Composition based on at least two glycosaminoglycans

Abstract

The present invention relates to a hyaluronic acid-based aqueous gel comprising crosslinked hyaluronic acid and 30-70% by mass of water-soluble hyaluronic acid relative to the total mass of hyaluronic acid. The present invention relates to a process for preparing an aqueous gel comprising at least one crosslinked hyaluronic acid and at least one non-crosslinked hyaluronic acid. The present invention also relates to an aqueous gel obtainable by the process composition, to a cosmetic and / or dermatological composition comprising said aqueous gel, and to a kit comprising a pre-filled syringe comprising said aqueous gel or said composition. These gels and compositions are particularly useful for treating changes in the surface appearance of the skin and / or for augmenting and / or filling soft tissues.

Description

Technical Field

[0001] This invention relates to an aqueous gel based on hyaluronic acid, the aqueous gel comprising at least cross-linked hyaluronic acid and at least one type of water-soluble hyaluronic acid. The invention further relates to a method for preparing a supplemented composition based on glycosaminoglycans, particularly hyaluronic acid, the supplemented composition being soft, stable, sterile, and injectable. The aqueous gel and composition thus obtained comprise at least two types of glycosaminoglycans, including at least one cross-linked glycosaminoglycan prepared from a non-cross-linked high-molar-weight glycosaminoglycan, particularly at least one cross-linked hyaluronic acid, and at least one non-cross-linked low-molar-weight glycosaminoglycan, particularly at least one cross-linked hyaluronic acid and at least one non-cross-linked low-molar-weight hyaluronic acid prepared from at least one non-cross-linked high-molar-weight hyaluronic acid. Background Technology

[0002] Polysaccharides such as glycosaminoglycans are widely used in the biomedical field. Specifically, most products marketed for aesthetic and / or cosmetic applications are based on hyaluronic acid.

[0003] Unmodified hyaluronic acid gels are attractive for improving skin quality due to their excellent biocompatibility. However, they degrade very rapidly after implantation. Therefore, the effects of such gels only last for a short time.

[0004] To increase its durability in vivo and resistance to degradation, hyaluronic acid is typically modified by cross-linking. Cross-linked hyaluronic acid gels can be obtained through various preparation methods. Polysaccharides such as hyaluronic acid can be cross-linked with different cross-linking agents such as epoxy reagents, aldehydes such as glutaraldehyde, divinyl sulfone (DVS), or polyamines. Currently, the most commonly used reagent for cross-linking hyaluronic acid is the epoxy reagent 1,4-butanediol diglycidyl ether (BDDE).

[0005] These cross-linking methods require two main steps: polysaccharide hydration and cross-linking.

[0006] Commercial products based on hyaluronic acid prepared in this way typically contain, and depending on their indications, fractions of cross-linked hyaluronic acid and degrees of modification ranging from 1% to 10%.

[0007] Nevertheless, increased cross-linking of hyaluronic acid leads to the preparation of more chemically modified gels, which may therefore have lower biocompatibility. On the other hand, the mechanical properties of these compositions are limited, resulting in very elastic and / or fragile final products.

[0008] However, for reasons of product safety and performance, there is interest in obtaining durable in vivo compositions containing biocompatible polysaccharides that are as natural as possible and as minimally modified and therefore as minimally crosslinked as possible.

[0009] Besides the degree of crosslinking, the concentration of glycosaminoglycans used is a key parameter determining the mechanical and resistance properties of the composition. Therefore, an alternative approach to increasing the in vivo durability and degradation resistance of polysaccharide-based compositions is to increase their total concentration in the final composition.

[0010] However, in addition to increasing its resistance and durability, the increased total concentration of the glycosaminoglycans leads to thickening of the composition, with a significant increase in viscosity. This increase in viscosity therefore makes the gel difficult to inject and prevents it from naturally integrating into the tissue. In particular, it is known that an increase in the total concentration of hyaluronic acid is a cause of post-injection papules.

[0011] Therefore, for all these reasons, most commercially available cross-linked hyaluronic acid products to date have a total hyaluronic acid concentration of only about 25 or 26 mg / g. However, while potentially useful for dermal filler applications, such gels are not suitable for skin regeneration and cosmetic improvement applications where mechanical filling is not the primary objective and injections are required in the superficial layers of the skin. For the latter type of injection, it is desirable to prepare gels with lower elastic (G') and viscous (G") moduli, i.e., soft, easy to inject, and easy to diffuse into the tissue.

[0012] In the ongoing pursuit of increasing the resistance and durability of glycosaminoglycan-based products (such as hyaluronic acid), various compounds with protective properties against hyaluronic acid have been proposed. Therefore, to address the thermal degradation of hyaluronic acid, application WO2014 / 032804 proposes the addition of magnesium ascorbate phosphate, application WO2013 / 186493 proposes the addition of sucrose octasulfate, and application WO2007 / 077399 proposes the addition of glycerol. However, the addition of such stabilizing compounds increases uncertainty regarding the biocompatibility of the product.

[0013] Therefore, there is still a need for sterile gels based on glycosaminoglycans and, more particularly, hyaluronic acid, which are soft, easy to inject and readily diffuse into tissues, while also possessing sufficient durability and effectiveness for skin regeneration and / or appearance improvement.

[0014] Specifically, there remains a need to increase the resistance and durability of glycosaminoglycan-based compositions (such as hyaluronic acid) to overcome the problems of the prior art. This invention addresses these needs. Invention Overview

[0016] According to a first aspect, the present invention relates to an aqueous gel based on hyaluronic acid, comprising cross-linked hyaluronic acid, characterized in that the gel comprises at least:

[0017] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.02 MDa to 0.30 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 30% to 70% by mass; and

[0018] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa is called "sHA HMW".

[0019] Furthermore, the water-soluble hyaluronic acid is characterized by having a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0020] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min.

[0021] This article describes a method for preparing an aqueous gel comprising at least one cross-linked glycosaminoglycan and at least one non-cross-linked glycosaminoglycan, the method comprising at least the following steps:

[0022] a) Obtain an aqueous solution comprising at least one cross-linked glycosaminoglycan, referred to as "cross-linked glycosaminoglycan HMW", which is formed from a non-cross-linked glycosaminoglycan having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa and from a cross-linking agent;

[0023] b) Obtaining an aqueous solution comprising at least one non-crosslinked glycosaminoglycan having a molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, and even more preferably 0.08 MDa to 0.10 MDa; and,

[0024] c) Forming a homogeneous mixture of all or part of the solutions from steps a) and b).

[0025] According to a second aspect, the present invention relates to a method for preparing an aqueous gel, the aqueous gel comprising at least one cross-linked hyaluronic acid and at least one non-cross-linked hyaluronic acid, the method comprising at least the following steps:

[0026] a) Obtain an aqueous solution comprising at least one cross-linked hyaluronic acid, referred to as "cross-linked HAHMW", which is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, and a cross-linking agent;

[0027] b) Obtain an aqueous solution comprising at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW," having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa; and,

[0028] c) Forming a homogeneous mixture of all or part of the solutions described in steps a) and b);

[0029] The characteristic is that it contains hyaluronic acid in a mass ratio of "non-crosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0030] Unexpectedly, the inventors have discovered that supplementing a composition containing at least one cross-linked glycosaminoglycan (which is prepared from at least one non-cross-linked high-molar-mass glycosaminoglycan) with at least one non-cross-linked low-molar-mass glycosaminoglycan, and in particular supplementing a composition containing at least one cross-linked hyaluronic acid (which is prepared from at least one non-cross-linked high-molar-mass hyaluronic acid) with at least one non-cross-linked low-molar-mass hyaluronic acid, enhances the resistance of the final composition.

[0031] As shown in the following examples, the result is:

[0032] Improved resistance to degradation compared to the same composition which does not contain added non-crosslinked low-molar mass glycosaminoglycans, and more specifically, does not contain added non-crosslinked low-molar mass hyaluronic acid; and

[0033] It exhibits better resistance to degradation compared to compositions having the same total concentration of glycosaminoglycans, more specifically hyaluronic acid, and the same degree of crosslinking of fractions of crosslinked glycosaminoglycans, more specifically crosslinked hyaluronic acid.

[0034] Due to this improved resistance to degradation, the compositions according to the invention are more heat-resistant for sterilization. Therefore, among two sterile and injectable compositions having similar rheological properties before sterilization, one prepared according to the invention and the other according to the teachings of the prior art, the former exhibits superior rheological properties after sterilization compared to the latter.

[0035] Due to their increased resistance to degradation, the compositions according to the invention are advantageously able to withstand varying temperatures, particularly during their transport and storage.

[0036] Furthermore, supplementing with at least one non-crosslinked low-molar-mass glycosaminoglycan, particularly at least one non-crosslinked low-molar-mass hyaluronic acid, makes it possible to prepare soft, injectable, and easily diffused compositions with an increased total glycosaminoglycan concentration, thus exhibiting one or more significant biomechanical and / or biological effects on skin physiology.

[0037] Furthermore, the use of the composition prepared according to the present invention allows for the simultaneous application of glycosaminoglycans, particularly hyaluronic acid, with different molar masses, which contributes to the diversification of the physiological effects of glycosaminoglycans, particularly hyaluronic acid, at the treatment site.

[0038] According to a third aspect, the present invention relates to aqueous gels of glycosaminoglycans, particularly hyaluronic acid, that can be obtained by the method according to the invention.

[0039] According to a fourth aspect, the present invention relates to a cosmetic and / or dermatological composition comprising an aqueous gel as defined herein.

[0040] The present invention also relates to compositions obtained according to the method of the present invention.

[0041] The present invention also relates to aqueous gels or compositions according to the present invention for the prevention and / or treatment of changes in the viscoelasticity or biomechanical properties of the skin.

[0042] The present invention also relates to aqueous gels or dermatological compositions according to the invention for the prevention and / or treatment of changes in the surface appearance of the skin, such changes being induced, for example, by external factors such as stress, air pollution, tobacco, or prolonged exposure to ultraviolet (UV) radiation.

[0043] Preferably, the present invention relates to an aqueous gel according to the invention or a dermatological composition according to the invention for soft tissue enlargement and / or filling.

[0044] It also relates to the non-therapeutic cosmetic use of aqueous gels or cosmetic compositions as defined in this invention for the prevention and / or treatment of changes in the surface appearance of the skin (e.g., signs of aging skin).

[0045] It also relates to the non-therapeutic cosmetic use of aqueous gels or cosmetic compositions as defined in this invention for enlarging and / or filling soft tissue, particularly for filling wrinkles. Brief description of the attached diagram

[0047] [ Figure 1 ] Figure 1 This is a graphical representation of the loss of elastic modulus (G′) (ΔG′(%)) of samples 1-1 to 1-3 after autoclaving, the concentration of non-crosslinked low molar mass hyaluronic acid, and the concentration of crosslinked and non-crosslinked high molar mass hyaluronic acid.

[0048] [ Figure 2 ] Figure 2 The figure shows the loss of elastic modulus (G′) (ΔG′(%)) after autoclaving for samples 3-5 without added non-crosslinked hyaluronic acid and samples 3-6 to 3-9 containing 0.04 MDa, 0.10 MDa, 0.20 MDa and 1.50 MDa molar masses of added non-crosslinked hyaluronic acid, respectively.

[0049] [ Figure 3 ] Figure 3 The figure shows the percentage improvement in elastic modulus (G′) loss after autoclaving for samples 3-5 without added non-crosslinked hyaluronic acid and samples 3-6 to 3-9 containing 0.04 MDa, 0.10 MDa, 0.20 MDa, and 1.50 MDa molar masses of added non-crosslinked hyaluronic acid, respectively.

[0050] [ Figure 4 ] Figure 4 This is a graphical representation of the loss of elastic modulus (G′) (ΔG′(%)) of samples 4-1 (without supplement) and 4-2 to 4-4 after autoclaving, the concentration of non-crosslinked low molar mass hyaluronic acid, and the concentration of crosslinked and non-crosslinked high molar mass hyaluronic acid.

[0051] [ Figure 5 ] Figure 5 The figure shows the difference (ΔG′(%)) in elastic modulus (G′) before and after hyaluronidase treatment for sample 6-1 without added non-crosslinked hyaluronic acid and samples 6-2 and 6-3 containing 0.10 MDa and 1.50 MDa molar masses of added non-crosslinked hyaluronic acid, respectively.

[0052] Detailed description

[0053] According to the definition of the terminology in this invention:

[0054] Glycosaminoglycans are defined as long chains composed of repeating disaccharide units, where one of the two carbohydrate residues is an amino carbohydrate (N-acetylglucosamine or N-acetylgalactosamine), and the second is a uronic acid (glucuronic acid or iduronic acid) or galactose. Glycosaminoglycans include hyaluronic acid, heparosan, chondroitin sulfate, dermatan sulfate, and keratin sulfate. A particularly preferred glycosaminoglycan is hyaluronic acid.

