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

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.061713v1 309/1/303    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 Citation Map 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 Mills, J. B. Articles by de Morais, S. M. F. Search for Related Content PubMed PubMed Citation Articles by Mills, J. B. Articles by de Morais, S. M. F.

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Induction of Drug Metabolism Enzymes and MDR1 Using a Novel Human Hepatocyte Cell Line

Exploratory Medicinal Sciences (J.B.M, S.M.F.de M.), Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut; and Department of Pharmacokinetics, Dynamics, and Metabolism (K.A.R., N.S., J.S.), Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan

Received October 15, 2003; accepted December 5, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 µM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6--hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 µM -naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by -naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.

P450s are involved in the metabolism of drugs, primarily in the liver. Induction of CYP3A gene expression is caused by a variety of marketed drugs, including rifampin, phenobarbital, clotrimazole, and dexamethasone (Meunier et al., 2000; Sahi et al., 2000; Luo et al., 2002; Madan et al., 2003) and represents the basis for a number of common drug-drug interactions. CYP1A2 is inducible by 3-methylcholanthrene, -naphthoflavone, and tetrachlorodibenzodioxin (Li et al., 1998; Breinholt et al., 1999; Meunier et al., 2000; Madan et al., 2003). CYP2C9 can be induced by rifampin and phenobarbital; however, the magnitude of induction is less than that for CYP3A4 (Li et al., 1997; Madan et al., 2003). Inducers of the UGT1A family include rifampin, chrysin, and -naphthoflavone (Abid et al., 1997; Li et al., 1997; Breinholt et al., 1999). The MDR1 gene product P-glycoprotein is an important drug efflux transporter. Inducers of P-glycoprotein include rifampin, phenobarbital, clotrimazole, and dexamethasone (Schuetz et al., 1996; Geick et al., 2001; Sahi et al., 2003).

The pregnane X receptor is the major determinant of CYP3A gene regulation by drugs and other xenobiotics (Bertilsson et al., 1998; Lehmann et al., 1998; Pascussi et al., 2003). In addition, pregnane X receptor mediates induction of P450s 2B6, 2C8/9, and 3A7, as well as the drug transporters MDR1, organic anion transporting polypeptide C, bile salt export protein, and multidrug resistance-associated protein 2 (Pascussi et al., 2003; Tirona et al., 2003). Other nuclear hormone receptors involved in induction of absorption, distribution, metabolism, excretion, and toxicity endpoints include glucocorticoid receptor (CYP2B6, CYP2C8/9, and CYP3A4/5), constitutive androstane receptor (UGT1A, CYP2B6, CYP3A4, and CYP2C9), and peroxisome proliferator-activated receptor (CYP4A) (Ferguson et al., 2002; Pascussi et al., 2003). A cytosolic receptor, the aryl hydrocarbon receptor, is involved in the induction of the CYP1A subfamily (Whitlock et al., 1996).

The ability to evaluate P450 induction in human hepatocytes is highly desirable because several drugs are known to induce P450 enzymes in humans but not rats, and vice versa (Bertilsson et al., 1998; Moore and Kliewer, 2000). For example, pregnenolone 16--carbonitrile induces CYP3A in rats but not humans, whereas rifampin is a known inducer of CYP3A in humans but not rats. Primary cultures of human hepatocytes have the distinct advantage of exhibiting species-specific induction of P450 isoforms, but are dependent on the availability of fresh cells and donor-to-donor variability. Cell lines such as HepG2, LS180, and LS174T, have been useful in studying induction of a limited subset of P450s and drug transporters (Schuetz et al., 1996; Li et al., 1998; Geick et al., 2001), but lack adequate response for other inducible targets (Silva and Nicoll-Griffith, 2002).

Induction of drug-metabolizing enzymes and drug transporters can be detected at the mRNA level (Schuetz et al., 1996; Abid et al., 1997; Li et al., 1998; Ferguson et al., 2002). The Invader assay (Kwiatkowski et al., 1999; Eis et al., 2001) quantifies transcript expression from total RNA extracted from cultured cells. It is an isothermal detection of RNA and does not require a polymerase chain reaction amplification step. An overlap between oligonucleotides consisting of an upstream invasive deoxyoligonucleotide and a downstream deoxynucleotide probe are both annealed to the RNA target, followed by cleavage by a 5' nuclease of the downstream probes. A second cleavage reaction utilizes a fluorescence resonance energy transfer oligonucleotide that further amplifies the signal. This assay can differentiate between closely related RNA transcripts, such as in P450 subfamilies (Eis et al., 2001).

