Pharmaceutical formula for cell and tissue healing and regeneration, and use thereof

Abstract

The present invention relates to a pharmaceutical composition comprising hyaluronic acid or a salt thereof, the molecular weight of which is greater than 2 MDa, and at least 8 amino acids which are essential for humans, selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine. The invention also relates to uses of the pharmaceutical composition for improving wound healing, for hydrating healthy tissue and / or a wound, for improving cell viability, for improving cartilage and / or joint regeneration, or for improving tissue or cell grafting and / or transplantation.

Description

DESCRIPTIONTITLE OF THE INVENTION: Pharmaceutical formula for cell and tissue healing and regeneration, and use thereofFIELD OF THE INVENTIONThe present invention relates to the field of human and / or veterinary medicine, in particular to a pharmaceutical formula for topical, transdermal, subcutaneous and / or intralesional administration, and to the use of the pharmaceutical formula in the treatment of wounds and / or for improving wound healing and / or for joint cartilage regeneration and / or for hydration of the skin and mucous membranes and / or for improving cell viability.PRIOR ARTAt the current time, wounds of all types remain one of the largest, most expensive and most common medical problems around the world. A wound is defined as a break in the protective function of the skin and a loss of continuity of the epithelium, with or without loss of the underlying tissues (i.e., muscles, bones, nerves). Thus, reference is made to a wound when the skin is injured or damaged, regardless of whether deeper tissues have been damaged. This skin damage may have different origins, for example from an accident or trauma, from infection, following a disease process, in particular an inflammatory disease process, or voluntary in the context of a surgical procedure. Wounds are generally categorized based on the duration of wound healing. A distinction is thus made between two major categories of wounds:- acute wounds, which are wounds that appear suddenly and heal within a period of time which is considered normal, i.e. less than 4 weeks, following the usual wound healing process. This category of wounds includes traumatic wounds (grazes, abrasions, lacerations, bites, penetrating wounds, etc.), burns of all degrees and surgical wounds, including grafts; and- chronic wounds, which are wounds for which the wound healing period is lengthened despite suitable management and treatment. Some wounds for which wound healing is not expected within a period of 4 to 6 weeks of progression are automatically considered chronic. This category of wound includes bedsores, diabetic foot wounds, leg ulcers, or amputation stumps, etc.Under normal circumstances, the wound healing process is broken down into 4 stages / phases, namely:- Phase 1: inflammatory (or wound cleaning-inflammatory) phase: a step of vasodilation with bleeding is followed by constriction of the severed vascular ends, with coagulation and production of an exudate rich in cells (granulocytes, macrophages, monocytes) that will eliminate (wound cleaning phase) bacteria, dead tissue and foreign microparticles lymphatically and / or by pus formation. At this stage, the wound has all the characteristic signs of inflammation: redness, swelling, heat and pain. The dilation of the blood capillaries is responsible for redness and heat. The increased permeability of these capillaries promotes plasma exudation, which is responsible for swelling and heat, while the pain is due to pressure on sensitive nerve endings. This reactive phase generally lasts between 3 and 6 days.- Phase 2: granulation or proliferative phase: this phase corresponds to the proliferation of fibroblasts, angiogenesis, and synthesis of the extracellular matrix. Immediately after the inflammatory phase, a granulation tissue with new capillary formation (neovascularization or angiogenesis), which will provide in situ the oxygen, nutrients and cells necessary for tissue repair, begins to become organized within a network of collagen and elastin (produced by fibroblasts). At this stage, macrophages still play an essential role by producing growth factors or cytokines that are capable of promoting fibroblast proliferation and collagen synthesis. At this stage, the wound is a young fibroplastic tissue containing many fibroblasts and a loose fibrillar framework at the periphery of the loss of substance. The granulation tissue is composed of fibroblasts, an inflammatory infiltrate (monocytes, lymphocytes, polynuclear cells), fibrin on the surface and newly formed vessels in an edematous fibrillar framework. The contraction of the wound to bring the edges thereof together is closely linked with the formation of granulation tissue and with the conversion of some fibroblasts into myofibroblasts which are able to contract and to transmit their contractile activity to the surrounding tissue by interaction between proteins in the cytoskeleton and in the extracellular matrix. This phase, which is highly active from day 7, can last up to 3 weeks.- Phase 3: epithelialization phase: after the tissue has been repaired, the wound shrinks and becomes gradually covered with new epithelium; this is the process of epithelialization. Epidermal cells that are capable of dividing (i.e. keratinocytes) multiply and begin to cover the granulation tissue, starting from the edges of the wound. In order to be able to migrate properly, these keratinocytes require healthy, moist and level granulation tissue. Following the formation of this first cell layer, the epithelium is thickened by cell division and soon becomes stronger. The wound is closed. This step lasts 1 to 3 weeks.- Phase 4: remodeling or maturation phase: this phase begins in the early days when a wound has been sutured, but can also last for months in the case of extensive wounds that are mostly open. This phase is characterized by remodeling of the connective tissue and the formation of a scar. The granulation tissue disappears to make way for fibrous connective tissue. Collagen fibres thicken, which increases resistance to tensile forces. The number of capillaries decreases, as does blood flow. The excess water and vessels then disappear, and the scar firms up. Nevertheless, in all events, scars are less strong and less elastic than normal skin, due in part to a deficit in elastin. This phase can last from several months to 2 years.The International Visual Colour Scale of the Wound describes the different phases of wound healing (see Table 1 below) and makes it possible to employ a shared language.[Table 1] BlackNecrotic tissue: wound covered with a blackish, dry or wet plaqueYellowFibrinous tissue: wound covered with yellowish or whitish tissue, more or less adherentRedGranulation tissue: red wound, vascularized, with isolated areas of granulationPinkEpithelialization tissue: wound covered with a thin epithelium. It is pink, pearly or shinyGreenInfected woundAt the cellular and molecular level, keratinocytes and fibroblasts have the property of producing and secreting different partners that are involved in the wound healing process, such as perlecan, laminin or collagen (collagen I and VII). Perlecan acts on keratinocyte proliferation and differentiation, is necessary for epidermis formation, and is involved in the presentation of growth factors to their receptors. The literature teaches that the administration of exogenous perlecan restores epidermis formation (Int J Cosmet Sci. 2020 Dec; 42(6): 529-535). Laminin plays an important role in normal tissues, which is maintaining epithelial-mesenchymal cohesion in tissues exposed to external forces, such as the skin.It therefore appears that laminin and perlecan are markers of interest that make it possible to monitor, or even optimize, dermal-epidermal adhesion during the wound healing process.In parallel, it is known that the main functions of the epidermis include water retention, thermal and pH homeostasis, and protection against the ingress of pathogenic microorganisms or toxic substances into the body. The formation of a suitable epidermal barrier relies on the gradual differentiation of keratinocytes, from the proliferating cells of the basal layer to the horny layer or stratum corneum, i.e. terminal differentiation. The physical properties of the stratum corneum are attributed to the formation of a specialized structure, the cornified envelope (CE) which is highly resistant to strong chemicals such as detergents, or highly alkaline chemicals. The process of strengthening the CE involves, in particular, loricrin, filaggrin or keratin intermediate filaments.It therefore appears that loricrin is a marker of interest which makes it possible to monitor the terminal differentiation of keratinocytes and, ultimately, the strengthening of the epidermis.Furthermore, beta-defensin (or β-defensin) is a natural molecule that acts as a physiological stimulator of cutaneous antibacterial activity (J Immunol August 1, 2008, 181 (3) 2103-2110). This molecule is therefore a good marker for monitoring the development of a wound towards healing or, conversely, towards becoming a chronic wound.A wound initially classified as an acute wound can develop into a chronic wound if it is inappropriately managed or if an unexpected pathological complication arises during the wound healing process. Any initially acute wound that has not healed within 4 weeks will therefore be considered a chronic wound. Chronic wounds remain in an inflammatory state for a longer period of time, resulting in a different treatment profile than for standard acute wounds.Nowadays, chronic wounds are a major public health problem. Their incidence continues to increase, in particular as a result of an aging population and an increase in the occurrence of underlying diseases such as diabetes. Furthermore, chronic wounds may never heal or their healing may take years. These wounds cause significant pain and stress in patients, both physically and emotionally. Therefore, suitable treatment is extremely important.Solutions are proposed by the prior art to improve the wound healing process, in particular for chronic wounds through the use of dressings which have a microarchitecture and composition that are adapted and / or improved in order to ensure better healing of the wound to be treated. Alternatively, the prior art proposes pharmaceutical compositions for wound healing or for promoting the regeneration of the skin’s cells, such as a biosimilar hydrogel based on hyaluronic acid crosslinked with L-lysine, described in document EP 3 666 278; a composition comprising recombinant genetically modified platelet-derived growth factor (PDGF) and dexamethasone as anti-inflammatory agent, described in document EP 0 575 484, or a composition comprising transforming growth factor β (TGF-β), as described in document US 5 981 606. However, these pharmaceutical products correspond to growth factors, cytokines or chemokines or hyaluronic acid, which are known for their adverse effects, in particular because they are not specific to a cell type. Regarding in particular the use of growth factors, their interaction with the cell membrane can cause a hypertrophic response (increase in the size of the cells) and a hyperplastic response (increase in the cell population), in other words clonal cell proliferation. The conventional cell growth factors used are of animal, cell and / or