Hyaluronic acid dermal filler compositions

Abstract

The present invention relates to dermal filler compositions comprising a crosslinked hyaluronic acid and a vitamin B3 compounds preventing in vivo oxidative degradation of hyaluronic acid, increasing in vivo stability of hyaluronic acid thereby increasing the duration of in vivo effects of the composition.

Description

[0001] HYALURONIC ACID DERMAL FILLER COMPOSITIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the cosmetic field, in particular to injectable hyaluronic acid implants.

[0004] BACKGROUND OF THE INVENTION

[0005] The human skin is subject to aging and environmental damage, leading to wrinkles, dryness, and loss of elasticity. Current treatments include the application of hyaluronic acid (HA) for its moisturizing and skin-rejuvenating properties.

[0006] Hyaluronic acid dermal fillers, also known as injectable implants, soft tissue fillers, lip and facial fillers, or wrinkle fillers, are defined by the FDA as medical device implants. They are approved for use in enhancing facial aesthetics by creating a smoother and / or fuller appearance in the face.

[0007] Natural HA is composed of repeating alternating units of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating P-(l— 4) and P-(l— 3) glycosidic bonds. It is synthesized as a compound of high molecular weight in both the epidermis and dermis but rapidly undergoes degradation induced by oxidative stress and hyaluronidases. The half-life (tl / 2) of unmodified HA in the skin is about 12 h. The fragmented HA of lower molecular size is a potent inducer of inflammation and angiogenesis, which can elicit adverse effects such as erythema, slight oedema, hematoma, itching, and pain (Slevin et al. Lab Invest. 1998 Aug;78(8):987-1003; Hoarau et al. Biomolecules. 2022 Apr 30; 12(5):658; Lee et al. Biomater Res. 2021 Aug 30;25(l):27; West et al. Science. 1985 Jun 14;228(4705): 1324-6; Hu et al. J Dermatol Sci. 2022 Jul;107(l):24-31; Termeer et al. J Immunol. 2000 Aug 15; 165(4): 1863-70).

[0008] Crosslinked HA hydrogels are significantly more stable than their non-crosslinked counterparts, because of the formation of a dense network by intermolecular bonds or bridges, that is much less susceptible to degradation by hyaluronidases. Therefore, HA used in dermal fillers is routinely cross-linked to improve mechanical properties and in vivo residence time (Kim et al. Polymers (Basel). 2021 Sep 16; 13( 18):3130; Picotti et al. Carbohydr Polym. 2013 Mar 1 ;93(l):273-8; Shu et al. Biomaterials. 2003 Sep;24(21):3825-34; Micheels et al. J Drugs Dermatol. 2016 May 1 ; 15(5):600-6). However, the degradation of HA fillers is not only mediated by specific catabolic enzymes (i.e., hyaluronidases) but also by reactive oxygen species (ROS) and, at the present time, traditional crosslinked HA formulations have limitations in terms of duration of effect because they do not effectively address the oxidative stress that contributes to HA degradation.

[0009] Thus, a need remains for novel formulations of hyaluronic acid, and in particular of crosslinked hyaluronic acid, exhibiting improved stability within the skin and thereby providing a longer lasting effects of the dermal filler.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention relates to an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, and a vitamin B3 compound,

[0012] The hyaluronic acid or the pharmaceutically acceptable salt thereof may have an average molecular mass from 10,000 to 6,000,000 Da, preferably from 100,000 to 3,500,000 Da.

[0013] The hyaluronic acid or the pharmaceutically acceptable salt thereof, is preferably crosslinked using 1,4-butanediol diglycidyl ether (BDDE).

[0014] The crosslinked HA or the pharmaceutically acceptable salt thereof may have a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0015] The concentration of hyaluronic acid may be in the range of 5 mg / mL to 30 mg / mL, preferably in the range of 10 mg / mL to 25 mg / mL, more preferably in in the range of 15 mg / mL to 20 mg / mL.

[0016] Preferably, the injectable composition of the invention comprises less than 10% non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0017] The vitamin B3 compound may be selected from the group consisting of nicotinic acid, niacinamide and nicotinamide riboside, and any combination thereof. Preferably, the vitamin B3 compound is niacinamide.

[0018] The vitamin B3 compound may be in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL. The composition may have a hyaluronic acid / vitamin B3 compound mass ratio between 0.5 and 20, preferably between 1 and 10, more preferably between 1 and 5. Preferably the composition has a hyaluronic acid / vitamin B3 compound mass ratio between 2 and 3.

[0019] In particular, the concentration of hyaluronic acid may be in the range of 10 mg / mL to 25 mg / mL and the concentration of vitamin B3 compound may be in the range of 5 mg / mL to 10 mg / mL.

[0020] In particular, the composition may comprise crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, less than 10% non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof, and a vitamin B3 compound, the composition having a hyaluronic acid / vitamin B3 compound mass ratio between 1 and 5, preferably between 2 and 3.

[0021] The injectable composition may further comprise a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof. In particular, the local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof, may be in the range of 1 mg / mL to 15 mg / mL, preferably in the range of 1 mg / mL to 10 mg / mL, more preferably in in the range of 1 mg / mL to 5 mg / mL.

[0022] The injectable composition may further comprise a pharmaceutically acceptable buffer system, preferably acetate, phosphate, citrate or borate buffer system. In particular, the composition may comprise 0.005 mg / mL to 0.5 mg / mL, preferably from 0.01 mg / mL to 0.1 mg / mL, more preferably from 0.03 mg / mL to 0.05 mg / mL, of sodium dihydrogen phosphate dihydrate and from 0.05 mg / mL to 1 mg / mL, preferably from 0.1 mg / mL to 0.5 mg / mL, more preferably from 0.2 mg / mL to 0.4 mg / mL, of disodium phosphate dihydrate.

[0023] The injectable composition may further comprise a tonicity agent, preferably selected from sodium chloride or potassium chloride, glycerin, mannitol or dextrose. In particular, the composition may comprise sodium chloride, preferably at a concentration in the range of 2 mg / mL to 10 mg / mL, preferably in the range of 4 mg / mL to 8 mg / mL, more preferably in the range of 5 mg / mL to 7 mg / mL.

[0024] In a particular embodiment, the composition comprises, or consists of,

[0025] - from 15 mg / mL to 20 mg / mL of hyaluronic acid or a pharmaceutically acceptable salt thereof, - from 5 mg / mL to 10 mg / mL of a vitamin B3 compound, preferably niacinamide,

[0026] - from 0.01 mg / mL to 0.1 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.1 mg / mL to 0.5 mg / mL of disodium phosphate dihydrate, and

[0027] - from 4 mg / mL to 8 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 1 mg / mL to 5 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0028] In a preferred embodiment, the composition comprises, or consists of,

[0029] - from 15 mg / mL to 20 mg / mL of hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0030] - from 7 mg / mL to 8 mg / mL of a vitamin B3 compound, preferably niacinamide,

[0031] - from 0.03 mg / mL to 0.05 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.2 mg / mL to 0.4 mg / mL of disodium phosphate dihydrate, and

[0032] - from 5 mg / mL to 7 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 2 mg / mL to 4 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0033] Preferably, the injectable composition of the invention is an injectable dermal filler composition.

