NOVEL ACTIVE IMMUNOMODULATORY AGENT AND COMPOSITION CONTAINING SAME

Abstract

Disclosed is a sulphated polysaccharide extracted from a red alga of the Haliptilon subulatum species or a salt thereof as active ingredient. More particularly, the present application relates to a sulphated polysaccharide extracted from a red alga of the Haliptilon subulatum species or a salt thereof as active ingredient, for its use in the modulation of the immune response in humans or animals, in particular in the prevention and/or treatment of inflammatory diseases, in particular of the skin.

Claims

1. Sulphated polysaccharide extracted from a red alga of the Haliptilon subulatum species or a salt thereof for its use in the prevention and/or treatment of inflammatory diseases in humans or animals.

2. Sulphated polysaccharide according to claim 1, wherein said sulphated polysaccharide is a VEGF antagonist.

3. Sulphated polysaccharide according to claim 1, wherein the sulphated polysaccharide has a molecular weight less than or equal to 500 kDa.

4. Sulphated polysaccharide according to claim 1, wherein the sulphated polysaccharide has a molecular weight comprised between 10 kDa and 250 kDa.

5. Composition comprising the sulphated polysaccharide according to claim 1 and at least one pharmaceutically or dermatologically or cosmetically acceptable excipient.

6. Composition according to claim 1, further comprising at least one other active agent, the active agent being in particular selected from anti-redness agents, decongestants, antibacterial agents, antiseptics and antimicrobials, anti-inflammatories, anti-irritant and/or soothing agents, cicatrizants and/or restructurants of the skin barrier, antioxidants, moisturizing and/or emollient agents, anti-aging agents, mineral or organic sun filters and screens and sun protection active agents.

7. Composition according to claim 5, suitable to be administered by oral, topical or injectable route or in the form of supplements and/or food products.

8. Composition according to claim 5, wherein the composition is a cosmetic composition or a dermatological composition.

9. Dermatological composition according to claim 8 for its use in the prevention and/or treatment of inflammatory diseases, in particular of the skin.

10. Dermatological composition according to claim 9, wherein the inflammatory disease is selected from the group comprising: psoriasis, atopical dermatitis, rosacea, couperose, acne, vulgar warts, bullous skin diseases, contact eczema, skin cancers, redness, erythema, telangiectasia, inflammations of the skin associated with UV exposure.

11. Sulphated polysaccharide according to claim 2, wherein the sulphated polysaccharide has a molecular weight less than or equal to 500 kDa.

12. Sulphated polysaccharide according to claim 2, wherein the sulphated polysaccharide has a molecular weight comprised between 10 kDa and 250 kDa.

13. Sulphated polysaccharide according to claim 3, wherein the sulphated polysaccharide has a molecular weight comprised between 10 kDa and 250 kDa.

14. Composition comprising the sulphated polysaccharide according to claim 2 and at least one pharmaceutically or dermatologically or cosmetically acceptable excipient.

15. Composition comprising the sulphated polysaccharide according to claim 3 and at least one pharmaceutically or dermatologically or cosmetically acceptable excipient.

16. Composition comprising the sulphated polysaccharide according to claim 4 and at least one pharmaceutically or dermatologically or cosmetically acceptable excipient.

17. Composition according to claim 2, further comprising at least one other active agent, the active agent being in particular selected from anti-redness agents, decongestants, antibacterial agents, antiseptics and antimicrobials, anti-inflammatories, anti-irritant and/or soothing agents, cicatrizants and/or restructurants of the skin barrier, antioxidants, moisturizing and/or emollient agents, anti-aging agents, mineral or organic sun filters and screens and sun protection active agents.

18. Composition according to claim 3, further comprising at least one other active agent, the active agent being in particular selected from anti-redness agents, decongestants, antibacterial agents, antiseptics and antimicrobials, anti-inflammatories, anti-irritant and/or soothing agents, cicatrizants and/or restructurants of the skin barrier, antioxidants, moisturizing and/or emollient agents, anti-aging agents, mineral or organic sun filters and screens and sun protection active agents.

19. Composition according to claim 4, further comprising at least one other active agent, the active agent being in particular selected from anti-redness agents, decongestants, antibacterial agents, antiseptics and antimicrobials, anti-inflammatories, anti-irritant and/or soothing agents, cicatrizants and/or restructurants of the skin barrier, antioxidants, moisturizing and/or emollient agents, anti-aging agents, mineral or organic sun filters and screens and sun protection active agents.

