Models of reconstructed sensitive skin

Abstract

The present invention relates to models of reconstructed sensitive skin reproducing the features of sensitive skin, as well as to processes for obtaining such models. The present invention further relates to in vitro processes for testing formulations or active ingredients for the prevention or treatment of sensitive skin.

Claims

1. A process for preparing a model of reconstructed sensitive skin, comprising the steps of: a) obtaining a skin sample from a subject; b) obtaining a reconstructed skin model from the skin sample of step a); c) contacting the reconstructed skin model from step b) with lactic acid for at least 48h, wherein the concentration of lactic acid used is about 0.6%; and d) measuring the expression level of a combination of biological markers, said combination comprising; skin inflammation markers comprising IL8, IL1?, ILR1A, ?-6 polyunsaturated fatty acids, ?-9 polyunsaturated fatty acids, prostaglandin E2, PLA2G2F, MGST1, and VEGF-A, barrier markers comprising involucrin, and defense markers comprising beta-defensin 2 and Toll-like receptor 2; wherein: (i) the expression level of the skin inflammation markers is higher in the reconstructed skin model from step d) than in normal skin; and (ii) the expression level of the barrier markers is lower in the reconstructed skin model from step d) than in normal skin; and (iii) the expression level of the defense markers is lower in the reconstructed skin model from step d) than in normal skin.

2. The process for preparing a model of reconstructed sensitive skin according to claim 1, wherein the concentration of lactic acid used is 0.6%.

3. The process for preparing a model of reconstructed sensitive skin according to claim 1, wherein said barrier markers further comprise, a ceramide selected from the group consisting of ceramides CER 1 to 9, and natural moisturizing factor (NMF).

4. The process for preparing a model of reconstructed skin according to claim 1, wherein said defense markers further comprise protein S100A7.

5. The process for preparing a model of reconstructed sensitive skin according to claim 1, wherein the reconstructed skin model of step b) is selected from the group consisting of suspended skin cell cultures, monolayer skin cell cultures, bilayer skin cell cultures, reconstructed skin cultures, and reconstructed mucosal cultures.

6. The process for preparing a model of reconstructed sensitive skin according to claim 1, wherein the skin sample of a subject from step a) comprises an explant of skin tissue or stem cells differentiated into skin cells.

7. The process for preparing a model of reconstructed sensitive skin according to claim 1, wherein said model of reconstructed skin from step b) comprises at least fibroblasts or keratinocytes.

Description

FIGURES

(1) FIG. 1: Hematoxylin/eosin staining of reconstructed epidermis.

(2) FIG. 2: Immunolabeling of involucrin (light gray) in reconstructed epidermis.

EXAMPLES

Example 1

Characterization of the Cellular, Molecular and Physiological Aspects of Sensitive Skin: Clinical Study

(3) A clinical study was evaluated on a panel of 97 subjects, including 77 children.

(4) Subjects were interviewed using a questionnaire to classify them into two categories: sensitive skin or normal skin. The results of this ranking are described in Table 1.

(5) TABLE-US-00001 TABLE 1 Ranking of the different subjects recruited for the clinical study, according to their age. Number of subjects (male/female) Age group Normal skin Sensitive skin 3-6 months N = 15 (6/9) N = 12 (8/4) 6-12 months N = 11 (5/6) N = 11 (4/7) 24-48 months N = 18 (7/11) N = 10 (6/4) 18-20 years old N = 10 (4/6) N = 10 (4/6)

(6) A clinical evaluation was performed on different parts of each subject's face and body. The scaling, roughness, redness and cracks (SRRC) criteria and the overall dry skin score (ODS) criteria including the evaluation of skin dryness, were measured. This clinical evaluation showed that sensitive skin is generally more scaly, rougher and erythematous than normal skin.

(7) Finally, this clinical evaluation and instrumental measurements were correlated with molecular analyses. Molecular analyses were performed on non-invasive biological samples for each subject (swab sampling): (i) Measurement of the levels cytokines IL1R1, IL1? and IL8; (ii) Measurement of the levels of polyunsaturated free fatty acids (arachidonic acid (C20:4 AA) and linoleic acid (C18:2 LA)).

