Method for evaluating the effects of dehydration on children's skin

Abstract

The invention relates to biomarkers in children's skin, in particular in the skin of infants, the expression of which changes when the skin is dry. Such markers are particularly advantageous in that they allow the skin's response to dehydration to be monitored. The inventors have developed methods for evaluating the in vitro efficacy of formulations in preventing the effects of dehydration on children's skin, using a skin model specifically capable of reproducing the characteristics of children's skin.

Claims

1. A method for evaluating the in vitro efficacy of an active agent or of a formulation in reducing the effects of dehydration on children's skin, characterized by said method comprising the following steps: a. incubating a reconstructed skin model under drying conditions, wherein the drying conditions comprises a relative humidity in the atmosphere of 25% or less; b. contacting said active agent or said formulation with the reconstructed skin model of step a; c. measuring the expression level of a combination of biological markers in the skin model of step b, wherein the biological markers comprise inflammation markers IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD, OSMR, PLA2G2F, and F2RL1; barrier function markers DSG, SCEL, POADI1, CASP14, LOR, TGM1, and CLDN1; and stem cell markers Tp63, KRT15, KRT19, BIRC5, and NOTCH1; d. measuring the amounts of natural moisturizing factors (NMFs) and ceramides in the skin model of step b; e. comparing the expression levels of the biological markers and the amounts of NMFs and ceramides to a control reconstructed skin model not exposed to drying conditions, wherein both the reconstructed skin model and the control reconstructed skin model are obtained from skin samples from a donor between newborn and 2 years, and determining that the agent or formulation is able to protect against dryness when the dried reconstructed skin model exposed to the agent or formulation has: i. the same or a lower expression level of the following inflammation markers: IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD, OSMR, PLA2G2F, and F2RL1 when compared to the control reconstructed skin model; ii. the same or a higher expression level of the following barrier function markers: DSG, SCEL, PAD1L, CASP14, LOR, TGM1, and CLDN1 when compared to the control reconstructed skin model; iii. the same or a higher expression level of the following stem cell markers: Tp63, KRT15, KRT19, BIRC5, and NOTCH1 when compared to the control reconstructed skin model; iv. the same or a higher amount of NMFs and ceramides.

2. The method of claim 1, wherein the reconstructed skin model comes from skin having a phototype I, II, III, IV, V, or VI.

3. The method of claim 1, wherein the reconstructed skin model is selected from suspended skin cell cultures, monolayer skin cell cultures, bilayer skin cell cultures, reconstructed skin cultures, and reconstructed mucosal cultures.

4. The method of claim 1, wherein the reconstructed skin model comes from a skin tissue explant or from differentiated cells.

5. The method of claim 1, wherein the reconstructed skin model comprises at least fibroblasts and keratinocytes.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the quantification of NMFs in the Normal Skin and Dry Skin panels, as well as the change in the amounts of NMFs as a function of age group.

(2) FIG. 2 shows the quantification of ceramides in the Normal Skin and Dry Skin panels, as well as the change in the amounts of ceramides as a function of age group.

EXAMPLES

Example 1: Characterization of Babies' and Children's Dry Skin In Vivo

(3) In order to characterize babies' and children's dry skin, an exploratory clinical study was carried out.

(4) 1. Materials and Methods

(5) A clinical study with paediatric and dermatological supervision was carried out on two panels (Normal Skin panel and Dry Skin panel; classification after clinical examination by the investigator) of 40 subjects each, divided into 4 age groups:

(6) Group 1: 1-28 days; Group 2: 3-6 months; Group 3: 1 year; Group 4: 2-4 years (Grp 1, Grp 2, Grp 3, Grp 4, respectively).

(7) Biological specimens were collected on the subjects' forearm using swabs. The measurement of NMFs (natural moisturizing factors) and of ceramides was carried out on the collected specimens by liquid chromatography coupled to mass spectrometry (LC/MS) for the ceramides or by liquid chromatography coupled to UV detection (LC/UV) for the NMFs. Analysis of ceramides

(8) The presence of ceramides having a sphingoid base of type sphingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] with an even-chain length of 16 to 22 carbon atoms was investigated by an LC/MS method.

