MXPA97010255A - Ribosomal fraction and composition that the conti - Google Patents

Abstract

The present invention relates to a ribosomal fraction of at least one non-photosynthetic filamentous bacterium as well as a cosmetic or pharmaceutical composition comprising the same.

Description

RIBOSOMAL FRACTION AND COMPOSITION CONTAINING IT DESCRIPTION OF THE INVENTION The present invention relates to a ribosomal fraction of at least one non-photo-synthetic filamentous bacterium. Ribosomes are corpuscular formations of 140 to 230 Angstroms formed by ribonucleic acids associated with proteins and which are present in the cells of all organisms, in particular bacteria. Ribosomes are the site of complex reactions that allow the synthesis of proteins thanks to the multienzyme complex that builds them. In prokaryotes, ribosomes have a constant sedimentation d to 1 order of 70S (Svedberg: International Unit that measures the sedimentation rate of bodies subjected to standard centrifugation). Morphologically, the ribosomes are divided into 2 sub-units of unequal sizes by a groove - perpendicular to its major axis. These 2 subunits have respectively a sedimentation constant of the order of 50 S and 30 S. The subunit 50 S contains two ribonucleic acids respectively of 23 S and 5 S and - about thirty proteins, while the sub-unit 30 S contains a ribonucleic acid of 16 S and a vein-ref: 26173 protein tena. It is thus understood that the term "rj_bosomal fraction" corresponds to the fraction of media enriched in ribosomes that can be obtained, for example after centrifugation, by separating the different organisates that constitute the cells of an organism. Thus, a ribosomal fraction can therefore contain, whatever its degree of purity, either - it is at least one whole ribosome, or at least one constituent element of a ribosome or else it is a constituent element of one of the subunits of a ribosome. Until now, ribosomal fractions have been used, for example in cosmetics, in the preparation of compositions intended to delay skin aging by stimulating cell growth and modulating the maturation of connective tissue (EP-A-631773). In medicine, compositions are known which are intended to reinforce the immune defenses in particular of patients who have experienced severe burns and are thus sensitive to opportunistic infections caused by bacteria, viruses or fungi (WO 91 / -11174) or even with regard to diseases of the otorhinolaryngological field (FR 2253499, FR - - - 2360314, FR 2388563, FR 2674755, ZA 8801071), allergies (US 4946945). The ribosomal fractions are also used in the preparation of adjuvants for vaccines (FR 2374911, DE 1617809). Generally, the ribosomal fractions used in these inventions are prepared from Gram-negative bacteria such as those of the Enterobacteriaceae family (for example Klebsiella pneumonias, Escherichia coli, Serratia marcessens), or from the family of the Pasteureaceae (for example Haemophilus influenzae), or Gram-positive bacteria such as, for example, those of Bacilus falale (for example Bacillus subtilis), of the Streptococcus family (for example Streptococcus pneumoniae, Streptococcus pyogenes Gr A), or from the family of Lactobacilli ceas (for example Acidoplu lus Bificum), or from yeasts such as, for example, Candida albicans or Candida tropicalis. Although the ribosomal fractions of the prior art are satisfactory, it is not surprising that it is interesting to investigate new ribosomal fractions prepared from organisms hitherto not used in this field. One of the purposes of the invention is therefore to propose a new ribosomal fraction capable of being used at least in the areas indicated above, possibly in new areas. The invention thus has as its object, as a new product, a ribose fraction prepared from at least one non-photosynthetic filamentous bacterium. The ribosomal fraction, as specified above, can contain, whatever its degree of purity, either at least one entire ribosome, or at least one constituent of a ribosome or an elem; constitutive of one of the sub-units of a riboso ^ ma. The immune system comprises a set of specialized cells subjected to multiple control mechanisms that ensure their renewal, activation and differentiation, essential for a normal level of immunocompetence. The role of the immune system is to discriminate whether or not to eliminate pathogens and spontaneous tumors. Any cellular decrease, any immune dysregulation or any functional deficit is likely to favor the production of pathological manifestations characterized by the disturbance of the mechanisms of recognition with respect to it that is not, and a greater sensitivity to assaults. microbial and neoplastic processes. The skin is the most important organ of the body and is recognized as one of the main active elements of the immune defense system. Three types of epidermal cells participate in this system: the keratinocytes, the melanocytes and the cells of Langerhans. These cells, which