Crosslinked hyaluronic acid hydrogels

Abstract

The present invention concerns a crosslinked polymer of hyaluronic acid, or a derivative thereof, as well as a novel process for preparing of the same. The invention also concerns uses of the crosslinked polymer, or compositions thereof, as cosmetic and / or medicinal products.

Description

[0001] ADV-00731PTITW0

[0002] “CROSSLINKED HYALURONIC ACID HYDROGELS”

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] The present invention concerns a crosslinked polymer of hyaluronic acid or a derivative thereof, and hydrogels thereof, as well as a novel process for preparing of the same. The invention also concerns uses of the crosslinked polymer as cosmetic and / or pharmaceutical products.

[0006] BACKGROUND ART

[0007] Hyaluronic acid (HA) is a linear sugar belonging to the glycosaminoglycan (GAG) family, formed by disaccharide units of N-acetylglucosamine (NAG) and glucuronic acid (GA) bound by pi— >4 and P 1 — 3 bonds.

[0008] HA is an endogenous molecule, that is widespread throughout the human body, as constituent of the extracellular matrix. Given its nature and ability to create hydrogen bonds within its chain, HA has remarkable properties: HA maintains the hydration, turgidity, viscosity and plasticity of the connective tissue.

[0009] In particular, HA is highly present in the dermis, contributing in skin moisture and tone with its unique capacity to bind and retain water molecules.

[0010] However, HA production tends to decrease over the years, with a consequent loss of filling and tone at the dermal level.

[0011] Exogenous administration of HA can help filling and supporting the dermal scaffold, together with other endogenous molecules, such as collagen and elastin. Therefore, HA is particularly used in aesthetic medicine, in particular as active component of dermal fillers for the treatment of wrinkles and of the signs of aging.

[0012] Its use has increased over years, even surpassing the use of other products such as botulin toxin (Botox).

[0013] In all tissues of the body, however, hyaluronidases (HAase) are present, i.e. specific enzymes that hydrolyze the pi— >4 bonds of HAase enzymes, on the one hand, degrade endogenous HA, reducing its viscosity and its structural power, and, on the other hand, reabsorb HA that may be supplied exogenously.

[0014] Although a certain grade of reabsorption of exogenous HA can be desirable, as this makes aesthetic treatments with HA reversible, the very fast action of HAase makes it necessary to ADV-00731PTITW0 supply HA very frequently to maintain the desired filling effect, with consequent increment of costs.

[0015] Therefore, methods to increase the residence time of HA in the body are widely studied.

[0016] In particular, the functionalization of HA (e.g. the functionalization of HA with amine groups) and the crosslinking of HA, preventing or slowing down the degradation of HA by HAase, are to date the most widely used strategies to prolong the half-life of HA-based dermal fillers.

[0017] The most widely used chemical agent for the crosslinking of HA is 1,4-butadienol diglycidyl ether (BDDE).

[0018] The crosslinking of HA with BDDE takes place easily in a basic environment, wherein the nucleophilic attack of the epoxy groups of BDDE on the hydroxyl groups of the sugar is favored. HA crosslinked with BDDE is then the most used HA in dermal fillers currently on the market.

[0019] However, crosslinking of HA with BDDE is not free of drawbacks.

[0020] In fact, when BDDE is reacted with HA, there can be three outcomes, optionally concurrent:

[0021] 1) both epoxy groups of BDDE react with two OH groups on two different HA polymers, creating a bridge between them;

[0022] 2) only one epoxy group of BDDE reacts with a HA polymer, so that a BDDE pendant is formed on the HA polymer chains;

[0023] 3) BDDE does not react.

[0024] In the first case there is a crosslinking reaction, changing the chemi cal -physical properties of HA gel, while in the second case the rheological properties of the gel are only slightly modified, without affecting the susceptibility of HA to hyaluronidases.

[0025] In the third case a considerable amount of unreacted BDDE, referred to as "residual" or “residue”, is present in the product.

[0026] Due to their high reactivity to biological structures (i.e. proteins), unreacted epoxy groups are toxic (in Drosophila BDDE has shown mutagenic effects).

[0027] Therefore, the Food and Drugs Administration (FDA) has imposed a legal limit of < 2 ppm of residual BDDE in the HA-based fillers; the same limit has been adopted for registering HA fillers crosslinked with BDDE in Europe.

[0028] New crosslinking agents have been proposed to substitute BDDE, such as polyethylene glycol (PEG) and divinyl sulfone (DVS), however their commercial use is limited for safety ADV-00731PTITW0 concerns.

[0029] In view of the above, the need arises to provide a novel HA crosslinking agent that is able to sufficiently increase the residence time of HA hydrogels in the body and that is safer than currently available agents, not producing toxic unreacted residues.

[0030] It is therefore an object of the present invention to provide new molecules for the crosslinking of HA, which are effective, have fewer side effects and better manageability than currently available crosslinking agents.

[0031] SUMMARY OF THE INVENTION

[0032] The limitations of the prior-art are overcome by the present invention, i.e. by using isosorbide diglycidyl ether (ISDE) as crosslinking agent to crosslink molecules of HA, or derivatives thereof.

[0033] Isosorbide diglycidyl ether (ISDE) molecule is characterized by a central portion of isosorbide, a sugar heterocycle, enclosed between two epoxy groups. The chemical structure of ISDE is shown in Formula I:

[0034] The presence of the two terminal epoxy groups makes ISDE extremely reactive.

[0035] In addition, the sugary internal structure creates a steric encumbrance and gives the molecule a specific spatial conformation that makes it stable and not easily attacked by degradation enzymes.

[0036] Due to its chemical nature, the use of ISDE as crosslinking agent of hyaluronic acid, or derivatives thereof, allows to obtain a cross-linked product that is entirely polysaccharide- based. Such a product is particularly desirable for cosmetic and / or dermatological applications. In fact, it is well known that polysaccharides have significant physiological capabilities. In fact they are biocompatible components that promote hydration maintenance and water up-take properties.

[0037] Furthermore, due to its saccharide nature, isosorbide has properties that can be advantageously exploited in cosmetic and / or dermatological applications. In fact, when used as a derivative in cosmetic products, isosorbide enhances hydration, penetration, and the delivery of active ingredients. ADV-00731PTITW0

[0038] Furthermore, in the crosslinked polymer, the rings of the ISDE molecule create greater steric hindrance compared to the crosslinkers of the prior art, making it more stable against HAase, thereby increasing the time required for its degradation. The cross-linked product of the invention has then a longer duration of action, compared to products crosslinked with other molecules of the art.

[0039] The higher stability of a crosslinking agent increases the half-life in tissues of polymers that include it.

[0040] Therefore, the use of polymers of HA, or derivatives thereof, crosslinked with ISDE, e.g. as dermal filler, represents an advantage also in terms of costs, as less frequent administrations of the crosslinked polymer is required to maintain the filler effect.

[0041] In addition, ISDE is of no hazard, unlike BDDE, being therefore safer and more manageable than currently available crosslinked polymers.

