Impact Of Nonspecific Binding On CYP3A4 Kinetics

In drug development, the capability of drug development is dramatically improved by the early ADME study, which makes it possible to predict the in vivo metabolism properties from in vitro dada. But the authenticity of in vitro data and the veracity of the prediction are affected by various in vitro factors. Therefore, it is very important to investigate these factors for developing drug with ideal pharmacokinetical properties. CYP3A4, one of the most important drug metabolizing enzymes, are responsible for the oxidative metabolism of the majority of clinical drugs, and the influence of CYP3A4 activities will affect the in vivo metabolism properties of these chemicals. In this paper, we aim to estimate the impact of nonspecific binding on CYP3A4 kinetics in order to getting exact pharmacokinetic parameters and predicting drug cleanance in vivo exactly. Testosterone (TS) and nifedipine (NIF), the specific probe substrates of CYP3A4, and their specific reactions, testosterone 6β-hydroxylation and oxidation of nifedipine, were selected as indicators of CYP3A4 activities. The nonspecific binding of TS and NIF to rat liver microsomes (RLM) and human liver microsomes (HLM) were studied using equilibrium dialysis and higy-pressure liquid chromatography (HPLC). 　Our results suggested that there are nonspecific binding between the two substrates and liver microsomes. In both of liver microsome systems, the nonspecific binding of TS and NIF to microsomes was insaturable and concentration-independent. Both TS and NIF exhibited an eaqual proportion in a range of substrate concentrations. The free frations of TS were 0.84±0.07 to RLM and 0.75±0.05 to HLM, respectively. And those of NIF were 0.87±0.04 and 0.83±0.03, respectively. The further kinetics studies showed that after removing the influence of nonspecific binding, the sigmodal substrate concentration-velocity curves were shifted left and Eadie-Hofstee plots tend to linear. Nonspecific binding also influenced the estimated kinetics properties and parameters of these two reactions. Although Vmax and Hill coefficent (n) were not changed, S50 were overestimated and CLmax were underestimated. In conclusion, the nonspecif binding is essential for both CYP3A4-mediated mechanism study and accurate estimation of CYP3A4 kinetics parameters. It is suggested that nonspecific binding, as an important factor of enzyme kinetics study, affects the authencity of in-vitro data and the veracity of the prediction of pharmacokinetical characters.