Whatever happened to cassette-dosing pharmacokinetics?

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<ul><li><p>1359-6446/04/$ see front matter 2004 Elsevier Ltd. All rights reserved. PII: S1359-6446(04)03137-X</p><p>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].</p><p>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).</p><p>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</p><p>Whatever happened to cassette-dosing pharmacokinetics?Prasarn Manitpisitkul and Ronald E. White</p><p>Prasarn ManitpisitkulJohnson &amp; Johnson</p><p>Pharmaceutical Research &amp;Development LLC920 US Route 202</p><p>RaritanNJ, USA</p><p>Ronald E. WhiteSchering-Plough Research</p><p>InstituteMS K-15-2-2745</p><p>2015 Galloping Hill RoadKenilworth</p><p>NJ 07033-1300, USAe-mail:</p><p>ronald.white@spcorp.com</p><p>reviews research focus</p><p>652</p><p>DDT Vol. 9, No. 15 August 2004</p><p>Cassette dosing is a procedure that is used for rapidly assessing the</p><p>pharmacokinetics of a series of discovery drug candidates by dosing a</p><p>mixture of compounds rather than a single compound. Cassette dosing</p><p>has advantages and disadvantages associated with its use, which leads </p><p>to controversy about how and if it should be used. To assess the current</p><p>practices of the pharmaceutical industry regarding cassette dosing, a</p><p>survey of several pharmaceutical companies was conducted. Analysis of</p><p>the survey revealed that opinion on this subject is divided within the</p><p>pharmaceutical industry. In addition, it was determined that approximately</p><p>only a half of those companies that perform in vivo pharmacokinetic</p><p>screening use cassette dosing for this purpose.</p><p>www.drugdiscoverytoday.com</p></li><li><p>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.</p><p>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).</p><p>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</p><p>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.</p><p>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</p><p>653</p><p>DDT Vol. 9, No. 15 August 2004 reviewsresearch focus</p><p>www.drugdiscoverytoday.com</p><p>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.</p><p>Drug Discovery Today </p><p>Conventional dosing Cassette dosing</p><p>A B C D E A B C D E</p><p>LCMSMSanalysis</p><p>LCMSMSanalysisAnalytical</p><p>queue</p><p>Onecompoundper animal</p><p>Many bloodsamples at each</p><p>time point</p><p>Fivecompoundsper animal</p><p>One bloodsample at each</p><p>time point</p><p>ReportReportReportReportReport</p><p>Report</p></li><li><p>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 the technique. In addition, two repre-sentatives of companies that are actively implementing CDimplied that this technique is only used because the direc-tor of the department has mandated it. In these two cases,the bench scientists themselves preferred to use CD as lit-tle as possible because of the unreliability of the data pro-duced. These views indicate that the industry is surpris-ingly polarized on the use of CD, with researchers eitherstrongly advocating or opposing the employment of thisapproach in the drug discovery process.</p><p>What is the problem with using cassette dosing?The diversity in opinion on the use of CD can be under-stood by examining the advantages and disadvantages ofthis technique. Features that make the CD approach attrac-tive to a drug discovery team include: Rapid analysis time resulting from a reduced analytical</p><p>sample load. Several respondents reported impressivenumbers of candidates being screened each week.</p><p> Reduction in interanimal variabilityin comparisons of compounds. Thetesting of several compounds sim-ultaneously in a single animal re-sults in the reduction or eliminationof individual differences among ani-mals; interanimal variability couldlead to ambiguous results with con-ventional dosing.</p><p> Consolidated report output. A singleexperiment performed on manycompounds leads to the generationof a single report. By contrast, con-ventional dosing could potentiallyresult in the production of numer-ous reports over several weeks.</p><p> Reduction in animal usage. In addi-tion to saving on the animal pur-chase and husbandry budget, CD canbe an important indication of good-faith efforts to an institutional ani-mal care and use committee.However, some serious practical dif-</p><p>ficulties with using CD were also re-ported by researchers. The three mostimportant problems encountered thatinfluenced the decision of PK scientists</p><p>not to perform investigations using CD or to abandon theuse of CD completely were: Drugdrug interactions. Data acquired from CD can sug-</p><p>gest PK properties for compounds that are different fromthe PK properties indicated by the discrete dosing of individual compounds. Several scientists told that PKvalues generated from a CD experiment had later beenidentified as erroneous and indicated that the inaccu-racy had been caused by the occurrence of a significantkinetic interaction.</p><p> Excessive time required by the liquid chromatographymass spectrometrymass spectrometry (LCMSMS) ana-lyst for the setting up of assay conditions for ten com-pounds. Often a cassette of five compounds (four un-knowns and one standard) is the compromise approach.However, in this scenario, the potential productivitygain of CD is never fully achieved.</p><p> Difficulties in preparing a usable dosing formulationfrom a mixture. The poor water solubility of many con-temporary discovery candidates requires their formula-tion as suspensions in a vehicle such as methylcelluloseor polyethylene glycol (PEG), which could make it difficultto obtain uniformity of the component compounds. Forintra venous dosing, which requires a solution formulation,</p><p>654</p><p>DDT Vol. 9, No. 15 August 2004reviews research focus</p><p>www.drugdiscoverytoday.com</p><p>Figure 2. Results of industry survey on the use of cassette dosing. Thirty-onepharmaceutical companies were polled about their experience of using CD. Of the 25respondents, 22 companies reported that they had tried CD, with 12 companiescontinuing to use CD and 10 completely abandoning the use of CD. Thus, approximatelyonly a half of respondents use CD for rapid in vivo pharmacokinetic screening.Abbreviation: CD, cassette dosing.</p><p>Drug Discovery Today </p><p>0Companiessurveyed</p><p>Respondents Discontinueduse of CD</p><p>Tried CD Continueto use CD</p><p>5</p><p>10</p><p>15</p><p>20</p><p>25</p><p>30</p><p>35</p><p>Num</p><p>ber o</p><p>f com</p><p>pani</p><p>es</p></li><li><p>the dissolution of a cassette of compounds might requirean organic solvent, even if doses are low.</p><p>What is the solution?It must be remembered that CD was invented to providerapid in vivo PK data to keep up with the fast pace of candi-date production and evaluation in drug discovery [9]. Thatneed has not changed. Therefore, there is a continued ac-tive publication of CD-based screening programs, but nowinvestigators are more aware of the dangers and designtheir studies more carefully to incorporate the precautionsoutlined [8]. For example, Hasegawa et al. [10] reportedgood results in evaluating the PK of antifungals in ratsusing CD. To avoid major problems with drugdrug inter-actions, Hasegawa and colleagues used only four unknowncompounds, plus a reference compound, per cassette. Inaddition, they limited the dose to 2 mg/kg/compound andused organic solvents to dissolve the compounds. However,the approach of dissolving compounds in organic solventscould produce misleading results for programs seeking anorally bioavailable drug. Similarly, Mallis et al. [11] appliedCD to the study of phytoestrogens in rats at low doses andused a cassette of only five compounds. Unfortunately,they reported several specific PK parameters, includingmaximal concentration in plasma (Cmax), half-life, areaunder curve (AUC) and bioavailability, as the definitivevalues, a step that most PK scientists are reluctant to carryout with the results of CD because of the possibility thatthe parameters contain substantial errors.</p><p>The CD method has been extended to measure brain up-take of a compoun...</p></li></ul>