plant origin, i.e. are obtained from a biological raw material. By way of example, widely used factors are cholera toxin, fetal calf serum (FCS), any extract of beef pituitary gland or else undefined milk or soya complexes or fractions. However, the use of these cell growth factors of variable, undefined formulation and of animal or plant origin is increasingly criticized, in particular because of the lack of traceability of these components, the variability in their quality, the risk of contamination, and the lack of reproducibility of the effects observed. In addition, the presence of allergens cannot be verified, since the formulations of these growth factors are undefined. There is therefore a risk of allergies developing in response to their use.In parallel, the prior art proposes other solutions for treating wounds or osteoarthritis, such as the solution of document US 2023 / 148647 which relates to a composition comprising 6 particular amino acids in a specific ratio and a divalent metal to promote collagen and tropoelastin synthesis, or also of document WO 2023 / 042120 which relates to a composition comprising 2 forms of sodium hyaluronate and a mixture of amino acids.In the field of cosmetics, numerous compositions have been developed to combat skin aging, as well as to promote the regeneration of skin cells, in particular in older people, for example document CN 108 618 997 which describes a cosmetic composition for use as an anti-aging and skin-whitening agent, said composition comprising sodium hyaluronate with a MW of less than 10 kDa, and the 21 essential amino acids. These various applications are generally classified under "anti-aging". In these "anti-aging" products, active ingredients are incorporated to directly or indirectly stimulate the growth of fibroblasts and / or keratinocytes, strengthen the dermal-epidermal junction, or promote the production of collagen and elastin. By way of example, retinoids are described for their ability to stimulate the growth of fibroblasts. However, their use is not recommended in cosmetics because of their phototoxic and potentially mutagenic action (“Photomutagenicity of retinyl palmitate by ultraviolet A irradiation in mouse lymphoma cells” Nan Mei et al. Toxicological sciences 88(1), 142-149 (2005)).The treatments currently used are not free of harmful side effects and do not make it possible to achieve effective therapeutic results, to reduce the incidence of chronic wounds or to effectively and durably relieve pain, discomfort or even aesthetic unpleasantness related to acute or chronic wounds from which patients are suffering. In addition, and to the Applicant's knowledge, there is no treatment that makes it possible to treat both an acute wound and a chronic wound indiscriminately.It is clear from the above that there remains a need to provide a composition capable of facilitating and / or improving and / or accelerating specifically the wound healing / recovery mechanism of acute and chronic wounds, in a safe, effective and durable manner (i.e. in the short, medium and long term), while guaranteeing an entirely traced and traceable composition.DESCRIPTION OF THE INVENTIONAfter a large amount of research, it is to the Applicant's credit to have identified, unexpectedly and surprisingly, a composition comprising a combination of safe, traceable compounds (or active principles) which are suitable for human use, in particular for therapeutic use, and which act, in particular synergistically, to meet the aforementioned needs.Thus, a first subject of the present invention relates to a composition, advantageously a pharmaceutical composition, comprising, as active principles:- hyaluronic acid or a salt thereof, having a molecular weight of greater than, advantageously strictly greater than, 2 MDa (megadaltons); and - at least 8 essential amino acids for humans, selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine.According to a particular embodiment, said at least 8 essential amino acids for humans contained in the composition according to the invention are lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine.The present invention offers a variety of advantages; in particular:(i) it enables simple and integrable routine use, by a doctor / medical staff, of an effective formula in which the compounds act synergistically, to improve the healing of a wound, in particular an acute or chronic wound;(ii) it is free of harmful side effects;(iii) the formula of the invention can be fully chemically identified and assayed; and(iv) the formula of the invention, and any degradation products thereof, are non-toxic, non-mutagenic, non-carcinogenic and non-allergenic; and (v) it is ready for use, in particular without any extemporaneous mixing step or other steps to be carried out beforehand.In the context of the invention, the articles “a” and “an” are used to denote one or more (for example at least one) of the grammatical objects of the article. By way of example, “an element” denotes at least one element, i.e. one or more elements.In the context of the invention, the terms “around” and “approximately” are used interchangeably and with reference to a measurable value such as an amount, a duration, and other similar values. These terms should be understood to cover measurement uncertainties of ± 20% or ± 10%, preferentially ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.In the context of the invention, the various characteristics of the invention may be presented in the form of ranges of values. It should be understood that the description of values in the form of a range is only for the purpose of facilitating reading and should not be interpreted as a rigid limitation of the scope of the invention. Therefore, the description of a range of values should be considered to specifically disclose all possible intermediate ranges as well as each of the values within that range. For example, the description of a range extending from 1 to 6 should be considered to specifically describe each of the ranges that it includes, such as ranges extending from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and also each of the values within that range, for example 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This definition applies regardless of the scope of the range.In the context of the invention, the terms “formula”, “composition” and “formulation” are used interchangeably.In the context of the invention, the expression “essential amino acid for humans” means an amino acid (i.e. an organic acid in which the molecule also bears an amine function) that is essential for humans and that cannot be synthesized by the body and must therefore be provided by food. The essential amino acids for humans are tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine and isoleucine, and for children this list also includes arginine and histidine.In the context of the invention, the expression “hyaluronic acid or a high-molecular-weight salt thereof” means a hyaluronic acid or a salt thereof, the molecular weight of which is greater than, advantageously strictly greater than, 2 MDa.In the context of the invention, the expressions “hyaluronic acid or a low-molecular-weight salt thereof” and “hyaluronic acid or one of its low molecular weight salts” are used interchangeably and denote a hyaluronic acid or a salt thereof, the molecular weight of which is less than 2 MDa, advantageously less than 1.8 MDa, preferably less than 1 MDa.In the context of the invention, the terms “active compound”, “active ingredient” and “active principle” are used interchangeably and denote a substance or a compound that has therapeutic properties underlying a physiological effect. A distinction must be made between the active compound, active ingredient or active principle and the at least one excipient which is preferentially present in the composition according to the invention.In the context of the invention, the term “excipient” means a substance or compound that has no therapeutic properties. The excipient is responsible in particular for creating a particular texture, viscosity, fragrance and / or color for a therapeutic formulation, and also for diluting it, preserving it, its storage stability, safety and shelf life, in accordance with regulations. A distinction should be made between the excipient and the at least one active compound, active ingredient or active principle present in the composition of the invention.In the context of the invention, the expression “pharmaceutical composition” denotes a composition for therapeutic and non-cosmetic use.In the context of the invention, the expression “pharmaceutically acceptable carrier” denotes a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, a diluent, an excipient, a solvent or an encapsulating material, involved in the transport of the agents in question from an organ or part of the body to another organ or part of the body. Each vehicle must be "acceptable" in the sense of being compatible with the other active ingredients and / or excipients of the composition; for example, the vehicle does not reduce the impact of the agent on the treatment. In other words, a carrier according to the invention is pharmaceutically inert.In the context of the invention, the expressions “wound”, “excoriation” and “lesion” are used interchangeably and denote what is referred to as an open injury, i.e. a break in the skin barrier, as opposed to what is referred to as closed trauma without a wound, i.e. a contusion. In particular, a wound is a lesion of the skin represented by a break in the continuity of the tissues and a break in the skin barrier requiring a complex dynamic process in order to be repaired or healed. It can be superficial, involving only the epidermis (erosion), a part of the dermis, or it can be deep with exposure of the subcutaneous tissue. The way the wound develops depends on its extent and depth but also on local or general factors that can hinder or prevent it from healing.In the context of the invention, the expression “acute wound” denotes a wound of sudden onset and short duration. It heals within the predicted and expected time frame and follows the normal healing process. For acute wounds, the physiological wound healing process lasts between 2 and 4 weeks. Acute wounds can affect any part of the body. They can be superficial scratches (or epithelial wounds), or deep wounds that damage blood vessels, nerves and / or muscles (vascular, nerve and / or muscle wounds). By way of example, they can include a surgical wound, i.e., an incision made voluntarily by a healthcare professional; a traumatic wound, i.e., a sudden and unforeseen injury such as an abrasion or bite; or a burn, i.e., a type of injury inflicted on the skin or other tissues and caused by heat, cold, electricity, chemical substances, radiation or friction. A distinction should be made between acute wounds and chronic wounds.In the context of the invention, the expression “chronic wound” denotes a wound that does not heal by following the sequence of steps of the wound healing process, and / or which takes longer to heal than the predicted time for the majority of other wounds referred to as acute wounds. The wound can become stuck in one of the phases of the wound healing process. For example, the inflammatory step often lasts too long. For chronic wounds, the physiological wound healing process lasts for more than 4 to 6 weeks. By way of example, mention may be made of venous and arterial ulcers, diabetic ulcers or