(9) The ceramides content was normalized to the amount of total proteins (BCA assay).

(10) Analysis of NMF Components

(11) Filaggrin catabolites were measured by an LC/UV method for screening the two isomers (cis and trans) of urocanic acid (UCA) as well as L-pyrrolidone carboxylic acid (PCA).

(12) The NMF content was normalized to the amount of total proteins (BCA assay).

(13) 2. Results and Conclusion

(14) The results presented below show that the subjects of the Dry Skin panel have significantly lower amounts of NMFs than those of the Normal Skin panel, regardless of age group (FIG. 1, Table 1). These observations show that low levels of NMFs constitute a biological marker of dry skin in newborns, infants and young children.

(15) Furthermore, the amounts of ceramides are lower for the subjects of the Dry Skin panel than for the subjects of the Normal Skin panel (FIG. 2, Table 1), showing that ceramides also constitute a marker of the dry skin condition in newborns, infants, and children with clinically dry skin.

(16) In conclusion, the amount of NMFs and of ceramides is lower in children's dry skin compared with children's normal skin.

(17) TABLE-US-00007 TABLE 1 Total content of NMFs and ceramides for all Normal Skin and Dry Skin panels - Student's t-test Comparison of Dry Normal Dry Skin vs Normal Skin Skin Skin Total NMFs 97.37 89.43 8.2% p < 0.05 (g/mg proteins) Total ceramides 92.68 84.44 8.9% p < 0.05 (g/100 mg proteins)

Example 2: Validation of the In Vitro Dry Skin Model

(18) In order to validate the modelling of a dry skin phenotype, the production of NMF (natural moisturizing factor) and of ceramides by epidermises incubated in dry atmosphere was evaluated.

(19) 1. Materials and Methods

(20) Reconstructed epidermises were prepared with keratinocytes from a donor aged 1 year.

(21) Reconstruction of the epidermises was carried out according to a model derived from the method of Poumay et al. (Arch Dermatol Res 2004; 296:203-11). After 2 days of immersion culture, the reconstructed human epidermises (RHE) were grown at the air/liquid interface for 11 days.

(22) On day 11, the epidermises were incubated for 48 h in a humid incubator for the control epidermises (normal condition: 37 C., 5% CO.sub.2 and >99% relative humidity) or in a dry incubator (37 C., 5% CO.sub.2 and <25% relative humidity).

(23) At the end of incubation, the amounts of NMF and of ceramides produced by the epidermises were evaluated.

(24) The experiments were repeated 3 times; for each these 3 tests, 3 replicates were prepared and analysed.

(25) Analysis of Ceramides

(26) The epidermal lipids were extracted by shaking the epidermises from a mixture of organic solvents for 2 h at room temperature. Solid/liquid extraction was then carried out to isolate the ceramides from the other constitutive epidermal lipids.

(27) The ceramide contents were analysed by LC/MS as described in Example 1.

(28) Analysis of NMF Components

(29) The reconstructed epidermises were extracted under shaking for 2 h at room temperature from an aqueous mixture in the presence of a non-ionic surfactant to promote extraction of the markers of interest.

(30) The NMF contents were analysed by LC/UV as described in Example 1.

(31) 2. Results and Conclusion

(32) Ceramides are important structural components of the epidermis. They are one of the constituents of the lipid matrix of the stratum corneum. The role of this matrix is essential in the regulation of water permeability. It indeed constitutes a hydrophobic barrier which regulates water circulation through the SC. The lipid matrix is composed of an equimolar mixture of ceramides (45 to 50% of the total weight), of cholesterol (20-25%) and of free fatty acids (10-15%). Ceramides derive from the transformation of sphingomyelin by sphingomyelinases and of glucoceramides by -glucocerebrosidase.