[0034] In a second aspect, the present invention relates to a cosmetic, non-therapeutic use of an injectable composition of the invention for cosmetic applications, in particular for reducing or filling wrinkles, improving the appearance of skin, improving the skin texture and / or elasticity, or contouring the face or body of a subject.

[0035] In a third aspect, the present invention relates to a cosmetic, non-therapeutic method of reducing or filling wrinkles, improving the appearance of skin, improving the skin texture and / or elasticity, or contouring the face or body of a subject, comprising injecting an injectable composition of the invention into the skin of a subject, preferably via subcutaneous and / or intradermal route.

[0036] In a fourth aspect, the present invention relates to a prefilled syringe, or a part thereof, filled with injectable composition of the invention.

[0037] In a fifth aspect, the present invention relates to a kit comprising (i) a container filled with injectable composition of the invention and optionally a sterile syringe or (ii) a prefilled syringe, or a part thereof, of the invention, and optionally a leaflet with instructions to use. In a sixth aspect, the present invention relates to a method of manufacturing an injectable composition, in particular an injectable composition of the invention, comprising a) mixing and reacting a crosslinking agent with hyaluronic acid or a pharmaceutically acceptable salt thereof in the non-crosslinked state so as to form a hydrogel comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof; b) mixing said hydrogel with a vitamin B3 compound, preferably niacinamide, so as to obtain a homogenous mixture; c) purifying the mixture so as to remove unreacted crosslinking agent; and d) sterilizing the mixture thereby obtaining an injectable composition.

[0038] The vitamin B3 compound may be added to the hydrogel in two steps: the first in step b) of the method and the second after step c) and before step d).

[0039] In another aspect, the present invention relates to the use of a vitamin B3 compound, preferably niacinamide, in an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, to prevent or reduce in vivo oxidative degradation of hyaluronic acid, to increase in vivo stability of hyaluronic acid and / or to increase the duration of in vivo effects of the composition.

[0040] In particular, the concentration of said vitamin B3 compound in the composition may be in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL.

[0041] Preferably, the hyaluronic acid or the pharmaceutically acceptable salt thereof, is crosslinked using 1,4-butanediol diglycidyl ether (BDDE).

[0042] The hyaluronic acid or the pharmaceutically acceptable salt thereof may have an average molecular mass from 10,000 to 6,000,000 Da, preferably from 100,000 to 3,500,000 Da.

[0043] Preferably, the crosslinked HA or the pharmaceutically acceptable salt thereof has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%, and / or the composition comprises less than 10%, preferably less than 9%, 8%, 7%, 6% or 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof. The composition may have a hyaluronic acid / vitamin B3 compound mass ratio between 0.5 and 20, preferably between 1 and 10, more preferably between 1 and 5, and even more preferably between 2 and 3.

[0044] In a further aspect, the present invention also relates to an injectable composition of the invention for use as a medicament. In particular, it relates to (i) an injectable composition of the invention for use in the therapeutic treatment of a skin condition, preferably a soft tissue condition, (ii) a method for treating a skin condition, preferably a soft tissue condition, in a subject in need thereof, comprising administering an effective amount of an injectable composition of the invention to the subject in need thereof, and (iii) the use of an injectable composition of the invention for preparing a medicament for treating a skin condition, preferably a soft tissue condition.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1: Results of accelerated stability studies expressed as endpoint residual fractions of rheological attributes for two formulations of the invention and Voluma from Juvederm exposed to strong oxidant sources (i.e., 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and xanthine-xanthine oxidase mixture) and hyaluronidases. After 10 minutes of exposure, the samples were analyzed in oscillatory rheology at 22°C with a frequency of 1 Hz. Data expressed as the residual fraction of the G' storage moduli and q* complex viscosity values are presented. Measurements were performed in triplicate and standard deviations were reported as error bars around mean values.

[0047] Figure 2: Comparative quantitative absolute values of the total antioxidant capacity in pM performed by trolox equivalent antioxidant capacity (TEAC) assay by measuring the absorbance after 10 minutes at 570 nm. Comparative quantitative absolute values of Nitric Oxide Synthase inhibitor (iNOS) results expressed in percentage of inhibition of the nitric oxide synthase (NOS) performed after 120 minutes of nitric oxide production and a measurement at 540 nm. A PBS solution containing 7.5 mg / ml of niacinamide and 3 mg / ml of lidocaine was used to mimic Formulation 1. A PBS solution containing 3 mg / ml of lidocaine was used to mimic Voluma product from Juvederm. A PBS solution containing 9 mg / ml of mannitol and 3 mg / ml of lidocaine was used to mimic Stylage M product from Vivacy. Measurements were performed in triplicate and standard deviations were reported as error bars around mean values. Figure 3: Comparative quantitative absolute values of the total collagen produced by human adult fibroblast after 96 hours of incubation with two formulations of the invention and Voluma from Juvederm. The measurements were realized following the instruction of a collagen assay kit using fluorescence. Comparative quantitative absolute values of the total protein of human adult fibroblast after 96 hours of incubation with the two formulations of the invention and Voluma from Juvederm. The measurements were realized at 562 nm following the instruction of a BCA assay kit. Measurements were performed four times and standard deviations were reported as error bars around mean values. Phosphate buffered saline solution (PBS) served as control.

[0048] Figure 4: Results of accelerated stability studies expressed as endpoint residual fractions of rheological attributes for Formulations 3, Cl, C2, C4 and Voluma product exposed to strong oxidant source (i.e., 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)). Data expressed as the residual fraction of the G' storage moduli values are presented. Measurements were performed in triplicate and standard deviations were reported as error bars around mean values.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The inventors herein demonstrated that niacinamide, a vitamin B3 compound, can efficiently prevent oxidation degradation of cross-linked hyaluronic acid induced by 2,2'- azobis(2-amidinopropane) dihydrochloride (AAPH) or xanthine-xanthine oxidase mixture. They further showed that niacinamide can inhibit the activity of nitric oxide synthase contrary to mannitol, a compound traditionally used in hyaluronic acid compositions as antioxidative agent.

[0051] Therefore, the inventors herein provide novel formulations of dermal fillers comprising cross-linked hyaluronic acid in combination with a vitamin B3 compound. This combination increases the residence time of hyaluronic acid in the skin by preventing its oxidative degradation while enhancing collagen production thereby offering a comprehensive approach to skin rejuvenation and protection.

[0052] In a first aspect, the present invention relates to an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, and a vitamin B3 compound. The composition of the invention is preferably a sterile injectable aqueous composition, in particular in the form of an hydrogel. The term “injectable” as used herein, refers to a composition that is adapted for injection and in particular for subcutaneous or intradermal injection, into a human being. Preferably, the composition only contains elements that are authorized by authorities monitoring the safety of medicinal products such as the FDA (Food and Drug Administration), European Medicines Agency (EMA) or the French national agency for the safety of medicinal and health products (Agence nationale de securite du medicament et des produits de sante - ANSM), as being adapted and safe for subcutaneous or intradermal injection.

[0053] The composition of the invention comprises crosslinked hyaluronic acid or a crosslinked pharmaceutically acceptable salt thereof.