bedsores. A distinction should be made between chronic wounds and acute wounds. Any initially acute wound that has not healed within 4 weeks will therefore be considered a chronic wound.In the context of the invention, the expressions “improve wound healing”, “increase wound healing”, “promote wound healing”, “encourage wound healing”, and “facilitate wound healing” are used interchangeably and refer to the mechanism by means of which the phases of wound healing are more effective (i.e. better cell survival, increase in the frequency and / or amount of cell cloning, etc.) and / or follow on from each other more quickly than the predicted period of time, i.e. faster and more stable wound healing; in other words, little, if any, risk of the wound reopening.In the context of the invention, the expressions “improve cell viability”, “increase cell viability”, “promote cell viability”, “encourage cell viability”, and “facilitate cell viability” are used interchangeably and denote an increase in cell adhesion and / or proliferation and / or metabolic activity and / or cell membrane integrity with respect to a reference value measured using the same measuring devices / tests (e.g. MTT assay; tetrazolium salt) and before the candidate composition / composition to be tested is applied to the cells.In the context of the invention, the expression “cell adhesion” denotes all the cellular and molecular mechanisms employed to cause the cells to adhere to each other or to the medium surrounding them. Cell adhesion is essential for the formation, maintenance and functioning of tissues.In the context of the invention, the expression “cell proliferation” denotes the fact that the cells multiply rapidly and abundantly. It is measured as the quantified value of the population of daughter cells. This mechanism is not the same as cell growth, which is the increase in the number and size of individual cells. It is used both in the context of cell development (i.e. proliferation with cell cycle) and cell division (i.e. reproduction).In the context of the invention, the expression “cell metabolic activity” denotes the sum of catabolism (breaking down substances) and anabolism (synthesizing substances). Catabolism breaks down complex molecules into basic molecules, either to produce energy or to produce molecules that can be directly used in anabolic pathways. Anabolism relates to all syntheses.In the context of the present invention, all the embodiments described above and below can be combined.According to a particular embodiment, the active principles included in the formula according to the invention consist of:- hyaluronic acid or a salt thereof, having a molecular weight of greater than 2 MDa; and - at least 8 essential amino acids for humans, selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine.According to a particular embodiment, the formula according to the invention consists of:- hyaluronic acid or a salt thereof, having a molecular weight of greater than 2 MDa; and - 8 essential amino acids for humans, consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine.Hyaluronic acid is a polysaccharide, in particular a glucosaminoglycan (GAG), the chain of which consists of a polymer of disaccharides linked together by alternating β-1,4 and β-1,3 glycosidic bonds, represented by the formula: Hyaluronic acid or a salt thereof according to the invention has a molecular weight of greater than 2 MDa, advantageously strictly greater than 2 MDa and / or less than 3 MDa, preferably between 2 MDa and 3 MDa.In combination with the amino acids as described above, hyaluronic acid or a salt thereof according to the invention brings about an unexpected synergistic technical effect that ensures an increase in the proliferation and / or in the rate of migration of skin cells, in particular of fibroblasts, which ultimately ensures faster and more stable wound healing. It is clear from the examples below that such an effect is surprising in that it is obtained only with a hyaluronic acid or a salt thereof according to the invention having a molecular weight of greater than 2 MDa, and not with a hyaluronic acid or a salt thereof having a lower molecular weight.According to a particular embodiment, the hyaluronic acid or a salt thereof is in a non-crosslinked or crosslinked, advantageously non-crosslinked, form.According to a particular embodiment, the hyaluronic acid or a salt thereof is in a non-hydrolyzed form. According to the invention, hyaluronic acid originates from biotechnology, in particular by bacterial fermentation. In other words, it is a product of non-animal origin.The hyaluronic acid salt according to the invention is pharmaceutically acceptable and is selected from the group consisting of sodium hyaluronate, potassium hyaluronate and mixtures thereof. In an advantageous implementation, the hyaluronic acid or a salt thereof included in the composition according to the invention is sodium hyaluronate.According to the invention, the hyaluronic acid or a salt thereof is an active compound and not an excipient, much less a solvent such as a dilution solvent or a viscosity excipient.According to one embodiment of the invention, the composition of the invention comprises said at least 8 essential amino acids for humans selected from the group consisting of L-lysine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-threonine and L-histidine.According to an advantageous embodiment, the composition of the invention comprises said 8 essential amino acids for humans selected from the group consisting of L-lysine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan and L-threonine.According to a particular embodiment, the composition of the invention further comprises, as excipient(s), one or more pharmaceutically acceptable pH adjusting compounds and / or buffer compounds, comprising acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate and sodium lactate; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers can be included in an amount required to maintain the pH of the composition within a pharmaceutically acceptable range. One or more pharmaceutically acceptable salts can be included in the composition in an amount sufficient to bring the osmolality of the composition to within a pharmaceutically acceptable range. Such salts include those containing sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions.In particular, the composition according to the invention further comprises, as excipient(s), at least one compound (with a pH buffer effect), advantageously all the compounds, selected from sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water, advantageously water for injectable preparations (or WFI). According to a preferred implementation, the composition according to the invention consists of:- sodium hyaluronate;- the following essential amino acids: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine;- Na2HPO4; - KH2PO4;- NaCl; and - WFI water.Advantageously,- hyaluronic acid or a salt thereof, advantageously sodium hyaluronate, represents between 6g / l and 8g / l;- lysine represents between 33 mg / l and 39 mg / l;- valine represents between 21 mg / l and 25 mg / l;- isoleucine represents between 5.7 mg / l and 6.6 mg / l;- leucine represents between 24.3 mg / l and 28.3 mg / l;- methionine represents between 6.8 mg / l and 8.2 mg / l;- phenylalanine represents between 15 mg / l and 18 mg / l;- tryptophan represents between 4.6 mg / l and 5.6 mg / l;- threonine represents between 21.1 mg / l and 25.2 mg / l;- Na2HPO4 represents between 1.35 g / l and 1.45 g / l;- KH2PO4 represents between 0.65 g / l and 0.75 g / l;- NaCl represents between 4.5 g / l and 5.5 g / l; and - WFI water is present in a sufficient amount to make up to 1 l.According to a preferred embodiment, the pH of the composition is between 5.0 and 8.0, advantageously between 5.5 and 7.9, preferably between 7.4 and 7.5, for example a value of 7.45, and / or the osmolarity is between 280 and 360 mosmol, advantageously between 300 and 350 mosmol.According to a particular embodiment, the composition according to the invention is sterile / made sterile.According to a particular embodiment, the composition according to the invention is in an administration form suitable for topical use and / or use by injection, which is in particular pharmaceutically acceptable, i.e. compatible with the skin, mucous membranes, blood vessels, cornea and / or muscles of the human body.According to a particular embodiment, the composition of the invention is in an administration form suitable for topical cutaneous or corneal administration.According to another particular embodiment, the composition according to the invention is in an administration form suitable for intradermal, intra-articular, intra-cartilaginous, subcutaneous, intralesional, intravenous, intramuscular and / or subcutaneous injection, advantageously for intradermal injection, preferably for intradermal injection into healthy tissues located at the periphery of a wound and / or injured tissues (i.e. a wound) and / or intra-articular injection and / or intra-cartilaginous injection.According to another specific embodiment, the composition of the invention is intradermally injected into the wound and / or into the healthy tissue close to the wound, i.e. into the healthy tissue at a distance of from 0.1 cm to 5 cm from the edge of the wound, advantageously from 0.1 to 4 cm, from 0.1 to 3 cm, from 0.1 to 2 cm, from 0.1 to 1 cm, or even from 0.1 to 0.5 cm. According to a particular embodiment, the composition according to the invention is in an administration form suitable for use in contact with cells and / or tissues in vivo, ex vivo or in vitro, in particular cells and / or tissues in culture or suspension, in a method for improving the viability of cells and / or grafts and / or transplants.Thus, the composition according to the invention is in the form of a cream, a lotion, a serum, a milk, an ointment, a gel, a foam, a spray / sprayable composition, eye drops, an impregnated dressing or in the form of an aqueous, aqueous-alcoholic, organic or oily solution, or even a suspension or a dispersion in solvents or fatty substances.Preferably, the pharmaceutical composition according to the invention contains a vehicle / carrier, considered as an excipient within the meaning of the invention, which is pharmaceutically acceptable for a formulation suitable for the use according to the invention, in particular an injectable formulation. The vehicle / carrier may in particular be isotonic, sterile or saline solutions (monosodium or disodium phosphate; sodium, potassium, calcium or magnesium chloride; etc., or mixtures of such salts), or dry compositions, in particular lyophilized compositions, which, when they are added, depending on the case, to sterilized water or physiological serum, enable injectable compositions to be made up.The doses used for administration can be adapted based on different parameters, and in particular based on the mode of administration used, the disease in question, or the desired duration of treatment and the surface area to be treated.For example, it is known from the prior art to start the treatment with doses of the composition at lower levels than those required to achieve the desired therapeutic effect, and to gradually increase the dose until the desired effect is achieved.The daily dosage of the formulation according to the invention, in particular of a formulation suitable for topical use, is 0.1 to 1 ml / cm2, advantageously 0.1 