[0055] "Hyaluronic acid" refers to hyaluronic acid or hyaluronan and its derivatives. Therefore, the term "hyaluronic acid" also includes hyaluronic acid salts such as sodium hyaluronate and chemically modified hyaluronic acid, for example, through oxidation, reduction, deacetylation, sulfation, or amidation. Hyaluronic acid is a linear polysaccharide with alternating D-glucuronic acid and N-acetyl-D-glucosamine units linked by alternating β-1,3-glycosidic and β-1,4-glycosidic bonds.

[0056]

[0057] It exists naturally in various tissues of the human body and is degraded by hyaluronidase, an enzyme also present in the body, and / or through oxidation mechanisms.

[0058] - "Water-soluble hyaluronic acid" or "soluble hyaluronic acid" or "extractable hyaluronic acid" (also known as "sHA") refers to hyaluronic acid that can be extracted from a hyaluronic acid-based gel when it swells in an excess of aqueous medium. The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min. In practice, those skilled in the art would appropriately adjust the concentration of the gel in the mobile phase during the dilution step and the injection volume in the SEC as the supernatant composition changes to maximize the signal-to-noise ratio and avoid column overload.

[0059] Conversely, "insoluble hyaluronic acid" or "non-extractable hyaluronic acid" (also known as "nsHA") refers to hyaluronic acid that cannot be extracted from a hyaluronic acid-based gel when it swells in an excess of aqueous medium.

[0060] The “mass-average molar mass” (expressed in g / mol or Da and abbreviated as Mw) for glycosaminoglycans, particularly hyaluronic acid, refers to the mass-average molar mass of the glycosaminoglycan molecules used. The mass-average molar mass of glycosaminoglycans can be determined by various methods known to those skilled in the art, such as by capillary electrophoresis, by size exclusion chromatography, or by measuring intrinsic viscosity. In this document, the terms “molar mass,” “average molar mass,” “mass-average molar mass,” or “mass-molar mass” are used interchangeably.

[0061] - Glycosaminoglycans, especially hyaluronic acid, have an "intrinsic viscosity" ([η]) (in mL / g or m 3 The intrinsic viscosity (in kg / sq.) represents the contribution of glycosaminoglycans to the solution viscosity. It can be measured by flow in a capillary viscometer. It represents the hydrodynamic specific volume of the polymer. The lower the intrinsic viscosity of a glycosaminoglycan, the lower its molar mass. For a given glycosaminoglycan, the intrinsic viscosity is related to the molar mass by an empirical relationship, namely the Mark-Houwink relationship, which can be understood by anyone skilled in the art, and its formula is as follows:

[0062] [η]=K x Mw α

[0063] in:

[0064] -K is a constant function of the polymer under study, the solvent used, and the temperature. In particular, K is related to the chain size of the polymer under study.

[0065] -α is a constant function of the polymer under study, the solvent used, and the temperature; in particular, α is related to the conformation of the polymer under study; and,

[0066] -Mw is the average molar mass of the polymer, expressed in Daltons (Da).

[0067] - "Low molar mass glycosaminoglycans", also known as "glycosaminoglycans LMW" or "LMW glycosaminoglycans", refer to glycosaminoglycans used as raw materials that have a mass-average molar mass in the range of 0.04 to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, and even more preferably 0.08 MDa to 0.10 MDa.

[0068] - "Low molar mass hyaluronic acid," also known as "hyaluronic acid LMW," "HA LMW," "LMW hyaluronic acid," or "LMW HA," refers to hyaluronic acid used as a raw material having a molar mass in the range of 0.04 to 0.30 MDa, preferably 0.08 to 0.20 MDa, and more preferably 0.08 to 0.15 MDa. This corresponds to hyaluronic acid with low intrinsic viscosity, i.e., having a molar mass in the range of 0.10 to 0.8 MDa as determined by the method specified in the European Pharmacopoeia (01 / 2017:1472). 3 / kg, preferably 0.2 to 0.5m 3 / kg, more preferably 0.2 to 0.4m 3 Inherent viscosity in the range of / kg.

[0069] - "Low molar mass water-soluble hyaluronic acid" or "low molar mass soluble hyaluronic acid," also known as "sHALMW" or "LMW sHA," refers to soluble hyaluronic acid having a molar mass in the range of 0.02 MDa to 0.30 MDa, preferably 0.04 MDa to 0.20 MDa, more preferably 0.04 MDa to 0.15 MDa, as measured on the final gel according to the method detailed in claim 1. That is, by adjusting the aqueous gel at a pH of 150 nM sodium nitrate containing 0.02% NaN3. 7.2 The solution was diluted internally at 25°C for 5 days and centrifuged at 4400 rpm for 10 minutes. The diluted gel was then filtered through a 0.45 mm filter to obtain the filtrate. The filtrate was analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the sodium nitrate mobile phase at pH 7.2 was 0.3 mL / min. In practice, those skilled in the art will appropriately adjust the gel concentration in the mobile phase during the dilution step and the injection volume in the SEC as the supernatant composition changes to maximize the signal-to-noise ratio and avoid column overload.

[0070] - "High molar mass glycosaminoglycans", also known as "glycosaminoglycans HMW" or "HMW glycosaminoglycans", refer to glycosaminoglycans used as raw materials that have a molar mass greater than 0.50 MDa, preferably greater than 1.00 MDa, and more preferably in the range of 1.00 MDa to 4.00 MDa.

[0071] - "High molar mass hyaluronic acid," also known as "HMW hyaluronic acid," "HA HMW," "HMW hyaluronic acid," or "HMW HA," refers to hyaluronic acid used as a raw material that has a molar mass greater than 0.50 MDa, preferably greater than 1.00 MDa, and more preferably in the range of 1.00 MDa to 4.00 MDa. This corresponds to hyaluronic acid with high intrinsic viscosity, i.e., having a molar mass greater than 0.8 MDa as determined by the method specified in the European Pharmacopoeia (01 / 2017:1472). 3 / kg, preferably greater than 1.2m 3 / kg, more preferably 1.2m 3 / kg to 3.5m 3 Inherent viscosity within the range of / kg.

[0072] - "High molar mass soluble hyaluronic acid" or "high molar mass water-soluble hyaluronic acid," also known as "sHAHMW" or "HMW sHA," refers to soluble hyaluronic acid having a molar mass greater than 0.30 MDa, preferably greater than 0.30 MDa and less than or equal to 4.00 MDa, as measured on the final gel according to the method detailed in claim 1. That is, by adjusting the aqueous gel at a pH of 150 nM sodium nitrate containing 0.02% NaN3. 7.2 The solution was diluted internally at 25°C for 5 days and centrifuged at 4400 rpm for 10 minutes. The diluted gel was then filtered through a 0.45 mm filter to obtain the filtrate. The filtrate was analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the sodium nitrate mobile phase at pH 7.2 was 0.3 mL / min. In practice, those skilled in the art will appropriately adjust the gel concentration in the mobile phase during the dilution step and the injection volume in the SEC as the supernatant composition changes to maximize the signal-to-noise ratio and avoid column overload.

[0073] - "Cross-linked glycosaminoglycans" refer to glycosaminoglycans that have been modified with a cross-linking agent during the cross-linking reaction. Conversely, "non-cross-linked glycosaminoglycans" refer to glycosaminoglycans that have not been modified with a cross-linking agent and therefore have not undergone a cross-linking reaction.

[0074] "Cross-linked hyaluronic acid" refers to hyaluronic acid that has been modified with a cross-linking agent during the cross-linking reaction. Conversely, "non-cross-linked hyaluronic acid" refers to hyaluronic acid that has not been modified with a cross-linking agent and therefore has not undergone a cross-linking reaction.

[0075] "Non-crosslinked" and "uncrosslinked" are synonymous terms and can be used interchangeably in this article.

[0076] The term "glycosaminoglycan HMW" or "HMW glycosaminoglycan" refers to the "crosslinked glycosaminoglycan HMW" component and the "non-crosslinked glycosaminoglycan HMW" component in an aqueous gel, if present.

[0077] - The designation "Hyaluronic Acid HMW", also known as "HAHMW" or "HMW HA", indicates the "crosslinked hyaluronic acid HMW" (also known as "crosslinked HA HMW" or "crosslinked HMW HA") component and, if applicable, the "non-crosslinked hyaluronic acid HMW" (also known as "non-crosslinked HAHMW" or "non-crosslinked HMW HA") component in the aqueous gel, if present.

[0078] In the preparation of "crosslinked glycosaminoglycans," particularly "crosslinked hyaluronic acid," the "crosslinking rate" corresponds to the ratio between the mass (or weight) of the crosslinking agent and the mass of the glycosaminoglycans, particularly hyaluronic acid, used to prepare the crosslinked glycosaminoglycans, particularly the crosslinked hyaluronic acid. It is expressed as a percentage.

[0079] Cross-linked glycosaminoglycans and compositions containing such cross-linked glycosaminoglycans, particularly cross-linked hyaluronic acid, can be defined by their “degree of modification” (MOD). This degree of modification corresponds to the molar amount of a modifier linked to the glycosaminoglycan via one or more of its ends, expressed in terms of disaccharide repeating units of glycosaminoglycan per 100 moles in the composition, particularly in terms of disaccharide repeating units of hyaluronic acid per 100 moles in the composition. A reference method for determining this degree of modification is nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 The degree of modification was determined by the molar ratio of the crosslinking agent signal to the glycosaminoglycan disaccharide unit signal (glycosaminoglycans that have participated in or have not participated in crosslinking).

[0080] "Cross-linked high molar mass (HMW) glycosaminoglycan" or "cross-linked high molar mass (HMW) glycosaminoglycan" refers to a cross-linked glycosaminoglycan prepared from a non-cross-linked high molar mass glycosaminoglycan. Preferably, the cross-linked HMW glycosaminoglycan has a cross-linking rate of less than or equal to 8%, preferably less than or equal to 5%, and more preferably less than 2%.

[0081] - "Cross-linked high molar mass (HMW) hyaluronic acid" or "cross-linked high molar mass (HMW) hyaluronic acid" refers to cross-linked hyaluronic acid prepared from non-cross-linked high molar mass hyaluronic acid. Preferably, the cross-linked HMW hyaluronic acid has a cross-linking rate of less than or equal to 8%, preferably less than or equal to 5%, and more preferably less than 2%.

[0082] - "The biomechanical and biological properties of glycosaminoglycans in skin physiology" particularly include hydration and fibroblast activation. For example, hyaluronic acid is involved in skin reconstruction, regeneration, and / or rejuvenation due to its biomechanical and biological properties.

[0083] - To "supplement" a composition means to add at least one non-crosslinked, low-molar-mass glycosaminoglycan to the composition. Preferably, supplementation corresponds to adding at least one non-crosslinked, low-molar-mass hyaluronic acid to the composition.

[0084] - "Total glycosaminoglycan concentration" refers to the sum of the concentrations of cross-linked high-molar mass glycosaminoglycans and optionally non-cross-linked high-molar mass glycosaminoglycans, plus the concentration of glycosaminoglycans used for supplementation. This concentration is expressed in milligrams of glycosaminoglycan per gram of gel.

[0085] - "Total hyaluronic acid concentration" refers to the sum of the concentrations of cross-linked high molar mass hyaluronic acid and optionally non-cross-linked high molar mass hyaluronic acid, plus the concentration of hyaluronic acid used for supplementation. This concentration is expressed in milligrams of glycosaminoglycan per gram of gel.

[0086] A solution is considered "homogeneous" when its color, visual appearance, and viscosity are perceived as uniform by the naked eye and touch.

[0087] - "Aqueous medium" refers to any liquid medium that contains at least water as a solvent and has the ability to dissolve glycosaminoglycans, especially hyaluronic acid.

[0088] - "Crosslinking agent" refers to any compound capable of introducing crosslinks between different glycosaminoglycan chains. Bifunctional or polyfunctional epoxy or non-epoxy crosslinking agents may be specifically mentioned as suitable crosslinking agents for carrying out the present invention. Among epoxy agents, butanediol diglycidyl ether (BDDE), diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, 1,2-bis(2,3-epoxypropyl)-2,3-emylene, and mixtures thereof may be mentioned. Among non-epoxy agents, endogenous polyamines such as spermine, spermidine and putrescine, aldehydes, carbodiimides, and divinyl sulfone may be mentioned.

[0089] - "Aqueous gel" means an aqueous gel that can be obtained by the method according to the invention or an aqueous gel according to the invention, unless otherwise stated.