(8) Molecular analyses showed that the expression levels of inflammation markers such as cytokines and the levels of polyunsaturated free fatty acids (PUFA) are higher in sensitive skin than in normal skin, as expected.

Example 2

Development and Validation of a Model of Reconstructed Sensitive Skin

(9) I. Materials and Methods

(10) a. Model ImplementedInduction of Lactic Acid Stress

(11) Reconstructed human epidermises were obtained from the keratinocytes of a 19-year-old donor using the method derived from Poumay et al. (Arch Dermatol Res 2004; 296:203-11). After 2 days of immersion culture, the reconstructed human epidermises were cultured at the air/liquid interface for 10 days.

(12) Epidermises at differentiation day 10 was treated topically with 0.6% lactic acid solution at 50 ?L/epidermis and incubated for 48 hours, with re-treatment at 24 hours. At the end of the incubation, different parameters were measured to evaluate the effect of lactic acid on the reactivity of the epidermises.

(13) b. Evaluation of the Viability of the Epidermises

(14) The viability of the epidermises was assessed by an MTT metabolism test (n=2). The optical density (OD) at 540 nm is proportional to the amount of living cells and their metabolic activity.

(15) c. Histological Analyses

(16) The epidermises were mounted in paraffin (n=2), their morphology was evaluated following hematoxylin/eosin staining.

(17) On the other hand, involucrin was stained by immunofluorescence (Alexa-488; green dye), the nuclei were stained by propidium iodide (red dye). The involucrin staining was quantified by measuring the fluorescence intensity reported on the surface of the living layers of the epidermis.

(18) d. Gene Analyses

(19) Gene expression of inflammatory markers and of markers of innate immunity and defense was evaluated by real-time quantitative PCR (qRT-PCR) on messenger RNAs extracted from epidermis (n=2). Gene expression of the innate immunity and defense markers, beta-defensin 2 hBD2, or Toll-like receptor 2, was also evaluated in the same way.

(20) The gene expression analysis was performed in duplicate using a PCR array. The inflammatory marker genes studied are presented in Table 2.

(21) The quantitative analysis of gene expression was based on the analysis of threshold cycles; relative expression (RE) was expressed in arbitrary units according to the following formula:
RE=(? number of cycles)?10.sup.6

(22) TABLE-US-00002 TABLE 2 Classification of the genes studied Marker group Gene Abbreviation Inflammation Interleukin 1 alpha IL1A (tissue Interleukin 1 beta IL1B component) Interleukin 1 receptor antagonist IL1RN Tumor Necrosis Factor TNF Interleukin 8 IL8 Prostaglandin-Endoperoxide PTGS2 Synthase 2 (=COX2) Phospholipase A2, group IIF PLA2G2F2F Inflammation Vascular Endothelial VEGF-A (vascular Growth Factor A component): Thrombospondin 1 THBS1 Vascularization Adenylate Cyclase Activating ADCYAP1 factors Polypeptide 1 (PACAP) Innate Immunity Beta-Defensin-2 (hBD2) DEFB4A and Defense Toll Like Receptor 2 TLR2
e. Determination of Inflammatory Markers
i. Determinations of Inflammatory Markers Released by the Epidermises

(23) The amount of inflammatory markers present in culture supernatants was assessed (n=6) using CBA kits (BD Biosciences) for IL-1 alpha (IL1?) and IL-8; and ELISA kits for VEGF (R&D Systems) and PGE2 (Enzo).

(24) ii. Assessment of Intracellular Inflammatory Markers

(25) Intracellular amounts of IL-1?, IL8 and IL1-RA were determined by ELISA (R&D Systems) in ground epidermis (n=2).

(26) iii. Determination of Poly-Unsaturated Fatty Acids (PUFAs)

(27) Screening of polyunsaturated fatty acids (PUFAs) expressed by the epidermis was carried out by GC/MS analysis of samples obtained after grinding the epidermises (n=2).

