Pharmaceutical formulations for cell and tissue healing and regeneration and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of human and / or veterinary medicine, and more particularly to a pharmaceutical formulation for topical, transdermal, subcutaneous and / or intralesional administration, and the use of the pharmaceutical formulation in treating wounds and / or improving wound healing and / or articular cartilage regeneration and / or moisturizing skin and mucous membranes and / or enhancing cell vitality. Background Technology

[0002] Currently, wounds of all types remain one of the most important, expensive, and common medical problems worldwide. A wound is defined as a disruption of the skin's protective function and loss of epithelial continuity, regardless of whether it is accompanied by damage to deeper tissues (such as muscles, bones, and nerves). Therefore, any injury or damage to the skin, regardless of whether deeper tissues are damaged, can be called a wound. Such skin injuries can be caused by a variety of factors, such as accidents or trauma, infection, disease processes (especially inflammatory disease processes), or human-induced trauma during surgery.

[0003] Wounds are typically classified based on their healing time. Therefore, wounds can be divided into two main categories: - Acute wounds refer to wounds that appear suddenly and heal within the normal healing time (i.e., within 4 weeks). These wounds include traumatic wounds (abrasions, lacerations, tears, bites, penetrating wounds, etc.), burns of all degrees, and surgical wounds (including skin grafts); and - Chronic wounds are those that take longer to heal despite proper management and treatment. Certain wounds are automatically considered chronic if they are not expected to heal within 4 to 6 weeks. These include bedsores, diabetic foot ulcers, leg ulcers, or amputation stumps.

[0004] Under normal circumstances, the wound healing process consists of four stages / steps: - Phase 1: Inflammatory Phase (or Wound Cleaning - Inflammatory Phase) The initial reaction phase involves vasodilation accompanied by bleeding, followed by constriction of the ruptured vessel ends. The blood clots, producing an exudate rich in cells (granulocytes, macrophages, monocytes). These cells clear bacteria, necrotic tissue, and foreign particles via the lymphatic system and / or pus formation (wound cleaning phase). During this stage, the wound exhibits all the typical symptoms of inflammation: redness, swelling, heat, and pain. Capillary dilation leads to redness and heat. Increased capillary permeability promotes plasma exudation, resulting in swelling and heat, while pain is due to compression of sensitive nerve endings. This reactive phase typically lasts 3 to 6 days.

[0005] - Second stage: Granulation tissue formation or proliferation stageThis stage corresponds to fibroblast proliferation, angiogenesis, and extracellular matrix synthesis. After the inflammatory phase, granulation tissue begins to form, accompanied by the formation of new capillaries (angiogenesis), which provide the necessary oxygen, nutrients, and cells for tissue repair. Granulation tissue begins to organize within a network of collagen and elastin (produced by fibroblasts). Macrophages continue to play a crucial role at this stage, producing growth factors or cytokines that promote fibroblast proliferation and collagen synthesis. At this point, the wound is composed of newly formed fibrous tissue with a large number of fibroblasts and a loose fibrous framework around the defect. Granulation tissue consists of fibroblasts, inflammatory infiltrating cells (monocytes, lymphocytes, multinucleated cells), surface fibrin, and newly formed blood vessels in edematous fibrous structures. Contraction at the wound edge is closely related to granulation tissue formation, and some fibroblasts transform into contractile myofibroblasts, transmitting contractile activity to surrounding tissues through the interaction of proteins in the cytoskeleton and extracellular matrix. This phase is most active starting from day 7 and can last up to 3 weeks.

[0006] - Third stage: Epithelialization stage After tissue repair, the wound contracts and is gradually covered by new epithelium; this is the process of epithelialization. Dividing epidermal cells (i.e., keratinocytes) proliferate and begin to cover the granulation tissue from the wound edges. These keratinocytes require healthy, moist, and smooth granulation tissue to migrate properly. After the first layer of cells forms, the epithelium thickens through cell division and quickly becomes more resilient. The wound closes. This stage lasts 1 to 3 weeks.

[0007] - Phase Four: Reshaping or Maturation This stage begins in the early post-suturing period, but for most open, large wounds, it can last for months. This stage is characterized by connective tissue remodeling and scar formation. Granulation tissue disappears, making way for fibrous connective tissue. Collagen fibers thicken, increasing tensile strength. The number of capillaries decreases, reducing blood flow. Excess fluid and blood vessels subsequently disappear, and the scar gradually hardens. However, regardless, the scar's strength and elasticity are lower than normal skin, partly due to a lack of elastin. This stage can last from several months to two years.

[0008] The International Wound Visual Color Scale describes different stages of wound healing (see Table 1 below) and is available in a common language.

[0009] [Table 1] At the cellular and molecular levels, keratinocytes and fibroblasts have the function of producing and secreting various factors involved in the wound healing process, such as globin, laminin, or collagen (type I and type VII collagen). Globin acts on the proliferation and differentiation of keratinocytes, is essential for epidermal formation, and participates in the presentation of growth factors to their receptors. Literature indicates that exogenous globin can promote epidermal formation (Int J Cosmet Sci. 2020 Dec; 42(6): 529-535). Laminin plays an important role in normal tissues, maintaining the adhesion of epithelial-mesenchymal tissues in tissues exposed to external forces (such as skin).

[0010] Therefore, laminin and lecithin appear to be promising biomarkers for monitoring and even optimizing dermal-epidermal adhesion during wound healing.

[0011] Meanwhile, the main functions of the epidermis are known to include retaining moisture, maintaining body temperature and pH balance, and preventing the invasion of pathogenic microorganisms or toxic substances. The formation of a suitable epidermal barrier depends on the gradual differentiation of keratinocytes, from proliferating cells in the basal layer to the stratum corneum, i.e., terminal differentiation. The physical properties of the stratum corneum are attributed to the formation of a special structure—the keratin capsule (EC)—which is highly resistant to strong chemicals (such as detergents) or strong alkaline chemicals. The strengthening process of the EC mainly involves keratin, filaggrin, or intermediate keratin filaments.

[0012] Therefore, keratin appears to be a noteworthy marker that can monitor terminal differentiation of keratinocytes and ultimately promote epidermal strengthening.

[0013] In addition, beta-defensin (or β-defensin) is a natural molecule that can serve as a physiological stimulant for the antibacterial activity of the skin (J Immunol 2008 Aug 1, 181(3) 2103-2110). Therefore, this molecule is a good marker for monitoring wound healing or the development of chronic wounds.

[0014] Tissue initially classified as an acute wound can develop into a chronic wound if not properly treated or if unexpected pathological complications occur during the wound healing process. Therefore, any wound that initially appeared acute but has not healed within 4 weeks is considered a chronic wound. The inflammatory state of chronic wounds lasts longer, so their treatment differs from that of ordinary acute wounds.

[0015] Chronic wounds have become a major public health problem. Their incidence continues to rise, particularly due to an aging population and the increasing prevalence of underlying diseases such as diabetes.

[0016] Furthermore, chronic wounds may never heal, or the healing process may take years. These wounds can cause patients immense physical and psychological pain and stress. Therefore, appropriate treatment is crucial.

[0017] Existing technologies offer solutions to improve the wound healing process, particularly for chronic wounds, by using dressings with adjusted and / or improved microstructures and compositions to ensure better wound treatment.

[0018] Furthermore, existing technologies have proposed several pharmaceutical compositions for wound healing or promoting skin cell regeneration, such as the bio-similar hydrogel based on L-lysine crosslinked hyaluronic acid described in document EP 3666278; the composition containing recombinantly modified platelet-derived growth factor (PDGF) and dexamethasone as an anti-inflammatory agent described in document EP0575484; or the composition containing transforming growth factor β (TGF-β) described in document US 5981606. However, these pharmaceutical products all belong to the categories of growth factors, cytokines, chemokines, or hyaluronic acid, and the adverse reactions of these substances are known, especially because they are not cell type specific.

[0019] Regarding the application of growth factors, their interactions with cell membranes can induce a mast response (increased cell volume) and a proliferative response (increased cell number), in other words, clonal cell proliferation. Traditionally used cell growth factors are derived from animals, cells, and / or plants—that is, extracted from biological raw materials. For example, widely used factors include cholera toxin, fetal bovine serum (SVF), bovine pituitary extract, or milk or soy complexes or components of unknown composition. However, the use of these cell growth factors of unknown composition, origin, and animal or plant origin is increasingly questioned, especially due to the lack of traceability, inconsistent quality, risk of contamination, and lack of reproducibility of observed effects. Furthermore, because the formulations of these growth factors are unknown, the presence of allergens cannot be determined. Therefore, there is a risk of allergic reactions when using these growth factors.