are only found at the level of the skin, play a key role in the immune response and, in particular, in the antigenic presentation. Healthy skin is able to defend itself against external aggressions thanks to the means placed at its disposal. However, it is subject to the - permanent environmental degradation, chemical products, radiation. In particular, Langerhans cells are the preferred targets of ultraviolet radiation. These aggressions are translated by a suppressive effect of the immune defenses producing a low resistance to the pathogens and an increase of the incidence to certain cancers. To help the skin to fulfill its immune function, the stimulation products of the cutaneous intnutary system are of great interest. It is also known that the immune system and more particularly that of the skin weakens during the course of chronological aging. This debilitation also occurs in the course of photo-induced aging. An immunostimulatory effect can then restore the unitary functions and more particularly those of the epidermis, reinforcing the natural defenses of the skin. Another purpose of the invention is therefore to propose a new immunostimulant, more particularly of the immune system of the skin. Another object of the invention relates to a cosmetic or pharmacological composition, characterized in that it comprises at least one ribosomal fraction of at least one non-photosynthetic filamentous bacterium. The ribosomal fraction of at least one non-photosynthetic filamentous bacterium has a remarkable power of stimulation of the immune system. Thus, the invention more specifically has as its object a cosmetic or pharmaceutical composition comprising, as an immunostimulant principle, at least one ribosomal fraction of at least one non-photosynthetic, non-latent bacterium. Preferably, the ribosomal fraction is designed to stimulate the immune system of the skin. This stimulation of the immune system of the skin is particularly interesting in the course of chronobiological aging and / or photo-aging. The ribosomal fraction of the invention comes from the ribosomal fraction of the invention comes from bacteria selected from non-photosynthetic filament bacteries such as those defined according to the Bergey's Manual of Systematic atic bacteriology classification (vol.3, sections 22 and 23, 9th edition, 1989), - among which we can mention the bacteria belonging to the genus of the Beggiatoales and more particularly the bacteria belonging to the genera Beggi toa, Vitreosci 1 la, Flexithríx or Leucothrix. Bacteria that have just been defined and of which several have already been described generally have an aquatic habitat and can be found particularly in marine waters or in hot springs. Among the usable bacteria, for example: Vitreoscilla filiformis (ATCC 15551) Vitreoscilla beggiatoides (ATCC 43181) Beggiatoa alba (ATCC 33555) Flexithrix dorotheae (ATCC 23163) Leucothrix ucor (ATCC 25107) Sphaerotilus natans (ATCC 13338) Preferably, it is used in accordance with invention a strain of Vitreoscilla filiformis. Any method of preparation of ribosomal fraction known to the person skilled in the art can be used in accordance with the invention. In particular, we can mention the methods described by Norris and Ribbons in "Me thods in Microbiology", 1973, (Academic Press). In the compositions according to the invention, the ribosomal fraction of at least one non-photosynthetic filamentous bacterium represents 0.0001% to 20% of the total weight of the composition and preferably 0.01 to 10% of the total weight of the composition. composition. Depending on the fraction, it is used in a composition that must be ingested, injected or applied to the skin (throughout the area of the skin of the body), the messes, nails or mucous membranes (buccal), malar, gingival, genital, anal, conjunctive) this composition can be presented in any of the galenic forms normally used. For a topical application on the skin, the composition can be in the form of, in particular, aqueous or oily solution or dispersion of the type of solution or serum, of emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous (H / E) or an inverse (E / H) phase, or in suspensions or emulsions of a soft or a cream or aqueous gel or - anhydrous type, or even of microcapsules or microparticles, or of vesicular dispersions of ionic and / or non-ionic type. These compositions are prepared according to the usual methods. They can also be used for the hair or scalp in the form of aqueous, alcoholic or hydroalcoholic solutions, or in the form of creams, gels, emulsions, foams or even in the form of aerosol compositions comprising also a pressure propellant. For injection, the composition may be in the form of an aqueous, oily lotion or in the form of a serum. For the eyes, it can be presented in the form of drops and for ingestion. It can be presented in the form of capsules, granules, syrups or tablets. The quantities of the different constituents of