FR2864445A1 - Use of a complex nutrient medium for skin treatment in humans and animals, e.g. for regenerating skin and promoting wound healing, which is free of growth factors and untraceable animal or cellular products - Google Patents

Abstract

Use of a complex nutrient base (A), in aqueous medium, to make or obtain a medical composition for topical application to humans or animals. Use of a complex nutrient base (A), in aqueous medium, to make or obtain a medical composition for topical application to humans or animals. (A) comprises many amino acids, vitamins, trace elements and metal salts but does not contain any cellular growth factors nor untraceable biological extracts of animal or cellular origin. It has at least one of the following characteristics: (a) glucose content at least 0.45, e.g. 0.1-0.6, % (sic); (b) L-hydroxyproline content at least 0.003, e.g. 0.001-0.01,% (sic); (c) ascorbic acid content at least 0.0001, e.g. 0.00001-0.001, % (sic) and (d) content of each of adenosine, guanine, deoxyribose and ribose at least 0.00001, e.g. 0.00001-0.001, % (sic). ACTIVITY : Dermatological; Vulnerary. MECHANISM OF ACTION : None given.

Description

The present invention relates to a complex nutrient base (abbreviated BNC)

  in an aqueous medium, and its various uses as a medicament for topical use, for the treatment of the skin, both in humans and in animals.

  By complex nutrient base is meant any composition or formulation, in an aqueous medium, differing, as specified below, from a cell culture medium, although it generally allows, like the latter, a viable in vitro culture. for at least 72 hours of an inoculum of certain predetermined cells.

  Such BNCs have been described in WO / 96/21421, which describes their use, alone or in combination with other components, as an active product or as an excipient.

  A BNC as considered according to the present invention, has neither, in its original composition nor in its implementation, cell growth factor, for example EGF (Epidermal Growth Factor), insulin or cholera toxin, non traceable.

  A BNC as considered according to the present invention, does not include any biological extract such as fetal calf serum (FCS) or any extract of beef pituitary stem, not traceable.

  By nature, these extracts have a variable composition, even indeterminate, sometimes with an indeterminate origin.

  By "tracer" or "traceable" is meant the characteristic that the origin and then the treatment of a biological material can be established and controlled.

  A BNC considered by the present invention does not include, for example, any chemically indefinite substance, such as biological substance, traceable or not.

  A BNC considered according to the present invention does not comprise any medicinal active principle, such as an antibiotic or a corticoid (hydrocortisone).

  Such BNCs are constituted in aqueous medium by at least a multiplicity of amino acids, including certain essential, different vitamins, trace elements, and metal salts.

  According to WO 96/21421, such a complex nutritional base has, for example, the following composition, according to Table 1 below:

Table 1

  COMPONENTS Concentration in mgll Water q.s.

  Amino acids L-Alanine 9 2 421 4 L-Asparagine (anhydrous 142 Laspartic acid 42 0 L-Glutamic acid 14.8 L-Glutamine 76 Glycine 0 LIsoleucine 1312 L-Leucine 54 0 L-Methionine 13 5 L-Phenylalanine 1O U LProline 126 1 L-Serine L-Threonine 24 0 9 3 L-Tryptophan 11 7 L-Tyrosine 2 Na 2H 2 O L-Valine 70.3 Vitamins

--_ _ -----

  d-Biotin O} 2 Folic acid 0 80 Nicotinamide 030 Pyridoxine HCl 0 06 Riboflavin 0 04 Thiamine HCl 0 30 I-Inositol 18 0 0 Thymidine 0.73 Adenine (Ha.) 24O DL-lipoic acid 0.20 Inorganic components Chloride Sodium 6800.0_ KCl 112.0 NmuHPO! 284.0 Sodium acetate 300.0 Sodium sulfate 3 4 Sodium bicarbonate 1160.0 The present invention relates to a BNC, as defined above, considered as a composition for topical use, for the therapeutic or non-therapeutic treatment of the skin in humans or animals, in particular improving the viability, growth and synthesis processes of the cells of the dermis and / or the epidermis, especially in case of aggression or stress or following lesions.

  The present invention more specifically aims to adapt or particularize a BNC as described above, to a therapeutic or clinical use on the skin.

  According to the present invention, so as to adapt a complex nutritional base as defined above, to a use as a medicament for topical use, for the treatment of the skin, at least one of the weight composition characteristics of said base , defined below, is used: (a) its glucose concentration is at least 0.45%, for example between 0.1% and 0.6% (b) its concentration of L-hydroxyproline is at least 0.003% , and for example between 0.001% and 0.1% (c) its ascorbic acid concentration is at least 0.0001%, and for example between 0.00001% and 0.001% (d) its concentration in each of the following compounds, namely adenosine , guanine, deoxyribose and ribose, is at least 0.00001%, and between 0.000001% and 0.0001%.

  Preferably, the BNC used according to the present invention as a medicament has all of the characteristics (a) to (d) above, of weight composition.

