547 Address all correspondence to Hamid Reza Moghimi, hrmoghimi@sbmu.ac.ir Terpene Conjugation: A Novel Approach for Topical Peptide Delivery SEYEDEH MARYAM MORTAZAVI, FARZAD KOBARFARD, ALI MANAFI, HOWARD I. MAIBACH AND HAMID REZA MOGHIMI Department of Pharmaceutics and Nanotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran (S.M.M, H.R.M.) Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran (F.K.) Department of Surgery, School of Medicine, Iran University of Medical Sciences, Tehran, Iran (A.M.) Department of Dermatology, School of Medicine, University of California, San Francisco, California, USA (H.I.M.) Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran (H.R.M) Accepted for publication August 2, 2021. Synopsis Peptides are poor candidates for skin permeation. KTTKS is not an exception and cannot permeate skin in therapeutic amounts. However, chemical modification could be a good approach to overcome this problem. In our study, we used citronellic acid and perillic acid terpenes to synthesize Cit-KTTKS and Per-KTTKS, and KTTKS and Pal-KTTKS for comparison. We studied the aqueous stabilities of the compounds. Peptides permeations through a membrane model of n-hexadecane and human epidermis permeations of KTTKS, Pal-KTTKS, and Cit-KTTKS were determined. The results revealed that the aqueous solution of compounds remained stable at 32°C for 48 h. The membrane model permeation studies showed that KTTKS and Per- KTTKS were not detected in receptor phases, but Pal-KTTKS and Cit-KTTKS passed across the membrane model and showed estimated kp of 17×10–4 cm/h and 2.9×10–4 cm/h, respectively. The epidermis studies showed that KTTKS and Pal-KTTKS did not pass across the epidermis in detectable amounts, whereas Cit- KTTKS permeated epidermis with kp of 7.3×10–4 cm/h. The donor phase assay and mass-balance studies showed that Pal-KTTKS was trapped by about 48% in the epidermis and 20% in the model membrane. Based on these results, terpenes appear to be good candidates for peptide conjugation to increase the skin permeation. INTRODUCTION Signal peptides are main categories of topical peptides. This type of peptide triggers the cellular process and consequently increases the dermal remodeling. KTTKS, a signal peptide, is recognized for its anti-wrinkle properties. KTTKS stimulates the extracellular J. Cosmet. Sci., 72, 547–556 (September/October 2021)

548 JOURNAL OF COSMETIC SCIENCE matrix biosynthesis and inhibits collagenase activities in the dermis (1,2) . However, this five-amino acid peptide (see Figure 1) does not permeate skin in a therapeutic amount. Different approaches have been previously applied to make KTTKS permeable the most prominent is the covalent attachment of palmitic acid, a sixteen-carbon hydrophobic moiety. Although palmitoylated derivative of KTTKS (Pal-KTTKS) is currently marketed by the Matrixyl® (Sederma, Le Perray en Yvelines, France) brand name and applied in anti- wrinkle products (3), unfortunately only one study has evaluated its skin permeation and it showed that neither KTTKS nor Pal-KTTKS permeated across the intact mouse skin (4). The skin permeation is affected by two main parameters: (1) diffusion coefficient, which is related to the molecular weight and shape and (2) partition coefficient, which is related to the permeant polarity. KTTKS possesses a molecular weight of 563.6 Da and clog P of −3.45, which is why this peptide is a poor candidate for permeation. Its palmitoylated derivative is more hydrophobic (clog P = 3.72) but it has a larger size (molecular weight of 802.0 Da). Other conjugates have been synthesized in previous studies such as lipoic acid- KTTKS (5), ascorbic acid-KTTKS (6), poly (ethylene glycol)-KTTKS (7), and ac-wahx– KTTKS (8). In the mentioned derivatives, although bioactivity and/or cytotoxicity of the conjugates have been evaluated previously (5–8), there is no data available on their skin permeation. All conjugates have shown bioactivity therefore, it seems that attaching a moiety to KTTKS does not considerably affect bioactivity of this signal peptide. Increased lipophilicity through conjugation is accompanied by increased molecular weight (9). To achieve suitable skin permeation through this mechanism, a balance between lipophilicity and molecular weight is needed. In our research, we synthesized conjugates with acceptable lipophilicity and low molecular weight. To achieve this goal, we selected Figure 1. The chemical structures of KTTKS, citronellic acid, and perillic acid.