AN HTS ASSAY FOR INDUCTION OF ENZYMES AND TRANSPORTERS USING A HUMAN HEPATOCYTE CLONAL LINE AND 
RNA DETECTION 
J.B. Mills1, R. Faris2, S. Cascio2, J. Liu2, and S. .M. de Morais1 
1Pfizer Global Research & Development, Groton Laboratories, Pfizer Inc, Groton, CT 06340, USA
2MultiCell Technologies, 55 Access Rd., Suite 700, Warwick, RI 02886, USA
ABSTRACT TABLE 1 FIGURE 3
Induction studies are usually done using primary hepatocyte cultures and 
Correlation between enzyme activity & mRNA 
measurement of enzyme activity. We investigated the use of a novel human 
(formation of 6 -b-OH-testosterone vs. Invaderฎ). 
Induction study with clone Fa2N-4 using 6-well plates (n=3). Treatments 
hepatocyte clone as an alternative reagent, which is readily available and 
include vehicle (0.1% DMSO), 10 m M rifampin, 1 mM phenobarbital , 
provides a consistent, reproducible system. For the majority of metabolic 
25 m M chrysin , & 10 m M b-naphthoflavone. 
enzymes and transporters, induction is a result of increased levels of mRNA 
ug 6-฿OH-testosterone amol CYP3A4 mRNA/pg 
Clone per mg protein total RNA 
Fa2N-4 pre-crisis 
2.1 
5.9 
Fa2N-4 post-crisis 
21.0 
52.1 
Ea1C-35 
10.9 
52.1 
Ea1C-41 
27.4 
29.3 
Ea1C-53 
3.3 
19.0 
Ea1C-22 
1.1 
11.7 
Ea1C-10 
1.5 
11.6 
Attomoles mRNA
transcripts. The Invaderฎ system is a robust, yet simple, high-throughput system 
4.0 
0.0 
Vehicle 
Rifampin 
Phenobarbital 
Chrysin 
B-Napthoflavoneb-
UGT1A CYP2C9 CYP3A4 MDR1 
FIGURE 4 
Induction study with clone Fa2N-4 using 24-well plates (n=3). 
Treatments include vehicle (0.1% DMSO), 10 m M rifampin , 1 mM 
phenobarbital, 25 m M chrysin, 10 m M b-naphthoflavone, 10 m M 
clotrimazole , & 50m M dexamethasone. 
3.5 
3.0 
2.5 
2.0 
1.5 
1.0 
0.5 
for quantification of mRNA transcripts. Levels of CYP1A2, CYP3A4, CYP2C9, 
UGT1A, and MDR1 transcripts were quantified from total RNA extracts from 
Fa2N-4 cells (MultiCellTechnologies) treated with a panel of known inducers and 
compared with vehicle controls. Results are displayed below: 
CYP3A4 CYP2C9 UGT1A MDR1 
Rifampin (m M )15 3 -1.9 
Phenobarbital (1 mM) 12 2.5 -1.7 
Chrysin (25 m M ) --1.6 10 
m M b-naphthoflavone exhibited a higher than 2-fold increase in the amount of 
Clones Ea1C-41, Ea1C-53, and Ea1C-10 did not 
CYP1A2 mRNA transcripts, and did not induce CYP3A4, CYP2C9, UGT1A, or 
survive crisis for further evaluation. Clone Ea1C -22 
MDR1. These data show that use of Fa2N-4 cells in conjunction with the 
survived crisis, however levels of CYP3A4 in post-crisis 
Invaderฎ technology is a potential high-throughput in-vitro method for assessment cells were below limits for mRNA detection. Post-crisis 
of induction of ADMET targets. FIGURE 1 clones Fa2N-4 and Ea1C-35 were assessed further. 
Clone screen: CYP3A4 mRNA in vehicle-treated 
versus 10 m M rifampin-treated clones. 
100
INTRODUCTION 
Ea1C-53 
Ea1C-41 
Ea1C-10 
Ea1C-35 
post-crisis 
Fa2N-4 
post-crisis 
Fa2N-4 pre-crisis 
Ea1C-22 
10 uM Rifampin 
Vehicle
TABLE 2 
Pfizer & MultiCell Technologies signed a 1 year collaboration agreement in 
Comparison of CYP3A4 mRNA in induction 
• 
November 2001. 
experiments: Ea1C-35 versus Fa2N-4. 
80 
• One objective of the collaboration is to find a suitable cell system to routinely 
monitor induction of ADMET endpoints. 