the compositions according to the invention are those conventionally used in the areas considered. For topical application on the skin, these compositions constitute particularly cleansing, protective, treatment or care creams for the face, hands, feet, for large anatomical or body wrinkles (for example, example day creams, night creams, make-up removal creams, foundation creams, anti-solar creams), make-ups, make-up milks, body care or care milks, sun-protection milks, lotions, gels or foams for skin care, such as cleansing lotions, anti-sun lotions, artificial-tanning lotions, bath compositions, deodorant compositions comprising a bactericidal agent, after-shave gels or lotions, depilatory eyelids , compositions against insect bites, anti-pain compositions, compositions for treating certain diseases of the skin such as eczema, acne rosacea, psoriasis, lichen, itching severe, dermatitis. The compositions can also be packaged in the form of an aerosol composition also including a pressurized propellant. The ribosomal fraction of at least one non-photosynthetic filamentous bacterium used according to the invention can also be incorporated into various compositions for hair care, and particularly shampoos, marking lotions, treating lotions, creams or gels for combing, dye compositions (particle oxidation dyes) eventually in the form of - coloring shampoos, restructuring lotions for hair, permanent compositions (particularly compositions for the first phase of a permanent), anti-hair loss lotions or gels, antiparasitic shampoos, etc. The compositions can also be of use - dental-dental, for example a toothpaste. In this case, the compositions may contain adjuvants and additives customary for compositions for oral use and particularly surface active agents, thickening agents, wetting agents, polishing agents such as silica, various active ingredients such as fluorides, in particular sodium fluoride and eventually sweetening agents such as sodium saccharinate. When the composition is an emulsion, the proportion of the fatty phase can range between 5% and 80% by weight, and preferably between 5% and 50% by weight relative to the total weight of the composition . The oils, waxes, emulsifiers and coemulsifiers used in the composition in the form of emulsification are chosen from those conventionally used in the cosmetic field. The emulsifier and the uluting component are present, in the composition, in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5 to 20% by weight relative to the - total weight of the composition. The emulsion may also contain lipid vesicles. When the composition is a solution or an oily gel, the fatty phase may represent more than 90% of the total weight of the composition. In a known way, any cosmetic composition - may also contain adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers , filters, odor absorbers and coloring matters. The amounts of these different adjuvants are those conventionally used in the cosmetic field, and for example from 0.01% to 10% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the aqueous phase and / or the lipid spherules. As oils or waxes which may be used in the invention, mention may be made of mineral oils (petrolatum oil), vegetable oils (liquid fraction of charcoal butter, sunflower oil), a-ni oils (perhydrosqualene), synthesis - (Purcellin oil), silicone oils or waxes (cycloTieticone) and fluorinated oils (perfluoro-polyethers), beeswax, carnauba or paraffin waxes. Fatty alcohols and fatty acids (stearic acid) can be added to these oils. As emulsifiers which can be used in the invention, mention may be made, for example, of glycerol stearate, polysorbate 60 and the mixture of PEG-6 / PEG-32 / Glycol-D Stéarate sold under the name of Tefose 63 by the company Gattefosse. As solvents usable in the invention, lower alcohols can be mentioned, particularly ethanol and isopropanol, propylene glycol. As the hydrophilic gelling agents which can be used in the invention, mention may be made of carboxyvinyl 1-cos polymers (carbomer), acrylic copolymers such as acrylate / alkylaryl copolymers, polyacrylamides, polysaccharides such as hydroxyprilcellulose. , natural gums and clays, and, as lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids such as aluminum stearates and hydrophobic silica, ethylcellulose, polyethylene. The composition may contain other hydrophilic active agents such as proteins or protein hydrolysates, amino acids, polyols, urea, allantoin, sugars and sugar derivatives, water-soluble vitamins, plant extracts and hydroxy acids. As lipophilic active substances, you can use -retinol (vitamin A) and its derivatives, tocopherol (vitamin E) and its derivatives, essential fatty acids, ceramides, essential oils, salicylic acid and its derivatives.According to the invention, it is possible, inter alia, to associate a ribosomal fraction of at least one non-photosynthetic filamentous bacterium with other active agents intended in