QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.018689v1 36/6/1172    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gupta, A. Articles by Unadkat, J. D. Search for Related Content PubMed PubMed Citation Articles by Gupta, A. Articles by Unadkat, J. D.

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

Department of Pharmaceutics, University of Washington, Seattle, Washington (A.G., K.E.T., J.D.U.); and Department of Pharmacokinetics and Biopharmaceutics, University of Cincinnati Medical Center, Cincinnati, Ohio (G.M.M., P.B.D.)

(Received August 31, 2007; accepted March 5, 2008)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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.

The intestine is a site of expression of P450s (especially CYP3A4/5) and multiple transporters (e.g., P-glycoprotein). As oral administration of drugs results in relatively high concentrations of the drugs in the intestinal lumen, the potential for clinically significant drug interactions (both inhibitory and inductive) at this site is high. Although a lot of attention has been paid to P450-based intestinal drug interactions, there is increasing evidence that transporter-based drug interactions can also occur at this site. Therefore, there is a need to identify models (e.g., cell lines) or systems that can be used to rapidly determine intestinal drug interactions.

Anti-human immunodeficiency virus protease inhibitors (PIs) are some of the most potent drugs available on the market with respect to their propensity to produce drug interactions (Barry et al., 1999; Malaty and Kuper, 1999; Back, 2006). Although a plethora of studies have shown their ability to produce clinically significant P450-based inhibitory drug interactions, their ability to produce inductive drug interactions is less well appreciated (Malaty and Kuper, 1999; Unadkat and Wang, 2000). In addition, there is evidence to suggest that PIs can inhibit and induce P-gp (encoded by MDR1) (Profit et al., 1999; Perloff et al., 2000, 2004; Huang et al., 2001; Storch et al., 2007). Therefore, as part of a larger study of in vitro to in vivo prediction of inductive drug interactions of the PIs, we quantified the induction of P450s and transporters by the PIs in an intestinal cell line, LS180 (this study), in human hepatocytes (Dixit et al., 2007), and in vivo (Kirby et al., 2006a).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. Protease inhibitors (ritonavir, nelfinavir, amprenavir, saquinavir, indinavir, lopinavir, tipranavir, and atazanavir) were obtained from the National Institutes of Health AIDS reagent program. Rifampin was purchased from Bedford Laboratories (Bedford OH). Midazolam and 1'-OH-midazolam were purchased from Cerilliant (Round Rock, TX). CITCO was purchased from BIOMOL International LP (Plymouth Meeting, PA). High-performance liquid chromatography-grade solvents (methanol, acetonitrile, and water) and DMSO were purchased from Fisher Scientific Co. (Pittsburgh, PA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific and were of highest purity available.

Cell Culture and Molecular Biology Reagents. LS180 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA). All of the cell culture reagents, as recommended for growth and maintenance of LS180 cells (e.g., culture medium and trypsin) were purchased from ATCC. Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT). Lipofectamine and Plus reagent and transfection medium (Opti-MEM) were obtained from Invitrogen (Carlsbad, CA). PCR Master Mix, reverse transcription reagents, and TaqMan probes were purchased from Applied Biosystems (Foster City, CA). Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). DNA and RNA extraction kits were purchased from QIAGEN (Valencia, CA). The Luciferase Reporter Assay System and the β-galactosidase assay kit were purchased from Promega (Madison, WI). Other cell culture reagents were purchased from Invitrogen unless otherwise noted.

Plasmid Constructs. The expression plasmid for human PXR (pSG5-hPXRATG) and the reporter plasmid (XREM-CYP3A4)-tk-luc reporter were kindly provided by Dr. Bryan Goodwin (GlaxoSmithKline, Research Triangle Park, NC). Details of the plasmid have been published previously (Goodwin et al., 1999). An expression vector containing β-galactosidase cDNA under T7 promoter, pCH110, was purchased from GE Healthcare (Piscataway, NJ). The expression plasmids for human CAR1, CAR3, and RXR and their respective controls were generously provided by Dr. Curtis Omiecinski (Pennsylvania State University, University Park, PA) (Auerbach et al., 2005). The CYP2B6-PBREM/XREM reporter plasmid was a kind gift from Dr. Hongbing Wang (University of Maryland, Baltimore, MD) (Wang et al., 2003).

Cell Culture. LS180 cells were grown as suggested by ATCC except that they were subcultured using 5 mM EDTA to minimize clumping. Briefly, the cells were maintained in Dulbecco's modified Eagle medium with 10% FBS, 2 mM L-glutamine, 50 units/ml penicillin G, and 50 µg/ml streptomycin.

Creating PXR Knockdown Cell Line in LS180 Cells. The PXR knockdown cell line was created by transfecting lentivirus containing shRNA for PXR. The primer pair designed for PXR shRNA was as follows: 5'-CCG GCG GCA TGA AGA AGG AGA TGA TCT CGA GAT CAT CTC CTT CTT CAT GCC GTT TTT G-3' and 5'-AAT TCA AAA ACG GCA TGA AGA AGG AGA TGA TCT CGA GAT CAT CTC CTT CTT CAT GCC G-3'.

In brief, lentivirus was produced by cotransfection of lentiviral plasmid (pKLO.1) containing shRNA for PXR along with packaging plasmid (ps-PAX2) and envelope plasmid (pMD2.G) in the ratio of 4:3:1, respectively, in HEK293T cells using the calcium phosphate transfection method. Twelve to 15 h after transfection virus was collected, filtered, and stored at –80°C until used. This virus filtrate was then added to adherent LS180 cells in the presence of 8 µg/ml Polybrene (Sigma-Aldrich). A pool of infected cells was selected in the presence of puromycin (1 µg/ml). Endogenous PXR mRNA levels were confirmed by quantitative real-time PCR using TaqMan probes from Applied Biosystems. This cell line was propagated in the presence of puromycin and called PXRsi. Similarly, the negative control cell line, called GFPsi, was created by using shRNA for GFP. Because of the lack of GFP in LS180 cells, shRNA for GFP should not affect the expression levels of any specific target gene in LS180 cells and would be a control for off-target silencing.