For the current studies, we have used the immortalized human hepatocyte cell line Fa2N-4. We have characterized these cells by studying their drug-metabolizing enzymes, both at the level of the transcript and enzyme activity. We have also studied the induction potential of the Fa2N-4 cells by treating them with a few prototypical inducers of the major drug-metabolizing enzymes and monitoring changes in mRNA and enzyme activities. The present work describes the utilization of the immortalized human hepatocytes Fa2N-4 in combination with the mRNA detection Invader assay as a potential method to predict clinical drug-drug interactions due to increase in the transcription of genes encoding drug metabolizing enzymes or transporters.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals
Phenobarbital (5-ethyl-5-phenyl-2,4,6-trioxohexahydropyrimidine), dexamethasone (9--fluoro-16--methylprednisolone), -naphthoflavone (5,6-benzoflavone), rifampin (3-[4-methylpiperazinyliminomethyl] rifamycin SV), clotrimazole (1-[o-chloro--,-diphenylbenzyl]-imidazole), and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-para-toluenesulfonate, testosterone, 6-hydroxytestosterone, 7-ethoxyresorufin, resorufin, hydrocortisone, and diclofenac were purchased from Sigma-Aldrich (St. Louis, MO). 4'-Hydroxydiclofenac was purchased from BD Gentest (Woburn, MA). [¹³C₆]-4'-OH-diclofenac was produced internally at Pfizer Global Research and Development.

Induction of Fa2N-4 Cells
This cell line originated from human hepatocytes isolated from a 12-year-old female donor and were immortalized via transfection with the simian virus 40 large T antigen. Fa2N-4 cells (Fig. 1) were obtained from MultiCell Technologies (Warwick, RI) and cultured as follows. For RNA analysis, multiwell plates were precoated with a rigid collagen complex composed of 2.75 mM 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-para-toluenesulfonate and 4% (v/v) Vitrogen 100 purified collagen (Cohesion, Palo Alto, CA) in sterile saline (0.9% NaCl). Excess collagen was removed before cell plating. For enzyme activity analysis, Biocoat type I collagen plates were used (BD Biosciences, Bedford, MA). Fa2N-4 cells were plated at confluence in MFE media (MultiCell Technologies) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Media was replaced with serum-free MFE media supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin after cell attachment (approximately 3 h). Cells were kept in an incubator set at 37°C, 5% carbon dioxide, and 95% relative humidity. Media were replaced with fresh serum-free MFE media supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin every 24 h. Treatment of cells with drug was initiated 48 h after plating. For RNA quantification, cells were exposed to drug for 48 h. For enzyme activity studies, cells were exposed to drug for 72 h.

View larger version (132K): [in this window] [in a new window]   Fig. 1. Fa2N-4 cells. Phase contrast image of confluent Fa2N-4 cells plated in 96-well Biocoat type I collagen plates (BD Biosciences) in MFE media (MultiCell Technologies) at 200x magnification.

RNA Analysis
Total RNA was extracted from cells using the mini RNeasy kit according to instructions provided by the manufacturer (QIAGEN, Valencia, CA). RNA (100 ng) was analyzed using the Invader RNA assay reagent kits according to instructions provided by the manufacturer (Third Wave Technologies, Madison, WI). Statistical analysis for increased levels of RNA in samples compared with vehicle-treatment was conducted using the two-sample, unpaired Student's t test with p < 0.05 indicating significant differences. Statistical analysis for increased levels of RNA to compare multiple treatments (more than two samples) was conducted using analysis of variance with p < 0.05 indicating significant differences, for the purpose of rank ordering multiple inducers.

