EP1165033A1

EP1165033A1 - Use of vitamin c or the like for stimulating skin cell synthesis

Info

Publication number: EP1165033A1
Application number: EP00909442A
Authority: EP
Inventors: André Rougier; Alain Richard
Original assignee: La Roche Posay Laboratoire Dermatologique SAS
Current assignee: La Roche Posay Laboratoire Dermatologique SAS
Priority date: 1999-03-24
Filing date: 2000-03-07
Publication date: 2002-01-02
Also published as: FR2791259B1; JP2002541074A; TWI225410B; CA2364608A1; UY26063A1; AU3172600A; WO2000056278A1; AR018230A1; FR2791259A1

Abstract

The invention concerns a method for increasing the differentiation and/or proliferation rate of skin fibroblasts and/or increasing the differentiation rate of skin keratinocytes by applying on the skin a composition comprising an efficient amount of ascorbic acid or of at least one of its analogues. The invention also concerns a method for stimulating cutaneous vimentin by applying on the skin a composition comprising an efficient amount of ascorbic acid or of at least one of its analogues. The invention further concerns a method for stimulating cutaneous keratin 10 synthesis by applying on the skin a composition comprising an efficient amount of ascorbic acid or of at least one of its analogues.

Description

USE OF VITAMIN C OR THE LIKE TO STIMULATE THE SYNTHESIS OF SKIN CELLS

The invention relates to a method for increasing the rate of differentiation and / or proliferation of skin fibroblasts and / or increasing the rate of differentiation of skin keratinocytes by applying to the skin a composition comprising an effective amount of ascorbic acid or at least one of its analogs. It also relates to a process for stimulating the synthesis of cutaneous vimentin by applying to the skin a composition comprising an effective amount of ascorbic acid or of at least one of its analogs. It further relates to a process for stimulating the synthesis of cutaneous keratin by applying to the skin a composition comprising an effective amount of ascorbic acid or of at least one of its analogs.

Human skin is made up of two compartments, namely a surface compartment, the epidermis, and a deep compartment, the dermis.

The natural human epidermis is mainly composed of three types of cells which are the keratinocytes, very majority, the melanocytes and the Langerhans cells. Each of these cell types contributes by its own functions to the essential role played in the body by the skin, in particular the role of protecting the body from external aggressions (climate, ultraviolet rays, tobacco, ...), called "function fence". Poor renewal of these cells and more particularly of keratinocytes, which is observed in particular with age, results in poor protection of the skin, the skin then having a dry and / or dull appearance.

The dermis provides the epidermis with solid support. It is also its nourishing element. It mainly consists of fibroblasts and an extracellular matrix itself composed mainly of collagen, elastin and a substance, known as the fundamental substance, components synthesized by the fibroblast. There are also leukocytes, mast cells or tissue macrophages. It is also crossed by blood vessels and nerve fibers.

Vimentin fibers are found in an important way in the dermis, since they correspond to the intermediate filament of fibroblasts. These vimentin fibers are also present in melanocytes and in Langerhans cells of the epidermis, they can also be present in keratinocytes when these are in a hyperproliferative state.

Keratins are the intermediate filaments of epithelial cells, such as keratinocytes in the skin. Thus, there are four types of keratin in the epidermis, including keratin 10, called K10, which is specific to the state of differentiation of keratinocytes.

With age, the quality of the skin decreases, in particular there is a thinning of the dermis. It is also recognized that extrinsic factors such as ultraviolet rays, tobacco or certain treatments (glucocorticoids, vitamin D and derivatives for example) also have a negative effect on the skin.

We therefore understand the importance of cell renewal and the quality of this renewal, both at the level of the epidermis and at the level of the dermis, so as to combat extrinsic aggressions which damage the skin, in particular by reducing its barrier function, and against the signs of skin aging, whether chronobiological or photo-induced.

One of the aims of the present invention is therefore to increase the rate of differentiation and / or proliferation of fibroblasts in the skin and / or to increase the rate of differentiation of keratinocytes in the skin so as to combat extrinsic aggressions, whether they either physical or chemical, which damage the skin, in particular by reducing its function barrier, and against skin aging, whether chronobiological or photo-induced.

The Applicant has now discovered that ascorbic acid applied topically to the skin increases the rate of differentiation and / or proliferation of skin fibroblasts and / or increases the rate of differentiation of keratinocytes in the skin by applying to the skin. a composition comprising an effective amount of ascorbic acid or at least one of its analogs.