20. The dermatological composition of claim 10, wherein the UV exposure comprises photoirritation, photosensitization, photoaging, photocarcinogenesis, venous lymphatic insufficiency, or heavy legs syndrome.

Description

[0120] The following figures and examples illustrate the invention without, however, limiting it.

FIGURES

[0121] FIG. 1: Effects of the sulphated polysaccharide on the formation of pseudotubes in the basal condition or stimulated with VEGF at 10 ng/ml according to Example 6.

[0122] FIG. 2: Effects of the sulphated polysaccharide on the viability of normal human keratinocytes according to Example 8.

[0123] FIG. 3: Effects of the sulphated polysaccharide on the release of VEGF by normal human keratinocytes, relative to the total quantity of proteins according to Example 9.

[0124] FIG. 4: Effects of the sulphated polysaccharide on the release of IL8 by normal human keratinocytes, relative to the total quantity of proteins according to Example 9.

EXAMPLES

Example 1

Extraction of the High-Molecular-Weight Polysaccharides (HMWP)

[0125] By high-molecular-weight polysaccharide or HMWP is meant a polysaccharide having a molecular weight comprised between 100 and 1,000 kDa.

[0126] The extraction of the high-molecular-weight polysaccharides is carried out by dispersing 100 grams of powder of the alga Haliptilon subulatum in 1 litre of water at 90 C. under vigorous stirring (500 rpm) for 4 hours. The mixture is then filtered hot using diatomaceous earth (100 g) on a frit glass (porosity 1, more precisely 100 to 160 m). The filtrate is then centrifuged (10,000 g, 30 minutes) at ambient temperature in order to obtain the alga extract enriched with polysaccharides. The Haliptilon subulatum extract is then precipitated from 3 volumes of ethanol 96% (at 4 C.) under stirring (500 rpm) for 2 hours.

[0127] The precipitate is recovered by filtration on frit glass (porosity 1 or 2, more precisely 100 to 160 m or 40 to 100 m respectively) or centrifugation (10,000 g, 30 minutes) at ambient temperature then washed with acetone (50 to 100 mL). The precipitate is then recovered by filtration on frit glass (porosity 2, more precisely 40 to 100 m) or centrifugation (10,000 g, 30 minutes) at ambient temperature then dried in an oven at 50 C. overnight. Finally, the precipitate is ground (in a blender) in order to obtain a fine powder of high-molecular-weight polysaccharides extracted from Haliptilon subulatum.

[0128] The yield of high-molecular-weight polysaccharides thus obtained is of the order of 10 to 20% with respect to the dry weight of powder of the alga Haliptilon subulatum used.

Example 2

Extraction of the Low-Molecular-Weight Polysaccharides (LMWP)

[0129] By low-molecular-weight polysaccharide or LMWP is meant a polysaccharide having a molecular weight comprised between 5 and 100 kDa.

[0130] The production of the low-molecular-weight polysaccharides is carried out by dispersing 2.5 grams of powder of high-molecular-weight polysaccharide (extracted from Haliptilon subulatum) in 125 mL of HCl (2M) at 100 C. under vigorous stirring (500 rpm) for 1 hour. The mixture is then cooled to ambient temperature then neutralized with soda (5 M). The medium is precipitated from 7 volumes of ethanol 96% (at 4 C.) under stirring (500 rpm) for 2 hours. The precipitate is recovered by filtration on frit glass (porosity 1 or 2, more precisely 100 to 160 m or 40 to 100 m respectively) or centrifugation (10,000 g, 30 minutes) at ambient temperature then washed with acetone (50 mL). The precipitate is then recovered by filtration on frit glass (porosity 2, more precisely 40 to 100 m) or centrifugation (10,000 g, 30 minutes) at ambient temperature then dried in an oven at 50 C. overnight. Finally, the precipitate is ground (in a blender) in order to obtain a fine powder of low-molecular-weight polysaccharides extracted from Haliptilon subulatum.

[0131] The yield of low-molecular-weight polysaccharides thus obtained is of the order of 70% with respect to the dry weight of powder of high-molecular-weight polysaccharides and 14% with respect to the dry weight of powder of the alga Haliptilon subulatum used.

