(T0930-12-79) Transdermal Permeability Assay of LK 467, a Selective and Potent Agonist of the Melanocortin 1 Receptor, Demonstrating the Efficacy of Topical Application

Purpose: Melanocytes are melanin-producing cells that determine the pigmentation of skin, hair and eyes. The melanocortin-1 receptor (MC1R) is a G_s-protein coupled receptor (GPCR) that is a central regulator of the synthesis of the dark form of melanin, eumelanin, which is the main determinant of the diversity of human pigmentation. The physiological agonist of MC1R is alpha-melanocyte stimulating hormone (α- MSH). Activation of MC1R upon binding of α-MSH increases eumelanin synthesis by human melanocytes, enhances repair of solar UV-induced DNA damage, and activates antioxidant defenses. Loss of function mutations in the MC1R gene are strongly associated with red hair and fair skin phenotype, due to reduced eumelanin synthesis, and with increased risk for melanoma, the most fatal form of skin cancer. There has been increasing interest in developing safe and effective analogs of α-MSH to induce sunless tanning and protect from sun-induced skin cancers. Novel small therapeutic analogs of α-MSH like LK467, LK 184, LK 513, LK514, and LK511 were synthesized at the University of Cincinnati. These peptides were shown to be highly selective for MC1R, and the tetrapeptides LK467 and LK 184 were found to be ten-fold more potent than α- MSH. Given their small size, these analogs are expected to permeate the stratum corneum and reach the viable epidermal layers, allowing for practical and ease of application as topical agents that can target the epidermal melanocytes Three different types of emulsions were developed to test the permeability of LK467 across the skin layers.

Methods: Three different water-in-oil (W/O) emulsions of LK467 were prepared for transdermal studies. The formulations were prepared by slowly dispersing their respective aqueous phase to the oil phase upon constant stirring at 45 ⁰C. Franz diffusion cells are used for determining transdermal permeability. Diffusion chambers were equilibrated to 37 ⁰C upon the addition of Phosphate Buffer Saline (PBS) to the receiver chamber, and the permeability of LK467 across porcine ear skin was tested. The formulation was added to the donor chamber of the Franz diffusion cell and the receiver solution was sampled after 24 hrs and immediately stored at -80°C until further analysis. A sensitive LC-MS method was developed to assay LK467 in the receiver solutions. The drug is separated on the Waters UPLC BEH C18 column (2.1X50 mm, 1.7µm). A mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile were used as mobile phases with a flow rate of 0.250 ml/min. Electrospray ionization (ESI) was used as an ionization source. Ions are separated using Quadrupole analyzers with electron multipliers as detectors. LK184 is a similar analyte to LK467 was used as an internal standard. Results were analyzed using Masslynx and Acquity UPLC software.

Results: Three W/O formulations were prepared at 45 ⁰C using ingredients shown in Table 1. Two different gradients were developed for LK-467 analysis and both assays exhibited good linearity (R²>0.994) in the concentration range of 100-20000 ng/ml as shown in Figure-1. The retention time of LK467 was observed as 1.4 (as shown in Figure-2) and 2.9 minutes in gradient 1 and gradient 3 respectively. The molecular mass of LK 467-2TFA is 1068 g/mol; the parent ion peak was observed at 840.45 m/z. Calibration curves ranged from 100 to 20000 ng/ml for LK-467. The total amount of LK-467 permeated across the skin in 24 hrs was observed to be 1551. 2 ± 803.1 ng/ml.

Conclusion: In the present study, we successfully formulated 3 emulsions and tested them for transdermal permeability. LC-MS method was developed. The results demonstrated that LK467 permeated the porcine ear skin well above the concentrations required to demonstrate biological efficacy.

References: 1. Koikov, L., Starner, R. J., Swope, V. B., Upadhyay, P., Hashimoto, Y., Freeman, K. T., Knittel, J. J., Haskell-Luevano, C., & Abdel-Malek, Z. A. (2021). Development of hMC1R Selective Small Agonists for Sunless Tanning and Prevention of Genotoxicity of UV in Melanocytes. The Journal of investigative dermatology, 141(7), 1819–1829. https://doi.org/10.1016/j.jid.2020.11.034
2. Swope, V. B., Abdel-Malek, Z.A. (2018) MC1R: Front and Centerin the Bright Side of Dark Eumelanin and DNA repair. International Journal of Moelcular Science. 19: 2667.

Table 1: Formulation Ingredients

Figure 1: Graph with concentration of LK-467 on the X-axis and area on the Y-axis. The R2 value was 0.994 indicating the accuracy of the analysis.

Figure 2 Chromatogram of pure LK-467: The tetrapeptide was prepared at different concentrations and analyzed in LC-MS. The method was developed, and the retention time of the peak was 1.42 min. A sharp peak was observed for LK-467 which was considered for further analysis.