Ascorbic acid (or vitamin C) is known to stimulate the synthesis of collagen, preventing, as a co-factor, the auto-inactivation of the enzymes lysine- and proline-hydroxylases and increasing the synthesis of mRNA of procollagens . Ascorbic acid (or vitamin C) is also known to stimulate the synthesis of elastin in the skin. We can cite in this regard US patents 5801192, US 4983382 and EP 0717983. We can also cite an article entitled "Pola to incorporate vitamin C in new cosmetics line for skin care" of the Japan Economy Journal of June 5, 1984 (page 15) . Thus, it has been described that ascorbic acid used in cosmetic compositions makes it possible to treat in particular wrinkles (Fragrance Journal, Vol.8, N ° 6 (45) (1980) pp38-43, "Cosmetic and vitamin -action and safety to dermatology ").

A subject of the invention is therefore the use of an effective amount of ascorbic acid or one of its analogs in a composition or in the preparation of a composition intended to be applied to the skin to increase the rate of differentiation and / or proliferation of skin fibroblasts and / or increase the rate of differentiation of skin keratinocytes.

A third object of the invention is the use of an effective amount of ascorbic acid or one of its analogs in a composition or in the preparation of a composition intended to be applied to the skin to stimulate the synthesis of cutaneous vimentin.

A fourth object of the invention is the use of an effective amount of ascorbic acid or one of its analogs in a composition or in the preparation of a composition intended to be applied to the skin to stimulate the synthesis of cutaneous keratin 10.

The invention further relates to a method for increasing the rate of differentiation and / or proliferation of fibroblasts of the skin in a person having an abnormally low rate of differentiation and / or proliferation of fibroblasts, comprising topical application to the skin with an effective amount of ascorbic acid or one of its analogs.

Another subject of the invention is a method for increasing the rate of differentiation of keratinocytes in the skin in a person having an abnormally low rate of differentiation of keratinocytes, comprising the topical application to the skin of an effective amount of ascorbic acid or one of its analogs.

Indeed, the Applicant has discovered that ascorbic acid applied topically to the skin makes it possible to increase the synthesis of mRNA of vimentin and thus to increase the rate of synthesis of vimentin. She also discovered that ascorbic acid applied topically to the skin increases the synthesis of keratin 10 mRNA and thus increases the rate of synthesis of keratin 10.

These proteins, intermediate filaments of skin cells, are therefore representative of the proliferating and / or differentiating state of skin cells, more particularly cells of the dermis and epidermis. More particularly, vimentin, which is the intermediate filament of fibroblasts, is representative of the proliferating and / or differentiating state of fibroblasts and keratin 10 is representative of the differentiating state of keratinocytes.

Thus, by topical application of an effective amount of ascorbic acid or its analogs, the renewal of these skin cells is faster, the appearance of the skin is improved, the skin is more radiant, less dull, firmer. , more toned, more elastic, wrinkles are reduced or their appearance is delayed, the skin signs of aging are reduced.

Advantageously, the ratio of the synthesis of mRNAs of vimentin to that of keratin 10 due to the topical application of ascorbic acid is comparable to that without topical application of ascorbic acid. This indicates that the skin condition, after topical application of ascorbic acid, is maintained in a normal state (without, for example, hyper-proliferation or -differentiation of one dermal or epidermal compartment relative to the other) .

The analogues of ascorbic acid are, more particularly, its salts, such as in particular sodium ascorbate, magnesium or sodium ascorbylphosphate, its esters, such as in particular its acetic, propionic or palmitic esters, or its sugars , such as in particular glycosilated ascorbic acid.

Ascorbic acid is usually in L-form because it is usually extracted from natural products.

The effective amount of ascorbic acid or its analogs which can be used according to the invention is of course that which is necessary to obtain the expected effects according to the invention. To give an order of magnitude, this quantity preferably represents from 0.001% to 20% of the total weight of the composition, preferably from 0.1% to 15% of the total weight of the composition. composition and advantageously from 3% to 10% of the total weight of the composition.

In addition, the composition of the invention is used for a time sufficient to obtain the expected effects according to the invention. To give an order of magnitude, this duration can be at least 15 days, but can also be more than 4 weeks, or even more than 8 weeks.

The composition of the invention intended for topical application contains a physiologically acceptable medium, that is to say compatible with the skin including the scalp, mucous membranes and / or the eyes and may in particular constitute a cosmetic or dermatological composition. .

