Bacterial secretome for use in the treatment of skin lesions

Abstract

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

Claims

1. A method of treatment of skin lesions which comprises administering to a patient in need thereof a bacterial secretome of a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, wherein the bacterium is the bacterium deposited with the CNCM on 8 Apr. 2010 under number 14920, and said secretome is obtained by a process comprising the steps of: a) culturing said bacterium in a culture medium and under conditions suitable for its growth, b) liquid/solid separation of the culture medium and recovery of the liquid phase, c) adding a basic buffer to the liquid phase obtained in step b) and incubating the buffered liquid phase obtained for 1 to 7 hours, optionally at a temperature between 1 and 6° C., and d) obtaining said secretome comprising biomolecules secreted by said bacterium in the liquid phase.

2. The method according to claim 1, wherein the basic buffer is selected from Tris buffer, arginine buffer and mixtures thereof.

3. The method according to claim 1, wherein step b) of liquid/solid separation is followed by a step b′) of clarification of the liquid phase or of the buffered liquid phase by filtration before step c).

4. The method according to claim 3, wherein the filtration is performed with a 0.2 μm cut-off.

5. The method according to claim 1, wherein said biomolecules comprise peptides, proteins and secondary metabolites secreted by said bacterium.

6. The method according to claim 1, wherein the skin lesions are selected from the group consisting of abrasions, burns, sunburns, scratches, cuts, scrapes, stitches.

7. The method according to claim 1, wherein the skin lesions follow trauma, surgery, a dermatological procedure, or a cosmetic medicine procedure.

8. The method according to claim 1, wherein the treatment of skin lesions comprises improving healing and skin repair.

9. The method according to claim 1, which further comprises administering to the patient a copper salt and/or a zinc salt.

10. The method according to claim 1, wherein the bacterial secretome is contained in a dermatological composition containing the bacterial secretome and a dermatological excipient suitable for topical application, for use in the treatment of skin lesions.

11. The method according to claim 1, wherein the bacterial secretome is contained in a dermatological composition containing the bacterial secretome, a copper salt and/or a zinc salt and a suitable dermatological excipient.

12. A method of treatment of skin lesions which comprises administering to a patient in need thereof a dermatological composition comprising a bacterial secretome of a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, a copper salt and/or a zinc salt and a suitable dermatological excipient, wherein the bacterium is the bacterium deposited with the CNCM on 8 Apr. 2010 under number 14920, and said secretome is obtained by a process comprising the steps of: a) culturing said bacterium in a culture medium and under conditions suitable for its growth, b) liquid/solid separation of the culture medium and recovery of the liquid phase, c) adding a basic buffer to the liquid phase obtained in step b) and incubating the buffered liquid phase obtained for 1 to 7 hours, optionally at a temperature between 1 and 6° C., and d) obtaining said secretome comprising biomolecules secreted by said bacterium in the liquid phase.

13. The method according to claim 12, wherein the basic buffer is selected from Tris buffer, arginine buffer and mixtures thereof.

Description

(1) The examples refer to the following figures

(2) FIG. 1: SDS-PAGE gel of secretome proteins.

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

(4) Lanes 2 to 5: (Sec, secretome before addition of arginine buffer)—10 μl, 15 μl, μl and 30 μl loads, respectively.

(5) Lanes 6 to 9: (Sec+Arg, secretome after addition of arginine buffer)—10 μl, 15 μl, 20 μl and 30 μl loads, respectively.

(6) FIG. 2: % stimulation of proliferation (mean±SEM) of normal human fibroblasts treated with the various products compared with control cells (n=3 donor pools).

(7) FIG. 3: % stimulation (mean±SEM) of migration of the HaCaT keratinocyte cell line treated with the various compounds compared with control cells (n=2).

(8) FIG. 4: Wound re-epithelialization (mean±SEM) after application for 48 h on explants of pig ears of the various products compared with a reference restorative cream (n=10 independent donors).

EXAMPLE 1: CULTURE OF BACTERIUM LMB64

(9) By way of non-limiting example, preferred culture media contain ammonium chloride, magnesium sulphate and yeast extract. It should also be noted that, as arises from application WO2012/085182, among others, other similar media may be used and must thus be regarded as forming an integral part of the present description. Any adaptation by persons skilled in the art must also be regarded as part of the invention.

(10) An exemplary culture process is described below. It should be recalled here that this example is for illustrative purposes only and must in no way be regarded as limiting.

(11) Strain LMB64 is grown in three steps, namely a first inoculum, a pre-culture (or pre-fermentation) in batch mode and finally a culture but in fed-batch mode (addition of glucose).