[0090] - "Soft gel" refers to a gel with the following characteristics: its viscoelastic distribution maintains moderate elasticity, that is, it has sufficient viscosity with a phase angle (δ) between 15° and 50° and a low elastic modulus G′, in other words, less than or equal to 150 Pa, preferably between 20 and 150 Pa, which is measured using a rheometer (TAInstruments DHR2) with a cone / plate geometry (cone angle 1° / plate diameter 40 mm) at room temperature with a vibrational stress oscillation at a frequency of 1 Hz and a stress of 5 Pa; and an extrusion force less than or equal to 18 N, preferably less than 15 N, which is measured in a syringe with an inner diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0091] - "Easy to inject" means that the gel has an average extrusion force of less than 18 N, preferably less than 15 N, when measured with a force gauge as follows: in a syringe with an outer diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0092] - A composition or gel that is "easily diffused into tissues" refers to a composition for skin application used for skin regeneration and appearance improvement that does not promote papule formation after being injected into the superficial areas of the skin (i.e., the upper and middle dermis).

[0093] - "In vivo durability" refers to the ability of a gel to remain at the treatment site over time after injection. An in vivo durable gel is a gel that can remain at the treatment site for at least 1 month, preferably at least 3 months, and more preferably at least 6 months, i.e., can maintain an overall aesthetic improvement for at least 1 month, preferably at least 3 months, and more preferably at least 6 months after application. In other words, it can maintain at least a grade 1 or 2 improvement on a subjective scale for evaluating overall aesthetic improvement, which includes 5 grades: 1. Very improved; 2. Improved; 3. No change; 4. Deteriorated; 5. Very deteriorated, and maintain this improvement for at least 1 month, preferably at least 3 months, and more preferably at least 6 months.

[0094] The term "stability" characterizes the ability of a composition to maintain its physicochemical properties (color, pH, homogeneity, turbidity, rheology, etc.) according to its specifications during a storage period of at least one year. The stability of a composition is typically monitored by observing and / or measuring one or more of its physicochemical parameters, and in particular one or more of its rheological parameters such as its elastic modulus (G′) or viscosity (η).

[0095] - "Sterile gel" refers to a gel that is safe in or through the superficial areas of the skin (i.e., the upper and middle dermis). In particular, gels to be applied via injection must be free of any contaminants that could trigger undesirable secondary reactions in the host organism.

[0096] The terms “additive,” “additional component,” and “excipient” are used interchangeably and refer to any component compatible with application within the skin and / or on the surface. The amount of additive used depends on the properties of the component, the desired effect, and the intended use of the composition according to the invention. The additive may be one of the following components or derivatives thereof: anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, or sodium chloride.

[0097] - As "anesthetics," the following can be mentioned: ampicillin, amocaine, pentacaine (Amylein), apralinidine, atrocaine, articaine, benzocaine, betocaine, bupivacaine, butacaine, butylamine, butancaine, chlorobutanol, chloroprocaine, cincocaine, chlordacine, cocaine, clarithromycin, cyclomethicaine, desicaine, diaminocaine, dipiperone, dacronin, eticaine, euprocine, febuerine, formocaine, guanificano, heptacaine, hecaine, hydroxyprolactin. Cocaine, hydroxybutylcaine, isobacterium, leucine, levobupivacaine, levosacral, lidamicin, lidocaine, lotusaine, monagricetide, mepivacaine, mepcocaine, myticaine, oxacaine, oxotoxane, oxoxicaine, oxabucaine, paraethoxycaine, pipedocaine, phenacaine, piperocaine, pidocaine, polydocaine, promocaine, prilocaine, procaine, pripacaine, pricapaine, prioxycaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, prilocaine, and their salts.

[0098] - As "antioxidants", glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, panthenol, ubiquinone, and alpha-lipoic acid can be mentioned.

[0099] - As for "amino acids", arginine, isoleucine, leucine, lysine, glycine, valine, threonine, proline, methionine, histidine, phenylalanine, tryptophan, and cysteine ​​can be mentioned.

[0100] - As “vitamins”, references may be made to vitamins E, A, C, B and especially vitamins B4, B5, B6, B8, B9, B7, B12 and preferably pyridoxine.

[0101] - As "minerals", salts of zinc, magnesium, calcium, potassium, manganese, sodium, and copper can be mentioned.

[0102] - As for "coenzyme", coenzyme Q10, CoA, NAD, and NADP can be mentioned.

[0103] - As "adrenergic derivatives", adrenaline and noradrenaline can be mentioned.

[0104] Finally, it should be understood that not all indicated values ​​are strictly considered, but rather approximated according to general convention; that is, the last digit of the indicated value corresponds to the accuracy of the measurement. For example, for a molar mass of 0.04 MDa, the error limit is 0.035–0.044 MDa.

[0105] According to the method of the present invention

[0106] As described above, this article describes a method for preparing an aqueous gel, which, on the one hand, requires an aqueous solution containing at least one cross-linked glycosaminoglycan (also referred to as "cross-linked glycosaminoglycan HMW") because it is formed from a high molar mass of non-cross-linked glycosaminoglycan and a cross-linking agent, and on the other hand, requires an aqueous solution containing at least one low molar mass of non-cross-linked glycosaminoglycan (referred to as "non-cross-linked glycosaminoglycan LMW"), and forms a homogeneous mixture of all or part of said solution.

[0107] The glycosaminoglycans used in the method according to the invention may be selected from hyaluronic acid, heparosan, chondroitin sulfate, dermatan sulfate, keratin sulfate, and mixtures thereof.

[0108] The glycosaminoglycans used in the methods of this invention may have the same or different chemical properties.

[0109] In one specific embodiment, the glycosaminoglycans used in the method of the present invention have the same chemical properties, and are preferably all hyaluronic acid.

[0110] More specifically, the present invention relates to a method for preparing an aqueous gel, which, on the one hand, requires an aqueous solution containing at least one cross-linked hyaluronic acid (also referred to as "cross-linked HA HMW"), as it is formed from a high molar mass of non-cross-linked hyaluronic acid and a cross-linking agent, and on the other hand, requires an aqueous solution containing a low molar mass of at least one non-cross-linked hyaluronic acid (which is referred to as "non-cross-linked HALMW"), and forms a homogeneous mixture of all or part of said solutions.

[0111] The characteristic is that it contains hyaluronic acid in a mass ratio of "non-crosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0112] In one specific embodiment, the hyaluronic acid used in the method of the present invention is not chemically modified.

[0113] As noted above, the method according to the invention uses these glycosaminoglycans and preferably these hyaluronic acids in the form of an aqueous solution, specifically as defined in steps a) and b) of the method according to the invention.

[0114] These solutions of glycosaminoglycans, especially hyaluronic acid, are advantageously homogeneous.

[0115] They can be prepared according to conventional methods, for example by dissolving and / or diluting glycosaminoglycans, particularly hyaluronic acid (crosslinked or uncrosslinked), in an aqueous medium (such as phosphate-buffered saline). These preparations are advantageously carried out at room temperature, preferably between 15°C and 25°C, under mechanical or manual stirring.

[0116] Step A)

[0117] Regarding the aqueous solutions of "crosslinked glycosaminoglycans HMW" and, in particular, "crosslinked HAHMW" considered in step a), they are first obtained as follows: at least one non-crosslinked glycosaminoglycan having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably from 1.00 MDa to 4.00 MDa is crosslinked with at least one crosslinking agent; and in particular, they are first obtained as follows: at least one non-crosslinked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably from 1.00 MDa to 4.00 MDa is crosslinked with at least one crosslinking agent.

[0118] Such cross-linking is clearly within the capabilities of technical personnel.

[0119] Advantageously, the crosslinking agent is introduced at a crosslinking rate of less than or equal to 8%, preferably less than or equal to 5%, and more preferably less than 2% by mass.

[0120] Advantageously, the crosslinking agent is introduced at a molar ratio of 0.001 to 0.25 relative to the total number of moles of glycosaminoglycan units, particularly relative to the total number of moles of hyaluronic acid units.

[0121] Therefore, according to a preferred embodiment, the "crosslinked HA HMW" is prepared with a crosslinking rate of less than or equal to 8%, preferably less than or equal to 5%, and more preferably less than 2% by mass.

[0122] This crosslinking is carried out at various temperatures and times to achieve the desired degree of modification.

[0123] Therefore, crosslinking at room temperature (i.e., a temperature varying from 15°C to 25°C) may require a reaction time of more than 5 hours or even 10 hours or longer.

[0124] On the other hand, by using stimulating elements such as heating, ultraviolet exposure, microwave exposure or catalysts, preferably by using crosslinking stimulated by heating, the degree of crosslinking can be significantly improved and the duration of crosslinking can be less than 5 hours.

[0125] In one specific embodiment, crosslinking of uncrosslinked glycosaminoglycans, particularly uncrosslinked hyaluronic acid, is carried out at a temperature greater than 40°C, preferably greater than 50°C, more specifically in the range of 45°C to 60°C or even better in the range of 50°C to 55°C, having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, and more preferably in the range of 1.00 MDa to 4.00 MDa.

[0126] Such crosslinking advantageously has a duration of 30-300 minutes, preferably 100-240 minutes.

[0127] In a particularly preferred embodiment, crosslinking of uncrosslinked hyaluronic acid with a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, and more preferably in the range of 1.00 MDa to 4.00 MDa is carried out at room temperature, i.e., at a temperature in the range of 15°C to 25°C.

[0128] Such crosslinking advantageously has a reaction time of more than 5 hours, or even more than 10 hours, particularly between 10 hours and 100 hours.

[0129] Preferably, the crosslinking agent used in the method according to the invention is an epoxy crosslinking agent, more preferably 1,4-butanediol diglycidyl ether (BDDE).

[0130] In specific embodiments where the crosslinking agent used is a bifunctional or multifunctional epoxy crosslinking agent such as BDDE, the pH of the aqueous medium is alkalized or acidified to activate the crosslinking reaction. According to one embodiment, the aqueous medium is typically alkalized with a solution containing sodium hydroxide, and preferably, the pH of the aqueous medium is greater than or equal to 11, more preferably greater than or equal to 12.

[0131] According to a preferred embodiment, the concentration of “crosslinked HMW glycosaminoglycan”, more specifically “crosslinked HMW HA” in the aqueous medium of step a) is adjusted to form an aqueous gel at the end of step c), wherein the concentration of “crosslinked HMW glycosaminoglycan”, more specifically “crosslinked HMW HA”, is at least 5 mg / g relative to the total mass of the aqueous gel.

[0132] Preferably, the method according to the invention uses an aqueous solution containing 5-15 mg / g of "crosslinked HA HMW".

[0133] Step B)

[0134] Step b) of the method of the present invention, in itself, requires obtaining an aqueous solution containing at least one "non-crosslinked glycosaminoglycan LMW", particularly at least one "non-crosslinked HALMW".

[0135] Preferably, the concentration of “non-crosslinked glycosaminoglycans LMW”, particularly “non-crosslinked HALMW”, considered in the aqueous solution of step b) is adjusted to form an aqueous gel at the end of step c), wherein the concentration of “non-crosslinked glycosaminoglycans LMW”, particularly “non-crosslinked HALMW”, is at least 3 mg / g, preferably 10 mg / g, relative to the total mass of the aqueous gel.

[0136] Preferably, the method according to the invention uses an aqueous solution containing 50-60 mg / g of "uncrosslinked HA LMW".

[0137] As detailed below, the method according to the invention may also include the use of glycosaminoglycans, particularly hyaluronic acid, which differ from those defined with respect to steps a) and b).

[0138] Step C)

[0139] As described above, step c) involves forming a homogeneous mixture from at least some or all of the solutions obtained in steps a) and b).

[0140] The formation of the homogeneous mixture considered in step c) advantageously involves at least one homogenization step.

[0141] Homogenization is advantageously carried out under mild conditions to prevent degradation of the glycosaminoglycan chains used.

[0142] According to conventional homogenization methods, such as three-dimensional stirring, paddle mixer stirring, scraper stirring, and / or extrusion through at least one mesh, step c) can be used to form a homogeneous mixture of at least one cross-linked glycosaminoglycan and at least one non-cross-linked glycosaminoglycan, particularly at least one cross-linked hyaluronic acid and at least one non-cross-linked hyaluronic acid.

[0143] According to a preferred embodiment, step c) includes at least one step of extruding the formed mixture through at least one mesh. The adjustment of the characteristics of such meshes is part of the general knowledge of those skilled in the art. Typically, they are selected according to the desired properties of the mixture to be formed, taking into account the individual characteristics of the glycosaminoglycan solutions, particularly the hyaluronic acid solutions, mixed together.

[0144] According to one specific embodiment, step c) therefore includes at least one homogenization step by means of a paddle mixer, followed by at least one step of extruding the mixture through at least one grid.

[0145] Homogenization is considered satisfactory when the resulting gel exhibits a macroscopically uniform color, consistent viscosity, and absence of agglomerates, both visually and tactilely. Those skilled in the art assess the duration of homogenization based on their general knowledge. The homogenization process may comprise several cycles, optionally separated by time-separated resting phases, particularly to assess the homogenization quality of glycosaminoglycans, especially hyaluronic acid, within the gel.

