Active for accelerated and long-lasting tanning

The genetically constitutive skin colour and tan is determined by the skin level of the melanin biopolymer pigment. The constitutive pigmentation determines to a large extent the skin pigmentary response to ultraviolet (UV) radiation to burn or tan, which has been used to classify skin phototypes (types I–VI).1 UV-stimulation of skin pigmentation over the basal constitutive level, commonly called tanning, involves an increase in the melanocyte activity and a stimulation of melanin neosynthesis and melanocyte dendricity, a crucial morphological feature required for melanin transfer to keratinocytes.2 Depending on the UV exposure time, tanning proceeds in three steps: immediate pigment darkening (IPD), persistent pigment darkening (PPD) and delayed tanning (DT) response. However, the only one resulting from the stimulation of melanin synthesis is DT, which also involves an increase in the tyrosinase activity and in functional melanocytes, dendricity, synthesis and transfer, as well as altered packaging of melanosomes.1 Skin pigmentation is the most important natural photoprotective factor present in the skin, as melanin acts as a broadband UV absorbent and presents antioxidant and radical scavenging properties.1 Melanin synthesis takes place in melanosomes, which are contained in specialised cells, called melanocytes, placed in the basal layer of epidermis.2 Melanosomes development involves matrix organisation, followed by formation and deposition of melanin until melanosomes are fully melanised.3 Melanin-containing melanosomes migrate towards the extremities of the melanocyte dendrites where they are transferred to the surrounding keratinocytes, ensuring a uniform distribution of melanin pigments in the epidermis. UV radiation induces the expression of the proopiomelanocortin (POMC) and melanocortin 1 receptor (MC1R) genes, which encode the ?-melanocytestimulating hormone (?-MSH) and its receptor, respectively, which play a crucial role in pigmentation.4 MC1R is a seven transmembrane domain G-protein-coupled receptor that binds the agonists ?-MSH and adrenocorticotropic hormone (ACTH) that stimulate the synthesis of melanin.2 Once MC1R is activated, G??dissociates from G??and ??subunits and stimulates the intracellular messenger adenylyl cyclase (AC) which, in turn, catalyses the conversion of cytoplasmic adenosine triphosphatase (ATP) to cyclic adenosine- 3’,5’-monophosphate (cAMP).5 Inside the cell, increased intracellular cAMP acts as the second messenger binding to the two sites of the regulatory subunits of protein kinase A (PKA), allowing the catalytic subunits to be liberated and activated.2 PKA is a serine/threonine kinase inactive tetramer consisting of two regulatory subunits and two catalytic subunits.6 PKA translocates to the nucleus where it activates the expression of microphthalmiaassociated transcription factor (MITF).5 MITF is a basic helix-loop-helix melanocyte-specific transcription factor that regulates transcription of genes encoding tyrosinase and TYRP-1 (tyrosine-related protein) and DCT (dopachrome tautomerase, also called TYRP-2).2 Tyrosinase is a binuclear coppercontaining enzyme regarded as the key enzyme in melanogenesis being absolutely necessary for pigmentation. cAMP can influence the transcription of tyrosinase via MITF and its central role in control of tyrosinase expression has been widely demonstrated.7 TYRP-1 and DCT are melanogenesis related proteins which also respond in a cAMP-dependent manner via MITF. Tyrosinase mediates the first and rate-limiting step of melanin formation. Tyrosinase along with TYRP-1 and TYRP-2 are the enzymes responsible for the conversion of tyrosine in eumelanin or pheomelanin in the presence of a sulphydryl donor (glutathione or cysteine), which are the two major types of melanin pigments in mammals.2  The capacity of melanocytes to synthesise melanin varies enormously. Melanocytes in dark skins or with the capacity to tan well have a great capacity to synthesise melanin and transfer it onto the surrounding keratinocytes, while in fair skin their capacity is very limited.8 Each melanocyte produces both eumelanin (black or brown) and pheomelanin (red or yellow) but the ratio varies greatly and determines the hue of the skin. Dark skin mostly has eumelanin but also smaller amounts of pheomelanin, while fair skin presents small amounts of melanin in the epidermis with large quantities of pheomelanin and a lesser amount of eumelanin.8 The main used active ingredient in commercial self-tanning sunless formulations is dihydroxyacetone (DHA). DHA and other self-tanning agents used in combination with DHA such as erythrulose, glyceraldehyde or hydroxymethylglyoxal are reducing sugars which react with free amino groups of the stratum corneum and the epidermis through the Maillard reaction to give melanoidins, which are responsible for the brown colour, and other Advanced Glycation End products (AGEs). AGEs can result in stiffening, photoageing, oxidative stress, lipid peroxidation products and protein cross-linking. In order to avoid a pronounced photoageing process, sun exposure should be avoided after the application of self-tanning products containing reducing sugars.9 A new patented peptide has shown to possess both tanning accelerating and long-lasting properties via the skin melanogenesis pathway. Melatime (proposed INCI name: Palmitoyl Tripeptide- 30) has proved in vitro its hyperpigmenting activity by increasing cAMP levels that stimulate melanin synthesis. Unlike reducing sugars, from which resulting tan comes together with AGEs, the peptide increases one’s own melanin content, providing the skin with a natural tan that fits its constitutive colour. In addition, as melanin presents antioxidant, radical scavenging and UV scattering and absorbent properties, the fact of increased melanin content implies also that the skin receives an extra protection which can help to prevent photoageing signs. Melatime is also regarded as a tanning accelerator, as it proved in vivo to provide a perfect sun-kissed skin in less time. In consequence, UV-exposure times shorten and also the free radical effects derived from the radiation exposition. Furthermore, the peptide also prolonged the tan after two weeks since the last irradiation, showing it creates a long-lasting effect.

Materials and methods

cAMP levels determination
The in vitro test to determine cAMP levels is based on the competition between free cAMP and cAMP Tracer (cAMPacetylcholinesterase conjugate) for a limited number of cAMP-specific antibody binding sites. The product of the enzymatic reaction between the substrate and the cAMP Tracer has a distinct yellow colour which can be determined spectrophotometrically, giving the proportional amount of cAMP Tracer bound to the well. Absorbance was read at 405 nm and values were normalised in respect to the control. Cellular cAMP levels were analysed in a G361 human caucasian melanocyte cell line, in presence of vehicle, 10 ?m Melatime or 40 ?m forskolin. The vehicle was used as negative control and forskolin, which is known to increase cAMP levels and to cause a progressive and robust darkening associated with dose-dependent accumulation of melanin in epidermic cells, was used as positive control.