BR112018071394B1 - GRAFTING POLYMER, METHODS FOR PREPARING A GRAFTING POLYMER AND FOR PREPARING A DISPENSING SYSTEM, COMPOSITION, SOFT TISSUE FILLER, USE OF A GRAFTING POLYMER, AND, KIT - Google Patents

Abstract

The present invention is directed to a new graft polymer of a hyaluronic acid polymer and N-isopropylacrylamide-based polymer, preparations, compositions and uses thereof. In particular, the invention relates to pH and/or temperature sensitive compositions capable of spontaneously forming nanoparticles useful as actives and agents and dispensing systems for at least one bioactive agent.

Description

FIELD OF INVENTION

[001] The present invention relates to graft polymers of a hyaluronic acid polymer and N-isopropylacrylamide-based polymer, preparations, compositions and use thereof.

BASICS OF THE INVENTION

[002] Hydrogels for biomedical applications have been developed extensively in recent years. They have become an important material of high interest due to their high water content, tissue-like elasticity and fairly good biocompatibility.

[003] Hydrogels are composed of hydrophilic homopolymers or copolymer networks and are capable of swelling in water or physiological fluids. Hyaluronic acid (HA), also called hyalurone, or its salt hyaluronate sodium, is a natural, linear heteropolysaccharide polymer typically found in the connective and epithelial tissues of vertebrates with a molecular weight range of 105-107 daltons. HA is composed of repetitive disaccharide units of β-1, 3-N-acetyl-glucosamine and β-1,4-glucuronic acid with excellent properties of viscoelasticity, high moisture retention capacity and high biocompatibility (LAVERTU ET AL., 2008, BIOMACROMOLECULES, 9, 640-65). Typically, HA was extracted from rooster combs and is currently essentially produced through the fermentation of microalgae or bacteria (LIU ET AL., 2011, MICROB. CELL FACT., 10, 99). HA is suitable for a wide variety of applications in medicine, cosmetics and nutraceuticals (FRASER ET AL., 1997, J. INTERN. MED., 242, 27-33; ROBERT, 2015, PATHOL BIOL, 63, 32-34) . Particularly, it is known for its role in cartilage lubrication and homeostasis. However, native HA does not persist for long in the human body and is cleared from the implant site because of its low retention capacity and its degradation due to the effect of hyaluronidase (Brown et al., 1991, Exp. Physiol., 76 , 125-134). Therefore, chemical modifications of HA, in particular cross-linking modifications, have been considered for effective applications (Schante et al., 2011, Carbohydrate Polymers, 85, 469-489).

[004] pH-sensitive or thermosensitive polymers have also been synthesized, formulated and formed a hydrogel matrix (Na, 2008, Tissue Engineering and Regenerative Medicine, 5, 482-487; Coronado et al., 2011, Polymer Bulletin, 67, 101 -124 and Lee et al., 2010, Soft Matter, 6, 977-983). However, these PH-sensitive or thermosensitive polymers, due to their physicochemical properties, only form a gel matrix and do not provide an effective damping effect. Additionally, the residence time in vivo is still limited by the rapid enzymatic degradation of HA.

[005] Poly(N-isopropylacrylamide) (variously abbreviated in the literature pNiPAM, PNIPA, PNIPAAm, NIPA, PNIPAA or PNIPAm, also called by IUPAC poly[1-(isopropylcarbamoyl)ethylene], also called homopolymer N-(1 -Methylethyl)-2-propenamide) is a highly pH-sensitive or thermosensitive polymer previously used to modify HA to impart pH or thermosensitivity (Schild, 1992, Progress in Polymer Science, 17, 163-249). pNiPAM represents a candidate with good biocompatibility to introduce physical cross-links through association of hydrophobic domains in situ forming hydrogel (Tan et al., 2009, Biomaterials, 30, 6844-6853; Cooperstein et al., 2013, Biointerphases, 8, 19). In the case of thermosensitivity below the minimum critical solution temperature (LCST) at about 32 °C, the N-substituted hydrophobic groups of pNiPAM are hydrated by water molecules to form a homogeneous solution. Above the LCST, the hydrophobic interaction between the N-substituted groups increases and exceeds the hydration energy of water, leading to an aggregation of hydrophobic polymer chains and hydrogel formation (Guan et al., 2011, Expert Opinion on Drug Delivery, 8, 991-1007). Although the pNiPAM monomer is non-biodegradable, it has been shown that low molecular weights of pure pNiPAM chains are eliminated by renal clearance (He et al., 2008, Journal of Controlled Release, 127, 189-207). Thermoreversible poly(N-isopropylacrylamide) hyaluronic acid hydrogels (HA-pNiPAM) have already been formulated for applications in multiple fields, such as tissue engineering, drug dispensing system, etc. and form a hydrogel matrix at body temperature similar to pure pNiPAM (Ohya et al., 2001, Biomacromolecules, 2, 856-863). In particular, thermoreversible poly(N-isopropylacrylamide) hyaluronan hydrogels (HA-pNiPAM) were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization and “click chemistry”, through the functionalization of HA with alkaline groups and the reaction of HA propargylamide formed with azide-terminated Poly(N-isopropylacrylamide) (N3-PNIPAM) in the presence of a chain transfer agent (CTA) and a copper-containing catalyst (WO 2010/099818; Mortisen et al., 2010, Biomacromolecules, 11, 1261-1272). The degradation products were found to be cytocompatible with hTERT-BJ1 fibroblasts at 35 x 103 g • mol-1 (Mortisen et al., 2010, supra). Disadvantages of these HA-pNiPAM gel matrices are that they do not provide long-term lubrication or damping effect and may additionally contain residual copper.

[006] Therefore, there is a need to find polymers derived from HA that can provide residence time in the body at the injection site with appropriate biomechanical properties, while being well tolerated and easy to synthesize.

SUMMARY OF THE INVENTION

[007] The present invention relates to the unexpected verification of HA graft polymers (HA-N-isopropylacrylamide-based conjugates, which are graft polymers capable of spontaneously forming nanoparticles by a change in temperature and/or pH. These new HA graft polymers comprise a linker molecule that is used to graft the N-isopropylacrylamide-based polymer onto the HA, giving the graft polymer the ability to form nanoparticles upon a change in temperature (above the LCST) and/or pH, which represents a significant improvement never achieved before with pH-sensitive and/or thermosensitive polymers. The present invention addresses medical, surgical and cosmetic needs in the field of tissue regeneration and may enable innovative uses/applications in the field of dispensing. These new graft polymers are pH-sensitive and/or thermosensitive polymer graft polymers that are injectable, stable, biocompatible or biodegradable with a long residence time in the body at the injection site due to the spontaneous formation of nanoparticles with the properties. potentials of self-lubricating nanosphere bearings (SLNBB), which can be used as a new viscosupplementation/lubrication material in medical or cosmetic applications.

[008] One aspect of the invention provides a graft polymer of a hyaluronic acid and (HA graft polymer) based on N-isopropylacrylamide according to the invention.

[009] Another aspect of the invention relates to a pharmaceutical comprising at least one HA graft polymer according to the invention and at least one pharmaceutically acceptable carrier.

[0010] Another aspect of the invention relates to nanoparticles comprising an HA graft polymer according to the invention.

[0011] Another aspect of the invention relates to a cosmetic composition comprising at least one HA graft polymer according to the invention.

[0012] Another aspect of the invention relates to a hydrogel comprising at least one HA graft polymer of the invention.

[0013] Another aspect of the invention relates to a soft tissue filler comprising at least one HA graft polymer of a composition thereof according to the invention.

[0014] Another aspect of the invention relates to a method for preparing an HA graft polymer, a composition thereof, a hydrogel, a soft tissue filler or nanoparticles thereof according to the invention.

[0015] Another aspect of the invention relates to an HA graft polymer according to the invention for use in in vivo drug dispensing, in vitro biological tissue or cell cultures and tissue engineering applications.

[0016] According to another particular embodiment, a biological cell or tissue culture medium comprising an HA graft polymer or a composition thereof according to the invention is provided.

[0017] According to another particular embodiment, a biological reconstruction tissue comprising an HA graft polymer or a composition thereof according to the invention is provided.

[0018] Another aspect of the invention relates to an HA graft polymer according to the invention for use in the prevention or treatment of a medical disorder, and in particular joint pathologies, joint diseases, ocular pathologies, defects or injuries of skin, thickening of urological tissue, any tissue deformation, improvement/modification and/or increase in volume of a part of the body for aesthetic or therapeutic reasons or for the treatment of a vascular malformation or tumor.

[0019] Another aspect of the invention relates to a use of an HA graft polymer according to the invention for preparing a pharmaceutical formulation for the prevention or treatment of a medical disorder, and in particular joint pathologies, diseases of joint, ocular pathologies, skin defects or lesions, enlargement of urological tissue, and any tissue deformation, improvement/modification and/or increase in volume of a part of the body for aesthetic, reconstruction or therapeutic reasons or for the treatment of a vascular or tumor malformation.

[0020] Another aspect of the invention relates to a use of an HA graft polymer according to the invention for the preparation of a cell or biological culture medium or a reconstruction tissue.

[0021] Another method of the invention relates to a method for preventing, treating or improving a medical disorder selected from joint pathologies, joint diseases, ocular pathologies, skin defects or lesions, thickening of urological tissue and any degeneration of tissue, a tumor or vascular malformation in a subject, said method comprising administering to a subject in need thereof an effective amount of at least one HA graft polymer according to the invention or a pharmaceutical formulation thereof.

[0022] Another aspect of the invention relates to a kit comprising at least one graft polymer of the invention or composition thereof, for example, in a lyophilized form.

[0023] Another aspect of the invention relates to a kit for the preparation of nanoparticles for encapsulation of material, for example, bioactive agent, drug substance, protein or antibody for prevention or treatment comprising at least one graft polymer of the invention or composition the same.

Description of the Figures

[0024] Figure 1 shows the 1H NMR spectra in D2O of graft polymers of the D-F invention as compared to those of the starting material. A: Hyaluronic acid (HA), B: HA-DBCO, C: azide-terminated pNiPAM, D: HA-B1, E: HA-B2 and F: HA-P2 as described in Example 1.

