USE OF AN OLIGOSACCHARIDE AS AN IMMUNOMODULATOR IN A DERMATO-COSMETIC COMPOSITION

Abstract: The invention relates to the use of at least one oligosaccharide comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or of a derivative of such an oligosaccharide substituted by a hydrophobic residue, for the preparation of an immunomodulating medicament. It also relates to a dermato-cosmetic composition and a cosmetic treatment of hyperreactive skins.

Description: USE OF AN OLIGOSACCHARIDE AS AN IMMUNOMODULATOR IN A DERMATO-COSMEΗC COMPOSITION The present invention relates to compounds useful in the field of immunology and in particular for the treatment of hyperεensitivity reactions responsible for allergies and variouε intolerance phenomena. Immunological defence against infectious agentε, toxins or neoplasms operates via the recognition of the foreign nature of an antigen (or allergen) and the activation of cellular or humoral mediators aiming to eliminate the foreign antigen. However, this mechanism can sometimes be undesirable, when it is too intense, or takes place on encountering environmental antigens which are not intrinsically noxious; this is likewise the case during organ transplantation or tisεue graftε. Immunological reactions at the root of allergy have been classified into 4 types: - Type I: The mast cells are bound to IgE antibodies by their F c receptor; the fixing of the antigen triggers the degranulation of the mast cells and the liberation of mediators (histamine, SRS-A, ECF-A) . - Type II: The specific antibodies (IgG or IgM) react with the antigen on the surface of target cells; this causes cytolysis, either by direct action of the K cells, or by activation of the complement. - Type III: The antibodies (IgG or IgM) form, with the antigen and the complement, immune complexes which are deposited in the tisεues and cause the production of chemotactic factors for polynuclear neutrophilε; local inflammation results. - τ YP e IV: The T lymphocytes sensitized to the antigen react with this and liberate lymphokines . The lymphokines induce an inflammatory reaction and cause the influx of macrophageε. The lymphocytes are thus key cells in immunity with cellular or humoral mediation, especially with the secretion of antibodies. Allergic ailments can be the expression of one or more types of immunological reactions. These reactions can likewise be at the root of intolerance reactions to hygiene and care products, observed in skins said to be "hyperreactive". These phenomena are due to intrinsic genetic factors and to acquired hypersensitivity caused by the environment. Unexpectedly, the applicant has now found that these hypersensitivity phenomena could be controlled by oligosaccharides comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or derivatives of such oligosaccharides substituted by a hydrophobic residue. The present invention relates to the use of oligosaccharides comprising from 2 to 6 oside residues and having a galactose residue in the non-reducing terminal position, or of their derivatives, for the preparation of immunomodulator compositions, and in particular of compositions intended for the treatment or prevention of hypersensitivity reactions . Particularly appropriate oligosaccharides could be chosen from the group formed by melibiose, lactose and their derivatives capable of being obtained by addition of a hydrophobic residue. Hydrophobic εubstituents are especially understood as meaning linear or branched Ci-Cis alkyls, Ci-Ciβ alkylamines, linear or branched, optionally substituted Cι-Cχ8 carboxylic acids, linear or branched, primary, secondary or tertiary Ci- Ci8 amides, and Cι-Cχ8 arylalkyls. The oligosaccharide derivatives suited for carrying out the invention can especially belong to one of the categories mentioned below, in which the oligosaccharide corresponds to the following general formula: galactose- n (α or β) - (Hex)p in which n represents the position 1, 2, 3, 4 or 6, Hex represents an α- or β-linked hexose or pentose, p is a number between 1 and 5; a) - glycosides corresponding to the formulae: • (I) oligosaccharide 1-O-R, in which R is a linear or branched alkyl residue of 1 to 18 carbon atoms, • (II) oligosaccharide 1-0-R-O-l-oligosaccharide in which R = (CH2)m m being between 2 and 10, b) - an osylamine acylated according to one of the following formulae, in which the oligosaccharide is preferably lactose, melibioεe or εtachiose: - oεylamines acylated corresponding to one of the following formulae : • (III) oligosaccharide 1-NH-CO-R, in which R is an alkyl residue of 2 to 18 carbon