[0146] More specifically, step c) according to the invention may include homogenization for a total duration of less than 200 minutes, preferably less than 150 minutes, or even for a duration in the range of 5-100 minutes.

[0147] Non-crosslinked HMW glycosaminoglycans, especially non-crosslinked HMW hyaluronic acid

[0148] In one specific embodiment, the method according to the invention also considers the use of at least one non-crosslinked glycosaminoglycan having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, referred to as "non-crosslinked HMW glycosaminoglycan", and in particular, the use of at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, referred to as "non-crosslinked HA HMW".

[0149] Preferably, the "non-crosslinked HMW glycosaminoglycan", particularly the "non-crosslinked HAHMW", is realized in an aqueous solution. This aqueous solution may be different from or similar to one of the aqueous solutions considered in steps a) and b).

[0150] According to an alternative embodiment, the “uncrosslinked glycosaminoglycan HMW”, in particular all or part of the “uncrosslinked HAHMW”, is used in the aqueous solution of step b).

[0151] According to another alternative embodiment, the "non-crosslinked HMW glycosaminoglycan," particularly the "non-crosslinked HAHMW," is used in the form of an aqueous solution different from the aqueous solutions of steps a) and b). According to this alternative embodiment, the aqueous solution containing all or part of the "non-crosslinked HMW glycosaminoglycan," particularly the "non-crosslinked HAHMW," and the aqueous solution of step b) are mixed simultaneously or sequentially with the aqueous solution of step a).

[0152] Preferably, in step b) of the method according to the invention, an aqueous solution containing 5-15 mg / g of "uncrosslinked HAHMW" is used.

[0153] additive

[0154] In one specific embodiment, the present invention relates to the method described above, which further comprises implementing at least one additive selected from anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate and sodium chloride.

[0155] sterilization

[0156] Therefore, according to one aspect of the invention, in order to maintain the sterility of the gel, the raw materials used are sterile, and the preparation of the gel includes packaging it in its application device under controlled atmospheric conditions.

[0157] According to another aspect, the present invention relates to a method for preparing a sterile and injectable aqueous gel, comprising at least one step of achieving an aqueous gel obtained by the method according to the invention or an aqueous gel as defined in the invention, and a sterilization step, particularly in an autoclave.

[0158] More specifically, a method for preparing an aqueous gel according to the present invention comprises at least the following steps:

[0159] a) Obtain an aqueous solution comprising at least one cross-linked glycosaminoglycan, referred to as "cross-linked glycosaminoglycan HMW", which is formed from a non-cross-linked glycosaminoglycan having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, and from a cross-linking agent.

[0160] b) Obtain an aqueous solution comprising at least one non-crosslinked glycosaminoglycan having a molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, and even more preferably 0.08 MDa to 0.10 MDa; referred to as "non-crosslinked glycosaminoglycan LMW".

[0161] c) Form a homogeneous mixture of all or part of the solutions from steps a) and b), and sterilize it, particularly in an autoclave.

[0162] More specifically, a method for preparing an aqueous gel according to the present invention comprises at least the following steps:

[0163] a) Obtain an aqueous solution comprising at least one cross-linked hyaluronic acid, referred to as "cross-linked HAHMW", which is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, and a cross-linking agent;

[0164] b) Obtain an aqueous solution containing at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW," having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa.

[0165] c) Forming a homogeneous mixture of all or part of the solutions described in steps a) and b), and sterilizing it, particularly in an autoclave.

[0166] The characteristic is that it contains hyaluronic acid in a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0167] The sterilization is preferably performed on the product already packaged in its application device (usually a syringe).

[0168] Therefore, advantageously, the aqueous gel obtained by the method according to the invention can be packaged in a syringe before sterilization.

[0169] Preferably, the method comprises a single sterilization step.

[0170] Preferably, the sterilization step is carried out by a thermal method, for example in an autoclave, particularly at a temperature between 120°C and 140°C.

[0171] Specifically, the sterilization step is performed in an autoclave under humid heat conditions at a platform temperature greater than or equal to 121°C, preferably between 121°C and 135°C, wherein F0 is greater than 15 (sterilization value). According to the invention, the sterilization value F0 corresponds, in minutes, the time required, at 121°C, to inactivate 90% of the microbial population present in the product to be sterilized.

[0172] Aqueous gel

[0173] As described above, one aspect of the present invention is an aqueous gel based on hyaluronic acid, comprising cross-linked hyaluronic acid, characterized in that the gel comprises at least:

[0174] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.02 MDa to 0.30 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 30% to 70% by mass; and

[0175] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa is called "sHA HMW".

[0176] The characteristic feature is that the total water-soluble hyaluronic acid has a modification degree of less than or equal to 5%, preferably less than or equal to 3%, more preferably less than or equal to 1%, and the percentages of soluble hyaluronic acid are determined as follows: the aqueous gel is diluted in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate at 25°C for 5 days, and centrifuged at 4400 rpm for 10 minutes. The diluted gel is then filtered through a 0.45 mm filter to obtain a filtrate, which is then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase is 0.3 mL / min.

[0177] In one specific embodiment, the aqueous gel according to the invention is a soft gel, i.e., a gel having the following characteristics: a phase angle (δ) between 15° and 50° and an elastic modulus G′ less than or equal to 150 Pa, preferably between 20 and 150 Pa, which is measured using a rheometer (TA Instruments DHR2) with a cone / plate geometry (cone angle 1° / plate diameter 40 mm) at room temperature with a vibrational stress oscillation at a frequency of 1 Hz and a stress of 5 Pa; and an extrusion force less than or equal to 18 N, preferably less than 15 N, which is measured in a syringe with an inner diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0178] In one specific embodiment, the aqueous gel according to the invention comprises 35% to 65% by weight of “sHA LMW” relative to the total mass of the aqueous gel.

[0179] In one specific embodiment, in the aqueous gel according to the invention, the water-soluble hyaluronic acid (referred to as "sHA LMW") has a mass-average molar mass in the range of 0.04 MDa to 0.20 MDa, preferably 0.04 MDa to 0.15 MDa.

[0180] In one specific embodiment, in the aqueous gel according to the invention, the water-soluble hyaluronic acid (referred to as "sHA HMW") has a mass-average molar mass greater than 0.30 MDa and less than or equal to 4.00 MDa.

[0181] In one specific embodiment, the aqueous gel according to the invention comprises 10% to 30% by weight, preferably 15% to 20% by weight, of “sHA HMW” relative to the total mass of the aqueous gel.

[0182] In one specific embodiment, in the aqueous gel according to the invention, the crosslinked hyaluronic acid is present in an amount of at least 5 mg / g relative to the total mass of the aqueous gel.

[0183] In one specific embodiment, in the aqueous gel according to the invention, the water-soluble hyaluronic acid (referred to as "sHA LMW") is present in an amount of at least 3 mg / g, preferably 10 mg / g, relative to the total mass of the aqueous gel.

[0184] In one specific embodiment, in the aqueous gel according to the invention, the total hyaluronic acid concentration is 10-40 mg / g, preferably 15-35 mg / g, and more preferably 20-30 mg / g, relative to the total mass of the aqueous gel.

[0185] In one specific embodiment, in the aqueous gel according to the invention, the degree of modification of total hyaluronic acid is less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0186] According to one specific implementation, in the aqueous gel defined in this invention:

[0187] - The cross-linked hyaluronic acid is present in an amount ranging from 6 mg / g to 12 mg / g relative to the total mass of the aqueous gel.

[0188] - The water-soluble hyaluronic acid (referred to as "sHA LMW") is present in an amount ranging from 12 mg / g to 20 mg / g relative to the total mass of the aqueous gel.

[0189] - The water-soluble hyaluronic acid (referred to as "sHA HMW") is present in an amount ranging from 3 mg / g to 6 mg / g relative to the total mass of the aqueous gel.

[0190] The aqueous gel contains a total hyaluronic acid concentration of 20-30 mg / g relative to the total mass of the aqueous gel.

[0191] In one specific embodiment, the aqueous gel according to the invention is subjected to a heat sterilization step, for example, in an autoclave.

[0192] Hyaluronic acid is known to be highly sensitive to strong acid and alkaline pH, high temperature (e.g. during heat sterilization), oxidation (e.g. reactive oxygen species) and enzyme activity (Stern R, Kogan G, Jedrzejas MJ, et al. The many ways to cleave hyaluronan. Biotechnol Adv 2007; 25:537-57).

[0193] Therefore, the preparation methods for producing compositions containing cross-linked hyaluronic acid, especially the cross-linking conditions, readily degrade the hyaluronic acid chains, thereby releasing LMW-soluble hyaluronic acid. Such LMW hyaluronic acid fragments are at least partially modified by the cross-linking agent used, for example, by BDDE.

[0194] Conversely, the soluble hyaluronic acid from the non-crosslinked LMW hyaluronic acid added during the preparation process was not modified by BDDE.

[0195] BDDE modification can be visualized using the degree of modification (MOD) measurement. In this context, a higher MOD for soluble hyaluronic acid corresponds to a higher proportion of LMW hyaluronic acid fragments generated in the gel preparation method studied, and a lower proportion of soluble hyaluronic acid from the LMW hyaluronic acid added in the preparation method.

[0196] This is explained in Example 7.

[0197] The above explanation of hyaluronic acid degradation explains why the molar mass of hyaluronic acid added to the gel before heat sterilization is higher than the molar mass of soluble hyaluronic acid measured in the sterilized gel.

[0198] This sensitivity also applies to other glycosaminoglycans.

[0199] In fact, "sHA" corresponds to:

[0200] - Uncrosslinked HALMW added in the gel preparation method after crosslinking step a); and,

[0201] - When present, uncrosslinked HAHMW is added in the gel preparation method after crosslinking step a); and,

[0202] - HA fragments released during a gel preparation method, wherein the HA fragments are at least partially modified by a crosslinking agent such as BDDE.

[0203] Therefore, the amount of "sHALMW" is at least equal to the following amounts of substance:

[0204] - Uncrosslinked HA LMW added in the gel preparation method after crosslinking step a); and,

[0205] - When present, uncrosslinked HAHMW is added in the gel preparation method after crosslinking step a).

[0206] - Conversely, "nsHA" corresponds to cross-linked HA.

[0207] According to one specific embodiment, the aqueous gel according to the present invention comprises at least:

[0208] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.20 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 30% to 70% by mass; and

[0209] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa and less than or equal to 4.00 MDa is called "sHA HMW".

[0210] Furthermore, the total water-soluble hyaluronic acid has a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0211] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, and centrifuged at 4400 rpm for 10 minutes. The diluted gel was then filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min. According to one specific embodiment, the aqueous gel according to the invention comprises at least:

[0212] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.15 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 30% to 70% by mass; and

[0213] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa and less than or equal to 4.00 MDa is called "sHA HMW".

[0214] Furthermore, the total water-soluble hyaluronic acid has a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0215] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min.

[0216] According to one specific embodiment, the aqueous gel according to the present invention comprises at least:

[0217] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.02 MDa to 0.30 MDa, referred to as "sHA LMW", with an amount relative to the total mass of hyaluronic acid ranging from 35% to 65% by mass; and

[0218] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa is called "sHA HMW".

[0219] Furthermore, the total water-soluble hyaluronic acid has a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0220] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min.

[0221] According to one specific embodiment, the aqueous gel according to the present invention comprises at least:

[0222] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.20 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 35% to 65% by mass; and

[0223] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa and less than or equal to 4.00 MDa is called "sHA HMW".

[0224] Furthermore, the total water-soluble hyaluronic acid has a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0225] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min.

[0226] According to one specific embodiment, the aqueous gel according to the present invention comprises at least:

[0227] - A water-soluble hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.15 MDa, referred to as "sHA LMW", with its amount relative to the total mass of hyaluronic acid being 35% to 65% by mass; and

[0228] -Optional, water-soluble hyaluronic acid with a mass-average molar mass greater than 0.30 MDa and less than or equal to 4.00 MDa is called "sHA HMW".

[0229] Furthermore, the total water-soluble hyaluronic acid has a modification level of less than or equal to 5%, preferably less than or equal to 3%, and more preferably less than or equal to 1%.

[0230] The percentages of soluble hyaluronic acid were determined as follows: the aqueous gel was diluted for 5 days at 25°C in a pH 7.2 solution containing 0.02% NaN3 and 150 nM sodium nitrate, centrifuged at 4400 rpm for 10 minutes, and the diluted gel was filtered through a 0.45 mm filter to obtain a filtrate. The filtrate was then analyzed using a size exclusion chromatography instrument equipped with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector (with a refractive index increment (dn / dc) set at 0.165 mL / g) and a liquid chromatography pump station equipped with two size exclusion columns (suitable for molecules with a mass-average molar mass in the range of 0.001 Da to 20 MDa), wherein the flow rate of the pH 7.2 sodium nitrate mobile phase was 0.3 mL / min. As described above, the present invention also relates to an aqueous gel obtainable by the method according to the invention.