(33) The major function of NMF is to maintain an optimal level of water in the stratum corneum. A well-known source of NMF constituents is filaggrin.

(34) Incubation of the 1-year-old reconstructed epidermises in a dry incubator induced significant inhibition of the ceramide and NMF content in the epidermises (Table 2).

(35) The decreased amount of NMF and of ceramides is one of the main features of dry skin, known and described in the literature. Moreover, Example 1 shows that the amount of NMF and of ceramides also decreases in children's dry skin. The model prepared consisting of incubation of reconstructed epidermises in dry atmosphere is thus well representative of the children's dry skin phenotype observed in vivo.

(36) TABLE-US-00008 TABLE 2 NMF and ceramide content in 1-year-old reconstructed epidermises incubated under normal conditions (control) in a dry incubator (dry atmosphere) Significance Control Epidermises in Inhibi- (Student's epidermises dry atmosphere tion t-test) NMF content 17.05 0.21 12.44 0.82 27% P < 0.001 (g/mg proteins) Ceramide content 173.6 3.86 149.4 2.62 14% P < 0.001 (AU/mg proteins)

Example 3: Evaluation of the Effect of Delipidation on the Amount of Ceramides in Reconstructed Epidermises

(37) In order to determine if other in vitro dry skin models made it possible reproduce the children's dry skin phenotype, the model of induction of dry skin by delipidation was tested. The amount of ceramides, one of the specific markers of children's dry skin, was analysed in this model.

(38) Reconstructed epidermises were prepared according to a model derived from the method of Poumay et al. (Arch Dermatol Res 2004; 296:203-11). After 2 days of immersion culture, the reconstructed human epidermises (RHE) were grown at the air/liquid interface for 10 days.

(39) On day 10, the epidermises were treated by topical application of a 30% 1:1 ether/acetone mixture in order to induce alteration of the barrier by delipidation, then incubated for 48 h (Miyamoto et al., 2002; Akiyama et al., 2010; Valvetcha et al., 2015).

(40) At the end of incubation, the amounts of ceramides produced by the epidermises were evaluated.

(41) Analysis of Ceramides

(42) The epidermal lipids were extracted by shaking the epidermises from a mixture of organic solvents for 2 h at room temperature. Solid/liquid extraction was then carried out to isolate the ceramides from the other constitutive epidermal lipids.

(43) The presence of ceramides having a sphingoid base of type sphingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] with an even-chain length of 16 to 22 carbon atoms was investigated by an LC/MS method.

(44) Results and Conclusion

(45) Delipidation of the epidermises by ether/acetone treatment does not induce a decrease in the amount of ceramides produced by the epidermises (Table 3).

(46) Therefore, surprisingly, delipidation does not allow modelling of the dry skin phenotype shown in vivo. Only the dry skin model obtained by incubation in a dry-atmosphere incubator designed by the present Inventors and described in Example 2 above makes it possible to reproduce the phenotype observed in vivo for children's dry skin.

(47) TABLE-US-00009 TABLE 3 Total ceramide content for the control epidermises and the delipidated epidermises - Student's t-test Comparison of Delipidated Control Delipidated epidermis vs epidermis epidermis Control epidermis Ceramides 234.3 26.0 227.9 3.9 2.8% ns (arbitrary units)

Example 4: Characterization of the Response of Infants' Epidermises Compared with Adult Epidermises

(48) 1. Materials and Methods

(49) Reconstructed epidermises were prepared, as described above, first with the keratinocytes of a 1-year-old donor and second with the keratinocytes of a 19-year-old donor; the keratinocytes of these two donors coming from a foreskin specimen.

(50) On day 10, the epidermises were incubated for 24 h to 48 h in a humid incubator for the control epidermises (normal condition: 37 C., 5% CO.sub.2 and >99% relative humidity) or in a dry incubator (37 C., 5% CO.sub.2 and <25% relative humidity).