[0054] As used herein, the term “hyaluronic acid”, “hyaluronic acid polymer”, “hyaluronan” or “HA” refers to a polymer of disaccharides which are composed of D- glucuronic acid and N-acetyl-D-glucosamine linked via alternating P-(l — 4) and P-( 1 — 3) glycosidic bonds. HA can be purified from animal sources or can be obtained by bacterial fermentation.

[0055] Any HA may be used in the compositions of the invention, in particular any pharmaceutically acceptable salt of HA. For example, pharmaceutically acceptable salts of HA may be selected from the group consisting of sodium hyaluronate, calcium hyaluronate, zinc hyaluronate, potassium hyaluronate, magnesium hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate, preferably from the group consisting of sodium hyaluronate, calcium hyaluronate, zinc hyaluronate and potassium hyaluronate. More preferably, the pharmaceutically acceptable salt of HA is sodium hyaluronate.

[0056] The hyaluronic acid or its pharmaceutically acceptable salt may have an average molecular mass from a few daltons to several million daltons. Preferably, the hyaluronic acid or its pharmaceutically acceptable salt has an average molecular mass from 10,000 to 6,000,000 Da, more preferably from 100,000 to 3,500,000 Da.

[0057] As used herein, the term "crosslinked" refers to intermolecular bonds which bind individual polymer molecules or monomer chains into a more stable structure such as gel. As such, crosslinked HA has at least one intermolecular bond linking at least one individual HA polymer molecule to another. The crosslinking of HA typically results in the formation of a hydrogel having high viscosity. Crosslinked HA used in the present invention may be crosslinked using any known crosslinking agent including, without limitation, dialdehydes and disulfides crosslinking agents such as multifunctional PEG-based crosslinking agents, divinyl sulfones, diglycidyl ethers, and bis-epoxides, bis-carbodiimide.

[0058] Preferably, HA or its pharmaceutically acceptable salt used in the present invention is crosslinked using a crosslinking agent selected from the group consisting of diglycidyl ethers, preferably 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), 1- ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC), O,O'-Bis(2- aminoethyl)polyethylene glycol, 1,2,7,8-diepoxyoctane, and glutaraldehyde (GA), and any combination thereof.

[0059] In preferred embodiments, HA or its pharmaceutically acceptable salt used in the present invention is crosslinked using a diglycidyl ether , preferably selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether, and any combination thereof. Preferably, HA or its pharmaceutically acceptable salt used in the present invention is crosslinked using 1,4-butanediol diglycidyl ether (BDDE).

[0060] The crosslinked HA or its pharmaceutically acceptable salt used in the present invention may have a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0061] As used herein, the term "degree of modification" or '^MoD" designates the ratio between the number of moles of the crosslinking agent (for example BDDE) linked to the HA polymer and the number of moles of disaccharide unis of the HA polymer, as determined by Kenne et al method (see Kenne et al. Carbohydr Polym. 2013 Jan 2;91(l):410-8, herein incorporated by reference). MoD may be determined via spectrometry and / or spectroscopy analytical methods, such as1H NMR.

[0062] The method for crosslinking HA into hydrogels are well-known by the skilled person. In particular, HA or its pharmaceutically acceptable salt may be crosslinked with BDDE under alkaline conditions to yield a stable covalent ether linkage between HA and the crosslinker. Typically, the crosslinked HA or pharmaceutically acceptable salt thereof may be obtained by mixing HA or its pharmaceutically acceptable salt with a crosslinking agent and an aqueous alkali solution, and reacting. Generally, the cross-linking of HA or its pharmaceutically acceptable salt ends when the reaction kinetics of bridging the hyaluronic acid chains by the cross-linker has reached a negligible level.

[0063] Preferably, in the composition of the invention, the concentration of hyaluronic acid is in the range of 5 mg / mL to 30 mg / mL, preferably in the range of 10 mg / mL to 25 mg / mL, more preferably in in the range of 15 mg / mL to 20 mg / mL.

[0064] The composition of the invention comprises crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, and optionally non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof.

[0065] In preferred embodiments, the composition of the invention comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof. Thus, the composition comprises at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0066] The composition of the invention further comprises a vitamin B3 compound.

[0067] As used herein, the term ‘‘‘’vitamin B3 compound" refers to one of the three forms of vitamin B3, namely niacin (or nicotinic acid), nicotinamide (or niacinamide), and nicotinamide riboside. In preferred embodiments, the vitamin B3 compound is niacinamide.

[0068] Preferably, in the composition of the invention, the concentration of the vitamin B3 compound, preferably niacinamide, is in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL. In particularly preferred embodiments, the concentration of the vitamin B3 compound, preferably niacinamide, is in the range of 7 mg / mL to 8 mg / mL, in particular is 7.5 mg / mL

[0069] The composition of the invention may have a hyaluronic acid / vitamin B3 compound mass ratio, preferably a hyaluronic acid / niacinamide ratio, between 0.5 and 20, preferably between 1 and 10, more preferably between 1 and 5, and even more preferably between 2 and 3.

[0070] The composition of the invention may further comprise a local anaesthetic. In particular, the local anaesthetic may be selected from the group consisting of lidocaine, ambucaine, amolanone, amylocaine, articaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and pharmaceutically acceptable salts thereof.

[0071] Preferably, the local anaesthetic is lidocaine or a pharmaceutically acceptable salt thereof.

[0072] The concentration of the local anaesthetic may be easily chosen by the skilled person depending on the nature of the anaesthetic and the amount required to provide an anaesthetic effect during or after injection to the subject.

[0073] Preferably, in the composition of the invention, the concentration of the local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof, is in the range of 1 mg / mL to 15 mg / mL, preferably in the range of 1 mg / mL to 10 mg / mL, more preferably in in the range of 1 mg / mL to 5 mg / mL. In particularly preferred embodiments, the concentration of the local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof, is in the range of 2 mg / mL to 4 mg / mL, in particular is 3 mg / mL.

[0074] The composition of the invention may also include other pharmaceutically acceptable components including, without limitation, buffers, tonicity agents, antioxidants, preservatives, salts, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like.

[0075] In particular, the composition of the invention may further comprise a pharmaceutically acceptable buffer system such as acetate, phosphate, citrate or borate buffer system. The buffer system and its concentration can be easily chosen by the skilled person. Preferably, the buffer system in the composition of the invention comprises sodium dihydrogen phosphate and disodium phosphate. Typically, the composition of the invention may comprise from 0.005 mg / mL to 0.5 mg / mL, preferably from 0.01 mg / mL to 0.1 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.05 mg / mL to 1 mg / mL, preferably from 0.1 mg / mL to 0.5 mg / mL of disodium phosphate dihydrate. More preferably, the composition of the invention may comprise from 0.03 mg / mL to 0.05 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.2 mg / mL to 0.4 mg / mL, of disodium phosphate dihydrate.

[0076] The pH of the composition of the invention is a physiologically acceptable pH, preferably in the range of pH 5.0 to pH 8.5.

[0077] The composition of the invention may also comprise a tonicity agent.

[0078] Tonicity agents useful in the composition include, without limitation, salts such as sodium chloride or potassium chloride, glycerin, mannitol or dextrose. Preferably, the tonicity agent is sodium chloride.