[0020] Meanwhile, existing technologies have proposed other solutions for treating wounds or osteoarthritis, such as the solution in document US2023 / 148647, which involves a composition containing a specific ratio of six specific amino acids and a divalent metal to promote the synthesis of collagen and elastin; or the solution in document WO 2023 / 042120, which involves a composition containing two forms of sodium hyaluronate and a mixture of amino acids.

[0021] In the cosmetics field, various compositions have been developed to combat skin aging and promote skin cell regeneration, particularly for the elderly. For example, document CN 108618997 describes a cosmetic composition for anti-aging and whitening, containing sodium hyaluronate with a molecular weight of less than 10 kDa and 21 essential amino acids. These different applications are generally categorized as "anti-aging." In these "anti-aging" products, active ingredients are added 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. For example, retinoids are mentioned because of their ability to stimulate fibroblast growth. However, due to their phototoxicity and potential mutagenicity, their use in cosmetics is not recommended ("Photomutability of retinyl palmitate in mouse lymphoma cells under UVA irradiation," Nan Mei et al., Science of Toxicology 88(1), 142-149 (2005)).

[0022] Current treatments are not without harmful side effects and cannot achieve effective therapeutic results, reduce the incidence of chronic wounds, or effectively and persistently relieve pain, discomfort, or even cosmetic problems caused by acute or chronic wounds. Furthermore, to the applicant's knowledge, there is currently no treatment that can simultaneously treat acute and chronic wounds.

[0023] In summary, there remains a need for a composition that can promote and / or improve and / or accelerate the healing / recovery mechanisms of acute and chronic wounds, and act in a safe, effective, and durable manner (i.e., short-term, medium-term, and long-term), while ensuring full traceability of the composition's components. Summary of the Invention

[0024] After extensive research, the applicant has made a surprising discovery: a composition containing a safe, traceable combination of compounds (or active ingredients) suitable for human use, particularly for therapeutic purposes, and these compounds can work synergistically to meet the aforementioned needs.

[0025] Therefore, the first subject of the present invention relates to a composition, preferably a pharmaceutical composition, comprising the following as active ingredients: - Hyaluronic acid or its salts, having a molecular weight greater than, preferably strictly greater than, 2 MDa (megada); and - Contains at least 8 essential amino acids, selected from lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, and histidine.

[0026] According to specific embodiments, the composition of the present invention contains at least eight essential amino acids for the human body: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and threonine.

[0027] This invention has many advantages, including in particular: (i) Doctors / medical staff can easily and routinely use an effective formula in which the compounds work synergistically to promote wound healing, especially for acute or chronic wounds; (ii) No harmful side effects; (iii) The formulation of this invention can be subjected to comprehensive chemical identification and analysis; (iv) The formulation of this invention and any of its degradation products are non-toxic, non-mutagenic, non-carcinogenic and non-sensitizing; (v) It can be used directly, especially without any temporary mixing steps or other preparatory steps.

[0028] In this invention, the articles "a" and "an" are used to indicate one or more (e.g., at least one) grammatical objects of the article. For example, "an element" means at least one element, that is, one or more elements.

[0029] In this invention, the terms "approximately" and "about" are used interchangeably and refer to measurable values, such as quantity, duration, and other similar values. These terms should be understood to cover measurement uncertainties of ±20% or ±10% of the specified value, preferably ±5%, more preferably ±1%, and particularly preferably ±0.1%.

[0030] In this invention, various features of the invention can be described in the form of numerical ranges. It should be understood that describing numerical values ​​in range form is merely for ease of reading and should not be construed as a strict limitation of the scope of the invention. Therefore, the description of numerical ranges should be considered as specifically disclosing all possible intermediate ranges and each numerical value within that range. For example, a description of a range from 1 to 6 should be considered as specifically describing each range it encompasses, such as 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 each numerical value within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This definition applies regardless of the specific range.

[0031] In this invention, the terms "formulation", "composition" and "preparation" can be used interchangeably.

[0032] In this invention, "essential amino acids" refers to amino acids that the human body must obtain from food (i.e., organic acids containing an amino group in their molecules) that are essential for human health and cannot be synthesized by the body itself. Essential amino acids include tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine, and isoleucine; for children, the list also includes arginine and histidine.

[0033] In this invention, "hyaluronic acid or its high molecular weight salt" refers to hyaluronic acid or its salt with a molecular weight greater than (preferably strictly greater than) 2 MDa.

[0034] In this invention, the expressions "hyaluronic acid or a low molecular weight salt thereof" and "hyaluronic acid or one of its low molecular weight salts" can be used interchangeably. They refer to hyaluronic acid or its salt with a molecular weight of less than 2 MDa, preferably less than 1.8 MDa, and more preferably less than 1 MDa.

[0035] In this invention, the terms "active compound," "active ingredient," and "active substance" are used interchangeably and all refer to substances or compounds that have therapeutic properties and can produce physiological effects. An active compound, active ingredient, or active substance differs from at least one excipient preferably present in the compositions of this invention.

[0036] In this invention, "excipient" refers to a substance or compound that does not possess therapeutic properties. Excipients are primarily responsible for imparting a specific texture, viscosity, aroma, and / or color to the therapeutic preparation, and for diluting and preserving the preparation in accordance with relevant regulations to ensure its storage stability, safety, and shelf life. Excipients differ from at least one active compound, active ingredient, or active substance present in the compositions of this invention.

[0037] In this invention, the term "pharmaceutical composition" refers to a composition intended for therapeutic rather than cosmetic purposes.

[0038] In this invention, a "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which participates in the delivery of a target drug from one organ or body site to another. Each carrier must be "acceptable," meaning it is compatible with other active ingredients and / or excipients in the composition; for example, the carrier does not diminish the therapeutic effect of the target drug. In other words, the carriers of this invention are pharmaceutically inert.

[0039] In this invention, the terms "wound," "abrasion," and "injury" are used interchangeably, all referring to open injuries, i.e., breaches of the skin barrier, as opposed to closed wounds (i.e., contusions) without open wounds. Specifically, a wound refers to damage to the skin, manifested as an interruption of tissue continuity and a breach of the skin barrier, requiring a complex and dynamic process for repair or healing. Wounds can be superficial, involving only the epidermis (erosion), or partially in the dermis, or deep, exposing subcutaneous tissue. Wound formation depends on its extent and depth, as well as on local or systemic factors that may hinder or prevent its healing.

[0040] In this invention, an "acute wound" refers to a wound that occurs suddenly and lasts for a short period. It heals within the expected timeframe and follows the normal healing process. For acute wounds, the physiological healing process typically lasts 2 to 4 weeks. Acute wounds can occur anywhere on the body. They can be epidermal abrasions (or epithelial wounds) or deep wounds that damage blood vessels, nerves, and / or muscles (vascular, nerve, and / or muscle wounds). For example, they can include surgical wounds, i.e., incisions made by healthcare professionals; traumatic wounds, i.e., sudden, accidental injuries such as abrasions or bites; or burns, i.e., damage to the skin or other tissues caused by heat, cold, electricity, chemicals, radiation, or friction. Acute wounds and chronic wounds should be distinguished.

[0041] In this invention, a "chronic wound" refers to a wound that fails to heal according to the steps of the wound healing process and / or takes longer than the expected healing time for most other wounds (i.e., acute wounds). A wound may stall at some stage of the wound healing process. For example, the inflammatory stage often lasts excessively long. For chronic wounds, the physiological wound healing process typically lasts 4 to 6 weeks or more. For example, venous ulcers, arterial ulcers, diabetic ulcers, or pressure sores are all chronic wounds. Chronic wounds should be distinguished from acute wounds. Therefore, any wound that was initially acute but has not healed within 4 weeks should be considered a chronic wound.

[0042] In this invention, the terms "improves wound healing", "promotes wound healing", "accelerates wound healing", "encourages wound healing" and "facilitates wound healing" can be used interchangeably. They refer to mechanisms that make the various stages of wound healing more effective (i.e., increase cell viability, increase the frequency and / or number of cell clones, etc.) and / or connect the stages faster than expected, i.e., faster and more stable wound healing; in other words, the risk of the wound reopening is very small or even non-existent.

[0043] In this invention, expressions such as "improving cell viability," "enhancing cell viability," "promoting cell viability," "encouraging cell viability," and "benefiting cell viability" can be used interchangeably to indicate an increase in cell adhesion and / or proliferation and / or metabolic activity and / or cell membrane integrity relative to reference values ​​measured before applying the candidate composition / test composition to cells using the same measuring equipment / test (e.g., MTT assay; tetrazolium salt).

