METHOD FOR PREPARING NON-COVALENT MOLECULAR COMPLEXES COMPRISING HYALURONIC ACID AND AT LEAST A PEPTIDE FRACTION, COSMETIC AND PHARMACEUTICAL COMPOSITIONS COMPRISING IT AND THEIR USES

Abstract

The invention relates to a method for preparing a molecular complex comprising at least one peptide fraction and hyaluronic acid with a molecular weight between 1 and 100 kDa, characterized in that the complex is not covalently bonded. The invention further relates to cosmetic compositions comprising such a molecular complex and their cosmetic uses for preventing or limiting the signs of skin aging. The invention also relates to compositions comprising such a molecular complex for use in aesthetic and regenerative medicine to improve wound healing, reduce inflammation, or for use in cartilage repair and the treatment of osteoarthritis. [Figure for the abstract: Figure 3]

Description

Title of the invention: METHOD FOR PREPARING NON-COVALENT MOLECULAR COMPLEXES COMPRISING HYALURONIC ACID AND AT LEAST ONE PEPTIDE FRACTION, COSMETIC AND PHARMACEUTICAL COMPOSITIONS COMPRISING THEREOF AND THEIR USES technical field

[0001] The present invention belongs to the field of ingredients comprising hyaluronic acid. It relates more particularly to a method for preparing covalently non-bonded molecular complexes, comprising hyaluronic acid and at least one peptide fraction composed of a peptide or a peptide extract, and their cosmetic and pharmaceutical uses. Technical background of the invention

[0002] Hyaluronic acid, or hyaluronan, is a linear polymer of glycosaminoglycans with a molar mass ranging from 100 to 10,000 kDa. It is naturally present in the extracellular matrix of epithelial, nervous, and connective tissues of vertebrates. It can form bonds with proteins, lipids, or carbohydrates to create supramolecular structures capable of trapping large quantities of water and ions.

[0003] In joints, hyaluronic acid contributes to lubrication, protects areas of friction, and maintains cartilage elasticity. In the skin, it contributes to hydration, turgor, and tissue cohesion. It also plays a role in controlling angiogenesis, inflammation, and wound healing.

[0004] These multiple properties, coupled with high biocompatibility and biodegradability, have made hyaluronic acid a widely used molecule in medicine, bioengineering and cosmetics.

[0005] However, the hyaluronic acid content decreases in the skin during aging, particularly in the epidermis, and its degradation is increased in certain inflammatory or degenerative pathologies such as osteoarthritis.

[0006] Peptides are also widely used in the cosmetics industry, primarily in the form of short-chain peptides of synthetic or natural origin. Examples include tripeptide-1, with sequence GHK, whether or not it is copper-bound, which stimulates collagen production, and certain peptides marketed by Ashland, such as peptide with sequence (Gly-Pro-Gln)2, marketed under the name Collaxyl™, described in European patent No. 406589, for its effectiveness in the treatment of skin and hair aging, the tripeptide with the trade name ATPeptide™ described in patent No. FR2846883), the hexapeptide with the trade name Peptide vinci™ 02 described in patent FR284178, or the dimerized dipeptide with the trade name Quintescine™ described in patent No. EP1799188B1.

[0007] Numerous peptide extracts have also been described for their beneficial properties on the skin. These are generally aqueous plant extracts or fermentation products that can be hydrolyzed, thereby improving their tolerance and bioavailability.

[0008] Many cosmetic formulas contain both peptides and hyaluronic acid, such as those described in patent documents EP1815843A2, KR2023134758 and CN116942584. However, nothing in these documents suggests that the formulations have been optimized to allow the formation of complexes between hyaluronic acid and peptides.

[0009] Hybrid molecules of hyaluronic acid and peptides, formed by the creation of covalent bonds between the two compounds, are also known in the prior art. These are widely used in the cosmetic industry as injectable dermal fillers or for topical application. This involves the use of crosslinking agents or the prior chemical modification of at least one of the two components. For example, document WO2017098474A1 describes a hyaluronic acid composition whose polymer chain has been modified to bear hydrophobic groups capable of covalently binding to an antimicrobial peptide.

[0010] One of the problems posed by these techniques is the toxicity, or even the carcinogenic effects, of the crosslinking agents that can be released into the body during the degradation of hyaluronic acid (Xue Y, et al. Synthesis of hyaluronic acid hydrogels by crosslinking the mixture of high-molecular-weight hyaluronic acid and low-molecular-weight hyaluronic acid with 1,4-butanediol diglycidyl ether. RSC Adv. 2020;10(12):7206-7213; Faivre J et al. Crosslinking hyaluronic acid soft-tissue fillers: current status and perspectives from an industrial point of view. Expert review of medical devices vol. 18,12(2021): 1175-1187).

[0011] Other documents describe the chemical functionalization of hyaluronic acid molecules to enable the creation of interchain amino covalent bridges, to improve the in vivo stability of a hydrogel for aesthetic use (US11224566B2), or the creation of disulfide covalent bridges to prepare complexes useful in skin care (CN116966114A).

[0012] Patent document EP3527194B1 describes, for its part, a method for the preparation of composite particles comprising hyaluronic acid (of high molecular weight, greater than 100 kDa) and at least one peptide not involving Chemical reactions. In this case, the peptide must be in its cationic form, at a pH of 5 or lower. This method allows for the production of particles of a defined size (less than 500 nm) intended to improve skin penetration. However, this technique requires adjusting the pH to a value lower than the pKa of each peptide to ensure that it is entirely in its cationic form.

