Effect of 1-aminobenzotriazole on the in vitro metabolism and single-dose pharmacokinetics of chlorzoxazone, a selective CYP2E1 substrate in Wistar rats

Abstract

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics (V_max and K_m) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486 pmol mg protein⁻¹ min⁻¹ and 345 µM), human liver microsomes (3194 pmol mg protein⁻¹ min⁻¹ and 335 µM) and pure isozyme (3423 pmol mg protein⁻¹ min⁻¹ and 403 µM), but K_I and K_inact values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 µM and 0.12 min⁻¹; human: 27.19 µM and 0.14 min⁻¹) and pure isozyme (3.18 µM and 0.29 min⁻¹). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was ∼71% when dosed alone and reached 100% under pre-treatment with ABT. The t_1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h post-treatment.

Keywords :

• Chlorzoxazone
• hydroxychlorzoxazone
• 1-aminobenzotriazole
• rats
• pharmacokinetics
• in vitro metabolism
• enzyme kinetics