FR3117012A1 - TOPICAL USE OF METFORMIN TO REDUCE INFLAMMATION IN THE SKIN - Google Patents

Abstract

The present invention relates to metformin, its salts, or its complexes, for its topical use to reduce inflammation of the skin, in particular to reduce protein glycation of the skin, more particularly of the dermis. The present invention relates to the topical use of metformin, its salts, or its complexes to reduce the load of AGEs, to inhibit the expression of the gene coding for the RAGE receptor and the protein abundance of RAGE and thus to reduce the secretion of IL-8. Finally, the present invention also relates to the topical use of metformin, its salts, or its complexes to activate and improve the reconstruction of the skin, in particular of the dermis, thus accelerating the healing of wounds.

Description

TOPICAL USE OF METFORMIN TO REDUCE INFLAMMATION IN THE SKIN

The present invention relates to metformin, its salts, or its complexes, for its topical use to reduce inflammation of the skin, in particular of the dermis. The present invention relates to the topical use of metformin, its salts, or its complexes to decrease protein glycation of the skin by decreasing the AGE load, the expression of the gene encoding the RAGE protein and the protein abundance of RAGE and thus by decreasing the secretion of IL-8.

Finally, the present invention also relates to the topical use of metformin, its salts, or its complexes to activate and improve the reconstruction of the skin, in particular of the dermis, thus accelerating the healing of wounds.

Inflammation of the skin

Acute inflammation develops quickly in response to a trigger, such as an allergen, the sun, or an infection. This type of inflammation is short term and usually goes away within a few weeks if the cause of the inflammation is treated. Acute inflammation does not cause permanent tissue damage.

Chronic inflammation is long-lasting inflammation that develops when the immune system releases sustained responses in the body. Over time, this can lead to chronic disease and tissue damage. As the inflammatory process occurs in the body, the symptoms are not always visible. Common chronic skin conditions are psoriasis, rosacea and eczema. Certain pathologies such as diabetes or obesity can also be responsible for chronic inflammation in the skin.

Common causes of skin inflammation include:
-Bacterial, fungal and viral infections can cause inflammation of the skin (such as Staphylococcus infections, viral infections such as warts and herpes simplex, fungal infections such as ringworm and athlete's foot) .
- Dysfunctions of the immune system: the immune cells begin to attack the body's own healthy cells by mistake, as is the case with psoriasis.
-allergic reactions: the immune system overreacts when it detects a foreign substance and sends cells to attack the foreign body. Foods, medications, and pollen can all trigger allergic reactions and cause skin redness, hives, and inflammation.
- photosensitivity: it is an extreme sensitivity to sunlight that can trigger an immune system response.
- wounds: cuts, scrapes, burns and surgical wounds cause redness, swelling and heat in the wound. The immune system sends a temporary inflammatory response to help heal damaged tissue.

Skin healing:

Wound healing is a natural biological phenomenon, mammals being able to repair localized lesions through their own repair and regeneration processes.

The speed and quality of wound healing depends on the general condition of the affected organism, the etiology of the wound, the condition and location of the wound, and whether or not infection, as well as genetic factors predisposing or not to wound healing disorders.

The natural healing of a wound takes place mainly in three successive phases, each of these phases being characterized by specific cellular activities which advance the repair process according to precise chronological phases: the inflammatory phase, the granulation phase (or proliferative) including in particular the epidermization step, and the maturation phase.

The second phase, the proliferative phase, thus comprises two stages. The first stage corresponds to the development of the granulation tissue while the second stage corresponds to the epidermization stage strictly speaking. The granulation phase allows the establishment of a transitory tissue which will fill the loss of substance resulting from the aggression at the origin of the wound. This transient tissue is called "granulation tissue". The latter consists of:
- Neo-vessels: starting from the peripheral vessels of the lesion focus, there will take place a multiplication then a migration of endothelial cells first of all in the form of solid cords which are hollow secondarily of vascular lumen leading to the reconstitution of new vessels.
- Fibroblasts - myofibroblasts synthesizing collagen and other elements of the extracellular matrix. They elaborate a new temporary conjunctive matrix; it is first of all fragile, rich in fibronectin and hyaluronic acid; it represents a scaffold allowing the migration of other fibroblasts and new vessels then forming the connective tissue constituting the dermis.

Subsequently, this colonization of the wound continues at the upper level by the proliferation of keratinocytes above this granulation tissue: proliferation and migration of said cells along the dermo-epidermal junction until they come into contact with cells from the opposite side (contact inhibition); Finally, the cells differentiate and stratify to reconstitute a complete and functional epidermis, this is called the epidermization stage.

