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3. Synergistic action of CYP3A4 and P-glycoprotein

CYP3A4 is the most prominent oxidative cytochrome P450 en-zyme present in human enterocytes. Despite the lower CYP3A4 con-tent in the intestine relative to the liver, first-pass metabolism in the intestine by CYP3A affects a large number of drugs. CYP3A4 has
conclusively been shown to be important in the disposition of midazolam [158] and cyclosporine [159] from studies in anhepatic patients. Drug interaction studies performed with grapefruit juice (inhibitor of intestinal CYP3A) have also shown significant increases in the oral bioavailability of many CYP3A4 substrates including felodipine [160]. As discussed above, drug absorption can also be im-paired by efflux transporters in the intestine [161].

The synergistic action of CYP3A and P-glycoprotein in limiting oral drug delivery is suggested by their joint presence in small intestine enterocytes, the significant overlap in their substrate specificities and the poor oral bioavailability of joint substrates for CYP3A and P-glycoprotein [162]. These proteins are induced or inhibited by many of the same compounds [163,164]. In the enterocyte, the spatial separation of P-gp, located on the apical plasma membrane, and that of CYP3A4, located in the endoplasmatic reticulum, supports the idea that P-gp may control the access of drugs to intracellular metabolism by CYP3A4. Drugs absorbed into the intestinal epithelium can interact with P-gp and be actively extruded back into the intestinal lumen. If this process of diffusion and active transport occurred repeatedly, the circulation of the drug from the lumen to the intracellular com-partment would potentially prolong the intracellular residence time of the drug, decrease the rate of absorption, and result in increased drug metabolism by CYP3A4 relative to the parent drug crossing the intestine (Fig. 2). Support for this hypothesis was obtained from a simulation model [165] as well as experimental studies examining the metabolism and transport of indinavir in vitamin D3-induced Caco-2 cells [166]. Further studies by Cummins et al. [161] were designed to identify the contribution of P-gp in determining the ex-tent of CYP3A4 drug metabolism using selective CYP3A4/P-gp sub-strates and inhibitors in CYP3A4-transfected Caco-2 monolayers. Two compounds were tested: one a dual P-gp and CYP3A4 substrate (K77: N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl) and the other only a CYP3A4 substrate (felodipine). The substrates were administered with the inhibitors cyclosporine (dual inhibitor of P-gp and CYP3A4) or GG918 (inhibitor of P-gp and not CYP3A4). When P-gp alone was inhibited, the extent of metabolism of the dual CYP3A4 and P-gp substrate was decreased, but there was no change in the extent of metabolism for the exclusive CYP3A4 sub-strate. These data indicate that P-gp, when active, can work in concert

with CYP3A4 to increase metabolism. Studies using the in vivo rat sin-gle pass intestinal perfusion model were performed to determine whether a similar drug metabolism–efflux alliance was present in vivo[167]. The results obtained were the first to show the specific in-teraction of P-gp with CYP3A in this isolated organ and support the proposed interplay between P-gp and CYP3A in the intestine [167].

As stated above, in the intestine, inhibition of the efflux transport-er P-gp with no effect on the enzyme will decrease metabolism. Thus, when both the enzyme and the efflux transporter are inhibited, a sig-nificant decrease in metabolism will be observed with the achieve-ment of higher AUCs. These results revealed that metabolism can be altered by changes that occur only in drug transport. Incorporating ef-flux, as well as uptake processes, affecting drug absorption and dispo-sition should lead to better predictions of drug clearances from in vitro systems [168].

While the interplay between P-gp and CYP3A4 has been extensively studied, this phenomenon may also occur with other enterocytic drug metabolizing enzymes (CYP3A4, CYP2C9, CYP2C19, CYP2C8, CYP2D6,

Fig. 3. The Biopharmaceutics Drug Disposition Classification System (BDDCS) after Wu and Benet.

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