This composition can be in all the galenical forms normally used in the cosmetic and dermatological fields, and it can in particular be in the form of an aqueous solution possibly gelled, of a dispersion of the lotion type possibly biphasic, of an emulsion obtained by dispersion of a fatty phase in an aqueous phase (O / W) or vice versa (W / O), or of a triple emulsion (W / O / W or O / W / O) or of a vesicular type dispersion ionic and / or non-ionic. These compositions are prepared according to the usual methods.

The composition of the invention can constitute, for example, a lotion, a gel, a cream or a milk, and for example a lotion or a cleansing or cleansing milk, a shampoo or a shower gel.

The following example illustrates the invention without limiting it in any way. In the compositions the proportions indicated are percentages by weight, unless otherwise stated. Example: 1. Method

We applied to the lower neck of 10 women between 55 and 60 years old for 3 months, once a day, on one side a water in oil emulsion (Vehicle or Placebo) and on the other side the same water emulsion in oil, but also including 5% vitamin C (= Composition or Active).

Composition: L-ascorbic acid 5.00%

Sodium hydroxide 1.83%

Citric acid, 1H20 1.24%

Disodium EDTA 0.05%

Apricot almond oil 3.00% Silicone oil 4%

Cyclopentasiloxane and dimethicone copolyol 20%

Dimethicone and dimethiconol 3%

Glycerin 23% Propylene glycol 4%

Charges 7%

Preservatives 0.30%

Water qs 100.00%

Biopsies of these treated surfaces are then carried out.

2. Extraction and purification of total RNA.

Biopsies are ground under liquid nitrogen in a Mikrodismembrator S (Braun). The powder obtained is collected in the teflon capsule with 2 ml of lysis solution (guanidine isothiocyanate 5M, mercaptoethanol, 0.1 M, lauryisucosyl of Na 0.017M, citrate Na 0.025M, pH7, antifoam 3 μl / ml). The suspension is transferred to a tube which is stirred at temperature room for 15 minutes. The lysate is deposited on the surface of a 1.4 ml cushion of 5.7 M cesium chloride, 0.1 M EDTA, pH 7 in a 3.8 ml polyallomer tube for the rotor SW60 (Beckman L70M ultracentrifuge ). Ultracentrifugation at 35,000 RPM is carried out for 18 hours at 20 ° C. The pellet is rinsed with absolute ethanol, centrifuged at 13,000 RPM, 4 ° C, 10 minutes and dissolved in 100 μl of distilled water.

3. Quantification of the concentration of total RNA and of specific mRNAs.

The amount of RNA harvested from biopsies is estimated by the optical density of the solution at 260 nm and then measured by amplifying the 28S ribosomal RNA by RT-PCR. The measurement of the specific mRNAs is carried out by quantitative RT-PCR on aliquots of the same dilution of total RNA, stored at -80 ° C. until their use.

Measurement of keratin 10 (K10) and vimentin mRNA

The specific oligonucleotide primers of the genes studied have 24 bases, have an A - T% close to 50% and are chosen from two different exons in order to avoid amplification of possible traces of DNA present in the samples. The optimal amplification conditions (temperature and number of cycles) were determined for each of the genes studied, taking into account their level of expression in the skin. RT-PCR is carried out using the Gen amp Amp rTth kit from Perkin Elmer or the Titam kit from Boehringer.

Each RT-PCR reaction is carried out in the presence of a known number of copies of a synthetic RNA created in the laboratory containing the sequences of the oligonucleotide primers specific for the mRNAs of interest and the amplification product of which has a molecular size allowing discriminate it from endogenous mRNA. This multistandard makes it possible to control and calculate the yield of the reverse transcription and of the amplification reaction. The amplification products are analyzed by electrophoresis in polyacrylamide gel followed by staining with CyberGreen. The intensity of the fluorescent signals is measured using a Fluoro S Multilmager. The results are corrected for the yield of RT-PCR and expressed in arbitrary units per unit of ribosomal 28 S RNA.

4. Statistical analysis

Statistical analysis was carried out using the unilateral Student t-Test on the ratios of Active (Vitamin C) / Placebo (= A / P) values.

t (n- 1) = (M A / P - 1) Vn M standard deviations

For a degree of freedom n-1 = 9, the A / P ratio is significantly greater than 1 with a probability greater than 95% for a value of t> 1.83 and a probability greater than 99% for a value of t> 2.82.

5. Results

Measurement of total RNA obtained from biopsies

The total quantity of RNA purified from biopsies is first evaluated by measuring the optical density at 260 nm and their quality estimated by measuring the OD ratio 260/290 nm.

