Abstract
Lack of an established cell line model to study induction of cytochromes P450 (P450s) and drug transporters poses a challenge in predicting in vivo drug-drug interactions. Although not well characterized, LS180 cells could be an excellent cell line to study induction of P450s and transporters because they express pregnane X receptor (PXR). Therefore, as part of a larger study of in vitro to in vivo prediction of inductive drug interactions, we determined induction of various P450s and drug transporters by the anti-human deficiency virus protease inhibitors (PIs) and the prototypic inducer, rifampin, in LS180 cells. Among these proteins, the various PIs significantly induced (n = 3–5) only CYP3A4 and multidrug resistance transporter 1 (MDR1) transcripts (2- to 50-fold). CYP3A4 activity (1′-hydroxymidazolam formation) was increased (2-fold) by rifampin (10 μM) but was reduced by the PIs (1.5- to 7-fold). Surprisingly, constitutive androstane receptor 1 (CAR1) was not found to be expressed in these cells. Additionally, using a reporter assay, we found that PIs did not activate CAR3 (the natural splice variant of CAR1) but significantly activated PXR (2- to 24-fold), which correlated well with induction of CYP3A4 and MDR1 transcripts (∼r = 0.9). Furthermore, in a PXR-knockdown stable LS180 cell line, induction of CYP3A4 and MDR1 mRNA after treatment with PIs and rifampin was significantly reduced (1.4- to 5-fold) compared with that in PXR nonsilenced cells. Based on these data, we conclude that LS180 cells could be used as a readily available, high-throughput cell line to screen for PXR-mediated induction of CYP3A4 and MDR1 transcripts. These data also indicate that the majority of the PIs are likely to produce intestinal drug-drug interactions by inactivating or inhibiting CYP3A enzymes even though they induce CYP3A4 and MDR1 transcripts via PXR.
Footnotes
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This work was supported by National Institutes of Health Grant GM032165. Parts of this work were presented in abstract form (Proceedings of the 14th North American ISSX Meeting; 2006 Oct 22–26, Rio Grande, Puerto Rico; International Society for the Study of Xenobiotics, Washington, DC; Proceedings of the American Association of Pharmaceutical Scientist Annual Meeting and Exposition; 2007 Nov 12–14, San Diego, CA; American Association of Pharmaceutical Scientists, Arlington, VA).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.018689.
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ABBREVIATIONS: P450, cytochrome P450; PI, anti-human immunodeficiency virus protease inhibitor; P-gp, P-glycoprotein; MDR1, multidrug resistance transporter 1; OH, hydroxy; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; DMSO, dimethyl sulfoxide; ATCC, American Type Culture Collection; PXR, pregnane X receptor; CAR, constitutive androstane receptor; CAR3, natural splice variant of CAR1; RXR, retinoid X receptor-α; FBS, fetal bovine serum; shRNA, short hairpin RNA; PCR, polymerase chain reaction; GFP, green fluorescent protein; qPCR, quantitative PCR; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistance protein; OATP, organic anion transporter; MDZ, midazolam; MTT, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; RTV, ritonavir; NFV, nelfinavir; APV, amprenavir; IDV, indinavir; SQV, saquinavir; LPV, lopinavir; TPV, tipranavir; ATV, atazanavir; RIF, rifampin; AUC, area under the plasma concentration-time curve.
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↵1 Current affiliation: AstraZeneca Pharmaceuticals LP, DMPK Department, Wilmington, DE.
- Received August 31, 2007.
- Accepted March 5, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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