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The Use of Immortalized Hepatocytes in Induction Studies

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The Use of Immortalized Hepatocytes in Induction Studies THE USE OF IMMORTALIZED HEPATOCYTES IN INDUCTION STUDIES Kevin Lyon, Maciej Czerwinski, Martin Perry, Paul Toren and Andrew Parkinson Kevin Lyon, Maciej Czerwinski, Martin Perry, Paul Toren and Andrew Parkinson XENOTECH XenoTech LLC, 1682516825 West 116th Street, LeneLenexa, KSKS 6621966219 INTRODUCTION RESULTS Figure 3: CONCLUSIONS CYP1A2: Phenacetin CYP2B6: Bupropion Primary cultures of human hepatocytes are widely used to 1. Fa2N-4 cells metabolize CYP-specific substrates. In Fa2N-4 Figure 2: Time course of CYP3A4 induction in Fa2N-4 cells 5 1. The Fa2N-4 cells respond like primary evaluate the cytochrome P450 (CYP) enzyme-inducing cells, activity of phenacetin O-dealkylase (CYP1A2) increased 1000 CYP induction 10 cultures of human hepatocytes to a wide 4 potential and/or toxicity of drug candidates. However, the 11 fold in response to three days' treatment with 100 µM in Fa2N-4 cells 800 range of xenobiotics. The Fa2N-4 cells nduction 5 availability of human hepatocytes for this purpose is limited 600 3 140 omeprazole. In response to a three-day treatment with 20 µM respond to prototypical PXR and AhR 400 2 old I and erratic, and there are large inter-individual differences in rifampin, the rates of midazolam 1´-hydroxylation (CYP3A4), 120 F 0 1 2 3 4 5 6 (pmol/incubation) (pmol/incubation) agonists in a manner that closely Acetaminophen 200 1 xylation the magnitude of induction (due to differences in both Hydroxybupropion Exposure Period (days) bupropion hydroxylation (CYP2B6), and diclofenac 4´- o 100 resembles the magnitude of induction "control" and "induced" CYP activities). Immortalized 0 0 dr hydroxylation (CYP2C9) increased by 4.9, 2.2, and 4.0 fold, y DMSO Omeprazole DMSO Rifampin 80 observed in freshly plated hepatocytes human hepatocytes, Fa2N-4, developed by Multicell -h 0.1% DMSO respectively. The activity of S-mephenytoin hydroxylase ´ but is largely free of the variability Technologies, (Warwick, RI) have the potential to overcome (CYP2C19) was not induced following the treatment with 20 µM CYP2C9: Diclofenac CYP2C19: S-Mephenytoin CYP3A4: Midazolam 60 these limitations. The cells, immortalized through 20 µM Rifampin associated with the use of primary 600 35 50 rifampin (fold induction < 2) (Figure 2). olam 1 40 (pmol/incubation) hepatocytes. transformation of human hepatocytes with SV40 T antigen, 500 30 40 2. A time-dependent increase in midazolam 1´-hydroxylase 25 20 400 display in vitro cell morphology that closely resembles Midaz 2. Fa2N-4 cells and human hepatocytes 20 30 primary human hepatocytes (Figure 1). The Fa2N-4 cells activity was seen in Fa2N-4 cells over the course of a six day- 300 0 15 respond similarly to several drugs and treatment with 20 µM rifampin, but not in the control (DMSO- 200 20 1 2 3 4 5 6 (pmol/incubation) (pmol/incubation) retain many of the characteristics of primary hepatocytes, (pmol/incubation) 10 -Hydroxydiclofenac -Hydroxymidazolam ´ ´ other xenobiotics, although some -Hydroxymephenytoin 4 100 10 Exposure Period (days) 1 treated) cells (Figure 3). On the last day of treatment CYP3A4 ´ 5 including the inducibility of multiple CYP mRNAs and 4 0 differences are noted. For example, 0 0 enzyme activities (Mills et al., 2002; Morris et al., 