Inhibitory Effects Of Amiodarone On Simvastatin Metabolism In Human Microsomes

Objective: The cytochrome P450s (CYPs) are a superfamily of heme-containing enzymes that catalyze the oxidation of a wide variety of compounds. An important role of these enzymes is to aid in the elimination of lipophilic xenobiotics, including environmental toxins and drugs. It is well known that many drugs can modulate the activity of the CYPs and thereby alter the pharmacokinetic profile of coadministered drugs. Such an effect is referred to as a metabolic drug-drug interaction (DDI). The consequences of these interactions can range from loss of therapeutic efficacy to the introduction of potentially lethal toxic effects. About 50% of drugs are oxidized by CYP3A4. The effects of multidrug on CYP450 may induce notable clinical drug-drug interactions. Due to the diversity and chronicity, the interaction among cardiovascular drugs will be increased. Simvastatin, which is metabolized by CYP3A4, is widely used in hypercholesterolemia and the prevention of cardiovascular events. Amiodarone is a potent antiarrhythmic drug. Amiodarone is mainly metabolized by CYP3A4. Previous studies have demonstrated that amiodarone increased the area under the plasma concentration-time curve (AUC) of simvastatin in vivo. The aim of present study is to investigate the interactions at a metabolic level between amiodarone and the substrate of CYP3A4 simvastatin in human liver microsomes and to account for the clinically observed interactions. The mechanism of the interaction between amiodarone and simvastatin has also been investigated and the prediction of the potentiality of interaction in vivo by using the in vitro-in vivo extrapolation.Methods: 1. Preliminary experiments were conducted to make sure all experiments were performed within the linear range of incubation time and protein concentration. The HPLC (High performance liquid chromatography) method to detect simvastatin and its metabolite was established. 2. The HLM was pretreated with amiodarone and then simvastatin was added. First, 50μM amiodarone preincubated with HLM in various time (0-30min). Used the software Origin 7.5 to fit the IC50 of amiodarone to CYP3A4. Second, fixed the concentration of amiodarone, to investigate the influence of preincubation time to the quantity of the metabolite of simvastatin. Peak heights of the metabolite were expressed as a ratio to the internal standard (Testosterone) peak height for each concentration of inhibitor. These peak height ratios represented the remaining CYP3A4 activity in the HLM and were expressed as a percentage of the time-matched control samples without inhibitor. The natural logarithm of relative inhibition values were plotted versus preincubation time for a series concentration of Amiodarone used (0, 2, 5, 25, 50μM), and the slopes determined by linear regression. These slope values represented the observed inactivation rate constants (kobs). Third, a double-reciprocal plot was then constructed using the reciprocal of the kobs (y-axis) and the reciprocal of the associated inhibitor concentration (x-axis) to estimated the values of kinact and KI , which were two principal kinetic constants that were specific for MBIs. They were the maximal rate constant of inactivation (kinact) and the inactivator concentration required for half-maximal inactivation (KI).3. Substituted the two parameters kinact and KI into equation (2) defined the fold reduction in intrinsic clearance (CLint) of a specific CYP caused by MBI.Results: 1. A NADPH-dependent component was evident for amiodarone, as the IC50 value was significantly reduced (i.e., more potent inhibition) when the inhibitor was preincubated with human liver microsomes. The IC50 value was still higher than 50μM with preincubation of the inhibitor. Therefore the inhibition of amiodarone to CYP3A4 was weak.2. With the increasing of concentration of amiodarone, the inhibition became stronger.3. At a fixed concentration the longer the preincubation time prolonged the stronger the inhibition became.4. The time-, concentration- and NADPH-dependent characteristics confirmed the mechanism of amiodarone inhibited CYP3A4 was mechanism -based inhibition, when simvastatin was the substrate of CYP3A4.5. The Double-reciprocal plot of mean data points indicated a KI value of 5.098μM, and a kinact value of 0.018min-1. The fold of reduction in CLint was 0.76-12.63, which was close to the result of in vivo study, the fold of AUC of simvastatin with the absence of amiodarone increased 0.76-12.63. Thus, the possibility of toxity of simvastatin will be increased.Conclusion: 1. The inhibition of metabolite of amiodarone was more potent than the parent drug.2. The main mechanism of interaction in vivo between amiodarone and simvastatin was mechanism-based inhibition.3. The recommended therapeutic dosage of simvastatin was 20mg daily, but clinically relevant DDI was possible via the mechanism-based inhibition of CYP3A4.