Human ATP-binding cassette transporter ABCB11 (SPGP/BSEP) mediates the elimination of bile salts from liver cells and thereby plays a critical role in the generation of bile flow. In the present study, we have developed in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis methods to investigate the interaction of ABCB11 with a variety of drugs. Plasma membrane vesicles prepared from insect cells overexpressing human ABCB11 were used to measure the ATP-dependent transport of [14C]taurocholate. Over 40 different drugs and natural compounds were tested to evaluate their interaction with ABCB11-mediated taurocholate transport. On the basis of the extent of inhibition, we have analyzed the QSAR to identify one set of chemical fragmentation codes closely associated with the inhibition of ABCB11. This approach can be used to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

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To produce a large amount of CYP3A4, we applied a jarfermenter (ABLE, BMJ-PI or BMS-PI) to culture the genetically engineered E. coli cells harboring CYP3A4 along with NADPH-cytochrome P450 reductase (OR). The jarfermenter is a stirred bacterial culture vessel in which the pH, the dissolved oxygen (DO) and the temperature of a culture medium can be controlled. The expression of CYP3A4 in E. coli cells in the 500 mL of culture medium contained in the BMJ-PI (1 L vessel) (JFM-1) was examined by altering the parameters mentioned above. The highest expression of CYP3A4 in E. coli cells was attained when cultured at pH 6.0, at 30 degrees C under the DO of 0.1 ppm. The incubation was performed 18 hr after the addition of 1.5 mM isopropyl beta-D(-)-thiogalactopyranoside. The expression levels of CYP3A4 and the OR in the membrane fraction of E. coli cells were 267 nmol/L culture and 552 units/L culture, respectively. The CYP3A4 level was about three times higher than that obtained by incubation in a 500 mL flask (100 mL of medium) (84 nmol/L culture). The testosterone 6beta-hydroxylase activity of CYP3A4 expressed in the membrane fraction of E. coli obtained with the JFM-1 was examined. The apparent K(m) and V(max) values were 66.4 microM and 57.8 nmol/min/nmol CYP, respectively. Expecting the mass production of the CYP3A4 by a culture of E. coli, the possibility of a scale up of the culture with the BMS-PI (10 L vessel) (JFM-10) was examined. The optimal culture condition to achieve the highest expression of CYP3A4 with JFM-1 was employed. The expression levels of CYP3A4 and the OR obtained with JFM-1 and JFM-10 were almost equal. The total level of CYP3A4 obtained by using JFM-10 (5 L of medium) was calculated to be about 1.4 mumol. Based on these results, we confirm that the jarfermenter is a useful tool to produce large amounts of CYP3A4.

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