Drug Treatment of LS180 or PXRsi or GFPsi Cells for mRNA Analysis. Cells (0.5 million) were seeded in six-well plates and allowed to attach at 37°C in a humidified atmosphere of 5% CO₂. Except for nelfinavir, stock solutions of the PIs or rifampin (500x prepared in DMSO) were diluted in medium before use. Because of limited solubility, nelfinavir stock solution (500x prepared in DMSO) was mixed with β-cyclodextrin (0.05%) before dilution in the medium. Therefore, β-cyclodextrin (0.05%) was added to the medium containing the other drugs as well. Twenty-four hours after cell seeding, media containing various drugs were added to the respective treatment wells at a final DMSO concentration of 0.2%. DMSO at this concentration had no effect on expression levels of various enzymes and was not cytotoxic. The media were not renewed for the duration of the experiment. Cells were harvested at different time points (24, 48, 72, or 96 h), as specified, after treatment, to determine the time point for maximal induction. To determine the level of induction of the target genes by the various PIs, cells were treated with the drug (10 µM) for 96 h followed by RNA extraction. Detailed concentration-response studies were performed for ritonavir, nelfinavir, or rifampin to obtain the EC₅₀ and E_max of induction of CYP3A and MDR1 transcripts. Cells were treated with various concentrations of the drug (0–75 µM) for 96 h and harvested in RNA extraction lysis buffer (provided in an RNeasy Mini Kit). RNA was extracted from the harvested cells using an RNeasy Mini Kit according to the manufacturer's protocol and stored at –80°C for mRNA analysis by real-time assay. The concentration of purified RNA was determined by a spectrophotometer (SmartSpec Plus Spectrophotometer; Bio-Rad, Hercules, CA), as was the purity, using a 260/280 absorbance ratio (ratio of 1.8 to 2.0).

Quantification of mRNA Induction. Of the total RNA, 2 µg (in a 50-µl reaction) was reverse-transcribed into cDNA using Applied Biosystems TaqMan reverse transcription reagents according to the manufacturer's instructions. The resulting cDNA was used for real-time qPCR analysis for all of the target genes.

qPCR assays for various P450 (CYP1A1, 1A2, 2B6, 3A4, 3A5, 2C8, 2C9, 2C19, and 2D6) enzymes and transporters (MDR1, MRP2, BCRP, and OATP1A2) were performed using gene-specific primers and FAM-labeled fluorescent MGB probes in an ABI 7000 Sequence Detection System (Applied Biosystems). β-Glucuronidase was used as the endogenous control. The real-time reaction contained 10 µl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 40 ng of RNA equivalent cDNA, and primers (200 nM) and probes (100 nM) in a final volume of 20 µl. The PCR reaction was as follows: 95°C hot start for 10 min, followed by 40 cycles at 95°C for 15 s and then 60°C for 60 s. Each sample was analyzed in triplicate. The mRNA levels of each test gene were normalized to β-glucuronidase, according to the following formula: C_T (test gene)–C_T (β-galactosidase) =C_T. Thereafter, the relative mRNA induction levels of each gene were calculated using the C_T method: C_T (test gene) –C_T (test gene in the DMSO control) = C_T (test gene). The fold changes of mRNA levels were expressed as the relative expression 2^–C_T.

CYP 3A Activity Assay. CYP3A activity was measured by estimating 1'-OH-midazolam (MDZ) formation in drug-treated intact cells. Cells (0.5 million) were seeded in a six-well plate in Dulbecco's modified Eagle's medium containing 10% FBS. After 24 h, cells were treated with DMSO (vehicle) or various drugs as described above. After a 96-h drug treatment, cells were washed twice with phosphate-buffered saline and then incubated in Dulbecco's modified Eagle's medium (without FBS) at 37°C for 4 h (time optimized in a time course study). After washing, the cells were incubated with media containing 8 µM MDZ. The media were sampled at 1 h and stored at 4°C pending analysis for 1'-OH-MDZ. In any given experiment, each reaction was performed in duplicate. A calibration curve ranging from 2.5 to 250 ng/ml of 1'-OH-MDZ was prepared in blank cell media. The samples were subjected to liquid-liquid extraction and the amount of 1'-OH-MDZ was measured by liquid chromatography-mass spectrometry as described previously (Kirby et al., 2006b) with the following minor modifications. To a 0.5-ml aliquot of cell lysate, a stable labeled internal standard (d₅-1'-OH-midazolam, m/z 346.9, 25 ng) was added. Samples were acidified with 100 µl of ammonium hydroxide and vortexed. Then, 5 ml of n-tert-butyl ether were used for extraction.

PXR, CAR1, or CAR3 Activation Assays. For the PXR activation assay, LS180 cells were seeded into 24-well plates at a density of 2 x 10⁵ cells/well. After 24 h of plating, overnight transfection was performed using Lipofectamine and Plus reagent, as per the manufacturer's protocol. Briefly, the transfection mixes contained 100 ng of human PXR expression vector (pSG5-humanPXR), 400 ng of luciferase reporter gene construct (XREM-CYP3A4 -tk-luc), and 400 ng of pCH110 (an expression vector containing β-galactosidase cDNA under T7 promoter). After transfection, plasmid-containing media were replaced with drug- or DMSO-containing media (replaced after 24 h) and incubated for 48 h. The final concentration of DMSO (0.2% v/v) was kept constant in each sample. In every experiment, rifampin (10 µM) was included as a positive control. At the end of 48 h, the cell layers were washed twice with ice-cold phosphate buffer saline (pH 7.4), scraped, and collected in 200 µl of reporter lysis buffer provided with the β-galactosidase assay system (Promega). Ten microliters of the cell lysate was used to determine the luciferase activity with a Luciferase Assay System (Promega). An aliquot of cell lysate (50 µl) was used to determine the β-galactosidase activity according to the manufacturer's recommendation. Luciferase activity was normalized to the β-galactosidase activity and expressed as fold activation with respect to the solvent (0.2% DMSO)-treated controls. The potency (EC₅₀) and maximal activation of PXR (E_max) by ritonavir, nelfinavir, or rifampin were determined by treating the transfected cells with various concentrations (0.1–50 µM) of the drug. PXR activation by the remaining PIs was studied at only 10 µM.

For CAR1 and CAR3 activation assays, the procedure was similar to the PXR activation assay with the following modifications. For CAR1 or CAR3 activation, 100 ng of human CAR1 (pCMV2-CAR1) or human CAR3 (pCMV2-CAR3) expression plasmid, respectively, was added to the transfection mix containing 200 ng of luciferase reporter plasmid (CYP2B6-PBREM/XREM) and 200 ng of pCH110 (β-galactosidase expression plasmid). To test for RXR-dependent activation, 100 ng of human RXR expression plasmid (pcDNA3.1-RXR) was added to the transfection mix. Negative controls for each experiment involved using the empty plasmid in the transfection mix. After overnight transfection with plasmids (expressing CAR1 or CAR3 along with RXR, 2B6 response elements, and β-galactosidase) using Lipofectamine 2000 (Invitrogen), the cells were treated with the drug-containing medium for 24 h followed by determination of the luciferase activity and β-galactosidase activity as explained above. The data for each set of transfections were normalized to the respective DMSO-treated controls.