(51) After 24 h of incubation, the gene expression of inflammation, hydration, barrier function and stem cell markers was evaluated by real-time quantitative PCR (qRT-PCR).

(52) After 48 h of incubation, the viability and the morphology of the epidermises were studied.

(53) Evaluation of the Viability of the Reconstructed Epidermises

(54) The viability of the epidermises was evaluated by an MTT reduction test (n=2): the epidermises were incubated in the presence of MTT (tetrazolium salt), the transformation of which into blue formazan crystals is proportional to succinate dehydrogenase (mitochondrial enzyme) activity. After dissociation of the cells and solubilization of the formazan by addition of isopropanol/HCl, the optical density (OD), representative of the number of living cells and their metabolic activity, was measured at 540 nm.

(55) Evaluation of the Morphology of the Reconstructed Epidermises

(56) The morphology of the reconstructed epidermises was evaluated (n=2) by histological analysis after haematoxylin/eosin staining.

(57) The epidermises were fixed and imbedded in paraffin, cross-sections were prepared with a microtome. The sections were deparaffinized and then stained with haematoxylin/eosin. Haematoxylin stains the cell nuclei blue/purple; eosin stains the cytoplasms varying intensities of pink.

(58) Microscopic analysis of the histological sections made it possible to evaluate and to quantify certain morphological parameters such as the thickness of the layer of living cells.

(59) Differential Gene Expression Analysis

(60) Expression of the markers was evaluated by qRT-PCR on messenger RNA extracted from the RHE of each treatment.

(61) The gene expression analysis was carried out (n=2) using a PCR array containing 30 genes of interest and 2 reference genes (housekeeping genes). Total RNA from each sample was extracted using TriPure Isolation Reagent according to the protocol recommended by the supplier. The amount and the quality of the RNA were evaluated by capillary electrophoresis (Bioanalyzer, Agilent). Complementary DNA (cDNA) was synthesized by reverse transcription of RNA in the presence of oligo(dT) and Transcriptor Reverse Transcriptase enzyme. The cDNA obtained was quantified by spectrophotometry and then the amounts of cDNA were adjusted.

(62) The PCR reactions were performed by quantitative PCR with the LightCycler system (Roche Molecular Systems Inc.) and according to the procedure recommended by the supplier. The reaction mixture for each sample was: cDNA, primers for the various markers used, reaction mixture containing Taq DNA polymerase enzyme, SYBR Green I marker (DNA intercalating agent) and MgCl.sub.2.

(63) Fluorescence incorporation in the amplified DNA is measured continuously during the PCR cycles.

(64) Quantitative analysis of the results is based on the collection of threshold cycles (Ct). The threshold cycle is the point at which the fluorescence emission signal is statistically and significantly higher than the background. The threshold cycle is directly correlated with the initial copy number of target DNA.

(65) Table 4 lists the genes studied.

(66) The relative gene expression value (RE) is expressed in arbitrary units according to the following formula:
RE=(.sub.cycle number)10.sup.6

(67) TABLE-US-00010 TABLE 4 Classification and name of the genes studied Abbre- Cluster name viation Gene name Housekeeping RPS28 Ribosomal protein 28S GAPDH Glyceraldehyde-3-phosphate dehydro- genase IL1A Interleukin 1 alpha IL8 Interleukin 8 Aspecific and PTGS2 Prostaglandin synthase 2 neurogenic NGFR Nerve growth factor receptor inflammation PLA2G2F Phospholipase A2 group IIF TAC1 Tachykinin precursor 1, or substance P TAC1R Tachykinin receptor 1, or substance P receptor TRPV1 Transient receptor potential cation channel, subfamily V, member 1 TRPV3 Transient receptor potential cation channel, subfamily V, member 3 MRGPRD MAS related GPR family member D F2RL1 Coagulation factor II (thrombin) receptor- like 1 or proteinase activated receptor 2 (PAR2) OSMR Oncostatin M receptor, interleukin 31 receptor Epidermal DSG Desmoglein 1 differentiation, SCEL Sciellin Barrier function, PADI1 Peptidylarginine deiminase 1 Hydration CASP14 Caspase 14 LOR Loricrin TGM1 Transglutaminase 1 CLDN1 Claudin 1 TP63 Tumour protein P63 KRT15 Keratin 15 Stem cells KRT19 Keratin 19 BIRC5 Baculoviral IAP repeat-containing 5 (survivin) NOTCH1 Notch homolog 1
2. Results

