whatever happened to cassette-dosing pharmacokinetics?

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  • 1359-6446/04/$ see front matter 2004 Elsevier Ltd. All rights reserved. PII: S1359-6446(04)03137-X

    Over the past decade, it has been widelyrecognized that the absorption, distribution,metabolism, excretion (ADME) and pharma-cokinetic (PK) properties of drug candidatesare as important as pharmacological activityand selectivity in the design of clinically use-ful therapeutic agents [1,2]. InappropriateADME and PK properties are among the majorreasons for the failure of a compound to reachthe market. This drives pharmaceutical com-panies to profile ADME and PK properties inthe early discovery phase to enable the selec-tion of compounds that have a greater proba-bility of success. With the advance of drug dis-covery technology, numerous in vitro and insilico methods have become available to helpdiscovery scientists predict the ADME proper-ties of compounds [3]. However, without invivo animal experiments, our understanding ofthe integration of biochemical and physiolog-ical process at the whole-body level remainsincomplete. Animal experiments are generallyexpensive and labor- and time-intensive.Therefore, to aid selection and add value todrug development candidates, the level ofthroughput of in vivo pharmacokinetic optimiz-ation studies must be increased [4].

    Cassette dosing (CD, also called N-in-onedosing) was introduced as a solution to theproblem of low-throughput screening and ex-ploits the capability of modern mass spec-trometers to measure simultaneously the indi-vidual concentrations of chemical compoundsin a plasma sample that contains multiple drugs[5]. A mixture of test compounds, which typi-cally comprises five to ten compounds, is dosedto a group of animals and the pharmacokinet-ics of all the compounds are determined inparallel, which substantially reduces the timerequired for the overall procedure comparedwith conventional serial determinations [6](Figure 1).

    The potential of CD to yield false PK infor-mation as the result of drugdrug interactionswas recognized immediately after its introduc-tion [5,7]. To minimize the errors observed,safeguards were introduced, including admin-istering only low doses of compounds andeliminating known inhibitors of drug-metabo-lizing enzymes. Furthermore, most scientistsbelieved that only false positives could occur,which means that drugdrug interactions, ifpresent, would result in deceptively enhancedPK properties for a compound, thus poorcompounds would appear to be better candi-dates than they actually are. In this case, theerror would be detected at the next stage whenthe compound is administered as a singledose. However, we performed a theoretical PKanalysis and evaluation of CD that demon-strated that the commonly used safeguardswere not necessarily effective and that theprobability of the occurrence of false negativeswas equivalent to that of false positives [8]. Forexample, if the compounds in a cassette com-pete for binding to plasma protein, then clear-ance of one or more of the drugs could behigher when that compound is dosed in combi-nation with other drugs, thus some drugs

    Whatever happened to cassette-dosing pharmacokinetics?Prasarn Manitpisitkul and Ronald E. White

    Prasarn ManitpisitkulJohnson & Johnson

    Pharmaceutical Research &Development LLC920 US Route 202

    RaritanNJ, USA

    Ronald E. WhiteSchering-Plough Research

    InstituteMS K-15-2-2745

    2015 Galloping Hill RoadKenilworth

    NJ 07033-1300, USAe-mail:

    ronald.white@spcorp.com

    reviews research focus

    652

    DDT Vol. 9, No. 15 August 2004

    Cassette dosing is a procedure that is used for rapidly assessing the

    pharmacokinetics of a series of discovery drug candidates by dosing a

    mixture of compounds rather than a single compound. Cassette dosing

    has advantages and disadvantages associated with its use, which leads

    to controversy about how and if it should be used. To assess the current

    practices of the pharmaceutical industry regarding cassette dosing, a

    survey of several pharmaceutical companies was conducted. Analysis of

    the survey revealed that opinion on this subject is divided within the

    pharmaceutical industry. In addition, it was determined that approximately

    only a half of those companies that perform in vivo pharmacokinetic

    screening use cassette dosing for this purpose.

    www.drugdiscoverytoday.com

  • would appear to be eliminated morerapidly than they would if dosed singly.This analysis also resulted in the devel-opment of several recommendations,including limiting cassette size andusing low doses of compounds. Sub-sequent to the publication of our analy-sis [8], we wondered if drug discoverycompanies would discontinue the useof CD or, at least, alter the way that it isused. Accordingly, we reviewed the cur-rent state of CD in the pharmaceuticalindustry.

