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Besançon, France

Guichard A.,Besancon University Hospital Center | Guichard A.,University of Franche Comte | Humbert P.,Besancon University Hospital Center | Humbert P.,University of Franche Comte | And 4 more authors.
International Journal of Pharmaceutics | Year: 2015

Topical-corticosteroids are mainly used for the treatment of inflammatory or hyperproliferative skin diseases. The in vivo assay to rank topical-corticosteroids potency, based on the skin blanching, is not adapted to compare their anti-proliferative efficacy. We have compared the antiproliferative effect of six topical-corticosteroids on a model of hyperproliferant keratinocytes (HaCaT). Betamethasone-dipropionate; clobetasol-propionate; betamethasone-valerate; desonide; hydrocortisone-butyrate and hydrocortisone-base, at different concentrations (10-8-10-4 M) have been compared. HaCaT proliferation has been evaluated by MTT-assay and the mechanism of the death was evaluated by annexin V/propidium iodide staining and cell cycle phases analysis. Topical corticosteroids reduced cell growth in a dose-dependent manner. At 10-4 M, betamethasone dipropionate was the most antiproliferative compound while hydrocortisone-butyrate was the less. Hydrocortisone-base which is usually considered as the less potent topical-corticosteroids showed a clear cytotoxic effect. Betamethasone-dipropionate and betamethasone-valerate induced more apoptosis than necrosis whereas the reverse has been observed for other topical-corticosteroids. All topical-corticosteroids, except clobetasol-propionate, arrested cell cycle mainly in G2-phase. Clobetasol-propionate arrested cell cycle in S-phase population. At 10-8 M, topical-corticosteroids induced HaCaT proliferation. In terms of antiproliferative effect at 10-4 M, we propose to rank topical corticosteroids as follow: betamethasone-dipropionate > desonide ≥ betamethasone-valerate = hydrocortisone-base = clobetasol-propionate > hydrocortisone-butyrate. This classification differs from the current ranking, based on the vasoconstrictive effect, but is more adapted for hyperproliferative disease treatment. © 2015 Elsevier B.V. All rights reserved.

Robin S.,SARL BIOEXIGENCE | Leveque N.,University Paris - Sud | Courderot-Masuyer C.,SARL BIOEXIGENCE | Humbert P.,University of Franche Comte
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2011

A simple, highly selective, sensitive and reproducible liquid chromatography-electrospray ionization mass spectrometry method has been developed for the direct and simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in microdialysis samples from human dermis. Chromatographic separation was carried out on an MODULO CART QS KROMASIL 5C18 (250 mm × 2 mm × 5. μm) analytical column at a flow rate of 0.25 ml/min. An isocratic mode was used and consisted of acidified water and acetonitrile (50/50, v/v). To improve the sensitivity, silver nitrate was added as post-column reagent. A trap mass spectrum was used equipped with an ESI interface. The limits of detection and quantification were respectively 0.12 and 0.4. ng/ml for GSH and 0.2 and 0.5. ng/ml for GSSG. Intra-day and inter-day accuracy and precision were determined and the variability was less than 6.2% (R.S.D.). © 2011 Elsevier B.V.

Robin S.,SARL BIOEXIGENCE | Courderot-Masuyer C.,SARL BIOEXIGENCE | Tauzin H.,SARL BIOEXIGENCE | Guillon S.,Valmont Group | And 5 more authors.
International Journal of Cosmetic Science | Year: 2012

Chronic sun exposure and especially UVA wavelengths are responsible for long-term clinical skin changes such as photoageing and photocancers. The objectives of the present study were to analyse the contractile activity of fibroblasts irradiated with several doses of UVA and to evaluate the preventive, protective and restoring effects of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. The forces generated by fibroblasts in tense collagen lattices were quantified using Glasbox device before and after UVA irradiation and the addition of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. The production of collagen was also evaluated before and after irradiation and with and without the presence of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. A dose of 3 J cm- of UVA showed more than 50% of mortality in fibroblast population after 48 h and significant decreases in contractile forces developed by irradiated fibroblasts and collagen I production. One percentage of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate protected fibroblasts from UVA irradiation and made it possible to restore their capacity to the same level as fibroblasts that were not irradiated. It also tended to restore the capacity to synthesize collagen I. These results show that the use of the new device Glasbox makes it possible to evaluate a possible preventive and repairing effect of a cosmetic functional active on photoageing. © 2012 The Authors. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Hhumbert P.,Besancon University Hospital Center | Hhumbert P.,University of Franche Comte | Fanian F.,Besancon University Hospital Center | Fanian F.,University of Franche Comte | And 13 more authors.
Clinical Interventions in Aging | Year: 2015