to 0.5 ml / cm2. In particular, said topical application corresponds to at least one application per day, for example 2 applications per day, or even 3 or 4 applications per day. The weekly dosage of the formulation according to the invention, in particular of a formulation suitable for use by injection, in particular by intradermal injection, depends on the surface area to be treated. Thus, - for 1 cm2: 0.25 ml to 1 ml;- for 2 cm2: 0.4 ml to 1.6 ml;- for 4 cm2: 0.65 ml to 2.6 ml;advantageously, injections are given for every cm or 0.5 cm once a week.The dosage, advantageously daily dosage, of the formulation according to the invention, in particular of a formulation suitable for cartilage and / or joint use, in particular for use by intra-articular injection, is from 0.1 to 5 ml / joint, advantageously from 0.5 to 5 ml / cm2, preferably from 1 to 5 ml / cm2.The dosage of the formulation according to the invention, in particular of a formulation suitable for use advantageously for improving the grafting and / or transplant of a tissue, in particular in a mixture with adipose tissue for a tissue graft, is from 1% to 50% of the volume of fat that will be grafted, advantageously from 5% to 40%, preferably from 10% to 30%.Regardless of the mode of administration, in the event of a cancerous lesion, administration is formally contraindicated. If a cancerous lesion is suspected, a biopsy should be performed and the result obtained before administering the formulation according to the invention. According to one embodiment, the composition used in the context of the invention is intended for human and / or veterinary use.According to a preferred embodiment, the composition used in the context of the invention is intended for a human subject. In the context of the invention, “human subject” means a man or a woman, in particular a child, an adult or an elderly person.According to another embodiment, the animal targeted by the present invention is advantageously a domestic animal, in particular dogs, cats, birds or rodents, and / or a farm animal, in particular poultry, cows, goats, sheep, pigs, horses, or even camelids or deer.According to one embodiment, the composition used in the context of the invention is intended for use in the fields of vascular medicine, gynecology, ophthalmology, endocrinology / metabolic medicine, and / or dermatology.According to the invention, the composition is intended for wound healing and cell and tissue regeneration.In particular, the composition of the invention is intended to be used:1- for healing acute wounds such as contused acute wounds; acute wounds in patients with a complicated healing situation (e.g. diabetes, arterial ischemia, venous ischemia, connective tissue disorders); postoperative acute cutaneous ischemia (arterial or venous): ischemia of the skin flaps, ischemia of the areolas and nipples after breast surgery, acute cutaneous ischemia after any type of plastic and esthetic surgery, acute cutaneous ischemia after any type of orthopedic surgery for the skin margins, acute cutaneous ischemia after any type of vascular surgery for the skin margins; postoperative cutaneous separation, regardless of the surgery and without exposure of joints, bones and vessels; or burns (in particular 1st and 2nd degree); and / or2 for healing chronic wounds such as: chronic arterial wounds; chronic venous wounds; uninfected grade I and grade II bedsores; chronic cutaneous separation; post-radiotherapy wounds; diabetic foot wounds or fibrinous wounds; and / or3- in addition to other skin repair techniques such as repair by direct suture; dermal-epidermal skin grafting or total skin grafting or in addition to skin flaps; and / or4- for healing and / or hydrating the cornea, in particular in the context of dry eye syndrome; for the treatment of corneal ulcers; in addition to corneal transplantation or as a treatment for the symptoms of corneal dryness; and / or5- for cartilage and / or joint hydration and / or regeneration, in particular intra-articular injection in the prevention and treatment of the symptoms of osteoarthritis / arthritis; injection into intervertebral discs for the treatment of the symptoms of osteoarthritis of the spinal column or injection into the muscles, ligaments, tendons and capsules; and / or6- for hydrating a mucous membrane, in particular to treat the symptoms of vaginal dryness, postmenopausal vaginal atrophy, dry nasal, buccal and anal mucous membranes, damaged mucous membranes (e.g. throat, vocal cords, esophagus, stomach, intestine, interstitial cystitis); and / or7- for tissue grafting and / or transplantation, in particular in addition to skin, tendon, fat or hair grafts; and / or 8- for cell grafting and / or transplantation.According to another aspect, the invention relates to the composition according to the invention as described above for use in a method for healing a wound, advantageously an acute or chronic wound. Similarly, the invention relates to the composition according to the invention as described above, for use in reducing the risk of wound reopening.In other words, the invention also targets:- the composition according to the invention for use in healing a wound, advantageously an acute or chronic wound; and / or- the use of the composition according to the invention for preparing a medicament for healing a wound, advantageously an acute or chronic wound; and / or - a method for healing a wound, advantageously an acute or chronic wound, comprising administering, advantageously administering by injection, the composition of the invention. According to a particular embodiment, the composition according to the invention for use as described above is for wound healing, in particular to improve, accelerate or promote wound healing, i.e. to increase the rate of migration and / or proliferation of cells such as fibroblasts and / or keratinocytes.It advantageously relates to accelerating at least one of the 4 phases of wound healing and / or the sequence of the phases of wound healing.In particular, the effect of the composition according to the invention is:- improving / accelerating the migration of fibroblasts and / or keratinocytes to the wound in order to reconstruct the dermis and / or epidermis; and / or - increasing / stimulating the production and / or secretion of at least one protein / peptide selected from perlecan, laminin, loricrin and beta-defensin; and / or- increasing / stimulating the activity of at least one protein / peptide selected from perlecan, laminin, loricrin and beta-defensin.According to another aspect, the invention relates to the composition according to the invention as described above, for use in reducing the risk of wound reopening.In other words, the invention also targets:- the composition according to the invention for use in reducing the risk of wound reopening; and / or- the use of the composition according to the invention for preparing a medicament for reducing the risk of wound reopening; and / or - a method for reducing the risk of wound reopening, comprising administering, advantageously administering by injection, the composition of the invention. According to another aspect, the invention relates to the composition according to the invention as described above for use in a method, advantageously a non-cosmetic method, for hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound.In other words, the invention also targets:- the composition according to the invention for hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound; and / or- the use of the composition according to the invention for preparing a medicament for hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound; and / or - a method for hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound, comprising administering, advantageously administering by injection, the composition according to the invention. According to another aspect, the invention relates to the composition according to the invention as described above for use in a method , advantageously an in vitro method, for maintaining, or even improving, the cell viability of cells, in particular of fibroblasts, keratinocytes, stem cells of the corneal epithelium (or corneal epithelial stem cells), and / or adipocyte stem cells, for example in a therapy that uses stem cells, in particular stem cell injections.In other words, the invention also targets:- the composition according to the invention for use, advantageously in vitro use, for maintaining or even improving cell viability, advantageously of fibroblasts, keratinocytes and / or stem cells of the corneal epithelium and / or adipocyte stem cells, for example in a therapy that uses stem cells, in particular stem cell injections; and / or- the use, advantageously in vitro use, of the composition according to the invention for preparing a medicament for maintaining or even improving cell viability, advantageously of fibroblasts, keratinocytes, stem cells of the corneal epithelium and / or adipocyte stem cells, for example in a therapy that uses stem cells, in particular stem cell injections; and / or - a method, advantageously an in vitro method, for maintaining or even improving cell viability, advantageously of fibroblasts, keratinocytes of the corneal epithelium and / or adipocyte stem cells, for example in a therapy that uses stem cells, in particular stem cell injections, comprising applying, advantageously administering by injection, the composition according to the invention. According to a particular embodiment, the composition for use as described above is in vitro, ex vivo and / or in vivo. According to a particular embodiment, the improvement in viability according to the invention is an improvement in the adhesion and / or proliferation and / or metabolic activity of the cells. According to a particular embodiment, the composition for use in improving cell viability should be distinguished from a cell culture medium, because it is not a medium which contains all the elements necessary for the viability and growth of cultured cells (e.g. glucose, trace elements, vitamins, etc.).According to another aspect, the invention relates to a method for administering the composition of the invention by intradermal or subcutaneous injection into the healthy / intact tissue located at the periphery of the wound and / or the injured tissue at the center of the wound. In particular, the edge of the wound determines the boundary between intact tissue and non-integrated tissue. It is important to treat intact tissue for which the biological function is or could be compromised, so as to minimize this compromise and to enable better vascularization and stimulation of the healing of tissues where there has been a break in their integrity (wound).At the wound level, the aim of the administration method according to the invention is to accelerate wound healing by virtue of optimizing all biological wound healing mechanisms. According to one embodiment, the edge of the wound is defined as the starting point for the treatment / intradermal or subcutaneous injection, which is continued toward the outside of the wound / toward healthy tissues.According to another embodiment, the edge of the wound is defined as the starting point for the treatment / intradermal injection, which is continued toward the inside of the wound / toward the damaged tissues.According to an alternative embodiment, the edge of the wound is defined as the starting point for the treatment / intradermal injection, which is continued toward