Novel hyaluronic acid derivatives as innovative fillers

Abstract

The present invention relates to novel hyaluronic acid derivatives conjugated with naturally occurring molecules having anti-inflammatory and / or antioxidant properties, and methods for their preparation.The covalent linkage of these bioactive molecules with hyaluronic acid and the degree of reticulation therewith provide unique characteristics in terms of controlled release of the bioactive molecules and viscoelastic properties, and the resulting hyaluronic acid derivatives are stabilized against chemical and enzymatic degradation.These novel hyaluronic acid derivatives are useful for the preparation of injectable dermal filler compositions that are effective as soft tissue fillers, for example dermal and subdermal fillers.

Description

[Technical field]

[0001] Technical Field The present invention relates to the field of polysaccharides. More specifically, the present invention relates to a novel method for crosslinking hyaluronic acid (HA) of various molecular weights with naturally occurring functionalized molecules with anti-inflammatory and / or antioxidant properties, and to a method for producing crosslinked HA products. The injectable monophasic gels containing hyaluronic acid derivatives obtained by these methods can be used as tissue fillers and for tissue augmentation in the fields of cosmetic surgery and aesthetic medicine.

[0002] 2. Background of the Invention Hyaluronic acid (HA) is a polysaccharide consisting of repeating monomers (sodium glucuronic acid and N-acetylglucosamine disaccharide units) linked together in a linear chain via β-1,4 glycosidic bonds and has the following structure (Formula 1): [ka] It belongs to the class of glycosaminoglycans having the structure

[0003] HA is a naturally occurring polymer found in the extracellular matrix, vitreous humor and cartilage. The total amount of HA found in a normal human (70 kg) is about 15 g, with an average turnover rate of 5 g / day. Approximately 50% of the total amount of HA in the human body is present in the skin, with a half-life of 24-48 hours. HA is one of the basic components of animal tissues, and at the dermal level it exists both in free and protein-bound form. HA moisturizes the skin due to its water-retaining capacity, and gives it firmness due to its cohesive properties of the extracellular matrix (the substance that "compacts" the dermis). A deficiency of HA leads to a weakening of the "scaffolding" of the skin, resulting in a decrease in tone, moisture and resistance. From a purely aesthetic criterion, it can be considered the source of "wrinkle formation".

[0004] Also, HA has no biomechanical properties because it is a liquid and not a gel in the presence of water. Injecting a solution of HA under a wrinkle to lift it would not be effective, and being a liquid, it would be absorbed by the tissue within a few hours. Pure HA is used as a so-called "biostimulant" injection; being a liquid, it is a stimulant in itself and is quickly absorbed by the tissue into which it is injected.

[0005] To obtain a gel capable of supporting and lifting the weight of tissues (not only in the case of skin wrinkles, but also in the case of deterioration of joints, e.g. the knee), HA must be chemically converted into a gel. The filler hyaluronic acid gels (sold and used as medical devices) are prepared through industrial processes so that they acquire biomechanical properties (viscosity and elasticity) and integrate with tissues.

[0006] During the manufacturing process of hyaluronic acid gel fillers, chemical linkers, also called crosslinkers, are used. One of the most used linkers is 1,4-butanediol diglycidyl ether (BBDE), which is able to form (more or less stable) bonds between hyaluronic acid filaments (Dermatol Surg 2013;39:1758-1766; DOI: 10.1111 / dsu.12301). The chains that are linked together become stable like a compact knit structure, which becomes a solid gel as a whole. Crosslinked hyaluronic acid is typically viscous, gelatinous to the touch, elastic, and has various degrees of hardness or softness that are "tailored" according to the intended injection site (i.e. cheek, lips, nasolabial fold, etc.). After injection, these fillers, which show various durations and are quantitative for several months, integrate with the tissue, thereby giving them "shape" and "long-lasting". Depending on the number of crosslinking molecules and the type of bonds they form, the gel will be softer, denser or harder. The stronger and more numerous the bonds, the more rigid and hard the gel will be, and conversely, the weaker and fewer the bonds, the softer the gel will be. The specific rheological properties of these crosslinked HA fillers can be measured by their elastic modulus (G'), their viscosity (G'') and their swelling coefficient (SwF) (Barnes HA; Handbook of Elementary Rheology, Institute of Non-Newtonian Fluid Mechanics, University of Wales, 2000). For this last rheological parameter, there are no clinical data linking the swelling coefficient to the swelling after the procedure, as the factors that may contribute to determining the swelling of the tissue can be diverse, such as the specific crosslinking technique, the injection technique, the quality of the tissue, etc.

[0007] Examples of hyaluronic acid gel fillers prepared using BBDE as a crosslinker are disclosed in International Patent Applications WO 2017 / 016917 and 2005 / 097218; 2012 / 062775, 2013 / 028904, 2013 / 040242, 2016 / 051219 and 2009 / 018076; 2017 / 001056, 2017 / 162676, 2016 / 074794, 2013 / 185934, 2017 / 001057, 2018 / 083195 and 2017 / 076495.

[0008] The metabolism of hydrolyzed BDDE has not been described in the literature, but is understood to proceed by cleavage of the ether bond by a family of enzymes called cytochrome P450. These enzymes are involved in the oxidative degradation of organic molecules and may catalyze the cleavage of the ether bond to an alcohol. After degradation, two main products may result: glycerol and 1-4-butanediol. Like all diol ethers, hydrolyzed BDDE is known to be excreted in the urine (Dermatol Surg 2013;39:1758-1766). 1,4-butanediol is known to be non-mutagenic, non-sensitizing, and slightly irritating (Ishikawa K. 1,4-butanediol. OECD SIDS CAS No 110-63-4 2000:1-60;NICNAS 1,4-butanediol. Existing chemical hazard assessment report ISBN 978-0-9803124-7-8 2009. pp. 1-25). No carcinogenic potential has been identified by studies performed on its metabolites. Neurotoxic adverse effects were observed in animals, and the no observed adverse effect level (NOAEL) was 100 mg / kg / day (determined by oral administration in mice). The median lethal dose (LD50) of 1,4-butanediol is 1,525 mg / kg (determined by oral administration in mice). However, the long-term effects of 1-4-butanediol, a synthetic compound used as an industrial solvent, are unknown. Upon ingestion, it is known to be converted to gamma-hydroxybutyrate, a drug of abuse that exerts depressant effects primarily on the central nervous system (N Engl J Med, Vol. 344, No. 2 January 11, 2001, 87-94).

[0009] Other crosslinkers utilized in the preparation of hyaluronic gel fillers are boronic acid derivatives belonging to the class of alkylboronic acid hemiesters that generate reversible bonds (WO 2018 / 024795); diamines and polyamines (hexamethylenediamine, lysine monomethyl ester and 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine) and carbodiimides (WO 20 13 / 040242); citric acid (WO 2018 / 087272); endogenous amines, such as spermine and spermidine, and as coupling agents, N-ethyl,N-(dimethylaminopropyl)-carbodiimide (WO 2014 / 064632); divinyl sulfone (WO 2005 / 066215); carboxyl groups are activated to react with alcohol groups present on the same polysaccharide chain or on other nearby polysaccharide chains. These include hyaluronic acid gels obtained by self-assembly (EP 0341745); multicomponent condensation products obtained by reacting carboxy and amino groups derived from partial N-deacetylation of HA or a derivative with aldehydes and isocyanides (WO 0218450); formaldehyde, glutaraldehyde, divinyl sulfone, polyanhydrides, polyaldehydes, polyhydric alcohols, carbodiimides, carboxylic acid chlorides, sulfonic acid chlorides, epichlorohydrin, ethylene glycol, butanediol diglycidyl ether, diglycidyl ethers, polyglycerol polyglycidyl ethers, polyethylene glycol diglycidyl ethers, polypropylene glycol diglycidyl ethers and preferably bis- or polyepoxides in the presence of butanediol diglycidyl ether or divinyl sulfone (EP 1837347).