[0013] To overcome the technical problems of the state of the art, the inventors have developed a process for preparing molecular complexes comprising at least one peptide or a peptide extract and non-crosslinked and non-functionalized hyaluronic acid, to produce a molecular complex exhibiting satisfactory stability and biological properties superior to molecules used in isolation.

[0014] An advantage of the process described in this application is that it eliminates the need to operate under pH conditions lower than the pKa of the peptide(s) used in order to form cations capable of self-assembly with anionic hyaluronic acid polymers.

[0015] Another advantage of the process described below is that it is simple to implement, more natural and therefore more environmentally friendly than prior art processes, since it does not involve prior chemical modifications of the compounds, nor the use of potentially toxic crosslinking agents.

[0016] To the inventors' knowledge, no prior art document describes the preparation process of the present application. Summary of the invention

[0017] The invention has as its first object a method for preparing a molecular complex comprising at least a peptide fraction and hyaluronic acid of a molecular weight between 1 and 100 kDa, characterized in that the molecular complex is not linked by covalent bonds.

[0018] The second object of the invention is the molecular complex that can be obtained according to the process of the invention.

[0019] The invention has as its third object a composition comprising an effective amount of the molecular complex of the invention as an active agent and a physiologically acceptable medium.

[0020] The invention has as its fourth object the cosmetic use of the composition of the invention to prevent or limit the signs of skin aging and in particular to firm the skin, improve its elasticity and hydration, reduce and limit the appearance of wrinkles and fine lines and also improve the homogeneity of the complexion.

[0021] The invention has as a fifth object a composition according to the invention for its use in aesthetic and regenerative medicine to improve healing, to limit inflammation, or for its use in repairing cartilage and treating osteoarthritis. Brief description of the figures

[0022] The following figures illustrate the advantages arising from the invention and the non-limiting embodiments presented in the description:

[0023] [Fig-1] Comparison of the molecular structures of the obtained molecular complexes according to example 1 and at different pH levels using FTIR

[0024] [Fig.2] Evaluation of the effectiveness of the molecular complex of example 1 on the expression of different types of collagen and the synthesis of hyaluronic acid in ex vivo skin biopsies

[0025] [Fig. 3] Demonstration of the synergistic properties of the molecular complex of Example 1 on the expression of collagen I in ex vivo skin biopsies. Detailed description of the invention. Definitions

[0026] All terms used in this description have their most widely understood meanings, unless otherwise stated. For the purposes of the invention, the following terms are defined as follows:

[0027] In this application, "hyaluronic acid" means an unbranched carbohydrate polymer formed by alternating units of glucuronic acid and N-acetylglucosamine, having a molecular weight between 1 kDa and 100 kDa.

[0028] When a range of values ​​is described, the bounds of that range must be understood as explicitly including the upper and lower bounds of said range, as well as all intermediate values ​​of the range. For example, a range of values ​​between 1% and 10% must be understood as including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all decimal values ​​between 1% and 10%.

[0029] Numerical values ​​in percentage and ppm (parts per million) are by weight, i.e. the weight of a compound in relation to the total weight of the mixture envisaged, unless otherwise specified.

[0030] The compositions described in this application may "include", "consist of" or "consist essentially of", the essential compounds or the optional ingredients.

[0031] “Consist essentially of” means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic characteristics or new characteristics of the composition or use described in this application.

[0032] The term “physiologically acceptable medium” means a solvent suitable for any route of administration in the body, for example oral route or injection, and more particularly for contact with the external layers of the skin, scalp or hair, without toxicity, irritation, undue allergic response and similar or intolerance reaction, and proportionate to a reasonable benefit / risk ratio, at the concentrations used.

[0033] By "effective quantity" is meant the minimum quantity of molecular complex according to the invention which is necessary to obtain at least one of the desired biological activities.

[0034] The term “Tripeptide-1” refers to the peptide with the sequence H-Gly-His-Lys-OH

[0035] The term “Hexapeptide-9” means the peptide consisting of the sequence (SEQ ID: No. 1), H-Gly-Pro-Gln-Gly-Pro-Gln-OH, marketed under the name Collaxyl™ biofunctional by Ashland.

[0036] In this application, "molecular complex", "molecular complexes" or "HA-peptide complex" means one or more complexes formed from several types of molecules not covalently linked to each other, obtained by self-assembly and comprising hyaluronic acid and at least one peptide fraction, i.e. a peptide or a peptide extract, or any mixture thereof.

[0037] In the present application, "self-assembly" means the phenomenon of spontaneous association of molecules of different nature to form supramolecular structures not covalently linked to each other.

[0038] In this application, the term “peptide fraction” means synthetic peptides or peptides purified from natural elements, or aqueous peptide extracts from plants, algae, fungi, or yeasts. These peptide extracts may contain one or more peptides with molecular weights of less than 5 kDa.

[0039] In the present application, "hybrid" means a particular formulation comprising 5% to 40% of the molecular complex of the invention, incorporated into a cosmetic vector composed of water, glycerin and 1% to 10% of a mixture of several carboxymethyl celluloses of different grades and degrees of substitution.