the outside of the wound / toward healthy tissues and then toward the inside of the wound / toward the damaged tissues, or vice versa in the treatment sequence (i.e., toward the inside and then the outside of the wound). According to one embodiment, the depth of the injection is between 1 mm and 3 mm, depending on the tissue (intracutaneous or subcutaneous in whole skin and immediately under the granulation or epithelialization tissue in wounds). According to one embodiment, the injection according to the invention is carried out by the injection technique of retrograde fanning or the point-by-point technique.The invention also relates to a kit for therapeutic purposes as described above. A kit according to the invention comprises the pharmaceutical composition according to the invention as described above, for example in pre-measured doses. The kit according to the invention may further comprise devices for administering said composition, and instructions for use.The devices comprise syringes, implantable pumps, such as mini-pumps and micro-pumps, and other devices for pharmaceutical use.In particular, the device included in the kit according to the invention is a syringe pre-filled with the composition of the invention.The examples below, without being limiting, form an integral part of the invention and any feature that appears to be novel over the prior art is claimed as such and as a general means.FIGURESFigure 1: Evaluation of the effect of the composition of the invention, (LUM 7 quater), the composition of the invention devoid of hyaluronic acid or a salt thereof (LUM 7 AA), and the composition of the invention devoid of amino acids (LUM 7 HA3), at 3 h, 24 h, 72 h and 6 days, on the viability of adipose tissue mesenchymal stem cells (ASC). The results came from ASCs from 3 specific donors and from an experiment with 10 wells per condition. The statistical tests are carried out against the "DMEM" condition (significance: *: p<0.05; **: p<0.01; ***: p<0.001).Figure 2: Evaluation of the effect of the composition of the invention, (LUM 7 quater), the composition of the invention devoid of hyaluronic acid or a salt thereof (LUM 7 AA), and the composition of the invention devoid of amino acids (LUM 7 HA3), at 3 h, 24 h, 72 h and 6 days, on the adhesion, proliferation and metabolic activity of adipose tissue mesenchymal stem cells (ASC). The results came from ASCs from 3 specific donors and from an experiment with 12 wells per condition. The statistical tests are carried out against the "CM" condition (significance: *: p<0.05; **: p<0.01; ***: p<0.001).Figure 3: In vitro evaluation of the effect of the composition of the invention at different concentrations (i.e. stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) in comparison with a control (DMEM) on the production / secretion of perlecan by cultured human primary fibroblasts.Figure 4: In vitro evaluation of the effect of the composition of the invention at different concentrations (i.e. stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) in comparison with a control (DMEM) on the production / secretion of laminin by cultured human primary fibroblasts.Figure 5: In vitro evaluation of the effect of the composition of the invention at different concentrations (i.e. stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) in comparison with a control (Celloneer KC / CC) on the production / secretion of loricrin by cultured human epidermal keratinocytes.Figure 6: In vitro evaluation of the effect of the composition of the invention at different concentrations (i.e. stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) in comparison with a control (Celloneer KC / CC) on the production / secretion of beta-defensin by cultured human epidermal keratinocytes.Figure 7: Monitoring of the effect of the composition of the invention at different concentrations (i.e. stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) in comparison with a control (DMEM) on the rate of migration of fibroblasts following an experimental injury.Figure 8: Photographs of a chronic wound on the lateral face of the foot, that has been developing for several years in a 58-year-old paraplegic patient before treatment (A) and after 2 months of treatment (B) by weekly injections into the wound (intra-lesional injections) and into the healthy tissue close to the wound (peri-lesional injections).Figure 9: Photographs of the monitoring of the migration of fibroblasts over 16 hours (i.e., at T0, T8 h, T12 h and T16 h) according to the compositions applied (Lum A: Composition devoid of hyaluronic acid; Lum H1: Composition devoid of amino acids and comprising a low-molecular-weight hyaluronic acid; Lum 7: composition of the invention) and dilutions (20% or 50%). Representative experiment of 6 replicates. Figure 10: Kinetics of fibroblast migration for 30 hours in the presence of 20% and 50% Lum 7, in comparison with the control Averages + / - standard deviation; statistical analysis using the Mann-Whitney test: *p<0.05; **p<0.01.Figure 11: Kinetics of fibroblast migration for 30 hours in the presence of 20% and 50% Lum H1, in comparison with the control Averages + / - standard deviation; statistical analysis using the Mann-Whitney test: *p<0.05; **p<0.01.Figure 12: Kinetics of fibroblast migration for 30 hours in the presence of 20% and 50% Lum A, in comparison with the control Averages + / - standard deviation; statistical analysis using the Mann-Whitney test: *p<0.05; **p<0.01.Figure 13: Evaluation of the cell viability of corneal epithelial cells exposed to the composition of the invention or 3 eye drops of the prior art for 3, 12 or 24 hours. Optical density results at 570 nm normalized with respect to the results obtained with the culture medium (CM): CMs normalized to 100% at all times.Figure 14: Raw results of optical densities (570 nm) and their change after 3, 12 or 24 hours of exposure to the composition of the invention or to the 3 eye drops of the prior art – 1 / 2 dilution in DMEM.Figure 15: Raw results of optical densities (570 nm) and their change after 3, 12 or 24 hours of exposure to the composition of the invention or to the 3 eye drops of the prior art – 1 / 2 dilution in PBS. EXEMPLARY EMBODIMENTSExample 1. Evaluation of the toxicity of the composition of the invention1. Aim of the study The purpose of this study is to demonstrate the lack of "cytotoxicity" of the composition of the invention at 4 different times (i.e. 3 h, 24 h , 72 h and 6 days) on adipose tissue stem cells (or adipose tissue mesenchymal stem cells or "Adipose derived Stem Cells" or ASC) from 3 donors using an MTT assay (or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).2. Materials and methods2.1. CellsThe ASCs from the 3 donors at a density of 10,000 cells / well (96-well plate).Donor 1: 11171 28 years (ampoule P2 - seeded in P4);Donor 2: 12025 28 years (ampoule P0 - seeded in P2); andDonor 3: 12106 40 years (ampoule P1 - seeded in P3)2.2. Preparation of the tested compositionsThe active principles used in the composition of the invention LUM 7 QUATER are illustrated in Table 2 below.[Table 2]ComponentsFor 10 lHigh-molecular-weight NaHA >2 MDa65 gNaCl50 gKH2PO47 gNa2HPO414 gisoleucine63 mgleucine263 mglysine362.5 mgmethionine75.5 mgphenylalanine165.1 mgvaline 234.3 mgthreonine238.2 mgtryptophan51 mgwater q.s. to10 l The active principles used in the composition LUM 7 HA3 are illustrated in Table 3 below.[Table 3]ComponentsFor 10 lHigh-molecular-weight NaHA >2 MDa65 gNaCl50 gKH2PO47 gNa2HPO414 gwater q.s. to10 l The active principles used in the composition LUM 7 AA are illustrated in Table 4 below.[Table 4]ComponentsFor 10 lNaCl50 gKH2PO47 gNa2HPO414 gisoleucine63 mgleucine263 mglysine362.5 mgmethionine75.5 mgphenylalanine165.1 mgvaline 234.3 mgthreonine238.2 mgtryptophan51 mgwater q.s. to10 l The preparation of the formula comprises 3 phases, namely:- producing the pH buffer solution: adding the total amount of water then stirring, adding the buffer salts and after complete dissolution, checking the pH is correct;- adding NaCl and amino acids: always with constant stirring, adding the NaCl, waiting for it to dissolve, and then adding the amino acids, checking they have dissolved before each addition; and- adding hyaluronic acid: always with constant stirring, adding hyaluronic acid in small fractions and waiting for it to completely dissolve before stopping stirring.Once the preparation of the formula is complete , checking the pH then distributing into vials or syringes and finally autoclaving the vials or syringes obtained.2.3. Conditions - LUM 7 Quater (composition of the invention): 6 vials of 5 ml- LUM 7HA3 (LUM 7 Quater composition devoid of amino acids): 8 vials of 5 ml- LUM 7 AA (LUM 7 Quater composition devoid of hyaluronic acid): 8 vials of 5 mlIt should be noted that only one dilution will be tested for each formula in the example (pure).Sodium dodecyl sulfate (SDS) is used as a negative control of viability (0.05% dissolved in the proliferation medium).An untreated control in contact only with DMEM will be used to determine 100% viability (positive control).A control in culture medium will also be carried out. The culture medium contains: “DMEM / F12 with GlutaMAX”, 10% FCS, 100 U / ml penicillin, streptomycin , 5 µg / ml fungizone, 10 ng / ml “FGF2 premium grade” (MiltenyiBiotec, Paris, France).2.4. Experimental protocolThe ASCs are thawed and seeded in flasks and cultured to confluence. The ASCs will be trypsinized, seeded in a 96-well plate at a density of 10,000 cells / well, and cultured in proliferation medium (DMEM) for 24 hours. The next day, the products are applied and left in contact for 3, 24, 72 hours and 6 days before performing an MTT assay. For each condition and each time, 12 wells will be treated. During the MTT assay, the percentage viability of each condition will be calculated relative to the positive control, which will represent 100% viability. The negative control, SDS at 0.05%, should be equal to 0% viability.The principle of the assay is based on the use of a tetrazolium salt, MTT ((3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which is reduced to formazan by a mitochondrial enzyme, succinate dehydrogenase, in living cells. The amount of formazan produced is proportional to the metabolic activity of the cells. This insoluble compound, which is purple in color, will have to be solubilized with DMSO to enable it to be spectrophotometrically assayed at 540 nm.2.5. Data AnalysisRaw data was analyzed using Microsoft Excel software. The intergroup comparisons were carried out using the unpaired Student’s t test. The differences are considered to be statistically significant from p<0.05. (NS: p>0.05; *: p≤0.05; **: p≤0.01; ***: p≤0.001).3. ResultsThe results are presented in Figure 1.It appears that:DMEM is a basic isotonic solution that does not contain either growth factor or calf serum. It makes it possible to maintain viable ASCs for 72 hours. It is used as a positive control.The culture medium (CM) is a very rich medium: it contains calf serum and a growth factor (FGF2), and cannot be administered to humans. The composition of the invention (LUM 7 QUATER) and the composition of the invention devoid of amino acids (LUM 7 HA3) have a similar viability over time.The viability of the cells in contact with the composition of the invention devoid of hyaluronic acid (LUM 7 AA) decreases rapidly.This data testifies to the synergistic effect between sodium hyaluronate and the amino acids of the composition of the