CA2262564A1 - Use of an oligosaccharide as an immunomodulator in a dermato-cosmetic composition - Google Patents
Use of an oligosaccharide as an immunomodulator in a dermato-cosmetic composition Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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
CA 02262~64 1999-01-29 USE OF AN OLIGOSACCHAR~DE AS AN ~MMUNOMODULAl~R ~N A DERMATO-COS~T[C
COMPOSmON
The present invention relates to compounds useful in the field of ~ lnology and in particular for the treatment of hypersensitivity reactions responsible for allergies and various intolerance phenomena.
Immunological defence against infectious agents, 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 mechani6m 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 tissue grafts.
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 Fc 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 Ig~) form, with the antigen and the complement, immune complexes which are deposited in the tissues and cause the production of chemotactic factor6 for polynuclear neutrophils; local inflammation results.
_ Type 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 macrophage6.
The lymphocytes are thus key cells in ;mmllnlty with cellular or humoral mediation, especially with the secretion of antibodies.
Allergic ailments can be the expression of one or more SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 PCTnB97/~951 types of immunological reaction~.
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 hyper~ensitivity 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 te ;nal po~ition, or of their derivatives, for the preparation of immunomodulator compo6itions, 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, lactoRe and their derivatives capable of being obtained by addition of a hydrophobic residue.
Hydrophobic substituents are especially understood as meaning linear or branched C1-C1g alkyls, C1-C1g alkylamines, linear or branched, optionally subQtituted Cl-C1g carboxylic acids, linear or branched, primary, secondary or tertiary C1-C1g amide~, and C1-C1g arylalkyls.
The oligosaccharide derivatives suited for carrying out the invention can e~pecially belong to one of the categories mentioned below, in which the oligosaccharide corresponds to the following general formula:
galactose-n (a or ~) - (Hex)p in which n represents the position 1, 2, 3, 4 or 6, SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 3 PCT~B97/00951 Hex represents an a- or ~-linked hexose or pentose, p is a number between l and 5;
a) - glycosides corresponding to the formulae:
(I) oligosaccharide l-O-R, in which R i8 a linear or branched alkyl residue of l to 18 carbon atoms, ~ (II) oligosaccharide l-O-R-O-l-oligosaccharide in which R = (CH2)m, m being between 2 and lO, b) - an osylamine acylated according to one of the following formulae, in which the oligosaccharide is preferably lacto~e, melibiose or stachiose:
- osylamines acylated corresponding to one of the following formulae:
~ (III) oligosaccharide l-NH-CO-R, in which R is an alkyl residue of 2 to 18 carbon atoms, containing 0, l or 2 double bonds, ~ (IV) oligofiaccharide l-NH-CO-R-CO-NH-l-oligosaccharide, in which R = (CH2)m, m being between 2 and 8, c) - an alkylamine 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 e~ning as in formula (III), 2~ d) - or a reduction product of Schiff bases formed by oligo6accharides 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, l or 2, X = l-NH2-hexitol, and R has the same meaning as in (III).
SUBSTITUTE StlEET (RULE 26) CA 02262~64 1999-01-29 WO g8/04270 PCT/IB97/00951 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 disorder6 associated with hyper6ensitivity can be correlated with the fixation, on a 6pecific lymphocyte receptor, of peptides re6ulting from the degradation of elastin, especially kappa-elastin.
The activation of these receptors triggers the liberation of lytic enzymes, ~-glucuronidase, elastases and free radicals such a6 the 6uperoxide ion, or hydrogen peroxide, which generates hydroxyl radical6. 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 de6quamation can take place.
All these symptoms will be ameliorated or even suppressed by the use of compositions cont~;n;ng 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 li~ewise inhibit the potentialization and liberation of lytic enzymes.
The compositions will preferably be formulated in vehicle6 not comprising any perfume or allergenic agents. The SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 5 PCT~B97/00951 compositions could be in the form of solutions, gels, lotions, creams, W/0 or 0/W 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 6uited to the treatment or to the prevention of intolerance and/or allergic reactions of the 6kin 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 derivative6 could be a6sociated with other agents enabling the skin to be protected and/or hydrated, such as hyaluronic acid, vitamin E, glycol extract of G. biloba, 60rbitol, glucosaminoglycans, alginates, etc.; they can likewise be a6sociated with vegetable oils to nourish the skin and/or with active emollient6 and salves 6uch as, for example, extracts of cat grape, althaea, oats, linden, camomile, sweet clover, Ruscus, procyanidols, a-bi6abolol, coconut oil, 18-~-glycyrrhetinic acid.
According to another aspect of the invention, the oligo6accharides and their derivatives such as defined above are used for the preparation of a compo6ition which additionally contains pharm~ceutically 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 ;mm--ne 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 eczema6.
The oligosaccharides having a galactose residue in the non-reducing tPrm;n~l position, and their derivatives SUBSTITUTE SHEE~ (RULE 26) CA 02262~64 1999-01-29 W098/04270 6 PCT~B97/00951 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 co6metic S treatment of hyperreactive skins, characterized in that a composition containing at least one oligosaccharide comprising from 2 to 6 oside re6idues, 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.
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 illu~trate the invention.
In these examples, reference will be made to the following figures:
Fig. l:Inhibition by lactose and melibiose of the activity of stimulation of lymphocyte proliferation by ~-elastin. The concentrations of lactose and of melibiose are l ~g/ml, lO
~g/ml, lO0 ~g/ml, l mg/ml and 2 mg/ml.
Fig. 2:Inhibition by lactose and melibio~e of the expre~ion of elastic activity by lymphocyte~, stimulated with 2 ~g/ml of ~-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 ~g/ml of K-elastin. Activity in the culture medium.
Example l l - Methods SeParation of lymPhocYtes SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W O 98/04270 rcTnBg7/00951 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 centri-fuged 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 se~im~nt is taken up in 10 ml of RPMI and the cells are counted. The monocytes and macrophages are eliminated by adhesion on a plastic surface (incubation for 2 hours in a CO2/O2 incubator).
