NOVEL COMPOSITIONS COMBINING HEMP DERIVATIVES AND AT LEAST ONE PEPTIDE AND USE THEREOF

Abstract

The present invention relates to an association comprising or consisting of a mixture of cannabidiol or cannahigerol and palinitoyl tripeptide-8, and to the use thereof for treating or soothing sensitive and/or reactive skins or as an after-sun ointment for soothing solar erythema.

Claims

1-13. (canceled)

14. Association comprising or consisting of a mixture of cannabidiol or cannabigerol and palmitoyl tripeptide-8.

15. Association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water.

16. Association according to claim 14, comprising or consisting of a mixture of cannabidiol and palmitoyl tripeptide-8.

17. Association according to claim 14 comprising a mixture of: cannabidiol, palmitoyl tripeptide-8, butylene glycol, dextran, and water.

18. Association according to claim 14 comprising or consisting of a mixture of cannabigerol and palmitoyl tripeptide-8.

19. Association according to claim 14 comprising a mixture of: cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water.

20. Association according to claims 14, in which: cannabidiol or cannabigerol is present in an amount of 0.01% to 5%, preferably 0.25% to 1% in percentage by weight, palmitoyl tripeptide-8 is present in an amount of 2 ppm to 20 ppm, preferably 4 ppm to 10 ppm by weight.

21. Association according to claim 14, additionally comprising acetyl hexapeptide-1

22. Association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water. additionally comprising acetyl hexapeptide-1

23. Association according to claim 14, comprising at least one oil containing α-linolenic acids, or one oil containing α-linolenic acids from Perilla Oil, Linseed Oil, Camelina Oil, Inca Inchi Oil, Chia Oil and Rosehip Oil from Chile.

24. Association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water. and at least one oil containing α-linolenic acids, or one oil containing α-linolenic acids from Perilla Oil, Linseed Oil, Camelina Oil, Inca Inchi Oil, Chia Oil and Rosehip Oil from Chile

25. Association according to claim 14, with an α-linolenic acid content of 33% to 66% relative to the total fatty acids of the oil and with an omega-3/omega-6 ratio of 0.20 to 1.0.

26. Association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water. with an α-linolenic acid content of 33% to 66% relative to the total fatty acids of the oil and with an omega-3/omega-6 ratio of 0.20 to 1.0.

27. Association according to claim 14, comprising a natural extract of Tasmannia Lanceolata.

28. Association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water and comprising a natural extract of Tasmannia Lanceolata

29. Cosmetic composition, comprising the association according to claim 14, in a cosmetically acceptable medium, said composition optionally comprising at least one other ingredient, chosen in particular from: moisturizing agents, chemical filters, sun filters, in particular UV A filters, inorganic mineral sun filters, in particular titanium dioxide, thermal waters, in particular Treignac® water.

30. Cosmetic composition, comprising the association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water in a cosmetically acceptable medium, said composition optionally comprising at least one other ingredient, chosen in particular from: moisturizing agents, chemical filters, sun filters, in particular UV A filters, inorganic mineral sun filters, in particular titanium dioxide, thermal waters, in particular Treignac® water.

31. Cosmetic composition, comprising the association according to claim 14, in a cosmetically acceptable medium, said composition optionally comprising at least one other ingredient, chosen in particular from: moisturizing agents, chemical filters, sun filters, in particular UV A filters, inorganic mineral sun filters, in particular titanium dioxide, thermal waters, in particular Treignac® waterCosmetic, said cosmetic composition being formulated for topical application, or topical application to the skin, or in the form of an emulsion, a cream, a gel, a dispersion, a serum, a mousse, a body milk or an anhydrous balm, a day cream, a night cream or an after-sun product.

32. Cosmetic composition, comprising the association according to claim 14 comprising a mixture of: cannabidiol or cannabigerol, palmitoyl tripeptide-8, butylene glycol, dextran, and water in a cosmetically acceptable medium, said composition optionally comprising at least one other ingredient, chosen in particular from: moisturizing agents, chemical filters, sun filters, in particular UV A filters, inorganic mineral sun filters, in particular titanium dioxide, thermal waters, in particular Treignac® water, said cosmetic composition being formulated for topical application, or topical application to the skin, or in the form of an emulsion, a cream, a gel, a dispersion, a serum, a mousse, a body milk or an anhydrous balm, a day cream, a night cream or an after-sun product.

