Peptide Approach to Enhance Anti-wrinkle Efficacy Between Injections

The face is a special target for internal and external aging, and wrinkles are one of the most common signs of progressing years. Although wrinkles can result from photoaging and intrinsic aging, expression lines or dynamic wrinkles can betray one’s chronological age by appearing sooner than expected. These lines are a result of repeated contractions exerted daily at the same anatomical site from facial expressions and movements while talking, smiling, drinking or smoking.1, 2 Frown lines, glabellar lines and crow’s feet wrinkles are examples that result from interactions between neuronal and muscle cells, i.e., the neuromuscular junction, in a process known as neuronal exocytosis.

Botulinum toxin type A (BoNT-A) injections are widely used to attenuate expression wrinkles, such as frown lines and crow’s feet, by paralyzing the muscles. A single dose provides a visible smoothing benefit within a short time, i.e., normally a few days, and it can last for weeks or even months. Immediacy and longevity make these injections an attractive option, although the effects eventually fade, leading to the reappearance of expression lines. Also, patients are advised to wait six months before the next BoNT-A injection due to its toxic nature and potential rejection by the immune system.

Therefore, topical cosmetic ingredients could complement such injections if they were able to enhance the BoNT-A anti-wrinkle benefits as well as prolong them until the next injection. Here, the development of a topical combination of acetyl hexapeptide-8 and pentapeptide-18a is described. It is shown to act on the key steps that attenuate muscle contraction, providing a non-invasive option to reduce expression lines and maintain the BoNT-A smoothing effects on skin.

Expression Wrinkle Mechanisms

At the neuromuscular junction, the neuron is initially at its resting potential, surrounded by an environment of high sodium (Na+) and low potassium (K+) concentrations. Its activation takes place when, after receiving a signal, Na+ channels open. This action implies the massive entry of sodium ions that positively charge its inside and cause membrane potential to rise fast. After a maintained period of Na+ ion entry, these channels close and inactivate to return the neuron to its resting potential. The opening of K+ channels helps to remove positive charges from the inside and consequently decreases membrane potential. At the end of the axon from which the impulse is sent, the membrane depolarizes, gated calcium channels open, and calcium ions (Ca2+) are allowed to enter the cell. In relation, enkephalins are endogenous compounds that can indirectly close Ca2+ channels to avoid the cascade of events that follows to cause muscle contraction.

The vesicle-associated membrane protein (VAMP), the membrane-associated protein syntaxin, and synaptosomal associated protein 25 (SNAP- 25) are collectively referred to as SNAP receptor or SNARE proteins.3 These form a crucial ternary structure known as the SNARE complex that acts as a cellular hook, capturing vesicles containing the acetylcholine (ACh) neurotransmitter. When Ca2+ ions enter the pre-synaptic terminal, the vesicles with ACh are induced to fuse with the neuronal membrane.3, 4 Muscle contractions occur when ACh traverses the synaptic space and binds to its receptors located in the post-synaptic membrane of muscle cells. As noted, when facial muscle contractions are highly repeated, expression wrinkles appear.

BoNT-A Injection

Injection with BoNT-A is among the most popular anti-wrinkle procedures performed. It was approved by the U.S. Food and Drug Administration in 2002 to temporarily smooth moderate to severe frown lines, and more than a decade later (2013), to smooth crow’s feet as well. A trained and qualified physician is required to inject this protein complex, which is produced by the bacterium Clostridium botulinum. Applied to the skin, a small sterile dose of this purified toxin blocks the release of ACh by nerve cells as it cleaves the SNAP-25 protein, thereby interfering with vesicle docking and exocytosis. This action prevents nerve impulses and ACh release at the neuromuscular junction that cause muscle contractions. The muscles are then paralyzed and the existing frown lines and crow’s feet are smoothed.

As stated previously, its effects are visible within a short period of time, i.e., 4-5 days, and they can last for weeks or months. According to the International Society of Aesthetic Plastic Surgeons, more than three million BoNT-A treatments were performed worldwide in 2011, which accounted for 38.1% of the total nonsurgical procedures. In the United States, BoNT-A procedures increased by 8% from 2011 to 2012, according to the 2012 report from the American Society of Plastic Surgeons. This data confirms that the concern for wrinkles and fine lines has increased, and that such injections are popular for treating them. Market data suggests this trend will continue growing in the coming years, especially in light of the FDA’s approval of BoNT-A to treat crow’s feet.

Topical Complement to Injections

Topical cosmetics represent another known option to smooth expression lines, for example, by reducing the formation of the SNARE complex and ACh release. Such products would provide similar smoothing benefits and prolong the effects of the injectable toxin. Acetyl hexapeptide-8 and pentapeptide-18 are two examples of such anti-expression wrinkle ingredients.

