USE OF OLIGOSACCHARIDES FOR THE TREATMENT OF CONNECTIVE TISSUES

Abstract: The present invention concerns a use of at least one oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose residue in a non-reducing terminal position, or of a derivative of such an oligosaccharide capable of being obtained by attachment of a hydrophobic radical, for the preparation of a composition intended for increasing the synthesis and/or decreasing the degradation of the proteoglycans of connective tissue.

Description: USE OF OLIGOSACCHARIDES FOR THE TREATMENT OF CONNECTIVE TISSUES The present invention relates to new uses of a class of oligosaccharides and their derivatives. It also relates to a cosmetic method which makes it possible to increase the glycosaminoglycan (GAG) content of connective tissues and to combat free radicals. The cohesion of the fibrous network of the connective tissues is effected by proteoglycans such as decorin and biglycan. The hydration and viscoelasticity of the tissues is largely attributable to hyaluronan, a glycosaminoglycan of high molecular weight (of the order of a million). In some tissues such as cartilage, this glycosaminoglycan (GAG) binds, via specific interactions (GAG- containing protein), a large number of aggrecan molecules which thus form very high molecular weight aggregates, imparting both hydration, resistance and elasticity to these tissues. Now, it has been shown that the biosynthesis of glycosaminoglycans falls with age. In cartilage, this is one of the major causes of osteoarthritis, which takes hold at different ages, depending on the joints, and progresses rapidly with age. Thinning of the skin and its progressive dehydration is also attributed to the progressive loss of hyaluronan. Hyaluronan is synthesized by means of hyaluronan synthetase, an enzyme located in the cell membrane. The hyaluronan synthesized surrounds the cell and thus regulates the movement of molecules in its vicinity, its migration, its association with other cells and macromolecules. Its degradation is effected by hyaluronidase, present in the testicles; similar enzymes (endogly- cosaminidases and endoglycuronidases) occur in the lysosomes, where the intracellular degradation of hyaluronan takes place after its endocytosis mediated by specific receptors. In addition, hyaluronan is very sensitive to attack by free radicals, which rapidly degrade it. This mechanism, coupled with the fall in synthesis, is the one which explains its progressive loss with age. For all these reasons, the stimulation of its biosynthesis, as well as its protection against free-radical degradation, are of great importance. Unexpectedly, the Applicant has now found that oligosaccharides having a galactose in a non-reducing terminal position can participate in these phenomena. Accordingly, the subject of the present invention is the use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose residue in a non-reducing terminal position, or of a derivative of such an oligosaccharide capable of being obtained by attachment of a hydrophobic radical, for the preparation of a composition intended for increasing the synthesis and/or decreasing the degradation of the proteoglycans of connective tissue. Such oligosaccharides have been described in Application WO 95/05155. It had been shown then that some compounds are capable of inhibiting the degradation of the elastin of connective tissue by enzymes of the elastase type, produced by the fibroblasts. In the context of the present invention, it has now been found that these oligosaccharides act on other constituents of the extracellular matrix, and on other mechanisms of degradation of this matrix. In effect, these oligosaccharides, especially lactose, melibiose or their hydrophobic derivatives, are useful for increasing the synthesis and/ or decreasing the degradation of glycosaminoglycans (GAGs), especially hyaluronan or hyaiuronic acid. Proteoglycans consist of a protein moiety combined via covalent bonding with a specific polysaccharide structure: glycosaminoglycans (GAGs); this polysaccharide fraction represents more than 95% of the total mass of the proteoglycan. Although there is always a disaccharide basic motif, the length and composition of the GAG chains is extremely variable. The polymerization of the disaccharide basic unit takes place via a β ( 1-4) bond. The classification of GAGs is based on the nature of the sugars of the disaccharide monomer, as well as on the number and position of the sulphate groups attached to the molecule. GAGs comprise, in particular, hyaiuronic acid, chondroitin sulphates, dermatan sulphate, keratan sulphate and heparan-sulphate proteoglycans, as well as heparin. Oligosaccharides which are especially suitable for the preparation of a composition according to the invention are chosen from the group consisting of melibiose, lactose and their derivatives which can be obtained by attachment of a hydrophobic residue. When an oligosaccharide derivative is used, the hydrophobic residue is preferably chosen from linear or branched Ci-Cis alkyls, Ci-Cis alkylamines, saturated or unsaturated, linear or branched Ci-Cis carboxylic acids which are optionally substituted, linear or branched