[0025] Figure 2 shows the injectability of the graft polymers of the invention measured as described in Example 2. A: The force in Newton (N) required to expel the fluid as a function of the stroke distance of the piston in the syringe with a needle 23G (distance %). The three forces were evaluated to characterize the injection profile. Fi (initial force), Fp (plateau force) and Ff (final force). B: Overview of injection profiles of HA graft polymers by a 23G (B1) and 29G (B2) needle-syringe systems at 20°C. Empty syringe, Ostenil®, and PBS were used as controls. C: The overview of injection profiles of HA graft polymers by a 29G needle at 37°C.

[0026] Figure 3 shows SEM micrographs of HA-B1 (A), HA-B2 (B), HA-P2 (C) and HA-pNiPAM graft polymer (negative control) (D). Scale bar = 1 μm and the percentage of nanoparticles formed as a function of temperature (from 40 to 25°C, 3 min/step) measured by Nanoparticle Tracking Analysis (E) measured as described in Example 4. Figure 4 shows the cumulative in vitro drug release profile of dexamethasone from saline, Ostenil®, HA-B2 and HA-P2 at 37°C as described in Example 7. After 28h, the polymer matrix was hydrolyzed by heating (121 °C) and the remaining dexamethasone content was measured.

[0027] Figure 5 shows the increased persistence/persistence of intravital HA-Cy5 and HA-P2-Cy5 fluorescence superimposed on white light images in mice after subcutaneous injection in healthy mice and intra-articular injection for 7 days in a model of mouse with osteoarthritis as described in Example 5.

Detailed Description

[0028] The term “polyethylene glycol (PEG) chain” refers to a chain comprising from two to 5,000 ethylene glycol units, preferably from 2 to 6 or 2 to 4 ethylene glycol units.

[0029] The term “hyaluronic acid” (HA) refers to linear heteropolysaccharide composed of D-glucuronic acid and D-N-acetylglucosamine, which are linked together through β-1,4 and β-1,3 glycosidic bonds. Suitable HA polymers according to the invention encompass polysaccharides composed of up to 12,500 disaccharide repeats, and range in size from about 500 to 5,000,000 Da depending on the origin, in particular from about 500 to 3,000,000 Da. Each repeat unit bears a carboxylic chemical group in the D-glucuronic acid motif that can potentially be linked to form a graft polymer according to the invention. According to a particular aspect, the degree of substitution of these carboxylic groups with a ligand of the invention is from about 0.5 to 50%, typically from 2 to about 10%.

[0030] The term “degree of cross-linking or substitution”, as used herein, means the amount of functional group pairs converted into cross-linking or grafting bonds relative to the total amount of functional group pairs initially present in the HA and the base polymer of N-isopropylacrylamide, expressed as a percentage.

[0031] Suitable HA polymers according to the invention can be of various origins, including natural (generally animal), semi-synthetic or biotechnological (such as non-animal, produced by fermentation of microorganisms).

[0032] The term “N-isopropylacrylamide-based polymer” includes all polymers called N-propan-2-ylprop-2-enamide by IUPAC (CAS number 2210,25.5). Suitable poly(N-isopropylacrylamide) polymers according to the invention include polymers with a molecular weight from about 500 to 35,000 Da, in particular from 2,000 to 25,000 Da.

[0033] The terms “conjugate” or “grafted” are used to indicate the formation of covalent bonds between HA and N-isopropylacrylamide-based polymers, leading to a graft polymer or conjugate of the invention, wherein such covalent bond occurs through of a binder according to the invention.

[0034] The term “click chemistry” refers to a variety of chemical reactions (e.g., azide-alkaline cycloadditions) that are distinguished by their high yield, regioselectivity, stability, and ability to proceed without the generation of offensive byproducts as described in Kolb et al., 201, Chem. Int. Ed Engl., 40(11), 2004-2021. Importantly, these reactions can occur in a benign solvent, are insensitive to water and oxygen, and have a high thermodynamic drive. The most common examples of these reactions are bond formation of carbon heteroatoms resulting from cycloadditions of unsaturated species, nucleophilic substitution chemistry, non-aldol carbonyl chemistry, and additions to multiple carbon-carbon bonds. The graft polymers of the invention can be synthesized by click chemistry, and examples of synthetic routes are provided herein, which can be slightly adapted by those skilled in the art using standard knowledge.

[0035] The term “alkyl”, when used alone or in combination with other terms, comprises a straight or branched C1C20 alkyl chain that also refers to monovalent alkyl groups with 1 to 20 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1-ethylpropyl, 2-methylbutyl, 3- methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methypentyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methyl- hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, tetrahydrogeranyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-octadecyl, n-nonadecyl, and n-eicosanil and the like. Preferably, these include C1-C9 alkyl, more preferably C1-C6 alkyl, especially preferably C1-C4 alkyl, which, by analogy, refer respectively to monovalent alkyl groups having 1 to 9 carbon atoms, monovalent alkyl groups having 1 to 6 carbon atoms and monovalent alkyl groups with 1 to 4 carbon atoms. Particularly, these include C1-C6 alkyl.

[0036] The term “C5-C10 cycloalkyl” refers to a saturated or unsaturated carbocyclic group of from 5 to 10 carbon atoms with a single ring such as C6-C8 cycloalkyl (e.g., cyclohexyl, cycloalkyl). octyl) or multiple condensed rings (e.g., norbornyl). C3-C8-cycloalkyl includes cyclopentyl, cyclohexyl, cyclopropyl, cyclobutyl, norbornyl and the like.

[0037] The term “heterocycloalkyl” refers to a C5-C10 cycloalkyl group according to the above definition, in which up to x carbon atoms are exchanged for heteroatoms chosen from the group consisting of O, S, NR, R, being defined by hydrogen or methyl. Heterocycloalkyl includes pyrrolidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, azacyclooctyl and the like.

[0038] The term “C8 cycloalkyl” refers to a saturated or unsaturated carbocyclic group of 8 carbon atoms with a single ring (e.g., cyclooctyl) or multiple condensed rings (e.g., dibenzocyclooctynyl, cyclooctyne monofluorinated, dibenzocyclo-octyl, difluorocyclo-octyl, biarylazacyclo-octinone, cyclooctyne, dibenzoazacyclo-octin, nonfluorocyclo-octyne, aryl-free cyclo-octin, bicyclonone and dimethoxyazacyclo-octin) or those as described in Debets et al., 2011, Accounts for chemical research, 44(9), 805-815.

[0039] The term “C8 heterocycloalkyl” refers to a C8-cycloalkyl group according to the above definition, in which up to 3 carbon atoms are exchanged for heteroatoms chosen from the group consisting of O, S, NR, R , being defined by hydrogen or methyl. C8 heterocycloalkyl optionally includes optionally substituted aza-dibenzocyclooctynyl and the like.

[0040] The term “alkoxycarbonyl” refers to the group -C(O)OR, where R includes “C1-C6 alkyl”, “aryl”, “heteroaryl”, “aryl C1-C6 alkyl”, “heteroaryl C1-C6 alkyl” or “heteroalkyl”.

[0041] The term “C1-C6 alkoxycarbonyl alkyl” refers to C1-C6 alkyl groups with an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl and the like.

[0042] The term “amino” refers to the group -NRR', where R and R' are independently H, “C1-C6 alkyl”, “aryl”, “heteroaryl”, “C1-C6 alkyl aryl”, “alkyl C1-C6 heteroaryl”, “cycloalkyl” or “heterocycloalkyl”, and where R and R', together with the nitrogen atom to which they are attached, may optionally form a 3 to 8 membered heterocyclocalkyl ring.

[0043] The term “aminoalkyl” refers to alkyl groups with an amino substituent, including 2-(1-pyrrolidinyl)ethyl and the like.

[0044] The term “aminocarbonyl” refers to the group -C(O)NRR', where R and R' are independently H, C1-C6 alkyl, aryl, heteroaryl, “aryl C1-C6 alkyl” or “heteroaryl C1- C6 alkyl”, including N-phenyl carbonyl and the like.

[0045] The term “aminocarbonyl C1-C6 alkyl” refers to alkyl groups with an aminocarbonyl substituent, including 2-(dimethylaminocarbonyl)ethyl, N-ethyl acetamidyl, N,N-diethyl-acetamidyl and the like.

[0046] The term “acylamino” refers to the group -NRC(O)R', where R and R' are independently H, “C1-C6 alkyl”, “C2-C6 alkenyl”, “C2-C6 alkynyl”, “cycloalkyl-C3-C8”, “heterocycloalkyl”, “aryl”, “heteroaryl”, “aryl C1-C6 alkyl”, “heteroaryl C1-C6 alkyl”, “aryl C2-C6 alkenyl”, “heteroaryl C2-C6 alkenyl ”, “aryl C2-C6 akynyl”, “heteroaryl C2-C6 alkynyl”, “cycloalkyl C2-C6 alkyl” or “heterocycloalkyl C1-C6 alkyl”, including acetylamino and the like.

[0047] The term “acylamino C1-C6 alkyl” refers to C1-C6 alkyl groups with an acylamino substituent, including 2-propionylamino)ethyl and the like.

[0048] The term “acyl” refers to the group -C(O)R, where R includes H, “alkyl”, preferably “C1-C6 alkyl”, “aryl”, “heteroaryl”, “C3-C8 cycloalkyl ”, “heterocycloalkyl”, “aryl C1-C6 alkyl”, “heteroaryl C1C6 alkyl”, “cycloalkyl-C3-C8 C1-C6 alkyl” or “heterocycloalkyl C1-C6 alkyl”, including acetyl and the like.

[0049] The term “C1-C6 acyl alkyl” refers to C1-C6 alkyl groups with an acyl substituent, including 2-acetylethyl and the like.

[0050] Unless otherwise limited by the definition of the individual substituent, the term “substituted” refers to groups substituted with from 1 to 5 substituents selected from the group consisting of “C1-C6 alkyl”, “C2-C6 alkenyl”, “C2-C6 alkynyl”, “C3-C8-cycloalkyl”, “heterocycloalkyl”, “C1-C6 alkyl aryl”, “C1-C6 alkyl heteroaryl”, “C1-C6 aryl alkyl”, “heteroaryl C1-C6 alkyl”, “alkyl C1-C6 cycloalkyl”, “alkyl heterocycloalkyl C1-C6”, “amino”, “aminosulfonyl”, “ammonia”, “acyl amino”, “amino carbonyl”, “aryl”, “heteroaryl”, “sulfinyl”, “sulfonyl”, “alkoxy”, “alkoxycarbonyl”, “carbamate”, “sulfanyl”, “halogen”, trihalomethyl, cyano, hydroxy, mercapto, nitro and the like.

