]. Cosmet. Sci.J 58, 157-171 (March/April 2007) Preparation and stability of cosmetic formulations with an anti-aging peptide M.A. RUIZ, B. CLARES, M. E. MORALES, S. CAZALLA, and V. GALLARDO, Departamento de Farmacia y Tecnologfa FarmaceuticaJ Facultad de FarmaciaJ Universidad de Granada! 10871 Granada, Spain. Accepted for publication November 21, 2006. Synopsis W tinkling of the skin is the most obvious sign of deterioration of the human body with age. This process involves a number of genetic, constitutional, hormonal, nutritional, and environmental factors, in addition to the influence of frequently repeated facial movements during laughing, smoking, etc. This article reviews the physiological basis and mechanism of action of the active cosmetic ingredient acetyl hexapeptide-8 (Argireline®). We prepared two formulations: an emulsion with an external aqueous phase for normal to dry skin, and a gel for oily skin. Laboratory analyses, rheology tests and in vitro release assays were used to evaluate the stability of these formulations for cosmetic treatment. INTRODUCTION The search for new compounds to prevent or attenuate skin aging and enhance self­ image (1) is a priority of current research on active cosmetics. Given the social impli­ cations surrounding physical appearance, we have undertaken work to investigate the treatment of facial expression wrinkles. Favorable results with botulin toxin infiltration led to the development of a new active principle with effects similar to the botox effect, named Argireline®, as an alternative to botulin um toxin. Unlike other creams developed to treat aging wrinkles, the formulas tested in this study are intended to treat expression wrinkles. Substances with a botox-like effect act upon the same phenomenon as botulin toxin, but via a different mechanism of action. To understand the mechanism of action of the formulas we tested, a brief review of how expression wrinkles are formed may be helpful. Expression wrinkles (2) form as a result of repeated muscle contraction caused by dermal atrophy and the appearance of hypodermal fibrosis (3). Facial movements cause cells of the dermis to contract and relax, and subject fibroblasts anchored by the network of Address all correspondence to M. A. Ruiz Martinez. 157

158 JOURNAL OF COSMETIC SCIENCE collagen and elastin fibers to similar stresses. As a result, the skin becomes contracted into a permanent expression wrinkle, where the extracellular matrix of collagen and elastin has been found to break down (4). Several processes are vulnerable to alteration from skin wrinkling in cosmetic terms (5 ): • Neuronal exocytosis involves neurotransmitter release from synaptic vesicles into the synaptic space. Synaptic vesicles bearing neurotransmitters are taken up by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and fused with the cell membrane, releasing neurotransmitters in the process. The receptor complex consists of three proteins: synaptobrevin (VAMP), syntaxin, and synaptosomal-associated protein (SNAP-25) (6). • Contraction and relaxation of fibroblasts, the cells that produce collagen and elastin and are responsible for maintaining the extracellular matrix, are transmitted to the connective tissue, where these forces successively stretch and relax the skin. Specifically, type A botulin toxin produced by Clostridium botulinum acts by irreversibly destroying SNAP-25 protein in the SNARE complex, thus preventing the release of acetylcholine and paralyzing the involved muscle (7). Between 15 and 20 days after infiltration, new nerve endings are formed, and these endings become active within two or three months. After three to six months, nerve signals to the muscle are completely restored (8). A novel aspect of Argireline® is its ability to act via topical application, which offers multiple advantages in comparison to formulations based on botulin toxin. The main advantage of Argireline® lies in its lower toxicity per unit weight. One gram of botulin toxin is enough to kill one million persons, whereas Argireline® is about 4000 times less potent, and thus constitutes a safer alternative for treating wrinkles (9). With the latter, injections to the face, which are potentially uncomfortable or painful, are unnecessary. Moreover, Argireline® can be used as an interim treatment between botulin toxin injections, since it prolongs the effects of Botox and reduces the frequency of microin­ jections. The synthetic peptide is cheaper, and is indicated for persons who have devel­ oped immunity to botulin toxin after prolonged use. Argireline® (acetyl hexapeptide-8), the active principle in the formulations studied here, is a hexapeptide formed from a chain of six amino acids linked via a synthetic process. This peptide has two main actions. One is muscle relaxation by inhibiting the SNARE complex, but unlike botulin toxin, Argireline® does not irreversibly destroy the SNAP- 25 protein, but modifies its conformation and competes with it for sites in the SNARE complex. The hexapeptide, an analog of the N-terminal end of the SNAP-25 protein, does not completely destroy the SNARE complex but only destabilizes it slightly, such that the synaptic vesicles cannot bind and release acetylcholine efficiently (10). As a result, a degree of neurotransmission is preserved in equilibrium with muscle relaxation, muscle contraction is attenuated, and wrinkle formation is prevented (11). Argireline® is also able to relax fibroblasts that relax the collagen and elastin matrix, through a mechanism involving calcium ion uptake. The main objective of this study was to develop a formulation that ensured transfor­ mation of the active principle (acetyl hexapeptide-8) into a cosmetically active product. We therefore studied stability, defined as the ability of the formulation to maintain its initial characteristics. The parameters we measured as relevant to structural changes that can occur in cosmetic formulations were changes in organoleptic characteristics (a fun-