[0044] In this invention, the term "cell adhesion" refers to all cellular and molecular mechanisms that enable cells to adhere to each other or to the surrounding medium. Cell adhesion is essential for tissue formation, maintenance, and function.

[0045] In this invention, "cell proliferation" refers to the process of rapid and massive cell reproduction, measured by a quantitative value of the number of daughter cells. This mechanism differs from cell growth, which refers to the increase in the number and volume of individual cells. The term "cell proliferation" can be used for both cell development (i.e., proliferation during the cell cycle) and cell division (i.e., reproduction).

[0046] In this invention, "cellular metabolic activity" refers to the sum of catabolism (or breakdown of substances) and anabolism (or synthesis of substances). Catabolism breaks down complex molecules into basic molecules to generate energy or produce molecules that can be directly used in anabolistic pathways. Anabolism, on the other hand, involves all synthetic processes.

[0047] In the context of this invention, all embodiments described above and below can be combined together.

[0048] According to a specific embodiment of the present invention, the active ingredients contained in the formulation of the present invention include: - Hyaluronic acid or its salts with a molecular weight greater than 2 MDa; and - Contains at least 8 essential amino acids, selected from lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, and histidine.

[0049] According to a specific embodiment of the present invention, the formula comprises: - Hyaluronic acid or its salts with a molecular weight greater than 2 MDa; and - It contains 8 essential amino acids, including lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and threonine.

[0050] Hyaluronic acid is a polysaccharide, specifically a glucosamine polysaccharide (GAG), whose chains are composed of disaccharides linked by alternating β-1,4 and β-1,3 glycosidic bonds. Its structural formula is as follows: [β-D-glucuronic acid + N-acetyl-D-glucosamine]n n According to the present invention, the molecular weight of hyaluronic acid or its salt is greater than 2 MDa, preferably strictly greater than 2 MDa and / or less than 3 MDa, and preferably between 2 MDa and 3 MDa.

[0051] As described above, the hyaluronic acid or its salts of the present invention, when used in combination with amino acids, can produce unexpected synergistic effects, thereby promoting the proliferation and / or migration of skin cells (especially fibroblasts), ultimately achieving faster and more stable wound healing. This effect is surprising, as it can only be obtained using hyaluronic acid or its salts with a molecular weight greater than 2 MDa, and cannot be obtained using hyaluronic acid or its salts with lower molecular weights.

[0052] According to one specific embodiment, hyaluronic acid or its salt is in a non-crosslinked or crosslinked form, preferably in a non-crosslinked form.

[0053] According to one specific implementation, hyaluronic acid or its salts are in a non-hydrolyzed form.

[0054] According to the present invention, hyaluronic acid is derived from biotechnology, particularly through bacterial fermentation. In other words, it is a product of non-animal origin.

[0055] The hyaluronic acid salts described in this invention are pharmaceutically acceptable and are selected from sodium hyaluronate, potassium hyaluronate, and mixtures thereof.

[0056] In a preferred embodiment, the hyaluronic acid or its salt contained in the composition of the present invention is sodium hyaluronate.

[0057] According to the present invention, hyaluronic acid or its salts are active compounds, not excipients, let alone solvents such as diluents or thickeners.

[0058] According to one embodiment of the present invention, the composition of the present invention comprises at least eight essential amino acids, wherein the amino acids are selected from L-lysine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-threonine and L-histidine.

[0059] According to a preferred embodiment of the present invention, the composition comprises the eight essential amino acids selected from L-lysine, L-valine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, and L-threonine.

[0060] According to one specific embodiment of the present invention, the composition further comprises one or more pharmaceutically acceptable pH-adjusting compounds and / or buffers as excipients. These compounds include acids, such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, and sodium lactate; and buffers, such as citrate / glucose, sodium bicarbonate, and ammonium chloride. The amounts of these acids, bases, and buffers can be adjusted as needed to maintain the pH of the composition within a pharmaceutically acceptable range. The composition may also contain one or more pharmaceutically acceptable salts, in an amount sufficient to bring the osmotic pressure of the composition within a pharmaceutically acceptable range. These salts include salts containing sodium, potassium, or ammonium cations, as well as chloride ions, citrate ions, ascorbate ions, borate ions, phosphate ions, bicarbonate ions, sulfate ions, thiosulfate ions, or bisulfite ions.

[0061] Specifically, the compositions of the present invention further comprise one or more compounds as excipients (having a pH buffering effect), preferably all compounds selected from the group consisting of: disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water, preferably water for injection (or WFI).

[0062] According to a preferred embodiment of the present invention, the composition comprises: - Sodium hyaluronate; - The following essential amino acids: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and threonine; - Na2HPO4; - KH2PO4; - NaCl; and - WFI water.

[0063] Preferably, - The content of hyaluronic acid or its salt (preferably sodium hyaluronate) is between 6 g / L and 8 g / L; - The lysine content ranges from 33 mg / L to 39 mg / L; - Valine content ranges from 21 mg / L to 25 mg / L; - Isoleucine content ranges from 5.7 mg / L to 6.6 mg / L; - Leucine content ranges from 24.3 mg / L to 28.3 mg / L; - Methionine content ranges from 6.8 mg / L to 8.2 mg / L; - Phenylalanine content ranges from 15 mg / L to 18 mg / L; - Tryptophan content ranges from 4.6 mg / L to 5.6 mg / L; - Threonine content ranges from 21.1 mg / L to 25.2 mg / L; - The Na2HPO4 content ranges from 1.35 g / L to 1.45 g / L; - KH2PO4 content ranges from 0.65 g / L to 0.75 g / L; - NaCl content is between 4.5 g / L and 5.5 g / L; and Add water for injection to a final volume of 1L.

[0064] According to a preferred embodiment, the pH of the composition is between 5.0 and 8.0, preferably between 5.5 and 7.9, more preferably between 7.4 and 7.5, for example 7.45, and / or the osmotic pressure is between 280 and 360 mmol, preferably between 300 and 350 mmol.

[0065] According to a specific embodiment of the present invention, the composition is sterile / sterilized.

[0066] According to specific embodiments of the present invention, the composition is administered in a form suitable for topical use and / or injection, and in particular has pharmaceutically acceptable properties, namely compatibility with human skin, mucous membranes, blood vessels, cornea and / or muscles.

[0067] According to one specific embodiment, the composition of the present invention is administered in a form suitable for topical skin or corneal administration.

[0068] According to another specific embodiment, the composition of the present invention is administered in a manner suitable for intradermal, intra-articular, intracartilaginous, subcutaneous, intralesional, intravenous, intramuscular, and / or subcutaneous injection, preferably for intradermal injection, more preferably for intradermal injection into healthy and / or damaged tissue (i.e., wound) located around a wound, and / or for intra-articular and / or intracartilaginous injection.

[0069] According to another specific embodiment, the composition of the present invention is injected intradermally into the wound and / or healthy tissue near the wound, i.e., injected into healthy tissue 0.1 cm to 5 cm from the edge of the wound, preferably into 0.1 cm to 4 cm, 0.1 cm to 3 cm, 0.1 cm to 2 cm, 0.1 cm to 1 cm, or even 0.1 cm to 0.5 cm.

[0070] According to specific embodiments of the present invention, the composition is a dosage form suitable for contact with in vivo, in vitro or ex vivo cells and / or tissues, particularly for contact with cultured or suspended cells and / or tissues to improve cell viability and / or promote transplantation and / or transplantation methods.

[0071] Therefore, the compositions of the present invention may be in the form of creams, lotions, serums, ointments, gels, foams, sprays / sprayable compositions, eye drops, impregnated dressings, or aqueous solutions, hydroalcoholic solutions, organic solutions or oily solutions, or even suspensions or dispersions in solvents or fatty substances.

[0072] Preferably, the pharmaceutical composition of the present invention comprises a carrier / transporter, which is considered an excipient in the sense of the present invention, and is pharmaceutically acceptable for formulations suitable for the use of the present invention, particularly injectable formulations.

[0073] Specifically, the carrier / transporter can be an isotonic, sterile, or saline solution (sodium monophosphate or disodium phosphate; sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or a dried composition, especially a lyophilized composition, which, depending on the specific circumstances, is added to sterile water or physiological serum to prepare an injectable composition.

[0074] The dosage can be adjusted according to different parameters, especially the route of administration, disease type, or required duration of treatment and the surface area to be treated.