invention on cell viability. In other words, the combination of hyaluronic acid or a salt thereof with the 8 amino acids of the invention has a significant synergistic effect in increasing cell viability.Example 2. Evaluation of the effect of the composition of the invention on the adhesion, proliferation and metabolic activity of adipose tissue mesenchymal stem cells1. Aim of the study The purpose of this study is to demonstrate the effect of the composition of the invention on the adhesion, proliferation and metabolic activity at 4 different times (i.e. 3 h, 24 h , 72 h and 6 days) on adipose tissue stem cells (or adipose tissue mesenchymal stem cells or "Adipose derived Stem Cells" or ASC) from 3 donors using an MTT assay (or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).2. Materials and methodsSee points 2.1. to 2.3. and 2.5. of Example 1.2.4. Experimental protocolThe ASCs are thawed into P0, P1 and P2 according to the donors, and seeded into flasks in P1, P2 or P3, and cultured to confluence.At confluence, the ASCs are trypsinized, then seeded into a 96-well plate. The products are applied as soon as the seeding has been done into the 96-well plate, and are left in contact for 24, 72, 96 hours and 7 days before performing an MTT assay. For each condition and each time, 8 wells were treated, giving a total of 1 96-well plate per time and per donor: 12 plates in total.During the MTT assay, the percentage viability of each condition will be calculated relative to the positive control, which will represent 100% viability. The negative control, SDS at 0.05%, should be equal to 0% viability.It should be noted that medium CM is used here since it is widely known to be a very rich medium because it contains calf serum and a growth factor (FGF2). It was used here for its function of improving the adhesion, viability and proliferation of ASCs in cell culture in order to be able to observe and quantify an effect of the composition of the invention.3. ResultsThe results are presented in Figure 2.It appears that the adipose tissue mesenchymal cells have similar behavior in the 3 formulations LUM 7 quater, LUM 7HA3 and LUM 7 AA. Example 3. In vitro evaluation of the effect of the composition of the invention on the production / secretion of perlecan and laminin by human primary fibroblasts (HPF), and also loricrin and beta-defensin by cultured human epidermal keratinocytes (HEK)1. Culture and maintenance of the HEK line HEKs are human keratinocyte cells from the skin of different donors, cultured in Celloneer KC / CC which is a basic medium for keratinocytes. The cells are kept in an incubator at 37°C in a humid atmosphere (85 to 90% humidity) containing 5% CO2. The medium is renewed 4 days after culturing, then twice a week.2. Culture and maintenance of the HPF line Fibroblasts from a 40-year-old woman (HPF) are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine containing the essential amino acids, salts, glucose and vitamins necessary for cell stimulation, and 1% penicillin-streptomycin. The addition of a volume corresponding to a concentration of 1% of penicillin-streptomycin makes it possible to prevent any bacterial growth. All the combined ingredients were filtered over a "stericup" (milliport). The cells are kept in an incubator at 37°C in a humid atmosphere (85 to 90% humidity) containing 5% CO2. The medium is renewed 4 days after culturing, then twice a week.3. Experimental protocolThe HEKs and HPFs are treated daily with the composition of the invention corresponding to the formula LUM 7 quater of Example 1, at different concentrations. In particular, a stock solution of LUM 7 quater is prepared as described in Example 1, then 20%, 30%, 40% and 50% dilutions are applied to this stock solution. The stock solution, and also the 20%, 30%, 40% and 50% diluted formulas, are applied to the cells in culture. 4. ResultsThe results are presented Fig. 3-6.It appears that:- fibroblasts cultured in the presence of the formula of the invention at different concentrations produce more laminin and perlecan than fibroblasts cultured in standard medium (DMEM); and- keratinocytes cultured in the presence of the formula of the invention at different concentrations produce more loricrin than keratinocytes cultured in standard medium (Celloneer KC / CC). Furthermore, the production of beta-defensin, linked to antimicrobial protection, is increased in keratinocytes in the presence of the composition of the invention. Example 4. In vitro evaluation of the effect of the composition of the invention on the rate of migration of fibroblasts following an experimental injuryThe wound healing test linked to injuring the cell layer consists in tearing the cells in a controlled manner over a precise trajectory in a Petri dish and observing at regular intervals, or filming, the way in which the cells adjacent to the "wound" colonize the empty space. The main component of the healing of the wound will be cell migration, but there may also be a component related to proliferation.1. Experimental protocol1.1. Preparation of culturesThe first step of the test consists in growing a confluent HPF monolayer. This monolayer represents the in vivo conditions of the tissue before the injury, for example an intact epithelium. 1.2. Creating the injuryOnce the cells have reached confluence, the next step consists in creating a cell-free space in the monolayer. The method consists in injuring the monolayer by mechanical scratching (or "scratch wound").1.3. Data acquisitionAfter creating the injury and treating or not treating with the stock solution or the 20%, 30%, 40% and 50% dilutions of the stock solution, optical microscopy is used to observe the cells migrating toward the wound area. Once the microscope has been configured, a series of accelerated images (snapshot method) is acquired as the cells migrate into the cell-free space. These time points are collected every hour for 72 hours. Precise measurements are also acquired manually using a digital camera installed on the microscope.1.4. Data analysisThe migration was analyzed using an inverted Nikon microscope at 20X magnification (Melville, NY). The time-lapse recording began after the HPF culture which had been subjected to an injury by scratching was treated with the composition of the invention at the different concentrations being tested. Three fields per well were imaged and monitored at 1-hour intervals for 72 hours with a Coolsnap HQ camera (Photo-metrics, Tucson, AZ) operated by the NIS-elements AR 2.30 software (Nikon). Manual monitoring of individual cells was carried out using the Metamorph software (Roper Scientific, Evry, France). The migration parameters calculated for each individual cell were determined from the time-lapse films. They include total migration distance, distance to origin, speed and directional persistence of cell migration. The total migration distance represents the sum of the distances between each measurement over a period of 72 h. For each condition, the results are expressed as the mean ± SD of at least 60 individual cells.It should be noted that a proliferation inhibitor, mitomycin C, was added to the medium in order to obtain data targeting only cell migration and not cell migration and / or proliferation. 2. ResultsThe results are presented in Figure 7.It appears that the fibroblasts in culture, stimulated by the composition of the invention at different concentrations, have a higher migration rate than the fibroblasts under the control condition. This means that the coverage of the wound, and therefore wound healing, is accelerated by administering the composition of the invention. This data is confirmed by Figure 8 which shows that in vivo administration of the composition of the invention (stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) leads to improved and accelerated wound healing compared to a conventional treatment protocol. Example 5. In vivo evaluation of the effect of the composition of the invention on the healing of a chronic woundA 58-year-old paraplegic patient suffering from a chronic wound on the lateral face of the foot that had been developing for several years was treated with weekly injections into the wound (intra-lesional injections) and into the healthy tissue close to the wound (peri-lesional injections) for 2 months.Figure 8 shows a photograph before treatment (A) and after 2 months of treatment (B).It is clear from these photographs that treatment with the composition of the invention, by intradermal injection, surprisingly improves the healing of a chronic wound that has been developing for several years.Example 6: In vitro comparative study of the effect of the composition of the invention on the rate of migration of fibroblasts following an experimental injuryThe aim of this study is to evaluate the effect of 3 compositions on the in vitro migration of human primary fibroblasts using the scratch-test technique.1.Experimental protocol1.1. Preparation of the cellsHuman neonatal fibroblasts (from LifeLine Cellsystems) are cultured in Fibrolife medium (LifeLine Cellsystems) in a 24-well plate (7x104 cells / well) for 3 days, to confluence, in an incubator at 37°C in a humid atmosphere with 5% CO2.1.2. Scratch assay techniqueIn each of the wells, the cell layer is injured using a 10 µl pipette tip. The culture medium is removed, then replaced with fresh medium containing the proportion of solution of each of the products, 20% or 50% final. The control is carried out with culture medium, in accordance with the customer's request. Each condition is carried out in triplicate.1.3. Imaging monitoring of cell migrationThe plate is then placed under a video microscope (CARLZEISS AXIO OBSERVER Z1) in a humid incubation chamber at 37°C, with 5% CO2. For each of the wells, two positions are pre-recorded and are then monitored over time. Images are recorded every 2 hours for 48 hours.1.4. Analysis of the resultsFor each of the positions, an analysis over time using the Image J software with the "Wound Healing Size Tool" PlugIn is carried out by quantifying the area of non-covered injury. The degree of coverage of the injury that evaluates cell migration is calculated using the following formula: The degree of coverage is calculated from the 6 values (3 replicates x 2 positions / well) for each of the conditions and for each time. The results are shown as an average (+ / - standard deviation) of the degree of coverage. A statistical study is performed using the non-parametric Mann-Whitney test and comparing each of the conditions to the control.2. Compositions testedControl: Fibrolife medium (LifeLine Cellsystems)LUM 7 Quater (or Lum 7; composition of the invention): composition according to Example 1LUM A (composition of the invention devoid of hyaluronic acid): corresponding to composition LUM 7 AA of Example 1LUM H1 (composition comprising a low-molecular-weight hyaluronic acid and the amino acids of the composition Lum 7 Quater), the active principles of which are presented in Table 5 below.