[0042] The present invention is then directed to the use of ISDE as crosslinking agent for the crosslink of hyaluronic acid or a derivative thereof, and to a crosslinked polymer comprising, or consisting of, hyaluronic acid, or a derivative thereof, crosslinked with isosorbide diglycidyl ether (ISDE).

[0043] Moreover, the present invention is directed to a process for the preparation of said crosslinked polymer, comprising the steps of: i) providing hyaluronic acid, or a derivative thereof, which is partially or totally in the non-crosslinked state; ii) providing isosorbide diglycidyl ether (ISDE) crosslinking agent; iii) reacting the hyaluronic acid, or a derivative thereof, with the ISDE crosslinking agent; and iv) obtaining a crosslinked polymer; optionally comprising also a subsequent step of: v) dialyzing the crosslinked polymer in a PBS solution.

[0044] In a further aspect, the present invention regards a pharmaceutical composition comprising the crosslinked polymer according to the invention and pharmaceutically acceptable excipients; optionally, the pharmaceutical composition further comprises at least one pharmacologically active substance and / or at least one substance having, optionally, a biological function.

[0045] The crosslinked polymer of the present invention is preferably in the form of a hydrogel. ADV-00731PTITW0

[0046] Hydrogels of HA or a derivative thereof, that are crosslinked with ISDE according to the present invention, feature chemi cal -physical and biological characteristics that are comparable to, or even superior to, the top-performing hyaluronic acid hydrogels currently on the market, in terms of degree of swelling and cross-linking, biocompatibility, rheological characteristics, and stability to enzymatic degradation.

[0047] The crosslinked polymer of the invention is therefore particularly advantageous for cosmetic or dermatological applications.

[0048] Therefore, in preferred aspects, the present invention is directed to the cosmetic use of the crosslinked polymer of the invention, preferably as dermal filler, e.g. for filling skin depressions.

[0049] The present invention is then directed also to a dermal filler composition comprising the crosslinked polymer of the invention and aqueous solutions, preferably saline solutions or distilled water.

[0050] Furthermore, in preferred aspects, the present invention is directed to the crosslinked polymer of the invention, or pharmaceutical compositions thereof, for use as medicine, in particular in the dermatological field, such as for treatment of a dermatological disease.

[0051] In addition, the outstanding rheological and biocompatibility properties of the crosslinked polymer of the invention makes it suitable also for the use in the orthopedic field with benefits for the patient in terms of safety and costs, in particular for intra-articular administration, and / or for tissue repair or reconstruction, and / or for the visco-supplementation, of bone cartilage or of joints.

[0052] Therefore, the present invention is also directed to the crosslinked polymer of the invention, or of pharmaceutical compositions thereof, for use as medicine in the treatment of a orthopedic disease.

[0053] In an even further aspect, the present invention concerns the use of the crosslinked polymer of the invention as biomaterial in plastic-aesthetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otorhinolaryngology, dentistry, gynecology, urology, dermatology, oncology, rheumatology, orthopedics; preferably for tissue repair.

[0054] BRIEF DESCRIPTION OF FIGURES

[0055] The characteristics and advantages of the present invention will become apparent from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the attached figures, wherein: ADV-00731PTITW0

[0056] Fig- 1 shows the chemical structure of ISDE in its crosslinked configuration according to the invention, where the 'H NMR peaks evaluated in de-DMSO are depicted at each atom, the peaks denoting the crosslinking being circled.

[0057] Fig. 2 exhibits the viability of BALB / 3T3 fibroblasts, clone A31 (CCL-163), in the presence of ISDE (diamonds) and BDDE (circles) as crosslinking agent for HA.

[0058] Fig- 3 exhibits the viability of BALB / 3T3 fibroblasts, clone A31, tested after 4 hours of contact with different ISDE-HA hydrogels and reference hydrogels.

[0059] Fig- 4 exhibits the degradation kinetics of ISDE-HA hydrogels and reference hydrogels, determined by carbazole assay measuring the glucuronic acid released in the supernatant by forced enzymatic degradation with HAase 50UI / ml after 4h (dark color) or 24 hours (light color).

[0060] Fig. 5 exhibits micrographs of BALB / 3T3 cells stained for live / dead visual viability assay at 24 hours: viable (light grey fluorescence) and dead (dark grey fluorescence) cells on 20X magnification are shown. A: cells in contact with crosslinked HA (HA with intrinsic viscosity of 1.2 m3 / kg, crosslinked with ISDE 13%); B: cells in contact with Reference 1. Scale bar corresponding to 20 mm, applicable to both micrographs.

[0061] Fig. 6 exhibits the viability of Synovial sarcoma SW982 cells after 4 hours of incubation with the hydrogel samples of the invention, and commercial products.

[0062] Fig. 7 exhibits the viability of Synovial sarcoma SW982 cells after 24 hours of incubation with the hydrogel samples and commercial products.

[0063] DETAILED DESCRIPTION OF THE INVENTION

[0064] The use of ISDE as crosslinker agent for crosslinking HA, or a derivative thereof, results in a crosslinked polymer with covalent crosslinks between free carboxylic and / or hydroxylic functional groups of separate molecules of hyaluronic acid, or derivative thereof, without the production of toxic unreacted residues. This means that the crosslinked polymer of the invention is the result of an intermolecular reaction by indirect crosslinking via ISDE.

[0065] The term "hyaluronic acid" or "HA", as used herein, includes hyaluronic acid, hyaluronate, and any of its salts, such as sodium hyaluronate, potassium hyaluronate, lithium hyaluronate, magnesium hyaluronate, calcium hyaluronate, iron hyaluronate, zinc hyaluronate, cobalt hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate, or a mixture thereof. Suitable derivatives of hyaluronic acid for the purposes of the present invention are preferably the following: ADV-00731PTITW0

[0066] - hyaluronic acid esters wherein a part or all the carboxyl groups are esterified with aliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclic series alcohols, as also described in EP0216453B1,

[0067] - auto-crosslinked hyaluronic acid esters wherein a part or all the carboxyl groups are esterified with alcoholic groups from the same polysaccharidic chain or other chains, as also described in EP0341745B1,

[0068] - crosslinked hyaluronic acid compounds wherein a part or all the carboxyl groups are esterified with aliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclic series polyalcohols, generating crosslinkings through spacer chains, as also described in EP0265116B1,

[0069] - semi-esters of the succinic acid or heavy metal salts of succinic acid with hyaluronic acid or with partial or total hyaluronic acid esters, as also described in WO96 / 357207,

[0070] - O-sulfated derivatives, as also described in WO95 / 25751, or N-sulfated derivatives, as also described in WO / 1998 / 045335.

[0071] The crosslinked polymer of the invention is obtainable from a starting hyaluronic acid, or a derivative thereof, that is partially or totally in the non-crosslinked state.

[0072] The term "non-crosslinked", as used herein, refers to molecules of HA, or a derivative thereof, that are linear (essentially un-crosslinked), or very lightly crosslinked, i.e. to molecules with a degree of modification (MoD, or crosslinking degree), i.e. the stoichiometric ratio between the sum of linked residues of the crosslinking agent to the HA disaccharide units, of less than 1% or less than 0.1 %. The degree of modification, can be measured by1H NMR spectrometry, as described in the present examples.

