SNAP-8 is an anti-wrinkle octapeptide which is the elongated version of the famous hexapeptide Argireline. SNAP-8 is the abbreviation of its full name Synaptosomoal-associated protein 25. As a octapeptide, this peptide is made up of a polypeptide chain that links 8 amino acids. It is also referred to as Acetyl Glutamyl Heptapeptide-1 or Acetyl Octapeptide and has a molecular weight of 1075.16 Daltons and its molecular formula is C14H70N16O16S1.SNAP-8 mimics the N-terminal end of SNAP-25 and as such it competes with SNAP-25 for a position within the SNARE complex resulting in modulation of its (SNARE complex) formation process. SNAP-25 belongs to the t-SNARE family of about 60 members and it accounts for the specificity of membrane fusion and particularly facilitates fusion by creating a tight complex that links the synaptic vesicle and plasma membranes together. Slight destabilization of the SNARE complex inhibits the efficient release of neurotransmitters by the vesicle and this brings about attenuation of muscle contraction, preventing the formation of wrinkles and lines.
Similarly to Argireline, SNAP-8 inhibits the formation of SNARE complex and it also inhibits the release of catecholamine, this brings about the decrease in the extent of existing facial wrinkles (it reduces the depth of facial wrinkles that are caused by the contraction of the facial expression muscles, particularly those in the forehead and in the eye region) and effectively prevents their development when used on regular basis. SNAP-8 operates as an inhibitor of muscle contractions and as such it allows the muscles to achieve a highly consistent means of achieving homeostasis. It also boosts the production and release of collagen from the cells, this strengthens the cells, making them to alleviate fine lines and furrows.
Based on scientific studies conducted on animals, researchers established that SNAP-8 works by enhancing the regulatory properties of the N-terminal end of SNAP-25. This enhancement enables the SNARE complex to remain in a stable state; as a result, the SNARE complex allows SNAP-25 to retain stability. Additionally, researchers have determined that SNAP-8 inhibits the release of catecholamine; a general class of neurotransmitters which are derivatives of the amino acid tyrosine containing amine and catechol groups. This inhibition also plays a role in the stabilization process in respect to the regulation of muscular contraction via the activation of the Ca2+ ion.
Scientific experiments that have been performed on animal test subjects have shown that SNAP-8 initiates activities that are similar to those initiated by acetyl hexapeptide-3 (a peptide derivative of a fragment of SNAP-25).These two peptides vary in structure whereby SNAP-8 is a octapeptide and SNAP-25 is a hexipeptide. Based on results obtained from animal based studies, researchers have established that the stabilizing function of SNAP-8 tends to offer more efficacy compared to SNAP-25.
Researchers have performed a number of studies using animal subjects; as a result, they established that the key role of SNAP-8 is inhibiting muscle contraction. With its inhibition properties, SNAP-8 ensures the muscles achieve and maintain consistent levels of homeostasis. Researchers noted that this effect was achieved as a result of its influence in the release of the neurotransmitters that stimulate homeostasis as it makes their release more efficiently.
Researchers have performed studies seeking to determine the safety profile of SNAP-8, so far, they have not observed any significant negative side effects following the administration of SNAP-8 to animal test subjects. In experiments performed using laboratory rats, researchers did not observe any acute oral toxicity as a result of treatment with the peptide in normal dosages. In a number of other animal based studies, researchers established that the peptide does not support citotoxicity or genotoxicity in vitro studies. Additionally, researchers have also observed that SNAP-8 does not bring about any side effects related to ocular-based irritation. Majority of these tests performed to determine the safety profile of SNAP-8 were all conducted in vitro and in vivo.
Researchers have also carried out clinical trials to determine the safety or toxicological profile of SNAP-8 for cosmetic uses. The clinical trials below were only in vitro and they were carried out on human volunteers in Italy. The researchers used solutions of SNAP-8 Powder which were at specific concentrations that were pre-determined by the researchers. The study results for citotoxicity tests showed no evidence of citotoxicity on human dermal fibroblasts as a result of SNAP-8. The researchers did not observe any signs of citotoxicity on human epidermal keratinocytes at the concentrations examined. In Ames test (Genotoxicity test), the researchers did not observe any genotoxicity under the examined conditions.SNAP-8 did also determined that the compound is not irritating to the eyes based on ocular irritation (NRU-Neutral Red Uptake test). Additionally, it did not show phototoxicity when 3T3 NRU Phototoxicity Test was performed. This results show that SNAP-8 is safe for use based on its remarkable toxicological profile.
In vivo tests were also performed on animal tests subjects. In acute oral toxicity test, the researchers concluded that DL50>2500 mg/Kg body weight in laboratory rats, therefore, SNAP-8 did not show any signs of acute oral toxicity at any of the doses tested. Acute oral toxicity tests must be performed on any new chemical entity as stipulated by the Dangerous Substances Directive 67/548/EC. In vivo tests were also performed to determine skin sensitization (Hpeoallergenicity). The researchers performed the Human Repeated Insult Patch Test (HRIPT) on 50 human volunteers between the ages of 18 to 70. They observed that 0.05% solution of SNAP-8 did not bring about sensitization in any of the study subjects. As such, it can be classified as Low Sensitization.
