Inhibitory Effects Of Macrolides On Nimodipine Metabolism In Human Microsomes

Objective To investigate the effect of macrolides on nimodipine metabolism in human microsomes.The result could provide a basis for the in-vivo studies,promote the right use of nimodipine.It would improve the treatment effect and reduce the drug adverse effect.Methods 1.To establish the determination of nimodipine with HPLC method in incubation system;2.To determine the enzyme kinetic parameters of nimodipine.Impacts of human microsomes with different concentrations,different incubation time,different concentrations of nimodipine on the metabolic rate of nimodipine were measured.To calculate enzyme kinetic parameters Vmax and Km with GraphPad Prism software;3.To find the effect of different concentrations of macrolides on nimodipine metabolism in human microsomes.1)To measurate IC50 values of macrolides on nimodipine.The incubation system includes:PBS(0.1 mol·L-1,pH 7.4),nimodipine(25 μmol·L-1),macrolides(0,2.5,5,10,25,50,100μmol·L-1),NADPH(1 mmol·L-1),human microsomes(0.5 mg·mL-1).The total volume was 200μL.To keep the incubation system on 37℃ for five minutes,then the reaction started.Twenty minutes later,acetonitrile was added to stop the reaction.To determine the rest weight of nimodipine,calculate IC50 values with GraphPad Prism software.2)To determine Ki values of macrolides on nimodipine.The incubation system included different concentrations of macrolides(0,10,25,50 μmol·L-1)and different concentrations of nimodipine(10,20,30μmol·L-1)To determine the weight of residual nimodipine when the reaction was over.Ki values was calculated by Dixon diagram based on the reciprocal of substrate reaction rate and inhibitor concentrations.Results 1.Established the determination of nimodipine with HPLC method in incubation system.Nimodipine concentration was used as abscissa,peak area ratio between nimodipine and carbamazepine was used as ordinate.The calibration curve was linear in the range from 0.622 to 27.99μg·mL-1 with r=0.9996 for nimodipine.2.The incubation system included:human microsomes(0.5mg·mL-1),nimodipine(3.75-60 μmol·L-1),incubation time was 10 minutes.Enzyme kinetic parameters Vmax and Km were 29.64 μmol·L-1,8.69 nmol respectively.3.Different concentrations of macrolides effected nimodipine metabolism in human microsomes.1)IC50 value can be called as half inhibition rate,it is the inhibition concentration when substrate metabolism rate reduced by half.IC50 values of clarithromycin and erythromycin were 46.23μmol·L-1,71.59μmol·L-1 respectively;2)Ki value is the dissociation constants between inhibition and enzyme.It represents the inhibition constant inhibitor acted on enzyme.The smaller Ki value is,the greater combination of inhibition and enzyme.Ki values of clarithromycin and erythromycin calculated by Dixon diagram were 43.02μmol·L-1,76.59 μmol·L-1 respectively.3)The straight lines of different concentrations of inhibitors plotted by Lineweaver-Burk equation intersected at the third quadrant.It can be judged that the inhibitory effects of clarithromycin and erythromycin on nimodipine metabolism were between non-competitive and anticompetitive inhibitions.Conclusion 1.Established the determination of nimodipine with HPLC method in incubation system;2.Nimodipine metabolism was linear with different concentration of human microsomes(0.25-1 mg·mL-1)between five and thirty minutes.Enzyme kinetic parameters Vmax and Km were 29.64 μmol·L-1,8.69 nmol respectively.3.Clarithromycin and erythromycin had weak inhibitions on nimodipine metabolism.Roxithromycin seldom inhibited nimodipine metabolism.4.[I]/Ki values of clarithromycin and erythromycin were about 0.1,the risk of drug interactions in vivo was low.5.The inhibitory effects of clarithromycin and erythromycin on nimodipine metabolism were between non-competitive and anticompetitive inhibitions.