FR2654342A1 - Pharmaceutical and/or cosmetic composition containing a hypophyseal growth factor (HDGF) stabilised with soluble elastin - Google Patents

Abstract

Pharmaceutical and/or cosmetic composition containing a stabilised growth factor as active substance. The growth factor is the growth factor derived from hypophyses, or HDGF, stabilised with soluble elastin. Use in skin treatment, in particular in the process of cicatrisation and of making the appearance of the skin more attractive.

Description

 The subject of the present invention is a pharmaceutical and / or cosmetic composition based on a stabilized pituitary growth factor and its use in the treatment of the skin, in particular in the healing process and in improving its aesthetic appearance.

 Growth factors are polypeptide molecules whose in vivo and in vitro studies have made it possible to demonstrate various properties as agents for the multiplication and differentiation of cells of origin and of different species.

 For some of them, the amino acid sequence could be determined as well as the structure of the corresponding gene.

Among the tissue growth factors, those which have been the subject of in-depth studies due on the one hand to the multiplicity of the target cells concerned, or to the biological effects observed, and on the other hand to the therapeutic impact that the doctors and in particular dermatologists can hope, we must particularly mention the epidermal growth factor or EGF (Epidermal Growth Factor), the fibroblast growth factor or FGF (Fibroblast Growth Factor) as well as the growth factor derived from the eye or EDGF (Eye Derived
Growth Factor).

 EGF exerts an activity stimulating the proliferation of epithelial cells and can also play an indirect role in the regeneration of nerve fibers, in the healing of tendons and in the formation of blood vessels. EGF versus FGF has been shown to be especially effective for superficial lesions while FGF would be especially effective for deep lesions.

 EDGF is a growth factor that has been identified and isolated from the retina of adult beef. In culture, it promotes the growth of a large number of target cells such as endothelial cells, fibroblasts, myoblasts, chondrocytes but also keratinocytes.

 As with EGF, EDGF also seems to play a non-negligible role in the healing phenomena, at least in that concerning superficial wounds on the epidermis.

 Among the studies that have focused on EGF, mention may in particular be made of the articles by G. CARPENTER et al, Ann. Rev. Biochem., 48, 193-216 (1979) and L. E. KING et al, Progress In Dermatology, Vol. 19, N01, 1-8 (1985).

 Among the studies that have focused on the F.G.F. mention may in particular be made of the articles by D. GOSPODAROWICZ et al, J. Cell. Physiol.

Supp., 5 / 15-26 (1987) and GARY D. SHIPLEY et al., J. Cell. Physiol.

138 / 511-518 (1989).

Among the studies relating to the EDGF one can cite the articles of D. BARRITAULT et al, Journal of Neuroscience Research, 8: 477-490 (1982) and of AY FOURTANIER et al, The Journal of Investigative Dermatology,
Flight. 87, N 1, 76-80 (1986).

 In addition, studies have been undertaken to prevent the inactivation of growth factors, in particular FGF, and thus to protect their biological activity.

 Given the great interest of cell growth factors in the pharmaceutical and cosmetic fields, in particular to influence the modalities of skin aging, studies have been continued on the means to stabilize, and therefore prevent the inactivation of growth factors. of the FGF type using various protein carriers.

 It has now been discovered, quite unexpectedly and surprisingly, that soluble elastin and in particular kappa-elastin (K-elastin) constitutes a support of choice which makes it possible to conserve the initial activity of growth factors of the FGF type.

 The present invention relates more particularly to the stabilization of growth factors derived from pituitaries or HDGF (Hypophysis Derived Growth Factor).

 The present invention therefore relates to a pharmaceutical or cosmetic composition containing, as active substance, a stabilized growth factor, characterized in that the growth factor is the growth factor derived from pituitary or HDGF, stabilized by soluble elastin.

 HDGF contains FGF but also other growth factors contained in the pituitary gland, having in particular an EGF-like activity (Epidermal growth Factor-Like).

