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 DOI: 10.1016/j.jala.2005.03.004

2005 10: 140Journal of Laboratory AutomationPaul V. Kaplita, Hanbo Hu, Lisa Liu, Thomas M. Farrell, Heather Grbic and Denice M. Spero

Automatable Formats of Higher Throughput ADMET Profiling for Drug Discovery Support  

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140 JALA Ju

Automatable Formats of HigherThroughput ADMET Profiling forDrug Discovery Support

Paul V. Kaplita,* Hanbo Hu, Lisa Liu,Thomas M. Farrell, Heather Grbic, and Denice M. Spero

Department of Drug Discovery Support,Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT

Original Report

ne

An evolving union of high throughput screening (HTS)

and absorption, distribution, metabolism, excretion,

and toxicology (ADMET) technologies have transformed

drug discovery. Human tissue-based, in vitro ADMET

assays can efficiently generate reliable profiles for

structure-activity or structure-property relationships of

compounds from screening ‘‘hit sets’’ or libraries. The

process of identifying discovery compounds with desirable

‘‘druglike’’ properties has consequently become

increasingly data-driven. Chemists and biologists initiate

the process by submitting requests to our laboratory

through an intranet database. A Caco-2 cell model,

automated on a Tecan Genesis workstation, evaluates

the intestinal absorption of drug candidates. Distribution

properties are determined with a high-throughput

equilibrium dialysis technique for measuring plasma

protein binding. Drug metabolism can be evaluated on

a Genesis workstation via measurements of metabolic

stability in liver microsomes. Drug-drug interactions can

be predicted with HTS techniques using human

recombinant hepatic CYP450 isoforms. A Genesis

workstation, integrated with a fluorescence plate reader,

executes CYP450 inhibition assays. Cell toxicity assays

*Correspondence: Paul V. Kaplita, Department of Drug DiscoverySupport, Boehringer Ingelheim Pharmaceuticals, Inc.,900 Ridgebury Rd, Ridgefield, CT 06877-0368; Phone:þ1.203.798.4601; Fax: þ1.203.837-4601; E-mail:pkaplita@rdg.boehringer-ingelheim.com

1535-5535/$30.00

Copyright �c 2005 by The Association for Laboratory Automation

doi:10.1016/j.jala.2005.03.004

2005

using human hepatocytes can serve as early,

high-throughput indicators of potential systemic drug

toxicity. The early availability of ADMET profiling data can

now enable discovery scientists to quickly evaluate the

factors that influence the pharmacodynamic and

pharmacokinetic properties of compounds in lead

optimization. ( JALA 2005;10:140–8)

INTRODUCTION

High throughput screening (HTS) has been routinelyemployed for the past 15 years as a strategic methodto discover compounds that elicit a ‘‘hit’’ activity ina particular molecular target or cellular screen. Asresearch organizations have entered more and morecompounds into development that were initiallydiscovered and optimized from specific moleculartargets, a trend towards higher rates of failure hasbeen observed. These failure rates cannot beattributed to one specific cause, but rather appearto be spread out among several factors includinglack of efficacy (30%) and poor pharmacokinetics ortoxicity (50%), which are actually all interrelated.1

With drug discovery and development costs nowrunning between $800–900 million over 10–15 years,these failure rates have become unacceptable to theindustry.1 A shift in strategy for the drug discoveryprocess has occurred, with the search for acceptablepharmacokinetics/pharmacodynamics (PK/PD) andtoxicity now assuming near-equal importance to thegoals of potency, efficacy, and selectivity.2 The goal

Keywords:

ADMET,

high throughput

screening,

drug discovery,

drug metabolism,

drug absorption

Original Report

now is to identify and design away potential liabilities withabsorption, distribution, metabolism, excretion, and toxicol-ogy (ADMET) early in the drug discovery process to reduceattrition later in drug development.