[0231] At the end of step c), a homogeneous aqueous gel is obtained containing at least one “non-crosslinked LMW glycosaminoglycan” and “HMW glycosaminoglycan”, including at least one “crosslinked HMW glycosaminoglycan”.

[0232] Advantageously, the aqueous gel obtained by the method according to the invention contains a total glycosaminoglycan concentration in the range of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g relative to its total mass.

[0233] The aqueous gel obtained by the method according to the invention can also be characterized by the mass ratio of "uncrosslinked glycosaminoglycan LMW" to "glycosaminoglycan HMW" in the range of 1:4 to 4:1, preferably 1:3 to 5:3, more preferably 1:2 to 2:1.

[0234] According to one specific embodiment, the aqueous gel has a degree of modification of the glycosaminoglycan of less than 5%, preferably less than 3%, and more preferably less than 1%.

[0235] More specifically, at the end of step c), a homogeneous aqueous gel is obtained containing at least one “uncrosslinked HALMW” and “HAHMW”, including at least one “crosslinked HA HMW”.

[0236] Advantageously, the aqueous gel obtained by the method according to the invention contains a total hyaluronic acid concentration in the range of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g relative to its total mass.

[0237] According to one embodiment, the aqueous gel obtained by the method according to the invention can also be characterized by the mass ratio of “uncrosslinked HA LMW” to “HA HMW” in the range of 1:3 to 5:3, preferably 1:2 to 5:3.

[0238] According to one specific embodiment, the aqueous gel has a degree of modification of the hyaluronic acid of less than or equal to 5%, preferably less than or equal to 3%, more preferably less than or equal to 1%.

[0239] According to another specific embodiment, the aqueous gel obtained by the method according to the invention is a soft gel, that is, a gel having the following characteristics: a phase angle (δ) between 15° and 50° and an elastic modulus G′ less than or equal to 150 Pa, preferably between 20 and 150 Pa, which is measured using a rheometer (TA Instruments DHR2) with a cone / plate geometry (cone angle 1° / plate diameter 40 mm) at room temperature with a vibrational stress oscillation at a frequency of 1 Hz and a stress of 5 Pa; and an extrusion force less than or equal to 18 N, preferably less than 15 N, which is measured in a syringe with an inner diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0240] According to one embodiment of the present invention, the aqueous gel is obtained at the end of the method comprising the following steps:

[0241] - In step a), an aqueous solution containing 5-15 mg / g of "crosslinked glycosaminoglycans (HMW)" is used;

[0242] - In step b), an aqueous solution containing 50-60 mg / g of "uncrosslinked glycosaminoglycan LMW" and 5-20 mg / g of "uncrosslinked glycosaminoglycan HMW", and,

[0243] The gel formed has a total glycosaminoglycan concentration of 20-30 mg / g relative to its total mass.

[0244] According to one embodiment of the present invention, the aqueous gel is obtained at the end of the method comprising the following steps:

[0245] - In step a), an aqueous solution containing 5-15 mg / g of "crosslinked HA HMW" is used;

[0246] - In step b), an aqueous solution containing 50-60 mg / g of "uncrosslinked HA LMW" and 5-20 mg / g of "uncrosslinked HAHMW" is provided, wherein...

[0247] The gel formed has a total hyaluronic acid concentration of 20-30 mg / g relative to its total mass.

[0248] Representative aqueous gels, obtainable by the method according to the invention and having a total glycosaminoglycan concentration of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g relative to their total mass, are derived, in particular, from the following:

[0249] - At least one cross-linked glycosaminoglycan formed from at least one non-cross-linked glycosaminoglycan having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, is referred to as "cross-linked glycosaminoglycan HMW".

[0250] - At least one non-crosslinked glycosaminoglycan having a molar mass of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, and even more preferably 0.08 MDa to 0.10 MDa, is referred to as "non-crosslinked glycosaminoglycan LMW"; and,

[0251] -Optionally, at least one non-crosslinked glycosaminoglycan having a molar mass greater than or equal to 0.50 MDa, preferably greater than 1 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, is referred to as "non-crosslinked glycosaminoglycan HMW".

[0252] A representative aqueous gel, obtainable by the method according to the invention and having a total hyaluronic acid concentration of 20-30 mg / g relative to its total mass, originates from, in particular, the following:

[0253] - In step a), an aqueous solution containing 5-15 mg / g of "crosslinked HA HMW" is used;

[0254] - In step b), an aqueous solution containing 50-60 mg / g of "uncrosslinked HA LMW" and 5-20 mg / g of "uncrosslinked HAHMW".

[0255] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0256] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than 1.00 MDa and a cross-linking agent;

[0257] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW", having a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa; and

[0258] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass greater than 1.00 MDa.

[0259] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0260] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0261] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent;

[0262] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW", having a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa; and

[0263] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa.

[0264] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0265] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0266] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa and a cross-linking agent;

[0267] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA LMW", having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa; and

[0268] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass greater than or equal to 0.50 MDa.

[0269] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 5:3.

[0270] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0271] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than 1.00 MDa and a cross-linking agent;

[0272] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW", having a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa; and

[0273] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass greater than 1.00 MDa.

[0274] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 5:3.

[0275] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0276] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent;

[0277] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW", having a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa; and

[0278] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa.

[0279] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 5:3.

[0280] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0281] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa and a cross-linking agent;

[0282] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA LMW", having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa; and,

[0283] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass greater than or equal to 0.50 MDa.

[0284] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:2 to 5:3.

[0285] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0286] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass average molar mass greater than 1.00 MDa and a cross-linking agent;

[0287] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA LMW", having a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa; and,

[0288] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass greater than 1.00 MDa.

[0289] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:2 to 5:3.

[0290] Representative aqueous gels obtainable by the method according to the invention are derived, in particular, from the following:

[0291] - At least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", is formed from non-cross-linked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent;

[0292] - At least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA LMW", having a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa; and,

[0293] -Optional, at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HA HMW", having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa.

[0294] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:2 to 5:3.

[0295] Aqueous gels with total hyaluronic acid of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g, relative to their total mass, obtainable by the method according to the invention, are representative examples derived from, in particular, the following:

[0296] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent, is referred to as "cross-linked HA HMW".

[0297] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, is referred to as "non-crosslinked HA LMW"; and,

[0298] -Optionally, at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, is referred to as "non-crosslinked HAHMW".

[0299] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0300] Representative aqueous gels, which can be obtained by the method according to the invention and have a total hyaluronic acid content of 10-40 mg / g relative to their total mass, are derived, in particular, from the following:

[0301] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass greater than 1.00 MDa and a cross-linking agent is called "cross-linked HA HMW".

[0302] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa, which is referred to as "non-crosslinked HA LMW"; and,

[0303] -Optional, at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than 1.00 MDa, which is referred to as "non-crosslinked HA HMW".

[0304] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0305] A representative aqueous gel, obtainable by the method according to the invention and having a total hyaluronic acid content of 20-30 mg / g relative to its total mass, originates from, in particular, the following:

[0306] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass greater than 1.00 MDa and a cross-linking agent is called "cross-linked HA HMW".

[0307] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa, which is referred to as "non-crosslinked HA LMW"; and,

[0308] -Optional, at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than 1.00 MDa, which is referred to as "non-crosslinked HA HMW".

[0309] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0310] Representative aqueous gels, which can be obtained by the method according to the invention and have a total hyaluronic acid content of 10-40 mg / g relative to their total mass, are derived, in particular, from the following:

[0311] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent is called "cross-linked HA HMW".

[0312] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa, which is referred to as "non-crosslinked HA LMW"; and,

[0313] -Optional, at least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa, which is referred to as "non-crosslinked HA HMW".

[0314] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0315] A representative aqueous gel, obtainable by the method according to the invention and having a total hyaluronic acid content of 20-30 mg / g relative to its total mass, originates from, in particular, the following:

[0316] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent is called "cross-linked HA HMW".

[0317] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa, which is referred to as "non-crosslinked HA LMW"; and,

[0318] -Optional, at least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa, which is referred to as "non-crosslinked HA HMW".

[0319] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 2:1.

[0320] Aqueous gels with total hyaluronic acid of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g, relative to their total mass, obtainable by the method according to the invention, are representative examples derived from, in particular, the following:

[0321] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent, is referred to as "cross-linked HA HMW".

[0322] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, is referred to as "non-crosslinked HA LMW"; and,

[0323] -Optionally, at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, is referred to as "non-crosslinked HAHMW".

[0324] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:3 to 5:3.

[0325] Aqueous gels with total hyaluronic acid of 10-40 mg / g, preferably 15-35 mg / g, more preferably 20-30 mg / g, relative to their total mass, obtainable by the method according to the invention, are representative examples derived from, in particular, the following:

[0326] - At least one cross-linked hyaluronic acid formed from at least one non-cross-linked hyaluronic acid having a mass average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent, is referred to as "cross-linked HA HMW".

[0327] - At least one non-crosslinked hyaluronic acid having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa, preferably 0.08 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.15 MDa, is referred to as "non-crosslinked HA LMW"; and,

[0328] -Optionally, at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than or equal to 0.50 MDa, preferably greater than 1.00 MDa, more preferably in the range of 1.00 MDa to 4.00 MDa, is referred to as "non-crosslinked HAHMW".

[0329] The hyaluronic acid was used at a mass ratio of "uncrosslinked HA LMW" to "HA HMW" of 1:2 to 5:3.

[0330] Of course, the aqueous gel obtained by the method according to the invention may also contain one or more additives conventionally considered in this type of formulation, and in particular one or more additives as defined above.

[0331] Of course, the selection and amount of additives can be adjusted to avoid increasing the risk of any incompatibility with other compounds used in the gel according to the invention, particularly with the hyaluronic acid, and to ensure compatibility with the uses described below. These adjustments are within the general capabilities of those skilled in the art.

[0332] In one specific embodiment, the invention also relates to a composition comprising at least one aqueous gel as defined according to the invention and, if desired, at least one additive selected from anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate and sodium chloride.

[0333] In a preferred embodiment, the additive is an anesthetic as defined above, and in particular lidocaine, mepivacaine or its salts such as hydrochloride.

[0334] The gel obtained according to the method of the invention and the aqueous gel according to the invention are particularly useful as compositions for preventing and / or treating changes in the appearance of the skin surface.

[0335] The compositions according to the present invention can be cosmetic and / or dermatological compositions.

[0336] These compositions can be applied topically, but can also be administered by injection, particularly in the superficial areas of the skin, i.e., in the upper and middle dermis.

[0337] When considering injection, it is of course necessary that the composition and / or aqueous gel be sterile and injectable.

[0338] When the composition according to the invention is a topical composition, it can be any galen formulation suitable for its application, such as cream, ointment, lotion, serum, gel or dispersion.

[0339] Any suitable excipient known to those skilled in the art, in an appropriate amount according to the intended indication, may be included in the composition according to the invention.

[0340] According to another aspect, the present invention relates to injectable aqueous gels sterilized according to the sterilization method described above.

[0341] Representative examples of such aqueous gels obtainable by the method according to the invention include, in particular, those containing the following substances:

[0342] - At least one "crosslinked glycosaminoglycan HMW" by mass, relative to the total mass of the aqueous gel;

[0343] - At least one "non-crosslinked glycosaminoglycan (LMW)" at 1.4-1.8% by mass relative to the total mass of the aqueous gel; and,

[0344] - At least one "non-crosslinked glycosaminoglycan HMW" by mass, relative to the total mass of the aqueous gel;

[0345] It also has a total glycosaminoglycan concentration between 20 and 30 mg / g, a phase angle (δ) between 15° and 50°, and a G′ between 20 and 150 Pa. This was measured using a rheometer (TAInstruments DHR2) with a cone / plate geometry (cone angle 1° / plate diameter 40 mm) at room temperature with a vibrational stress oscillation at a frequency of 1 Hz and a stress of 5 Pa. The extrusion force is less than or equal to 15 N when measured with a force gauge as follows: in a syringe with an outer diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0346] Representative examples of such aqueous gels obtainable by the method according to the invention include, in particular, those containing the following substances:

[0347] - At least one "crosslinked HAHMW" by mass, relative to the total mass of the aqueous gel;

[0348] - At least one "non-crosslinked HALMW" at 1.4-1.8% by mass relative to the total mass of the aqueous gel; and,

[0349] - At least one "non-crosslinked HAHMW" at 0.2-0.6% by mass relative to the total mass of the aqueous gel;

[0350] It also has a total hyaluronic acid concentration between 20 and 30 mg / g, a phase angle (δ) between 15° and 50°, and a G′ between 20 and 150 Pa. This was measured using a rheometer (TAInstruments DHR2) with a cone / plate geometry (cone angle 1° / plate diameter 40 mm) at room temperature with a vibrational stress oscillation at a frequency of 1 Hz and a stress of 5 Pa. The extrusion force is less than or equal to 18 N when measured with a force gauge as follows: in a syringe with an outer diameter greater than or equal to 6.3 mm, with a needle having an outer diameter less than or equal to 0.3 mm (30 G) and a length of 1 / 2″, at a fixed speed of about 12.5 mm / min at room temperature.