Nevertheless, certain types of wounds do not heal properly, with certain key stages of the process (including the epidermization phase) taking place abnormally, despite the implementation of the best possible physico-chemical and biological conditions. Indeed, the speed and quality of wound healing depend on intrinsic and extrinsic factors. This repair process can therefore be abnormally prolonged depending on:
- the etiology of the wound;
- its condition and location;
- the occurrence of an infection caused by the presence of certain infectious agents such as Staphylococcus aureus or Pseudomonas aeruginosa;
- the existence of a pre-existing pathology (such as diabetes, immune deficiency, venous insufficiency, etc.);
- the external environment; Or
- genetic factors predisposing or not to healing disorders.

Among these wounds, we find chronic wounds such as venous ulcers, bedsores or wounds characteristic of diabetic subjects. Chronic wounds are defined by a lack of healing after a period of 6 weeks from the appearance of the wound, regardless of the treatment applied. To treat these types of wounds, it can be crucial to speed up the healing process at any of these stages.

Protein glycation of the skin:

During the normal aging process, many metabolic alterations occur, including an imbalance of intracellular glucose metabolism. This generates increased production of oxidizing products such as the highly reactive dicarbonyls glyoxal and methylglyoxal. This process, called non-enzymatic glycation, induces in the skin the covalent bonding of the hydroxyl groups of reducing sugars to the free amino acids (lysine and arginine) of proteins such as collagens and elastin, lipids and nucleic acids. Glycation leads to the irreversible formation of complex compounds called advanced glycation end products (AGEs), which damage protein structure and function, alter enzyme activity, and reduce the elimination of damaged proteins, leading to disruption of metabolism cell and homeostasis. Glycation of proteins can cause cell death, cell differentiation, or reduced cell adhesion and migration.

AGEs present various structures such as N-ε-carboxymethyllysine (CML) which is one of the best characterized AGEs in humans and the AGE mainly produced by glyoxal, pyrraline, pentosidine or other lines cross-linked according to the precursor molecule, which are known to be associated with degenerative and aging processes, diabetes, atherosclerosis and renal failure (Goh and Cooper, 2008; Kyung et al., 2013; Lee et al., 2016; Nowotny et al. , 2015a).

The accumulation of AGEs, which can be accelerated under hyperglycaemic and/or diabetic conditions, modifies the physical and mechanical properties of human tissues, including the dermis. AGEs exert their deleterious actions through their intrinsic biological properties and through their interaction with specific receptors. After binding of AGEs to their RAGE membrane receptors, intracellular oxidative stress and pro-inflammatory status are induced, which are implicated in the pathogenesis of various aging-related disorders such as complications of diabetes, atherosclerosis, Alzheimer's disease. Alzheimer's and cancer (Danby, 2010; Gkogkolou and Böhm, 2012; Goldin et al., 2006; Kyung et al., 2013; Lee et al., 2015b; Leibold et al., 2013; Lohwasser et al., 2006 Sadowska-Bartosz and Bartosz, 2016; Sejersen and Rattan, 2009). Interestingly, several research studies have reported the critical role played by the formation and accumulation of AGEs in the skin, which are increased in diabetic patients due to increased circulating glucose concentration. Their cross-linking to macromolecules in the dermis and activation of different signaling pathways via their binding to their cell surface receptor RAGE affect extracellular matrix (ECM) remodeling and immune response, leading to skin conditions associated with diabetes (delayed wound healing , predisposition to infections, etc.) and accelerated skin aging (Goh and Cooper, 2008; Lohwasser et al., 2006; Monnier et al., 2005; Pageon et al., 2015; Serban et al., 2016a; Sorci et al. ., 2013).

Lifestyle factors, such as diets high in fat, sugar, and salt, play a key role in the development and progression of chronic diseases, such as type 2 diabetes, cardiovascular disease, and kidney disease chronicles. The modern Western diet is made up of highly processed foods that are not only high in fat, sugar and salt, but also contain EFAs (Clarke et al., 2016). AGEs are formed when foods are processed at high temperatures (frying, broiling, roasting). They are also found in cigarette smoke.

Summary

Quite surprisingly, the Applicant has demonstrated that metformin, its salts or its complexes, made it possible to reduce the inflammation of the skin in a context of abnormal inflammation, and in particular in a context of inflammation linked to too high glycation in the patient (for example a context of diabetes) and this, by reducing the protein glycation of the skin, in particular of the dermis. Metformin, its salts, or its complexes make it possible to reduce the load of AGEs, to reduce the expression of the gene coding for the RAGE protein and thus to reduce the secretion of IL-8. All of these properties therefore participate in activating and improving the reconstruction of the skin, in particular of the dermis, thus improving the healing of wounds. By reducing the load of AGEs, metformin allows a reduction in inflammation of the skin, in particular of the dermis, more particularly when said inflammation is linked to excessive glycation (as opposed to inflammation linked to a germ or irritation ).