[0073] For the purposes of the present invention, the average molecular mass of HA is preferably determined by viscometry via the Mark-Houwink equation. The Mark-Houwink equation gives a relation between intrinsic viscosity (q) and the viscosity average molecular weight and allows determination of the average molecular weight of a polymer from data on the intrinsic viscosity and vice versa.

[0074] For calculation of the average molecular weight of HA from intrinsic viscosity, the following Mark-Houwink equation is thus used:

[0075] [q] = K x Ma, wherein [q] = intrinsic viscosity in m3 / kg, M = viscosity average molecular weight, K = 2.26 x 10'5, and a = 0.796. ADV-00731PTITW0

[0076] Within the context of the present invention, the intrinsic viscosity is preferably measured according to the procedure defined in European Pharmacopoeia 7.0 (Hyaluronic Acid monograph No. 1472, 01 / 201 1).

[0077] Preferably, the crosslinked polymer of the invention is obtained from a starting HA, or a derivative thereof, having intrinsic viscosity of 0.5 m3 / kg to 3 m3 / kg, more preferably of 1 m3 / kg to 2 m3 / kg, most preferably of 1.2 to 1.8 m3 / kg.

[0078] Preferably, the non-crosslinked hyaluronic acid, or derivative thereof, is provided in the form of a solution (starting solution), more preferably an aqueous solution, comprising the noncrosslinked hyaluronic acid, or derivative thereof, to which ISDE is added, optionally dissolved in a solution, and mixed until obtaining a homogeneous solution (reaction solution).

[0079] Preferably, the reaction solution comprises non-crosslinked HA, or derivative thereof, in a concentration of 5-15% w / w, more preferably 7-14% w / w, most preferably 10-12% w / w, based on the weight of reaction solution.

[0080] Preferably, the reaction solution comprises ISDE in a concentration of 1-15% w / w, more preferably 2-14% w / w, most preferably 5-10% w / w, based on the weight of the reaction solution.

[0081] Preferably, the non-crosslinked HA, or a derivative thereof, is reacted with ISDE in a weight ratio of non-crosslinked HA (or a derivative thereof) to ISDE of 1 : 1 to 10: 1, more preferably of 2: 1 to 10: 1, most preferably of 2: 1 to 5:1, wherein the weight ratio of non-crosslinked HA (or a derivative thereof) to ISDE is the ratio of the weight amount of non-crosslinked HA, or derivative thereof, to the weight amount of ISDE, in the crosslinking reaction.

[0082] In some embodiments of the invention, the non-crosslinked HA, or a derivative thereof, is reacted with ISDE in a weight ratio of non-crosslinked HA (or a derivative thereof) to ISDE of from 5: 1 to obtain cross-linked product, which is only slightly cross-linked. This product can be used as dermal filler or in orthopedic applications.

[0083] Cross-linked hydrogels are flexible, biodegradable, and have excellent water retention properties, making them highly suitable for biomedical and environmental applications such as tissue engineering and soft biomedical devices. These hydrogels represent a promising platform for next-generation materials that blend structural versatility with biocompatibility and sustainability In some embodiments of the invention, a crosslinked polymer suitable for use as dermal filler is preferably obtained by reacting, in a reaction solution, a starting ADV-00731PTITW0 solution comprising HA, or derivative thereof, with an intrinsic viscosity of 0.6 m3 / kg to 2 m3 / kg, more preferably of 1 to 2 m3 / kg, most preferably of 1.2 to 1.8 m3 / kg.

[0084] In other embodiments of the invention, a crosslinked polymer suitable for use in orthopedic applications is preferably obtained by reacting, in a reaction solution, a starting solution comprising HA, or derivative thereof, with an intrinsic viscosity of 0.2 m3 / kg to 2 m3 / kg, more preferably of 0.6 to 1.8 m3 / kg, most preferably of 0.6 to 1.2 m3 / kg.

[0085] Preferably, the step iii) of reacting the hyaluronic acid, or a derivative thereof, with the ISDE crosslinking agent is carried out at a temperature of 20°C to 40°C, more preferably of 25°C to 35°C, most preferably 28°C to 32°C.

[0086] Preferably, the step iii) of reacting the hyaluronic acid, or a derivative thereof, with the ISDE crosslinking agent is carried out for a reaction time of 1 to 10 hours, more preferably of 3 to 8 hours, most preferably of 4 to 7 hours.

[0087] Optionally, the reaction is stopped by adding a neutralized solution (HC1 IM, H3PO4 10% v / v and H2O) or by extruding the hydrogel obtained from the reaction in phosphate buffer solution (PBS).

[0088] Preferably, after the crosslinking reaction, in step v), the hydrogel is dialyzed in PBS solution with physiological pH and osmolarity, for a dialysis time of 1 to 10 days, more preferably of 2 to 8 days, most preferably of 2 to 6 days.

[0089] During the dialysis phase, the hydrogel is purified from excess crosslinker, resulting in a product with a higher degree of purity. Additionally, this step promotes homogeneity between batches, as the pH of the hydrogel is buffered by the neutral environment of the dialysis.

[0090] The crosslinked polymer of the invention is generally in the form of a three-dimensional network or "gel "structure. The term "gel", as used herein, usually refers to a material having fluidity at room or body temperature between that of a liquid and solid. Since it is generally capable of absorbing water it may also be referred to as "hydrogel" herein.

[0091] Therefore, the crosslinked polymer of the invention is preferably in the form of a hydrogel. Preferably, the crosslinked polymer of the invention has a degree of modification (MoD%) of at least 20%, more preferably 25% to 99.9%, most preferably 30% to 99.8%.

[0092] Preferably, the crosslinked polymer of the invention is characterized by the peaks at 3.30 ppm due to four proton of ISDE, as indicated with a circle in Fig 1, and the peak at 2 ppm due to three proton of N-methyl group of hyaluronic acid. ADV-00731PTITW0

[0093] The crosslinked polymer of the invention is preferably the main component of a dermal filler. The term "dermal filler" or "dermal filler composition", as used herein, is intended to refer to a material designed to add volume to, or replace or augment volume of, soft tissue areas of skin. A dermal filler composition according to the invention comprises the crosslinked polymer of the invention and a physiological saline solution (i.e. a solution of 0.90% w / v of NaCl, 308 mOsm / L or 9.0 g per liter) or distilled water.

[0094] A dermal filler according to the invention is normally sterile and in the form of a gel, in particular in the form of a hydrogel.

[0095] Preferably, a dermal filler according to the invention, further to the crosslinked polymer, comprises at least one anesthetic, such as lidocaine.

[0096] More generally, the crosslinked polymer of the invention can be the main component of a pharmaceutical composition.

[0097] Therefore, the present invention also regards a pharmaceutical composition comprising the crosslinked polymer of the invention and pharmaceutically acceptable excipients.

[0098] Pharmaceutically acceptable excipients are for example pH regulators, isotone regulators, solvents, stabilisers, chelating agents, diluents, binding agents, disintegrants, lubricants, glidants, colorants, suspending agents, surfactants, cryoprotection agents, preservatives, and antioxidants.

