▼ Preclinical drug development teams are strug-

gling to deal with the increased rate at which

new, active molecules are being created.This dis-

tress is a natural consequence of the scientific

heritage of pharmacokineticists and drug-

metabolism experts: slow and careful analysis

that has attempted to describe in detail the intri-

cate processes governing the distribution and

disposition of drugs.

New challenges in preclinical drugdevelopmentNeed for high-throughput methodsThe traditional pace of analysis is wholly inad-

equate to deal with the enormous volume of new

chemical entities being produced by modern

chemical and biological techniques. Not only are

compounds being made in their millions by

combinatorial chemistry, corporate libraries of

existing compounds are being screened with

high-speed robots.The application of genetic en-

gineering to fundamental questions of whole-

body and cellular physiology continues to reveal

tempting new targets for research teams. Thus,

our greater knowledge of cell physiology has

resulted in the identification of many new targets

to be subjected to high-throughput screening.

Progress in genetic engineering has facilitated

the cloning and expression of newly discovered

genes, further speeding the use of their corre-

sponding proteins in robotic screens. The

synergistic, multiplicative interactions of these

advances has made this a very exciting and pro-

ductive time for scientists in the pharmaceutical

industry, but we are faced with this question:

how can we characterize these numerous com-

pounds, so that we can narrow the field of candi-

dates to the best few?

In vitro methods can helpOne approach to this problem has been to model

various aspects of the physiology of drug distri-

bution and elimination with in vitro systems. A

good example of such a model is the attempt to

replicate enzymatic routes of clearance by expos-

ing chemicals to subcellular fractions of organs,

such as liver, that are rich in some types of these

enzymes1. Organ culture systems have also been

developed, so that drug candidates can be ex-

posed to more-or-less intact pieces of liver2.

Although these in vitro metabolism screens are

useful, not all metabolic enzymes are active in

these systems, and not all compounds are cleared

solely by metabolism. Permeation across cell

membranes is a key feature of several processes

that are important to the behavior of chemicals

in animals, such as oral bioavailability and pen-

etration of the blood–brain barrier. Cultures of

Caco2 cells have been used to measure the ability

of drugs to be absorbed by the intestine3, and

bovine brain endothelial cells have been used to

model the passage across the blood–brain bar-

rier4–6. All of these systems offer the advantages

common to in vitro models: relatively high

Cassette dosing: rapid in vivoassessment of pharmacokineticsLloyd W. Frick, Kimberly K. Adkison, Kevin J. Wells-Knecht, Patrick Woollard and David M. Higton

Lloyd W. Frick Kimberly K. Adkison and

Kevin J. Wells-Knecht Glaxo Wellcome, Inc., 5 Moore

Drive, Research Triangle ParkNC 27709, USA

tel: +1 919 483 9475 fax: +1 919 315 8011

Patrick Woollard and David M. Higton

Glaxo WellcomeResearch and Development

Gunnels Wood RoadStevenage, UK SG1 2NY

reviews research focus

12

PSTT Vol. 1, No. 1 April 1998

Cassette dosing, combining many test chemicals into one dose

solution, is an attractive method for increasing the throughput of in

vivo pharmacokinetic experiments. This dosing technique depends on

the sensitivity and selectivity of modern analytical techniques, par-

ticularly HPLC/MS/MS. Cassettes vary in size, but even relatively

small ones greatly increase the numbers of compounds investigated

by reducing the effort devoted to animal handling, sample process-

ing and sample analysis. The major drawback of cassette dosing is the

potential for drug–drug interactions.

Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1461-5347/98/$19.00. PII: S1461-5347(98)00010-8

throughput and the clean matrices favored by analytical

chemists. Another advantage of in vitro models is their ability to

isolate a single process or group of processes, so that it can be

studied free from interferences caused by other factors.

However, no matter how elegant the model, at some point in

drug development a chemical must encounter the blood,

lungs, liver, intestines and kidneys of an intact animal. There

really is no substitute, yet, for the dynamics of blood flow, the

exquisitely maintained balances of biochemistry, the subtle

interplay between chemical and physical forces, and the

awesome complexity that is a living creature.