The subject of the invention is therefore metformin, its salts, or its complexes, for its topical use to reduce the inflammation of the skin by reducing the protein glycation of the skin, in particular of the dermis. In particular, the subject of the invention is metformin, its salts, or its complexes, for its topical use to reduce the inflammation linked to abnormally high glycation. The context of abnormal glycation leading to the type of inflammation referred to in the context of the present application is observed in particular in very specific populations, such as in particular patients suffering from diabetes, atherosclerosis or renal insufficiency, patients on a diet food high in fat, sugar and salt, or aging populations (over 60). This decrease in glycation is done via the decrease in the production of AGEs and the expression of the gene coding for the RAGE protein allowing the decrease in the secretion of pro-inflammatory cytokines.

The invention finally relates to metformin, its salts, or its complexes for its topical use to activate and improve the reconstruction of the skin, in particular of the dermis, thus accelerating the healing of wounds.

The invention finally relates to metformin, its salts and its complexes as described above, for its use to accelerate the healing of a chronic wound, in particular a diabetic foot ulcer.

Other characteristics, details and advantages will appear on reading the detailed description below, and on analyzing the appended drawings, in which:

Fig. 1

represents the quantification of the content of AGEs in protein extracts collected from NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of glyoxal. Aminoguanidine (10mM) was used as an anti-glycation reference compound.

Fig. 2

represents the quantification of mRNA levels coding for the fl-RAGE receptor in NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of glyoxal at 0.6 mM. Aminoguanidine (10 mM) was used as an anti-glycation reference compound.

Fig. 3

represents the quantification of the protein abundance of the RAGE receptor in NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of glyoxal at 0.5 mM. Aminoguanidine (10 mM) was used as an anti-glycation reference compound.

Fig. 4

represents the quantification of IL-8 secretion performed by ELISA assay in culture supernatants after treatment of NHDF fibroblasts with metformin (0.1, 0.3 and 1 mg/ml) alone during the first 24 h, followed by 72 hours in the presence of 0.6 mM of glyoxal.

By the expression "activating and improving the reconstruction of the skin and the dermis", we mean any positive stimulation of the migration or the proliferation of the fibroblasts at the level of the dermis, so as to improve the general appearance of the skin, to increase the rate of healing in damaged skin, resulting in accelerated wound closure and improved scar appearance.

RAGE is an approximately 50 kDa transmembrane receptor belonging to the immunoglobulin superfamily. RAGE is expressed in several cell types, including skin cells; fibroblasts, dendritic cells and keratinocytes (Lee et al., 2016; Lohwasser et al., 2006; Metz et al., 2012; Ott et al., 2014).

RAGE has a vast repertoire of ligands including in particular the AGEs which give it its name. Recognition of ligands by RAGE (V-like domain) leads to receptor activation and triggers various downstream signaling cascades primarily via the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of a factor transcription factor, nuclear factor kappa-B (NFκB), leading to various cellular responses, including altered expression of certain genes, widely reported to be associated with inflammation (Kierdorf and Fritz, 2013; Sorci et al. , 2013). AGEs have been shown to play an important role as stimuli that activate intracellular signaling pathways via their binding to the cell surface receptor RAGE, promoting the secretion of cytokines and growth factors, which in turn accelerate the chronic inflammation and enhance oxidative stress (Brings et al., 2017; Nowotny et al., 2015; Ott et al., 2014).

Moreover, the activation of RAGE by its ligands upregulates its expression via an NFκB-dependent mechanism. Indeed, after RAGE activation, NFκB is translocated to the nucleus and binds to the promoter region of the gene encoding RAGE, enhancing the translation of RAGE mRNA (Gkogkolou and Böhm, 2012; Goldin et al., 2006; Kierdorf and Fritz, 2013; Serban et al., 2016; Xie et al., 2013). Thus, NFκB induces increased expression of RAGE, which itself further stimulates NFκB secretion, forming a vicious circle of self-renewal and perpetuation of RAGE signaling (Gkogkolou and Böhm, 2012).

This positive feedback mechanism can be interrupted by decreasing the amount of AGEs and therefore the activation of the receptor. (Gkogkolou and Böhm, 2012; Kierdorf and Fritz, 2013; Sorci et al., 2013).

As shown above, the ligand-RAGE interaction plays a major role in numerous signaling pathways (Ott et al., 2014) and activates several intracellular transduction cascades leading to the activation of the transcription factor NFκB, the production of reactive oxygen species (ROS), pro-inflammatory mediators as well as modification of the composition of the extracellular matrix.