(12) Inoculum:

(13) A tube of WCB LMB64 is used to inoculate an Erlenmeyer flask containing 1000 mL of sterile medium. The Erlenmeyer flask is then placed in the incubator shaker and shaken. When the cell density of the broth is sufficient, the culture is stopped. The cells are then cooled until transfer to the pre-fermenter.

(14) Pre-Culture:

(15) The pre-fermenter is then filled with roughly 16 L of medium and then fully sterilized.

(16) Two satellite flasks are connected to the pre-fermenter after sterilization of the tank and then of the addition blocks: a flask containing a sterile 50% glucose solution. This solution (Glucose batch pre-culture) is immediately transferred to the culture medium to attain the initial glucose concentration of 20 g/L. The Erlenmeyer flask containing the inoculum described above in the Inoculum step is inoculated in the pre-fermenter.

(17) The pre-culture is launched and then regulated automatically. By way of example, the following parameters may be mentioned: Temperature, Shaking speed, Pressure, Air flow rate, or PO.sub.2.

(18) Cell growth is monitored by measurement of the optical density at 620 nm. The pre-culture is stopped by cooling when it reaches a sufficient density.

(19) Culture:

(20) The fermenter is then filled with 127 L of medium adjusted to pH 7.0 and then fully sterilized. Three satellite flasks are used: a flask containing a sterile glucose solution. This solution is immediately transferred to the culture medium to attain the initial glucose concentration of 20 g/L. a flask of antifoam. This antifoam will be added automatically during the culture to control the level of foam in the tank. a flask of fed-batch glucose. This solution will be added during the culture to promote cell growth.

(21) The culture is launched and then regulated automatically. By way of example, the following parameters may also be mentioned: Temperature, pH, Shaking, Pressure, air flow rate, PO.sub.2.

(22) After exhaustion of the glucose initially present in the medium (rise in PO.sub.2), addition of the fed-batch glucose solution is triggered, thus enabling high-density cell growth. Fermentation is stopped after total consumption of the glucose. At this stage, the fermentation must is automatically cooled. Throughout the culture, cell growth is monitored by measurement of the optical density at 620 nm. The amount of dry biomass (g/L) obtained at the end of the culture is determined using a weight method.

EXAMPLE 2: EXTRACTION OF THE SECRETOME

(23) The example below is given by way of illustration of a preferred embodiment, but must not be regarded as limiting.

(24) The secretome is obtained, generally, after centrifugation of the result of the culture step in order to remove cells, surface proteins and proteins located in the periplasmic space of the bacterium. This centrifugation step is followed by an addition of a Tris-arginine basic buffer to the supernatant. A last step of filtration of the supernatant is also possible. Any adaptation by persons skilled in the art must also be regarded as part of the invention.

(25) Centrifugation:

(26) The transfer line from the fermenter to the centrifuge is sterilized. The fermentation must is then separated by continuous centrifugation on a centrifuge. Centrifugation is performed at 150 L/h (±30 L/h) with a bowl rotation speed of 10900±1000 rpm. The supernatant is collected in a container fitted with a disposable pouch. The weight of the supernatant is measured on the balance platform.

(27) Addition of Arginine:

(28) The Tris arginine extraction buffer is sterilized and then transferred to the disposable pouch containing the culture supernatant. The contact time necessary is between 1 and 7 hours. The target concentration of Tris and arginine after addition to the disposable pouch is roughly 0.3 M L-arginine and 20 mM Tris.

(29) The total volume after addition of the extraction buffer is roughly 240 L.

(30) Filtration:

(31) Two filtration steps are performed on-line, in order to clarify the supernatant and to produce a clear, germ-free secretome. Filtration is controlled by the filtration/distribution system. The disposable depth-filtration cartridge is placed in its filter housing. All of the filtered product is recovered sterilely in a container fitted with a disposable pouch. The pouch of filtered product is weighed on the balance platform and then stored at +5° C. until distribution.

(32) Gel Electrophoresis of Secretome Proteins: See FIG. 1

(33) Bis-Tris SDS-PAGE gel (4-12%), under denaturing and reducing conditions, detection by Coomassie Blue of the loads of various volumes of culture supernatants (10, 15, 20, 30 μl, respectively) before and after addition of arginine buffer. In both cases, various protein bands of between 14 and 100 kDa in size are observed, including a major band of roughly 38 kDa.

EXAMPLE 3: EFFECT OF THE SECRETOME ON PROLIFERATION OF NORMAL HUMAN FIBROBLASTS

(34) The technique used is that of incorporation of a nucleotide, 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue, in the DNA of S-phase cells, at 37° C. This technique allows quantification of the cells whose progression in the cell cycle is characteristic of a proliferative cell (S or DNA synthesis phase).

