Cassette Dosing for Pharmacokinetic Screening in DrugDiscovery: Comparison of Clearance, Volume of Distribution,Half-Life, Mean Residence Time, and Oral BioavailabilityObtained by Cassette and Discrete Dosing in Rats

RAKESH NAGILLA,1 MELANIE NORD,2 JEFF J. MCATEE,2 LARRY J. JOLIVETTE2

1Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Center of Excellence in Drug Discovery,GlaxoSmithKline, Collegeville, Pennsylvania 19426

2Heart Failure Discovery Performance Unit, Metabolic Pathways Center of Excellence in Drug Discovery, GlaxoSmithKline,King of Prussia, Pennsylvania 19406

Received 3 November 2010; revised 24 January 2011; accepted 2 February 2011

Published online 1 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22525

ABSTRACT: The purpose of this investigation was to compare selected pharmacokinetic (PK)parameters obtained by cassette and discrete dosing of compounds in rats. The concordanceof PK properties obtained by the two dosing strategies was evaluated for 116 compounds rep-resenting various therapeutic programs and diverse chemical structures. The correspondencebetween cassette- and discrete-dosing-derived PK properties was examined semiquantitativelyand qualitatively. For semiquantitative comparison, compounds with cassette-to-discrete PKparameter ratios between 0.5 and 2 (inclusive) were considered to be in agreement. For qual-itative comparison, compounds were divided into three categories (low, moderate, and high)based on the value of the PK parameter; compounds that fell into the same category follow-ing cassette and discrete dosing were considered to be in agreement. Of the 116 compoundsevaluated, 89%, 91%, 80%, and 91% of the compounds were semiquantitatively equivalentfor the intravenous PK parameters of clearance (CL), volume of distribution (Vdss), termi-nal elimination plasma half-life (HL), and mean residence time (MRT), respectively, whereas79%, 80%, 79%, and 72% were qualitatively similar for CL, Vdss, MRT, and terminal elim-ination plasma HL, respectively. Following oral administration, bioavailability concordancewas 72% when assessed qualitatively and 78% when determined semiquantitatively. Resultsfrom these analyses indicate that a cassette dosing strategy is a viable approach to screencompounds for PK properties within a drug discovery setting. © 2011 Wiley-Liss, Inc. andthe American Pharmacists Association J Pharm Sci 100:3862–3874, 2011Keywords: clearance; cassette dosing; N-in-one dosing; pharmacokinetics; drug discovery;volume of distribution at steady state; half-life; oral bioavailability; liquid chromatography—tandem mass spectrometry

INTRODUCTION

The successful and rapid identification of a moleculelikely to possess clinically favorable characteristics isan important factor within an early drug discoverysetting. Significant causes of clinical failure of new

Correspondence to: Rakesh Nagilla (Telephone: +610-917-5859;Fax: +610-917-4178; E-mail: rakesh.2.nagilla@gsk.com)

This work was presented in part at the 22nd Annual AmericanAssociation of Pharmaceutical Scientists (AAPS) Meeting; Novem-ber 11–15, 2007; San Diego, CaliforniaJournal of Pharmaceutical Sciences, Vol. 100, 3862–3874 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

chemical entities have been ascribed to insufficientpharmacokinetics (PKs), lack of pharmacological effi-cacy, and toxicity.1 In view of this, the pharmaceuticalindustry has increased its focus on drug metabolismand PK issues within the early stage of drug discov-ery to quickly separate potential development candi-dates that are likely to demonstrate suitable clinicalPK properties from chemicals that possess inherentPK liabilities.2 By virtue of this change in the drugdiscovery setting, the pharmaceutical industry hasexperienced some success in reducing the magnitudeof attrition attributable to poor clinical PK.1

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The processes of absorption, distribution,metabolism, and excretion of compounds can beassessed with various in vitro and in silico models.3,4

However, because of the uncertainty of these modelsto accurately project the effect of an intact physi-ological process or system on a given compound,2

determining PKs with an in vivo model remains asan essential step during the lead optimization phaseof drug discovery. Advances in medicinal chemistrytechniques have greatly increased the number ofcompounds that interact specifically and potentlywith potential drug targets.5 Therefore, the abilityto rapidly generate the PK properties for a largenumber of compounds has become very importantduring lead optimization. Several approaches such aspooling of plasma samples,6–8 truncating PK sampleperiod collection,9 cassette-accelerated rapid ratscreen,10 snapshot PK,11 automation of extractionprocesses, and column switching12–14 have beensuggested to increase throughput.