Ci-Cis primary, secondary or tertiary amides and Ci-Cis arylalkyls. The oligosaccharide derivatives suitable for carrying out the invention can belong, in particular, 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 position 1 , 2, 3, 4 or 6, Hex represents a pentose or a hexose linked in α or β fashion, p is a number between 1 and 5; a) glycosides corresponding to the formulae: • (I) oligosccharide 1-O-R in which R is a linear or branched alkyl residue having 1 to 18 carbon atoms, • (II) oligosccharide 1-O-R-O- l -oligosaccharide in which R = (CH2J..1, m being between 2 and 10, b) an acylated glycosylamine according to one of the following formulae, in which the oligosaccharide is preferably lactose, melibiose or stachyose: - acylated glycosylamines corresponding to one of the following formulae: • (III) oligosccharide 1-NH-CO-R, in which R is an alkyl residue having 2 to 18 carbon atoms, containing 0, 1 or 2 double bonds, • (IV) oligosaccharide 1-NH-CO-R-CO-NH- l-oligo- saccharide in which R = (CH )m, m being between 2 and 8, c) an alkylamine acylated with an aldonic acid obtained by oxidation of an oligosaccharide • (V) oligosaccharide -CO-NH-R, in which R has the same meaning as in the formula (III), • (VI) oligosccharide CO-NH-R-NH-CO-oligosaccharide, where R has the same meaning as in the formula (IV), d) or a reduction product of the Schiffs 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-NH 2 -hexitol, and R has the same meaning as in (III) or (IV). According to an embodiment of the present invention, the oligosaccharide or its derivative as defined above will be used for the preparation of a composition containing, in addition, cosmetically or pharmaceutically acceptable excipients suitable for oral administration. According to an other embodiment of the present invention, the oligosaccharide or its derivative as defined above will be used for the preparation of a composition containing, in addition, cosmetically or pharmaceutically acceptable excipients suitable for internal topical administration. According to an especially advantageous embodiment, the oligosaccharide or its derivative as defined above will be used for the preparation of a composition containing, in addition, cosmetically or pharmaceutically acceptable excipients suitable for external topical administration. In effect, such oligosaccharides, and more especially melibiose and its derivatives, stimulate the biosynthesis of giycoconjugates in general and more specifically of hyaluronan, by fibroblasts, and in particular old fibroblasts, at and above very low doses. They are hence capable of improving the turgor, hydration and cohesion of the connective tissues in general and of the skin in particular. According to one of the aspects of the invention, oligosaccharides possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or hydrophobic derivatives as defined above, will be used for the preparation of a composition intended for combating the effects of UV radiation, namely the skin aging induced by UV radiation, and in particular the effects of free radicals. Free radicals are, by definition, neutral or charged chemical species which possess an unpaired electron. This "celibate electron" endows them with special chemical properties and a short lifetime. They are reaction intermediates which become stabilized by fusion or transfer, and can be the source of a chain reaction. Among free radicals, the superoxide anion 0 2 ", the hydroxyl radical OH ' and the nitric oxide radical NO- may be mentioned. The peroxides generated during metabolic reactions, and also during the neutralization of the superoxide anion by SOD (superoxide dismutase), cause the same damage. They are involved in the degradation of numerous physiological components, and in particular of GAGs. The activity of the oligosaccharides according to the invention, and more especially of melibiose, in relation to this can be explained, in particular, by two mechanisms, by which the invention is in no way limited. On the one hand, they block the liberation of free radicals, mediated by the elastin receptor. The radicals liberated are superoxide and nitric oxide, which can combine to form the toxic peroxynitrite anion. On the other hand they inhibit the degradation of hyaluronan by free radicals, probably by a "suicide" effect and preferential scavenging of the free radicals. Hence the invention also relates to the use of oligosaccharides as defined above, and in particular of melibiose, as free radical scavengers. In effect, these oligosaccharides, especially lactose and melibiose, are capable of exerting a favourable effect against the free-radical attack involved in the degradation of cells and macromolecules of the dermis. Confirmation of these results has been obtained using the effect of UV on the skin. The presence of free radicals in human skin after UV irradiation was demonstrated by the electron spin resonance technique. The irradiation sustained causes an oxidative stress and, under normal physiological conditions, the free radicals produced are dealt with by the various natural defence systems, namely: - reduced glutathione, an effective free radical scavenger - glutathione peroxidase (GPX), responsible for protection against peroxides - elastases originating from the tissues and the infiltrating mononuclear cells (PMN, monocytes, lymphocytes). The elastase activity which can be extracted from the skin increases under the effect of UV. Two types of activity are found in the dermis: one of the metalloendopeptidase type originating from the fibroblasts and sensitive to EDTA, and the other, of leukocytic origin, a PMN serine protease which is not inhibited by EDTA. Monocytes-macrophages also produce a metalloelastase. Treatment with these active principles, such as lactose and/or melibiose, increases the body's, and more especially the skin's, defences against free radicals and decreases oxidative stress, as is evidenced by measurement of the corresponding markers. Melibiose and its derivatives are especially effective, since they are only slightly degraded in the body which does not contain clivage enzyme (melibiase) which today would only have been detected in bacteria. The subject of the invention is also a cosmetic treatment method for combating the effects of free radicals and /or for increasing the synthesis or decreasing the degradation of glycosaminoglycans in a mammal in good health, comprising the administration of at least one oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of at least one derivative of the latter capable of being obtained by attachment of a hydrophobic radical. Advantageously, this administration will be carried out on the skin, the mucosae or the exoskeleton. The oligosaccharides according to the invention are also useful in compositions intended for combating alopecia, for improving the hydration of the skin and for improving the cutaneous microcirculation. The compositions according to the invention can take the form, in particular, of solutions, gels, lotions, creams, W/O or O/W emulsions or multiple-phase emulsions, or the form of liposomes or any vesicular form. They will be formulated in a manner known to a person skilled in the art with texturing agents, emulsifiers, •thickeners, preservatives, and the like. The concentration of oligosaccharides, in particular of melibiose, in the compositions according to the invention will advantageously be between 0.01 and 10% w/w, and preferably between 1 and 4% w/w. The examples which follow are intended to illustrate the invention without limiting its scope. In these examples, reference will be made to the following figures: Figure 1 Figure LA. : Variation of the amount of hyaiuronic acid as a function of the melibiose concentration in the fibroblast extracellular medium at the 5th passage. Figure B.: Variation of the amounts of hyaiuronic acid as a function of the melibiose concentration in the fibroblast extracellular medium at the 10th passage. Figure l .C: Variation of the amounts of hyaiuronic acid present in the fibroblast extracellular medium at the 15th passage. Figure 2 Figure 2. A.: Variation of the amount of hyaiuronic acid in the fibroblast intracellular medium as a function of the melibiose concentration at the 5th passage. Figure 2.B.: Variation of the amount of hyaiuronic acid in the fibroblast intracellular medium as a function of the melibiose concentration at the 10th passage. Figure 2.C.: Variation of the amounts of hyaiuronic acid in the fibroblast intracellular medium as a function of the melibiose concentration at the 15th passage. Figure 3: Incorporation of [ 3 H]glucosamine into the total glycoconjugates in the intra- and pericellular compartment. Effect of increasing doses of melibiose. Abscissa: Melibiose concentration (μg/ml). Ordinate: [ 3 H]Glucosamine incorporated into the glycoconjugates, expressed as cpm/ million cells. The results are expressed as Mean ± Standard deviation. Statistical analysis: Student's t test, * p < 0.01 , " p < 0.005, * " p < 0.001. Figure 4: Incorporation of [ 3 H]glucosamine into the total glycoconjugates in the extracellular compartment. Effect of increasing doses of melibiose. Abscissa: Melibiose concentration (μg/ml). Ordinate: [ 3 H]Glucosamine incorporated into the glycoconjugates, expressed as cpm/million cells. The results are expressed as Mean ± Standard deviation. Statistical analysis: Student's t test, ' p < 0.01 , " p < 0.005, " * p < 0.001. Figure 5: Incorporation of [ 3 H]glucosamine into hyaluronan in the intra- and pericellular compartment. Effect of melibiose at a concentration of 1 μg/ml. Abscissa: Melibiose concentration C: 0; M L 1 (μg/ml) . Ordinate: [ 3 H]Glucosamine incorporated into hyaluronan, expressed as cpm/million cells. Figure 6: Incorporation of [ 3 H]glucosamine into hyaluronan in the fibroblast extracellular compartment. Effect of melibiose at a concentration of 1 μg/ml. Abscissa: Melibiose concentration C: 0; M L 1 (μg/ml). Ordinate: [ 3 H]Glucosamine incorporated into hyaluronan, expressed as cpm/million cells. Figure 7: Viscosity of hyaluronan alone or mixed with 1 / 1,000 Udenfriend's reagent (ascorbic acid/ iron/ EDTA; generates OH-radicals) in the presence or absence of melibiose at different concentrations. Figure 8: The figure shows the effect of treatment with the active principles on the selenium-containing glutathione peroxidase content of rats' skin. In