[0099] In further aspects the present invention regards a pharmaceutical composition comprising at least one crosslinked polymer and at least one pharmacologically active substance and / or at least one substance having, optionally, a biological function.

[0100] Suitable pharmacologically active substances are antibiotics, anti-infectives, antimicrobials, antivirals, cytostatics, cytotoxics, anti-tumour agents, anti-inflammatory agents, cicatrizants, anaesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotics, anticoagulants, haemostatics, fibrinolytics, thrombolytics, proteins and their fragments, peptides, polynucleotides, factors of growth, enzymes, vaccines, or a combination thereof.

[0101] Preferably, said substance having, optionally, a biological function is selected from collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, gelatine, albumin, poly(glycolide-co-caprolactone), poly(glycolide-co- ADV-00731PTITW0 trimethylene carbonate), polyhydroxyalkanoates, hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralised bone matrix and mixtures thereof.

[0102] Preferably, the dermal filler or the pharmaceutical composition of the invention comprise up to 10 wt% of said crosslinked polymer, based on the weight of the pharmaceutical composition or dermal filler, more preferably, up to 5 wt% of said crosslinked polymer. Particularly preferable are pharmaceutical compositions or dermal fillers wherein said crosslinked polymer amounts to 0.1-5 wt%, more preferably to 1-3 wt%, based on the total weight of the pharmaceutical composition or dermal filler.

[0103] In preferred embodiments, the dermal filler or the pharmaceutical composition of the present invention comprises a mixture of two or more crosslinked HA polymeric materials that differ from each other by their mean molecular weight.

[0104] For example, the crosslinked HA, or derivative thereof, may comprise a mixture of high molecular weight HA, or derivative thereof, having a molecular weight of, e.g., 1.0 x 106Da to 4.0 x 106Da, and a low molecular weight HA, or derivative thereof, having a molecular weight of, e.g., 5 x 104Da to 50 x 104Da, said molecular weight referring to the molecular weight of the starting molecules, i.e. of HA, or derivative thereof, not yet crosslinked.

[0105] In some embodiments of the present invention, the crosslinked polymer of the invention, or a dermal filler or pharmaceutical composition comprising the same, is for cosmetic or medical use, preferably for dermatological use or for orthopedical used, as previously disclosed.

[0106] Preferably, the dermal filler or the pharmaceutical composition of the present invention is injectable, i.e. can be dispensed from syringes or similar devices under normal conditions under normal pressure to the desired target site (e.g., into the dermis and hypodermis or into bone cartilage or joints). In some embodiments, the pharmaceutical composition is in a form which is injectable into the body’s hard or soft tissues, such as organs, adipose, mucous membrane, gum, cartilage, and bone tissues, preferably by intradermal, subcutaneous, intramuscular, intra-articular or intraocular means.

[0107] The pharmaceutical composition comprising the crosslinked polymer of the invention can also be administered by inhalation, by mouth, or by intramuscular, venous, intra-articular, or surgical administration.

[0108] The dermal filler comprising the crosslinked polymer of the invention is preferably administered by transdermal, sub-cutaneous, or external or internal topical administration. ADV-00731PTITW0

[0109] In some embodiments, the crosslinked polymer of the invention is a biomaterial, preferably a bio-resorbable biomaterial, preferably for use as medicine, optionally in conjunction with other pharmacologically active and / or bioactive substances.

[0110] Said biomaterial can be in the form of microspheres, nanospheres, membranes, sponge, wire, film, gauze, guide ways, pads, gel, hydrogels, fabrics, non-woven fabrics, cannulas, or a combination thereof.

[0111] The crosslinked polymer can also be employed as a biomaterial for coating objects used in both the medical field and in others sectors of industry, providing the surface of the object employed with new biological characteristics. Therefore, the present invention is also directed to an object coated with a biomaterial comprising or consisting of the crosslinked polymer of the invention.

[0112] The objects which can be coated include, for example, catheters, cannulas, probes, heart valves, soft tissue prostheses, prostheses of animal origin, artificial tendons, bone and cardiovascular prostheses, contact lenses, artificial oxygenators for blood, kidneys, heart, pancreas, liver, blood bags, syringes, surgical instruments, filtration systems, laboratory instruments, containers for cultures and for the regeneration of cells and tissues, media for peptides, proteins and antibodies.

[0113] It should be understood that all the possible combinations of the preferred aspects of the present invention are also described, and therefore similarly preferred.

[0114] Examples of preferred embodiments of the present invention and analyses of their efficacy are provided below for illustrative and non-limiting purposes.

[0115] EXAMPLES

[0116] Materials

[0117] Non-crosslinked hyaluronic acid was purchased from CMed Aesthetics (Italy).

[0118] Isosorbide diglycidyl ether (ISDE, d= 1.3 g / ml) was obtained from Specific Polymers (France).

[0119] Hyaluronidase 400-100 Ul / mg (H3506) was obtained from Sigma-Aldrich (USA).

[0120] Nine commercial dermal fillers were purchased from the market and used as comparative products, shortly referred to as “Reference 1” to “Reference 9”. Those comparative products contain respectively:

[0121] Reference 1 : 15 mg / ml HA crosslinked with BDDE;

[0122] Reference 2: 23 mg / mL HA crosslinked with BDDE; ADV-00731PTITW0

[0123] Reference 3 : 24 mg / ml HA crosslinked with BDDE;

[0124] Reference 4: 20 mg / mL HA crosslinked with BDDE;

[0125] Reference 5: 20 mg / ml HA crosslinked with BDDE;

[0126] Reference 6: 20 mg / mL HA crosslinked with BDDE;

[0127] Reference 7: 24 mg / ml HA crosslinked with BDDE;

[0128] Reference 8: 24 mg / mL HA crosslinked with BDDE;

[0129] Reference 9: 24 mg / mL HA crosslinked with BDDE;

[0130] The MoD (degree of modification) was investigated through1H NMR spectroscopy and the data reveal the MoD of all the comparative products above ranges between 9-25%.

[0131] Methods

[0132] Synthesis of hydrogels

[0133] An amount of HA is dissolved in deionized water and NaOH 1 N up to obtaining a homogeneous HA solution, then ISDE is added to the HA solution and mixed until the solution is homogeneous. The crosslinking reaction proceeds for 4 hours at 35°C. After the incubation period, the hydrogel is dialyzed against PBS for 2 days in a dialysis bag. At the end of the procedure, the gels are weighed, and the osmolarity and pH are evaluated. The gels are then diluted and left to hydrate for days. Subsequently, the samples are placed in syringes and autoclaved.

[0134] Modification degree (MoD) determination

[0135] A volume of hyaluronidase (HAase) 100 Ul / ml is added to the hydrogel in weight ratio 1 : 1. The mixture is hydrolyzed overnight or for three days at 37°C, then the mixture is keep at 100°C for 10 minutes to inactivate the enzymes. The hydrolase hydrogel is dried under vacuum and the obtained pellet is dissolved in 0.5 ml of deuterium oxide (D2O). 'H-NMR spectra are acquired on Bruker Ultrashield™ spectrometer, working at 400 MHz for 'H, at 25 ± 0.1 °C on 2 w.t% in D2O.