33. Method for the cosmetic treatment of sensitive skin, comprising the topical application to healthy skin of an association according to one of claims 14 to reduce the sensations of stinging, tingling, itching or pruritus, burning, overheating, discomfort or tightness of the skin.

Description

LEGENDS OF FIGS.

[0189] FIG. 1 represents the dose-response curves of the inhibition of NO production by nonlinear regression of 3 series of experiments, part a) for different concentrations of cannabidiol and part b) for different concentrations of Neutrazen™

[0190] FIG. 2 shows the dose-response curves of cell viability by non-linear regression of 3 series of experiments, part a) for different concentrations of cannabidiol and part b) for different concentrations of Neutrazen™.

[0191] FIG. 3 represents for mixtures containing 0.01% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), in part a) the dose-response of the inhibition of NO production and partly b) those of cell viability, the curves being obtained by non-linear regression of 3 series of experiments.

[0192] FIG. 4 represents for the mixtures containing 0.025% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), in part a) the dose-response curves of the inhibition of NO production and in part b) those of cell viability, the curves being obtained by non-linear regression of 3 series of experiments.

[0193] FIG. 5 represents for the mixtures containing 0.05% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), in part a) the dose-response curves of the inhibition of NO production and in part b) those of cell viability, the curves being obtained by non-linear regression of 3 series of experiments.

[0194] FIG. 6 represents for the mixtures containing 0.01% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), in part a) the dose-response curves of the inhibition of NO production and partly b) those of cell viability by non-linear regression of 3 series of experiments.

[0195] FIG. 7 reports the isobolograms, in part a) for inflammatory activity and in part b) for cell viability; in the figures the theoretical line observed for the additive effects is represented by a dotted line. The following examples illustrate the invention, without limiting its scope.

EXAMPLE 1

Formulation of an Emulsion

[0196]

TABLE-US-00004 TABLE 4 Formulation of an emulsion w/w Phase Ingredient INCI % A Montanov ™68 Cetearyl Alcohol (and) Cetearyl 1.50 Glucoside Montanov ™202 Arachidyl Alcohol (and) Behenyl 1.50 Alcohol (and) Arachidyl Glucoside Lanol ™1688 Cetearyl Ethylhexanoate 15.00 B Water Water 77.70 Simugel ™ EG Sodium Acrylate/Acryloyldimeth- 1.00 ylaurate Copolymer (and) Isohexadecane (and) Polysorbate 80 Neutrazen ™ Water (and) Butylene Glycol (and) 2.50 Dextran (and) Palmitoyl Tripeptide-8 Cannabidiol Cannabidiol 0.50 Sepicide ™ HB Phenoxyethanol (and) Methylparaben 0.30 (and) Ethylparaben (and) Propylparaben (and) Butylparaben

EXAMPLE 2

Cream Gel Containing an α-Linolenic Oil

[0197]

TABLE-US-00005 TABLE 5 Formulation of a Cream gel containing an α-linolenic oil w/w Phase Ingredient INCI % A Water Water 58.88 Carbopol ® Ultrez-21 Acrylate/C10-30 Alkyl Acrylate 0.60 Crosspolymer B Polyethylene Glycol 400 PEG-8 5.00 Sepicide ™ HB Phenoxyethanol (and) 0.30 Methylparaben (and) Ethylparaben (and) Propylparaben (and) Butylparaben C Cutina ® MD Glyceryl Stearate 5.00 Cetearyl Alcohol Cetearyl Alcohol 2.00 Cetiol ® CC Carbonate Dicaprylyl 3.00 Mineral Oil USP Mineral Oil 2.00 Perilla oil Perilla Ocymoides Seed Oil 2.00 Cannabidiol Cannabidiol 0.50 D Parfum Fragance 0.02 Triethanolamine 99 Triethanolamine 0.70 Water Water 20.00 Neutrazen ™ Water (and) Butylene Glycol 2.50 (and) Dextran (and) Palmitoyl Tripeptide-8