Effective acceleration of this new paradigm for leadoptimization requires a rapid turnaround of hundreds ofrequests for ADMET data per week from multiple projects,all the while maintaining the high level of data qualitypreviously provided for a relatively limited number ofcompounds. A collaborative union of HTS and ADMETtechnologies in recent years has transformed the hit-to-leadand lead optimization stages of drug discovery.3–5 Technol-ogies and industrialization techniques, originally developedfrom HTS, have successfully provided pharmaceuticalcompanies with early ADMET data. Assay throughputs,higher than previously achieved, have been realized by adapt-ing the assay protocols to microplate formats, includingthe use of scaled-down volumes and benchtop automation.High-throughput assay platforms and laboratory automa-tion have been applied to established methods for CYP450inhibition,6,7 liver metabolic stability,7–9 plasma proteinbinding,10,11 drug absorption,12,13 and cell toxicity.14

The contemporary HTS paradigm now routinely encom-passes the profiling of compounds in vitro for ADMETproperties. Human or animal tissue-based, in vitro ADMETscreens can efficiently generate reliable profiles for structure-activity or structure-property relationships. These assays hadbeen traditionally performed manually on a very limitednumber of compounds in drug development for candidateselection. Now, data profiles can be assembled usingautomatable HTS assays that successfully balance quality(i.e., reproducibility and predictive value), quantity (i.e.,throughput and turnaround time), and cost (i.e., sample size,supplies, capital equipment, and manpower). These earlydata have been used to rank compounds on a relative scalefor acceptable druglike characteristics. Such profiles can beused prospectively for prediction of PK/PD properties, orretrospectively for ad hoc problem solving with compoundsalready in lead optimization. The process of identifying dis-covery compounds with desirable druglike properties hasconsequently become more data driven than ever before.

We have employed commercially available HTS assaytechnologies, laboratory automation, and user-friendly in-formation processing systems to assemble and operatea comprehensive panel of in vitro ADMET assays for cost-effective use in the hit-to-lead and lead optimization stages ofdrug discovery.

MATERIALS AND METHODS

Caco-2 Cell Permeability Assay

A Tecan Genesis CPW 150 workstation (Tecan US,Durham, NC) was used as the automation platform. Thisrobot has two arms: a liquid handling (LiHa) arm anda robotic manipulator (RoMa) arm. The LiHa is comprised

of eight, independently controlled pipetting tips. The RoMacan grasp objects off the surface of the deck (e.g., micro-plates) and carry them to associated peripherals, such as theincubator or the microplate hotels. The workstation wasprogrammed with Gemini software (Version 3.4), and allprocedures (i.e., liquid handling, plate manipulation, andsample collection) were custom-written.

Caco-2 cells (American Type Culture Collection, Rockville,MD) were maintained at 37 �C in complete Dulbecco’smodified Eagle’s medium, containing 10% fetal bovine serum,1.1% nonessential amino acids, 100 units/mL penicillin, and100 mg/mL streptomycin, in an atmosphere of 5% CO2 and90% relative humidity. For permeability experiments, cellswere seeded at a density of 80,000 cells/cm2 in Costar 12-or 24-well plates (Corning, Inc., Corning, NY) on Transwellpolycarbonate filters (12 or 6.5 mm diameter, respectively,and with 0.4 mmpore size). Themediumwasmanually changedevery other day for the first seven days and every day thereafter.The cells were allowed to grow and differentiate for 21–25 days.

Culture medium was aspirated from the cell monolayersprior to permeability experiments. All cell monolayers wererinsed once with 1X Hank’s balanced salt solution (HBSS),pH 7.4, and allowed to equilibrate at 37 �C for 30 min.Monolayers with transepithelial electrical resistance (TEER)values greater than 400 U$cm2 were used in assays.

Dosing solutions for test compounds were prepared in 1XHBSS, pH 7.4, at a nominal concentration of 25 mM, andallowed to equilibrate overnight at room temperature whilebeing mixed on a magnetic stirrer. Prior to the experiment,the dosing solutions were filtered through a 0.45 mm poly-vinylidene fluoride filter. The actual concentration wasdetermined by analyzing the dose solutions for parentcompound.

For the experiment, the dosing solution was added to thedonor side of the monolayers, the apical side for an A/Bexperiment, and the basolateral side for a B/A experiment.1X HBSS, pH 7.4, was added to the receiver side of themonolayers. At each time point, samples were collected fromthe receiver side of the monolayers. Bidirectional (A/B andB/A) permeability studies were conducted in duplicate overthe course of 150 min. Samples were collected at 15, 30, 60,90, 120, and 150 min. Volumes in the apical and basolateralcompartments were maintained constant by replenishing the1X HBSS after each sampling.