[0010] KR 20180010361 discloses crosslinked hyaluronic acid obtained by reacting hyaluronic acid with 1,4-butanediol diglycidyl ether (BDDE) and catechin. The crosslinks are of the ether type.

[0011] KR 20160031081 discloses hyaluronic acid functionalized with polyphenols, where the polyphenol moiety does not act as a crosslinker.

[0012] Another characteristic of HA as a dermal filler is its rapid degradation under physiological conditions. The degradation of HA can be described as a depolymerization process mediated by the cleavage of glycosidic bonds. This depolymerization may precede the dissociation (dissolution and diffusion) of the polymer chains at the macromolecular level. The depolymerization of HA has been well characterized in the literature and involves two main mechanisms: enzymatic degradation and free radical degradation. A large class of enzymes collectively referred to as hyaluronidases mediates the enzymatic degradation of HA, and several reports in the literature have also shown that the free radical-mediated degradation of HA proceeds via the cleavage of glycosidic bonds. Catabolism of HA occurs in situ (e.g., in the extracellular matrix), within cells or after translocation to lymph nodes, and involves the conversion of long HA chains (polysaccharides) into smaller HA units (oligosaccharides). Two separate studies using various BDDE-crosslinked HA fillers with different physicochemical properties demonstrated that BDDE modification does not interfere with the native enzymatic degradation mechanisms of HA (Jones D, et al. Dermatol Surg 2010;36:804-9, Sall I et al. Polym Degrad Stab 2007;92:915-9).

[0013] In recent years, the use of dermal fillers has increased significantly, from 650,000 cases per year in 2000 to over 2.4 million cases in 2015, resulting in an increase in complications (American Society of Plastic Surgeons, 2014 Plastic surgery statistics report. https: / / www.plasticsurgery.org / news / plastic-surgery-statistics ?sub=2014+Plastic+Surgery+Statistics, accessed June 1, 2017). In most cases, fillers are used without causing clinically significant complications to patients, but the overall number of complications is increasing due to increased use and large variations in the training and experience of clinicians (Haneke E. Managing complications of fillers: rare and not-so-rare.J Cutan Aesthet Surg. 2015;8(4): 198-210). According to the US Food and Drug Administration's (FDA) manufacturer and user device experience (MAUDE) database, complications of the HA-based fillers Restylane®, Belotero®, Juvederm®, and Juvederm Voluma® include swelling, infection, and nodule formation.

[0014] Even if these complications are estimated to be 0.01% of all injections for HA fillers, there is a need for safer HA-based fillers and the possibility to develop new crosslinked HA fillers with improved properties of safety, stability against depolymerization, and tailored rheological properties.In fact, the long-term effects of some known metabolites of some fully synthetic crosslinkers, such as 1-4 butanediol that may be generated by BDDE, are not fully understood, prompting the search for safer, naturally derived crosslinkers.

[0015] Description of the Invention The present invention discloses a novel method for producing a cross-linked hyaluronic acid (HA) gel product, which meets the following requirements: efficient incorporation of a cross-linking agent with anti-inflammatory and / or antioxidant properties, sufficient gel strength to resist deformation and migration upon implantation, and improved stability against sterilization heat treatments and enzymatic hydrolysis relative to the native HA utilized. Thus, the present invention allows the production of gels with enhanced strength and limited swelling relative to non-cross-linked HA, with surprisingly low chemical modification of HA.

[0016] It is a further object of the present invention to provide a method that has a modular efficiency of the cross-linking reaction. It is a further object of the present invention to minimize the degree of modification required to obtain an HA gel product with a desired gel strength. It is a further object of the present invention to obtain an HA gel product that has improved durability in vivo relative to non-cross-linked HA, while at the same time having a limited degree of structural modification. It is also an object of the present invention to obtain an HA gel product that has useful implantation properties, including viscoelastic gel properties and purity from by-products and residues.

[0017] The claimed crosslinked hyaluronic acid (HA) gel products of the present invention were prepared from three types of hyaluronic acid having the following molecular weights: · Low molecular weight fraction: 8-15 kDa - used preferentially for conjugation and formation of novel non-crosslinked derivatives or as part of a crosslinked matrix imparting more viscous properties. Mid molecular weight fractions: 500-750 kDa - these fractions were sometimes subjected to targeted purification by cross-flow filtration to narrow the molecular weight range and get as close to the upper end as possible, or were used directly for cross-linking. High molecular weight fractions: 1.5-3.0 MDa - These fractions were used to complete the formulation which function to support the structure of the filler itself giving it more elastic properties.

[0018] The preparation of this novel trimodal (or ternary) filler utilized natural and safe bioactive agents such as polydatin, gallic acid, chlorogenic acid and phloridzin as derivatives or crosslinkers.

[0019] These compounds share the common features of being natural molecules commonly found in foods and beverages, having anti-inflammatory and / or antioxidant properties, being water soluble, and having suitable functional groups (i.e., hydroxyl and / or carboxyl functionalities) useful for subsequent modification and subsequent conjugation with hyaluronic acid.

[0020] In particular, polydatin (chemical name: β-D-glucopyranoside, 3-hydroxy-5-[2-(4-hydroxyphenyl)ethenyl]phenyl; formula 2), the main component of grape juice, is the most abundant form of resveratrol in nature. This molecule exhibits a wide range of bioactivities, including anti-inflammatory, antioxidant, anticancer, neuroprotective, hepatoprotective, nephroprotective and immunostimulatory activities (Didem Sohretoglu et al., Recent advances in chemistry, therapeutic properties and sources of polydatin. Phytochemistry Reviews volume 17, 973-1005 (2018)). [ka]

[0021] This molecule is a stilbenoid, trans-resveratrol substituted at position 3 by a β-D-glucoside residue. Polydatin has six hydroxyl groups, two of which are phenolic with various reactivities, which can be used as anchor points for subsequent derivatization. The presence of the double bond directs the activity, since the trans form, unlike the cis form, is bioactive. Derivatization was undertaken to modify two hydroxyl groups (phenolic moiety) or all hydroxyl groups, so that this molecule can be used both as a crosslinker and to derivatize hyaluronic acid chains.

[0022] Gallic acid (chemical name: 3,4,5-trihydroxybenzoic acid; formula 3) is a naturally occurring secondary metabolite found in a variety of plants, vegetables, nuts and fruits, such as gall nut, sumac, witch hazel, tea leaves and oak bark. [ka]

[0023] Gallic acid is a compound with anti-inflammatory and / or antioxidant activity, and based on available literature data, it has shown little toxicity in animal or clinical trials, making it potentially useful for long-term use in inflammation-related diseases (Nouri, F. Heibati, E. Heidarian, Gallic acid exerts anti-inflammatory, anti-oxidative stress, and nephroprotective effects against paraquat-induced renal injury in male rats, Naunyn Schmiedebergs Arch. Pharmacol. 2020). Literature toxicity data confirms that gallic acid is safe for most cells at low concentrations and only toxic at relatively high concentrations. The acute toxicity of gallic acid in albino mice was shown to have an LD50 of over 2000 mg / kg (BC Variya, et al., Acute and 28-days repeated dose sub-acute toxicity study of gallic acid in albino mice, Regul. Toxicol. Pharmacol 101 (2019) 71-78).