Example 3

Determination of the Molecular Weights of the High- (HMWP) and Low- (LMWP) Molecular-Weight Polysaccharides

[0132] The high- and low-molecular-weight polysaccharides are prepared according to the conditions described previously (Examples 1 and 2). The molecular weights of the sulphated polysaccharides are determined according to the following protocol: [0133] a) Solubilization of the polysaccharides powder at a level of 0.5 to 10 g/L in an aqueous solution of ultrapure quality at a temperature ranging from 4 C. to 60 C. and under stirring for 30 minutes to 48 hours; [0134] b) Filtration of the samples on membrane with a porosity of 0.45 m; [0135] c) Injection and analysis by size-exclusion chromatography coupled with light scattering (SEC/MALLS); [0136] d) The technique makes it possible to access number average molecular weights (Mn) and weight average molecular weights (Mw) and also provides information on the shape and dimension of the chains and the polydispersity (PI).

TABLE-US-00001 TABLE 1 Summary of the weight and number average molecular weights observed for HMWP and LMWP. Mn Mw PI (g/mol) (g/mol) (Mw/Mn) HMWP 133300 213500 1.6 LMWP 12840 36640 2.8

[0137] This example shows that the molecular weight of the high-molecular-weight polysaccharides is of the order of 214 kDa and that the molecular weight of the low-molecular-weight polysaccharides is of the order of 37 kDa.

Example 4

Determination of the Sulphates and 3,6-anhydrocialactopyranose Residues Content of the Polysaccharides of the Invention

1. Determination of the Sulphates Content

Assay by Turbidimetry (BaCl.SUB.2./Gelatin)

[0138] The sulphate ions released during the hydrolysis of the polysaccharides will form, in the presence of barium chloride (BaCl.sub.2, 2H.sub.2O) and of gelatin, a precipitate of barium sulphate, the appearance of which is measured at 550 nm, as described in the publication by Dodgson & Price (Dodgson & Price, 1962, Biochemical Journal 84: 106-110).

[0139] 150 mg of gelatin is dissolved in 50 mL of milli-Q water at 70 C. After cooling for 16 hours at 4 C., 0.5 g of BaCl.sub.2 is added to the gelatin solution. 120 mg of lyophilized polysaccharide is hydrolyzed using 3 mL of 2 M HCl for 2 hours at 100 C. The mixture is centrifuged at 13,000 g for 30 minutes. 1 mL of supernatant is mixed with 9 mL of milli-Q water, 1 mL of 0.5 M HCl and 0.5 mL of BaCl.sub.2/gelatin reagent. After 30 minutes at ambient temperature, the mixture is stirred and the absorbance is read immediately at 550 nm. The standard range is produced using a stock solution of K.sub.2SO.sub.4 at 3 mg/mL.

Assay Using Azure A

[0140] The quantity of sulphates was determined using the colorimetric assay method developed by Jaques et al. (Jaques L. B. et al., 1968, Canadian Journal of Physiology and Pharmacology 46, pages: 351-360). In the aqueous phase, 3-amino-7-(dimethylamino)phenothizin-5-ium chloride (Azure A) complexes the sulphates that may be present, in particular within the polysaccharides composing the SPE fractions. The medium then develops a pink-violet colour absorbing at =535 nm, due to the formation of a chromophore in the presence of sulphates. The assay is semi-quantitative and gives an order of magnitude (mg) of the concentration of sulphates in a sample. 200 L of solution to be assayed is introduced into plastic spectrophotometer cuvettes. 2 mL of aqueous solution of Azure A at 10 mg/L is added, then the sample is stirred. The absorbance is measured at =535 nm.

[0141] The quantification of the sulphates is determined from the calibration range of dextran sulphate (17% sulphated) and correction of the degree of sulphation of the latter (17 mg of sulphates per 100 mg of dextran sulphate).

TABLE-US-00002 TABLE 2 Summary of the sulphates content for HMWP and LMWP. Sulphates content Standard (% m/m) deviation HMWP 13.5 0.09 LMWP 15.7 0.26

[0142] This example shows that the sulphates content of the high-molecular-weight polysaccharides is of the order of 13.5% and that the sulphates content of the low-molecular-weight polysaccharides is of the order of 15.7%.

2. Determination of the 3,6-anhydrodalactopyranose Residues Content

[0143] The most reproducible colorimetric method for assaying the 3,6-anhydrogalactose residues is that which uses a reagent based on resorcinol (see Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). The pink colouring which develops during the reaction is monitored at 555 nm. Three solutions are necessary for carrying out this assay: (i) a solution of acetaldehyde prepared by diluting 1 mL of acetaldehyde in 100 mL of ultrapure water (stable for approximately 1 month); (ii) a solution of resorcinol prepared by dissolving 150 mg of resorcinol in 100 mL of ultrapure water (stable for 7 days, away from light) and (iii) a solution of 10 M HCl.