[0075] For example, it is known in the prior art that the dose of the composition used at the start of treatment should be lower than the dose required to achieve the desired therapeutic effect, and then the dose should be gradually increased until the desired effect is achieved.

[0076] The daily dosage of the formulations of the present invention, particularly those suitable for topical application, is 0.1 to 1 mL / cm². 2 Preferably 0.1 to 0.5 mL / cm 2 Specifically, the topical medication is equivalent to using it at least once a day, for example, twice a day, or even three or four times a day.

[0077] The weekly dose of the formulations of the present invention, particularly the weekly dose of formulations suitable for injection (especially intradermal injection), depends on the surface area to be treated. Therefore, - 1 cm 2 0.25 mL to 1 mL; - 2 cm 2 : 0.4 ml to 1.6 ml; - 4 cm 2 : 0.65 ml to 2.6 ml; Preferably, injections are given once a week at a rate of 1 cm or 0.5 cm.

[0078] The dosage (preferably daily dose) of the formulation of the present invention, particularly suitable for cartilage and / or joints, especially for intra-articular injection, is 0.1 to 5 mL / joint, preferably 0.5 to 5 mL / cm. 2 Preferably 1 to 5 mL / cm 2 .

[0079] The dosage of the formulation of the present invention, particularly suitable for improving tissue transplantation and / or transplantation surgery, especially when mixed with adipose tissue for tissue transplantation, is 1% to 50% of the volume of fat to be transplanted, preferably 5% to 40%, and more preferably 10% to 30%.

[0080] Regardless of the route of administration, this product is strictly prohibited once a cancerous lesion is detected. If a cancerous lesion is suspected, a biopsy should be performed first to obtain the results before using the formulation described in this invention.

[0081] According to one embodiment, the compositions used in this invention are intended for human and / or veterinary use.

[0082] According to a preferred embodiment of the present invention, the compositions used in the present invention are intended for use in the human body. In the present invention, "human body" refers to males or females, and more particularly to children, adults, or the elderly.

[0083] According to another embodiment, the animals targeted by the present invention are preferably livestock, especially dogs, cats, birds or rodents, and / or farm animals, especially poultry, cattle, goats, sheep, pigs, horses, or even camels or deer.

[0084] According to one embodiment, the compositions used in this invention are intended for use in the fields of vascular medicine, gynecology, ophthalmology, endocrinology / metabolic medicine, and / or dermatology.

[0085] According to the present invention, the composition is intended for wound healing and cell and tissue regeneration.

[0086] Specifically, the compositions of the present invention are intended for use in: 1- Treatment of acute wounds, such as acute contusion wounds; acute wounds in patients with complex healing conditions (e.g., diabetes, arterial ischemia, venous ischemia, connective tissue diseases); postoperative acute skin ischemia (arterial or venous): flap ischemia, areola and nipple ischemia after breast surgery, acute skin ischemia after any type of plastic and cosmetic surgery, acute skin ischemia after any type of marginal skin orthopedic surgery, acute skin ischemia after any type of marginal skin vascular surgery; postoperative skin separation, regardless of the type of surgery, without exposure of joints, bones, and blood vessels; or burns (especially first- and second-degree burns); and / or 2. Treatment of chronic wounds, such as: chronic arterial wounds; chronic venous wounds; uninfected grade I and II pressure ulcers; chronic skin separations; post-radiation therapy wounds; diabetic foot wounds or fibrous wounds; and / or 3- In addition to other skin repair techniques, such as direct suture repair; dermal-epidermal grafting or full skin grafting, or in addition to flaps; and / or 4- Used for healing and / or moisturizing the cornea, especially in cases of dry eye; used to treat corneal ulcers; except for corneal transplantation or as a means of treating symptoms of dry cornea; and / or 5- For moisturizing and / or regenerating cartilage and / or joints, particularly intra-articular injections for the prevention and treatment of symptoms of osteoarthritis / arthritis; intervertebral disc injections for the treatment of symptoms of spinal osteoarthritis; or injections into muscles, ligaments, tendons, and joint capsules; and / or 6- Used to moisturize mucous membranes, especially for the treatment of symptoms of vaginal dryness, postmenopausal vaginal atrophy, dryness of the nasal, oral and anal mucous membranes, and damaged mucous membranes (such as pharynx, vocal cords, esophagus, stomach, intestines, and interstitial cystitis); and / or 7- Used for tissue transplantation and / or transplantation surgery, especially for transplantation of skin, tendons, fat or hair; and / or 8- Used for cell transplantation and / or transplantation.

[0087] On the other hand, the present invention relates to the above-described compositions of the present invention, which can be used in wound healing methods, particularly suitable for acute or chronic wounds. Similarly, the present invention relates to the above-described compositions of the present invention, which can be used to reduce the risk of wound re-opening.

[0088] In other words, the present invention also relates to: - The compositions of the present invention are used to treat wounds, preferably acute or chronic wounds; and / or - Use of the compositions of the present invention in the preparation of medicaments for treating wounds (preferably for treating acute or chronic wounds); and / or - A method for treating wounds (preferably acute or chronic wounds), comprising applying (preferably by injection) the composition of the present invention.

[0089] According to specific embodiments, the composition of the present invention used as described above is for wound healing, particularly for improving, accelerating or promoting wound healing, i.e., increasing the migration and / or proliferation rate of cells such as fibroblasts and / or keratinocytes.

[0090] It helps to accelerate at least one of the four stages of wound healing and / or the sequence of the wound healing stages.

[0091] Specifically, the function of the composition of the present invention is: - Improves / accelerates the migration of fibroblasts and / or keratinocytes to the wound to rebuild the dermis and / or epidermis; and / or - Increase / stimulate the production and / or secretion of at least one protein / peptide selected from globin, laminin, keratin, and β-defensin; and / or - Increase / stimulate the activity of at least one protein / peptide selected from globin, laminin, keratin and β-defensin.

[0092] On the other hand, the present invention relates to the composition described above for reducing the risk of wound dehiscence.

[0093] In other words, the present invention also relates to: - The compositions of the present invention are used to reduce the risk of wound dehiscence; and / or - Use of the compositions of the present invention in the preparation of medicaments that reduce the risk of wound dehiscence; and / or - A method for reducing the risk of wound dehiscence, comprising applying (preferably by injection) the composition of the present invention.

[0094] According to another aspect, the present invention relates to the composition of the invention as described above, which can be used in a method (preferably a non-cosmetic method) to moisturize skin and / or wounds, preferably acute or chronic and / or dry or exudative wounds.

[0095] In other words, the present invention also relates to: - The compositions of the present invention are used for moisturizing skin and / or wounds, preferably for acute or chronic wounds and / or dry or exudative wounds; and / or - The use of the compositions of the present invention in the preparation of medicaments for moisturizing skin and / or wounds, preferably for acute or chronic wounds and / or dry or exudative wounds; and / or - A method for moisturizing skin and / or wounds, preferably for acute or chronic wounds and / or dry or oozing wounds, the method comprising applying (preferably by injection) the composition of the present invention.

[0096] According to another aspect, the present invention relates to the composition of the invention as described above, for use in a method (preferably an in vitro method) to maintain or even improve the cell viability of cells, particularly fibroblasts, keratinocytes, corneal epithelial stem cells (or corneal epithelial stem cells) and / or adipose stem cells, for example in stem cell-based therapies, particularly in stem cell injections.

[0097] In other words, the present invention also relates to: - The compositions of the present invention are preferably used in vitro to maintain or even enhance cell viability, preferably for fibroblasts, keratinocytes and / or corneal epithelial stem cells and / or adipose stem cells, for example in stem cell therapy, particularly in stem cell injection therapy; and / or - The use of the compositions of the present invention in the preparation of medicaments for maintaining or even enhancing cell viability, preferably in vitro, and preferably for use with fibroblasts, keratinocytes, corneal epithelial stem cells and / or adipose stem cells, for example in stem cell therapy, particularly in stem cell injection therapy; and / or - A method for maintaining or even enhancing cell viability, preferably an in vitro method, preferably for the viability of fibroblasts, corneal epithelial keratinocytes and / or adipose stem cells, for example in stem cell-based therapies, particularly in stem cell injections, including the application (preferably by injection) of the composition according to the invention.

[0098] According to specific embodiments, the above composition can be used in vitro, in vitro and / or in vivo.

[0099] According to a specific embodiment of the present invention, the increase in vitality is an increase in cell adhesion and / or proliferation and / or metabolic activity.