Activation of NFκB increases the expression of pro-inflammatory cytokines such as interleukin (IL)-6 (IL-6) and IL-8, and chemokines such as MCP-1 as well as the RAGE receptor itself thereby intensifying the inflammatory response (Nowotny et al., 2015; Rasheed et al., 2011; Serban et al., 2015, 2016; Sparvero et al., 2009; Xie et al., 2013). Exposure to AGEs is therefore known to induce a maladaptive immune response, which may contribute to the plethora of complications of diabetes, including difficulty in skin healing and susceptibility to infections (Serban et al., 2016).

Besides its pro-inflammatory effect, the AGEs-RAGE axis is associated with alterations in the extracellular matrix (ECM) leading to increased vascular permeability, contractility, ECM synthesis, cell growth, disrupted cell-matrix interactions, altered cell adhesion and apoptosis (Serban et al., 2016). The dermal extracellular matrix (ECM) and its associated glycation-modified proteins alter matrix-matrix interactions as well as matrix-cell interactions, affect fibroblast growth, differentiation and motility, and metalloproteinase activity ( Crisan et al., 2013; Nowotny et al., 2015).

Metformin

Metformin is an oral antidiabetic from the normoglycemic biguanide family used in the treatment of type 2 diabetes. Its role is to reduce the insulin resistance of the organism intolerant to carbohydrates and to reduce hepatic gluconeogenesis. Metformin is administered orally. Metformin is absorbed in the small intestine, circulates in the blood in an unbound way and is excreted, unchanged, by the kidneys. Its mechanism of action is complex and has not yet been fully elucidated. Metformin is a normoglycemic agent: it does not affect insulin secretion or the insulin sensitivity of glucose-using tissues (muscles, adipose tissue). Metformin also has a role in the inhibition of gluconeogenesis, by inhibiting mitochondrial glycerophosphate dehydrogenase, and in the membrane transport of glucose (reduction of its intestinal resorption). It also increases the release of Glucagon-like peptide-1, inhibits the glucagon pathway, increases the production of lactates by enterocytes.

According to a preferred embodiment, the metformin used is in the form of a metformin salt, preferably in the form of metformin hydrochloride.

Composition

A subject of the invention is also a pharmaceutical composition comprising the metformin described above, at a concentration of 0.01 to 10 mg/mL of composition, preferably 0.05 to 5 mg/mL, and more preferably 0.08 to 2 mg/mL, for its topical use for reducing skin inflammation, in particular by reducing protein glycation of the skin, in particular reducing protein glycation of the dermis.

Additional active substance

In general, the metformin according to the invention may be used alone or in combination with one (or more) other active ingredient(s).

In general, the active agents are chosen from antibacterials, antiseptics, painkillers, anti-inflammatories, active agents promoting healing, depigmenting agents, antipruritics, UV filters, soothing agents, moisturizers, antioxidants, and mixtures thereof.

In general, the assets are chosen from:
- anti-bacterials such as Polymyxin B, penicillins (Amoxycillin), clavulanic acid, tetracyclines, Minocycline, chlorotetracycline, aminoglycosides, Amikacin, Gentamicin, Neomycin, silver and its salts (Silver Sulfadiazine), Probiotics, Silver Salts;
- antiseptics such as sodium mercurothiolate, eosin, chlorhexidine, phenylmercury borate, hydrogen peroxide, Dakin's liquor, triclosan, biguanide, hexamidine, thymol, Lugol, Povidone iodine, Merbromine, Benzalkonium and Benzethonium Chloride, ethanol, isopropanol;
- painkillers such as Paracetamol, Codeine, Dextropropoxyphene, Tramadol, Morphine and its derivatives, Corticosteroids and derivatives;
- anti-inflammatory drugs such as Glucocorticoids, non-steroidal anti-inflammatory drugs, Aspirin, Ibuprofen, Ketoprofen, Flurbiprofen, Diclofenac, Aceclofenac, Ketorolac, Meloxicam, Piroxicam, Tenoxicam, Naproxen, Indomethacin, Naproxcinod, Nimesulide, Celecoxib, Etoricoxib, Parecoxib, Rofecoxib, Valdecoxib, Phenylbutazone, niflumic acid, mefenamic acid;
- active ingredients promoting healing such as Retinol, Vitamin A, Vitamin E, N-acetyl-hydroxyproline, extracts of Centella Asiatica, papain, silicones, essential oils of thyme, niaouli, rosemary and sage, hyaluronic acid, Allantoin, -Hema'tîte (gattefossé), Vitamin C, TEGO Pep 4-17 (evonik), Toniskin (silab), Collageneer (Expanscience), Timecode (Seppic), Gatuline skin repair (gattefossé), Panthenol, PhytoCellTec Alp Rose (Mibelle Biochemistry), Erasyal (libragen), Serilesine (Lipotec), Heterosides of Talapetraka (beyer), Stoechiol (codif), macarose (Sensient), Dermaveil (Ichimaru Pharcos), Phycosaccaride AI ( Codif), polysulfated oligosaccharides;
- depigmenting agents such as kojic acid (Kojic Acid SL® - Quimasso (Sino Lion)), Arbutin (Olevatin® - Quimasso (Sino Lion)), the mixture of sodium palmitoylpropyl and white water lily extract (Sepicalm® - Seppic), undecylenoyl phenylalanine (Sepiwhite® - Seppic),
- antipruritics: hydrocotisone, enoxolone, diphenyhydramine, antihistamine with local application anti H1
- moisturizing active ingredients such as xpermoist (lipotec), hyaluronic acid, urea, fatty acids, glycerine, waxes, exossine (unipex)
- UV filters such as Parsol MCX, Parsol 1789
-soothing agents such as chamomile, bisabolol, xanthalene, glycyrrhebenic acid, tanactin (CPN), Calmiskin (Silab),
- antioxidants, such as vitamin E.