[0079] The concentration of this agent, preferably sodium chloride, may be easily adjusted by the skilled person and typically is comprised in the range of 2 mg / mL to 10 mg / mL, preferably in the range of 4 mg / mL to 8 mg / mL, more preferably in the range of 5 mg / mL to 7 mg / mL.

[0080] In a particular embodiment, the composition comprises, or consists of,

[0081] - crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, and optionally non-crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0082] - a vitamin B3 compound, preferably niacinamide,

[0083] - a buffer system, preferably comprising sodium dihydrogen phosphate and disodium phosphate, and

[0084] - a tonicity agent, preferably sodium chloride, and optionally a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0085] Preferably, the crosslinked HA or its pharmaceutically acceptable salt has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0086] Preferably, the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0087] In a more particular embodiment, the composition comprises, or consists of,

[0088] - from 5 mg / mL to 30 mg / mL, preferably from 10 mg / mL to 25 mg / mL, of hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0089] - from 1 mg / mL to 25 mg / mL, preferably from 1 mg / mL to 15 mg / mL, of a vitamin B3 compound, preferably niacinamide,

[0090] - from 0.005 mg / mL to 0.5 mg / mL, preferably from 0.01 mg / mL to 0.1 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.05 mg / mL to 1 mg / mL, preferably from 0.1 mg / mL to 0.5 mg / mL of disodium phosphate dihydrate, and

[0091] - from 2 mg / mL to 10 mg / mL, preferably from 4 mg / mL to 8 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 1 mg / mL to 15 mg / mL, preferably from 1 mg / mL to 10 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0092] Preferably, the crosslinked HA or its pharmaceutically acceptable salt has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0093] Preferably, the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0094] In a preferred embodiment, the composition comprises, or consists of,

[0095] - from 10 mg / mL to 25 mg / mL, preferably from 15 mg / mL to 20 mg / mL, of hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0096] - from 5 mg / mL to 10 mg / mL of a vitamin B3 compound, preferably niacinamide,

[0097] - from 0.01 mg / mL to 0.1 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.1 mg / mL to 0.5 mg / mL of disodium phosphate dihydrate, and

[0098] - from 4 mg / mL to 8 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 1 mg / mL to 5 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0099] Preferably, the crosslinked HA or its pharmaceutically acceptable salt has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%. Preferably, the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0100] In a particularly preferred embodiment, the composition comprises, or consists of,

[0101] - from 10 mg / mL to 25 mg / mL, preferably from 15 mg / mL to 20 mg / mL, of hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0102] - from 7 mg / mL to 8 mg / mL of a vitamin B3 compound, preferably niacinamide,

[0103] - from 0.03 mg / mL to 0.05 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.2 mg / mL to 0.4 mg / mL of disodium phosphate dihydrate, and

[0104] - from 5 mg / mL to 7 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 2 mg / mL to 4 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

[0105] Preferably, the crosslinked HA or its pharmaceutically acceptable salt has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0106] Preferably, the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0107] In preferred embodiments, the composition of the invention is a dermal filler composition. As used herein, the term “ dermal filler ' refers to a material or composition designed to add volume to areas of soft tissue deficiency. This term should not be construed as imposing any limitations as to the location and type of injection. Dermal fillers are generally used in the dermis and / or hypodermis area and may be thus injected subcutaneously and / or intradermally. In the context of cosmetics, injectable dermal fillers are often used as "volumizers”, to enhance facial shaping, plump lips and reduce the sight of fine lines and wrinkles, as well as reduce the appearance of deep wrinkles and folds.

[0108] In another aspect, the present invention also relates to a prefilled syringe, or a part thereof, filled with the composition of the present invention. The prefilled syringe may by a ready-to-use syringe with a tip cap or a pre-mounted needle. All the embodiments described above for the composition of the invention are also contemplated in this aspect.

[0109] In a further aspect, the present invention relates to a kit comprising (i) a prefilled syringe, or a part thereof, of the present invention or (ii) a container filled with the composition of the present invention and optionally a sterile syringe. The kit may further comprise a leaflet with instructions to use.

[0110] All the embodiments described above for the composition and the prefilled syringe of the invention are also contemplated in this aspect.

[0111] In another aspect, the present invention further relates to a method of manufacturing an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, in particular a composition of the invention, said method comprising a) mixing and reacting a crosslinking agent with hyaluronic acid or a pharmaceutically acceptable salt thereof in the non-crosslinked state so as to form a hydrogel comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof; b) mixing said hydrogel with a vitamin B3 compound, preferably niacinamide, so as to obtain a homogenous mixture; c) purifying the mixture so as to remove unreacted crosslinking agent; and d) sterilizing the mixture thereby obtaining an injectable composition.

[0112] All the embodiments described above for the composition, the prefilled syringe and the kit of the invention are also contemplated in this aspect.

[0113] In step a) the crosslinking agent may be as defined above, preferably is a diglycidyl ether, more preferably is 1,4-butanediol diglycidyl ether (BDDE). The conditions to mix and react hyaluronic acid with a crosslinker are well known by the skilled person and can easily be adjusted depending on the crosslinker.

[0114] The aqueous mixture can be provided in any way and is not limited to a particular sequence of steps. For example, hyaluronic acid may be combined with an aqueous solution (e.g. water, phosphate buffer or phosphate buffered saline) to obtain an aqueous mixture of hydrated hyaluronic acid in the non-crosslinked state, followed by contacting the aqueous mixture comprising a crosslinker. Alternatively, dry hyaluronic acid may be combined with an aqueous solution comprising a crosslinker.

[0115] In preferred embodiments, said aqueous mixture comprises a buffer system, preferably comprising sodium dihydrogen phosphate and disodium phosphate, and a tonicity agent, preferably sodium chloride.

[0116] Optionally, after step a) and before step b), the hydrogel obtained from step a) may be neutralized by adding an appropriate amount of an aqueous solution containing an acid or base. To this extent, a solution of NaOH or HCI in water, phosphate buffer or phosphate buffered saline may be used. Alternatively, the neutralizing step may also simultaneously occur with purification, such as in the course of dialysis.

[0117] In step b), the hydrogel is mixed with a vitamin B3 compound, preferably niacinamide, so as to obtain a homogenous mixture. The mixing can be done during and / or after the addition of the vitamin B3 compound. The mixing operation is performed using techniques well known to those skilled in the art, such as by mechanical mixing within a mixing tank. The mixing can be carried out at a temperature above room temperature and / or under pressure and / or ultrasound to optimize the homogenization of the composition.

[0118] In step c), the homogenous mixture comprising the hydrogel and the vitamin B3 compound is purified. This purification step allows for the removal of undesirable molecules, in particular unreacted crosslinking residues. This purification is performed according to techniques well known to those skilled in the art, such as by dialysis baths, washing by continuous water flow, or precipitation.

[0119] In step d), the homogenous mixture is sterilized to obtain a sterile injectable composition. This step is usually carried out by subjecting the mixture to sterilization by moist heat. Sterilization may be, for example, accomplished by autoclaving the mixture under appropriate conditions. Preferably, moist heat sterilization is carried out at a temperature above 100°C, advantageously above 110°C, advantageously above 120°C. Generally, the sterilization duration can range from a few seconds to several minutes. For example, the following moist heat sterilization cycles can be mentioned: 121 °C for 20 minutes or 126°C for 7 minutes or 130°C for 3 minutes. Other suitable conditions are readily apparent to those skilled in the art.