Cassette dosing, also known as N-in-1 dosing,13

represents an attractive way to rapidly screen rela-tively large sets of compounds for their PK propertieswhile minimizing the number of animals otherwiseneeded, using a traditional discrete dosing paradigm.Cassette dosing was proposed in the late 1990s whenBerman et al.6 evaluated the correlation between cas-sette and discrete dosing in dogs for five compoundsand demonstrated that comparable PK parametersare obtained with the two study designs. Advances inmass spectrometric technology, such as the advent ofmultiple reaction monitoring and newer more capablemass spectrometric equipment, have increased capac-ity, sensitivity, and selectivity. This has enabled thequantification of multiple compounds in a sample inrelatively less time and made the analytical capacityrequired for PK studies conducted under a cassette-dosing regimen more practical.15 Advantages of cas-sette dosing compared with discrete dosing includea reduction in the number of studies and animalsneeded to support lead optimization, and the genera-tion of fewer samples for quantitative analysis; thus,cassette dosing may increase the efficiency and pro-ductivity of the industrial PK scientist in a drug dis-covery setting. Nonetheless, the reliability and mer-its of cassette dosing have been debated in the sci-entific community.13,16–18 Potential drawbacks of thecassette-dosing paradigm include increased time anddifficulties sometimes encountered during analyticalmethod development and dose formulation,17 and thepotential for drug–drug interactions upon coadminis-tration of multiple compounds.6,15,19 On the basis ofa recent publication that surveyed the use of cassettedosing in the pharmaceutical industry, it has beensuggested that about 50% of pharmaceutical compa-nies have tried and abandoned the use of some form ofa cassette-dosing regimen.20 About 80% of those who

discontinued the use of cassette dosing listed unrelia-bility as a primary reason; the complexity associatedwith the development of the liquid chromatography—tandem mass spectrometry (LC–MS/MS) analyticalmethods was a common secondary reason. Anothersurvey reported by Ackermann21 indicated that 25%of the pharmaceutical companies never employed cas-sette dosing, 8% employed the dosing paradigm asthe default for exposure screening, and the remain-ing 67% used cassette dosing depending on projectrequirements.

Recently, He et al.22 investigated the correlation be-tween cassette- and discrete-dosing regimens for se-lected PK parameters in rats, dogs, and chimpanzeesand concluded that cassette dosing is a useful tool toquickly obtain reliable PK parameters. However, thedataset used for reaching the conclusion contained alimited number of compounds, that is, 13 in rats, 11in dogs, and eight in chimpanzees. To the authors’knowledge, there is no publication that has comparedPK parameters obtained with cassette- and discrete-dosing paradigms in a comprehensive manner utiliz-ing a large dataset. Therefore, the objective of thisinvestigation was to conduct a retrospective evalua-tion of the correlation of PK data obtained with cas-sette and discrete dosing in rats for 116 compoundsrepresenting various therapeutic areas and diversechemical structures.

METHODS

Materials

All test compounds for PK studies were synthesizedby the Department of Medicinal Chemistry at Glax-oSmithKline (King of Prussia, Pennsylvania). 11-$-Hydropropyl cyclodextrin (Cavitron R© or Encapsin R©)was obtained from Cerestar (Hammond, Indiana) orCargill (Minneapolis, Minnesota). Dimethylsulfoxide(DMSO) was purchased from Sigma–Aldrich chemicalcompany (St. Louis, Missouri).

Data Collection

The dataset used for the analysis represents diversechemical structures from various research programsacross several therapeutic areas. More than 10,000compounds had been profiled for the different pro-grams with cassette-dosing protocols. This databasewas thoroughly searched and 116 compounds wereidentified for which PK data had been generated withboth cassette- and discrete-dosing regimens.

Animals

Adult male or female Sprague–Dawley rats (n = 3 perstudy) weighing between 0.25 and 0.45 kg were usedfor PK (cassette and discrete) studies. All animalswere housed according to the National Institutes of

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Health Guide for the Care and Use of Laboratory An-imals in temperature- and humidity-controlled roomswith 12-h light and dark cycles. Filtered tap waterwas available ad libitum. Animals were fed a stan-dard animal diet; food was available ad libitum ex-cept for overnight periods before the oral-dosing studydays. Whenever overnight fasting was employed, foodwas provided after the 2-h blood sample was obtained.All animal use was conducted according to protocolsreviewed and approved by the Institutional AnimalCare and Use Committee.