According to a preferred embodiment, the metformin according to the invention can be used in combination with potassium sucrose octasulfate.

Galenic

The metformin used in the context of the present invention can be administered topically, and in particular implemented within a galenic formulation, such as for example a gel, a solution, an emulsion, a cream, granules or capsules. of variable size ranging from nano or micrometer to millimeter, which will allow their application directly to the wound. Alternatively, the metformin used in the context of the present invention can be implemented within a solution for subcutaneous injection.

If it is used in combination with one or more other active substances, these compounds may be incorporated in the same galenic formulation or in separate galenic formulations.

Of course, the quantity of metformin according to the invention used in the galenic formulation is adapted according to the kinetics sought as well as the specific constraints linked to its nature, solubility, resistance to heat, etc.

Bandage

Preferably, the metformin used in the context of the present invention, or a composition containing it, will be administered by means of a medical device such as a dressing, allowing in particular direct application to the wound.

Metformin, and in particular metformin hydrochloride or a composition containing it, may be incorporated into any element of the structure of a dressing provided that the metformin can come directly or indirectly into contact with the surface of the wound.

Preferably and in order to promote rapid action, the metformin (or a composition containing it) will be incorporated into the layer of the dressing which comes into contact with the wound or deposited on the surface of the dressing which comes into contact with the wound.

Advantageously, metformin (or a composition containing it) may thus be deposited, continuously or discontinuously, on the surface intended to come into contact with the wound:
- either in liquid form, for example by vaporization of a solution or suspension containing it;
- Either in solid form, for example by sieving a powder containing it.

The layer or surface coming into contact with the wound may consist, for example, of an absorbent material such as a hydrophilic polyurethane absorbent foam; a textile material such as a compress, such as for example a nonwoven, a film, a web of fibers; an adhesive material, absorbent or not; an adherent interface structure or not.

In general, it is possible to play on the galenic or the structure of the dressing to obtain a specific metformin release profile, rapid or delayed, depending on the needs.

Of course, the quantity of metformin used in the galenic formulation or in the dressing will be adapted according to the kinetics sought as well as the specific constraints linked to its nature, solubility, resistance to heat, etc.

By dressing is meant, within the meaning of the present application, all types of dressings used for the treatment of wounds.

Typically, a dressing comprises at least one layer or matrix, adhesive or not.

The metformin according to the invention, or a composition containing it, can be incorporated into any element of the structure of a dressing, for example into the matrix.

Preferably, and in order to promote rapid action, metformin (or a composition containing it) can be incorporated into the layer of the dressing which comes into contact with the wound or deposited on the surface of the layer of the dressing which comes into contact with the wound.

Such deposition techniques are well known to those skilled in the art and some are for example described in patent application WO 2006/007814.

Very often, when applying these dressings, the nursing staff holds them in place with a bandage or covers them with a secondary element such as a second absorbent dressing or a compression bandage. It is therefore useful for the dressing to remain attached to the wound so that the nursing staff keeps their hands free to position these secondary elements. In general, any type of adhesive commonly used in dressings can be used for this purpose.

In order not to alter the healthy tissues or the edges of the wound, in particular during the removal of the dressing, an adhesive having the property of adhering to the skin without adhering to the wound is preferred.

By way of example of such an adhesive, mention may thus be made of adhesives based on silicone or polyurethane elastomers, such as silicone or polyurethane gels, and hydrocolloid adhesives.

Such hydrocolloid adhesives consist in particular of an elastomeric matrix based on one or more elastomers chosen from poly(styrene-olefin-styrene) block polymers in combination with one or more compounds chosen from plasticizers, such as mineral oils , tackifying resins and, if necessary, antioxidants, in which is incorporated a quantity, preferably small, of hydrocolloids (from 3 to 20% by weight) such as for example sodium carboxymethylcellulose or superabsorbent polymers such as the products marketed under the name LUQUASORB® by the company BASF.

