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

[0001] DESCRIPTION

[0002] TITLE OF THE INVENTION: Pharmaceutical formula for healing and cell and tissue regeneration and its use

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the human and / or veterinary medical field, in particular to a pharmaceutical formula for topical, transdermal, subcutaneous and / or intralesional administration and the implementation of the pharmaceutical formula in the treatment of wounds and / or for improving wound healing and / or for articular cartilage regeneration and / or for skin and mucous membrane hydration and / or in improving cell viability.

[0005] STATE OF THE PRIOR ART

[0006] Wounds of all types remain one of the most important, costly and common medical problems in the world today. A wound is defined as a breakdown in the protective function of the skin and a loss of epithelial continuity, with or without loss of underlying tissues (skin, muscle, bone, nerves). We therefore speak of a wound as soon as the skin is injured or damaged, whether or not there is damage to deeper tissues. This skin damage can have different origins, for example, accidental / traumatic, infectious, following a pathological process, including inflammatory, or voluntary as part of a surgical procedure.

[0007] Wounds are generally categorized according to their healing time. There are two main categories of wounds:

[0008] - the acute wound which is a wound of sudden appearance healing in a period of time considered normal, i.e. less than 4 weeks, following the usual healing process. This category of wound includes in particular traumatic wounds (grazes, abrasions, lacerations, bites, penetrating wounds, etc.), burns of all degrees and surgical wounds, including grafts; and

[0009] - chronic wounds, which are wounds whose healing time is prolonged despite appropriate care and treatment. Some wounds for which healing is not expected within 4 to 6 weeks of development are automatically considered chronic. This category of wound includes bedsores, diabetic foot wounds, leg ulcers or amputation stumps, etc. Under standard circumstances, the wound healing process is broken down into 4 stages / phases, namely:

[0010] - Phase 1: inflammatory (or detersive-inflammatory) phase: a stage 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) which will eliminate (detersion phase), via the lymphatic route and / or by the formation of pus, bacteria, dead tissue and foreign microparticles. At this stage the wound shows all the characteristic signs of inflammation: redness, swelling, heat, pain. The dilation of the blood capillaries is responsible for the redness and heat. The increased permeability of these capillaries promotes plasma exudation, which is responsible for the swelling and heat, while the pain is due to pressure on the sensitive nerve endings. This reactive phase generally lasts between 3 and 6 days.

[0011] - 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, granulation tissue with neoformation of capillaries (neovascularization or angiogenesis), which will provide in situ oxygen, nutrients and cells necessary for tissue repair, begins to organize itself within a network of collagen and elastin (produced by fibroblasts). Macrophages at this stage still play an essential role by producing growth factors or cytokines capable of promoting fibroblast proliferation and collagen synthesis. At this stage, the scar is a young fibrosis containing numerous fibroblasts and a loose fibrillar framework at the periphery of the loss of substance.The fleshy bud is composed of fibroblasts, an inflammatory infiltrate (monocytes, lymphocytes, polymorphonuclear cells), superficial fibrin and neovascularization in an edematous fibrillar framework. Contraction of the wound to bring the edges together is closely linked to the formation of granulation tissue and the transformation of certain fibroblasts into myofibroblasts capable of contracting and transmitting their contractile activity to the surrounding tissue by interaction between the proteins of the cytoskeleton and the extracellular matrix. This phase, very active from the 7th day, can last up to 3 weeks.

[0012] - Phase 3: Epithelialization phase: After tissue repair, the wound shrinks and gradually becomes covered with new epithelium; this is the epithelialization process. Epidermal cells capable of dividing (keratinocytes) multiply and begin to cover the granulation tissue, starting from the edges of the wound. In order to migrate properly, these keratinocytes need 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 stage lasts 1 to 3 weeks.

[0013] - Phase 4: Remodeling or maturation phase: This phase begins within the first few days in the case of a sutured wound, but can also last for months in the case of extensive and widely open wounds. It 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. The collagen fibers thicken, increasing resistance to tensile forces. The number of capillaries decreases, as does blood flow. Excess water and vessels then disappear, and the scar firms up. However, in all cases, scars are less resistant and less elastic than normal skin, partly due to a certain elastin deficiency. This phase can last from several months to 2 years.

[0014] The International Visual Wound Colour Scale describes the different phases of wound healing (see Table 1 below) and provides a common language.

[0015] [Table 1]

[0016] At the cellular and molecular level, keratinocytes and fibroblasts have the property of producing and secreting different partners that participate in the wound healing process such as perlecan, laminin or collagen (collagens I and VII). Perlecan acts on the proliferation and cellular differentiation of keratinocytes, it is necessary for the formation of the epidermis and participates in the presentation of growth factors to their receptors. The literature teaches that the administration of exogenous perlecan restores the formation of the epidermis (Int J Cosmet Sci. 2020 Dec; 42(6): 529-535). Laminin plays a main role in normal tissues which is that of 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 which make it possible to monitor, or even optimize, dermo-epidermal adhesion during the healing process.

[0017] In parallel, it is known that the primary functions of the epidermis include water retention, thermal and pH homeostasis, and protection against the entry of pathogenic microorganisms or toxic substances into the body. The formation of an appropriate epidermal barrier relies on the progressive differentiation of keratinocytes, from the proliferating cells of the basal layer to the horny layer or stratum corneum, ze, terminal differentiation. The physical properties of the stratum corneum are attributed to the formation of a specialized structure, the cell envelope (CE), which is highly resistant to strong chemicals such as detergents or those with high alkalinity. The process of strengthening the CE incorporates, among others, loricrin, filaggrin or keratin intermediate filaments.

[0018] 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.

[0019] Furthermore, beta-defensin (or P-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 progression of a wound towards healing or, conversely, towards chronicity of the wound.

[0020] It turns out that a wound initially classified as an acute wound can develop into a chronic wound if its management is not appropriate or if an unexpected pathological complication occurs during the healing process. Any initially acute wound that does not heal within 4 weeks should therefore be considered a chronic wound. Chronic wounds remain in an inflammatory state for a longer period, which is the reason for a different treatment profile than standard acute wounds.

[0021] Today, chronic wounds are a major public health problem. Their incidence continues to increase, particularly due to an aging population and an increase in the occurrence of underlying diseases such as diabetes.

[0022] Additionally, chronic wounds may never heal, or may take years to heal. These wounds cause significant pain and stress for patients, both physically and emotionally. Therefore, proper treatment is extremely important.

[0023] Solutions are proposed by the prior art to improve the healing process, in particular of chronic wounds via the use of dressings whose microarchitecture and composition are adapted and / or improved to ensure better healing of the wound to be treated.

[0024] Alternatively, the prior art provides pharmaceutical compositions for healing or for promoting the regeneration of skin cells such as a biosimilar hydrogel based on hyaluronic acid crosslinked with L-lysine, described by document EP 3 666 278; a composition comprising genetically modified recombinant platelet-derived growth factor or PDGF and dexamethasone as an anti-inflammatory agent, described by document EP 0 575 484 or a composition comprising transforming growth factor P or TGF-P, as described by document US 5 981 606. However, these pharmaceutical products correspond to growth factors, cytokines or chemokines or hyaluronic acid, known for their adverse effects, in particular due to the fact that they are not specific to a cell type.

[0025] Concerning the implementation of growth factors in particular, their interaction with the cell membrane is at the origin of a hypertrophic response (increase in cell size) and a hyperplastic response (increase in cell population), i.e. a clonal proliferation of cells. The conventional cell growth factors used are of animal, cellular and / or plant origin, i.e. obtained from a biological raw material. For example, widely used factors are cholera toxin, fetal calf serum (FCS), any extract of bovine pituitary gland or undefined complexes or fractions of milk or soy.However, the use of these cell growth factors of variable, undefined formulation and of animal or plant origin is increasingly criticized, particularly due to the lack of traceability of these components, the variability of their quality, a risk of contamination and the lack of reproducibility of the observed effects. In addition, the presence of allergens cannot be verified since the formulations of these growth factors are undetermined. There is therefore a risk of allergies developing in response to their use.