atoms, containing 0, 1 or 2 double bonds, • (IV) oligosaccharide 1-NH-CO-R-CO-NH-l-oligosaccharide, in which R = (CH2)m m being between 2 and 8, c) - an alkyla ine acylated by an aldonic acid obtained by oxidation of an oligosaccharide • (V) oligosaccharide -CO-NH-R, in which R has the same meaning as in formula (III) , • (VI) oligosaccharide CO-NH-R-NH-CO-oligosaccharide, where R has the same meaning as in formula (III) , d) - or a reduction product of Schiff bases formed by oligosaccharides with aliphatic mono- or diamines, and corresponding to one of the following formulae: • (VII) Gal- (Hex) n -X-HN-R, • (VIII) Gal- (Hex) n -X-HN-R-NH-X- (Hex) n -Gal, in which: Hex is a hexose or a pentose, n = 0, 1 or' 2, X = l-NH2-hexitol, and R has the same meaning as in (III) . According to a particularly advantageous method, the oligosaccharide or its derivative such as defined above will be used to prepare a composition containing, in addition, pharmaceutically acceptable excipients suited to administration by the external topical route. In fact, the applicant has found that the reactions involved in the disorders associated with hypersensitivity can be correlated with the fixation, on a specific lymphocyte receptor, of peptides resulting from the degradation of elastin, especially kappa-elastin. The activation of these receptors, triggers the liberation of lytic enzymes, β-glucuronidase, elastases and free radicals such as the superoxide ion, or hydrogen peroxide, which generates hydroxyl radicalε. These products can involve the degradation of macromolecules of the extracellular matrix, fibronectin, collagen and hyaluronan. The fixing of the elastin peptides likewise stimulates the proliferation of lymphocytes. These phenomena play an important part in the immunoallergic reactions of atopic origin, especially of atopic dermatitides or anaphylactic reactions, urticaria and allergic contact dermatitides. The subjects having skins said to be "sensitive" or hyperreactive are more and more numerous in the population. Such a skin reddens or smarts easily, and its reactivity threshold is lowered with respect to other skins. The subject experiences cutaneous discomfort, associated with erythema, and a fine desquamation can take place. All these symptoms will be ameliorated or even suppressed by the use of compositions containing oligosaccharides according to the invention; melibiose, lactose, or their derivatives, partially or totally suppress cellular proliferation, in particular of the previously stimulated lymphocytes; they can likewise inhibit the potentialization and liberation of lytic enzymes . The compositions will preferably be formulated in vehicles not comprising any perfume or allergenic agents . The compositions could be in the form of solutions, gels, lotions, creams, W/O or O/ emulsions, or multiple emulsions or in liposomal form. They will be adapted by the person skilled in the art, preferably using emollients or mild surfactants. The compositions are particularly suited to the treatment or to the prevention of intolerance and/or allergic reactions of the skin and/or of the mucous membranes. In particular, the compositions according to the invention are intended to prevent or to decrease the formation of free radicals. The oligosaccharides or their derivatives could be associated with other agents enabling the skin to be protected and/or hydrated, such as hyaluronic acid, vitamin E, glycol extract of G. JbiloJba, εorbitol, glucosaminoglycans, alginateε, etc.; they can likewise be associated with vegetable oils to nourish the skin and/or with active emollients and salves such as, for example, extracts of cat grape, althaea, oats, linden, camomile, sweet clover, Ruscuε, procyanidols, α-bisabolol, coconut oil, 18-β-glycyrrhetinic acid. According to another aspect of the invention, the oligosaccharides and their derivatives such as defined above are used for the preparation of a composition which additionally contains pharmaceutically acceptable excipients, suited for administration by the parenteral or enteral route. The efficacy of the oligosaccharides according to the invention is based on their surprising activity on cell- mediated immune reactions. The compositions prepared according to the invention are particularly useful for the treatment or prevention of hypersensitivity reactions mediated by lymphocytes. They are particularly intended for the treatment or for the prevention of symptoms of an ailment chosen from amongst atopy, polymorphous erythemae, xerodermititides, lupus erythematosus, pemphigus, dermatitides, psoriasis and