- SeParation of the PolYnuclear cells (PMN) After the first centrifugation at 3000 rpm (say 600 g), the residue cont~in;ng 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 i~ 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 ~hock 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 CA 02262~64 1999-01-29 W 098/04270 PCT~B97/00951 centrifugation for 20 min at 0~C. The supernatant con-tains the enzymes.
Preparation of lYmPhocytes startinq from human tonsil~
Freshly remo~ed tonsils are cut into small pieces under sterile conditionR, in 10 ml of ~PMI. The tissue suspension is filtered on a coarse filter in order to remove the largest tissue debris. The suspension i8 placed in a test tube and allowed to s~im~nt for 15 min at A~hient temperature. The supernatant is placed on Ficoll-paque plus and centrifuged as described for lymphocytes derived from blood. The monocytes are elimin-ated as described for the lymphocytes derived from blood.
The sediment containing the lymphocytes is re~uspended 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~o of type T and 20% of type B.
Culture of the lYmphocytes The lymphocytes isolated as described a~ove are cultured in 24-well Costar plates, 500 ~1 of cell suspension/well (5 x 105 cells/ml or 2.5 x 105 cells) in RPMI 1640 medium (Eurobio) made up with 10% foetal calf serum (ATGC), 2 mM glutamine (Gibco), penicillin and streptomycin (500 ~/ml, 0.25 mg/ml), and phyto-haemagglutinin (SIGMA) 5 ~g/ml. After incubation for 4 days under cell culture conditions (37~C, CO2/O2 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 CA 02262~64 1999-01-29 W O 98/04270 PCT~B97/00951 g 24-well Costar plate.
Action of the elastin peptides A sterile solution of K-elastin (75 kD, Solabia) is added at the concentration indicated (for example 2 ~g/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 ~y pipetting and counted. The suspension is centrifuyed 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 ~1 per tube and kept at -40~C if it is not u6ed immediately for enzymatic determinations. The cell sediment is resuspended in extraction buffer (0.1% Triton X-100, 1 M NaCl, 0.02% NaN3 0.01% 8rij 35, pH 8). 1 ml per 106 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 det~r~;n~tions.
Determination of the enzymatic activity of leucocyte elastase-type .
The synthetic substrate used for determining the enzymatic activity of elastase-type i~ Me-O-Suc-Ala-Ala-Pro-Val-pNA. 50 ~1 of culture medium or 20 ~1 of cell lysate are mixed with the buffer (100 mM tris-HCl, 0.05%
CaCl2, 0.02% NaN3, 0.01% Brij 35, pH 8) up to 190 ~1, then with 10 ~1 of 85 mM substrate solution (in N-methylpyr-rolidone) for a total volume of 200 ~1. The optical density is read immediately at 410 nm and then after 4, 24, 48 and 72 hours' incubation at 37~C. The elastase activity i8 expre6sed in nM of substrate hydrolysed per 106 cells and per hour.
Determination of the enzymatic activitY of cathepsin G
25 mg of the su~strate, Me O-Suc-Ala-Ala-Pro-Met-W O 98/04270 PCT~B97/0095 pNA in 1 ml of N-methylpyrrolidone (40 nM) are used under the same conditions as above.
2 - Re~ults 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 i8 the entry of cells into proliferation. It ha6 been shown that thi~ was the case for human skin fibroblasts in the presence of elastin peptides (Ghuysen-Itard et al, C.R. Acad. Sci., 1992, 315 : 473-478). The result~ reported in Table 1 show that a stimulation analogous to the proliferation of lymphocytes was observed.
Table 1 Concentration Number of experiments Percentage ~timulation, of K-ela~tin mean + SD
2 ~g/ml 6 62.67 + 37.90 10 ~g/ml 13 35.76 + 29.50 A m~i mllm Rtimulation is observed at 2 ~g/ml of elastin peptides and a little weaker ~timulation with 10 ~g/ml of elastin peptides.
B) Liberation of proteolytic activity by the elastin peptides When PMNs or lymphocytes are incubated under culture condition~ in the presence of 2 ~g/ml of K-elastin, a gradual increase in the elastase and CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 cathepsin activity can be determined in the cell extract6 and the cell lysates. Thi6 increa6e i6 much more marked in the cell6 derived from elderly athero6clerotic 6ubject6. Such an increaBe iB not observed in the absence of K-elastin. Addition of elastin peptides increa6e6 the proteolytic activity 6imultaneously 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) ~ffect of elastin peptides on cell survival Thi6 experiment is carried out under the culture condition6 such a6 described above, in the pre6ence of phytohaemaglutinin (PHA).
S ml of aliquots of lymphocyte suspension (2.5 x 106 cell6 in 5 ml) obtained starting from human tonsil6 - are di6tributed in te~t tubes and increa6ing concentra-tion6 of ~-elastin (BPM, PM c 10 kDa) are added:
0.1 ~g/ml, 2 ~g/ml, 10 ~g/ml, 500 ~g/ml, 1 and 2 mg/ml.
After 4 day6' incubation under the culture conditions the cell6 are recovered and counted. Table 2 gives the cell 1O6s a6 a function of the concentration of elastin peptide6 added.
W098/04270 PCT~B97/00951 Table 2 Concentration of elastin peptide~ Percentage of cell 1O8~
~g/ml det~rmined by Trypan Blue exclusion 0 5.3 1 5.0 2 2.5 17.4 lO0 l9.6 500 28.9 0 lO00 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 ~ ted by the elastin receptor of l~ ,hocytes 1) Inhibition of cell proliferation As shown in Figure l, 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 (l 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 CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 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 ~g/ml of K-elastin. The stimulation of the liberation of a lytic enzymatic activity i~ inhibited effecti~ely by melibiose starting from 1 ~g/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 ~ n ly, hocytes Methods Ant; hoAi es used:
- 1st antibody: 67 kD elastin/l~m;n;n 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 Tmmllnoresearch 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 ~g/ml of phytohaemagglutinin (to acti~ate them) and for certain cultures with 2 ~g/ml of kappa-elastin of high CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 molecular weight (75 kD~ in an RPMI 1640 medium with 10%
foetal calf serum (FCS). After 48 to 120 hours' incuba-tion at 37~C, the lymphocytes are washed three times with ~PMI medium contA;ning 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' cells/ml with RPMI medium cont~;n;ng 2% FCS.