EXAMPLE 3

Composition Containing Acetyl Hexapeptide-1 and an Extract of Tasmannia Lanceolata

[0198]

TABLE-US-00006 TABLE 6 composition containing acetyl hexapeptide-1 and an extract of Tasmannia Lanceolata w/w Phase Ingredient INCI % A Water Water 80.35 Chlorphenesin Chlorphenesin 0.30 Phenoxyethanol Phenoxyethanol 0.60 Dermofeel ™PA-3 Sodium Phytate (and) Water 0.10 (and) Alcohol B Lecigel ™ Sodium Acrylate Copolymer 1.75 (and) Lecithin C Dermofeel ™ BGC Butylene Glycol Dicaprylate 3.00 (and) Dicaprate DUB ININ Isononyl Isononanoate 5.00 Myritol ® 318 Caprylic (and) Capric Triglyceride 3.00 Vitapherole ® E1000 Tocopherol (and) Helianthus 0.20 Annuus (Sunflower) Seed Oil D Tazman Pepper ™ Glycerin (and) Water (and) 2.00 Tasmannia Lanceolata Fruit/ Leaf Extract Neutrazen ™ Water (and) Butylene Glycol 2.50 (and) Dextran (and) Palmitoyl Tripeptide-8 Cannabidiol Cannabidiol 0.50 Melitane ™ Glycerin (and) Water (and) 0.50 Dextran (and) Acetyl Hexapeptide-1

EXAMPLE 4

[0199] In-vitro analysis of the anti-inflammatory activity of the cannabidiol/palmitoyl tripeptide-8 association on murine macrophages.

I. Aim of the Study

[0200] Low-grade inflammation is strongly implicated in degenerative processes in human skin such as photoaging and atopy. The reduction of low-intensity inflammatory reactions by topical products may be necessary in the event of cutaneous aggression, to obtain optimal healing and restore the physiological balance of human skin. In order to identify new anti-inflammatory candidates, an in vitro anti-inflammatory test was developed with a murine cell line: the murine macrophage test RAW 267.4. This test was used to monitor the inhibitory effects of chemical compounds or natural extracts on the low-intensity inflammatory cascade leading to overproduction of nitrogen oxide (NO) in the endothelial wall of blood vessels.

[0201] The study was carried out to evaluate the ability of cannabidiol, palmitoyl tripeptide-8 (active ingredient in Neutrazen™), and their mixture to inhibit the pro-inflammatory cascade leading to the production of NO in murine macrophages stimulated by liposaccharides (LPS).

II. Materials and Methods.

Materials

[0202] Cannabidiol is used in the form of a white powder, for example marketed by PHYTOGRASE.

[0203] A Neutrazen™ solution containing palmitoyl tripeptide-8 is used.

[0204] Neutrazen™ comes in the form of a translucent liquid. It is marketed by the Lucas Meyer company. It is composed of water, butylene glycol, dextran and palmitoyl tripeptide-8.

[0205] The INCI name for Neutrazen™ is: Water (and) Butylene Glycol (and) Dextran (and) Palmitoyl Tripeptide-8.

Principle of the Tests

[0206] The in vitro anti-inflammatory test is based on the ability of murine macrophages to generate a strong inflammatory response when stimulated by antigens such as LPS. Immobilized murine macrophages (RAW 267.4 cell line) are stimulated with LPS from E. coli and exposed for 24 hours. At the end of the incubation period, the production of NO is evaluated indirectly by measuring the accumulation of nitrite/nitrate, stable final products of the oxidation of NO, in the culture medium using a method spectrophotometric based on the Griess reaction.

Cell Line

[0207] MURINS RAW 267.4 Macrophage cell line (Sigma-Aldrich, No. P6110401, Lot. 091006), low number of passages (less than 50).