Amply sufficient quantities of RNA were obtained from each of the biopsies (between 2.1 and 6.3 μg) with a satisfactory degree of purity (D.O. report 260/280).

The concentration of total RNA is brought by dilution to a calculated value of 4 nanograms per μl. This process makes it possible to carry out the reactions of reverse transcription and amplification on similar amounts of total RNA for all samples. The amount of total RNA present in the diluted solution is quantitatively determined by measurement of the 28S ribosomal RNA, carried out in tripiicate. This same RNA solution will be used for all measurements of specific RNAs whose results are expressed per unit of 28S RNA.

Measurement of the equilibrium rate of vimentin and keratin mRNAs 10

The results expressed in arbitrary units per unit of 28S RNA are detailed in Tables 1 and 2.

Table 1: Vimentin mRNA

Unilateral Student test: ^* t = 2.02, P <0.05

Seven out of 10 subjects have an equilibrium level of vimentin mRNA increased by ascorbic acid. Table 2: keratin 10 (K10) mRNA

Unilateral Student test: ** t = 2.95, p <0.01

Eight out of ten subjects have an equilibrium level of keratin 10 mRNA increased with ascorbic acid.

The vimentin / keratin 10 ratio is detailed in Table 3.

Table 3: Vimentin / keratin 10 ratio (VIM / KIO)

VIM / K10 subject Active Placebo a 7.22 6.05 b 6.88 [14.10] c 5.27 5.63 d 4.53 2.68 e 4.82 4.18 f 2.65 4.31 g 3.59 6.34 h 3.77 5.36 i 3.21 4.48 j 4.19 4.93

Average 4.61 4.88

These results indicate that the biopsies contain a proportion of mRNA of keratin 10 and of vimentin, comparable on the side treated with ascorbic acid and placebo. The results also indicate that the biopsies were performed uniformly in the different individuals. The fa-placebo sample is outside the norm.

When the measurements of vimentin are compared to the equivalent measurements made for the procollagen mRNA I or III, the average value of these reports calculated on the series of the treated samples and of the placebo samples is very close indicating a coordinated modulation of the expression of the procollagens and vimentin. In addition, if we consider that vimentin is representative of the dermal compartment, since it is the intermediate filament of fibroblasts, then the increase in the expression of procollagens I and III is accompanied by a parallel increase in expression of vimentin and suggests that ascorbic acid induces either an increase in the number of connective cells of the dermis or the activation of their biosynthetic phenotype.

Claims

1. Use of an effective amount of ascorbic acid or one of its analogs in the preparation of a composition intended to be applied to the skin to increase the rate of differentiation and / or proliferation of skin fibroblasts.

2. Use of an effective amount of ascorbic acid or one of its analogs in the preparation of a composition intended to be applied to the skin to increase the rate of differentiation of the keratinocytes of the skin.

3. Use of an effective amount of ascorbic acid or one of its analogs in a composition or in the preparation of a composition intended to be applied to the skin to stimulate the synthesis of cutaneous vimentin.

4. Use of an effective amount of ascorbic acid or one of its analogs in a composition or in the preparation of a composition intended to be applied to the skin to stimulate the synthesis of cutaneous keratin.

5. Use according to one of the preceding claims, characterized in that the analogs of ascorbic acid are chosen from its salts, its esters, and its sugars.

6. Use according to the preceding claims, characterized in that the analogs of ascorbic acid are chosen from sodium ascorbate, magnesium and sodium ascorbylphosphate, its acetic, propionic, palmitic esters and glycosylated ascorbic acid .

7. Use according to any one of the preceding claims, characterized in that the amount of ascorbic acid or its analogs represents from 0.001% to 20%, preferably from 0.1% to 15%, and advantageously from 3% to 10% of the total weight of the composition.

8. A method for increasing the synthesis of skin vimentin in a person deficient in skin vimentin comprising the topical application to the skin of an effective amount of ascorbic acid or one of its analogs.

9. A method for increasing the synthesis of cutaneous keratin 10 in a person deficient in cutaneous keratin 10 comprising the topical application to the skin of an effective amount of ascorbic acid or one of its analogs.

10. A method of increasing the rate of differentiation and / or proliferation of skin fibroblasts in a person having an abnormally low rate of differentiation and / or proliferation of fibroblasts, comprising the effective topical application to the skin ascorbic acid or one of its analogs.

11. A method for increasing the rate of differentiation of keratinocytes in the skin in a person with an abnormally low rate of differentiation of keratinocytes, comprising the topical application to the skin of an effective amount of ascorbic acid or a product thereof analogues.