[0040] The term “peptide” means a molecule composed of 2 to 20 amino acids linked together by peptide bonds, of natural or synthetic origin, linear or cyclic, substituted or unsubstituted. Preparation process

[0041] The present invention relates to a new method for preparing molecular complexes of hyaluronic acid and at least one peptide fraction, capable of self-assembling in a non-covalent manner.

[0042] The invention thus has as its first object a method for preparing a molecular complex comprising at least a peptide fraction and hyaluronic acid of a molecular weight between 1 kDa and 100 kDa, characterized in that the molecular complex is not linked by covalent bonds.

[0043] The spatial organization of hyaluronic acid is controlled by the combination of factors such as molecular weight and concentration of the biopolymer, with physical factors such as temperature, pH and method of dissolution.

[0044] Hyaluronic acid of intermediate or high molecular weight (e.g. greater than 100 kDa), at a concentration less than Img / ml, is in the form of long supercoiled chains, stabilized by a high number of inter-chain interactions (De Souza A. et al., Carbohydrate Polymers 222 (2019), 115001). This spatial conformation is not very favorable to the formation by self-assembly of complexes with peptides or peptide extracts.

[0045] The inventors have shown that it is advantageous, in order to implement the process of the present invention, to use hyaluronic acid of low molecular weight, that is to say less than 100 kDa, and to place oneself in conditions which favor the spatial conformation of hyaluronic acid in a double helix.

[0046] The process is based on the rearrangement of the spatial architecture of hyaluronic acid in solution in the form of short antiparallel double helices, to promote their non-covalent self-assembly with peptides to form molecular complexes.

[0047] Advantageously, the peptide fraction comprises at least one peptide comprising from 2 to 20 amino acids or at least one peptide extract, or any mixture thereof. Preferably, the peptide(s) have a length of between 3 and 12 amino acids and more preferably between 3 and 9 amino acids.

[0048] In another advantageous embodiment, the peptide fraction comprises at least one peptide extract and the peptides contained in this extract have a molecular weight of less than 5 KDa.

[0049] The preparation process comprises the following steps:

[0050] Step a) Solubilize hyaluronic acid, initially in powder form, with a molecular weight between 1 and 100 kDa, preferably between 3 kDa and 10 kDa, and more preferably between 3 kDa and 5 kDa, in an aqueous solvent which may optionally contain different types of solutes. Preferably, the solvent is pure water. Advantageously, the concentration of hyaluronic acid is between 500 ppm and 50,000 ppm, preferably between 1,000 ppm and 10,000 ppm, and more preferably between 5,000 ppm and 10,000 ppm. Solubilization is carried out under moderate stirring, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C. The stirring method may include any known method. from a person skilled in the art, such as magnetic stirring. The solubilization time should be between 5 and 30 minutes and is proportional to the molecular weight and concentration of the hyaluronic acid used.

[0051] Step b) Add the peptide fraction to the hyaluronic acid solution obtained in the previous step. The mass ratio of hyaluronic acid to peptide fraction should be between 10:1 and 2:1, preferably between 4:1 and 2:1. The peptide fraction is in powder form when it is synthetic or purified peptides, but can also be in liquid form if it is a peptide extract. Solubilization, if necessary, is carried out under moderate stirring at a temperature between 20°C and 50°C, preferably between 20°C and 30°C. The stirring method may include any method known to those skilled in the art, such as magnetic stirring. This step is continued until all components are completely dissolved.

[0052] Steps a) and b) can be carried out in either the reverse order indicated above.

[0053] Step c) Adjust the pH to a value between 2 and 3.5, preferably between 2.5 and 3, using an acid such as citric, lactic or hydrochloric acid, preferably citric acid.

[0054] Indeed, at pH values ​​close to 2.5 to 3, the literature reports significant structural changes in hyaluronic acid, with the formation of antiparallel double helices (Snetkov, Petr et al. “Hyaluronic Acid: The Influence of Molecular Weight on Structural, Physical, Physico-Chemical, and Degradable Properties of Biopolymer.” Polymers vol. 12, 8, 1800. 11 Aug. 2020). The process of the invention thus exploits this particular structure of hyaluronic acid at pH values ​​close to 2.5 to 3, allowing the formation of molecular complexes with peptide fractions (peptides or peptide extracts), which was not suggested in the prior art. At a pH below 2.5, hyaluronic acid is in a single-stranded form, which is very rapidly hydrolyzed.

[0055] Step d) Leave under medium stirring for a period of between 30 min and 3 hours, preferably 1 hour, at a temperature between 20°C and 50°C, preferably at a temperature between 20°C and 30°C. The stirring method may include any method known to a person skilled in the art, such as magnetic stirring.

[0056] Step e) Adjust the pH to a value between 4.5 and 6.5, preferably between 4.5 and 5.5, by adding a basic solution, preferably concentrated sodium hydroxide. This increase in pH allows the peptide fractions to be trapped by the abrupt change in the spatial organization of the small doublet structures. hyaluronic acid helices formed in step c). This step allows higher order molecular self-assembly, and the formation of stable molecular complexes.

[0057] The solution obtained in step e) constitutes the molecular complex.

[0058] To carry out the process described above, when the peptide fraction is composed of one or more peptides, their concentration can be between 10 ppm and 25,000 ppm, even more preferably between 30 ppm and 5,000 ppm.

[0059] In a particular embodiment, the peptide fraction is composed of at least one synthetic peptide with a length of 3 to 9 amino acids.

