Composition for Prevention or Treatment of Cutaneous Disorder

Abstract

The present invention relates to a pharmaceutical composition or medical device comprising a fucoidan and at least one compound of formula A or B:

##STR00001##

wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of —OH, —CH.sub.3, —CF.sub.3CH.sub.2O—, and CH.sub.3O—. Such pharmaceutical composition or medical device is notably useful for the treatment and/or prevention of a skin disorder involving excessive angiogenesis and/or fibrogenesis, such as in skin fibrosis, angiofibromas, hamartomas and periungual fibromas, skin manifestation occurring with rosacea, acne, atopic dermatitis, scleroderma, psoriasis and lupus erythematosus, and especially of Tuberous Sclerosis Complex and skin manifestation occurring with Tuberous Sclerosis Complex.

Claims

1. A pharmaceutical composition or medical device comprising: at least one fucoidan; and at least one compound of formula A or B: ##STR00007## wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of —OH, —CH.sub.3, —CF.sub.3CH.sub.2O—, and CH.sub.3O—.

2. The pharmaceutical composition or medical device according to claim 1, wherein said compound of formula B is dimethylmethoxy chromanol.

3. The pharmaceutical composition or medical device according to claim 1, wherein said pharmaceutical composition or medical device further comprises resveratrol.

4. The pharmaceutical composition or medical device according to claim 1, wherein said pharmaceutical composition or medical device further comprises pharmaceutically active ingredients selected from the group consisting of algae flavonoids, algae oligosaccharides, oligosaccharides of entenomorpha, Tea tree extract (cyclodextrine), vectorized Tea tree extract, flavonoids and polyphenols of green tea extract, EGCG, curcumin, cardamom (cardamomum) essential oil, perilla (perilla frutescens) essential oil, common St.-John's wort (Hypericum perforatum) essential oil, lemongrass (Cymbopogon) essential oil, compact oregano (Origanum compactum) essential oil, clove (Syzygium aromaticum) essential oil, nutmeg (Myristica fragrans—without safrole) essential oil, frankincense (olibanum) essential oil, holy basil (Ocimum tenuiflorum) essential oil, Curcuma (Curcuma) essential oil, and combinations thereof.

5. The pharmaceutical composition or medical device according to claim 1, wherein said pharmaceutical composition or medical device is a topical formulation selected from a cream, lotion, serum, balm, gel for topical application, food supplement, solid formulation, tablet, effervescent tablet, sublingual tablet, orally-disintegrating tablet, capsule, granule, powder, paste, liquid formulation, syrup, solution, emulsion, and a suspension.

6. The pharmaceutical composition or medical device according to claim 1, wherein said pharmaceutical composition or medical device is a topical pharmaceutical composition or a medical device for topical application.

7. A method of therapeutic treatment comprising co-administering, concomitantly or in sequence, at least one fucoidan and at least one compound of formula A or B: ##STR00008## wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of —OH, —CH.sub.3, —CF.sub.3CH.sub.2O—, and CH.sub.3O—.

8. The method of claim 7, wherein said method treats or prevents a cutaneous disorder.

9. The method of claim 7, wherein said method treats or prevents a cutaneous disorder involving excessive angiogenesis and/or fibrogenesis.

10. The method of claim 7, wherein said method treats or prevents skin fibrosis, angiofibromas, hamartomas, periungual fibromas, and combinations thereof.

11. The method of claim 7, wherein said method treats or prevents Tuberous Sclerosis Complex.

12. The method of claim 7, wherein said method treats or prevents a skin manifestation occurring with Tuberous Sclerosis Complex.

13. The method of claim 7, wherein said method treats or prevents a skin manifestation occurring with rosacea, acne, atopic dermatitis, scleroderma, psoriasis, lupus erythematosus, or a combination thereof.

14. A method of treatment or prevention of a skin disorder involving excessive angiogenesis and/or fibrogenesis, said method comprising administering to a subject in need thereof an effective amount of a combination of a fucoidan and resveratrol.

