In Vitro Metabolism And Transport Study Of Zolmitriptan

Cytochrome P450 (CYP) is one of the most important representatives of drug metabolism enzymes, and plays a key role in the metabolic inactivation/activation of numerous exogenous and endogenous. CYP exists mainly in liver microsomes. CYP superfamily shows wide range, poor selectivity, large structure difference, inter-individual variation, overlapping substrate specificities in exogenous metabolism and can be easily induced and inhibited by a number of chemical compounds. There is, therefore, a great probability of competition between drugs and endogenous compounds for the same enzyme, between different enzymes for the same substrate and between two drugs for the same enzyme. Meanwhile, many drugs are not only the substrate of the enzyme, but also the inducer or inhibitor of the enzyme or others. This may change the pharmacological and toxicological effects of drug itself or other drugs, which is one of the reasons resulting in drug interaction in pharmacokinetics.Caco-2 cell line was originally isolated from a human colon adenocarcinoma, and was widely used as an in vitro model of the human intestinal mucosa. And Caco-2 cells also can be used to characterize the absorption of oral drugs, because the cells exhibit both morphological and functional similarities to the human small intestinal epithelial cells such as cytochrome p450 enzymes and p-glycoprotein.Zolmitriptan is a novel and highly selective 5-HT₁b/1d receptor agonist used as an acute oral treatment for migraine. The in vitro study of zolmitriptan was very limited. So this paper has investigated both metabolism and absorption of zolmitriptan in vitro. This research can provide some scientific evidences, which can instruct clinical secure and efficient medication.This study was divided into two chapters and four parts. Chapter one:Part one: To investigate the in vitro metabolism mechanism of zolmitriptan and the possible drug-drug interactions at enzyme level, and to instruct the reasonable drug administration in clinic. METHODS: Using HPLC method to assay the concentration of zolmitriptan, and using pretreated rat liver microsomal and human liver microsomal models to study the metabolism and interaction of zolmitriptan in vitro. RESULTS: 1) Zolmitriptan was intensely metabolized in rat liver microsomes induced with BNF, but was slightly metabolized in rat liver microsomes induced with DEX and PB.The enzyme kinetics for the formation of N-demethylzolmitriptan from zolmitriptan in rat liver microsomes pretreated with BNF were 96±22umol-L"1(#m), 11 ±3 pmolmin^-mg protein"1 (Vmax) and O.^^.t^ulmin^-mg protein"1 (CLint) . Fluvoxamine and diphenytriazol inhibited zolmitriptan iV-demethylase activity catalyzed by CYP1A2 {K\ = 3.8 ± 0.3 and 3.2 ± O.lumol-L1, respectively). Diazepam and propranolol elicited a slight inhibitory effect on the metabolism of zolmitriptan (K{ = 70±ll and 90±18umol-L"1, respectively). Cimetidine and moclobemide produced no significant effect on the metabolism of zolmitriptan. Fluvoxamine is a mechanism-based inactivator of CYP1A2. 2) i. Propafenone enantiomer, niguldipine enantiomer, propranolol entiomer and verapamil enantiomer can all inhibite the N-demethylate metabolism of zolmitriptan. The inhibited effects were all stereoselectivity. ii. The study has established a chiral high-performance liquid chromatography method to detect the concentration of zolmitriptan enantiomers. The results indicated that the metabolism rate of S-zolmitriptan was high than that of R-zolmitriptan. There was significant difference between the IC5o of R- to S-zolmitriptan and S- to R-zolmitriptan (IC5os/r/IC5or/s=45.2). iii. The metabolism of zolmitriptan enantiomer in human liver microsomes was also stereoselective. The metabolism rate of S-zolmitriptan was high than that of R-zolmitriptan. 3) There were no significant inhibitive interactions between zolmitriptan and several major human cytochrome P450, CYP1A2, 2B, 2C9, 2C19, 2D6 and 3A4. (IC5o?100 umol-L1). CONCLUSION: The metabolism of zolmitriptan was involved in CYP1A2 both in rat liver microsomes and human liver microsomes,and the main metabolite was N-demethylzolmitriptan. The metabolism rate of zomitriptan enantimer was stereoselective, and the disposition process favored the S-form of zolmitriptan. Fluvoxamine, diphenytriazol, diazepam, propranolol, propafenone, niguldipine and verapamil can all inhibite the metabolism of zolmitriptan. It suggests that the pharmacokinetics and pharmacodynamics of zolmitriptan may be changed when zolmitriptan was coadministrated with these drugs in clinic, so we must consider it.Part two: To study the effect of zolmitriptan