[0351] Reagent test kit

[0352] Specifically, the present invention relates to a pre-filled syringe comprising an aqueous gel obtainable by the method according to the invention, or an aqueous gel as defined in the invention, or a cosmetic and / or dermatological composition according to the invention.

[0353] According to another aspect, the present invention relates to a kit comprising a pre-filled syringe and instructions for use, said pre-filled syringe comprising an aqueous gel obtainable by means of the present invention, or an aqueous gel as defined in the present invention, or a cosmetic and / or dermatological composition according to the present invention.

[0354] use

[0355] According to another aspect, the present invention relates to an aqueous gel or a dermatological composition according to the present invention for the prevention and / or treatment of changes in the appearance of the skin surface. Preferably, the present invention relates to an aqueous gel or a dermatological composition as defined herein for enlarging and / or filling soft tissue.

[0356] Aqueous gels or dermatological compositions according to the invention, as defined herein, with viscoelastic or biomechanical properties for the prevention and / or treatment of skin alterations are also described.

[0357] Also described are aqueous gels or dermatological compositions according to the invention, as defined herein, for the prevention and / or treatment of skin signs induced by external factors such as stress, air pollution, tobacco, or prolonged exposure to ultraviolet (UV) radiation.

[0358] The present invention also relates to non-therapeutic cosmetic uses of aqueous gels or cosmetic compositions as defined herein for the prevention and / or treatment of changes in the surface appearance of the skin, such as for the prevention and / or treatment of signs of aging.

[0359] The present invention also relates to the non-therapeutic cosmetic use of aqueous gels as defined herein or cosmetic compositions according to the present invention for enlarging and / or filling soft tissue, particularly for filling wrinkles.

[0360] The present invention also relates to a method for preventing and / or treating changes in the surface appearance of the skin, comprising at least one step of topically applying an effective amount of an aqueous gel as defined in the present invention or a dermatological composition according to the present invention to a subject, such as a patient with such need, or by injecting an effective amount of an aqueous gel as defined in the present invention or a dermatological composition according to the present invention into the body of a subject, such as a patient with such need.

[0361] The present invention also relates to a method for enlarging and / or filling soft tissue, particularly for filling wrinkles, comprising at least one step of topically applying an effective amount of an aqueous gel as defined in the present invention or a cosmetic and / or dermatological composition according to the present invention to a subject, such as a patient with such need, or applying an effective amount of an aqueous gel as defined in the present invention or a cosmetic and / or dermatological composition according to the present invention by injection into the body of a subject, such as a patient with such need. Example

[0362] Material

[0363] Separate sample sets were prepared for the following different examples. Unavoidable differences exist between these series, especially due to the use of different batches of hyaluronic acid or different sterilization cycles. Therefore, it is important to note that comparisons between these series of examples are inappropriate.

[0364] method

[0365] The following scheme was used to study the viscoelastic properties of the compositions analyzed in the examples.

[0366] Rheological characterization of samples

[0367] The viscoelastic properties of the composition were measured at room temperature using a rheometer (TA Instruments DHR2) with a cone / plate geometry (1° cone angle / 40 mm plate diameter). Vibrational stress oscillation measurements were performed at a frequency of 1 Hz. The elastic modulus (G′, in Pa), phase angle (δ, in °), and complex viscosity (η*) were measured under a stress of 5 Pa.

[0368] Characterization of extrusion force of the sample

[0369] The sample extrusion force was determined using a force gauge as follows: a 25-mm drop path, in a 1mL COC syringe with a 6.4mm inner diameter, at a fixed speed of 12.5mm / min at room temperature using a 30G 1 / 2″ needle.

[0370] Characterization of sample degradation resistance over time and under heat

[0371] Hyaluronic acid injectable gels are usually sterilized using an autoclave (moist heat sterilization method).

[0372] The storage stability of these compositions can be evaluated in an accelerated manner through the high-temperature forced degradation test (ASTM F1980-16).

[0373] The degradation of the gel was characterized by a significant loss of elastic modulus (G′).

[0374] In this context, the percentage loss of G' after sterilization (ΔG′(%)) is a relevant indicator of the gel's resistance to degradation over time and under heat sterilization.

[0375] Therefore, the change in G' during sterilization is calculated as follows:

[0376] ΔG′(%)=[(G′ after sterilization - G′ before sterilization) / G′ before sterilization] x 100

[0377] In addition, the percentage improvement of G' is defined as follows:

[0378] G′ Improvement (%) = [(ΔG′Reference Sample - ΔG′Study Sample) / ΔG′Reference Sample] x 100

[0379] Characterization of the sample's resistance to enzymatic degradation

[0380] To investigate the resistance of the samples to enzymatic degradation, 2g of the test gel was contacted with and homogenized with a hyaluronidase (HA) solution (0.5U / mL gel) in phosphate-buffered saline, and then placed in an oven at 37°C for 24h.

[0381] Each sample was characterized by rheological measurements before and after enzyme treatment, and the change in G' before / after degradation was calculated as follows: ΔG′=[(final G′-initial G′) / initial G′]x 100. This change in G' is a direct indicator of the degree of gel degradation in the presence of HA enzyme.

[0382] Characterization of the size and distribution of water-soluble and insoluble hyaluronic acid in the sample

[0383] The Mw and amount of sHA released from HA gel were determined by size exclusion chromatography (SEC) equipped with a multi-angle laser scattering detector (Dawn Neon MALS, Wyatt Technology Corp) and a refractive index detector (Optilab, Wyatt Technology Corp). The instrument used an Agilent Infinity LC system equipped with two sets of size exclusion columns (OHpak LB-806M, Shodex) maintained in tandem flow, covering a wide range of mass-average molar masses (from 0.001 MDa to 20 MDa). A mobile phase of 150 mM sodium nitrate (containing 0.02% NaN3) at pH 7.2 was used as the eluent at a flow rate of 0.3 mL / min. Under these conditions, the refractive index increment (dn / dc) was set to 0.165 mL / g. Chromatograms were obtained and analyzed using ASTRA software (Wyatt Technology Corp).

[0384] Sample preparation involved diluting the aqueous gel in a pH 7.2 solution containing 150 nM sodium nitrate with 0.02% NaN3 at 25°C for 5 days, centrifuging it at 4400 rpm for 10 minutes, and filtering it at 0.45 mm to obtain a filtrate. The filtrate was then analyzed using an SEC apparatus prepared as described above.

[0385] Adjust the gel concentration in the mobile phase used for the dilution step and the injection volume in the SEC appropriately based on changes in the supernatant composition to maximize the signal-to-noise ratio and avoid column overload.

[0386] The determined sHA percentage corresponds to the ratio of the mass of water-soluble HA to the total mass of HA in the sample.

[0387] pass 1 H NMR determined the degree of modification of water-soluble hyaluronic acid present in the sample.

[0388] 3 g of each sample was incubated with 10 mL of water for injection at room temperature for 5 days. The samples were then centrifuged briefly (4400 rpm, 10 min) to separate the gel from the supernatant (containing water-soluble HA) and filtered through a 0.45 mm filter to remove insoluble gel aggregates. The supernatant was freeze-dried and dissolved in 1 mL of D₂O. The gel was then analyzed on a 500 MHz Bruker Avance spectrometer. 1 1H NMR analysis. The degree of modification was determined by the molar ratio of the cross-linking agent signal to the HA disaccharide unit signal (HA is cross-linked or non-cross-linked).

[0389] Example 1

[0390] The effect of supplementing non-crosslinked LMW hyaluronic acid to crosslinked HMW hyaluronic acid compositions

[0391] The samples studied are shown below.

[0392] Comparative sample 1-1 is a cross-linked hyaluronic acid gel that was sterilized in an autoclave in a syringe and prepared from HMW hyaluronic acid (4.00 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade) and BDDE (Sigma Aldrich, ≥95%), with a total hyaluronic acid concentration of 20 mg / g and a degree of modification of 2%.

[0393] According to the present invention, Sample 1-2 is a cross-linked hyaluronic acid gel, which is sterilized in a syringe in an autoclave, has a total hyaluronic acid concentration of 20 mg / g, and is prepared from a 1:1 mixture of Sample 1-1 before sterilization with a 20 mg / g solution of uncross-linked LMW sodium hyaluronate (0.10 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade).

[0394] Comparative samples 1-3 are cross-linked hyaluronic acid gels that were sterilized in an autoclave in a syringe, have a total hyaluronic acid concentration of 10 mg / g, and were prepared from a 1:1 mixture of sample 1-1 prior to sterilization and a phosphate-buffered saline solution containing no non-cross-linked hyaluronic acid.

[0395] The characteristics of the samples, their G′, and their G′ loss after sterilization are summarized in Table 1 below. Figure 1 middle.

[0396] Table 1

[0397]

[0398] The results showed that the composition containing LMW hyaluronic acid (Sample 1-2 according to the present invention) had better degradation resistance compared with the composition having the same total hyaluronic acid concentration but not containing LMW hyaluronic acid (Sample 1-1 (reference)).

[0399] Furthermore, it appears that the higher the concentration of cross-linked HMW hyaluronic acid, the greater the stability of the composition. Therefore, compared to sample 1-1 (comparative) containing 20 mg / g HMW hyaluronic acid (ΔG′ = -10.8%), sample 1-3 (comparative, 10 mg / g HMW hyaluronic acid) showed a greater G′ loss (ΔG′ = -21.6%). Nevertheless, this increase in stability is significantly lower than that seen in sample 1-2 (of the present invention) containing non-cross-linked LMW hyaluronic acid (ΔG′ = -2.4%).

[0400] Therefore, the increase in degradation resistance of the composition is not associated with a simple increase in the total hyaluronic acid concentration, but rather with the supplementation of the relevant HMW hyaluronic acid composition.

[0401] Example 2

[0402] The effect of supplementing LMW hyaluronic acid in compositions based on cross-linked HMW hyaluronic acid, optionally containing non-cross-linked HMW hyaluronic acid.

[0403] Sample 2-1 was prepared as follows.

[0404] BDDE-crosslinked hyaluronic acid gels were prepared from uncrosslinked HMW hyaluronic acid (HTL, 4.00 MDa, European Pharmacopoeia pharmaceutical / medical grade) in a base medium. The resulting crosslinked fractions were neutralized and swollen in phosphate-buffered saline to obtain a final gel with 15 mg / g NaHA. This gel was then dialyzed. At this stage, the gel had an elastic modulus G′ of approximately 80 Pa to 120 Pa and a phase angle δ of 7° to 10°.

[0405] A 15 mg / g solution of uncrosslinked HMW sodium hyaluronate (4.00 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade) was added to the crosslinked fraction to achieve a ratio of 30% by mass relative to the total mass of hyaluronic acid in the composition. The gel was homogenized, extruded through a mesh, and filled into 1 mL COC syringes. Finally, the gel was autoclaved.

[0406] The preparation of Sample 2-2 was similar to that of Sample 2-1, but the composition of the uncrosslinked hyaluronic acid gel used was changed so that the final gel further included 10 mg / g of uncrosslinked LMW hyaluronic acid (0.10 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade).

[0407] Samples 2-3 were prepared as in Sample 2-1, but without non-crosslinked 4.00 MDa hyaluronic acid.

[0408] Samples 2-4 were prepared as in Sample 2-1, but with 0.10 MDa hyaluronic acid instead of the non-crosslinked 4.00 MDa hyaluronic acid.

[0409] For each of these samples, the change in G′ (in percentage) was calculated. The results are summarized in the table below.

[0410] Table 2 shows the stabilization of mixtures of cross-linked and non-cross-linked hyaluronic acid.

[0411] Table 2

[0412]

[0413] Table 3 shows the stabilization of individually cross-linked hyaluronic acid.

[0414] Table 3

[0415]

[0416] The results showed that the protective effect of non-crosslinked LMW hyaluronic acid was observed in compositions containing only crosslinked HMW hyaluronic acid (sample 2-4 vs. sample 2-3) and compositions containing a mixture of crosslinked and non-crosslinked HMW hyaluronic acid (sample 2-2 vs. sample 2-1) during autoclaving.