[0051] The term “pharmaceutically acceptable” refers to a carrier composed of a material that is not biological or otherwise undesirable and not especially toxic.

[0052] The terms “two-phase hydrogel” is used here to describe the state above the minimum critical solution temperature (LCST), distinguished by a suspension of gel nanoparticles condensed into a low-viscosity, gel-like continuous phase.

[0053] The terms “single-phase hydrogel” and “single-phase graft polymer solution” can be used interchangeably to describe a flowable single-phase state below the LCST. According to one aspect of the invention, an HA graft polymer for the invention undergoes a transition between a single-phase hydrogel and two-phase hydrogel at an LCST that is from about 25°C to about 32°C, e.g. body temperature.

[0054] The term “carrier” refers to any component present in a pharmaceutical formulation that is not an active agent and therefore includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, buffering agents, pH, preservatives and the like.

[0055] The term “bioactive agent” is used to describe any agent with biological activity to be incorporated into a graft polymer composition of the invention. It may be natural, synthetic, semi-synthetic or derivatives thereof which may include hydrophobic, hydrophilic, soluble or insoluble compounds. More specifically, it may be any bioactive agent useful for the treatment and/or prevention and/or diagnosis of conditions in any known therapeutic area in mammals, such as animals and humans, particularly humans, which include, but are not limited to, diseases. inflammatory diseases (including arthritis, osteoarthritis and rheumatoid arthritis), ocular pathologies, tumors and infections. Bioactive agents can be selected from a macromolecular compound or a small molecular compound, such as peptides, proteins, oligo- or polynucleotides, antibiotics, antimicrobials, growth factors, enzymes, antitumor drugs, anti-inflammatory drugs, antiviral drugs, antibacterials, antifungal drugs, antineoplastic drugs, analgesics, anticoagulants, hemostatic drugs and antibodies. More specifically, at least one bioactive agent can be selected from the group consisting of corticosteroids and p38 mitogen-activated kinase inhibitors.

[0056] The term “skin disorders” or “skin diseases” includes skin damage where the surface of the skin has a painful depression without necessarily a cut on the surface such as age-related tissue damage (e.g., wrinkles). , wounds and scars such as, for example, acne and rubella scars. These disorders additionally include wounds, scars, psoriasis, acne, eczema and rosacea. The term “wounds” includes any damaged tissue, for example after trauma or surgery. Wounds in mammals include, for example, abrasions, lacerations, contusions, concussions, stab wounds, skin cuts, surgical wounds, gunshot wounds, thermal wounds, chemical wounds, bites and stings, and electrical wounds. It additionally includes chronic skin disorders such as ulcers.

[0057] The term “ocular pathologies or disorders” are disorders or injuries that affect the eye and in particular the cornea. Such disorders include corneal abrasion, corneal scratches, corneal alkali burns, age-related macular degeneration, bulging eyes, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, floaters and flash vision, glaucoma, keratoconus, amblyopia, low vision, ocular hypertension, retinal detachment, eyelid spasms, uveitis, keratoconjunctivitis sicca (KCS) and dry eye syndrome.

[0058] The term “joint pathologies” includes osteoarthritis, arthritic pain, rheumatoid arthritis, pain due to infection and inflammation, traumatic events in the knees leading to damage to the cartilage, bone, ligaments or synovial capsule.

[0059] The term “reconstructive tissue” refers to a biological tissue (endogenous or exogenous) or a synthetic or semi-synthetic material useful for repairing damaged body tissues such as epidermal, neurological, cartilaginous or bone tissues.

[0060] As used herein, “treatment” and “treat” and the like generally mean obtaining a pharmacological and/or physical and/or physiological effect. The effect may be prophylactic in terms of prevention or partial prevention of a disease, symptom or condition thereof and/or may be in therapeutic terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but who has not yet been diagnosed as having it; (b) inhibit the disease, that is, stop its development; or alleviate the disease, that is, cause a regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage.

[0061] The term “subject” as used here refers to mammals. For example, mammals contemplated by the present invention include humans, primates and domesticated animals, such as cattle, sheep, pigs, horses and particularly racehorses, laboratory rodents and the like.

[0062] The term “efficacy” of a treatment according to the invention can be measured based on changes in the course of the disease in response to use according to the invention. For example, the effectiveness of the treatment according to the invention can be measured by decreased joint pain, improved joint lubrication, improved mobility of a subject, improved eye surface or reduced pressure in the eyes.

Graft polymers according to the invention

[0063] According to one embodiment, a graft polymer of a hyaluronic acid polymer is provided and an N-isopropylacrylamide-based polymer in which the hyaluronic acid polymer and the N-isopropylacrylamide-based polymer are conjugated through hair. least one ligand L of Formula (II): wherein one of Ra and Rb is covalently linked to the N-isopropylacrylamide-based polymer and one of Ra and Rb is covalently linked to the hyaluronic acid polymer and when Ra or Rb is covalently linked to the N-isopropylacrylamide-based polymer , will be equal to R1 and when Ra or Rb is covalently linked to the hyaluronic acid polymer, it will be equal to R3, where R1 is a group selected from - (CH2)x-NH-(CH2)yS-, -( CH2)x-NH-OC(O)-(CH2)yS-, -(CH2)x-NH-(CH2)y- C(O)-NH-(CH2)zS-, -(CH2)xOC(O )-CR7R8-, -C(O)-(CH2)xC(O)-NH- (CH2)yS and a polyethylene glycol (PEG) chain in which R7 and R8 are optionally substituted C1-C6 alkyl such as methyl, R3 is a group -EG-L1- in which E is either absent or selected from -C(O)-NR9-, -C(O)-O- and - C(O)-, G is a group of linker selected from optionally substituted C1-C20 alkyl such as ethyl, optionally substituted polyethylene glycol (PEG) chain, optionally substituted C1-C6 acylamino alkyl, optionally substituted C1-C6 acyl alkyl, optionally substituted C1-C6 alkyl aminocarbonyl and optionally substituted C1-C6 alkoxy and L1 is selected from -NR10C(O)-, -C(O)-NR10-, -C(O)- O- and -C(O)-, where R9 and R10 are independently selected from H and optionally substituted C1-C6 alkyl such as methyl; A is an optionally substituted C5-C10-cycloalkyl or optionally substituted heterocycloalkyl ring, such as an optionally substituted C8 heterocycloalkyl ring or optionally substituted C8-cycloalkyl ring, wherein R5 represents one or more substituent(s) independently selected from H , optionally substituted alkoxy such as methoxy and optionally substituted C1-C6 alkyl such as methyl; x, y and z are integers independently selected from 1 to 20 or any pharmaceutically acceptable salts thereof.

[0064] According to a particular embodiment, an HA graft polymer of Formula (I) is provided: where M is a group selected from a portion of Formula (M1) and Formula (M2): , R2 is a -B-(CH2)Í-D group, in which B is either absent or selected from -SC(S)-S- and -S-; D is a group selected from optionally substituted C1-C15 alkyl such as optionally substituted methyl, ethyl, butyl, propyl, hexyl, heptyl, octyl, nonyl, decanyl (e.g., optionally substituted C1-C4 alkyl such as methyl or ethyl optionally substituted), optionally substituted C1-C4 alkyl alkoxycarbonyl, optionally substituted amino C1-C4 alkyl, optionally substituted C1-C6 alkyl aminocarbonyl, -COOH and NH2; p is an independently selected integer from 1 to 500; i is an independently selected integer from 0 to 500; R6 is a group selected from - COOH and R3, depending on the degree of substitution; L is a ligand of Formula (II) as defined herein and n is an integer selected from 1 to 7,000 or any pharmaceutically acceptable salts thereof.

[0065] According to a particular embodiment, a conjugate of Formula (I) is provided, in which the ligand L is linked to the HA portion of the conjugate through its substituent Rb.

[0066] According to a particular embodiment, a conjugate of Formula (I) is provided, in which the ligand L is linked to the HA portion of the conjugate through its Ra substituent.

[0067] According to a particular embodiment, a conjugate of Formula (I) is provided, in which R1 is a group -(CH2)x-O-C(O)-CR7R8- in which R7 and R8 are optionally substituted C1-C6 alkyl such like methyl.

[0068] According to another embodiment, a conjugate of Formula (I) is provided in which R1 is a group -C(O)-(CH2)x-C(O)-NH- (CH2)y-S.

[0069] According to a particular embodiment, a conjugate of Formula (I) is provided in which x is 3.

[0070] According to another particular embodiment, a conjugate of Formula (I) is provided in which x is 2.

[0071] According to another particular embodiment, a conjugate of Formula (I) is provided in which y is 2.

[0072] According to an additional particular embodiment, an HA graft polymer of Formula (Ia) is provided: or any pharmaceutically acceptable salts thereof, wherein L, R2, R6 and p are as defined in the present description.

[0073] According to a particular embodiment, a conjugate of Formula (I) is provided, wherein A is an optionally substituted C8 cycloalkyl or optionally substituted C8 heterocycloalkyl ring.

[0074] According to an additional particular embodiment, a conjugate of Formula (I) is provided in which A is an optionally substituted aza-dibenzocyclooctynyl.

[0075] According to a particular embodiment, a conjugate of Formula (I) is provided in which

[0076] p is from about 50 to 200.

[0077] According to a particular embodiment, a conjugate of Formula (I) is provided in which

[0078] R7 and R8 are optionally substituted C1-C6 alkyl, in particular methyl.

[0079] According to a particular embodiment, a conjugate of Formula (I) is provided in which B is -S-C(S)-S-.

[0080] According to a particular embodiment, a conjugate of Formula (I) is provided in which B is absent.

[0081] According to a particular embodiment, a conjugate of Formula (I) is provided in which i is from 0 to 20, in particular 0 to 15 such as 0 to 22, in particular 11.

[0082] According to a particular embodiment, a conjugate of Formula (I) is provided in which D is optionally substituted C1-C4 alkyl, such as optionally substituted methyl (for example, methyl optionally substituted by C1-C12 alkyl).

[0083] According to a particular embodiment, a conjugate of Formula (I) is provided in which L is a group according to Formula (IIa): wherein A, R1, R3 and R5 are as described herein.

[0084] According to a particular embodiment, a conjugate of Formula (I) is provided in which L is a group according to Formula (IIb): wherein A, R1, R3 and R5 are as described herein.

[0085] According to a particular embodiment, a conjugate of Formula (I) is provided in which L is a group according to Formula (II'): wherein R11 and R12 represent one or more substituents independently selected from H, optionally substituted alkoxy such as methoxy and optionally substituted C1-C6 alkyl such as methyl and Rae Rb are as described herein.