eczemas. The oligosaccharides having a galactose residue in the non-reducing terminal position, and their derivatives substituted by a hydrophobic residue, can be utilized as adjuvants limiting the hypersensitivity reactions capable of being provoked by another active principle. The invention likewise relates to a method of cosmetic treatment of hyperreactive skins, characterized in that a composition containing at least one oligosaccharide comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or a derivative of such an oligoεaccharide substituted by a hydrophobic residue, is applied by the topical route in a cosmetically acceptable vehicle. Particularly preferred compounds for putting this method into practice are melibiose, lactose and their derivatives capable of being obtained by addition of a hydrophobic residue. The examples which follow are intended to illustrate the invention. In these examples, reference will be made to the following figures : Fig. 1: Inhibition by lactose and melibiose of the activity of stimulation of lymphocyte proliferation by K-elastin. The concentrations of lactose and of melibiose are 1 μg/ml, 10 μg/ml, 100 μg/ml, 1 mg/ml and 2 mg/ml. Fig. 2 inhibition by lactose and melibiose of the expression of elastic activity by lymphocytes, stimulated with 2 μg/ml of K- elastin. Activity in the culture medium. Fig. 3: Inhibition by lactose and melibiose of the expression of cathepsin G activity of lymphocytes stimulated with 2 μg/ml of K-elastin. Activity in the culture medium. Example 1 Methods Separation of lymphocytes The lymphocytes used for these experiments were obtained starting from circulating human blood and likewise from human tonsil after tonsillectomy . The isolation of the peripheral lymphocytes is carried out as follows: 5 ml of blood were deposited (in 15 ml centrifugation tubes) on Ficoll-paque plus (Pharmacia) with care before separating the two phases, then centrifuged at 3000 rpm (say 600 g) for 40 min at ambient temperature (20 °C) . The layer containing the lymphocytes and the monocytes is recovered and mixed with 10 ml of RPMI medium and centrifuged at 2200 rpm (say 400 g) for 10 min at 20 °C. The sediment is resuspended in 0.5 ml of RPMI, then an additional 4.5 ml of RPMI are added and a new centrifugation is carried out at 1500 rpm (say 400 g) for 10 min at 20°C. The final sediment is taken up in 10 ml of RPMI and the cells are counted. The monocytes and acrophages are eliminated by adhesion on a plastic surface (incubation for 2 hours in a C0 2 /O a incubator) . Separation of the poly iclear cells (PMN) After the first centrifugation at 3000 rpm (say 600 g) , the residue containing the red corpuscles and the PMNs are resuspended in 1% polyvinyl alcohol (PVA) in DPBS (Dulbecco's modified phosphate-buffered saline), (two volumes per volume of sediment) , then allowed to sediment for 20 min at ambient temperature. The supernatant is then centrifuged for 5 min at 400 g at 4°C, the sediment is washed with DPBS in order to eliminate the PVA by gentle agitation, without activating the PMNs, followed by centrifugation at 400 g at 4°C. The supernatant is eliminated. The red corpuscles are lysed by osmotic shock then an excess of DPBS is added in order to reestablish osmotic equilibrium. After centrifugation for 5 min at 400 g at 4°C, the sediment, containing the PMNs, is taken up in a small volume of RPMI. The cells are counted and can be lysed to liberate the lytic enzymes (elastase, cathepsin) by addition of 0.1% Triton X-100 in 1 M NaCl at 0°C for 20 min, followed by centrifugation for 20 min at 0°C. The supernatant contains the enzymes . Preparation of lymphocytes starting from human tonsils Freshly removed tonsils are cut into small pieces under sterile conditions, in 10 ml of RPMI. The tissue suspension is filtered on a coarse filter in order to remove the largest tissue debris. The suspension is placed in a test tube and allowed to sediment for 15 min at ambient temperature. The supernatant is placed on Ficoll-paque plus and centrifuged as described for lymphocytes derived from blood. The monocytes are eliminated as described for the lymphocytes derived from blood. The sediment containing the lymphocytes is resuspended and the cells are counted after two centrifugations as described. The lymphocytes obtained starting from tonsils are approximately 50% of the type T and 50% of the type B. The lymphocytes of blood origin are approximately 80% of type T and 20% of type B. Culture of the lymphocytes The lymphocytes isolated as described above are cultured in 24-well Costar plates, 500 μl of cell