[0120] Conveniently, the homogenous mixture may be packaged in a container or a syringe prior to step d) and may be therefore sterilized in said container or syringe. Optional compounds such as local anaesthetic, preferably lidocaine or a acceptable salt thereof, may be added to the hydrogel at any appropriate process stage before step d), but are preferably added after step c). Any addition to the hydrogel is accompanied by a mixing operation intended to homogenize the mixture.

[0121] In a particular embodiment, the vitamin B3 compound is added to the hydrogel in two steps: the first in step b) of the method and the second after step c) and before step d). In particular, the proportion of vitamin B3 compound added in step b) may represent from 0.1% to 50%, preferably from 0.1% to 10%, of the total amount of vitamin B3 compound present in the final product. On the other hand, the proportion of vitamin B3 compound added after step c) and before step d) may represent from 50% to 99.9%, preferably from 90% to 99.9%, of the total amount of vitamin B3 compound present in the final product.

[0122] In another aspect, the present invention also relates to an injectable composition, preferably a dermal filler composition, obtainable or obtained by the method of the invention.

[0123] In another aspect, the present invention also relates to a cosmetic, non-therapeutic use of an injectable composition, prefilled syringe or kit of the invention for cosmetic applications. In particular, the composition of the invention may be used to replace or fill a biological tissue or increase the volume of a biological tissue for cosmetic purposes.

[0124] Preferably, for cosmetic, non-therapeutic uses and methods as disclosed herein, the subject has healthy skin.

[0125] Examples of non therapeutic cosmetic applications include, but are not limited to, reducing or filling wrinkles and lines of the skin, in particular of facial lines and facial wrinkles (e.g., glabellar lines, nasolabial folds, chin folds, marionette lines, buccal commissures, peri-oral wrinkles, and crow's feet),

[0126] - improving the appearance of skin, the skin texture and / or elasticity, in particular in the skin of the perioral region, infraorbital region, cleavage, neck, hands, and upper arms, and

[0127] - contouring the face or body of a subject, in particular redefining the shape of the cheeks and enhancing the cheekbones, curving the lips and redefining the contour of the mouth, or adding volume to the temples, dark circles and eye area. The present invention also relates to a cosmetic, non-therapeutic method of reducing or filling wrinkles and lines of the skin, improving the appearance of skin, the skin texture and / or elasticity, and / or contouring the face or body of a subject comprising injecting an effective amount of an injectable composition of the invention into the skin of a subject in need thereof, preferably via subcutaneous and / or intradermal route.

[0128] The composition of the invention is generally administered by injection, more specifically by subcutaneous or intradermal injection, using techniques known in the art such as the serial puncture technique. In particular, the composition may be injected into the dermis and / or the subcutis, preferably into the deep dermis and / or upper subcutis. The term "effective amount", as used herein, is generally intended to refer to the amount of the composition sufficient to effect beneficial or desired non therapeutic cosmetic (aesthetic) results. The subject is typically a human being.

[0129] All the embodiments described above for the composition, the prefilled syringe and the kit of the invention are also contemplated in this aspect.

[0130] In another aspect, the present invention also relates to an injectable composition, prefilled syringe or kit of the invention for use as a medicament. In particular, it relates to an injectable composition, prefilled syringe or kit of the invention for use in the therapeutic treatment of a skin condition, preferably a soft tissue condition. It also relates to a method for treating a skin condition, preferably a soft tissue condition, in a subject in need thereof, comprising administering an effective amount of an injectable composition of the invention to the subject in need thereof. It further relates to the use of an injectable composition of the invention for preparing a medicament for treating a skin condition, preferably a soft tissue condition.

[0131] All the embodiments described above for the composition, the prefilled syringe and the kit of the invention are also contemplated in this aspect.

[0132] As used herein, the term "treating," refers to reducing or eliminating in a subject a clinical symptom of a soft tissue condition characterized by a soft tissue imperfection, defect, disease, and / or disorder; or delaying or preventing in a subject the onset of a clinical symptom of a condition characterized by a soft tissue imperfection, defect, disease, and / or disorder.

[0133] The composition and methods disclosed herein are useful in treating a soft tissue condition. Soft tissue conditions include, but are not limited to, breast reconstruction, micromastia, thoracic hypoplasia, Poland's syndrome, defects due to implant complications like capsular contraction and / or rupture, facial reconstruction, reconstruction of another body part, Parry-Romberg syndrome, lupus erythematosus profundus, and defects post-liposuccion.

[0134] The amount of a composition used with any of the methods as disclosed herein will typically be determined based on the alteration and / or improvement desired, the reduction and / or elimination of a soft tissue condition symptom desired, the clinical and / or cosmetic effect desired by the subject and / or physician, and the body part or region being treated.

[0135] In a last aspect, the present invention also relates to the use of a vitamin B3 compound in an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, to prevent or reduce in vivo oxidative degradation of hyaluronic acid, to increase in vivo stability of hyaluronic acid and / or to increase the duration of in vivo effects of the composition.

[0136] In preferred embodiments, the vitamin B3 compound is niacinamide.

[0137] The crosslinked hyaluronic acid may be as defined above. Preferably, the hyaluronic acid or its pharmaceutically acceptable salt is crosslinked using 1 ,4-butanediol diglycidyl ether (BDDE).

[0138] Preferably, the crosslinked HA or its pharmaceutically acceptable salt has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

[0139] Preferably, the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0140] Preferably, the concentration of the vitamin B3 compound in the composition is in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL.

[0141] Preferably, the composition has a hyaluronic acid / vitamin B3 compound mass ratio, preferably a hyaluronic acid / niacinamide ratio, between 0.5 and 20, preferably between 1 and 10, more preferably between 1 and 5, and even more preferably between 2 and 3. The following examples are given for purposes of illustration and not by way of limitation.

[0142] EXAMPLES

[0143] EXAMPLE 1

[0144] MATERIALS AND METHODS

[0145] Degradation studies

[0146] Accelerated oxidative degradation of the HA fillers was assessed by means of rheology. A volume of 300 pL of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) (2%) or a volume of 300 p L of xanthine-xanthine oxidase mixture was added to 300 pL of the sample which were incubated at 37°C under gentle shaking during 10 minutes before rheological measurement. The storage modulus (G') was measured on a HR 10 rheometer (TA Instruments, Guyancourt, France) equipped with a Peltier plateplate measuring geometry on 600 pL samples at a constant oscillatory frequency of 1 Hz and a temperature of 22°C. A sample hood was used during the measurements to minimize evaporation. Shear stress was set to 3 N / m2in all experiments to respect the linear viscoelastic region (LVE).

