WO2001007006A1 - Association of fibrillin and a cyanophyta extract, preparation method and use as medicine - Google Patents

Abstract

The invention concerns an association of fibrillin with a cyanophyta extract and a composition containing said association and more particularly containing a fibrillin having a concentration ranging between 0.0001 and 20 wt. % relative to the composition total weight. Said composition can be used as medicine, in particular for healing stretch marks. Said composition may also be used in cosmetics to improve the appearance of the skin and/or its appendages, to reduce disorders related to skin ageing and/or complications of certain healing processes such as stretch marks.

Description

 Association of fibrilin and a cyanophycea extract, process for its preparation and its use as a medicament

The present invention relates to a combination of fibrillin and an extract of cyanophyceae, its use as a medicament in particular for the treatment of induced skin aging and / or for the treatment of certain scarring processes such as stretch marks and a cosmetic treatment process. using the application of a composition comprising a combination of fibrillin and an extract of cyanophyceae. Aging is a multi-factorial process.

In addition to intrinsic skin aging, known as chronological aging, which is genetically programmed, there is an induced skin aging in which various extrinsic factors participate. Among these, UV and oxidative stress play an important role (Aging mechanisms: from genetics to epigenetics; application of new concepts to skin aging - L. Robert, AM Robert, J. Med. Esthe. And Chir. Derm., Vol XXIV, June 97, 75-78, 1994).

In the dermis, we observe a decrease in fibroblast activity with age (decrease in the rate of proliferation, biosynthesis and exchange with the extra-cellular matrix), as well as an alteration of the support fibers (both qualitative than quantitative) (A. CHUTTANI, B. GILCHREST, Handbook of physiology-aging, chapter 11, p. 312-313).

At the dermo-epidermal junction, there is a flattening which results in a decrease in cohesion between these two zones (dermis and epidermis), with rarefaction of the elements of the dermis and as well as anchoring fibers, including collagen VII (YQ CHEN , A. MAUVIEL J. Ryynänen, S. Sollberg J. UITTO: Type VII collagen _ge no term by human skin fibroblasts and keratinocytes in culture Influence of donor age and cytokine responses, J. Invest Dermatol (1994).. , 102, (2), 205-209). This results in a decrease in metabolic exchanges and the formation of wrinkles. Stretch marks appear to result from an attack on the fibroblastic cell associated with a predominant damage to the collagen tissue and structural glycoproteins. The origin of the stretch mark formation seems to be a modification of the fibroblast metabolism (resulting in poor healing) as well as a break in contact between the cell and the matrix. A therapeutic approach to stretch marks consisting in improving or activating fibroblastic metabolism was then suggested (Stretch marks: fibroblast disease? Etiopathogenic theories, Cohen-Letessier, Nouv. Dermatol., 17, 431-435, 1998).

We find in the prior art numerous proposals for combating skin aging.

One of the most recent proposed by Sanofi Concept is an active ingredient capable of stimulating cellular communication through interleukin-6, thereby improving cell renewal.

A combination of retinol and oligosaccharide is used for its anti-aging properties, with in particular a synergistic activity on the synthesis of collagen (ROC patent n ° WO 98/38980).

An extract of Bertholletia (family Lecythidaceae) is proposed to stimulate collagen VII of the dermo-epidermal junction (LVMH patent n ° FR 2752527).

The present patent application proposes to find a solution to the problem of induced aging, which results in particular in the reduction of the activity of the fibroblast as well as the reduction of exchanges between the cell and its environment.

Surprisingly, it has been demonstrated that a combination of fibrillin and an extract of cyanophyceae stimulates the metabolic activity of cells, improves cell-matrix exchanges and the organization of support fibers (in particular the elastin). These two active ingredients have been shown to act in synergy. The present invention relates to a (synergistic) association of fibrillin and an extract of cyanophyceae, the fibrillin possibly being present in form associated with collagen, a pharmaceutical composition containing this association and a cosmetic composition containing it. The invention relates more particularly to a composition containing said combination of fibrillin and an extract of cyanophyceae and more particularly to a composition containing a solution of fibrillin with a concentration of between 0.0001% and 20% by weight relative to the total weight of the composition and a solution of an extract of cyanophyceae with a concentration of between 0.0001% and 20% by weight relative to the total weight of the composition.

Said pharmaceutical composition may be in the form of a cream, a serum, a mask, a body milk of a food supplement, a transdermal device.