Cell Cytotoxicity (MTT) assay. Cell viability was quantified by the colorimetric MTT assay. Cytotoxicity of drugs in LS180 cells was tested in a collagen-coated 96-well plate. Cells (5 x 10⁴/well) were seeded overnight followed by addition of rifampin (0–100 µM) or various PIs (0–50 µM) for 96 h as described above (see Drug Treatment of LS180 or PXRsi or GFPsi Cells for mRNA Analysis). The MTT assay was performed at the end of 96 h as per the manufacturer's instructions.

Cell viability was also tested in LS180 cells after transfection and drug treatment. Collagen-coated 24-well plates (2 x 10⁵ cells/well) were used for toxicity determination. After transfection (as described for the PXR activation assay), cells were treated with various drugs (as described above) for 24 or 48 h. At the end of the treatment, the MTT assay was performed as per the manufacturer's instructions. Values are expressed as a percentage of the control.

Statistical and Data Analysis. Results from real-time assays, reporter gene assays, and the CYP3A activity assay are expressed relative to those observed in the vehicle control. Data are presented as mean ± S.D. of three to five different experiments (triplicate determinations in each experiment unless otherwise indicated). Where appropriate, statistical comparisons were made on log-transformed data (Figs. 1, 2, 4, 5, and 6) using analysis of variance followed by the Bonferroni correction. The criterion of significance was set at p < 0.05 (or as indicated). For concentration-dependent mRNA induction experiments (Fig. 3), E_max and EC₅₀ were determined using WinNonlin (Pharsight Corporation, Mountain View, CA).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Induction of CYP3A4 and MDR1 transcripts by 10 µM RIF, RTV, or NFV was significant at 24, 48, 72, and 96 h. For RIF, induction of CYP3A4 transcripts was constant over 24 to 72 h and then increased at 96 h (p < 0.05), whereas those of RTV and NFV showed a similar pattern but induction levels at 96 h were not significantly different from that at 24 to 72 h. MDR1 transcripts remained unchanged from 24 to 96 h. Data are presented as mean ± S.D. (n = 4).

View larger version (18K): [in this window] [in a new window]   FIG. 2. When expressed relative to DMSO (A) or 10 µM rifampin (B), PIs (10 µM) significantly induced CYP3A4 () transcripts with RIF, TPV, APV, RTV, and LPV producing >10-fold induction, NFV and SQV producing >5-fold induction, and IDV and ATV producing <5-fold induction. The induction of MDR1 () transcripts was significant (p < 0.05) for rifampin and the PIs (except IDV) with RIF, APV, RTV, LPV, and TPV producing >10-fold induction, NFV and ATV producing >5-fold induction, and SQV producing <5-fold induction. Data are presented as mean ± S.D. (n = 3–5). LS180 cells were treated with various PIs (10 µM) for 96 h, and mRNA levels were measured as described under Materials and Methods.

View larger version (12K): [in this window] [in a new window]   FIG. 4. PIs affect CYP3A activity as measured by 1'-OH-midazolam formation (picograms per microgram of protein). LS180 cells were treated with PIs or rifampin at various concentrations for 96 h, cells were washed for 4 h, and MDZ activity was measured as described under Materials and Methods. Rifampin treatment at 10 µM resulted in a 2-fold increase in CYP3A activity, whereas PI treatment resulted in decreased activity in the following order: ritonavir (1, 5, and 10 µM) > nelfinavir (1, 5, and 10 µM) > saquinavir (10 µM) > lopinavir (10 µM) = tipranavir (10 µM) = atazanavir (10 µM). Indinavir (10 µM) and amprenavir (10 µM) treatment did not affect CYP3A activity. Data are presented as mean ± S.D. (n = 4).

View larger version (9K): [in this window] [in a new window]   FIG. 5. PIs or rifampin activates PXR. LS180 cells were transfected with PXR expression plasmid and CYP3A4-PXRE/XREM followed by treatment with the test drugs for 48 h. Luciferase activity was measured after treatment, and data were normalized to β-galactosidase activity. PIs (10 µM) activated PXR in the following order: ritonavir amprenavir > lopinavir tipranavir saquinavir atazanavir indinavir. Data are expressed relative to DMSO control treatment and presented as mean ± S.D. (n = 3).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Induction of CYP3A4 and MDR1 transcripts is reduced in PXRsi cells when compared with GFPsi cells. PXRsi (PXR levels 20% of control cells) cells were treated for 96 h with moderate to strong inducers (fold induction >5) namely RIF, RTV, NFV, APV, LPV, or TPV. For all drugs, the magnitude of induction of CYP3A4 () and MDR1 () transcripts in PXRsi cells was 20 to 40% of that in control cells. Data are presented as mean ± S.D. (n = 4).

View larger version (9K): [in this window] [in a new window]   FIG. 3. Induction of CYP3A4 and MDR1 transcripts by RIF, RTV, or NFV is concentration-dependent. LS180 cells were treated with RIF (, 0–50 µM), RTV (, 0–25 µM), or NFV (, 0–15 µM) for 96 h, and mRNA levels were measured as described under Materials and Methods. Data (mean ± S.D., n = 3–5) were expressed relative to DMSO. EC50 and Emax were estimated by WinNonlin. The EC50 was comparable among the compounds for MDR1 (1.5 µM) and CYP3A4 (7 µM). The Emax of induction differed in the following order: CYP3A4, RIF > RTV and MDR1, RIF > RTV > NFV. The Emax and EC50 of CYP3A4 transcript induction by nelfinavir could not be estimated because of cytotoxicity at concentrations greater than 15 µM. n.d., could not be determined.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Time Course of Induction of CYP3A4 and MDR1 Transcripts by Ritonavir, Nelfinavir, or Rifampin. At 10 µM, the three drugs induced CYP3A4 mRNA to different extents (3- to 16-fold). This induction reached a plateau between 24 and 72 h and then increased (10- to 30-fold) at 96 h (statistically significant for rifampin but not for ritonavir or nelfinavir) (Fig. 1). In contrast, MDR1 mRNA was maximally induced (4.5- to 14-fold) by all the three drugs at 24 h, and this induction remained stable until 96 h. Because the 96-h time point showed the highest level of induction of CYP3A4 mRNA, all further induction studies were performed over this period. We did not investigate longer time points because of the concern about viability of the cells beyond 96 h.