[0147] Accelerated enzymatic degradation of the HA fillers was assessed by means of rheology. A volume of 300 pL of hyaluronidase (100 U / mL) was added to 300 pL of the sample which were incubated at 37 °C under gentle shaking during 10 minutes before rheological measurement. The storage modulus (G') was measured on a HR 10 rheometer (TA Instruments, Guyancourt, France) equipped with a Peltier plate-plate measuring geometry on 600 pL samples at a constant oscillatory frequency of 1 Hz and a temperature of 22°C. A sample hood was used during the measurements to minimize evaporation. Shear stress was set to 3 N / m2in all experiments to respect the linear viscoelastic region (LVE). Antioxidant studies

[0148] The Trolox equivalent antioxidant capacity (TEAC) of the various products was determined using a colorimetric Total Antioxidant Capacity Assay Kit, following the instructions of the manufacturer. Briefly, the samples were transferred to 96-well microtitration plates (i.e., 20 pL per well). Volumes of 100 p L of the reaction mix were added to each well and the plates were incubated at ambient temperature for 10 min. Absorbance values were determined at a wavelength of 570 nm and the TEAC values were calculated based on an experimental Trolox standard curve. iNOS (Nitric Oxide Synthase inhibitor) assay was conducted using an assay kit (Nitric Oxide Synthase Inhibitor Screening Kit, Sigma Aldrich, USA). In a clear, flat bottom 96 well plate, 10 pL of NOS solution (Nitric Oxide Synthase, 12.5 U / mL) were transferred in each well. Then, 5 pL of test compound or PBS for the Blank and Control wells were added with 25 pL Assay Buffer to all wells. After 15 minutes of incubation at 25°C, the appropriate reaction mixes were introduced in the sample and blank wells. Afterwards, an incubation at 37°C, which allows the formation of Nitric Oxide (NO), was performed once for one hour, and for a second plate for two hours. Following this incubation step, 200 pL of nitric oxide NO detection reagent were immediately added in all wells and another incubation step was performed at 37 °C for one hour. The measurements were performed in triplicate using a microplate reader to determine the absorbance at a wavelength of 540 nm.

[0149] Biological evaluation

[0150] Human adult fibroblast was grown in culture flasks containing Dulbecco's Modified Eagle Medium (DMEM), supplemented with a 10% fetal bovine serum (FBS), a 1% antibiotic-antimycotic solution at 37 °C in a humidified 5% CO2 atmosphere and monitored daily using an inverted microscope. Subcultures were performed twice a week when a confluence of 80% was observed. For 96 hours collagen quantification, cells were seeded into 24-well culture plate (flat bottom, Costar, Corning, Inc.) at a density of 1 x 105cells per well. After 24 h, the culture media were replaced with a 1000 pL of a mix sample: media at a ratio (1:1). The assay was carried out in triplicate. All plates were incubated at 37 °C in a humidified 5% CO2 atmosphere, and After 96 hours of incubation, the supernatant was removed. The cells were then detached and hydrolyzed through cycles of freezing and thawing in order to measure the collagen by fluorescence with the Collagen Assay Kit (Collagen Assay Kit MAK322 Sigma Aldrich, USA). In the first step of this procedure, collagen in the sample is enzymatically digested into peptides. Subsequently, the N-terminal glycine containing peptides react with the dye reagent to form a fluorescent complex. The fluorescence intensity of this product, measured at Xex = 375 / Xem = 465 nm, is directly proportional to collagen concentration in the sample. Following the same schema protein concentration after 96 hours were determined using a BCA assay (BCA assay, Pierce). Bovine serum albumin (BSA) protein standards consisting of known concentrations of BSA were prepared in a range of 200-1000 pg / mL. Proteins present in the samples change from green to purple when they react with the BCA solution. The colour intensity is highest at 562 nm and is determined spectrophotometrically by comparing it with a known protein standard.

[0151] RESULTS

[0152] Niacinamide prevents oxidation degradation of cross-linked hyaluronic acid

[0153] Oxidative degradation of cross-linked hyaluronic acid was assessed in three different formulations:

[0154] - Formulation 1 comprised 15 mg / mL of hyaluronic acid crossed linked with 0.089 eq. COO- of BDDE (MoD of about 3.8%), 7.5 mg / mL Niacinamide and 3 mg / ml of lidocaine. Formulation 1 comprised less than 10% non-crosslinked hyaluronic acid by total weight of hyaluronic acid;

[0155] - Formulation 3 comprised 20 mg / mL of hyaluronic acid crossed linked with 0.091 eq. COO- of BDDE (MoD of about 4.2%), 7.5 mg / mL Niacinamide and 3 mg / ml of lidocaine. Formulation 3 comprised less than 10% non-crosslinked hyaluronic acid by total weight of hyaluronic acid; and

[0156] - Voluma from Juvederm is a commercial formulation comprising 20mg / mL of cross-linked hyaluronic acid and 3 mg / ml of lidocaine.

[0157] As shown in Figure 1, when comparing oxidative degradation induced by AAPH, a free radical generator, Formulations 3 and 1 surprisingly exhibited significantly higher remaining fractions compared to Voluma, indicating better stability against AAPH exposure. When analyzing the remaining fraction of complex viscosity of the tested formulations, once again, Voluma exhibited significantly lower complex viscosity values compared to Formulation 1 and Formulation 3.

[0158] Similarly, in Figure 1, when comparing oxidative degradation induced by Xanthine oxidase, Formulation 1 and Formulation 3 showed significantly higher remaining fractions of storage modulus and complex viscosity, indicating superior stability against Xanthine oxidase compared to Voluma (i.e., close to 1.7 times).

[0159] On the other hand, when comparing degradation induced by hyaluronidases, no significant differences were highlighted (see Figure 1).

[0160] In conclusion, formulations 1 and 3 comprising niacinamide exhibited similar remaining fraction values when incubated with a high dose of ROS. Both Formulations 1 and 3 showed remaining fractions ranging between 78 % and 91 % with two different ROS . In contrast, the formulation Voluma without niacinamide displayed considerable disparity in degradation linked to ROS, with values dropping as low as approximately 50%. Formulations 1 and 3 therefore demonstrated better resistance to oxidative stress-induced degradation.

[0161] These results show that niacinamide has a protective effect against the degradation of cross-linked hyaluronic acid due to oxidative stress but not to hyaluronidases. Niacinamide prevents oxidation degradation of cross-linked hyaluronic acid.

[0162] Niacinamide provides a higher level of protection against oxidative degradation than mannitol

[0163] In order to limit biases related to hyaluronic acid, the TEAC and iNOS assays were conducted with a PBS solution containing 7.5 mg / ml of niacinamide and 3 mg / ml of lidocaine to mimic Formulations 1 or 3, a PBS solution containing 3 mg / ml of lidocaine to mimic Voluma product from Juvederm, and a PBS solution containing 9 mg / ml of mannitol and 3 mg / ml of lidocaine to mimic Stylage M product from Vivacy.

[0164] The TEAC test that was used is a colorimetric test based on the reduction of copper. As shown in Figure 2, the results did not reveal the presence of measurable TEAC values for the solution mimicking Voluma formulation. The average of TEAC values for the solution mimicking Formulation 1 were significantly higher than for the solution mimicking Stylage M (i.e., 23.8 pM vs 18.8 pM respectively).