15. The method of claim 14, wherein the skin disorder involving excessive angiogenesis and/or fibrogenesis is selected from the group consisting of skin fibrosis, angiofibromas, hamartomas, periungual fibromas, rosacea, acne, atopic dermatitis, scleroderma, psoriasis, lupus erythematosus, Tuberous Sclerosis Complex, and combinations thereof.

16. A cosmetic treatment comprising applying on skin of a subject a cosmetic composition comprising: at least one fucoidan; and at least one compound of formula A or B: ##STR00009## wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of —OH, —CH.sub.3, —CF.sub.3CH.sub.2O—, and CH.sub.3O—.

17. The cosmetic treatment according to claim 16, wherein said cosmetic composition further comprises resveratrol.

18. The cosmetic treatment according to claim 16, wherein said cosmetic treatment is limiting skin redness and said skin area is affected by a cutaneous disorder involving excessive angiogenesis and/or fibrogenesis.

19. The cosmetic treatment according to claim 16, wherein said cosmetic composition is a foundation perfector and said skin area is affected by a cutaneous disorder involving excessive angiogenesis and/or fibrogenesis.

20. A method for inhibiting pseudotube formation in endothelial cells and/or fibroblasts, said method comprising administering to a subject in need thereof an effective amount of fucoidan.

21. A method for inhibiting neovascularization observed in case of a skin disorder involving excessive angiogenesis and/or fibrogenesis, said method comprising administering to a subject in need thereof an effective amount of fucoidan.

22. The method of claim 21 wherein said disorder is skin fibrosis, angiofibromas, hamartomas, or periungual fibromas.

Description

EXAMPLES

Abbreviations

[0091] TSC Tuberous Sclerosis Complex [0092] DMEM Dulbecco's modified Eagle's medium [0093] EBM Endothelial cell basal medium [0094] EGF Epidermal growth factor [0095] FCS Fetal calf serum [0096] FGF Fibroblast growth factor [0097] GAM Goat anti-mouse [0098] GAR Goat anti-rabbit [0099] HMVEC Human microvascular endothelial cell [0100] IGF Insulin Like Growth Factor [0101] NHDF Normal human dermal fibroblast [0102] OD Optical density [0103] RT Room temperature [0104] Sd Standard deviation [0105] sem Standard error of the mean [0106] TGF Transforming growth factor [0107] VEGF Vascular Epidermal growth factor

Data Management

[0108] Raw data were analyzed using Microsoft Excel software.

[0109] The inter-group comparisons were performed by an unpaired Student's t-test. The statistical analysis can be interpreted if n 5, however for n<5 the statistical values are for information only.

Formulas Used in this Report:

[0110] Standard error of the mean: sem=Sd/n

[0111] The standard error of the mean (sem) is a measure of how far the sample mean is likely to be from the true population mean. The sem is calculated as the Sd divided by the square root of sample size.

[0112] Percentage of inhibition: Inhibition (%)

[00001]$\mathrm{Inhibition}.Math..Math.\left(\%\right)=\frac{\mathrm{Stimulated}.Math..Math.{\mathrm{Control}}^{\prime }.Math.s.Math..Math.\mathrm{Mean}-\mathrm{Value}}{\mathrm{Stimulated}.Math..Math.{\mathrm{Control}}^{\prime }.Math.s.Math..Math.\mathrm{Mean}-\mathrm{Non}.Math.\text{-}.Math.\mathrm{stimulated}.Math..Math.{\mathrm{Control}}^{\prime }.Math.s.Math..Math.\mathrm{Mean}}\times 100$

Example 1—Inhibition of Collagen-I

[0113] Analysis of collagen I expression in NHDF stimulated with vitamin C+TGF-beta by in situ immunolabeling. Activated HMVEC release TGF-beta which stimulates extracellular matrix synthesis by NHDF. TGF-beta stimulates the synthesis of collagen and vitamin C its release, creating experimental conditions close to TSC pathology conditions.