the lower diagram, the values are given in moles of enzyme per mg of fresh weight of skin, in the upper the results are shown as percentages, the values for the control skins having been taken as 100%. Figure 9: The histogram in the figure shows as ordinates the elastase activity of the skins on both the control and the treated sides of 3 groups of rats. It is seen that, with the exception of the group treated with the excipient alone (No. 3), the treated sides (No. 1: prep. RoC; No. 2: L + M) contain less elastase activity than the control sides. Example 1: Effect of melibiose on the amount of hyaluronan in fibroblast cultures Hyaiuronic acid or hyaluronan (HA) is assayed by an ELSA technique derived from that developed by Delpech et al. (Anal. Biochem., 1985, 149: 555-565) . This technique is based on the affinity of hyaluronectin (HN) for hyaluronan (HA). HN coupled to alkaline phosphatase binds to HA adsorbed on the titration plate. The bound HN level is measured by the phosphatase activity. When HN is preincubated in the presence of HA, the binding of HN to the plate is inhibited. The change in absorbance at 405 nm is proportional to the inhibition by the HA contained in the samples. This technique possesses several advantages: it is specific, has high affinity (threshold of detection: 1 μg/1) and is fast (assay within the day). 1 - Materials and Methods a) preparation of hyaluronectin This comprises three main steps: extraction of hyaluronectin from lamb's brain by grinding and centrifugation; preparation of an HA-Sepharose 4 B column by attachment of HA ( 100 mg) to Sepharose (4 g) in the presence of carbodiimide ( 125 mg). The pH is adjusted to between 4.5 and 5 with 0. 1 N HCl every 15 minutes for 4 hours. After incubation at room temperature, the HA-Sepharose gel is washed with 1 M NaCl and then with 1 M acetic acid at 4°C; the gel is thereafter - washed with 200 ml of 0.2 M glycine/HCl buffer pH 2, then 100 ml of 4 M guanidine and physiological saline; purification of HN by affinity on the HA-Sepharose 4 B column and coupling with alkaline phosphatase: this is performed on the extract obtained at the end of the first step, removing the contaminating HA-HN complex; after coupling to alkaline phosphatase (AP) in the presence of glutaraldehyde, the uncoupled AP is removed by gel filtration. The dilution of HN-AP to be used for the HA assay is determined on an ELISA plate, so as to obtain an OD = 1.5 at 405 nm in the absence of HA. b) hyaluronan assay The assay of HA by the ELSA technique comprises 5 principal steps. 1. Preparation of reference HA Commercial HA (Sigma) isolated from rooster comb has to be purified prior to its use. In effect, this HA is contaminated with proteins and proteoglycans. The HA is dissolved in PBS ( 1 mg/ml). The contaminating proteoglycans are removed by treatment with chondroitinase ABC (0.5 U/ 10 mg HA) for 1 hour at 37°C. The chondroitinase and the contaminating proteins are destroyed by protease treatment (Streptomyces vulgaris type XIV protease, 1 U/mg HA) for 18 hours at 37°C. The protease is denaturated by heat (20 minutes at 100°C). The HA is then dialysed against 0.2 M glycine/HCl buffer pH 2.2 and thereafter against PBS. The HA is quantified by assaying the hexuronic acids according to the technique of Bitter and Muir ( 1 μg of hexuronic acid = 1.766 μg HA), and the HA concentration is then readjusted to 500 μg/ml in TBS/0.2% Az. 2. Sensitization of plates The sensitization step consists in adsorbing HA in the plates. Commercial HA ( 1 mg/ml) is dissolved in 0.1 M bicarbonate buffer, 0.2 g/1 azide, and 250 μl are then distributed in each well of the ELISA plate. The plates are sensitized at least overnight at 4°C and can be stored for 1 month at + 4°C. 3. Sample preparation The hyaluronectin which may be contaminating the sample is removed by protease treatment (Proteus vulgaris type XIV). The sample is diluted to 1/ 10 in a protease solution ( 1 mg/ml) in TBS/0.2 g azide/ 1 for 18 hours at 37°C. The protease activity is denatured by heat (20 minutes at 100°C). 4. Preincubation of samples The preincubation of HA with HN enables the sensitivity of the assay to be increased. 300 μl of each point (sample and HA reference series) is treated with 300 μl HN-AP (diluted to 1 /50 in TBS/Az/BSA) for 1 hour at 4°C. The reference HA is diluted in TBS/Az ( 160, 80, 40, 20, 10, 5, 2.5 μl final). The series is prepared in duplicate and the samples are assayed in duplicate at 3 different dilutions. The assay of the samples was carried out on not less than 2 plates. The intra-assay variation is less than 5% and the inter- assay variation varies from 10 to 15%. 5. Incubation and visualization treatment of the plate The sensitized plate is washed with tap water and 0. 