Then a volume (100 ~1) of cell suspension of human lymphocytes (approximately 106 cells) is incubated with 10 ~1 of solution of the first antibody (diluted to 1:100) for 30 minutes in an ice bath. 1.5 ml of cold ~PMI
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 su~pension, 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 centri-fuged 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 S minutes, the 61ides 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 brightnes6 of the ;mml~nofluorescence.
To identify the labelled cells, microscopic fields are ~mined 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:
CA 02262~64 1999-01-29 W O 98/04270 PCT~B97/00951 - total T lymphocytes (CD3+) - "helper" T lymphocytes (CD4+) - suppressor T lymphocytes (CD8+) - B lymphocytes (CD20+) 5 - T activated lymphocytes (CD25+) - memory T lymphocytes (CDA+/CD45RO+) - granulocytes (CD15+) Description of the labels used The CD 3 complex is expressed by all mature hl7m~n T cells. It has molecular weights of between 16 and 28 ~D
composed of 5 chains (~ ) a6sociated with the T-cell receptor in a non-co~alent manner. It i~ involved in the transmission of activation signals.
CD 4 (T4) recognizes the class II MHC molecules - during the interaction of CD4+ cells with cells present-ing the antigen or target cells. It is a glycoprotein of 59 kD belonging to the ;~llnoglobulin superfamily. It is found in the "helper/inducer~ subpopulation of T
lymphocytes (45% of the lymphocytes of peripheral blood).
The CD 8 molecule (T8, 30/32 kD) is a glycoprotein formed of two peptide ch~in~ It iB 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.
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 CA 02262~64 1999-01-29 t30-60%). This antigen is absent from the 6urface of normal lymphocytes.
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.
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.
This is a tr~n~ ~ hrane 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 CD45R0 are memory T lymphocytes (or primed T cells) (45%
of the T lymphocytes of the peripheral blood).
The CD4+/CD45R0+ cells produce "helper" signals.
These are early producers of IL-2 and IFN-r.
For labelling with anti-CDx antibodies (Sigma products, excluding anti-CD25 antibody, from Serotec), 100 ~l of the lymphocyte su~pension (already labelled or not) are taken with the 67 kD anti-receptor antibody of elastin at a cell density of 107 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 i~ 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 ;mm--noglobulins (non-speci-CA 02262~64 1999-01-29 fic myeloma proteins) (for control).
R~S~LTS
a) Fluoresce~ce microscopy Demonstration of the elastin reCePtor on activated lymphocytes Part of the analysed lymphocytes - those which express the 67 kD elastin/l~;n;n receptor - show a specific ;~mllnofluorescence under the experimental conditions described above (positive cells).
The Percentaqe of positive cells on the 4th day of culture is evaluated bY fluorescence microscoPY:
- cells cultured in the presence of 2 ~g/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 PXA is a sufficient stimulus to make the elastin receptor express.
The percentaqe of Positive cells on the 5th daY of culture:
- cells cultured in the presence of 2 ~g/ml of kappa-elastin: 77.2% + 6.7%.
Effect of washinq cells with lactose or melibiose:
- cells cultured in the presence of 2 ~g/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 pre~ence of 2 ~g~ml of kappa-elastin + washed at the end of cell culture with a solution of 1 mg/ml of melibiose: lB.3% + 9.7% (p~0.001).
Fluorescent cells incubated only with the second CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 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 deRorbing 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 1 lymphocytes expressing the elastin receptor on different culture days D0: on the day of separation of the lymphocytes;
D2, D3, D5: on the second; third; fifth day after separa-tion of the lymphocytes.
% of positive cells Day without number of with 2 ~g/ml of P
kappa-elastin experiments kappa-elastin D01.12 + 0.2% 6 1.15+ 0.1%
D222.33 + 5.2% 5 23.02 + 3.2%
D329.70 + 0.8% 7 32.40 + 3.5% 0.178 D559.91 + 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 (266%) express the elastin receptor on their surface.
- Results of double lab~ll;n~8 CA 02262~64 l999-0l-29 W O 98/04270 PCT~B97/009~1 Table 4 Double lab~ll i n~ iment of 1~ ~hocytes to detr ;~ the nature of the subpopulations expressing the elastin receptor 5% of cells 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+R67kD = 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 expre6s 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 e~m;ned 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 CD45RO+ lymphocytes which in the presence of CA 02262~64 1999-01-29 W 098/04270 rCT~B97/00951 - 20 -elastin peptides 6trongly increased the expression of the elastin receptor. It is a matter in that case of a subpopulation of cells considered as helper and memory cell~. The coupling of the elastin receptor by positive retroaction to its own synthesis after its activation would thus be specific to these cells.
A le 4: Protection against the cytotoxic effect of ~appa-elastin of low ~ lecular weight on ly, hr~Cyte8 1) Isolation of the ly ~hs~ytes Isolation of the lymphocytes according to the method described in Example 1.
The cell suspension is diluted with RPMI contain-ing 5 mM glutamine and 10% FCS such that the concentra-tion is 106 cells/2 ml.
2) Cell culture (D0) The lymphocytes are put into culture in 24-well Costar plates (500 ~1 of cell suspension/well at a concentration of 106 cells/2 ml) in complemented RPMI
1640 medium. The distribution of the cells is as follows:
- 10 ml of cell suspension without K-elastin;
- 10 ml of cell suspension with 2 ~g/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 su~pension with 1 mg/ml lactose W098/04270 PCTnB97/00951 3) Counting of the cell~ in the pre~ence of Trypan Blue to determine the percentage of dead cell~
On the fifth day of culture (D4), the cell ~u~pension i~ reco~ered, the cells are counted in a Mala~sez cell in the pre~ence of 0.1% Trypan Blue (Sigma) (only the dead cells are ~tained ~lue with Trypan Blue).