[0026] In parallel, the prior art proposes other solutions for treating wounds or osteoarthritis such as that of document US 2023 / 148647 which concerns a composition comprising 6 particular amino acids in a specific ratio and a divalent metal to promote the synthesis of collagen and tropoelastin, or document WO 2023 / 042120 which concerns a composition comprising 2 forms of sodium hyaluronate and a mixture of amino acids.

[0027] In the field of cosmetics, many compositions have been developed to combat skin aging, as well as to promote the regeneration of skin cells, particularly in the elderly, for example document CN 108 618 997 which describes a cosmetic composition for its use as an anti-aging and skin whitening agent and which comprises sodium hyaluronate with a MW less than 10 kDa, the 21 essential amino acids. These different applications are generally grouped under the indication "anti-aging". In these "anti-aging" products, active ingredients are integrated to directly or indirectly stimulate the growth of fibroblasts and / or keratinocytes, strengthen the dermo-epidermal junction, or promote the production of collagen and elastin. For example, retinoids are described for their ability to stimulate the growth of fibroblasts.However, their use is not recommended in cosmetics due to their phototoxic and potentially mutagenic action (“Photomutagenicity of retinyl palmitate by ultraviolet Airradiation in mouse lymphoma cells” Nan Mei et al. Toxicological sciences 88(1), 142-149 (2005)).

[0028] The treatments currently used are not without harmful side effects and do not achieve effective therapeutic results, reduce the incidence of chronic wounds or effectively and sustainably relieve the pain, discomfort or even aesthetic inconveniences associated with acute or chronic wounds suffered by patients. Furthermore, to the Applicant's knowledge, no treatment can treat an acute wound and a chronic wound indifferently.

[0029] It is clear from the above that there remains a need to provide a composition capable of specifically facilitating and / or improving and / or accelerating the healing mechanism of acute and chronic wounds, in a safe, effective and sustainable manner ( / .«., in the short, medium and long term), while guaranteeing a fully traceable and traceable composition.

[0030] DESCRIPTION OF THE INVENTION

[0031] After extensive research, it is to the Applicant's credit that he has identified, unexpectedly and surprisingly, a composition comprising a combination of safe, traceable compounds (or active ingredients), suitable for human use, in particular therapeutic use, and which act, in particular in a synergistic manner, to meet the aforementioned needs.

[0032] Thus, a first object of the present invention relates to a composition, advantageously pharmaceutical, comprising, as active principles:

[0033] - hyaluronic acid or one of its salts whose molecular weight is greater, advantageously strictly greater, than 2MDa (for megadalton); and

[0034] - at least 8 essential amino acids for humans selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine.

[0035] According to a particular embodiment, said at least 8 essential amino acids for humans included in the composition according to the invention are lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine.

[0036] The present invention provides various advantages, including:

[0037] (i) it allows the simple and routinely integrable use by a doctor / medical personnel of an effective formula whose compounds act in synergy, to improve the healing of a wound, in particular an acute or chronic wound;

[0038] (ii) it is free from deleterious side effects;

[0039] (iii) the formula of the invention is fully chemically identifiable and assayable; and

[0040] (iv) the formula of the invention and its possible degradation products are non-toxic, non-mutagenic, non-carcinogenic and non-allergenic; and

[0041] (v) ready to use, in particular without any extemporaneous or other mixing step to be carried out beforehand.

[0042] In the context of the invention, the articles "a" and "an" are used to refer to one or more (e.g., at least one) units of the grammatical object of the article. For example, "an element" designates at least one element, i.e., one or more elements.

[0043] Within the scope of the invention, the terms "about" and "approximately" are used interchangeably and in reference to a measurable value such as a quantity, a time, and the like. These terms should be understood as encompassing measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value. Within the scope of the invention, the various features of the invention may be presented in the form of a range of values. It should be understood that the description of values ​​in the form of a range is solely for the purpose of making reading easier and should not be interpreted as a rigid limitation of the scope of the invention.Accordingly, the description of an interval of values ​​should be considered to specifically disclose all possible intermediate intervals as well as each of the values ​​within that interval. For example, the description of an interval from 1 to 6 should be considered to specifically describe each of the intervals it comprises, such as the intervals 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., as well as each of the values ​​within that interval, e.g., 1; 2; 2.7; 3; 4; 5; 5.3 and 6. This definition holds regardless of the scope of the interval.

[0044] In the context of the invention, the terms "formula", "composition" and "formulation" are used interchangeably.

[0045] In the context of the invention, the expression "amino acid essential for humans" means an amino acid (i.e., an organic acid, the molecule of which also carries an amine function) essential for humans which 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, to which arginine and histidine must be added for children.

[0046] In the context of the invention, the expression "hyaluronic acid or one of its high molecular weight salts" denotes a hyaluronic acid or one of its salts whose molecular weight is greater, advantageously strictly greater, than 2MDa.

[0047] In the context of the invention, the expressions "hyaluronic acid or one of its low molecular weight salts" and "hyaluronic acid or one of its low molecular weight salts" are used interchangeably and designate a hyaluronic acid or one of its salts whose molecular weight is less than 2 MDa, advantageously less than 1.8 MDa, preferably less than 1 MDa.

[0048] In the context of the invention, the expressions "active compound", "active ingredient" and "active principle" are used interchangeably and designate a substance or a compound which has therapeutic properties which underlie a physiological effect. The active compound, active ingredient or active principle is to be distinguished from at least one excipient, preferably present in the composition according to the invention. In the context of the invention, the term "excipient" designates a substance or a compound which does not have any therapeutic properties. The excipient guarantees in particular the creation of a particular texture, viscosity, fragrance and / or color for a therapeutic formulation, but also its dilution, preservation, storage stability, safety and shelf life, in compliance with the regulations. The excipient is to be distinguished from the at least one active compound, the active ingredient or the active principle present in the composition according to the invention.

[0049] In the context of the invention, the expression “pharmaceutical composition” means a composition for therapeutic and non-cosmetic use.

[0050] For the purposes of the invention, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in the transport or delivery of the agents in question from one organ, or part of the body, to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other active ingredients and / or excipients of the composition, for example the carrier does not diminish the impact of the agent on the treatment. In other words, a carrier according to the invention is pharmaceutically inert.

[0051] In the context of the invention, the expressions "wound", "excoriation" and "lesion" are used interchangeably and designate a so-called open trauma, that is to say a rupture of the skin barrier, as opposed to a so-called closed trauma without a wound, that is to say a contusion. In particular, a wound is a lesion of the skin represented by a rupture of the continuity of the tissues and a breach of the skin barrier requiring a complex dynamic process to be repaired or healed. It can be superficial, affecting only the epidermis (erosion), a part of the dermis or be deep with exposure of the subcutaneous tissue. Its evolution depends on its extent and depth but also on local or general factors which can slow down or prevent its healing.

[0052] In the context of the invention, the expression "acute wound" means an injury of sudden onset and short duration. It heals within the foreseeable, expected time frame and in accordance with the normal healing process. For acute wounds, the physiological 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 damaging blood vessels, nerves and / or muscles (vascular, nerve and / or muscle wounds). For example, it can be a surgical wound, i.e., an incision made voluntarily by a healthcare professional; a traumatic wound, i.e., a sudden and unexpected injury such as an abrasion or a bite; or a burn, i.e., a type of injury to the skin or other tissues caused by heat, cold, electricity, chemicals, radiation, or friction. An acute wound is distinguished from a chronic wound.