particular for the prevention and / or treatment of skin conditions. Among these active agents, mention may be made, by way of example: the agents that modulate the differentiation and / or the proliferation and / or the pigmentation of the skin such as retinoic acid and its isomers, retinol and -sus esters, vitamin D and its derivatives, estrogens such as estradiol, cojic acid or hydroquinone; - antibacterials, such as indamycin phosphate, erythromycin or antibiotics of the tetracycline class; - antiparasitic agents, in particular metronidazole, crotamiton or pyrethrinoids; - antifungals, in particular compounds belonging to the class of imidazoles such as econazole, ketoconazole or miconazole or their -salts, polyene compounds, such as amphotericin B, compounds of the allylamines family , such as terbinafine, or also octopirox; - antiviral agents such as acyclovir; - anti-steroidal anti-inflammatory agents, such as hydrocortisone, betamethasone valerate or clobetasol propionate, or non-steroidal anti-inflammatory agents such as ibuprofen and its salts, diclofenac and its salts, acid acetyl salicylic acid, acetaminophen or glycyrrhetinic acid; - anesthetic agents such as lidocaine hydrochloride and its derivatives; - antipruritic agents such as tenaldi-na, trimeprazine or cyproheptadine; keratolytic agents such as alpha- and beta-hydroxycarboxylic acids or beta-ketocarbo-xylic acids, their salts, amides or esters and more particularly hydroxy acids such as glycolic acid, lactic acid, salicylic acid , citric acid and generally fruit acids, and n-octanoyl-5-salicylic acid; - anti-free radical agents, such as alpha-tocopherol or its esters, superoxide dismutase, certain metal chelators or ascorbic acid and its esters; - anti seborrheic drugs such as progesterone; - antiplatelet agents such as octopirox or zinc pyridine; - anti-acne drugs such as retinoic acid or benzoyl peroxide. Thus, according to a particular mode, the invention relates to a composition containing at least one ribosomal fraction of at least one non-photosynthetic filamentous bacterium and at least one agent selected from antibacterial, antiparasitic, antifungal, antiviral, anti-viral agents. inflammatories, anti-prurig_i_ nos, anesthetics, ikitic keratol, anti-free radicals, anti-seborrheic, antipelicular, anti-acne and / or agents that modulate differentiation and / or --proliferation and / or cutaneous pigmentation. The subject of the present invention is also a method of cosmetic treatment with a view to stimulating the immune system and in particular the immune system and in particular the skin's immune system, characterized in that it is applied to the skin, over the hair, and / or on the mucous membranes, a composition as described above. The cosmetic treatment process of the invention can be carried out in particular by applying the hygienic or cosmetic compositions such as those defined above, according to the customary use technique of these compositions. For example; - application of creams, gels, serums, lotions, make-up milks or anti-sun compositions on the skin or on dry hair, application of a lotion for hair on wet hair, de-shampoos, or also application of toothpaste on the gums. The following examples and compositions illustrate the invention without limiting it to any node. In the compositions the indicated proportions are percentages in step, unless otherwise mentioned. Example 1: Preparation of a ribosomal fraction of Vitreoscilla -fi 1 iformis: A culture of Vitreoscilla filiformis was scraped according to the procedure described by the applicant in the French patent application published under No. 2700172. 100 ml were then centrifuged. I cultivate 10 minutes at 13000 revolutions per minute to 4! C. The supernatant was recovered. Ammonium sulfate was added at a pH of 7.5 at a rate of 210 mg / ml. A new centrifugation was performed in the same conditions as before. The supernatant was dialysed (dialysis-spectra membranes, porosity 1000 MWCO (approximately -1000 Daltons) against a Tris-HC1 buffer at a pH of 7.6, 70 ml of ribosome-enriched medium were obtained. of a ribosomal fraction of Vitreoscilla fili-form on the growth and on the differentiation-of the 7-week Balb / c mouse splenocytes: The ribosomal fraction of Vitreoscilla filiformis of example 1 was tested at different concentrations in a model representative of the activity of a product on the immune system: growth and differentiation of mouse splenocytes --Balb / c at an age of 7 weeks.The potential activity of the ribosomal fraction was evaluated; a) by the measurement of the incorporation of the iodine into the deoxyribonucleic acid (process index); The negative control is represented by the effect of the culture msdio on the splenocytes and the positive control by the effect of 1 ipopol isaccharides - (LPS) of reference to 0.001%. The LPS used of the LPS of Escherichia coli, sold by the Society -Signa. Tritiated thymidine incorporation procedure.