PK Studies

Compounds with “favorable” PK properties, as deter-mined by cassette-dosed PK studies, were typicallyprogressed into discrete-dosing regimens with similarstudy conditions as used for the initial cassette study.Briefly, the studies were conducted with a crossoverdesign on two study days with a 1-day recovery periodbetween each study day. The dosing regimen consistedof administering a mixture of up to five test com-pounds (cassette, typically five) or a single test com-pound (discrete) at doses ranging from 1 to 4 mg/kg(intravenous or i.v.) or from 2 to 8 mg/kg (oral). Alldoses were prepared as aqueous solutions in either20% (i.v.) or 6% (oral) 11-$-hydroxypropyl cyclodextrinand contained 5% or less DMSO. All i.v. doses were ad-ministered as 30-min infusions to conscious animalsand oral doses were administered either by gavage orbolus through a gastric catheter. Blood samples wereobtained at regular intervals for 8 or 24 h followingdose administration. The majority (95%) of the cas-sette studies employed 8-h sampling schemes. Com-pounds selected for cassette dosing were screened,a priori, for cytochrome P450 (CYP450) inhibition po-tential with a recombinant human CYP450 system.Compounds with one or more CYP450 isozymes witha half maximal inhibitory concentration of 1:M orless were typically excluded from the cassette-dosingstudy. To avoid potential interference during sam-ple analysis, compounds in the dosing cassettes hadmolecular weights that were separated by at least2 atomic mass units.

For a majority of the discrete studies, the sam-pling duration was longer than that employed forthe cassette-dosing study. Otherwise, study designconsiderations, vehicle pH, sample sizes, and so on,were similar to that employed for the cassette studies.Blood samples under both regimens were obtainedfrom either a lateral tail vein or a femoral arterialcatheter and plasma was harvested. Plasma sampleswere frozen at −80◦C till further analysis, which typ-ically was within 24–48 h after the final sample, wasobtained.

All plasma samples were analyzed for test com-pound content with LC–MS/MS methodologies bythe bioanalytical group within the Department of

Drug Metabolism and Pharmacokinetics at Glaxo-SmithKline. Specific assay conditions were depen-dent upon the structural characteristics of test com-pounds. Briefly, quantitative analysis was performedon plasma samples and dose solutions for eachtest compound using a LC–MS/MS detection methodoptimized for each individual analyte in the ap-propriate biological matrix. Plasma samples werethawed and each analyte was isolated from 50:Lof plasma using a method based upon protein pre-cipitation with 95:5 acetonitrile/buffer (10 mM am-monium formate, pH 3.0) containing an appropri-ate mass spectral internal standard, and the result-ing mixture was vortex mixed for 2 min followed bycentrifugation for 30 min at more than 2000 × g.The resulting supernatant was injected by an HTSPAL autosampler (CTC Analytics, Zwingen, Switzer-land) into an appropriate analytical liquid chro-matography column eluting to either a Sciex API365 or API 4000 triple quadrupole mass spectrom-eter (Applied Biosystems, Foster City, California).The eluent was subjected to Turbo IonSpray ion-ization multiple reaction monitoring and eachanalyte was characterized by an appropriate massspectral transition of the parent precursor ion to anappropriate product ion generated at optimized colli-sion energy. Data were reported as quantitative drugconcentrations as determined by standard calibra-tion curve analysis, using linear fitting of a weightedplot of the analyte/internal standard peak area ra-tio versus analyte concentration. Using these opti-mized conditions, the typical lower limit of quantita-tion achieved was 1.00 ng/mL and, in certain cases,was 5.0 or 10 ng/mL for certain compounds.

Data Analysis

Pharmacokinetic parameters, that is, clearance (CL),volume of distribution at steady state (Vdss), meanresidence time (MRT), area under the plasma con-centration versus time curve (AUC), and terminalelimination half-life (HL), were estimated by non-compartmental analysis with Winnonlin R© software(v3.3–v5.1; Pharsight Corporation, Mountain View,California). The terminal phase rate constant wasdetermined by choosing at least, but not limited to,three points from the log-linear regression of the ter-minal portion of the plasma concentration versus timecurve. Oral bioavailability (%F) was calculated by di-viding the dose-normalized AUC from the oral studysegment by the dose-normalized AUC from the i.v.study segment and multiplying by 100.