[0086] According to an additional particular embodiment, a conjugate of Formula (I) is provided in which L is a group according to Formula (IIa'): wherein R11 and R12 represent one or more substituents independently selected from H, optionally substituted alkoxy such as methoxy and optionally substituted C1-C6 alkyl such as methyl and R1 and R3 are as described herein.

[0087] According to another additional particular embodiment, a conjugate of Formula (I) is provided in which L is a group according to Formula (IIb'): wherein R11 and R12 represent one or more substituents independently selected from H, optionally substituted alkoxy such as methoxy and optionally substituted C1-C6 alkyl such as methyl and R1 and R3 are as described herein.

[0088] According to a particular embodiment, a conjugate of Formula (I) is provided, wherein R3 is an -E-G-Li- group as described herein, wherein the linker group G is optionally substituted acylamino C1-C6 alkyl , in particular a C1-C6 alkyl acylamino group substituted with a PEG group, such as, for example, a group of the following formula: -C1-C6 alkyl-NH-C(O)-(CH2-CH2-O)j - CH2-CH2-, where j is selected from 1 to 10, in particular from 1 to 8 or 1 to 6, such as, for example, j is 1, 2, 3 or 4, in particular 4.

[0089] According to a particular embodiment, there is provided a conjugate of Formula (I), wherein R3 is a group -E-G-L1- as described herein, wherein the linker group G is C1-C20 alkyl such as methyl optionally substituted with an optionally substituted polyethylene glycol (PEG) chain, such as, for example, a group of the following formula: -C1-C6 alkyl-(CH2-CH2-O)j-O-CH2-, where j is selected from 1 to 10, in particular from 1 to 8 or 1 to 6, such as, for example, j is 1, 2, 3 or 4, in particular 4.

[0090] According to a particular embodiment, there is provided a conjugate of Formula (I), wherein R3 is a group -E-G-L1- as described herein, wherein the linker group G is optionally substituted C1-C20 alkyl, such as methyl.

[0091] According to a particular embodiment, a conjugate of Formula (I) is provided, in which R3 is a group -E-G-L1- as described here, in which E is absent.

[0092] According to a particular embodiment, a conjugate of Formula (I) is provided, in which R3 is a group -E-G-L1- as described here, in which E is -CO)-.

[0093] According to a particular embodiment, a conjugate of Formula (I) is provided, in which R3 is a group -E-G-L1- as described here, in which L is -NR10C(O)-.

[0094] According to a particular embodiment, R10 is H.

[0095] According to a particular embodiment, a conjugate of Formula (I) is provided, in which L is a group according to Formulas (II), in particular (IIa') with Formula (II''') Next: wherein Q is absent or is a group of the following formula: - NH-C(O)-(CH2-CH2-O)1-4-CH2-CH2- and R11 and R12 are as described herein.

[0096] According to a particular embodiment, a conjugate of Formula (I) is provided, wherein L is a group according to Formulas (II), in particular (IIb') with Formula (II''') Next: wherein G1 is a PEG and, in a more particular embodiment, a group of Formula: - (CH2-CH2-O)1-4-, such as, for example, -(CH2-CH2- O)2-, and R11 and R12 are as described herein.

[0097] According to a particular embodiment, R11 and R12 are H.

[0098] According to a particular embodiment, a conjugate of Formula (I) selected from the following group is provided: having a degree of substitution between about 0.5 and 50, preferably about 2 and 10, or any pharmaceutically acceptable salts thereof.

[0099] According to a particular embodiment, the pharmaceutically acceptable salts of conjugates of the invention include such as sodium, potassium, ammonia and calcium.

[00100] According to a particular embodiment, there is provided a hyaluronic acid polymer graft polymer according to the invention, in which R3 is a group -E-G-L1- as described herein, in which E is absent.

[00101] According to a particular embodiment, there is provided a hyaluronic acid polymer graft polymer according to the invention, wherein R3 is a group -E-G-L1- as described herein, wherein E is -C(O )-.

[00102] According to a particular embodiment, there is provided a hyaluronic acid polymer graft polymer according to the invention, wherein R3 is a group -E-G-L1- as described herein, wherein E is a C1- alkyl C20 optionally substituted, for example selected from methyl, ethyl and propyl.

[00103] According to a particular embodiment, there is provided a hyaluronic acid polymer graft polymer according to the invention, wherein R3 is a group -E-G-L1- as described herein, wherein Li is -C(O )-O-.

[00104] According to a particular embodiment, a hyaluronic acid polymer graft polymer according to the invention is provided, in which R3 is a group -O-(CH2)-C(O)-O-.

[00105] According to a particular embodiment, a hyaluronic acid polymer graft polymer according to the invention is provided, in which R3 is a group -(CH2)3-C(O)-O-.

[00106] It is understood that the molecular weight of the conjugate of the invention can be adapted by selecting the starting material and its choice will depend on the intended function of the graft polymer composition, formulation and hydrogel formed therefrom. For example, the degradation rate of the desired graft polymer composition, the desired release rate of the bioactive agent optionally incorporated therein, the therapeutic condition to be treated will influence the choice of molecular weight for the conjugate of the invention.

[00107] In one embodiment, the molecular weight of the graft polymers of the invention for use in lubricant/filler compositions can be chosen from about 2,000 to about 250,000,000 Da, preferably from about 500,000 to about 10,000 ,000.

[00108] In another embodiment, the molecular weight of the graft polymers of the invention for use in or as a drug delivery system can be chosen from about 2,000 to about 250,000,000 Da, preferably from about 500,000 and about 10,000,000.

1. Preparation of conjugates according to the invention

[00109] The new HA conjugate according to the invention can be prepared from readily available starting materials by the use of copper-free “click” chemistry and the following general methods and procedures. It will be recognized that where typical or preferred experimental conditions (i.e., reaction temperatures, time, moles of reactants, solvents, etc.) are available, other experimental conditions may also be used unless otherwise indicated. Optimal reaction conditions may vary with the particular reagents or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures. A general synthetic approach to obtain conjugates of Formula (I) is to react an azide-bearing N-isopropylacrylamide-based polymer with an HA polymer bearing a cyclooctin moiety or an azide-bearing HA polymer with an N-based polymer. - isopropylacrylamide carrying a cyclooctin moiety.

[00110] According to one aspect of the invention, there is provided a method for preparing a graft polymer of a hyaluronic acid polymer and an N-isopropylacrylamide-based polymer, wherein the hyaluronic acid polymer and the hyaluronic acid-based polymer of N-isopropylacrylamide are conjugated by at least one ligand L of Formula (II), comprising the steps of: (a) providing an HA polymer with at least one carboxylic acid group that is conjugated to at least one first functional group capable of participate in a “click chemical” reaction; (b) providing an N-isopropylacrylamide-based polymer with at least one second complementary functional group capable of participating in a “click chemical” reaction with the first functional group, wherein said one of the functional groups is an azide moiety and the other functional group is an alkyne-bearing precursor of said ligand L; (c) reacting the at least one first functional group of the HA polysaccharide with the at least one second complementary functional group of the N-isopropylacrylamide-based polymer by means of a “click chemistry” reaction to obtain a graft polymer composition of the invention ; and (d) isolating the graft polymer composition.

[00111] According to an additional particular embodiment, a method of the invention may comprise additional steps to physically disperse and/or covalently bind and/or add at least one bioactive agent into the composition of the graft polymer obtained in step d).

[00112] A general synthetic approach to obtaining conjugates of Formula (I), in particular Formula (Ic1) is illustrated in Scheme 1 below. The HA conjugates according to Formula (I), in which R1, R2, R3, R5, R6 and p are defined in the detailed description, can be prepared in a single chemical step by click chemistry in an appropriate solvent, such as, for example, an aqueous mixture of DMSO at a volume ratio of 1-1, from custom-made or commercially available starting materials.

[00113] Intermediate (Ia1) can be commercially available or prepared by reacting a compound in which one of the functional groups is an azide moiety with an N-isopropylacrylamide-based polymer by amidation, esterification, thioetherification, oxidation or Ugi condensation.

[00114] The intermediate (Ib1) can be commercially available or prepared by reacting the ligand precursor of Formula (Lprec1) with hyaluronic acid or any pharmaceutically acceptable salts thereof by amidation, esterification, thioetherification or maleimide-sulfhydryl reaction, oxidation or Ugi condensation as described in Schanté et al., 2011, Carbohydrate Polymers, 85, 469-489. wherein A and R5 are as defined herein and R3' is a group - EG-L1' wherein E and G are as defined herein and L1' is an amino group. Scheme 1

[00115] Another general synthetic approach to obtaining conjugates of Formula (I), in particular Formula (Ic2), is depicted in Scheme 2 below, in a single chemical step by click chemistry in an appropriate solvent, such as water and/or dimethyl sulfoxide, etc., from custom-made or commercially available starting materials. Scheme 2

[00116] Intermediate (Ia2) can be commercially available or prepared by reacting the ligand precursor of Formula (Lprec2) with an N-isopropylacrylamide-based polymer by amidation, esterification, thioetherification, oxidation or Ugi condensation. wherein A and R5 are as defined herein and R1' is an alcohol, carboxylic acid, carbonyl acid, azide, thioester, maleimide, acyl phosphate, acid anhydride, acyl chloride, amino carboxylate group or ester.

[00117] The intermediate (Ib2) can be commercially available or prepared by reacting a compound in which one of the functional groups is an azide moiety with hyaluronic acid or any pharmaceutically acceptable salts by amidation, esterification, thioetherification, oxidation or Ugi condensation.

[00118] According to a particular embodiment, an intermediate (Ia2) is provided wherein A, R1, R2, R3 are as defined herein.

Compositions

[00119] The invention provides pharmaceutical or therapeutic agents as compositions, or medical devices comprising the same and methods for treating a subject, preferably a mammalian patient, and more preferably a human patient who is suffering from a medical disorder, and in particular pathology. joints, joint diseases, ocular pathologies (such as age-related macular degeneration, bulging eyes, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, floaters and flash vision), glaucoma, keratoconus, amblyopia, low vision, ocular hypertension, retinal detachment, eyelid spasms and uveitis), skin defects or lesions (such as wounds, scars, psoriasis, acne, eczema and rosacea) and any tissue degeneration (such as stress urinary incontinence, lupus systemic erythematosus, rheumatoid arthritis, scleroderma, Sjogren's syndrome, mixed connective tissue disease, psoriatic arthritis, Marfan's syndrome, Peyronie's disease, Ehlers-Danlos syndrome, Stickler's syndrome, Alport's syndrome, congenital contractural arachnodactyly, Loeys' syndrome -Dietz and scurvy).