[0417] Therefore, the addition of non-crosslinked LMW hyaluronic acid to compositions containing only crosslinked HMW hyaluronic acid (samples 2-4) or compositions containing a mixture of crosslinked HMW hyaluronic acid and non-crosslinked HMW hyaluronic acid (sample 2-2) advantageously increases their resistance to degradation.

[0418] Example 3

[0419] Rheological and stability studies of hyaluronic acid gels containing different molar masses of non-crosslinked hyaluronic acid

[0420] Rheology of hyaluronic acid solutions with different molar masses

[0421] Solutions of non-crosslinked hyaluronic acid at 10 mg / g (1%) in different molar masses (0.04 MDa, 0.10 MDa, 0.20 MDa, and 1.50 MDa, HTL, European Pharmacopoeia Pharmaceutical / Medical Grade) were prepared in phosphate-buffered saline. The elastic modulus (G′) and complex viscosity (η*) of each of these fractions were determined and detailed in Table 4 below.

[0422] Table 4

[0423]

[0424] **ND is not measurable under 5 Pa stress (Sample 3-1: η* = 2.55 mPa.s and G′ = 0.65 Pa to 0.6 Pa; Sample 3-2: η* = 5.2 mPa.s and G′ = 0.0057 Pa to 1 Pa).

[0425] The above results indicate that uncrosslinked hyaluronic acid with a molar mass of less than or equal to 0.20 MDa, in a 1% concentrated gel, does not exhibit significant elastic properties and has very low viscosity.

[0426] Mechanical contribution

[0427] A mixture of crosslinked and non-crosslinked HMW hyaluronic acid, i.e., samples 3-5 (reference), was prepared as follows. BDDE-crosslinked hyaluronic acid gel was prepared in a base medium from uncrosslinked HMW hyaluronic acid (4.00 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade). The resulting crosslinked fraction was neutralized and swollen in phosphate-buffered saline to obtain a final gel with 15 mg / g NaHA. It was then dialyzed. At this stage, the gel had an elastic modulus G′ of approximately 80-120 Pa and a phase angle δ of 7-10°. A 15 mg / g solution of uncrosslinked HMW sodium hyaluronate (4.00 MDa, European Pharmacopoeia pharmaceutical / medical grade) was added to the crosslinked fraction to achieve a ratio of 30% by mass relative to the total mass of hyaluronic acid in the composition. The gel was homogenized, extruded through a mesh, and filled into a 1 mL COC syringe. Finally, the gel was autoclaved.

[0428] Samples 3-6 to 3-9 were prepared similarly to samples 3-5, but with a non-crosslinked hyaluronic acid gel, which further contained an additional fixed concentration of non-crosslinked hyaluronic acid (1%) relative to the total mass of the composition of 10 mg / g.

[0429] For this addition, non-crosslinked hyaluronic acid of different molar masses was used, namely, for samples 3-6 to 3-9, in the following order: 0.04 MDa, 0.10 MDa, 0.20 MDa, and 1.50 MDa (HTL, European Pharmacopoeia Pharmaceutical / Medical Grade).

[0430] The rheological characteristics of each sample were determined in six syringes before and after sterilization. The results after sterilization are summarized in Table 5 below. Figure 2 middle.

[0431] Table 5

[0432]

[0433] *The mean is significantly different from the mean of the reference sample, p<0.001, two-sample t-test.

[0434] Due to the limited mechanical contribution of uncrosslinked LMW hyaluronic acid, samples 3-6 to 3-8 remained soft gels, i.e., gels with δ between 15° and 45°, G′ less than or equal to 150 Pa, and extrusion force less than or equal to 18 N.

[0435] It should be noted that the presence of additional LMW hyaluronic acid and thus the increased hyaluronic acid concentration in the gel according to the invention does not affect their flexibility.

[0436] On the other hand, comparative samples 3-9 containing additional HMW hyaluronic acid showed a very significant increase in rheological characteristics and were no longer considered as soft gels (G′>150Pa).

[0437] Resistance to degradation

[0438] The resistance of each sample to thermal degradation was studied by calculating the percentage of G' loss after undergoing the same autoclave cycle l.

[0439] The results are summarized in Table 6 below. Figure 2 and Figure 3 middle.

[0440] Table 6

[0441]

[0442] As in Figure 2 As shown, under the sterilization conditions implemented (which were the same for all test samples), reference samples 3-5 suffered approximately 20% loss.

[0443] The addition of non-crosslinked HMW hyaluronic acid (1.50 MDa, samples 3-9) did not improve the loss of G' after sterilization, and it was still higher than the loss of G' (20%) in reference samples 3-5.

[0444] Conversely, according to a supplement of the invention, the addition of hyaluronic acid having a molar mass in the range of 0.04 MDa to 0.20 MDa, more preferably 0.08 MDa to 0.10 MDa, is a source of reduced G′ loss after sterilization (comparison of samples 3-6 to 3-8 with sample 3-5), i.e., a source of increased degradation resistance of the composition.

[0445] Therefore, compared with compositions having the same total hyaluronic acid concentration and the same crosslinking rate of HMW hyaluronic acid fraction, but without supplementation, compositions according to the present invention supplemented with LMW hyaluronic acid have better degradation resistance.

[0446] The improvement is minor, but noteworthy at the lowest molar mass of hyaluronic acid used (0.04 MDa) (samples 3-6). The improvement is significant for 0.10 MDa and 0.20 MDa hyaluronic acid (samples 3-7 and 3-8).

[0447] Therefore, comparing the average of six measurements taken from six syringes of each product after sterilization showed that the G' values ​​of samples 3-7 and 3-8 were significantly higher than those of sample 3-5. Figure 2 and 3 ).

[0448] This increase in G′ is related to the interaction between LMW HA and HMW hyaluronic acid used in the formulation, and in particular to the effect of improved resistance to degradation (which is related to the presence of LMW HA in the composition, as can be seen from the improvement in G′ loss value after sterilization).

[0449] Therefore, while the addition of non-crosslinked HMW hyaluronic acid (e.g., 1.50 MDa) increases the mechanical properties of the composition without significantly improving its resistance to degradation, the addition of non-crosslinked LMW HA increases the gel's resistance to degradation while maintaining the mechanical properties of the soft gel.

[0450] Example 4

[0451] Impact of changes in the supplement

[0452] For this embodiment, the comparative sample 4-1 is the same as the sample 3-5 (comparative) prepared in Example 3. Different samples were prepared to study the effect of different concentrations of non-crosslinked LMW HA.

[0453] Sample 4-1 was compared with Samples 4-2 to 4-4, which were prepared in a similar manner, but the composition of the non-crosslinked hyaluronic acid gel used in Samples 4-2 to 4-4 further included an increasing concentration of non-crosslinked LMW HA (0.10 MDa HTL, European Pharmacopoeia pharmaceutical / medical grade).

[0454] The proportion of non-crosslinked LMW HA (0.10 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade) corresponds to the ratio (%) of the mass of non-crosslinked LMW hyaluronic acid to the mass of crosslinked HMW hyaluronic acid in the composition.

[0455] The rheological characteristics of the samples and their G′ loss ΔG′ (%) after sterilization are summarized in Table 7 below. Figure 4 middle.

[0456] Table 7

[0457]

[0458]

[0459] The supplementary stabilizing effect can be seen in all samples 4-2 to 4-4 according to the present invention.

[0460] Therefore, it may be possible to obtain the beneficial effect of supplementing a gel containing at least one cross-linked HMW hyaluronic acid with a larger or smaller amount of non-cross-linked LMW HA.

[0461] Furthermore, the above results indicate that when a low proportion of non-crosslinked LMW HA (approximately 50%) or a higher proportion of non-crosslinked LMW HA (approximately 150%) is achieved, a highly concentrated hyaluronic acid composition (up to 30 mg / g total hyaluronic acid) is obtained, which remains soft and easy to inject.

[0462] Example 5

[0463] The effect of HMW hyaluronic acid concentration

[0464] For this embodiment, comparative sample 5-1 was prepared in the same manner as samples 3-5 (comparison) prepared in Example 3. To investigate the effect of a fixed concentration of non-crosslinked LMW hyaluronic acid on compositions of different concentrations of crosslinked and non-crosslinked HMW hyaluronic acid, samples 5-2 and 5-3 according to the present invention were prepared, and they contained a lower concentration of HMW hyaluronic acid than in the previous embodiments.

[0465] The preparation of Sample 5-2 is similar to that of Sample 5-1, but the target concentration of the cross-linked hyaluronic acid fraction is 12 mg / g and the target concentration of the non-cross-linked HMW hyaluronic acid solution is 12 mg / g. Sufficient amounts of 0.10 MDa non-cross-linked hyaluronic acid (HTL, European Pharmacopoeia pharmaceutical / medical grade) are added to achieve a final composition concentration of 16 mg / g of 0.10 MDa non-cross-linked hyaluronic acid.

[0466] Sample 5-3 was prepared similarly to the reference sample, but the target concentration of the cross-linked HMW hyaluronic acid fraction was 9 mg / g and the target concentration of the non-cross-linked HMW hyaluronic acid solution was 9 mg / g. Sufficient amounts of 0.10 MDa non-cross-linked LMW hyaluronic acid (HTL, European Pharmacopoeia pharmaceutical / medical grade) were added to achieve a final composition concentration of 16 mg / g of 0.10 MDa non-cross-linked hyaluronic acid.

[0467] The rheological characteristics of samples 5-1 to 5-3, the proportion (%) of their non-crosslinked LMW hyaluronic acid, and their G′ loss (ΔG′(%)) after sterilization are summarized in Table 8 below.

[0468] Table 8

[0469]

[0470] Due to the addition of non-crosslinked LMW hyaluronic acid, the ratio of crosslinked and non-crosslinked HMW hyaluronic acid may be reduced from 15 mg / g to 12 mg / g, and only a slight decrease in G' was observed (sample 5-1 (comparison) vs. sample 5-2 (invention)).

[0471] Furthermore, due to the addition of non-crosslinked LMW hyaluronic acid, it is possible to change from a gel containing 15 mg / g of hyaluronic acid to a gel containing 28 mg / g of hyaluronic acid while maintaining the mechanical properties of the soft gel (Sample 5-1 (comparative) vs. Sample 5-2 (invention)).

[0472] Samples 5-3 prepared with very small amounts of cross-linked and non-cross-linked HMW hyaluronic acid showed minimal loss of G′ (only -4%) after sterilization.

[0473] The presence of non-crosslinked LMW hyaluronic acid advantageously reduces the amount of crosslinked hyaluronic acid required to prepare the soft gel. It may also increase the total hyaluronic acid concentration. In other words, this supplement thus makes it possible to obtain a soft gel with potentially increased biocompatibility (due to the reduced proportion of crosslinked hyaluronic acid used), but which is also effective from a biomechanical and / or biological perspective.

[0474] Example 6

[0475] Resistance to enzymatic degradation

[0476] Sample 6-1 was prepared using the same method as samples 3-5 (comparison) prepared in Example 3.

[0477] Sample 6-2 was prepared in a similar manner, but the composition of the uncrosslinked hyaluronic acid gel used was changed so that the final gel further contained 10 mg / g of uncrosslinked LMW hyaluronic acid (0.10 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade).

[0478] Sample 6-3 was prepared in a similar manner to Sample 6-1, but the composition of the uncrosslinked hyaluronic acid gel used was changed so that the final gel further contained 10 mg / g of uncrosslinked HMW hyaluronic acid (1.50 MDa, HTL, European Pharmacopoeia pharmaceutical / medical grade).

[0479] All of these samples underwent enzymatic degradation assays using hyaluronidase (HA enzyme).

[0480] The rheological characteristics of the samples and their G' loss due to enzymatic degradation are summarized in Table 9 below. Figure 5 middle.

[0481] Table 9

[0482]

[0483] It should be noted that samples 6-2 and 6-3 (containing hyaluronic acid with molar masses of 0.10 MDa and 1.50 MDa, respectively) have very different starting properties. The properties of sample 6-3 are not suitable for skin regeneration and / or skin improvement applications (see δ, G′, and F).

[0484] The G′ loss due to enzymatic degradation in the presence of non-crosslinked LMW hyaluronic acid is lower than that in the absence of non-crosslinked LMW hyaluronic acid (Sample 6-2 vs. Sample 6-1), and even lower than that in the presence of non-crosslinked HMW hyaluronic acid (Sample 6-2 vs. Sample 6-3). Therefore, the increased resistance exhibited in the composition conforming to the present invention (Sample 6-2) is not related to the simple addition of non-crosslinked hyaluronic acid, but is particularly related to the addition of non-crosslinked LMW hyaluronic acid.

[0485] Therefore, compared with a composition having the same total hyaluronic acid concentration, the same crosslinking rate of the hyaluronic acid fraction, but containing only HMW hyaluronic acid, the composition supplemented according to the present invention has better resistance to enzymatic degradation.