The mouse splenocytes were distributed in 96-well NUNC round-bottom culture dishes at 250,000 cells per well in a final volume of 200 ml. The products to be tested were added in the wells at different concentrations. Cell proliferation was evaluated by the incorporation of tritiated thymidine (1 iCi / pot 1 lo). 18 hours before the end of the culture, 10 joule of tritiated thymidine was added at 100 μCi / ml in cac, to the culture well. 18 hours later, the cells were recovered on a filter with the help of a Tontee type cell harvester. The filter was then dried and covered with a scintillation liquid. The radioactivity deposited on the filter was determined by compaction in a tip a counter. Proliferation was calculated by means of an index-stimulation according to the following formula: Radioactivity assay - Control radioactivity (cells + medium) Radioactivity control The results are presented in the table given below: (value of stimulation indices calculated according to the indicated formula previously) 24h 48h 72h 96h Ribosomal fraction 5% 1, 02 4.08 3.10 1.97 2.5% 1.84 8.18 3.03 2.87 1.25% 1.64 4.80 2.03 0.89 0, 63% 1, 61 4.56 3.81 1, 33 0.31% 1.63 3.97 4.66 4.16 LPS 0.001% 1, 50 5.47 13.64 16.48 0.0001% 1 , 03 1, 03 2.47 5.32 The ribosomal fraction increases the rates of proliferation very significantly. The maximum effect is obtained at the concentration of 2.5% and after 48 hours of culture. The effect is comparable to that obtained with the positive control. b) by measuring the production of immunoglobulins after differentiation of total splenocytes in B cells (B lymphocytes). The negative control was represented by the effect of the culture medium on the splenocytes and the positive control by the effect of 1 ipopol isaccharides (LPS) of reference to 0.001% and 0.0001%. The LPS used are the LPS of Escherichis coli, sold by the company - Sigma. Dosing of imunoglobulins: The cells were placed in culture in the same conditions as those described above. After 2 days of culture, the supernatants were extracted and dosed for the presence of immunoglobulins. (ig). This dosage was made with the help of a -Kit Pharmingen, according to the supplier's protocol. Immunoglobulins are proteins present in the blood that combine with the antigens for the purpose of the latter being recognized and identified as foreign bodies by B lymphocytes. Immunoglobulins are divided into different types and subtypes in function of the structure of the constant leg of its heavy chain. In the mouse, 5 classes are distinguished (Ig, M, G, A, D and E). Immunoglobulin G is the most abundant immunoglobulin in bodily fluids particularly extravascular where - it combats microorganisms and toxins. Immunoglobulins G in 4 sub-classes (1, 2, 3 and 4) can be classified. Immunoglobulin A is the most important immunotlobulin in seromucosal secretions where it defends the outer surfaces of the body. Immunoglobulin M is a very effective binding agent produced very early in the immune response. It is the first line of defense against bacteremia. Only Ig Gl, G2a, G2b, G3, M and A have been tested in this assay. The results are presented in the table given below: IgG 1 IgG 2a IgG 2b IggGG 33 IiggMM IgA Witness 1 1 1 1 1 1 LPS 0.001 3.4 3.7 3.2 3.4 2.9 3.5 LPS 0.0001 3 3.3 3.1 3.2 3 3 F.R. 5% 2.1 2.2 2.2 2.1 2.2 2.3 F.R. 2.5% 3.3 3.3 3 3.1 2.9 3.4 F.R. 1.25% 3.8 3.9 3.4 3.6 3.3 4 F.R. - ribosomal fraction. The ribosomal fraction sigificantly increases the production of immunoglobulins from all the classes and sub-classes studied. The optimal active concentration is 1.25%. These results are comparable to those obtained with the positive control. Example 3: Example of compositions according to the invention. These compositions were prepared by the classical methods known to the person skilled in the art. Cleaning cream Ribosomal fraction of example 1 0.50 Cetyl Alcohol 2.00 Glycerol stearate 2.00 Stearic Acid 2.00 Pol igl iceri 1-3 Hydroxy lauri 1 Ether 5.0 Codex Mineral Oil 12.00 Carbomer 0.35 Sodium hydroxide 0.15 Perfume with Meth Paraben 0.20 Sterile demineralized water 100, oo Cleaning milk Ribosomal fraction of example 1 0.50 Carbomer 0.40 Sodium hydroxide 0.10 Codex mineral oil 5.00 glycerol stearate l.oo Cetyl alcohol 0.50 PEG 100 Stearate 0.80 Methyl Paraben 0.20 Spray Perf sterile demineralized water esp 100.00 Lotion of care Ribosomal fraction of example 1 1, 00 Gl icerol 2, 00 Methyl Paraben 0. 1 5 Perfume e s p Sterile demineralized water e sp 100, 00 Care cream Ribosomal fraction of example 1 2.00 Glycerol stearate 1.00 PEG 100 Stearate 1.00 Stearic acid 1, 00 Cetyl alcohol 2.00 Soybean Oil 3.00 Palm oil 2,00 Cyclomethicone 2,00 Dimethicone 1, 00 Pol iacri lick 0.20 Gl icerol 3.00 Methyl Paraben 0.20 Spray Perf Sterile demineralized water esp 100.00 It is noted that in relation to this date, the best method known by the applicant to bring to the practice said invention, is that which is clear from the present description of the invention. The invention having been described as above, Property is claimed as contained in the following:

Claims (10)

R E I V I N D I C A C I O N S

1. - Ribosomal fraction, characterized because it is prepared from at least one filamentous bacterium not fotosiji thetica.

2. Ribosomal fraction according to claim 1, characterized in that said bacterium belongs to the genus - of the Beggiatoales.

3. Ribosomal fraction according to any of the preceding claims, characterized in that the indicated bacteria belongs to the genus Beggiatoa, Vitreoscilla, Flexi-thrix or Leucothrix.

4. Ribosomal fraction according to any of the preceding claims, characterized in that the bacterium indica is chosen among the strains of Vitreoscilla filiformis. 5.- Cosmetic or pharmaceutical composition, characterizes; da because it comprises at least one ribosomal fraction as defined according to any one of claims 1 to 4. 6. Composition according to claim 5, characterized in that the ribosomal fraction is an immunostimulating agent. 7. Composition according to claim 6, characterized in that the ribosomal fraction is an immunostimulating agent of the skin's immune system. 8. Composition according to any one of claims 5 to 7, characterized in that the ribosomal fraction represents 0.0001% to 20% of the total weight of the composition. 9. Composition according to claim 8, characterized in that the robosomal fraction represents 0.01% -10% of the total weight of the composition. 10. Cosmetic treatment procedure, characterized in that at least one cosmetic composition as defined according to any of claims 5 to 9 is applied to the skin, on the head and / or mucous membranes.