[0136] Swelling degree determination

[0137] 100 mg of hydrogel is weighted in a pre-calibrated Transwell® 3 times, successively 3.5 ml of Dulbecco’s Modified Eagle’s Medium (DMEM) without calf bovine serum, L-glutamine and penicillin / streptomycin are added to apical and basal chamber and the plate is incubated at 37°C for 24 hours. The Transwell® is moved in a new plate and centrifugated at 1000 g for 5 minutes to remove excess of medium. The excess of surface water on the bottom of the Transwell® inserts is removed by blotting, then the swollen gel is weighed (n=3). ADV-00731PTITW0 residue determination

[0138] 10 pl of nicotinamide solution 125 mM is mixed with 20 pl of hydrogel first hydrolyzed for 24h with 300 Ul / ml hyaluronidase and incubated at 37°C for 2 hours. Then, 100 pl of acetophenone solution 15% (in absolute ethanol) and 100 pl of NaOH IM is added and incubated for 5 minutes in ice bath. Successively, 500 pl of formic acid is added and kept for 8 minutes at 60°C. The fluorescence is evaluated at excitation wavelength of 384 nm and emission wavelength at 428 nm. The stability of the complex is about 15 minutes. Residue value is determined by using ISDE standard curve (5-80 pg / ml; y=l 1.6x+3.09, R2= 0.949, n=3).

[0139] Rheological proprieties test

[0140] Rheological properties are tested with a modular compact rheometer (MCR 302, Anton Paar) equipped with plate-plate geometry (diameter of 5 cm) 1 mm of gap. The strain value is 0.1% and oscillation frequency sweep test are carried out over a frequency 0.01 - 10 Hz at 25°C. The elastic modulus (G’), viscous modulus (G”) and complex shear modulus are measured at 0.1 Hz.

[0141] Crosslinker-Cytotoxicitv test

[0142] 104fibroblast BALB / 3T3 clone A31 (CCL-163) are seeded in each well and incubated at 37 °C in 5% CO2 and left to proliferate for 24 hours before incubation with samples. The culture medium is removed from each well and replaced with culture medium containing the crosslinker and incubated for 4 hours. After incubation the samples are removed and substituted with fresh culture medium containing 10% WST-1 reagent solution and incubated at 37°C for 2 hours, 5% CO2. Successively, formazan dye absorbance is evaluated at 450 nm with reference wavelength 650 nm with multilabel reader (BioTek 800 / TS, Thermo Scientific, Walthman, MA, USA).

[0143] Cell viability on fibroblast BALB / 3T3 - Direct method

[0144] In each well of a 96-well plate, 5xl03fibroblast cells (BALB / 3T3 clone A31) are seeded per well and incubated at 37°C in 5% CO2 and made growing for 24 hours. Subsequently, 10 pl of hydrogel samples conditioned with the culture medium was added to cells and incubated for 4 hours at 37°C in a 5% CO2 atmosphere.

[0145] To assess cell viability, the samples are removed, and the cells are washed three times with PBS to remove any apoptotic bodies. Fresh medium containing 10% WST-1 reagent solution is added and kept for 2 h at 37°C in 5% CO2. The absorbance is quantified at 450 nm with ADV-00731PTITW0 reference wavelength of 630 nm by using multilabel reader (BioTek 800 / TS, Thermo Scientific, Walthman, MA, USA).

[0146] Stability to enzymatic degradation

[0147] 20 pl of hydrogel previously incubated at 37°C is mixed with 50 pl of PBS and successively 25 pl of enzymatic solution (Sodium phosphate 0.02M, NaCl 0.077M, Bovine Serum Albumin 0.01% w / v, pH 7 at 37°C) with hyaluronidase (HAase) 50 Ul / ml (temperature about 37°C) are added to the mixture and incubated for necessary time.

[0148] The mixture is heated at 100°C for 10 minutes to stop the enzymatic activities and kept at room temperature. The residual hydrogel is washed with 50 pl of PBS and then centrifugated at 14’000 rpm for 10 minutes. The entire process was repeated three times.

[0149] The total supernatant is filtered on 0.45 pm filter to remove hydrogel that is not hydrolyzed.

[0150] The stability to enzymatic degradation is evaluated by carbazole assay: enzymes break the glycosidic bonds between the di saccharide units and between disaccharide monomers, leading to the release of N-acetyl glucosamine and glucuronic acid; the carbazole assay quantifies free glucuronic acid in the supernatant from enzymatic hydrolysis.

[0151] Briefly, 50 pl of supernatant is placed in a 96-well plate, then 200 pl of 25 mM sodium tetraborate decahydrate in sulfuric acid 98% are added. The plate is heated for 10 minutes at 100°C in oven and then cooled at room temperature for 15 minutes. Successively, 50 pl of 0.125% carbazole in absolute ethanol are carefully added and heated in oven for 10 minutes at 100°C and cooled at room temperature for 15 minutes. The plate is read with a microplate reader at X=562 nm.

[0152] Live / Dead assay

[0153] Preliminary qualitative cell culture viability is assessed by the Live / Dead assay dye (Invitrogen, Italy). BALB / 3T3 cells (5xl03 / 100 ml) are seeded directly onto the hydrogel to be tested, each sample in 96 well plates, and allowed to proliferate for 24 hours.

[0154] In each well 80 pl extra of medium are added to avoid the swelling of the hydrogel.

[0155] After 24 hours both samples are washed with PBS to remove extracellular esterase activity prior to incubating with calcein-AM (2 mM) and EthD-1 (4 mM) in PBS for 30 min at room temperature following the producer’s protocol. The incubating solution is added to each well to completely cover the hydrogels. The excited scaffold fluorophores at 488 nm and 514 nm, respectively, are observed by fluorescence laser scanning microscope (Nikon Eclipse Ts2R). ADV-00731PTITW0

[0156] EXAMPLE 1

[0157] Synthesis of crosslinked hyaluronic acid hydrogels

[0158] Hyaluronic acids with different intrinsic viscosities (0.6m3 / kg, 1.2m3 / kg e 1.8m3 / kg) were provided, to be crosslinked with four different concentrations of ISDE crosslinking agent, to obtain the weight ratios of HA to ISDE summarized in Table 1 : Table 1.

[0159] For the cross-linking reaction, 11.5% w / w of HA was dissolved in H2O and NaOH IM until a homogeneous solution was obtained. The required amount of ISDE was added, homogenized, and the solution was sonicated for 10 minutes to remove any bubbles. Each reacting solution was kept at 30°C for 6h.

[0160] At the end of incubation, each hydrogel obtained was extruded from a syringe and washed in the dialysis medium for 10 minutes, then placed in MWCO 14-16kDa dialysis stocking and dialyzed in PBS for 3 days.

[0161] At the end of the process, the gels were weighed, and osmolality and pH were evaluated.

[0162] All the hydrogels were set on a final hyaluronic acid concentration of 24 mg / ml.

[0163] The samples were packed in syringes and sterilized by moist heat (autoclave).

[0164] HA with intrinsic viscosity between 1.2 and 1.8 m3 / kg displayed the best crosslinking. Crosslinking of HA with low viscosities (0.6 m3 / kg) resulted in gels that tend to brown or decrease in viscosity after the sterilization process.