INGREDIENTS

  CaCl2.2H2O ZnSO4.7H2O (NH4) 6_MoO24.4H2O MnCl2.4H2O SnCl2.2H2O NH4 VO3 Concentration in mg / I 1.39 120.0 13.0 to 22.05 0.144 0.122 0.00002 0.00011 Preferably, the BNC comprises peptides, for example extracted from milk, at a concentration of between 0.1% and 1% by weight, and for example 0.5%.

  Preferentially, the BNC comprises at least 0.07% by weight of sodium hyaluronate.

  Preferably, the BNC comprises at least 0.20% by weight of Lglutamine.

  According to a preferred embodiment of the present invention, the pH of the BNC is adjusted between 7.4 and 7.5, for example at a value of 7.45, and / or the osmolarity of said BNC is adjusted to at most between 300 and 350 mOsm. .

  By way of example of a BNC used as a topical composition according to the invention, the weight composition according to Table 2 is used:

Table 2

NAME DENOMINATION CONCENTRATION

INTERNATIONAL LINE In mgA

NOMENCLATURE

COSMETIC

  INGREDIENT (INCI NAME) 1 WATER q.s. (1000mI) 2 CHLORIDE OF 6085

SODIUM

  3 GLUTAMINE 2043.2 4 SODIUM BICARBONATE 1160 GLUCOSE 4500 6 ARGININE HCL 421.4 7 SODIUM ACETATE 300 8 DISODIUM PHOSPHATE 284 9 LEUCINE 131.2 SERINE 136.6 11 CHLORIDE Mg 120.0 12 CHLORIDE K 112 13 VALINE 70.3 14 SODIUM PYRUVATE 55 LYSINE HCL 54 16 HISTIDINE HCL 50 17 CYSTEINE HCL 42 18 ADENINE 24 19 THREONIN 24 CHLORIDE FROM 13.0 to 22.05 21 INOSITOL 18 22 GLUTAMIC ACID 29.5 23 ASPARAGINE 29.2 24 METHIONINE 13.5 TYROSINE 11.7 26 PHENYLALANINE 10.0 27 TRYPTOPHAN 9.3 28 ALANINE 18.1 29 GLYCINE 15.1 ISOLEUCIN 6.0 31 ASPARTIC ACID 17.3 32 SODIUM SULFATE 3,4

NAME DENOMINATION CONCENTRATION

INTERNATIONAL LINE In mgll

NOMENCLATURE

COSMETIC

  INGREDIENT (INCI NAME) 33 FERROUS SULFATE 1.4 34 FOLIC ACID 1.8 THYMIDINE 0.73 36 CYANOCOBALAMINE 0.41 37 CALCIUM 13

PANTOTHENATE

  38 THIAMINE HCL 1.3 39 THIOCTIC ACID 0.3 ZINC SULFATE 0.144 41 SODIUM SILICATE 0.144 42 PYRIDOXIN HCL 1.06 43 NIACINAMIDE 1.04 44 RIBOFLAVIN 0.4 BIOTIN 0.02 46 COPPER SULPHATE 0.003 47 AMMONIUM 0.00120

Molybdate

  48 AMMONIUM VANADATE 0.003 49 CHLORIDE Mn 0.00002 HYALURONATE 70

SODIUM

  51 HYDROXYPROLINE 30 52 PROLINE 46 53 ASCORBIC ACID 1 54 ADENOSINE 0.1 GUANINE 0.1 56 DEOXYRIBOSE 0.1 57 RIBOSE 0.1 58 "MILK PEPTIDE 1000 to 10000 COMPLEX" 59 CHOLINE CHLORIDE 1 MYO INOSITOL 2 A BNC according to the present invention has no indication or application ophthalmic or ophthalmic, in particular by application to the outside of the cornea, for example locally, in humans or animals. A BNC adapted according to the present invention for topical use generates within the skin the following joint beneficial effects: - supply of exogenous energy to the cutaneous cells - stimulation of collagenous and elastic fibrillogenesis - and the stimulation of cell renewal.

  This is particularly evidenced by the experimental protocol described hereinafter.

  The in vitro efficacy of a BNC according to the invention, having the composition according to Table 2, is objectified with respect to a BNC according to the prior art (WO96 / 21421), having the composition according to Table 1, on the one hand by normal human fibroblast assays (Run 1), and by human keratinocyte line assays (Run 2).

  Test n 1: test on normal human fibroblasts.

  The growth capacity of normal human fibroblasts cultured for 9 days in BNC according to Table 2 versus BNC according to Table 1 is evaluated.

  1. Purpose of the Study The objective of this study is to evaluate the growth of normal human fibroblasts seeded at low density in a BNC according to Table 2.

  The study is performed in comparison with the BNC according to Table 1.

  The study is conducted with BNCs lacking cell growth factors (absence of FCS and pituitary beef stem extract), and other chemically undefined or drug substances.

  2. Technique The fibroblasts are inoculated at a low density in a 96-well plate in a standard medium (DMEM + 10% FCS) and grow for 24 hours after seeding in this medium.

  At the 2nd day, the cells are placed under the different conditions studied. The BNC medium according to Table 2 is enriched extemporaneously in MPC complex - complex of milk peptides (lot n 3035626 CLR) - tested at 2 concentrations: 1 and 5 mg / ml.