Advent of high-throughput in vivo methodsThe difficulties with the traditional, one-at-a-time approach to

characterizing the pharmacokinetics of compounds eventually

became severe enough to force a re-evaluation of the process

used to conduct these studies. In a series of experiments ‘N-in-

One dosing’ was shown to be a useful way to increase the

speed with which compounds are studied7,8. This technique

was applied successfully to speed the progress of an

a1A-adrenoceptor antagonist to the clinic9. A survey of the lit-

erature revealed that others10–13 had at least considered using

some form of cassette dosing. This review will discuss each

stage of the cassette dosing process in roughly the sequence

that they are encountered in the laboratory. We will also

describe experiments by us and others to validate its utility,

extend its application and explore its problems.

Advances in analytical technology enable cassette dosingMost pharmacokineticists are, at least in part, analytical

chemists. Before the widespread availability of high-perfor-

mance liquid chromatography–mass spectrometry

(HPLC–MS), pharmacokineticists typically quantitated com-

pounds with UV monitors following HPLC. Even with the ad-

vent of diode-array detectors, method development focussed

on discovering a way to make the analyte, and perhaps a key

metabolite or two, elute from the column in ‘empty’ regions of

the chromatogram. Many hours could be spent testing sample

clean-up methods and exploring the effects of slight changes

in pH, gradient composition and packing material on the re-

tention times of the analytes and the interfering peaks. Given

the difficulty of resolving even one or two analytes from

plasma matrix (urine is even worse) it should not be surpris-

ing that cassette dosing awaited the development of more-

sensitive and selective methods.

Mass spectrometry is renowned for its sensitivity and selec-

tivity.The combination of MS with HPLC was, thus, a major ad-

vance in analytical chemistry, adding the resolving power of

HPLC to the sensitivity and selectivity of MS (Ref. 14). A key

technique in HPLC–MS is the use of multiple-reaction

monitoring, or MRM (Refs 15,16). MRM adds another level of

selectivity by isolating the precursor ion of the analyte in the

first quadrupole of the instrument, fragmenting this ion in a

collision chamber, and then isolating a selected product ion of

the precursor in the third quadrupole. Although it is conceiv-

able that different compounds might have the same precursor

ion mass/charge ratio (m/z), it is highly unlikely that they will

also share a common product ion.The use of MRM has largely

eliminated selectivity as an issue in assay development. A se-

quential series of precursor ion isolations, fragmentations and

re-isolations may be used to measure many compounds simul-

taneously. However, because each reaction to be monitored

takes about one tenth of a second, issues of dwell-time can be-

come critical for larger cassettes.The level of selectivity achiev-

able with MRM makes the analysis of samples from cassette

dosing studies possible routinely.

New mass spectrometers may be able to overcome the dwell-

time limitations mentioned above.Time-of-flight machines op-

erate by scanning through a molecular-weight range, instead of

sampling different m/z values sequentially17.Therefore, they do

not have a dwell-time that is dependent on how many com-

pounds are being measured. Reasonably priced time-of-flight

mass spectrometers are not usually capable of MRM because

they cannot introduce a fragmentation step: the required speci-

ficity might be achievable with the high mass-resolution of

some of these instruments (which allows them to determine

molecular composition), but this has not yet been established.

Thus, a cassette dosing study will start with a series of ex-

periments on the MS, seeking to establish that the compound

series is suitable for assay. Next, one or two of the most potent

and selective compounds, the ‘leads’, will be administered as

discrete compounds to animals and the pharmacokinetic pa-

rameters determined. These data will be combined with the

earlier MS experiments to show that the levels that can be as-

sayed are within the range of those expected during the study

and to design the pharmacokinetic study. Finally, the MRM con-

ditions for each compound will also be determined. All of this

information is vital for the next stage of the experiment: cas-

sette construction.