Culture Medium

[0208] Complete medium: DMEM with stabilized L-glutamine (Dulbecco's Minimum Essential Medium, PAN BIOTECH. Lot 974251) supplemented with Penicillin 100 IU/ml and streptomycin 100 μg/m1 (PAN BIOTECH, Lot 225614), and 10% inactivated calf serum (PAN BIOTECH, Lot P460518), pH 7.2, freshly prepared, stored for less than 3 weeks.

Dilution of Materials

[0209] Cannabidiol (CBD) is diluted in DMSO (10 mg/mL storage solution), and subjected to an ultrasonic bath for 20 minutes. The Neutrazen™ solution is diluted in PBS (Phosphate buffer saline, Sigma-Aldrich).

Controls

[0210] The negative control: DMSO 1% The positive control: Dexamethasone (Sigma-Aldrich) 1-5-10-50-100 μM.

Test Procedure

[0211] RAW 267.4 murine macrophages were inoculated into 48-well tissue culture plates at a concentration of 1.10.sup.5 cells/ml (200 μl/well) and incubated for 24 hours at 37° C. (5% CO.sub.2).

[0212] At the end of the incubation period, the culture medium was replaced with 200 μl of medium containing the appropriate concentrations of the material to be tested, and the cells were incubated at 37° C. (5% CO.sub.2) for one hour.

[0213] At the end of the incubation period, pro-inflammatory E. coli LPS was added to the cell cultures (1 μg/ml. Then the cells were incubated at 37° C. (5% CO.sub.2) for 24 hours.

Evaluation of the Level of NO Released

[0214] The amount of NO released was measured in the culture supernatant by the Griess reaction. 100 μl of the supernatants were transferred into the wells of a 96-well tissue culture plate, and modified Griess reagent (SIGMA-ALDRICH) were added to each well. After a period of 15 min at room temperature, the Optical Density (OD) of each well was read at a wavelength of 540 nm by an Infinite M200 Pro fluorescence-luminescence reader (TECAN). The results obtained for the wells treated with the test material were compared with those of the untreated control wells (PBS, 100% NO production) and converted into percentage values.

Evaluation of the Cell Viability

[0215] In parallel with the evaluation of the release of NO, the cell viability was measured to validate the tests. Vital Stain Reagent WST-1 was used to measure cellular mitochondrial respiration. For this, the culture medium was decanted and 100 μl of WST-1 reagent ( 1/10 dilution) were added to each well. After an incubation period of 30 min at 37° C. (5% CO2), the Optical Density (OD) of each well was read at a wavelength of 450 nm by an Infinite M200 Pro fluorescence-luminescence reader (TECAN). The results obtained for the wells treated with the test material were compared with those of the untreated control wells (DMSO, 100% viability) and converted into percentage values.

Calculation of the IC50 for NO Release and the IC50 for Cell Viability

[0216] Inhibition of NO release and inhibition of cell viability were expressed as a percentage relative to the negative controls:

[00001]$\mathrm{Percentage}\mathrm{of}\mathrm{NO}\mathrm{release}=\frac{100\times \left(\mathrm{test}OD-\mathrm{control}OD\right)}{\mathrm{DMSO}\mathrm{control}OD-\mathrm{control}OD}$$\mathrm{Percentage}\mathrm{of}\mathrm{cell}\mathrm{viability}=\frac{100\times \left(\mathrm{test}OD-\mathrm{control}OD\right)}{\mathrm{DMSO}\mathrm{control}OD-\mathrm{control}OD}$

[0217] The concentrations of test material causing a 50% decrease in NO release (IC50 (NO release)) and a 50% decrease in cell viability (IC50 (cell viability)) respectively were calculated using TableCurve software Version 2.0.

[0218] The anti-inflammatory ratio corresponds to the ratio between anti-inflammatory activity and toxicity. It is expressed as follows:

[00002]$\mathrm{Anti}-\mathrm{inflammatory}\mathrm{ratio}=\frac{\mathrm{IC}50\left(\mathrm{cell}\mathrm{viability}\right)}{\mathrm{IC}50\left(\mathrm{NO}\mathrm{release}\right)}$

EXAMPLE 5

[0219] Analysis of the anti-inflammatory activities of cannabidiol and Neutrazen™ (containing the palmitoy tripeptide-8)

[0220] The results concerning the anti-inflammatory activity of cannabidiol and Neutrazen™ (containing the palmitoy tripeptide -8) are reported in Table 7. FIG. 1 represents the dose-response curves of the inhibition of NO production by nonlinear regression of 3 series of experiments for different concentrations of cannabidiol (figure la) and for different concentrations of Neutrazen™ (FIG. 1b).