Counting i~ carried out immediately after having added the ~tain on account of the toxicity of Trypan Blue.
.... .
W098/04270 PCT~B97100951 R~S~LTS
T~R~ - number of % of dead cell6 cells/ml (Trypan Blue) without ~rr~-elastin 5.8 _ 0.6 9.2 + 0.4 with 2 ~g/ml ~appa- lO.23 _ 3.5 6.6 ~ l.3 5 elastin (control) with 2 mg/ml ~appa- 3.6 + l.0 41.3 + 3.l elastin with l mg/ml lactose 7.7 + l.5 14.5 _ l.9 with l mg/ml -lihiose 7.26 _ 1.3 15.4 _ 1.6 10 with 2 mg/ml ~appa-elastin + 1 mg/ml 8.8 + O.7 lO.6 + 2.3 lactose with 2 mg/ml ~appa-elastin ~ 1 mg/ml 8.8 + 1.6 10.5 + 0.9 l5 meli~iose The table Rhows the percentage of dead cells in the presence of elastin peptides and the protection against cell death by lactose and melibiose. This protec-tion 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 l.3% (96% protection).
COMPOSmON
The present invention relates to compounds useful in the field of ~ lnology and in particular for the treatment of hypersensitivity reactions responsible for allergies and various intolerance phenomena.
Immunological defence against infectious agents, 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 mechani6m 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 tissue grafts.
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 Fc 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 Ig~) form, with the antigen and the complement, immune complexes which are deposited in the tissues and cause the production of chemotactic factor6 for polynuclear neutrophils; local inflammation results.
_ Type 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 macrophage6.
The lymphocytes are thus key cells in ;mmllnlty with cellular or humoral mediation, especially with the secretion of antibodies.
Allergic ailments can be the expression of one or more SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 PCTnB97/~951 types of immunological reaction~.
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 hyper~ensitivity 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 te ;nal po~ition, or of their derivatives, for the preparation of immunomodulator compo6itions, 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, lactoRe and their derivatives capable of being obtained by addition of a hydrophobic residue.
Hydrophobic substituents are especially understood as meaning linear or branched C1-C1g alkyls, C1-C1g alkylamines, linear or branched, optionally subQtituted Cl-C1g carboxylic acids, linear or branched, primary, secondary or tertiary C1-C1g amide~, and C1-C1g arylalkyls.
The oligosaccharide derivatives suited for carrying out the invention can e~pecially belong to one of the categories mentioned below, in which the oligosaccharide corresponds to the following general formula:
galactose-n (a or ~) - (Hex)p in which n represents the position 1, 2, 3, 4 or 6, SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 3 PCT~B97/00951 Hex represents an a- or ~-linked hexose or pentose, p is a number between l and 5;
a) - glycosides corresponding to the formulae:
(I) oligosaccharide l-O-R, in which R i8 a linear or branched alkyl residue of l to 18 carbon atoms, ~ (II) oligosaccharide l-O-R-O-l-oligosaccharide in which R = (CH2)m, m being between 2 and lO, b) - an osylamine acylated according to one of the following formulae, in which the oligosaccharide is preferably lacto~e, melibiose or stachiose:
- osylamines acylated corresponding to one of the following formulae:
~ (III) oligosaccharide l-NH-CO-R, in which R is an alkyl residue of 2 to 18 carbon atoms, containing 0, l or 2 double bonds, ~ (IV) oligofiaccharide l-NH-CO-R-CO-NH-l-oligosaccharide, in which R = (CH2)m, m being between 2 and 8, c) - an alkylamine 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 e~ning as in formula (III), 2~ d) - or a reduction product of Schiff bases formed by oligo6accharides 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, l or 2, X = l-NH2-hexitol, and R has the same meaning as in (III).
SUBSTITUTE StlEET (RULE 26) CA 02262~64 1999-01-29 WO g8/04270 PCT/IB97/00951 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 disorder6 associated with hyper6ensitivity can be correlated with the fixation, on a 6pecific lymphocyte receptor, of peptides re6ulting from the degradation of elastin, especially kappa-elastin.
The activation of these receptors triggers the liberation of lytic enzymes, ~-glucuronidase, elastases and free radicals such a6 the 6uperoxide ion, or hydrogen peroxide, which generates hydroxyl radical6. 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 de6quamation can take place.
All these symptoms will be ameliorated or even suppressed by the use of compositions cont~;n;ng 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 li~ewise inhibit the potentialization and liberation of lytic enzymes.
The compositions will preferably be formulated in vehicle6 not comprising any perfume or allergenic agents. The SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W098/04270 5 PCT~B97/00951 compositions could be in the form of solutions, gels, lotions, creams, W/0 or 0/W 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 6uited to the treatment or to the prevention of intolerance and/or allergic reactions of the 6kin 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 derivative6 could be a6sociated with other agents enabling the skin to be protected and/or hydrated, such as hyaluronic acid, vitamin E, glycol extract of G. biloba, 60rbitol, glucosaminoglycans, alginates, etc.; they can likewise be a6sociated with vegetable oils to nourish the skin and/or with active emollient6 and salves 6uch as, for example, extracts of cat grape, althaea, oats, linden, camomile, sweet clover, Ruscus, procyanidols, a-bi6abolol, coconut oil, 18-~-glycyrrhetinic acid.
According to another aspect of the invention, the oligo6accharides and their derivatives such as defined above are used for the preparation of a compo6ition which additionally contains pharm~ceutically 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 ;mm--ne 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 eczema6.