Enzyme Activity
CYP3A4. Activity was determined by measuring the extent of 6-hydroxytestosterone formation from testosterone by mass spectrometry, essentially as described by Wood et al. (1983), Sonderfan et al. (1987), and Sonderfan and Parkinson (1988), with the following modifications. Test drugs were washed from cells by removing dosing media, replacing with fresh media, and incubating cells for 1 h. After removing wash media, reactions were started with the addition of 250 µl of MFE media containing 200 µM testosterone to the tissue culture well. At 30 min, aliquots were removed for analysis via HPLC or LC/MS/MS. For LC/MS/MS analysis, the aliquot was mixed with 1 volume of acetonitrile spiked with 250 ng/ml hydrocortisone. Mass spectrometry was carried out with a PerkinElmer 200 HPLC system and a Micromass Quattro II detector. Samples were injected and were ionized using the electrospray positive ion mode in a mobile phase of 70:30 methanol/trifluoroacetic acid 0.02% (v/v) at 0.20 ml/min (isocratic) and a Keystone Aquasil C18, 100 x 2.1-mm, 5-µm particle size column. Some of the studies used an HPLC-UV assay for testosterone metabolism, as follows: 200 µl of medium was mixed with 5 µl of internal standard solution (20 µg/ml prednisolone in acetonitrile) and evaporated to approximately 50 µl. Samples (20 µl) were then injected on an Agilent 1100 HPLC system using an Agilent Zorbax Eclipse XDB-C8 column (4.6 x 150 mm) with UV detection at 254 nm. Mobile phase A consisted of 10 mM ammonium phosphate in water, and mobile phase B consisted of 100% acetonitrile. Initial conditions were 35% B for 3 min, increasing to 65% B over 2 min, and then at 10 min, returning to 35% B, for a total run time of 15 min. Retention times were 2.8, 3.0, and 7.0 min for 6--hydroxy-testosterone, prednisolone, and testosterone, respectively. The standard curve for 6--hydroxy-testosterone was linear from 25 ng/ml to at least 1000 ng/ml. Peak area for 6--hydroxy-testosterone was normalized to internal standard, and reported as fold-change from DMSO-treated cells.

CYP2C9. Activity was determined by measuring the extent of 4'-hydroxydiclofenac formation using the method of Leemann et al. (1993), modified as follows. Test drugs were washed from cells by removing dosing media, replacing with fresh media, and incubating cells for 15 min. After removing wash media, reactions were started with the addition of 250 µl of MFE media containing 7.5 µM diclofenac to each well. Aliquots were removed at 60 min for LC/MS/MS analysis. Mass spectrometry was carried out with a PerkinElmer 200 HPLC system and a Micromass Quattro II detector. Samples were injected and were ionized using the electrospray positive ion mode in a mobile phase of 50:50 acetonitrile/0.1% formic acid in water (v/v) at 0.27 ml/min (isocratic) and a Phenomenex, Synergi Max RP, 50 x 2.0-mm, 4-µm particle column.

CYP1A2. Activity was determined by measuring the extent of O-dealkylation of 7-ethoxyresorufin using the fluorometric method of Burke et al. (1985), with minor modifications (Rodrigues and Prough, 1991). Test drugs were washed from cells by removing dosing media, replacing with fresh media, and incubating cells for 15 min. After removing wash media, reactions were started with the addition of 250 µl of MFE media containing 7-ethoxyresorufin (20 µM) to each well. Aliquots were removed at 15 min for fluorometric analysis.

Metabolites were quantified by comparing measurements to standard curves. The concentration of protein for each cell treatment was determined with Bio-Rad DC reagents (Hercules, CA) according to instructions provided by the manufacturer, using bovine serum albumin as standard. Values were used to calculate enzyme activities as picomoles of metabolite per milligram protein per minute of incubation.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Inductive Response of Fa2N-4 Cells to Known Inducers of Drug-Metabolizing Enzymes and Drug Transporters. As illustrated in Fig. 2, Fa2N-4 cells are useful for monitoring several endpoints, including CYP1A2, CYP2C9, CYP3A4, UGT1A, and MDR1 using the known inducers rifampin (induces CYP2C9, CYP3A4, UGT1A, and MDR1), phenobarbital (induces CYP2C9, CYP3A4, and MDR1), dexamethasone (induces CYP3A4 and MDR1), and -naphthoflavone (induces CYP1A2 and UGT1A). Increases in transcripts can be observed for all positive controls. In comparison with the vehicle control, CYP1A2 transcript was increased 15-fold after treatment with 10 µM -naphthoflavone, but not significantly increased with other inducers. CYP2C9 transcript was increased 3.8-fold with 10 µM rifampin, 2.6-fold with 1 mM phenobarbital, and not induced by treatment with 50 µM dexamethasone, nor 10 µM -naphthoflavone. CYP3A4 transcript was increased 17-fold with 10 µM rifampin, 9.2-fold with 1 mM phenobarbital, and 1.3-fold with 50 µM dexamethasone. UGT1A transcript was increased 2.1-fold with 10 µM -naphthoflavone, and not induced by treatment with 1 mM phenobarbital, nor 50 µM dexamethasone. Rifampin induction of UGT1A was not statistically significant (p = 0.08). MDR1 transcript was increased 3.1-fold with 10 µM rifampin, 2.3-fold induction with 1 mM phenobarbital, 1.3-fold induction with 50 µM dexamethasone, and there was no MDR1 induction by 10 µM -naphthoflavone. Table 1 summarizes the induction data in Fa2N-4 cells for three P450s expressed as fold increase in mRNA compared with published data in primary hepatocytes.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Induction of CYP1A2, CYP2C9, CYP3A4, UGT1A, and MDR1 transcripts in Fa2N-4 cells. Fa2N-4 cells were plated in 24-well plates and exposed to 0.1% DMSO vehicle (open columns), 10 µM rifampin (red columns), 1000 µM phenobarbital (blue columns), 50 µM dexamethasone (green columns), and 10 µM -naphthoflavone (black columns) for 48 h. The levels of transcripts were quantified from total RNA isolated from the treated cells. Plot represents the mean ± S.D. from the data of quadruplicate samples. Asterisk denotes statistically significant increase in transcript versus vehicle control treatment (Student's t test, p < 0.05). CYP3A4 inducers were significantly different from each other using analysis of variance (p < 0.05).