[0114] Normal Human Dermal Fibroblasts (NHDF) [0115] Cell type: NHDF, used at the 8th passage [0116] Culture conditions: 37° C., 5% CO.sub.2 [0117] Culture medium: DMEM supplemented with L-glutamine 2 mM, Penicillin 50 U/ml—Streptomycin 50 μg/ml Fetal calf serum (FCS) 10% [0118] Assay medium: DMEM supplemented with L-glutamine 2 mM, Penicillin 50 U/ml, Streptomycin 50 μg/ml FCS 1%.

Culture and Treatment

[0119] The fibroblasts were seeded in 96-well plates and cultured in culture medium for 24 hours. The medium was then replaced with assay medium containing or not (control) the test compounds or associations in presence or not (non-stimulated control) of the association vitamin C+TGF-beta (20 μg/ml+10 ng/ml). The cells were incubated for 72 hours. All experimental conditions were performed in n=3.

Active Compound Tested

[0120] Dimethylmethoxy chromanol: LIPOCHROMAN® synthetic molecule from Lipotec.

Expression of Collagen I in Fibroblasts—In Situ Immunolabeling

[0121] After incubation, culture media were discarded and the cells were rinsed, fixed and permeabilized. The cells were then labeled using a primary antibody anti-collagen I. The primary antibody was then revealed using a fluorescent secondary antibody (GAR-Alexa 488) and the cell nuclei were stained using Hoechst 33258 solution (bis-benzimide) in parallel.

[0122] The acquisition of the images (5 photos per well) was performed using INCell Analyzer™ 1000 (GE Healthcare). Representative images for each experimental condition were included in the report.

[0123] The labeling was quantified by the measurement of the fluorescence intensity normalized to the total number of cells (Integration of numerical data with the Developer Toolbox 1.5, GE Healthcare software).

[0124] Results are given in Table 1.

TABLE-US-00001 TABLE 1 Basic data Collagen I % Normalized data Treatment branching Stimulated sem % sem Concentration points Mean sem control (%) p(1) inhibition (%) p(1) Control — 241679 233484 4774 62 1 ** 100 3 ** 233629 225143 Control — 335648 374343 20025 100 5 — 0 14 — 384747 402634 Dimethyl- 50 μg/ml 250891 274529 11940 73 3 * 71 8 * methoxy 283414 chromanol 289284

Effect on Collagen I Expression

[0125] The treatment of the normal human dermal fibroblasts (NHDF) by the mix vitamin C+TGF- (20 μg/ml+10 ng/ml), induced a strong expression of collagen I by the cells. This effect was expected and validated the assay.

[0126] Under the experimental conditions of the assay, dimethylmethoxy chromanol, tested at 50 μg/ml, induced a significant inhibition of collagen I expression in NHDF stimulated by the mix vitamin C+TGF-beta (71% of inhibition).

Example 2—Inhibition of Pseudotube Formation

[0127] Analysis of pseudotube formation in a HMVEC/NHDF co-culture by in situ immunolabeling.

[0128] Human Microvascular Endothelial Cells (HMVEC) [0129] Cell type: HMVEC, used at the 7th passage

[0130] HMVEC/NHDF Co-Culture [0131] Cell type: HMVEC+NHDF (50:50 ratio) [0132] Culture conditions: 37° C., 5% CO2 [0133] Co-culture medium: Endothelial Cell Basal Medium 2 (EBM-2) supplemented with Fetal calf serum (FCS) 5%, rhEGF, rhFGF, R3 IGF-1, hydrocortisone, vitamin C and gentamycin
+

[0134] DMEM supplemented with L-glutamine 2 mM, Penicillin 50 U/ml, Streptomycin 50 μg/ml FCS 10%.