• A clonal cell system provides advantages over primary and cryopreserved 60 
100 ng total RNA 
amol 3A4 
Fa2N-4 
amol 3A4 
Ea1C-35 
fold-induction 
Fa2N-4 
fold-induction 
Ea1C-35 
DMSO 0.243 0.973 
Rifampin 3.728 2.141 15.4 2.2 
b -Naphthoflavone 0.177 0.102 none none 
Phenobarbital 2.946 1.749 12.1 3.8 
Chrysin 0.104 0.276 none none 
Attomoles mRNA
hepatocytes: 
• Primary hepatocytes are not readily available. 
Cryopreserved hepatocytes exhibit variable plating efficiency, are 
• 
40 
expensive, and supply is exhaustible. 
• Donor-to-donor variability in induction response is problematic for 
both primary and cryopreserved hepatocytes. 20 
• MultiCell immortalized primary human hepatocyte cells (via SV40) and 
produced approximately 100 clonal cell lines. 
0 
2.5 
0.0 
CYP 1A2 CYP 2C9 CYP 3A4 MDR1 
2.0 
Control 
Rifampin 
Phenobarbital 
Chrysin 
B-Naphthoflavone 
Clotrimazole 
Dexamethasone 
b-
1.5 
1.0 
0.5 
Ea1C-41Ea1C-53Ea1C-10 
CONCLUSIONS 
• Human hepatocyte clones will provide a readily available, consistent cell 
Ea1C-35 post-crisis 
METHODS FIGURE 2 
system for induction. 
• PART 1: SCREEN CLONES Best clones from screen: CYP3A4 mRNA in vehicle-treated versus 10 m M Clone Fa2N-4 Induction Study • Out of ~ 100 clones, clone Fa2N-4 is the best clone for induction. 
– Treat clones with vehicle or 10 uM Rifampin for 72 hours (@ 3 days post-rifampin -treated clones versus primary human hepatocytes (pool of 5 • Measurement of induction with Invader technology correlates well with 
confluency). donors). • Plate cells at confluencyin 6-well or 24-well plate classic enzyme activity assessment. 
– Assess induced clones for 6 -b hydroxytestosterone. 
0 
50 
100 
150 
200 
250 
300 
Primary Fa2N-4 
pre-crisis 
Fa2N-4 
post-crisis 
Ea1C-35 
post-crisis 
Ea1C-41 
pre-crisis 
Ea1C-53 
pre-crisis 
Ea1C-22 
pre-crisis 
Ea1C-10 
pre-crisis 
amol 3A4 RNA/pg total RNARifampin 
Vehicle 
(Vitrogen-coated) on Friday. • Fa2N-4 responded to a panel of inducers for CYP3A4, CYP1A2, 
• Treat cells with inducers on Monday and Tuesday. 
CYP2C9, UGT1A & MDR-1 as expected. 
– Harvest total RNA. 
– Vehicle (0.1% DMSO) 
Basal level of CYP3A4 in Fa2N-4 is less than in primary human 
Assess CYP3A4 with Invaderฎ assay. Run follow-up Invader assays for 
•
– 
– 10 m M Rifampin (CYP2C9, CYP3A4) 
hepatocytes, the dynamic range for induction is superior. 
other DMEs for inducible clones. 
– 50 m M Dexamethasone (CYP3A4) 
Future plans are for optimization/validation.
Select best clones for follow-up studies. Follow clones through crisis to 
•
– 
– 10 m M Clotrimazole (CYP3A4) 
true immortalized cell line. 
– Optimize miniaturization (e.g. 96-well). 
– 10 m M B-Naphthoflavone (CYP1A2) 
PART 2: SECOND TIER SCREEN 
Assess multiple inducer concentrations, earlier timepoints , use of 
• 
– 
– 25 m M Chrysin (UGT1A, CYP1A2) 
lysates, & reproducibility. 
– Treat clone with panel of inducers for 48 hours 
–1 m M Phenobarbital (CYP2C9, CYP3A4) 
(@ 3 days post-confluency). 
– Harvest total RNA. Assess DMEs (3A4, 1A2, Pgp, 2C9, UGT1A) with 
• Harvest cells and extract total RNA with Qiagen Mini-
Prep Kit on Wednesday. Store @ –80C. 
Invader. 
– Determine top 1-2 clones that can show expected induction and can rank 
order (eg. RIF>DEX). 
• Run Invader assays for CYP1A2, CYP2C9, CYP3A4, 
UGT1A, and MDR-1. 
PART 3: OPTIMIZATION AND VALIDATION (still to be done) 
ACKNOWLEDGEMENTS
• 
– Optimize miniaturization (e.g. 96-well). 
Assess multiple inducer concentrations, earlier timepoints , use of lysates, 
HENRY SANTANGINI, KATHY GARCIA & PAUL SILVA of MultiCell 
– 
& reproducibility. 
Technologies 
KELLY LONGO of Pfizer Strategic Alliances 
PEGGY EIS & JAY SELOVER of Third Wave Technologies