[0165] The spectra of the hydrogels after purification, verified by 'H-NMR as described in the methods, showed a congested region between 3.29-4.21 ppm due to the proton signals of the isosorbide and hyaluronic acid of monomer rings. Moreover, the lack of the epoxy ring proton ADV-00731PTITW0 signals of ISDE between 2.64 and 2.85 ppm confirmed the opening of the ring and the reaction between the OH group of the HA and the epoxy groups of ISDE. The signal due to CH3 of N-acetyl groups present in hyaluronic acid is visible at 2.0 ppm, while signals at 3.28- 3.35 ppm, spaced from the cluttered portion, are absent in the native hyaluronic acid but still present in spectra of crosslinked-hyaluronic acid. These signals are due to four different protons of ISDE, confirmed from native ISDE 'H NMR spectra. A prediction of 'H NMR spectra of ISDE generated by ChemDraw software is shown in Fig. 1, where the protons which contribute to those signals are highlighted.

[0166] In order to calculate the degree of modification (MoD %), the area (I 5H) of 2.0 ppm hyaluronic acid signal and the area of 3.28-3.35 ppm peaks of ISDE were calculated, then equation 1 is used to evaluate the MoD %.

[0167] The 'H NMR analysis thus confirms the cross-linking reaction carried out by epoxy groups, demonstrating that ISDE can be used as a crosslinker to produce hydrogels. The degrees of modification were calculated according to equation 1 for different ISDE concentration used. The modification degrees and peak ppm values are presented in Table 2.

[0168] Table 2.

[0169] EXAMPLE 2

[0170] The swelling degree expresses the gel’s ability to store water and swell

[0171] The hydrogels were weighed in a Traswell® then placed in a well containing cell culture medium (Dulbecco's Modified Eagle's Medium (DMEM) without the addition of calf serum, L-glutamine and penicillin / streptomycin) to mimic the physiological environment. The samples were incubated at 37°C for 24 hours, the Transwell® were removed from the medium, the gels were dabbed on paper towels, after which they were weighed. ADV-00731PTITW0

[0172] The entire process was carried out on the samples of Example 1 obtained by reacting HA with ISDE in a weight ratio of HA: ISDE of 1 : 1 and 2: 1, in triplicate.

[0173] The degree of swelling for cross-linked gels with BDDE on the market today is reported to be within 80-130% in 48h.

[0174] The hydrogels samples according to the present invention showed a degree of swelling much higher than 100% in the first 24 hours, in particular of 228.4 ± 3.5%, for hydrogels obtained by reacting HA and ISDE in a weight ratio of HA: ISDE of 2: 1, and of 172.3 ± 5.8%, for hydrogels obtained by reacting HA and ISDE in a weight ratio of HA: ISDE of 2: 1

[0175] The high water-uptake by the cross-linked polymer of the invention, much higher than the water uptake by the polymers of the art, is especially advantageous when using the polymers as dermal filler, because the same result of prior art products can be achieved with less product. This reduces the injection time and consequently side effects of injections, such as oedema, resulting in an overall amelioration of compliance.

[0176] EXAMPLE 3

[0177] Rheological properties

[0178] The rheological properties of hydrogel samples according to the present invention, sterilized by autoclave, and of sterile commercial samples were evaluated comparing the values of G’ and G’ ’ .

[0179] The samples tested and the results obtained are shown in Table 2 that follows, wherein each HA-ISDE hydrogel sample is identified by the intrinsic viscosity of the starting HA and by the weight ratio of HA:ISDE in the crosslinking reaction.

[0180] Table 2. ADV-00731PTITWO

[0181] The rheological proprieties of a hydrogel depend on the crosslinking grade, on the concentration of the crosslinked polymer and on the formulation type (monophasic or biphasic). Monophasic fillers consist of a homogeneous gel of hyaluronic acid, offering a smooth and uniform texture, while biphasic fillers are composed of two phases of hyaluronic acid formed by a gel phase and a particulate phase which make them more resistant to enzymatic degradation.

[0182] In general, comparative products have a G’ ranging from 46.8 (Reference 1) to 211.3 (Reference 3).

[0183] Among the samples of the invention, those obtained by reacting HA and ISDE in a weight ratio of 10: 1 showed poorer rheological properties after sterilization, regardless of the intrinsic viscosity of the HA used. Hydrogels obtained by reacting HA and ISDE in a weight ratio of 5: 1 showed instead rheological properties comparable to those of the commercial products, with G’ between 60-80 Pa e G” of about 17 Pa.

[0184] There are no significant differences when using hyaluronic acids of medium or high molecular weights, while samples produced with low molecular weight HA (intrinsic viscosity = 0.6 m3 / kg) are liquid after autoclave.

[0185] However, this shortcoming of low molecular weight polymers can be overcome by increasing ADV-00731PTITW0 the amounts of the crosslinking agent. In fact, when reacting HA and ISDE in a weight ratio of 2: 1 the molecular weight of HA is less relevant: the elastic module is very high (436.8 Pa), while G” stays stable (9.9 Pa).

[0186] Last, when using higher amounts of ISDE crosslinking agent, in a weight ratio of HA:ISDE of 1 : 1, the resulting polymers have very high G’ and G’ ’ (> 2000 Pa G’).

[0187] Such polymers, obtained with higher amounts of ISDE, can be more suitable for intraarticular applications, especially for treating chronic articular diseases.

[0188] Optionally, such polymers with high G’ and G” values can be used for producing biphasic products, e.g. mixing the crosslinked polymer with a linear hyaluronic acid.

[0189] Advantageously, this can reduce the concentration of crosslinked HA, so to reduce costs, still obtaining hydrogels with the desired rheological properties.

[0190] Furthermore, such biphasic products polymers can be particularly suitable for use in the orthopedic field, to improve joint lubrication especially in chronic inflammatory diseases, where the presence of hyaluronidases is limited and the use of crosslinked hydrogels with high rheological properties cha improve the patient’s benefits and compliance.

[0191] EXAMPLE 4

[0192] Cytotoxicity of ISDE was evaluated and compared to cytotoxicity of BDDE

[0193] The cytotoxicity test was carried out as described in the methods, contacting the cells with 1500, 1000, 750, 500, 250, 100, 75, 50 and 25 ppm of ISDE or with the same amounts of BDDE, for 4h at 37°C. The results are shown in Fig. 2.

[0194] Cell viability is inversely proportional to the concentration of crosslinker. This trend is visible for both the cross-linking agents.

[0195] However, interestingly, cells treated with ISDE have a much higher viability than cells treated with BDDE. Calculating the IC of ISDE, this turns out to be IC5o=6OOpmm, i.e. a logarithm higher than that of BDDE.

[0196] In conclusion, the experiment showed that ISDE is safer than BDDE.

[0197] EXAMPLE 5

[0198] Residue of crosslinking agent quantified by a fluorometric assay

[0199] Samples were incubated with hyaluronase 300UI / ml and maintained at 37°C overnight.