[0100] According to specific embodiments, the composition used to improve cell viability is different from cell culture medium because it does not contain all the elements (such as glucose, trace elements, vitamins, etc.) required for cell viability and growth.

[0101] On the other hand, the present invention relates to a method for delivering the composition of the present invention to healthy / intact tissue around a wound and / or damaged tissue at the center of a wound by intradermal or subcutaneous injection.

[0102] Specifically, the wound margins define the boundary between intact and unintegrated tissue. It is important that intact tissue, whose biological function has been or may be impaired, be treated to minimize this damage and promote vascularization and healing at sites of impaired tissue integrity (wounds).

[0103] At the wound level, the purpose of the drug delivery method of this invention is to accelerate wound healing by optimizing all biological wound healing mechanisms.

[0104] According to one implementation, the wound edge is defined as the starting point for treatment / intradermal or subcutaneous injection and extends outward toward the wound / healthy tissue.

[0105] According to another embodiment, the wound edge is defined as the starting point of treatment / intradermal injection and extends toward the inside of the wound / damaged tissue.

[0106] According to another embodiment, the wound edge is defined as the starting point for treatment / intradermal injection, and then continues towards the outer side of the wound / healthy tissue, and then towards the inner side of the wound / damaged tissue, or vice versa in the treatment sequence (i.e., first towards the inner side of the wound, then towards the outer side of the wound).

[0107] According to one implementation, the injection depth is 1 mm to 3 mm, depending on the tissue type (intradermal or subcutaneous injection of the whole body skin, and injection under granulation tissue or epithelialized tissue in a wound).

[0108] According to one embodiment, the injection of the present invention is performed by a reverse fan-shaped injection technique or a point-by-point injection technique.

[0109] The present invention also relates to a kit for therapeutic purposes as described above.

[0110] The kit of the present invention comprises the pharmaceutical composition of the present invention as described above, for example, in a pre-measured dose. The kit of the present invention may also comprise a device for administering the composition and instructions for use.

[0111] These devices include syringes, implantable pumps (such as micropumps and ultramicropumps), and other pharmaceutical devices.

[0112] Specifically, the device included in the kit of the present invention is a syringe pre-loaded with the composition of the present invention.

[0113] The following embodiments are not limiting, but constitute an integral part of the present invention. Any feature that is novel relative to the prior art is declared as a novel feature and general method. Attached Figure Description

[0114] Figure 1 This study evaluated the effects of the composition of the present invention (LUM 7 quater), the composition without hyaluronic acid or its salts (LUM 7AA), and the composition without amino acids (LUM 7 HA3) on the viability of adipose tissue mesenchymal stem cells (ASCs) at 3 hours, 24 hours, 72 hours, and 6 days. Results were obtained from ASCs from three specific donors, with 10 replicates for each condition. Statistical tests were performed relative to the “DMEM” condition (significance level: *: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0115] Figure 2 This study evaluated the effects of the present invention's composition (LUM 7 quater), the present invention's composition without hyaluronic acid or its salts (LUM 7 AA), and the present invention's composition without amino acids (LUM 7 HA3) on the adhesion, proliferation, and metabolic activity of adipose tissue mesenchymal stem cells (ASCs) at 3 hours, 24 hours, 72 hours, and 6 days. Results were obtained from ASCs from three specific donors, with 12 replicates for each condition. Statistical tests were performed relative to the "CM" condition (significance level: *: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0116] Figure 3 The effect of the composition of the present invention at different concentrations (i.e., stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) on the production / secretion of bead-like proteoglycans in cultured human primary fibroblasts was evaluated in vitro compared with the control group (DMEM).

[0117] Figure 4 The effects of the composition of the present invention at different concentrations (i.e., the stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) on the production / secretion of laminin in cultured human primary fibroblasts were evaluated in vitro compared with the control group (DMEM).

[0118] Figure 5 The effect of the composition of the present invention at different concentrations (i.e., the stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) on the production / secretion of keratin by cultured human epidermal keratinocytes was evaluated in vitro compared with the control group (Celloneer KC / CC).

[0119] Figure 6 The effect of the composition of the present invention at different concentrations (i.e., stock solution and 20%, 30%, 40% and 50% dilutions of the stock solution) on the production / secretion of β-defensins in cultured human epidermal keratinocytes was evaluated in vitro compared with the control (Celloneer KC / CC).

[0120] Figure 7 The effect of the composition of the present invention at different concentrations (i.e., the mother liquor and 20%, 30%, 40% and 50% dilutions of the mother liquor) on the migration rate of fibroblasts after experimental injury was monitored compared with the control group (DMEM).

[0121] Figure 8 Photographs of a 58-year-old paraplegic patient with a chronic wound on the lateral side of the foot that had developed over several years. The photos show the wound before treatment (A) and two months after treatment (B). The treatment involved weekly injections into the wound (intralesional injection) and injections into healthy tissue near the wound (perilesional injection).

[0122] Figure 9 Photographs of fibroblast migration monitored over 16 hours (i.e., T0, T8, T12, and T16 hours) using different compositions (Lum A: composition without hyaluronic acid; Lum H1: composition without amino acids but containing low molecular weight hyaluronic acid; Lum 7: composition of the present invention) and dilutions (20% or 50%). Representative experiments were repeated 6 times.

[0123] Figure 10 Fibroblast migration kinetics over 30 hours in the presence of 20% and 50% Lum 7, compared with a control group. Mean + / - standard deviation; statistical analysis was performed using the Mann-Whitney test: *p<0.05; **p<0.01.

[0124] Figure 11 Fibroblast migration kinetics over 30 hours in the presence of 20% and 50% Lum H1, compared with a control group. Mean + / - standard deviation; statistical analysis was performed using the Mann-Whitney test: *p<0.05; **p<0.01.

[0125] Figure 12 Fibroblast migration kinetics over 30 hours in the presence of 20% and 50% Lum A, compared with a control group. Mean + / - standard deviation; statistical analysis was performed using the Mann-Whitney test: *p<0.05; **p<0.01.

[0126] Figure 13 The study evaluated the cell viability of corneal epithelial cells after exposure to the composition of this invention or three prior art eye drops for 3, 12, or 24 hours. Absorbance results at 570 nm were normalized to the results of the culture medium (MC): MC at all time points was normalized to 100%.

[0127] Figure 14 The original results of optical density (570 nm) and their changes after exposure to the composition of the present invention or three prior art eye drops (diluted 1 / 2 in DMEM) for 3, 12 or 24 hours.

[0128] Figure 15 The original results of optical density (570 nm) and their changes after exposure to the composition of the present invention or three prior art eye drops (diluted 1 / 2 in PBS) for 3, 12 or 24 hours.

[0129] Example Example 1. Toxicity assessment of the composition of the present invention 1. Research Objectives The aim of this study was to demonstrate, using MTT assay (or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide), that the composition of the present invention was non-cytotoxic to adipose tissue stem cells (or adipose tissue mesenchymal stem cells or "adipose-derived stem cells" or ASCs) from three donors at four different time points (i.e., 3 hours, 24 hours, 72 hours, and 6 days).

[0130] 2. Materials and Methods 2.1. Cells ASCs were obtained from 3 donors at a density of 10,000 cells / well (96-well plate).

[0131] Donor 1: 11171, 28 years old (ampoules P2 - inoculated at P4); Donor 2: 12025, 28 years old (ampoules P0 - inoculated at P2); and Donor 3: 12106, 40 years old (ampoules P1 - administered at P3) 2.2. Preparation of the test composition The active ingredients used in the LUM 7 QUATER composition are shown in Table 2 below.

[0132] [Table 2] The active ingredients used in composition LUM 7 HA3 are shown in Table 3 below.

[0133] [Table 3] Table 4 below lists the active ingredients used in composition LUM 7 AA.

[0134] [Table 4] The preparation of this formula involves three stages: - Prepare pH buffer solution: Add all the water and stir, add buffer salt, and after it is completely dissolved, check if the pH value is correct; - Adding sodium chloride and amino acids: Keep stirring constantly. Add sodium chloride, wait for it to dissolve, then add amino acids, checking for dissolution before each addition; and - Add hyaluronic acid: Keep stirring constantly, add hyaluronic acid in small amounts several times, and wait for it to dissolve completely before stopping stirring.

[0135] After the formula is prepared, check the pH value, then dispense it into vials or syringes, and finally autoclave the resulting vials or syringes.

