any one of the gel- forming formulations or kits of parts as disclosed herein for sustaining or prolonging the release of a glycosaminoglycan from the formulation. A related aspect provides any one of the gel-forming formulations or kits of parts as disclosed herein for use as a sustained release formulation. Further provided is any one of the gel- forming formulations or kits of parts as disclosed herein for use in sustaining the release of a glycosaminoglycan from the formulation. Another related aspect provides a method for sustaining the release of a glycosaminoglycan comprising formulating the glycosaminoglycan in a gel- forming formulation comprising the glycosaminoglycan and a monoglyceride. Also provided is the use of a monoglyceride for sustaining the release of a glycosaminoglycan, wherein the glycosaminoglycan and the monoglyceride are formulated in any one of the gel-forming formulations or kits of parts as described herein. A related aspect provides a monoglyceride for use in sustaining the release of a glycosaminoglycan, wherein the glycosaminoglycan and the monoglyceride are formulated in any one of the gel-forming formulations or kits of parts as disclosed herein. In a non- limiting example where the formulation or the kit of parts embodying the principles of the invention comprises hyaluronic acid, the formulation or the kit of parts may provide for sustaining the release of the hyaluronic acid.

As noted, the gel-forming formulations taught herein comprise a glycosaminoglycan and a monoglyceride. 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 gycosaminoglycans. 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 terms "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 comprising 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 P-(l,4)-linked N-acetylglucosamine.

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

The glycosaminoglycan can be employed in a therapeutically effective amount. Without limitation, the present formulations can comprise the glycosaminoglycan in a concentration ranging between 0.1 and 15% by weight (w/w). For example, the formulations can comprise the glycosaminoglycan 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 glycosaminoglycan in a concentration ranging between 0.7 and 1.0% by weight. Preferably, the formulations can comprise the glycosaminoglycan in a concentration ranging between 0.7 and 1.5% by weight (w/w). More preferably, the formulations can comprise the glycosaminoglycan in a concentration of 0.75% by weight (w/w).

In preferred embodiments, the glycosaminoglycan 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 glycosaminoglycan can be for instance but is not limited to sodium hyaluronate characterized by a high molecular weight of about 1.9 MDa.

In preferred embodiments, the 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 a derivative thereof in a concentration ranging between 0.7 and 1.0% by weight (w/w). Preferably, the formulations as taught herein can comprise the hyaluronic acid or derivative thereof in a concentration ranging between 0.7 and 1.5% by weight (w/w).

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 biopolymers such as for instance suspensions, emulsions or 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 or gel-forming formulations providing sustained release of the glycosaminoglycan over an extended period of time.

Further, the present formulations allow to protect the glycosaminoglycan from the physical environment, thereby improving the stability of the glycosaminoglycan in vivo, such as but without limitation through protecting the glycosaminoglycan against enzymatic degradation.

The formulations according to the present invention further advantageously have mechanical and/or rheological properties close to healthy synovial fluid. The formulations therefore allow to improve the articular function by their lubricating action on the joint.

In particularly preferred formulations the glycosaminoglycan is sodium hyaluronan and the monoglyceride is glycerol monooleate. Without limitation, such formulations can comprise sodium hyaluronan in a concentration ranging between 0.5 and 15% by weight (w/w) and glycerol monooleate in a concentration ranging between 5 and 85%> by weight (w/w). Preferably, the formulations can comprise sodium hyaluronan in a concentration ranging between 0.7 and 1.5% by weight (w/w) and glycerol monooleate in a concentration ranging between 45 and 65% by weight (w/w).

The formulations as disclosed herein may further comprise one or more excipients selected from a solvent, a vegetable 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. In addition, the one or more solvents may allow the sterilization of the lipidic phase of the formulations by filtration.

In certain embodiments, such formulations may comprise ethanol in a concentration ranging between 0 and 95% by weight (w/w). For example, the formulations can comprise ethanol in a concentration ranging between 0 and 20% by weight (w/w), for example, between 5%> and 15%> by weight. Preferably, the formulations 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 formulations 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 formulations 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 formulations 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 coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil (Ground nut oil), rapeseed oil, safflower oil, sesame oil, soybean 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 glycosaminoglycan from the formulations. Preferably, the formulations may further comprise one or more vegetable oils. The formulations can comprise the vegetable oil in a concentration ranging between 0 and 95% by weight (w/w). For example, the formulations can comprise the vegetable oil in a concentration ranging between 0 and 10%) by weight (w/w), for example, the formulations can comprise the vegetable oil in a concentration ranging between 2.5 and 7.5% by weight (w/w). Preferably, the formulations can comprise the vegetable oil in a concentration ranging between 2.5 and 5% by weight (w/w).