Comparative Analysis

Correlation analysis of the PK parameters (CL, Vd,MRT, HL, and %F) between cassette and discretedosing was performed using both qualitative (“right”vs. “wrong” box approach) and semiquantitative

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Table 1. Classification Scheme for Clearance (CL), Volumeof Distribution (Vdss), Half-Life (HL), Mean Residence Time(MRT), and Oral Bioavailability (%F)

CL (mL/min/kg) Low Moderate High

<26 26 ≤ CL < 60 ≥60

Vdss (L/kg) Small Moderate Large<0.7 0.7 ≤ Vdss < 3.5 ≥3.5

HL and MRT (h) Short Moderate Long<1 1 ≤ HL or MRT < 3 ≥3

%F Low Moderate High<10 10 ≤ %F < 33 ≥33

CL, Vdss, HL, and MRT following intravenous administration.

(twofold error) methods. For qualitative analysis, theparameters were classified into three categories as de-scribed in Table 1 based on the PK parameter values.Compounds that fell into the same category follow-ing cassette and discrete dosing were considered asqualitatively equivalent. For semiquantitative com-parison, compounds with cassette-to-discrete PK pa-rameter ratios between 0.5 and 2 (inclusive), that is, atwofold error, were considered as semiquantitativelyequivalent. In addition, quantitative comparison ofthe PK parameters was performed under the assump-tion that discrete dosing yielded the “correct” PK pa-rameter value and that cassette dosing was a means ofprediction. Average-fold error and root mean squareerror in prediction of each of the PK parameter wereestimated as measures of precision and bias, respec-tively, using the following equations:23

Root mean square error

=√∑

(Cassette parameter value − Discrete parameter value)2

Number of observations

Physicochemical properties [acid/base class, calcu-lated log of the octanol–water partition coefficient(clogP), total polar surface area, and number of ro-tatable bonds; data not shown] were calculated within-house in silico models. These data were manuallyinterrogated for trends with cassette- and discrete-dosing-derived PK similarities and dissimilarities.

RESULTS

The individual PK parameters obtained after cassetteand discrete dosing are listed in Appendix 1. The 116compounds represented structurally diverse data setfrom different research efforts and chemical classes.The average molecular weight was 508 ± 102 (me-dian 527, ranged from 242 to 918), with an average

clogP value of 4.6 ± 1.8 (median 4.5, ranged from 0.6to 8.8) and an average topological polar surface areavalue of 92 ± 40Å 2 (median 86; ranged from 33 to347). No clear trends were obvious between any of thephysicochemical properties described above to differ-entiate between compounds that demonstrated “good”agreement in PK parameters obtained with cassetteand discrete dosing from those that did not (data notshown).

Clearance

The comparison of CL values obtained followingcassette- and discrete-dosing regimens are shownin Figures 1a and 1b. For the qualitative approach(Fig. 1a), cassette- and discrete dose administrationyielded CL values that were in the same category fora total of 92 of the 116 compounds (∼79%). Of the 24compounds with CL values that were not in the sameCL category, eight compounds (33%) demonstrated ahigher CL category with cassette dosing than withdiscrete dosing. On the basis of the semiquantitativeapproach (Fig. 1b), a concordance between cassetteand discrete dosing was observed for 103 (89%) of the116 test compounds. Of the 13 compounds that didnot fall within the twofold window, three (23%) of thecompounds demonstrated CL values that were higherwith the cassette study design than that was obtainedwith the discrete study design, and 10 (77%) of thecompounds demonstrated lower CL values from cas-sette dosing compared with discrete administration.

Volume of Distribution

Figures 2a and 2b depict the comparison of cassetteand discrete dosing with respect to Vdss. On the ba-sis of qualitative analysis approach (Fig. 2a), cassetteand discrete dosing yielded Vdss values that were inthe same category for 93 of the 116 compounds (80%).Of the 23 compounds for which Vdss were not in thesame category, eight compounds (35%) demonstratedlarger Vdss following cassette dosing compared withdiscrete dosing. By the semiquantitative approach(Fig. 2b), cassette and discrete dosing resulted in Vdssvalues that were within twofold of each other for 105(90%) of the 116 compounds. Of the 11 compoundsfor which Vdss values were not within twofold, threecompounds (27%) had a larger Vdss following cassettedosing compared with that obtained with discretedosing.