[00120] In a particular embodiment, the invention provides a pharmaceutical formulation comprising at least one conjugate or composition according to the invention for use as a medicament.

[00121] In another particular embodiment, the compositions of the invention are parenteral formulations.

[00122] In a particular embodiment, the compositions of the invention are injectable formulations, such as intra-articular, intra-arterial, intravenous, intrasynovial, intradermal, subdermal, submucosal, interstitial and intramuscular formulations.

[00123] In another particular embodiment, the compositions of the invention are oral formulations.

[00124] In another particular embodiment, the compositions of the invention are topical formulations.

[00125] In another particular embodiment, the compositions of the invention are ophthalmological formulations.

[00126] Alternatively, the invention provides compositions that can be used in another mammal, therefore, humans, for the same uses as described above.

[00127] The invention further provides medical compositions or devices useful for cosmetic applications, reconstructive surgeries (e.g., tissue reconstruction) and cell or biological tissue culture (e.g., stem cell culture). These compositions additionally respectively comprise cosmetically acceptable carriers or cell or biological tissue culture.

[00128] Compositions according to the invention comprise soft tissue filler compositions, for example, dermal or subdermal fillers, comprising at least one conjugate of the invention or hydrogel or nanoparticles thereof. The preparation of HA-based soft tissue filler compositions is well known to those skilled in the art and can be carried out, for example, as described in Jeon et al., 2007, Carbohydrate Polymers, 2007. 70(3): p. 251-257, Iannitti et al, 2013, Int. J. Pharm., 456:583-592.

[00129] According to a particular embodiment, the compositions of the invention have a viscosity of between about 0.001 Pa*s and about 100 Pa*s, and preferably from about 0.1 to 10 Pa.s when measured at around 100 Hz in oscillating mode at 20°C.

[00130] According to a particular embodiment, the compositions of the invention have an extrusion force between about 0.01 N and about 20 N, and preferably from about 1 N and about 5 N at 25°C with an extrusion rate of about 1 mm/minute through a 29 gauge needle.

[00131] According to a particular embodiment, after solubilization in an aqueous solvent, the graft polymer compositions of the invention can form a pH-sensitive and/or thermosensitive hydrogel showing a monophasic to biphasic phase transition with temperature change.

[00132] According to the particular aspect, the graft polymer compositions of the invention form a single-phase hydrogel and “single-phase graft polymer solution” below the LCST, which is below from about 25°C to about 32°C. According to another particular aspect, the graft polymer compositions of the invention form a biphasic hydrogel above such a critical minimum solution temperature.

[00133] In a particular embodiment, the graft polymer compositions of the invention can spontaneously form nanoparticles above from about 25°C to about 32°C, for example body temperature.

[00134] Additionally, the graft polymer compositions of the invention have the advantage of undergoing this nanoparticle formation, which is reversible and, therefore, potential temperature effects during storage and transport would not irreversibly affect the physical state of the polymer.

[00135] In a particular embodiment, nanoparticles of graft polymer compositions of the invention are provided with an average size ranging from about 40 to 500 nm.

[00136] According to another particular embodiment, the compositions of the invention may be in the form of a pH-reversible and/or thermoreversible hydrogel that is biodegradable and biocompatible. In particular, the compositions of the invention may be in the form of a pH-reversible and/or thermoreversible hydrogel that is in a single-phase (solution or liquid) state at or about room temperature and in a gelled state at or about , physiological (or in vivo) temperature or greater.

[00137] The compositions of this invention may additionally comprise one or more additional pharmaceutically acceptable ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants and the like.

[00138] The compositions according to the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient can be placed in the form of pharmaceutical compositions and can be employed as solids, such as tablets or filled capsules, or liquids such as solutions , suspensions, ointments, emulsions, elixirs or capsules filled with the same, films or gels, all for oral use. The compositions may also be formulated as a dry product for reconstitution with water or any other suitable vehicle prior to use.

[00139] The compositions of this invention as liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups and elixirs.

[00140] Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, sugar/glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include, but are not limited to, poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®.

[00141] Additional materials, as well as formulation processing techniques and the like, are set forth in The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), 22nd Edition, 2012, , , which is incorporated herein by way of reference. Lloyd, Ed. Allen http://www.openisbn.com/author/Lloyd,_Ed._Allen/ Pharmaceutical Press http://www.openisbn.com/publisher/PHARMACEUTICAL_PRESS/

[00142] The solid compositions of this invention can be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[00143] In another particular embodiment, the compositions or hydrogels of the invention additionally comprise a bioactive agent, which is either dispersed in or covalently connected to the graft polymer composition.

[00144] In another particular embodiment, the compositions or hydrogels of the invention are in the form of a bioactive agent dispensing system.

[00145] In one embodiment, the at least one bioactive substance may be present in an amount of between about 0.001 to 40% by weight, preferably about 0.01 to 30% by weight based on the total amount of polymer composition graft or hydrogel of the invention.

[00146] According to another particular aspect, the compositions according to the invention comprise a biological cell or tissue culture medium comprising an HA graft polymer or a composition thereof. This culture medium may further comprise cell nutrients such as glucose, vitamins, growth factors, metal ions and the like.

[00147] According to another aspect, the compositions according to the invention comprise a biological tissue (endogenous or exogenous) or a synthetic or semi-synthetic material useful for repairing damaged tissues of the body such as epidermal, neurological, cartilaginous or bone tissues.

[00148] In another particular embodiment, a method is provided for preparing a culture medium comprising a step of mixing an HA graft polymer according to the invention with cell culture nutrients, such as glucose, vitamins, growth factors, metal ions and the like.

[00149] In another particular embodiment, there is provided a method for preparing a reconstruction tissue comprising a step of combining an HA graft polymer or a composition thereof according to the invention with materials, tissues or cells useful for repair damaged tissues of the body, such as stem cell or epidermal, neurological, cartilaginous or bone tissues.

[00150] In a particular embodiment, a method is provided for preparing a graft polymer composition, or hydrogel of the invention, in particular a dispensing system for an active ingredient comprising the steps of: - providing a graft polymer or a composition of the same or hydrogel of the invention in a gel state; - provide an active ingredient to be dispensed; - mixing said graft polymer, or composition thereof, or hydrogel of the invention with said active ingredient (e.g., drug); - optionally inducing nanoparticle formation by increasing the temperature of the mixture (typically from about 25 to about 40°C); - collect the composition thus obtained, hydrogel and nanoparticles loaded with the active ingredient (e.g. drug).

[00151] According to a particular aspect, the compositions, hydrogels or nanoparticles of the invention may be useful, once loaded with an active ingredient, for in situ administration and release of said active ingredient injection.

Administration mode

[00152] The compositions of this invention can be administered in any way orally, including the mucosal surfaces of the oral cavity, including the gums, floor of the oral cavity, cheeks, lips, tongue, teeth.

[00153] The compositions of this invention can also be administered in any way by injection, such as subcutaneous, intrasynovial, intra-arterial, intravenous, intra-articular, intramuscular, subdermal, submucosal and interstitial injections.

[00154] The compositions of this invention can also be administered topically to the skin, various mucous membranes or the eye.

Combination

[00155] According to one aspect, the polymers of the invention or any suitable pharmaceutically acceptable thereof and pharmaceutical formulations thereof can be administered alone or in combination with at least one coagent.

[00156] According to a particular aspect, the coagents according to the invention include pure HA, chondroitin sulfate or other intra-articular drug composition, as well as bioactive agents according to the invention.

[00157] The invention encompasses the administration of conjugates of the invention and pharmaceutical formulations thereof to an individual simultaneously or sequentially with at least one coagent useful in the treatment of inflammatory diseases (including arthritis, osteoarthritis and rheumatoid arthritis), ocular pathologies, tumors and infections . These coagents can be selected from antibiotics, antimicrobials, growth factors, enzymes, antitumor drugs, anti-inflammatory drugs such as corticosteroids, antiviral drugs, antibacterial drugs, antifungal drugs, anesthetics, antineoplastic drugs, analgesics, anticoagulants and hemostatics.

[00158] The polymers, nanoparticles or hydrogel of the invention or a pharmaceutical formulation thereof that is administered simultaneously with said coagent can be administered in the same or different composition(s) and by the same or different route(s) of administration.

[00159] The concentration range of a polymer of the invention, nanoparticles or hydrogel thereof in compositions of the invention may vary depending on the circumstances of each case, the underlying disease, the clinical indication and the desired intensity of pain relief that is sought and /or necessary.

[00160] According to one embodiment, there is provided a pharmaceutical formulation comprising at least one conjugate of the invention or any suitable pharmaceutically acceptable thereof combined with at least one coagent described herein, and at least one pharmaceutically acceptable carrier.

[00161] The dosage administered, as single or multiple doses, to an individual will vary depending on a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health and size), extent of symptoms , concomitant treatments, frequency of treatment and desired effect.

Patients

[00162] In one embodiment, patients according to the invention are subjects suffering from, or at risk of suffering from, some tissue degeneration, joint diseases, ocular pathologies, skin defects or injuries.

[00163] In a further embodiment, patients according to the invention are subjects suffering from, or at risk of suffering from, joint pathologies and articular diseases, such as osteoarthritis, arthritis, infection, rheumatoid arthritis and traumatic events in the knee leading to to damage to cartilage, bone, ligament or synovial capsule.

[00164] In another additional embodiment, patients according to the invention are subjects suffering from, or at risk of suffering from, ocular pathologies, such as age-related macular degeneration, bulging eyes, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, floaters and flash vision, glaucoma, keratoconus, amblyopia, low vision, ocular hypertension, retinal detachment, eyelid spasms and uveitis.

[00165] In another additional embodiment, patients according to the invention are subjects suffering from, or at risk of suffering from, skin defects or injuries, such as scars, abrasions, lacerations, contusions, concussions, stab wounds, cuts in the skin, surgical wounds, gunshot wounds, thermal wounds, chemical wounds, bites and stings, and electrical wounds. It additionally includes chronic skin disorders such as ulcers.

[00166] In another additional embodiment, patients according to the invention are subjects who desire or require enhancement in volume in parts of the body or anatomical changes in shape, such as thickening of urological tissue, any enhancement/modification and /or increasing the volume of a part of the body for aesthetic or therapeutic reasons.