Induction of CYP3A4 and MDR1 Transcripts by the PIs or Rifampin. Treatment of LS180 cells for 96 h with 10 µM concentrations of various PIs (RTV, NFV, APV, IDV, SQV, LPV, TPV, and ATV) or RIF significantly (n = 3–5) induced CYP3A4 (2- to 28-fold) in the order, RIF TPV APV > RTV LPV > NFV SQV IDV ATV and MDR1 (4- to 20-fold) in the order RIF APV > RTV LPV TPV > NFV ATV SQV (Fig. 2A). Indinavir did not significantly induce MDR1 transcripts. When these data were expressed with respect to induction by 10 µM rifampin, tipranavir and amprenavir were as potent as rifampin (p > 0.05) in inducing CYP3A4 transcripts followed by RTV LPV > NFV > SQV > IDV > ATV. For induction of MDR1 transcripts, ritonavir and amprenavir were as potent as rifampin followed by TPV LPV > NFV ATV > SQV (Fig. 2B). At 10 µM, the transcripts of none of the other P450s and transporters tested were significantly induced by the PIs. This observation occurred despite the fact their transcript expression was robust and easily detected (threshold cycle, C_T ranging from 29–35 as estimated in cDNA synthesized from 40 ng of mRNA in real-time qPCR) and followed the order 2B6 > 2D6 > 2C9 > 1A1 = 1A2 > 3A5 > 3A4 = 2C19 or MDR1 > MRP2 > BCRP. However, the expression of CYP2C8 and OATP1A2 transcripts was low or undetectable. At the concentrations used, the cytotoxicity (determined by the MTT assay) of the drugs was minimal with estimated cell survival of 85 to 100%.

Concentration-Dependent Induction of CYP3A4 and MDR1 by Rifampin, Ritonavir, or Nelfinavir in LS180 Cells. To allow future in vitro to in vivo prediction of P450- and transporter-based inductive drug interactions by ritonavir and nelfinavir, we conducted a concentration-dependent study to estimate the E_max and EC₅₀ of induction of CYP3A4 and MDRI transcripts by these PIs and rifampin. Transcript levels of CYP3A4 and MDR1 were determined in cells treated with varying concentrations of ritonavir (0–25 µM), nelfinavir (0–15 µM), or rifampin (0–50 µM). The concentration ranges for the three drugs were based on obtaining greater than 85% cell survival (estimated by MTT assay) in the presence of the drugs. All three drugs induced CYP3A4 and MDR1 transcripts in a concentration-dependent manner (Fig. 3). E_max and EC₅₀ of CYP3A4 transcript induction by nelfinavir could not be estimated because of cytotoxicity observed at the higher concentrations (>15 µM) of the drug. The EC₅₀ values of induction of CYP3A4 (7 µM) or MDR1 (1.5 µM) transcripts by ritonavir and rifampin were remarkably similar, with MDR1 transcripts being more inducible at a lower concentration of the drugs than CYP3A transcripts. The maximal induction of transcripts, as determined by E_max (Fig. 3), differed between the drugs and decreased in the order RIF > RTV for CYP3A4 and RIF > RTV > NFV for MDR1. Maximal induction of CYP3A4 transcripts was higher than for MDR1 transcripts by rifampin (49- and 24-fold, respectively) at 50 µMorby nelfinavir (15- and 6-fold, respectively) at 15 µM concentration. However, maximal induction of CYP3A4 and MDR1 transcripts by ritonavir (25 µM) was comparable (13 fold).

CYP3A Activity in LS180 Cells in the Presence of Various PIs or Rifampin. To test whether the above induction in CYP3A4 transcripts translated into activity, we optimized a CYP3A activity assay in LS180 cells using midazolam as a substrate. Because the inducers could act as CYP3A inhibitors, after induction and before measuring CYP3A activity, we incubated the cells with drug-free media for 4 h to promote drug efflux (a time determined to be optimal in a time course experiment ranging from 0 to 12 h). Rifampin induced CYP3A activity by 2-fold (compared with the DMSO control) at 10 µM but not at other concentrations tested (1 and 5 µM) (Fig. 4). At the concentrations tested (1, 5, and 10 µM), ritonavir decreased CYP3A activity by 7-fold, whereas nelfinavir decreased CYP3A activity by approximately 4-fold. At 10 µM, saquinavir decreased CYP3A activity by 3-fold, whereas lopinavir, tipranavir, and atazanavir decreased this activity by only 1.5-fold. At 10 µM, amprenavir and indinavir had no effect on CYP3A activity.

Role of PXR in the Induction of CYP3A4 and MDR1 Transcripts in LS180 Cells. To gain insight into the mechanisms involved in induction of CYP3A4 and MDR1 transcripts by the PIs, we measured the activation of PXR as well as the induction of CYP3A4 and MDR1 transcripts in PXR-competent and PXR-knockdown LS180 cells by the PIs or rifampin.

PXR activation by PIs or rifampin in a reporter gene assay in LS180 cells. At 10 µM, the order of PXR activation was as follows: RIF (20-fold) > RTV (11-fold) APV (12-fold) > LPV (5-fold) TPV (5-fold) SQV (4-fold) ATV (4-fold) IDV (2.4-fold) (Fig. 5). Statistically significant activation was observed with all of the PIs except 10 µM nelfinavir because nelfinavir was significantly more toxic to the transfected cells (10% cell survival) than the other PIs for which the cytotoxicity was minimal (>90% cell survival observed). When nelfinavir was studied in a concentration-response study, significant PXR activation (2.7- to 11.7-fold) was observed at lower concentrations (0.1–5 µM). This result demonstrates that nelfinavir was more toxic in this assay than in the induction assay possibly because of treatment of the cells with Lipofectamine combined with 48 h of drug exposure. Concentration-response studies for PXR activation were performed by treating transfected cells with various concentrations of ritonavir (0.1–50 µM), nelfinavir (0.1–5 µM), or rifampin (0.1–50 µM). All three drugs activated PXR in a concentration-dependent manner (data not shown). The EC₅₀ values of PXR activation (0.4 µM) by ritonavir and nelfinavir were remarkably similar but lower than those for rifampin (0.9 µM). The EC₅₀ of PXR activation by rifampin was comparable with that reported previously (0.72 µM) (Lemaire et al., 2006). The maximal PXR activation, as determined by E_max, was similar between ritonavir and nelfinavir (13-fold) and slightly higher for rifampin (16-fold).