[0024] Chlorogenic acid (chemical name: 3-[[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]oxy]-1,4,5-trihydroxy-cyclohexanecarboxylic acid, (1S,3R,4R,5R); formula 4) is a cinnamate ester obtained by the formal condensation of the carboxy group of trans-caffeic acid with the 3-hydroxy group of quinic acid, and was first isolated from green coffee beans (Freudenberg, Ber. 53, 237, 1920). This compound scavenges free radicals, which inhibits DNA damage and protects against the induction of carcinogenesis. In addition, this agent may upregulate the expression of genes involved in the activation of the immune system, promoting the activation and proliferation of cytotoxic T lymphocytes, macrophages and natural killer cells. [ka]

[0025] Phlorizin (chemical name: 1-[2-(β-D-glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone; Formula 5) is a phytochemical belonging to the class of polyphenols. Phlorizin is a glucoside found in the stems, roots, and bark of plants in the Rosaceae family, including apples, cherries, and pears. Potential and investigational uses for phlorizin include adjuvant treatment of type 2 diabetes, weight loss agent for obesity, and acute management of hyperglycemia (Diabetes Metab Res Rev 2005; 21: 31-38). [ka] [Brief description of the drawings]

[0026] [Figure 1] UV analysis used to quantify PODG in the reticulation reaction of HA with PODG. [Diagram 2] UV analysis was used to quantify POca in the reticulation reaction of HA with POca. [Diagram 3] UV analysis was used to quantify POca in the reticulation reaction of HA with POca. [Figure 4] 1H-NMR spectra 1 are reported in FIG. F for HA in NaOD solution, polydatin in NaOD solution, and the filler PR032D, from top to bottom. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention These compounds have been derivatized with epichlorohydrin or 2-chloroacetic anhydride to obtain low molecular weight HA derivatives in solution conjugated with the bioactive agents mentioned above. The covalent bond with low molecular weight HA does not result in a three-dimensional network, and the final derivatives in water do not show the characteristics of a gel, but rather show the appearance of a homogeneous solution that is used as one component of the final trimodal (or ternary) structure of the final filler.

[0028] To obtain crosslinks between medium molecular weight HA chains, the bioactive agents are modified by introducing at least two reactive groups, epichlorohydrin or 2-chloroacetic anhydride, to facilitate the crosslinking reaction with the subsequent hyaluronan chains. In this case, polydatin, gallic acid, chlorogenic acid and phlorizin covalently bound to medium molecular weight HA have a dual role: bioactive molecule and reticulating agent.

[0029] The preparation of glycidyl polydatin derivatives is carried out by using epichlorohydrin (EP) as a reagent, a solvent, and an organic ammonium salt as a phase transfer catalyst. Specifically, tetrabutylammonium chloride (TBACl) or benzyltriethylammonium chloride (BTEACl) was used. The synthetic scheme used to obtain the diglycidylated derivatives of polydatin is reported below (Scheme 1). [ka]

[0030] To obtain the diglycidyl derivative of polydatin (chemical name: (2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-(3-(oxiran-2-ylmethoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol), the following experimental conditions were utilized. The reaction proceeds at a temperature of 100° C. using 20 molar equivalents of EP and 0.1 equivalents of TBACl (or BTEACl) per mole of polydatin. Moreover, under these conditions, a reaction time of 3 hours is sufficient to reach the maximum conversion. After this time, the reaction mixture is cooled at room temperature and then an aprotic organic solvent, preferably di-isopropyl ether, is added under vigorous stirring to obtain a white solid, which is recovered. The resulting crude reaction product can be further purified by column chromatography on silica gel to give polydatin diglycidate.

[0031] To the best of the inventors' knowledge, (2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-(3-(oxiran-2-ylmethoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol; polydatin diglycidate) is novel. The reaction raw material contains a by-product identified as (2S,3R,4S,5S,6R)-2-(3-(3-chloro-2-hydroxypropoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, so chromatographic purification is essential to obtain a pure product.

[0032] Polydatin derivatives were prepared using 2-chloroacetic anhydride according to the following synthesis scheme (Scheme 2). [ka]

[0033] Various solvents were evaluated to obtain the 2-chloroacetylated product. The reaction in ethyl acetate (AcOEt) was found to be the most advantageous since it does not require a solvent exchange and it is possible to purify the molecule or mixture of molecules directly from the reaction mixture. The reaction was carried out under anhydrous conditions and under an inert atmosphere (nitrogen). Polydatin (1 eq.) was suspended in AcOEt and then 6-12 eq. monochloroacetic anhydride was added under stirring at room temperature. The reaction mixture was refluxed for 5-10 h, then cooled and left under stirring at room temperature for 24 h. The reaction mixture was then diluted with water to give a white solid precipitate that was collected by suction. This was washed with water and dried under vacuum at room temperature to give the hexa-chloroacetyl derivative of polydatin in 91% yield. To the best of the inventors' knowledge, this compound is novel. The mixture of mono-, di-, tri- and tetra-2-chloroacetylpolydatin esters that could be obtained is 17±3.4% / 44.3±8.8% / 17.7±3.6% / 1.9±0.4%.

[0034] The reaction of gallic acid with 2-chloroacetic anhydride gave the corresponding 3,4,5-tris(2-chloroacetoxy)benzoic acid, as reported in the scheme below (Scheme 3). [ka]

[0035] The general synthesis procedure involves reacting gallic acid with 4-8 milliequivalents, preferably 6 milliequivalents, of 2-chloroacetic anhydride in ethyl acetate. The reaction temperature ranges from 10-30°C, preferably 20-25°C. The reaction is usually complete in about 24 hours. The organic phase is first washed with an acidic aqueous solution, preferably 0.5M HCl, then with brine, followed by drying over Na2SO4, filtration, and evaporation of the filtrate under vacuum to give an oily residue. The oil is then treated with water to give a white solid, which is then dried under vacuum. The overall molar yield is 79%. To the best of the inventors' knowledge, this gallic acid derivative is novel.

[0036] The reaction of gallic acid with epichlorohydrin is carried out according to the following synthetic scheme (Scheme 4). [ka]

[0037] The preparation of glycidyl derivatives was carried out by mixing gallic acid (GA) and EP in the presence of an organic salt (tetrabutylammonium chloride, TBACl) as a phase transfer catalyst. In this reaction, EP functions as both a reagent and a solvent. The relative molar ratio between GA and EP is comprised in the range of 1 / 14 to 1 / 18, preferably 1 / 16. The procedure involves sequential addition of GA, TBACl and EP, followed by leaving the mixture at a temperature range of 60 to 100°C, preferably at 100°C, for 6 hours. The reaction mixture is then cooled to room temperature and treated with a 20% w / w NaOH solution (2 molar equivalents / OH) and 0.1 molar equivalent TBACl. The resulting white suspension is vigorously shaken at room temperature, then the reaction mixture is diluted four times with water and extracted three times with AcOEt. The combined organic phases are washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude product. This can be further purified by chromatography to give the desired product.

[0038] Similarly, the glycidyl and 2-chloroacetyl derivatives of chlorogenic acid and phlorizin were obtained.

[0039] Next, a functional matrix was prepared by conjugating the above-mentioned polydatin derivative with medium molecular weight hyaluronic acid.

[0040] The important parameters for the preparation of these novel hyaluronic acid derivatives are reported below: Depending on the type of reaction medium utilized, the reaction temperature and time, and the relative molar ratio between the activated molecules (i.e. polydatin, gallic acid, chlorogenic acid and phloridzin derivatized with epichlorohydrin and 2-chloroacetic acid) and hyaluronic acid, different properties of viscosity, elasticity and stability (thermal and enzymatic) were obtained.

[0041] Solvent Type Derivatization of the auxiliary molecules (polydatin, gallic acid, chlorogenic acid, and phlorizin) significantly reduces their water solubility, necessitating the use of organic solvents for their solubilization. In our experiments, the solvent used was dimethyl sulfoxide (DMSO), a polar, water-soluble solvent with low toxicity. On the other hand, it has been demonstrated that hyaluronic acid, in its sodium salt form, is completely soluble in water, with a moderate and limited solubility in DMSO. Based on these considerations, various reaction conditions were evaluated based on the use of DMSO, H2O, and DMSO / H2O mixtures. Experimental evidence indicates that the use of water alone does not promote the conjugation reaction. The use of DMSO as the intrinsic reaction solvent allows the reaction to proceed by generating conjugates that form solutions once dissolved in water.