[0144] For the assay, 50 to 100 L of the polysaccharide solution to be assayed) is introduced into glass tubes. The volume is topped up to 200 L using milli-Q water. The resorcinol reagent is prepared extemporaneously by adding 9 mL of the resorcinol solution and 1 mL of the acetaldehyde solution diluted to 1/25, to 100 mL of 10 M HCl. This reagent is only stable for 3 hours away from light. 1 mL of the resorcinol reagent is added to 200 L of the polysaccharide solution to be assayed. After stirring, the tubes are left to rest for 4 minutes, then placed in a water bath at 80 C. for 10 minutes. They are then transferred to an ice bath for 1 minute 30 seconds. The absorbance must be read within the following 15 minutes at 555 nm.

[0145] D-fructose (solutions from 10 to 70 g/mL) is used as standard. In fact it has been demonstrated that the absorbance curves at 555 nm depending on the monosaccharide concentration of the D-fructose and of the 3,6-anhydrogalactose are identical (see Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148).

TABLE-US-00003 TABLE 3 Summary of the 3,6-anhydrodalactopyranose (anhydroG) residues content for HMWP. Content of (3,6) anhydroG Standard (% m/m) deviation HMWP 1.32 0.013

[0146] This example shows that the 3,6-anhydrogalactopyranose content of the high-molecular-weight polysaccharides is 1.32%.

Example 5

Determination of the Composition in Terms of Monosaccharides and of the Structure of the Polysaccharides According to the Invention

[0147] 10 mg of polysaccharides is dissolved in 1 mL of 2 M trifluoracetic acid for 90 minutes at 120 C., with manual stirring every 30 minutes. The samples are evaporated under a stream of nitrogen in order to remove excess traces of acid. 1 mL of methanol is added, then the sample is vortexed and evaporated under a stream of nitrogen. This step is repeated twice in order to remove residual traces of acids. Derivatization is carried out using BSTFA:TMCS (99:1). 400 L of pyridine and 400 L of N,O-bis(trimethylsilyl) trifluoroacetamide:trimethylchlosylane (BSTFA:TMCS) (99:1) are added per 2 mg of monosaccharides. The samples are then mixed, then placed at ambient temperature for 2 hours under stirring (450 rpm).

[0148] The samples are evaporated under a stream of nitrogen, then the trimethylsilyl-O-glycoside residues are taken up in 500 L of dichloromethane. In this step, it is possible to dilute the sample to a greater or lesser extent. The standards (L-Rha, L-Fuc, L-Ara, D-Xyl, D-Man, D-Gal, D-Glc, D-GlcA, D-GalA) are prepared under the same conditions, in at least three different concentrations.

[0149] The trimethylsilylated derivatives are analyzed by gas chromatography coupled with mass spectrometry on an OPTIMA-1 MS column (30 m, 0.32 mm, 0.25 m) with a helium flow rate of 2.3 mL/minute (up to 3 mL/min). The helium pressure is fixed at 8.8 psi, i.e. 60673.9 Pa, and the injection ratio at 25:1 (or 50:1). The temperature rise is 8 C./minute up to 100 C., over 3 minutes. Another temperature rise of 8 C./minute up to 200 C., maintained for 1 minute is programmed. The procedure concludes with a temperature rise of 5 C./minute up to 250 C. The ionization is carried out by Electron Impact (EI, 70 eV), the temperature of the trap is fixed at 150 C. and the targeted ions between 40 and 800 m/z.

TABLE-US-00004 TABLE 4 Composition of the HMWP sample in terms of monosaccharides. Monosaccharides (mol %)* Gal Ara Xyl GlcA Glc 94.1 1.51 1.88 1.51 1.0 *Composition in terms of monosaccharides estimated by GC/MS-IE. Gal: Galactose; Ara: Arabinose, Xyl: Xylose, GlcA: Glucoronic acid, Glc: Glucose.

Example 6

Effect of the High-Molecular-Weight Sulphated Polysaccharide According to the Invention on the Formation of Pseudotubes in an HMVEC+NHDF Co-Culture

[0150] The effects of the high-molecular-weight sulphated polysaccharide (HMWP) on the formation of pseudotubes was studied in a co-culture of human dermal endothelial cells (HMVECs) and normal human dermal fibroblasts (NHDFs), in basal condition or stimulated by VEGF (analysis by immunolabelling in situ).

Culture and Treatment

[0151] The HMVEC and NHDF cells in co-culture were seeded in 96-well plates and cultured for 24 hours in culture medium.