Said present invention also relates to a process for the preparation of said combination or of said composition, characterized in that at least fibrillin and an extract of cyanophyceae are mixed. Said composition can be used as a medicament, in particular intended to combat skin aging, or intended to aid in healing, in particular in the case of stretch marks. It then preferably comprises at least one pharmaceutically acceptable adjuvant chosen from excipients, thickeners, preservatives, perfumes, dyes, collagens (including collagen V), chemical or mineral filters, hydrating agents, thermal waters. , hormone substitutes.

Said composition can also be used as a cosmetic composition, it then preferably comprises at least one cosmetically acceptable adjuvant chosen from excipients, thickeners, preservatives, perfumes, dyes, collagens (including collagen V), chemical or mineral filters, hydrating agents, thermal waters, hormone substitutes.

The present invention also relates to a cosmetic treatment process comprising the administration of the cosmetic composition according to the present invention, this cosmetic treatment process being in particular intended to improve the appearance of the skin and / or integuments or also intended to reduce disorders related to skin aging, and / or complications of certain scar processes such as stretch marks. Fibrillin is a glycoprotein found at the dermal level

(Fibrillin, a new 350 kD glycoprotein, is a component of extracellular microfibrils, Lynn Y. Sakai, Douglas R. Keene and Eva Engvall, the journal of Cell. Biology., Vol. 103, n ° 6 Pt 1, 2499-2509 , Dec. 1986). It forms microfibrils of 10 nm in diameter participating in particular in the assembly of elastic fibers. These microfibrils appear before elastin in tissue development and form a scaffold on which the elastin molecules are deposited. They are also found isolated at the dermo-epidermal junction.

Marfan syndrome, which results in the risk of rupture of connective tissues rich in elastic fibers (wall of the aorta, ...) is due to a mutation in the fibrillin gene.

UV, responsible for photoaging, modulates the expression of fibrillin (UVB irradiation alters cellular responses to cytokines: role in extracellular matrix gene expression, VP Werth, KJ Williams, EA Fisher, M. Bashir, S. Rosensloom and Xiaomei Shi, J. of Invest. Dermat, vol. 108, no.3, March 1997).

In addition, a decrease in fibrillin in stretch marks has been observed (Fibrillin microfibrils are reduced in skin exhibiting striae distensae, REB Watson, EJ Parry, JD Humphries, CJP Jones, DW Poison, CM., Kielty, and CEM Griffiths, British J. of Dermat, 138, 931-937, 1998). In the prior art, the gene encoding fibrillin has been introduced into fibroblasts to aid in the healing phenomenon (UNIV.JEFFERSON THOMAS patent WO 9704647-A1).

The structure and function of fibrillin have been preserved during the evolution of the species (An extracellular matrix protein ofjellyfish homologous to the fibrilline forms different fibήls depending on the life stage of the animal, S. Reber-Muller, T. Spissinger , P. Schuchert, J. Spring

& V. Schmid, Dev. Bioi, 169, 662-672, 1995).

Fibrillin can be obtained by extraction from cnidarians, especially jellyfish, where it is responsible for the elasticity of the parasol. In this type of extraction, the fibrillin obtained is associated with collagen related to the type V of vertebrates in a weight ratio of fibrillin 15 / collagen 85. It is also possible, by various methods, to increase the quantity of fibrillin and modify this ratio. Pure fibrillin can also be used.

The fibrillin used in the composition according to the present invention can be in the form of pure fibrillin or associated with collagen, the collagen is then not purified and it contains a non-negligible proportion of fibrillin. The fibrillin used in the composition according to the present invention can be obtained by implementing the process for preparing fibrillin described in patent application FR 98 07 078 filed on June 5, 1998. It can also be used in the form of collagen prepared according to the process described in patent FR 2 714 063, analyzes carried out on the collagen obtained according to this process revealed the presence of fibrillin in sufficient proportions for the preparation of the composition according to the present invention (fibrillin / collagen weight ratio: 15 / 85). The fibrillin, in the form of collagen, used in the composition according to the present invention can also be obtained from the company JAVENECH, this product is sold under the reference "Fibriline-Javenech". In the present invention, fibrillin is used in dermo-cosmetic preparations for its organizational properties of the connective tissue, with surprisingly synergies of activity in the presence of cyanophycea extract, as shown in the work which follows .