[0053] In the context of the invention, the expression "chronic wound" refers to a wound that does not heal in accordance with the order of the stages of the healing process and / or within a period of time greater than the foreseeable period, like most other wounds known as acute wounds. Sometimes one or other phase of the healing process becomes blocked. For example, the inflammatory stage often lasts too long. For chronic wounds, the physiological healing process lasts for more than 4 to 6 weeks. Examples include venous and arterial ulcers, diabetic ulcers, and pressure ulcers. A chronic wound must be distinguished from an acute wound. Any initially acute wound that does not heal within 4 weeks must therefore be considered a chronic wound.

[0054] In the context of the invention, the expressions "improve healing", "increase healing", "promote healing", "encourage healing", "promote healing" and "facilitate healing" are used interchangeably and designate the mechanism by which the phases of healing are more efficient (i.e., better cell survival, increased frequency and / or quantity of cell cloning, etc.) and / or follow one another more quickly than the foreseeable time frame, i.e., faster and more stable healing, i.e. little, if any, risk of the scar reopening.

[0055] In the context of the invention, the expressions "improve cell viability", "increase cell viability", "promote cell viability", "encourage cell viability", "promote cell viability" and "facilitate cell viability" are used interchangeably and designate an increase in cell adhesion and / or proliferation and / or metabolic activity and / or integrity of the cell membrane compared to a reference value measured with the same measuring apparatus / tests (e.g., MTT test; tetrazolium salt) and before application to the cells of the candidate / test composition.

[0056] In the context of the invention, the expression "cell adhesion" refers to all the cellular and molecular mechanisms used to make cells adhere to each other or to the environment surrounding them. Cell adhesion is essential for the formation, maintenance and functioning of tissues.

[0057] In the context of the invention, the expression "cell proliferation" refers to the fact that cells multiply rapidly and abundantly. It is measured as the quantified value of the population of daughter cells. This mechanism is to be dissociated from 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).

[0058] In the context of the invention, the expression "cellular metabolic activity" means the sum of catabolism (or degradation activity) and anabolism (or synthesis activity). Catabolism degrades complex molecules into basic molecules, either to produce energy or to produce molecules directly usable by the anabolism pathways. Anabolism concerns all syntheses.

[0059] Within the scope of the present invention, all the embodiments described above and below can be combined.

[0060] According to a particular embodiment, the active ingredients included in the formula according to the invention consist of:

[0061] - hyaluronic acid or one of its salts with a molecular weight greater than 2MDa; and

[0062] - at least 8 essential amino acids for humans selected from the group consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine and histidine.

[0063] According to a particular embodiment, the formula according to the invention consists of:

[0064] - hyaluronic acid or one of its salts with a molecular weight greater than 2MDa; and

[0065] - 8 essential amino acids for humans consisting of lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine.

[0066] Hyaluronic acid is a polysaccharide, in particular a glucosaminoglycan (GAG), whose chain is made up of a polymer of disaccharides, linked together by alternating P-1,4 and P-1,3 glycosidic bonds, represented by the formula:

[0067] [p-D-glucuronic acid + N-acetyl D-glucosamine]n n The hyaluronic acid or one of its salts according to the invention has a molecular weight greater than 2 MDa, advantageously strictly greater than 2 Mda and / or less than 3 MDa, preferably between 2 MDa and 3 MDa.

[0068] Hyaluronic acid or one of its salts according to the invention induces, in combination with the amino acids as described above, an unexpected technical synergistic effect ensuring an increase in the proliferation and / or migration speed of skin cells, in particular fibroblasts which, ultimately, ensures in particular faster and more stable healing of a wound. It is clear from the examples below that such an effect is surprising in that it is obtained only with a hyaluronic acid or one of its salts according to the invention having a molecular weight greater than 2 MDa and not with a hyaluronic acid or one of its salts of lower molecular weight.

[0069] According to a particular embodiment, the hyaluronic acid or one of its salts is in a non-crosslinked or crosslinked form, advantageously non-crosslinked.

[0070] According to a particular embodiment, the hyaluronic acid or one of its salts is in a non-hydrolyzed form.

[0071] According to the invention, hyaluronic acid is derived from biotechnology, in particular by bacterial fermentation. In other words, it is a product of non-animal origin.

[0072] 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.

[0073] According to an advantageous embodiment, the hyaluronic acid or one of its salts included in the composition according to the invention is sodium hyaluronate.

[0074] According to the invention, hyaluronic acid or one of its salts is an active compound and not an excipient, even less a solvent such as a dilution solvent or a viscosity excipient.

[0075] 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.

[0076] 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.

[0077] 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 buffering compounds, including 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 may be included in an amount required to maintain the pH of the composition within a pharmaceutically acceptable range. One or more pharmaceutically acceptable salts may be included in the composition in an amount sufficient to bring the osmolality of the composition 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.

[0078] In particular, the composition according to the invention further comprises, as excipient(s), at least one compound (with pH buffer effect), advantageously all the compounds, chosen from sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water, advantageously water for injections (or ppi).

[0079] According to a preferred embodiment, the composition according to the invention consists of:

[0080] - sodium hyaluronate;

[0081] - the following essential amino acids: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and threonine;

[0082] - Na2HPÛ4;

[0083] - KH2PO4;

[0084] - NaCl; and

[0085] - water ppi.

[0086] Advantageously,

[0087] - hyaluronic acid or one of its salts, advantageously sodium hyaluronate, represents between 6 g / L and 8 g / L;

[0088] - lysine represents between 33 mg / L and 39 mg / L;

[0089] - valine represents between 21 mg / L and 25 mg / L; - isoleucine represents between 5.7 mg / L and 6.6 mg / L;

[0090] - leucine represents between 24.3 mg / L and 28.3 mg / L;

[0091] - methionine represents between 6.8 mg / L and 8.2 mg / L;

[0092] - phenylalanine represents between 15 mg / L and 18 mg / L;

[0093] - tryptophan represents between 4.6 mg / L and 5.6 mg / L;

[0094] - threonine represents between 21.1 mg / L and 25.2 mg / L;

[0095] - Na2HPÛ4 represents between 1.35 g / L and 1.45 g / L;

[0096] - KH2PO4 represents between 0.65 g / L and 0.75 g / L;

[0097] - NaCl represents between 4.5 g / L and 5.5 g / L; and

[0098] - the ppi water represents a sufficient quantity for IL.

[0099] 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 at a value of 7.45 and / or the osmolarity is between 280 and 360 mOsmole, advantageously between 300 and 350 mOsmole.

[0100] According to a particular embodiment, the composition according to the invention is sterile / made sterile.

[0101] According to a particular embodiment, the composition according to the invention is in a galenic form suitable for topical use and / or by injection, in particular pharmaceutical, acceptable, that is to say compatible with the skin, mucous membranes, blood vessels, cornea and / or muscles of the human body.

[0102] According to a particular embodiment, the composition according to the invention is in a galenic form suitable for topical cutaneous or corneal administration.

[0103] According to another particular embodiment, the composition according to the invention is in a galenic 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., wound) and / or intra-articular injection and / or intra-cartilaginous injection.

[0104] According to another specific embodiment, the intradermal injection of the composition of the invention is carried out in the wound and / or in the healthy tissue close to the wound, that is to say in the healthy tissue at a distance of 0.1 cm to 5 cm from the edge of the wound, advantageously 0.1 to 4 cm, 0.1 to 3 cm, 0.1 to 2 cm, 0.1 to 1 cm, or even 0.1 to 0.5 cm.

[0105] According to a particular embodiment, the composition according to the invention is in a galenic form suitable for use in contact with cells and / or tissues in vivo, ex vivo or in vitro, in particular in culture or in suspension, in a method for improving the viability of cells and / or grafting and / or transplantation.