Culture and Treatment

[0135] The HMVEC and NHDF were seeded in 96-well plates and co-cultured in co-culture medium for 24 hours. The medium was then removed and replaced by co-culture medium containing or not (control) the test compounds, associations or the reference, suramine at 5 μM, in presence or not (non-stimulated control) of the inducer, VEGF at 100 ng/ml. The cells were incubated for 10 days with treatment renewal after 72 hours of incubation. All experimental conditions were performed in n=3.

Active Compound Tested

[0136] Dimethylmethoxy chromanol: LIPOCHROMAN® synthetic molecule from Lipotec, Spain; [0137] Fucoidan: PHYTELENE EG 755 BG Wakame from Greentech, France; Resveratrol: Grape Glycolic Extract from Greentech, France.

Determination of Pseudotube Branching Points—In Situ Immunolabeling

[0138] After incubation, culture media were discarded and the cells were rinsed, fixed and permeabilized. The cells were then labeled using a primary antibody anti-Von Willebrand Factor. The primary antibody was then revealed using a fluorescent secondary antibody (GAM-Alexa 488) and the cell nuclei were stained using Hoechst 33258 solution (bis-benzimide) in parallel.

[0139] The formation of pseudotubes was observed using a NIKON Diaphot 300 microscope (lens×4). The images were captured (1 photo per well) using a NIKON DS-Fi1 camera and NIS-Elements 3.10 software. Then the number of branching points within the pseudotube network was counted on each image.

[0140] Representative images for each experimental condition were included in the report. The image references of each condition are presented in Table 2.

TABLE-US-00002 TABLE 2 Effect of active compounds on the number of branching points in HMVEC/NHDF co-culture Treatment Test Basic data Normalized data compound Number of % % Test Concen- codifi- branching stimulated sem inhibi- sem compound tration cation points Mean sem control (%) p(1) tion (%) p(1) Non- Control — T − 3 0 0 0 0 0 *** 100 0 *** stimulated T − 5 0 condition T − 6 0 Control — T + 7 122 125 5 100 4 — 0 4 * T + 12 118 T + 17 135 Suramine 5 μM  R1 72 78 4 63 3 ** 37 3 ** R6 85 R7 79 dimethylmethoxy 6 μg/ml M3 M31 83 98 8 79 6 ns 21 6 ns chromanol M32 104 M33 109 Fucoidan +  30 μg/ml + M4 M41 3 5 1 4 1 *** 96 1 *** Resveratrol 2 μg/ml M42 6 M43 6 Fucoidan 30 μg/ml  M5 M51 9 8 1 6 1 *** 94 1 *** M52 8 M53 6 Resveratrol 2 μg/ml M6 M61 139 141 9 113 7 ns −13 7 ns M62 157 M63 126 Fucoidan +  30 μg/ml + M9 M91 17 21 3 17 3 *** 83 3 *** dimethylmethoxy 6 μg/ml M92 28 chromanol M93 19 (1)Threshold for statistical significance ns: >0.05, Not significant * 0.01 to 0.05, Significant ** 0.001 to 0.01, Very significant *** <0.001, Extremely significant

Effect on Pseudotube Formation

[0141] Under the non-stimulated control condition, the human microvascular endothelial cells (HMVEC) did not form pseudotubes and then no branching points were observed. The stimulation of the HMVEC with VEGF, tested at 100 ng/ml, induced the formation of pseudotubes, and a high number of branching points among this capillary network was counted. The reference suramine, tested at 5 μM, induced a significant decrease of pseudotube network and of the quantity of branching points (37% of inhibition). These results were expected and validated the assay.

[0142] Under the experimental conditions of the assay, compound Fucoidan, tested at 30 μg/ml, and associations Fucoidan+Resveratrol, tested at 30 μg/ml+2 μg/ml, and Fucoidan+dimethylmethoxy chromanol, tested at 30 μg/ml+6 μg/ml, induced a strong inhibition of pseudotube formation, characterized by the disorganization of network architecture and by the strong decrease of the number of branching points (94%, 96%, and 83% of inhibition, respectively).