According to a preferred embodiment, the metformin used in the context of the present invention, or a composition containing it, will be integrated into a dressing comprising a hydrocolloid adhesive, said metformin being incorporated into said adhesive preferably in an amount between 0, 5 and 20% by weight, more preferably between 2 and 10% by weight, relative to the weight of the adhesive.

The formulation of such hydrocolloid adhesives is well known to those skilled in the art and described for example in patent applications FR 2 783 412, FR 2 392 076 and FR 2 495 473.

The use of a net of adhesive on the nonwoven makes it possible in a particularly advantageous way to reduce or avoid the risk that small fibrils of the textile material come into contact with the wound and cling to the tissues, in thus causing a painful sensation on removal, or even an obstacle to the healing process of the wound.

According to a preferred embodiment variant of the present invention, the metformin according to the invention is incorporated into such an adhesive at a concentration compatible with its solubility and its resistance to heat.

On the basis of these criteria, the metformin according to the invention is preferably used in an amount of between 0.5 and 20% by weight, and more preferably between 2 and 10% by weight, relative to the total weight of the 'adhesive.

If one wishes to increase the absorption of this non-woven dressing, it can be associated with an additional absorbent layer, and preferably an absorbent layer which does not gel, such as in particular a compress such as that used in the product URGOTUL ® Duo or URGOTUL® Trio, an absorbent hydrophilic foam, preferably a hydrophilic polyurethane foam having an absorption capacity greater than that of the nonwoven such as that used in the CELLOSORB® product.

According to a preferred embodiment, the metformin according to the invention is incorporated into a nonwoven dressing, associated with an additional absorbent layer, and preferably an absorbent layer which does not gel, such as in particular a compress.

According to another preferred embodiment, the metformin according to the invention is incorporated into a nonwoven dressing, associated with an additional absorbent layer, and preferably an absorbent layer which does not gel, such as in particular an absorbent hydrophilic foam, preferably a hydrophilic polyurethane foam with an absorption capacity greater than that of the nonwoven.

The nonwoven and the foam can be combined by techniques well known to those skilled in the art, for example by hot calendering using a thermofusible powder based on TPU/polycaprolactone polymers.

This technique is commonly used for bonding together nonwovens intended for the medical market.

Finally, this foam or the nonwoven (when the latter is used alone) can be covered with a support to protect the wound from the outside.

This support can be larger in size than that of the other layers and made adhesive continuously or discontinuously on its side coming into contact with the wound in order to optimize the maintenance of the dressing during its use, in particular if the wound is located on uneven body areas.

This support and its adhesive are preferably impermeable to fluids but highly permeable to water vapor in order to allow optimal management of the exudates absorbed by the dressing and to avoid problems of maceration.

Such supports are well known to those skilled in the art and consist for example of breathable and impermeable films such as polyurethane films, foam/film or nonwoven/film complexes.

Additives

In addition to the active agents, the metformin according to the invention may be used in combination with one (or more) additives commonly used in the preparation of dressings. These additives can in particular be chosen from perfumes, preservatives, vitamins, glycerin, citric acid, etc.

The activity of metformin according to the invention has been demonstrated in the following non-limiting examples.

Examples

Evidence of the effect of metformin on inflammation in the skin

The choice of using normal human dermal fibroblasts (NHDF) derived from the foreskin cultured in 2D is a well-suited model to demonstrate the direct activity of metformin on the dermis when exposed to AGEs. The use of a less complex cellular model makes it possible to more specifically characterize the effects of metformin on the dermal component, by limiting possible interference with other cellular compartments.

Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco/Life Technologies, 31885) supplemented with 10% fetal bovine serum (FBS, Gibco/Life Technologies, 10270) and antibiotics (penicillin/streptomycin, Gibco / Life Technologies, 15140). Cells were maintained in a humidified incubator at 37°C with a 5% _CO2 atmosphere during the treatment period.

After one day, NHDFs were cultured in serum-depleted medium (1% fetal bovine serum (FBS)). Indeed, FBS being an extremely complex and indefinite mixture of plasma proteins, growth factors, hormones and inhibitors, some components of FBS could negatively affect the response of NHDF cells to the treatment of interest. In addition, the use of a lower serum concentration allows for better control of the amount of glycated products, since serum proteins are frequent targets of modifications by reactive carbonyl compounds such as glyoxal (Nowotny et al. ., 2015).

Glycation inducer
In order to induce cell glycation, glyoxal (0.6 mM), a highly reactive precursor of AGEs, was applied to cultured NHDF fibroblasts. Aminoguanidine (AMG 10 mM), known as an anti-glycation agent (Thornalley, 2003; Thornalley et al., 2000), was used as a reference molecule to prevent treated NHDF fibroblasts from producing AGEs. AMG was added to the culture medium and its efficacy on glyoxal-induced glycation was evaluated in parallel with metformin.