suspension/well (5 x 10 s cells/ml or 2.5 x 10 s cells) in RPMI 1640 medium (Eurobio) made up with 10% foetal calf serum (ATGC) , 2 mM glutamine (Gibco) , penicillin and streptomycin (500 U/ml, 0.25 mg/ml), and phyto- haemagglutinin (SIGMA) 5 μg/ml. After incubation for 4 days under cell culture conditions (37°C, C0 2 /0 2 incubator) the cell suspension is recovered with a pipette, centrifuged at 400 g for 10 min at 20°C and the cell sediment is taken up in 0.5 ml of RPMI (without FCS) , dispersed by agitation and then after addition of 10 ml of RPMI without FCS, centrifuged again at 1500 rpm (say 400 g) for 10 min at 20°C. After two more washings as described above, the sediment is taken up in 10 ml of RPMI without FCS and the cells are again cultured in a 24-well Costar plate. Action of the elastin peptides A sterile solution of x-elastin (75 kD, Solabia) is added at the concentration indicated (for example 2 μg/ml) to cells put back into culture in the medium without FCS for 2.5 hours at 37 °C. After incubation, the plates are gently agitated, and the cells are recovered by pipetting and counted. The suspension is centrifuged for 10 min at 4°C at 1500 rpm (say 400 g) , and the supernatant is distributed in 1 ml Eppendorf tubes at a rate of 500 μl per tube and kept at -40°C if it is not used immediately for enzymatic determinations. The cell sediment is resuspended in extraction buffer (0.1% Triton X-100, 1 M NaCl, 0.02% NaN 3 0.01% Brij 35, pH 8) . 1 ml per 10 s cells, agitated for 15 min at 1600 g at 0°C and the supernatant obtained by centrifugation as described above is redistributed in Eppendorf tubes and kept at - 40 °C for the enzymatic determinations. Determination of the enzymatic activity of leucocyte elastase- type The synthetic substrate used for determining the enzymatic activity of elastase- type is Me-O-Suc-Ala- Ala-Pro-Val-pNA. 50 μl of culture medium or 20 μl of cell lysate are mixed with the buffer (100 mM tris-HCl, 0.05% CaCl 2 , 0.02% NaN 3 , 0.01% Brij 35, pH 8) up to 190 μl, then with 10 μl of 85 mM substrate solution (in N-methylpyr- rolidone) for a total volume of 200 μl . The optical density is read immediately at 410 n and then after 4, 24, 48 and 72 hours' incubation at 37°C. The elastase activity is expressed in nM of substrate hydrolysed per 10 6 cells and per hour. Determination of the enzymatic activity of cathepsin G 25 g of the βubstrate, Me O-Suc-Ala-Ala-Pro-Met- pNA in 1 ml of N-methylpyrrolidone (40 nM) are used under the same conditions as above. 2 - Results A) Effect of elastin peptides on the proliferation of lymphocytes The passages of calcium produced by the addition of elastin peptides to the white cells of the blood can be considered as an indication of the intracellular signal triggering a set of cellular functions. One of these is the entry of cells into proliferation. It has been shown that this was the case for hviman skin fibroblasts in the presence of elastin peptides (Ghuysen- Itard et al, CR. Acad. Sci., 1992, 315 : 473-478). The results reported in Table 1 show that a stimulation analogous to the proliferation of lymphocytes was observed. Table 1 Concentration Number of experiments Percentage stimulation, of K -elastin mean ± SD 2 μg/ml 6 62 . 67 ± 37 . 90 10 μg/ml 13 35 .76 ± 29 . 50 A maximum stimulation is observed at 2 μg/ml of elastin peptides and a little weaker stimulation with 10 μg/ml of elastin peptides . B) Liberation of proteolytic activity by the elastin peptides When PMNs or lymphocytes are incubated under culture conditions in the presence of 2 μg/ml of K -elastin, a gradual increase in the elastase and cathepsin activity can be determined in the cell extracts and the cell lysates. This increase is much more marked in the cells derived from elderly atherosclerotic subjects. Such an increase is not observed in the absence of K -elastin. Addition of elastin peptides increases the proteolytic activity simultaneously in the culture medium (salted-out enzyme) and in the cell extract (enzyme bound to the cell) for elastase as well as for cathepsin G, although this effect may be much more pronounced for the liberation of enzymatic activity in the culture supernatant . C) Effect of elastin peptides on cell survival This experiment is carried out under the culture conditions such as described above, in the presence of phytohaemaglutinin (PHA) . 