In modern society, wrinkles are a matter of major concern as our faces are our calling card. Since a healthy young appearance is a sign of success, the beauty industry is constantly researching new cosmetic anti-ageing solutions. Wrinkles, as a sign of cutaneous ageing, may cause cosmetic disability and psychological distress.
Its formation can occur naturally over time and this process is identified by certain biochemical, histological and physiological changes that are enhanced by environmental exposure. There are, however, other secondary factors that can cause characteristic folds, furrows and creases of the face, such as 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 which particularly create expression wrinkles. From a physiological point of view, the formation of expression wrinkles is due, at least partly, to the excessive stimulation of the muscle fibres in the face, which pull inwards on the skin, giving rise to the wrinkles. The dynamic expression lines progressively become permanent, always oriented in a stereotyped pattern according to the forces imposed by facial muscles. The frown lines, the glabellar lines and the crow’s feet wrinkles are typical examples.
Their anatomical support is in the hypodermal connective tissue strands harbouring fascicles of muscle cells. Repeated contractions exerted at the same anatomical sites by facial expression are accompanied by thickening and shortening of these structures, maintaining a strong hide binding of the dermis to the underlying muscles beneath the hypodermis.1 It has been clearly established that the conformational changes in the skin and the disturbance of the perfect packing of the lipid matrix can be significantly avoided by modulating muscle contraction. Thus, targeting the number and strength of these contractions would be of major interest in avoiding and/or minimising expression wrinkles’ formation, which are the most macroscopically noticeable ones. Each type of wrinkles usually develops on specific skin regions exhibiting distinct micro anatomical characteristics. This is of importance because the different types of wrinkles respond differently to cosmetic and dermatological treatments.1 Expression wrinkles, as an external sign of ageing, are undesired features that can be minimised with the appropriate treatment. As they are linked to facial muscle contraction, its modulation is the most reliable approach to fight this unpleasant feature. A cosmetic treatment can smooth the skin surface if it targets the right mechanism entailed in muscle contraction. Muscle contraction is a process where both the nerve and the muscle are involved in a synapse named neuromuscular Junction (NMJ), where pre-synaptic and post-synaptic mechanisms take place. The pre-synaptic process requires a series of actors, each one playing their specific role. Due to the local excitation in the nerve terminal, the motor neuron releases the neurotransmitter acetylcholine (ACh) into the synapse.
Then, ACh diffuses along the synapse until it reaches the AChRs on the post-synaptic membrane of the muscle fibre to trigger the signal. Nicotinic acetylcholine receptors (AChRs) are pentamers activated by the agonist acetylcholine, which are found in the NMJ. These AChRs are crucial in communication from brain to muscle: they mediate synaptic transmission by triggering endplate potentials that lead to action potentials and contraction of skeletal muscle fibres. When muscle fibres are innervated by motor axons, AChR clustering is increased dramatically at synaptic contact sites because of a nerve-secreted factor, named agrin, which associates with the synaptic basal lamina.2 At the NMJ, release of neural agrin causes MuSK dimerisation and stimulates MuSK tyrosine phosphorylation.3 Agrin is the nerve-secreted heparansulfate proteoglycan that activates MuSK (Muscle-Specific Kinase). It is a receptor tyrosine kinase selectively expressed in muscle cells that triggers redistribution of previously unlocalised proteins, including AChR, to synaptic sites when activated. MuSK is essential for a functional post-synaptic apparatus and plays a key role in muscular contraction. Agrin is thus the molecule orchestrating the overall post-synaptic function in muscular contraction. Once the interaction between agrin and MuSK has occurred, rapsyn induces aggregation of AChRs. Rapsyn (Receptorassociated protein at the synapse) is a cytoplasmic peripheral membrane protein associated with AChRs in the post-synaptic membrane. Agrin may act in part by promoting interactions of AChRs with rapsyn. Agrin also stimulates tyrosine phosphorylation of the AChR beta- and delta-subunits, and the time course of AChR phosphorylation precedes AChR clustering.4 Recent studies suggest that tyrosine phosphorylation of AChR betasubunit regulates AChR clustering to form AChR clusters at NMJ.2 When activated MuSK has recruited enough rapsyn/AChR, the cluster is formed and is then able to interact with the neurotransmitter. ACh binds with its receptors and activates them, then a flux of sodium passes through AChRs’ sodium channels and, if it is powerful enough, generates the action potential that will lead to muscle contraction. Clustering of AChRs at the post-synaptic apparatus of the NMJ is essential to reach the potential needed to perform contraction. If AChRs were not recruited by MuSK, the ACh signal would not be strong enough to trigger contraction. Clustering of AChRs in the post-synaptic membrane is critical for synaptic function, as a high density of synaptic AChRs is required to generate a synaptic potential of sufficient magnitude to initiate an action potential in the myofibre. This action potential propagates through the muscle and originates the contraction. There are two main strategies to minimise muscle contraction relaxing the muscle and helping to avoid the appearance of expression wrinkles: presynaptic and post-synaptic. A pre-synaptic pathway involves different mechanisms that restrain the release of the neurotransmitter acetylcholine (ACh) from the motor neuron. On the other hand, a post-synaptic route avoids or diminishes the ligand (ACh)-receptor (AChR) binding responsible for the formation of the action potential.