Design of the cassetteHow many compounds should be put in the cassette, and if

more than one cassette is to be made, which compounds

should be grouped together? The answers to these questions

depend on the particulars of the problem faced by the project

team. How many compounds are interesting enough to study?

Are the compounds all of one series, or several? What is the

maximum dose of active compounds that can be administered

to an animal without adverse effects? How sensitive is the MS

assay? Is there evidence for non-linear kinetics in the compound

13

PSTT Vol. 1, No. 1 April 1998 reviews research focus

series (suggesting that the potential for drug–drug interactions

is high)?

Factors to consider when grouping compoundsWhen large numbers of diverse compounds are to be investi-

gated, merely grouping compounds into a cassette and then

proceeding with the study is unlikely to be a satisfactory ex-

perience. Several important points need to be considered when

deciding which compounds are to be put into the cassette.

First, are there any isobaric pairs of compounds? Isobars need

to be avoided because of the use of MS. Common daughter

ions, even when they are a minor component of the total ions

produced, can result in analytical interferences when operating

the MS in the MRM mode, so it is best not to rely solely on

MRM, if it can be avoided. The possibility of isobaric metab-

olites should also be considered.These metabolites will not be

known beforehand, so it is not possible to predict their MRM

patterns. MRM offers great selectivity; however, this can be

achieved less routinely when the analytes are drawn from a

group of closely related compounds prepared by combinator-

ial chemistry. Such compounds and their metabolites are likely

to have common structural features and, consequently, similar

fragmentation patterns. A source of uncertainty can therefore

be lessened by eliminating compounds from a cassette if they

differ by 16 mass units (equivalent to an oxidation), or by

some other amount equal to known or suspected metabolic

pathways.

Second, do the compounds have similar physicochemical

properties? Many compounds are not very soluble, and need to

be coaxed into solution by the addition of either acids or bases,

but these maneuvers are unlikely to be successful when used

together. Additionally, sample preparation and the HPLC

method can be difficult to work out if the range of compound

properties is very diverse.Third, some compounds are best ion-

ized by the mass spectrometer in the positive ion mode, and

some in the negative ion mode.These compounds should not

be mixed. Finally, has a compound with known properties been

included? This reference compound can be used as a positive

control to validate that the results of the cassette dosing study

are in line with expectations.

Factors influencing the size of the cassetteHow many compounds should be put in each cassette? On the

one hand, the increased throughput realized with larger cass-

ettes tempts us to make them as big as possible. On the other

hand, highly complicated experiments are more likely to fail.

For one thing, large cassettes are difficult to assay well. The

dwell-time of the MS operated in the MRM mode is about a

tenth of a second for each fragment to be monitored. One hun-

dred compounds in a cassette would therefore mean that each

would be monitored only once every ten seconds. Infrequent

sampling of the chromatographic peaks will result in large

errors in estimating the areas, especially when the peaks are

sharp. One way around this problem is to use shallower gradi-

ents to broaden the peaks, but this means that sensitivity will

be reduced. Sensitivity is of paramount importance when

using big cassettes because the total dose should be held as

constant and as low as possible to avoid overdose: the more

compounds that are included in the cassette, the more the dose

of each one is reduced.The problem of sensitivity is mitigated

somewhat by the fact that the analyst is usually interested in

those compounds with ‘good’ kinetics, typified by high plasma

levels long after dosing. Even so, it is reassuring to be able to

see compound in at least the first few samples taken: the total

absence of observable compound in the plasma gives rise to

the suspicion that something is amiss. Finally, the problem of

encountering a serious drug–drug interaction is increased

with larger cassettes, as discussed below.

The in-life portion of the study: mitigating the effects ofdrug–drug interactionsNature of drug–drug interactionsSome of the biggest savings of cassette dosing are realized in

the animal room.Although saving time on an expensive instru-

ment like a mass spectrometer is important, MS assays can be

automated, whereas dosing and bleeding animals cannot (yet).

