EP3607049A1

EP3607049A1 - Bacterial secretome for use in the treatment of skin lesions

Info

Publication number: EP3607049A1
Application number: EP18716267.2A
Authority: EP
Inventors: Nathalie Castex-Rizzi; Marie Florence GALLIANO; Hélène HERNANDEZ-PIGEON
Original assignee: Pierre Fabre Dermo Cosmetique SA
Current assignee: Pierre Fabre Dermo Cosmetique SA
Priority date: 2017-04-06
Filing date: 2018-04-06
Publication date: 2020-02-12
Also published as: SG11201909144VA; RU2019134658A3; JP2020513007A; FR3065009B1; JP7334119B2; US11484555B2; US20200030387A1; CA3059380A1; AU2018248899A1; AU2018248899B2; MX2019011951A; FR3065009A1; RU2019134658A; WO2018185324A1; TW201841647A; BR112019020954A2; KR20190138640A; KR102585934B1; CN111630152A; CN111630152B

Abstract

The present invention relates to a new use of a bacterial secretome in the treatment of skin lesions and more particularly in wound healing. The invention also relates to cosmetic or dermatological compositions comprising such a bacterial secretome as active agent.

Description

Bacterial secretonoma for use in the treatment of skin lesions

FIELD OF THE INVENTION

The present invention relates to a new use of a bacterial secretome in the field of the treatment of cutaneous lesions and more particularly of cicatrization. The invention also relates to cosmetic or dermatological compositions comprising such a bacterial secretome as active agent.

PRIOR ART

Wound healing is a complex and dynamic biological process that involves the interaction of many local and systemic factors in normal tissue repair. There are three interdependent phases to healing progression: hemostasis and inflammation, proliferation and remodeling (Witte MB, Barbul A. Clin Clin North Am., 1997 Jun; 77 (3): 509). -28.). Proliferation involves three observable processes: granulation, contraction and re-epithelialization.

During granulation, there is proliferation and migration to the wound bed of the cells that will be involved in the rest of the repair process. Thus, macrophages, fibroblasts and endothelial cells are found. Macrophages are constantly releasing chemotactic factors and growth factors. The fibroblasts construct the new cell matrix necessary for the growth of cells at the bottom of the wound. This scaffold promotes cell migration. Finally, the endothelial cells trigger the formation of vascular buds that will form new capillaries, which will restore the perfusion and ensure the supply of oxygen and nutrients essential to the metabolic activity of cells in the wound.

The contraction of the wound is a mechanism for reducing the size of the wound and the fibroblasts play a leading role in this contraction. Reepithelialization is the regeneration of an epidemic that covers a wound to reform an effective barrier against the external environment, capable of pigmentation and regain its sensory and immune functions. It therefore involves the cellular processes of migration and proliferation of keratinocytes, but also the differentiation of this neoepithelium and the restoration of a basement membrane that reconnects the dermis and the epidermis. When the migration of basal cells towards the wound center allows both edges of the wound to meet, a wave of cellular mitosis occurs to fill the spaces left by the migration and provide cells for epithelial tissue in three-dimensional regeneration. .

The proliferation steps of keratinocyte cells, fibroblasts or endothelial cells can be considered as one of the functional phenomena testifying to the healing activity of an active ingredient. An increase in the proliferation of fibroblasts would participate in the healing of a deep wound (damage to the dermis), whereas the increase in proliferation and / or migration of keratinocytes would participate in reepithelialization.

Healing also causes cosmetic problems from the moment when defects in healing or poor healing may be responsible for unsightly and almost permanent visible marks such as thick scars or keloids.

All surgical procedures, aesthetic medicine, dermatology as well as non-medical (scrapes, cuts, scrapes, burns) or pathological traumatic lesions cause scarring. One of the main skills of plastic surgeons is to control the formation of scars.

For example, pressure has been used for scars caused by burns for many years and has more recently been combined with silicone sheets placed directly on the scar.

There is still a need to provide new compositions to promote the healing of injured skin as well as to improve the aesthetic appearance of scars.

For the first time, and surprisingly, the Applicant has demonstrated the beneficial properties in the tissue regeneration and healing of cutaneous lesions of a bacterial secretome derived from a bacterial strain (or bacteria) LMB64 isolated from a water table.

This bacterium has been described by the Applicant in the patent application WO2012 / 085182. More particularly, the bacterium was described as such for its anti-inflammatory activities. Even more particularly, the extracts designated S0, E0 and ESO, respectively consisting of the culture supernatant separated from the biomass, the biomass of lysed cells, and the supernatant after incubation of the basic pH culture for several hours were described and exemplified. . Only the pharmacological activities of the EO and ESO extracts were tested and it was shown that these EO and ESO extracts had the ability to induce cytokines and Langerhans cell maturation (for EO) as well as TLR2 / TLR4 receptor activation. / TLR5, PARs receptor antagonists and induction of antimicrobial peptides (for ESO). These results allowed to consider the use of such extracts in the treatment of inflammatory diseases such as pruritus, psoriasis, eczema or atopic dermatitis.