P450 Enzyme Activity in Fa2N-4 Cells. CYP3A4 activity increased 8.9-fold and 2.1-fold, as assessed by increases in formation of the 6--hydroxytestosterone with 10 µM rifampin and 50 µM dexamethasone, respectively, compared with vehicle-treated control (Fig. 3A). Formation of 4'-hydroxydiclofenac for assessment of CYP2C9 activity was increased approximately 2-fold for treatments with 10 µM rifampin and 1 mM phenobarbital (Fig. 3B). Fold changes in the ethoxyresorufin O-dealkylase assay for CYP1A2 were 27-fold with 10 µM -naphthoflavone (Fig. 3C).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Measurement of induction by cytochromes 450 enzyme activity. Induction of CYP3A4, CYP2C9, and CYP1A2 enzyme activity in Fa2N-4 cells after 72 h exposure to 0.1% DMSO vehicle (VEH) or inducer. Study was conducted using a 12-well plate format. Data represents enzyme activity in terms of metabolite formed per milligram of total Fa2N-4 protein per minute of incubation with parent compound. Data are from duplicate assays denoted by open and closed columns. A, measurement of CYP3A4 activity by formation of the testosterone metabolite 6--hydroxytestosterone in cells induced with vehicle, 10 µM rifampin (RIF), and 50 µM dexamethasone (DEX). B, measurement of CYP2C9 activity by formation of the diclofenac metabolite 4'-hydroxydiclofenac in cells induced with vehicle, 10 µM RIF, and 1000 µM phenobarbital (PB). C, measurement of CYP1A2 activity by O-dealkylation of 7-ethoxyresorufin in cells induced with vehicle or 50 µM -naphthoflavone (BNF).

In addition to examining the inductive effect of a single concentration of drug, the Fa2N-4 cells can also be used to look at dose-response relationships. For example, EC₅₀ values were calculated based on the response of Fa2N-4 cells dosed with multiple concentrations of rifampin ranging from 100 nM to 50 µM. Figure 4 contains EC₅₀ plots for Fa2N-4 cells using increased CYP3A4 transcript values (Fig. 4A), as well as increased CYP3A4 enzyme activity (Fig. 4B). The calculated EC₅₀s were 0.43 µM (r² = 92) and 0.77 µM (r² = 94), for the transcript and enzyme activity, respectively. In addition, the calculated maximum induction (I_max) values were 13-fold for the transcript endpoint and 9.7-fold for the enzyme activity endpoint.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Dose-response dependence of CYP3A4 induction by rifampin in Fa2N-4 cells. Measurement of induction of CYP3A4 was performed in Fa2N-4 cells treated with 100 nM to 50 µM rifampin. Data were fitted using SigmaPlot (version 8) using a 3-parameter sigmoidal curve. A, total RNA was analyzed to determine level of CYP3A4 transcript and then compared with vehicle control to determine fold induction. Data represents mean ± S.D. from the data of triplicate samples. B, CYP3A4 activity was measured by formation of the testosterone metabolite 6--hydroxytestosterone and then compared with vehicle control to determine fold induction. Data represents mean ± S.D. from the data of triplicate samples.