[0152] The culture medium used is as follows: [0153] EBM-2 (Endothelial cell basal medium 2) supplemented with 5% foetal calf serum (FCS), rhEGF, rhFGF, R3 IGF-1, hydrocortisone, vitamin C, gentamycin, [0154] DMEM supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin and 10% FCS.

[0155] The medium was then replaced with test medium containing or not containing (control) the compound to be tested and/or the VEGF inductor reference tested at 100 ng/ml; the compound was tested simultaneously in basal and stimulated condition (in the absence and in the presence of VEGF).

[0156] The test medium used was as follows: [0157] EBM-2 (Endothelial cell basal medium 2) supplemented with rhEGF, rhFGF, R3 IGF-1, hydrocortisone, vitamin C, gentamycin, [0158] DMEM supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin and 1% FCS.

[0159] The cells were then incubated for 7 days, repeating the treatment after incubating for 72 hours. All the experimental conditions were produced in triplicate (n=3).

In Situ Immunolabelling of the Pseudotubes

[0160] After incubation, the culture medium was removed and the cells were rinsed, fixed and permeabilized. The cells were then labelled with anti-VWF (Von Willebrand Factor) primary antibody. This antibody was revealed by a secondary antibody coupled with a fluorochrome (GAR-Alexa 488). Simultaneously, the cell nuclei were coloured with Hoechst 33258 (bis-benzimide).

[0161] The formation of the pseudotubes was observed using a NIKON Diaphot 300 microscope (4 objective lens). The digital images (1 photo per well) were then recorded with a NIKON DS-Ri1 camera and the NIS-Elements 4.13.04 software.

[0162] The labelling was quantified by measuring the entire surface of the pseudotubes using ImageJ software. The results of the labelling are presented in FIG. 1.

Results

[0163] The formation of pseudotubes in the co-culture of endothelial cells (HMVECs) and dermal fibroblasts (NHDFs) after incubating for 7 days was measured by image analysis following immunolabelling with an anti-vWF antibody, the vWF being specifically expressed by the HMVEC cells.

[0164] The stimulation percentage is calculated according to the following formula:

[00001]$\mathrm{Stimulation}.Math..Math.\left(\%\right)=\left(\frac{\mathrm{Value}}{\mathrm{Average}.Math..Math.\mathrm{of}.Math..Math.\mathrm{the}.Math..Math.\mathrm{control}}100\right)-100$

[0165] The inhibition percentage is calculated according to the following formula:

[00002]$\mathrm{Inhibition}.Math..Math.\left(\%\right)=\frac{\mathrm{Average}.Math..Math.\mathrm{stimulated}.Math..Math.\mathrm{control}-\mathrm{Value}}{\mathrm{Average}.Math..Math.\mathrm{stimulated}.Math..Math.\mathrm{control}-\mathrm{Average}.Math..Math.\mathrm{basal}.Math..Math.\mathrm{control}}100$

TABLE-US-00005 TABLE 5 Effect of the HMWP sulphated polysaccharide according to the invention on the formation of the pseudotubesBasal condition. Basic data Surface Treatment of the Average Standardized data Compounds pseudotubes Surface sem % sem % sem tested Concentration (mm.sup.2) (mm.sup.2) (mm.sup.2) Control (%) p.sup.(1) Stimulation (%) p.sup.(1) Control T 1 0.16 0.12 0.02 100 16 0 16 medium T 2 0.09 without T 3 0.12 VEGF Medium 100 ng/ml T + 1 2.15 with T + 2 2.52 2.17 0.20 1759 159 1659 159 VEGF T + 3 1.84 Medium 10 g/ml P1 1 0.15 with P1 2 0.15 0.15 0.00 119 3 ns 19 3 ns HMWP P1 3 0.14 sulphated polysaccharide .sup.(1)Statistical significance threshold ns: >0.05, not significant *: 0.01 to 0.05, significant **: 0.001 to 0.01, Very significant ***: <0.001, Extremely significant sem: standard error of the mean

[0166] Inter-group comparisons were carried out using the unpaired bilateral Student's t test.