Researchers have also performed in vitro tests to evaluate the efficacy of SNAP-8 in inhibiting the formation of SNARE complex. The researchers carried out an in vitro test to monitor the formation and thermal stability of the reconstituted SNARE protein complex so as to assess the degree by which small peptides influence the stability of the SNARE complex. The in vitro method was based on evaluation of the antagonistic competitive efficacy of the small peptides sampled after the SNAP-25 N-terminal domain together with the wild type protein on its ability to come together with the synaptobrevin and syntaxin to form the SNARE complex. The study results showed that short peptides obtained from the N-terminal end of the SNAP-25 tend to compete with the local protein and inhibit the creation of the SNARE complex by means of affecting its stability. The study results were as follows in respect to the formation of the SNARE complex, control -about 95%, heat -about 5%, argireline- about 50%, DD02116 -about 35%, and SNAP-8 -about 30%.
Researchers also evaluated the modulation of catecholamine release in chromaffin cells. They determined how the release of catecholamines was inhibited by monitoring the neurotransmitters Noradrenaline and Adrenaline. They incubated chromaffin cells using tritiated adrenaline/noradrenaline and SNAP-8. They then used liquid scintillation counting to establish the total cell content and the release of catecholamines. They observed a significant modulation of the two neurotransmitters at µM concentrations of the octapeptide SNAP-8, and this was a clear indication of its potent anti-wrinkle property.
In another study, researchers compared the Antiwrinkle Activity Units (AAUs) of four synthetic peptides in relation to their catecholamine inhibition.they performed a number of catecholamine release tests at various concentrations of peptide so as to plot the dose-response curves required to calculate the IC50 values for each peptide. They then quantified and contrasted the exocytosis-blocking property, which has a direct correlation to the antiwrinkle powder. The IC50 is the concentration of active which inhibits 50% catecholamine secretion (i.e. it is the 50% inhibitory concentration). Lower IC50 measures mean that smaller amounts of the peptide are required to inhibit secretion 50%, and that the compound is more active. Based on the study results; Argireline IC50 was 110µM and AAC was 0.0028; SNAP-8 IC50 was 55µM and AAC was 0.0056; ESUP E IC50 was 0.310 µM and AAC was 1; BoNT A IC50 was ~0.0260 µM and AAC was 12. Despite ESUP E being regarded as the most potent of all synthetic peptides in terms of catecholamine inhibition, it is not suitable for cosmetic application being a long peptide. It is therefore used as a reference in defining AAUs.
Researchers also evaluated the modulation of glutamate release in a neuron cell culture. Depolarized neuron cells inhibition of glutamate release is an accepted cell assay for determining the potential activity of substances/compounds on the inhibition of neuronal exocytosis. Glutamate is released when hippocampal cultures depolarization is stimulated by K+ in the presence of extracellular Ca2+ leads to the release of glutamate. Glutamate happens to be the most excitatory neurotransmitter in the nervous system. The researchers incubated a primary cell culture of neurons in tritiated glutamate for 3 hours so as to load them with radiolabelled glutamate. They then rinsed off the excess of glutamate and incubated the cell culture with the test compounds for 1 hour at 37ºC. The researchers observed that the release of radiolabelled glutamate was initiated by depolarization induced by buffered 75mM KCI/2mM CaCl2 in a physiologic buffer for a period of 10 minutes and a temperature of 37ºC. in the study, untreated neuron cultures were the negative controls while neuron cultures treated with Botulinum Toxin A (BoNT A) were the positive controls. The researchers retrieved the culture media and used a scintillation counter to measure the quantity of radiolabelled glutamate. They obtained values that were the average of the 6 determinations. The six determinations included control, SNAP-8 (0.75mM), Leuphasyl (0.75mM), SNAP-8 +Leuphasyl (both at 0.75mM each), SNAP-8 (1.5mM),Leuphasyl (1.5mM), and BoNTA (50nM).By measuring the release of glutamate by the neurons, researchers were able to contrast the in vitro activities of SNAP-8 and Leuphasyl which are both anti-wrinkle expression peptides. Combined-addition of SNAP-8 and Leuphasyl indicated a higher inhibitory potential of glutamate release compare to the inhibitory potential observed when each single product was added. The researchers concluded that the two peptides had a synergistic effect in vitro, they had independent mechanisms of action and their effects can be added.
In vivo anti-wrinkle test was performed in healthy volunteers. The researchers carried out skin topography analysis to measure the effectiveness of a cream with 10% SNAP-8 Solution whereby silicon imprints were obtained from the region surrounding the eyes from 17 healthy women subjects. The silicon imprints were carried out before the test began and after 28 days use of the cream which was to be applied twice a day over the entire study period. The collected silicon imprints were analysed using the confocal laser scanning microscopy to determine the evolution of the skin surface pre- and post- treatment. The depth of wrinkles was observed to significantly decrease after 28 days of therapy which backed the biochemical mechanism hypothesis. The researchers compared results of this test with previous results obtained in a similar test performed on 10 women volunteers using a cream that had 10% Argireline Solution. In the results, the placebo had a wrinkle reduction of -2.99%, Argireline had a reduction of -27.05%, and SNAP-8 had a reduction of -34.98%. Based on the study results, 10% SNAP-8 Solution had a maximum wrinkle reduction value of -63.13%. Based on the in vitro tests results, the researchers concluded that the two extra amino acids derived from the SNAP-25 sequence are able to moderately boost the anti-wrinkle activity.