Fa2N-4 Inductive Response over Multiple Passages. Multiple passages of the Fa2N-4 cells have been tested for CYP3A4 induction. Figure 5 shows response of multiple passages of Fa2N-4 cells to a CYP3A4 inducer with a weak response (50 µM dexamethasone) and a CYP3A4 inducer that exhibits a strong response (10 µM rifampin). Treatment with dexamethasone increased CYP3A4 transcripts, 1.6-fold and 1.5-fold at passages 21 and 36, respectively. Treatment with 10 µM rifampin increased CYP3A4 transcripts, 17-fold and 16-fold at passages 21 and 36, respectively (Fig. 5A). CYP3A4 enzyme activity was increased 2.1-fold and 2.0-fold for dexamethasone and 8.9-fold and 4.9-fold for 10 µM rifampin at passages 28 and 36, respectively (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Induction of CYP3A4 in different passages of Fa2N-4 cells. Various passages of Fa2N-4 cells were plated in 24-well plates and exposed to 0.1% DMSO vehicle (open columns), 50 µM dexamethasone (striped columns), and 10 µM rifampin (black columns). A, levels of CYP3A4 transcripts were quantified from isolated total RNA. Plot represents the mean ± S.D. from the data of quadruplicate samples. B, CYP3A4 activity was measured by formation of the testosterone metabolite 6--hydroxytestosterone. Plot represents the mean of duplicate samples. All compounds showed statistically significant increase in transcript versus vehicle control treatment (Student's t test, p < 0.05).

Capacity for HTS with Fa2N-4 Cells and Invader Assay. Fig. 6 compares various multiwell plate formats. Regardless of the plate format, Fa2N-4 cells exhibit substantial CYP3A4 inductive response to rifampin. Fold changes in CYP3A4 transcript were 17.1-fold when using a 24-well plate, 6.6-fold when using a 24-well plate, and 5.7-fold for when using a 96-well plate.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Comparison of CYP3A4 induction in various multiwell plate formats. Induction of CYP3A4 transcript in Fa2N-4 cells after 48-h exposure to 10 µM rifampin (closed columns) in comparison with vehicle (open columns). Data are from studies conducted in each multiwell plate format as indicated. Plot represents the mean ± S.D. from the data of quadruplicate samples. All compounds showed statistically significant increase in transcript versus vehicle control treatment (Student's t test, p < 0.05).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The Fa2N-4 cells have the ability to induce CYP1A2, CYP2C9, CYP3A4, UGT1A, and MDR1 mRNA in response to known inducers. Using CYP3A4 transcript as an endpoint, we have demonstrated the ability of the assay to rank inducers according to potency and demonstrate dose-response for rifampin as observed previously in primary human hepatocytes (Li et al., 1997; Sahi et al., 2000). In addition to distinguishing inducers from noninducers, this assay has a wide dynamic range for some endpoints such as CYP3A4 and CYP1A2, enabling rank ordering for induction potency. The same decreasing potency for CYP3A4 inducers (rifampin > phenobarbital > dexamethasone) has been previously reported in the literature for studies in primary human hepatocytes using both mRNA and enzyme activity endpoints (Luo et al., 2002). Our results using mRNA induction in Fa2N-4 cells are in good agreement with the publication by Madan et al. (2003), who reported the effects of prototypical inducers for CYP1A2, CYP2C9, and CYP3A4 in cultured primary human hepatocytes by measuring enzyme activity in microsomes preparations from treated cells (Table 1). The rank order for CYP3A4 induction potency for rifampin and phenobarbital expressed as fold induction over vehicle controls was the same in both studies. CYP2C9 was also induced, albeit to a lesser extent than CYP3A4, and CYP1A2 had the highest response in both systems. Another study (Sahi et al., 2000) using primary human hepatocytes reported the EC₅₀ for rifampin in CYP3A4 induction, using an activity assay. Those results are also in good agreement with our own EC₅₀ results using the Fa2N-4 cells and mRNA measurements.

This immortal hepatocyte clone was identified in a screen of several of clones where the best response to rifampin induction of CYP3A4 was the selection criteria. Hence, the most appropriate utilization of this assay is for CYP3A4 induction. Further characterization of this clone indicated that it had high response to a CYP1A2 inducer, enabling also the detection of this endpoint in a screening format. The dynamic range of the responses to CYP2C9 and MDR1 were smaller, but they were in the same proportion as the inductive response found in fresh hepatocytes (Li et al., 1997; Madan et al., 2003; Sahi et al., 2003)

Although the average UGT1A transcript was higher in rifampin-treated Fa2N-4 cells than in vehicle-treated cells, the level of induction was not statistically significant. Previous induction studies in primary human hepatocyte cite interindividual variation in the effects of rifampin, using 1-naphthol glucuronidation as an endpoint. Abid et al. (1997) reported that the variability may be attributed to differential induction of two UGT1A isoforms. The Invader UGT1A oligos used here span a common region in the RNA among all isoforms, and measurement of mRNA is a sum of all UGT1A isoforms. Thus, the inductive effect on rifampin could have been minimized by the noninduced UGT1A isoforms. It is likely that probes designed for individual UGT1A isoforms would be able to detect significant increases in their mRNA.