The preparation of HDGF can be carried out according to the method of
LEMMON and BRADSHAW (J. of Cell. Biochem. 21/3, 195-208, 1983) from pituitaries. In a particularly preferred embodiment of the invention, the HDGF is prepared from bovine pituitaries according to the following steps
1 ") Washing of the frozen pituitary glands in physiological liquid at around 40C,
- homogenization in an electric mill at low speed in an ammonium sulphate solution containing protease inhibitors, in particular leupeptin and pepstatin,
- agitation of the ground material at 40C,
- first centrifugation to recover a first extract,
- second centrifugation after recovery of the first residue in an ammonium sulphate solution containing protease inhibitors to recover the second extract,
20) Mixing of the two extracts and precipitation at 65% saturation with ammonium sulfate,
- recovery of the precipitate by centrifugation,
- solubilization in an ammonium sulphate solution at 25% saturation,
- dialysis of the supernatant against physiological fluid,
- clarification of the solution by high speed centrifugation,
- determination of the "growth factor" activity of the sterile solution (membrane sterilization) by biological test.

HDGF features
10 / Physical and chemical characteristics.

 - colorless to yellowish and odorless, slightly translucent aqueous solution, pH 6.5.

 - protein concentration: <500g / ml.

 2 / Analysis of the peptide composition by electrophoresis in polyacrylamide gel 6.25% -12.5%.

 This analysis shows that the HDGF preparation does not contain proteins with a molecular weight greater than 66 KD. The majority of peptides have a molecular weight of + 20 KD compared to the proteins of known molecular weight used as references as well as peptides of smaller sizes.

 3 / Determination of biological activity.

 The assay is carried out by measuring the stimulation of cell multiplication on a line of mouse embryonic fibroblasts, the line Balb 3T3.

 The biological unit is defined by the amount of RDGF that must be added to obtain 50% of the maximum stimulation obtained with 5% of fetal calf serum.

 The details of these studies on the biological activity of HDGF alone or associated with soluble telastin will be described below.

 By the method as described above, in general, from 1 kg of bovine pituitary glands, 5 to 10 million units of mitogenic activity are obtained.

 Soluble elastin or kappa-elastin (K-elastin) is a mixture of peptides obtained by solubilization of fibrous or polymeric elastin by a process of degradation which is managed either by elastases, for example pancreatic elastase, or by solvents such as potash in an aqueous-organic medium, for example 1M potash in 80% ethanol.

 The size of the peptides which compose it and which are coacervable have a molecular weight generally between 10 RD and 60 RD.

For its preparation and its properties, one can usefully refer to the following articles
L. ROBERT and N. POULAIN "Studies on the structure of telastin and the traction mode of elastase. I. New method for the preparation of soluble derivatives of telastin". Bull. Soc. Chim. Biol. (Paris), 1963, 45, 1317-1326,
L. ROBERT "The connective tissue: I'elastine", published in "Précis de Physiologie cutané" Ed. Meynadier J. Editions de la Porte Verte, Paris, 1980, 155-173, and
M. MENASCHE, MP JACOB, and L. ROBERT "Pharmacological studies on elastin peptides (K. elastin)". Path. Biol. 1981.29, no 9.548-554.

 Its preparation has also been described in French Patent No. 76.16602 (2,353,282).

 This lyophilized soluble elastin is in the form of a fine, light yellow, slightly hygroscopic powder having no particular odor.

 The aqueous and alcoholic solutions from 1 to 30% are perfectly clear at room temperature and at + 40C.

 The pH of a 5% solution is around 6.5 ~ 0.5 at 200C.

 As soluble elastin, it is possible to use according to the invention that sold by the company SODICHIMIC S.A. under the name of "Kappa-elastin SCD".

 Although the stabilization of HDGF by soluble telastin can be carried out extemporaneously during the manufacture of pharmaceutical or cosmetic compositions, it is preferred, according to the invention, to proceed to the preliminary mixing of the soluble telastin and the HDGF solution, then to carry out a lyophilization followed by sterilization.

 The studies that have been carried out have shown that lyophilization does not significantly alter the biological activity of HDGF.

 EDGF solution after freezing: 16.1 U / ml.

 HDGF solution after lyophilization: 14.1 U / ml: loss of activity of 12%.

 HDGF and elastin solution soluble after freezing 17.0 U / ml.

 HDGF and elastin solution soluble after lyophilization 14.6 U / ml: loss of activity of 9%.

 The ratio of the solution of HDGF and soluble elastin is generally between 500 and 100,000 units of mitogenic activity per gram of soluble elastin and preferably between 5,000 and 50,000 units, the preferred ratio being 10,000 mitogenic units.