Induction of CYP3A4 and MDR1 transcripts by various protease inhibitors and rifampin in PXR-knockdown LS180 cells. To confirm the predominant involvement of PXR in PI-mediated induction of CYP3A4 and MDR1 transcripts in LS180 cells, we established a PXR-knockdown LS180 cell line (PXRsi) using RNA interference. When compared with the negative vector-transfected control cell line, GFPsi, we consistently achieved greater than 80% knockdown of PXR transcripts in this PXRsi cell line. The basal levels of CYP3A4 and MDR1 decreased (1.5- to 3-fold; p > 0.05) in PXRsi cells compared with the control cells. When these cells were treated with various PIs or rifampin (10 µM) for 96 h, induction of CYP3A4 and MDR1 transcripts by rifampin or the more potent PI inducers (RTV, NFV, APV, LPV, and TPV; induction levels greater than 5-fold) was reduced by 60 to 80% in PXRsi cells compared with the control cells (Fig. 6). As expected, weak inducers (IDV, ATV, and SQV) did not affect the induction of CYP3A4 and MDR1 transcripts in PXRsi cells compared with control cells (data not shown).

Role of CAR in the Induction of CYP3A4 and MDR1 Transcripts by the PIs in LS180 Cells. Although PXR appeared to be the major player in the induction of CYP3A4 and MDR1 transcripts in LS180 cells, we also investigated the role of CAR1 in the induction of these transcripts because it has been shown previously to be involved in the transcriptional regulation of CYP3A4 (Goodwin et al., 2002) and intestinal MDR1 (Burk et al., 2005).

Expression of CAR1 transcript in LS180 cells. Before creating the CAR1-knockdown cell line, we determined the expression of CAR1 in LS180 cells. To our surprise, we discovered that CAR1 was not expressed in LS180 cells. CITCO is a known activator of CAR and induces the prototypical CAR target gene, CYP2B6 (Maglich et al., 2003). To confirm the absence of CAR1 in LS180 cells, we treated LS180 cells with CITCO (1 and 5 µM) for 96 h. Consistent with our observation that CAR1 is not expressed in LS180 cells, CITCO did not induce CYP2B6 transcripts. These data indicate that the PIs or rifampin induce CYP3A4 and MDR1 transcripts in LS180 cells by PXR and not CAR1 activation.

CAR1 and CAR3 activation by PIs or rifampin. Even though CAR1 is absent in LS180 cells, it is present in human hepatocytes in which the PIs and rifampin are potent inducers of CYP2B6 (Dixit et al., 2007). Therefore, we determined whether the PIs could activate CAR1. Transfection of LS180 cells with CAR1 resulted in high basal activity even in the absence of ligands (e.g., 5 µM CITCO), thus making it difficult to determine whether PIs are ligands of CAR1. A similar phenomenon has been reported by other groups in immortalized hepatic cell lines such as HepG2 (Honkakoski et al., 1998). For this reason, we used a naturally occurring splice variant of CAR1, namely CAR3. This variant has low basal activity and has been successfully used to identify CAR ligands (Auerbach et al., 2003). None of the PIs activated CAR3, and CITCO activated CAR3 only 2.3- to 4-fold. The poor activation of CAR3 by CITCO led us to test whether coexpression of RXR was required for CAR3 activation and indeed it was. When RXR (pcDNA3.1-RXR) was coexpressed with CAR3 (CMV2-CAR3), we observed a 35- to 48-fold activation by CITCO (5 µM) of the CAR reporter (data not shown). This magnitude of CAR3 activation is consistent with CITCO-mediated RXR-dependent CAR3 activation observed in COS-1 cells (Auerbach et al., 2005). However, even in this refined activation assay, PIs (10 µM) did not activate CAR3 (data not shown).

Correlation in Induction of Transcripts. The correlation between the three drugs (RTV, NFV, and RIF) in induction of CYP3A4 transcripts was excellent (r = 0.8–0.86) as was that of the MDR1 transcripts (r = 0.91–0.92) (Table 1). In addition, the induction of CYP3A4 transcripts was highly correlated (r = 0.88) with that of MDR1 transcripts across all eight PIs and rifampin. Similarly, across all the drugs (nelfinavir was not included because of cytotoxicity), the correlation between PXR activation and induction of CYP3A4 (r = 0.93) or MDR1 transcripts (r = 0.93) was excellent and similar (Table 1).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
LS180 cells are an intestinal colon carcinoma cell line that expresses some characteristics of the small intestine (Tom et al., 1976). We chose to use these cells as they have been used previously to study induction of CYP3A and P-gp and because they constitutively express CYP3A, P-gp, and PXR (Pfrunder et al., 2003).

Although LS180 cells expressed transcripts of many of the P450s at levels comparable to that of CYP3A4, only CYP3A4 transcripts were inducible by the various PIs and rifampin (Fig. 2). This finding is surprising as we have previously shown that RTV, NFV, and RIF are inducers of CYP2B6, CYP2C9, and CYP2C19 transcripts in human hepatocytes (Dixit et al., 2007). This observation suggests that LS180 cells lack the necessary transcriptional factors (or other cellular machinery) for induction of these P450 transcripts. As discussed below, one such factor is CAR1, which is particularly important in the induction of CYP2B6 (Wang et al., 2004). Interestingly, tissue-specific induction of CYP3A expression and activity has been observed in vivo. Mouly et al. (2002) observed induction of in vivo hepatic CYP3A4 activity by efavirenz as measured by the erythromycin breath test without a change in intestinal CYP3A4 or P-glycoprotein expression. Similarly, chronic administration of NFV to rats induced hepatic but not intestinal CYP3a expression (Huang et al., 2001).

RTV, APV, LPV, and TPV were more potent inducers of CYP3A4 and MDR1 transcripts (>50% of induction produced by RIF) than NFV, which was a modest inducer, or SQV, IDV, and ATV, which were weak inducers (Fig. 2). Among the transporters studied, the PIs and rifampin induced MDR1 transcripts (Fig. 2) but not BCRP, MRP2, or OATP1A2.