[0106] 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 mousse, a spray / sprayable composition, an eye drop, a soaked dressing or even an aqueous, hydroalcoholic, organic or oily solution, or even a suspension or a dispersion in solvents or fatty substances.

[0107] Preferably, the pharmaceutical composition according to the invention contains a vehicle / support, 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 a formulation capable of being injected.

[0108] These may include isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts), or dry compositions, particularly lyophilized, which when added, depending on the case, to sterilized water or physiological serum, allow the constitution of injectable compositions.

[0109] The doses used for administration can be adapted according to different parameters, and in particular according to the method of administration used, the pathology concerned, or even the desired duration of treatment and the surface area to be treated.

[0110] For example, it is known from the prior art to start treatment with doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

[0111] The daily dosage of the formulation according to the invention, in particular of a formulation suitable for topical use, is 0.1 to 1.5 ml / cm 2 , advantageously from 0.1 to 0.5 mL / cm 2 . 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 to be treated. Thus,

[0112] - for 1 cm 2 : 0.25mL to ImL;

[0113] - for 2 cm 2 : 0.4mL to 1.6mL;

[0114] - for 4 cm 2 : 0.65mL to 2.6mL; advantageously, injections are performed every cm or 0.5cm once a week.

[0115] The dosage, advantageously daily, of the formulation according to the invention, in particular of a formulation suitable for cartilaginous and / or articular use, in particular for use by intra-articular injection, is from 0.1 to 5 mL / joint, advantageously from 0.5 to 5 mL / cm 2 , preferably 1 to 5 mL / cm 2 .

[0116] The dosage of the formulation according to the invention, in particular of a formulation suitable for use to advantageously improve the grafting and / or transplantation of a tissue, in particular for in a mixture with adipose tissue for tissue grafting, is from 1% to 50% of the volume of fat which will be grafted, advantageously from 5% to 40%, preferably from 10% to 30%.

[0117] Regardless of the method of administration, administration is strictly contraindicated in the case of a cancerous lesion. If a cancerous lesion is suspected, a biopsy should be performed and the results obtained before administering the formulation according to the invention.

[0118] According to one embodiment, the composition used within the framework of the invention is intended for human and / or veterinary use.

[0119] 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, the term "human subject" means a man or a woman, in particular a child, an adult or an elderly person.

[0120] According to another embodiment, the animal targeted by the present invention is advantageously a domestic animal, in particular felines, canines, avians or even rodents and / or a farm animal, in particular poultry, bovines, goats, sheep, pigs, equines, even camelids or deer. According to one embodiment, the composition used in the context of the invention is intended for use in the field of vascular medicine, gynecology, ophthalmology, endocrinology / metabolic medicine and / or dermatology.

[0121] According to the invention, the composition is intended for healing and cellular and tissue regeneration.

[0122] In particular, the composition of the invention is intended to be implemented:

[0123] 1- for the healing of acute wounds such as acute contuse wounds; acute wounds in patients with difficult healing conditions (eg, diabetes, arterial ischemia, venous ischemia, connective tissue diseases); post-operative acute cutaneous ischemia (arterial or venous): ischemia of skin flaps, ischemia of the areolas and nipples after breast surgery, acute cutaneous ischemia after any type of plastic and aesthetic surgery, acute cutaneous ischemia after any type of orthopedic surgery for cutaneous approaches, acute cutaneous ischemia after any type of vascular surgery for cutaneous approaches; post-operative skin detachments regardless of the surgery, without exposure of joints, bones and vessels; or burns (especially 1st and 2nd degree); and / or

[0124] 2 for the healing of chronic wounds such as: chronic arterial wounds; chronic venous wounds; non-infected grade I and grade II pressure ulcers; chronic skin dehiscence; post-radiotherapy wounds; diabetic foot wounds or fibrinous wounds; and / or

[0125] 3- in addition to other skin repair techniques such as direct suture repair; dermo-epidermal skin grafting or total skin grafting or in addition to skin flaps; and / or

[0126] 4- for the healing and / or hydration of the cornea, particularly in the context of dry eye syndrome; for the treatment of corneal ulcers; in addition to corneal grafting or as symptomatic treatment of corneal dryness; and / or

[0127] 5- for hydration and / or cartilage and / or joint regeneration, in particular an intra-articular injection in the prevention and symptomatic treatment of osteoarthritis / arthritis; an injection into the intervertebral disc for the symptomatic treatment of osteoarthritis of the spine or an injection into the muscles, ligaments, tendons and capsules; and / or

[0128] 6- for the hydration of a mucous membrane, in particular to treat the symptoms of vaginal dryness, postmenopausal vaginal atrophy, dry nasal, oral and anal mucous membranes, damaged mucous membranes (eg throat, vocal cords, esophagus, stomach, intestine, interstitial cystitis); and / or

[0129] 7- for tissue grafting and / or transplantation, in particular in addition to skin, tendon, fat or hair grafts; and / or

[0130] 8- for cell grafting and / or transplantation.

[0131] According to another aspect, the invention relates to the composition according to the invention as described above for use in a method of healing a wound, advantageously an acute or chronic wound. In the same way, the invention relates to the composition according to the invention as described above for use in reducing the risk of reopening of the scar.

[0132] In other words, the invention also aims at:

[0133] - the composition according to the invention for use in healing a wound, advantageously an acute or chronic wound; and / or

[0134] - 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

[0135] - a method for healing a wound, advantageously an acute or chronic wound, comprising the administration, advantageously the administration by injection, of the composition according to the invention.

[0136] According to a particular embodiment, the composition according to the invention for its use as described above is for healing, in particular to improve, accelerate or promote healing, i.e. to increase the speed of migration and / or proliferation of cells such as fibroblasts and / or keratinocytes.

[0137] Advantageously, it is a question of accelerating at least one of the 4 phases of healing and / or the sequence of phases of healing.

[0138] In particular, the composition according to the invention has the effect of:

[0139] - to improve / accelerate the migration of fibroblasts and / or keratinocytes at the wound level to reconstruct the dermis and / or epidermis; and / or

[0140] - to increase / stimulate the production and / or secretion of at least one protein / peptide chosen from perlecan, laminin, loricrin and beta-defensin; and / or

[0141] - to increase / stimulate the activity of at least one protein / peptide chosen 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 scar reopening.

[0142] In other words, the invention also aims at:

[0143] - the composition according to the invention for use in reducing the risk of reopening of the scar; and / or

[0144] - the use of the composition according to the invention for preparing a medicament for reducing the risk of reopening of the scar; and / or

[0145] - a method of reducing the risk of scar reopening comprising the administration, advantageously the administration by injection, of the composition according to the invention.

[0146] According to another aspect, the invention relates to the composition according to the invention as described above for use in a method, advantageously non-cosmetic, of hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound.

[0147] In other words, the invention also aims at:

[0148] - the composition according to the invention for use in moisturizing the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound; and / or

[0149] - the use of the composition according to the invention for preparing a medicament for moisturizing the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound; and / or

[0150] - a method of hydrating the skin and / or a wound, advantageously an acute or chronic and / or dry or exudative wound, comprising the administration, advantageously the administration by injection, of the composition according to the invention.

[0151] According to another aspect, the invention relates to the composition according to the invention as described above for use in a method, advantageously in vitro, for maintaining, or even improving, the cellular viability of cells, in particular fibroblasts, keratinocytes, corneal epithelial stem cells (or corneal epithelial stem cells), and / or adipocyte stem cells, for example in a therapy using stem cells, in particular stem cell injections.

