# In Vitro and Clinical Evaluation of the Anti-Wrinkle Efficacy of Medipep-6PN, a Novel Peptide Identified by Phage Display

## TL;DR

A new peptide, Medipep-6PN, was developed to reduce wrinkles by targeting muscle contractions and collagen breakdown, and it showed promising results in both lab and clinical tests.

## Contribution

The novel peptide Medipep-6PN was developed to simultaneously target muscle nAChR and MMP-1, addressing both dynamic and static wrinkles.

## Key findings

- Medipep-6PN inhibited muscle nAChR activity by 80% at 30 μM and showed an IC50 of 4.2 ppm for MMP-1 inhibition.
- The peptide increased collagen I production in human fibroblasts and reduced wrinkle depth and volume in a clinical trial.
- No significant toxicity was observed in cytotoxicity tests, confirming its safety.

## Abstract

Face wrinkles caused by skin aging can be classified into dynamic wrinkles, which are caused by repetitive contraction of facial expression muscles, and static wrinkles, which are related to extracellular matrix damage and collagen breakdown caused by ultraviolet and oxidative stress. These two mechanisms are closely related, and prolonged, repetitive muscle contractions act as mechanical stress that promotes extracellular matrix degradation within the dermis, accelerating wrinkle formation. In this study, we used phage display to develop a novel peptide, Medipep-6PN, that targets both muscle-type nicotinic acetylcholine receptor (muscle nAChR), a major cause of dynamic wrinkles, and matrix metalloproteinase-1 (MMP-1), a cause of static wrinkles. In this study, the kinetic analysis of Medipep-6PN using surface plasmon resonance analysis showed that the equilibrium dissociation constant (KD) for muscle nAChR α1 was 9.56 × 10−6 M, and the KD for MMP-1 was 1.25 × 10−6 M. Calcium imaging analysis in TE671 cells expressing the muscle nAChR pentamer determined that Medipep-6PN inhibited muscle nAChR channel activity in a concentration-dependent manner, and in particular, it was confirmed that about 80% of muscle nAChR channel activity was inhibited under 30 μM of Medipep-6PN. In addition, in an in vitro test performed to evaluate MMP-1 activity, Medipep-6PN inhibited MMP-1 activity in a concentration-dependent manner, and the IC50 was 4.2 ppm. When measuring MMP-1 gene expression in UVB-induced human fibroblasts, 1 ppm of Medipep-6PN showed a 52.3% decrease compared to UVB irradiation alone. When measuring type I procollagen synthesis in human fibroblasts, Medipep-6PN increased procollagen Iα1 production in a concentration-dependent manner, and concentrations between 5 and 10 ppm of Medipep-6PN significantly increased collagen I production. No significant toxicity was observed in cytotoxicity tests, demonstrating its safety. Furthermore, in a clinical study evaluating wrinkle improvement efficacy in 25 adults over a four-week period, the Medipep-6PN group demonstrated statistically significant reductions in wrinkle depth (by 10.16%) and wrinkle volume (by 13.00%), demonstrating efficacy comparable to that of commercially available functional anti-wrinkle ingredients. In conclusion, this study demonstrates that Medipep-6PN, developed to target two mechanisms—the relaxation of muscle contraction and the inhibition of collagen degradation—is a functional peptide effective in improving skin wrinkles, confirmed through in vitro evaluation and clinical studies.