A comprehensive in vitro and in silico analysis of antibiotics that activate pregnane X receptor and induce CYP3A4 in liver and intestine

Abstract

We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6beta-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (> 10-fold) by rifampin and troleandomycin; moderately (> or = 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (> or = 4-fold) erythromycin; and weakly (> 2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (> 2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.

Fig 1

Effect of penicillins and sulfonamides on CYP3A4 protein expression and activity in human hepatocytes and in LS180 cells. Primary human hepatocytes (HH) from donors 1180, 1183, 1188 and 1201 were treated with vehicle (DMSO), RIF, penicillins, or sulfonamides at the indicated concentrations for 48 h. HH were incubated with testosterone immediately before harvest, and the medium was analyzed for the formation of 6β-hydroxytestosterone. The fold change (mean ± S.D. (top panels a,c) in 6β-hydroxytestosterone formation rate (nmol/min/mg protein) for drug vs vehicle treated cells (in triplicate) of a representative experiment are shown. The same cells were lysed and 5 µg analyzed by immunoblot for CYP3A4 (bottom panels a, c). LS180 cells stably expressing PXR.1 were treated for 48 h with the indicated concentrations of drug or 0.1% DMSO as the vehicle control (CT). Total cell lysate (25 µg) was analyzed by immunoblot analysis for CYP3A4 protein (panels b,d). Abbreviations for drugs are indicated in Supplemental Table 1.

Fig 2

Effect of cephems and macrolides on CYP3A4 protein expression and activity in human hepatocytes and in LS180 cells. Primary human hepatocytes (HH) from donors 1180, 1183, 1188 and 1201 were treated with vehicle (DMSO), RIF, penicillins, or sulfonamides at the indicated concentrations for 48 h. HH were incubated with testosterone immediately before harvest, and the medium was analyzed for the formation of 6β-hydroxytestosterone. The fold change (mean ± S.D. (top panels a,c) in 6β-hydroxytestosterone formation rate (nmol/min/mg protein) for drug vs vehicle treated cells (in triplicate) of a representative experiment are shown. The same cells were lysed and 5 µg analyzed by immunoblot for CYP3A4 (bottom panels a, c). LS180 cells stably expressing PXR.1 were treated for 48 h with the indicated concentrations of drug or 0.1% DMSO as the vehicle control (CT). Total cell lysate (25 µg) was analyzed by immunoblot analysis for CYP3A4 protein (panels b,d). Abbreviations for drugs are indicated in Supplemental Table 1.

Fig 3

Effect of tetracyclines, clindamycin, and griseofulvin on CYP3A4 protein expression and activity in human hepatocytes and in LS180 cells. Primary human hepatocytes (HH) from donors 1180, 1183, 1188 and 1201 were treated with vehicle (DMSO), RIF, penicillins, or sulfonamides at the indicated concentrations for 48 h. HH were incubated with testosterone ilmediately before harvest, and the medium was analyzed for the formation of 6β-hydroxytestosterone. The fold change (mean ± S.D. (top panels a,c) in 6β-hydroxytestosterone formation rate (nmol/min/mg protein) for drug vs vehicle treated cells (in triplicate) of a representative experiment are shown. The same cells were lysed and 5 µg analyzed by immunoblot for CYP3A4 (bottom panels a, c). LS180 cells stably expressing PXR.1 were treated for 48 h with the indicated concentrations of drug or 0.1% DMSO as the vehicle control (CT). Total cell lysate (25 µg) was analyzed by immunoblot analysis for CYP3A4 protein (panels b,d). Abbreviations for drugs are indicated in Supplemental Table 1.

Fig 4

Molecules mapped to PXR pharmacophores. A. nafcillin B. dicloxacillin mapped to the original PXR pharmacophore; C. nafcillin D. dicloxacillin mapped to the diverse (n=31) PXR pharmacophore; E. dicloxacillin, F. clindamycin, G. griseofulvin mapped to the steroidal (n=30) PXR pharmacophore. Spheres represent: Hydrophobic features (cyan), hydrogen bond acceptor and vector (green), hydrogen bond donor and vector (purple).