  Different media are tested: BNC according to Table 2 (+ MPC 1 and 5 mg / ml) versus BNC according to Table 1, in the absence of FBS and any other chemically undefined or medicinal substance.

  Each condition is made in triplicate. BNCs are not renewed during the experiment.

  The cell density is evaluated 24h after seeding of the cells, before contacting the various study conditions (= TO), then fibroblast growth is evaluated at the 2nd, 4th, 7th and 5th days of culture by the method. converting the WST-1 (reading at 450 nm).

  3. Results Cell growth is objectified by the measurement of cell viability at different times of the experiment. The results obtained show the percentage of cell viability calculated with respect to the initial cell density at TO, for which it is estimated to have a cell density equal to 100%.

  The results obtained are summarized as follows: In BNC medium according to Table 1, one observes, on the 2nd day of culture, a decrease in the cell viability due to the passage of the cells in a medium without serum. A slight resumption of cell growth is observed until the 4th day (57% cell viability / T0), then a stabilization and a slow decrease from the 7th day.

  In BNC medium according to Table 2 containing 0.1% MPC complex, stimulation of cell growth is observed. Cell growth increases steadily until the 7th day, then slows down.

  In a BNC medium according to Table 2 containing 0.5% of MPC complex, a very strong stimulation of cell growth is observed. Until the 7th day, cell growth is very strongly stimulated compared to culture with BNC. according to Table 1 (+ 360% at T7). Subsequently, a slowing down of cell growth is observed, which is explained by the non-renewal of the media during the experiment.

  4. Conclusion Under the experimental conditions thus defined, it appears that the BNC medium according to Table 2 allows the growth of normal human fibroblasts. The extemporaneous addition of the MPC Complex stimulates very strongly cell growth in a dose-dependent manner, with a maximum effect for a concentration of 5 mg / ml.

  The cell growth is of low intensity in BNC medium according to Table 1.

  Test No. 2: Test on a line of human keratinocytes.

  The growth capacity of an immortalized line of normal human keratinocytes, the HaCaT cells, cultured for six days in the BNC according to Table 2, versus the BNC according to Table 1, is evaluated: 1. Objective of the study The objective of this study is to evaluate the growth of an immortalized line of human keratinocytes, HaCaT cells, seeded at low density in a BNC according to Table 2.

  The study is carried out in comparison with the BNC according to Table 1.

  Each of the BNCs is used without the addition of cell growth factor (absence of fetal calf serum and pituitary beef stem extract), and other chemically undefined or drug substances.

  2. Technique HaCaT cells are seeded at low density in 96-well plate in standard medium (DMEM + 10% FCS) and grow for 24 hours after seeding in this medium.

  At day 2, the cells are placed under the different conditions studied. The BNC medium according to Table 2 is enriched extemporaneously in MPC Complex - Milk Peptide Complex (Lot No. 3035626 CLR) tested at 2 concentrations: 1 and 5 mg / ml.

  Different media are tested: BNC according to Table 2 (+ MPC 1 and 5 mg / ml) versus BNC according to Table 1, in the absence of FBS and any other chemically undefined or medicinal substance.

  Each condition is made in triplicate. BNCs are not renewed during the experiment.

  The cell density is evaluated 24h after seeding of the cells, before contacting the various study conditions (= TO), then the growth of HaCaT cells is evaluated at the 2nd, 4th and 6th days of culture by the method of WST-1 conversion (reading at 450 nm).

  3. Results Cell growth is objectified by the measurement of cell viability at different times of the experiment. The results obtained show the percentage of cell viability calculated with respect to the initial cell density at TO, for which it is estimated to have a cell density equal to 100%.

  The results obtained can be summarized as follows: In BNC medium according to Table 1, there is observed, on the 2nd day of culture, a strong decrease in cell viability due to the passage of the cells in a serum-free medium (15% cell viability). / T0). We then observe a resumption of cell growth until the fourth day (42% cell viability / TO), then a stabilization until the 6th day.

  In BNC medium according to Table 2 containing 0.1% of MPC complex, there is observed on the 2nd day of culture, as for the previous medium, a strong decrease in cell viability due to the passage of the cells in a serum-free medium. Cellulability Vi% OT). Then there is a strong recovery in cell growth, which continues until the 6th day of culture.

  In a BNC medium according to Table 2 containing 0.5% of MPC Complex, there is observed on the 2nd day of culture a decrease in cell viability when the cells pass in a serum-free medium, but this is of less intensity compared to that observed for the BNC according to Table 1 (43% cellulose viability). Subsequently, a very strong recovery of cell growth is observed, higher than that obtained in the BNC medium according to Table 1 and that obtained with the BNC medium according to Table 2 containing 0.1% MPC Complex. Growth continues until the 6th day when a plateau is reached at the cell confluence.

  4. Conclusion Under the experimental conditions thus defined, it appears that the BNC medium according to Table 2 allows the growth of an immortalized line of normal human keratinocytes, HaCaT cells. The extemporaneous addition of the MPC Complex stimulates cell growth very strongly, in a dose-dependent manner with a maximum effect at a concentration of 5 mg / ml.

