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Meyer, Imkea, Marco Massironib, Gabriele Vielhaberc, Paolo Pertiled, Michele Massironid
a Symrise GmbH & Co. KG, Mühlenfeldstraße 1, 37603 Holzminden, Germany b SCoutech/Cutech srl, via San Marco 9M, 35129 Padova, Italy c Symrise SA, rue Mozart 15, 92110 Clichy, France d Cutech srl, via San Marco 9M, 35129 Padova, Italy Corresponding author: Marco Massironi, Phone: +39 049 762 8532, E-mail: INTRODUCTION
The demand for innovative in vitro screening tools for development process. Models targeting the epidermis and the Therefore we developed an ex vivo excised porcine full skin cosmetic actives is increasing. The possibility of providing dermis are available on the market. However, there is an model that, by including the subcutaneous tissue, is an preclinical information in connection to both the bioavailability unmet need for a model targeting the subcutaneous tissue, appropriate model to gain information on the lipolytic effect of and the long-term effect of an active ingredient is of pivotal which is required for the proof of efficacy of anti-cellulite an active ingredient and to assess the modulation of adipo- importance in order to save money and speed up the cyte size by lipolytic as well as adipogenic compounds [1]. MATERIALS AND METHODS
After 24 h incubation the glycerol in the surrounding medium Adipocyte size was determined by image analysis software. was quantified using Free Glycerol Reagent (Sigma, The histological sections have been digitally recorded and The pig skin organ culture model (PSOCM) was derived from converted into a binary format (Fig. 1). A check of the binary slaughtered animals dedicated to meat production [2]. Skin against the original images was performed, before the cross- pieces of 7 mm diameter and a minimum layer (3 mm) of sectional area of each adipocyte was calculated and subcutis were excised from the dorsal part of a young pig. Hydrodispersion gel with or without anti-cellulite active (i.e. expressed as pixels [3]. Values less than 300 pixels were For each treatment four skin specimens have been used for BIO1617) was applied topically to the pig skin model. The assumed not to be adipocytes and excluded from analysis. By the lipolysis assay and six skin specimens for the adipocyte application has been renewed daily. After 10 days incubation allowing the measurement of a significant number of the skin models have been fixed in formalin, embedded in adipocytes (i.e. 60-80) in a relatively small skin biopsy (i.e. 7 paraffin and cut with the microtome followed by staining of the mm diameter) the model permits to obtain reliable, histological sections with hematoxylin and eosin. Caffeine (Acros): Anti-Cellulite active with lipolytic activity; BIO1617 (Symrise): Anti-cellulite active with triple activity, stimulator of lipolysis, inhibitor of adipogenesis as well as lipogenesis; BIO1750 (Symrise): Anti-cellulite active without lipolytic activity, but inhibitor of adipogenesis as well as representations. A: Hematoxylin and eosin-stained sections of subcuta- Hydrodispersion gel with or without anti-cellulite active (i.e. generated binary images of A. C: Computer analysis of image B. BIO1617) was applied topically to the pig skin model. A formulation containing caffeine served as a positive control. for the lipolytic actives caffeine and BIO1617. On PSOCM the activity was applied in different concentrations to evaluate the adipocyte size was reduced after 10 days by 35% and 36% long-term effect of the active dose-dependently. The On PSOCM the lipolysis was stimulated by 21% and 15% relative to placebo for 0.1% BIO1617 and 0.1% caffeine, adipocyte size was slightly reduced by 6% and 9% relative to relative to placebo by formulations containing 0.1% BIO1617 respectively (Figure 2). Both actives showed as a long-term placebo by 0.1% BIO1750 and 0.2% BIO1750, respectively, and 0.02% caffeine, respectively (data not shown). effect a shrinkage of adipocytes inter alia due to their lipolytic In the next step the reduction of adipocyte size was evaluated Subsequent the anti-cellulite active BIO1750 without lipolytic 0.1% BIO1617
0.2% BIO1750
0.1% BIO1617
average area size (pixel)
* p<0,05, ** p<0,01 calculated versus vehicle Fig. 2: Measurement of adipocytes size in ex vivo porcine skin model at day 10 (n=4) (A) applied as DMSO Fig. 3: Alterations in adipocyte size by anti-cellulite active BIO1750 after 6 days applied in O/W formulation solutions, (B) applied as formulations; computer image analysis after hematoxylin and eosin staining. relative to placebo (n=6); A: Comparison of different concentrations of BIO1750; B: Comparison of placebo with 0.2% BIO1750. DISCUSSION
Targeting the subcutaneous adipose tissue is a challenging The presented ex vivo model is the ideal tool to study the
cosmetic active. The culture conditions enable a long-term approach as topically applied actives have to penetrate deep effect of topically applied finished formulations targeting
cultivation and thus an efficacy testing up to 10 days. [5] into the skin. The bioavailability of actives is the major cellulite.
uncertainty for the transfer of in vitro to in vivo anti-cellulite Read-out parameters are the short-term effect of glycerol It allows to evaluate the effect of ingredients including
release as well as the long-term effect of adipocyte size optimization of formulations regarding bioavailability as
reduction. Thus, the model is not limited to lipolytic cosmetic well as adaptation of effective concentrations in a
An ex vivo porcine skin model was developed that is suited actives like caffeine. Cosmetic ingredients with adipogenic preclinical
for systemic as well as topical application of anti-cellulite and/or lipogenic efficacy result also in a reduction of requirements.
actives, including finished formulations containing the adipocyte size when appropriately formulated. REFERENCES
[2] Massironi, M. et al., SOFW, 135 (2009) 5, 15-20 [4] Corino, C. et al., J Nutr, 135 (2005) 6, 1444-1450 [1] Novakofski, J., J Anim Sci, 82 (2004) 3, 905-915 [3] Chen, H.C. et al., J Lipid Res, 43 (2002) 6, 986-989


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