Pre- and post-synaptic strategies fight wrinkles

Repeated facial muscle contraction is responsible for the appearance of these unpleasant lines in the top half of the face (crow’s feet, frown wrinkles etc.), but also around the mouth and nose which can start showing as early as the age of 30. Both surgery and botulinum toxin injections are invasive and expensive methods compared to cosmetic alternatives to fight against ageing. However, if a cosmetic treatment targets the right mechanism involved in muscle contraction, it can help to minimise these undesirable lines. Muscle contraction is a process where both the nerve and the muscle are involved in a synapse named Neuromuscular Junction (NMJ). The motor neuron releases the neurotransmitter acetylcholine (ACh) which travels through the synapse to activate the ACh receptors (AChRs) on the fibre muscle surface, creating an action potential and leading to muscle contraction. However, contraction is a complex mechanism formed by presynaptic and post-synaptic mechanisms involving several proteins, channels and vesicles. At the pre-synaptic pathway, a localised change in membrane potential, known as action potential, is transmitted along the neuron to the terminal where it triggers the entry of Ca2+ into the neuron. Natural enkephalins are endogenous opioids that modulate Ca2+ channels by binding to their receptors located on the outside of neurons. When calcium ions enter the pre-synaptic terminal, it is induced by the fusion of vesicles containing ACh with the membrane, releasing the neurotransmitter into the synapsis and leading to muscle contraction. On the other hand, the combination of SNARE (SNAp REceptor) proteins (VAMP, Syntaxin and SNAP-25) forms a complex which acts like a cellular hook capturing vesicles and fusing them with the membrane. This complex is essential for ACh release at the synapsis and mediates the final steps of the exocytosis. Released acetylcholine diffuses along the synapse to reach the AChRs receptors on the neuromuscular junction, thus starting the post-synaptic route. At the NMJ, release of the nerve-secreted proteoglycan agrin activates MuSK (Muscle-Specific Kinase) and induces the formation of AChR clusters. AChR clustering is essential to reach the sufficient magnitude of potential to initiate an action potential which will propagate through the muscle and originate muscle contraction. If AChRs were not recruited by MuSK, the ACh signal would not be strong enough to trigger contraction. Thus, modulation of MuSK activation leads to attenuation of muscle contraction. In order to target different parts of the expression wrinkles formation, Lipotec have designed four peptides which act in pre-synaptic and post-synaptic mechanisms to relax the muscle. Pre-synaptic strategy involves different mechanisms that restrain or inhibit the release of the neurotransmitter (acetylcholine) from the motor neuron. The most widely-known example of a presynaptic treatment is botulinum toxin, which cleaves the protein SNAP-25 irreversibly, preventing SNARE complex assembly and finally paralysing the muscle. However, Lipotec has developed safer cosmetic alternatives to botulinum toxin such as the well-known peptides Leuphasyl (INCI name: Pentapeptide-18), Argireline (INCI name: Acetyl Hexapeptide-8) and SNAP-8 (INCI name: Acetyl Octapeptide-3). The enkephalin-like pentapeptide-18 couples to the enkephalin receptor decreasing the neuron excitability and resulting in ACh release modulation. Acetyl Hexapeptide-8 and Acetyl Octapeptide-3 target the same protein complex as botulinum toxin A. Both actives are a mimic of the N-terminal end of SNAP-25 which competes with SNAP-25 for a position in the SNARE complex, thereby modulating its formation. If the SNARE complex is slightly destabilised, the vesicle cannot release neurotransmitters efficiently. Consequently, muscle contraction is attenuated and the muscle is relaxed rather than paralysed, preventing the formation of lines and wrinkles. In post-synaptic strategy, although the neurotransmitter is released into the synapse, the formation of the action potential is reduced. Inyline (INCI name: Acetyl Hexapeptide-30) is a peptide that acts as a competitive antagonist of agrin, thus preventing AChR clustering which is necessary for ACh to trigger the action potential that leads to muscle contraction. A novel post-synaptic approach that helps to relax the muscle to minimise the appearance of expression wrinkles.

Materials and methods

Modulation of SNARE complex formation