Articles 283 Potent inhibition of CYP3A4 by the endomorphin-2 analogues Kaloyan Georgiev 1 , Maya Radeva 1 , Tamara Pajpanova 2 1 Medical University of Varna, Bulgaria 2 Bulg arian Academy of Sciences, Bulg aria https://doi.org/10.17952/35EPS.2018.283 Introduction Endomorphins(endomorphin-1,EM-1, Tyr-Pro-Trp-Phe-NH 2 , endomorphin-2,EM-2, Tyr-Pro-Phe-Phe- NH 2 ) are endogenous peptides, which are very potent and highly selective μ-opioid receptor agonists. Moreover, they possess a potent analgesic activity comparable to that of morphine but are devoided of its’ undesirable effects [1, 2]. However, their use as therapeutic agents is limited because of their pharmacokinetic features, such as stability and permeability via blood-brain barrier (BBB). In our previous developments, modifications in the structure of endomorphine-2have shown an improvement in stability and permeability through the membranes [3]. In the current study, the goal was to investigate another pharmacokinetic feature, namely the risk of drug interactions at the level of cytochrome enzymes. CYP3A4 is the most important drug metabolizing enzyme in humans. It is highly expressed in liver and gastrointestinal mucosa and is involved in the metabolism of more than 50% of the used drugs. Therefore, the risk of drug interactions is greatest in the drugs modifying the activity of CYP3A4. Materials and Methods Peptide synthesis and analysis Peptides in this study were synthesized by manual solid-phase procedures using techniques for Fmoc-protected amino acids on Wang or MBHA Rink-Amide peptide resins respectively. 20% Piperidine in DMF was used for deprotection of Fmoc-groups and DIC and HBTU were employed as a coupling agent. Simultaneous deprotection and cleavage from the resin was accomplished by treatment with TFA/TIS/water (95:2.5:2.5) for 3 h at room temperature. Crude peptides were purified by preparative TLC and their purity was checked by analytical HPLC. Measurement of CYP3A4 activity in vitro The test compound was mixed with a master pre-mix comprising CYP450 BACULOSOMES ® reagent and regeneration system, which contained glucose-6-phosphate and glucose-6-phosphate dehydrogenase. The mixture was incubated at room temperature for 20 min. Following incubation, CYP enzyme-specific substrate (Vivid DBOMF for CYP3A4) and NADP + were added and the mixture was incubated at room temperature for 30 min. CYP activity was evaluated by measuring the fluorescence of the fluorescent metabolite generated from CYP3A4 enzyme-specific substrate. The fluorescence was measured using a microplate reader BioTek Synergy. Results and Discussion For the purpose of our study, we used four endomorphin-2analogues, two of them were modified at third position - Phe(pF) (1, Tyr-Pro-Phe(pF)-Phe-1,2-ethylenediamine) and conjugated at forth position with 1,2-ethylenediamine (2, Tyr-Pro-Phe-Phe-1,2-ethylenediamine). The other two, was conjugated at first position with deoxycholic acid and modified at third position - Phe(pF) (3, Deoxycholic-Tyr-Pro-Phe(pF)-Phe- OH)and Phe (pCl) (4, Deoxycholic-Tyr-Pro-Phe(pCl)-Phe-OH)(Figure 1). Proceedings of the 35th European Peptide Symposium Patrick B. Timmons, Chandralal M. Hewage, Michal Lebl (Editors) European Peptide Society & PSP, 2018