Preferably, the 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 formulation can comprise soybean oil in a concentration ranging between 0 and 10%> by weight (w/w), for example, the present formulations can comprise soybean oil in a concentration ranging between 2.5 and 7.5% by weight (w/w). Preferably, the present formulations can comprise soybean oil in a concentration ranging between 2.5 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, sulfites, 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 formulations can comprise the antioxidant 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 present formulations 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 may prevent oxidation of the lipidic compounds present in the formulations.

Preferably, the present formulations may comprise alpha-tocopherol acetate as an antioxidant. The formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0 and 3% by weight (w/w). For example, the formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.01 and 1% by weight (w/w), for example, the formulations can comprise alpha-tocopherol acetate in a concentration ranging between 0.1 and 0.6% by weight (w/w). Preferably, the formulation 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 stability of the glycosaminoglycan, such as sodium hyaluronate, in the formulations, for instance, during storage 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 examples 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 water uptake of different formulations illustrating the invention in function of time.

Figure 2 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 3 represents a graph illustrating the chromatograms obtained by gel permeation chromatography (GPC) analysis showing the rate of degradation by 2 Ul/ml of ovine hyaluronidase of a solution of 1 mg/ml of sodium hyaluronate at pH 7 and 37°C.

Figure 4 represents a graph illustrating the rate of degradation by 2 Ul/ml of ovine hyaluronidase at pH 7 and 37°C of a solution of 1 mg/ml of sodium hyaluronate, a commercial product (Structovial ^® ) and a formulation illustrating the invention (26K10/2 = Fl 1).

Figure 5 represents a graph illustrating the chromatograms obtained by gel permeation chromatography (GPC) analysis showing the protection of sodium hyaluronan 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 of a formulation illustrating the invention (gel).

Figure 6 represents an agarose gel illustrating 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 formulation illustrating the invention at day 0, (5) at day 7, (6) at day 14 and (7) at day 21. Figure 7 represents a graph illustrating the release of hyaluronic acid (as a percentage) in function of time from a formulation illustrating the invention (HA-GMO) and a commercial product (Structovia )(«=l).

Figure 8 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 9 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)

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 and a monoglyceride, as well as to related kits, uses and methods. In the present formulations, the glycosaminoglycan can be conveniently considered to represent a therapeutically active principle, i.e., an active ingredient. 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.

In embodiments, the gel- forming formulation comprises a glycosaminoglycan (i.e., one or more glycosaminoglycans) as an active ingredient and further comprises one or more other active ingredients.

In preferred embodiments, the glycosaminoglycan (i.e., one or more glycosaminoglycans) may be the sole (i.e., the only or the exclusive) active ingredient in the present formulations.

The present formulations advantageously allow sustained release of the active ingredient following parenteral administration, for instance following injection of the formulation to a damaged and/or inflamed joint. The terms "sustained release" or "prolonged release" as used herein broadly refer 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 reference formulation such as a formulation know in the prior art. As used herein, the sustained release refers to the prolonged release of a glycosaminoglycan from the present formulations. For instance, it is know from the prior art 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, thus refers to the extended release of 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.

The present formulations are suitable for use as a protectant against a glycosaminoglycanase. The term "protectant" refers to the ability 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 formulation of the invention can be a pharmaceutical formulation. Accordingly, the invention encompasses pharmaceutical formulations as taught herein. A pharmaceutical formulation may comprise in addition to the herein particularly specified components 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.). 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 active ingredients. 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. In particular, a medical device is disclosed comprising a gel-forming formulation comprising a glycosaminoglycan and a monoglyceride.

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 and a monoglyceride; 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 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 spondyloarthritis, 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-articular route.

Also 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 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 intraarticular 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 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 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 particularly 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 a 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 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 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.

EXAMPLES

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

Note that whereas some formulations described in the following examples may contain an additional active ingredient, in particular the alpha-2 adrenergic receptor agonist, clonidine, the results obtained using such formulations are considered to be adequately representative of formulations not containing this additional active ingredient, and in particular of formulations containing a glycosaminoglycan as the sole active ingredient.

Example 1: Preparation of a glycerol monooleate (GMO) based gel-forming formulation comprising sodium hyaluronate

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

A formulation as described in Table 1 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 sterile and apyrogenic water was added under stirring till sodium hyaluronate was completely dissolved. Table 1 : Composition of a formulation according to an embodiment of the invention

B. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate 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, 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 sterile and apyrogenic water was added under stirring till sodium hyaluronate was completely dissolved.