[0060] To carry out the above process, the peptides may have a linear or cyclic structure, and may be of natural or synthetic origin. When the peptides are synthetic, they may or may not carry chemical modifications well known to those skilled in the art, such as one or more amidations, carboxylations, myristoylations, palmitoylations, or farnesylations, or any combination of such modifications. When the peptides are synthetic, they may or may not be associated with a vitamin, a trace element, or any other compound to form associations well known to those skilled in the art.

[0061] The process for preparing molecular complexes described in this application can also be carried out with natural peptides, from yeast or plant extracts, or with low molecular weight peptide extracts, preferably less than 5 kDa, such as plant peptide extracts of flax (marketed by Ashland under the name lipigenine™ biofunctional and described in patent FR2956818), yeast extracts (marketed by Ashland under the name dynagen™ biofunctional and described in patent FR2951946, or under the name actopontine™ biofunctional, and described in patent FR2944526), ​​extracts of soy, spelt, rapeseed, flax, rice, corn or peas. Molecular complexes

[0062] The second object of the invention is the molecular complex that can be obtained according to the process described above.

[0063] In an advantageous embodiment, the molecular complex comprises hyaluronic acid of a molecular weight between 1 and 100 kDa, preferably between 3 kDa and 10 kDa, and more preferably between 3 kDa and 5 kDa, and at least one peptide fraction selected from a peptide comprising 2 to 20 amino acids, preferably 3 to 12 amino acids and more preferably 3 to 9 amino acids, and a peptide extract of less than 5 kDa, or any mixture thereof.

[0064] In a particular embodiment, the molecular complex comprises at least tripeptide-1 (of sequence GHK) as a peptide fraction.

[0065] In a particular embodiment, the molecular complex comprises, as at least one peptide fraction, the peptide of general formula (I) comprising the sequence (Gly-Pro-Gln)n, in which n is between 1 and 3 and the carboxyl end is amidated or not.

[0066] In a particular embodiment, the molecular complex comprises as a peptide fraction at least of the hexapeptide-9 of sequence Gly-Pro-Gln)2 (SEQ ID No 1).

[0067] In another particular embodiment, the molecular complex comprises a peptide fraction comprising hexapeptide-9 (SEQ ID: No. 1) and tripeptide-1.

[0068] In this last particular embodiment, tripeptide-1 is present at a concentration between 500 ppm and 5000 ppm, preferably at a concentration between 1000 ppm and 3000 ppm.

[0069] In this same particular embodiment, hexapeptide-9 is present at a concentration between 20 ppm and 500 ppm, preferably at a concentration between 50 ppm and 300 ppm.

[0070] In this same particular embodiment, hyaluronic acid is present at a concentration between 5,000 ppm and 10,000 ppm.

[0071] Comparative analysis by FTIR (Fourier Transform Infrared Spectrometry) showed that the molecular complex of the invention has a specific structure.

[0072] Stability studies have shown that the molecular complex of the invention exhibits remarkable stability after 15 days at 50 °C. Compositions

[0073] A third object of the invention is a composition comprising, as an active agent, an effective amount of the molecular complex described above and a physiologically acceptable medium.

[0074] Advantageously, the molecular complex is present in the composition at a concentration of between 0.01% and 10% by weight relative to the total weight of the composition, preferably at a concentration of between 0.05% and 5% by weight relative to the total weight of the composition, and even more preferably at a concentration of between 0.1% and 2% by weight relative to the total weight of the composition.

[0075] The composition of the present application is formulated to be administered by any suitable route, including injection, oral route, or external topical route, and the formulation of the compositions will be adapted by a person skilled in the art.

[0076] Preferably, the composition of the present application is in a form suitable for topical application and comprises a physiologically acceptable medium, particularly suitable for this type of application.

[0077] The compositions may in particular be in the form of an aqueous, hydroalcoholic or oily solution or gel, an oil-in-water, water-in-oil emulsion or multiple emulsions; they may also be in the form of suspensions, or even powders, suitable for application to the skin, mucous membranes, lips and / or hair.

[0078] The composition may be more or less viscous and may also have the appearance of a cream, lotion, fluid, milk, serum, ointment, gel, paste, balm or mousse. It may also be in solid form, such as a stick, or be applied to the skin as an aerosol.

[0079] Examples of physiologically acceptable media commonly used in the intended field of application include formulation aids such as solvents, thickeners, gelling agents, diluents, emulsifiers, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, esters, vegetable oils or butters, etc.

[0080] In all cases, a person skilled in the art will ensure that these adjuvants and their proportions are chosen in such a way as not to harm the advantageous properties sought from the composition according to the invention.

[0081] In an advantageous embodiment, the molecular complex may be pre-included in a particular cosmetic carrier such as a mixture of water, glycerin, and cellulose derivatives. The cellulose derivatives used may be sodium carboxymethyl celluloses, marketed under the name blanose™ carboxymethylcellulose (CMC) by Ashland. For example, several CMCs of different grades and degrees of substitution may be mixed. The total concentration of the CMCs may be between 1% and 10%, and preferably between 1% and 3%. The molecular complex may be incorporated at a concentration of between 5% and 40%, preferably between 5% and 15%, in this cosmetic carrier, by weight relative to the total weight of the composition.

[0082] For example, 10% of the molecular complex may be pre-included in 90% of a cosmetic vector comprising water, glycerin, and 1% to 3% of a mixture of several carboxymethyl celluloses. This particular mixture is described as a hybrid in this application.