[Table 5]ComponentsFor 10 lLow-molecular-weight NaHA 1.6 MDa65 gNaCl50 gKH2PO47 gNa2HPO414 gisoleucine63 mgleucine263 mglysine362.5 mgmethionine75.5 mgphenylalanine165.1 mgvaline 234.3 mgthreonine238.2 mgtryptophan51 mgwater q.s. to10 l 3. ResultsThe results are presented Fig. 9-12.It appears from the photographs in Figure 9 that the injuries are all completely covered at around 30 hours, whether for the different conditions or for the control, and therefore the analysis was carried out on the times from 0 to 30 hours. The photographs depict, for each of the conditions, the time T0 and the times T8 h, T12 h and T16 h where the differences between the treatment condition and the control are mainly observed when these differences exist.From the time T0, the cells gradually cover the injury to the cell layer. From the time T8 h, the cells cover significantly more surface area with the 20% or 50% Lum 7 treatment compared to the control and to the other conditions.Regarding the kinetics of fibroblast migration, it appears that:Lum 7 activates the migration of fibroblasts in the early phase at 50%, and in a later phase at 20%. The effect is significant for 50% Lum 7 at the times 6 h and 8 h, with +22% and +25% increase, respectively, compared to the control, and for 20% Lum 7 at the times 12 h, 16 h to 22h, with a maximum increase of 23% at 12 h. There is no significant difference when comparing 20% Lum 7 and 50% Lum 7;Lum H1 activates the migration of fibroblasts at 50%, with a significant effect for 50% Lum H1 at 16 h to 22 h, with a maximum increase of 14% at 16 h. The increase observed for 20% Lum H1 was not significant compared to the control. There is no significant difference when comparing 20% Lum H1 and 50% Lum H1; and20% or 50% Lum A has no significant effect on the migration of fibroblasts compared to the control. There is no significant difference when comparing 20% Lum A and 50% Lum A.4. Conclusion Lum 7 is the most active and accelerates the migration of fibroblasts with the scratch assay technique, with a maximum 25% increase compared to the control. The 2 concentrations, 20% and 50%, are active but at different times: 50% Lum 7 is more active at the start of the assay, 20% Lum 7 is more active at later times. Lum H1 is also active, but only at a concentration of 50%, with a maximum increase of 14% compared to the control. Lum A, in contrast, exhibits no effect on fibroblast migration using this technique.In other words, it is clear from this data that the high-molecular-weight hyaluronic acid combined with the amino acids of the invention act synergistically to improve wound healing. Example 7: Evaluation of the effect of the composition of the invention on the viability of corneal epithelial cells (COEP) The aim of this study is to verify the non-cytotoxicity of the composition of the invention, and also to evaluate the impact of its administration on COEPs in comparison with eye drops of the prior art.1. Study of cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The reagent used is the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The tetrazolium ring that it contains is reduced to formazan by mitochondrial succinate dehydrogenase in living and active cells. This forms a purple-colored precipitate in the mitochondria. The amount of precipitate formed is proportional to the amount of living cells. Assay of the optical density at 570 nm by spectroscopy gives the relative amount of living cells and metabolically active cells.1.%2. Materials The cells used are corneal epithelial cells (COEP) at a density of 10,000 cells / well (48-well plate) COEP 38230536G P3The compositions tested are:LUM 7 Quarter composition (composition of the invention; see Example 1): 2 vials of 5 ml Prior art eye drops: artificial tears that have the status of first-line medicaments for dry eyes and mild epithelial ulcer, namely Dulcilarmes 1.5%; Hylovis multi15; and Thealose.The eye drops and the composition of the invention are tested at ½ dilution with DMEM or with PBS.Cell lysis buffer: negative control of viability DMEM: diluent onlyPBS: diluent only Culture medium: used for seeding the COEPs and as a control for the MTT assay.2.%2. Methods The COEPs are thawed and amplified in a flask for 1 week, then they are seeded at a density of 10,000 cells / well in a 48-well plate previously covered with an irradiated nutrient layer. The COEPs are cultured for 4 days with 500 µl of culture medium at the start, the medium being changed after 3 days of culture with 300 µl of medium and EGF. After the 4 days of culture, the culture medium is removed and each condition is brought into contact with the COEPs: - Cell lysis buffer 300 µl- Culture medium: 300 µl - DMEM: 300 µl- PBS: 300 µl- Diluted formulation: 300 µl- Diluted eye drops: 300 µlEach condition is left in contact with the COEPs for 4, 12 and 24 hours before performing the MTT assay. Regarding the performance of the MTT assay, each well was aspirated, then rinsed with PBS and reaspirated before adding 300 µl of MTT at 1 mg / ml. After 2 hours of incubation at 37°C, the plates were inverted and 300 µl of acidified solution were added to lyse the crystals. Finally, each well was analyzed using a spectrophotometer. The percentage viability of each condition is calculated relative to the positive control, which represents 100% viability, and the negative control must be equal to 0% viability. The cells were cultured for 4 days before being brought into contact with LUM 7 Quater and the eye drops to be tested for 4 hours, 12 hours and 24 hours. The results were normalized to the results of the control culture medium, which represents 100% of all the times analyzed.2. Results3.%2. MTT assayThe results are presented in Figure 13.The data show the non-"cytotoxicity" of the formulation of the invention at 3 different times (4, 12 and 24 hours), in particular in comparison with the eye drops of the prior art tested. 4.%2. ½ dilution DMEMThe results are presented in Figure 14.After 4 hours of contact: The optical densities (OD) measured via the MTT reaction are similar for all the conditions tested (eye drops and formulations diluted in DMEM as well as in the culture medium). Mean ODs range between 0.414 and 0.448.After 12 hours of contact: For all the conditions tested with dilution in DMEM, cell activity at 12 hours is greater than that observed at 4 hours, suggesting cell proliferation and / or increased metabolic activity. The conditions tested therefore appear to be favorable for cell activity.The rich and optimized culture medium is distinguished by a higher cell activity (proliferation and / or metabolic activity) than all other conditions.• Two commercial eye drops (Hylovis Multi 15 and ThéaLose) and the LUM 7 Quarter formulation (composition of the invention) exhibit similar results that are slightly lower than those of the culture medium (activities between 90.4% and 91.2% relative to the culture medium), and are comparable to those of the diluent alone (DMEM).• Commercially available eye drops (Dulcilarmes) exhibit lower cell activity (82%) than all other conditions, including the diluent.After 24 hours of contact: While cell activity continues to increase in the culture medium, it stagnates or decreases slightly for the cells exposed to Hylovis multi 15, ThéaLose, LUM 7 quater formulation diluted in DMEM, and in the diluent alone. However, the cell activity for Hylovis multi 15, ThéaLose, and the LUM 7 quater formulation remains higher than that observed in the diluent alone.The cell activity for Dulcilarmes eye drops diluted in DMEM remains lower than that of the other conditions.5.%2. ½ dilution PBSThe results are presented in Figure 15.After 4 hours of contact: In the dilutions performed in PBS, the results are more dispersed than for those in DMEM. The culture medium exhibits the strongest cell activity. The activities are decreasing in the following order: Hylovis Multi 15, ThéaLose, the formulation, and the diluent (PBS). The cell activity in Dulcilarme is almost zero, probably due to precipitates observed during the dilution. This lack of activity persists at the times of 12 hours and 24 hours.After 12 hours of contact: For the eye drops and the LUM 7 quater formulation diluted in PBS, and also for the culture medium, the cell activity at 12 hours exceeds that measured at 4 hours, indicating cell proliferation and / or increased metabolic activity. These conditions are therefore favorable for cell activity. Conversely, the activity of the cells kept in the diluent alone (PBS) stagnates.The rich and optimized culture medium exhibits higher cell activity than all other conditions.• The cell activities under the conditions Hylovis Multi 15, ThéaLose and the LUM 7 quater formulation diluted in PBS are very similar (OD between 0.376 and 0.413)• The activity in the diluent alone is significantly lower: it is halved (OD of 0.182).After 24 hours of contact: Cell activity decreases for all conditions except the culture medium. This decline can be explained by the lack of nutrients (amino acids, glucose) in the diluent, resulting in cell death.3. ConclusionThe composition of the invention (LUM 7 Quater) makes it possible to maintain corneal epithelial cells and enables the activity of said cells. This maintenance and activity are similar to the Hylovis multi 15 and Théalose eye drops of the prior art. Therefore, this data demonstrates the therapeutic effects of the composition on the treatment of the cornea, in particular for dry eyes, ulceration treatment, etc. GENERAL CONCLUSIONThis data testifies to the synergistic effect between a specific hyaluronic acid or a salt thereof, the molecular weight of which is greater than 2 MDa, and the amino acids of the composition of the invention on cell viability, adhesion, and proliferation and cellular metabolic activity, and ultimately on wound healing and cell and tissue regeneration. In other words, the combination of hyaluronic acid or a salt thereof with the 8 amino acids of the invention has a significant synergistic effect in increasing these phenomena.It also appears that the composition of the invention (LUM 7 QUATER) enables the cell survival over a period of 6 days that is greater than that achieved with the composition of the invention that is devoid of hyaluronic acid or a salt thereof (LUM 7 AA).In addition, this data clearly shows that the different components of the basal lamina are stimulated by the formula of the invention, tested at different concentrations. This is reflected by a positive effect on:- the dermal-epidermal junction: the composition of the invention reinforces this junction and acts on the mechanical strength of the epidermis;- dermal and epidermal tissues: the composition of the invention increases the interactions between the cells and their environment;- terminal keratinocyte differentiation: the composition of the invention stimulates different effectors related to the mechanical, immunological and bacterial protection of the epidermis; and- fibroblast functionality: increases the rate of migration of fibroblasts, induces a greater number of myofibroblasts as well as the synthesis of collagen I and III, thereby facilitating wound healing / repair of the dermis.Consequently, it is clear from the above that the composition of the invention acts on the synthesis of the constituents of the basal lamina / dermal-epidermal junction by modifying the synthesis expression of keratinocytes and fibroblasts.The composition of the invention contributes greatly to keratinocyte stimulation for the synthesis of receptors and molecules associated with protection of the epidermis, and accelerates the migration of fibroblasts while increasing the myofibroblast phenotype in an experimental injury model.These conclusions make it possible to confirm the beneficial effects of the invention in a context of using the composition of the invention for healing the cornea, a joint or cartilage, improving cell viability, hydrating the mucous membranes or in a tissue or cell graft.  