Metformin was applied at 3 concentrations in the culture medium supplemented with 1% FBS. After the first 24 hours of pretreatment, metformin was applied at the same 3 concentrations in combination with glyoxal (0.6 mM) for the next 72 hours. The total incubation time was 96 h with metformin, including 72 h under glyoxal.

Untreated cells were used as a control, illustrating the basal level of the different cellular events studied. Cells treated with glyoxal, without metformin or AMG, were used as reference conditions for induction of cellular glycation. Each treatment was compared to the respective control.

At the end of the treatment, the morphology and the cell proliferation were evaluated as well as the intracellular content of AGE, the expression of the gene coding for the RAGE receptor as well as the protein quantity of the receptor were measured, and finally the secretion of cytokine proinflammatory IL-8 was quantified.

Example 1: Evaluation of the effect of metformin on AGE synthesis by cultured fibroblasts under glycation conditions

In order to study and further understand the potential of metformin in protecting NHDF fibroblasts from glyoxal-induced stress, intracellular AGE content was quantified by ELISA assay after application of metformin before and during treatment with glyoxal on NHDF cultures.

There represents the quantification of the content of AGEs in protein extracts collected from NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of glyoxal at 0.6mM. Aminoguanidine (10mM) was used as an anti-glycation reference compound. The means of the data obtained from 3 cultures for each condition (n=3) ± standard deviation (SD) are related to the control condition treated with glyoxal arbitrarily set at 100%. Statistical analysis was performed using Student's t-test (*0.01<P<0.05;**0.001<P<0.01;***P<0.001, with p values in italics) for comparison of all conditions to the glyoxal control condition.

Treatment with glyoxal for 72 hours significantly increased the production of AGEs compared to the untreated control, reflecting the property of this dicarbonyl compound to stimulate the glycation reaction of proteins.

Addition of AMG significantly inhibited glyoxal-mediated AGE production, supporting its ability to scavenge dicarbonyl and thereby inhibit AGE formation.

Cellular pretreatment with metformin followed by its application in the presence of glyoxal induced a decrease in the production of AGEs compared to the glyoxal condition alone.

This reduction was significant when NHDF fibroblasts were treated with metformin at all 3 concentrations.

2. Evaluation of the effect of metformin on the expression of RAGE

As previously reported, recognition of the ligand by the cell surface RAGE receptor (full-length (fl)-RAGE receptor) leads to its activation, which in turn activates downstream signaling cascades that drive various responses contributing to the cell dysfunction and inflammatory disorders as well as increased expression of the gene encoding the receptor itself through a positive autoregulatory loop.

The effects of metformin on RAGE gene expression as well as RAGE protein abundance were analyzed.

Cells were treated with glyoxal used at 0.5 (protein abundance) or 0.6 mM (gene expression) for 72h to induce upregulation of RAGE expression, reflecting axis activation AGEs-RAGE leading to cell damage and inflammatory state related to glycation (Kierdorf and Fritz, 2013; Serban et al., 2015, 2016; Xie et al., 2013). As mentioned previously, NHDF fibroblasts were treated with AMG as a positive anti-glycation control or with metformin alone for the first 24 hours, then in combination with glyoxal for the next 72 hours.

2.1. RAGE gene expression analysis

NHDF fibroblasts were seeded 24 h before adding metformin at 3 concentrations in the culture medium (DMEM) with 1% FBS. After the first 24 hours, the addition of metformin was combined with 0.6 mM glyoxal for the next 72 hours. The aminoguanidine used as the reference anti-glycation compound was applied in parallel with metformin. After 96 hours of treatment with the molecules of interest, the total RNAs were extracted, quantified, and validated for their quality/integrity before being processed by RT-qPCR. The means of the data obtained from 3 cultures for each condition ± the standard deviations (SD) are related to the untreated control arbitrarily set at 100%.

Data are normalized against the reference gene GAPDH and analyzed using the quantitative ΔΔ cycle threshold method. Statistical analysis was performed using Student's t-test for comparison of untreated control to each condition. The values P * 0.01 < P < 0.05; ** 0.001<P<0.01; *** P < 0.001, are considered significant, very significant and very highly significant respectively. P values >0.05 are considered insignificant (ns); P values are shown in italics.

The results are presented in the

There represents quantitation of fl RAGE mRNA levels. The NHDF fibroblasts were seeded 24 hours before the addition of metformin at 3 concentrations in the culture medium (DMEM) with 1% FBS.

Glyoxal treatment slightly, but not significantly, increased fl-RAGE expression.