[00167] In another additional embodiment, patients according to the invention are subjects suffering from a tumor or vascular malformation.

Use according to the invention

[00168] According to a particular embodiment, the HA conjugates of the invention and compositions thereof are useful for various applications, such as in vitro cell or biological tissue culture, as tissue fillers, for cosmetic applications and for engineering applications. tissue.

[00169] According to another particular embodiment, there is provided a use of a graft polymer or a hydrogel or nanoparticle according to the invention for the preparation of a dispensing system, in vitro cell culture or biological tissue or materials for tissue engineering, such as neurosurgical, bone, cartilaginous or epidermal reconstruction tissues.

[00170] According to another particular embodiment, the HA conjugates of the invention and compositions thereof are useful for use in the prevention or treatment of a medical disorder, and in particular joint pathologies, joint diseases, ocular pathologies, defects or skin lesions, thickening of urological tissue, any tissue deformation, improvement/modification and/or increase in volume of a part of the body for aesthetic or therapeutic reasons or for the treatment of a vascular malformation or tumor.

[00171] According to an additional particular embodiment, the formulations of the invention can be used in the form of a monophasic hydrogel, below the LCST, for injection into the capillary bed of a tumor or a vascular malformation, wherein said formulations form particles of small hydrogel in situ after exposure to body temperature (above LCST), thereby allowing vascular embolization. Embolization resulting from said vascular or tumor malformation results in blockage of blood and nutrient access, and could be useful either to directly kill the tumor or to prepare for resection or additional surgical treatment, such as in combination with radiotherapy or chemotherapy.

[00172] According to a further particular aspect of the invention, there is provided an HA conjugate of the invention and compositions thereof for use in vascular embolization.

[00173] According to another additional particular aspect of the invention, there is provided an HA conjugate of the invention and compositions thereof for cosmetic use and for aesthetic or reconstructive surgeries.

[00174] According to another additional particular aspect, there is provided an HA conjugate of the invention and compositions thereof for use in in vivo drug dispensing.

[00175] According to a particular embodiment, there is provided a pH-sensitive and/or thermosensitive biodegradable hydrogel comprising at least one HA conjugate of the invention, which is useful in many medical applications, such as a dispensing system for at least a bioactive agent or cell delivery system in tissue engineering, cell culture systems or biological tissue, and the like.

[00176] According to a particular aspect, the use of a hydrogel according to the invention is provided as a dispensing system for at least one bioactive agent.

[00177] In another embodiment of the invention, there is provided a use of at least one HA conjugate of the invention or any suitable pharmaceutically acceptable composition thereof alone or in combination with at least one coagent for the preparation of a pharmaceutical formulation for the prevention or treatment of tissue degeneration and related disorders.

[00178] According to a particular aspect, there is provided an HA conjugate of the invention and compositions thereof in which said conjugate is capable of forming nanoparticles in situ spontaneously.

[00179] Examples illustrating the invention will be described from now on in more detail and with reference to the embodiments represented in the Figures.

EXAMPLES

[00180] The following abbreviations refer specifically to the definitions below: DMSO (dimethyl sulfoxide), MWCO (Molecular Weight Cutoff).

Example 1: Synthesis of hyaluronic acid conjugates of the invention

[00181] The conjugates of the invention were synthesized as described in Scheme 1 above, where the starting materials (ligand precursors) used to create the ligands were:

[00182] Dibenzocyclo-octin-Amine (DBCO-amine) (Formula (L-prec1), where A is a C8 heterocycloalkyl ring being dibenzocyclo-octin, R5 is H and R3' is an -E-G-L1' group, in which E is -C(O)-, G is ethyl and L1' is -NH2) purchased from Sigma-Aldrich (St. Louis, USA). Dibenzocyclo-octin-PEG4- Amine (DBCO-PEG4-amine) Formula (L-prec1), where A is a C8 heterocycloalkyl ring being dibenzocyclo-octin, R5 is H and R3' is a -E- G-L1' group , where E is -C(O)-, G is -NH-C(O)-(CH2-CH2-O)4- CH2-CH2- and Li' is -NH2) purchased from Click Chemistry Tools (Scottsdale, Arizona, USA).

[00183] Alternatively, the DBCO-amine and DBCO-PEG4-amine ligand precursors can be prepared as described in Pola et al., 2014, Polym. Chem., 5, 1340 and Poly(N-isopropylacrylamide) azide terminated (pNiPAM-N3) can be prepared as described in Xu et al., 2007, Macromolecules, 40, 9103-9110.

[00184] Azido-PEG3-Amine (N3-PEG4-amine) purchased from Click Chemistry Tools (Scottsdale, Arizona, USA).

[00185] Dibenzocyclo-octin-NHS Ester (DBCO-NHS Ester) (Formula (L-prec2), where A is a C8 heterocycloalkyl ring being dibenzocycloctin, R5 is H and R1' is an -E-G-R1' group , where E is -C(O)-, G is ethyl and R1' is -COOH activated with N-hydroxysuccinimide/ethyl(dimethylaminopropyl) carbodiimide (NHS/EDC)).

[00186] Alternatively, N3-PEG4-amine ligand precursors can be prepared as described in Hiki et al., 2007, Bioconjugate Chem., 18, 2191-2198 and DBCO-NHS Ester can be prepared as described in Liu et al ., 2012, J. Am. Chem. Soc., 134, 18886-18888.

[00187] Additional starting materials used are as follows:

[00188] Hyaluronic Acid (HA) was purchased from Soliance (Pomacle, France) (MW: 1.85 MDa); N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Poly(N-isopropylacrylamide) azide terminated in (pNiPAM-N3) (MW: 15kDa) (Formula (Ia1 ), where R1 is as described in the detailed description with x being 3, R7 and R8 are methyl, p is 132 and R2 is as described in the detailed description with B being -S-C(S)-S-, i being 11 and D being methyl) and Poly(N-isopropylacrylamide)-terminated amine (pNiPAM-amine) were purchased from Sigma-Aldrich (St. Louis, USA). Cyanine5-azide (Cy5-N3) was provided by Lumiprobe (Hannover, Germany).

HA-B1 synthesis

[00189] The title conjugate of the invention was synthesized according to Scheme 1A below.

[00190] HA (1) is dissolved in distilled water at 1% (w/v). After dissolution of the polymer, EDC was added (1 eq. COO-) and 5 minutes later NHS was dissolved (1 eq.) under magnetic stirring (5 minutes at 1,200 RPM). The pH was adjusted to 5.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl. DBCO-amine (2) was used as a precursor of Formula (Lprec1) as defined above and was added (1 eq.) with DMSO to a final volume ratio of 40:60 of aqueous DMSO mixture. The reaction was stirred overnight at room temperature and was precipitated in ethanol to obtain the intermediate of Formula (Ib1), where A is a C8 heterocyclealkyl ring being dibenzocycloctyne, R5 is H and R3 is - C(O )-(CH2)2-NH-C(O)- (3). The precipitate (3) was lyophilized, redissolved in 1% (w/v) distilled water and pNiPAM-N3 (1 eq.) (4) used as an intermediate of Formula (Ia1) as described above was added (2h at 1,200 RPM ). HA-P1 was dialyzed (MWCO 25,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 1,500 RPM and was precipitated in ethanol and lyophilized and stored at 4 °C.

[00191] The structure of the conjugate resulting from the HA-B1 invention was determined by 1H NMR spectra (Figure 1D) Gemini 300 MHz from Varian (Grenoble, France) at room temperature using D2O as a solvent. Scheme 1

[00192] The degree of substitution (DS) of HA-DBCO was determined as (1 eq. COO-) using the ratio of the integration of aromatic proton peaks of DBCO (δ 7.67 ppm) to the integration of acetyl proton peak of HA (δ 2.00 ppm) and the degree of substitution (DS) of HA-B1 was finally quantified as 7.13% (1 eq. COO-) using the integration ratio of the aromatic proton peaks DBCO (δ 7.67 ppm) to the integration of pNiPAM methyl proton peaks (δ 1.56 ppm).

HA-P2 synthesis

[00193] The conjugate of the title of the invention was synthesized according to Scheme 1B below:

[00194] HA (1) is solubilized in 1% (w/v) distilled water. After dissolution of the polymer, EDC was added (0.5 eq. COO-) and 5 minutes later NHS was dissolved (0.5 eq.) under magnetic stirring (5 minutes at 800 RPM). The pH was adjusted to 5.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl. DBCO-PEG4-amine (2P) was added (0.5 eq.) with DMSO to a 40:60 final volume ratio of DMSO aqueous mixture. The reaction was stirred overnight at room temperature and was dialyzed (MWCO 12-14,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 900 RPM and then in distilled water 3 times for 3h at 900 RPM to obtain an intermediate of Formula (Ib1), where A is a heterocycle-alkyl ring C8 being dibenzocyclooctine, R5 is H and R3 is -C(O)-(CH2 )2-NH-C(O)-(CH2-CH2-O)4)-(CH2)2-NH-C(O)- (3P). The mixture was redissolved in 1% (w/v) distilled water and pNiPAM-N3 (0.5 eq.) (4) was added (2h at 1500 RPM). The HA conjugates were dialyzed (MWCO 25,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 900 RPM and then in distilled water 3 times for 3h at 900 RPM and freeze-dried and stored at 4°C. The conjugate of the invention HA-B2 was also synthesized by this method by using DBCO-amine (2) instead of DBCO-PEG4-amine (2P) to lead to the same conjugate as (HA-B1), but with different ratios. (pNiPAM/HA). After lyophilization, the structure and DS of the obtained conjugates were determined by 1H NMR spectra in D2O as described above (Figures 1F and 1E). The DS of HA-B2 and HA-P2 were respectively 7.47% and 9.53% (0.5 eq. COO-). Scheme 1B

HA-B3 synthesis

[00195] The conjugate of the title of the invention was synthesized according to general Scheme 2, in particular Scheme 2A below. Scheme 2A

[00196] HA (1) is solubilized in 1% (w/v) distilled water. After dissolution of the polymer, EDC was added (1 eq. COO-) and 5 minutes later NHS was dissolved (1 eq.) under magnetic stirring (5 minutes at 1,200 RPM). The pH was adjusted to 5.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl and N3-PEG4-amine (5) was added (1 eq.) with DMSO to a ratio of final volume 40:60 of DMSO aqueous mixture. HA-N3 was dialyzed (MWCO 1,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 1,500 RPM and was precipitated in ethanol and lyophilized to lead to HA -N3 (6) (Intermediate Ib2, where R3 is a group -C(O)-NH-CH2-O- (CH2-CH2-O)2- CH2- CH2-).