It is therefore not surprising that many of the difficulties with

cassette dosing are encountered in the in-life portion of the

study. These difficulties are largely due to the possibility of en-

countering serious drug–drug interactions. Such interactions

are not rare: any journal containing pharmacokinetic papers is

sure to be a rich source of information about clinically impor-

tant drug interactions. In most cases, these interactions are ob-

served when the clearance of one drug is reduced by co-

administration of a second drug.The reduction of clearance is

usually caused by the inhibition of an enzyme. Inhibition of

the cytochrome P450 3A4 by the antiviral protease inhibitor

ritonavir is a good example18. Administration of this protease

inhibitor can have a profound effect on clearance and oral

bioavailability of other drugs. Patients infected with the human

immunodeficiency virus are often highly medicated. In those

who take numerous drugs, including ritonavir, the probability

of encountering a serious, clinically relevant drug–drug inter-

action can be greater than 50% (Ref. 19).

The concentration of a compound in the plasma is governed

by the ratio of the dose to the summation of all of the rates

of the various routes of elimination. Therefore, it is harder to

affect the plasma level of a compound that is eliminated by a

variety of renal, biliary and metabolic processes, because even

if one route is completely inhibited, the others will still be able

14

PSTT Vol. 1, No. 1 April 1998reviews research focus

to eliminate the compound. For example, if a compound is

eliminated with equal rates by two processes, completely in-

hibiting one of them will result in only a twofold increase in

the plasma concentration. Compounds that are only eliminated

by one route, however, will be exquisitely sensitive to interfer-

ence with that process. The routes of clearance of many com-

mon drugs have been reviewed recently20.

Statistics of drug–drug interaction probabilitiesThe key drug interaction questions for cassette dosing are: how

does the likelihood of encountering a significant interaction

change as the size of the cassette is increased?; and how much

more good data will be obtained by increasing the cassette

size? The probability that the clearance of a particular com-

pound will be affected is a function of the cassette size and the

frequency with which inhibitors occur in the population of

compounds being studied. To reduce this problem to a com-

prehensible level of statistics, it is similar to flipping a coin

(50% frequency of interaction) N times (where N is the cass-

ette size), and having it come up heads every time (no interac-

tion from any of the other compounds in the cassette). This

probability can be calculated using the binomial distribution.

Table 1 shows the probability of encountering a drug–drug

interaction as a function of cassette size and frequency of oc-

currence of inhibitors. The amount of useful data generated

can be calculated easily by multiplying the frequency with

which accurate data are obtained by the number of com-

pounds in the cassette. This analysis suggests that big cassettes

are not a good idea when a chemical series is known or sus-

pected to contain metabolic inhibitors, because the proportion

of useful data quickly declines with cassette size.

Another consideration is the possibility of including a really

‘bad’ compound in a cassette, one whose effect is severe

enough that it destroys the whole experiment.This destructive

effect does not need to be due to a drug–drug interaction.The

likelihood of including an acutely toxic compound in the

cassette also increases with the cassette size. A chemist once

challenged one of us during a presentation on cassette dosing,

explaining (in faintly accusatory tones) that a dog given a cass-

ette of five compounds orally had promptly vomited them all

up, ruining the experiment and sending her back to the bench

to synthesize more. When a series contains chemicals that are

likely to be acutely toxic (or emetic), large cassettes may be

counterproductive.

Reasons for persevering with cassette dosingSeveral factors mitigate the dire state of affairs described above.

First, most interactions are not that severe. As discussed in the

next section, cassette data are usually nicely correlated with

data on discrete compounds, and even slightly inaccurate data

are still very useful if the range in compound properties is

great. Evidently, enough alternate pathways of clearance exist

so that inhibiting one of them does not have a big effect on

clearance. Second, most interactions will lower clearance, so

that ‘good’ compounds will not be incorrectly eliminated from

further consideration. The bad ones that get included by mis-

take will be weeded out when they are retested as discretes.