[0200] At the end of the incubation, the supernatant was taken and analyzed using nicotinamide as described previously. In brief, lOpl of 125mM nicotinamide was added to 20pl of sample and incubated at 37°C for 2h, then lOOpl of IM NaOH and lOOpl of acetophenone (15% w / v ADV-00731PTITW0 in EtOH) were added and incubated at 0°C for 10 mins. Then, 0.5 mL of formic acid was added to each sample and incubated at 60°C for 8 minutes. Fluorescence was read by a UV- fluorescence spectrophotometer (Variuskan LUX, ThermoFisher) using kexcitation=384nm and ^emission- 428nm .

[0201] After purification, samples crosslinked with ISDE 10: 1 have a residue of 12.1 ± 2.8 ppm, while samples crosslinked with ISDE 5:1 have a residue is 7.27 ± 1.2 ppm in a hydrogel having a final concentration of HA of 24 mg / ml and 3.48 ± 0.9 ppm in a hydrogel having a final concentration of HA of 15 mg / ml.

[0202] Due to the high cytocompatibility, as shown in Example 4, these amounts of ISDE residue are considered harmless.

[0203] EXAMPLE 6

[0204] Biocompatibility assessment

[0205] The biocompatibility of the hydrogels of the invention, in accordance with ISO-10993-5, was verified, assessing cells viability in the presence of the hydrogels of the invention or of commercial hydrogels available in the market.

[0206] Briefly, 5xl03BALB / 3T3 clone A31 fibroblasts were seeded in each well of a multi-well and kept in culture at 37°C in 5% CO2 for 24 h, until obtaining a monolayer of cells.

[0207] Cell culture medium was then substituted with hydrogel samples, conditioned with culture medium. Cells were incubated for 4 hours at 37°C in 5% CO2.

[0208] At the end of incubation, samples were removed and the cells were washed three times with PBS to remove any apoptotic body, then fresh medium containing 10% WST-1 reagent solution was added and kept for 2 h at 37°C in 5% CO2.

[0209] Cell viability was measured, by quantifying absorbance at 450nm normalized at 630nm.

[0210] Fig. 3 shows the results obtained incubating the cells with 10 mg of each sample.

[0211] Viability of cells incubated with all the hydrogels was comparable or even greater than viability of cells incubated with commercial hydrogels, as summarized in Table 3 that follows, wherein each HA-ISDE hydrogel sample is identified by the intrinsic viscosity of the starting HA and by the weight ratio of HA:ISDE in the crosslinking reaction.

[0212] Table 3. ADV-00731PTITWO

[0213] The results confirm that the biocompatibility of the hydrogels of the invention is comparable or even greater than the biocompatibility of dermal fillers that are available in the market. The selected references were determined based on the concentration of HA and its rheological properties, ensuring coverage of a broad spectrum of values and a diverse range of products.

[0214] EXAMPLE 7

[0215] Stability assessment

[0216] The stability of hydrogel samples to enzymatic degradation was evaluated at 4h and 24h as described in the methods. Briefly, a solution with a high concentration of HAase (50 lU / ml) was added to the hydrogels to be tested and incubated at 37°C for 4 or 24 hours.

[0217] Once the incubation time was over, the mix was kept at 100°C for 10 minutes to deactivate the enzymatic activity, then the samples were centrifuged at 14000 rpm for 10 minutes and filtered with 0.45 pm filters to remove non-hydrolyzed HA residue. Carbazole test was then carried out on the supernatants as described in the methods.

[0218] Fig. 4 shows the results obtained at 4h (dark color) and 24 h (light color) of incubation.

[0219] After 4 hours of incubation with enzymes, the % of free glucuronic acid in commercial products is: 25.6% for Reference 7, 24.2% for Reference 5, and 16.1% for Reference 1. ADV-00731PTITW0

[0220] Hydrogels obtained by crosslinking HA with ISDE show a similar stability: about 16% of free glucuronic acid for hydrogel crosslinked with ISDE 1 : 1 and 2: 1 (HA to ISDE weight ratio), 20% and 25% of free glucuronic acid for hydrogel crosslinked with ISDE 10: 1 and 5: 1.

[0221] After 24 hours of incubation with enzymes, the % of free glucuronic acid in commercial products is around 35%.

[0222] Hydrogels obtained by crosslinking HA with an ISDE in a weight ratio of HA: ISDE between 10: 1 and 5: 1 show a stability that is comparable or even greater than stability of commercial products, making them particularly suitable for use as dermal fillers, due to the high concentration or hyaluronidases in dermis. Except HA 0.6 m3 / kg - ISDE 2: 1 and HA 1.8 m3 / kg - ISDE 1 : 1, all samples do not show significant difference between the values at 4 hours and 24 hours, suggest their stability against enzymatic degradation. The data are probably due to the presence of a higher extractable HA value, which is release in the short- time but show long-term biostability. In contrast, commercial products exhibit a similar behaviour regarding the fraction released after 4 hours, but the long-term degradation is less stable compare to prototypes, as the fraction released at 24 hours is significantly higher than that at 4 hours.

[0223] Furthermore, it is noted that, although crosslinked polymers obtained by crosslinking HA with higher concentrations of ISDE showed a lower stability, the presence of hyaluronidases in the intraarticular tissues is very low, confirming suitability of such polymers for their use in the orthopedic field.

[0224] EXAMPLE 8

[0225] Live / dead assay

[0226] A live / dead assay was carried out by using the two-color fluorescence assay (grey stain for live cells and red for dead cells).

[0227] Preliminary analyses were performed on cells treated with crosslinked HA according to the invention (HA with intrinsic viscosity of 1.2 m3 / kg, crosslinked with ISDE in a weight ratio of HA to ISDE of 1 : 1) and with cells treated with the commercial product Reference 1, at 24 hours, crosslinked HA according to the invention showed a high live / dead ratio (A), while dead cells were more prevalent after treatment with Reference 1 (B) (see Fig. 5). EXAMPLE 9

[0228] Comprehensive validation report on the application of ISDE as a cross-linking agent in the ADV-00731PTITW0 manufacturing of cross-linked hyaluronic acid systems for intra-articular orthopaedic therapies

[0229] Hyaluronic acid has long been an important tool in the non-surgical management of joint disorders, but the introduction of cross-linked formulations has further expanded therapeutic possibilities in orthopaedics. Cross-linking modifies the structure of hyaluronic acid, making it more resistant, more viscous, and more similar - mechanically - to natural synovial fluid. As a result, these formulations exhibit prolonged intra-articular residence time and a longer- lasting therapeutic effect.

[0230] In orthopaedics, cross-linked hyaluronic acid is used primarily in the treatment of knee osteoarthritis, where its role is to improve joint lubrication, reduce pain, and contribute to smoother recovery of mobility. In recent years, its application has also been extended to other joints - such as the hip, shoulder, ankle, and the trapeziometacarpal joint - thus offering a therapeutic option for patients with degenerative or post-traumatic conditions in less traditional anatomical sites. Although not a curative therapy, cross-linked hyaluronic acid represents a valuable resource within the therapeutic pathway of osteoarthritis and other joint diseases, often enabling the postponement of more invasive treatments and supporting a more conservative and personalized clinical approach.