[0152] In other words, the invention also aims at:

[0153] - the composition according to the invention for use, advantageously in vitro, to maintain, or even improve, the cellular viability of cells, advantageously fibroblasts, keratinocytes and / or stem cells of the corneal epithelium and / or adipocyte stem cells, for example in a therapy using stem cells, in particular stem cell injections; and / or

[0154] - the use, advantageously in vitro, of the composition according to the invention for preparing a medicament for maintaining, or even improving, the cellular viability of cells, advantageously fibroblasts, keratinocytes, corneal epithelium stem cells and / or adipocyte stem cells, for example in a therapy using stem cells, in particular stem cell injections; and / or

[0155] - a method, advantageously in vitro, for maintaining, or even improving, the cellular viability of cells, advantageously fibroblasts, keratinocytes of the corneal epithelium and / or adipocyte stem cells, for example in a therapy using stem cells, in particular stem cell injections, comprising the application, advantageously the administration by injection, of the composition according to the invention.

[0156] According to a particular embodiment, the composition for its use as described above is in vitro, ex vivo and / or in vivo.

[0157] According to a particular embodiment, the improvement in viability according to the invention is the improvement in adhesion and / or proliferation and / or metabolic activity of the cells.

[0158] According to a particular embodiment, the composition for its use in improving the viability of cells is to be distinguished from a cell culture medium in that it is not a medium in which all the elements necessary for the viability and growth of cultured cells are found (eg, glucose, trace elements, vitamins, etc.).

[0159] According to another aspect, the invention relates to a method of 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 in the center of the wound.

[0160] In particular, the wound edge determines the boundary between intact and unintegrated tissue. It is important to treat intact tissue whose biological function is or may be compromised so that this compromise is minimized and allows for better vascularization and stimulation of tissue healing with disruption of integrity (wound).

[0161] At the wound level, the objective of the administration method according to the invention is to accelerate healing by optimizing all biological healing mechanisms. According to one embodiment, the edge of the wound is delimited as the starting point of the treatment / intradermal or subcutaneous injection which is continued towards the outside of the wound / healthy tissue.

[0162] According to another embodiment, the edge of the wound is delineated as the starting point of the treatment / intradermal injection which is continued towards the interior of the wound / damaged tissue.

[0163] According to an alternative embodiment, the edge of the wound is delimited as the starting point of the treatment / intradermal injection which is continued towards the outside of the wound / healthy tissues then towards the inside of the wound / damaged tissues, or vice versa in the treatment sequence (ze, towards the inside then the outside of the wound).

[0164] According to one embodiment, the depth of the injection is between 1 mm and 3 mm depending on the tissue (intra or subcutaneous in the entire skin and immediately under the granulation or epithelialization tissue in wounds).

[0165] According to one embodiment, the injection according to the invention is carried out by the fan-shaped retrograde injection technique or the point-by-point technique.

[0166] The invention also relates to a kit for therapeutic purposes as described above.

[0167] 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.

[0168] Devices include syringes, implantable pumps, such as mini- and micro-pumps, and other devices for pharmaceutical use.

[0169] In particular, the device included in the kit according to the invention is a syringe pre-filled with the composition of the invention.

[0170] The following examples, without being limiting, form an integral part of the invention and any characteristic which appears to be new compared to the state of the prior art is claimed as such and as a general means.

[0171] FIGURES 1 Evaluation of the effect of the composition of the invention (LUM 7 quater), the composition of the invention devoid of hyaluronic acid or one of its salts (LUM 7 AA) and the composition of the invention devoid of amino acids (LUM 7 HA3), at 3h, 24h, 72h and 6 days, on the viability of adipose tissue mesenchymal stem cells (ASC). The results are from ASCs from 3 specific donors and from an experiment with 10 wells per condition. Statistical tests are performed versus the “DMEM” condition (significance: *: p<0.05; **: p<0.01; ***: p<0.001).

[0172] Evaluation of the effect of the composition of the invention (LUM 7 quater), of the composition of the invention devoid of hyaluronic acid or one of its salts (LUM 7 AA) and of the composition of the invention devoid of amino acids (LUM 7 HA3), at 3h, 24h, 72h and 6 days, on the adhesion, proliferation and metabolic activity of adipose tissue mesenchymal stem cells (ASC). The results are from ASCs from 3 specific donors and from an experiment with 12 wells per condition. Statistical tests are performed versus the “MC” condition (significance: *: p<0.05; **: p<0.01; ***: p<0.001).

[0173] In vitro evaluation of the effect of the composition of the invention, at different concentrations ( / .«., stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution) in comparison with a control (DMEM), on the production / secretion of perlecan by primary human fibroblasts in culture.

[0174] Figure 4 In vitro evaluation of the effect of the composition of the invention, at different concentrations ( / .«., stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution) in comparison with a control (DMEM), on the production / secretion of laminin by primary human fibroblasts in culture.

[0175] In vitro evaluation of the effect of the composition of the invention, at different concentrations ( / .«., stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution) in comparison with a control (Celloneer KC / CC), on the production / secretion of loricrin by human epidermal keratinocytes in culture.

[0176] In vitro evaluation of the effect of the composition of the invention, at different concentrations ( / .«., stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution), in comparison with a control (Celloneer KC / CC), on the production / secretion of beta-defensin by human epidermal keratinocytes in culture. 7 Monitoring of the effect of the composition of the invention, at different concentrations (ze, stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution), in comparison with a control (DMEM), on the migration speed of fibroblasts following an experimental injury.

[0177] Photographs of a chronic wound on the lateral side 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 (intralesional injections) and into the healthy tissue near the wound (perilesional injections).

[0178] Figure 9 Photographs of the monitoring of fibroblast migration for 16 hours (i.e., at T0,

[0179] T8h, T12h and T16h) depending on 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.

[0180] Kinetics of fibroblast migration for 30 hours in the presence of Lum 7

[0181] 20% and 50% compared to control Means + / - standard deviation; statistical analysis with Mann Whitney test: *p<0.05; **p<0.01.

[0182] Kinetics of fibroblast migration for 30 hours in the presence of Lum

[0183] H1 20% and 50% compared to control Means + / - standard deviation; statistical analysis with Mann Whitney test: *p<0.05; **p<0.01.

[0184] Kinetics of fibroblast migration for 30 hours in the presence of Lum A

[0185] 20% and 50% compared to control Means + / - standard deviation; statistical analysis with Mann Whitney test: *p<0.05; **p<0.01.

[0186] Figure 13 Evaluation of 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 to the results obtained with the culture medium (CM): CM normalized to 100% at all times.

[0187] Raw results of optical densities (570 nm) and their evolution after 3, 12 or 24 hours of exposure to the composition of the invention or to the 3 eye drops of the prior art - dilution 1 / 2 in DMEM. Figure 15: Raw results of optical densities (570 nm) and their evolution after 3, 12 or 24 hours of exposure to the composition of the invention or to the 3 eye drops of the prior art - dilution 1 / 2 in PBS.

[0188] EXAMPLES OF ACHIEVEMENT

[0189] Example 1. Evaluation of the toxicity of the composition of the invention

[0190] 1. Objective of the study

[0191] The aim of this study is to demonstrate the non-cytotoxicity of the composition of the invention at 4 different times (ze, 3h, 24h, 72h and 6 days) on adipose tissue stem cells (or mesenchymal stem cells of adipose tissue or "Adipose derived Stem Cells" or ASC) from 3 donors by an MTT test (or 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide).

[0192] 2. Materials and methods

[0193] 2.1, Cells

[0194] ASCs from the 3 donors at a density of 10,000 cells / well (96-well plate).

[0195] Donor 1: 11171 28 years old (P2 bulb - seeded in P4);

[0196] Donor 2: 12025 28 years (PO bulb - seeded in P2); and

[0197] Donor 3: 12106 40 years old (PI bulb - seeded in P3)

[0198] 2.2, Preparation of tested compositions

[0199] The active ingredients used in the composition of the invention LUM 7 QUATER are illustrated in Table 2 below.