  The cell growth is of low intensity in BNC medium according to Table 1.

  A BNC according to the present invention has been tested in vivo and ex vivo (on skin explants) as described below.

  All the tests described below are made with a BNC according to Table 2.

  According to test No. 3 described below, the efficacy of a BNC according to the invention in 12 subjects was evaluated by an immunohistochemical study of laminin and a morphometric analysis of collagen and elastic fibers at the level of skin biopsies, and this by studying comparatively the treated area with a BNC according to the invention and the untreated area.

  This study demonstrates an action of BNC according to the invention on the stimulation of fibroblast metabolism in patients over 60 years old, namely, increase of laminin deposition at the dermo-epidermal junction, densification of elastic fiber networks in the middle dermis and collagen in the superficial and middle dermis.

  Test No. 3: Evaluation of laminin, collagen and elastic fibers at cutaneous biopsies performed in 12 subjects, with comparative study between the zone treated with a BNC according to the invention, and an untreated zone.

  Introduction and purpose The aim of this study is to demonstrate the efficacy of a BNC in 12 subjects by an immunohistochemical study of laminin and a morphometric analysis of collagen and elastic fibers at cutaneous biopsies of the treated area. comparison with the untreated area.

  MATERIALS AND METHODS Immunohistochemical evaluation of dermal changes was performed from skin biopsies after fixation in Bouin fluid and paraffin embedding.

  (a) Analysis of Laminin From the paraffin-embedded fragments, analysis of the dermal-epidermal junction was performed with the anti-laminin antibody (Clinisciences).

  After unmasking of the antigenic sites by heat (microwave) then enzymatic action by pepsin, the immunodetection is carried out using a technique of indirect immunoperoxidase in 3 layers (ABC Peroxidase kit, Vector Laboratories) and revealed in AEC. This unmasking is responsible for an artefactual detachment of the junction between dermis and epidermis.

  The intensity of immunohistochemical staining was evaluated using a semi-quantitative histological score (0 to 3) at magnification 40 (5 to 6 fields per section).

  (b) Histological quantification of elastic fibers by computerized image analysis.

  The elastic fibers were revealed by (+) catechin staining.

  Morphometric quantification of the elastic fibers by computer-assisted image analysis was performed. The percentage of surface area of the superficial and average dermis occupied by the elastic fibers was thus calculated.

  (c) Histological quantification of collagen by computerized image analysis Collagen was demonstrated by sirius red staining and quantified morphometrically by computer-assisted image analysis. The percentage of superficial and average dermal surface occupied by collagen was calculated.

  (d) Statistics An average was obtained from the results obtained on the 12 skins.

  A comparative study was thus carried out between the BNC side and the untreated side. An average was also calculated by age group, 6 subjects between 60 and 64 years (subjects 1, 3, 6, 7, 9 and 12) and 6 subjects between 44 and 49 years (subjects 2, 4, 5, 8 , 10 and 11).

  Statistical analysis was performed by Student's test of the reduced gap or paired test, with a risk of 5%.

  An iconography illustrates the results in the form of examples.

  Results a) Laminin analysis The quality of the dermoepidermal junction tends to be increased in the skins treated with BNC. The difference between the treated side (average score of 2.15) and the untreated side (average score of 1.75) is clear in subjects in the age group 40 years.

  b) Histological quantification of elastic fibers by computerized image analysis The analysis of elastic fibers shows a tendency to improve the network of these fibers, in particular at the level of the average dermis, 8.35% of fibers treated side against 7, 7% untreated side and more discreet in the superficial dermis: 4.77% versus 4.5%. In the superficial dermis, it is at the 60-year age group that the improvement on the treated side is the clearest: 5.2% of elastic fibers against 4.6% of the untreated side. We did not observe a significant change in the elastic fiber network in the age group 40 years after treatment with BNC. The lack of significance is likely related to the small number of subjects.

  c) Histological quantification of collagen by computerized image analysis The analysis of collagen shows a tendency to improve this network for the age group around 60 years of the side treated by the BNC, both in the dermis superficial (collagen network increased by 1.9%) than the average dermis (network increased by 1.95%). This interesting result is to correlate with those of the literature observing a collagen loss with the age of the order of 1% every 5 years.

  We did not observe any changes in collagen bundles in the age group around 40 years after BNC treatment, whereas the collagen network is larger at age 40 than at age 60.

  Case-by-case analysis in the age group around 40 years of age made it possible to verify that the nature of the treated arm (right or left) is not responsible for a possible change in the amount of elastic tissue and therefore does not influence the results.

  Conclusion We have demonstrated an action of the BNC according to the invention on the stimulation of fibroblast metabolism, essentially in the 20 subjects of age around the sixties.

  Test No. 3 thus illustrates the beneficial properties of a BNC according to the invention with regard to cicatrization and anti-aging effect.

  According to the test No. 4 described below, the proliferation, differentiation and cell cohesion in cutaneous biopsies carried out in 12 patients were evaluated immunohistochemically by studying, by comparison, the zone treated with a BNC according to the invention and the untreated area.