CONCLUSION

[0143] Under the experimental conditions of the assay, Fucoidan, tested alone or in association with resveratrol or dimethylmethoxy chromanol, had a strong inhibitory effect on pseudotube formation by HMVEC.

[0144] Dimethylmethoxy chromanol induced a moderate inhibition of collagen I expression by the NHDF stimulated with vitamin C and TGF-beta.

[0145] Overall, the results of this study indicated that compound Fucoidan had strong anti-angiogenic properties. Compound dimethylmethoxy chromanol had anti-fibrosis properties. The above results support that a combination of fucoïdan and dimethylmethoxy chromanol inhibits angiogenesis and fibrosis especially in case of a disorder involving excessive angiogenesis and/or fibrogenesis.

[0146] Accordingly the results support the use of a combination of fucoïdan and dimethylmethoxy chromanol in a pharmaceutical composition or medical device, notably for the treatment or prevention of skin fibrosis, angiofibromas, hamartomas and periungual fibromas, skin manifestation occurring with rosacea, acne, scleroderma, psoriasis and lupus erythematosus, and especially of Tuberous Sclerosis Complex and skin manifestation occurring with Tuberous Sclerosis Complex.

[0147] Also the above results support that dimethylmethoxy chromanol inhibits collagen-I expression, especially in fibroblasts, and more particularly in conditions simulating a cutaneous disorder involving excessive angiogenesis and/or fibrogenesis, such as in skin fibrosis, angiofibromas, hamartomas and periungual fibromas, skin manifestation occurring with rosacea, acne, scleroderma, psoriasis and lupus erythematosus, and especially inTuberous Sclerosis Complex and skin manifestation occurring with Tuberous Sclerosis Complex.

[0148] In addition, the above results support that dimethylmethoxy chromanol inhibits pseudotube formation in endothelial cells and/or fibroblasts. Accordingly, dimethylmethoxy chromanol is considered to inhibit neovascularization observed in case of a skin disorder involving excessive angiogenesis and/or fibrogenesis, such as in skin fibrosis, angiofibromas, hamartomas and periungual fibromas, skin manifestation occurring with rosacea, acne, scleroderma, psoriasis and lupus erythematosus, and especially in Tuberous Sclerosis Complex and skin manifestation occurring with Tuberous Sclerosis Complex.

Example 3—Example of Formulation

[0149] The table below is an example of a formulation according to the present invention without any limitation to the ingredients.

[0150] The following gel formulation including at least one fucoidan, and at least dimethylmethoxy chromanol was prepared according to the skills of one ordinary in the art:

TABLE-US-00003 Weight % by weight of the total INGREDIENTS composition Fucoidan (Phytelene EG 755 BG Wakame) 4.00% Resveratrol (Grape Glycolic Extract from Greentech) 1.00% dimethylmethoxy chromanol (Lipochroman ®) 0.05% Mineral powder (Silice) 0.75% Butylene Glycol 4.00% Oligosaccharide - (algae extract (entenomorpha)) 1.00% Vectorized concentrated Tea tree (Melaleuca alternifolia) 3.00% extract (cyclodextrine) Flavonoids and polyphenols of Green tea extract 1.00% Other excipients (moisturizers, humectant, etc.) About 4% Water Qsp Qsp: quantity sufficient to 100%

Example 4—Example of Formulation

[0151]

TABLE-US-00004 Ingredients % Association comprising: FUCOIDAN  0.5% RESVERATROL   1% LIPOCHROMAN 0.05% IMPERFECTIONS CORRECTOR 2.50% (Betapur) Boldo Extract (leaf of Chile wild minth) ACIDE SALICYLIQUE 0.10% (Synthèse Organique) MINERAL POWDER  0.5% (Silica) SODIUM HYALURONATE 0.01% GLYCOL DERIVATIVE  7.1% Butylène Glycol ALLANTOIN 0.25% Syringa vulgaris lilac extract -  0.5% Malodextrin Perfum 0.25% Pure wheat alcool of vegetal origin  9.% Symbiol 68  0.5%

[0152] The formulation of example 4 is useful as anti-redness of skin and as foundation perfector.