(28) The amounts were normalized by C16:0 (palmitic acid), which is representative of the overall lipid content of the epidermis.

(29) II. Results

(30) a. Morphology and Viability of Reconstructed Epidermises

(31) Lactic acid induced a moderate decrease in the viability of the epidermises (Table 3) and did not induce any major alteration in their morphology (FIG. 1).

(32) TABLE-US-00003 TABLE 3 Viability of reconstructed epidermises (MTT) Reconstructed epidermises Viability Control 100% Lactic acid 0.6% 66%
b. Evaluation of Lactic Acid-Modulated Gene Markers

(33) The application of lactic acid to the surface of reconstructed epidermis induced stimulation of markers of inflammation of the tissue and vascular components, suggesting greater skin sensitivity (Table 4).

(34) TABLE-US-00004 TABLE 4 Gene expression in reconstructed epidermises (qRT-PCR) Lactic acid Marker group Genes Control 0.6% Inflammation IL1A 100 240 (tissue IL1B 100 195 component) IL1RN 100 144 TNF 100 105 IL8 100 649 PTGS2 100 182 PLA2G2F2F 100 129 Inflammation VEGF-A 100 138 (vascular THBS1 100 181 component): ADCYAP1 100 117 Vascularization factors Innate immunity DEFB4A 100 6 and defense TLR2 100 54

(35) Lactic acid treatment of reconstructed skins induced a decrease in the expression of innate immunity and defense markers (beta-defensin 2 hBD2-DEFB4A, Toll-like receptor 2 TLR2), suggesting an alteration in defense capabilities.

(36) c. Expression of Involucrin

(37) Lactic acid induced a decrease in the expression level of involucrin (?22%; FIG. 2), showing an alteration of the epidermal barrier.

(38) d. Assessment of the Inflammatory Response

(39) Lactic acid treatment induced an increase in the intracellular amount (Table 4) and the amount released into the supernatant (Table 5) of early markers of inflammation, such as inflammatory cytokines IL1-alpha (Interleukin 1-alpha, primary cytokine, main trigger of the inflammatory cascade) and IL8 (Interleukin 8, chemokine induced by de novo synthesis in stimulated keratinocyte).

(40) At the intracellular level, the amount of IL1-RA (Interleukin 1 Receptor Antagonist, a member of the IL1 family that is induced in response to irritation, secondary to IL1) was also increased in response to lactic acid treatment, as was the IL1-RA/IL1-alpha ratio (Table 5).

(41) The balance of the IL1-RA/IL1-alpha ratio allows skin homeostasis to be maintained. The increase in this ratio as well as the increase in IL1-RA are sensitive markers of the level of skin inflammation.

(42) Lactic acid treatment induced induction of release of PGE2 (Prostaglandin E2, a lipid marker of the tissue component of inflammation) and VEGF (Vascular Endothelial Growth Factor, a vascular growth factor and marker of the vascular component of inflammation; Table 6).

(43) TABLE-US-00005 TABLE 5 Intracellular determinations of inflammatory markers (ELISA) Lactic Change vs. Control acid 0.6% control IL8 (pg/mg protein) 10.6 52.1 +390% IL1-alpha (pg/mg protein) 88 794 +806% IL1-RA (pg/mg protein) 2328 31243 +1242% IL1-RA/IL1-alpha ratio 26.6 39.3 +48%

(44) TABLE-US-00006 TABLE 6 Determinations of released inflammatory markers (ELISA) Lactic Change vs. Control acid 0.6% control IL1-alpha (pg/ml) 7 ? 1 23 ? 7 +221% ns IL8 (pg/ml) 65 ? 4 120 ? 18 +84% p < 0.05 PGE2 (pg/ml) 48 ? 4 149 ? 36 +207% p < 0.05 VEGF (pg/ml) 433 ? 17 1109 ? 45 +156% p < 0.001
e. Poly-Unsaturated Fatty Acid (PUFA) Analysis

(45) Screening of PUFAs reveals an inducing effect of lactic acid on proinflammatory omega-6 fatty acids involved in the arachidonic acid (AA) and linoleic acid (LA) pathways as well as proinflammatory omega-9 fatty acids involved in the oleic acid pathway (C18:1; Table 7).