Third, interactions are dose-dependent, so lowering the dose

of compounds as far as possible will tend to weaken the inter-

actions and reduce the frequency of occurrence of a com-

pound that is potent enough to affect clearance. Finally, the in-

clusion of a reference standard with known pharmacokinetic

properties can lend some assurance that things are more-or-

less normal. The use of a reference compound involves the as-

sumption that it and other members of the cassette are cleared

by the same mechanism. Nevertheless, such compounds are

useful, particularly if they are the lead compound for the se-

ries: co-administration with potential competitors eliminates

inter-animal variability from the comparison. Therefore, de-

spite the dangers, cassette dosing has been steadily gaining a

wider acceptance.

Post-dose poolingA very useful variant of cassette dosing is the post-dose pool-

ing of samples. As the name implies, this method combines

samples only when they are ready to be assayed, thereby avoid-

ing the problem of drug–drug interactions. This method is

most valuable when generation of samples is relatively easy,

as is the case for some in vitro screens. However, it does not

provide some of the most important advantages of cassette

dosing, namely the reduction in animal handling and use.

Practical experienceCorrelation with singly dosed compoundsThe most often-quoted validation of cassette dosing is that the

data gained using it are correlated with data on individual

compounds. Several groups, including those at Merck21,

15

PSTT Vol. 1, No. 1 April 1998 reviews research focus

Table 1. The probability (%) of a compound experiencing adrug–drug interaction as a function of cassette size and thefrequency of metabolic inhibitors in the population ofcompounds being studied.

Frequency of occurrence of metabolic inhibitors Cassette size 10% 3% 1% 0.3%

05 041 14 05 0110 065 26 10 0320 088 46 18 0640 099 70 33 1180 100 91 55 21

Proctor and Gamble22,23, Schering-Plough24 and SmithKline

Beecham25 have published reports recently, describing the suc-

cessful use of cassette dosing. Most have been able to show at

least a modest correlation of discretes and cassettes. Our own

experience has confirmed this observation. Figure 1 shows the

correlation in total body clearance (determined by noncom-

partmental methods) of intravenous doses of 21 a1A-adreno-

ceptor compounds given to dogs either individually or in cass-

ettes of about 15 compounds. Similar data have been obtained

with many other series of compounds. Although some scatter

is evident, even in this log–log plot, those compounds with

the slowest rate of clearance were clearly identified in the cass-

ette dosing studies, with the expenditure of much less time

and effort. Experiments such as these account for the rapid

adoption of cassette dosing.

Cassette of N590We have tried to push the limits of cassette dosing by adminis-

tering a total of 90 a1A-adrenoceptor antagonists to a single

dog.All of these compounds had been given previously to dogs

in smaller cassettes of N<22. This experiment was extremely

useful for illustrating the obstacles that must be overcome to

extend the technique to bigger cassettes, but also illustrates the

power of using large cassettes to screen large numbers of com-

pounds. First, because of the accumulative pharmacological ef-

fects of the compounds, the total dose had to be kept low, so

that the levels of the individual compounds were low relative

to the detection limits of the MS assay. Dividing the dose into

four different cassettes and giving them at one-hour intervals

circumvented this problem. Four of the compounds were not

present in the dosing solutions, and are presumed to have pre-

cipitated before administration.The preparation for the experi-

ment was quite labor-intensive. However, the correlation

between half-lives in the N590 and in smaller cassettes was

good: these data are shown in Figure 2. Note that many com-

pounds fall along the line of identity of the two data sets, but

that some in the N590 set are scattered above this line, indi-

cating that these compounds tended to be eliminated more

slowly than they were in smaller cassettes. Note also that there

were many compounds with ‘negative half-lives’: the concen-

trations were apparently increasing over the latter part of the

experiment (the last four samples, taken 5–8 h post-dose, were

used to calculate half-life). This problem is not a serious one.