We compared the concentration-dependent induction of CYP3A4 and MDR1 transcripts by ritonavir and nelfinavir with that produced by rifampin. These two PIs were chosen as they differ in their frequency and magnitude of clinical drug interactions. Ritonavir produces more frequent and profound drug interactions than nelfinavir. In addition, in vivo, nelfinavir appears to be a less potent inducer of P450 enzymes than ritonavir. The relative induction of CYP3A4 and MDR1 transcripts by the three drugs was concentration-dependent. The EC₅₀ values for induction of MDR1 and CYP3A4 transcripts by the inducers were remarkably consistent, whereas the E_max values differed (Fig. 3). These data support the notion that these three drugs share a common mechanism, most likely PXR, in the induction of CYP3A4 and MDR1 transcripts (see below for further discussion). The EC₅₀ value for CYP3A4 mRNA induction by rifampin obtained in our study is higher (8 µM) than those found in similar studies in either human hepatocytes [0.37 µM (Faucette et al., 2004)] or the hepatic cell lines [1.9 µM in Fa2N-4 cells (Ripp et al., 2006) and 0.25 µM in HepaRG cells (Kanebratt and Andersson, 2008)]. The reasons for this discrepancy are not clear. It may be attributable to differences in the expression of transcription factors such as CAR1 (HepaRG and human hepatocytes express CAR1) (Aninat et al., 2006). Also, differences in the cytotoxicity profile of some drugs (e.g., ritonavir) between the cell lines have been observed, suggesting differences in drug permeability between the cells (e.g., because of differences in expression of transporters). These could result in differences in EC₅₀ values between the cell lines. The same may be true for rifampin, resulting in different EC₅₀ values between cell types. Because we did not replenish the medium with drug during the 96-h treatment, there might have been some drug depletion during the period, resulting in a shift in the concentration-response curve.

In contrast to the magnitude of induction of CYP3A4 transcripts by the three drugs, the magnitude of induction of CYP3A activity by rifampin was modest (2-fold), whereas this activity was reduced in the presence of the majority of the PIs. For rifampin, the reason for the discrepancy between induction of CYP3A4 transcripts and CYP3A activity is not clear. The LS180 cells do not translate the increased CYP3A transcripts into activity either because they lack the necessary machinery to do so or because the protein may be unstable in these cells. In addition, our assay for quantification of CYP3A activity did not distinguish between CYP3A4 and CYP3A5. Although CYP3A5 transcripts are expressed in LS180 cells at levels similar to those in CYP3A4 transcripts, CYP3A5 transcripts are less inducible by rifampin and negligibly induced by the PIs (data not shown), which could have dampened the overall induction in CYP3A activity but is unlikely to completely explain the modest induction of this activity. Also, despite washing of the cells for 4 h, residual amounts of rifampin in the cells could also have inhibited CYP3A activity. In contrast, the reduction in CYP3A activity by the majority of the PIs was probably due to inactivation of the protein as has been previously shown in vitro (Granfors et al., 2006) and in vivo (Fellay et al., 2005; Yeh et al., 2006). In addition, we cannot rule out the possibility that despite washing of the cells for 4 h, residual intracellular PIs may have inhibited CYP3A activity because these drugs are potent inhibitors of CYP3A activity (Ernest et al., 2005). Consistent with the previous data (Ernest et al., 2005), ritonavir was the most potent inhibitor of CYP3A activity whereas indinavir did not inhibit CYP3A activity. Despite our efforts to test whether P-gp activity is induced by the PIs or rifampin, we were unable to measure P-gp activity in these cells possibly because of low levels of P-gp protein expression at the cell surface. Although Perloff et al. (2003) observed induction of P-gp activity (measured by RH123 accumulation) in the LS180 cells by ritonavir, they did so in cells selected (using vinblastine) for elevated expression of P-gp.

Rifampin has been shown to induce CYP3A and P-gp activity by activating PXR (Lehmann et al., 1998; Geick et al., 2001). Dussault et al. (2001) have reported that ritonavir is an excellent activator/ligand of PXR (comparable with rifampin) but not of CAR or vitamin D receptor (Dussault et al., 2001). Interestingly, none of the other PIs tested in their study (saquinavir or indinavir) activated PXR. In contrast, we observed statistically significant PXR activation with all the PIs. We suspect that the discrepancy in the two studies could be due to Dussault et al. (2001) not accounting for the poor solubility of the PIs. We used β-cyclodextrin to improve the solubility of the PIs (data not shown). The significant correlation between PXR activation and induction of MDR1 or CYP3A4 transcripts (Table 1) supports the notion that these drugs induce CYP3A4 and MDR1 transcripts in LS180 cells by PXR activation.

The above correlation is not proof. To directly test the hypothesis that PXR was involved in the induction of CYP3A4 and MDR1 transcripts in LS180 cells, we knocked-down PXR expression in these cells. In PXR knocked-down cells, the induction of CYP3A4 and MDR1 transcripts by the PIs and rifampin was significantly reduced, and this reduction was proportionate to the reduction in PXR transcripts (Fig. 6). These data unequivocally show that PIs and rifampin induce CYP3A4 and MDR1 transcripts in LS180 cells by PXR activation. Because the CAR is also important in inducing CYP3A4 and MDR1 (Goodwin et al., 2002; Burk et al., 2005; Cerveny et al., 2007), we investigated whether CAR1 is expressed in LS180 cells. Surprisingly, we found that CAR1 transcripts were not expressed in LS180 cells. These data further support our conclusion that induction of CYP3A4 and MDR1 transcripts by the PIs and rifampin in LS180 cells is PXR- and not CAR1-mediated.

Although CAR1 is not expressed in LS180 cells, it is expressed in the human liver where it may play a role in the in vivo induction of CYP3A4 and MDR1 transcripts by the PIs. It is difficult to study CAR1 activation in hepatic-derived immortalized cells, as CAR1 spontaneously translocates to the nucleus in the absence of ligand (Honkakoski et al., 1998), resulting in high constitutive CAR activity. The most frequently used cell system to study CAR1 activation is primary human hepatocytes. Their limited availability, variability, and lack of suitability for high-throughput assays prompted us to test whether LS180 cells could potentially be used for CAR1 activation. Unfortunately, results from our experiments suggest that CAR1 translocates to the nucleus in the absence of ligand in LS180 cells similar to what occurs in HepG2 cells, and, hence, LS180 cells cannot be used for CAR1 activation studies. To overcome this problem, measurement of activation of CAR3, a splice variant of CAR1, has been proposed. CAR3 has low basal activity (Auerbach et al., 2005) and does not spontaneously translocate to the nucleus. Therefore, we determined the ability of the PIs to activate CAR3 in LS180 cells. None of the PIs tested was able to activate CAR3, indicating negligible involvement of CAR in PI-mediated induction of CYP3A4 and MDR1. These data further support our conclusion that PIs and rifampin induce CYP3A4 and MDR1 transcripts by PXR- and not CAR-mediated activation.