[0221] Results for macrophage viability of murine cannabidiol and Neutrazen™ (containing Palmitoy tripeptide-8) are reported in Table 8.

[0222] FIG. 2 shows the dose-response curves of cell viability by nonlinear regression of 3 series of experiments for different concentrations of cannabidiol (FIG. 2a) and for different concentrations of Neutrazen™ (FIG. 2b).

[0223] The results of the nonlinear regression analyzes to determine the IC50s and the anti-inflammatory ratio are reported in Table 9.

TABLE-US-00007 TABLE 7 Anti-inflammatory activity of cannabidiol and Neutrazen ™ (containing palmitoyl-tripeptide-8) Concentrations Optical Densities observed for NO production (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.061 0.065 0.063 — — — Non-stimulated control 0.097 0.095 0.096 — — — Stimulated control 0.260 0.252 0.254 100 100 100 CBD 0.01% 0.259 0.253 0.258 99.39 100.64 102.53 0.025%  0.255 0.248 0.251 96.93 97.45 98.10 0.05% 0.256 0.235 0.243 97.55 89.17 93.04  0.1% 0.252 0.251 0.253 95.09 99.36 99.37 0.25% 0.224 0.221 0.225 77.91 80.25 81.65  0.5% 0.101 0.103 0.1 2.45 5.10 2.53   1% 0.098 0.098 0.099 0.61 1.91 1.90  2.5% 0.097 0.099 0.098 0.00 2.55 1.27 NeutraZen 0.01% 0.251 0.258 0.25 94.48 103.82 97.47 0.025%  0.255 0.256 0.255 96.93 102.55 100.63 0.05% 0.254 0.248 0.251 96.32 97.45 98.10  0.1% 0.257 0.25 0.246 98.16 98.73 94.94 0.25% 0.249 0.252 0.241 93.25 100.00 91.77  0.5% 0.251 0.249 0.244 94.48 98.09 93.67   1% 0.249 0.236 0.231 93.25 89.81 85.44  2.5% 0.244 0.233 0.235 90.18 87.90 87.97

TABLE-US-00008 TABLE 8 Cell Viability of cannabidiol and Neutrazen ™ (containing palmitoyl-tripeptide-8) Concentrations Optical Densities observed for Cell viability (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.07 0.07 0.071 — — — Non-stimulated control 0.075 0.071 0.075 — — — Stimulated control 0.730 0.726 0.721 100 100 100 CBD 0.01% 0.726 0.724 0.722 99.39 99.69 100.15 0.025%  0.719 0.725 0.716 98.32 99.85 99.23 0.05% 0.716 0.690 0.681 97.86 94.50 93.81  0.1% 0.423 0.406 0.431 53.13 51.15 55.11 0.25% 0.129 0.108 0.112 8.24 5.65 5.73  0.5% 0.106 0.097 0.096 4.73 3.97 3.25   1% 0.094 0.084 0.087 2.90 1.98 1.86  2.5% 0.088 0.085 0.078 1.98 2.14 0.46 NeutraZen 0.01% 0.725 0.726 0.720 99.24 100.00 99.85 0.025%  0.728 0.727 0.718 99.69 100.15 99.54 0.05% 0.715 0.723 0.720 97.71 99.54 99.85  0.1% 0.718 0.712 0.717 98.17 97.86 99.38 0.25% 0.721 0.706 0.714 98.63 96.95 98.92  0.5% 0.713 0.710 0.715 97.40 97.56 99.07   1% 0.708 0.708 0.705 96.64 97.25 97.52  2.5% 0.706 0.704 0.705 96.34 96.64 97.52