  This study shows an increase in the mitotic index of keratinocytes, that is to say cell renewal within the epidermis, in the zones treated with BNC according to the invention, in the 12 patients, compared to untreated areas, potentially beneficial property for the promotion of healing or slowing of skin aging.

  Test No. 4. Immunohistochemical evaluation of proliferation, differentiation and cell cohesion in cutaneous biopsies performed in 12 patients, with comparative study between the zone treated with a BNC according to the invention, and an untreated zone.

  Introduction and purpose The purpose of this study is to demonstrate the efficacy of the BNC according to the invention in 12 patients by an immunohistochemical study of differentiation, proliferation and cell cohesion at skin biopsies of the zone. treated, in comparison with the untreated area.

  Material and Methods Immunohistochemical evaluation of epidermal changes was performed from skin fragments fixed in Bouin's liquid and embedded in paraffin.

  a) Immunohistochemical Analysis of the Mitotic Activity of Keratinocytes Epithelial proliferation was analyzed by immunohistochemistry using an anti-Ki67 antibody (cell marker in M, S, G1 and G2 phases of the cell cycle). The immunodetection was carried out using a technique of indirect immunoperoxidase in 3 layers, amplified (DAKO kit) and revealed in DAB.

  The number of labeled cells is evaluated in relation to a total of 200 to 400 cells. The percentage of cells undergoing proliferation at the level of the basal layer has thus been calculated.

  b) Immunohistochemical Analysis of Epithelial Differentiation Epithelial differentiation is demonstrated with an anti-total cytokeratin antibody (Novocastra).

  The immunodeteclion is performed using a 3-layer indirect immunoperoxidase technique (ABC Peroxidase kit, Vector Laboratories) and revealed in DAB (Diaminobenzidine).

  The intensity, topography, and distribution of immunohistochemical staining were assessed using the following semiquantitative scores: E Marked Intensity - Negative: Score 0 - Moderate: I Score - Moderate: Score 2 - Important: Score 3 Topography of the staining (over the entire height of the epithelium) - negative: score 0 - stratum corneum and granular layer marked: score 1 - stratum corneum, granular layer and upper part of the mucous body: score 2 - all the epithelium except basal layer: score 3 E Distribution of immunolabeling - Inhomogen: score 0 (some areas of the epidermis only are positively marked).

  - Homogeneous: score 1 (the whole of the epithelium is concerned).

  An overall score of differentiation is calculated by summing the intensity, topography, and distribution scores of immunostaining and an average is calculated. A high score indicates good epithelial differentiation.

  c) Demonstration of cellular cohesion The interkeratinocyte cohesion as well as the cohesion between the keratinocytes of the basal layer and the underlying connective tissue can be demonstrated using an anti-integrin VLA antibody (31 ( Immunotech, CD 29, 13OdD).

  Immunodetection was performed using a 3-layer indirect immunoperoxidase technique (ABC Peroxidase kit, Vector Laboratories) and revealed in DAB (Diaminobenzidine).

  Intensity and distribution of immunohistochemical staining were assessed using the following semi-quantitative scores: E Tagging intensity - negative: score 0 - mild: score 1 - mild to moderate: moderate score 2: score 3 - moderate to important: score 4 E Distribution of Immunolabeling - very inhomogeneous (some cells positively labeled) score 1 - inhomogeneous (rare areas marked positively): score 2 - several areas marked positively: score 3 - homogeneous with labeling of the entire basal layer An overall score of cell cohesion is calculated by summing the intensity and distribution scores of immunostaining and a mean is calculated. A high score shows good cell cohesion.

  Results a) Immunohistochemical Analysis of the Mitotic Activity of Keratinocytes The treatment with BNC according to the invention made it possible to significantly increase the renewal of the epithelial cells (p <0.05): indeed, after treatment, 16.8% of the keratinocytes are labeled with the anti-Ki67 antibody, against 10.5% of the untreated side.

  b) Immunohistochemical Analysis of Epithelial Differentiation Epithelial differentiation tends to be of better quality in the skins treated with BNC according to the invention in comparison with untreated skins. The overall score obtained is 6.54 against 4.96. We did not obtain a statistically significant difference, the case-by-case analysis nevertheless allows to observe a much better differentiation in 7 out of 12 treated cases, compared with the untreated sides.

  c) Demonstration of cell cohesion The anti-VLA antibody [31 revealed inter-keratinocyte cohesion at the level of the basal layer (score of 1 in all the cases analyzed, treated or untreated), as well as the cohesion between the basal layer and the dermal-epidermal junction. Cellular cohesion tends to be of better quality at the level of

  skin treated with BNC according to the invention, compared with untreated skin: the overall score obtained is 5.5 against 4.6. We did not obtain a statistically significant difference, case-by-case analysis nevertheless allowing to observe a superior cellular cohesion in 8 treated cases out of 12, in comparison with the untreated sides.

  Conclusion Treatment with BNC according to the invention makes it possible to obtain in 12 subjects a statistically significant increase in the mitotic index. Epithelial differentiation and cell cohesion are improved in the treated zone, but without any significant character.