[0042] Surprisingly, it has been found that the use of DMSO / HO mixtures is advantageous for preparing both water-soluble conjugates and true gels, depending on the proportion of HO used. In particular, the use of solvent mixtures in a 1:1 ratio favors the formation of gels.

[0043] Reaction temperature The reaction temperature dictates the degree of substitution of the polysaccharide chains. The range considered is comprised between 30 and 80°C. At the lower end of this range, no conjugation occurs, whereas at 50°C the reaction starts to occur. A temperature of 50°C was chosen as the best temperature to carry out this reaction, since at this temperature there is evidence of conjugation and gel formation. Working at the upper limit of 80°C results in a conjugate structure that also has gel properties, while the structure may change.

[0044] Reaction time In the early stages of setting the reaction conditions, in-process control (IPC) was performed to evaluate the progress of the reaction under the aforementioned conditions. The reaction rate was fairly slow, with the first evidence of conjugation occurring after 3 hours, with significant displacement occurring around 15 hours. As mentioned above, the use of a solvent mixture in a 1:1 ratio allowed the reaction rate process to be accelerated within 2 hours.

[0045] Molar ratio between the derivatized auxiliary molecule and hyaluronic acid The amount used in the reaction, especially the molar ratio between them, regulates the degree of substitution on polymer chain.The ratio used is 1 / 1, 1 / 5 and 1 / 10, which is expressed between the mole number of derivatized auxiliary molecule (e.g. polydatin derivative) and the mole number of dimer unit that constitutes hyaluronic acid chain.The inventors have found that the ratio of 1 / 5 and 1 / 1 increases the loading of conjugated auxiliary molecule.

[0046] Hyaluronic acid of three different molecular weights (HMW, MMW, and LMW) and diglycidyl polydatin (PO DG The reaction conditions and characterization utilized for the preparation of gels using ) are described below.

[0047] These reaction conditions were applied, with minor modifications, to all the glycidyl derivatives of the auxiliary molecules of the present invention (ie, gallic acid, chlorogenic acid and phloridzin).

[0048] Degree of crosslinking The degree of crosslinking is preferably selected to give a ratio of viscous modulus (G'') to elastic modulus (G') of less than 1.0. When using 2-chloroacetylated polydatin as crosslinking agent, the obtained degree of crosslinking is comprised between 70 and 80% when the molar ratio of 2-chloroacetylated polydatin to the number of moles of repeating units of HA is comprised between 1:5 and 1:10. When using diglycidated polydatin as crosslinking agent, the obtained degree of crosslinking is comprised between 15 and 55% when the molar ratio of diglycidated polydatin to the number of moles of repeating units of HA is comprised between 1:5 and 1:1.

[0049] The following important reaction parameters related to the crosslinking reaction were investigated: ·PO DG and hyaluronic acid; the following molar ratios were investigated: 5 / 1; 1 / 1; 1 / 5; 1 / 10. Three fractions of hyaluronic acid were tested: HMW, MMW and LMW. Reaction times of 1 hour and 4 hours, preferably 2 hours, were investigated.

[0050] Other process parameters were kept constant: the reaction temperature was always at 50° C. and the solvent used was a constant H2O / DMSO mixture with a v / v ratio of 1:3 or 1:1.

[0051] A preliminary general cross-linking procedure involves the use of 0.25 M NaOH aqueous solution to solubilize hyaluronic acid of a defined molecular weight at room temperature (a balance is involved between the use of basic solutions and temperature in order not to degrade the hyaluronic acid). Thus, in the preparation step, hyaluronic acid was placed in a basic environment at room temperature for variable times (1 h for HMW to 30 min for LMW, depending on the molecular weight).

[0052] In parallel, PO in DMSO DG A solution was prepared. When polydatin was derivatized with epoxy groups, its solubility in water was significantly reduced, and its solubility itself was quite low. Therefore, it was necessary to use DMSO as a co-solvent for the reaction. Both components (hyaluronic acid and PO DG Once PO is dissolved, the DMSO solution is poured into the hyaluronic acid solution in 0.25M NaOH and the temperature is raised to 50°C. The reaction is maintained under stirring for 1 or 4 hours. At the end of the reaction, the polymer must be precipitated and the excess reagent removed. DGWhen crosslinking with , the progress of the reaction requires the addition of a low molecular weight alcohol, preferably ethanol. Under these experimental conditions, the polymer appears as a precipitate and can be isolated by centrifugation. In the final step, the precipitate is hydrated in deionized water (MilliQ water) and then the salts still present are purified by dialysis. The final isolation of the hyaluronic acid derivative is then carried out by lyophilization.

[0053] When HA with MMW and LMW was used under the same reaction conditions, PO DG To obtain a gel containing DG It was confirmed that the / HA ratio was 1 / 1, the reaction temperature was 50° C., and the reaction time was comprised between 1 and 4 hours.

[0054] Preliminary tests on heat sterilization of the gel samples obtained provide further insights relevant for the development of stable sterile gel formulations. Indeed, gels made with poly(2-chloroacetylated) polydatin blends do not tolerate thermal stress. This treatment produces a solution at the end of the sterilization cycle that is no longer a gel. This process induces deconstruction in these gels, probably due to hydrolysis of the particularly heat-labile ester bonds that give rise to crosslinks. On the other hand, diglycidylated polydatin (PO DG ) are not affected by such extensive thermal stresses since the ether bonds which give rise to the crosslinks are hardly susceptible to thermal degradation.

[0055] Based on these sterilization results, gel formulations based exclusively on glycidylated crosslinkers were developed for moist heat sterilization, whereas 2-chloroactylated co-crosslinkers were preferred for gamma radiation sterilization.

[0056] The crosslinked hyaluronic acid of the present invention is useful in a dermal filler composition in an amount between 1 mg / ml and 50 mg / ml, optionally in the presence of an anesthetic, preferably lidocaine, at a final concentration comprised between 0.1 and 0.4% weight / volume, which composition may be used in a method for replacing or filling biological tissue for cosmetic purposes or for increasing the volume of biological tissue.

[0057] The injectable compositions of the present invention are administered intradermally or intraarticularly in the form of a sterile gel. Mixtures of cross-linked hyaluronic acid of various molecular weights can be used.

[0058] The compositions can be provided in the form of a kit, including instructions for use and, optionally, other useful agents.

[0059] The invention will now be described in detail with reference to the following examples.

[0060] Example 1 - Preparation of Polydatin Diglycidylates (2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-(3-(oxiran-2-ylmethoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol [ka]

[0061] The reaction was carried out under an inert atmosphere (argon). 58 mg BTEACl (0.256 mmol, 0.1 molar equivalents) and 4 mL epichlorohydrin (51.2 mmol, 20 molar equivalents) were added to 1.0 g polydatin (2.56 mmol). The resulting suspension was heated to 100°C under stirring. After 30 min at 100°C, the suspension became a clear pale yellow solution, heating was continued for 3 h and cooled to room temperature. Before the reaction mixture solidified, 40 mL diisopropyl ether was added under vigorous stirring. The white solid was immediately separated and filtered by washing with 5 mL of the same solvent. By TLC analysis (CH2Cl2 / MeOH 90 / 10), the resulting solid (1.18 g) consisted of two compounds.