Induction in the Fa2N-4 cells is not limited to mRNA and can also be assessed at the enzyme activity level. The extent of induction using mRNA quantified with Invader correlated well with enzyme activity data as indicated by similar rank order for several prototypical inducers. The ability to induce P450 enzyme activity provides further evidence on the expression of a comprehensive array of P450s in the Fa2N-4 cells. In addition, it shows the potential of these cells for alternative applications, such as P450 inhibition or metabolite generation.

The Invader assay can adequately quantify induction based on mRNA level increases. Advantages of the mRNA endpoint include increased throughput and target specificity. For enzyme activity, a separate well in a multiwell plate must be used for each enzyme activity endpoint, whereas a single well can be used to assess multiple mRNA targets. The recovery of RNA from each sample (24-well plate) is high enough to run up to 50 separate mRNA endpoints. The Invader assay is able to discriminate among closely related P450s, whereas enzyme activity assays are not always specific. For example, O-dealkylation of 7-ethoxyresorufin characterizes the combination of CYP1A1 and CYP1A2, and 6--hydroxytestosterone can also be formed by both CYP3A4 and CYP3A5 (Williams et al., 2002).

In contrast to fresh human hepatocytes, Fa2N-4 cells are readily available. Because accessibility to fresh human hepatocytes is reliant on availability of a suitable liver tissue donor, it can take a long time to conduct experiments using hepatocytes isolated from three different livers to verify that a certain compound is an inducer. In addition, plating efficiency of fresh hepatocytes is unpredictable, so it is not uncommon to have a suitable donor, but find that the cells are not usable due to poor plating efficiency or substandard cell health. Fa2N-4 cells can be passaged and used over several passages while retaining activity of the major drug-metabolizing enzymes. With fresh human hepatocytes, cells can only be used one time, making it difficult to compare data between studies. Plateable cryopreserved primary human hepatocytes are an improvement by theoretically allowing multiple experiments at different times from a single donor, or potentially the use of multiple donors at one time. However, plateable cryopreserved primary human hepatocytes are in limited supply. Both fresh primary human hepatocytes and plateable cryopreserved primary human hepatocytes have donor-to-donor variability, based on the influence of genetics, the environment, and comedications. There are vast differences seen in the drug-metabolizing enzyme profile of donors, leading to the current recommendation of obtaining data from three donors before reaching a conclusion for induction potential of a chemical. In addition, some authors cite the necessity for potency indexes to compare data between donors (Silva and Nicoll-Griffith, 2002). The potency index standardizes data between donors by reporting the ratio of induction response (i.e., fold induction) of the test compound to that of a prototypical inducer.

Thus, our preliminary data using a few prototypical inducers demonstrates that Fa2N-4 cells can be a suitable substitute for fresh human hepatocytes in induction studies, and provide the additional attribute of being amenable for higher throughput studies. Fa2N-4 cells are superior to previously published immortal cell lines, as they show induction of a varied number of genes. These cells can be used to determine the induction potential of a drug, with findings consistent with monitoring increased enzyme activity in primary human hepatocytes. Higher throughput cell culturing and analysis via mRNA endpoint enables more compounds to be tested and reduces the cost per compound; two favorable traits for drug discovery assays. Future studies will focus on expanding the number of inducers and absorption, distribution, metabolism, and excretion endpoints tested. Pending more extensive evaluation, this induction assay has the potential of becoming a useful tool for pharmaceutical companies to eliminate compounds with drug-drug interaction potential and to understand the likelihood and extent of drug-drug interaction for compounds in development.

	Acknowledgements

 
We thank Ronald A. Faris, Jin Liu, Stephanie Cascio, Henry Santangini, Kathy Garcia, and Paul Silva of MultiCell Technologies for the Fa2N-4 clone and the scientific collaborations; Sharon Ripp of Pfizer Groton Laboratories for contribution to CYP3A4 enzyme activity data; Teresa Jenkinson and Mike Banker of Pfizer Groton Laboratories for the photomicrograph of the Fa2N-4 cells; Kelly Longo of Pfizer Strategic Alliances for support; and Bo Feng, Ronald Scott Obach, and Ted Liston of Pfizer Groton Laboratories for suggestions and critical reading of this manuscript.

	Footnotes

 
DOI: 10.1124/jpet.103.061713.

