j. Cosmet. sci., 53, 209-218 (July/August 2002) An in vitro, ex Lit, o, and in L, iL, O demonstration of the lipolytic effect of slimming liposomes' An unexpected ot-adrenergic antagonism L. THOLON, G. NELIAT, C. CHESNE, D. SABOUREAU, E. PERRIER, and J.-E. BRANKA, Co/etica, 32 rue Saint Jean de Dieu, 69007 Lyon (L.T., E.P., J.-E.B.), Cerep, Le Bois /'Ev•que, 86660 Celles L'Evescau/t (G.N., J.-E.B.), Biopredic, 14-18 rue Jean Pecker, 35000 Rennes (C.C.), and Decs, 1 rue du Golf Parc' Innolin, 33 700 Mdrignac (D.S.), France. Accepted for publication March 15, 2002. Synopsis Most of the slimming products already developed for cosmetic applications did not result from strategies that integrate whole lipolysis-regulating mechanisms. We thus focused our attention on a more complete integration of these mechanisms and we developed slimming liposomes (SLC) containing two micro- circulation activators, i.e., esculoside and Centella asiatica extracts, one phosphodiesterase inhibitor, i.e., caffeine, and one fatty acid-lB oxidation activator, i.e., L-carnitine. The validity of our approach was assessed through (a) in vitro tests demonstrating that SLC induced a dramatic increase in the cyclic adenosine monophosphate (cAMP) content in human adipocytes, with a subsequent rise in the nonesterified fatty acids (NEFA) content of human adipocyte incubation medium, and (b) in vivo studies showing that SLC could provide an actual potent slimming effect on human volunteers. Moreover, we give here, through binding experiments, the unambiguous demonstration that SLC is able to antagonize the %-adrenergic receptor that is known to reduce intracellular AMPc content and, subse- quently, to down-regulate lipolysis. This %-adrenergic antagonism has never been reported for any com- ponent of SLC, and this work is the first demonstration of the o•2-adrenergic antagonism of such a combination of active liposome compounds. INTRODUCTION In order to develop potent slimming products we have to (a) enhance the external skin grain (smoothness) and (b) produce a lipolytic effect on human adipocytes acting through main mechanisms regulating lipolysis. Address all correspondence to G. Neliat. 209

210 JOURNAL OF COSMETIC SCIENCE To reach the first goal, i.e., enhance skin smoothness, we can resort to microcirculation activators, which present some draining properties. Esculin, for example, which presents anti-inflammatory properties (1), is able to stimulate venous resistance (2,3) and to enhance capillary resistance (1). Esculin is also active in quenching free radicals (4). It can therefore protect compounds especially prone to oxidation from this phenomenon, notably epinephrine, a •3-adrenergic receptor agonist that is known to play an impor- tant role in lipolysis regulation. Ce,tell• •si•tic• extracts are potent veinotonics as well. Their activities are mediated via effects on metabolic pathways implicated in vascular wall connective tissue homeostasis (5). Such extracts have also been found to stimulate the synthesis of collagen, fibronectin, and proteoglycans by normal dermal human fibroblasts in tissue culture (6). This double activity makes these extracts ideal for (a) stimulating the rate of capillary blood flow and (b) improving the texture of the skin. This regeneration of the cutaneous extracellular matrix implies enhanced elasticity and subsequent reduction of the "orange peel" aspect. For the second part of our approach one needs to well understand lipolysis regulation. Two enzymes are involved in this process: a hormone-sensible lipase (HSL) and a monoglyceride lipase (MGL) these two enzymes catalyze successive hydrolysis reactions leading to the breakdown of lipids stored in adipose tissue (7,8). The final step of this process, which results in the release of free monoglycerides and glycerol, is rapid and occurs spontaneously, but the initial step is dependent on the status of HSL, which exists in two different forms, where only one is active. An understanding of intracellular mechanisms controlling the interconversion of this enzyme between active and inactive forms is essential to developing active fat-reducing products. In other words, HSL represents the key enzyme in the control of lipolysis. The HSL-modulating mechanisms are now well characterized. This enzyme is activated by a phosphorylation catalyzed by protein kinase A (PKA) (9). Phosphatase enzymes can subsequently inactivate the HSL by removing the phosphate group. The regulation of HSL activity can be viewed as the control of the balance between the amounts of active phosphorylated and inactive dephosphorylated enzymes present in the cell. Certain compounds have the capacity to modulate the activity of PKA and thereby modify this balance. Such compounds act by altering the intracellular concentration of the molecule that controls PKA activity, i.e., the cyclic adenosine monophosphate (cAMP) (10). There are two ways of increasing intracellular concentration of cAMP: (a) by increasing the activity of the enzyme responsible for its production, i.e., adenylate cyclase (AC), or (b) by inhibiting the enzyme responsible for its degradation, i.e., phosphodiestarase (PDE) (11). Any factor that tends to increase the activity of AC will lead to the activation of PKA and, thereby, of HSL. Similarly, any factor that inhibits PDE will have the same ultimate effect. Caffeine, for instance, is a PDE inhibitor that can diffuse across cell membranes and directly interact with its target (12). A third efficient way coexists with the two preceding ones: in order to increase lipolysis it is possible to act on the intracellular transport of free fatty acids derived from the breakdown of triglyceride. As enzyme-catalyzed reactions inducing triglyceride break- down are reversible, the balance between triglyceride degradation and/or triglyceride synthesis depends on the intracellular free fatty acid concentration. In the presence of a high level of free fatty acids, the balance will be shifted to favor triglyceride synthesis. In these conditions, L-carnitine, which stimulates free fatty acid transfer to mitochondria