2003; activity was induced 8.3 fold. A time-dependent increase in DMSO Rifampin DMSO Rifampin DMSO Rifampin Table 2: Cellular response of Fa2N-4 cells and Fa2N-4 cells are more sensitive than Czerwinski et al., 2003). In this study, we further phenacetin O-dealkylase activity was seen in Fa2N-4 cells over primary hepatocytes human hepatocytes to the toxic effects of characterized the ability of these cells to respond to enzyme- the course of six day-treatment with 100 µM omeprazole (and to primary hepatocytes inducing xenobiotics. Additionally, we examined the toxicity a much lower extent, in the control cultures). On day 5 of Figure 4: Effects of efavirenz, hyperforin and probenecid on Compound: troglitazone, and benzo[a]pyrene, profile of multiple enzyme inducers in primary hepatocytes treatment, induction of CYP1A2 activity reached 8.6 fold, based Fa2N-4 cells and primary human hepatocytes Non-toxic Toxic whereas they are less sensitive than human hepatocytes to menadione. and Fa2N-4 cells. on amount of acetaminophen formed/incubation (data not Efavirenz Rifampin Probenecid 3-Methylcholanthrene shown). Fa2N-4 100 Human hepatocytes 250 Phenobarbital Felbamate Methotrexate 100 150 3. The Fa2N-4 hepatocyte-based analysis MATERIALS AND METHODS (% positive control**) (% positive control**) Phenytoin Acetaminophen Rotenone LDH or Resazurin LDH or Resazurin 3. Fa2N-4 cells plated in 96-well plates correctly differentiated 80 80 200 Carbamazepine Ciglitazone Efavirenz of enzyme induction and cellular toxicity The Fa2N-4 cells were propagated in MFE media (Multicell 100 Same Troleandomycin Sulfinpyrazone has been carried out in 6-, 12-, 24- and compounds that induce CYP3A4 (e.g. efavirenz, hyperforin) in 60 CYP3A4 60 150 Lansoprazole Simvastatin Technologies) on plasticware coated with Vitrogen LDH primary hepatocytes from those that do not (e.g. probenecid) 40 40 100 Omeprazole Fexofenadine 96-well plates. Data from the different (Cohesion Technologies, Palo Alto, CA) and maintained at 50 Resazurin Troglitazone (A) plating formats is highly reproducible. (Figure 4). Both toxicity markers used in this study, LDH release 20 20 50 37°C, 5% CO , 95% humidity. Immortalized hepatocytes CYP3A4 Activity CYP3A4 Activity Benzo[a]pyrene (A) 2 (% positive control*) (% positive control*) The use of 96-well plates with and reduction of resazurin, identified toxic concentration of 0 0 Hyperforin (A) were grown to confluency in 24 or 96-well plates and treated 0 0 Cellular response: selected xenobiotics. Fa2N-4 cells plated in 24-well plates 0.1 1 10 100 1000 1 10 100 1000 Different Menadione (B) immortalized hepatocytes bodes well for with enzyme inducers for up to 6 consecutive days with daily Efavirenz (µM) Efavirenz (µM) correctly identified those compounds that induce CYP1A2 or (A) = Fa2N-4 cells more sensitive than human hepatocytes the development of the higher throughput changes of medium. All enzyme inducers were dissolved in (B) = Fa2N-4 cells less sensitive than human hepatocytes DMSO (final concentration of solvent 0.1%, v/v). Primary CYP3A4 in primary hepatocytes (Figure 5). Hyperforin screening of CYP induction and toxicity Fa2N-4 (% positive control**) Human hepatocytes (% positive control**) in a reproducible human-based cell LDH or Resazurin LDH or Resazurin Figure 5: Effect of enzyme inducers on human hepatocytes were isolated and cultured as described 4. Cultures of both primary and immortalized hepatocytes were 