ABBREVIATIONS: P450, cytochrome P450; MDR1, multidrug resistance 1; HPLC, high-performance liquid chromatography; LC/MS/MS, liquid chromatography/tandem mass spectrometry; DMSO, dimethyl sulfoxide.

Address correspondence to: Dr. Sonia M. F. de Morais, Pfizer Global Research and Development, Groton Laboratories, Eastern Point Rd., Groton, CT 06340. E-mail: demoraissm{at}groton.pfizer.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Abid A, Sabolovic N, and Magdalou J (1997) Expression and inducibility of UDP-glucuronyltransferases 1-naphthol in human cultured hepatocytes and hepatocarcinoma cell lines. Life Sci 60: 1943-1951.[CrossRef][Medline]

Baciewicz AM, Self TH, and Bekemeyer WB (1987) Update on rifampin drug interactions. Arch Intern Med 147: 565-568.[Abstract]

Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P, and Berkenstam A (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci USA 95: 12208-12213.[Abstract/Free Full Text]

Breinholt V, Lauridsen ST, and Dragsted LO (1999) Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat. Xenobiotica 29: 1227-1240.[CrossRef][Medline]

Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparant T, and Mayer RT (1985) Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol 34: 3337-3345.[CrossRef][Medline]

Eis PS, Olson MC, Takova T, Curtis ML, Olson SM, Vener TI, Ip HS, Vedvik KL, Bartholomay CT, Allawi HT, et al. (2001) An invasive cleavage assay for direct quantitation of specific RNAs. Nat Biotechnol 19: 673-676.[CrossRef][Medline]

Ferguson SS, Lecluyse EL, Nagishi M, and Goldstein JA (2002) Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site. Mol Pharmacol 62: 737-746.[Abstract/Free Full Text]

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]

Kwiatkowski RW, Lyamichev V, de Arruda M, and Neri B (1999) Clinical, genetic and pharmacogenetic applications of the Invader assay. Mol Diagnosis 4: 353-364.[CrossRef][Medline]

Leemann T, Transon C, and Dayer P (1993) Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4'-hydroxylation in human liver. Life Sci 52: 29-34.[CrossRef][Medline]

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 Investig 102: 1016-1023.[Medline]

Li AP, Reith MK, Rasmussen A, Gorski JC, Hall SD, Xu L, Kaminski DL, and Cheng LK (1997) Primary human hepatocytes as a tool for the evaluation of structure-activity relationship in cytochrome P450 induction potential of xenobiotics: evaluation of rifampin, rifapentine and rifabutin. Chem Biol Interact 107: 17-30.[CrossRef][Medline]

Li W, Harper PA, Tang BK, and Okey AB (1998) Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells. Biochem Phamacol 56: 599-612.[CrossRef][Medline]

Luo G, Cunningham M, Kim S, Burn T, Lin J, Sinz M, Hamilton G, Rizzo C, Jolley S, Gilbert D, et al. (2002) CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metab Dispos 30: 795-804.[Abstract/Free Full Text]

Madan A, Grahm RA, Carroll KM, Mudra DR, Burton LA, Krueger LA, Downey AD, Czerwinski M, Forster J, Ribadeneira MD, et al. (2003) Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab Dispos 31: 421-431.[Abstract/Free Full Text]

Meunier V, Bourrie M, Julian B, Marti E, Guillou F, Berger Y, and Fabre G (2000) Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up. Xenobiotica 30: 589-607.[CrossRef][Medline]

Moore JT and Kliewer SA (2000) Use of nuclear receptor PXR to predict drug interactions. Toxicology 153: 1-10.[CrossRef][Medline]

Pascussi JM, Gerbal-Chaloin S, Drocourt L, Maurel P, and Vilarem MJ (2003) The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors. Biochim Biophys Acta 1619: 243-253.[Medline]

Rodrigues AD and Prough RA (1991) Induction of cytochromes P450IA1 and P450IA2 and measurement of catalytic activities. Methods Enzymol 206: 423-431.[Medline]

Sahi J, Hamilton G, Sinz M, Barros S, Huang SM, Lesko LJ, and LeCluyse EL (2000) Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes. Xenobiotica 30: 273-284.[CrossRef][Medline]

Sahi J, Milad MA, Zheng X, Rose KA, Wang H, Stilgenbauer L, Gilbert D, Jolley S, Stern RH, and LeCluyse EL (2003) Avasimibe induces CYP3A4 and multiple drug resistance protein 1 gene expression through activation of the pregnane X receptor. J Pharmacol Exp Ther 306: 1027-1034.[Abstract/Free Full Text]

Schuetz EG, Beck WT, and Schuetz JD (1996) Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Mol Pharmacol 49: 311-318.[Abstract]

Silva JM and Nicoll-Griffith DA (2002) In vitro models for studying induction of cytochrome P450 enzymes, in Drug-Drug Interactions (Rodrigues AD ed) pp 189-216, Marcel Dekker, New York, NY.