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

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

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

A formulation as described in Table 3 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,lg of purified soybean oil at 45°C. The obtained solution was filtrated through a 0,22μιη 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.

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

B. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate and purified Soybean oil

A formulation as described in Table 4 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 at 45°C. The obtained solution was filtrated through a 0.22μιη 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 sterile and apyrogenic water was added under stirring till sodium hyaluronate was completely dissolved.

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

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

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

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, 0.3 g of PG, 0.1 g of purified soybean oil and 600 μg of acetate alpha- tocopherol at 45°C. The obtained solution was filtrated through a 0.22 μηι filter 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.

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

B. Preparation of a GMO based gel-forming formulation comprising 30 mg of sodium hyaluronate, purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 6 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, 0.1 g of purified soybean oil and 600μg of acetate alpha- tocopherol 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 sterile and apyrogenic water was added under stirring till sodium hyaluronate was completely dissolved.

Table 6: Composition of a formulation according to an embodiment of the invention

Example 4: 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 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 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 7: 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 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 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, 0.3g 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 8: 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 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 (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 9: 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 and 1.35 mg of clonidine

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

Example 5: 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 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 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 11 : 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 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 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 till sodium hyaluronate was completely dissolved.

Table 12: 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 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 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 13 : 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 14 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 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 14: Composition of a formulation according to an embodiment invention

Example 6: Preparation of a GMO based gel-forming formulation comprising sodium hyaluronate, clonidine, purified Soybean oil and acetate alpha-tocopherol

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

A formulation as described in Table 15 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 acetate alpha-tocopherol 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 15: 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 acetate alpha-tocopherol

A formulation as described in Table 16 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 acetate alpha-tocopherol 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 16: 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 acetate alpha-tocopherol

A formulation as described in Table 17 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 acetate alpha-tocopherol 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 17: 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 acetate alpha-tocopherol

A formulation as described in Table 18 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 acetate alpha- tocopherol 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 18: Composition of a formulation according to an embodiment of the invention

Example 7: Water uptake profiles of GMO based gel-forming formulations

A. Formulations

Different formulations which illustrate the invention (Fl -Fl 1 , Table 19) were prepared as described in Examples 4 to 6.

Table 19: Composition of formulations Fl to Fl 1 as examples of the invention

Abbreviations: CLO: clonidine; HA: sodium hyaluronate; PG: propylene glycol; GMO: glycerol monooleate B. 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 calculed by the equation of Formula (I),

(I)

wherein W _f is the final weight and W; is the initial weight.

As shown in Figure 1 , 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 sodium hyaluronate (Fl , F2, F3 and F4) or polyethylene glycol (PG) (F2 and F7), the higher the water uptake of the forming gels (Figure 1). On the other hand, soybean oil induced a decrease in the water uptake of the forming gels (F9, F10 and Fl 1 in Figure 1). Indeed, 5% by weight of soybean oil replacing PG considerably decreased the water uptake of the formulation (Figure 1). This could possibly be explained by an increase of the hydrophobicity of the formulation. Formulation Fl 1 showed a low water uptake of the forming gel in function of time.

Example 8: 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 illustrating the invention 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 (II), is widely used as a model for non- Newtonian fluids,

= kf

(Π)

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 20: Determination of the flow behavior and viscosity of different formulations which illustrate the invention (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 20). On the other hand, the higher the concentration of sodium hyaluronate, the higher was the viscosity and pseudoplasticity of the formulations (Fl , F2, F3 and F4, Table 20).

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 22) 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 10000s ^"1 .

The use of PG at 40% by weight (F7) seems to influence the viscosity but not the pseudoplasticity of the formulations (Table 20). The use of soybean oil in a concentration of 2.5% and 5% by weight (F9 and F10, respectively) seems to influence the viscosity and also the pseudoplasticity of the formulations compared to a formulation without soybean oil (F2) (Table 20).

The flow behavior and viscosity of different formulations illustrating the invention was compared with a commercially available product Structovial ^® (Croma-Pharma GmbH, Austria). Structovial ^® is an aquous solution comprising 1% sodium hyaluronate and is currently used in the treatment of arthritis.

As illustrated in Table 20, 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 F10 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 2). As shown in Figure 2, in contrast with Structovial ^® , formulation Fl l (26K10/2) did not present any cross-over which means that the structure of this formulation was conserved in the range of frequency studied. Moreover, the elastic function predominated which means that potential impacts could be strongly absorbed (Figure 2).

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., 1 157-1 163, 2002). This value of crossover frequency is confirmed by Fam et al., 2007, Biorheology, 44, 59-74. 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 21 : Rheological properties of formulation Fl 1 which illustrates the invention in comparison with Structovial® and healthy synovial fluid

As shown in Table 21, in contrast to Structovial , formulation Fl 1 had rheological properties close to the healthy synovial fluid.