[00197] pNiPAM-amine (7) is dissolved in 1% (w/v) distilled water under magnetic stirring (5 minutes at 1,200 RPM). The pH was adjusted to 7.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl and DBCO-NHS Ester (8) was added (1 eq.), was used as a precursor of Formula (Lprec2) as defined above, wherein A is aza-dibenzocyl-ocynyl, R5 is H and R1' is (2,5-dioxopyrrolidin-1-yl)4-oxobutanoyl, with DMSO for a final volume ratio of 40:60 aqueous DMSO mixture to lead to pNiPAM-DBCO (9) (Intermediate Ia2 in which A is aza-dibenzocyclo-octynyl, R5 is H, R3 is -C(O)-(CH2)2-C(O )-NH-(CH2)2-S and R2 is a -B-(CH2)i-D group, where B is absent, i is 0 and D is methyl). pNiPAM-DBCO was dialyzed (MWCO 1,000, Spectra/Por® membrane, Rancho Dominguez, USA) against distilled water 3 times for 3h at 1,500 RPM and lyophilized.

[00198] HA-N3 (6) is then solubilized in 1% (w/v) distilled water and pNiPAM-DBCO (9) (1 eq.) was added (2h at 1,500 RPM). The HA conjugates were dialyzed (MWCO 25,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 900 RPM and then in distilled water 3 times for 3h at 900 RPM and lyophilized and stored at 4°C to lead to the conjugate of the invention HA-B3, in particular a conjugate of Formula (I), where M is M2.

Synthesis of HA-pNiPAM (comparative control)

[00199] A comparative ligand-free HA-pNiPAM conjugate was synthesized using the method described below: HA is solubilized in 1% (w/v) distilled water. After dissolution of the polymer, EDC was added (1 eq. COO-) and 5 minutes later NHS was dissolved (1 eq.) under magnetic stirring (5 minutes at 1,200 RPM). The pH was adjusted to 5.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl and pNiPAM-amine was added (1 eq.). The mixture was stirred overnight at room temperature. HA-pNiPAM was dialyzed (MWCO 25,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 1,500 RPM and was precipitated in ethanol and lyophilized and stored at 4 °C.

Synthesis of HA-P2-Cy5 (fluorescent HA conjugate)

[00200] Conjugates of HA and a fluorescent azide dye were coupled with the same synthesis parameters as HA-P2, except 0.1 wt% Cy5-N3, as an example, was added before the addition of pNiPAM-N3 .

Synthesis of HA-Cy5 (fluorescent comparative control)

[00201] HA (1) is solubilized in distilled water at 1.5% (w/v). After dissolution of the polymer, EDC was added (1.1 eq. COO-) and 5 minutes later NHS was dissolved (1.1 eq.) under magnetic stirring (5 minutes at 900 RPM). The pH was adjusted to 5.0 with a Metrohm gel pH electrode (Herisau, Switzerland) with 0.1 M NaOH/HCl. DBCO-amine was added (1 eq.) with DMSO to a final volume ratio of 1:1 aqueous DMSO mixture. The reaction was stirred overnight at room temperature and was dialyzed (MWCO 12-14,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 900 RPM and then in distilled water 3 times for 3h at 900 RPM. The mixture was redissolved in 1% (w/v) distilled water and 0.1% (w/w) Cy5-N3 was added (2 h at 1500 RPM). The HA conjugates were dialyzed (MWCO 12-4,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 5% (w/v) NaCl in distilled water 3 times for 3h at 900 RPM and then in distilled water 3 times for 3h at 900 RPM and freeze-dried and stored at 4°C.

Example 2: Injectability of compounds of the invention

[00202] The maximum percentage (w/v) of the HA conjugate below an injectability force of 5 N was determined in order to test the injectability of the compounds of the invention.

[00203] A 1 mL Norm-ject® syringe was filled with a 23G needle and a 1Ml insulin syringe with a 29G BD Micro-Fine™ fixed needle were used. Injection force was measured at 20 and 37°C using a Texture Analyzer TA.XT. plus (Tracomme AG, Switzerland). The conjugates were measured with 2 syringe-needle systems and showed an injection profile influenced by the syringe material (Fig. 2B). In fact, the BD Micro-Fine™ 29G Fixed Needle is made with a plunger lock with double seal ring for slow aspiration and injection. Fi (initial force) and Fp (plateau force) were similar and greater than Ff (final force) with a 23G syringe and Fi>Fp>Ff with a 29G syringe, as shown in Figure 2. The concentrations for HA- B1, HA-B2 and HA-P2 were respectively 7.5%, 0.5% and 0.75% (w/v) were considered easy to inject (<5 N). These results showed the excellent injectability of the HA conjugates of the invention and the possible increase in the maximum concentration according to the injection system.

Example 3: Rheological behavior of the compounds of the invention

[00204] The rheological behavior of the compound of the invention was investigated as follows:

[00205] rheological measurements were carried out on samples of 0.4 of HA-B1 (7.5% w/v), HA-B2 (0.5% w/v), HA-P2 (0.75% w/v /v) and Ostenil® (1 % w/v) with a Haake 1 Rheometer using a cone-plate geometry with a 35/2° Ti cone (Vreden, Germany) and a shear rate ranging from 0.1 s-1 at 100 s-1 at 20 °C in a controlled humidity chamber.

[00206] The results indicated that all HA graft polymers of the eOstenil® invention were non-Newtonian and pseudoplastic fluids. The viscosity of pseudoplastic fluids decreased with increased shear, which is an injection with advantageous facilitating properties.

[00207] The Minimum Critical Solution Temperature (LCST) was also investigated by dynamic rheometry modules G1 (elasticity) and G'' (viscosity) as a function of heating temperature at 1°C/min. The LCST was distinguished by the intersection of modules G’ and G’’. All HA conjugates showed a critical temperature below which the mixture was miscible (LCST): 24 to 25°C for HA-P1 and 31 to 32°C for HA-B2 and HA-P2. All HA graft polymers of the invention were solutions (G''<G'), unlike Ostenil®, and in the case of HA-P2, the G' and G'' values increased rapidly, especially the elastic properties (G'), unlike HA-B1. The grafting of pNiPAM and the linker influenced nanoparticle formation and, in turn, changed the rheological properties above the LCST, indicating a biphasic nanoparticle-solvent mixture.

Example 4: Particle size and morphology of the conjugates of the invention

[00208] The HA graft polymers of the invention were solubilized in distilled water and the HA graft polymer solutions were incubated at 37°C, thus forming nanoparticles.

[00209] The average particle size of the thus formed HA nanoparticles was determined above the LCST by three methods. Scanning Electron Microscopy (SEM) (Jeol Microscope, JSM-7001TA, Tokyo, Japan) micrographs by measuring diameters of 50 particles using the analysis software version 1.45s Image J (NIH, USA), Dynamic Light Scattering (DLS ) (Nanosizer, Malvern, England) and Nanoparticle Tracking Analysis (NTA) (NanoSight LM10, Malvern, England). SEM analysis showed that the nanoparticle morphologies of the HA conjugates after incubation at 37 °C as shown in Figure 3. The HA-B1 nanoparticles were smaller and more spherical compared to the other HA conjugates. The high graft ratio of pNiPAM/HA influenced the spherical formation (Fig. 3 ABC) of the nanoparticles and also the viscosity/elasticity as a function of temperature. For HA-P2 (Fig. 3B), the size of the nanoparticles indicated the influence of the PEG4 ligand. Also, HA-P2 nanoparticles were slowly resolubilized below the LCST (31 to 32°C), unlike the rapid reversible transition of HA-B2 (Fig. 3E). In a different way, the PEG4 ligand influenced the solubility of the HA conjugates and the size of the nanoparticles formed (Table 1). Such a beauty 1

[00210] In contrast, the Ostenil® hydrogel and the unliganded HA-pNiPAM polymer (negative control) (Fig. 3D) did not form any nanoparticles after heating.

Example 5: Intravital fluorescence

[00211] The in vivo residence time of the conjugates of the invention was measured as follows: intravital fluorescence in mice was measured for HA-P2-Cy5 and HA-Cy5 (comparative control) after administration by two injection routes in mice ( healthy hypodermis and intra-articular injection in knee osteoarthritis). HA-P2-Cy5 and HA-Cy5 solutions (1% w/v) were injected into joints (n=7) and subcutaneously (n=4).

[00212] In the case of HA-Cy5, significant differences were observed. After 7, the total signal decreased in intra-articular injection and the area in pixels increased for the subcutaneous site. HA-Cy5 showed a short residence time at the injection site (Figure 5). HA-P2-Cy5 spontaneously formed nanoparticles at body temperature. The increase in pixel area was balanced by the decrease in average signal. Clearly, no significant difference for the total HA-P2-Cy5 signal was found. The HA conjugate showed a long residence time in the body at the injection site due to the spontaneous formation of nanoparticles (Figure 5).

Example 6: Biocompatibility

[00213] The biocompatibility of the conjugates of the invention was measured as follows:

[00214] after 7 days (n=4) and 21 days (n=4), subcutaneous injection into healthy mice of HA-P2-Cy5, pieces of skin were removed, where Cyanine 5 fluorescence was detected, and were cut in sagittal plane. After embedding in paraffin, 4 μm sections were cut with a microtome. Sections were stained with hematoxylin-eosin (HE). The biocompatibility of HA-P2-Cy5 at injection sites is controlled by tissue/material interaction. The interaction and biocompatibility of HA-P2-Cy5 in subcutaneous injections after 21 days was confirmed. Macrophages, larger cells in the tissue reaction were not larger in surrounding tissues compared to the control group (Ostenil®).