100 120 100 140 120 system. (LeCluyse et al., 2000, Madan et al., 2003). Enzymatic exposed to several known toxicants and their effects were 80 100 80 CYP1A2 and CYP3A4 activity in Fa2N-4 cells 80 CYP3A4 100 60 activities were determined by incubating the cells with CYP- monitored by LDH release into the medium and by changes in LDH 60 80 60 REFERENCES Omeprazole, 100 µM specific substrates. Metabolite formation was measured by 40 Resazurin 40 60 mitochondrial respiration (based on resazurin reduction). The 40 40 3-Methylcholanthrene, 0.4 µM Czerwinski M, Lyon K, Perry M, Toren P, Steen D, 20 LC-MS as summarized in Table 1. CYP3A4 Activity 20 CYP3A4 Activity 20 toxicity profiles of Fa2N-4 and primary hepatocytes were similar 20 (% positive control*) (% positive control*) Lansoprazole, 10 µM Settle K and Parkinson A (2003) Induction of major 0 0 0 0 Leakage of lactate dehydrogenase (LDH) into the medium, for most compounds (Table 2). Few quantitative differences in 1,2 Benzanthracene, 10 µM cytochrome P450 enzymes in immortalized 0.01 0.1 1 10 0.01 0.1 1 10 β hepatocytes. Drug Metabolism Reviews 35: suppl. an indicator of cellular toxicity, was measured with the the response of the two cell types were observed. The Fa2N-4 Hyperforin (µM) Hyperforin (µM) -naphthoflavone, 2 µM 2 #462. Cytotoxicity Detection Kit (LDH) (Roche Diagnostics Corp., cells were more sensitive than primary hepatocytes to Resveratrol, 10 µM Probenecid Indianapolis, IN). Another marker of toxicity, mitochondrial hepatotoxins such as troglitazone, hyperforin, and Probenecid, 50 µM LeCluyse E, Madan A, Hamilton G, Carroll K, Fa2N-4 Benzo[a]pyrene, 2 µM 100 120 (% positive control**) Human hepatocytes 150 (% positive control**) DeHaan R and Parkinson A (2000) Expression and respiration, was determined by fluorescent monitoring of the benzo[a]pyrene, but less sensitive than the primary hepatocytes LDH or Resazurin 100 LDH or Resazurin Oltipraz, 10 µM regulation of cytochrome P450 enzymes in primary to a mitochondrial respiration inhibitor, menadione. 80 100 reduction of resazurin (Alamar Blue) to resorufin. 80 100 Phenytoin, 10 µM cultures of human hepatocytes. J Biochem Mol 60 CYP3A4 60 LDH 50 Benzo[e]pyrene, 0.5 µM Toxicol 12:177-188. 5. Cultures of Fa2N-4 immortalized hepatocytes, representing 40 Figure 1: Fa2N-4 cells 40 Resazurin 50 cell passages 32 - 47, were treated with 100 µM omeprazole or 20 0 2 4 6 8 10 12 14 16 Madan A, Graham R, Carroll K, Mudra D, Burton CYP3A4 Activity 20 CYP3A4 Activity (% positive control*) (% positive control*) Phenacetin O-dealkylation (CYP1A2) L, Krueger L, Downey A, Czerwinski M, Forster J, 20 µM rifampin and analyzed for induction of CYP1A2 and 0 0 0 0 (fold induction) 0.1 1 10 100 1000 1 10 100 1000 Ribadeneira M, Gan L, LeCluyse E, Zech K, CYP3A4, respectively. Regardless of passage number, the cells Probenecid (µM) Probenecid (µM) Robertson P, Jr., Koch P, Antonian L, Wagner G, Yu responded consistently to the inducers by increasing the activity µ Rifampin, 20 µM L and Parkinson A (2003) Effects of prototypical * cells treated with 20 M rifampin Dexamethasone 250 µM of both enzymes to the extent that has been observed in extensive **cells treated with 0.1% v/v DMSO Hyperforin, 0.4 µM microsomal enzyme inducers on cytochrome P450 series of primary hepatocyte cultures (Figure 6, Madan et al., Sulfinpyrazone, 50 µM expression in cultured human hepatocytes.
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