Analysis was done by liquid chromatography-massspectrometry-mass spectrometry (LC-MS-MS), and the ap-parent permeability coefficients (cm/s) were determined as

P ¼ dQ

dt$A$C0$60

where dQ/dt$60 is the slope of the permeation profile acrossthe Caco-2 cell monolayers, A is the surface area of the insert(1.13 cm2), and C0 is the initial dosing concentration of thedonor.

JALA June 2005 141

Original Report

Plasma Protein Binding Assay

The approximate in vitro fractional binding of testcompounds to human and animal plasma proteins wasdetermined in 96-well equilibrium dialysis plates (Harvard/Amika, Holliston, MA). Each well consists of a top (plasma)chamber and a bottom (buffer) chamber, separated by anultrathin, semipermeable membrane with a 10 kDa molec-ular weight cutoff. Stock solutions for each test compoundwere prepared in acetonitrile at 200 mg/mL. The subsequentdialysis suspensions were prepared in pooled plasma (with Kethylenediamine tetraacetic acid) from male and femaledonors at 1000 ng/mL and dispensed into the plasmachambers in quadruplicate aliquots of 250 mL. The reciprocalbuffer chambers were filled with of 250 mL of 100 mMpotassium phosphate buffer, pH 7.4. Dialysis was performedat 37 �C for 5 h, with the plates mounted on an automatedrotator (Harvard Bioscience, Holliston, MA) set at 20 rpm.

Samples of 150 mL were collected at the end of the dialysisperiod from all chambers. Each sample was diluted 1:1 withthe reciprocal matrix to give a plasma-to-buffer ratio of 1:1.Sample extraction was performed by protein precipitationwith 3 volumes of acetonitrile and centrifugation at 3000 rpmfor 20 min. The supernatant was removed for analysis byLC-MS-MS. Percent bound is calculated with the formula:

%Bound ¼�1� buffer concentration

plasma concentration

�� 100%

Liver Microsomal Metabolic Stability Assay

A Tecan Genesis 150 workstation (Tecan US, Durham,NC), programmed with Gemini software (Version 3.4) wasused as the automation platform. This robot has one liquidhandling (LiHa) arm and an orbital shaker that is equippedwith a water-jacketed aluminum heating manifold. Proce-dures for liquid handling and incubation were custom-written.

Test compounds were incubated in duplicate MatrixMultiScreen minitubes (Matrix Technologies, Hudson, NH)with liver microsomes (Xenotech, Lenexa, KS) to measurethe first order rate of consumption. Each assay wasperformed in 50 mM potassium phosphate buffer, pH 7.4,and 2.5 mM NADPH. Compounds were tested at a finalassay concentration of 1.0 mM. The protein concentration inthe reaction mix was 1 mg/mL. Compounds were preincu-bated for 5 min at 37 �C, and the metabolic reactions wereinitiated by the addition of NADPH. Aliquots of 80 mL wereremoved from the incubation mix at 0, 3, 6, 10, 15, 20, 40,and 60 min after the start of the reaction for profiling data,or at 0 and 30 min after the start of the reaction for screeningdata. Each aliquot was added to 160 mL acetonitrile forextraction by protein precipitation. These samples weremixed for 1 min by vortexing, and a volume of the mixturewas filtered through wells in 0.25 mm glass fiber filter platesby centrifugation at 3000 rpm for 5 min. Sample extracts

142 JALA June 2005

were analyzed by LC-MS-MS to determine parent com-pound levels. Percent loss of parent compound wascalculated from the peak area at each time point to determinethe half-life (t1/2, min) for test compounds.

LC-MS-MS Instrumentation

Sample analyses were performed by LC-MS-MS. In-cubation plates were transferred off-line for quantitativeanalysis. High-performance liquid chromatography wasaccomplished on an Agilent 1100 Series HPLC (AgilentTechnologies, Waldbronn, Germany). The system wasinterfaced with a Gilson 215 liquid handler and a Gilson819 injection valve actuator (Gilson, Inc., Middleton, WI) toinject samples onto a reverse-phase column. Mobile phase A,with 0.1% (v/v) formic acid in water, and mobile phase B,with 0.1% (v/v) formic acid in acetonitrile were used in theHPLC gradient conditions.