Half-Life

Figures 3a and 3b represent the comparison of plasmaHL values obtain with cassette and discrete dosing.On the basis of the qualitative approach (Fig. 3a), 84 ofthe 116 compounds (72%) had plasma HL values fol-lowing cassette and discrete dosing that were in the

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Figure 1. Qualitative (a) and semiquantitative (b) comparisons of clearance (CL) values ob-tained with cassette and discrete dosing of compounds in rats. Closed circles (�) representcompounds with the same CL classification and open triangles (�) represent compounds withdifferent CL classifications. Dotted lines represent the limits of each category (for qualitative)and the upper and lower limit of the ratio (for quantitative), respectively.

same category. Of the 32 compounds for which the HLvalues were not in the same category, 19 (60%) com-pounds demonstrated a shorter HL following cassettedosing compared with discrete dosing. The semiquan-titative approach yielded a similar outcome, that is, 93(80%) of the 116 compounds had HLs that were withintwofold of each other with cassette and discrete doseadministration (Fig. 3b). Of the 23 compounds that

were not within twofold, 14 (61%) compounds had ashorter HL following cassette dosing compared withdiscrete dosing.

Mean Residence Time

Figures 4a and 4b represent the assessments ofMRT values obtained with cassette- and discrete-dose

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Figure 2. Qualitative (a) and semiquantitative (b) comparisons of volume of distribution atsteady state (Vdss) values obtained with cassette and discrete dosing of compounds in rats.Closed circles (�) represent compounds with the same Vdss classification and open triangles(�) represent compounds with different Vdss classifications. Dotted lines represent the limitsof each category (for qualitative) and the upper and lower limit of the ratio (for quantitative),respectively.

administration. Qualitatively, MRT values for 92 ofthe 116 compounds (79%) were in the same categorywith both the cassette- and discrete-dosing regimens(Fig. 4a). Of the 24 compounds for which MRT wasnot in the same category, 20 compounds (83%) demon-strated a longer MRT with cassette dosing compared

with discrete dosing. By the semiquantitative ap-proach, 106 (91%) of the 116 compounds had cassette-and discrete-dosing-derived MRT values that werewithin twofold of each other (Fig. 4b). Of the 10 com-pounds for which the MRT values were more thantwofold apart, four compounds (40%) had a longer

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Figure 3. Qualitative (a) and semiquantitative (b) comparisons of half-life (HL) values ob-tained with cassette and discrete dosing of compounds in rats. Closed circles (�) representcompounds with the same HL classification and open triangles (�) represent compounds withdifferent HL classifications. Dotted lines represent the limits of each category (for qualitative)and the upper and lower limit of the ratio (for quantitative), respectively.d

MRT following cassette dosing compared with dis-crete dosing.

Bioavailability

Figures 5a and 5b represent the assessment of %Fvalues following cassette and discrete dosing. Thedataset for %F evaluation contained three fewer com-

pounds (113 vs. 116) than that employed for CL, Vdss,HL, and MRT because three compounds were eval-uated in an i.v. only PK screen, and therefore, oralPK data were not available for these compounds. Onthe basis of qualitative analysis (Fig. 5a), %F for 81(72%) of the 113 compounds evaluated were withinthe same qualitative category following both cassette

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Figure 4. Qualitative (a) and semiquantitative (b) comparisons of mean residence time (MRT)values obtained with cassette and discrete dosing of compounds in rats. Closed circles (�) repre-sent compounds with the same MRT classification and open triangles (�) represent compoundswith different MRT classifications. Dotted lines represent the limits of each category (for quali-tative) and the upper and lower limit of the ratio (for quantitative), respectively.

and discrete dosing. Of the 32 compounds that werenot within the same category, there were six instances(19%) when the %F from cassette was lower than thatfrom discrete dosing. With the semiquantitative ap-proach, %F for 88 (78%) of the 113 compounds werewithin twofold of each other following cassette anddiscrete dosing (Fig. 5b). Of the 25 compounds that

were not within twofold, three (12%) compounds hadlower bioavailability following cassette dosing com-pared with discrete dosing.

Bias and Precision

Table 2 lists the bias and precision in comparisonof the PK parameters between cassette and discrete

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Figure 5. Qualitative (right box) comparison of oral bioavailability (%F) values obtained withcassette and discrete dosing of compounds in rats. Closed circles (�) represent compoundswith the same %F classification and open triangles (�) represent compounds with different %Fclassifications. Dotted lines represent the limits of each category (for qualitative) and the upperand lower limit of the ratio (for quantitative), respectively.

dosing. There was minimum bias in the estimationof CL, Vdss, MRT, HL, and %F from cassette dosing(when discrete PK values were considered as the truevalue). In terms of precision, the predictions for Vdss,HL, and MRT were more accurate as compared withCL and bioavailability.