[0486] This increased resistance to hyaluronidase degradation indicates increased in vivo durability of the compositions according to the invention.

[0487] Finally, the above results highlight the protective effect of non-crosslinked LMW hyaluronic acid, thus emphasizing the beneficial effects of supplementing compositions based on at least one crosslinked glycosaminoglycan.

[0488] Example 7

[0489] Characterization of water-soluble and water-insoluble hyaluronic acid present in the sample

[0490] As described above, the water-soluble and water-insoluble hyaluronic acid (sHA and nsHA) of several sterilized samples were characterized. HA raw materials were obtained from HTL for pharmaceutical / medical grade according to the European Pharmacopoeia.

[0491] The following compositions are considered as references:

[0492] - Sample 7-1: Cross-linked HA HMW: Cross-linked HA gel prepared in phosphate buffer with HA alone 4,00 MDa; the cross-linked HA was prepared as the cross-linked HA portion of Sample 2-1, but was swollen to obtain a gel with a NaHA concentration of 10.5 mg / g relative to the total gel mass.

[0493] - Sample 7-2: Non-crosslinked HA HMW: Non-crosslinked HA prepared in phosphate buffer with HA 4,00 MDa alone, having a NaHA concentration of 4.5 mg / g relative to the total mass of the gel;

[0494] - Sample 7-3: Non-crosslinked HA LMW: Non-crosslinked HA prepared in phosphate buffer with 0.10 MDa of HA alone, having a NaHA concentration of 10 mg / g relative to the total mass of the gel.

[0495] The following three samples had different proportions of HMW HA and LMW HA added during the process.

[0496] - Sample 7-4: A gel, as previously presented as Sample 2-4, comprising, prior to sterilization, cross-linked HAHMW prepared with HA 4.00 MDa alone and non-cross-linked HALMW prepared with HA 0.10 MDa;

[0497] - Sample 7-5: A gel containing, prior to sterilization, cross-linked HA prepared with HA HMW 4.00MDa alone and non-cross-linked HA LMW prepared with HA 0.10MDa and non-cross-linked HA HMW prepared with HA 4.00MDa; the gel corresponds to Sample 2-1 and further contains an additional fixed concentration (1%) of non-cross-linked HA LMW 0.10MDa at 10 mg / g relative to the total mass of the gel;

[0498] - Sample 7-6: A gel, as previously presented in Sample 2-1, comprising cross-linked HAHMW prepared with HA 4.00MDa and uncross-linked HA HMW 4.00MDa.

[0499] All samples 7-1 to 7-6 were sterilized by autoclaving (moist heat) for F0 > 15 min.

[0500] In addition, a product commercialized by Allergan, Juvederm Volift was tested as sample 7-7. This product is manufactured using Vycross crosslinking technology, which is known to refer to crosslinked hyaluronic acid (including a high proportion of LMW HA) prepared from hyaluronic acid with different average molar masses. The product contains lidocaine hydrochloride, has a total HA concentration of 17.5 mg / g, and is sterilized by moist heat.

[0501] Table 10

[0502]

[0503]

[0504] Sample 7-1 (100% HA involved in the crosslinking reaction) showed sHA with a MOD of 17%, while samples 7-2 and 7-3 (sterilized, uncrosslinked HA alone) logically had no MOD. The crosslinked sample 7-1 released sHA fragments (primarily LMW) that were highly modified by the crosslinking agent. Sample 7-2, prepared using 4.00 MDa uncrosslinked HA feedstock, exhibited its largest sHA proportion above 0.30 MDa, while Sample 7-3, prepared using 0.10 MDa uncrosslinked HA feedstock, showed a total sHA proportion below 0.30 MDa.

[0505] Because uncrosslinked LMW (0.10 MDa)HA increases the molar amount of HA disaccharide units in sHA, thereby reducing the MOD value, the supplemented compositions according to the invention contain sHA with a lower MOD (samples 7-4 vs. 7-1, 7-5 vs. 7-6) compared to compositions prepared without supplementation. Notably, the gels according to the invention (samples 7-4 and 7-5) exhibit a higher proportion of LMW sHA below 0.3 MDa compared to samples 7-6 and 7-7. In particular, commercial sample 7-7, manufactured using a high proportion of uncrosslinked LMW HA for crosslinking, did not exhibit such a high amount of LMW sHA.

[0506] We can theoretically expect that a composition containing cross-linked HAHMW supplemented with non-crosslinked HALMW will have a total sHA percentage equal to the sum of the following:

[0507] - The percentage of non-crosslinked HALMW relative to the total mass of HA in the composition, and

[0508] - The percentage of total sHA relative to the total mass of HA, measured separately for the crosslinked portion.

[0509] Sample 7-1 (100% HA involved in the crosslinking reaction) showed a total sHA of 20.6% relative to the total mass of HA in the composition. Therefore, we can theoretically expect that Sample 7-3 (based on the same crosslinked HA as Sample 7-1, further containing non-crosslinked HALMW) will have approximately 30 + 20.6 = 50.6% sHA relative to the total mass of HA in the composition.

[0510] However, the measured total sHA percentage for sample 7-3 was approximately 10 points lower (40.5%). This indicates that the supplement according to the invention protects the HA chains during the manufacturing process, thereby reducing the release of LMW sHA; in other words, it increases the composition's resistance to degradation.

[0511] In summary,

[0512] SEC and 1 1H NMR technology can be successfully used to characterize gel sHA and help distinguish standard gels from gels according to the present invention.

[0513] Example 8

[0514] The effect of the mass ratio of "HALMW non-crosslinked" to "HAHMW"

[0515] The samples used in the study are presented below.

[0516] Sample 8-1 according to the present invention is a cross-linked hyaluronic acid gel sterilized in a syringe by autoclaving (F0>15 minutes), having a total hyaluronic acid concentration of 20 mg / g, and obtained by mixing the following substances in a 1:1 ratio before sterilization:

[0517] - A cross-linked hyaluronic acid gel prepared using non-cross-linked HA HMW (4.00 MDa, HTL, pharmaceutical / medical European Pharmacopoeia grade) and BDDE (Sigma-Aldrich, ≥95%), having a total hyaluronic acid concentration of 20 mg / g and a cross-linking rate of 2% in phosphate buffer; and,

[0518] -20 mg / g of non-crosslinked HA LMW (0.10 MDa, Altergon, pharmaceutical / medical European Pharmacopoeia grade) non-crosslinked hyaluronic acid solution.

[0519] Sample 8-2 is a comparative gel prepared as in Sample 8-1, but with the 20 mg / g non-crosslinked hyaluronic acid solution of non-crosslinked HALMW (0.10 MDa, Altergon, pharmaceutical / medical European Pharmacopoeia grade) in phosphate buffer replaced by a non-crosslinked hyaluronic acid solution of 2 mg / g non-crosslinked HALMW (0.10 MDa, Altergon, pharmaceutical / medical European Pharmacopoeia grade).

[0520] Sample 8-3 is a reference gel with a crosslinked HMW HA concentration of 10 mg / g relative to the total mass of the gel. It was prepared as in Sample 8-1, but with a separate phosphate buffer solution instead of a 20 mg / g solution of non-crosslinked HALMW (0.10 MDa, Altergon, pharmaceutical / medical European Pharmacopoeia grade) of non-crosslinked hyaluronic acid.

[0521] Table 11

[0522]

[0523] Sample 8-1 showed a good percentage improvement in G', while sample 8-2 showed a lower percentage improvement in G'. Therefore, it can be inferred that a sufficient proportion of LMW HA is required to obtain a significant percentage improvement in G'.

Claims

1. A method for preparing an aqueous gel, said aqueous gel comprising at least one cross-linked hyaluronic acid and at least one non-cross-linked hyaluronic acid, said method comprising at least the following steps: a) Obtain an aqueous solution containing at least one cross-linked hyaluronic acid, referred to as "cross-linked HA HMW", which is formed from non-cross-linked hyaluronic acid having a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa and a cross-linking agent; b) Obtain an aqueous solution containing at least one non-crosslinked hyaluronic acid, referred to as "non-crosslinked HALMW," having a mass-average molar mass in the range of 0.04 MDa to 0.30 MDa; and, c) Forming a homogeneous mixture of all or part of the solutions from steps a) and b). The characteristic is that it contains hyaluronic acid in a mass ratio of "non-crosslinked HA LMW" to "HA HMW" of 1:3 to 2:1, and The characteristic is that the aqueous gel has a total hyaluronic acid concentration in the range of 20-40 mg / g relative to the total mass of the aqueous gel. The characteristic is that the "crosslinked HA HMW" is prepared with a crosslinking rate of less than or equal to 8% by mass.

2. The method according to claim 1, characterized in that the non-crosslinked HA LMW has a mass-average molar mass in the range of 0.08 MDa to 0.20 MDa; and the "crosslinked HA HMW" is prepared with a crosslinking rate of less than or equal to 5% by mass.

3. The method according to claim 1, characterized in that the non-crosslinked HA LMW has a mass-average molar mass in the range of 0.08 MDa to 0.15 MDa; and the "crosslinked HA HMW" is prepared with a crosslinking rate of less than or equal to 2% by mass.

4. The method according to any one of claims 1-3, characterized in that the crosslinking agent used to form the "crosslinked HA HMW" is a bifunctional or multifunctional epoxy crosslinking agent.

5. The method according to claim 4, characterized in that the crosslinking agent used to form the "crosslinked HA HMW" is 1,4-butanediol diglycidyl ether.

6. The method according to any one of claims 1-3, the method further comprising using at least one non-crosslinked hyaluronic acid having a mass-average molar mass greater than or equal to 0.50 MDa, referred to as "non-crosslinked HAHMW".

7. The method according to claim 6, characterized in that the at least one non-crosslinked hyaluronic acid has a mass-average molar mass greater than 1.00 MDa.

8. The method according to claim 6, characterized in that the at least one non-crosslinked hyaluronic acid has a mass-average molar mass in the range of 1.00 MDa to 4.00 MDa.

9. The method according to claim 6, characterized in that all or part of the "non-crosslinked HA HMW" is used in the aqueous solution of step b).

10. The method according to claim 9, characterized in that, in step c), the aqueous solution of the "non-crosslinked HA HMW" and the aqueous solution of step b) are simultaneously mixed with the aqueous solution of step a).

11. The method according to any one of claims 1-3, characterized in that step c) comprises at least one step of extruding the mixture formed by at least one grid.

12. The method according to any one of claims 1-3, characterized in that it comprises using "crosslinked HA HMW" at a concentration of at least 5 mg / g relative to the total mass of the aqueous gel.

13. The method according to any one of claims 1-3, characterized in that it comprises using the "non-crosslinked HA LMW" at a concentration of at least 3 mg / g relative to the total mass of the aqueous gel.

14. The method of claim 13, characterized in that it comprises using the "non-crosslinked HA LMW" at a concentration of at least 10 mg / g relative to the total mass of the aqueous gel.

15. The method according to any one of claims 1-3, characterized in that the aqueous gel has a total hyaluronic acid concentration in the range of 20-35 mg / g relative to the total mass of the aqueous gel.

16. The method according to claim 15, characterized in that the aqueous gel has a total hyaluronic acid concentration in the range of 20-30 mg / g relative to the total mass of the aqueous gel.

17. The method according to any one of claims 1-3, characterized in that it comprises using hyaluronic acid at a mass ratio of "non-crosslinked HA LMW":"HA HMW" of 1:3 to 5:

3.

18. The method of claim 17, characterized in that it comprises using hyaluronic acid at a mass ratio of "non-crosslinked HA LMW":"HA HMW" of 1:2 to 5:

3.

19. The method according to any one of claims 1-3, characterized in that the aqueous gel has a degree of modification of the hyaluronic acid of less than or equal to 5%.

20. The method according to claim 19, characterized in that the aqueous gel has a degree of modification of less than or equal to 3% of the hyaluronic acid.

21. The method according to claim 19, characterized in that the aqueous gel has a degree of modification of less than or equal to 1% of the hyaluronic acid.

22. The method according to any one of claims 1-3, characterized in that it uses: - In step a), an aqueous solution containing 5-15 mg / g of "crosslinked HA HMW" is used; - In step b), an aqueous solution comprising 50-60 mg / g of "non-crosslinked HA LMW" and 5-20 mg / g of "non-crosslinked HAHMW" is characterized in that, The gel formed has a total hyaluronic acid concentration of 20-30 mg / g relative to its total mass.

23. The method according to any one of claims 1-3, the method for preparing a sterile and injectable gel, characterized in that it comprises at least one step of using an aqueous gel obtained according to any one of claims 1-3, and a heat sterilization step.

24. The method of claim 23, wherein the heat sterilization step is performed in an autoclave.

25. The method of claim 23, characterized in that the gel is packaged in a syringe before sterilization.

26. The method of claim 24, characterized in that the gel is packaged in a syringe before sterilization.

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