Chromatography for samples collected from the perme-ability and protein binding assays was done on Polaris C18

columns (30 � 2 mm i.d., 5 mm; Varian, Walnut Creek, CA)at a flow rate of 0.4 mL/min, with 10 mL injection volumes.Gradient conditions were varied to accommodate a variety ofcompounds. Mass spectrometry for these samples wasperformed by a Waters Micromass Quattro micro withelectrospray ionization in either multireaction monitoring(MRM), or selected reaction monitoring (SRM), foroptimum sensitivity.

The chromatography for liver microsome metabolicstability samples was carried out with injection volumes of10 mL, typically added to Zorbax SB C8-A (30 � 4.6 mm i.d.,3.5 mm; Phenomenex, Torrance, CA) or Polaris C18 (20 � 2mm i.d., 5 mm; Varian, Walnut Creek, CA) columns at flowrates of 1.5 mL/min, or an Aquasil C18 (30 � 2 mm i.d.,3 mm; ThermoHypersil, Keystone, PA) column at a flow rateof 0.5 mL/min. Mass spectrometry was usually performedin positive mode with a Waters Micromass Quattro Ultima(Waters Corp., Milford, MA). MS methods for testcompounds were automatically developed using MassLynxsoftware (Version 4.0) with QuanOptimize, which selectedthe most intense precursor and product ion (ESIþ, ESI–),and optimized the cone voltage and collision energy formultiple reaction monitoring (MRM) conditions. Com-pounds that failed this method development were analyzedwith atmospheric pressure chemical ionization using selectedreaction monitoring (SRM).

Cytochrome P450 lnhibition Assay

A Tecan Genesis 150 workstation (Tecan US, Durham,NC), programmed with Gemini software (Version 4.0) wasused as the automation platform. This robot has one liquidhandling (LiHa) arm and one robotic manipulator (RoMa)arm, and was integrated with a Tecan Genios Promultifunctional plate reader using Magellan (Version 4.00)software. The procedures for liquid handling, plate manip-ulation, and incubation were custom-written.

Original Report

This assay measured the IC50 of test compounds to inhibitisoforms of the human hepatic xenobiotic metabolizingenzymes CYP1A2, 2C9, 2C19, 2D6, and 3A4. Eightconcentrations (0.01–30 mM in half-log intervals) of eachtest compound were tested in 200 mM KPO4, 1.3 mMNADPþ, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and0.4 units/mL glucose-6-phosphate dehydrogenase. Test com-pounds that were previously dissolved in acetonitrile wereseparately diluted in assay buffer. Duplicate 100 mL aliquotsof diluted test compound were dispensed into FalconMicrotest 96-well assay plates (BD Biosciences, FranklinLakes, NJ), and the plates were preincubated for at least 10min at 37 �C. A 100 mL volume of human recombinant CYPisoforms (Supersomes, BD Gentest, Woburn, MA; 2.5–15.0pmol/mL) and substrate (1.5–75 mM) were then addedto each test well. The reaction was incubated for 15–45 minat 37 �C. Standard inhibition curves using assay-specificreference inhibitors (BD Gentest, Woburn, MA) were runwith each assay plate. The reaction was terminated with theaddition of 75 mL 80% acetonitrile/20% 0.5 M Tris base. Theassay plate was read with the fluorescent plate reader atproduct-specific excitation and emission wavelengths.

Multiparameter Toxicity Panel

All cell toxicity assays were performed with the ACTIV-Tox human hepatocyte cell line (Amphioxus Cell Technol-ogies, Houston, TX). Following receipt of cell plates fromAmphioxus Cell Technologies, the cells were acclimated to37 �C and the medium in the wells was replaced. After a 24 hincubation at 37 �C in an atmosphere of 5% CO2 and 90%relative humidity, test compounds were incubated withthe cells for 24–48 h, depending on the particular assayconditions.