[0083] When incorporated into the above cosmetic vector, the molecular complex of the invention forms a composition which exhibits rheological parameters and a texturizing effect particularly sought after in a cosmetic composition.

[0084] Advantageously, the composition may include, in addition to the active agent, i.e. the molecular complex, at least one other active agent having cosmetic effects similar and / or complementary to those of the invention.

[0085] Such additional active agents may also be selected according to their chemical composition, from the group comprising: amino sugars, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, vitamin B3 and its derivatives, niacinamide, sodium dehydroacetate, dehydroacetic acid and its salts, phytosterols, salicylic acid compounds, hexamidines, dialkanoyl dihydroxyproline compounds, soy extracts and derivatives, equol, isoflavones, flavonoids, phytantriol, farnesol, geraniol, bisabolol, peptides and their derivatives, di-, tri-, tetra-, penta-, and hexapeptides and their derivatives, lys-thr-thr-lys-ser, palmitoyl-lys-thr-thr-lys-ser, camosine, N-acyl amino acid compounds, retinoids, retinyl propionate, retinol, retinyl palmitate, retinyl acetate, retinal, retinoic acid, water-soluble vitamins, ascorbates, vitamin C, ascorbyl glucoside, ascorbyl palmitate,magnesium ascorbyl phosphate, sodium ascorbyl phosphate, vitamins and their salts and derivatives, provitamins and their salts and derivatives, panthenol ethyl, vitamin A and its derivatives, vitamin B and its derivatives, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin F, vitamin K and its derivatives, pantothenic acid and its derivatives, pantothenyl ethyl ether, panthenol and its derivatives, ethyl panthenol, dexpanthenol, biotin, amino acids and their salts and derivatives, water-soluble amino acids, asparagine, alanine, indole, glutamic acid, water-insoluble vitamins, beta-ionol, cedrol, and their derivatives, water-insoluble amino acids, tyrosine, tryptamine, particulate materials, butylated hydroxytoluene, butylated hydroxyanisole, allantoin, tocopheryl nicotinate, tocopherol, esters of tocopherol, palmitoyl-glycerol, phytosterol, hydroxy acids, glycolic acid, lactic acid, lactobionic acid, keto acids, pyruvic acid, phytic acid,lysophosphatidic acid, stilbenes, cinnamates, resveratrol, kinetin, zeatin, dimethylaminoethanol, natural peptides, soy peptides, acidic sugar salts, manganese gluconate, zinc gluconate, piroctone olamine, 3,4,4'-trichlorocarbanilide, triclocarban, zinc pyrithione, hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucoside, pyridoxine, aloe vera, terpene alcohols, allantoin, bisabolol, dipotassium glycyrrhizate, glycerol, sorbitol, pentaerythritol, pyrrolidone and its salts, dihydroxyacetone, erythrulose, glyceraldehyde, tartaraldehyde, clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate, eicosene and vinyl pyrrolidone copolymer, iodopropyl butylcarbamate, a polysaccharide, an essential fatty acid, a salicylate, glycyrrhetinic acid, carotenoids, ceramides and , pseudoceramides, a complex lipid, oils in general of natural origin such as shea butter, apricot oil, evening primrose oil, prune oil, palm oil, monoi oil, kahai oil, hydroquinone, HEPES, procysteine, 1'-Octanoyl-6-D-maltose, disodium salt of methylglycine diacetyl, steroids such as diosgenin and DHEA derivatives, DHEA dehydroepiandrosterone and / or a chemical or biological precursor or derivative, N-ethylcarbonyl-4-para-aminophenol, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolytic enzymes, plant extracts, phytohormones, yeast extracts, a metalloproteinase inhibitor, enzymes, inhibitors enzymatics, enzyme inducers, coenzymes, chelating agents, plant extracts and plant derivatives, essential oils, marine extracts,Agents derived from a biofermentation and / or biotechnology process, mineral salts, cell extracts.

[0086] By way of examples, we can also mention the peptides marketed by Ashland under the names Chronogen™, Laminixyl™ IS, Peptide Q10™, Collaxyl™ (patent FR2827170, Ashland), Peptide vinci™ 01 (patent FR2837098, Ashland), Peptide vinci™ 02 (patent FR2841781, Ashland), ATPeptide™ (patent FR2846883, Ashland) and the extract of Artemia salina, marketed under the name GP4G™ (FR2817748, Ashland). Uses

[0087] The fourth object of the invention is the cosmetic use of one of the compositions comprising the molecular complex described above to prevent or limit the signs of skin aging and in particular to firm the skin, improve its elasticity and hydration, reduce and limit the appearance of wrinkles and fine lines and also improve the homogeneity of the complexion.

[0088] The term “cosmetic use” means that the use is intended for individuals with healthy skin, scalp or hair.

[0089] By "signs of skin aging" we mean any change in the external appearance of the skin due to aging such as, for example, the appearance of wrinkles and fine lines at the corners of the lips and crow's feet, sagging, loss of elasticity, firmness and / or tone of the skin, the appearance of a duller and less homogeneous complexion but also any internal changes in the skin which do not systematically result in a changed external appearance such as, for example, thinning of the skin, or any internal degradation of the skin resulting from external aggressions producing free radicals, such as pollution and solar radiation including UV.

[0090] The extracellular matrix is ​​one of the primary determinants of facial morphology; therefore, the molecular complex of the invention was tested on the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid and showed a positive effect on the expression of these compounds.

