0022-3565/98/2842-0460$03.

00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2
Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 284:460 466, 1998

Pharmacokinetic-Pharmacodynamic Modeling of the

Anticonvulsant and Electroencephalogram Effects of Phenytoin
in Rats

O. E. DELLA PASCHOA, J. W. MANDEMA, R. A. VOSKUYL and M. DANHOF

Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, University of Leiden, University of Leiden, P.O. Box. 9503, 2300 RA
Leiden, The Netherlands (O.E.D.P., M.D.), Stanford University School of Medicine, Department of Anesthesia, Palo Alto, California (J.W.M.) and
Instituut voor Epilepsiebestrijding, Heemstede, The Netherlands (R.A.V.)
Accepted for publication September 9, 1997 This paper is available online at http://www.jpet.org

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 22, 2017

ABSTRACT
In this study a pharmacokinetic-pharmacodynamic model is
proposed for drugs with nonlinear elimination kinetics. We ap-
plied such an integrated approach to characterize the pharma-
cokinetic-pharmacodynamic relationship of phenytoin. In par-
allel, the anticonvulsant effect and the electroencephalogram
(EEG) effect were used to determine the pharmacodynamics.
Male Wistar-derived rats received a single intravenous dose of
40 mg z kg21 phenytoin. The increase in the threshold for gen-
eralized seizure activity (TGS) was used as the anticonvulsant
effect and the increase in the total number of waves in the 11.5
to 30 Hz frequency band was taken as the EEG effect measure.
Phenytoin pharmacokinetics was described by a saturation
kinetics model with Michaelis-Menten elimination. Vmax and Km
values were, respectively, 386 6 31 mg z min21 and 15.4 6 2.2
mg z ml21 for the anticonvulsant effect in the cortical stimulation
model and 272 6 31 mg z min21 and 5.9 6 0.7 mg z ml21 for the
EEG effect. In both groups, a delay to the onset of the effect
was observed relative to plasma concentrations. The relation-
ship between phenytoin plasma concentrations and effect site
was estimated by an equilibration kinetics routine, yielding
mean ke0 values of 0.108 and 0.077 min21 for the anticonvul-
sant and EEG effects, respectively. The EEG changes in the
total number of waves could be fitted by the sigmoid Emax
model, but Emax values could not be estimated for the nonlinear
relationship between concentration and the increase in TGS. An
exponential equation (E 5 E0 1 Bn z Cn) derived from the sig-
moid Emax model was applied to describe the concentration-
anticonvulsant effect relationship, under the assumption that
Emax values cannot be reached within acceptable electric stim-
ulation levels. This approach yielded a coefficient (B) of 2.0 6
0.4 mA z ml z mg21 and an exponent (n) of 2.7 6 0.9. The derived
EC50 value of 12.5 6 1.3 mg z ml21 for the EEG effect coincides
with the therapeutic range in humans.

Phenytoin (diphenylhydantoin) has been used extensively
in the treatment of seizure disorders. Although its pharma-
cokinetics has been well reported both in animals and in
humans (Olanow and Finn, 1981; Bauer and Blouin, 1983;
Shavit et al., 1984; Jones and Wimbish, 1985; Levine and
Chang, 1990) the concentration-anticonvulsant effect profile
of phenytoin is not thoroughly understood. Consequently,
prediction of the accurate PK-PD relationship for both clini-
cal and preclinical scopes is still a difficult undertaking. This
can be attributed largely to the lack of adequate quantitative
measures of the effect of AED in vivo (Danhof et al., 1992b).
The nonlinear pharmacokinetics of phenytoin represents an
additional complicating factor (Theodore, 1992).
To date, the numerous models developed to delineate the
Received for publication March 10, 1997.
anticonvulsant properties of antiepileptic drugs in preclinical
investigation, such as the kindling and maximal electroshock
models, have limitations which prohibit the assessment of
the PK-PD relationship (Rundfeldt et al., 1990; Rundfeldt
and Loscher, 1993; Mulzac and Scott, 1993; Dimmock and
Baker, 1994). A major limitation is the impossibility to de-
termine repeatedly the anticonvulsant effect intensity within
individual rats.
Furthermore, approaches in which the use of antagonists
might fully explain the effect of the agonist are not applicable
in vivo because of the mechanism of action of phenytoin. The
neuropharmacological and biochemical effects of phenytoin
are vast. Some of the reported actions of phenytoin in various
systems include: (1) changes in the conductance of ionic chan-
nels (Jones and Wimbish, 1985); (2) inhibition of calcium
uptake and calcium-dependent protein phosphorylation