Against all expectations, the Applicant demonstrates here that a secretome consisting not of the same elements as the extracts ESO and EO mentioned above, but mainly constituted bio molecules secreted and extemalised in the culture supernatant by said bacterium LMB64, presents surprising healing properties.

DETAILED DESCRIPTION According to a first embodiment (1), the subject of the present invention is a bacterial secretome of a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, comprising a 16S rRNA comprising the sequence SEQ ID No. 1, or any sequence having at least 80% identity with the sequence SEQ ID No. 1, for its use in the treatment of cutaneous lesions,

said secretome being obtainable or obtained by a process comprising the steps of: a) culturing said bacterium in a culture medium and conditions adapted to its growth,

b) liquid / solid separation and recovery of the liquid phase,

c) obtaining said secretome comprising the bio molecules secreted by said bacterium in the liquid phase.

According to a second embodiment of the present invention, the bacterial secretome for its use in the treatment of cutaneous lesions according to the invention is characterized in that a basic buffer is added to the liquid phase obtained in step b) and the buffered liquid phase obtained is optionally left in contact for 1 to 7 hours, preferably at a temperature of between 1 and 6 ° C.

The bacterium LMB64 has been characterized and defined as belonging to the class Beta-proteobacteria, subfamily of Neisseriaceae, and probably of a new genus not yet defined. Analysis of the 16S ribosomal RNA (rRNA) gene sequence allowed us to locate this bacterium close to the genera Chromobacterium, Paludimonas, Lutelia and Glubenkiana, with which it shares 95% sequence similarity.

This non-pathogenic bacterium is a Gram-negative bacterium that has been isolated from the water table.

More particularly, the bacterium LMB64 is in the form of a rod with a length around 2.3μιη (+/- 0.3) and a width around Ι, Ομιη (+/- 0, 1). A peculiarity of this bacterium is the presence of a polar flagellum.

The gene coding for the 16S rRNA was almost completely sequenced (1487 bp, corresponding to the sequence SEQ ID No. 1). The bacterium LMB64 has a circular plasmid of 10948 bp. This plasmid was completely sequenced and the sequence is represented in the sequence SEQ ID No. 2.

According to another embodiment, a bacterium from which the secretome according to the invention is derived comprises at least one plasmid comprising the sequence SEQ ID No. 2, or any sequence having at least 80% identity with the sequence SEQ ID No. 2, advantageously at least 85%, at least 90%, at least 95%, at least 97% and even more preferably at least 98% identity with the sequence SEQ ID No. 2. Also, a bacterium from which the secretome is derived is a non-pathogenic gram-negative bacterium belonging to the class Betaproteobacteria, subfamily of Neisseriaceae, said bacterium comprising a 16S rRNA comprising the sequence SEQ ID No. 1, or any sequence presenting at least 80%, at least 90%>, at least 95% o, at least 97% identity with the sequence SEQ ID No. 1 and said bacterium comprising at least one plasmid comprising the sequence SEQ ID No. 2, or any sequence having at least 80% identity with SEQ ID No. 2, advantageously at least 85%, at least 90%, at least 95%, at least 97% and more preferably at least 98% identity with the sequence SEQ ID No. 2.

By way of example, such a bacterium is represented by the LMB64 strain which has been deposited in the name of the applicant at the National Collection of Microorganism Cultures (CNCM), Institut Pasteur, Paris, on April 8, 2010 under the reference I-4290. .

Having this genotypic information, associated with the growth characteristics in a non-sulphurized medium, of the non-filamentous nature of this bacterium, a person skilled in the art would not have difficulty in finding / identifying another bacterium making it possible to obtain a secretome according to the invention. . Such an identification of another bacterium which is admittedly slightly different genotypically but which brings together the phenotypic criteria of the invention as regards the secretome can be achieved after a selection work which is by no means insurmountable and on the basis of the information contained in the invention. present application as well as those contained in the application WO2012 / 085182 associated with the general knowledge of the skilled person. By "percent identity" between two nucleic acid sequences in the sense of the present invention, is meant a percentage of identical nucleotides between the two sequences to be compared, obtained after the best alignment (optimal alignment), this percentage being purely statistics and the differences between the two sequences being randomly distributed over their entire length. The sequence comparisons between two nucleic acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being possible by segment or by "comparison window". The optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).

The percentage identity between two nucleic acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid sequence to be compared may comprise additions or deletions with respect to the reference sequence for alignment. optimal between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

For example, one may use the BLAST program, "BLAST 2 sequences" (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett., 174: 247-250) available on the site. http://www.ncbi.nlm.nih.gov/gorf/bl2.html, The parameters used are those given by default (in particular for the parameters "open gap penalty": 5, and "extension gap penalty": 2 the matrix chosen is for example the matrix "BLOSUM 62" proposed by the program). The percentage of identity between the two sequences to be compared is calculated directly by the program. It is also possible to use other programs such as software "ALIGN" or "Megalign" (DNASTAR).