Example 5—Example of Formulation

[0153]

TABLE-US-00005 Ingredients Mass ratio % Mixture of ingredients comprising: 43% Fucoidan - 0.5% (Phytelene EG 755 BG Wakame) Resveratrol - 1% (Grape Glycolic Extract from Greentech) Dimethylmethoxy chromanol - 0.05% (Lipochroman ®) Glycol derivative 16% Perfum 0.3% 
Such a formulation is particularly dedicated to provide a cosmetic benefit. This formulation corrects immediately skin imperfections, and is comfortable when applied on skin. This formulation also provides a mattifying effect.

Example 6—Example of Formulation

[0154]

TABLE-US-00006 FORMULATION F12 100% Ingredients Phase % (mass) g Osmosed water A 63.09 126.18 EDTA Bisodico A1 0.15 0.3 Allantoin EP A2 0.25 0.5 Butylene glycol A6 3.1 6.2 Covacryl MV60 A6 2 4 Osmosed water A4 2 4 MSS 500 A4 0.5 1 Green tea C2 0 Betapur A00067 C2 2.5 5 (Aqua, Butylene Glycol, Peumus Boldus Leaf Extract, Xanthan Gum) Osmosed water A3 2 4 Sodium Hyaluronate A3 0.01 0.02 Symdiol 68 A5 0.5 1 Geogard ECT D 1 2 Salicylic Acid D 0.1 0.2 Perfum B 0.15 0.3 Massocare HC0 40 B 1.5 3 FDC BLUE N.sup.o1 à 0.1% D 0.55 1.1 Tinogard TL B 0.05 0.1 Fucoidan C4 5 10 Butylene glycol C1 4 8 Alcohol C1 9 18 resveratrol C1 1 2 Lipochroman B 0.05 0.1 Pronalen sport re-energizer C2 1 2 Sebuless C3 0.5 1
The formulation is prepared based on the different phases (A, B, C, and D) according to the common knowledge of the skilled person. The formulation of example 6 is particularly dedicated to provide a cosmetic benefit. Such a formulation is useful as anti-redness of skin and as foundation perfector. This formulation corrects immediately skin imperfections, and is comfortable when applied on skin. This formulation also provides a mattifying effect. Such formulation comprises a fucoidan, lipochroman and resveratrol which are active on pseudotube formation and collagen I expression.

Example 7—Evaluation of the Cutaneous Tolerance and Anti-Redness Effect of a Composition According to the Invention

[0155] The study was performed under dermatological control. Under the conditions of the study, it has been evaluated the tolerance and efficacy of a composition according to the invention (“the product”) after 28 days of use.

[0156] The study was carried out on 21 subjects with rosacea type I and II (type I—11 subjects, type II—10 subjects) with irritable and reactive skin on the face. In the panel of subjects were 2 male who represent the 10% of population and 19 female who represent the 90% of population. The mean age of the panel was 41±3 years (between 23 and 61 years old). Due to the hard recruitment process in the study were included three subjects with a higher age than anticipated in the protocol: 56, 57, 61 (instead of up to 55).

[0157] The primary objective was to evaluate the cutaneous tolerance of the product after 28 days of twice daily use.

[0158] Dermatologist assessment was done by the same investigator for each subject according to his extensive knowledge of the skin and types of diseases associated with it. Assessment was done in pursuance of the established scheme, which includes the main clinical signs such as erythema, oedema, dryness, desquamation, roughness, vesicles and other in scale: none, very slight, slight, moderate, severe. In opinion of dermatologist, the products reduced the redness in a large degree. The reactivity of the skin was also reduced. Additionally, was carried out an assessment of functional signs commonly occurring in subjects (such as stinging, burning sensation, warm sensation, tightness, etc.) performed on the basis of subjective feelings of subjects. During the study six subjects reported some discomfort reactions after products applications. Most of them were judged as usual signs and not relevant. However, one clinical sign (subject #21) was observed on D28 by the dermatologist and was judged relevant. Based on this evaluation the dermatologist rated the product as well tolerated on the cutaneous level.