[0165] The protocol outlined by the commercial kit for iNOS assay consists of two distinct incubation steps. The initial two-hours incubation period facilitates the synthesis of nitric oxide by nitric oxide synthase, while the subsequent one-hour incubation period allows for the detection of the synthesized nitric oxide. The nitric oxide production is evaluated by monitoring the conversion of nitrate to nitrite.

[0166] The solution mimicking Formulation 1 achieved an average inhibition of 22 % while the solutions mimicking Voluma and Stylage M products had no effect on the inhibition of nitric oxide synthase.

[0167] These results demonstrate that niacinamide (i.e., Formulation 1) exhibits superior antioxidant activity compared to mannitol (i.e., the solution mimicking Stylage M product). Niacinamide also demonstrates inhibition of NOS activity, unlike mannitol.

[0168] Human adult fibroblasts were seeded into 24-well flat bottom culture plate at a density of 1 x 105cells per well. Then, 24 hours later, 4 wells were filled with Voluma product, 4 wells with Formulation 1, 4 wells with Formulation 3, and 4 wells with PBS as a control. Formulations 1 and 3 and Voluma product are as defined above. After 96 hours of incubation, the cells and products were gently removed from each well and cleaned multiple times with PBS. Then, the adherent cells were detached and lysed using freeze-thaw cycles to quantify total proteins and specifically the collagen produced by the cells. Total protein quantification was performed using the bicinchoninic acid reagent (i.e., BCA assay). Collagen quantification involved an enzymatic digestion step followed by a second step of fluorescent quantification. In Figure 3, in the tested conditions, Formulation 1 and Formulation 3 stimulated protein production and in particular collagen production, more effectively than Voluma product and PBS. The results demonstrated that niacinamide present in formulations 1 and 3 not only prevents oxidation degradation of cross-linked hyaluronic acid but can also stimulate the production of proteins and in particular the production of collagen. EXAMPLE 2

[0169] MATERIALS AND METHODS

[0170] Tested formulations

[0171] Six formulations of HA with different HA / Niacinamide mass ratio were prepared according to Table 1.

[0172] Table 1: Compositions of tested formulations

[0173] In formulations 3, C2, C3 and C4, 20 mg / mL of hyaluronic acid were crossed linked with 0.091 eq. COO- of BDDE (MoD of about 4,2%). These formulations comprised less than 10% non-crosslinked hyaluronic acid by total weight of hyaluronic acid.

[0174] Cytocompatibility / toxicity assays

[0175] In order to first assess the cytocompatibility / toxicity of the formulations of Table 1, these formulations were incubated in direct contact with primary cells. In detail, primary dermal fibroblasts were retained for the study, as they are the most relevant target cellular population for dermal fillers (i.e., cells present in dermal tissues). Human primary dermal fibroblasts were grown in vitro in culture flasks containing Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. The cell cultures were incubated at 37 °C in a humidified atmosphere under 5% CO2. Subcultures were performed when a confluency level of 80% was observed. The confluent fibroblasts were transferred to a 24-well plate (i.e., 104cells / well). After 24 h, the culture medium was replaced with a mix of formulation: medium at a 1:1 ratio. The cells were treated with the tested formulations for up to 4 days of incubation. Then, the adherent cells were rinsed and tested for viability / metabolic activity using the cell proliferation reagent WST-1 according to the specifications of the manufacturer (ROCHE Cell Proliferation Reagent WST-1. Roche, Basel, Switzerland). The assay was carried out four times, using four experimental replicates.

[0176] Degradation studies

[0177] Accelerated oxidative degradation of the formulations of Table 1 was assessed by means of rheology. A volume of 300 pL of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) (2%) was added to 300 p L of the sample which were incubated at 37 °C under gentle shaking during 10 minutes before rheological measurement. The storage modulus (G') was measured on a HR 10 rheometer (TA Instruments, Guyancourt, France) equipped with a Peltier plate-plate measuring geometry on 600 pL samples at a constant oscillatory frequency of 1 Hz and a temperature of 22°C. A sample hood was used during the measurements to minimize evaporation. Shear stress was set to 3 N / m2in all experiments to respect the linear viscoelastic region (LVE).

[0178] RESULTS

[0179] Cytocompatibility / toxicity assays showed that formulations 3, Cl, C2 and C4 and Voluma product did not significantly alter the cellular viability of primary dermal fibroblasts after 4 days of incubation. On the contrary, Formulation C3 induced a decrease of cellular viability of about 40%. This formulation was thus excluded from accelerated stability studies.

[0180] The results of accelerated stability studies for formulations 3, Cl, C2, C4 and Voluma product are presented in Figure 4.

[0181] Formulation C2 (HA / Niacinamide mass ratio = 20) and Formulation C4 (HA / Niacinamide mass ratio=10), which contained crosslinked HA performed similarly to Voluma product (without niacinamide), with an average remaining G' of 72.70% (Formulation C2) and 75.19% (Formulation C4). One the other side, Formulation 3 comprising crosslinked HA and exhibiting a HA / Niacinamide mass ratio of 2,67, achieved the highest remaining G' (average: 86.07%) thereby demonstrating the important of this ratio to achieve oxidative stability.

[0182] Despite having the same HA / Niacinamide mass ratio (2.67) as Formulation 3, Formulation Cl (non cross-linked HA) was the most vulnerable, with an average remaining G' of only 24.14%, confirming that niacinamide alone cannot compensate for the lack of crosslinking.

[0183] These findings confirm that crosslinking is the dominant factor in HA stability, while niacinamide provides significant additional benefits when used at a specific ratio.

Claims

CLAIMS1. An injectable composition comprising- crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof,- less than 10% non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof, and- a vitamin B3 compound, wherein the composition has a hyaluronic acid / vitamin B3 compound mass ratio between 1 and 5.

2. The injectable composition of claim 1, wherein hyaluronic acid or pharmaceutically acceptable salt thereof comprised in the composition has an average molecular mass from 10,000 to 6,000,000 Da, preferably from 100,000 to 3,500,000 Da.

3. The injectable composition of claim 1 or 2, wherein the crosslinked hyaluronic acid or the pharmaceutically acceptable salt thereof, is crosslinked using 1 ,4-butanediol diglycidyl ether (BDDE).

4. The injectable composition of any one of claims 1 to 3, wherein the crosslinked hyaluronic acid or the pharmaceutically acceptable salt thereof has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

5. The injectable composition of any one of claims 1 to 4, wherein the concentration of hyaluronic acid is in the range of 5 mg / mL to 30 mg / mL, preferably in the range of 10 mg / mL to 25 mg / mL, more preferably in in the range of 15 mg / mL to 20 mg / mL.

6. The injectable composition of any one of claims 1 to 5, comprising less than 5% non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

7. The injectable composition of any one of claims 1 to 6, wherein the vitamin B3 compound is selected from the group consisting of nicotinic acid, niacinamide and nicotinamide riboside, and any combination thereof.

8. The injectable composition of any one of claims 1 to 7, wherein the vitamin B3 compound is niacinamide.

9. The injectable composition of any one of claims 1 to 8, wherein the concentration of vitamin B3 compound is in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL.

10. The injectable composition of any one of claims 1 to 9, wherein the concentration of hyaluronic acid is in the range of 10 mg / mL to 25 mg / mL and the concentration of vitamin B3 compound is in the range of 5 mg / mL to 10 mg / mL.