The presence of metformin in the culture medium appeared to decrease in a dose-dependent manner the level of gene expression of fl-RAGE with a significant reduction (ranging from 5 to 7 times lower) observed in cells cultured with metformin at 1mg/ml. In the presence of metformin at 1 mg/ml, the expression of fl-RAGE was detected at a lower level than that recorded under basal conditions, suggesting the inhibition or significant disruption by metformin of the cellular mechanisms involved in the AGEs axis. -RAGE.

These data therefore suggest that metformin applied at its highest dose for 96 h could alter the cellular mechanisms involved in the regulation of the expression of RAGE (trapping of AGEs, inhibition of signaling pathways, epigenetic changes, modulation of the machinery of translation, etc.).

2.2. RAGE Protein Abundance Analysis

Quantification of RAGE protein abundance in NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of 0.5 mM glyoxal a been carried out. Aminoguanidine (10 mM) was used as an anti-glycation reference compound. .

At the end of the treatment, the cells were fixed in order to carry out an immunolabeling of the RAGE protein. The cells were then visualized by conventional fluorescence microscopy.

RAGE abundance was then quantified based on 9 representative images per condition.

There represents the quantification of the protein abundance of RAGE in the NHDF fibroblasts treated with metformin (0.1; 0.3 and 1 mg/l) alone during the first 24 hours, followed by 72 hours in the presence of glyoxal at 0.5 mM .

The results of the are expressed relative to the untreated control (means +/- standard deviations from 3 cultures) arbitrarily set at 100%.

A t-student statistical analysis was performed to compare the glyoxal control to each condition (“untreated, metformin, or aminoguanidine”).

P values between 0.01 and 0.05, and those <0.001 (***) are considered significant and very highly significant, respectively (p values are shown in italics).

P values between 0.01 and 0.05, and those <0.001 (***) are considered significant and very highly significant, respectively (p values are shown in italics).

Thus, treatment with glyoxal for 72 h significantly increased the protein abundance of RAGE, compared to the untreated control. The presence of metformin significantly reversed this glyoxal-induced RAGE accumulation with a greater effect when applied at its two highest doses.

A similar effect was observed after exposure of cells to AMG.

3. Analysis of the effect of metformin on IL8 secretion

AGEs are involved in the maturation and release of cytokines, triggering a local inflammatory response.

Among the cellular processes studied for their sensitivity to glycation, IL-8 has been shown to be strongly induced in response to glycation-induced stress (Rasheed et al., 2011; Serban et al., 2015; Serban et par).

The quantification of the extracellular quantities of IL-8 represented in was performed by ELISA assay in culture supernatants after treatment of NHDF fibroblasts with metformin (0.1, 0.3 and 1 mg/ml) alone for the first 24 hours, followed by 72 hours in the presence of 0.6 mM of glyoxal.

Aminoguanidine (10 mM) was used as an anti-glycation standard.

The means of the data obtained from the 3 cultures for each condition (n=3) ± the standard deviations (SD) are related to the untreated control condition arbitrarily set at 100%.

Statistical analysis was performed using Student's t-test (*0.01<P<0.05; **0.001<P<0.01; ***P<0.001, with p values in italics) for the comparison of all conditions to the glyoxal control.

After 3 days of culture in the presence of glyoxal, metformin seems to induce protection against the release of IL-8 induced by AGEs (in particular at a concentration of 1 mg/mL).

Thus, metformin, particularly when used at 1 mg/ml, reduces the inflammatory response of NHDF fibroblasts induced by glyoxal, thus demonstrating the protective effect against the pro-inflammatory response induced by glycation stress.

Claims (9)

Metformin, its salts and its complexes for topical use for the reduction of inflammation of the skin, in particular, inflammation related to abnormally high glycation. Metformin, its salts and its complexes according to claim 1, for its use in decreasing the glycation of skin proteins. Metformin, its salts and its complexes according to Claim 2, characterized in that the skin proteins are the proteins of the dermis. Metformin, its salts and its complexes according to claim 1, for its use in reducing the load of AGEs. Metformin, its salts and its complexes according to any one of the preceding claims, for its use in inhibiting the expression of the gene encoding the RAGE receptor and the protein abundance of the RAGE receptor, thereby down-regulating the IL8 secretion. Metformin, its salts and its complexes according to any one of the preceding claims, for its use in the activation and improvement of the reconstruction of the skin. Metformin, its salts and its complexes according to any one of the preceding claims, for its use in the activation and improvement of the reconstruction of the skin, in particular of the dermis. Metformin, its salts and its complexes according to any one of the preceding claims, characterized in that it is in the form of a composition such as a gel, a solution, an emulsion, a cream, granules, or a medical device such as a dressing. Metformin, its salts and its complexes according to any one of the preceding claims, for its use in accelerating the healing of a chronic wound, in particular a diabetic foot ulcer.