The oligosaccharides having a galactose residue in the non-reducing tPrm;n~l position, and their derivatives SUBSTITUTE SHEE~ (RULE 26) CA 02262~64 1999-01-29 W098/04270 6 PCT~B97/00951 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 co6metic S treatment of hyperreactive skins, characterized in that a composition containing at least one oligosaccharide comprising from 2 to 6 oside re6idues, 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.
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 illu~trate the invention.
In these examples, reference will be made to the following figures:
Fig. l:Inhibition by lactose and melibiose of the activity of stimulation of lymphocyte proliferation by ~-elastin. The concentrations of lactose and of melibiose are l ~g/ml, lO
~g/ml, lO0 ~g/ml, l mg/ml and 2 mg/ml.
Fig. 2:Inhibition by lactose and melibio~e of the expre~ion of elastic activity by lymphocyte~, stimulated with 2 ~g/ml of ~-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 ~g/ml of K-elastin. Activity in the culture medium.
Example l l - Methods SeParation of lymPhocYtes SUBSTITUTE SHEET (RULE 26) CA 02262~64 1999-01-29 W O 98/04270 rcTnBg7/00951 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 centri-fuged 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 se~im~nt is taken up in 10 ml of RPMI and the cells are counted. The monocytes and macrophages are eliminated by adhesion on a plastic surface (incubation for 2 hours in a CO2/O2 incubator).
- SeParation of the PolYnuclear cells (PMN) After the first centrifugation at 3000 rpm (say 600 g), the residue cont~in;ng 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 i~ 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 ~hock 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 CA 02262~64 1999-01-29 W 098/04270 PCT~B97/00951 centrifugation for 20 min at 0~C. The supernatant con-tains the enzymes.
Preparation of lYmPhocytes startinq from human tonsil~
Freshly remo~ed tonsils are cut into small pieces under sterile conditionR, in 10 ml of ~PMI. The tissue suspension is filtered on a coarse filter in order to remove the largest tissue debris. The suspension i8 placed in a test tube and allowed to s~im~nt for 15 min at A~hient temperature. The supernatant is placed on Ficoll-paque plus and centrifuged as described for lymphocytes derived from blood. The monocytes are elimin-ated as described for the lymphocytes derived from blood.
The sediment containing the lymphocytes is re~uspended 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~o of type T and 20% of type B.
Culture of the lYmphocytes The lymphocytes isolated as described a~ove are cultured in 24-well Costar plates, 500 ~1 of cell suspension/well (5 x 105 cells/ml or 2.5 x 105 cells) in RPMI 1640 medium (Eurobio) made up with 10% foetal calf serum (ATGC), 2 mM glutamine (Gibco), penicillin and streptomycin (500 ~/ml, 0.25 mg/ml), and phyto-haemagglutinin (SIGMA) 5 ~g/ml. After incubation for 4 days under cell culture conditions (37~C, CO2/O2 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 CA 02262~64 1999-01-29 W O 98/04270 PCT~B97/00951 g 24-well Costar plate.
Action of the elastin peptides A sterile solution of K-elastin (75 kD, Solabia) is added at the concentration indicated (for example 2 ~g/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 ~y pipetting and counted. The suspension is centrifuyed 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 ~1 per tube and kept at -40~C if it is not u6ed immediately for enzymatic determinations. The cell sediment is resuspended in extraction buffer (0.1% Triton X-100, 1 M NaCl, 0.02% NaN3 0.01% 8rij 35, pH 8). 1 ml per 106 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 det~r~;n~tions.
Determination of the enzymatic activity of leucocyte elastase-type .
The synthetic substrate used for determining the enzymatic activity of elastase-type i~ Me-O-Suc-Ala-Ala-Pro-Val-pNA. 50 ~1 of culture medium or 20 ~1 of cell lysate are mixed with the buffer (100 mM tris-HCl, 0.05%
CaCl2, 0.02% NaN3, 0.01% Brij 35, pH 8) up to 190 ~1, then with 10 ~1 of 85 mM substrate solution (in N-methylpyr-rolidone) for a total volume of 200 ~1. The optical density is read immediately at 410 nm and then after 4, 24, 48 and 72 hours' incubation at 37~C. The elastase activity i8 expre6sed in nM of substrate hydrolysed per 106 cells and per hour.
Determination of the enzymatic activitY of cathepsin G
25 mg of the su~strate, Me O-Suc-Ala-Ala-Pro-Met-W O 98/04270 PCT~B97/0095 pNA in 1 ml of N-methylpyrrolidone (40 nM) are used under the same conditions as above.
2 - Re~ults 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 i8 the entry of cells into proliferation. It ha6 been shown that thi~ was the case for human skin fibroblasts in the presence of elastin peptides (Ghuysen-Itard et al, C.R. Acad. Sci., 1992, 315 : 473-478). The result~ reported in Table 1 show that a stimulation analogous to the proliferation of lymphocytes was observed.
Table 1 Concentration Number of experiments Percentage ~timulation, of K-ela~tin mean + SD
2 ~g/ml 6 62.67 + 37.90 10 ~g/ml 13 35.76 + 29.50 A m~i mllm Rtimulation is observed at 2 ~g/ml of elastin peptides and a little weaker ~timulation with 10 ~g/ml of elastin peptides.
B) Liberation of proteolytic activity by the elastin peptides When PMNs or lymphocytes are incubated under culture condition~ in the presence of 2 ~g/ml of K-elastin, a gradual increase in the elastase and CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 cathepsin activity can be determined in the cell extract6 and the cell lysates. Thi6 increa6e i6 much more marked in the cell6 derived from elderly athero6clerotic 6ubject6. Such an increaBe iB not observed in the absence of K-elastin. Addition of elastin peptides increa6e6 the proteolytic activity 6imultaneously 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) ~ffect of elastin peptides on cell survival Thi6 experiment is carried out under the culture condition6 such a6 described above, in the pre6ence of phytohaemaglutinin (PHA).