[0159] The product provided: [0160] a significant decrease of parameter “visible redness” of 25% on average on D28. Less visible redness was observed in 90% of the subjects. [0161] a significant decrease of parameter “unhealthy skin look” of 26% on average on D28. More healthy skin look was observed in 95% of the subjects. [0162] a significant decrease of parameter “visible imperfections” of 27% on average on D28. Less visible imperfections were observed in 71% of the subjects. [0163] a significant decrease of parameter “uneven skin” of 26% on average on D28. More uniform skin was observed in 86% of the subjects. [0164] a significant decrease of parameter “dull skin” of 26% on average on D28. More luminous skin was observed in 76% of the subjects.

[0165] The product induced a significant decrease of the cutaneous microcirculation of 6% on average on D28. This effect was observed in 86% of the subjects.

[0166] The secondary objectives in the study were to evaluate the efficacy of tested products. To achieve this purpose have been used several biometrological methods.

[0167] First of them was clinical score which evaluated the efficacy after 28 days of the product. It consists of the five items: redness, healthy skin look, imperfections, unified skin, luminous skin, which were evaluated in eleven point scale from 0 to 10. This visual assessment was done by the same investigator for each subject according to his extensive knowledge as well.

[0168] After 28 days in 90% of subjects was observed decrease in redness parameter of 25% on average in comparison to the initial state. More healthy skin look of 26% on average in comparison to the initial state was observed in 95% of subjects. 71% of subjects were judged to have less visible imperfections of 27% on average in comparison to the initial state. 86% of subjects, in opinion of dermatologist, have more unified skin of 26% on average in comparison to the initial state. More luminous skin of 26% on average in comparison to the initial state was observed in 76% of subjects.

[0169] These results provide that the product presented an improvement of the efficacy clinical score and thus improved skin condition after 28 days of twice daily use.

[0170] The immediate clinical score after first application at the laboratory of the product enabled to evaluate the immediate efficacy of the product. Improvement of skin conditions in the subjects was observed. The dermatologist judged that 52% of subjects had more unified skin of 10% on average in comparison to the initial state. Moreover, in 33% of subject were observed less visible imperfections and in 67% of subject less redness, of 7% on average in comparison to the initial state. The product presented an improvement of the immediate efficacy clinical score and thus improved skin condition after first application at the laboratory.

[0171] The effect on the cutaneous microcirculation measured by Tissue Viability Imager (TiVi) on D28 was evaluated. Tissue viability Imaging gives information about the skin microcirculation by using subsurface polarization light spectroscopy. The technique utilizes a digital camera equipped with perpendicular polarization filters. When light flashes from the camera, white light gets polarized by the polarization filter. The reflected light from the skin contains both the same polarized light and randomly polarized light. When the light falls on the skin, a part of the light gets reflected directly by the superficial layers of the skin. The major part of the light gets randomly polarized due to the backscattering of other dermal tissues and part of the light is absorbed. Directly reflected light from the skin is filtered out using a second polarizing filter on the camera lens. The depth of measurement was 300 μm so the measurement was done in part of dermis. Red Blood Cells in the skin absorb light in the 500-600 nm spectral range (green light region) to much higher extent than light in the red wave-length region (about 600-700 nm). The surrounding tissue components of the dermis, in comparison, absorb lesser light, and this absorption is not as wave-length dependent as that of red blood cells. The TiVi-technology takes advantage of this difference in absorption by separating the images into their different color planes. Hence the TiVi method can distinguish the images according to their wavelength absorption ranges. The processed image is color-coded map in which red and blue color represent high and low content of red blood cells, respectively. The value of resulting matrix, referred to as TiVi-values(index), scale linearly with the momentary red blood cells concentration in the actual tissue volume. Due to the selection of wavelength range the system was relatively insensitive to the oxygen state of red blood cells and the velocity of red blood cells. One zone on the face (generally on the cheeks) was chosen to perform the measurement by TiVi device where the red color was the most intensive on inclusion visit and after treatment and represent the effect in subject. The analysis parameter (TiVi value-index) is described in arbitrary units. After 28 days of use the product induced a significant decrease of the cutaneous microcirculation of 6% on average on D28 and this effect was observed in 86% of the subjects.