5 ml of aliquots of lymphocyte suspension (2.5 x 10 6 cells in 5 ml) obtained starting from human tonsils are distributed in test tubes and increasing concentrations of K -elastin (BPM, PM < 10 kDa) are added: 0.1 μg/ml, 2 μg/ml, 10 μg/ml, 500 μg/ml, 1 and 2 mg/ml. After 4 days' incubation under the culture conditions the cells are recovered and counted. Table 2 gives the cell lose as a function of the concentration of elastin peptides added. Table 2 Concentration of elastin peptides Percentage of cell loss μg/ml determined by Trypan Blue exclusion 0 5.3 1 5.0 2 2.5 10 17.4 100 19.6 500 28.9 1000 39.3 2000 39.1 It appears that elastin peptides exert a cytotoxic activity at high concentrations (a thousand times higher than the concentration stimulating cell proliferation and the salting-out of the lytic enzymes) . Example 2 : Effect of lactose and of melibiose on reactions mediated by the elastin receptor of lymphocytes 1) Inhibition of cell proliferation As shown in Figure 1, growing concentrations of lactose and of melibiose progressively suppress the growth stimulation effect of elastin peptides, followed by a net inhibition of proliferation at the strongest concentrations tested (1 and 2 mg/ml) , equivalent to 5.84 x 10 ~3 mM) . The strongest inhibition of proliferation is of the order of 16 to 30% for lactose and 26 to 58% for melibiose. 2) Effect of lactose and of melibiose on the salting-out of proteolytic enzyme triggered by the elastin receptor As shown above, an increase of 40 to 120% in the activity of proteolytic enzymes is observed in the culture medium as well as in the cell lysate of the cultivated human lymphocytes in the presence of 2 μg/ml of K -elastin. The stimulation of the liberation of a lytic enzymatic activity is inhibited effectively by melibiose starting from 1 μg/ml. This inhibition can be demonstrated as well for the elastase activities as for cathepsin G. Lactose likewise has an efficacy but it is less strong than that of melibiose (Figures 2 and 3) . Example 3 : Quantification of the elastin receptor in subpopulations of human lymphocytes Methods Antibodies used: - 1st antibody: 67 kD elastin/laminin anti-receptor antibody (bovine) (type: IgM) produced in mice (Elastin Products Company) . - 2nd antibody: mouse anti-IgG + IgM (H + L) antibody produced in goats, labelled by rhodamine (TRITC) (Jackson I munoresearch Laboratories) (for fluorescence microscopy) . 2nd antibody: anti-IgM antibody (F(ab') 2 of mice produced in goats, labelled by r-phycoerythrin (Chemicon) ) (for flow cytometry) . PROTOCOL: a) Fluorescence microscopy Human lymphocytes are isolated from tonsils (as described in Example 1) and cultured in the presence of 5 μg/ml of phytohaemagglutinin (to activate them) and for certain cultures with 2 μg/ml of kappa-elastin of high molecular weight (75 kD) in an RPMI 1640 medium with 10% foetal calf serum (FCS) . After 48 to 120 hours' incubation at 37 °C, the lymphocytes are washed three times with RPMI medium containing 2% FCS and then centrifuged (400 g, 10 minutes at 4°C) . Next, the cells are counted and the cell density is adjusted to 10 7 cells/ml with RPMI medium containing 2% FCS. Then a volume (100 μl) of cell suspension of human lymphocytes (approximately 10 6 cells) is incubated with 10 μl of solution of the first antibody (diluted to 1:100) for 30 minutes in an ice bath. 1.5 ml of cold RPMI are added to 2% FCS and then the cells are washed in a refrigerated centrifuge at 4°C (at 400 g for 10 minutes) . The solution of the second antibody (conjugated to TRITC) (diluted in cold RPMI to 1:200) is added to the plug. After gentle agitation of the suspension, it is incubated at 4°C for 30 minutes. Next, 1.5 ml of cold RPMI are added to 2% FCS and the suspension is centrifuged at 400 g at 4°C for 10 minutes. At the end of this last washing, the lymphocytes are resuspended in the residual medium after having removed the supernatant by decantation. Smears are then made on slides and dried in the air. After fixing with absolute ethanol for 5 minutes, the slides are rehydrated by immersion in several baths of PBS and mounted in buffered glycerol (9 volumes of glycerol and 1 volume of PBS). This post-fixation procedure increases the brightness of the immunofluorescence . To identify the labelled cells, microscopic fields are examined alternatively under ultraviolet illumination and in visible light. b) Fluorimetry At the end of the last washing, the lymphocytes are suspended in 1 ml of PBS and the cells are analysed by cytofluorimetry. The cell populations characterized are the following: - total T lymphocytes (CD3+) - "helper" T lymphocytes (CD4+) - suppressor T lymphocytes (CD8+) - B lymphocytes (CD20+) - T activated lymphocytes (CD25+) - memory T lymphocytes (CDA+/CD45RO+) - granulocytes (CD15+) Description of the labels used CD 