[0200] [Table 2]

[0201] The active ingredients used in the LUM 7 HA3 composition are illustrated in Table 3 below.

[0202] [Table 3]

[0203] The active ingredients used in the LUM 7 AA composition are illustrated in Table 4 below.

[0204] [Table 4] The preparation of the formula involves 3 phases, namely:

[0205] - preparation of the pH buffer solution: add the total quantity of water then stir, add the buffer salts and after complete dissolution, check the pH conformity; - addition of the NaCL and amino acids: still with constant stirring, add the NaCL, wait for it to dissolve then add the amino acids, checking their dissolution before each addition; and

[0206] - adding hyaluronic acid: still stirring constantly, add the hyaluronic acid in small portions and wait for complete dissolution before stopping stirring.

[0207] Once the preparation of the formula is complete, check the pH then divide into bottles or syringes and finally autoclave the bottles or syringes obtained.

[0208] 2.3. Conditions

[0209] - LUM 7 Quater (composition of the invention): 6 vials of 5ml

[0210] - LUM 7HA3 (LUM 7 Quater composition devoid of amino acids): 8 vials of 5mL

[0211] - LUM 7 AA (LUM 7 Quater composition without hyaluronic acid): 8 vials of 5ml Please note that only one dilution will be tested for each formula in the example (pure).

[0212] Sodium dodecyl sulfate (SDS) is used as a negative viability control (0.05% dissolved in the proliferation medium).

[0213] An untreated control in contact only with DMEM will be used to determine 100% viability (positive control).

[0214] A control in culture medium will also be carried out.

[0215] The culture medium contains: “DMEM / F12 with GlutaMAX”, 10% SVF, 100 / mL penicillin, streptomycin, 5 pg / mL fungizone, 10 ng / mL “FGF2 premium grade” (MiltenyiBiotec, Paris, France).

[0216] 2.4. Experimental protocol

[0217] ASCs are thawed and seeded into flasks and cultured to confluence. ASCs will be trypsinized, seeded into 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 test. For each condition and time, 12 wells will be treated. During the MTT test, the percentage of viability of each condition will be calculated against the positive control which will represent 100% viability. The negative control 0.05% SDS should be equal to 0% viability.

[0218] The principle of the test is based on the use of a tetrazolium salt, MTT ((3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium 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, purple-colored compound must be solubilized with DMSO to allow its spectrophotometric determination at 540 nm.

[0219] 2.5. Data analysis

[0220] Raw data were analyzed using Microsoft Excel software.

[0221] Intergroup comparisons were performed by unpaired Student's t-test. Differences were considered statistically significant at p<0.05. (NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001).

[0222] 3. Results

[0223] The results are shown in Figure 1.

[0224] It appears that:

[0225] DMEM is an isotonic base solution containing neither growth factor nor calf serum. It allows ASCs to be maintained viable for 72 hours. It is used as a positive control.

[0226] The culture medium (CM) is a very rich medium: it contains calf serum and a growth factor (FGF2) and cannot be administered to humans.

[0227] 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.

[0228] The viability of cells in contact with the composition of the invention devoid of hyaluronic acid (LUM 7 AA) decreases rapidly.

[0229] These data demonstrate 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 one of its salts with the 8 amino acids of the invention has a significant synergistic effect of increasing cell viability. Evaluation of the effect of the of the invention on adhesion. and activity stem cell research fabric

[0230] 1. Objective of the study This study aims to demonstrate the effect of the composition of the invention on adhesion, proliferation and metabolic activity at 4 different times (ze, 3h, 24h, 72h and 6 days) on adipose tissue stem cells (or mesenchymal stem cells of adipose tissue or "Adipose derived Stem Cells" or ASC) from 3 donors by an MTT (or 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test.

[0231] 2. Materials and methods

[0232] See points 2.1. to 2.3. and 2.5. of Example 1.

[0233] 2.4. Experimental protocol

[0234] ASCs are thawed in PO, PI or P2 depending on the donors and seeded in flasks in Pl, P2 or P3 and cultured until confluence.

[0235] At confluence, the ASCs were trypsinized and then seeded into a 96-well plate. The products were applied immediately upon seeding into the 96-well plate and left in contact for 24, 72, 96 hours, and 7 days before performing an MTT test. For each condition and time, 8 wells were treated, for a total of 1 96-well plate per time and per donor, 12 plates in total.

[0236] In the MTT test, the percentage of viability for each condition will be calculated relative to the positive control, which will represent 100% viability. The negative control, 0.05% SDS, should be equal to 0% viability.

[0237] It should be noted that MC medium is used here since it is widely known to be a very rich medium, in that it contains calf serum and a growth factor (FGF2). It is 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 according to the invention.

[0238] 3. Results

[0239] The results are shown in Figure 2.

[0240] It appears that mesenchymal cells of adipose tissue have a similar behavior in the 3 formulations LUM 7 quater, LUM 7HA3 and LUM 7 AA. tion in vitro of the effect of the of the invention on the of perlecan and laminin by human primary fibroblasts as well as loricrin and beta-defensin by human culture

[0241] 1. Cultivation and maintenance of the HEK line

[0242] HEK cells are human keratinocyte cells derived from skin from different donors, cultured in Celloneer KC / CC, a basal medium for keratinocytes. The cells are maintained 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.

[0243] 2. Cultivation and maintenance of the FPH line

[0244] Fibroblasts from a 40-year-old woman (FPH) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-Glutamine containing essential amino acids, salts, glucose, and vitamins necessary for cell stimulation, and 1% penicillin-streptomycin. The addition of a volume corresponding to a 1% concentration of penicillin-streptomycin prevented possible bacterial growth. The mixture was filtered through a stericup (milliport). The cells were maintained in an incubator at 37°C in a humid atmosphere (85 to 90% humidity) containing 5% CO2. The medium was renewed 4 days after culturing and then twice a week.

[0245] 3. Experimental protocol

[0246] The HEK and FPH are treated daily with the composition of the invention corresponding to the LUM 7 quater formula of Example 1 at different concentrations. In particular, a stock solution of LUM 7 quater is prepared as described in Example 1 and then dilutions of 20%, 30%, 40% and 50% are applied to this stock solution. The stock solution as well as the formulas diluted to 20%, 30%, 40% and 50% are applied to the cells in culture.

[0247] 4. Results

[0248] The results are presented in Figures 3 to 6.

[0249] It appears that:

[0250] - 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). In addition, the production of beta-defensin, linked to antimicrobial protection, is increased in keratinocytes in the presence of the composition of the invention.

[0251] 4. In vitro evaluation of the effect of the of the invention on the speed of fibroblasts following injury

[0252] The cell mat wound healing assay involves pulling cells in a controlled manner along a specific path in a Petri dish and observing at regular intervals / filming how cells surrounding the "wound" colonize the empty space. The main component of wound healing will be cell migration, but there may also be a proliferation component.

[0253] 1. Experimental protocol

[0254] 1.1. Preparation of cultures

[0255] The first step of the test is to culture a monolayer of confluent FPH. This monolayer represents the in vivo conditions of the tissue before injury, for example, an intact epithelium.

[0256] 1.2. Creation of the injury

[0257] Once the cells have become confluent, the next step is to create a cell-free space within the monolayer. The method involves injuring the monolayer by mechanical scratching (or "scratch wound").

[0258] 1.3. Data acquisition

[0259] After wound application and treatment or not 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 to the wound area. Once the microscope is set up, a series of time-lapse images (snapshot method) are acquired as the cells migrate into the cell-free space. These time points are collected every hour, for 72 hours. Accurate measurements are also acquired manually using a digital camera installed on the microscope.

