Intestinal Human Colon Adenocarcinoma Cell Line LS180 Is an Excellent Model to Study Pregnane X Receptor, but Not Constitutive Androstane Receptor, Mediated CYP3A4 and Multidrug Resistance Transporter 1 Induction: Studies with Anti-Human Immunodeficiency Virus Protease Inhibitors

Abstract

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 M) 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 (ϳr ‫؍‬ 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.To date, much of the focus on clinically significant drug interactions has been on metabolic-based inhibitory drug interactions. However, there is increasing appreciation that inductive drug interactions are more common than previously thought, and such interactions can also extend to transporters. For this reason, the U.S. Food and Drug Administration has recently issued draft guidance on models to study inductive drug interactions. This draft guidance states that "At this time, the most reliable method to study a drug's induction potential is to quantify the enzyme activity of primary hepatocyte cultures following treatments including the potential inducer drug, a positive control inducer drug [rifampin for CYP3A4], and vehicle-treated hepatocytes (negative control), respectively"

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

“…As well, results of MTT cell viability assays suggest that the concentrations of these agents used do not affect cell viability (data not shown). In the present study, P-gp induction in hCMEC/D3 cells mediated by treatment with ritonavir, atazanavir, lopinavir, darunavir, nevirapine, and efavirenz supports previous findings demonstrating that these drugs were able to induce P-gp expression in lymphocytes and intestinal and hepatic tissues (24,(40)(41)(42)(43)(44)(45)(46). Interestingly, efavirenz is the only drug that appears to serve as a ligand of both hPXR and hCAR.…”

“…In addition, efavirenz, a nonnucleotide reverse transcriptase inhibitor (NNRTI), is capable of inducing the promoter activity of the phase I drug-metabolizing cytochrome P450 enzyme 3A4 when transfected with a full-length hPXR plasmid (23). Moreover, Svärd et al used a panel of antiretroviral drugs to screen for CYP3A4 and CYP2B6 promoter activation mediated by hPXR and hCAR; however, they reported several discrepancies in receptor activation by several HIV PIs (i.e., nelfinavir, ritonavir, and atazanavir) compared to previously published reports (22,24). The aims of this study were (i) to determine whether several antiretroviral drugs currently used in first-line and alternative regimens during HIV pharmacotherapy are ligands for hPXR or hCAR and (ii) to examine P-gp function and expression in human brain microvessel endothelial cells following treatment with antiretroviral drugs identified as ligands of hPXR and hCAR.…”

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

“…In fact, RTV treatment decreased intestinal CYP3A activity by ϳ78% (f Cl int GI ϭ 0.22, 90% CI of 0.10 -0.48) (Table 4), consistent with that expected on the basis of normal intestinal enzyme recovery over the 12-h dosing interval. RTV is known to activate PXR, thereby inducing transcription of CYP3A and other genes (Gupta et al, 2008). If synthesis of CYP3A was induced as little as 3-fold, recovery of ϳ90% of baseline CYP3A activity would be expected (f Cl int GI of 0.9).…”

Bortezomib, carfilzomib and ixazomib do not mediate relevant transporter-based drug-drug interactions

“…The LS180 human colon adenocarcinoma cell line (American Type Culture Collection, Manassas, USA) is one model frequently used for investigating pregnane-X-receptor (PXR) and aryl hydrocarbon receptor mediated induction (22)(23)(24), and was used as an induction model in the present study. LS180 cells were cultured under standard cell culture conditions at 37˚C, 5% CO 2 and 100% humidity in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 0.1 mM nonessential amino acids.…”