A bacterium according to the invention comprises the bacterium LMB64 which comprises at least one plasmid comprising the sequence SEQ ID No. 2, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity. with said sequence SEQ ID No. 2. Other features of said LMB64 bacterium will be detailed later in the examples. In addition, the bacterium LMB64 was deposited in the name of the plaintiff at the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, Paris, April 8, 2010 under the reference 1-4290. The present invention also relates to another embodiment of a bacterial secretome for its use in the treatment of cutaneous lesions according to the second embodiment, characterized in that the basic buffer is selected from Tris buffer, Arginine buffer and mixtures thereof. In another embodiment, the invention relates to a bacterial secretome for its use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that step b) of liquid / solid separation is followed by a step b ') of clarifying the liquid phase or the liquid phase buffered by filtration before step c).

In another embodiment, the invention relates to a bacterial secretome for its use in the treatment of cutaneous lesions according to the preceding embodiment, characterized in that the filtration is carried out with a threshold of 0.2 μιη. According to another embodiment, the invention also relates to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that said bio molecules consist of peptides, proteins and proteins. secondary metabolites secreted by said bacterium. It is also another embodiment of the present invention to provide a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that the bacterium comprises at least one plasmid comprising the sequence SEQ ID No. 2, or any sequence having at least 80% identity with the sequence SEQ ID No. 2.

According to another embodiment, the invention also relates to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the methods of previous embodiment, characterized in that the bacterium is the bacterium deposited at the CNCM April 8, 2010 under the reference 1-4920.

In general, the term "secretome" is used to describe all the secreted bio-molecules excreted in the culture medium and solubilized in this medium, by a cell, a tissue or an organism. In the present case, it is necessary to understand by secretome all the secreted and outsourced bio-molecules by the bacterium LMB64, without modification, alteration or lysis of the bacterial cell. More particularly, these biomolecules consist mainly of proteins, peptides and secondary metabolites secreted and externalized by the bacterium in the culture medium.

The bacteria multiply by binary fission, that is to say that each bacterium grows and then divides into two daughter cells separated by a septum of division formed by the cell wall. During division, the DNA is duplicated as are the other constituents. Various enzymatic systems of synthesis and degradation participate in cell division.

Bacterial growth is the orderly growth of all components of the bacteria. It results in an increase in the number of bacteria. During the growth, there is, on the one hand, a depletion of the nutrient culture medium and, on the other hand, an enrichment in biomolecules secreted and excreted in the culture medium by the bacterium and solubilized in this medium. as well as by-products of metabolism.

By the expression "culture medium" or "medium" is meant any medium comprising at least the nutrients necessary for the growth and multiplication of bacteria. The bacteria can be cultured in a liquid, solid or semi-liquid medium. Preferably, the culture medium is a liquid medium for recovering the culture supernatant containing the secretome.

An adequate culture medium contains nutrients that promote the growth and multiplication of bacteria. In general, a suitable culture medium may include water, a carbon source, a nitrogen source and salts. By way of illustration, a particular example of a culture medium is described below in the examples. By "secondary metabolites", it is necessary to understand the small molecules that a bacterium according to the invention, in particular the bacterium LMB64, produced, secreted and excreted in the culture medium. By way of illustration, such secondary metabolites may be soluble molecules such as bacteriocins, non-ribosomal peptides, siderophores, lipopeptides or polyketides or molecules such as terpenes, pyrazines, indoles or sulphide derivatives. . The proteins present in the secretome have sizes of between 10 Kd and 100 Kd, preferably between 18 Kd and 98 Kd, and preferably a majority protein whose size is approximately 38 kDa.

In practice, the secretome of a bacterium according to the invention, for example and in particular the bacterium LMB64, can be obtained from a culture in a culture medium allowing the growth, development and multiplication of the bacterium ( as for example that described below) by simple recovery of the culture supernatant which contains the secreted biomolecules constituting the secretome according to the invention. Concretely, because these biomolecules are secreted and externalized by the bacterium and solubilized in the medium, it is not necessary to perform a step of treating said bacteria (mechanical or chemical inducing lysis total or partial cells or protein release or components of the wall or membrane or the periplasmic space). The recovery of the components of the culture supernatant (hereinafter the secretome) of the insoluble solid cell components can be by any liquid / solid separation means. More particularly, it is therefore possible, by the techniques known to those skilled in the art, to isolate the liquid fraction containing the solubilized bio molecules from the solid fraction containing predominantly whole cells, cell debris comprising surface proteins. and / or proteins located in the periplasmic space of the bacterium.

As an illustration, the liquid / solid separation can be carried out by a technique chosen from: centrifugation, sedimentation, filtration, ultrafiltration, decantation, dewatering for example. Preferably, the liquid / solid separation is carried out by centrifugation of the bacterial culture in order to separate, on one side, the solid phase, ie the pellet, containing cells and cellular debris and, on the other hand, the liquid phase, ie the supernatant, comprising the soluble molecules, i.e. the secretome.