The process for the preparation of cyanophycea extracts and their growth factor-type activity are described in patent FR 2643559; they are extracts of filamentous cyanobacteria, in particular of the Phormidium species, obtained by fractionation of their cytosolic content.

Study of the adhesion of human fibroblasts to their matrix support: Synergy of activity in the presence of cyanophycea extract

This potentiation has been demonstrated on an in vitro cell model: the test of adhesion of human fibroblasts to a matrix support (Majewski et al., Journal of Investigative Dermatology vol. 98 (1): 86-91, 1992). The extracellular matrix is a primordial and essential medium surrounding the cells; as an adhesion support, it generates, via cellular receptors, cell / matrix or cell / cell interactions, thus governing certain biological functions of our organism (Aumalley et ai, Exp. Cell Research vol.181: 463 -474, 1989). Molecular adhesion phenomena are therefore of capital importance, contributing to the structural integrity of tissues and organs.

The fibroblasts are preincubated with the cyanophycea extract and, thus treated, are put to adhere to a collagen support supplemented with fibrillin or not. The adhesion rate is evaluated by cell count. A statistical analysis by the Student test was performed to compare the effect of fibrillin and cyanophycea extract in synergy with the additive effect fibrillin + cyanophycea extract. The following table shows that the combination of fibrillin (in the form of solutions of concentrations: 1 μg / ml; 2.5 μg / ml; 5, μg / ml) and of cyanophycea extract in the form of solution of concentration equal to 50 μg / ml has a potentiating effect significant, increase in the rate of adhesion of fibroblasts to their matrix support.

Fibrillin alone

Cyanophyceae extract alone

Fibrillin / Cyanophyceae extract (50 ug / ml)

Effect on cell metabolism in single-layer culture: Potentiation in combination with an extract of cyanophyceae.

The protocol followed is that of the metabolism of XTT (J. of Immunological Methods, 1993, 157, 225-231) in EM medium on a fixed population of fibroblasts line L929. The proliferation of cells in the presence of the combination according to the invention was compared with the proliferation of cells when the latter are subjected to fibrillin alone or to an extract of cyanophyceae alone. The cells studied are an L 929 line, the increase in cell proliferation was measured 72 hours after adding the fibrillin and or the cyanophycea extract.

Figure 1 illustrates the increase in cell proliferation: the first measurement represents the percentage of cell proliferation when an aqueous solution of an extract of cyanophyceae at a concentration of 1 mg per 100 g is added to the medium. The second measurement represents the percentage of proliferation of the cells when an aqueous solution of fibrillin of concentration 1 g per 100 g is added to the medium. The third measurement represents the percentage of proliferation of the cells when an aqueous solution of an extract of cyanophyceae of concentration 1 mg per 100 g and a solution of fibrillin of concentration 1 g per 100 g is added to the medium.

At a concentration of 1 mg per 100 g of cyanophycea extract, an increase of + 17% in the cell population is observed. An aqueous solution of fibrillin, at a concentration of 1 g per 100 g, has an action of + 49%.

When the cyanophycea extract is combined with fibrillin, at the same doses as above, the activity is increased by + 81%, which reflects the significant potentiating effect of this association on cell proliferation.

The preparation formulas based on a combination of fibrillin and cyanophycea extract presented below are given for information; they are not exhaustive and therefore in no way limit the scope of the present invention. The components of these preparation formulas presented are expressed in percentages by weight. They can be extended to all categories of cosmetic products, oral supplements, transdermal devices and pharmaceutical products requiring a marketing authorization.

MOISTURIZING DAY CREAM

- Cyanophyceae extract 0.0001 to 20%

- Fibrillin 0.0001 to 20% - Pyrrolidone carboxylic acid 0.2 to 0.5%

- Ethoxylated hydrogenated castor oil 1 to 5%

- Fluid paraffin oil 1 to 5%

- Cyclopentasiloxane oil 5 to 12%

- Squalane 1 to 5% - Carboxyvinyl polymer 0.1 to 0.5%

- Triethanolamine 0.2 to 0.5%

- Glycerin 2 to 4%

- Curator q.s.