S ml of aliquots of lymphocyte suspension (2.5 x 106 cell6 in 5 ml) obtained starting from human tonsil6 - are di6tributed in te~t tubes and increa6ing concentra-tion6 of ~-elastin (BPM, PM c 10 kDa) are added:
0.1 ~g/ml, 2 ~g/ml, 10 ~g/ml, 500 ~g/ml, 1 and 2 mg/ml.
After 4 day6' incubation under the culture conditions the cell6 are recovered and counted. Table 2 gives the cell 1O6s a6 a function of the concentration of elastin peptide6 added.
W098/04270 PCT~B97/00951 Table 2 Concentration of elastin peptide~ Percentage of cell 1O8~
~g/ml det~rmined by Trypan Blue exclusion 0 5.3 1 5.0 2 2.5 17.4 lO0 l9.6 500 28.9 0 lO00 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 ~ ted by the elastin receptor of l~ ,hocytes 1) Inhibition of cell proliferation As shown in Figure l, 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 (l 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 CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 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 ~g/ml of K-elastin. The stimulation of the liberation of a lytic enzymatic activity i~ inhibited effecti~ely by melibiose starting from 1 ~g/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 ~ n ly, hocytes Methods Ant; hoAi es used:
- 1st antibody: 67 kD elastin/l~m;n;n 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 Tmmllnoresearch 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 ~g/ml of phytohaemagglutinin (to acti~ate them) and for certain cultures with 2 ~g/ml of kappa-elastin of high CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 molecular weight (75 kD~ in an RPMI 1640 medium with 10%
foetal calf serum (FCS). After 48 to 120 hours' incuba-tion at 37~C, the lymphocytes are washed three times with ~PMI medium contA;ning 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' cells/ml with RPMI medium cont~;n;ng 2% FCS.
Then a volume (100 ~1) of cell suspension of human lymphocytes (approximately 106 cells) is incubated with 10 ~1 of solution of the first antibody (diluted to 1:100) for 30 minutes in an ice bath. 1.5 ml of cold ~PMI
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 su~pension, 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 centri-fuged 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 S minutes, the 61ides 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 brightnes6 of the ;mml~nofluorescence.
To identify the labelled cells, microscopic fields are ~mined 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:
CA 02262~64 1999-01-29 W O 98/04270 PCT~B97/00951 - total T lymphocytes (CD3+) - "helper" T lymphocytes (CD4+) - suppressor T lymphocytes (CD8+) - B lymphocytes (CD20+) 5 - T activated lymphocytes (CD25+) - memory T lymphocytes (CDA+/CD45RO+) - granulocytes (CD15+) Description of the labels used The CD 3 complex is expressed by all mature hl7m~n T cells. It has molecular weights of between 16 and 28 ~D
composed of 5 chains (~ ) a6sociated with the T-cell receptor in a non-co~alent manner. It i~ involved in the transmission of activation signals.
CD 4 (T4) recognizes the class II MHC molecules - during the interaction of CD4+ cells with cells present-ing the antigen or target cells. It is a glycoprotein of 59 kD belonging to the ;~llnoglobulin superfamily. It is found in the "helper/inducer~ subpopulation of T
lymphocytes (45% of the lymphocytes of peripheral blood).
The CD 8 molecule (T8, 30/32 kD) is a glycoprotein formed of two peptide ch~in~ It iB 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.
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 CA 02262~64 1999-01-29 t30-60%). This antigen is absent from the 6urface of normal lymphocytes.
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.
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.
This is a tr~n~ ~ hrane 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 CD45R0 are memory T lymphocytes (or primed T cells) (45%
of the T lymphocytes of the peripheral blood).
The CD4+/CD45R0+ cells produce "helper" signals.
These are early producers of IL-2 and IFN-r.
For labelling with anti-CDx antibodies (Sigma products, excluding anti-CD25 antibody, from Serotec), 100 ~l of the lymphocyte su~pension (already labelled or not) are taken with the 67 kD anti-receptor antibody of elastin at a cell density of 107 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 i~ 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 ;mm--noglobulins (non-speci-CA 02262~64 1999-01-29 fic myeloma proteins) (for control).
R~S~LTS
a) Fluoresce~ce microscopy Demonstration of the elastin reCePtor on activated lymphocytes Part of the analysed lymphocytes - those which express the 67 kD elastin/l~;n;n receptor - show a specific ;~mllnofluorescence under the experimental conditions described above (positive cells).
The Percentaqe of positive cells on the 4th day of culture is evaluated bY fluorescence microscoPY:
- cells cultured in the presence of 2 ~g/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 PXA is a sufficient stimulus to make the elastin receptor express.
The percentaqe of Positive cells on the 5th daY of culture:
- cells cultured in the presence of 2 ~g/ml of kappa-elastin: 77.2% + 6.7%.
Effect of washinq cells with lactose or melibiose:
- cells cultured in the presence of 2 ~g/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 pre~ence of 2 ~g~ml of kappa-elastin + washed at the end of cell culture with a solution of 1 mg/ml of melibiose: lB.3% + 9.7% (p~0.001).
Fluorescent cells incubated only with the second CA 02262~64 1999-01-29 W098/04270 PCT~B97/00951 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 deRorbing 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 1 lymphocytes expressing the elastin receptor on different culture days D0: on the day of separation of the lymphocytes;
D2, D3, D5: on the second; third; fifth day after separa-tion of the lymphocytes.
% of positive cells Day without number of with 2 ~g/ml of P
kappa-elastin experiments kappa-elastin D01.12 + 0.2% 6 1.15+ 0.1%
D222.33 + 5.2% 5 23.02 + 3.2%
D329.70 + 0.8% 7 32.40 + 3.5% 0.178 D559.91 + 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 (266%) express the elastin receptor on their surface.