[0062] The resulting crude solid was dissolved in 20 mL of MeOH (with slight heating), 7 g SiO2 was added to the solution, and the solvent was evaporated in a rotavap. The powder obtained after complete removal of MeOH by a mechanical vacuum pump was loaded onto a SiO2 flash chromatography column and eluted with CHCl / MeOH 90 / 10 v / v. The first product to elute was the desired diglycidylated derivative of polydatin 3, of which 449 mg (molar yield 35%) was crystallized from 25 mL of ethanol. The compound had an mp of 167–170 °C, [α] D 25℃=-50℃(c0.5 in MeOH) and [α] D The compound had a temperature of 25°C = -36°C (c1 in DMSO). 1 H, 13 C, H, H COSY, ETCORR). These allowed assignment of all signals and confirmation of the structure. Numbers refer to the formula reported in Eq. 6.

[0063] [ka]

[0064] 11H NMR (500 MHz, DMSO-d6): δ 7.51 (2H, d, J = 8.8 Hz, H-10 and 14), 7.19 (1H, d, J = 16.4 Hz, H-8), 7.00 (1H, J = 16.4 Hz, H-7), 6.98 (2H, d, J = 8.8 Hz, H-11 and 13), 6.88 (1H, br s, H-6), 6.82 (1H, br s, H-4), 6.55 (1H, m, H-2), 5.31 (1H, d, J = 4.9 Hz, OH on C-2’), 5.12 (1H, d, J = 4.4, OH on C-3’), 5.05 (1H, d, J = 5.2 Hz, OH on C-4’), 4.87 (1H, d, J = 7.0 Hz, H-1’), 4.67 (1H, dd, J = 5.1 and 5.1 Hz, OH on C-6’), 4.36 (2H, dd, J = 11.4 and 2.6 Hz, H-1a” x 2), 3.84 (2H, dd, J = 11.4 and 6.6 Hz, H-1b” x 2), 3.74 (1H, dd, J = 10.4 and 5.2 Hz, H6a’), 3.46 (1H, m, H-6b’), 3.40 - 3.32 (3H, overlapping, H-5’ and H-2” x 2), 3.31 - 3.22 (2H, overlapping, H-2’ and H-3’) 3.15 (1H, dd, J = 8.8 and 5.1 Hz, H-4’), 2.85 (2H, m, H-3a”x 2), 2.71 (2H, m, H-3b” x 2); 13C NMR (125 MHz, DMSOd6): δ 159.3 (C-12), 158.7 (C-1), 157.9 (C-3), 139.3 (C-5), 129.8 (C-9), 128.6 (C-8), 127.8 (C-10 e C-14), 126.0 (C-7), 114.7 (C-11 e C-13), 106.9 (C-6), 106.1 (C-4), 102.0 (C-2), 100.5(C-1'), 77.1 (C-5'), 76.7 (C-3'), 73.2 (C-2'), 69.8 (C-4'), 68.9(C-1" x 2), 60.7 (C-6'), 49.6 (C-2” x 2), 43.7 (C-3” x 2). MS spectroscopy (ESI) shows a peak at m / z=525.8. This is the Na ion to molecular ion. + This is equivalent to the addition of

[0065] The second product to elute was a by-product (261 mg). This was crystallized from 15 mL of isopropyl alcohol, mp 138-141 °C (dec.), [α] D A solid was given which showed 25°C = -43° (c1 in MeOH).

[0066] This compound was analyzed by NMR ( 1 H, 13 C, H,H COSY, ETCORR) and was found to consist of a mixture of two very similar (and chromatographically inseparable) compounds (ratio of about 3:1) that differed in the location of the glycidyl and chloroalcohol substituents. The NMR data for major component 4A are reported below. The numbering system is reported in formula 7 (chemical name: (2S,3R,4S,5S,6R)-2-(3-(3-chloro-2-hydroxypropoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol).

[0067] [ka]

[0068] 1 1H NMR (500 MHz, DMSOd6): δ 7.53 (2H, d, J = 8.5 Hz, H-10 and 13), 7.19 (1H, d, J = 16.4 Hz, H-8), 7.02 (1H, d, J = 16.4 Hz, H-7), 6.98 (2H, d, J = 8.5 Hz, H-11 and 13), 6.89 (1H, br s, H-6), 6.81 (1H, br s, H-4), 6.52 (1H, br s, H-2), 5.58 (1H, d, J = 4.0 Hz, OH at 3”’), 5.30 (1H, d, J = 4.7 Hz, OH at 2’), 5.11 (1H, d, J = 4.0 Hz, OH at 3’), 5.05 (1H, d, J = 4.9 Hz, OH at 4’), 4.88 (1H, d, J = 7.3 Hz, H-1’), 4.67 (1H, dd, J = 5.0 and 5.0 Hz), 4.35 (1H, dd, J = 11.4 and 2.1 Hz, H-1”a), 4.03 (1H,m, H-2”’), 4.01 (2H, m, H-1”’a and 1”’b), 3.85 (1H, dd, J = 11.4 and 6.5 Hz, H”b), 3.75 (1H, dd, J = 11.1 and 4.4 Hz, H-3”’a), 3.73 (1H, m, H-6’a), 3.68 (1H, dd, J = 11.1 and 5.1 Hz, H-3”’b), 3.47 (1H, dd, J = 11.7 and 5.9 Hz, H-6’b), 3.38 - 3.33 (2H, overlapping, H-2” and 5’), 3.28 (1H, m, H-3’), 3.26 (1H, m, H-2’), 3.15 (1H, m, H-4’), 2.85 (1H, m. H-3”a), 2.71 (1H, m, H-3”b); 13C NMR (125 MHz, DMSOd6): δ 159.5 (C-3), 158.7 (C-1), 157.9 (C-12), 139.3 (C-%), 129.8 (C-9), 128.6 (C-8), 127.8 (2C, C-10 e 14), 126.0 (C-7), 114.7 (2C, C-11 e 13), 106.8 (C-6), 106.2 (C4), 102.1 (C-2), 100.6 (C-1'), 77.1 (C-5'), 76.6 (C-3'), 73.2 (C-2'), 69.8(C-4'), 69.1 (C-1''), 68.9(C-1''), 68.6 (C-2”), 60.7 (C-6’), 49.6 (C-2”), 46.7 (C-3”), 43.7 (C-3”).

[0069] Example 2 - Preparation of polydatin hexa-2-chloroacetyl derivative [ka]

[0070] The reaction was carried out under anhydrous conditions and inert atmosphere (nitrogen). Polydatin (2.0 g, 5.13 mmol) was suspended in AcOEt (16 ml) and then monochloroacetic anhydride (8.7 g, 51.2 mmol) was added with stirring at room temperature. The reaction was refluxed and after 45 min dissolution was observed. The reaction mixture was refluxed for an additional 6 h 15 min, then cooled and stirred at room temperature for 24 h. Then 20 mL of H2O was added and the resulting mixture was stirred for 30 min. The formation of a white solid precipitate was observed. The solid was suction filtered and washed with water (20 mL x 3). The white solid was dried under vacuum at room temperature for 48 h to give 4.3 g product (91% yield).