[0091] The invention also relates to the cosmetic use of the composition comprising the molecular complex described above to increase the expression of collagens I, III, V, VI, VIII, XI, XIIX, XXIV, XXVII, involved in skin aging and more generally in skin laxity and the appearance of wrinkles and fine lines and to increase the expression of collagens IV, VII, XVII present in the dermo-epidermal junction and also to increase the expression of hyaluronic acid.

[0092] The invention also relates to the cosmetic use of a composition comprising between 0.1% and 5%, and preferably between 0.3% and 1% of the molecular complex comprising a) hyaluronic acid of a molecular weight between 3 kDa and 10 kDa, and preferably of a molecular weight between 3 kDa and 5 kDa, present at a concentration between 5,000 ppm and 10,000 ppm, b) tripeptide-1 at a concentration between 500 ppm and 5,000 ppm, preferably at a concentration between 1,000 ppm and 3,000 ppm, c) haxapeptide-9 of sequence (SEQ ID: No. 1) at a concentration between 20 ppm and 500 ppm, preferably at a concentration between 50 ppm and 300 ppm, and d) a physiologically acceptable medium.

[0093] The above composition was tested on the expression of collagen I and showed a positive effect superior to the effect obtained using each compound separately, which demonstrates a synergistic effect between the different components of the molecular complex.

[0094] The invention also relates to pharmaceutical compositions comprising such a molecular complex, which may be in the form of injectable compositions, for oral administration or for topical application, for their uses to stimulate the expression of hyaluronic acid and collagens in various applications in aesthetic and regenerative medicine.

[0095] In particular, the invention relates to a composition comprising the molecular complex for its use to improve healing, limit inflammation, or for its use to repair cartilage and treat osteoarthritis. Examples

[0096] The present invention is illustrated by means of the following non-limiting examples:

[0097] Example 1: Preparation of a molecular complex comprising hyaluronic acid with a molecular weight of 3 kDa to 5 kDa and two peptides

[0098] Principle: the aim of this experiment is to obtain a molecular complex made up of hyaluronic acid with peptides whose molecular interactions are as strong as possible.

[0099] Protocol: The hyaluronic acid used is marketed under the name hyalurotech™ 5 by Ashland. It is derived from bacterial fermentation, is in powder form and has a molecular weight between 3 kDa and 5 kDa.

[0100] a) Hyaluronic acid at a concentration of 6,000 ppm is solubilized in 1 liter of demineralized water, at room temperature (20 °C) under medium intensity magnetic stirring for 5 minutes.

[0101] b) Tripeptide-1 at a concentration of 2000 ppm and hexapeptide-9 at a concentration of 200 ppm, in powder form, are solubilized at room temperature under medium stirring for 20 minutes. The initial pH of this solution is 7.2.

[0102] c) The pH is adjusted by adding 50% diluted citric acid to reach the value of pH 2.8.

[0103] d) The solution is maintained for 1 hour under medium magnetic stirring, at room temperature, at acidic pH 2.8.

[0104] e) The pH is readjusted to a value of 5.0 by the addition of concentrated sodium hydroxide.

[0105] Results: the solution containing the molecular complex obtained is colorless and clear.

[0106] Optionally, the molecular complex obtained above can be diluted to a concentration of 10% in 90% of a cosmetic carrier itself composed of water, glycerin, and 1% to 3% of a mixture of several carboxymethyl celluloses of different grades and degrees of substitution. This particular mixture has the advantage of being easily incorporated into a finished cosmetic formulation and is described in this application as a "hybrid".

[0107] Example 2: FTIR characterization of molecular complexes obtained according to the process of Example 1 and at different pH

[0108] Principle: The aim of this experiment is to validate the interest of the process of example 1, and in particular the role of pH adjustments, by comparing the molecular structures of the molecular complexes obtained according to different preparation processes, by FTIR (Fourier Transform Infrared Spectrometry).

[0109] The infrared spectrum obtained is between 600 and 4,000 cm-1 and can be divided into two:

[0110] The part of the "functional groups" from -4000 cm-1 to 1500 cm-1 is a region of the spectrum that is easily analyzable.

[0111] The "footprint region" goes from 1500 cm-1 to 600 cm-1. This is a complex area to interpret, having only a comparative value.

[0112] Protocol: The three processes for preparing molecular complexes carried out are presented below in Table 1:

[0113] [Tables 1] Process 1 (example 1) Process 2 Process 3 Steps a) and b) of dissolving hyaluronic acid, hexapeptide-9, and tripeptide-1 Yes Yes Yes Step c) adjusting the pH to 2.8 Yes No Yes Step d) stirring for 1 hour Yes Yes Yes Step e) readjusting the pH to 5 Yes No No

[0114] The solutions obtained according to the 3 processes above were frozen at -80°C and then lyophilized to obtain a powder that can be analyzed by FTIR spectrometry on a Perkin Elmer Spectrum 100 FT-IR spectrometer equipped with a diamond ATR module which allows the analysis of powders without special preparation.

[0115] The powders were deposited so as to completely cover the diamond of the FT-IR spectrometer. The samples were then pressed with a pressure force of 80 using a gauge, in order to remove the air trapped between the grains.