ABBREVIATIONS: CSM, cortical stimulation model; E0, base-line effect; Emax, maximal effect; EC50, concentration at half maximal effect; EEG,
electroencephalogram; fu, free fraction; Km, Michaelis constant; PK-PD, pharmacokinetic-pharmacodynamic; TGS, threshold for generalized
seizure activity; TLS, threshold for localized seizure activity; Vd, volume of distribution; Vmax, maximum metabolic rate; TNW, total number of
waves.

460
1998
(Twombly et al., 1988); (3) elevation of membrane potential
and changes in the amplitudes of synaptic potentials; (4)
suppression of bursting activity; (5) increase in cortical levels
of g-aminobutyric acid (Griffith and Taylor, 1988).
With respect to the pharmacokinetics, it is important to
consider that the decay in plasma concentration is not linear.
Because the rate at which phenytoin is hydroxylated is dose
dependent, elimination can follow both first and zero-order
processes, depending on the concentration range. A model
with Michaelis-Menten elimination is therefore required to
describe the pharmacokinetics of phenytoin (Gibaldi and Per-
rier, 1982).
In addition, one has to consider that the bulk of PK-PD
modeling theory and methodology pertains to linear pharma-
cokinetics with specific drug-receptor interaction systems
(Danhof et al., 1992a, 1993). Mathematical models and meth-
ods for such nonlinear systems are much less developed
(Ritschel and Hussain, 1984; van Rossum and Burgers, 1984;
Gillespie, 1993).
The intent of this report was to develop an integrated
approach for the assessment of the time course of the effect of
phenytoin by use of the EEG and CSM models. The former is
based on the use of quantitative EEG parameters as a mea-
sure of the effect on brain electric activity (Mandema and
Danhof, 1992). The latter consists of the induction of mild
convulsive activity, which is evoked by applying electric
pulse trains directly to the cortex. The TLS and TGS can be
used as measures of the anticonvulsant effect (Voskuyl et al.,
1992). In this way, a realistic estimate of the anticonvulsant
effect intensity is obtained (Hoogerkamp et al., 1994).
Methods
Study Design
The study protocol was approved by the Ethical Committee for
Animal Experimentation. The anticonvulsant activity and the EEG
effect of phenytoin were determined in two groups of rats according
to a parallel group design. Two groups of male Wistar-derived rats
(Sylvius Laboratory Breeding Facility, Leiden, The Netherlands)
(225300 g) were used throughout the study. The animals were
housed individually in plastic cages under constant temperature
(21C) and 12-hr light/dark cycle. Laboratory chow (Standard Labo-
ratory Rat, Mouse and Hamster Diets, RMH-TM, Hope Farms,
Woerden, The Netherlands) and water were available ad libitum,
except during the experimental procedures.
Surgical Procedure
Seven chronic cortical EEG electrodes were implanted in the skull
of the animals (Mandema and Danhof, 1990) 1 week before the
experiments for the measurement of EEG signals (group I). Implan-
tation of two permanent electrodes over the motor area of the fron-
toparietal cortex (Voskuyl et al., 1989) allowed the assessment of the
anticonvulsant effect (group II). One day before the measurements,
indwelling cannulae were implanted into the right jugular vein (for
drug administration) and femoral artery (for blood sampling).
Drug Dosage, Blood Sampling and Pharmacokinetics
A 40 mg z kg21 dose of phenytoin was infused intravenously at a
rate of 0.1 ml z min21 for 5 min. Phenytoin sodium (Sigma Chemical
Co., St. Louis, MO) was dissolved in water alkalinized with 0.1 N
sodium hydroxide. Arterial blood samples (100 or 200 ml) were col-
lected in heparinized tubes before and 5, 10, 15, 20, 30, 40, 60, 90,
120, 150, 180, 240 and 300 min after drug administration. Blood
PK-PD Modeling of Phenytoin 461
samples were then centrifuged for 10 min at 5000 rpm and plasma
