Automated 384-Well Cell-Based Cytochrome P450 Inhibition Assays Using Cryopreserved Human Hepatocytes in Suspension

A p p l i c a t i o n N o t e

ADME/Tox (Drug Metabolism)

Introduction

Drug-drug interactions (DDI) are a serious concern to pharmaceutical manufacturers and regulatory agencies. As such, ADME-Tox studies including DDI are tested early in the xenobiotic life cycle. Cytochrome P450 (CYP) enzymes are key drug me-tabolizers in the body, and therefore play a major role in DDI research and testing.

Numerous formats are used to monitor drug com-pound effects on CYP enzymes. Recombinant CYP isoforms use a host cell transfected with specific CYP-expressing DNA. Microsomes are sub-cellular vesicles derived from the endoplasmic reticulum. Finally, immortalized cell lines may be created with hepatocyte-like function. While each has advan-tages, these formats do not provide an accurate account of how a drug compound will react in vivo1.

Primary hepatocytes, or liver cells, are a better op-tion when testing lead drug compounds against enzymes such as CYP because they posses the full complement of enzymes, nuclear factors and co-factors at physiological levels. Data from primary hepatocytes is closely correlated to in vivo condi-tions, and in order to use these cells most efficient-ly, high-throughput, easy-to-use and robust assay chemistries with automated instrumentation have been developed.

Here we demonstrate an automated assay system, including a luminescent CYP inhibition assay, pri-mary hepatocytes and automated microplate in-strumentation, to test lead compound inhibitory effects on CYP isoforms. Validation and pharmacol-ogy data, including a comparison to data gener-ated using human liver microsomes, prove that the combination of cells, assay, and instrumentation provide rapid, dependable information on CYP-based drug inhibition in a cell-based format.

Materials and Methods Materials Suspension HepatocytesHepatocytes are the gold standard when determining drug metabolism and safety profiling. As the most abundant liver cells, hepatocytes are involved in many critical body functions, including the majority of endogenous and exogenous substance metabolism. Though liver-derived cells lines such as HepG2 and HepaRG are available, they lack the full complement of enzymes and transporters at physiologically relevant expression levels. Additionally, non-human hepatocytes are used in new chemical entity ADME studies, however, species-specific differences make human correlations difficult. Human in vitro hepatocytes retain most of their in vivo function at physiologically relevant levels, especially Phase I and Phase II metabolism and transport activities.

Suspended hepatocytes may be cryopreserved or freshly isolated, and are used for studies lasting less than six hours. These studies may include metabolic stability, metabolic enzyme inhibition or uptake transporter assays. Cryopreserved human hepatocytes allow flexible experimental scheduling, and use of the same donor in repeat studies. This is ideal for human-specific metabolic profiling, and critical for drug discovery screening assays. Liverpool™ cryopreserved human suspension hepatocytes (Cat. No. X008052), and InVitroCYP™ M-class 50-donor Mixed Gender Pooled Human Liver Microsomes were obtained from Celsis In Vitro Technologies (Baltimore, MD).

P450-Glo AssayThe P450-Glo™ Screening Systems incorporate human CYP enzymes with luminogenic CYP substrates. The sensitivity and simple “add-and-read” protocol enables small CYP enzyme concentrations per reaction, and makes the assay amenable to profiling applications in high-density plates.

BioTek Instruments, Inc.P.O. Box 998, Highland Park, Winooski, Vermont 05404-0998 USATel: 888-451-5171 Outside the USA: 802-655-4740 E-mail: customercare@biotek.com www.biotek.comCopyright © 2012

Key Words:

Cytochrome P450

CYP

CYP450

Drug Metabolism

Inhibition

Human Liver Microsomes

Hepatocytes

Profiling

Drug Safety

Instrumentation

Luminescence

Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, VT Timothy Moeller, Celsis IVT, Baltimore, MD Tracy Worzella, Mary Sobol, Dongping Ma, James J. Cali, Promega Corporation, Madison, WI

Methods P450-Glo Assay ProcedureCryopreserved hepatocytes were thawed and resuspended in InVitroGro HT Medium. The cultures were centrifuged, supernatant was removed, and the cells were resuspended to 1.0 x 106 cells/mL in Krebs-Ringer bicarbonate buffer. All other assay components were prepared according to the manufacturer’s protocol.

All compounds were titrated in 100% DMSO to create an 11-point dose-response curve. The compounds were then diluted from the 200X stock in water or Krebs-Ringer bicarbonate buffer for the microsome- and hepatocyte-based assays, respectively, to a final 4X concentration before addition to the assay plates.