[0136] 2.3 Conditions - LUM 7 Quater (Composition of the Invention): 6 bottles, 5 ml per bottle - LUM 7 HA3 (ALM 7 Quater composition without amino acids): 8 vials, 5 ml per vial - LUM 7 AA (LUM 7 Quater composition without hyaluronic acid): 8 vials, 5 ml per vial It should be noted that each formulation (pure product) in the examples was tested at only one dilution.

[0137] Sodium dodecyl sulfate (SDS) was used as a negative control for cell viability (dissolved in proliferation medium at a concentration of 0.05%).

[0138] 100% viability (positive control) will be determined using an untreated control group that has only been exposed to DMEM.

[0139] A culture medium control experiment will also be conducted.

[0140] The culture medium consisted of: "DMEM / F12 with GlutaMAX", 10% SVF, 100 U / ml penicillin, streptomycin, 5 µg / ml amphotericin B, and 10 ng / ml "FGF2 premium grade" (Miltenyi Biotec, Paris, France).

[0141] 2.4 Experimental Scheme After thawing, ASCs were seeded into culture flasks and cultured until confluence. ASCs were then digested with trypsin and seeded at a density of 10,000 cells / well in 96-well plates and cultured in DMEM for 24 hours. The following day, appropriate assay reagents were added, and MTT assays were performed after incubation for 3, 24, 72 hours, and 6 days. Twelve wells were set up for each treatment and time point. In the MTT assay, cell viability in each treatment group was calculated against the positive control group, which had a cell viability of 100%. The negative control group (0.05% SDS) had a cell viability of 0%.

[0142] The principle of this detection method is based on the tetrazolium salt MTT ((3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide). In living cells, the mitochondrial enzyme succinate dehydrogenase can reduce MTT to formazan. The amount of formazan generated is directly proportional to the cellular metabolic activity. This insoluble compound is purple and needs to be dissolved in DMSO before it can be spectrophotometrically measured at a wavelength of 540 nm.

[0143] 2.5 Data Processing Use Microsoft Excel software to analyze the raw data.

[0144] Unpaired Student's t-test was used for comparisons between groups. p < 0.05 was considered statistically significant. (NS: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

[0145] 3. Results The results are as follows Figure 1 As shown.

[0146] Therefore: DMEM is a basal isotonic medium that does not contain growth factors or fetal bovine serum. It keeps ASCs active for 72 hours and is therefore used as a positive control.

[0147] The medium (MC) is a very rich medium: it contains calf serum and growth factor (FGF2) and is not suitable for human use.

[0148] The composition of the present invention (LUM 7 QUATER) and the composition of the present invention without amino acids (LUM 7 HA3) have similar long-term viability.

[0149] Cells exposed to the composition without hyaluronic acid showed a rapid decline in survival (LUM 7 AA).

[0150] These data demonstrate the synergistic effect of sodium hyaluronate and the amino acids in the composition of this invention on cell viability. In other words, the combination of hyaluronic acid or its salts with the eight amino acids of this invention has a significant synergistic effect in improving cell viability.

[0151] Example 2. Evaluation of the adhesion, proliferation, and metabolic activity of the composition of the present invention on adipose tissue mesenchymal stem cells. Impact 1. Research Objectives The aim of this study was to investigate the effects of the composition of the present invention on the adhesion, proliferation, and metabolic activity of adipose tissue stem cells (or adipose tissue mesenchymal stem cells or "adipose-derived stem cells" or ASCs) from three donors at four different time points (i.e., 3 hours, 24 hours, 72 hours, and 6 days) using the MTT assay (or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide).

[0152] 2. Materials and Methods See points 2.1 to 2.3 and 2.5 of Example 1.

[0153] 2.4. Experimental Procedure According to the donor, ASCs are thawed into P0, P1, and P2, and then inoculated into P1, P2, or P3 culture flasks and cultured until confluence.

[0154] After cell confluence, ASCs were digested with trypsin and then seeded into 96-well plates. The test product was added immediately after seeding, and MTT assays were performed after 24, 72, 96 hours, and 7 days of incubation. Eight wells were processed at each time point under each condition, therefore one 96-well plate was used per time point and per donor, for a total of 12 wells.

[0155] In the MTT assay, the percentage of cell viability under each condition was calculated against the positive control group, which had a cell viability of 100%. The cell viability of the negative control group (0.05% SDS) should be 0%.

[0156] It should be noted that MC culture medium is used here because it is rich in nutrients, containing fetal bovine serum and growth factor (FGF2). The purpose of using MC is to improve the adhesion, viability, and proliferation of ASCs in cell culture, thereby observing and quantifying the effects of the composition of the present invention.

[0157] 3. Results The results are as follows Figure 2 As shown.

[0158] Adipose tissue mesenchymal cells exhibited similar behavior in the three formulations: LUM 7 quater, LUM 7 HA3, and LUM 7 AA.

[0159] Example 3. In vitro evaluation of the effect of the composition of the present invention on the production / secretion of beaded proteins by human primary fibroblasts (HPF). Leucoglucan and laminin, as well as keratinized glucan produced / secreted by cultured human epidermal keratinocytes (HEK) and Effects of β-defensin 1. Culture and maintenance of HEK cell lines HEK cells were obtained from skin samples from different donors and cultured in Celloneer KC / CC medium, which is the basal medium for keratinocytes. Cells were cultured in an incubator at 37°C, 85%–90% humidity, and 5% CO2. The medium was changed after 4 days of culture, and then twice a week thereafter.

[0160] 2. Culture and maintenance of HPF cell lines Fibroblasts (HPF) from a 40-year-old woman were cultured in Duchenne Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine (containing essential amino acids, salts, glucose, and vitamins required for cell stimulation), and 1% penicillin-streptomycin. The addition of the equivalent of 1% penicillin-streptomycin effectively inhibited bacterial growth. All mixtures were filtered through a Stericup filter. Cells were cultured in an incubator at 37°C, 85%–90% humidity, and 5% CO2. The medium was changed after 4 days of culture, and then twice a week thereafter.

[0161] 3. Experimental Design HEK cells and HPF cells were treated daily with different concentrations of the composition of the present invention (corresponding to the LUM 7 quater formulation in Example 1). Specifically, a stock solution of LUM 7 quater was first prepared according to the method described in Example 1, and then the stock solution was diluted by 20%, 30%, 40%, and 50%, respectively. The stock solution and the above-mentioned 20%, 30%, 40%, and 50% dilutions were all applied to the cultured cells.

[0162] 4. Results The results are as follows Figure 3-6 As shown.

[0163] The results show that: - Fibroblasts cultured in the presence of different concentrations of the compositions of the present invention produced more laminin and chondroitin sulfate than fibroblasts cultured in standard medium (DMEM); and - Keratinocytes cultured in the presence of different concentrations of the compositions of the present invention produced more keratinocytes than those cultured in standard medium (Celloneer KC / CC). Furthermore, the production of β-defensins associated with antimicrobial protection was also increased in keratinocytes in the presence of the compositions of the present invention.

[0164] Example 4. In vitro evaluation of the effect of the composition of the present invention on the migration rate of fibroblasts after experimental injury. Wound healing assays associated with cell layer damage involve: tearing cells along a precise trajectory in a controlled manner in a culture dish and periodically observing or photographing how cells fill the gaps near the "wound." The primary component of wound healing is cell migration, but proliferation may also be involved.

[0165] 1. Experimental Design 1.1. Preparation of Culture The first step in the test is to culture a confluent, high-power field monolayer of cells. This monolayer represents the in vivo state of tissue before damage, such as intact epithelial tissue.

[0166] 1.2. Creating a wound Once the cells have reached a confluence state, the next step is to create a cell-free region within the monolayer. This is achieved by mechanically scratching (or “scratch trauma”) the monolayer.

[0167] 1.3 Data Acquisition After creating damage, cells were treated with either the mother liquor or dilutes of the mother liquor at 20%, 30%, 40%, and 50%, or left untreated. Cell migration to the wound area was then observed using an optical microscope. Once the microscope was set up, a series of images were acquired using a rapid imaging (snapshot) method as cells migrated to the cell-free area. Images were acquired hourly for 72 hours. In addition, precise measurement data were manually acquired using a digital camera mounted on the microscope.

[0168] 1.4 Data Analysis Cell migration was analyzed at 20x magnification using a Nikon inverted microscope (Melville, NY). High-power field (HPF) cultures with scratch damage were treated with different concentrations of the composition of this invention before time-lapse imaging began. Three fields of view were selected for each well, and imaging and monitoring were performed using a Coolsnap HQ camera (Photo-metrics, Tucson, Arizona) and NIS-elements AR 2.30 software (Nikon), recording every hour for 72 hours. Individual cells were manually tracked using Metamorph software (Roper Scientific, Evry, France). Migration parameters for each cell, including total migration distance, distance from the origin, migration velocity, and persistence of migration direction, were determined from the time-lapse films. The total migration distance was the sum of distances measured each time over 72 hours. Results for each condition are expressed as mean ± standard deviation of at least 60 individual cells.