(50 or 100 ml) separated and stored at 230C until analysis.
EEG Measurements and Cortical Stimulation
Group I. The output from bipolar leads was continuously moni-
tored by a Nihon Kohden EEG recorder. During the course of the
experiment, animals were kept in motion in a slow-speed rotating
drum (10 rph) to control the vigilance level. Base-line activity was
monitored for 30 min. After drug administration, EEG recordings
were continued for 5 hr. The frontocentral lead on the left hemi-
sphere was subjected to on-line aperiodic analysis for quantification
of the effect. The aperiodic analysis algorithm calculates the ampli-
tudes of each EEG signal on a wave-by-wave basis. The analyzed
EEG data were stored on a magnetic floppy disk. The drug-induced
change in the total number of waves in the 12.0- to 30.0-Hz frequency
band was applied as effect measure.
Group II. The seizure thresholds were determined as described
previously (Voskuyl et al., 1989). Convulsive activity was induced by
a single train of bipolar pulses (total pulse duration, 2 msec, 50 Hz)
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of increasing amplitude (0 1000 mA in 15 sec) applied to the elec-
trodes. The TLS and TGS were defined as the minimal current
intensity necessary to induce clonic movements of the forelimbs and
generalized clonic activity, respectively. Stimulation was continued
until the TGS was reached. Seizure activity was induced five times
before the infusion started, at intervals of 5 min between each
stimulation, to determine the base-line threshold values. Thereafter,
drug effect was assessed up to 5 hr after administration, at intervals
between stimulations, varying from 5 min immediately after the
infusion to 15 min 2 hr later. The effect was determined subse-
quently by use of a video-recording device. The elevation of the
thresholds above their average base line represents the anticonvul-
sant effect.
Phenytoin Assay
Samples were analyzed by the high-performance liquid chroma-
tography technique essentially in the manner outlined by Ratnaraj et
al.(1989). A solution of 1.5 mg mephenytoin (internal standard) in
150 ml acetonitrile was added to a 50- or 100-ml plasma sample. To
separate the organic phase, 100 ml saturated NaH2PO4 solution were
added and then whirl-mixed for 15 sec. After 10 min centrifugation
at 5000 rpm, 75 ml were transferred from the supernatant and 25 ml
were injected into the chromatographic system. The mobile phase
consisted of a mixture of 0.067 M phosphate buffer (pH 5 5.6) and
acetonitrile in a 70:30 ratio with a flow rate of 1.7 ml z min21. The
high-performance liquid chromatography system consisted of a Kra-
tos solvent delivery system, a WISP-710B automatic sample injector
and a Spectroflow 757 Kratos spectrophotometer (wave length, 215
nm). The analytical column was a 25 cm 3 4.6 mm i.d. Altex column
filled with Ultrasphereo-ODS 5 m. Data processing was performed by
a Chromatopack C-R3A reporting integrator. Phenytoin retention
time was 8.0 min. Within-day precision was 1.7% for a 10 mg z ml21
control sample (n 5 8). Limit of detection was 0.25 mg z ml21 and the
assay was linear in the range from 1 to 100 mg z ml21.
Protein Binding
Residual blood was collected by aorta puncture after completion of
the experiments and centrifuged for 10 min at 5000 rpm. Plasma was
separated and stored at 230C until assay. The protein binding was
determined for each individual animal by ultrafiltration at 37C,
with use of the Amicon Micropartition System (Amicon Division,
Danvers, MA). Three 0.5-ml aliquots of a plasma sample were spiked
with phenytoin to a concentration of 10, 50 and 100 mg z ml21. Total
plasma concentration was measured by the described method in a
50-ml sample of the spiked plasma. Separation of free drug from
protein-bound drug was carried out at 37C by filtration of a 400-ml
plasma through a YMT ultrafiltration membrane (Amicon) at 1090 3
462 Paschoa et al. Vol. 284