(35) Fibroblasts are seeded in a 96-well plate and then incubated for 72 h in the presence of the compounds to be tested, EGF as positive control and 0.4, 0.6 and 1% secretome. BrdU incorporation is performed during the last 24 h and is quantified using the BrdU ELISA kit (item no. 11647229001, Roche Diagnostics).

(36) The % of the control is calculated according to the formula:
(Value/mean of the control)×100

(37) Stimulation in % is calculated according to the formula:
Mean Stimulation in %=(Mean(Value/mean of the control)×100)−100

(38) SEM=standard dev/√n

(39) Statistical analyses are performed using Student's t-test. The studies were carried out on 3 donor pools.

(40) The positive control, EGF, stimulated fibroblast proliferation strongly and significantly and in a reproducible manner 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 proliferation of normal human fibroblasts. Table 1 below summarizes the results of BrdU incorporation (mean±SEM) by normal human fibroblasts (by ELISA) after 72 h of incubation in the presence of the tested compounds (n=3 donor pools). The results are shown in FIG. 2.

(41) TABLE-US-00001 TABLE 1 EGF Secretome Secretome Secretome Compound control 10 ng/ml 0.4% 0.6% 1% Mean % 100  195  138 134 127 control SEM 2 10 6 4 3 Mean % 0 95 38 34 27 stimulation SEM 2 10 6 4 3 Student's — *** *** *** *** t-test

EXAMPLE 4: EFFECT OF THE SECRETOME ON MIGRATION OF THE HACAT KERATINOCYTE CELL LINE

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

(43) Cell migration is evaluated by photographing the wells and measuring the migration surface area in mm.sup.2.

(44) The % of the control is calculated according to the formula:
(Value/mean of the control)×100

(45) Stimulation in % is calculated according to the formula:
Mean Stimulation in %=(Mean(Value/mean of the control)×100)−100

(46) SEM=standard dev/√n

(47) Statistical analyses are performed using Student's t-test.

(48) The studies are performed on 6 technical replicas and in n=2 independent experiments.

(49) The positive controls, TGF-b and foetal calf serum (FCS), stimulated cell migration strongly and significantly and in a reproducible manner (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 HaCaT keratinocyte migration. Table 2 below summarizes the results of HaCaT cell migration after 24 h of incubation with the various compounds (n=2). The results are shown in FIG. 3.

(50) TABLE-US-00002 TABLE 2 FCS TGF-b Secretome Secretome Secretome compound control 10% 10 ng/ml 0.4% 0.6% 1% Mean % 100 276 151 267 266 272 control SEM 5 9 7 8 10 9 Mean % 0 176 51 167 166 172 stimulation SEM 5 9 7 8 10 9 Student's — *** *** *** *** *** t-test

EXAMPLE 5: EFFECT OF THE SECRETOME ON WOUND CLOSING ON AN EX VIVO MODEL OF HEALING

(51) Skin explants of pig ears are maintained in survival conditions. Six-millimetre biopsies are performed at the centre of which a circular, 3 mm wound is made. The wound damages the entire epidermis and the upper dermis. The products to be evaluated are applied topically. Forty-eight hours later, the biopsies are frozen and haematoxylin-eosin staining is performed on sections. The kinetics of re-epithelialization is evaluated according to the following process:

(52) 1) Attribution of a score defined from 0 to 3 according to the protocol developed by J. Brandner (Pollok et al., 2010, J Cell Mol Med).

(53) Precise measurement in m of the extent of the re-epithelialized zone from the wound edge. Student's t-test is applied for significance.

(54) 2) The morphological appearance at the wound edge is also evaluated in order to assess tissue preservation quality.

(55) Preparations of the products to be applied: the secretome was added at 0.4, 0.6 and 1 wt % to the same base formulation as that of a reference restorative cream (Cicalfate® cream) tested in parallel. Re-epithelialization is evaluated in comparison with this reference cream.

(56) The results show a dose effect with the secretome on the progression of wound re-epithelialization. The formulation containing 1% secretome significantly accelerates the progression of wound closing compared with the reference restorative cream (p=0.043). Furthermore, morphological analysis of the tissues shows an excellent tolerance of the formulations containing the secretome. The results are shown in FIG. 4.

(57) Exemplary Composition: Oil-in-Water Emulsion

(58) Secretome according to Example 2: 0.1-5%

(59) Glycerine: 1-30%

(60) Hexylene glycol: 0.1-7%

(61) Cetearyl glucoside: 0.1-1%

(62) Cetearyl alcohol: 0.9-4%

(63) Stearic acid: 0.5-4%

(64) Glyceryl stearate: 0.1-4%

(65) Vegetable oil: 0.1-10%

(66) Water qs 100%