J. Cosmet. Sci., 62, 469–482 (September/October 2011) 469 Diguanoside tetraphosphate (Gp 4 G) is an epithelial cell and hair growth regulator DIVINOMAR SEVERINO, TELMA M.T. ZORN, GUSTAVO A. MICKE, ANA C. O. COSTA, JOSÉ ROBERTO M.C. SILVA, LEANDRO F. NOGUEIRA, ALICA J. KOWALTOWSKI, and MAURÍCIO S. BAPTISTA, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (D.S., A.J.K., M.S.B.), Instituto de Ciências Biomédicas, Universidade de São Paulo (T.M.T.Z., J.R.M.C.S.), Universidade Federal de Santa Catarina (G.A.M.), Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo (A.C.O.C.), and Universidade Estadual de Mato Grosso (L.F.N.), Brazil. Accepted for publication June 7, 2011. Synopsis Our goal was to study the effect of Gp4G on skin tissues and unravel its intracellular action mechanisms. The effects of Gp4G formulation, a liposomic solution of Artemia salina extract, on several epidermal, depmal, and hair follicle structures were quantifi ed. A 50% increase in hair length and a 30% increase in the number of papilla cells were explained by the changes in the telogen/anagen hair follicle phases. Increasing skin blood vessels and fi broblast activation modifi ed collagen arrangement in dermal tissues. Imunohistochemical stain- ing revealed expressive increases of versican (VER) deposition in the treated animals (68%). Hela and fi bro- blast cells were used as in vitro models. Gp4G enters both cell lines, with a hyperbolic saturation profi le inducing an increase in the viabilities of Hela and fi broblast cells. Intracellular ATP and other nucleotides were quantifi ed in Hela cells showing a 38% increase in intracellular ATP concentration and increases in the intracellular concentration of tri- , di- , and monophosphate nucleosides, changing the usual quasi-equilib- rium state of nucleotide concentrations. We propose that this change in nucleotide equilibrium affects several biochemical pathways and explains the cell and tissue activations observed experimentally. INTRODUCTION Hair growth and loss in mammalian species is controlled by a specifi c follicular cell cycle that includes periods of growth (anagen phase), regression (catagen phase), and rest (telogen phase) (1,2). In the earlier stages of the anagen phase, dermal papillae are restimulated to produce new hair shafts. Matrix bulb cells die by apoptosis in the catagen phase, and fol- licles remain in a resting state in the telogen phase until signaling factors activate them. Address all correspondence to Maurício S. Baptista at baptista@iq.usp.br.

JOURNAL OF COSMETIC SCIENCE 470 Understanding this cycle is highly important to control hair loss, and several authors have published comprehensive articles on this subject (3–6). Several drugs interfere with different steps of the follicle cycle, affecting hair growth, stimulating vascularization and cell proliferation, and providing energy resources for the follicles. However, there are few studies aiming to detect the effects of bioextracts that facilitate hair elongation (7). The indirect effects of antioxidants, which bring general benefi ts to skin health, have been shown to improve hair growth (7). Apoptosis has also been shown to allow a shift from anagen to catagen phases in the fol- licle cycle (2,3,8). Apoptosis can be initiated by the activation of death domain receptors, present in the intracellular domain of the TGF-β receptor. Antagonists of TGF-β cause delays in the outcome of the catagen phase and consequent extension of the anagen phase, which can elongate the life cycle of a hair shaft (3,8). Some plant extracts have also been shown to suppress TGF-β2 (9). Interesting initial results suggest that adenosine can pro- mote hair growth through the activation of specifi c receptors in the follicular bulb, in- creasing the release of fi broblast growth factor 7, which activates hair growth (10). Topical applications of minoxidil were widely used to induce hair growth, desite its col- lateral effects (11). Although its action mechanism has been thoroughly studied, it is not yet totally understood. Minoxidil is known to be a nitric oxide (NO) agonist promoting opening of K+channels and vasodilatation. Such actions may increase follicular irrigation, facilitating oxidative metabolism in the follicles (5,11,12). Hair follicles are highly active structures distinguished by intense cell proliferation and protein synthesis, required during the long period of hair growth. Adequate function of the follicle cycle uses large amounts of intracellular ATP and, consequently, effi cient ATP synthesis is required. Therefore, the bioenergetics of bulb cells, as well as the correct ho- meostasis of dermal tissues, is fundamental to allow cycles of hair growth and involution (4,5). ATP levels can be decreased by a relative depletion of oxygen and/or nutrients in the follicle cells, fi broblasts, and keratinocytes, causing hair loss. Diguanoside tetraphosphate (Gp4G) is a symmetrical bis-diphospho nucleoside (Scheme 1) that is found in large concentrations in the cysts of Artemia salina (CAS). Finamore and Warner have shown that after the contact of CAS with water, Gp4G is enzymatically hydrolyzed and increases intracellular ATP around 30 minutes after rehydration (12,13). Small rates of Gp4G hydrolysis provide a free energy fl ow and allow the encysted embryos to survive for several years of continuous harsh conditions, including continuous anoxia and hydrolytic stress (14). A similar molecule, which is widely found in mammalian cells, diadenosine tetraphosphate, has been shown to be a pleiotypic activator because its con- centration is highly correlated with tissue activity (15). The increase in intracellular ATP concentrations after Gp4G hydrolysis has been claimed to cause a stimulating effect that could be helpful in several cosmetic industry Scheme 1. Molecular of p1, p4 diguanoside 5′ tetraphosphate (Gp4G).