Lactate dehydrogenase (LDH) release, inhibition of cellproliferation, cellular adenosine triphosphate depletion, andcaspase activation were determined with commercial assaykits (CytoTox96, CellTiter 96, CellTiter-Glo and Apo-ONE,respectively, Promega Corp., Madison, WI) that weresupplied with the cells.

Data Analysis

Compound tracking and the analysis of data were accom-plished with ActivityBase Version 5.2.2.49 (ID BusinessSolutions, Guilford, United Kingdom), which incorporatedeither the XLFit software (Version 4.1.1) or custom-writtenMicrosoft Excel 2002 macros to process compound dispen-sary worklists and data from instrument output files, andautomatically upload analyzed data to a central corporateR&D database.

RESULTS AND DISCUSSION

HTS assay technologies, integrated with laboratory automa-tion and information processing, can be effective in address-ing ADMET issues early in drug discovery. In particular, theattrition rate due to failure of compounds with in vitro

potency to exhibit in vivo efficacy, or potential liability toelicit drug-drug interactions, or systemic toxicity may bereduced with early in vitro profiling.

Chemists and biologists initiate the process on behalf ofproject teams by submitting requests to our laboratorythrough a corporate intranet database (eRoom Software(Version 7), Documentum Inc., Pleasanton, CA). A stan-dardized request form enables a time-stamped request forassays to be entered compound-by-compound, with support-ing physicochemical data (e.g., salt form, molecular weight,structure, solubility). The form is updated as assay requestsare fulfilled, with a final time stamp when the entire requestis completed. This permits designated team members tomonitor the progress of the request in real time, and allowsthe data to be previewed prior to upload to the centralcorporate database.

It is possible to request data from all available assays inorder to obtain a broad profile of activity or property dataon a particular compound or series of compounds. Alterna-tively, assays may be intentionally staggered in an iterativedesign to provide additional or higher resolution data asa compound successfully progresses along the work plan.This strategy allows for certain assays to be performed ina truncated, high throughput screening format (e.g., reducednumber of time points or test concentrations; refer to thediscussion of the metabolic stability assays below) for rapidfiltering out of compounds with obvious undesirableproperties. These findings would then be followed up onrequest with more definitive, high-content profiling assayformats. These options may be specifically requested; the ex-perimental design and data collection are then performedfollowing distinct templates.

Drug absorption considers the path of a molecule fromits site of administration to the systemic circulation. Themechanism of absorption will govern the onset and durationof drug action, as well as the level of systemic exposure. Fororally administered drugs, this path passes through gastro-intestinal epithelial cells and into the enterohepatic circula-tion. The predominant route is via passive diffusion throughcells, although paracellular diffusion along the tight junctionsbetween cells and carrier-mediated active transport are alsosignificant. Drug absorption may also be limited by effluxtransporters, such as p-glycoprotein. Consequently, drugabsorption is a complex process dependent upon solubilityand permeability, formulation variables, and physiologicalfactors including regional differences in gastrointestinal pH,permeability, and motility.

Monolayers of the human colon carcinoma cell line,Caco-2, are regarded as the industry standard in vitro modelsystem for studying mechanisms of drug transport across theintestinal epithelium.2,12,13,15,16 These human intestinal enter-ocytes express transporter and efflux pumps and possess tightjunctions and brush border enzymes (Fig. 1). For moleculesthat are largely absorbed through passive diffusion, a goodcorrelation exists between apparent permeability coefficientand lipophilicity (measured by the distribution coefficient

JALA June 2005 143

Original Report

between aqueous and organic solvents). An assay thatmeasures bidirectional flux from one side of the Caco-2 cellmonolayer to the other, readily automated on a TecanGenesis CPW 150 workstation (Fig. 2), can effectively profilehundreds of compounds per month.

Additional data on the effect of transporter mechanismsmay be subsequently collected by testing particular com-pounds for A/B and B/A flux in the absence and presenceof known transporter inhibitors.17 We have observed, forexample, that the apparent permeability coefficients ofthe anticancer agent vinblastine were modulated by a p-glycoprotein inhibitor, suggesting that this drug and itstherapeutic effect can be attenuated by efflux transport(Table 1). The antihistamine chlorpheniramine, on the otherhand, was unaffected by the p-glycoprotein inhibitor.