  According to test N 5 described below, the effects of a BNC according to the invention on the maintenance of survival and differentiation of skin fragments are evaluated. This study shows the ability of a BNC according to the invention to maintain in survival human skin fragments in vitro culture, as well as its action on the regeneration of epidermal cells, potentially beneficial property for the promotion of healing or slowing down. skin aging.

  Test n 5: Evaluation of a BNC on the maintenance of survival and differentiation of skin fragments.

  Introduction and purpose The aim of this study is to demonstrate the efficacy of a BNC on the ex vivo survival and cutaneous differentiation of human skin fragments.

  The evaluation is histological (cell viability analysis) and immunohistochemical (antibody to analyze proliferation, differentiation and cell cohesion) with semi-quantitative scores.

  Materials and Methods a) Maintenance of human skin in survival Fragments of normal human skin of 4 different donors were deposited in inserts themselves positioned on culture wells. The BNC according to the invention is deposited at the bottom of the wells, a passage being carried out by slow diffusion between the two compartments by means of a porous membrane (12pm). B) Culture medium to be tested The effectiveness of the BNC according to the invention on the survival and differentiation of the skin fragments was compared with that obtained in the presence of a phosphate buffer type PBS (with or without EGF, "Epithelial Growth Factor"). Thus, the following comparisons were made: - PBS buffer - PBS + EGF buffer

- BNC

  The media changes were performed daily for 3 days, then the skin fragments were collected for histological and immunohistochemical analysis.

  c) Analyzes Histological and immunohistochemical evaluation of the epidermal changes The skin fragments were fixed in Bouin's liquid and included in paraffin.

  - Histological evaluation on the viability of the epidermis The epidermis is observed from sections stained by hemalunéosine. Cell viability is assessed at the nucleus (dye affinity to haematin) and cytoplasm (eosin dye affinity). In fact, the nuclear alterations testifying to the suffering and the cellular necrosis correspond to a condensation of the chromatin (pycnosis) or conversely to a clarification of the nucleus. The alterations of the cytoplasm also correspond to a clarification or, more rarely, to an eosinophilic condensation of the cytoplasm.

  A count of the number of altered cells was performed under an optical microscope at the level of the epidermis on 10 fields (objective 40).

  When the percentage of necrotic cells exceeds 50%, acantholysis (cleavage of the union bridges between cells) is usually noted with sometimes detachment at the dermal-epidermal junction.

  The appearance of chromatin was examined after Feulgen staining. This evaluation made it possible to complete the histological analysis and to create an iconography. The nuclear alterations are evaluated using semi-quantitative scores: - very important alteration (massive necrosis with alterations concerning 60 to 100% of the cells): score 0 important alteration (nuclear alterations concerning 25 to 60% of the cells): score 1 - slight to moderate alteration (nuclear alterations involving 5 to 25% of cells with clarification or pycnosis): score 2 no nuclear alteration or <5%: score 3 - Evaluation of mitotic activity At the end of the 3 days of culture the skin fragments are incubated for 1 hour with 5-bromo-2'-deoxyuridine, a thymidine analogue capable of being incorporated into the DNA of the cells being replicated. The dividing cells are then revealed by immunoperoxidase (Amersham Cell Proliferation kit).

  A count of the number of labeled cells is made and related to the length of the epithelium measured on the section (mm).

  Immunohistochemical Analysis of Epithelial Differentiation Epithelial differentiation is demonstrated with total anticytokeratin antibody (Novocastra).

  Immunodetection was performed using a 3-layer indirect immunoperoxidase technique (ABC Peroxidase kit, Vector Laboratories) and revealed in DAB (Diaminobenzidine).

  Intensity, topography and distribution of immunohistochemical staining were assessed using the following semiquantitative scores: E Intensity of labeling 35 - mild: moderate score 1: score 2 - significant: score 3 É Topography of the mark (on all the height of the epithelium) - negative: score 0 - stratum corneum and granular layer marked: score 1 - stratum corneum, granular layer and upper part of the mucous body: score 2 - all the epithelium except basal layer: score 3 Distribution of Immunolabeling - Inhomogen: score 0 (some areas of the epidermis only are positively labeled) Homogeneous: score 1 (all epithelium is concerned) An overall score of differentiation is calculated by summing the intensity, topography and distribution of immunostaining and an average is calculated. A high score indicates good epithelial differentiation.

  - Demonstration of cellular cohesion The interkeratinocyte cohesion as well as the cohesion between the keratinocytes of the basal layer and the underlying connective tissue can be demonstrated using an anti-integrin antibody VLA 131 (Novocastra, CD 29, 130 kD).

  Immunodetection was performed using a 3-layer indirect immunoperoxidase technique (ABC Peroxidase kit, Vector Laboratories) and revealed in DAB (Diaminobenzidine).