DISCUSSION

A cassette-dosing strategy may be useful in the drugdiscovery setting for the screening of a relatively largenumber of new chemical entities for potential PK lia-bilities while minimizing animal usage; however, the

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Table 2. Bias and Precision of the PharmacokineticParameters Obtained with Cassette and Discrete Dosing ofCompounds in Rats

ParameterAverage-Fold

ErrorRoot Mean

Square Error

Clearance 1.4 25Volume of distribution atsteady state

1.4 4.6

Half-life 1.5 2.2Mean residence time 1.4 1.8Bioavailability 1.6 22

utility of this strategy is dependent on the reliabil-ity of the cassette-derived data to dependably yieldreasonably good estimates of PK parameters. Intu-itively, cassette-dosing-derived PK parameters maymisrepresent the “true” PK properties of test com-pounds due to “drug–drug interactions,” likely re-sulting in underestimation of CL and overestima-tion of HL, and for orally dosed compounds, reducedgastrointestinal and hepatic first-pass effects result-ing in overestimation of systemic exposure and %F.The merit of cassette dosing has long been debatedin the literature, with researchers reaching contra-dicting conclusions.6,13,16,24,25 A summary of the pub-lished cassette studies has been recently compiled bySmith et al.,2 which implies that cassette dosing hasbeen used successfully to test wide range of chemicalstructures in multiple species including rats, dogs,and mice. The current report is derived from a ret-rospective analysis of cassette- and discrete-dosing-derived PK data generated over a period of about10 years for 12 drug discovery programs. The analy-ses included data for 116 compounds wherein the PKdata were generated with study design considerationssuch as dosing vehicles, sampling times and tech-nique, bioanalytical methodologies, and so on thatwere similar for all compounds; therefore, the sizeof the dataset and similarity of the manner in whichthe data were generated makes these data useful forassessing the impact of cassette dosing on PK prop-erties compared with those generated with discretelydosed compounds.

Overall, the comparative cassette-to-discrete PKfindings of this investigation were consistent with therecent findings by He et al.,22 who concluded with 31compounds that cassette dosing yields PK data thatare useful for differentiating PK properties betweencompounds in a drug discovery setting.

The current investigation included cassette anddiscrete administration of compounds as solutionsby both i.v. and oral routes, which enabled the com-parison of key PK parameters often sought in theearly drug discovery settings, namely, CL, Vdss,HL, and %F. The evaluation of i.v. PK propertiesdifferentiates this investigation from previous in-vestigations, which have focused on comparisons oforal exposure data.14,26 The results reported herein

indicated that PK parameters obtained with thecassette-dosing screening approach were comparableto those derived by traditional discrete-compound-administration study designs, and thus, confirm theutility of the cassette-dosing regimen in the drug dis-covery setting. The dataset represented compoundswith diverse PK values for CL (0.1−210 mL/min/kg), Vdss (0.1−57 L/kg), HF (0.25−14 h), and %F(4%–100%). The dataset was somewhat skewed to-ward low and moderate CL compounds (based on clas-sification of discrete-dosing data), which made up 42%and 39%, respectively, of the total dataset comparedwith high CL compounds (19%).

Qualitatively and semiquantitatively, CL and Vdssvalues obtained by cassette dosing were indicative ofthose obtained by discrete dosing (Figs. 1 and 2). Sim-ilarly, the concordance between cassette and discretedosing for the secondary PK parameters HL and MRTwas also good (Figs. 3 and 4). In the drug discoverysetting, one of the common concerns with screeningtechniques such as cassette dosing is the potential forfalse negatives or false positives, wherein the progres-sion of a compound may be continued or discontinuedbased on “incorrect” PK obtained under a cassette-dosing regimen.22 These results demonstrate that theincidence of either false negatives or false positives,qualitatively and semiquantitatively, is not inherentwith cassette-dosing administration.

A weaker concordance was observed for %F (Fig. 5)compared with the i.v. PK parameters. The mecha-nism behind this observation cannot be derived fromthese data, but may be investigated in subsequentinvestigations. Nonetheless, in cases wherein %F val-ues were categorically different following cassette anddiscrete dosing, the %F value obtained with discretedosing tended to be lower. Noteworthy differences inthe i.v. PK properties following cassette and discretedosing were not observed for these compounds, whichsuggest that reduction of gastrointestinal or hepaticfirst-pass effect when administering compounds as acassette may be at least partially responsible for this%F observation. Additionally, in some cases, the per-centage of DMSO in the dose formulations differedbetween cassette and discrete doses, which may haveled to differences in absorption rates, permeability, orboth.