1 M acetic acid, then twice in demineralized water and lastly once in PBS. 250 μl of sample are applied per well. The plate is incubated for 4 hours at 4°C, then emptied and washed 3 times in PBS. 250 μl of visualizing buffer (para-nitrophenyl phosphate 1 g/1, 1 M diethanolamine pH 9.8 + 1 mM MgCl 2 ) are added to each well. The plate is incubated for 1 hour at 37°C, the optical density is then determined at 405 nm in an automatic plate reader (Dynatech). The inhibition of the binding of HN to the HA adsorbed on the plate is proportional to the HA added during the preincubation. To facilitate the calculations, the HA/OD 405 nm ratio is linearized by expressing the HA concentration on a logarithmic scale. The optical density, which is proportional to the amount of HA to be assayed, varies relatively little for a large variation in HA. In order to increase the reliability, the assay is always carried out in duplicate at 3 different sample concentrations. The intra-assay variation is less than 5% and the inter-assay variation varies from 10 to 15%. c) fibroblast culture The material is a culture of human skin fibroblasts. The source skin came from a 13-year-old child, and was removed during plastic surgery for resetting of the ears. These cells are cultured on the basis of 3 x 10 5 cells per dish (nunc, 6 cm in diameter) in 5 ml of DMEM medium with 10% of foetal calf serum, 1% of penicillin/ streptomycin and 1% of fungizone, in a humid atmosphere containing 5% of C0 2 and at 37°C. Melibiose is added to the medium at different concentrations: 0.1 μg/ml, 1 μg/ml and 5 μg/ml final ( 1 μg/ml 3 μM). Effect of the number of passages In order to test for a possible modulation of the effect of age on the biosynthesis of hyaluronan, the assays are carried out with cells from different passages, the dilution ratio (split ratio) being 1 /2: a young or early passage: 5th passage or cell population doubling (CPD); a medium-term passage: 10th passage; - a more advanced passage: 15th passage. 2 - Results Figures 1 and 2 show the effect of increasing concentrations of melibiose on the amount of hyaluronan assayed in the culture medium and in the pericellular layer of the fibroblasts. At the 5th passage with 1 μg/ml, a modest but significant increase is observed in the extracellular medium. 5 μg/ml doubles the amount of hyaluronan excreted into the culture medium. Already at the 10th passage, 0.1 μg/ml melibiose produces a large increase in hyaluronan in the culture medium, with a maximal effect at 1 μg/ml. At the 15th passage, a large drop is noted in the synthesis of hyaluronan excreted by unstimulated fibroblasts. Melibiose at a concentration of 1 μg/ml and 5 μg/ml strongly stimulates these biosyntheses. Similar stimulations are observed in the peri- and intracellular medium; the optimal concentration varies according to the passage, from 1.0 μg/ml at the 5th passage to 0. 1 μg/ml at the 10th passage and 15th passage. These results collectively show that melibiose strongly stimulates the biosynthesis of hyaluronan in the culture medium and in the fibroblasts and increases the content thereof. Example 2: Effect of melibiose on the biosynthesis of glycoconjugates - glycoproteins and glycos aminoglycans - in fibroblast cultures 1 - Materials and Methods Culture of human skin fibroblasts The material used is a culture of human skin fibroblasts. The effects of increasing doses of melibiose on the biosynthesis of glycoconjugates, and more especially that of hyaluronan, are evaluated. Melibiose is added to the culture medium at different concentrations: 0.1 μg/ml, 1 μg/ml and 5 μg/ml final. In order to test for a possible age-linked modulation of the biosynthesis of glycoconjugates, the assays are carried out with cells from two different passages, the dilution ratio (split ratio) being 1 /2: an early passage: 5th passage or cell population doubling (CPD); - a later passage: 15th passage. Biosynthesis of glycoconjugates Glucosamine is an amino sugar which participates in the composition of three families among the 4 main families of macromolecules of the extracellular matrix which possess glycanic chains, namely the collagens, the structural glycoproteins, the proteoglycans and the glycosaminoglycans (GAG chains), and in particular hyaluronan. Hence [ 3 H]glucosamine added to the culture medium of fibroblasts ( 1 μCi/ml) permits radioactive labelling of all the newly synthesized glycoconjugates. Skin fibroblasts are cultured on the basis of 1.5-3 x 10 5 cells per dish (nunc, 6 cm in diameter) in 5 ml of DMEM medium with 10% of foetal calf serum, 1% of penicillin/ streptomycin and 1% of fungizone, in a humid atmosphere containing 5% of CO2 and at 37°C. 24 hours later, the medium is replaced by a medium containing increasing doses of melibiose (0.1 , 1 or 5 μg/ml). The cells are cultured for 48 hours at 37°C with agitation. [ 3 H]Glucosamine ( 1 μCi/ml), permitting radioactive labelling of all the newly synthesized glycoconjugates, is added. 24 hours later, the culture media are collected after two washes with 1 ml of OBS, 2 mM EDTA/PMSF, and the detached in the presence of 1 M urea. The intra- and extracellular compartments are dialysed so as to remove non-incorporated radioactivity. The radioactivity associated with [ 3 H]glucosamine is counted by liquid scintillation. Biosynthesis of hyaluronan Measurement of the radioactivity associated with the [ 3 H]glucosamine incorporated into hyaluronan is carried out after selective degradation with hyaluronidase (Streptomyces hyaluronidase 0.1 TRU/ml) at 60°C overnight. The extracts are then ultrafiltered (cut-off threshold 10 kDa) . The radioactivity is measured in the filtrate containing the degraded fragments of hyaluronan. 