  Intensity, topography and distribution of immunohistochemical staining were assessed using the following semiquantitative scores: E Marked intensity - negative: score 0 - mild: score 1 - mild to moderate: score 2 - moderate: score 3 - Moderate to important: score 4 É Topography of the marking - negative: score 0 - The basal layer: score 1 - The basal and suprabasal layer: score 2 - The basal layer and the mucous body: score 3 É Distribution of immunostaining - Very inhomogeneous (some cells marked positively): score 0 Inhomogeneous: (rare sectors marked positively): score 1 - Homogeneous: score 2 An overall score of cellular cohesion is calculated by summing the scores intensity, topography and distribution of immunostaining and an average is calculated. A high score shows good cell cohesion.

  Results 1) Histological evaluation of the viability of the epidermis Examination of the epidermis from sections stained by the hemalun-eosin made it possible to demonstrate a good viability of the skins maintained in the presence of a BNC according to the invention. This epidermal viability is significantly better compared with that observed in the presence of PBS alone. Thus, the skins maintained in survival for 3 days in the presence of the different media can be classified according to their histological quality: 1st: BNC according to the invention 2nd: PBS + EGF 3rd: PBS The appearance of chromatin was examined after Feulgen staining. The visible cellular DNA is colored pink. Altered cells result in decreased visualization of DNA. The analysis using scores confirms the previous result. As for the skins maintained in survival in the presence of PBS, with or without EGF, moderate to significant nuclear alterations are observed: score of 1.4.

  A classification can therefore be made: 1st: BNC according to the invention 2nd: PBS + EGF similar to PBS 2) Evaluation of mitotic activity The dividing cells revealed by immunoperoxidase are visualized at the level of the basal layer and at the level of 2 to 3 supra-basal layers.

  The proliferating epithelia are those which have been cultured in the presence of the BNC according to the invention: approximately 10 cells / mm of epidermis are counted in the proliferation phase S. There is a statistically significant difference between skins cultured in the presence of BNC and skins cultured in the presence of PBS (with or without EGF) (p <0.05). Indeed, the skins cultured in the presence of PBS + EGF for 3 days show very few proliferating cells (on average 2.3 cells / mm). Skins cultured in the presence of PBS alone show practically no proliferating cells (0.45 cells / mm).

  3) Immunohistochemical Analysis of Epithelial Differentiation Epithelial differentiation is demonstrated with the aid of anti-total cytokeratin antibodies.

  Due to the small number of samples and the standard deviations obtained, we did not obtain any significant difference as regards the epithelial differentiation, whatever the applied culture medium. The differentiation nevertheless tends to be of better quality in the skins cultured in the presence of the BNC according to the invention (score of 5.9) in comparison with those cultured in the presence of PBS, with or without EGF (respectively score of 4). and 5.1). Case-by-case analysis shows significantly better differentiation in 2 out of 4 cases in the presence of BNC, compared with PBS, with or without EGF.

  4) Demonstration of the cellular cohesion The anti-VLA 131 antibody makes it possible to highlight the interkeratinocyte cohesion at the level of the basal layer and at the level of 2 to 3 supra-basal layers, as well as the cohesion between the basal layer and the dermal-epidermal junction.

  Good cell cohesion is noted in the skins cultured in the presence of the BNC according to the invention, compared with those cultured in the presence of PBS, with or without EGF (scores of 3.5 and 3.9, respectively). The small number of cases analyzed and the standard deviations do not show any significant difference.

  CONCLUSION: The survival of human skin in the presence of a BNC according to the invention makes it possible to obtain good epidermal viability, with an increase in the mitotic index, an epithelial differentiation and an increased cellular cohesion, in comparison with skin maintained in vitro. survival in the presence of PBS.

  Test No. 6: In Vitro Objectivation Tests for the Efficacy of a BNC According to the Invention The cytocompatibility and the performances of the BNC according to Table 2 were tested on cultures of human keratinocytes in monolayer and on reconstituted total epidermides. in vitro, compared with buffered saline (PBS).

  This BNC allows a culture of keratinocytes monolayer under optimal conditions of viability, for at least 36 hours, without the slightest cytotoxic effect. In contrast, a conventional survival solution such as PBS is cytotoxic, especially after 12 hours of incubation.

  In the same way, this BNC makes it possible to maintain in vitro replenished human epidermis with intact, orthokeratotic basal, spinous, spinous, granular and corneal cell layers of regular and normal stratification. On the other hand, the use of PBS induces the appearance of end-stage differentiating keratinocytes at basal and spinous bases, with more or less pronounced signs of necrosis.

  We also studied the effects of this BNC on the growth of a transformed keratinocyte line. No significant difference in cell proliferation rate was noted until confluence of cultures. We can therefore conclude that there are no components that stimulate the proliferation of transformed cells.

  A BNC according to the invention recreates an extracellular environment characterized by: an optimized nutritional intake, as well as vitamins, trace elements and essential amino acids, pH and osmolarity close to physiological conditions, without resorting to usual animal substances not traced acting as cell growth factors.

  This BNC, manufactured and packaged under very strict aseptic conditions, is devoid of preservatives.

  Many topical pharmaceutical applications are implemented as follows: E sterile solution (uni-doses) intended for the maintenance and grafting of burn victims and especially the "decontamination" (depletion) process of the epidermis of culture substances of biological and medicinal origin, present in commercial culture media, before they are grafted to burn victims.