Example 7: in vitro drug release

[00215] To determine the drug release profile of the HA graft polymer nanoparticles of the invention, in vitro drug release was conducted as follows. The saline solution (NaCl 0.9%), Ostenil®, the graft polymers of the invention HA-B2 and HA-P2 were saturated by dexamethasone base (drug). Ten mL of the above solutions were placed in a dialysis bag (MWCO 1,000, Spectra/Por® membrane, Rancho Dominguez, USA) against 400 mL of 0.9% NaCl (w/v) and incubated at 37°C and 80 RPM . After 28 h, the polymer matrix was hydrolyzed by heating (121°C), releasing the remaining dexamethasone content, and DMSo was added to a final volume ratio of 1:1 aqueous DMSO. Release from each time point was quantified by reverse phase UHPLC using a C18 Hypersil Gold column (bead particle size 50/2.1, 1.9 μm Thermo Scientific, Waltham, USA). The mobile phase composed of 0.1% v/v formic acid in water (A) and 0.1% v/v trifluoroacetic acid (TFA) in acetonitrile (B) and a gradient elution was used with 30 to 95% A (0 to 3 min), 95 to 10% A (3 to 4 min), 10 to 30% A (4 to 4.5 min) and 30% A (4.5 to 5 min), at a flow rate of 400 μL.min-1.

[00216] HA graft polymers took about 2.5 times more dexamethasone compared to controls (saline and Ostenil®). As shown in figure 4, after destruction (t=30h), 40% of the dexamethasone was still inside the HA conjugate nanoparticles.

[00217] Above the LCST, the HA conjugate particles were loaded with hydrophobic drug substance such as dexamethasone base. This study demonstrated the high efficiency of HA conjugated nanoparticles of the invention as a new drug delivery system.

Example 8: Stability

[00218] The stability of the graft polymers of the invention was measured by rheology and pH/zeta potential measurements as follows: the HA graft polymers were stored at 4°C and the viscosity was checked over time (t0, t7 and t30). Rheological measurements were performed on 0.4 samples of HA-B2 (0.5% w/v) and HA-P2 (0.75% w/v) with a Haake 1 Rheometer using a cone-plate geometry with a cone 35/2° Ti (Vreden, Germany) and a logarithmic shear rate mode ranging from 0.1 s-1 to 100 s-1 at 20°C in a controlled humidity chamber. pH was measured with a Metrohm gel pH electrode (Herisau, Switzerland) and Zeta potential was measured using a Zetasizer NanoZS (Malvern, Worcestershire, UK).

[00219] No difference in viscosity was observed, which indicates that the HA-B2 and HA-P2 graft polymers were stable for 1 month at 4°C. The HA graft polymers showed an acidic pH with high zeta potential, ensuring colloidal stability (Table 2). In order to neutralize pH, HA graft polymers were solubilized in phosphate-buffered saline (PBS) for in vitro/in vivo experiments. Table 2

Example 9: Cell viability assay

[00220] Viability tests were performed using human synovial fibroblasts. Cells were plated at a density of 20,000 cells/wells in 96 well plates. After 24 hours, the HA conjugates (HA-B2 and HA-P2) were solubilized in PBS and incubated for 24h at 37°C. 50 μL of 0.5% MTT solution was added to each well for 3 h. All wells were incubated for 45 min with 100 μL DMSO and absorbance was measured at 570 nm (8 points per well) by BioTeK Synergy Mx (Winooski, USA).

[00221] HA-B2 0.75% (w/v) and HA-P2 0.5% (w/v) in PBS did not decrease cell viability compared to untreated controls.

Claims (19)

1. Graft polymer of a hyaluronic acid polymer and an N-isopropylacrylamide-based polymer, characterized in that the hyaluronic acid polymer and the N-isopropylacrylamide-based polymer are conjugated through at least one linker L of Formula (II): wherein one of Ra and Rb is covalently linked to the N-isopropylacrylamide-based polymer and one of Ra and Rb is covalently linked to the hyaluronic acid polymer and when Ra or Rb is covalently linked to the N-isopropylacrylamide-based polymer , will be equal to R1 and when Ra or Rb is covalently linked to the hyaluronic acid polymer, it will be equal to R3, where R1 is a group selected from - (CH2)x-NH-(CH2)yS-, -( CH2)x-NH-OC(O>(CH2)yS-, -(CH2)x-NH-(CH2)y- C(O)-NH-(CH2)zS-, -(CH2)xOC(O) -CR7R8-, -C(O)-(CH2)xC(O)-NH- (CH2)yS and an optionally substituted polyethylene chain in which R7 and R8 are optionally substituted C1-C6 alkyl, R3 is an -EG group -L1- where E is either absent or selected from -C(O)-NR9-, -C(O)-O- and - C(O)-, G is a ligand group selected from optionally substituted C1-C20 alkyl, optionally substituted polyethylene glycol (PEG) chain, optionally substituted C1-C6 acylamino alkyl, optionally substituted C1-C6 acyl alkyl, optionally substituted C1-C6 alkyl aminocarbonyl and optionally substituted C1-C6 alkoxy and L1 is selected from -NR10C(O)-, -C(O)-NR10-, -C(O)-O- and -C(O)-, where R9 and R10 are independently selected from H and alkyl C1 -C6 optionally replaced; A is an optionally substituted C5-C10-cycloalkyl or optionally substituted heterocycloalkyl ring, wherein R5 represents one or more substituent(s) independently selected from H, optionally substituted alkoxy and optionally substituted C1-C6 alkyl; x, y and z are integers independently selected from 1 to 20 or any pharmaceutically acceptable salts thereof. 2. Graft polymer according to claim 1, characterized by the fact that it is of Formula (I): where M is a group selected from a portion of Formula (M1) and Formula (M2): , R2 is a -B-(CH2)iD group, in which B is either absent or selected from —SC(S)-S- and -S-; D is a group selected from optionally substituted C1-C15 alkyl, optionally substituted C1-C4 alkoxycarbonyl alkyl, optionally substituted C1-C4 amino alkyl, optionally substituted C1-C6 alkyl aminocarbonyl, -COOH and NH2; p is an independently selected integer from 1 to 500; i is an independently selected integer from 0 to 500; R6 is a group selected from - COOH and R3, depending on the degree of substitution; L is a ligand of Formula (II) as defined in claim 1 and n is an integer selected from 1 to 7,000 or any pharmaceutically acceptable salts thereof. 3. Graft polymer according to any one of claims 1 or 2, characterized by the fact that it has Formula (Ia): or any pharmaceutically acceptable salts thereof, wherein L, R2, R6 and p are as defined in any one of the preceding claims. 4. Graft polymer according to any one of claims 1 to 3, characterized in that the linker L is linked to the HA portion of the graft polymer through its substituent Rb. 5. Graft polymer according to any one of claims 1 to 3, characterized in that the linker L is linked to the HA portion of the graft polymer through its Ra substituent. 6. Graft polymer according to any one of claims 1 to 5, characterized in that B is absent or is -S- C(S)-S-. 7. Graft polymer according to any one of claims 1 to 6, characterized by the fact that L is a group according to Formula (II'): wherein R11 and R12 represent one or more substituents independently selected from H, optionally substituted alkoxy and optionally substituted C1-C4 alkyl; Ra and Rb are as defined in any of the preceding claims. 8. Graft polymer according to any one of claims 1 to 7, characterized in that L is a group according to Formula (II) with a formula selected from Formula (II'') and (II'''): wherein Q is absent or is a group of the following formula: - NH-C(O)-(CH2-CH2-O)1-4- CH2-CH2-; G1 is a PEG, in particular (CH2-CH2-O)1-4- and R11 and R12 are as defined in any one of the preceding claims. 9. Graft polymer according to any one of claims 1 to 3 and 5 to 7, characterized in that said graft polymer is selected from the following group: having a degree of substitution between about 0.5 and 50 or any pharmaceutically acceptable salts thereof. 10. Method for preparing a graft polymer of a hyaluronic acid polymer and an N-isopropylacrylamide-based polymer, characterized in that the hyaluronic acid polymer and the N-isopropylacrylamide-based polymer are conjugated by at least a linker L of Formula (II) as defined in any one of the preceding claims comprising the steps of: (a) providing an HA polymer with at least one carboxylic acid group that is conjugated to at least one first functional group capable of participating in a “click chemical” reaction; (b) providing an N-isopropylacrylamide-based polymer with at least one second complementary functional group capable of participating in a “click chemical” reaction with the first functional group, wherein said one of the functional groups is an azide moiety and the other functional group is an alkyne-bearing precursor of said ligand L; (c) reacting the at least one first functional group of the HA polysaccharide with the at least one second complementary functional group of the N-isopropylacrylamide-based polymer by means of a “click chemistry” reaction to obtain a graft polymer composition of the invention ; and (d) isolating the graft polymer composition. 11. Composition, characterized in that it comprises at least one graft polymer as defined in any one of claims 1 to 9 or obtainable by a process as defined in claim 10 and at least one carrier, wherein, in particular said composition is a pharmaceutical composition and said carrier is a pharmaceutically acceptable carrier or a cosmetic composition and said carrier is a cosmetically acceptable carrier. 12. Composition according to claim 11, characterized by the fact that said composition is a hydrogel or nanoparticle composition. 13. Composition according to claim 11 or 12, characterized in that said composition is a cell or tissue culture medium additionally comprising cell or tissue nutrients. 14. Composition according to any one of claims 11 to 13, characterized by the fact that said composition is a reconstruction tissue. 15. Soft tissue filler, characterized in that it comprises at least one graft polymer as defined in a graft polymer as defined in any one of claims 1 to 9 or obtainable by a process as defined in claim 10 or a composition as defined in claim 11. 16. Method for preparing a system for dispensing an active ingredient, characterized in that said method comprises the steps of: a) providing a graft polymer as defined in any one of claims 1 to 9 or a hydrogel as defined in claim 12 in a gel state; b) provide an active ingredient to be dispensed; c) mixing said graft polymer or hydrogel composition with said active ingredient; d) optionally inducing the formation of nanoparticles by increasing the temperature of the mixture; e) collect the obtained composition, hydrogel or nanoparticles loaded with the active ingredient. 17. Use of a graft polymer as defined in any one of claims 1 to 9 or obtainable by a process as defined in claim 10 or a composition as defined in claim 11, characterized in that it is for manufacturing a medicament for prevention or treatment of a medical disorder selected from tissue degeneration, joint pathology, joint disease, eye pathology, a tumor or vascular malformation, skin defects or lesions, enlargement of urological tissue, and vascular or tumor malformation. 18. Use of a graft polymer as defined in any one of claims 1 to 9 or a hydrogel or a composition as defined in claim 12, characterized in that it is for the preparation of a drug delivery system, a cell culture or in vitro biological tissue or a tissue engineering material. 19. Kit, characterized in that it comprises at least one graft polymer as defined in any one of claims 1 to 9 or obtainable by a process as defined in claim 10 or a composition as defined in claim 11 and instructions for use.