[0116] Results: The spectra of the three molecular complexes obtained by FTIR, presented in [Fig. 1], show different profiles in both the functional group region and the "imprint" region. The molecular complexes obtained by the three methods differ in their molecular structure and also have their own "imprint" region. Furthermore, the correlation factor between the spectra of the molecular complex obtained by methods 1 and 3 is low (0.163234), which demonstrates significant differences in the structure of the molecular complexes obtained. Indeed, identical spectra have a correlation of 1, and the lower this correlation, the more different the spectra, and therefore the molecular structures, become.

[0117] Conclusion: pH adjustments cause significant changes in the structure of the molecular complexes obtained.

[0118] Example 3: Evaluation of the stability of molecular complexes by spectrophotometry and colorimetry

[0119] Principle: The aim of this experiment is to evaluate the stability of the molecular complexes obtained according to Example 1 (Procedure 1) and Procedure 2 described in the table 1, and in particular to assess the impact of pH changes on the stability of molecular complexes.

[0120] Protocol: The solutions obtained by processes 1 and 2 (described in Table 1) were placed at 50°C for 15 days. The differences in appearance of the solutions were then analyzed by spectrophotometry by measuring the parameters L*, a*, and b*. L* represents the luminance of the solution, a* and b* express the difference in color compared to a colorless solution, with a* representing the value on a green-red axis and b* on a blue-yellow axis.

[0121] The colour of the solutions was measured according to the Gardner scale, which allows the colours of liquids to be compared on a scale from 0 to 18; 0 being a colourless liquid and 18 being a dark brown liquid.

[0122] Result: On day 0, both solutions are clear and colorless. After 15 days at 50°C, the molecular complex from Example 1 (process 1) has colorimetric parameters similar to those on day 0, whereas the molecular complex obtained by process 2, i.e., without pH adjustment, has turned yellow. The results are presented in Table 2.

[0123] [Tables2] L*a*b* Gardner Molecular complex obtained by process 2 L*=93.16; a*=-2.54; b*=16.44 3.1 Molecular complex obtained by process 1 (example 1) L*=95.94; a*=-0.37; b*=3.11 0.5

[0124] Conclusion: Process 1 (described in Example 1) allows obtaining a stable molecular complex after 15 days at 50 °C, unlike the molecular complex obtained without pH adjustment.

[0125] Example 4: Evaluation of the effect of the hybrid comprising the molecular complex on the expression of different types of collagen and hyaluronic acid

[0126] Principle: The aim of this experiment is to demonstrate an effect of the hybrid obtained according to example 1 and therefore comprising 10% of molecular complex on the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid in ex vivo human skin biopsies.

[0127] Protocol: The expression of the different collagens and hyaluronic acid is evaluated by indirect immunofluorescence on skin biopsies, pre-treated by topical application of the hybrid obtained according to Example 1 at a concentration of 5% diluted in PBS, for 48 hours (twice a day). Control biopsies incubated in parallel under the same conditions receive Phosphate Buffer Saline (PBS). At the end of incubation, the biopsies are fixed and embedded in paraffin and in mounting medium for cryostats, for the preparation of histological sections.

[0128] The detection of different collagens and hyaluronic acid is performed by incubation with antibodies against Collagen I (Proteintech), Collagen III (Proteintech), Collagen IV (Merck Millipore), Collagen V (Proteintech), Collagen VI (Proteintech), Collagen VII (Sigma-Aldrich), Collagen XVII (Abcam), and a biotinylated HABP (hyaluronan-binding protein) probe (Merck Millipore). After two hours of incubation followed by rinsing, the sections are incubated in the presence of a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagens I, III, V, VI, and XVII. The sections are incubated in the presence of the anti-mouse secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagens IV and VIL. A biotin / streptavidin system coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) is used to detect hyaluronic acid.The sections are then examined under an epifluorescence microscope (Nikon Eclipse Ni), controlled by NIS-Element software (Nikon). Expression levels are quantified by image analysis (Volocity® software, Improvision).

[0129] Results: When biopsies were treated with the 5% hybrid, the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid increased significantly after 48 hours of treatment compared with control samples. The results are shown in [Fig. 2].

[0130] Conclusion: The hybrid obtained according to Example 1, comprising 10% of molecular complex, showed a positive effect on the expression of collagens I, III, IV, V, VI, VII, XVII and the synthesis of hyaluronic acid in ex vivo human skin biopsies.

[0131] Example 5: Evaluation of the synergistic effect of the hybrid comprising the molecular complex on the expression of collagen I

[0132] Principle: The aim of this experiment is to demonstrate a synergistic effect of the hybrid obtained according to example 1 and therefore comprising 10% of molecular complex on the expression of collagen I in ex vivo human skin biopsies.

[0133] Protocol: The hybrid processing of example 1 is carried out as in example 4.

[0134] The treatments under control conditions are carried out as follows:

[0135] Stock solutions of hyaluronic acid at 600 ppm, hexapeptide-9 at 20 ppm and tripeptide-1 at 200 ppm, were diluted to 5% in PBS.

[0136] Detection of collagen I is performed by incubation with anti-Collagen I antibody (Proteintech). After two hours of incubation followed by rinsing, the Sections are incubated in the presence of a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagen I. The sections are then examined using an epifluorescence microscope (Nikon Eclipse Ni), controlled by NIS-Element software (Nikon). Finally, expression levels are quantified by image analysis (Volocity® software, Improvision).