The supernatant, i.e. the liquid phase, can be used as it is, or after simple filtration to rid it of any cell debris present and sterilize it. It can also be envisaged to concentrate it for example by diafiltration. The proteins constituting the secretome may also be recovered by ammonium sulfate precipitation.

As indicated above, the process for preparing a bacterial secretome according to the invention is characterized in that a basic buffer is added to the liquid phase obtained in step b) and that the buffered liquid phase obtained is optionally left in contact with such a buffer for 1 to 7 hours, in particular at a low temperature, preferably at a temperature of between 1 and 6 ° C.

The addition of a basic buffer to the supernatant makes it possible to improve the solubility and / or the stability of the proteins and peptides in solution.

This basic buffer is preferably a Tris buffer or an arginine buffer or a Tris-arginine mixture. Preferably it is a Tris-arginine buffer.

The amount of basic buffer, preferably Tris-arginine added to the liquid phase is such that the buffered liquid phase obtained has an arginine concentration of between 100 mM and 500 mM and / or a tris concentration of between 10 mM and 90 mM.

The pH of the buffered liquid phase may be between 8 and 12, and preferably between 9 and 11.

The use of a basic buffer (Tris, Arginine or Tris-arginine) makes it possible to stabilize the proteins in solution and to avoid their aggregation over a long period of storage.

A preferred culture mode of the bacterium according to the invention, in particular the LMB64 strain, may comprise three steps. • A first inoculum can be prepared in Erlenmeyer flask in a suitable medium containing mineral substances, protein and carbohydrates suitable for this preculture.

• A second pre-fermentation culture in batch mode in a second medium near or identical to the first medium (or any equivalent medium) is then performed.

• Finally, a fermentation in fed-batch mode in an identical medium or close to the second medium in order to obtain a high cell density. A centrifugation step then allows the removal of cell and cell debris and recovery of the culture supernatant.

This buffered supernatant, or buffered liquid phase, can then be clarified by filtration.

As indicated above, the liquid / solid separation step b) is followed by a step b ') of clarifying the liquid phase or the buffered liquid phase, for example by filtration, before step c). The filtration may be carried out by any suitable means for clarification of the liquid phase, or buffered liquid phase where appropriate. Such clarification by filtration allows the removal of suspended particles that would not have been removed in the liquid / solid separation step and aims to obtain a clear secretome.

The filtration can be carried out by any means of filtration, ultrafiltration or diafiltration.

Advantageously, the filtration is carried out by filtration on a filter or filter cartridge having a threshold of 0.4 μιη, preferably 0.2 μιη. In this case, the bacterial secretome is characterized in that the peptides, proteins and secreted secondary metabolites have a size less than or equal to 0.2μιη. As a preference, electrostatically charged filters or prefilters can be used to avoid any absorption of the biomolecules responsible for all or part of the activity.

The exact nature of the media as well as the different steps will be described in more detail in the examples. It is to be understood that any modification of the process, the media or the sequence of steps which appears obvious to a person skilled in the art with regard to the present description should be considered as falling within the scope of the present patent application. As mentioned above, the invention also relates, in another embodiment, to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that the cutaneous lesions are chosen from the group including abrasions, burns, sunburns, scratches, cuts, scrapes, sutures.

The invention also relates to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments for its use for the attenuation of healing defects. The invention also relates, in another embodiment, to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that the cutaneous lesions are post-traumatic lesions, post -surgical, posterior to an act of dermatology, posterior to an act of aesthetic medicine.

Advantageously, the invention also relates to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the preceding embodiments, characterized in that it is associated with a copper salt or a zinc salt. In another embodiment, the invention also relates to a bacterial secretome for use in the treatment of cutaneous lesions according to one of the methods of previous embodiment, characterized in that the treatment of skin lesions is the improvement of healing and skin repair.

In another embodiment, the invention also provides a method of cosmetic treatment for attenuating skin healing defects, comprising the application of an effective amount of a composition comprising an effective amount of a bacterial secretome as defined in one of the preceding embodiments in combination with a cosmetically suitable excipient and formulated in a form suitable for topical application.

Advantageously, the invention also relates to a bacterial secretome for use in the treatment of cicatricial defects according to one of the preceding embodiments, characterized in that it is associated with a copper salt and / or a salt. of Zinc. Thus, the invention also aims in another embodiment, the use of a bacterial secretome as defined in one of the preceding embodiments for the cosmetic treatment of attenuation of skin healing defects. According to a particular embodiment, the secretome according to the invention is used in combination with a copper salt and / or a zinc salt.

In another embodiment, the invention is directed to a dermatological composition comprising a bacterial secretome as defined in one of embodiments 1 to 9 and a dermatological excipient suitable for topical application. In an advantageous embodiment, the dermatological composition further comprises a zinc salt and / or a copper salt.