Sonderfan AJ, Arlotto MP, Dutton DR, McMillen SK, and Parkinson A (1987) Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch Biochem Biophys 255: 27-41.[CrossRef][Medline]

Sonderfan AJ and Parkinson A (1988) Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch Biochem Biophys 265: 208-218.[CrossRef][Medline]

Spina E, Pisani F, and Perucca E (1996) Clinically significant pharmacokinetic drug interactions with carbamazepine: an update. Clin Pharmacokinet 31: 198-214.[Medline]

Tirona RG, Leake BF, Wolkoff AW, and Kim RB (2003) Human organic anion transporting polypeptide-C (SLC21A6) is a major determinant of rifampin-mediated pregnane X receptor activation. J Pharmacol Exp Ther 304: 223-228.[Abstract/Free Full Text]

Whitlock JP, Okino ST, Dong L, Ko HP, Clarke-Katzenberg R, Ma Q, and Li H (1996) Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. FASEB J 10: 809-818.[Abstract]

Williams AJ, Ring BJ, Cantrell VE, Jones DR, Eckstein J, Ruterbories K, Hamman MA, Hall SD, and Wrighton SA (2002) Comparative metabolic capabilities of CYP3A4, CYP3A5 and CYP3A7. Drug Metab Dispos 30: 883-891.[Abstract/Free Full Text]

Wood AW, Ryan DE, Thomas PE, and Levin W (1983) Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes. J Biol Chem 258: 8839-8847.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. Hariparsad, B. A. Carr, R. Evers, and X. Chu Comparison of Immortalized Fa2N-4 Cells and Human Hepatocytes as in Vitro Models for Cytochrome P450 Induction Drug Metab. Dispos., June 1, 2008; 36(6): 1046 - 1055. [Abstract] [Full Text] [PDF]

				R. Callaghan, E. Crowley, S. Potter, and I. D. Kerr P-glycoprotein: So Many Ways to Turn It On J. Clin. Pharmacol., March 1, 2008; 48(3): 365 - 378. [Abstract] [Full Text] [PDF]

				M. Weiner, W. Burman, C.-C. Luo, C. A. Peloquin, M. Engle, S. Goldberg, V. Agarwal, and A. Vernon Effects of Rifampin and Multidrug Resistance Gene Polymorphism on Concentrations of Moxifloxacin Antimicrob. Agents Chemother., August 1, 2007; 51(8): 2861 - 2866. [Abstract] [Full Text] [PDF]

				K. A. Youdim, C. A. Tyman, B. C. Jones, and R. Hyland Induction of Cytochrome P450: Assessment in an Immortalized Human Hepatocyte Cell Line (Fa2N4) Using a Novel Higher Throughput Cocktail Assay Drug Metab. Dispos., February 1, 2007; 35(2): 275 - 282. [Abstract] [Full Text] [PDF]

				S. L. Ripp, J. B. Mills, O. A. Fahmi, K. A. Trevena, J. L. Liras, T. S. Maurer, and S. M. de Morais Use of Immortalized Human Hepatocytes to Predict the Magnitude of Clinical Drug-Drug Interactions Caused by CYP3A4 Induction Drug Metab. Dispos., October 1, 2006; 34(10): 1742 - 1748. [Abstract] [Full Text] [PDF]

				C. Aninat, A. Piton, D. Glaise, T. Le Charpentier, S. Langouet, F. Morel, C. Guguen-Guillouzo, and A. Guillouzo EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS Drug Metab. Dispos., January 1, 2006; 34(1): 75 - 83. [Abstract] [Full Text] [PDF]

				D. Hallifax, H. C. Rawden, N. Hakooz, and J. B. Houston PREDICTION OF METABOLIC CLEARANCE USING CRYOPRESERVED HUMAN HEPATOCYTES: KINETIC CHARACTERISTICS FOR FIVE BENZODIAZEPINES Drug Metab. Dispos., December 1, 2005; 33(12): 1852 - 1858. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.061713v1 309/1/303    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 Citation Map 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 Mills, J. B. Articles by de Morais, S. M. F. Search for Related Content PubMed PubMed Citation Articles by Mills, J. B. Articles by de Morais, S. M. F.