- Perfume q.s. - Purified water q.s.p. 100g

NUTRITIVE CREAM FOR VERY DRY SKIN

- Cyanophyceae extract 0.0001 to 20%

- Fibrillin / Collagen (15/85) 0.0001 to 20%

- Polysaccharide derivative 0, 1 to 4%

- Stearic acid 1 to 3%

- Polyoxyethylene glycol stearate 2 to 6% - Vegetable wax 1 to 3%

- Synthetic whale white 1 to 2%

- Jojoba oil 2 to 8%

- Glycerin 2 to 5%

- Preservative, perfume, qs - Purified water qs 100 g FIRMING ANTI-WRINKLE MASK

- Cyanophyceae extract 0.0001 to 20% - Fibrillin 0.0001 to 20%

- Hyaluronic acid (Na salt) 0.01 to 0.2%

- Sucrose stearate 1 to 3%

- Sucrose distearate 2 to 5%

- Capric and carprylic triglycerides 2 to 8% - Liquid paraffin 5 to 12%

- Polyoxyethylene glycol 600 2 to 10%

- 0.2 to 1% carboxyvinyl polymer

- Triethanolamine 0.5 to 1%

- Perfume q.s. - Preservatives q.s.

- Purified water q.s.p. 100g

FIRMING ANTI-WRINKLE SERUM

- Cyanophyceae extract 0.0001% to 20%

- Fibrillin / Collagen (90/10) 0.0001% to 20%

- Wheat protein 3%

- Carrageenane (chondrus Crispus) 3% - Colorant q. s.

- Preservatives q. s.

- Demineralized water qs 100 g ANTI-STRETCH TREATMENT

Cyanophyceae extract 0.0001 to 20% - Fibrillin 0.0001 to 20%

Glycolic acid 10%

Guanidine carbonate 10%

Dimethicone copolyol / cyclomethicone 5% Decamethyl cyclopentasiloxane 10g - Glycerol 20g

Sodium chloride 1 g

Preservatives q. s. Dyes q. s. Perfume q. s. - Drinking water q.s.p. 100g

ANTI STRENGTHENING BODY MILK

- Cyanophyceae extract 0.0001% to 20% - Fibrillin / Collagen (15/85) 0.0001% to 20%

- Sorbitan ester 1 to 2%

- Ethoxylated sorbitan ester 1 to 3%

- Vaseline oil 4 to 6%

- Fatty ester 1 to 5% - Carboxyvinyl polymer 0.2 to 0.5%

- Glycerin 3 to 10%

- Vitamin E acetate 0.2 to 1%

- Curator q.s.

- Colorant q.s. - Perfume q.s.

- Purified water q.s.p. 100g

Claims

1) Association of fibrillin and an extract of cyanophyceae. 2) Association according to claim 1, characterized in that the fibrillin is present in the form associated with collagen. 3) Composition characterized in that it contains a combination according to claim 1 or 2. 4) Composition according to claim 3 characterized in that it contains a fibrillin solution of concentration between 0.0001% and 20% by weight relative to the total weight of the composition and a solution of an extract of cyanophyceae of concentration between 0.0001% and 20% by weight relative to the total weight of the composition. 5) Composition according to one of claims 3 to 4 characterized in that it is in the form of a cream, a serum, a mask, a body milk of a food supplement, d '' a patch. 6) A method of preparing the combination according to claim 1 or 2 or the composition according to one of claims 3 to characterized in that at least fibrillin and an extract of cyanophyceae are mixed. 7) Composition according to one of claims 3 to 5 as a medicament. 8) Composition according to claim 7 characterized in that it comprises at least one pharmaceutically acceptable adjuvant chosen from excipients, thickeners, preservatives, perfumes, dyes, collagens (including collagen V), chemical or mineral filters, hydrating agents, thermal waters, hormone substitutes. 9) Composition according to claim 7 or 8 as a medicament intended to combat skin aging. 10) Composition according to claim 7 or 8 as a medicament intended to aid in healing, in particular in the case of stretch marks. 11) Composition according to one of claims 3 to 5 as a cosmetic composition. 12) Cosmetic composition according to claim 11 characterized in that it comprises at least one cosmetically acceptable adjuvant chosen from excipients, thickeners, preservatives, perfumes, dyes, collagens (including collagen V), chemical filters or minerals, hydrating agents, thermal waters, hormone substitutes. 13) Cosmetic treatment method characterized in that a composition according to one of claims 11 or 12 is administered. 14) Cosmetic treatment method according to claim 13 for improving the appearance of the skin and / or integuments. 15) Cosmetic treatment method according to claim 13 intended to reduce the disorders linked to skin aging, and / or to the complications of certain scarring processes such as stretch marks.