In another embodiment, the invention also provides a dermatological composition according to one of the preceding embodiments for its use in the treatment of cutaneous lesions. In an advantageous embodiment, the dermatological composition further comprises a zinc salt and / or a copper salt.

In another embodiment, the invention provides a cosmetic composition comprising a bacterial secretome as defined in one of the embodiments of the invention. and a cosmetic excipient suitable for topical application. The cosmetic composition may further comprise a zinc salt and / or a copper salt.

Finally, in another embodiment, the present invention provides a use of a cosmetic composition according to a previous embodiment for the cosmetic treatment of attenuation of cutaneous healing defects.

According to one embodiment, the subject of the invention is a bacterial secretome as defined in one of the preceding embodiments, for a topical use intended to promote the proliferation of fibroblasts.

According to one embodiment, the subject of the invention is a bacterial secretome as defined in one of the preceding embodiments, for a topical use intended to promote proliferation and / or migration of keratinocytes.

According to one embodiment, the subject of the invention is a bacterial secretome as defined in one of the preceding embodiments, for a topical use intended to treat cutaneous lesions.

According to one embodiment, the subject of the invention is a bacterial secretome as defined in one of the preceding embodiments, for a topical use intended to improve cutaneous cicatrization and recovery.

Another object of the invention relates to a composition for accelerating cutaneous repair in order to restore the integrity and the quality of the skin comprising as active principle the secretome defined in one of the preceding embodiments. Still advantageously, the composition may further comprise a copper salt and / or a zinc salt.

Preferably, the concentration of the secretome in a composition according to the invention is between 0.05% and 10%. More preferably, it is between 0.1% and 5%.

The composition according to the invention further comprises one or more dermatologically and / or cosmetologically compatible excipients known to those skilled in the art in order to obtain a composition for topical application, such as emulsifiers, thickeners, gelling agents, water fixatives, leveling agents, stabilizers, colorants, perfumes and preservatives.

In the present invention, the term "dermatologically or cosmetically acceptable" is intended to mean that which is useful in the preparation of a dermatological or cosmetic composition, which is generally safe, non-toxic and neither bio-logical nor otherwise undesirable and which is acceptable for dermatological or cosmetic use, and in particular dermatological or dermo-cosmetic use, especially by topical application.

The compositions according to the invention are advantageously intended for topical application, in particular on the skin.

The composition according to the invention may be prepared in the form of an emulsion, a dispersion or an aqueous or oily lotion.

In another particular embodiment, the dermatological or cosmetic composition according to the invention comprises at least one other active ingredient, more particularly at least one hydrating active ingredient.

This other active ingredient can thus be selected especially in the group comprising:

• Well-known healing or repair agents: panthenol, madecassoside, allantoin, aloe vera, honey;

• Anti-infectious agents: trace elements like copper-zinc, honey

• Moisturizing and nourishing agents: glycerine, shea butter, natural waxes, hyaluronic acid, fatty acids

• Soothing agents: allantoin, bisabolol, panthenol, enoxolone

• UV filters.

Preferably, the second active ingredient will be a trace element copper-zinc type. In this preferred embodiment, the composition of the invention will therefore comprise the secretome and copper-zinc type microelement.

As mentioned, a dermatological or cosmetic composition according to the invention comprising the secretome may comprise a copper salt or a salt of zinc. Preferably the copper salt will be copper sulfate. Advantageously, the zinc salt will be chosen from zinc oxide and zinc sulphate.

The amount of copper sulfate in a composition according to the invention may vary between 0.05 and 0.5% by weight of the composition, preferably between 0.1 and 0.25% by weight of the composition, more preferably still about 0.2% by weight. of the composition.

The amount of zinc sulphate in a composition according to the invention may vary between 0.05 and 0.5% by weight of the composition, preferably between 0.1 and 0.4% by weight of the composition, more particularly between 0.1 and 0.2% by weight. weight of the composition.

The amount of zinc oxide in a composition according to the invention may vary between 0.5 and 10%, by weight of the composition, preferably between 1 and 5%, by weight of the composition, more particularly about 2 to 4% by weight. of the composition.

According to one embodiment of the invention, it is envisaged a composition according to one of the preceding embodiments characterized in that it further comprises at least one other active ingredient chosen from the cicatrisants, the anti-infectives, moisturizing agents, soothing agents and mixtures thereof. According to another particular embodiment, at least one other active ingredient will be chosen from hydrating agents, healing agents, soothing agents and mixtures thereof, preferably moisturizing agents. Such an active ingredient will preferably be used in dermatological compositions.

The present invention also relates to a method for treating and healing skin lesions comprising administering, preferably topically, an effective amount of a composition comprising as active ingredient a secretome according to the invention.

The compositions according to the invention are intended in particular for the care of skin having undergone lesions.