- Results of double lab~ll;n~8 CA 02262~64 l999-0l-29 W O 98/04270 PCT~B97/009~1 Table 4 Double lab~ll i n~ iment of 1~ ~hocytes to detr ;~ the nature of the subpopulations expressing the elastin receptor 5% of cells 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+R67kD = 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 expre6s 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 e~m;ned 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 CD45RO+ lymphocytes which in the presence of CA 02262~64 1999-01-29 W 098/04270 rCT~B97/00951 - 20 -elastin peptides 6trongly increased the expression of the elastin receptor. It is a matter in that case of a subpopulation of cells considered as helper and memory cell~. The coupling of the elastin receptor by positive retroaction to its own synthesis after its activation would thus be specific to these cells.
A le 4: Protection against the cytotoxic effect of ~appa-elastin of low ~ lecular weight on ly, hr~Cyte8 1) Isolation of the ly ~hs~ytes Isolation of the lymphocytes according to the method described in Example 1.
The cell suspension is diluted with RPMI contain-ing 5 mM glutamine and 10% FCS such that the concentra-tion is 106 cells/2 ml.
2) Cell culture (D0) The lymphocytes are put into culture in 24-well Costar plates (500 ~1 of cell suspension/well at a concentration of 106 cells/2 ml) in complemented RPMI
1640 medium. The distribution of the cells is as follows:
- 10 ml of cell suspension without K-elastin;
- 10 ml of cell suspension with 2 ~g/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 su~pension with 1 mg/ml lactose W098/04270 PCTnB97/00951 3) Counting of the cell~ in the pre~ence of Trypan Blue to determine the percentage of dead cell~
On the fifth day of culture (D4), the cell ~u~pension i~ reco~ered, the cells are counted in a Mala~sez cell in the pre~ence of 0.1% Trypan Blue (Sigma) (only the dead cells are ~tained ~lue with Trypan Blue).
Counting i~ carried out immediately after having added the ~tain on account of the toxicity of Trypan Blue.
.... .
W098/04270 PCT~B97100951 R~S~LTS
T~R~ - number of % of dead cell6 cells/ml (Trypan Blue) without ~rr~-elastin 5.8 _ 0.6 9.2 + 0.4 with 2 ~g/ml ~appa- lO.23 _ 3.5 6.6 ~ l.3 5 elastin (control) with 2 mg/ml ~appa- 3.6 + l.0 41.3 + 3.l elastin with l mg/ml lactose 7.7 + l.5 14.5 _ l.9 with l mg/ml -lihiose 7.26 _ 1.3 15.4 _ 1.6 10 with 2 mg/ml ~appa-elastin + 1 mg/ml 8.8 + O.7 lO.6 + 2.3 lactose with 2 mg/ml ~appa-elastin ~ 1 mg/ml 8.8 + 1.6 10.5 + 0.9 l5 meli~iose The table Rhows the percentage of dead cells in the presence of elastin peptides and the protection against cell death by lactose and melibiose. This protec-tion 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 l.3% (96% protection).
Claims (13)
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, said oligosaccharide having the following general formula:
galactose-n (.alpha. or .beta.) - (Hex)p in which n represents the position 1, 2, 3, 4 or 6, Hex represents an .alpha.- or .beta.-linked hexose or pentose, p is a number between 1 and 5;
and the derivative of said oligosaccharide being chosen in the following categories:
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-O-R-O-1-oligosaccharide in which R = (CH2)m m being between 2 and 10, b) - an osylamine acylated according 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-1-oligosaccharide, in which R = (CH2)m m being between 2 and 8, c) - an alkylamine 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, in which 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 = 1-NH2-hexitol, and R has the same meaning as in (III), for the preparation of an immunomodulating medicament involved in cellular immune reactions.
galactose-n (.alpha. or .beta.) - (Hex)p in which n represents the position 1, 2, 3, 4 or 6, Hex represents an .alpha.- or .beta.-linked hexose or pentose, p is a number between 1 and 5;
and the derivative of said oligosaccharide being chosen in the following categories:
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-O-R-O-1-oligosaccharide in which R = (CH2)m m being between 2 and 10, b) - an osylamine acylated according 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-1-oligosaccharide, in which R = (CH2)m m being between 2 and 8, c) - an alkylamine 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, in which 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 = 1-NH2-hexitol, and R has the same meaning as in (III), for the preparation of an immunomodulating medicament involved in cellular immune reactions.
2. Use according to claim 1, for the preparation of an immunomodulating medicament involved in cellular immune reactions, said medicament confering a protection against the cytotoxic effect of kapa-elastin of low molecular weight on lymphocytes.
3.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, said oligosaccharide and derivative thereof being as defined in claim 1, for the preparation of a composition intended for the treatment or the prevention of hypersensitivity reactions and involved in cellular immune reactions.
4.Use according to one of claims 1 to 3, characterized in that the oligosaccharide is chosen from the group formed of melibiose and lactose, the derivative of said oligosaccharide being as defined in claim 1.
5.Use 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 administration by the parenteral or enteral route.
7.Use 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 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, characterized 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, said oligosaccharide and derivative thereof being as defined in claim 1.
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, said oligosaccharide and derivative thereof being as defined in claim 1, is applied by the topical route in a cosmetically acceptable vehicle.