11. The injectable composition of any one of claims 1 to 10, wherein the composition has a hyaluronic acid / vitamin B3 compound mass ratio between 2 and 3.

12. The injectable composition of any one of claims 1 to 11, further comprising a local anaesthetic.

13. The injectable composition of any one of claims 1 to 11, further comprising lidocaine or a pharmaceutically acceptable salt thereof.

14. The injectable composition of claim 12 or 13, wherein the local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof, is in the range of 1 mg / mL to 15 mg / mL, preferably in the range of 1 mg / mL to 10 mg / mL, more preferably in in the range of 1 mg / mL to 5 mg / mL.

15. The injectable composition of any one of claims 1 to 14, further comprising a pharmaceutically acceptable buffer system, preferably acetate, phosphate, citrate or borate buffer system.

16. The injectable composition of any one of claims 1 to 14, further comprising 0.005 mg / mL to 0.5 mg / mL, preferably from 0.01 mg / mL to 0.1 mg / mL, more preferably from 0.03 mg / mL to 0.05 mg / mL, of sodium dihydrogen phosphate dihydrate and from 0.05 mg / mL to 1 mg / mL, preferably from 0.1 mg / mL to 0.5 mg / mL, more preferably from 0.2 mg / mL to 0.4 mg / mL, of disodium phosphate dihydrate.

17. The injectable composition of any one of claims 1 to 16, further comprising a tonicity agent, preferably selected from sodium chloride or potassium chloride, glycerin, mannitol or dextrose.

18. The injectable composition of claim 17, wherein the tonicity agent is sodium chloride, preferably at a concentration in the range of 2 mg / mL to 10 mg / mL, preferably in the range of 4 mg / mL to 8 mg / mL, more preferably in the range of 5 mg / mL to 7 mg / mL.

19. The injectable composition of any one of claims 1 to 18, wherein the composition comprises, or consists of,- from 15 mg / mL to 20 mg / mL of hyaluronic acid or a pharmaceutically acceptable salt thereof,- from 5 mg / mL to 10 mg / mL of a vitamin B3 compound, preferably niacinamide,- from 0.01 mg / mL to 0.1 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.1 mg / mL to 0.5 mg / mL of disodium phosphate dihydrate, and- from 4 mg / mL to 8 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 1 mg / mL to 5 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

20. The injectable composition of any one of claims 1 to 18, wherein the composition comprises, or consists of,- from 15 mg / mL to 20 mg / mL of hyaluronic acid or a pharmaceutically acceptable salt thereof,- from 7 mg / mL to 8 mg / mL of a vitamin B3 compound, preferably niacinamide,- from 0.03 mg / mL to 0.05 mg / mL of sodium dihydrogen phosphate dihydrate and from 0.2 mg / mL to 0.4 mg / mL of disodium phosphate dihydrate, and- from 5 mg / mL to 7 mg / mL of a tonicity agent, preferably sodium chloride, and optionally from 2 mg / mL to 4 mg / mL of a local anaesthetic, preferably lidocaine or a pharmaceutically acceptable salt thereof.

21. The injectable composition of any one of claims 1 to 20, which is an injectable dermal filler composition.

22. A cosmetic, non-therapeutic use of an injectable composition as defined in any one of claims 1 to 21 for cosmetic applications, in particular for reducing or filling wrinkles, improving the appearance of skin, improving the skin texture and / or elasticity, or contouring the face or body of a subject.

23. A cosmetic, non-therapeutic method of reducing or filling wrinkles, improving the appearance of skin, improving the skin texture and / or elasticity, or contouring the face or body of a subject, comprising injecting an injectable composition as defined in any one of claims 1 to 21 into the skin of a subject, preferably via subcutaneous and / or intradermal route.

24. A prefilled syringe, or a part thereof, filled with injectable composition as defined in any one of claims 1 to 21.

25. A kit comprising (i) a container filled with injectable composition as defined in any one of claims 1 to 21 and optionally a sterile syringe or (ii) a prefilled syringe, or a part thereof, of claim 24, and optionally a leaflet with instructions to use.

26. A method of manufacturing an injectable composition, in particular an injectable composition as defined in any one of claims 1 to 21, comprising a) mixing and reacting a crosslinking agent with hyaluronic acid or a pharmaceutically acceptable salt thereof in the non-crosslinked state so as to form a hydrogel comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof;b) mixing said hydrogel with a vitamin B3 compound, preferably niacinamide, so as to obtain a homogenous mixture; c) purifying the mixture so as to remove unreacted crosslinking agent; and d) sterilizing the mixture thereby obtaining an injectable composition.

27. The method of claim 26, wherein the vitamin B3 compound is added to the hydrogel in two steps: the first in step b) of the method and the second after step c) and before step d).

28. Use of a vitamin B3 compound, preferably niacinamide, in an injectable composition comprising crosslinked hyaluronic acid or a pharmaceutically acceptable salt thereof, to prevent or reduce in vivo oxidative degradation of hyaluronic acid, to increase in vivo stability of hyaluronic acid and / or to increase the duration of in vivo effects of the composition, wherein the composition has a hyaluronic acid / vitamin B3 compound mass ratio between 1 and 5.

29. The use of claim 28, wherein the concentration of said vitamin B3 compound in the composition is in the range of 1 mg / mL to 25 mg / mL, preferably in the range of 1 mg / mL to 15 mg / mL, more preferably in in the range of 5 mg / mL to 10 mg / mL.

30. The use of claim 28 or 29, wherein the cross-linked hyaluronic acid or the pharmaceutically acceptable salt thereof, is crosslinked using 1,4-butanediol diglycidyl ether (BDDE).

31. The use of any one of claims 28 to 30, wherein the hyaluronic acid or the pharmaceutically acceptable salt thereof comprised in the composition has an average molecular mass from 10,000 to 6,000,000 Da, preferably from 100,000 to 3,500,000 Da.

32. The use of any one of claims 28 to 31, wherein the crosslinked hyaluronic acid or the pharmaceutically acceptable salt thereof has a degree of modification (MoD) in a range of 1% to 7%, preferably in a range of 3% to 5%, more preferably in a range of 3.5% to 4.5%.

33. The use of any one of claims 28 to 32, wherein the composition comprises less than 10%, preferably less than 9%, less than 8%, less than 7%, less than 6% or less than 5%, non-crosslinked hyaluronic acid or pharmaceutically acceptable salt thereof, by total weight of hyaluronic acid or pharmaceutically acceptable salt thereof.

34. The use of any one of claims 28 to 33 , wherein the composition has a hyaluronic acid / vitamin B3 compound mass ratio between 2 and 3.

35. An injectable composition of any one of claims 1 to 21 for use as a medicament.

36. An injectable composition of any one of claims 1 to 21 for use in the therapeutic treatment of a skin condition, preferably a soft tissue condition.

37. A method for treating a skin condition, preferably a soft tissue condition, in a subject in need thereof, comprising administering an effective amount of an injectable composition of any one of claims 1 to 21 to the subject in need thereof.

38. Use of an injectable composition of any one of claims 1 to 21 for preparing a medicament for treating a skin condition, preferably a soft tissue condition.