Claims

1. A pharmaceutical composition comprising:- hyaluronic acid or a salt thereof, having a molecular weight of greater than 2 MDa; and - at least 8 essential amino acids for humans, selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine.

2. The composition according to claim 1, characterized in that the at least 8 essential amino acids for humans are lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine.

3. The composition according to claim 1 or 2, characterized in that the hyaluronic acid or a salt thereof is in a non-crosslinked form.

4. The composition according to any one of the preceding claims, characterized in that the hyaluronic acid or a salt thereof is sodium hyaluronate, potassium hyaluronate or mixtures thereof, advantageously sodium hyaluronate.

5. The composition according to any one of the preceding claims, characterized in that it further comprises sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water, advantageously water for injectable preparations.

6. The composition according to any one of the preceding claims, characterized in that it consists of:- sodium hyaluronate;- the following essential amino acids: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine;- Na2HPO4; - KH2PO4;- NaCl; and - water.

7. The composition according to any one of the preceding claims, characterized in that:- hyaluronic acid or a salt thereof, advantageously sodium hyaluronate, represents between 6g / l and 8g / l;- lysine represents between 33 mg / l and 39 mg / l;- valine represents between 21 mg / l and 25 mg / l;- isoleucine represents between 5.7 mg / l and 6.6 mg / l;- leucine represents between 24.3 mg / l and 28.3 mg / l;- methionine represents between 6.8 mg / l and 8.2 mg / l;- phenylalanine represents between 15 mg / l and 18 mg / l;- tryptophan represents between 4.6 mg / l and 5.6 mg / l;- threonine represents between 21.1 mg / l and 25.2 mg / l;- Na2HPO4 represents between 1.35 g / l and 1.45 g / l;- KH2PO4 represents between 0.65 g / l and 0.75 g / l;- NaCl represents between 4.5 g / l and 5.5 g / l; and - water is present in a sufficient amount to make up to 1 l.

8. The composition according to any one of the preceding claims, for use as a medicament.

9. The composition according to any one of claims 1 to 7, for use in healing a wound, advantageously an acute or chronic wound.

10. The composition for use thereof according to claim 9, characterized in that wound healing is improved.

11. The composition according to any one of claims 1 to 7, for use in reducing the risk of wound reopening.

12. The composition according to any one of claims 1 to 7, for use in hydrating a tissue, advantageously the skin, cornea, a mucous membrane, a cartilage, a bone, and / or a wound, advantageously an acute or chronic wound.

13. The composition according to any one of claims 1 to 7, for use in cartilage and / or joint regeneration.

14. The composition according to any one of claims 1 to 7, for use in the grafting and / or transplant of a tissue or a cell.

15. An in vitro method for maintaining, or even improving, the cell viability of cultured cells by applying the composition according to any one of claims 1 to 7 to said cells; wherein the cells are advantageously fibroblasts, keratinocytes, stem cells of the corneal epithelium, or adipocyte stem cells.

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