Obviously, the compounds don’t really have negative half-lives

of elimination. The important point is that the rate of elimi-

nation is slow. Probably, the half-lives were prolonged to the

point where our experiment was not able to measure them ac-

curately (to be expected in an eight-hour experiment). In fact,

most of these compounds had good kinetics when tested in

smaller cassettes, so that compounds with desirable properties

were usually identified successfully. More importantly, most of

the rapidly eliminated compounds were identified. Only one

promising compound of the top 20 would have been missed.

It had a half-life of >20 hours in a smaller cassette, and a half-

life of 3.4 hours in the N590 experiment. Thus, despite the

problems, very large cassettes can be useful as a crude filter to

eliminate compounds.

Oral dosing of cassettesAlthough some researchers, particularly those at Merck and

Proctor and Gamble21–23, have been very successful with oral

dosing of cassettes, our own experience has been less success-

ful. One interesting situation arose during cassette dosing of a

group of compounds known to have dose-dependent kinetics

in rats. One of these compounds had an oral bioavailability of

more than 300% when the oral cassette data were compared

with the corresponding intravenous cassette data. When dosed

16

PSTT Vol. 1, No. 1 April 1998reviews research focus

Figure 1. Comparison of total body clearances of 21 a1A-adrenoceptorcompounds administered intravenously to dogs as discretes or incassettes of 12–22. The line is the power function trendline fromExcel®.

100

10

1

0.10.1 1

Clearance, discrete (ml min–1 kg–1)

Cle

aran

ce, c

asse

tte(m

l min

–1 k

g–1)

10 100

Figure 2. The half-lives of 90 a1A-adrenoceptor antagonists given insmaller cassettes (N<22), or in a large cassette (N=90). The line ofequivalence is shown.

40

20

–20

0

–400 105

Half-life in cassette N < 22 (hours)

Hal

f-lif

e in

cas

sette

N =

90

(hou

rs)

15 20

singly, the oral bioavailability was <10%. The hypothesis was

that these compounds were saturating the mechanism of clear-

ance when given orally as a cassette, possibly due to a first-pass

effect on metabolism by one or more of them.To test this idea,

a single rat was first dosed orally with all the members of the

cassette but one.After four hours, the same rat was dosed intra-

venously with the missing compound. The resulting plasma

concentration–time curve compared with that of the same

compound dosed intravenously, individually and in a cassette in

other rats, is shown in Figure 3 (concentrations are corrected

for the different doses given). Even the compound dosed intra-

venously in a cassette seems to suffer from drug–drug interac-

tions, but this effect is magnified after dosing with the oral

cassette. The increased deviation from the data obtained on

the intravenously administered, discrete compound could be

caused by the higher concentration of the drugs in the liver

after oral dosing. Another possibility is that a metabolite with

more of an inhibitory effect was produced after oral dosing.

This experience underscores the necessity of confirming

promising results by a more traditional approach. Oral dosing

is also more difficult to deal with because of the nature of the

data. Total body clearance can vary over an infinite range,

whereas oral bioavailability is confined to within 0–100%.

Therefore, it is not valid to log-transform oral bioavailability

values, whereas we can for clearance.Thus, extra vigilance may

be required with oral cassette dosing.

In-life standardsThe use of in-life standards is nearly universal. Most workers

surely anticipate that they will discard data from a cassette

when the in-life standard indicates a problem. However, how

far off does the standard have to be? Examination of other’s

data reveals that repeatedly measured values of in-life stand-

ards, such as clearance, tend to follow a log-normal distribu-

tion. For example, we have plotted data from Olah et al.21 in

Figure 4. These are the log-transformed, averaged area-under-

the-curve (AUC) values for an in-life standard given on 19 oc-

casions to two dogs, dosed with cassettes of about ten com-

pounds each. Note that the arithmetic mean and standard

deviation of the AUCs was 5 ± 2 h mg ml21: the range of AUC

values was from 2 to nearly 11 h mg ml21. Since the frequency

distribution is fairly broad, only interactions with a major ef-

fect will be detected with this in-life standard. Some of this

variation could be caused by the oral route of administration,

and some to drug interactions that are judged to be mild.