3 The CD 3 complex is expressed by all mature human T cells. It has molecular weights of between 16 and 28 kD composed of 5 chains (γ, σ, e, ζ , η ) associated with the T-cell receptor in a non-covalent manner. It is involved in the transmission of activation signals. CD 4 CD 4 (T4) recognizes the class II MHC molecules during the interaction of CD4+ cells with cells presenting the antigen or target cells. It is a glycoprotein of 59 kD belonging to the immunoglobulin superfamily. It is found in the "helper/inducer" subpopulation of T lymphocytes (45% of the lymphocytes of peripheral blood) . CD 8 The CD 8 molecule (T8, 30/32 kD) is a glycoprotein formed of two peptide chains. It is found in the cytotoxic/suppressor subpopulation of the T lymphocytes (20-35% of the lymphocytes of the peripheral blood) . It is also present on NK cells and on 30% of the "null" cells of the peripheral blood. CD 15 The CD 15 antigen (3FAL, X-haptene, SSEA) is a lacto-N-fucopentose III (200-185 kD) . At least 5 major antigens of CD 15 are present on the surface of poly- nuclear cells (approximately 90% of the circulating human granulocytes) and on part of the circulating monocytes (30-60%) . This antigen is absent from the surface of normal lymphocytes . CD 20 The CD20 antigen is a phosphoprotein of 35/37kD. It is present on all normal B cells of the peripheral blood, of the tonsil and of the bone marrow. CD25 The CD25 molecule corresponds to the low-affinity receptor of interleukin-2. It is a glycoprotein of 55 kD expressed by activated lymphocytes (T and B) but also by activated macrophages. CD45RO This is a transmembrane glycoprotein of 180 kD (the isoform of low molecular weight of the leucocyte common antigen) (LCA) . It is present on the surface of T lymphocytes, thymocytes, granulocytes, monocytes (but is absent on the surface of macrophages) and on a small population of B lymphocytes. The T lymphocytes expressing CD45RO are memory T lymphocytes (or primed T cells) (45% of the T lymphocytes of the peripheral blood) . The CD4+/CD45RO+ cells produce "helper" signals. These are early producers of IL-2 and IFN-γ. For labelling with anti-CDx antibodies (Sigma products, excluding anti-CD25 antibody, from Serotec) , 100 μl of the lymphocyte suspension (already labelled or not) are taken with the 67 kD anti-receptor antibody of elastin at a cell density of 10 7 cells/ml. 10 ml of the anti-CDx antibody are added and the suspension is incubated for 30 minutes at 20°C (except with anti-CD25 antibody: incubation takes place at 0°C). Next, 2 ml of PBS are added. After two washings (two centriguations at 400 g for 10 minutes at 20°C), the cell plug is taken, 0.5 ml of PBS is added to it and the suspension is analysed by cytofluorimetry . The labellings of the cells are likewise carried out by isotype-similar mouse immunoglobulins (non-speci- fie myeloma proteins) (for control) . RESULTS a) Fluorescence microscopy Demonstration of the elastin receptor on activated lymphocytes Part of the analysed lymphocytes - those which express the 67 kD elastin/laminin receptor - show a specific immunofluorescence under the experimental conditions described above (positive cells) . The percentage of positive cells on the 4th day of culture is evaluated by fluorescence microscopy: - cells cultured in the presence of 2 μg/ml of kappa- elastin: 28.52 ± 12.60% - cells cultured without kappa-elastin: 28.09 ± 10.91% There is no significant difference between the two series, activation by PHA is a sufficient stimulus to make the elastin receptor express. The percentage of positive cells on the 5th day of culture: - cells cultured in the presence of 2 μg/ml of kappa- elastin: 77.2% + 6.7%. Effect of washing cells with lactose or melibiose: - cells cultured in the presence of 2 μg/ml of kappa- elastin + washed at the end of cell culture with a solution of 1 mg/ml of lactose: 26.6% ± 6.7% (p<0.001). Before washing, 77.2% of cells are positive - cells cultured 5 days in the presence of 2 μg/ml of kappa-elastin + washed at the end of cell culture with a solution of 1 mg/ml of melibiose: 18.3% ± 9.7% (p<0.001) . Fluorescent cells incubated only with the second antibody (negative controls) and not with the first antibody do not show any fluorescence. It thus appears that melibiose is more effective than lactose for desorbing the 67 kD subunit of the lymphocyte elastin receptor: lactose desorbs 66^ of the receptor and melibiose 76% under the experimental conditions used. b) Cytofluorimetry Table 3 Percentage of human lymphocytes expressing the elastin receptor on different culture days DO: on