[0071] Characterization: [α] D 25 =-12.00(c=1.0;CHCl3); 11H NMR (500 MHz, CDCl3): δ = 7.51 (doublet, 2H, H-10 and H-14), 7.16 (doublet, 2H, H-11 and H-13), 7.07 (d, J 7,8 = 16.2 Hz, 1H, H-7), 7.04 (s, 1H, H-6), 7.01 (s, 1H, H-8), 6.97 (d, J 7,8 = 16.2 Hz, 1H, H-8), 6.78 (s, 1H, H-2), 5.43 (t, J = 9.3 Hz, 1H, H-3’), 5.37 (t, J = 9.3 Hz, 1H, H-2’), 5.25 (t app, J = 9.3 Hz, 1H, H-4’), 5.20 (d, J = 7.6 Hz, 1H, H-1’), 4.39 - 4.35 (doublet, 2H; H-6a’ and H-6b’), 4.33 - 4.29 (doublet, 4H, 2 x CH2Cl), 4.10 (s, 2H, CH2Cl), 4.07 (s, 2H, CH2Cl), 4.06 - 4.00 (doublet, 5H, 2 x CH2Cl and H-5); 13C NMR (125 MHz, CDCl3): δ = 166.9 (2C, CO at C6' and CO at C3'), 166.2 (CO at C4'), 160.0 (CO at C2'), 165.8 and 165.7 (2C, 2 x CO at Ph), 157.1 (C1), 151.3 (C3), 150.1 (C12), 140.0(C5), 134.7 (C9), 129.9 (C7 or C8) 127.8 (2C, C10 and C14), 127.3 (C7 or C8), 121.5 (2C, C11 and C13), 114.3 (C6), 113.6 (C4), 109.2 (C2), 98.5 (C1'), 73.6 (C3'), 72.1 (C2'), 71.6 (C5'), 69.5 (C4'), 63.2 (C6'), 40.8 (2C, CH2Cl), 40.5 (CH2Cl), 40.3(CH2Cl), 40.2 (CH2Cl), 40.1 (CH2Cl). MS(ESIポジティブ):m / z:851.0[M+H] + .

[0072] Example 3-Preparation of gallic acid からの3,4,5-トリス(2-クロロアセトキシ) benzoic acid

change

[0073] To a suspension of gallic acid (0.5 g; 2.94 mmol) in ethyl acetate (3 ml) was added 2-chloroacetic anhydride (3 g; 17.6 mmol) under stirring at room temperature. The reaction proceeded at room temperature and was complete after 24 h. The reaction mixture was then treated with 0.5 M aqueous HCl (6 ml) and stirred for 0.5 h to decompose excess anhydride. The organic phase was separated and washed with brine (3 times). The organic phase was dried over Na2SO4, filtered and the filtrate was evaporated under vacuum to give an oily residue. The oil was then treated with water to give a white solid. This was dried under vacuum (25°C for 10 h and 50°C for 3 h) to give 0.925 g product as a white solid (2.3 mmol; molar yield 79%). Physicochemical properties: mp 158-159°C; 1 13C NMR (125 MHz, DMSO-d6): δ 165.2 (2C, COCH2Cl at C4 and C6), 164.9 (C1), 164.3 (COCH2Cl at C5), 142.6 (2C, C4 and C6), 137.2 (C5), 129.7 (C2), 122.3 (C3 and C7), 40.9 (2C, COCH2Cl at C4 and C6), 40.3 (COCH2Cl at C5). By mass spectrometry, C 13 The molecular weight was confirmed to correspond to H9Cl3O8. MS (ESI negative) [MH] - m / z:396.6(100%), 398.7(100%), 400.3(45%).

[0074] Example 4 - Preparation of oxiran-2-ylmethyl 3,4,5-tris(oxiran-2-ylmethoxy)benzoate from gallic acid [ka]

[0075] The reaction was carried out under an inert atmosphere (argon) to avoid possible oxidation of gallic acid. To a mixture of 2.0 g (11.76 mmol) gallic acid and TBACl (266 mg, 1.18 mmol, 0.1 molar equivalents), 14.68 mL epichlorohydrin (187.6 mmol, gallic acid / epichlorohydrin molar ratio 1:16) was added and the resulting white suspension was stirred at 100°C. After 30 min, a pale yellow solution was obtained and stirring was continued at 100°C for 6 h. After cooling to room temperature, 15.4 mL 20% w / w NaOH solution (2 molar equivalents / OH) and 266 mg TBACl were added. The resulting white suspension was shaken vigorously at room temperature for 90 min, then 60 mL water was added and extracted with AcOEt (3 x 60 mL). The combined organic phase was washed with saturated NaCl solution (2×80 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure at 70° C. to give 2.50 g crude as a pale yellow oil. This crude was purified by chromatography (flash chromatography). Elution with petroleum ether / AcOEt 20 / 80 gave the desired product (1.01 g; 22% yield). 13 The C-NMR spectral data are consistent with published literature data (Aouf, Chahinez; Tetrahedron 2013, 69(4),1345-1353).

[0076] Example 5 - Preparation of the hepta-chloroacetyl derivative of phlorizin [ka]

[0077] The system was dried under argon, phlorizin (200 mg, 0.51 mmol) and monochloroacetic anhydride (0.7 g, 4.09 mmol) were suspended in AcOEt and the reaction was brought to reflux (80° C.) giving a solution after a few minutes. It was left under stirring at reflux for 3 h and at room temperature overnight. After this period, monochloroacetic anhydride (0.3 g, 1.75 mmol) was added and the reaction was refluxed for 4 h. 6 mL of 0.5 M HCl was added and stirred for 30 min. The organic phase was extracted three times with AcOEt (10 mL×3) and the combined organic phase was washed with saturated NaCl solution (brine, 12 mL). The organic phase was dried over Na2SO4 and concentrated under vacuum. 6 mL of H2O was added to the oily residue and left on ice, after 20 min, an additional 6 mL of water was added. After further washing with NaHCO3 then brine, the organic phase was dried over sodium sulfate and concentrated in vacuo to give the desired product as a white solid residue (345 mg, 79%). mp 145-148 °C; [α] D 25 =-19.00(c=1.0;CHCl3); 1 H NMR (500 MHz, CDCl3): δ = 7.27 (overlapping, 2H, H-11 and H-14), 7.05 (overlapping, 2H, H-12 and H-14), 6.96 (d, J 2,4 = 1.9 Hz, 1H, H-2), 6.82 (d, J 4,2 = 1.9 Hz, 1H, H-4), 5.39 (t app, J 3’,2’ = 9.4 Hz, 1H, H-3'), 5.31 (dd, J 2’,1’ = 7.8, J 2’,3’ = 9.4 Hz, 1H, H-2'), 5.17 (t app, J = 9.6 Hz, 1H, H-4'), 5.07 (d, J = 7.8 Hz, 1H, H- 1'), 4.35 (dd, J 3’,2’ = 2.5, J 6a’,6b’= 12.4 Hz, 1H, H-6a’), 4.33 - 4.28 (doublet, 5H, 2 x CH2Cl and H-6b’), 4.14 (s, 2H, CH2Cl), 4.05 - 4.00 (doublet, 5H, 2 x CH2Cl and H-5), 4.04 (s, 2H, CH2Cl), 3.98 (s, 2H, CH2Cl), 3.13 - 3.04 (m, 2H, H8a and H8b), 3.04 - 2.87 (m, 2H, H9a and H9b); 13 13C NMR (125 MHz, CDCl3): δ = 199.9 (C7), 166.9, 166.7 (2C, CO at C6’ and CO at C3’), 166.2, 160.0 (2C, CO at C4’ and CO at C2’), 165.1 and 165.0 (2C, 2 x CO at Ph), 154.0 (C1), 151.5 (C3), 148.6 (C13), 147.3 (C5), 139.0 (C10), 129.8 (2C, C11 and C15), 123.7 (C6), 121.1 (2C, C12 and C14), 111.5 (C4), 107.7 (C2), 99.3 (C1’), 73.1 (C3’), 71.9 (C5’), 71.3 (C2’), 69.3 (C4’), 63.2 (C6’), 46.0 (C8), 40.9 (CH2Cl), 40.7 (CH2Cl), 40.5 (CH2Cl), 40.3(CH2Cl), 40.2 (2C, CH2Cl), 40.1 (CH2Cl), 28.4 (C9). MS(ESI positive): m / z 973.1[M + H] + 。

[0078] Example 6 - Preparation of penta(chloroacetyl) derivative of chlorogenic acid