Per each assay, 5 μL of each titrated compound was added to the 384-well assay plate in quadruplicate. The assay plates were then placed in a 5% CO2/37o C incubator for 20 minutes. This warmed the compounds to 37o C. After incubation, microsome-based assay plates received 5 μL of microsomes and 5 μL of NADPH per well, while hepatocyte-based assay plates received 10 μL of suspended hepatocytes per well.

All assay plates were then incubated using the aforementioned environmental conditions for 10 minutes. Following this second incubation, 5 of μL substrate was added to each well, and the plates were returned to the same incubator for the appropriate time listed in Table 2.

2

Application Note

The luminescent assay incorporates a stabilized firefly luciferase formulation to minimize interfer-ence from compounds, and provides a stable sig-nal for assay plate batch processing. The assay is performed in two steps. In step one, CYP enzymes convert a luminogenic substrate to a luciferin product. In step two, luciferin detection reagent is added. This simultaneously stops the CYP reac-tion and initiates a luminescent signal that is di-rectly proportional to the luciferin produced in step one, an indication of CYP activity amounts.

In this protocol, the substrates are Luciferin-IPA, Luciferin-H and Luciferin-1A2 for CYP3A4, CYP2C9 and CYP1A2, respectively. P450-Glo™ Assay Sys-tems (Cat. Nos. V9002, V8791, and V8421) were donated by Promega Corporation (Madison, WI).

Other ReagentsKrebs-Ringer Bicarbonate Buffer (Cat. No. K4002) was purchased from Sigma-Aldrich Company (St. Louis, MO). Sulfaphenazole (Cat. No. S0758), Di-clofenac (Cat. No. D6899), Ketoconazole (Cat. No. K1003), Verapamil (Cat. No. V4629), Alpha-Nap-thoflavone (Cat. No. N5757), Furafylline (Cat. No. F124), Fluvoxamine (Cat. No. F2802), and Propran-olol (Cat. No. P0884) were purchased from Sigma-Aldrich Company. Fluconazole (Cat. No. 3764) and Fluoxetine (Cat. No. 0927) were purchased from Tocris Bioscience (Ellisville, MO). Troleando-mycin (Cat. No. BML-EI249) was purchased from Enzo Life Sciences (Farmingdale, NY), and Ritona-vir (Cat. No. SRP013031r) was purchased from Se-quoia Research Products Ltd., (Pangbourne, UK).

Instrumentation

The Precision™ Microplate Pipetting System (BioTek Instruments, Inc.) combines an 8-chan-nel pipetting head and an 8-channel bulk re-agent dispenser in one instrument. The instru-ment was used to serially titrate across a 96-well polypropylene plate, dilute compounds, and transfer compounds in quadruplicate from the 96-well compound plate to a 384-well assay plate.

The MultiFlo™ Microplate Dispenser (BioTek Instru-ments, Inc.) combines two peristaltic pumps and two syringe pumps in one unit for fast and accurate microplate-based liquid dispensing. The instrument was used to dispense hepatocytes and microsomes, and to dispense reagents to the 384-well assay plates.

The Synergy™ H4 Hybrid Multi-Mode Microplate Reader combines a filter-based and monochroma-tor-based detection system in one unit for a wide range of fluorescence-based assays, as well as high-performance luminescence detection. The instrument was used to read the luminescent sig-nal from each assay well in the P450-Glo™ assays. Optimized instrument settings are listed in Table 1.

ADME/Tox (Drug Metabolism)

Table 1. Synergy H4 Optimized Instrument Settings.

Table 2. Recommended Substrate Incubation Times.

After the third incubation, 20 μL of Luciferin Detection Reagent was added to each microplate well, the assay plates were incubated a final time at room temperature for 20 minutes, and the luminescent signal was read using the aforementioned Synergy H4 settings.

CYP Compound Profiling Setup Each of the three CYP enzymes was profiled against twelve inhibitors, using human liver microsomes and suspension hepatocytes and the previously detailed processes. The known inhibitors for each enzyme are detailed in Table 3. Eleven point dose responses were generated for each compound, including a no compound control. The entire process was repeated three times each with microsomes and hepatocytes on separate days and with fresh compound aliquots.

3

Application Note

Table 3. Known inhibitors chosen for the three CYP enzymes included in the panel.

Results

Z’-Factor ValidationA Z’-Factor assay was performed to validate the automat-ed CYP2C9, -3A4, and -1A2 assay procedures using human liver microsomes and suspension hepatocytes. Known in-hibitors (Sulfaphenazole:CYP2C9; Ketoconazole:CYP3A4; Alpha Napthoflavone:CYP1A2) were used at 0 μM or 10 μM concentrations for the positive and negative controls, respectively. Per Figure 1, all Z’-Factor values are greater than 0.5; indicative of an excellent assay2.