Because the PIs inactivate CYP3A enzymes, both in vitro and in vivo, their ability to induce CYP3A4 transcripts will not translate into induction of CYP3A activity. Therefore, such induction will not correlate with in vivo CYP3A activity after chronic administration of these drugs. However, does the ability of PIs to induce MDR1 transcripts in LS180 cells correlate with their ability to induce P-gp activity in vivo? In humans, single dose administration of ritonavir or lopinavir/ritonavir increased the plasma AUC (2.2- to 4-fold) and C_max of the P-gp substrate, fexofenadine. However, a moderate induction of P-gp activity was suspected after 12 days of lopinavir/ritonavir 400/100 mg twice daily, although a net inhibitory effect on P-gp activity was still maintained (van Heeswijk et al., 2006). Additionally, when digoxin (0.4 mg), a P-gp substrate, was administered orally before and after ritonavir treatment (200 mg twice daily for 14 days), digoxin AUC_0–72 was increased by 22% and oral digoxin clearance was reduced by approximately 30% (Penzak et al., 2004). In another study, 300 mg of ritonavir twice daily for 11 days resulted in increased AUC_0– (86%) and volume of distribution (77%) of digoxin (0.5 mg) administered i.v. on day 3 of ritonavir administration. Furthermore, the nonrenal (48%) and renal clearance (35%) of digoxin was reduced (Ding et al., 2004). Collectively, these data suggest that with chronic administration of the drug, ritonavir is a net inhibitor, not inducer, of intestinal P-gp.

In conclusion, this is the first comprehensive study of PI-mediated induction of CYP3A4 and MDR1 transcripts in LS180 cells. We show for the first time that nelfinavir and several other PIs such as amprenavir, tipranavir, lopinavir, atazanavir, and saquinavir induce CYP3A4 and MDR1 transcripts by PXR (but not CAR) activation. In addition, our results indicate that LS180 cells do not express CAR1 and therefore can serve as an excellent, inexpensive cell line to screen for PXR-mediated induction of CYP3A4 and MDR1 gene products.

	Acknowledgments

 
We thank Andrei Mikheev for technical help in creating the PXR knocked-down cell line, Mary Blonski for the CAR activation assay, Peng Hsiao for the P-gp activity assay, and Dale Whittington for liquid chromatography mass spectrometry analysis of 1'-OH-midazolam. We acknowledge Brian J. Middleton (AstraZeneca Pharmaceuticals, Alderley Park, UK) for his help with the statistical analysis. We thank Dr. Hongbing Wang (Department of Pharmaceutical Sciences, University of Maryland) for generously providing us with the CYP2B6-PBREM/XREM firefly luciferase reporter plasmid and Dr. Curtis J. Omiecinski (Department of Veterinary and Biomedical Sciences, Pennsylvania State University) for generously providing us with CMV2-CAR1, CMV2-CAR3, and pcDNA3.1-RHR constructs and their respective vector control plasmids. We thank Brian Kirby and Vaishali Dixit for helpful scientific discussions and help in troubleshooting technical problems.

	Footnotes

 
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).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.018689.

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-; 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.

¹ Current affiliation: AstraZeneca Pharmaceuticals LP, DMPK Department, Wilmington, DE.

Address correspondence to: Dr. Jashvant D. Unadkat, Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195. E-mail: jash{at}u.washington.edu

	References

Top Abstract Materials and Methods Results Discussion References

 


Aninat C, Piton A, Glaise D, Le Charpentier T, Langouët S, Morel F, Guguen-Guillouzo C, Guillouzo A (2006) Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 34: 75–83.[Abstract/Free Full Text]

Auerbach SS, Ramsden R, Stoner MA, Verlinde C, Hassett C, and Omiecinski CJ (2003) Alternatively spliced isoforms of the human constitutive androstane receptor. Nucleic Acids Res 31: 3194–3207.[Abstract/Free Full Text]

Auerbach SS, Stoner MA, Su S, and Omiecinski CJ (2005) Retinoid X receptor--dependent transactivation by a naturally occurring structural variant of human constitutive androstane receptor (NR1I3). Mol Pharmacol 68: 1239–1253.[Abstract/Free Full Text]

Back DJ (2006) Drug-drug interactions that matter. Top HIV Med 14: 88–92.[Medline]

Barry M, Mulcahy F, Merry C, Gibbons S, and Back D (1999) Pharmacokinetics and potential interactions amongst antiretroviral agents used to treat patients with HIV infection. Clin Pharmacokinet 36: 289–304.[CrossRef][Medline]

Burk O, Arnold KA, Geick A, Tegude H, and Eichelbaum M (2005) A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression. Biol Chem 386: 503–513.[CrossRef][Medline]

Cerveny L, Svecova L, Anzenbacherova E, Vrzal R, Staud F, Dvorak Z, Ulrichova J, Anzenbacher P, and Pavek P (2007) Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways. Drug Metab Dispos 35: 1032–1041.[Abstract/Free Full Text]

Ding R, Tayrouz Y, Riedel KD, Burhenne J, Weiss J, Mikus G, and Haefeli WE (2004) Substantial pharmacokinetic interaction between digoxin and ritonavir in healthy volunteers. Clin Pharmacol Ther 76: 73–84.[CrossRef][Medline]

Dixit V, Hariparsad N, Li F, Desai P, Thummel K, and Unadkat J (2007) Cytochrome P450 enzymes and transporters induced by anti-HIV protease inhibitors in human hepatocytes: implications for predicting clinical drug interactions. Drug Metab Dispos 35: 1853–1859.[Abstract/Free Full Text]

Dussault I, Lin M, Hollister K, Wang EH, Synold TW, and Forman BM (2001) Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. J Biol Chem 276: 33309–33312.[Abstract/Free Full Text]

Ernest CS 2nd, Hall SD, and Jones DR (2005) Mechanism-based inactivation of CYP3A by HIV protease inhibitors. J Pharmacol Exp Ther 312: 583–591.[Abstract/Free Full Text]

Faucette SR, Wang H, Hamilton GA, Jolley SL, Gilbert D, Lindley C, Yan B, Negishi M, and LeCluyse EL (2004) Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metab Dispos 32: 348–358.[Abstract/Free Full Text]

Fellay J, Marzolini C, Decosterd L, Golay KP, Baumann P, Buclin T, Telenti A, and Eap CB (2005) Variations of CYP3A activity induced by antiretroviral treatment in HIV-1 infected patients. Eur J Clin Pharmacol 60: 865–873.[CrossRef][Medline]

Geick A, Eichelbaum M, and Burk O (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem 276: 14581–14587.[Abstract/Free Full Text]

Goodwin B, Hodgson E, D'Costa DJ, Robertson GR, and Liddle C (2002) Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor. Mol Pharmacol 62: 359–365.[Abstract/Free Full Text]

