Vol. 28, Issue 10, 1238-1243, October 2000

Selective Inhibition of Human Cytochrome P450 3A4 by 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 and P-glycoprotein by Valspodar in Gene Transfectant Systems

Iichiro Kawahara, Yukio Kato, Hiroshi Suzuki, Meguru Achira, Kiyomi Ito, Charles L. Crespi, and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (I.K., Y.K., H.S., M.A., K.I., Y.S.); and Gentest Corporation, Woburn, Massachusetts (C.L.C.)

Our previous report showed that L754.394 and valspodar (PSC833) are potent inhibitors of midazolam hydroxylation in human jejunum microsomes and vectorial transport of vinblastine in Caco-2 cells, respectively. In the present study, to directly examine the interactions of these compounds as well as other substrates with CYP3A4 and P-glycoprotein (P-gp), we performed in vitro inhibition studies using recombinant CYP3A4-expressed microsomes and an MDR1-transfected cell line, LLC-MDR1, respectively. In CYP3A4-expressed microsomes, both L754.394 and ketoconazole, at a concentration less than 0.5 µM, are the most potent inhibitors of the formation of 1'-hydroxymidazolam, a major metabolite of midazolam formed by CYP3A4. The greatest inhibitory effect on the transcellular transport of digoxin in LLC-MDR1 cells was observed in the presence of valspodar (<0.1 µM), followed by verapamil. From a comparison of the IC₅₀ values, it was shown that L754.394 and valspodar exhibited the highest selectivity for CYP3A4 and P-gp, respectively. To demonstrate such specificity, both midazolam hydroxylation and digoxin transport were observed in CYP3A4 transfected Caco-2 cells, which coexpress both P-gp and CYP3A4, in the presence or absence of L754.394 (0.5 µM) and valspodar (1.0 µM). L754.394 almost completely inhibited midazolam hydroxylation, but not digoxin transport, whereas almost complete inhibition of digoxin transport was observed in the presence of valspodar, but inhibition of the hydroxylation was minimal. Thus, the present study has demonstrated that L754.394 has a specific inhibitory effect on CYP3A4, whereas valspodar is specific for P-gp.