Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes

Medetomidine is a potent and selective α 2‐adrenergic agonist. The activation of α 2‐adrenergic receptor mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia. However, our main interest is the sedative effects of medetomidine when used as a premedicant prior surgery in companion animals, especially in dogs. Recently, data suggested that following intravenous infusion at six dosing regiments non‐linear pharmacokinetics was observed. Major causes of non‐linear pharmacokinetics are the elimination of the drug not following a simple first‐order kinetics and/or the elimination half‐life changing due to saturation of an enzyme system. The goal of this study was to establish the metabolic stability and determine the metabolic pathway of medetomidine in dog liver microsomes. Consequently, Michaelis–Menten parameters ( V max , K m ), T 1/2 and CL i were determined. The incubations were performed in a microcentrifuge tube and containing various concentrations of medetomidine (10–5000 n m ), 1 mg/mL of microsomal proteins suspended in 0.1  m phosphate buffer, pH 7.4. Microsomal suspensions were preincubated with NADPH (1 m m ) for 5 min at 37°C prior to fortification with medetomidine. Samples were taken at various time points for kinetic information and the initial velocity ( v i ) was determined after 10 min incubation. The reaction was stopped by the addition of an internal standard solution (100 ng/mL of dextrometorphan in acetone). Medetomidine concentrations were determined using a selective and sensitive HPLC‐ESI/MS/MS method. Using non‐linear regression, we determined a K m value of 577 n m , indicating relatively low threshold enzyme saturation consistent with previous in vivo observation. The metabolic stability was determined at a concentration of 100 n m (≪ K m ) and the observed T 1/2 was 90 min with a CL i of 0.008 mL/min indicating moderately low clearance in dog liver microsomes, also consistent with previous in vivo data. Moreover, results suggest that principally medetomidine is metabolized by the CYP3A with a small contribution from CYP2D and CYP2E. The participation of CYP3A is an important discovery since medetomidine is used as a premedicant in combination with fentanyl, ketamine and/or midazolam. These findings combined with a low K m value may indicate that medetomidine can competitively inhibit the metabolism of these drugs and consequently significantly impair metabolic clearance. Copyright © 2009 John Wiley & Sons, Ltd.