- Anti-wrinkle Octapeptide SNAP-8™
SNP-8™: Elongation of ARGIRELINE®
The anti wrinkle octapeptide SNAP-8™ is an elongation of the famous hexapeptide ARGIRELINE®. The study of the basic mechanism of anti-wrinkle activity led to the revolutionary hexapeptide which has taken the cosmetic world by storm. Those same studies have been applied to bring another addition to the Botulinum Toxin inspired family of peptides.
It has been clearly established that these conformational changes and the disturbance of the perfect packing of the lipid matrix can be significantly avoided by modulating muscle contraction.
Cause of Facial Wrinkling
One of the most striking signs of skin aging is increased wrinkling of the face. This can occur naturally over time and is identified by certain biochemical, historical and physical changes that are enhanced by environmental exposure. These are other secondary factors that can b cause characteristic folds, furrows and creases of the face. These include the constant pull of gravity, frequent and constant positional pressure of the skin of the face (e.g. during sleep) or repeated facial movements caused by the contraction of the muscles of facial expression. In molecular mechanism involved in face aging is directly related to changes in the conformation of the collagen triple helix, degradation of the elastin polypeptides and certain disorder in the packing of lipidic matrix of the skin.
Muscles are contacted when they receive neurotransmitter released from inside a vesicle. The SNARE (SNAp REcetor) complex is essential for this neurotransmitter release at the synapsis. It is temary complex formed by the proteins VAMP, Syntaxin and SNAP-25 (SyNatosomal Associated Protein). This complex is like cellular hook which captures vesicles and fuses them with the membrane for the release of neurotransmitter.
SNAP-8™ is a mimic of the N-terminal end of SNAP-25 which completes with SNAP-25 for a position in the SNARE Complex, thereby modulating its formation. If the SNARE complex is slightly destabilized, the vesicle can not release neurotransmitters efficiently and therefore muscle contraction is attenuated , preventing the formation of lines and wrinkles.
SNAP-8™ reduces the depth of wrinkles on the face caused by the contraction of muscles of facial expression, especially in the forehead and around eyes. SNAP-8™ is a safer, cheaper, and milder alternative to Botulinum Toxin, topically targeting the same wrinkle formation mechanism in a very different way.
In VIVO Testing
Inhibition of SNARE complex formation
To assay the efficacy of small peptides on the stability of the SNAR complex we developed an in vitro method that enabled us to follow the formation and thermal stability of the reconstituted SNARE protein complex. The rational of the method evaluates the antagonistic competitive efficacy of small peptides patterned after the SNAP-25 N terminal domain with the wild type protein on its capacity to assemble with syntaxin and synaptobrevin forming by the SNARE complex.
The results prove that short peptides from the N-terminal end of SNP-25 compete with the native protein and inhibit the formation of the SNARE complex by affecting its stability.
EGTA is metal chelator used as a negative control, because it captures Ca2+ which is essential for vesicle fusion and catecholamine release.
Anti-wrinkle Activity Units (AAUs)
One of the problem of working with peptides with similar effects but different potency is to compare their activities. Several catecholamine release tests at different concentrations of peptides were performed, in order to plot the dose response curves needed to calculate the IC50 values for the different peptides. We can therefore quantity and compare the exocytosis blocking activity, which is directly related to the anti wrinkle power. The IV50 is the 50% inhibitory concentration, in this case, the concentration of active that inhibits 50% of catecholamine secretion. The lower IC50, the smaller the amount needed to inhibit secretion 50%, and the more active the compound is. This facilitates the definition of an anti wrinkle activity unit .
AAU sample = [IC50] esup e/ [IC50] sample
ESUP-E is the most potent synthetic peptide in terms of catecholamine inhibition. It is a long peptide, not suitable for cosmetic application, but is used as reference to define AAUs.
In VIVO Testing
Anti wrinkle test on healthy volunteers
Skin topography analysis for measuring the effectiveness of an cream containing 10%of SNAP-8 solution were performed obtaining silicon imprints from around the eyes from 17 healthy women volunteers. Silicon imprints were obtained pre-test and after 28 days of twice a day applications. Analysis of imprints were performed by control laser scanning microscopy to assess the evolution of the skin surface before and after the treatment. Skin topography images from the three dimensional reconstruction of optical sections are shown. It can be observed that the depth of the wrinkle has significantly decreased after 28 days of treatment which confirms the validation of the biochemical mechanism hypothesis.
Skin topography images before (left) and after (right) a 28 days treatment with a cream containing 10% SNAP-8 solution.
Results are compared to a previous test (Oct, 2001) performed by the same company using the same technique, testing a cream with 10% ARGIRELINE® solution on 10 women volunteers. The maximum reduction value found for 10% SNAP-8™ solution was -63.18%.
An observed in vitro, the two extra amino acid from SNAP-25 sequence seems to moderately increases the anti-wrinkle activity.
The construction is that, both in vivo and in vitro testing suggests that SNAP-8™ is approximately 30% more active than its parent peptide ARGIRELINE®.