the day of separation of the lymphocytes; D2 , D3 , D5 : on the second; third; fifth day after separation of the lymphocytes . % of positive cells Day without number of with 2 μg/ml of P kappa-elastin experiments kappa-elastin DO 1.12 ± 0.2% 6 1.15+ 0.1% - D2 22.33 ± 5.2% 5 23.02 ± 3.2% - D3 29.70 ± 0.8% 7 32.40 ± 3.5% 0.178 D5 59.91 ± 4.9% 10 66.42 ± 1.9% 0.006 It is evident from this experiment that the incubation of cells with PHA as stimulant progressively induces the expression of the elastin receptor. This induction is again stimulated in the presence of elastin peptides. However, this stimulation does not become significant until the start of the 5th day of culture. Under these conditions approximately two thirds of the lymphocytes (≥66%) express the elastin receptor on their surface . - Results of double labellings Table 4 Double labelling experiment of lymphocytes to determine the nature of the subpopulations expressing the elastin receptor % of cell B expressing the elastin receptor (67 kD) + the label CDx without kappa elastin with kappa elastin R67kD+ 59.9 66.4 CD4+R67kD+ 26.5 45.7 CD8+R67kD+ 11.9 16.1 CD15+R67kD+ 0.0 0.0 CD20+R67kD+ 36.7 36.3 CD25+R67kD+ 39.6 46.9 CD45RO+R67kD+ 26.6 41.1 CDx+R57kD = cells labelled simultaneously with the anti-CDx antibody and with anti-subunit antibody of the 67 kD elastin receptor. Note: The sum of the percentages does not give 100% since a cell can simultaneously express several receptors . In this experiment, the nature of the lymphocyte subclass expressing the elastin receptor in the presence and in the absence of elastin peptides was determined by double labelling. It appears that the majority of the lymphocyte subclasses examined express the elastin receptor, with the exception of CD15+ cells which remain negative. However, this CD15 label corresponds to the PMNs and to a part of monocytes . This expression is only stimulated in an import- ant fashion by the presence of elastin peptides on the CD4+ and CD45R0+ lymphocytes which in the presence of elastin peptides strongly increased the expression of the elastin receptor. It is a matter in that case of a subpopulation of cells considered as helper and memory cells. The coupling of the elastin receptor by positive retroaction to its own synthesis after its activation would thus be specific to these cells. Example 4 : Protection against the cytotoxic effect of kappa-elastin of low molecular weight on lymphocytes 1) Isolation of the lymphocytes Isolation of the lymphocytes according to the method described in Example 1. The cell suspension is diluted with RPMI containing 5 mM glutamine and 10% FCS such that the concentra- tion is 10 β cells/2 ml. 2) Cell culture (DO) The lymphocytes are put into culture in 24-well Costar plates (500 μl of cell suspension/well at a concentration of 10 6 cells/2 ml) in complemented RPMI 1640 medium. The distribution of the cells is as follows: - 10 ml of cell suspension without κ-elaεtin; - 10 ml of cell suspension with 2 μg/ml K -elastin, and 10 ml of cell suspension with 2 mg/ml of K -elastin (controls with kappa-elastin, without lactose and melibiose) ; - 10 ml of cell suspension with 2 mg/ml K -elastin + 1 mg/ml of melibiose; - 10 ml of cell suspension with 2 mg/ml K -elastin + 1 mg/ml of lactose - 10 ml of cell suspension with 1 mg/ml melibiose - 10 ml of cell suspension with 1 mg/ml lactose 3) Counting of the cells in the presence of Trypan Blue to determine the percentage of dead cells On the fifth day of culture (D4) , the cell suspension is recovered, the cells are counted in a Malassez cell in the presence of 0.1% Trypan Blue (Sigma) (only the dead cells are stained blue with Trypan Blue) . Counting is carried out immediately after having added the stain on account of the toxicity of Trypan Blue. RESULTS TREATMENT number of % of dead cells cells/ml (Trypan Blue) without kappa-elastin 5.8 ± 0.6 9.2 ± 0.4 with 2 μg/ml kappa- 10.23 ± 3.5 6.6 ± 1.3 elastin (control) with 2 mg/ml kappa- 3.6 + 1.0 41.3 + 3.1 elastin with 1 mg/ml lactose 7.7 ± 1.5 14.5 + 1.9 with 1 mg/ml melibiose 7.26 ± 1.3 15.4 ± 1.6 with 2 mg/ml kappa- elastin + 1 mg/ml 8.8 + 0.7 10.6 + 2.3 lactose with 2 mg/ml kappa- elastin + 1 mg/ml 8.8 + 1.6 10.5 + 0.9 melibiose The table shows the percentage of dead cells in the presence of elastin peptides and the protection against cell death by lactose and melibiose. This protection is close to 100%: the overmortality (compared with the control without K -elastin) is 32.1% with 2 mg/ml of elastin peptides. In the presence of lactose or of melibiose this overmortality is 1.3% (96% protection).