In summary, the present investigation presents aninterrogation of the concordance of PK properties ob-tained with cassette and discrete dosing. The findingsindicate that PK data derived with a cassette-dosingparadigm were comparable with those generated witha discrete-dosing study design. The cassette-dosingtechnique is recommended for use in early PK screen-ing situations because fewer animals are needed, com-pared with traditional discrete-dosing study designs,to generate PK data of sufficient quality to guide leadoptimization efforts.

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Appendix 1.

Cassette Discrete

CompoundClearance (CL,

(mL/min/kg)Volume of Distribution atSteady State (Vdss, L/kg)

Half-Life( HL, h)

Oral Bioavailability(%F)

CL(mL/min/kg) Vdss (L/kg) HL (h) %F

1 19 1.6 2.5 75 24 1.5 3.2 1002 14 1.2 3.7 70 27 1.7 4.1 673 0.69 0.19 3.9 100 0.82 0.18 3.3 1004 4.5 1.0 3.5 100 6.4 1.8 5.5 1005 14 0.71 1.7 90 10 0.50 1.9 456 2.9 0.46 2.9 100 5.6 0.71 2.8 1007 99 22 3.8 16 134 22 2.8 5.78 33 0.56 0.34 8.1 34 1.3 1.6 4.39 70 0.61 0.25 4.2 117 0.65 0.06 17

10 15 0.26 0.34 41 18 0.37 0.52 2011 37 0.71 0.80 6.7 24 0.67 0.64 5.912 15 1.2 1.6 16 21 1.1 0.83 1313 8.5 0.24 0.86 18 5.1 0.21 0.59 1214 5.6 0.29 0.75 41 11 0.39 0.53 3115 0.65 0.22 18 74 0.15 0.13 25 1716 5.7 0.65 4.4 82 15 0.35 0.79 3917 6.3 0.33 0.94 15 7.6 0.31 0.64 1718 8.1 0.95 1.7 30 24 2.5 2.1 3719 21 1.6 1.4 16 19 0.79 0.73 1520 39 6.4 3.2 15 16 5.3 8.0 2521 4.1 0.37 0.95 22 7.0 0.34 0.86 2222 42 1.1 0.68 23 33 0.71 0.55 3.223 3.3 43 0.71 53 2.1 10 1.7 5424 53 1.2 0.47 22 52 1.6 0.55 3225 26 1.4 1.6 65 80 3.3 0.71 2626 34 1.3 0.54 99 61 1.6 0.34 3127 32 3.3 6.5 30 27 2.3 5.5 1628 34 2.5 1.7 29 50 2.8 1.5 2329 44 1.6 0.39 43 38 1.1 0.49 3630 7.4 0.22 0.54 32 6.6 0.23 0.84 5031 25 0.46 0.46 19 23 0.44 0.89 7.332 26 0.63 0.55 33 29 0.73 0.78 4033 34 0.87 0.79 37 29 0.65 0.59 2134 28 2.1 2.2 100 36 1.6 1.2 8235 28 2.8 4.0 69 26 2.0 3.8 2636 121 16 2.1 24 133 22 2.8 2237 29 4.2 0.9 6.0 96 5.6 1.0 7.938 9.0 3.4 6.7 36 9.0 4.5 5.1 6339 4.0 0.87 2.7 –a 3.0 0.58 2.6 –a

40 13 2.1 2.5 –a 9.1 1.5 2.7 –a

41 11 0.86 2.2 –a 9.8 1.0 2.1 –a

43 16 19 14 17 11 12 14 1144 87 16 3.2 18 59 17 6.3 7.245 38 1.4 0.91 58 32 1.8 1.3 3646 25 0.82 0.66 27 46 1.6 1.2 4247 54 3.5 2.9 43 27 15 17 2048 44 4.4 2.2 100 31 3.7 4.1 10049 23 2.0 2.3 67 24 1.9 1.8 7150 32 2.6 1.9 62 29 2.4 2.5 3851 15 1.2 2.9 40 15 1.2 3.4 4052 21 3.8 1.8 100 47 5.4 1.5 10053 28 3.0 1.5 100 48 4.5 1.5 10054 15 1.4 0.95 95 30 2.1 1.2 10055 19 3.4 2.6 100 46 4.9 1.6 10056 76 4.7 0.93 64 107 5.9 0.92 10057 88 26 4.6 11 118 15 1.6 2658 20 3.6 2.6 100 28 3.4 2.6 10059 8.1 4.8 7.6 100 11 5.1 5.7 10060 57 3.8 1.9 100 84 3.1 1.1 100

(Continued)

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 DOI 10.1002/jps

CASSETTE DOSING FOR PHARMACOKINETIC SCREENING IN DRUG DISCOVERY 3873

Appendix 1. (Continued)