2 - Results a) biosynthesis of total glycoconjugates Control group: Virtually all of the glucosamine incorporated is to be found in the extracellular compartment (90-95%). Only a small fraction is present in the in tra/ pericellular compartment. Intra/ pericellular compartment: (Figure 3). No modification observed with the number of passages. Extracellular compartment: (Figure 4). The [ 3 H]glucosamine incorporated is multiplied by a factor of 2.5 between passages 5 and 15. Hence a stimulation of the biosynthesis of the total glycoconjugates is observed during successive passages. Effect of increasing doses of melibiose Intra/ pericellular compartment: (Figure 3). For the late passage (P15) , a statistically significant stimulation of 14% is observed at a dose of 0. 1 μg/ml, 50% at a dose of 1 μg/ml and 35% at a dose of 5 μg/ml. Extracellular compartment: (Figure 4). A slight decrease is observed for the incorporation of [ 3 H]glucosamine at the early passage P5 at doses of 0.1 and 1 μg/ml ( 14% and 10%), and a slight increase ( 12%) at a dose of 5 μg/ml. In contrast, a significant increase is observed at the late passage P15 ( 14% at 0. 1 μg/ml, 35% at 1 μg/ml and 15% at 5 μg/ml). b) biosynthesis of hyaluronan Control group Intra/pericellular compartment: (Figure 5) . The [ 3 H]glucosamine incorporated is multiplied by a factor of 4.2 between passages 5 and 15. Extracellular compartment: (Figure 6) . The [ 3 H]glucos- amine incorporated is multiplied by a factor of 3.5 between passages 5 and 15. Hence a stimulation of the biosynthesis of hyaluronan, as well as its retention in the intra- and pericellular spaces, is observed during successive passages. Effect of melibiose Intra/pericellular compartment: (Figure 5) . The [ 3 H]glucosamine incorporated is multiplied by a factor of 1.3 at the early passage P5 and by a factor of 7.6 at the late passage P15 in the presence of melibiose at a dose of 1 μg/ml. Extracellular compartment: (Figure 6). An increase of 15% at the early passage P5 and of 75% at the later passage P15 is observed for the incorporation of [ 3 H]glucosamine in the presence of melibiose at a dose of 1 μg/ml. It is hence apparent that melibiose simultaneously stimulates the biosynthesis and inhibits the post-synthetic degradation of glycoconjugates, and of hyaluronan in particular, by skin fibroblasts. It is thus a cumulative double effect for increasing namely the amount of hyaluronan. This stimulation is more marked with "old" fibroblasts (late passages); very strong stimulations are obtained with very low concentrations of melibiose, of the order of 0.1 to 1 μg/ml. Example 3: Anti-free-radical effect of melibiose The degradation of hyaluronan by free radicals is determined by the fall in viscosity of a solution of Na hyaluronate at 37°C. In the presence of Udenfriend's reagent (ascorbic acid/ EDTA/ iron), which liberates OH- radicals, there is rapid degradation of hyaluronan. In the presence of melibiose at a concentration of 5, 10 or 30 mg/ml, the same reagent degrades hyaluronan increasingly slowly. At 3% (30 mg/ml), protection is complete (Fig. 7). Example 4: Study on hairless rats of the anti-free-radical effect of melibiose or of lactose The defences against oxidative stress, reduced glutathione, glutathione peroxidase and also elastase, were measured after treatment with lactose and melibiose. A Solar Light type 1 148 lamp was used as UV source. The hairless rats were males of initial weight between 150 and 180 g. Three groups of 5 rats received on their right side 1 g of a product, the penetration of which was promoted by massage for approximately one minute. The first group received melibiose in the form of a composition marketed by the company ROC (Anti-ageing melibiose), the second a lactose (L)/ melibiose (M) mixture containing 3% of each, and finally the third group received only the excipient. The left side of the rats was not treated. The treated surface area was of the order of 15 cm 2 . Hence the rats receive approximately 2 mg/cm 2 of each active principle. The treatment lasted two weeks at the rate of 5 applications weekly. At the end of the treatment, the rats were anaesthetized and irradiated on both sides with the UV lamp at 6 spots at a dose of 3 MED. On the day after irradiation, the rats are anaesthetized again and the spots of irradiated skin are dissected, grouped in threes and weighed. The mean fresh weight of the 3 spots was 266 ± 25 mg. The skins are then finely cut up and are ground in a Potter homogenizer with a buffer solution suitable for each assay, and the extracts are thereafter collected by centrifugation in Eppendorf tubes. The assays are performed on the extracts thereby obtained and the results are expressed with reference to the fresh weight of skin. 1 - Assay of glutathione peroxidases (GPX) Selenium-containing GPX effects the conversion: - of hydroperoxides according to the following reaction: ROOH + 2 GSH > GSSG + ROH + H 2 0 - of hydrogen peroxide: H 2 0 2 + 2 GSH > GSSG + 2 H 2 0 according to non-Michaelis kinetics. The method used for the assays is adapted from the method of Paglia and Valentine (D.E. Paglia and