TABLE-US-00006 TABLE 6 Effect of the HMWP sulphated polysaccharide according to the invention on the formation of the pseudotubesCondition stimulated by the VEGF Basic data Surface of the Average % Standardized data Treatment pseudotubes Surface sem Stimulated sem % sem Compounds tested (mm.sup.2) (mm.sup.2) (mm.sup.2) control (%) p.sup.(1) inhibition (%) p.sup.(1) Non- Control T 1 0.16 0.12 0.02 6 1 *** 100 1 *** stimulated T 2 0.09 condition T 3 0.12 Stimulated Control T + 1 2.15 2.17 0.20 100 9 0 10 conditions: T + 2 2.52 VEGF- T + 3 1.84 100 ng/ml Sulphated P1 + 1 0.70 0.59 0.06 27 3 ** 77 3 ** polysaccharide P1 + 2 0.54 according to P1 + 3 0.53 the invention (10 g/ml) .sup.(1)Statistical significance threshold ns: >0.05, not significant *: 0.01 to 0.05, Significant **: 0.001 to 0.01, Very significant ***: +<0.001, Extremely significant sem: standard error of the mean

[0167] Inter-group comparisons were carried out using the unpaired bilateral Student's t test.

[0168] In the basal condition, only diffuse and weak labelling could be observed, indicating absence of organization of the endothelial cells. The treatment with the reference VEGF (100 ng/ml) clearly led to an organization of the endothelial cells into pseudotubes.

[0169] Tested in basal condition, the HMWP compound, at 10 g/ml, had no significant effect compared with the non-stimulated control condition and did not therefore induce the formation of pseudotubes in the HMVEC/NHDF co-culture.

[0170] Tested in stimulated condition, the HMWP compound, at 10 g/ml, clearly and significantly inhibited the formation of pseudotubes induced by the VEGF (77% inhibition).

Example 7

Complete Transcriptome Analysis of the Effects of the HMWP Sulphated Polysaccharide

[0171] Complete transcriptome analysis of the effects of the HMWP sulphated polysaccharide was carried out on normal human epidermal keratinocytes (NHEKs) and on normal human dermal fibroblasts (NHDFs) at two points in time during incubation: 4 hours and 24 hours.

Compound Tested

[0172] Compound tested: HMWP sulphated polysaccharide [0173] Concentrations tested: [0174] 0.1 mg/ml on the NHEKs [0175] 3 mg/ml on the NHDFs

Cultures and Treatments

[0176] The keratinocytes were seeded in 24-well plates and the fibroblasts in 12-well plates, then cultured in culture medium for 48 hours.

[0177] The culture medium used is as follows: [0178] Keratinocyte-SFM supplemented with 0.25 ng/ml EGF (epidermal growth factor), 25 g/ml pituitary extract (PE) and 25 g/ml gentamycin.

[0179] The culture medium was then replaced with test medium and the cells were cultured for another 24 hours. The test medium used is as follows: Keratinocyte-SFM supplemented with 25 g/ml gentamycin.

[0180] The cells were then treated or not treated (control) with the compound to be tested and incubated for 4 or 24 hours. All the experiments were carried out in triplicate (n=3).

[0181] At the end of the incubation, the culture supernatants were removed and the cell layers were rinsed with a solution of phosphate-buffered saline (PBS). The plates were immediately frozen dry at 80 C.

RNA Extraction

[0182] Before the extraction, the culture replicates were pooled. The total RNA from each sample was extracted using the NucleoSpin RNA Plus kit (Macherey-Nagel) according to the protocol recommended by the supplier.

Differential Expression Analysis

[0183] The quantity and quality of the RNAs were evaluated by capillary electrophoresis (Bioanalyzer 2100, Agilent). Synthesis of the biotinylated anti-sense RNAs (aRNA) was carried out using the GeneChip 3IVT Express kit (Affymetrix). For each biotinylated aRNA sample, an electrophoretic profile was produced (Bioanalyzer 2100, Agilent) before and after fragmentation. The hybridization of the labelled and fragmented aRNAs on the Affymetrix U219 chip (36,000 transcripts and variants) was carried out on the GeneAtlas fluidics Affymetrix hybridization station for 20 hours at 45 C. The U219 chips were then scanned using the GeneAtlas Imaging station (Affymetrixresolution 2 Inn) in order to generate the signal intensity data.

Processing of the Data

[0184] The signal intensity data are standardized using the Expression Console software (Affymetrix), based on the RMA algorithm. Quality control of the labelling as well as of the hybridization is then carried out.

Results Obtained

[0185] The results obtained are presented in Table 7.