The inhibition of the agrin/MuSK pathway to lessen muscle contraction in the treatment of expression wrinkles is a novel cosmetic approach. The new molecular cosmetic peptide, Inyline, (INCI name: Acetyl Hexapeptide-30) was designed according to the predicted structure of agrin’s binding domain of MuSK’s tyrosine kinase domain by molecular modelling. The new hexapeptide acts as a competitive antagonist of MuSK at the agrin binding site, which is essential for muscle contraction, in a novel postsynaptic strategy in anti-wrinkle cosmetics. Thus, modulation of muscle contraction helps to avoid the formation of expression lines showing a tighter skin and much fewer wrinkles, furrows and creases. In vitro and in vivo tests were performed with acetyl hexapeptide-30 which confirmed the mechanism of action of the peptide and proved to diminish wrinkles caused by facial expression. Acetyl hexapeptide-30 is a post-synaptic active ingredient designed to diminish expression wrinkles. That is, a molecular solution for a macroscopic effect.
Materials and methods
Inhibition of acetylcholine receptor clustering
Efficacy of acetyl hexapeptide-30 was evaluated in an assay of competition with the C-terminal fragment of agrin for the induction of the aggregation of AChRs in cultured differentiated myoblasts. C2C12 cells were grown in appropriate medium and differentiation of myotubes was induced. After differentiation, cells were incubated overnight. NaCl and LiCl were used as negative and positive controls respectively. After incubation, recombinant C-terminal agrin were added to culture dishes and cells were incubated for an additional five hours at 37°C. AChR clusters were visualised by treatment of treated differentiated myoblasts with ?-bungarotoxin (a toxin which competitively binds to the AChR in an irreversible way) conjugated to tetramethylrhodamine (a fluorophore). Differentiated myoblasts were examined on a fluorescence microscope with a 40X oil immersion lens and several pictures were taken. Photographs were analysed with appropriate software. Reduction of AChR aggregation was calculated for acetyl hexapeptide-30 according to a defined formula. Inhibition of AChR clustering was established by counting the number of AChR aggregates in all the pictures recorded for all treatments. Values were normalised regarding the negative control.
Anti-wrinkle efficacy
The anti-wrinkle efficacy of acetyl hexapeptide-30 was assessed on a panel of 20 female volunteers aged 41 to 50. The subjects applied a cream containing 0.0025% acetyl hexapeptide-30 in the crow’s feet area twice daily for 28 days. Measurements of the wrinkle depth were taken before the test, to assess the initial condition, and after 28 days of treatment. Wrinkle depth was determined by means of PRIMOS technique (Fig. 5), which is a non-contact measurement device that allows for real-time 3D in vivo measurement of the microtopography of human skin based on the technology of active image triangulation. Images taken were further processed and the data obtained was appropriately treated. Statistical analysis of the data included descriptive statistics and the Wilcoxon Rank Test, which yielded the final results.
Results
Inhibition of acetylcholine receptor clustering
Acetyl hexapeptide-30 highly reduced AChR clustering induced by agrin C-terminal fragment. The hexapeptide induced an inhibition comparable to that of the positive control (LiCl) at 100 ?M (Fig. 2). The novel peptide proved to be able to reduce the formation of AChR clusters induced by the addition of the C-terminal region of agrin with a dose-response efficacy, with values that reached 72% of inhibition at 100 ?M. This in vitro assay undoubtedly proved that acetyl hexapeptide-30 specifically avoids AChR clustering, which is a key step in the post-synaptic functionality of the NMJ.
Anti-wrinkle efficacy
After 28 days of treatment, a significant decrease in wrinkle depth of almost 15% was observed in the tested area as compared to the initial condition (Fig. 4) and a positive effect due to acetyl hexapeptide-30 treatment could be detected in 90% of the volunteers. A mean decrease in wrinkle depth of 14.9% was observed after 28 days of treatment with acetyl hexapeptide-30, which demonstrates the ability of this novel cosmetic ingredient to attenuate expression wrinkles. The anti-wrinkle efficacy of the hexapeptide could also be appreciated in the PRIMOS images.
Conclusions
Acetyl hexapeptide-30 is a new peptide that has been specifically designed to target the agrin/MuSK post-synaptic pathway, a novel approach in the cosmetic treatment of expression wrinkles. The hexapeptide acts as a competitive antagonist of MuSK at the agrin binding site and its mechanism of action causes modulation of muscle contraction leading to muscle relaxation. The novel active ingredient notably reduces the formation of AChR clusters in vitro with a dose-response efficacy thus preventing ACh from triggering the contraction signal. Its anti-wrinkle properties were proved in vivo showing significant decrease in wrinkle depth, which demonstrates its ability to attenuate expression lines. Inyline proved to be an excellent active ingredient to diminish expression wrinkles. That is, a molecular solution for a macroscopic effect.
InylineTM is a registered trademark of Lipotec S.A. (Gavà, Spain).
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