W.N. Valentine, J. Lab. Clin. Med., 70: 158 ( 1967)). It is based on the measurement of the rate of formation of oxidized glutathione (GSSG), which will be reduced by an excess of glutathione reductase. The substrate chosen is t-butyl hydroperoxide. It reacts according to the equation: ROOH + 2 GSH > GSSG + H 2 0 + ROH The reduced glutathione (GSH) needed for the GPX-catalysed reaction is supplied at the same time as NADPH and the glutathione reductase in order to permit recycling of the oxidized glutathione obtained after consumption of the GSH, according to the following reaction: GSSG + 2 NADPH > 2 GSH + 2 NADP The oxidation of NADPH is monitored by spectro- photometry at 340 nm. This method permits the assay of total glutathione peroxidases. The assay of glutathione peroxidase is carried out under the following conditions: Buffer: 50 nM Tris 0.1 mM EDTA, pH = 7.6 with HCl Mercaptosuccinic acid Reduced glutathione: Sigma C_oH _ 7 N 3 θ6S Glutathione reductase: Sigma NADPH: nicotinamide-adenine dinucleotide monophosphate reduced, in the form of the tetrasodium salt, Sigma t-Butyl hydroperoxide: Sigma C.H 10O2 Wavelength: 340 nm Reading time: 3 min. Results Figure 8 shows the effect of the treatment on glutathione peroxidase, a selenium-containing enzyme, which plays an important part in protection against free radicals. It is apparent that the treatment with the active principles has greatly increased the glutathione peroxidase activity of the skin, confirming the anti- free-radical effect of lactose and melibiose. 2 - Glutathione assay Glutathione or γ-glutamylcysteinylglycine tripeptide displays nucleophilic reducing properties which enable it to play an essential part in antioxidant protective systems. It is an important antioxidant since it is necessary for the activity of glutathione peroxidases. To assay it, a colorimetric method which is exploited by the Bioxytech kit GSH-400 and which brings the sample into contact with a reagent Rl which reacts with all mercaptans, was used. This step is followed by an alkalinization of the chromophoric medium which is specific for glutathione with a highly characteristic absorbance. Wavelength: 400 nm. Results Table 1 shows the increase in reduced glutathione (GSH) content of the skin irradiated with UV and treated as above: Table 1 Group No. No. 1 No. 2 No. 3 % incr. GSH 109 96 10 No. 1 : prep. RoC; No. 2: L + M; No. 3: control It is apparent that the treatment with the active principles has caused a large increase in the reduced glutathione content (factor 10 increase in view of the control). Now, GSH represents one of the most effective protective agents against free radicals. 3 - Elastase activity A preliminary experiment was carried out in order to define the optimal conditions for conducting the studies. This work consisted in assaying the elastase activities of unirradiated skin and skin irradiated with 3 MED. The skins were removed 24 hours after irradiation and treated for the extraction of elastase activities. Parallel samples were incubated before measuring the optical density at 37°C for variable times from 2.5 to 20 hours, with and without EDTA. EDTA is a selective inhibitor of metalloendopeptidases. When its effect on the results of the assays is considered, it is observed in the irradiated skins that, initially (2.5 and 4.5 hours of incubation), only a small part of the elastase activity is inhibited, indicating that virtually all of the measured activity relates to PMN (polymorphonuclear leukocyte) elastase. On the unirradiated side, a relatively high activity is also observed, attributable to the effect of cytokines released at the sites of irradiation and acting at a distance. At these points, the addition of EDTA increases the measured activity by 55%, which may be attributed to an activation by EDTA, by complexing of trace metals, of the PMN serine proteases which are partially inhibited by these metals. After prolonged incubation times, 6.5 and 20 hours, it is observed in the irradiated and unirradiated skin that the proportion of elastase activity which can be inhibited by EDTA increases and finally reaches approximately 50% of the total activity. The increase in the relative value of the elastase activity which is attributable to metalloendopeptidases is probably due to their greater resistance to inactivation than those of the serine elastases. Another mechanism envisaged is the activation of the latent metalloendopeptidase of fibroblasts by PMN elastase. From these results, the incubation time of 6 hours was adopted for the subsequent assays of elastase activation, since, under these conditions, there is a total activity which is inhibited by the EDTA added to the reaction mixture, and hence originates from the fibroblasts. Results Figure 9 gives the inhibition of elastase activities measured in the extracts of skin irradiated and treated with melibiose (RoC "Anti-ageing melibiose"): group No. 1 ; or with a mixture of 3% lactose/ melibiose in an excipient: group No. 2; or with the excipient alone: group No. 3. The preparations containing lactose and/or melibiose produced a protection of the order of 30% against the increase in elastase activity induced in the skin by UV irradiation.