Goodwin B, Hodgson E, and Liddle C (1999) The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol 56: 1329–1339.[Abstract/Free Full Text]

Granfors MT, Wang JS, Kajosaari LI, Laitila J, Neuvonen PJ, and Backman JT (2006) Differential inhibition of cytochrome P450 3A4, 3A5 and 3A7 by five human immunodeficiency virus (HIV) protease inhibitors in vitro. Basic Clin Pharmacol Toxicol 98: 79–85.[CrossRef][Medline]

Honkakoski P, Zelko I, Sueyoshi T, and Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18: 5652–5658.[Abstract/Free Full Text]

Huang L, Wring SA, Woolley JL, Brouwer KR, Serabjit-Singh C, and Polli JW (2001) Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors. Drug Metab Dispos 29: 754–760.[Abstract/Free Full Text]

Kanebratt K and Andersson T (2008) HepaRG cells as an in vitro model for evaluation of cytochrome P450 induction in humans. Drug Metab Dispos 36: 137–145.[Abstract/Free Full Text]

Kirby B, Collier A, Thummel K, Kharasch E, and Unadkat J (2006a) Effect of multiple dose administration of nelfinavir or rifampin on in-vivo CYP1A2, 2C9, 2D6, 3A, and P-gp activity In HIV-negative volunteers. Drug Metab Rev 38: 110.

Kirby B, Kharasch ED, Thummel KT, Narang VS, Hoffer CJ, and Unadkat JD (2006b) Simultaneous measurement of in vivo P-glycoprotein and cytochrome P450 3A activities. J Clin Pharmacol 46: 1313–1319.[Abstract/Free Full Text]

Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, and Kliewer SA (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102: 1016–1023.[Medline]

Lemaire G, Mnif W, Pascussi JM, Pillon A, Rabenoelina F, Fenet H, Gomez E, Casellas C, Nicolas JC, Cavailles V, et al. (2006) Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system. Toxicol Sci 91: 501–509.[Abstract/Free Full Text]

Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, Stoltz CA, Kliewer SA, Lambert MH, Willson TM, et al. (2003) Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem 278: 17277–17283.[Abstract/Free Full Text]

Malaty LI and Kuper JJ (1999) Drug interactions of HIV protease inhibitors. Drug Saf 20: 147–169.[CrossRef][Medline]

Mouly S, Lown KS, Kornhauser D, Joseph JL, Fiske WD, Benedek IH, and Watkins PB (2002) Hepatic but not intestinal CYP3A4 displays dose-dependent induction by efavirenz in humans. Clin Pharmacol Ther 72: 1–9.[CrossRef][Medline]

Penzak SR, Shen JM, Alfaro RM, Remaley AT, Natarajan V, and Falloon J (2004) Ritonavir decreases the nonrenal clearance of digoxin in healthy volunteers with known MDR1 genotypes. Ther Drug Monit 26: 322–330.[CrossRef][Medline]

Perloff MD, Stormer E, von Moltke LL, and Greenblatt DJ (2003) Rapid assessment of P-glycoprotein inhibition and induction in vitro. Pharmacol Res 20: 1177–1183.[CrossRef]

Perloff MD, von Moltke LL, Fahey JM, Daily JP, and Greenblatt DJ (2000) Induction of P-glycoprotein expression by HIV protease inhibitors in cell culture. AIDS 14: 1287–1289.[CrossRef][Medline]

Perloff MD, von Moltke LL, and Greenblatt DJ (2004) Ritonavir and dexamethasone induce expression of CYP3A and P-glycoprotein in rats. Xenobiotica 34: 133–150.[CrossRef][Medline]

Pfrunder A, Gutmann H, Beglinger C, and Drewe J (2003) Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines. J Pharm Pharmacol 55: 59–66.[Medline]

Profit L, Eagling VA, and Back DJ (1999) Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors. AIDS 13: 1623–1627.[CrossRef][Medline]

Ripp SL, Mills JB, Fahmi OA, Trevena KA, Liras JL, Maurer TS, and de Morais SM (2006) Use of immortalized human hepatocytes to predict the magnitude of clinical drug-drug interactions caused by CYP3A4 induction. Drug Metab Dispos 34: 1742–1748.[Abstract/Free Full Text]

Storch CH, Theile D, Lindenmaier H, Haefeli WE, and Weiss J (2007) Comparison of the inhibitory activity of anti-HIV drugs on P-glycoprotein. Biochem Pharmacol 73: 1573–1581.[CrossRef][Medline]

Tom BH, Rutzky LP, Jakstys MM, Oyasu R, Kaye CI, and Kahan BD (1976) Human colonic adenocarcinoma cells. I. Establishment and description of a new line. In Vitro 12: 180–191.[Medline]

Unadkat JD and Wang Y (2000) Antivirals, in Metabolic Drug Interactions (Levy RH, Thummel KE, Trager WF, Hansten PD and Eichelbaum M eds), pp 421–434, Lippincott Williams & Wilkins, Baltimore.

van Heeswijk RP, Bourbeau M, Campbell P, Seguin I, Chauhan BM, Foster BC, and Cameron DW (2006) Time-dependent interaction between lopinavir/ritonavir and fexofenadine. J Clin Pharmacol 46: 758–767.[Abstract/Free Full Text]

Wang H, Faucette S, Moore R, Sueyoshi T, Negishi M, and LeCluyse E (2004) Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin. J Biol Chem 279: 29295–29301.[Abstract/Free Full Text]

Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M, and LeCluyse EL (2003) A novel distal enhancer module regulated by pregnane X receptor/constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. J Biol Chem 278: 14146–14152.[Abstract/Free Full Text]

Yeh RF, Gaver VE, Patterson KB, Rezk NL, Baxter-Meheux F, Blake MJ, Eron JJ Jr, Klein CE, Rublein JC, et al. (2006) Lopinavir/ritonavir induces the hepatic activity of cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP1A2 but inhibits the hepatic and intestinal activity of CYP3A as measured by a phenotyping drug cocktail in healthy volunteers. J Acquir Immune Defic Syndr 42: 52–60.[Medline]

This article has been cited by other articles:

				E. Y. H. Yeung, T. Sueyoshi, M. Negishi, and T. K. H. Chang Identification of Ginkgo biloba as a Novel Activator of Pregnane X Receptor Drug Metab. Dispos., November 1, 2008; 36(11): 2270 - 2276. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.018689v1 36/6/1172    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gupta, A. Articles by Unadkat, J. D. Search for Related Content PubMed PubMed Citation Articles by Gupta, A. Articles by Unadkat, J. D.