(68) a. Morphology and Viability of the Epidermises

(69) Incubation in dry atmosphere did not heavily alter the morphology of the epidermises observed under the microscope after haematoxylin/eosin staining (data not shown); furthermore, the viability of the 1-year-old epidermises and of the adult epidermises was impacted in the same manner (30% and 29% inhibition, respectively, Table 5). However, the measurement of the thickness of the layer of living cells was more heavily impacted in the 1-year-old epidermises (18%) compared with the adult epidermises in which no decrease in this parameter is observed (Table 5).

(70) These observations tend to show an increased sensitivity of the 1-year-old epidermises to dehydration stress.

(71) TABLE-US-00011 TABLE 5 Analysis of the viability and the thickness of the layer of living cells Viability (MTT) Control Dry atmosphere Viability (MTT) 1-year-old 100% 70% Adult 100% 71% Thickness of the layer of 1-year-old 100% 82% living cells Adult 100% 100%

(72) b. Analysis of Expression of Gene Markers of Aspecific and Neurogenic Inflammation

(73) Incubation in dry atmosphere induced an increase in the expression level of markers of aspecific and neurogenic inflammation, both in the 1-year-old reconstructed epidermises and in the adult reconstructed epidermises (Table 6).

(74) However, this stimulation is much greater in the case of infants' reconstructed epidermises (+355%) compared with adult epidermises (+114%).

(75) This suggests an increased sensitivity of infants' epidermises, the inflammatory and neurosensory response of which is exacerbated in response to dehydration stress.

(76) TABLE-US-00012 TABLE 6 Level of gene expression of inflammation markers (relative expression in % compared with the control grown under normal conditions) 1-year-old Adult Control epider- Control epider- 1-year-old mises adult mises epider- in dry epider- in dry mises atmosphere mises atmosphere IL1A 100 177 100 155 IL8 100 314 100 147 PTGS2 100 1421 100 666 NGFR 100 223 100 100 TRPV1 100 675.5 100 125 TRPV3 100 606 100 150 MRGPRD 100 246 100 351 F2RL1 100 171 100 119 OSMR 100 263 100 113 Mean expression 100 455 100 214 of inflammation markers (%) Increase relative 355% 114% to the control (%)

(77) c. Analysis of Expression of Stem Cell Marker Genes

(78) The stem cells of tissues undergoing permanent renewal are traditionally defined as being rare and relatively quiescent cells. They have a unique capacity of self-renewal and of tissue regeneration which enables them to provide homeostasis and integrity to the tissue in which they reside.

(79) Among the epidermal stem cells, the interfollicular stem cells located in the basal layer constitute the chief epidermal stem cell reservoir. These cells reside in an anatomical and functional microenvironment, the niche, which helps maintain their characteristics, notably when the physiological conditions change. Interfollicular stem cells and their niches are involved in maintaining skin integrity and regeneration. Stem cells are identifiable only by following several markers. We thus evaluated the expression level of various gene markers characteristic of stem cells in the dry skin model.

(80) Incubation in dry atmosphere induced a substantial decrease (45%) in the mean expression level of the stem cell marker pool studied (Table 7) in the 1-year-old epidermises, whereas no inhibition of expression of these markers was observed in the adult epidermises (+12%).

(81) This tends to show an increased vulnerability of this cell capital in the 1-year-old epidermises.