Claims: CLAIMS 1. Use of at least one oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose residue in a non-reducing terminal position, or of a derivative of such an oligosaccharide capable of being obtained by attachment of a hydrophobic radical, for the preparation of a composition intended for increasing the synthesis and/or decreasing the degradation of the proteoglycans of connective tissue. 2. Use of an oligosaccharide according to Claim 1 , characterized in that the composition is intended for increasing the synthesis and/or decreasing the degradation of glycosaminoglycans (GAGs). 3. Use of an oligosaccharide according to either of Claims 1 and 2, characterized in that the glycosaminoglycan is hyaluronan or hyaiuronic acid. 4. Use of an oligosaccharide according to one of Claims 1 to 3, characterized in that the oligosaccharide is chosen from the group consisting of melibiose, lactose and their derivatives which can be obtained by attachment of a hydrophobic residue. 5. Use of an oligosaccharide according to one of Claims 1 to 4, characterized in that the hydrophobic residue is chosen from linear or branched Ci-Cis alky Is, Ci-Cis alkylamines, saturated or unsaturated, linear or branched Ci-Cis carboxylic acids which are optionally substituted, linear or branched Cι-C_ 8 primary, secondary or tertiary amides and Ci-Cis arylalkyls. 6. Use according to one of Claims 1 to 5, characterized in that the composition contains, in addition, cosmetically or pharmaceutically acceptable excipients suitable for oral administration. 7. Use according to one of Claims 1 to 5, characterized in that the composition contains, in addition, cosmetically or pharmaceutically acceptable excipients suitable for internal topical administration. 8. Use according to one of Claims 1 to 5, characterized in that the composition contains, in addition, cosmetically or pharmaceutically acceptable excipients suitable for external topical administration. 9. Use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of a derivative of such an oligosaccharide as defined in one of Claims 1 , 4 and 5, for the preparation of a composition intended for combating the effects of UV radiation. 10. Use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of a derivative of such an oligosaccharide as defined in one of Claims 1, 4 and 5, for the preparation of a composition intended for combating the skin aging induced by UV radiation. 1 1. Use according to Claim 9 or 10, characterized in that the composition is intended for combating the effects of free radicals. 12. Use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of a derivative of such an oligosaccharide as defined in one of Claims 1 , 4 and 5, for the preparation of a composition intended for combating alopecia. 13. Use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of a derivative of such an oligosaccharide as defined in one of Claims 1 , 4 and 5, for the preparation of a composition intended for improving the hydratation of the skin. 14. Use of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of a derivative of such an oligosaccharide as defined in one of Claims 1 , 4 and 5, for the preparation of a composition intended for improving the cutaneous microcirculation. 15. Cosmetic method for combating the effects of free radicals and/ or for increasing the synthesis or decreasing the degradation of glycosaminoglycans in a mammal in good health, comprising the administration of an oligosaccharide possessing from 2 to 6 glycoside residues and having a galactose in a non-reducing terminal position, or of at least one derivative of the latter capable of being obtained by attachment of a hydrophobic radical.

Inventors: CASTELLI DOMINIQUE, ROBERT ALEXANDRE MICHEL, ROBERT LADISLAS, CASTELLI, DOMINIQUE, ROBERT, ALEXANDRE, MICHEL, ROBERT, LADISLAS
Applicants: CASTELLI DOMINIQUE, ROBERT ALEXANDRE MICHEL, ROBERT LADISLAS, ROC SA, CASTELLI, DOMINIQUE, ROBERT, ALEXANDRE, MICHEL, ROBERT, LADISLAS, ROC
Application year: 1998
Main classification: A61K7
IPC codes: A61K8, A61K31, A61P17, A61Q19, C07H3