13.Method of cosmetic treatment, characterized in that a composition containing melibiose or a derivative of melibiose capable of being obtained by addition of a hydrophobic residue, is applied by the topical route in a cosmetically acceptable vehicle.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9609649A FR2751876B1 (en) | 1996-07-31 | 1996-07-31 | USE OF AN OLIGOSACCHARIDE AS AN IMMUNOMODULATOR, DERMATO-COSMETIC COMPOSITION AND METHOD OF COSMETIC TREATMENT |
| FR96/09649 | 1996-07-31 | ||
| PCT/IB1997/000951 WO1998004270A1 (en) | 1996-07-31 | 1997-07-30 | Use of an oligosaccharide as an immunomodulator in a dermato-cosmetic composition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2262564A1 true CA2262564A1 (en) | 1998-02-05 |
Family
ID=9494678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002262564A Abandoned CA2262564A1 (en) | 1996-07-31 | 1997-07-30 | Use of an oligosaccharide as an immunomodulator in a dermato-cosmetic composition |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0954322A1 (en) |
| JP (1) | JP2000516591A (en) |
| KR (1) | KR20000029754A (en) |
| CN (1) | CN1228705A (en) |
| AU (1) | AU3457697A (en) |
| BR (1) | BR9710630A (en) |
| CA (1) | CA2262564A1 (en) |
| CZ (1) | CZ29599A3 (en) |
| FR (1) | FR2751876B1 (en) |
| HU (1) | HUP9903904A3 (en) |
| NZ (1) | NZ333985A (en) |
| PL (1) | PL331478A1 (en) |
| RU (1) | RU99104398A (en) |
| SK (1) | SK12199A3 (en) |
| WO (1) | WO1998004270A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19836339B4 (en) | 1998-08-11 | 2011-12-22 | N.V. Nutricia | carbohydrate mix |
| CN100528169C (en) * | 2002-05-31 | 2009-08-19 | 天野酶株式会社 | Antiinflammatory agent, agent for preventing/ameliorating allergic diseases and functional food |
| EP2223691A1 (en) * | 2003-10-24 | 2010-09-01 | N.V. Nutricia | Immunemodulating oligosaccharides |
| JP2005281298A (en) * | 2004-03-04 | 2005-10-13 | Fancl Corp | T cell immunomodulator comprising melibiose as active ingredient |
| EP1597978A1 (en) | 2004-05-17 | 2005-11-23 | Nutricia N.V. | Synergism of GOS and polyfructose |
| EA200802038A1 (en) * | 2006-04-10 | 2009-06-30 | Александр Владимирович ДИКОВСКИЙ | PHARMACEUTICAL COMPOSITION OF ENTEROSORBENT AND PREBIOTICS, MEDICAL FORMS AND METHOD OF PREVENTION AND TREATMENT OF DISEASES OF THE GASTROINTESTINAL TRACT |
| GB0915315D0 (en) | 2009-09-03 | 2009-10-07 | Univ Manchester | Use of non-digestible oligosaccharides |
| GB2500585A (en) | 2012-03-23 | 2013-10-02 | Univ Manchester | Use of oligosaccharides to reduce skin pigmentation |
| CN105764518A (en) * | 2013-10-25 | 2016-07-13 | 红艇有限公司 | Dietary regime for treatment of acne and other inflammatory skin conditions |
| US11328621B2 (en) | 2013-10-25 | 2022-05-10 | Red Pinnace Limited | Dietary regime for treatment of acne and other inflammatory skin conditions |
| KR101689875B1 (en) | 2014-08-28 | 2016-12-26 | (주)셀아이콘랩 | Cosmetic composition for treating atopic dermatitis containing mixtures of peptide and amino acid |
| KR101689877B1 (en) | 2014-08-28 | 2016-12-26 | (주)셀아이콘랩 | Cosmetic composition for treating atopic dermatitis |
| KR102046566B1 (en) | 2018-08-09 | 2019-11-19 | 박정혜 | cosmetic composition for improving pruritus of skin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| FR2545087B1 (en) * | 1983-04-29 | 1985-12-27 | Malte Oeuvres Hospit Fses Ordr | NOVEL CARBOHYDRATE DERIVATIVES OF 5-HYDROXYTRYPTOPHANE, OBTAINMENT AND APPLICATION AS MEDICAMENTS |
| GR860379B (en) * | 1985-02-22 | 1986-06-11 | Akzo Nv | Novel disaccharide and trisaccharide derivatives of the lipid a type |
| CA2118405A1 (en) * | 1992-05-26 | 1993-09-12 | Robert Maurice Ippolito | Immunosuppressive and tolerogenic modified lewisc and lacnac compounds |
-
1996
- 1996-07-31 FR FR9609649A patent/FR2751876B1/en not_active Expired - Fee Related
-
1997
- 1997-07-30 EP EP97930717A patent/EP0954322A1/en not_active Withdrawn
- 1997-07-30 WO PCT/IB1997/000951 patent/WO1998004270A1/en not_active Application Discontinuation
- 1997-07-30 CA CA002262564A patent/CA2262564A1/en not_active Abandoned
- 1997-07-30 RU RU99104398A patent/RU99104398A/en unknown
- 1997-07-30 CZ CZ99295A patent/CZ29599A3/en unknown
- 1997-07-30 AU AU34576/97A patent/AU3457697A/en not_active Abandoned
- 1997-07-30 JP JP10508650A patent/JP2000516591A/en active Pending
- 1997-07-30 CN CN97197506A patent/CN1228705A/en active Pending
- 1997-07-30 PL PL97331478A patent/PL331478A1/en unknown
- 1997-07-30 HU HU9903904A patent/HUP9903904A3/en unknown
- 1997-07-30 KR KR1019997000867A patent/KR20000029754A/en not_active Application Discontinuation
- 1997-07-30 SK SK121-99A patent/SK12199A3/en unknown
- 1997-07-30 BR BR9710630-5A patent/BR9710630A/en unknown
- 1997-07-30 NZ NZ333985A patent/NZ333985A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000516591A (en) | 2000-12-12 |
| RU99104398A (en) | 2001-02-20 |
| SK12199A3 (en) | 2000-01-18 |
| AU3457697A (en) | 1998-02-20 |
| CZ29599A3 (en) | 1999-06-16 |
| CN1228705A (en) | 1999-09-15 |
| HUP9903904A2 (en) | 2000-04-28 |
| KR20000029754A (en) | 2000-05-25 |
| HUP9903904A3 (en) | 2000-05-29 |
| EP0954322A1 (en) | 1999-11-10 |
| FR2751876A1 (en) | 1998-02-06 |
| PL331478A1 (en) | 1999-07-19 |
| BR9710630A (en) | 2000-01-11 |
| NZ333985A (en) | 2000-09-29 |
| FR2751876B1 (en) | 1998-12-31 |
| WO1998004270A1 (en) | 1998-02-05 |
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