[0231] Cross-linking with isosorbide diglycidyl ether (ISDE) enhances resistance to hyaluronidases and improves the rheological properties required to reduce mechanical microtrauma and ensure efficient lubrication of articulating surfaces.

[0232] To validate the use of ISDE as a suitable cross-linking agent for intra-articular applications, the rheological behaviour and biocompatibility on synoviocytes were investigated. The resulting data were compared with three commercially available products currently used for similar indications, here referred to as Reference 10 to Reference 12. Specifically, Reference 10 and Reference 11 are cross-linked with BDDE, whereas Reference 12 is based on functionalized hyaluronic acid. Their characteristics are listed below:

[0233] • Reference 10: 20 mg / mL hyaluronic acid cross-linked with BDDE

[0234] • Reference 11: 30 mg / mL hyaluronic acid cross-linked with BDDE

[0235] • Reference 12: 8 mg / mL functionalized hyaluronic acid

[0236] Methods

[0237] Rheological evaluation

[0238] Rheological properties were evaluated by a rheometer (HAAKE™ MARS™ iQ Air ADV-00731PTITW0

[0239] Rheometer) equipped with a rotor C25 17TI. After determining the LVE region, the test was performed at 20°C in control stain (CS) in the strain range of 1-100 Pa. Successively frequency sweep was performed in the LVE region, in particular the test was performed at 20°C with a constant strain of 0.05 % in a frequency range of 0.05-10 Hz. Storage modulus (G’), loss modulus (G”) and viscosity (r|) were elaborated at 2 Hz through HAAKE RheoWin 4.93.00 Data Manager software. The composition of the hydrogels evaluated are reported in Table 4, moreover Reference 10, Reference 11 and Reference 12 were evaluated.

[0240] Table 4.

[0241] Cell viability

[0242] The biocompatibility of the hydrogels of the invention was verified, assessing cells viability in the presence of the hydrogels of the invention or commercially available hydrogels.

[0243] Synovial sarcoma SW982 cells (American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. For the experiments, cells from passages 4 were utilized. SW982 cells were seeded in 96-well culture plates at a density of 104and 5* 103cells per well for the assessment of cell viability at 4 and 24 hours (h), respectively. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 and allowed to proliferate for 24 h before exposure to the samples. Subsequently, 10 pl of hydrogel was added to cells and incubated for 4 and 24 h at 37°C in a 5% CO2 atmosphere. WST-1 reagent was added and kept for 2 h at 37°C in 5% of CO2. The absorbance is quantified at 450 nm with reference wavelength of 630 nm by using multilabel reader (BioTek 800 / TS, Thermo Scientific, Walthman, MA, ADV-00731PTITWO

[0244] USA). Cell viability values were compared to cells cultured without gels, while absorbance signals from cell-free gels (blanks) were subtracted from all measurements.

[0245] Results

[0246] Rheological Properties The rheological properties of hydrogel samples according to the present invention, sterilized by autoclave, and of sterile commercial samples were evaluated comparing the values of G’ and G”. The samples tested and the results obtained are shown in Table 5 that follows, wherein each HA-ISDE hydrogel sample is identified by the intrinsic viscosity of the starting HA and by the weight ratio of HATSDE in the crosslinking reaction. Table 5. ADV-00731PTITWO

[0247] Cell viability

[0248] Cell viability in synoviocytes was assessed after 4 and 24 hours of incubation with the test samples, and the corresponding data are presented in Figures 6 and 7, respectively. Overall, the cell viability of the hydrogel samples was comparable to — or higher than — that observed for the commercial prototypes. The only exception was the hydrogel HA 1.8 m3 / kg ISDE 1 :1, which showed reduced biocompatibility. This is likely due to the combination of high molecular weight hyaluronic acid and a high degree of cross-linking, resulting in a gel with excessively elevated rheological properties.

[0249] Conclusions ISDE is an effective cross-linking agent for producing hyaluronic acid hydrogels for orthopaedic use. Rheological testing showed that HA: ISDE hydrogels can be tuned to achieve a wide range of stiffness, from values comparable to commercial products to higher moduli suitable for specific intra-articular applications.

[0250] Biocompatibility studies demonstrated that most ISDE-based hydrogels maintain synoviocyte viability comparable to or higher than commercial references.

[0251] Overall, ISDE enables the development of hydrogels with both suitable mechanical properties and good cellular tolerance, supporting their potential use in viscosupplementation and other orthopaedic therapies.

Claims

ADV-00731PTITW0CLAIMS1. A crosslinked polymer comprising or consisting of hyaluronic acid, or a salt or derivative thereof, crosslinked with isosorbide diglycidyl ether (ISDE) crosslinking agent, the crosslinked polymer being in the form of hydrogel.

2. The crosslinked polymer of claim 1, having a degree of modification (MoD%, or crosslinking degree) of at least 20%, more preferably 25% to 99.9%, most preferably 30% to 99.8%.

3. A dermal filler composition comprising the crosslinked polymer of claim 1 or 2 in a physiological saline solution or distilled water.

4. A pharmaceutical composition comprising the crosslinked polymer of claim 1 or 2 and pharmaceutically acceptable excipients.

5. The dermal filler composition of claim 3 or the pharmaceutical composition of claim 4 comprising up to 10 wt% of the crosslinked polymer, preferably comprising up to 5 wt% of the crosslinked polymer, more preferably comprising 0.1-5 wt% of the crosslinked polymer, based on the weight of the pharmaceutical or dermal filler compositions.

6. Cosmetic use of the crosslinked polymer of claim 1 or 2, or of the dermal filler of claims 3 or 5, or of the pharmaceutical composition of claim 4 or 5.

7. The crosslinked polymer of claim 1 or 2, or the pharmaceutical composition of claim 4 for use as medicine, preferably for use in the treatment of a dermatological or orthopedic disease.

8. The crosslinked polymer of claim 1 or 2, or the pharmaceutical composition of claim 4 or 5, for use as a biomaterial in plastic-aesthetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otorhinolaryngology, dentistry, gynecology, urology, dermatology, oncology, rheumatology, or orthopedics.ADV-00731PTITWO9. A process for preparing a crosslinked polymer, comprising the steps of: i) providing hyaluronic acid, or a derivative thereof, which is partially or totally in the non-crosslinked state; ii) providing isosorbide diglycidyl ether (ISDE) crosslinking agent; iii) reacting the hyaluronic acid, or a derivative thereof, with the ISDE crosslinking agent; and iv) obtaining the crosslinked polymer of claim 1.

10. The process of claim 9, wherein the hyaluronic acid, or derivative thereof, provided in step i) has an intrinsic viscosity of 1 to 2 m3 / kg, preferably of 1.2 to 1.8 m3 / kg.

11. The process of claim 9 or 10, wherein hyaluronic acid, or a derivative thereof, is reacted in the crosslinking reaction with ISDE crosslinking agent in a weight ratio of hyaluronic acid, or a derivative thereof, to ISDE of 1 : 1 to 10 : 1 , more preferably of 2 : 1 to 10 : 1 , most preferably of2:l to 5:l.

12. Use of ISDE as a crosslinking agent for crosslinking hyaluronic acid or a derivative thereof.

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