(46) TABLE-US-00007 TABLE 7 Determination of polyunsaturated fatty acids (PUFAs) in reconstructed epidermises (GC/MS); amounts normalized to C16:0 Lactic Change vs. PUFAs Control acid 0.6% control C18:1 0.189 0.226 +20% C18:2 LA 0.181 0.216 +19% C20:4 AA 0.035 0.050 +42%

Conclusions

(47) Treating the reconstructed skin models with lactic acid induced a significant increase in the expression of the different protein markers of the tissue and vascular components of inflammation, both at the transcript and protein level, as well as lipid markers of inflammation. This treatment of reconstructed skin models also induced a significant decrease in the expression of innate immunity and defense markers and barrier markers.

(48) These data are correlated with the results obtained in the clinical study described in section 1 above.

(49) The treatment of reconstructed skin models with lactic acid therefore makes it possible to reproduce the features of sensitive skin.

Example 3

Evaluation of the Effectiveness of Active Ingredients in the Reconstructed Sensitive Skin Model (Presented in Example 2)

(50) I. Materials and Methods

(51) a. Compounds EvaluatedDermocosmetic Active Ingredients

(52) The ingredients described below were evaluated in the reconstructed Sensitive Skin model obtained from Example 2 described above. The ingredients tested are cosmetic active ingredients with recognized anti-inflammatory properties. Cycloceramide (OX100) [Laboratoires Expanscience]: tested at 10 and 100 ?M

(53) Cycloceramide is an active ingredient obtained by synthesis. It is a PKC inhibitor with anti-inflammatory properties (EP 2 266 530 B1; WO2006/11443 A1; WO 03/055463 A1; Piccardi et al., 2005, JID, vol 124, no. 4, suppl, abstract 216; Staquet et al., Int Arch Allergy Immunology, 2004, April; 133(4):348-56). Peptide and sugar extract of Schizandra (SC) [Laboratoires Expansciencetrade name Sweetone?]: tested at 0.05% and 0.1%

(54) This extract has anti-inflammatory and anti-redness properties demonstrated in vitro and clinically (WO2011/012612; WO2011/012615 A3; EP2015/077795; Br?dif et al., 2013, JID, Volume 133, Issue S1, page S162, Abstract 953). Dipotassium glycyrrhizinate (GLY) [Maruzen Pharmaceuticals]: tested at 0.005% and 0.01%

(55) Dipotassium glycyrrhizinate is an extract of licorice with anti-inflammatory properties.

(56) b. Protocol

(57) Reconstructed human epidermises were obtained from the keratinocytes of a 2.5-month-old donor using the method derived from Poumay et al. (Arch Dermatol Res 2004; 296:203-11). After 2 days of immersion culture, the reconstructed human epidermises were cultured at the air/liquid interface for 10 days.

(58) Epidermises at differentiation day 10 was treated or not (control) systemically with the test compounds and incubated for 24 hours. After incubation (day 11), treatment with 0.6% lactic acid (topical application of 50 ?L/epidermis) was performed and the medium replaced by medium containing the test compounds and then the epidermises were incubated for 48 hours, with renewal of treatment at 24 hours (topical lactic acid and systemic test compounds).

(59) At the end of incubation, the inflammatory parameters described below were measured to evaluate the effect of lactic acid on skin reactivity. Determinations of inflammatory markers released by the epidermises (n=8)

(60) The amount of inflammatory mediators IL-1alpha (IL1?), IL-8 and PGE2 present in skin culture supernatants was evaluated using ELISA kits (R&D Systems). Evaluations of intracellular inflammatory markers IL-1?, IL8 and IL1-RA (n=2)

(61) Same protocol as described in Example 2. Determination of Poly-Unsaturated Fatty Acids (PUFAs) (n=2)

(62) Same protocol as described in Example 2.