[0169] It is worth noting that, in order to obtain data on cell migration only, rather than cell migration and / or proliferation, the proliferation inhibitor mitomycin C was added to the culture medium.

[0170] 2. Results The results are as follows Figure 7 As shown.

[0171] Experiments have shown that, under stimulation with different concentrations of the composition of the present invention, the migration rate of cultured fibroblasts in vitro is higher than that of the control group. This means that application of the composition of the present invention can accelerate wound coverage, thereby promoting wound healing.

[0172] Figure 8This data is confirmed by the figure, which shows that in vivo application of the composition of the present invention (the stock solution and 20%, 30%, 40% and 50% stock solution dilutions) improves and accelerates wound healing compared to conventional treatment regimens.

[0173] Example 5. In vivo evaluation of the effect of the composition of the present invention on chronic wound healing A 58-year-old paraplegic patient had a chronic wound on the lateral side of his foot that had persisted for several years. He received a two-month treatment consisting of weekly injections into the wound (intralesional injection) and injections into healthy tissue near the wound (perilesional injection).

[0174] Figure 8 Photos are shown before treatment (A) and two months after treatment (B).

[0175] These photos clearly demonstrate that treatment with intradermal injection of the composition of the present invention has surprisingly improved the healing of chronic wounds that have persisted for many years.

[0176] Example 6: In vitro comparative study of the effect of the composition of the present invention on the migration rate of fibroblasts after experimental injury. This study aimed to evaluate the effects of three components on the in vitro migration of human primary fibroblasts using scratch assay technology.

[0177] 1. Experimental Design 1.1 Cell Preparation Human neonatal fibroblasts (from LifeLine Cellsystems) were seeded in Fibrolife medium and placed in 24-well plates (7 x 10⁴ cells / well) and cultured for 3 days at 37°C and 5% CO₂ until the cells confluenced.

[0178] 1.2 Scratch Testing Technique In each well, damage the cell layer using a 10 µl pipette tip. Remove the culture medium and replace it with fresh medium containing different proportions (final concentrations of 20% or 50%) of each product solution. Perform control experiments using culture medium as requested by the customer. Perform three replicates for each condition.

[0179] 1.3 Imaging monitoring of cell migration The culture plates were then placed in a humidified incubator containing 5% CO2 at 37°C and observed using a video microscope (CARLZEISS AXIO OBSERVER Z1). Two positions of each well were pre-recorded and continuously monitored. Images were recorded every 2 hours for 48 hours.

[0180] 1.4 Results Analysis For each location, analysis over time was performed using ImageJ software and its "Wound Healing Size Tool" plugin to quantify the unhealed lesion area. The lesion healing rate, assessing cell migration, was calculated using the following formula: The healing rate was calculated from six values ​​(3 replicates x 2 sites per well) at each time point under each condition. Results are expressed as the mean (± standard deviation) of the healing rate. Statistical analysis was performed using the nonparametric Mann-Whitney test, and each condition was compared with the control group.

[0181] 2. The composition tested - Control group: Fibrolife culture medium (LifeLine Cellsystems) -LUM 7 Quater (or Lum 7; the composition of the present invention): the composition according to Example 1 -LUM A (Composition of the present invention, hyaluronic acid-free): Corresponds to composition LUM 7 AA of Example 1. -LUM H1 (composed of low molecular weight hyaluronic acid and amino acids from the Lum 7 Quater composition), its active ingredients are shown in Table 5 below.

[0182] [Table 5] 3. Results The results are as follows Figure 9-12 As shown.

[0183] from Figure 9 The images show that, regardless of the treatment or control groups, the wounds were completely healed after approximately 30 hours; therefore, the analysis time range is 0 to 30 hours. The images show each condition at T0, as well as at T8, T12, and T16 hours, where the differences between the treatment and control groups were most significant.

[0184] Starting at time T0, cells gradually covered the damaged areas of the cell layer. From time T8, compared with the control group and other treatment groups, the surface area covered by cells was significantly increased after treatment with 20% or 50% Lum 7.

[0185] Regarding fibroblast migration kinetics, the results show: - Lum 7 activated fibroblast migration in both the early (50%) and late (20%) stages. 50% Lum 7 showed significant effects at 6 and 8 hours, increasing fibroblast migration by 22% and 25% compared to the control group, respectively; 20% Lum 7 showed significant effects at 12, 16, and 22 hours, with the largest increase at 12 hours (23%). There was no significant difference between 20% and 50% Lum 7.

[0186] - A 50% concentration of Lum H1 activated fibroblast migration, with significant effects over 16 to 22 hours, reaching a maximum increase of 14% at 16 hours. A 20% concentration of Lum H1 did not show a significant increase compared to the control group. There was no significant difference between the 20% and 50% concentrations of Lum H1.

[0187] - Compared with the control group, 20% or 50% Lum A had no significant effect on fibroblast migration. There was also no significant difference between 20% Lum A and 50% Lum A.

[0188] 4. Conclusion Lum 7 showed the highest activity, accelerating fibroblast migration in the scratch assay, with a maximum increase of 25% compared to the control group. Both 20% and 50% concentrations of Lum 7 were active, but at different times: 50% Lum 7 showed stronger activity in the early stages of the assay, while 20% Lum 7 showed stronger activity in the later stages. Lum H1 also showed activity, but was only effective at a 50% concentration, with a maximum increase of 14% compared to the control group. In contrast, Lum A had no effect on fibroblast migration in this assay.

[0189] In other words, these data clearly show that high molecular weight hyaluronic acid, combined with the amino acids of this invention, has a synergistic effect and can improve wound healing.

[0190] Example 7: Evaluation of the effect of the composition of the present invention on corneal epithelial cell (COEP) viability The purpose of this study is to verify the non-cytotoxicity of the compositions of the present invention and to evaluate the effect of their administration on COEPs, compared with prior art eye drops.

[0191] 1. Cell viability was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT). The reagent used was MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide). The tetrazolium ring contained in MTT is reduced to formazan by mitochondrial succinate dehydrogenase in living and metabolically active cells. Formazan forms a purple precipitate in the mitochondria. The amount of precipitate is directly proportional to the number of living cells. The relative number of living and metabolically active cells can be determined by spectrophotometric measurement of the absorbance at 570 nm.

[0192] 1.1. Material The cells used were corneal epithelial cells (COEP), seeded at a density of 10,000 cells / well (48-well plate), COEP38230536G P3. The tested components are as follows: - LUM 7 Quarter Composition (Composition of the present invention; see Example 1): 2 bottles per mL - Existing eye drops: artificial tears, which are used as first-line treatments for dry eye and mild epithelial ulcers, namely... o Dusilames 1.5%; o Hylovis multi15; and o Theophylline.

[0193] The eye drops and compositions of the present invention were tested with DMEM or PBS at a 1 / 2 dilution.

[0194] - Cell lysis buffer: a negative control for cell viability - DMEM: Contains diluent only - PBS: Contains diluent only - Culture medium: used for inoculating COEP and as a control for MTT assay.

[0195] 1.2. method After thawing, COEP cells were amplified in flasks for one week, and then seeded at a density of 10,000 cells / well into 48-well plates pre-coated with an irradiated nutrient layer.

[0196] COEP was cultured for 4 days. 500 µl of medium was added at the beginning, and after 3 days of culture, the medium was changed and 300 µl of medium and EGF were added.

[0197] After 4 days of incubation, remove the culture medium and contact each condition with COEPs: - Cell lysis buffer 300 µL Culture medium: 300 µL -DMEM: 300 µL -PBS: 300 µL - Diluent: 300 µL - Diluted eye drops: 300 µL Before performing MTT testing, expose each condition to COEP for 4, 12, and 24 hours.

[0198] For MTT assay, the liquid in each well was first aspirated, then rinsed with PBS buffer, aspirated again, and 300 µl of 1 mg / ml MTT solution was added. After incubation at 37°C for 2 hours, the culture plate was inverted, and 300 µl of acidifying solution was added to dissolve the crystals. Finally, cell viability in each well was analyzed using a spectrophotometer. Cell viability in each group was calculated against the positive control group, with a cell viability of 100% in the positive control group and 0% in the negative control group.