[0172] On each kinetic were performed photographs on determined lesion to illustrate their expected visual effect. The measurement performed on each kinetic was the analysis the vascularization index by Visia Complexion Analysis System and concerned the 10 subjects with rosacea type II. In the device there are three types of lightening. Standard light IntelliFlash, Cross Polarized flash and Ultraviolet lighting are used to record and measure surface and subsurface skin conditions. The skin image captured by the digital camera is composed of red (R), green (G) and blue (B) channels and is presented in camera's native RGB space. RGX technology transforms this RGBimage into the RBX color-space where the Red and Brown channels represent hemoglobin and melanin distribution, respectively. In our situation Red areas referred to hemoglobin concentration. For accurate imaging of hemoglobin it is essential that the re-emitted light from the skin is free of spectacular reflections. The skin images were captured under polarized illumination with a pair of orthogonally-polarized filters placed over the flash and on the camera lens. Cross-polarization eliminated spectacular reflections from the skin surface, improving visibility of re-emitted light in the part of skin where hemoglobin resides. Hemoglobin occurs within the vascular structure at the papillary dermis (first layer between the epidermis and dermis with lot of papillas which overlap in epidermis) in oxygenated and deoxygenated forms and is responsible for the red color-action of the skin tone. Skin condition such as acne, rosacea, telangiectasia can cause changes in vascular structures and elevate the level of hemoglobin. The red images were processed to detect vascular features. The zone of analysis for this method was face in which software inscribed the mask which covers almost whole face. Due to this fact, technology detected all vascular features on the face connected with all conditions which can cause this redness, so not only rosacea. Additionally the mapping of places with hemoglobin depends also on oxygenation of red blood cells. Oxygenation of red blood cells may also be genetically determined and depends on age of the subject. Further this method can also analyse the vessels, which are permanently dilated. The reason why we could observe the positive and significant results on the Tivi device in contrast to Visia, where we did not observed any significant change, could be primarily connected with zone of measurement and probably also with the mechanism of this measurement. In Tivi this result depends on the amount of red blood cells which reflect the red light (this method is also more precise) and in Visia on the content of hemoglobin. Moreover, in Tivi device the measurement was performed on the dermis layer and in Visia on papillary dermis layer. From this can be inferred that the products can act on the deeper parts. Additionally, in Visia analysis there were only small panel analyzed so the obtained results are unequivocal and there is no statistical global result. At the end of the study, each subject filled in a subjective evaluation questionnaire on D28 (after the product's use) to subjectively evaluate the properties, the efficacy, the tolerance and the future use of the studied product. The product was evaluated in a general appreciation as a very pleasant by 76% of subjects. The combination of the product was evaluated in a general appreciation as a very pleasant by 100% of subjects. The subjects appreciated the properties of the product, such as aspect, texture. All of the subjects concluded that the product is pleasant to apply and penetrated quickly. The subjects appreciated also the efficacy of tested product. Most of the subjects agreed that the skin after 28 day of use is soft, moisturized, comfortable and protected by the product. They also decided that the product soothes and heals the skin and did not leave the skin greasy and sticky. 86% of the subject rated that product decreases the redness after 28 days of use. 33% of the subject noted an improvement already after 7 days of use the product and another 29% of them after 14 day of use.