TABLE-US-00007 TABLE 7 Transcriptome analysis of the HMWP and effects on the process of cell proliferation and angiogenesis Incubation time: Incubation time: Treatments Effects observed 4 hours 24 hours Treatment Effects on the cell Inhibition of cell Inhibition of cell of the proliferation proliferation by proliferation by keratinocytes process inhibiting the inhibiting the (NHEK) with expression of the expression of the HMWP genes coding for the genes coding for the at 0.1 mg/ml growth factors and growth factors and the genes involved the genes involved in the regulation of in the regulation of cell proliferation cell proliferation Effects on Inhibition of the Inhibition of the angiogenesis expression of genes expression of genes involved in the involved in the angiogenesis angiogenesis process: JAG1, process: JAG1, VEGFA, CYR61 VEGFA, CYR61 Treatment Effects on the cell Increase in the Increase in the of fibroblasts proliferation expression of the expression of the (NHDF) process genes involved in genes involved in with HMWP cell proliferation cell proliferation at 3 mg/ml Effects on Increase in the Increase in the angiogenesis expression of genes expression of genes involved in the involved in the angiogenesis angiogenesis process: C3, process: C3, MMP14 MMP14

[0186] This example shows that the HMWP sulphated polysaccharide is capable of inhibiting the cell proliferation process and angiogenesis at a concentration of 0.1 mg/ml on the keratinocytes, whereas it rather tends to stimulate the cell proliferation process and angiogenesis at a concentration of 3 mg/ml on the fibroblasts.

[0187] This makes it a good active ingredient for the prevention and/or treatment of redness.

[0188]

Example 8

Evaluation of the HMWP Sulphated Polysaccharide on the Cell Viability of Normal Human Keratinocytes

[0189] Human keratinocytes are seeded in 96-well microplates at a density of 20,000 keratinocytes per well (equivalent to 60,000 cells/cm.sup.2, then left to adhere/proliferate for 24 hours at 37 C. with 5% CO.sub.2 in complete KSFM medium (containing added antibiotics and growth supplements, Gibco 17005).

[0190] The human keratinocytes are treated with the HMWP sulphated polysaccharide to be tested in non-supplemented medium (without growth supplements) for 48 hours and incubated at 37 C. with 5% CO.sub.2. Each concentration of the product is evaluated in triplicate. Two positive controls are used: one of cytotoxicity, 10% dimethylsulphoxide (DMSOSigma D4540) and one of proliferation, the complete medium (i.e. with growth supplements).

[0191] A cell viability/cytotoxicity test (XTT test) is carried out in order to determine the non-cytotoxic doses. The XTT test is carried out by means of the Cell Proliferation Kit II (XTT) (Sigma/Roche Diagnostics, 11465015001).

[0192] After the 48 hours of treatment, the wells are carefully rinsed with a phosphate-buffered saline (PBS, Introvogen). The keratinocytes are then brought into contact with a tetrazolium sodium salt solution (XTT) at 0.3 mg/mL. The plates are incubated at 37 C. with 5% CO.sub.2 in darkness. The tetrazolium sodium salt solution (XTT) is also deposited in wells without cells (medium with or without product) in order to produce blanks.

[0193] After incubating for 3 hours, the absorbance is measured at 450 nm with a reference at 650 nm. For each condition, the optical density values (OD, absorbance) are averaged.

[0194] The viability of the cells treated is expressed as a percentage with respect to the control (untreated cells):

[00003]$\%.Math..Math.\mathrm{viability}.Math..Math.\mathrm{sample}=\frac{\mathrm{OD}.Math..Math.\mathrm{sample}}{\mathrm{Average}.Math..Math.\mathrm{OD}.Math..Math.\mathrm{untreated}.Math..Math.\mathrm{control}}100$

[0195] A treatment leading to a reduction in viability, below the threshold value of 80% mitochondrial activity with respect to the control, is considered as cytotoxic for the cells. Conversely, an increase in the value evidences an increase in mitochondrial activity, or even in cell proliferation.

[0196] The significance of the results is evaluated by comparison of the values with those obtained for the control condition, by the Student's t-test with the following criteria:

TABLE-US-00008 Criterion of the p-value Significance (of the difference Graphical (Student's t-test) between the values compared) notation p > 0.05 not significant 0.01 < p 0.05 significant (at 95%) * 0.001 < p 0.01 very significant (at 99%) ** p 0.001 very highly significant (at 99.9%) ***

[0197] The results obtained are presented in FIG. 2.

Conclusions

[0198] The HMWP sulphated polysaccharide is non-cytotoxic to the keratinocytes after 48 hours of application, at concentrations of 0.3 and 1 g/mL (above the 80% viability threshold), whereas it appears to be cytotoxic in the strongest doses tested, i.e. from 10 to 600 g/mL.