(82) TABLE-US-00013 TABLE 7 Expression level of stem cell marker genes (relative expression in % compared with the 1-year-old control) 1-year-old Adult Control epider- Control epider- 1-year-old mises adult mises epider- in dry epider- in dry mises atmosphere mises atmosphere KRT19 100 65 84 120 BIRC5 100 64 85 94 TP63 100 13 86 80 NOTCH1 100 77 45 43 Mean expression 100 55 75 84 of the stem cell marker pool (%) Modulation relative 45% +12% to the control (%)

(83) d. Analysis of Expression of Barrier and Hydration Marker Genes

(84) Incubation in dry atmosphere induced a decrease in the expression level of barrier markers, both in the 1-year-old reconstructed epidermises and in the adult reconstructed epidermises (Table 8).

(85) However, this inhibition is greater in the case of infants' reconstructed epidermises (57%) compared with adult epidermises (38%).

(86) This suggests a greater vulnerability of the barrier function markers in the infants' epidermises.

(87) TABLE-US-00014 TABLE 8 Expression level of barrier and hydration marker genes (relative expression in % compared with the 1-year-old control) 1-year-old Adult Control epider- Control epider- 1-year-old mises adult mises epider- in dry epider- in dry mises atmosphere mises atmosphere DSG1 100 56 153 92 SCEL 100 20 62 34 PADI1 100 51 117 78 CASP14 100 47 112 74 Mean expression of 100 43 111 69 barrier markers (%) Inhibition relative 57% 38% to the control (%)

(88) Desmoglein 1 (DSG1) is a constitutive protein of corneodesmosomes which provides corneocyte cohesiveness within the corneal layer.

(89) Sciellin (SCL) is a precursor of the cornified envelope.

(90) PADI1 (peptidylarginine deiminase 1) and caspase 14 (CASP14) are two enzymes involved in the processing of filaggrin to obtain NMF.3. Conclusion

(91) Comparative analysis of the behaviour of the 1-year-old and the adult (19-year-old) reconstructed epidermises in the dry skin induction model shows a greater vulnerability and susceptibility of the infants' epidermises to dehydration stress induced by incubation in dry atmosphere.

(92) This greater vulnerability justifies the development of cosmetic products for dry skin specifically adapted to babies' skin.

Example 5: Evaluation of Products for Dry Skin

(93) The babies' dry skin model previously prepared, consisting in incubating 1-year-old infants' reconstructed epidermises in dry atmosphere, was used for a comparative evaluation of the biological efficacy of 4 cosmetic products for dry skin.

(94) 1. Materials and Methods

(95) Reconstructed epidermises were prepared, as described above, from keratinocytes of a 1-year-old donor.

(96) On day 11, the epidermises were treated by topical application of the test products in the amount of 5 mg/cm.sup.2 and then incubated for 6 h, 24 h or 48 h in a dry incubator (dry atmosphere condition: 37 C., 5% CO.sub.2 and <25% relative humidity), except for the control epidermises in a humid incubator (37 C., 5% CO.sub.2 and >99% relative humidity) and analysed as described above:

(97) After 6 h and 24 h of incubation, the gene expression of markers of aspecific and neurogenic inflammation, of hydration, of the barrier function and of stem cells (Table 1) was evaluated (n=2) by real-time quantitative PCR (qRT-PCR).

(98) After 48 h of incubation, the viability (MTT) and the morphology (histology, haematoxylin/eosin staining) of the epidermises were studied (n=2) and the production of NMF and ceramides was analysed (n=3).

(99) Test products=reference formulations as cited above: Product A Product B Product C Product D
2. Results

(100) a. Morphology and Viability of the Epidermises

(101) Just as during the preceding model development step, incubation in dry atmosphere did not significantly alter the morphology and the viability of the epidermises (Table 9).

(102) TABLE-US-00015 TABLE 9 Analysis of the viability of the epidermises Viability (MTT) Control epidermis 100% Dry incubator 87% control Product A 90% Product B 89% Product C 75% Product D 97%

(103) b. Analysis of Expression of Neurogenic and Aspecific Inflammation Marker Genes

(104) Products A, B and C inhibit the inflammation markers expressed under conditions of incubation in dry atmosphere (Table 10). These products thus offer protection against the skin reactivity which can be exacerbated under these conditions. Only product D has no inhibitory effect on these markers.