Drug distribution considers the movement of drug fromthe systemic circulation to the extravascular space, which isoften the location of the therapeutic target. The process ofdrug distribution is largely a function of passive diffusionand protein binding in blood and tissues. Drug moleculesthat are protein bound have their distribution restricted andare therefore pharmacologically inactive. Equilibrium di-alysis is the most common method in the industry formeasuring drug-protein binding and thus estimating effective

Figure 2. Tecan Genesis CPW 150 workstation supportingCaco-2 cell in vitro drug permeability assays.

Tight Junction

Apical

BasolateralParacellular Transcellular

CarrierMediated

CarrierLimited

Figure 1. Schematic overview of the various parallel mechanismsof drug transport across a prototypical Caco-2 cell monolayer.

144 JALA June 2005

drug concentration at the pharmacological target.2,10,11 Thistechnique measures binding at equilibrium, and unlikeultrafiltration or column chromatography, is less susceptibleto artifacts of nonspecific binding to filter membranes andother experimental surfaces, or changes in plasma proteinconcentration during ultracentrifugation.

The throughput of the equilibrium dialysis was effectivelyscaled up with 96-well microplate technology and benchtopautomation to profile hundreds of compounds per month.Our in vitro assay produced binding data (Fig. 3) thatcorrelated well with human clinical data for a test set ofthirteen drugs. The assay was particularly effective forbinning test compounds as poorly (! 80%), moderately,and highly (O 95%) protein bound. The assay was alsorobust enough to accommodate plasma samples fromcommon experimental animal species (Table 2). Thiscapability can thus provide for species scaling and compar-ison of data among experimental pharmacological ortoxicological models.

Table 1. Effect of a p-glycoprotein inhibitor on the apparentpermeability coefficients for test compounds across Caco-2 cellmonolayers. Data are from duplicate measurements collected inthe absence and presence of 2 mM inhibitor.

Papp A/B Papp B/A PDR(10�6 cm/s)

Vinblastine 0.7 15.5 22.0

With inhibitor 3.7 8.0 2.2

Chlorpheniramine 16.8 11.1 0.7

With inhibitor 15.9 11.3 0.7

0

10

20

30

40

50

60

70

80

90

100

110

0 10 20 30 40 50 60 70 80 90 100 110

% bound (equilibrium dialysis microplate)

% b

ound

(cl

inic

al)

Figure 3. Comparison of human plasma protein bindingmeasurements from the in vitro equilibrium dialysis microplatemethod and clinical data for quadruplicate determinations with 13test compounds (amitriptyline, desipramine, methotrexate, nevir-apine, ontazolast, pirenzepine, prednisone, propranolol, quinine,rifampicin, warfarin, compound 7, and compound 8).

Original Report

Drug metabolism considers the biotransformation ofdrugs, usually in the liver, to hydrophilic derivatives thatmay be more rapidly eliminated by the kidneys. Metabolicreactions that add or uncover polar groups, such asoxidation and hydrolysis, generally receive the most atten-tion in drug discovery. Drug metabolism is a significantcomponent of potential ADMET issues in drug develop-

Table 2. High-throughput equilibrium dialysis plasma proteinbinding measurements for a poorly bound drug (pirenzepine),a moderately bound drug (nevirapine), and a highly bound drug(ontazolast)24 from quadruplicate measurements with plasma fromvarious species

% Protein BoundPirenzepine Nevirapine Ontazolast

Human (clinical) 12 60 95.6

Human 7.5 55.9 98.9

Cynomolgus 28.7 50.2 99.9

Rhesus 19.2 54.7 99.9

Beagle 5.2 56.5 99.8

Guinea pig 56.2 51.8 99.8

Rat 6.5 49.1 99.8

Mouse 14.8 63.1 99.9

ment, including metabolic stability, drug-drug interactions,and drug toxicity.