    Current cassette dosing industrialpracticesTo address the current status of the cas-sette-dosing pharmacokinetic technique,we decided to poll industry-based col-leagues directly. Thirty-one interna-tional pharmaceutical companies (rang-ing from small to large companies) werequeried about their past experience of and present use of CD. Twenty-fivecompleted and returned the question-naire, giving an overall response rate of81%. Therefore, the data should pres-ent an accurate representation of thepharmaceutical industry. Twenty-two(88%) of the companies surveyed havetried CD, but only 12 of these continueto use CD (48%). Although these find-ings indicate that CD remains a viableapproach, the discontinued use of CDby ten (40%) companies suggests thatthere has been a substantial decline inthe popularity of this technique (Figure 2).

    The frequency of the implementation of CD ranged fromrarely to six times per week, with each cassette compris-ing between two and ten compounds (average number ofcompounds per cassette was five). Hence, despite someearly publications demonstrating the analytical feasibilityof much larger cassettes, most companies prefer to usemodest-sized cassettes, even though these cassettes wouldappear to limit the productivity gain. The majority of com-panies recognize the importance of keeping dose size smalland the dose size that was most frequently chosen was ~1 mgof compound per kg of body weight for each compound(mg/kg/compound). However, as a result of assay-sensitiv-ity limitations with smaller doses, two companies still usedose sizes of 10 mg/kg/compound. Companies that routinely

    employ cassette dosing appear satisfied with the resultsand report an average reliability of 80% for derived PK pa-rameters (range 50100%). Although the exact meaning ofreliability was generally not well-defined by our respon-dents, it could be taken qualitatively to mean that the re-sults obtained using CD were comparable to the resultsthat would have been generated ~80% of the time with adiscrete dosing approach.

    It is interesting to consider the responses of the 13 (52%)companies that do not use CD today. The majority of thesecompanies (ten, 40%) have tried CD and abandoned it.These organizations reported opposite experiences withCD to those that continue to use this method. The primaryreason cited for abandoning CD (80%) was that the resultsgenerated had proven to be unreliable; other reasons cited

    653

    DDT Vol. 9, No. 15 August 2004 reviewsresearch focus

    www.drugdiscoverytoday.com

    Figure 1. Conventional dosing versus cassette dosing in the pharmacokinetic screening offive compounds. In conventional (or discrete) dosing, five formulations containing onecompound each (A, B, C, D and E) are separately dosed to five animals. At selected time points, blood samples are drawn from each animal and assayed byLCMSMS for the particular compound that the animal received. The analysis of onecompound per sample permits the use of a simple assay method. The data are analyzedand a report of the calculated PK parameters is issued for each compound. As aconsequence of there being only one compound present in a particular animal, theseparameters are the true values for that animal. However, five animals have to be dosedand five sets of samples have to be analyzed. In cassette dosing, the five compounds aremixed to produce a single formulation for dosing (usually a solid-liquid suspension orsolution), which is then dosed to a single animal. At selected time points, blood samplesare drawn from the animal and assayed by LCMSMS for all five compounds. The assayof five compounds simultaneously typically requires a complex assay method. The dataare analyzed and a single report containing the calculated PK parameters for all fivecompounds is issued. The simultaneous presence of five compounds in the animal couldpotentially result in the distortion of the parameters by PK interactions among thecompounds. However, this approach requires the use of only one animal and the analysisof only one set of samples, which lowers the total number of animals required andpotentially leads to a reduction in costs. In addition, cassette dosing is generallysignificantly faster than conventional dosing. Abbreviations: LCMSMS, liquidchromatographymass spectrometrymass spectrometry; PK, pharmacokinetic.

    Drug Discovery Today

    Conventional dosing Cassette dosing

    A B C D E A B C D E

    LCMSMSanalysis

    LCMSMSanalysisAnalytical

    queue

    Onecompoundper animal

    Many bloodsamples at each

    time point

    Fivecompoundsper animal

    One bloodsample at each

    time point

    ReportReportReportReportReport

    Report

  • included that the analytical requirements were too de-manding of analyst time and that CD took too long com-pared with other available rapid PK screening techniques(Figure 3). The remaining three companies (12%) indicatedthat they had never tried CD, with one stating that theyhad never trusted t