The syringeability of the formulations which illustrate the invention 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 22: 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 illustrating the invention 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 15 mg sodium hyaluronate/g of formulation (F4) showed a syringeability of about 62.4N ± 1.6N and 142.5N ± 3.2N, respectively (Table 22). For instance, the syringeability of formulation Fl l was significantly higher than the syringeability of the commercially available product Structovial ^® (Table 22). This could certainly be explained by the advantageous higher rheological properties of formulation Fl 1 (Tables 20 and 21).

The pH 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 formulations illustrating the invention demonstrate the superiority of such formulations in the treatment of osteoarticular diseases.

Example 9: 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 3 for the degradation with 2 Ul/ml of ovine hyaluronidase). 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 H 7 and extraction of sodium hyaluronate was performed at 100°C during 10 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 4 and 5, the formulation Fl l which is an example of the present invention 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 for treatment of osteoarticular diseases.

Example 10: Release profiles of hyaluronic acid from glycerol monooleate (GMO) based gel- forming formulations

A. Release profile of HA from HA-GMO in vitro

In order to determine the protective role of GMO in the sustained release of HA, the HA release from a formulation of the invention, hereinafter referred to as HA-GMO formulation, is compared with the compounds (HA and GMO) alone and with comparators in the presence of degrading enzymes (hyaluronidases). The HA-GMO formulation is prepared as described in Example 3A. The HA release from the HA-GMO formulation is compared with the HA release form: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators.

The HA release is studied by agarose gel electrophoresis (qualitative), ELISA (quantitative) and indirect quantification by measuring the degradation time by the hyaluronidases.

In a preliminary experiment, a formulation illustrating the invention was prepared as described in Example 2A. The HA release from said formulation was evaluated in complete cell culture medium comprising 10% of serum and compared with the HA release from HA alone. 500mg of the formulation was incubated at 37°C during 7, 14, or 21 days in 0,5ml of complete cell culture medium.

After incubation, supernatant was retrieved and diluted in charging buffer comprising Stain- All® (Sigma Aldrich) and charged on a 1% agarose gel. The gel migrated in Tris-acetate buffer at 100V during 4 to 5 hours. After migration, the gel was stained in Stain-All® overnight at room temperature under agitation followed by a destaining by natural light. HA was colored in blue and quality or molecular weight was evaluated by comparison to HA alone and to an HA standard ladder.

The results indicated that the release of HA from the formulation illustrating the invention (Figure 6, lanes 4-7) is qualitatively similar to non-formulated HA (Figure 6, lanes 2, 3).

The results further indicated that the release of HA from the HA-GMO formulation was not immediate (Figure 6, lane 4) but was induced over time (Figure 6, lanes 5, 6 and 7).

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 (HA-GMO in Figure 6) prepared as described in Example 2A. Structovial® was used as a comparator.

The preliminary results indicated that the release of HA was progressive. After 21 days, all HA was probably released from said formulation illustrating the invention (Figure 7). The release of hyaluronic acid from the comparator Structovial® was probably underestimated due to technical imprecision.

In healthy synovial fluid, the concentration of hyaluronic acid may vary from 0.35 to 4.22mg/ml (Fam et al., 2007, Biorheology 44:59-74). However, this concentration seems to be decreased in osteoarthritis. Relying on the above-mentioned ELISA data on the release of HA in vitro (Figure 7), 20% of the HA is released at 5.5 days which corresponds to 3 mg of HA. As viscosupplementation is injected after joint lavage, the volume of synovial fluid present in the joint is around 2 to 4 ml which means that at day 5.5 post-injection of the formulation illustrating the invention, the HA concentration in the joint is around 1 mg/ml which is in the range of HA concentration in a healthy synovial fluid.

B. Release profile of HA from HA-GMO in vivo

In order to determine the protective role of GMO in the sustained release of HA, an indirect evaluation of the HA release is performed by the evaluation of the remaining HA content in the formulation. Subcutaneous injection in healthy mice is performed with the HA-GMO formulation as described in Example 3A and compared with: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators. The general behavior and clinical sign are studied. Histology is studied. The residual HA content is extracted from the remaining formulation and evaluated. To retrieve HA from the formulations illustrating the present invention, the gel-based formulations are dissolved in an equal amount of All-Stain® solution (Sigma Aldrich) for at least 24h prior to deposition on an agarose gel. Evaluation on agarose gel is performed as described in Example 10A. The release profiles may demonstrate a prolonged release of HA from the formulations of the invention compared with the release of HA from HA or GMO alone, from a commercially available product (Structovial®) and/or from other comparators.