[0260] 1.4. Data Analysis Migration was analyzed using a Nikon inverted microscope at 20X magnification (Melville, NY). Time-lapse recording began after treatment with the composition of the invention at the different concentrations tested, of the scratch-wounded FPH culture. Three fields per well were imaged and tracked at 1-hour intervals for 72 hours with a Coolsnap HQ camera (Photo-metrics, Tucson, AZ) operated by NIS-elements AR 2.30 software (Nikon). Manual tracking of individual cells was performed using Metamorph software (Roper Scientific, Evry, France). Calculated migration parameters for each individual cell were determined from the time-lapse movies. They include total migration distance, distance to the origin, velocity, and directional persistence of cell migration.The total migration distance represents the sum of the distances between each measurement over a 72-h period. For each condition, results are expressed as the mean ± SD of at least 60 individual cells.

[0261] Note 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.

[0262] 2. Results

[0263] The results are shown in Figure 7.

[0264] It appears that cultured fibroblasts, stimulated by the composition of the invention at different concentrations, exhibit a higher migration speed than fibroblasts in control conditions. This means that wound recovery and therefore healing is accelerated by the administration of the composition of the invention.

[0265] These data are confirmed by Figure 8 which shows that the in vivo administration of the composition of the invention (stock solution and dilutions at 20%, 30%, 40% and 50% of the stock solution) leads to improved and accelerated healing compared to a conventional treatment protocol.

[0266] Example 5. In vivo evaluation of the effect of the composition of the invention on the healing of a chronic wound

[0267] A 58-year-old paraplegic patient with a chronic wound on the lateral aspect of the foot that had been developing for several years was treated with weekly injections into the wound (intralesional injections) and into healthy tissue near the wound (perilesional injections) for 2 months. Figure 8 shows a photograph before treatment (A) and after 2 months of treatment (B).

[0268] 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.

[0269] Example 6: Comparative in vitro study of the effect of the composition of the invention on the migration speed of fibroblasts following an experimental injury

[0270] This study aims to evaluate the effect of 3 compositions on the in vitro migration of primary human fibroblasts using the Scratch-test technique.

[0271] 1. Experimental protocol

[0272] 1.1. Cell preparation

[0273] Human neonatal fibroblasts (from LifeLine Cellsystems) are cultured in Fibrolife medium (LifeLine Cellsystems) in 24-well plates (7x104 cells / well) for 3 days, until confluence, in an incubator at 37°C in a humid atmosphere with 5% CO2.

[0274] 1.2. Scratch test technique

[0275] In each well, a wound is made in the cell mat using a 10 μL cone. The culture medium is removed and 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.

[0276] 1.3. Imaging monitoring of cell migration

[0277] The plate is then placed under a video microscope (CARLZEISS AXIO OBSERVER Zl) in a humid incubation chamber at 37°C, with 5% CO2. For each well, two positions are pre-recorded to be monitored over time. Images are recorded every 2 hours for 48 h.

[0278] 1.4. Analysis of results

[0279] For each position, over time, an analysis by Image J software with the “Wound Healing Size Tool” Plugin is carried out by quantifying the extent of the uncovered wound. The wound recovery rate, which evaluates cell migration, is calculated using the following formula: Area at time TO — Area at time T

[0280] — - - x 100 = Recovery rate (%)

[0281] Area at time T0

[0282] The recovery rate is calculated from the 6 values ​​(3 replicates x 2 positions / well) for each condition and for each time. The results are represented as the mean (+ / - standard deviation) of the recovery rate. A statistical study is carried out using the non-parametric Mann-Whitney test and comparing each condition to the control.

[0283] 2. Compositions tested

[0284] - Control: Fibrolife medium (LifeLine Cellsystems)

[0285] - LUM 7 Quater (or Lum 7; composition of the invention): composition according to example 1

[0286] - LUM A (composition of the invention devoid of hyaluronic acid): corresponding to the composition LUM 7 AA of example 1

[0287] - LUM H1 (composition comprising a low molecular weight hyaluronic acid and the amino acids of the Lum 7 Quater composition) whose active ingredients are shown in Table 5 below.

[0288] [Table 5]

[0289] 3. Results

[0290] The results are presented in Figures 9 to 12. It is clear from the photographs in Figure 9 that the wounds are all completely covered at approximately 3 Oh, whether for the different conditions or for the control, so the analysis was carried out over times 0 to 30 hours. The photographs represent for each of the conditions time T0 and times T8h, T12h and T16h where the differences between the treatment condition and the control are mostly observed when there are any.

[0291] From time T0, cells gradually cover the wound with the cell mat. From time T8h, cells cover significantly more surface area with the Lum 7 20% or 50% treatment compared to the control and other conditions.

[0292] Concerning the kinetics of fibroblast migration, it appears that:

[0293] - Lum 7 activates fibroblast migration in the early phase at 50%, in the later phase at 20%. The effect is significant for Lum 7 at 50% at times 6h and 8h with +22% and +25% increase compared to the control respectively, and for Lum 7 at 20% at times 12h, 16h to 22h with a maximum increase of 23% at 12h. There is no significant difference if we compare Lum 7 20% and Lum 7 50%;

[0294] - Lum-Hl activates fibroblast migration at 50%, with a significant effect for Lum H1 at 50% at times 16h to 22h with a maximum increase of 14% at 16h. The increase observed for Lum H1 at 20% is not significant compared to the control. There is no significant difference when comparing Lum H1 20% and Lum H1 50%; and

[0295] - Lum A at 20% or 50% has no significant effect on fibroblast migration compared to the control. There is no significant difference when comparing Lum A 20% and Lum A 50%.

[0296] 4. Conclusion

[0297] Lum 7 is the most active and accelerates fibroblast migration with the Scratch Test technique, with a maximum increase of 25% compared to the control. The two concentrations, 20% and 50%, are active but at different times: Lum 7 50% is more active at the beginning of the test, Lum 7 20% more active for later times. Lum H1 is also active but only at the 50% concentration with a maximum increase of 14% compared to the control. Lum A, on the other hand, does not show any effect on fibroblast migration with this technique.

[0298] In other words, it is clear from these data that high molecular weight hyaluronic acid combined with the amino acids of the invention act synergistically to improve wound healing. 7: Evaluation of the effect of the invention on the viability of enitheli cells corneans

[0299] The aim of this study is to verify the non-cytotoxicity of the composition of the invention as well as to evaluate the impact of its administration on COEP in comparison with eye drops of the prior art.

[0300] 1. Study of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test

[0301] The reagent used is the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). The tetrazolium ring it contains is reduced by mitochondrial succinate dehydrogenase in active living cells to formazan. This forms a purple-colored precipitate in the mitochondria. The amount of precipitate formed is proportional to the amount of living cells. An optical density measurement at 570 nm by spectroscopy allows the relative amount of living and metabolically active cells to be determined.

[0302] 1.1. Materials

[0303] The cells used are corneal epithelial cells (COEP) at a density of 10,000 cells / well (48-well plate) COEP 38230536G P3

[0304] The tested compositions are:

[0305] - The LUM 7 Quarter composition (composition of the invention; see example 1): 2 vials of 5 mL

[0306] Prior art eye drops: artificial tears which have the status of drugs used as first-line treatment in dry eye and mild epithelial ulcer, namely o Dulcilarmes 1.5%; o Hylovis multi 15; and o Thealose.

[0307] The eye drops and the composition of the invention are tested diluted to U with DMEM or with PBS.

[0308] Cell lysis buffer: Negative viability control

[0309] DMEM: diluent only

[0310] PB S: diluent alone - Culture medium: used for seeding COEPs and as a control for the MTT test

[0311] 1.2. Methods

[0312] The COEPs are thawed and amplified in a flask for 1 week and then they are seeded at a density of 10,000 cells / well in a 48-well plate previously covered with an irradiated feeder layer.