Chemical Structure

[0079] The system was dried under argon, chlorogenic acid (200 mg, 0.56 mmol) and monochloroacetic anhydride (0.677 g, 3.96 mmol) were suspended in AcOEt, the reaction was refluxed (bath 80 °C) to give a solution after 15 min. The mixture was left under stirring for 6 h, then 6 mL of 0.5 M HCl was added and the mixture was stirred for another 30 min. The reaction mixture was extracted three times with AcOEt (10 mL x 3) and the pooled organic phase was washed with saturated NaCl solution (12 mL). The organic phase was dried over Na2SO4 and concentrated under vacuum. 6 mL of H2O was added to the oily residue and cooled to 0 °C to give the desired product as a powder (224 mg, 40% yield). mp 118-122 °C; [α] D 25 =-21.00(c=1.0;CHCl3); 1 H NMR (500 MHz, CDCl3): δ = 7.81 (d, J 5',9' = 1.8 Hz, 1H, H-5'), 7.74 (dd, J 9',5' = 1.8, J 9',8' = 8.8 Hz, 1H, H-9'), 7.63 (d, J 3',2' = 16.0 Hz, 1H, H-3'), 7.43 (d, J 8',9' = 8.8 Hz, 1H, H-8'), 6.62 (d, J 2',3' = 16.0 Hz, 1H, H-2'), 5.55-5.52 (m, 1H, H-6), 5.42-5.36 (overlapping, 2H, H-4 and H-5), 7.78-7.71 (overlapping, 4H, 2 x CH2Cl), 4.60-4.23 (overlapping, 6H, 3 x CH2Cl), 2.62 (dd, J 7a,6 = 3.4, J 7a,7b = 16.0 Hz, 1H, H-7a), 2.56-2.51 (overlapping, 2H, H-3a and H-7b), 2.19 (dd, J 3b,4 = 9.9, J 3b,3a = 12.9 Hz, 1H, H-3b); 13C NMR (125 MHz, CDCl3): δ = 170.3 (C1), 166.9, 166.4, 166.1, 165.4, 165.3, 164.8 (6C, C1', CO at C2, CO at C6, CO at C5, CO at C6', CO at C7'), 143.2 (C3'), 142.7 (C7'), 141.6 (C6'), 133.2 (4'), 127.7 (9'), 124.0 (8'), 123.0 (5'), 118.9 (C2'), 80.0 (2), 72.1 (C4), 69.5 (C6), 66.5 (C5), 41.4 (CH2C), 40.9 (CH2Cl), 40.8 (CH2Cl), 40.7 (2C, 2 x CH2Cl), 35.8 (C3), 31.3 (C7);MS(ESI negative): m / z 734.9(100%)[MH] - , 737.0(80%)[MH] - , 733.1(65%)[MH] - .

[0080] A general procedure for cross-linking using glycidated compounds: Cross-linking of hyaluronic acid with glycidated polydatin 100 mg hyaluronic acid sodium salt (HANa) is added to 2 or 4 mL 0.25 M NaOH (Col A Table 1), the mixture is vortexed and left at rt for 15 min. Then, 0, 25, 50 or 78.2 mg (Col C Table 1) polydatin diglycidylates (PO DG ) is added. PO PO DG The molar ratio of HANa to HANa (repeating unit) is given in Col F. The mixture is heated at 50° C. for 2 h under stirring. It is then neutralized (pH approx. 7) with 1 M HCl (approx. 0.95 mL for 4 mL 0.25 M NaOH and approx. 0.4 mL for 2 mL 0.25 M NaOH). The polymer is precipitated by adding 5 or 10 mL EtOH (Col G), vortexed for 2 min, and centrifuged at 4000 g for 10 min. The supernatant (surn1) is analyzed by UV spectrophotometry to determine the amount of PO present. DG(PO unbound to hyaluronic acid and crosslinked with hyaluronic acid) DG The concentration of 100 mg of 10 ...

[0081] [Table 1]

[0082] The gel precipitated during centrifugation is washed by vortexing with 5 mL of EtOH / HO (4:1). Centrifuge as before to obtain the supernatant (surn2). The wash is repeated multiple times with 4 mL of EtOH to obtain the corresponding supernatant. This supernatant is analyzed by UV to determine the amount of unbound PO present. DG The amount (% in Col M) is measured. If the supernatant after washing appeared slightly opaque, indicating a possible colloidal dispersion unsuitable for UV analysis, 30 mg finely ground NaCl was added and centrifuged again at 4000 g for 10 min, then UV analyzed. After the final wash, the gel was hydrated overnight with 5 mL H2O. It was then frozen at -20°C and lyophilized. The results of the UV analysis of the supernatant are shown in Table 2.

[0083] [Table 2]

[0084] [Table 3]

[0085] [Table 4]

[0086] HA and PO DG PO in the crosslinking reaction with DG UV analysis used to quantify Readings were taken at λ=322 nm and for surns 1 and 2 readings were taken against a 4:1 mixture of EtOH / HO and for surns 3 onwards readings were taken against EtOH using calibration curves reported below (Figures A-C).

[0087] General procedure for cross-linking reactions using chloroacetylated compounds: Cross-linking of hyaluronic acid with polydatin hexachloroacetyl derivative (perchloroacetylated polydatin) Hyaluronic acid-sodium salt (HA) of medium molecular weight (MMW, 500-750 kDa) or low molecular weight (LMW, 8-15 kDa) was used. Both cross-linking reactions were carried out using hyaluronic acid at concentrations of 25 and 50 mg / mL. The tests carried out are reported in detail below under "General Procedure" and in Table 1. The supernatant (surn) after washing and centrifugation was analyzed to assess the total amount (mg) of perchloroacetylated polydatin (POca) present, which indicates the amount of unbound cross-linker, and by difference, the cross-linked POca on HA was assessed. The results are shown in Table 2.

[0088] General Procedure 100 mg hyaluronic acid sodium salt (HA) is added to 2 or 4 mL H2O, the mixture is vortexed and left at 70°C for 15 min. Then a solution of 0, 21 or 42 mg perchloroacetylated polydatin (POca) dissolved in 2 or 4 mL DMSO is added. The molar ratio of POca:HANa (repeating units) is given in column F (1:5 or 1:10). The mixture is heated at 70°C under stirring for 15 h. Then it is cooled to room temperature and the polymer is precipitated by adding 5 or 10 mL EtOH or 10 mL EtOH+2CH3CN (Col I), vortexed for 2 min and centrifuged at 4000g for 10 min. The supernatant (surn1) is analyzed by UV spectrophotometer to determine the mg POca present. The gel is washed by vortexing with 5 mL of EtOH / H2O 4:1 or directly with 4 mL of CN3CN. Centrifuge as before and obtain the supernatant (surn2). This wash is repeated multiple times with 4 mL of EtOH to obtain the corresponding supernatant. This supernatant is analyzed by UV to measure the amount of unbound POca present (% in column P of Table 1'). In some cases, it is washed with 4 mL of CH3CN where POca is more soluble. If the wash supernatant appears slightly opaque, indicating a possible colloidal dispersion unsuitable for UV analysis, 30 mg of finely ground NaCl is added and centrifuged again at 4000g for 10 minutes, then analyzed by UV. After the last wash, the gel is hydrated overnight with 5 or 10 or 20 mL of H2O. It is then frozen in a freezer at -20°C and lyophilized.

[0089] The results of quantitative UV analysis of the supernatant for POca are shown in Table 2'.

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7] TIFF2025502169000025.tif98161

[0093] HA and PO ca PO in the crosslinking reaction with ca UV analysis used to quantify Readings were taken at λ=313 nm and for surn1 and 2 readings were taken against a 4:1 EtOH / H2O mixture, for surn3 onwards against EtOH, using calibration curves shown below, or in the cases indicated in the table against EtOH:CH3CN 10:2 or CH3CN (see Figures A'-E').