Background: Loss of mechanical tension appears to be the major factor underlying decreased collagen synthesis in aged skin. Numerous in vitro studies have shown the impact of mechanical forces on fibroblasts through mechanotransduction, which consists of the conversion of mechanical signals to biochemical responses. Such responses are characterized by the modulation of gene expression coding not only for extracellular matrix components (collagens, elastin, etc.) but also for degradation enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). A new device providing a mechanical stimulation of the cutaneous and subcutaneous tissue has been used in a simple, blinded, controlled, and randomized study. Materials and methods: Thirty subjects (aged between 35 years and 50 years), with clinical signs of skin sagging, were randomly assigned to have a treatment on hemiface. After a total of 24 sessions with Mécano-Stimulation™, biopsies were performed on the treated side and control area for in vitro analysis (dosage of hyaluronic acid, elastin, type I collagen, MMP9; equivalent dermis retraction; GlaSbox®; n=10) and electron microscopy (n=10). Furthermore, before and after the treatment, clinical evaluations and self-assessment questionnaire were done. Results: In vitro analysis showed increases in hyaluronic acid, elastin, type I collagen, and MMP9 content along with an improvement of the migratory capacity of the fibroblasts on the treated side. Electron microscopy evaluations showed a clear dermal remodeling in relation with the activation of fibroblast activity. A significant improvement of different clinical signs associated with skin aging and the satisfaction of the subjects were observed, correlated with an improvement of the sagging cheek. Conclusion: Mécano-Stimulation is a noninvasive and safe technique delivered by flaps microbeats at various frequencies, which can significantly improve the skin trophicity. Results observed with objective measurements, ie, in vitro assessments and electron microscopy, confirm the firming and restructuring effect clinically observed. © 2015 Humbert et al.

Vasquez N.P.,University Paris - Sud | Vasquez N.P.,SARL BIOEXIGENCE | Crosnier de bellaistre-Bonose M.,University Paris - Sud | Leveque N.,University Paris - Sud | And 9 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2015

The main objective of this work was to evaluate a comprehensive two-dimensional gas chromatographic (GCxGC) coupled to quadrupole mass spectrometry (qMS) method in the field of biomarker candidates' discovery. To this purpose we developed a GCxGC-qMS method suitable for the separation of organic acids and other classes of compounds with silylable polar hydrogen such as sugars, amino-acids, and vitamins. As compared to those obtained by a widely used 1D-GC method, the urinary chromatographic profiles performed by the proposed 2D-GC method exhibit higher resolution and sensitivity, leading to the detection of up to 92 additional compounds in some urine samples including some well-known biomarkers.In order to validate the proposed method we focused on three metabolites of interest with various functional groups and polarities including CH3-malonic acid (MMA: biomarker of methylmalonic acidemia), 3-hydroxy-3-methyl-glutaric acid (3-OHMGA: biomarker of 3-hydroxy-3-methylglutaric acidemia), and phenylpiruvic acid (PhPA: marker of phenylketonuria). While these three metabolites can be considered as representative of organic acids classically determined by 1D-GC, they cannot be representative of new detected metabolites. Thus, we also focused on quinolic acid (QUIN), taken as an example of biomarker not detected at basal levels with the classical 1D GC-qMS method. In order to obtain sufficient recoveries for all tested compounds, we developed a sample preparation protocol including a step of urea removal followed by two extraction steps using two solvents of different polarity and selectivity. Recoveries with the proposed method reached more than 80% for all targeted compounds and the linearity was satisfactory up to 50μmol/L. The CVs of the within-run and within-laboratory precisions were less than 8% for all tested compounds. The limits of quantification (LOQs) were 0.6μmol/L for MMA, 0.4μmol/L for 3-OHMGA, 0.7μmol/L for PhPA, and 1μmol/L for QUIN. The LOQs of these metabolites obtained by a classical GC-MS method under the same chromatographic conditions were 5μmol/L for MMA, 4μmol/L for 3-OHMGA, 6μmol/L for PhPA while QUIN was below the limit of detection. As compared to 1D-GC, these results highlight the enhanced detectability of urine metabolites by the 2D-GC technique. Our results also show that for each new detected compound it is necessary to develop and validate an appropriate sample preparation procedure. © 2015 Elsevier B.V.

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