  E serums or creams for the treatment of delays and disorders of healing (pressure ulcers, varicose ulcers, prevention of keloids and stretch marks ...), and slowing of skin aging.

  Different ways can be implemented to apply a BNC according to the invention topically on all or part of the body surface.

  A first way, "traditional", is to formulate a BNC in the form of a cream (emulsion water in oil or oil in water, multiple emulsions EIHIE or H / E / H), ointment, gel, cream, or spray, with one or more excipients suitable for contact with the skin.

  A second way is to associate a BNC according to the invention, in one way or another, a dressing or any equivalent medical device or the like, intended to come into contact with the skin.

  A traditional adhesive dressing comprises, for example: an external part, for example a non-woven, often hydrophobic, and gas-permeable, an absorbent intermediate part, for example a defibrated cellulose part, and an internal part, in external and superficial contact, the skin, for example non-adherent textile veil.

  From this traditional structure, a BNC according to the invention can be incorporated into the absorbent intermediate part in three ways: - at the time of application of the dressing, for example on a wound, a dose of BNC is applied beforehand on this intermediate part, for its impregnation, - the BNC impregnates permanently, that is to say at the beginning and ready to use, this same intermediate part, - the BNC is incorporated, in the dry state, for example under lyophilized form, this same intermediate part, and the moisture of the skin, or the exudate of the wound reconstitutes the BNC, for its action on the skin.

  Other dressings, less traditional, consist of an adhesive plate composed of a hydrocolloid, or gel (for example carboxymethyl sodium cellulose).

  In this case, as before, the BNC is incorporated and dispensed, dry or in an aqueous medium, into the matrix of the adhesive plate.

  By way of example, a BNC is incorporated in a hydroactive film-forming gel, obtained from a sodium or calcium alginate, or from any other film-forming polymer. This gel forms, after drying, a film on the surface of the skin, permeable to air, acting as a bacteriological barrier, for example by promoting cicatrization with autolysis and elimination of necrotic tissue, if the film-forming gel is applied to a wound.

  From the results of the tests above, a BNC according to the invention can be used as a dermatological medicament, in particular according to at least one of the following indications, namely: - regeneration of the skin in the elderly - slowing of skin aging - promotion of healing - repair or reconstitution of the epidermis and / or dermis, treatment of post-surgical burns, actinic keratoses, burns "post-peeling" or post-dermabrasion.

  A BNC according to the invention can be used as a dressing, particularly as a compressive dressing, for the prevention or treatment of keloids. In such a case, the BNC can be reconstituted or released from the dressing.

  A BNC according to the invention can be used for the depletion of substances of biological or medicinal origin of cultured human epidermis, before transplanting them.

Claims (12)

  1. Use of a complex nutrient base in an aqueous medium, for manufacturing or obtaining a medicament for topical use in humans or animals, said base being constituted at least by a multiplicity of amino acids, vitamins, oligo -elements, and metal salts, said base excluding any cell growth factor, any biological extract of animal or cellular origin, not plotted, characterized in that the complex nutritive base has at least one of the following composition characteristics: (a) its glucose concentration is at least 0.45%, for example between 0.1% and 0.6%, (b) its concentration of L-hydroxyproline is at least 0.003%, and for example between 0.001% and 0.01 %, (c) its concentration of ascorbic acid is at least 0.0001%, and for example between 0.00001% and 0.001%, (d) its concentration in each of the following compounds, namely adenosine, guanine, deoxyribose and ribose, is 0.00001% at the m oins, and between 0.000001% and 0.0001%.   2. Use according to claim 1, characterized in that the complex nutritive base has all the characteristics of weight composition (a) to (d).   3. Use according to claim 1 or 2, characterized in that the complex nutritional base comprises peptides, for example extracted from milk, at a concentration of between 0.1% and 1% by weight.   4. Use according to claim 1, 2 or 3, characterized in that the complex nutritive base comprises at least 0.07% by weight of sodium hyaluronate.   5. Use according to claim 1 or 2, characterized in that the complex nutritive base comprises at least 0.20% by weight of L-glutamine.   6. Use according to claim 1, characterized in that the pH of the complex nutrient base is adjusted between 7.4 and 7.5.   7.Use according to claim 1, characterized in that the osmolarity of the complex nutritive base is adjusted to at most between 300 and 350 mOsm.   8. Use of a complex nutritional base according to claim 1, as a dermatological drug, in particular according to at least one of the following indications, namely: - regeneration of the skin in the elderly - slowing of skin aging - promotion of healing - repair or reconstitution of the epidermis and / or dermis, treatment of post-surgical burns, actinic keratoses, burns "post-peel" or post-dermabrasion.   9. Use of a complex nutritional base according to claim 1 as a dressing.   10. Use according to claim 9 as a compressive dressing for the prevention or treatment of keloids.   Use according to claim 9, wherein the complex nutritional base is reconstituted or released from the dressing 12. Use of a complex nutritional base according to claim 1, for the depletion of substances of biological or medicinal origin, cultured human epidermis, before transplanting them.