[0137] Results: When biopsies were treated with the 5% hybrid, collagen I expression increased more significantly than when biopsies were treated separately with 5% tripeptide-1, 5% hexapeptide-9 or 5% hyaluronic acid, as shown in [Fig. 3].

[0138] Conclusion: The hybrid comprising 10% of the molecular complex of the example 1 showed a synergistic effect on collagen I expression, since the effects of the hybrid are greater than the average increase in collagen I expression specific to each of the compounds taken separately.

[0139] Example 6: Formulation of the hybrid of Example 1 in an aqueous gel

[0140] [Table 3]: List of Ingredients for the formulation of an aqueous gel

[0141] [ref.. # 12951] Ingredient (trade name) Ingredient (INCI) %p / p Supplier Phase A purified water Water / Aqua Qsp. 100 Local natrathix™ bio cellulose Cellulose Gum 0.70 Ashland Sodium citrate tribasic dihydra te Sodium citrate 0.30 Local Phase B Hybrid according to example 1 10.00 Ashland sensiva™ sc 80 multifunctiona 1 Propanediol (and) Capryl yl Glycol (and) Caprylhy droxamic acid 0.80 Ashland hyalurotech™ 1000 / 1800 sodi um hyaluronate Sodium Hyaluronate 0.10 Ashland effisin™ pg natural multifunctional Pentylene Glycol 3.00 Ashland

[0142] Description: transparent gel

[0143] pH: 5.5-6.5

[0144] viscosity: 3.000 - 6.000

Claims

Demands

1. A method for preparing a molecular complex comprising at least one peptide fraction and hyaluronic acid of a molecular weight between 1 and 100 kDa, characterized in that the molecular complex is not linked by covalent bonds.

2. A method according to claim 1 wherein the peptide fraction is selected from a peptide comprising 2 to 20 amino acids, preferably 3 to 12 amino acids, and more preferably 3 to 9 amino acids and a peptide extract, or any mixture thereof.

3. A process according to claim 1 or 2 characterized in that when the peptide fraction comprises at least one peptide extract, then the peptides contained in this extract have a molecular weight of less than 5 KDa.

4. A process according to any one of the preceding claims characterized in that it comprises the following steps: a. solubilizing hyaluronic acid powder of a molecular weight between 1 kDa and 100 kDa, preferably between 3 kDa and 10 kDa, and more preferably between 3 kDa and 5 kDa, in an aqueous solvent, preferably water, at a concentration between 500 ppm and 50,000 ppm, preferably at a concentration between 1,000 ppm and 10,000 ppm, and even more preferably between 5,000 ppm and 10,000 ppm under moderate stirring, for a time between 5 min and 30 min, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C; b.add the peptide fraction to the hyaluronic acid solution obtained in the previous step to obtain a mass ratio between hyaluronic acid and peptide fraction of between 10 / 1 and 2 / 1, and preferably between 4 / 1 and 2 / 1, under moderate stirring, at a temperature between 20°C and 50°C; preferably between 20°C and 30°C; c. adjust the pH to a value between 2 and 3.5, preferably between 2.5 and 3; d. leave under medium stirring for a time of between 30 min and 3 hours, preferably 1. hour, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C; e. adjust the pH to a value between 4.5 and 6.5, preferably between 4.5 and 5.

5.

5. A process according to any one of claims 1 to 4 characterized in that when the peptide fraction comprises at least one peptide, its concentration is between 10 ppm and 25,000 ppm and even more preferably between 30 ppm and 5,000 ppm.

6. Molecular complex capable of being obtained by the process of any one of claims 1 to 5.

7. Molecular complex of claim 6, wherein the peptide fraction comprises at least tripeptide-1.

8. Molecular complex of claim 6, wherein the peptide fraction comprises at least of the peptide of SEQ ID No 1.

9. Molecular complex of claims 7 and 8, wherein hyaluronic acid is present at a concentration of between 5,000 ppm and 10,000 ppm, wherein the peptide fraction comprises tripeptide-1 at a concentration of between 500 ppm and 5,000 ppm, preferably between 1,000 ppm and 3,000 ppm, and sequence hexapeptide-9 (SEQ ID: No. 1) at a concentration of between 20 ppm and 500 ppm, preferably between 50 ppm and 300 ppm.

10. Composition comprising an effective amount of the molecular complex of claim 6 as the active agent and a physiologically acceptable medium.

11. Composition of claim 10, characterized in that it comprises between 0.01% and 10%, preferably between 0.05% and 5% and even more preferably between 0.1% and 2% of the molecular complex by weight relative to the total weight of the composition.

12. Composition of any one of claims 10 or 11, characterized in that 10% of molecular complex is pre-included in 90% of a cosmetic vector comprising water, glycerin and 1% to 3% of a mixture of several carboxymethyl celluloses of different grades and degrees of substitution to form a hybrid.

13. Cosmetic use of the composition of any one of claims 10 to 12 to prevent or limit the signs of skin aging and in particular to firm the skin, improve its elasticity and Its hydration, reducing and limiting the appearance of wrinkles and fine lines, and also improving the evenness of the complexion.

14. Cosmetic use according to claim 13, wherein the expression of collagens I, III, IV, V, VI, VII, XXVIIXVII and XXIV, as well as the expression of hyaluronic acid is increased.

15. Composition of any one of claims 10 or 11 for its use in aesthetic and regenerative medicine to improve wound healing, limit inflammation, or for its use to repair cartilage and treat osteoarthritis.

Images