LIPOLYTIC EFFECT OF SLIMMING LIPOSOMES 211 (13), is able to reduce the intracytosolic free fatty acid concentrations favoring the breakdown rather than the synthesis of the triglycerides. As a consequence, L-carnitine acts as a major stimulator of the lipolysis. The rationale to evaluate the lipolytic effect of SLC through in vitro and ex vivo models to test its slimming activity in human subjects is provided by (a) the demonstration by Deguercy et al. in 1995 (14) that human adipocytes in suspension represent a good predictive model to evaluate the in vivo activity of slimming products and (b) the relationship existing between the adrenergic control of lipolysis and the weight loss observed in humans in in vivo studies (15). We have therefore demonstrated in a first series of experiments that SLC can act through the main lipolysis regulating mechanisms to show its actual potent slimming effect in human subjects. Moreover, and in an interesting way, we demonstrate for the first time, the o•2-adrenergic antagonism of such a combination of encapsulated active compounds. MATERIALS AND METHODS DRUGS, CHEMICALS, AND REAGENTS All the culture media came from GIBCO-BRL (France). The [3H]RX821002 (specific activity 67.0 Ci/mmol) was purchased from Amersham Pharmacia Biotech Europe (Saclay, France). The (-)epinephrine bitartrate, clonidine hydrochloride, prazosin hy- drochloride, propanolol hydrochloride, and indomethacin were obtained from Sigma (St. Quentin Fallavier, France). Phospholipon 90 G came from Nattermann (Cologne, Ger- many). Centella asiatica came from Indena. L-carnitine was purchased from Quimdis S.A. (Levallois-Perret, France). Esculoside was purchased from Zileco (Evry, France). Pheno- nip came from Clariant (Puteaux, France). Butylene glycol came from Lambert-Riviere (Pierre-Benite, France). Carbopol was purchased from Gattefosse (Genevilliers, France). All other reagents came from Aldrich (St. Quentin Fallavier, France), Carlo Erba (Val de Reuil, France), Merck (Nogent sur Marne, France), or Sigma. SLC PREPARATION To prepare one kilogram of SLC, 2 g of Centella asiatica extract was dissolved in 150 g of butylene glycol at 80øC. Twenty grams of caffeine and 113 ml of deionized water were then added to the mixture under agitation until complete dissolution. One hundred grams of L-carnitine and 2 g of esculoside were then solubilized in the blend before addition of 30 g of phospholipon 90 G. When phospholipon 90 G was completely dissolved, the mixture was left to cool overnight under agitation. Added to the mixture at room temperature were 125.8 ml of deionized water, 7.5 g of a phenonip/butylene glycol mixture (10-2.5), 2 g of preservatives, and 250 g of a collagen/chondroYtine sulfate solution. After homogenization, liposomes were formed by strong agitation (10 min). Finally, 166.7 g of a 3% carbopol solution was added, and the pH of the mixture was adjusted to 5.7 with citric acid.

212 JOURNAL OF COSMETIC SCIENCE ISOLATION OF HUMAN ADIPOCYTES Adipose tissue samples from abdominal fat deposits were obtained after plastic surgery. Tissue samples were rinsed in physiological medium to remove adhering blood. Visible fibrous material and blood vessels were carefully dissected. The remaining adipose tissue was cut into small fragments with a scalpel and then incubated in the dissociation medium [MEM medium without phenol red added, with 50 IU/ml of penicillin, 50 pg/ml of streptomycin, and 0.1% (w/v) collagenase], at 37øC under slow shaking for 60 min. The ratio of adipose tissue mass to digestion solution was approximately 4 g for 10 mi. The dispersed tissue was then filtered onto a 250-pm nylon filter to discard non- digested pieces. The suspension was allowed to stand for 10 rain at room temperature to allow adipocytes to float. The medium under the cells was removed and replaced twice by the incubation medium [MEM medium without phenol red added, with 50 IU/ml of penicillin, 50 pg/ml of streptomycin, and 0.5% (w/v) lipid-free bovine serum albu- min]. The integrity of the adipocytes was checked by microscopic observation. cAMP AND NEFA MEASUREMENTS Adipocyte suspensions were incubated 2 hr at 37øC in a water bath in the absence (control) or in the presence of increasing concentrations of SLC. At the end of the incubation period, cell lysates were obtained by sonication and the intra-adipocyte cAMP contents were quantified using a sensitive and specific enzyme immunoassay (EIA) (Amersham, Saclay, France). The NEFA were measured in the incubation medium using a colorimetric enzymatic test (Wako Chemicals, Japan). BINDING EXPERIMENTS Human ot2-adrenoreceptor binding assay. Competition binding experiments were performed as described by Bylund et al. (16), with slight modifications. Briefly, membranes from HT-29 human adrenocarcinoma cells (40 lag protein) were incubated for 20 min at 22øC with 0.7 nM of [3H]RX821002 in the presence of increasing concentrations of SLC in 250 lal of assay buffer containing 1 mM of EDTA, 2 mM of MgC12, and 50 mM of Tris-HC1 (pH 7.4). The reaction was terminated by rapid filtration under vacuum through glass fiber filters (Filtermat A, Wallac), using a 48-sample cell harvester (Much IV, Tomtec). The filters were cooled with 50 mM of ice-cold Tris-HC1 (pH 7.4), dried, and counted for radioactivity in a scintillation counter (1204 Betaplate, Wallac), using a solid scintillant (MeltiLex B/HS, Wallac). Non-specific binding was determined in the presence of 100 laM of (-)epinephrine. The specific binding was defined as the difference between total and non-specific binding. Assays were performed in duplicate and the results expressed as a percent of the control specific binding (mean values). IC50 values and Hill slopes (nil) were calculated by computer-assisted nonlinear regression analysis of the competition curves. ISOLATED ORGAN EXPERIMENTS--o•2-ADRENORECEPTOR ANTAGONISM In vitro ot2-adrenoreceptor bioassay. The bioassay was performed using the rabbit isolated ear vein as described by Duly et al. (17). Male New Zealand white rabbits weighing