Metabolic stability is a key determinant of the bio-availability and clearance of a drug. Liver microsomes arewidely used as an in vitro model for drug metabolism, owingto their ease of preparation and use, high content of drugmetabolizing enzymes, high rate of substrate turnover, androbustness.18 Typically, a single concentration of test sub-strate (at levels well below the apparent KM of drugmetabolizing enzymes) is incubated with microsomes andthe appropriate cofactors so that the first order disappear-ance of the parent compound may be measured over multipletime points to calculate a half-life.19 Microsomal metabolicstability can be a reliable predictor of in vivo drugmetabolism20 if one assumes that oxidative metabolism inthe liver is the major route of drug clearance.

Our metabolic stability assay was performed on a TecanGenesis 150 workstation (Fig. 4) and can test thousands ofcompounds per month with microsomes from human,monkey, dog, rat, and mouse. This versatility can thusenable species scaling and comparison of metabolism dataamong experimental pharmacological or toxicological mod-els. The test method was designed to measure percentremaining parent compound at a single time point for rapidscreening21 or at multiple time points for follow-up profiling.

3,000 rpm

Receiver plate

Co

mp

ou

nd

s

Time points

Extraction plate

80 µL

Co

mp

ou

nd

s

Assay Minitubes

NADPH

200 µL

Figure 4. Tecan Genesis 150 workstation supporting in vitro liver microsome metabolic stability assays, and representation of the decklayout of the incubation, extraction, and filtration plates when the assay is conducted in duplicate with eight time points (0, 3, 6, 10, 15, 20,40, 60 min).

JALA June 2005 145

Original Report

Table 3. Comparison of multiple time point and single time pointmethods for determining mouse liver microsome metabolicstability, expressed as first order half-lives for disappearance ofparent compound from duplicate measurements

Mouse liver Microsome half-life (min)

Multi-pointincubation

Single-pointincubation

Compound 1 3 4

Compound 2 13 18

Compound 3 20 29

Compound 4 54 46

Compound 5 64 160

Compound 6 95 300

146 JALA June 2005

The half-life data were comparable between the two methods(Table 3), which allows for quick discrimination of com-pounds with either brief or prolonged half-lives. Generally,low metabolic stability or a short half-life suggests high-hepatic clearance and consequently poor in vivo exposure.

The most important drug metabolizing enzymes area superfamily of heme-containing, membrane-bound mono-oxygenases, cytochrome P450 (CYP450).2,18 The majorhuman isoforms are designated 1A2, 2C9, 2C19, 2D6, and3A4; over half of the drugs in current use are metabolized byCYP3A4. These enzymes have broad substrate specificities,catalyze multiple sites of oxidation, invoke very reactiveoxygenating species, and are differentially expressed amongvarious human populations. CYP450s may be inhibited (orinduced) by drugs, which create potential liabilities for drug-drug interactions. On occasions when drugs are coadminis-tered with CYP450 inhibitors, their plasma concentrationmay become elevated as a result of decreased clearance,which could result in overdose and toxicity.

Ketoconazole7-Benzoloxy-4-(trifluoromethyl)coumarin (BFC)CYP3A4

Ketoconazole7-benzyloxyquinoline (BQ)CYP3A4

Quinidine3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC)CYP2D6

Tranylcypromine3-cyano-7-ethoxycoumarin (CEC)CYP2C19

Sulfaphenazole7-methoxy-4-(trifluoromethyl) coumarin (MFC) CYP2C9

Furafylline3-cyano-7-ethoxycoumarin (CEC)CYP1A2

InhibitorFluorogenic Enzyme SubstrateIsozyme

concentrations (0.01 – 30 µM)

Sample Plate

Assay Plate

com

poun

ds

com

poun

ds

6 µL

Figure 5. Tecan Genesis 150 workstation supporting in vitro human CYP450 inhibition assays, and representation of the deck layout of theincubation plates when the assay is conducted in duplicate with eight test compound concentrations (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 mM).

Original Report

Cell Proliferation

0

0.5

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1 10 100 1000 10000 100000

1 10 100 1000 10000 100000 1 10 100 1000 10000 100000

1 10 100 1000 10000 100000

concentration ( M)

abso

rban

ce

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concentration ( M)

abso

rban

ce

acetaminophen

amiodarone

diclofenac

rosiglitazone

valproate

tacrine

tamoxifen

acyclovir

acetaminophen

amiodarone

diclofenac

rosiglitazone

valproate

tacrine

tamoxifen

acyclovir

acetaminophen

amiodarone

diclofenac

rosiglitazone

valproate

tacrine

tamoxifen

acyclovir

acetaminophen

amiodarone

diclofenac

rosiglitazone

valproate

tacrine

tamoxifen

acyclovir

ATP content

0

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lum

ines

cen

ce

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0

5000

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15000

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concentration ( M)

flu

ore

scen

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Figure 6. Concentration-dependent effect of test compounds on toxicity biomarkers after 24–48 h exposure to HepG2/C3A hepatocytes.Data are from duplicate measurements.