Skin lesions can occur as a result of trauma or as a result of medical or surgical procedures, invasive or non-invasive, curative or aesthetic. As invasive medical or surgical procedures may be mentioned: surgical procedures (exeresis, shavings) with or without suture, cryotherapy, ablative laser, medium or strong peels, suture, mesotherapy or curettage for example.

As post-traumatic skin lesions, for example following a slight external damage of the skin, there may be mentioned cuts, scratches, scratches and superficial burns or sunburn for example.

As a non-invasive medical procedure requiring a superficial healing product that accelerates skin recovery, usable in the long run (until complete repair of the skin) include light peels, laser hair removal, superficial vascular treatments (rosacea), for example.

The treatment of lesions of the skin and mucous membranes according to the invention may in particular include the treatment of cuts, sutures, abrasions, scratches, scratches, scars post-surgery or post-act of cosmetic dermatology, superficial burns , sunburns.

The present invention further relates to the use of a cosmetic composition according to the invention for the improvement of scarring and skin repair and in particular for the attenuation of healing defects.

The invention will be better understood on reading the examples below illustrate it without limiting its scope.

The examples refer to the following figures

FIGURE 1: SDS-PAGE gel of secreto proteins.

Lane 1 (M, standard molecular weight markers, Sea Blue).

Lanes 2 to 5: (secret secret before addition of Arginine buffer) - deposits of 10 μΐ, 15 μΐ, 20 μΐ and 30 μΐ, respectively.

Lanes 6 to 9: (Sec + Arg, secretome after addition of Arginine buffer) - deposits of 10 μΐ, 15 μΐ, 20 μΐ and 30 μΐ, respectively. FIG. 2:% Proliferation stimulation (mean ± sem) of normal human fibroblasts treated with the different compared to the control cells (n = 3 pools of donors). FIG. 3:% stimulation (mean ± sem) of the migration of the HaCaT keratinocyte line treated with the various compounds as compared to the control cells (n = 2).

FIGURE 4: Reepithelialization (mean ± wk) of the wound after application for 48 h on porcine earplants of the different products compared to a reference repair cream (n = 10 independent donors).

Example 1 Culture of the LMB64 bacterium

By way of nonlimiting example, preferred culture media contain ammonium chloride, magnesium sulfate and yeast extract. Note also, as is apparent inter alia from the application WO2012 / 085182, other similar media can be used and should therefore be considered as an integral part of the present description. Any adaptation of the person skilled in the art must also be considered as part of the invention.

An example of a culture method is described below. It should be remembered here that this example has only illustrative value and should in no way be considered as limiting.

The LMB64 strain is cultured in three stages, namely a first inoculum, a preculture (or pre-fermentation) in batch mode and finally a culture but in fed-batch mode (addition of glucose). Inoculum: A tube of the WCB LMB64 is used to seed a

Erlenmeyer flask containing 1000 mL of sterile medium. The Erlenmeyer flask is then placed in the shaker incubator with agitation. When the cell density of the broth is sufficient, the culture is stopped. The cells are then cooled until transfer to the prefermenter.

Preculture: The prefermenter is then filled with approximately 16 L of medium then sterilized to full. Two satellite bottles are connected to the pre-fermenter after sterilization of the tank then add blocks:

• one vial containing a sterile 50% glucose solution. This solution (Glucose batch preculture) is transferred immediately into the culture medium to reach the initial glucose concentration of 20 g / L

• The Erlenmeyer flask containing the inoculum described above in the Inoculum stage is seeded in the prefermenter.

The preculture is started and then regulated automatically. By way of example, the following parameters may be mentioned: temperature, stirring speed, pressure, air flow, or even P0 ₂ .

The growth of the cells is followed by a measurement of the optical density at 620 nm. The preculture is stopped by cooling when it reaches a sufficient density.

Culture: The fermenter is then filled with 127 L of medium adjusted to pH 7.0 and sterilized to full. Three satellite bottles are used:

• a vial containing a sterile glucose solution. This solution is transferred immediately into the culture medium to reach the initial glucose concentration of 20 g / L.

• a bottle of antifoam. This antifoam will be added automatically during the crop to control the level of foam in the tank.

• a bottle of glucose fedbatch. This solution will be added during culture to support cell growth.

The culture is launched and then regulated automatically. By way of example, the following parameters can also be mentioned: Temperature, pH, agitation, pressure, air flow P0 ₂ .

After depletion of glucose initially present in the medium (rise of P0 ₂ ), the addition of fedbatch glucose solution is triggered and allows growth of high density cells The fermentation is stopped after total consumption of glucose. At this stage, the fermentation broth is automatically cooled. All along of the culture, cell growth is followed by a measurement of the optical density at 620 nm. The amount of dry biomass (g / L) obtained at the end of culture is determined using a weight method. Example 2 Extraction of the Secretome

The example below is given for illustrative purposes of a preferred embodiment, but should not be considered as limiting.