g for 10 min. The ultrafiltrate was then analyzed for free drug where E(Ce) is the observed effect at concentration C, E0 is the
concentrations. base-line effect value, Emax is the maximal effect, EC50 is the con-
centration at half-maximal effect and n is a constant expressing the
Data Analysis shape of the concentration-effect relationship.
Finally, based on the sigmoid Emax model we have derived an
The pharmacokinetics and pharmacodynamics of phenytoin were equation to describe the exponential profile of the anticonvulsant
quantified for each individual rat. First, a two-compartment model effect. This was performed under the assumption that both EC50 and
with Michaelis-Menten elimination was used to describe the plasma Emax values are very high and cannot be determined experimentally,
concentration-time profile (Gibaldi and Perrier, 1982): without lesion of the brain or animal harm. When drug concentra-
dC 1 V max z C 1 R tions are small in relation to EC50, the equation tends to a constant
52 1 2 k 12 z C 1 1 k 21 z C 2 (1) value in the denominator. In practice, the combined parameter Emax/
dt Km 1 C1 V1 EC50n may be used to describe drug effect in the range observed:

dC 2
5 k 12 z C 1 2 k 21 z C 2 (2) E max n
dt E ~ C e! 5 E 0 1 n zC when C ,, EC50 (6)
EC50
with the error model
Assuming that Emax/EC50n 5 Bn, the equation can be rewritten as:
log~ C m! 5 log~ C pi! 1 i (3)
n n
where dC1/dt is the rate of decline of drug concentration at time t, V1 E ~ C e! 5 E 0 1 B z C (7)

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the distribution volume, Vmax the theoretical maximum rate of the
process, Km the Michaelis-Menten constant and kij the transfer rate
constant from compartment i to j; 1 and 2 refer to the first and second
compartments, respectively. Cmi is the ith measured concentration
and Cpi is the predicted concentration according to the PK model.
The model was programmed into SIMULINK (The MathWorks Inc.,
Natick, MA) and a fifth-order Runge-Kutta method was used for
integration. The data were fitted to the model by use of the nonlinear
least squares routine fmins in MATLAB (The MathWorks Inc.,
Natick, MA), which uses the simplex method for the minimization.
The log-transform is approximately equivalent to a constant CV
model.
The hysteresis was collapsed with a hysteresis minimization rou-
tine written for MATLAB based on the COLAPS program described
previously (Veng-Pedersen et al., 1991). Ce was calculated by SIMU-
LINK according to the following link model (see fig. 1):
k e0 t
C e 5 k e0 z e z Cp (4)

where *Cp indicates convolution of the concentration in the central

compartment.
The sigmoid Emax model was used to describe the relationship
between drug concentration and EEG effect (Holford and Sheiner,
1982):
n
E max z C
E ~ C e! 5 E 0 1 n n (5)
EC50 1 C

Fig. 2. Time course of phenytoin plasma concentration (n $ 7) after i.v.

infusion of 40 mg z kg21 phenytoin. The markers denote the mean 6 S.E..
Fig. 1. Schematic representation of the fitting model used for kinetics The solid line represents the fit to all individual data. The bars indicate
and hysteresis minimization. the duration of infusion.
1998 PK-PD Modeling of Phenytoin 463
TABLE 1
Comparison of the pharmacokinetics and pharmacodynamics of
phenytoin in the EEG model (group I) and the CSM (group II)a
EEG CSM

Pharmacokinetics
Vmax (mg z min21) 272 6 31 386 6 31b
Km (mg z ml21) 5.9 6 0.7 15.4 6 2.2c
V1 (ml) 406 6 32 184 6 13d
k12 (min21) 0.197 6 0.018 0.255 6 0.019c
k21 (min21) 0.060 6 0.004 0.072 6 0.002b
fu (%) 30.2 6 2.4 25.3 6 3.0
Pharmacodynamics
ke0 (min21) 0.077 6 0.020 0.108 6 0.017
E0 (TNW or mA) 6.0 6 0.2 685 6 14
Emax (TNW) 2.6 6 0.3
B 2.0 6 0.4
(mA z ml z mg21)
Hill factor 4.6 6 0.9 n 5 2.7 6 0.9
EC50 (mg z ml21) 12.6 6 1.3
a
n $ 7; mean 6 S.E. Statistical significance: b
P , .05; c
P , .01; d
P , .001
(ANOVA).