TABLE-US-00009 TABLE 9 Results of non-linear regression analysis Anti- inflammatory Samples IC.sub.50-NO release IC.sub.50-cell viability ratio Dexamethasone .sup. 2.98 ± 0.18 μM 192.65 ± 4.25 μM 64.65 CBD 0.319 ± 0.006% .sup. 0.101 ± 0.006% <1 NeutraZen >2.5% >2.5% —

EXAMPLE 6

[0224] Analysis of the anti-inflammatory activity and of the cell viability of the cannabidiol/palmitoyl tripeptide-8 (Neutrazen™) association in a mixture by isobologram analysis. For each concentration of Neutrazen™ (0.01, 0.025, 0.05 and 0.1%), different concentrations of CBD are tested (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%) to determine both the production of NO and cell viability.

[0225] The results of the anti-inflammatory activity and cell viability assays are reported in Tables 10-13 and FIGS. 3-6.

[0226] Tests on anti-inflammatory activity and cell viability for mixtures containing 0.01% Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%) are reported in the table 10. FIG. 3 represents for the mixtures containing 0.01% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), the dose-response curves of the inhibition of the production of NO (FIG. 3a) and cell viability (FIG. 3b) by nonlinear regression of 3 sets of experiments.

[0227] Tests on anti-inflammatory activity and cell viability for mixtures containing 0.025% Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%) are reported in the table 11. FIG. 4 represents for the mixtures containing 0.025% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), the dose-response curves of the inhibition of the production of NO (FIG. 4a) and cell viability (FIG. 4b) by nonlinear regression of 3 sets of experiments.

[0228] Tests on anti-inflammatory activity and cell viability for mixtures containing 0.05% Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%) are reported in the table 12. FIG. 5 represents for the mixtures containing 0 05% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), the dose-response curves of the inhibition of the production of NO (FIG. 5a) and cell viability (FIG. 5b) by nonlinear regression of 3 series of experiments.

[0229] Tests on anti-inflammatory activity and cell viability for mixtures containing 0.01% Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%) are reported in the table 13. FIG. 6 represents for the mixtures containing 0.01% of Neutrazen™ and different concentrations of CBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2.5%), the dose-response curves of the inhibition of the production of NO (FIG. 6a) and cell viability (FIG. 6b) by nonlinear regression of 3 sets of experiments.

TABLE-US-00010 TABLE 10 Anti-inflammatory activity and cell viability for mixture of NeutraZen 0.01% and cannabidiol. Concentrations Optical Densities observed for NO production (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.061 0.065 0.063 — — — Non-stimulated control 0.097 0.095 0.096 — — — Stimulated control 0.260 0.252 0.254 100 100 100 CBD 0.01% 0.251 0.250 0.255 94.48 98.73 100.63 0.025%  0.239 0.235 0.242 87.12 89.17 92.41 0.05% 0.235 0.230 0.236 84.66 85.99 88.61  0.1% 0.228 0.216 0.231 80.37 77.07 85.44 0.25% 0.184 0.191 0.192 53.37 61.15 60.76  0.5% 0.089 0.091 0.097 0 0 0.63   1% 0.085 0.089 0.093 0 0 0  2.5% 0.089 0.089 0.089 0 0 0 Concentrations Optical Densities observed for Cell viability (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.07 0.07 0.071 — — — Non-stimulated control 0.075 0.071 0.075 — — — Stimulated control 0.730 0.726 0.721 100 100 100 CBD 0.01% 0.721 0.723 0.731 98.63 99.54 101.55 0.025%  0.728 0.721 0.726 99.68 99.24 100.77 0.05% 0.715 0.719 0.725 97.73 98.93 100.62  0.1% 0.741 0.726 0.738 101.66 100.00 102.63 0.25% 0.679 0.659 0.681 92.20 89.77 93.81  0.5% 0.116 0.118 0.119 6.34 7.18 6.81   1% 0.112 0.110 0.111 5.66 5.95 5.57  2.5% 0.122 0.108 0.103 7.21 5.65 4.33