The secretome is generally obtained after centrifugation of the result of the culturing step to remove cells, surface proteins and proteins located in the periplasmic space of the bacterium. This centrifugation step is followed by addition of a basic Tris-Arginine buffer to the supernatant. A last step of filtration of the supernatant is also possible. Any adaptation of the person skilled in the art must also be considered as part of the invention.

Centrifugation: The transfer line from fermenter to centrifuge is sterilized. The fermentation must is then separated by continuous centrifugation on a centrifuge. The centrifugation is conducted at 150 L / h (+/- 30 L / h) with a bowl rotation speed of 10900 ± 1000 rpm. The supernatant is recovered in a container equipped with a disposable pocket. The weight of the supernatant is measured on the balance plate.

Arginine addition: The Tris Arginine extraction buffer is sterilized and transferred to the disposable bag containing the culture supernatant. The required contact time is between 1 and 7 hours. The target concentration of Tris and arginine after addition to the single-use bag is around 0.3 M L-arginine and 20 mM Tris.

The total volume after addition of the extraction buffer is about 240 L.

Filtration: Two filtration steps are performed in-line in order to clarify the supernatant and result in a clear, germ-free secretome. Piloting the filtration is achieved through the filtration / distribution system. The disposable depth filtration cartridge is placed in its filter housing. The entire filtered product is recovered sterilely in a container equipped with a disposable pocket. The filtered product bag is weighed on the weighing pan and stored at + 5 ° C until distribution.

Gel electrophoresis of secrete proteins: see Figure 1

SDS-PAGE gel (4 - 12%) Bis-Tris, under denaturing and reduced conditions, revealing to Blue de Comassie deposits of different volumes of culture supernatants (10, 15, 20, 30 μΐ respectively) before and after addition Arginine buffer. In the two cases, we observe different protein bands of size between 14 and 100 Kda, of which a major band at about 38 Kda.

EXAMPLE 3 Effect of the Secretome on the Proliferation of Normal Human Fibroblasts

The technique used is that of the incorporation of a thymidine-like nucleotide, 5-bromo-2'-deoxyuridine (BrdU), into the DNA of S-phase cells at 37 ° C. This technique makes it possible to quantify the cells whose advance in the cell cycle is characteristic of a proliferative cell (S phase or DNA synthesis).

The fibroblasts are inoculated into a 96-well plate and then incubated for 72 hours in the presence of the test compounds, the EGF as a positive control and the secretome at 0.4, 0.6 and 1%. The incorporation of BrdU is carried out during the last 24 hours. BrdU incorporation is quantified using the BrdU ELISA kit (11647229001, Roche Diagnostic).

The% of the control is calculated according to the formula:

(Value / average of control) x 100

The stimulation in% is calculated according to the formula:

Average Stimulation in% = (Mean (Value / Average Control) x 100) - 100

SEM = standard dev / ^ ⁿ Statistical analyzes are done using the Student's test. The studies were performed on 3 donor pools.

The positive control EGF stimulated strongly and significantly the proliferation of fibroblasts and reproducibly on the 3 donor pools (95% stimulation). This validates the experiments. The results show that the secretome tested at 0.4%, 0.6% and 1% significantly stimulated the proliferation of normal human fibroblasts. Table 1 below groups the results of BrdU incorporation (average ± sem) by normal human fibroblasts (by ELISA assay) after 72 hours of incubation in the presence of the compounds tested (n = 3 donor pools). The results are shown in Figure 2.

Table 1

EXAMPLE 4 Effect of the secretome on the migration of the keratinocyte HaCaT line

The studies were performed using the Oris Cell Migration Assay Cell Migration Kit (Platypus Technologies) according to the standard protocol recommended by the manufacturer. Mainly, the cells of the HaCaT line (human cell line of keratinocytes) are seeded in a 96-well plate Oris ™ stoppers are removed and the compounds to be evaluated are added to the culture medium. Incubation is continued for 24 hours to allow cell migration. A cell-permeable fluorescent tracer, calcein is then added to allow visualization of the cells in the area delimited by the stoppers.

Cell migration is assessed by imaging wells and measuring the migration area in mm ² .

The% of the control is calculated according to the formula:

(Value / average of control) x 100 The stimulation in% is calculated according to the formula:

Average Stimulation in% = (Mean (Value / Average Control) x 100) - 100

SEM = standard dev / n

Statistical analyzes are done using the Student's test.

The studies are carried out on 6 technical replicas and in n = 2 independent experiments.

The positive controls, TGF-b and fetal calf serum (FCS), stimulated strongly and significantly cell migration and reproducibly (51% and 176% stimulation, respectively). This validates the experiments. The results show that the secretome tested at 0.4%, 0.6% and 1% significantly stimulated the migration of HaCaT keratinocytes. Table 2 below groups the results of the migration of HaCaT cells after 24 h of incubation with the various compounds (n = 2). The results are illustrated in Figure 3.