[0313] The COEPs are cultured for 4 days with 500 pL of culture medium initially and a change of medium after 3 days of culture with 300 pL of medium and EGF.

[0314] After the 4 days of culture, the culture medium is removed and each condition will be put in contact with the COEP:

[0315] Cell lysis buffer 300 pL

[0316] Culture medium: 300 pL

[0317] DMEM: 300 pL

[0318] PBS: 300pL

[0319] Diluted formulation: 300 pL

[0320] Diluted eye drops: 300pL

[0321] Each condition is left in contact with the COEPs for 4, 12 and 24 hours before performing the MTT test.

[0322] Regarding the MTT test, each well was aspirated, then rinsed with PBS and re-aspirated before adding 300 pL of MTT at 1 mg / mL. After 2 hours of incubation at 37°C, the plates were inverted and 300 pL of acidified solution was added to lyse the crystals. Finally, each well was analyzed with a spectrophotometer. The percentage of viability for each condition was calculated relative to the positive control, which represents 100% viability, and the negative control should be equal to 0% viability.

[0323] The cells were cultured for 4 days before being placed in contact for 4 hours, 12 hours and 24 hours with LUM 7 Quater and the eye drops to be tested.

[0324] The results were normalized to the results of the culture medium control which represents 100% of all times analyzed.

[0325] 2. Results

[0326] 2.1. MTT test The results are shown in Figure 13.

[0327] The data show the non-cytotoxicity of the formulation of the invention at 3 different times (4, 12 and 24 hours), particularly in comparison with the eye drops of the prior art tested.

[0328] 2.2.Dilution U DMEM

[0329] The results are shown in Figure 14.

[0330] After 4 hours of contact: Optical densities (OD) measured via the MTT reaction are similar for all tested conditions (eye drops and formulations diluted in DMEM as well as in the culture medium). Average ODs vary between 0.414 and 0.448.

[0331] After 12 hours of contact: For all conditions tested with dilution in DMEM, cellular activity at 12 hours is higher than that observed at 4 hours, suggesting cell proliferation and / or an increase in metabolic activity. The conditions tested therefore appear favorable to cellular activity.

[0332] The rich and optimized culture medium is distinguished by higher cellular activity (proliferation and / or metabolic activity) than all other conditions.

[0333] • Two commercial eye drops (Hylovis Multi 15 and ThéaLose) and the LUM 7 Quarter formulation (composition of the invention) present similar results and slightly lower than those of the culture medium (activities between 90.4% and 91.2% compared to the culture medium), and comparable to those of the diluent alone (DMEM).

[0334] • A commercially available eye drop (Dulcilarmes) shows lower cellular activity (82%) than all other conditions, including the diluent.

[0335] After 24 hours of contact: While cell activity continues to increase in the culture medium, it stagnates or decreases slightly for cells exposed to Hylovis multi 15, ThéaLose, the LUM 7 quarter formulation diluted in DMEM, and in the diluent alone. However, cell activity in Hylovis multi 15, ThéaLose, and the LUM 7 quarter formulation remains higher than that observed in the diluent alone.

[0336] Cell activity in Dulcilarmes eye drops diluted in DMEM remains lower than in other conditions.

[0337] 2.3. Dilution U PBS

[0338] The results are shown in Figure 15. After 4 hours of contact: In the dilutions made in PB S, the results are more dispersed than for those in DMEM. The culture medium shows the strongest cellular activity. The activities decrease in the following order: Hylovis Multi 15, ThéaLose, the formulation, and the diluent (PB S). The cellular activity in Dulcilarme is almost zero, probably because of precipitates observed during dilution. This lack of activity persists at the 12-hour and 24-hour times.

[0339] After 12 hours of contact: For eye drops and the LUM 7 quarter formulation diluted in PBS, as well as for the culture medium, cell activity at 12 hours exceeds that measured at 4 hours, indicating cell proliferation and / or an increase in metabolic activity. These conditions are therefore conducive to cell activity. In contrast, the activity of cells maintained in the diluent alone (PBS) stagnates.

[0340] The rich and optimized culture medium shows superior cellular activity compared to all other conditions tested.

[0341] • Cellular activities under Hylovis Multi 15, ThéaLose and LUM 7 quarter formulation diluted in PBS are very close (OD between 0.376 and 0.413).

[0342] • The activity in the diluent alone is significantly lower, reduced by half (OD of 0.182).

[0343] 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, leading to cell death.

[0344] 3. Conclusion

[0345] The composition of the invention (LUM 7 Quarter) allows the maintenance and activity of corneal epithelial cells. This maintenance and activity are similar to the prior art Hylovis multi 15 and Théalose eye drops. Therefore, these data demonstrate the therapeutic effects of the composition on the treatment of the cornea, in particular for dry eye, ulceration treatment, etc.

[0346] GENERAL CONCLUSION

[0347] These data demonstrate the synergistic effect between a specific hyaluronic acid or one of its salts with a molecular weight greater than 2MDa and the amino acids of the composition of the invention on viability, adhesion, cell proliferation and cellular metabolic activity, ultimately on healing and cellular and tissue regeneration. In other words, the combination of hyaluronic acid or one of its salts with the 8 amino acids of the invention has a significant synergistic effect of increasing these phenomena.

[0348] It also appears that the composition of the invention (LUM 7 QUATER) allows cell survival for a period of 6 days longer than the composition of the invention without hyaluronic acid or one of its salts (LUM 7 AA).

[0349] Furthermore, these data clearly show that the different components of the basal lamina are stimulated by the formula of the invention, tested at different concentrations. This results in a positive effect on:

[0350] - the dermo-epidermal junction: the composition of the invention strengthens this junction and acts on the mechanical firmness of the epidermis;

[0351] - dermal and epidermal tissues: the composition of the invention increases the interactions between cells and their environment;

[0352] - terminal differentiation of keratinocytes: the composition of the invention stimulates different effectors linked to the mechanical, immunological and bacterial protection of the epidermis; and

[0353] - the functionality of fibroblasts: increases the speed of fibroblast migration, induces a greater number of myofibroblasts as well as the synthesis of collagen I and III, thus facilitating the healing / repair of the dermis.

[0354] Therefore, it is clear from the above that the composition of the invention acts on the synthesis of the constituents of the basal lamina / dermo-epidermal junction by modifying the expression of synthesis of keratinocytes and fibroblasts.

[0355] The composition of the invention strongly participates in the stimulation of keratinocytes, for the synthesis of receptors and molecules linked to the protection of the epidermis and accelerates the migration of fibroblasts while increasing the myofibroblast phenotype in an experimental injury model.

[0356] These conclusions make it possible to confirm the beneficial effects of the invention in a context of use of the composition of the invention to heal the cornea, a joint or cartilage, improve the viability of cells, the hydration of mucous membranes or even in an indication of tissue or cell grafting.

Claims

CLAIMS 1. Pharmaceutical composition comprising: - hyaluronic acid or one of its salts with a molecular weight greater than 2MDa; 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. 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. Composition according to one of claims 1 or 2, characterized in that the hyaluronic acid or one of its salts is in a non-crosslinked form.

4. Composition according to any one of the preceding claims, characterized in that the hyaluronic acid or one of its salts is sodium hyaluronate, potassium hyaluronate or their mixtures, advantageously sodium hyaluronate.

5. 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 injections.

6. 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. Composition according to any one of the preceding claims, characterized in that: - hyaluronic acid or one of its salts, advantageously sodium hyaluronate, represents between 6 g / L and 8 g / 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; - Na2HPÛ4 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 represents a sufficient quantity for IL.

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

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

10. Composition for its use according to claim 9, characterized in that healing is improved.

11. Composition according to any one of claims 1 to 7, for use in reducing the risk of scar reopening.

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

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

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

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

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