LIPOLYTIC EFFECT OF SLIMMING LIPOSOMES 213 about 3 kg (Charles River, St. Aubin les Elbeuf, France) were sacrificed, and the main ear veins were removed quickly, cleaned of connective tissue, and cut into rings 3 mm in length. These preparations were suspended in a 20-ml organ bath containing Krebs solution of the following composition (in mM): NaC1, 118.0 KC1, 4.7 MgSO4, 1.2 CaC12, 2.5 KH2PO 4, 1.2 NaHCO3, 25.0, and glucose 11.0 (pH 7.4). It also contained 1 pM of prazosin, 1 pM of propranolol, and 1 pM of indomethacin to block o•- and [3-adrenergic receptor activation and prostanoid release, respectively. This solution was continuously aerated with a mixture of 95% 0 2 and 5% CO 2 and maintained at 37øC. The tissues were connected to isometric force transducers (UF1, Phymep, Paris, France) for tension recordings, stretched to a resting tension of 1 g, and allowed to equilibrate for 60 min. During this time, they were washed repeatedly and the resting tension was readjusted at 15-min intervals. For the evaluation of o•2-receptor antagonistic activity, the tissues were exposed to a submaximal concentration of clonidine (0.1 pM) to obtain a sustained contraction. Thereafter, they were exposed to cumulative increasing concentrations of SLC. Each concentration was added after the response to the previous one had stabilized, and the resulting change in the clonidine-induced contraction was measured. SLC was thereby investigated on two tissues and the results expressed as a percent of the clonidine- induced contraction (mean values). IN VIVO EXPERIMENTS Eighteen healthy human volunteers were included in this study. All the volunteers applied daily a 3% SLC formulation on one of their thighs their other thigh served as a control. Before and at the end of the study, twenty-eight days later, the volunteers' thigh circumferences were measured. STATISTICAL ANALYSIS Results were presented as means _+ standard error (SE). The level of significance was assessed using a one-way analysis of variance (ANOVA 1), followed by a Dunnett's test. RESULTS AND DISCUSSION The product we have developed consists of a liposome containing esculoside, Centella asiatica triterpenes, caffeine, and L-carnitine. The liposome form allows the enhancement of the penetration of active compounds through the skin (18). In order to assess the magnitude or the SLC lipolytic effect, we first conducted some in vitro experiments on primary normal human adipocytes in suspension. The capability of SLC to induce lipolysis in this model was evaluated by measuring NEFA liberation from adipocytes. As shown in Figure 1, SLC induced a dose-related increase in the NEFA content of the adipocyte incubation media. The SLC effect was detectable for a concen- tration as low as 0.1% (v/v) and reached a maximum at 1% (v/v). To decipher the intracellular signaling pathway underlying this lipolytic effect and because the main intracellular messenger regulating lipolysis is cAMP, we measured

214 JOURNAL OF COSMETIC SCIENCE •-- 25o0 ,• 20oo z lOOO Control SLC 0.1% SLC 1% SLC 2.5% SLC 5% Figure 1. Effect of SLC on the concentration of NEFA present in the human adipocyte incubation medium. ***p 0.001 vs control value. under the same experimental conditions the intracellular cAMP content of adipocytes. As shown in Figure 2, SLC increased the cAMP content of adipocytes in a dose- dependent manner. The SLC effect regarding this endpoint was detectable for a con- centration as low as 0.1% (v/v) and reached a maximum at 5% (v/v). It should be noted here that the rate of NEFA appearance increased in parallel with the increase in intracellular cAMP content. This result strongly suggests that these two phenomena are causally interrelated. Therefore, at concentrations over 1%, although SLC continued to induce higher intracellular cAMP levels, the rate of lipolysis did not further Control SLC 0.1% SLC 1% SLC 2.5% SLC 5% Figure 2. Effect of SLC on the intracellular cAMP concentration of human adipocytes. **p 0.01 and ***p 0.001 vs control value.