Yuichi Sugiyama

It has been suggested that cytochrome P450 3A4 (CYP3A4) and MDR1 P-glycoprotein (P-gp) act synergistically to limit the bioavailability of orally administered agents. In order to determine the relative role of these proteins, it is essential to identify a selective inhibitor for either P-gp or CYP3A4. In the present investigation, comparative studies were performed to examine the effect of inhibitors on the function of these proteins. The IC₅₀ of P-gp function, determined by examining the inhibition of the transcellular transport of vinblastine across Caco-2 monolayers, was in the order PSC833 ≪ ketoconazole, verapamil ≪ N-(2(R)-hydroxy-1(S)-indanyl)-5-(2(S)-(1,1-dimethylethylaminocarbonyl)-4-(furo(2,3-b)pyridin-5-yl) methyl)piperazin-1-yl)-4(S)-hydroxy-2(R)-phenylmethylpentanamide (L-754,394). In contrast, the IC₅₀ of CYP3A4 function, determined by examining the inhibition of the metabolism of midazolam by intestinal and liver microsomes, was in the order L-754,384 ≪ketoconazole≪ PSC 833 and verapamil. The ratio of IC₅₀ for P-gp to that for CYP3A4 was more than 200 for L-754,394,60 ∼ 150 for ketoconazole, 1.5 for verapamil, and 0.05 for PSC 833. Collectively, it was demonstrated that PSC 833 and L-754,394 can be used as selective inhibitors of P-gp and CYP3A4, respectively.

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB‐3‐1 derived cell lines, C‐A500 and KCP‐4, was examined; the former expresses multi‐drug resistance‐associated protein (MRP1), whereas the latter is resistant tocis‐diammine‐dichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P‐glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was over‐expressed on KCP‐4. Membrane vesicles isolated from C‐A500 and KCP‐4, but not from KB‐3‐1, exhibited the ATP‐dependent uptake of glutathione conjugates (GS‐X) such as leukotriene C₄and 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), indicating the presence of GS‐X pumps on these cells. The uptake of these GS‐X by membrane vesicles from C‐A500 was approximately twice that in the case of KCP‐4. Kinetic analysis indicated that theK_mandV_maxvalues for DNP‐SG uptake were 2.56 and 1.43 μM, and 570 and 160 pmol/min/mg protein for C‐A500 and KCP‐4, respectively. In marked contrast, significant ATP‐dependent uptake of glutathione‐platinum complex was observed only in membrane vesicles from KCP‐4, but not those from KB‐3‐1 and C‐A500. The transport properties of estradiol‐17β‐d‐glucuronide (E₂17βG) were also different between the two cell lines. This was reflected in the findings that the ATP‐dependent uptake of this conjugated metabolite in membrane vesicles from C‐A500 (K_m= 2.33 μM,V_max= 34 pmol/min/mg protein) was much more extensive than that in the case of KCP‐4 (K_m= 5.5 μM,V_max= 35 pmol/min/mg protein), and that comparable uptake was observed between KCP‐4 and KB‐3‐1. Overall, a clear difference in substrate specificity among GS‐X pump family members expressed on resistant tumor cells was demonstrated.

Purpose. To evaluate the effects of gut metabolism and efflux on drug absorption by simulation studies using a pharmacokinetic model involving diffusion in epithelial cells. Methods. A...