Figure 2. Compound inhibition curves generated with the CYP2C9, CYP3A4 and CYP1A2 microsome-based assays.

ADME/Tox (Drug Metabolism)

Table 4. CYP known inhibitor IC50 values using microsome- and hepatocyte-based assays. Data shows average and standard deviations of three runs for each compound, and values represent µM concentrations.

Figure 1. Z’-Factor validation data for the human liver microsome- and suspension hepatocyte-based assays.

Compound ProfilingPer compound profiling data using human liver micro-somes, the automated method provides inhibition data for numerous compounds (Figure 2) with multiple CYP assays in a single run. The titration process and assays are repeatable over multiple runs as witnessed by the inhibition curve and IC50 value similarities in Figure 3. Table 4 also illustrates the automated profiling process repeatability and pharmacology data accuracy for each compound. Microsome IC50 values are within one-half log of existing literature values, taken from the 2006 FDA Draft Guidance for Drug Interaction Studies3 or other previously published values. Literature references are IC50 values with the exception of Diclofenac and Pro-pranolol, which are substrates of CYP2C9 and CYP1A2, respectively. The values listed here represent Km values for the substrate, although one would expect these values to be similar when these compounds are used as competitive inhibitors, as they are for this project.

Figure 3. Multi-run individual compound inhibition curves generated with the microsome-based assay.

Per compound profiling data using suspension he-patocytes, Figure 4 and Figure 5 demonstrate the effective adaptation and validation of automated profiling to the cell-based assay format. IC50 and Km standard deviations across multiple runs remain small (Table 4) and agree with existing literature values.

4

Application Note

Figure 4. Compound inhibition curves generated with the CYP2C9, CYP3A4 and CYP1A2 hepatocyte-based assays.

Summary

The P450-Glo assay allows rapid and accurate compound inhibitory assessment with multiple CYP enzymes in a profiling format. The assay may be used with human liver microsomes and suspension hepatocytes; however, comparative results illustrate the importance of using hepatocytes and cell-based assays to perform CYP inhibition assessments. Finally, regardless of method, automated instrumentation improves CYP profiling throughput, yet still provides a robust assay process.

References

1. Brown, H.S.; Wilby, A.J.; Alder, J.; Houston, J.B. Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay. Drug Metab Dispos., 2010 38(12): 2139-46.

2. Zhang, J.H.; Chung, T.D.; Oldenburg, K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen., 1999 4(2): 67-73.

3. Guidance for Industry: Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling, Draft Guidance. U.S. Department of Health and Human Services, FDA, 2006.

4. McGinnity, D.F.; Zhang, G.; Kenney, J.R.; Hamilton, G.A.; Otmani, S.; Stams, K.R.; Haney, S.; Brassil, P.; Stresser, D.M.; Riley, R.J. Evaluation of Multiple in Vitro Systems for Assessment of CYP3A4 Induction in Drug Discovery: Human Hepatocytes, Pregnane X Receptor Reporter Gene, and Fa2N-4 and HepaRG Cells. Drug Metab Dispos., 2009 37(6): 1259-68.

5. Brøsen, K.; Skjelbo, E.; Rasmussen, B.B.; Poulsen, H.E.; Loft, S. Fluvoxamine is a potent inhibitor of Cytochrome P4501A2. Biochem Pharmacol., 1993 45(6): 1211-14.

6. Johnson, J.A.; Herring, V.L.; Wolfe, M.S.; Relling, M.V. CYP1A2 and CYP2D6 4-hydroxylate propranolol and both reactions exhibit racial differences. J Pharmacol Exp Ther., 2000 294(3): 1099-105.

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ADME/Tox (Drug Metabolism)

Figure 5. Multi-run individual compound inhibition curves generated with the hepatocyte-based assay.

Figure 6. Compound inhibition comparison curves using human liver microsomes and suspension hepatocytes.

Additionally, Table 4 and Figure 6 show the differences that may be seen when comparing microsome-based data generated using a biochemical format to cell-based data using hepatocytes. These differences, highlighted in yellow (Table 1) may be up to 10-fold. One main rea-son for this difference may be because cells contain all of the enzymes that can affect the availability of a com-pound to cytochrome P450 enzymes, including trans-porter enzymes. For example, Verapamil IC50 is lower in hepatocytes, presumably due to the transporter effect of increasing intracellular inhibitor, compared to microsomal activity. This supports the notion that processes within an intact, functioning cell, such as uptake and efflux drug transport, may affect a compound’s inhibitory properties.