Example 11: Resistance of GMO based gel-forming formulations to degradation in situ

A. Residual HA from HA-GMO in vitro

In order to determine the protective role of GMO in the lifetime of the formulation, the residual HA in the remaining formulation is evaluated in presence of hyaluronidases. The residual HA is studied in the HA-GMO formulation as described in Example 3A and compared with: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators. The residual HA content is extracted from the remaining formulation and evaluated by the same techniques as described in Example 4A.

B. Residual HA from HA-GMO in vivo

In order to determine the protective role of GMO in the lifetime of the formulation, subcutaneous injection in healthy mice is performed with the HA-GMO formulation as described in Example 3 A and compared with: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators. The general behaviour and clinical sign are studied. Histology is studied. The residual HA content is extracted from the remaining formulation and evaluated by the same techniques as described in Example 4A.

Furthermore, intra-articular injection in healthy rabbits is performed with the HA-GMO formulation as described in Example 3A and compared with: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators. The general behaviour and clinical sign are studied. Histology is studied. The residual HA content is extracted from the remaining formulation and evaluated by the same techniques as described in Example 4A.

The protection of HA from the physical environment such as from hyaluronidases may be increased in the formulations of the invention compared with the protection of HA in a commercially available product (Structovial®) and/or in other comparators. In this regard, the protection of HA of the present formulations may demonstrate the superiority of the formulations of the invention in the treatment of osteoarticular diseases compared with the compounds HA and GMO alone, a commercially available product (Structovial®) and other comparators.

Example 12: Rheological properties of GMO based gel-forming formulations

In order to test the viscosity and elasticity of the formulations, the rheological properties of the HA- GMO formulation as described in Example 3A are studied as described in Example 8 and compared with the rheological properties of: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators.

The rheological and mechanical properties of the present formulations may demonstrate to be superior compared with the properties of the compounds HA and GMO alone, a commercially available product (Structovial®) and other comparators. In this regard, the rheological and mechanical properties of the present formulations may demonstrate the superiority of the formulations of the invention in the treatment of osteoarticular diseases compared with the compounds HA and GMO alone, a commercially available product (Structovial®) and other comparators.

Example 13: Efficacy of GMO based gel-forming formulations in a rat model of osteoarthritis

A. Efficacy of HA-GMO formulation in vivo

In order to determine the efficacy of the HA-GMO formulations of the invention, intra-articular injection is performed in an osteoarthritis induced rat model with the HA-GMO formulation as described in Example 3A and compared with: (i) hyaluronic acid alone, (ii) GMO alone, (iii) a commercially available product, Structovial® and (iv) prior art comparators prepared. The efficacy e.g. protection, regeneration of cartilage is determined. The general behaviour and clinical signs are studied. Histology and (blood) biomarkers are studied.

Efficacy studies of the present formulations in an osteoarthritis induced rat model may demonstrate the superiority of the formulations of the invention in the treatment of osteoarticular diseases compared with the compounds HA and GMO alone, a commercially available product (Structovial®) and other comparators.

Conclusions

Collectively, the above examples show that formulations of the invention allow to prolong the release of a glycosaminoglycan such as hyaluronic acid in the joint over an extended period of time such as over a few weeks. The formulations illustrating the invention further allow to protect the glycosaminoglycan against enzymatic degradation. The present formulations show rheological properties close to healthy synovial fluid. The present formulations allow to improve the articular function by their extended lubricating action on the joint.

Example 14: Preparation of a GMO based gel formulation comprising sodium hyaluronate 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

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 and 0.4 g of PG 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 23: Composition of a formulation according to an embodiment of the invention

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

A formulation as described in Table 24 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 and 0.4 g of PG 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 24: Composition of a formulation according to an embodiment of the invention

It was 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 for instance of 5, 25 or 30°C. Indeed, the rheological properties of the developed formulation decreased at least after 1 month of storage at 25 and 30°C. Nevertheless, 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.

Figure 8 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 Figure 9, 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 9. 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.

Example 15: Preparation of a GMO based gel formulation comprising sodium hyaluronate, 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; purified Soybean oil and acetate alpha-tocopherol

A formulation as described in Table 25 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 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 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 25: Composition of a formulation according to an embodiment of the invention

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

A formulation as described in Table 26 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, 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 26: Composition of a formulation according to an embodiment of the invention

It was 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 for instance of 5, 25 or 30°C. Indeed, the rheological properties of the developed formulation decreased at least after 1 month of storage at 25 and 30°C. Nevertheless, 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.

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.