(63) II. Results

(64) a. Assessment of the Inflammatory Response

(65) The results confirm the observations described in Example 2 demonstrating the effect of lactic acid on the induction of an increase in intracellular (Table 8) and released (Table 9) amount of inflammation markers: IL8, IL1?, IL1RA, IL1RA/IL1? ratio, PGE2.

(66) The active ingredients evaluated significantly inhibited these markers of inflammation:

(67) Dipotassium Glycyrrhizinate (referred to as GLY in the results tables) significantly inhibited the increase in intracellular IL8, IL1? and IL1RA levels and the release of IL1? and PGE2 induced by lactic acid treatment. In addition, Dipotassium Glycyrrhizinate also maintained the IL1RA/IL1? ratio.

(68) Peptide and sugar extract of Schizandra (SC) significantly inhibited the intracellular amount of IL1? and IL1RA, also preserving the balance of the IL1RA/IL1? ratio. Schizandra extract also inhibited the induction of PGE2 release.

(69) Cycloceramide (OX100) significantly inhibited intracellular IL1RA increase and maintained the balance of the IL1RA/IL1? ratio. In addition, OX100 significantly inhibited the release of IL1?, IL8 and PGE2 induced by lactic acid.

(70) TABLE-US-00008 TABLE 8 Intracellular measurements of inflammatory mediators (ELISA): IL8, IL1?, IL1RA, IL1RA/IL1? ratio In bold: Change vs. control; In italics: Change vs. lactic acid Single-factor Analysis of Variance followed by Tukey test IL8 Mean ? SD IL8 (pg/mg protein) Change Control 37.23 ? 6.484 Lactic acid 0.6% 79.20 ? 7.715 +113% p < 0.01 GLY 0.01% 50.17 ? 3.366 ?37% p < 0.05 GLY 0.005% 78.39 ? 6.350 ?1% ns SC 0.1% 53.00 ? 9.291 ?33% ns SC 0.05%. 70.57 ? 3.825 ?11% ns OX100 100 ?M 56.95 ? 7.460 ?28% ns OX100 10 ?M 76.24 ? 8.309 ?4% ns IL1-alpha Mean ? SD IL1 alpha (pg/mg protein) Change Control 277.0 ? 46.67 Lactic acid 0.6% 1044 ? 123.0 +277% p < 0.001 GLY 0.01% 587.0 ? 63.64 ?44% p < 0.01 GLY 0.005% 987.5 ? 101.1 ?5% ns SC 0.1% 448.0 ? 76.37 ?57% p < 0.01 SC 0.05% 984.0 ? 116.0 ?6% ns OX100 100 ?M 713.5 ? 113.8 ?32% ns OX100 10 ?M 919.5 ? 53.03 ?12% ns IL1RA Mean ? SD IL1RA (pg/mg protein) Change Control 5217 ? 688 Lactic acid 0.6% 25750 ? 1974 +394% p < 0.001 GLY 0.01% 11244 ? 1341 ?56% p < 0.001 GLY 0.005% 25195 ? 1305 ?2% ns SC 0.1% 8605 ? 1527 ?67% p < 0.001 SC 0.05% 22739 ? 1312 ?12% ns OX100 100 ?M 14581 ? 1989 ?43% p < 0.001 OX100 10 ?M 23566 ? 1624 ?8% ns IL1RA/IL1-alpha ratio Mean ? SD IL1RA/IL1a ratio (pg/mg protein) Change Control 18.93 ? 0.7142 Lactic acid 0.6% 24.73 ? 1.011 +31% p < 0.001 GLY 0.01% 19.14 ? 0.2121 ?23% p < 0.001 GLY 0.005% 25.58 ? 1.287 +3% ns SC 0.1% 19.20 ? 0.1556 ?22% p < 0.001 SC 0.05% 23.20 ? 1.407 ?6% ns OX100 100 ?M 20.46 ? 0.4808 ?17% p < 0.001 OX100 10 ?M 25.63 ? 0.2758 +4% ns