on the expression of cytochrome P450 in rat liver. METHODS: Using probe substrates to determine the change of several major cytochrome P450 activities and using RT-PCR to evaluate the degree of mRNA induction by zolmitriptan. RESULTS: Zolmitriptan can't improve the activities of CYP1A2, 2B, 2C and 2E1 in both male and female Sprague-Dawley rat liver, and can't improve the activities of CYP3A1 and CYP3A2 in female Sprague-Dawley rat liver. But the male rat liver microsome induced with zolmitriptan, as that induced with dexamethasone, can potently metabolize testosterone, diazepam and nifedipine. Both of the expression level of CYP3A1 and CYP3A2 mRNA was significantly improved in male rat liver pretreated by zolmitriptan, but not improved in female rat liver pretreated by zolmitriptan. CONCLUSIONS: Zolmitriptan was an inducer of CYP3A1 and CYP3A2 in male rat liver.Chapter two:Part one: To investigate the characteristic of uptake of zolmitriptan, a novel andhighly selective 5-HT ib/id receptor agonist, and the interaction between some drugs and zolmitriptan on human epithelial cell line (Caco-2). METHODS: Caco-2 cell was used. RESULTS: The study demonstrated that zolmtriptan uptake significantly depended upon the extracelluar temperature and pH. The zolmitriptan uptake at 39 °C was 2.1 fold as that at 23 °C and the zolmitriptan uptake at pH 8.0 was 2.7 fold as that at pH 6.0. The initial uptake rates from the apical side (AP) to the basolateral side (BL)at the concentration from 1 to 25mmol-L"1 were saturable with a V^ of (75.6±26.4) pinol-h'1 ^"'(protein), Km of (15.3±4.5) mmolL"1 and Kd of (1.3±0.4) ml-h'g'Cprotein), but the uptake rates of zolmitriptan from the BL to AP were linear with increasing zolmitriptan concentration. The uptake rates of zolmitriptan from BL to AP were 3-7 times as that from AP to BL. Verapamil, nimodipine, nifedipine, flunarizine, amiloride and sumatriptan significantly increased the uptake rates of zolmitriptan from AP to BL. Propafenone significantly inhibited the uptake rate of zolmitriptan both from AP-BL and from BL-AP. Propranolol and aspirin have no significant effect. There was no stereoselective uptake of the enantiomers of zolmitriptan on both sides of the Caco-2 cells monolayers. Cetirizine and atenolol enantiomers significantly inhibited the uptake rate of zolmitriptan on both sides of the Caco-2 cells monolayers, but there was no stereoselective between them. There was stereoselective between S- and R-propranolol to the uptake of zolmitriptan on both sides of the Caco-2 cells monolayers. CONCLUSION: The zolmitriptan uptake in Caco-2 cell was temperature, pH and concentration dependent, and was partially conteracted by the action of an out wardly directed efflux pump, presumably P-glycoprotein.Part two: To investigate the characteristic of transport of zolmitriptan, a novel and highly selective 5-HT ib/id receptor agonist, and the interaction between some drugs and zolmitriptan on human epithelial cell line (Caco-2). METHODS: Caco-2 cell was used. RESULTS: The study demonstrated that zolmtriptan transport significantly depended upon the extracelluar pH. The apparent permeability coefficient (PapP) of zolmitriptan at pH 8.0 was 3.8 fold greater than that at pH 6.0. The flux of zolmitriptan across the Caco-2 cell layer (apical to basolateral side or basolateral to apical side) was linear with time for up to 2.5 hr and linear with concentration for up to 10 mmol-L"1. The PapP from the basolateral to the apical side was 1.4-2.6 times as that from the apical to the basolateral side. Verapamil, nimodipine and nifedipine can significantly increase the transport rate of zolmitriptan from apical to basolateral side and significantly reduce the transport rate of zolmitriptan from basolateral to apical side, indicating thatP-glycoprotein participates in the transport of zolmitriptan. Flunarizine and propranolol significantly inhibited the transport rate both from apical to basolateral side and from basolateral to apical. The transport of S- and R-zolmitriptan was stereoselective, and S-zolmitriptan was the eutomer. Cetirizine, propranolol and atenolol enantiomers significantly inhibited the transport rate of zolmitriptan on both sides of the Caco-2 cells monolayers, but only propranolol enantiomers had stereoselective inhibit effect. CONCLUSION: The transport of zolmitriptan in Caco-2 cell was pH and concentration dependent and rapid passive diffusion was the major pathway for zolmitriptan absorption. The absorption of zolmitriptan was partially counteracted by the action of an out wardly directed efflux pump, presumably P-glycoprotein. The transport of R- and S-zolmitriptan was stereoselective.