[0199] After culturing the cells for 4 days, they were exposed to LUM 7 Quater and eye drops for 4 hours, 12 hours, and 24 hours, respectively.

[0200] The results were standardized using the control culture medium, which represents 100% of all analytical time points.

[0201] 2. Results 2.1 MTT Testing The results are as follows Figure 13 As shown.

[0202] Data shows that the formulation of this invention has no "cytotoxicity" at three different time points (4, 12 and 24 hours), especially compared with existing eye drops.

[0203] 2.2 1 / 2 diluted DMEM The results are as follows Figure 14 As shown.

[0204] Four hours after exposure: Optical density (OD) measured by the MTT reaction was similar under all test conditions (eye drops, formulation diluted with DMEM, and culture medium). The mean OD values ​​ranged from 0.414 to 0.448.

[0205] At 12 hours of exposure: Cell viability was higher at 12 hours than at 4 hours under all test conditions using DMEM dilution, indicating enhanced cell proliferation and / or metabolic activity. Therefore, the tested conditions appear to favor cell viability.

[0206] Compared to all other conditions, nutrient-rich and optimized culture media exhibit higher cell activity (proliferative and / or metabolic activity).

[0207] • Two commercially available eye drops (Hylovis Multi 15 and ThéaLose) and the LUM 7 Quarter formulation (the composition of the present invention) showed similar results, slightly lower than those from the culture medium (activity relative to the culture medium was between 90.4% and 91.2%), and comparable to those from the diluent alone (DMEM).

[0208] • The cell activity of commercially available eye drops (Dulcilarmes) is lower (82%) than under all other conditions, including diluents.

[0209] 24 hours after exposure: Although cell viability continued to increase in the culture medium, the viability of cells exposed to Hylovis multi 15, ThéaLose, LUM 7 quater formulations (dissolved in DMEM medium) and those treated with diluent alone stagnated or decreased slightly. However, the viability of cells treated with Hylovis multi 15, ThéaLose, and LUM 7 quater formulations was still higher than that of cells treated with diluent alone.

[0210] Cell activity of Dulcilarmes eye drops diluted with DMEM remained lower than that under other conditions.

[0211] 2.3 1 / 2 diluted PBS The results are as follows Figure 15 As shown.

[0212] Four hours after exposure: Dilution experiments in PBS buffer showed more dispersed results than those in DMEM medium. The medium exhibited the strongest cell viability. Viability decreased in the following order: Hylovis Multi 15, ThéaLose, formulation, and diluent (PBS). Cell viability in Dulcilarme was almost zero, likely due to precipitation observed during dilution. This lack of activity persisted at 12 and 24 hours.

[0213] At 12 hours of exposure: For both the eye drops and the LUM 7-quarter formulation diluted with PBS, as well as the culture medium, cell viability was higher at 12 hours than at 4 hours, indicating enhanced cell proliferation and / or metabolic activity. Therefore, these conditions favored cell viability. Conversely, cell viability remained stagnant when stored solely in the diluent (PBS).

[0214] Compared to all other conditions, the nutrient-rich and optimized culture medium exhibited higher cell viability.

[0215] • Cell viability was very similar under PBS-diluted Hylovis Multi 15, ThéaLose and LUM 7 quarter formulations (OD values ​​between 0.376 and 0.413).

[0216] • The activity was significantly reduced when the diluent was used alone: ​​the activity was reduced by half (OD was 0.182).

[0217] 24 hours after exposure: Cell viability decreased under all conditions except the culture medium. This decrease can be attributed to the lack of nutrients (amino acids, glucose) in the diluent, leading to cell death.

[0218] 3. Conclusion The composition of this invention (LUM 7 Quater) maintains the integrity and activity of corneal epithelial cells. This maintenance and activity are similar to those of prior art Hylovis multi 15 and Théalose eye drops. Therefore, these data demonstrate the efficacy of this composition in the treatment of corneal diseases, particularly dry eye and corneal ulcers.

[0219] Overall Conclusion These data demonstrate a synergistic effect between specific hyaluronic acids or their salts with a molecular weight greater than 2 MDa and the amino acids in the compositions of this invention. This synergistic effect promotes cell viability, adhesion, proliferation, and metabolic activity, ultimately promoting wound healing and cell and tissue regeneration. In other words, the combination of hyaluronic acid or its salts with the eight amino acids of this invention has a significant synergistic effect, enhancing the aforementioned phenomena.

[0220] Furthermore, the composition of the present invention (LUM 7 QUATER) appears to enable cells to survive for 6 days, a longer survival time than that achieved by the composition of the present invention (LUM 7 AA) which does not contain hyaluronic acid or its salts.

[0221] Furthermore, these data clearly demonstrate that the formulation of this invention can stimulate different components of the basement membrane at various concentrations. This is reflected in the following positive effects: - Dermal-epidermal junction: The composition of the present invention enhances the junction and provides mechanical strength to the epidermis; - Dermal and epidermal tissues: The compositions of this invention enhance the interaction between cells and their environment; - Terminal differentiation of keratinocytes: The compositions of this invention stimulate different effector factors associated with epidermal mechanical, immune, and antimicrobial protection; and - Functional effects on fibroblasts: It increases the migration rate of fibroblasts, induces the generation of more myofibroblasts and the synthesis of type I and type III collagen, thereby promoting wound healing / repair in the dermis.

[0222] Therefore, it can be clearly seen from the above content that the composition of the present invention acts on the synthesis of components in the basal layer / dermal-epidermal junction by altering the synthetic expression of keratinocytes and fibroblasts.

[0223] The composition of this invention greatly promotes the stimulation of keratinocytes to synthesize epidermal protection-related receptors and molecules, accelerates fibroblast migration, and increases the phenotype of myofibroblasts in experimental injury models.

[0224] These conclusions confirm the beneficial effects of the present invention. The compositions of the present invention can be used to treat the cornea, joints or cartilage, improve cell vitality, moisturize mucous membranes, or be used for tissue or cell transplantation.

Claims

1. A pharmaceutical composition comprising: - Hyaluronic acid or its salts with a molecular weight greater than 2 MDa; and - At least 8 essential amino acids for humans, selected from the following group: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, and histidine.

2. The composition according to claim 1, characterized in that, The at least eight 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 its salt is in a non-crosslinked form.

4. The composition as claimed in any of the preceding claims, characterized in that, The hyaluronic acid or its salt is sodium hyaluronate, potassium hyaluronate or a mixture thereof, preferably sodium hyaluronate.

5. The composition as claimed in any of the preceding claims, characterized in that, It also contains sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water, preferably water for injection.

6. The composition as claimed in any of the preceding claims, characterized in that, It is composed of the following substances: - Sodium hyaluronate; - The following essential amino acids: lysine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and threonine; - Na2HPO4; - KH2PO4; - NaCl; and - water.

7. The composition as claimed in any of the preceding claims, characterized in that: - Hyaluronic acid or its salt, preferably sodium hyaluronate, in a concentration of 6 g / L to 8 g / L; - Lysine content ranges from 33 mg / L to 39 mg / L; - Valine content is 21 mg / L to 25 mg / L; - Isoleucine content ranges from 5.7 mg / L to 6.6 mg / L; - Leucine content ranges from 24.3 mg / L to 28.3 mg / L; - Methionine content ranges from 6.8 mg / L to 8.2 mg / L; - Phenylalanine content is 15 mg / l to 18 mg / l; - Tryptophan content ranges from 4.6 mg / L to 5.6 mg / L; - Threonine content ranges from 21.1 mg / L to 25.2 mg / L; - The Na2HPO4 content is 1.35 g / L to 1.45 g / L; - KH2PO4 content is 0.65 g / L to 0.75 g / L; - NaCl content is 4.5 g / L to 5.5 g / L; and Add water to 1L.

8. The composition as claimed in any of the preceding claims, used as a medicine.

9. The composition according to any one of claims 1 to 7, for treating wounds, preferably for acute or chronic wounds.

10. The composition for use as described in claim 9, characterized in that, The wound healing has improved.

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

12. The composition according to any one of claims 1 to 7, for tissue moisturizing, preferably for skin, cornea, mucous membrane, cartilage, bone and / or wounds, preferably for acute or chronic wounds.

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

14. The composition according to any one of claims 1 to 7, for transplantation and / or implantation of tissues or cells.

15. An in vitro method for maintaining or even enhancing cell viability of cultured cells by applying the composition of any one of claims 1 to 7 to cultured cells; wherein, The cells are preferably fibroblasts, keratinocytes, corneal epithelial stem cells, or adipose stem cells.

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