Reliable, low-cost microplate-based assays for CYP450inhibition have been developed using baculovirus-expressedrecombinant human enzyme systems and fluorogenic probesubstrates.6,22 These probes undergo O-dealkylations, andtheir corresponding product fluorescence is not usuallysubject to interference from any background fluorescenceor quenching by cofactors (e.g., NADPH) or testcompounds. Multiple probe substrates are available for usewith each CYP450, which can be helpful in accommodatingthe complex, non-Michaelis-Menten inhibition kineticsoften observed with these enzymes.23 Our CYP450 inhibi-tion assays were fully automated on a Tecan Genesis 150workstation (Fig. 5) and were capable of profiling thousandsof compounds per month.

Drug-induced organ toxicity is a function of the intrinsictoxicity of the drug substance, the concentration of the drugat the target organ, and the relative influence of systemicdetoxification (or bioactivation) mechanisms. Since intrinsictoxicity can be most directly related to chemical structure, itreceives considerable attention in drug discovery. The livercan be a particularly sensitive end organ for toxicity asa consequence of the first-pass effect and the liver’spredominant role in metabolic detoxification and activation.A suite of cell toxicity assays are frequently used to generatean ‘‘early warning’’ sentinel profile.2,4 Toxicity profiling withhepatocytes can provide particular insights to metabolism-based toxicity.

Prototypical in vitro toxicity assays include measurementof the release of cytoplasmic enzymes, such as lactate

dehydrogenase (LDH), as an indicator of cytoplasmicmembrane integrity, measurement of mitochondrial activityor ATP levels as indicators of cellular viability and energyconsumption, and measurement of caspase activity as amarker for the induction of apoptosis.14 These assays can beminiaturized and automated in 96-well microplate formats,with fluorescent, luminescent, or colorimetric readouts.Figure 6 illustrates the effects of a test set of eight com-pounds on toxicity biomarkers in an immortalized line ofHepG2/C3A hepatocytes. Hepatotoxicants such as amiodar-one, diclofenac, tacrine, and tamoxifen were notably effectivein most assays, while the negative control compound,acyclovir, was comparatively inactive. The patterns ofresponse in these assays can provide a broad picture of thepotential toxic interactions with test compounds.

CONCLUSION

Pharmacokinetic, drug metabolism, pharmaceutics, andtoxicology information on pharmacologically active com-pounds can now be acquired very shortly after synthesis, orafter selection from screening hit sets or libraries. This workdescribes the use of commercially available individualworkstations, hardware, and software to support a panel ofin vitro ADMET assays. The automation and informationprocessing technology take full advantage of the sensitivity,selectivity, and throughput available from microplate-compatible instrument readouts and contemporary liquid

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Original Report

chromatography coupled with tandem mass spectrometry.The robust, miniaturized techniques improve assay repro-ducibility, reduce potential hazards from handling humanbiological media, and save time and money. The resultingearly ADMET data serve as valuable predictors of pharma-cokinetics and pharmacodynamics. Universal access to theseprofiling data via a central R&D database can accelerate theselection of druglike predevelopment candidates from cur-rent drug discovery programs and accrue added value forsubsequent programs by enriching the data content of thecorporate compound collection.

ACKNOWLEDGMENTS

The authors would like to thank Carol Ann Homon, Mehran Yazdanian,

Stephen Norris, Guanfa Gan, Donald Tweedie, Odette Fahmi, Scott

Leonard, David Pilloise, Qihong Huang, and Supriya Jayadev from

Boehringer Ingelheim Pharmaceuticals, and Norman Sussman and James

Kelly from Amphioxus Cell Technologies for their collaboration and helpful

discussions.

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