Cassette Discrete

CompoundClearance (CL,

(mL/min/kg)Volume of Distribution atSteady State (Vdss, L/kg)

Half-Life( HL, h)

Oral Bioavailability(%F)

CL(mL/min/kg) Vdss (L/kg) HL (h) %F

61 37 3.3 2.2 100 66 3.6 1.3 10062 52 7 3.7 100 69 6.9 2.9 10063 210 34 2.4 100 420 35 1.3 10064 110 30 4.0 100 110 15 1.8 10065 75 12 2.8 100 79 5.9 1.0 10066 1.3 0.58 5.7 84 2.2 0.81 3.7 8767 0.15 0.11 9.5 19 0.14 0.094 8.4 9.768 1.4 0.21 2.0 42 1.4 0.18 2.6 4569 8.2 1.7 2.5 69 7.6 1.3 2.2 7370 28 1.5 0.89 17 45 1.8 0.97 1371 20 5.1 3.3 76 26 6.6 3.1 4672 25 1.9 1.9 20 16 0.75 1.4 1473 69 4.4 1.0 28 80 5.8 1.8 3374 22 1.5 0.93 76 25 1.4 1.0 6175 43 3.5 1.2 100 52 2.5 0.57 10076 27 3.7 4.8 100 27 4.3 2.3 10077 9.2 2.2 3.4 100 16 2.9 2.5 10678 33 1.8 0.93 48 37 1.8 0.59 5179 38 4.7 1.7 37 26 3.9 2.0 1980 13 0.98 1.7 53 9.6 1.3 2.5 6.481 42 11 2.8 53 29.8 2.7 2.8 1382 81 5.0 0.65 37 53 4.1 1.4 3383 90 2.3 0.42 8.7 67 3.4 0.92 7.084 66 3.7 0.94 34 82 3.5 0.71 2.686 60 2.1 0.43 23 83 2.9 0.35 3.387 77 2.5 0.57 70 49 1.6 0.51 8.788 78 4.0 0.66 87 62 2.7 0.37 3889 34 1.0 0.54 35 45 1.5 1.5 2290 28 1.2 0.46 84 44 1.5 0.67 5191 30 1.2 0.94 51 31 1.3 2.7 2392 64 2.0 0.34 26 44 2.0 0.75 2893 48 1.1 0.49 27 48 1.7 1.3 1494 43 1.1 0.51 37 63 1.8 0.63 1295 45 2.1 0.67 45 58 2.9 0.94 3296 44 1.9 0.53 50 87 2.9 0.41 4397 48 1.2 0.52 62 47 1.5 0.39 3.298 17 3.4 4.1 61 35 29 16 2799 37 6.6 3.3 63 39 11 5.5 46100 36 2.4 1.7 34 94 4.0 0.64 45101 41 1.8 0.67 42 44 2.6 1.4 46102 60 13 3.3 59 46 9.3 4.2 14103 0.62 0.28 5.6 81 0.95 0.37 5.1 108104 3.8 1.6 5.2 72 1.7 0.95 6.3 84105 3.0 1.5 6.5 58 7.2 2.9 5.2 111106 1.2 0.50 5.2 100 2.3 0.53 2.6 100107 9.3 2.6 7.4 46 8.4 6.3 9.7 63108 22 3.7 2.8 89 21 3.7 2.4 93109 65 6.2 4.9 59 57 5.3 6.9 26110 50 3.3 0.86 76 53 1.8 1.3 67111 2.3 0.45 2.8 52 3.1 1.1 4.6 100112 29 3.4 2.4 100 41 7.3 2.9 100113 2.9 0.57 4.6 100 4.7 1.2 2.7 87114 1.7 0.70 2.2 88 1.5 0.79 5.7 103115 2.0 0.34 7.2 79 4.4 0.43 3.4 84116 2.2 1.3 7.2 70 1.2 0.67 6.5 80

All data represented here are an average from n = 3 animals. The experimental conditions are similar, for a given compound, between cassette and discretedosing.

aData from intravenous-only screen.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011

3874 NAGILLA ET AL.

ACKNOWLEDGMENTS

The authors would like to express their sincere grat-itude for the personnel of the GlaxoSmithKline DrugMetabolism and Pharmacokinetics department in-volved in the conduct of the PK studies. Additionally,the authors express their sincere thanks to Dr. KeithW. Ward and Dr. Mike Reilly for their suggestions andcritical evaluation of the manuscript.

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011 DOI 10.1002/jps