 The conservation tests made it possible to show that in aqueous solution, at room temperature, the association was perfectly stable for several months.

 The lyophilized preparation is perfectly stable, provided moisture is avoided.

Measurement of the biological activity of HDGF incorporated in a gel in the presence and in the absence of soluble elastin
Technical
An HDGF solution containing 800 U is lyophilized in the absence and in the presence of 50 mg of soluble elastin. The lyophilized material is taken up in 0.5 ml of distilled water which is incorporated into 5 g of the following neutral gel
Crosslinked polyacrylic acid sold under the name of
"CARBOPOL 940" by the company GOODRICli .. 50%
Dehydroacetic acid 0.4%
0.1% sodium methyl parahydroxybenzoate
0.25% sodium propyl parahydroxybenzoate
Triethanolamine qs pH = 7.4
Purified water qs 100 g
This incorporation therefore leads to a gel containing 1% soluble elastin and 16,000 U / 100 g. The gel is dissolved in a culture medium containing 0.1% fetal calf serum (quiescent medium) at a rate of 10 mg / ml. After dissolving the gel, two dilutions are made to test in culture 0 , 5 and 2.5 mg of gel.

 - At the concentrations tested (0.5 and 2.5 mg), the gel alone does not have cytotoxic activity with respect to fibroblasts in culture.

Soluble elastin alone has no activity. The gel containing the
HDGF alone (2.5 mg of gel: 0.4 U of HDGF) has a mitogenic activity measured at 0.065 U while the gel containing HDGF and soluble telastin has a mitogenic activity measured at 0.12 U.

The measurements are made in duplicate with a deviation of + 10% compared to the average.

 In conclusion, it is possible to find a biological activity after incorporation of HDGF in a gel. The presence of soluble elastin protects this activity.

 The pharmaceutical and cosmetic compositions, intended to be applied to the skin, preferably contain from 5 to 1000 mitogenic units per gram of composition and preferably from 20 to 100.

 These compositions are preferably aqueous compositions whose pH should preferably be between 5.5 and 8.5.

 These compositions can be in the form of lotions, gels, creams, ointments or even aerosol sprays.

 Preferably the compositions are in the form of aqueous lotions or oil-in-water or water-in-oil emulsions or vesicular systems.

 These compositions make it possible to combat the degradation and aging of skin tissues, to act against redness, burns caused by the sun or can also promote the healing of wounds or burns.

 They may of course also contain other usual pharmaceutical or cosmetic ingredients such as preservatives, perfumes, oils, alcohols, fatty esters or vitamins.

 We will now give by way of illustration and without any limiting character, an example of preparation of the association of HDGF and soluble elastin, the HDGF being obtained from bovine pituitaries, as well as several examples of pharmaceutical compositions. and dermocosmetics.

Example of preparation
A - Frozen bovine pituitaries (1ka,) are washed several times in physiological liquid at 40C. They are then homogenized in an electric mill with 2 liters of a 0.15M ammonium sulphate solution containing protease inhibitors (ethylene diaminotetracetate lmM, leupeptin 4 mg, pepstatin A 4 mg).

 The ground pituitary is stirred at 40C for 2 hours. The first extract is recovered by centrifugation for 20 minutes at 7,000g. The pituitary residue is then reextracted a second time for 18 hours at 40C, with 1 liter of the same solution. The second extract is recovered by centrifugation at 7,000g.

 The two extracts are combined and brought to 65% saturation with ammonium sulfate. After a few hours of precipitation, the insoluble material is recovered by centrifugation at 4 "C at 12,000 g. The material precipitated with 65% ammonium sulfate is then partially dissolved in a solution of ammonium sulfate at 25% final saturation The insoluble material is removed by centrifugation at 12,000 g The supernatant is then dialyzed against a large volume of physiological liquid at 40C with a Spectrapor dialysis membrane with a cut-off of 3,500 daltons.

 After two changes of the dialysis bath, the solution is clarified by high speed centrifugation (20,000 RPM for 30 min.) And sterilized on a 0.22 micron EK membrane.

 A biological test is carried out on increasing volumes of this solution (from 0.05 to 5 ′).

 B - To 1 liter of the HDGF solution obtained above (corresponding to 10 million units of mitogenic activity), 1 kg of soluble elastin (Kappa-elastin SCD) is added in the form of sterile powder.