(105) TABLE-US-00016 TABLE 10 Level of gene expression of markers of neurogenic and aspecific inflammation (relative expression in % compared with the dry incubator control) Inhibition Mean relative expression to the dry of markers incubator PLA2G2F TAC1 TAC1R (%) control Dry incubator 100 100 100 100 control Product A 90.5 81.5 70 81 19% Product B 75.5 66 55 65 35% Product C 103.5 75.5 75 85 15% Product D 87.5 95.5 105.5 96 4%

(106) c. Analysis of Expression of Stem Cell Marker Genes

(107) Only product A fully restores (96% restoration) the expression level of stem cell markers inhibited by incubation in dry atmosphere with an efficacy superior to the other products tested (Table 11).

(108) Product A thus protects the cell capital in the dry skin model.

(109) TABLE-US-00017 TABLE 11 Expression level of stem cells marker genes (relative expression in % compared with the 1-year-old control) Mean expression of the stem cell marker pool % KRT15 KRT19 BIRC5 TP63 (%) restoration Control 100 100 100 100 100 Dry 25 74 45 47 48 incubator control Product A 97 130 78 90 98 96% Product B 80 68 32 62 61 61% Product C 86 131 21 70 77 77% Product D 86 74 19 71 63 63%

(110) d. Analysis of Expression of Barrier Marker Genes

(111) Under conditions of incubation in dry atmosphere, products A and B restored the expression level of barrier marker genes and show a restoration efficacy (140% and 113%, respectively) superior to the other formulas tested (Table 12).

(112) Therefore, product A, and to a lesser extent product B, protect the expression of barrier markers altered by incubation in dry atmosphere.

(113) TABLE-US-00018 TABLE 12 Expression level of barrier marker genes (relative expression in % compared with the 1-year-old control) Mean expression of barrier % LOR TGM1 CLDN1 DSG1 markers (%) restoration Control 100 100 100 100 100 Dry 88 93 93 68 85 incubator control Product A 103 109 111 103 106 140 Product B 97 109 110 91 102 113 Product C 78 91 92 114 94 60 Product D 72 87 86 101 87 13 Loricrin (LOR) is a late marker of epidermal differentiation involved in formation of the cornified envelope. Transglutaminase 1 (TGM1) is an enzyme responsible for cross-linking various proteins during production of the cornified envelope, ensuring the impermeability/solidity of the stratum corneum. Claudin 1 (CLDN1) is one of the constitutive proteins of tight junctions, involved in the skin barrier function, notably controlling water flow. Desmoglein 1 (DSG1) is a constitutive protein of corneodesmosomes which provides corneocyte cohesiveness within the corneal layer. Corneodesmosin is a constitutive protein of corneodesmosomes, junctions which form between corneocytes to maintain the structure of the stratum corneum and thus to take part in proper desquamation of the skin [10].

(114) e. Analysis of NMF and Ceramide Production

(115) Products A and B made it possible to restore the ceramide and NMF production inhibited in the dry skin model (Table 13) whereas the two other products tested (C and D) show lesser efficacy: practically no effect on ceramide production, a small increase in NMF production.

(116) TABLE-US-00019 TABLE 13 NMF and ceramide content in 1-year-old reconstructed epidermises incubated under normal conditions (control) in a dry incubator (dry atmosphere) NMF content Ceramide content (g/mg proteins) (AU/mg proteins) Control 16.6 1.8 179.5 Dry incubator 11.7 8.8 30% 138.25 23% control Product A 17.3 3.6 +48% 178.25 +24% Product B 18.3 8.1 +56% 183.25 +27% Product C 13.9 2 +19% 127.3 12% Product D 14.5 1.7 +24% 150.95 +5%