Table 2

EXAMPLE 5 Effect of the Secretome on the Closure of the Wound on an Ex Vivo Model of Healing

Cutaneous explants of pork ears are kept alive. 6mm biopsies are performed at the center of which a wound is formed of 3 mm circular. The wound affects the entire epidermis as well as the upper dermis. The products to be evaluated are applied topically. Forty-eight hours later, the biopsies are frozen and haemalun-eosin staining is performed on section. The kinetics of reepithelialization is evaluated according to the following method: 1) Assigning a defined score from 0 to 3 according to the protocol developed by J. Brandner (Pollok et al., 2010, J Cell Mol Med).

Precise measurement in μηι of the extent of the reepithelialized area from the edge of the wound. A Student test is carried out for the signifcativity.

2) The morphological aspect at the edge of the wound is also evaluated and allows to account for the quality of the preservation of the tissue.

Preparations of the products to be applied: the secretome was incorporated in 0.4%, 0.6% and 1% by weight in the same formulation base as that of a repair cream, reference (cream Cicalfate®) tested in parallel . The evaluation of the reepithelialization is made by comparison with this reference cream.

The results show a dose effect with the secretome on the progression of the reepithelialization of the wound. The formulation containing the 1% secretome significantly accelerates the closure progress of the wound relative to the restorative repair cream (p = 0.043). Moreover, the morphological analysis of the tissues show an excellent tolerance of the formulations containing the secretome. The results are shown in Figure 4.

Example of composition: oil-in-water emulsion

Secretory according to Example 2: 0.1-5%

Glycerin: 1-30%

Hexylene glycol: 0.1 - 7%

Cetearyl glucoside: 0.1 - 1%

Cetearyl alcohol: 0.9 - 4%

Stearic acid: 0.5-4%

Glyceryl stearate: 0.1-4%

Vegetable oil: 0.1-10%

Water qs 100%

Claims

A bacterial secretecome of a non-pathogenic gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, comprising a 16S rRNA comprising the sequence SEQ ID No. 1, or any sequence having at least 80% identity with the sequence SEQ ID No. 1, for its use in the treatment of cutaneous lesions,

said secretome being obtained by a process comprising the steps of:

a) culturing said bacterium in a culture medium and conditions adapted to its growth,

b) liquid / solid separation and recovery of the liquid phase,

A bacterial secretome for use in the treatment of skin lesions according to claim 1, characterized in that a basic buffer is added to the liquid phase obtained in step b) and the buffered liquid phase obtained is optionally left in contact during 1 to 7 hours, preferably at a temperature of between 1 and 6 ° C.

Bacterial secretonoma for use in the treatment of cutaneous lesions according to claim 2, characterized in that the basic buffer is selected from Tris buffer, Arginine buffer and mixtures thereof.

Bacterial secrete for its use in the treatment of cutaneous lesions according to one of Claims 1 to 3, characterized in that the liquid / solid separation step b) is followed by a liquid phase clarification step b '). or the filtration buffered liquid phase before step c).

Bacterial secreted for its use in the treatment of cutaneous lesions according to claim 4, characterized in that the filtration is carried out with a threshold of 0.2 μιη.

6. bacterial secretome for use in the treatment of cutaneous lesions according to one of claims 1 to 5, characterized in that said biomolecules consist of peptides, proteins and secondary metabolites secreted by said bacterium.

7. bacterial secretome for use in the treatment of cutaneous lesions according to one of claims 1 to 6, characterized in that the bacterium comprises at least one plasmid comprising the sequence SEQ ID No. 2, or any sequence having at least 80 % identity with the sequence SEQ ID No. 2.

8. bacterial secreton for use in the treatment of cutaneous lesions according to one of claims 1 to 7, characterized in that the bacterium is the bacterium deposited at the CNCM April 8, 2010 under the reference 1-4920.

9. A bacterial secretome for use in the treatment of cutaneous lesions according to one of claims 1 to 8, characterized in that the cutaneous lesions are selected from the group consisting of abrasions, burns, sunburns, scratches, cuts, scratches, sutures.

10. A bacterial secretome for use in the treatment of cutaneous lesions according to one of claims 1 to 9, characterized in that the cutaneous lesions are post-traumatic lesions, post-surgical, posterior to a dermatological act, posterior to an act of aesthetic medicine.

11. bacterial secretome for use in the treatment of skin lesions according to one of claims 1 to 10, characterized in that the treatment of cutaneous lesions is the improvement of healing and skin repair.

12. bacterial secretome for use in the treatment of skin lesions according to one of claims 1 to 11 characterized in that it is associated with a copper salt and / or a zinc salt.

13. Dermatological composition comprising a bacterial secretome as defined in one of claims 1 to 8 and a dermatological excipient suitable for topical application, for its use in the treatment of cutaneous lesions.

14. Dermatological composition comprising a bacterial secretome as defined in one of claims 1 to 8, a copper salt and / or a zinc salt and a suitable dermatological excipient.

15. Dermatological composition according to claim 14, for its use in the treatment of cutaneous lesions.