After homogenization of this mixture maintained at 40C, one proceeds, according to conventional methods, to its lyophilization.

Examples of compositions
1 - Aqueous lotion
"HDGF + Kappa-elastin SCD" at 10,000 mitogenic units / g of
Kappa-elastin 0.5 g
Lavender essence ........................................ 0.1 g
Witch hazel water 10.0 g
Sorbitol at 70% ............................................ 4, 0 g
Sodium salt of ethylenediamine tetracetic acid 0.1 g
Polyoxyethylene hydrogenated castor oil with 60 moles of ethylene oxide 0.3 g
Methyl parahydroxybenzoate ............................ 0.15 g
Distilled water ........................................ qs 100 g
2 - O / W emulsion "HDGF + Kappa-elastin SCD" at 10,000 mitogenic units / g of
Kappa-elastin 1 g
Polyethylene glycol 400 .................................... 3 g
Propylene glycol ........................................... 4 g
Triethanolamine 0.6 g
Sweet almond oil ....................................... 2 g
Isopropyl myristate ..................................... 1 g
Esters of capric / caprylic acids and C12-Cl8 fatty alcohols sold under the name of "Cetiol LC-WD"
By the company HENKEL ...................................... 1 g
Cetyl alcohol ........................................... 3 g
Etearic acid ............................................ 3 g
Non-self-emulsifiable glycerol mono-di-stearate 3 g
Perfume 0.1 g
Methyl parahydroxybenzoate ............................ 0.2 g Water qs 100 g 3 - W / O emulsion "HDGF + kappa- elastin SCD "at 10,000 mitogenic units / g of
Kappa-elastin 2 g
Magnesium lanolate (50% in oil) 14.4 g
Lanolin alcohol ......................................... 3.6 g
Sunflower oil ......................................... 15 g
Isopropyl myristate ..................................... 4 g
Codex white petrolatum ..................................... 10 g
Vitamin F ................................................ 0.5 g
Ascorbic acid 0.5 g
Perfume 0.8 g
Methyl parahydroxybenzoate ............................. 0.15 g
Propyl parahydroxybenzoate ............................. 0.15 g Water qs 100 g

Claims (9)

 1. Pharmaceutical or cosmetic composition containing, as active substance, a stabilized growth factor, characterized in that the growth factor is the growth factor derived from pituitary or HDGF, stabilized by soluble elastin.  2. Composition according to claim 1, characterized in that the HDGF is obtained from bovine pituitaries.  3. Composition according to any one of claims 1 and 2, characterized in that the HDGF is obtained according to the following steps  i) Washing of bovine pituitary glands in physiological fluid,  - homogenization in an ammonium sulfate solution  containing protease inhibitors,  - agitation of the ground material,  - first centrifugation to recover a first extract,  - second centrifugation after recovery of the first residue in  ammonium sulfate solution containing inhibitors  protease to recover the second extract,  ii) Mixing of the two extracts and precipitation,  - recovery of the precipitate,  - solubilization in an ammonium sulphate solution,  - dialysis of the supernatant against physiological fluid,  - clarification by centrifugation, and  - determination of the "growth factor" activity of the  sterile solution.  4. Composition according to any one of claims 1 to 3, characterized in that the HDGF is stabilized by soluble lelastin, prior to its incorporation into said composition, by mixing soluble lelastin and a solution of HDGF, followed by '' lyophilization and sterilization.  5. Composition according to any one of claims 1 to 4, characterized in that the ratio of the solution of HDGF and soluble elastin is between 500 and 100,000 mitogenic units per gram of soluble elastin.  6. Composition according to claim 5, characterized in that the ratio of the solution of HDGF and soluble elastin is between 5,000 and 50,000 mitogenic units per gram of soluble elastin.  7. Composition according to any one of claims 1 to 6, characterized in that it contains from 5 to 1000 mitogenic units per gram of composition and preferably from 20 to 100 mitogenic units.  8. Composition according to any one of claims 1 to 7, characterized in that it is in the form of a lotion, an oil-in-water or water-in-oil emulsion d '' a gel, a cream, an ointment, an aerosol spray or vesicular systems.  9. Composition according to any one of claims 1 to 8, characterized in that it also contains at least one pharmaceutical or cosmetic ingredient taken from the group consisting of a preservative, a perfume, an oil, an alcohol, a fatty ester or a vitamin