Although some of our own in-life standards for particular

chemical series have been as variable, standards for other series

that have been administered intravenously have had coeffi-

cients of variation of <20%. Perhaps a more useful aspect of

the in-life standard is that it facilitates comparisons among

compounds by lessening the importance of inter-animal vari-

ability. Note that the standard does not have to be the same

compound each time, it just has to have been dosed alone at

least once.

Urinary recovery after cassette dosingUsually, we have assayed plasmas. However, scientists at Proctor

and Gamble have successfully applied the cassette dosing tech-

nique to studies of urinary recovery22. For compounds that are

renally eliminated, urine is an attractive biomatrix because it

tends to contain high concentrations of the analyte, and be-

cause only a few samples are generated. These scientists used

cassettes of about six compounds to estimate oral bioavailabil-

ity with some success, even with a compound series that had

non-linear kinetics. Another interesting example of measuring

urinary excretion of drugs given in combination is represented

17

PSTT Vol. 1, No. 1 April 1998 reviews research focus

Figure 3. Inhibition of clearance by a first-pass effect. The drug–druginteraction responsible for the erroneous oral bioavailability data wasexacerbated when the animals were pre-treated with an oral cassette.

2.5

2

1.5

1

0.5

00 1 2 3 4 5 6 7 8

Time (h)

Pla

sma

conc

entr

atio

n (µ

M)

9

IV discreteIV cassetteIV (PO cassette)

Figure 4. Histogram of the frequency distribution of log-transformedarea-under-curve (AUC) values from an orally dosed in-life standard.The standard was administered on at least 19 occasions. These datawere obtained from Ref. 21.

7

6

5

4

3

2

1

00.75

Natural logarithm of AUC

Fre

quen

cy

1.1 1.45 1.8 2.15 2.5

by a technique developed to determine the metabolic pheno-

types of humans13,26. A cocktail of up to five ‘indicator’ drugs,

each one cleared by a different route, was given to volunteers

and plasma pharmacokinetics and urinary recoveries deter-

mined. Co-administration did not appear to affect the extent of

metabolism (as would be expected, because all are cleared by

different enzymes).

Brain penetrationAnother tissue of interest besides plasma is the brain. We have

been able to show for a group of 89 triazines that concen-

trations of compounds at one hour post-dose in the brains of

mice following cassette dosing were correlated with those ob-

served after dosing with discrete compounds. A single individ-

ual was used in each branch of the experiment.These data are

shown in Figure 5.Although some scatter is evident, the agree-

ment was generally good. This in vivo method for estimating

brain penetration could, thus, be an excellent addition to high-

throughput in vitro methods.

ConclusionMuch of this review has been devoted to the problems associ-

ated with cassette dosing. This is not meant to convey a sense

of pessimism, but merely of realism. Using cassette dosing,

large numbers of compounds can be put through the preclini-

cal evaluation process much more quickly than by traditional

methods. For this reason, it has been gaining wide acceptance

in the pharmaceutical industry. New developments in analyti-

cal methodologies and in computerized data analysis should

advance the utility of this technique further.

AcknowledgementsJ. Berman, J. Shaffer and K. Halm played critical roles in con-

ceptualizing cassette dosing and in reducing it to practice. M.

Tarbit and J. Harrelson provided support and guidance.All pro-

vided critical review of the manuscript.The excellent technical

assistance of M. Cockman is gratefully acknowledged. The re-

search complied with national legislation and with company

policy on the Care and Use of Animals and with related codes

of practice.

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PSTT Vol. 1, No. 1 April 1998reviews research focus

Figure 5. Concentrations of Triazine compounds in the brains of singlemice, dosed either intravenously with individual compounds, or withcassettes of nine compounds. The concentrations of most compoundsin the brains at one hour post-dose were similar when dosed by eithermethod.

1000

0.01

0.1

1

10

100

0.01 0.1 1 10 100

Concentration in brain, discrete (ng g–1)

Con

cent

ratio

n in

bra

in, c

asse

tte (

ng g

–1)

1000