Claims: CLAIMS 1. Use of at least one oligosaccharide comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or of a derivative of such an oligosaccharide substituted by a hydrophobic residue, for the preparation of an immunomodulating medicament. 2. Use of at least one oligosaccharide comprising from 2 to 6 oside residues, and having a galactose residue in the non-reducing terminal position, or of a derivative of such an oligosaccharide substituted by a hydrophobic residue, for the preparation of a composition intended for the treatment or the prevention of hypersensitivity reactions. 3. Use of an oligosaccharide according to either of Claims 1 and 2, characterized in that the oligosaccharide is chosen from the group formed of melibiose, lactose and their derivatives capable of being obtained by addition of a hydrophobic residue. 4. Use of an oligosaccharide according to one of Claims 1 to 3 , characterized in that a derivative substituted by a hydrophobic residue chosen from amongst: linear or branched C j -C^ alkyls, C ι _ C ι β alkylamines, linear or branched, optionally substituted C 1 -C 18 carboxylic acids, linear or branched, primary, secondary or tertiary C 1 -C 18 amides, and C^C^ arylalkyls is used. 5. Use of an oligosaccharide according to one of Claims 1 to 4, characterized in that the composition contains, in addition, pharmaceutically acceptable excipients suited to administration by the external topical route. 6. Use according to one of Claims 1 to 4, characterized in that the composition contains, in addition, pharmaceutically acceptable excipients suited to admin- istration by the parenteral or enteral route. 7. Use of an oligosaccharide according to one of Claims 1 to 6 for the preparation of a composition intended for the treatment or for the prevention of hypersensitivity reactions mediated by lymphocytes. 8. Use of an oligosaccharide according to one of Claims 1 to 7, characterized in that the composition is intended for the treatment or for the prevention of intolerance and/or allergic reactions of the skin and/or of the mucous membranes . 9. Use according to one of Claims 1 to 8, characterized in that the composition is intended to prevent or to decrease the formation of free radicals. 10. Use according to one of Claims 1 to 8, charac- terized in that the composition is intended for the treatment or for the prevention of symptoms of an ailment chosen from amongst atopy, psoriasis, polymorphous erythemas, xerodermatitides, lupus erythematosus, pemphigus, dermatitides and eczemas. 11. Dermatocosmetic composition, characterized in that it contains at least one active principle in association with an adjuvant limiting the hypersensitivity reactions to the active principle, and in that the adjuvant is an oligosaccharide comprising from 2 to 6 oside residues and having a galactose residue in the non- reducing terminal position, or a derivative of such an oligosaccharide substituted by a hydrophobic residue. 12. Method of cosmetic treatment of hyperreactive skins, characterized in that a composition containing at least one oligosaccharide comprising from 2 to 6 oside residues and having a galactose residue in the non- reducing terminal position, or a derivative of such an oligosaccharide substituted by a hydrophobic residue, is applied by the topical route in a cosmetically acceptable vehicle. 13. Method of cosmetic treatment according to Claim 12, characterized in that the oligosaccharide is chosen from the group formed of melibiose, and its derivatives capable of being obtained by addition of a hydrophobic residue.

Inventors: CASTELLI DOMINIQUE, ROBERT ALEXANDRE, ROBERT LADISLAV, CASTELLI, DOMINIQUE, ROBERT, ALEXANDRE, ROBERT, LADISLAS
Applicants: CASTELLI DOMINIQUE, ROBERT ALEXANDRE, ROBERT LADISLAS, ROC SA, CASTELLI, DOMINIQUE, ROBERT, ALEXANDRE, ROBERT, LADISLAS, ROC
Application year: 1997
Main classification: A61K31
IPC codes: A61K8, A61K31, A61P17, A61P37, A61Q19, C07H3, C08B37