TABLE-US-00011 TABLE 11 Anti-inflammatory activity and cell viability for mixture of NeutraZen 0.025% and cannabidiol Concentrations Optical Densities observed for NO production (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.061 0.065 0.063 — — — Non-stimulated control 0.097 0.095 0.096 — — — Stimulated control 0.260 0.252 0.254 100 100 100 CBD 0.01% 0.258 0.255 0.254 98.77 101.91 100.00 0.025%  0.248 0.246 0.249 92.64 96.18 96.84 0.05% 0.235 0.233 0.234 84.66 87.90 87.34  0.1% 0.227 0.222 0.226 79.75 80.89 82.28 0.25% 0.174 0.172 0.178 47.24 49.04 51.90  0.5% 0.09 0.092 0.091 0 0 0   1% 0.085 0.089 0.088 0 0 0  2.5% 0.084 0.081 0.087 0 0 0 Concentrations Optical Densities observed for Cell viability (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.07 0.07 0.071 — — — Non-stimulated control 0.075 0.071 0.075 — — — Stimulated control 0.730 0.726 0.721 100 100 100 CBD 0.01% 0.745 0.729 0.733 102.29 100.46 101.86 0.025%  0.756 0.731 0.728 103.92 100.76 101.08 0.05% 0.705 0.701 0.723 96.20 96.18 100.31  0.1% 0.732 0.702 0.716 100.24 96.34 99.23 0.25% 0.655 0.648 0.651 88.53 88.09 89.16  0.5% 0.120 0.119 0.123 6.95 7.33 7.43   1% 0.109 0.106 0.107 5.19 5.34 4.95  2.5% 0.102 0.102 0.104 4.12 4.73 4.49

TABLE-US-00012 TABLE 12 Anti-inflammatory activity and cell viability for mixture of NeutraZen 0.05% and cannabidiol Concentrations Optical Densities observed for NO production (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.061 0.065 0.063 — — — Non-stimulated control 0.097 0.095 0.096 — — — Stimulated control 0.260 0.252 0.254 100 100 100 CBD 0.01% 0.26 0.258 0.259 100.00 103.82 103.16 0.025%  0.25 0.252 0.251 93.87 100.00 98.10 0.05% 0.234 0.231 0.235 84.05 86.62 87.97  0.1% 0.227 0.22 0.228 79.75 79.62 83.54 0.25% 0.174 0.178 0.171 47.24 52.87 47.47  0.5% 0.09 0.102 0.098 0 4.46 1.27   1% 0.089 0.095 0.092 0 0 0  2.5% 0.088 0.087 0.086 0 0 0 Concentrations Optical Densities observed for Cell viability (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.07 0.07 0.071 — — — Non-stimulated control 0.075 0.071 0.075 — — — Stimulated control 0.730 0.726 0.721 100 100 100 CBD 0.01% 0.717 0.738 0.731 98.02 101.83 101.55 0.025%  0.724 0.734 0.726 99.08 101.22 100.77 0.05% 0.737 0.73 0.725 101.04 100.61 100.62  0.1% 0.733 0.726 0.721 100.46 100.00 100.00 0.25% 0.721 0.72 0.723 98.56 99.08 100.31  0.5% 0.126 0.131 0.124 7.76 9.16 7.59   1% 0.126 0.125 0.123 7.74 8.24 7.43  2.5% 0.128 0.122 0.12 8.14 7.79 6.97