Widely known, acetyl hexapeptide-8 is a replica of the N-terminal end of SNAP-25 that competes with this natural protein for a position in the SNARE complex, modulating its formation without breaking any of its components. If such a complex is destabilized, the vesicles cannot release ACh efficiently and the muscle remains relaxed. This topical ingredient thus targets the same protein complex as the toxin injection without paralyzing the muscle.

Pentapeptide-18 is a modified enkephalin that modulates ACh release from neuron cells. This molecule imitates the family of endogenous opioids that internally inhibits neuronal activity by joining enkephalin receptors outside of nerve cells that are coupled to G proteins. Such an interaction results in the release of G protein subunits to close Ca2+ channels. Thus, a decrease of the neuronal excitability is induced, and vesicle fusion and ACh release towards the synapse is prevented, therefore attenuating muscle contraction.

Considering these effects, the authors’ company developed an active ingredient based on acetyl hexapeptide-8 and pentapeptide-18 to fight expression wrinkles. In vitro and in vivo studies, described here, were implemented to test its efficacy. In addition, a novel assay performed between two BoNT-A injections assessed the ability of the active to benefit the toxin injections by extending their visible effects.

Glutamate Release

The inhibition of glutamate release by depolarized neuron cells is a validated cell assay for measuring the potential activity of compounds to inhibit neuronal exocytosis. The K+-induced depolarization of hippocampal cultures in the presence of extracellular calcium ions results in the release of glutamate, which is the most abundant excitatory neurotransmitter in the nervous system.5-7

Primary neuron cells were incubated with L-[3H]-glutamine to charge them with L-[3H]-glutamate, after which the excess L-[3H]-glutamine was rinsed off and the cells were incubated with acetyl hexapeptide-8, pentapeptide-18, or both. The release of L-[3H]-glutamate was carried out by depolarization in a physiologic buffer. The culture media was then collected and the quantity of L-[3H]-glutamate was determined by a scintillation counter. The results were normalized regarding the release of L-[3H]-glutamate in absence of the test items (control), and corrected from the basal release in absence of calcium.

Compared with the control, acetyl hexapeptide-8 and pentapeptide-18 induced a 20.0% and 11.0% decrease on glutamate release, respectively, while their combination caused a 40.0% reduction, indicating a synergistic activity (see Figure 1). Thus, the main constituents of the peptide complex offer a complementary effect in reducing neuronal exocytosis.

Anti-wrinkle Effects

A novel in vivo study was designed to evaluate the anti-wrinkle effects of the peptide complex. A panel of 22 Caucasian females, with an average age of 51, received a 50-IU BoNT-A injection in the periorbital, i.e., crow’s feet, and frontal regions of the face. Immediately following the injection, volunteers applied either an active formulation containing 10% active ingredient or a placebo formulation (control) twice daily for six months. Skin silicon replicas were obtained before the injection and at different times during the treatments to analyze the effects. The skin relief was evaluated by confocal profilometry to quantify the reduction of the average surface roughness (Ra) versus baseline. Macroscopic photographs were also taken at baseline and after two, four and six months.

Regarding treatment with the toxin alone, a maximum reduction in skin roughness was observed one month after the injection in the frontal region, drastically diminishing afterward, and just two weeks after the injection in the periorbital region, gradually decreasing afterward.

With topical treatment, in the frontal region, the anti-wrinkle effect of the injection was highly intensified at all times, prolonging its benefit for several months. The topical active nearly tripled the anti-wrinkle effect vs. the control treatment in the frontal region (see Figure 2a); in the periorbital region, it potentiated the smoothing effect on wrinkles induced by the toxin over the course of the six months (see Figure 2b). As images of the volunteers show, the topical active visibly potentiated the effect of a BoNT-A treatment and extended its anti-wrinkle benefit on the skin (see Figure 3 and Figure 4).


Surgical and nonsurgical procedures to address the undesired signs of aging, especially expression wrinkles and lines induced by repeated muscular contractions, are widespread. BoNT-A injections are one of the most demanded treatments because they are effective and less invasive than surgery, although with some restrictions. To complement the effect of this well-known toxin, the described peptide blend was designed to diminish expression lines by acting on the SNARE complex and calcium channels, both key factors in their development.

The ingredient was found to modulate glutamate release, inducing a 40.0% reduction in the neurotransmitter. In vivo, it proved to maintain and prolong the anti-wrinkle effect of the BoNT-A injection in the frontal and periorbital regions, in all cases resulting in effects more beneficial than the control treatment. In fact, it nearly tripled the anti-wrinkle effect of the control treatment in the frontal region after six months, and it clearly decelerated the reappearance of expression lines in the crow’s feet area. Thus, the ingredient is presented as an ideal topical complement to BoNT-A injections, with demonstrated efficacy in visibly prolonging their anti-expression wrinkle effects.


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