Example 9

Effects of the HMWP Sulphated Polysaccharide on the Release of VEGF and Interleukin (IL8) by Normal Human Keratinocytes

[0199] Human keratinocytes are seeded in 96-well microplates at a density of 20,000 cells/well (i.e. 60,000 cells/cm.sup.2), in complete KSFM medium and left to adhere/proliferate at 37 C. under 5% CO.sub.2, 24 hours before treatment.

[0200] The HMWP sulphated polysaccharide, tested in 3 non-cytotoxic concentrations (0.1 g/ml, 0.3 g/ml and 1 g/ml), is brought into contact with the keratinocytes for 24 hours at 37 C. with 5% CO.sub.2 (in unsupplemented KSFM medium). The HMWP sulphated polysaccharide is then re-applied for another 24 hours (i.e. 48 hours in total), in the presence or absence of stimulation with IL1 at 20 ng/mL (Bio-Techne/R&Dsystems, 201-LB). Each unstimulated condition is tested in triplicate (n=3 cell wells), and the same applies for each stimulated condition. The assays are then carried out in duplicate.

[0201] Positive controls of inhibition of the production of VEGF and of IL8 are used (at 1 M): EGCG (Epigallocatechin gallateTocris 4524) and staurosporine (Sigma S4400) respectively.

Assay of IL8 and of VEGF

[0202] The targets of interest, IL8 and VEGF are assayed by means of kits supplied by Bio-Techne/R&Dsystems (D8000C) and Thermo/Fisher Scientific (EH2VEGF) respectively. The results of the assays are obtained by measuring the absorbance (OD) at a wavelength of 450 nm, with 550 or 570 nm as reference wavelength.

Assay of the Total Proteins

[0203] The total proteins are assayed using the BCA method. The BCA assay kit (Sigma BCA1) is composed of a bicinchoninic acid solution (BCA) (Sigma B9643) and copper sulphate (CuSO.sub.4Sigma C2284). The standard range is prepared based on BSA (Bovine Serum AlbuminSigma A9418). The cell pellets are kept dry at 20 C. while awaiting this assay. In order to lyse the cells and alkalinize the reaction medium, the cell pellets are equilibrated at ambient temperature, then placed in alkaline medium for a minimum of 30 minutes. The assay is carried out by adding a mixture of the reagents bicinchoninic acid and CuSO.sub.4. The plate is incubated at 37 C. and the reaction is stopped by placing the plate for a few minutes at 4 C. The assay reading is then carried out at a wavelength of 570 nm.

Results

[0204] The results obtained by the BCA method are presented in FIGS. 3 and 4.

[0205] With the HMWP sulphated polysaccharide, in the case of inflammatory stress, a potential inhibitory effect is observed on the syntheses of VEGF and IL8, at the lowest doses tested.

[0206] In fact, following the treatment with the HMWP sulphated polysaccharide, in the two concentrations tested, the lowest of 0.1 and 0.3 g/mL, a significant reduction in the release of keratinocytes from the VEGF induced by the inflammatory stress (IL1), of 29% and 33% respectively, with respect to the untreated but stimulated control (significant * or highly significant ** respectively) is obtained. This inhibition is comparable to that obtained with the positive control.

[0207] In the strongest concentration tested, 1 g/mL, the effect of the HMWP sulphated polysaccharide is slight (9%) and not significant.

[0208] As regards IL8, a significant reduction (*) is observed in the release of keratinocytes after induction (inflammatory stress), of 14% after application of 0.1 g/mL of the product, with respect to the stimulated control. This inhibitory effect is of the same order of magnitude as that recorded with the positive control.

[0209] At the dose of 0.3 g/mL, the effect of the extract is slight (6%) and not significant and at 1 g/mL there is no effect.

[0210] In the absence of stimulation, there is no significant inhibition of the basal releases of VEGF or of IL8, with any of the three doses tested: [0211] At the doses of 0.1 and 0.3 g/mL, a slight reduction in the VEGF released is recorded (5% and 10% respectively, in a non-significant manner). Conversely, at the strongest concentration tested (1 g/mL,) a stimulation of this production of +31% (significant *) is observed. [0212] At the concentration of 0.1 g/mL, a slight reduction in IL8 is recorded (7%, in a non-significant manner). Conversely, at 0.3 and 1 g/mL, a slight stimulation of this production appears (+8% and +19% respectively, in a non-significant or significant manner **).

Conclusions

[0213] The HMWP sulphated polysaccharide makes it possible: [0214] at the doses of 0.1 and 0.3 g/mL, to counter the release of the VEGF in an inflammatory stress context, [0215] at the dose of 0.1 g/mL, to also combat the production of IL8 (under inflammatory stress).