(71) TABLE-US-00009 TABLE 9 Determinations of released inflammatory mediators (ELISA) IL1-alpha IL8 PGE2 Mean ? Mean ? Mean ? Standard Standard Standard deviation deviation deviation (pg/ml) Change (pg/ml) Change (pg/ml) Change Control 11 ? 2 78 ? 10 29 ? 22 Lactic acid 68 ? 34 +511% 209 ? 40 +169% 197 ? 79 +584% 0.6% p < 0.001 p < 0.001 p < 0.001 GLY 0.01% 35 ? 16 ?49% 244 ? 138 +17% 137 ? 83 ?31% p < 0.05 ns ns GLY 28 ? 10 ?59% 211 ? 41 +1% 94 ? 36 ?52% 0.005% p < 0.01 ns p < 0.05 SC 0.1% 91 ? 68 +34% 293 ? 49 +40% 127 ? 56 ?36% ns ns ns SC 0.05% 84 ? 59 +24% 261 ? 50 +25% 113 ? 39 ?43% ns ns p < 0.05 OX100 at 31 ? 19 ?54% 146 ? 24 ?30% 100 ? 48 ?49% 100 ?M p < 0.05 p < 0.01 p < 0.01 OX100 at 27 ? 13 ?61% 175 ? 30 ?16% 98 ? 28 ?50% 10 ?M p < 0.01 ns p < 0.01 In bold: Change vs. control; In italics: Change vs. lactic acid Single-factor Analysis of Variance followed by Tukey test
b. Poly-Unsaturated Fatty Acid (PUFA) Analysis

(72) The screening of PUFAs confirmed the inducing effect of lactic acid on proinflammatory omega-6 (C18:2; C20:4) and omega-9 (C18:1) fatty acids (Table 10).

(73) The active ingredients evaluated showed an inhibitory effect of proinflammatory fatty acid induction.

(74) TABLE-US-00010 TABLE 10 Determination of polyunsaturated fatty acids (PUFAs) in reconstructed epidermises (GC/MS); amounts normalized to C16:0 C18:1 C18:2 LA C20:4 AA Mean ? SD Change Mean ? SD Change Mean ? SD Change Control 0.1850 ? 0.09100 ? 0.0270 ? 0.02404 0.01697 0.008485 Lactic acid 0.3880 ? +110% 0.2130 ? +134% 0.0575 ? +113% 0.6% 0.07212 p < 0.01 0.04808 p < 0.05 0.01626 ns GLY 0.01% 0.2680 ? ?31% 0.1440 ? ?32% 0.0395 ? ?31% 0.01697 ns 0.01273 ns 0.009192 ns GLY 0.005% 0.2785 ? ?28% 0.1600 ? ?25% 0.0435 ? ?24% 0.02475 ns 0.01414 ns 0.009192 ns SC 0.1% 0.2245 ? ?42% 0.1190 ? ?44% 0.0330 ? ?43% 0.01202 p < 0.05 0.01273 ns 0.002828 ns SC 0.05% 0.2520 ? ?35% 0.1400 ? ?34% 0.0350 ? ?39% 0.02404 ns 0.01697 ns 0.007071 ns OX100 0.2065 ? ?47% 0.0970 ? ?54% 0.0265 ? ?54% 100 ?M 0.007778 p < 0.05 0.008485 p < 0.05 0.002121 ns OX100 0.2795 ? ?28% 0.1515 ? ?29% 0.0380 ? ?34% 10 ?M 0.03323 ns 0.02192 ns 0.005657 ns In bold: Change vs. control; In italics: Change vs. lactic acid Single-factor Analysis of Variance followed by Tukey test
c. Conclusion

(75) In the model of sensitive skin on reconstructed epidermis, the active ingredients evaluated modulated the inflammatory response induced by lactic acid, by acting on the inflammatory mediators produced by skin cells (cytokines and prostaglandins) as well as on the inflammatory pathways derived from omega-6 and omega-9 fatty acids.

(76) These results confirm the validity of the sensitive skin model. These results also confirm the validity of the sensitive skin model for use in methods for evaluating the effectiveness of cosmetic active ingredients in preventing or treating sensitive skin.