Inhibitory effects of CYP3A4 substrates and their metabolites on P-glycoprotein-mediated transport

Abstract

It is generally known that the substrates and/or inhibitors of cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp) overlap with each other. In intestinal epithelial cells, it is surmised that the metabolites coexist with their parent drug. However, most studies on P-gp did not take the effects of those metabolites into consideration. Therefore, in the present study, we investigated the inhibitory effects of five substrates of CYP3A4 (nifedipine, testosterone, midazolam, amiodarone, and azelastine) and their metabolites on the P-gp-mediated transcellular transport. The transcellular transports of [³H]daunorubicin or [³H]digoxin by monolayers of LLC-GA5-COL150 cells in which P-gp was overexpressed were measured in the presence or absence of the CYP3A4 substrates and their metabolites. Nifedipine, testosterone, midazolam, and their metabolites exhibited no effects on the P-gp-mediated transport of [³H]daunorubicin and [³H]digoxin. On the other hand, the transport of [³H]daunorubicin was strongly inhibited by amiodarone, desethylamiodarone, azelastine, and desmethylazelastine, with IC₅₀ values of 22.5, 15.4, 16.0 and 11.8 μM, respectively. The transport of [³H]digoxin was also strongly inhibited by these compounds, with IC₅₀ values of 45.6, 25.2, 30.0 and 41.8 μM, respectively. Another metabolite of azelastine, 6-hydroxyazelastine, exhibited no effects on these transports. It was suggested that the CYP3A4 metabolites of which their parent drug exhibited inhibition on the P-gp-mediated transport are possibly also inhibitors. It would be possible more complicated drug–drug interactions would be caused by the metabolites as well as their parent drugs in the liver and the intestine via the inhibition of CYP3A4 and P-gp.

Introduction

Oral drug absorption has been recognized to be determined by intestinal metabolism and active extrusion of the absorbed drug (Benet et al., 1996). In the small intestine, P-glycoprotein (P-gp) is co-localized in a polarized fashion at the apical membrane of cells in which cytochrome P450 (CYP) 3A4 also expressed (Gottesman and Pastan, 1993). P-gp is a transporter responsible for the efflux of drugs such as chemotherapeutic agents (Gottesman and Pastan, 1993). The efflux runs countercurrent to the absorptive transport or diffusion of drugs, thereby restricting the extent of oral absorption. CYP3A4 is an important enzyme in drug metabolism in humans accounting for approximately 30% of hepatic CYP (Shimada et al., 1994), and is predominant in small intestinal CYP (Zhang et al., 1999). Moreover, it is well-known that about 50% of drugs currently in the market are substrates for CYP3A4 (Guengerich, 1996). The intestinal CYP3A4 presents a metabolic barrier to drug absorption. It has been surmised that P-gp and CYP3A4 act synergistically to presystemic drug metabolism, although the mechanisms of restricting absorption may be different (Gan et al., 1996, Watkins, 1997, Wacher et al., 1998, Ito et al., 1999). Therefore, a substrate of P-gp would repeatedly circulate between the lumen and epithelial cells, leading to prolonged exposure to CYP3A4, resulting in reduced absorption of the drug into the blood.

It has been reported that the substrates and/or inhibitors of CYP3A4 and P-gp overlap (Wacher et al., 1995). In the intestinal epithelial cells, it is expected that the metabolites coexist with their parent drug. The purpose of this study was to investigate the inhibitory effects of the metabolites as well as substrates of CYP3A4 on the P-gp-mediated transport. Five substrates of CYP3A4 of which structures are diverse were selected. Nifedipine, testosterone, and midazolam were used as well-known substrates of CYP3A4. Amiodarone and azelastine were also selected, since these drugs have been clarified to be mainly metabolized by CYP3A4 in our laboratory.