TABLE-US-00013 TABLE 13 Anti-inflammatory activity and cell viability for mixture of NeutraZen 0.1% and cannabidiol. Concentrations Optical Densities observed for NO production (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.061 0.065 0.063 — — — Non-stimulated control 0.097 0.095 0.096 — — — Stimulated control 0.260 0.252 0.254 100 100 100 CBD 0.01% 0.262 0.258 0.251 101.23 103.82 98.10 0.025%  0.265 0.254 0.255 103.07 101.27 100.63 0.05% 0.227 0.228 0.226 79.75 84.71 82.28  0.1% 0.215 0.212 0.214 72.39 74.52 74.68 0.25% 0.18 0.164 0.175 50.92 43.95 50.00  0.5% 0.091 0.097 0.092 0 1.27 0   1% 0.088 0.09 0.091 0 0 0  2.5% 0.09 0.089 0.088 0 0 0 Concentrations Optical Densities observed for Cell viability (% as compared Samples (weight %) the 3 sets of experiments to the negative control) Blank 0.07 0.07 0.071 — — — Non-stimulated control 0.075 0.071 0.075 — — — Stimulated control 0.730 0.726 0.721 100 100 100 CBD 0.01% 0.736 0.726 0.724 100.96 100.00 100.46 0.025%  0.728 0.722 0.722 99.68 99.39 100.15 0.05% 0.749 0.723 0.721 102.89 99.54 100.00  0.1% 0.711 0.715 0.718 97.10 98.32 99.54 0.25% 0.702 0.709 0.712 95.73 97.40 98.61  0.5% 0.131 0.133 0.128 8.56 9.47 8.20   1% 0.131 0.126 0.124 8.58 8.40 7.59  2.5% 0.133 0.121 0.116 8.93 7.63 6.35

[0230] Table 14 reports the calculated values of the IC50 of NO release, the IC50 of cell viability and the anti-inflammatory ratio for dexamethasone, cannabidiol, Neutrazen and mixtures combining cannbidiol/Neutrazen.

[0231] The results are obtained by a non-linear regression analysis of the curves obtained in FIGS. 1 to 6 of the different series of experiments.

TABLE-US-00014 TABLE 14 Calculated values of the IC50 of NO release, the IC50 of cellular viability and the anti-inflammatory ratio for dexamethasone, cannabidiol, Neutrazen and mixtures associating cannabidiol/Neutrazen. IC50-NO IC50-cell Anti-inflammatory Samples release viability ratio Dexamethasone .sup. 2.98 ± 0.18 μM 192.65 ± 4.25 μM.sup.  64.65 CBD 0.319 ± 0.006% 0.101 ± 0.005% <1 Neutrazen >2.5% >2.5% — CBD/Neutrazen (0.01% weight) 0.272 ± 0.005% 0.328 ± 0.003% 1.20 CBD/Neutrazen( 0.025% weight) 0.234 ± 0.004% 0.343 ± 0.005% 1.46 CBD/Neutrazen (0.05% weight) 0.233 ± 0.005% 0.372 ± 0.003% 1.59 CBD/Neutrazen (0.1% weight) 0.218 ± 0.005% 0.434 ± 0.003% 1.99

[0232] The calculated results of the anti-inflammatory ratio in Table 14 show that the presence of Neutrazen, namely palmitoyl tripeptide-8, induces an improvement in the anti-inflammatory ratio of cannabidiol. Indeed cannabidiol has an anti-inflammatory ratio of less than 1, but associated with Neutrazen, the ratio is improved. A ratio of 1.99 is notably observed when cannabidiol is combined with a 0.1% weight Neutrazen solution.

[0233] FIG. 7 reports the isobolograms for inflammatory activity and cell viability.

[0234] The results obtained with the different mixtures of CBD and Neutrazen™ made it possible to calculate the pairs of concentrations corresponding to a 50% reduction in the production of NO and a 50% reduction in cell viability. These data have been transferred to FIG. 7, which represents the isobolograms corresponding to the experiment.

[0235] In the isobologram in FIG. 7a concerning the anti-inflammatory activity, the experimental curve obtained with the cannabidiol/Neutrazen™ concentration pairs representing a 50% decrease in NO production are below the observed theoretical line for additive effects (shown in dotted line).

[0236] In parallel with the isobologram in FIG. 7b concerning cell viability, the experimental curve obtained with the cannabidiol/Neutrazen™ concentration pairs representing a 50% decrease in cell viability is greater than the theoretical line of additive effects shown in dotted line.

[0237] These results demonstrate that the association of cannabidiol and Neutrazen™ (containing Palmitoyl hexapeptide-8) in a mixture involves synergistic anti-inflammatory activity and a concomitant decrease in cytotoxicity, resulting in an improved anti-inflammatory ratio.

REFERENCES

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