[0094] HPLC_SEC analysis of the obtained fillers: PO crosslinked with hyaluronic acid DG Hyaluronic acid, diglycidylated polydatin and packing materials of batch numbers PR019, PR015 and PR008 were analyzed by size exclusion chromatography (SEC) using a HPLC Column BioSep (5um SEC-s3000 290A) with HO as mobile phase; flow rate 1mL / min. Medium molecular weight HA was injected (1uL, 5uL, 10uL, 25uL) at a concentration of 0.5mg / mL and the spectrum was recorded at 322nm (at this wavelength, PO DG The absorbance was taken at 220 nm and 215 nm (where the absorbance of HA is maximal and that of HA is almost nonexistent) and at 220 nm and 215 nm (where the absorbance of HA is maximal). 25 uL of these solutions were injected, followed by injections of several samples that had been appropriately solubilized and prepared at a concentration of 0.5 mg / mL.

[0095] [Table 8]

[0096] From the results reported in Table 1'', POs of batch numbers PR015, PR008 and PR019 DGIt was confirmed that cross-linked derivatives of hyaluronan by 100% polydatin contained fractional amounts of polydatin, further supporting the observed percentage of polydatin present in the filler reported in Table 2.

[0097] NMR evaluation of the degree of reticulation of fillers obtained by reaction of hyaluronic acid (various molecular weights) with polydatin hexachloroacetyl derivative (POca). The reticulation of fillers obtained by reaction of hyaluronic acid (various molecular weights) with polydatin hexachloroacetyl derivatives (POca) was also investigated using NMR spectroscopy of the fillers after hydrolysis in deuterated sodium hydroxide (NaOD). The results showed that the CH3-related signals (d1.80±0.5 ppm) of N-acetylglucosamine of hyaluronic acid (HA) and the trans (J) double bond of polydatin (PO) were strongly correlated with each other. H,H =16.0) (6.93±0.5ppm e 6.67±0.5ppm) (for packing material PR032D) 1 The results are expressed as the ratio between the percentage of polydatin bound to hyaluronic acid and the percentage of polydatin bound to hyaluronic acid (shown in H-NMR spectrum 1). The data reported in Table 2' further support the results of the percentage of polydatin bound to hyaluronic acid reported in Table 1'.

[0098] From the NMR data, The resulting filler is crosslinked with highly acetylated polydatin (PO) since strong basic hydrolysis releases PO and HA from the filler in relative ratios (% bound PO) consistent with those reported in Table 1'. A higher degree of reticulation of these fillers is obtained when using a relative molar ratio between POca and HA (the molar ratio of HA is considered to refer to the repeating disaccharide unit of sodium glucuronic acid and N-acetylglucosamine) of 1:5 versus 1:10, both for MMW and LMW hyaluronic acids; The hypothesis is confirmed.

[0099] Materials and Methods 3 mg of bulk material was solubilized in 0.7 mL of 0.5 M NaOD (in some experiments samples were also solubilized in 0.25 M NaOD with no substantial difference. When working in more dilute conditions solubilization times were several minutes longer). NaOD was prepared at a concentration of 1 M by solubilizing Na metal in D2O in a suitable anhydrous container on ice under argon (to avoid possible interference from non-deuterated water). The 1 M NaOD solution was then diluted 1:1 v / v with D2O to give a 0.5 M final solution or 1:4 v / v to give a 0.25 M final solution. 1 H-NMR (500 MHz) spectra were acquired with at least 16 scans. The spectra of the polymers were compared to those of medium molecular weight hyaluronic acid alone in 0.25M or 0.5M NaOD, PR030A in 0.5M NaOD (reactions were performed without the addition of perchloroacetylated polydatin; see Table 1'), polydatin alone or perchloroacetylated polydatin alone in 0.5M NaOD (the latter two are clearly identical since perchloroacetylated polydatin in 0.5M NaOD is hydrolyzed to free polydatin).

[0100] The relationship between the CH3-related signals of N-acetylglucosamine of hyaluronic acid and the aromatic protons of polydatin was evaluated.

[0101] 1H-NMR spectra 1 are reported in FIG. F for HA in NaOD solution, polydatin in NaOD solution, and the filler PR032D, from top to bottom.

[0102] result [Table 9]

Claims

1. Crosslinked with activated derivatives of plant polyphenols selected from polydatin, gallic acid, chlorogenic acid, ellagic acid and phloridzin, Hyaluronic acid.

2. 2. The crosslinked hyaluronic acid of claim 1, wherein the activated derivative of the plant polyphenol is a glycidyl ether (oxiran-2-yl-methyl ether) or a 2-chloroacetyl ester.

3. 3. The crosslinked hyaluronic acid of claim 2, wherein the activated derivative is selected from the group consisting of diglycidated polydatin, hexa-chloroacetyl polydatin, a mixture of mono-, di-, tri-, and tetra-2-chloroacetyl polydatin esters, 3,4,5-tris(2-chloroacetyl)ester of gallic acid, oxiran-2-ylmethyl 3,4,5-tris(oxiran-2-ylmethoxy)benzoate, the hepta-2-chloroacetyl derivative of phlorizin, the penta-chloroacetyl derivative of chlorogenic acid, oxiran-2-ylmethyl 3,4,5-tris(oxiran-2-ylmethoxy)benzoate, and 3,4,5-tris(2-chloroacetoxy)benzoic acid.

4. 4. The crosslinked hyaluronic acid of claim 3, wherein said activated derivative is a glycidyl ether or 2-chloroacetyl ester of polydatin or gallic acid.

5. 4. The crosslinked hyaluronic acid of claim 3, wherein the relative molar ratios of mono-, di-, tri-, and tetra-2-chloroacetylpolydatin esters are 17±3.4% / 44.3±8.8% / 17.7±3.6% / 1.9±0.4%.

6. 2. The crosslinked hyaluronic acid of claim 1, obtained from hyaluronic acid having an average molecular weight Mn of 80 to 110 kDa.

7. 2. The crosslinked hyaluronic acid of claim 1, obtained from hyaluronic acid having an average molecular weight Mn of 250 to 450 kDa.

8. 2. The crosslinked hyaluronic acid of claim 1, obtained from hyaluronic acid having an average molecular weight Mn of 1.5 to 3.0 MDa.

9. 2. The crosslinked hyaluronic acid of claim 1, having a degree of crosslinking such that it has a ratio of viscous modulus (G") to elastic modulus (G') of less than 1.

0.

10. reacting crosslinked hyaluronic acid in aqueous solution with activated derivatives of plant polyphenols in dimethyl sulfoxide solution at a temperature ranging from 30 to 80°C; A method for producing crosslinked hyaluronic acid according to any one of claims 1 to 9.

11. 11. The method of claim 10, wherein the molar ratio of activated polyphenol derivative to hyaluronic acid is in the range of 1:1 to 1:

10.

12. 10. A composition comprising cross-linked hyaluronic acid according to any one of claims 1 to 9, in the form of a sterile gel. A composition for intradermal or intraarticular injection.

13. 13. The injectable composition of claim 12, comprising a mixture of cross-linked hyaluronic acid of various molecular weights.

14. 14. The injectable composition of claim 13, comprising the cross-linked hyaluronic acid of claim 6.

15. 13. The injectable composition of claim 12, comprising 1 mg / ml to 50 mg / ml of cross-linked hyaluronic acid, optionally in the presence of an anesthetic agent at a concentration of 0.1 to 0.4% weight / volume.

16. formula 【Chemistry 1】 The compound represented by the formula: (2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-(3-(oxiran-2-ylmethoxy)-5-((E)-4-(oxiran-2-ylmethoxy)styryl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol.

17. formula 【Chemistry 2】 Polydatin hexa-2 chloroacetyl represented by the formula:

18. formula 【Transformation 3】 Hepta-2-chloroacetylphlorizin represented by the formula:

19. formula 【Chemistry 4】 Penta-2-chloroacetylchlorogenic acid represented by the formula:

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