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where E(Ce) is the observed effect at concentration C, E0 is the
base-line effect value, B represents the quotient between Emax and
EC50 and n is a constant expressing the steepness of the curve.
In both cases the pharmacokinetic model was used to generate
drug concentrations at the times of effect measurement. The equa-
tions were fitted to the data by the nonlinear least squares regres-
sion routine in MATLAB. After applying the Bartletts test for non-
homogeneity of variances, statistical analysis was carried out by a
one-way analysis of variance or the nonparametric Kruskal-Wallis
test.

Results
Pharmacokinetics
The kinetic disposition of phenytoin followed a two-com-
partment model with Michaelis-Menten elimination. The av-
eraged concentration profile is plotted against time in figure
2. Vmax and Km presented somewhat higher values in group
II (table 1). Because protein binding was similar in both
groups, the lower values of volume of distribution apparently
compensated for such differences, without additional conse-
quences for biophase equilibration and pharmacodynamics.
Hysteresis
The effect onset was characterized by a temporal delay
relative to plasma concentrations in both groups. As shown
in figure 3, the hysteresis was minimized successfully by the
link model. ke0 values of the same magnitude were obtained
for the EEG and anticonvulsant effects.
Pharmacodynamics
Group I. Intravenous administration of phenytoin in-
duced changes in both amplitudes and TNW in the beta
frequency band (12.0 30.0 Hz) of the EEG spectral power.
The increase in TNW was selected as the EEG effect measure
(fig. 4). Because an Emax value was reached, the concentra-
tion-EEG effect relationship could be characterized by the
sigmoid Emax model (fig. 5).
Group II. The anticonvulsant effect of phenytoin was re-
flected by the elevation of the TGS without any significant
alteration in the TLS. As depicted in figure 4, the effect-time
course profile followed a pattern remarkably similar to the
changes observed in the EEG. However, fitting these data to
the sigmoid Emax model was not possible. It seems that Emax
Fig. 3. The hysteresis between plasma concentration and EEG effect of
an individual rat is normalized after minimization with a monoexponen-
tial conductance function. The same is observed for the hysteresis be-
tween plasma concentration and the anticonvulsant effect (TGS). The
arrow indicates the time sequence of the observations.
cannot be reached at the administered dose. The exponential
equation 7 was used to describe the nonlinear profile of the
anticonvulsant effect (fig. 5). The pharmacokinetic and phar-
macodynamic parameters generated from the PK-PD model-
ing are summarized in table 1.
Discussion
In this study we demonstrate the application of an inte-
grated PK-PD approach to characterize the biophase equili-
bration kinetics and the concentration-effect relationship of
phenytoin both in the EEG and in the cortical stimulation
models.
The nonlinear pharmacokinetic profile of phenytoin, de-
scribed by a two-compartment model with Michaelis-Menten
elimination, presented high variability. Our data agree with
published data, however (Jones and Wimbish, 1985; Itoh et
al., 1988). Km (Michaelis constant) and Vmax (maximum met-
464 Paschoa et al.
Fig. 4. Time course of averaged EEG (F) and anticonvulsant (DTGS, l)
effects (n $ 7) after i.v. administration of phenytoin (40 mg z kg21). Plots
show changes relative to base-line values and the markers indicate the
mean 6 S.E.. The TLS () was not affected by drug administration. The
solid bars represent the duration of infusion.
abolic rate) estimates in different populations range from 1.2
to 24.2 mg z ml21 and 108 to 568 mg z min21, respectively
(Rambeck et al., 1979). The nonlinear phase of decay in
plasma concentration (concentrations greater than the Km
values) persisted up to 2 hr after administration in both
groups (fig. 2). Because the effect predominated over the
same time span, modeling of the concentration-effect profile
occurred essentially in the nonlinear phase. Plasma protein
binding presented only minor variations within the concen-
tration range investigated. The free fraction ranged from 25
to 30%, which is consistent with data published previously
(Levine and Chang, 1990).
To exert its anticonvulsant effect, phenytoin needs to reach
its site of action in the central nervous system. Phenytoin in
the unbound form distributes into transcellular fluids and is
present in various tissue compartments, including liver, fat,
muscle and brain (Jones and Wimbish, 1985). In principle,
one could expect peak brain concentrations after i.v. admin-
istration to be reached immediately, because of the high
Vol. 284
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Fig. 5. PK-PD relationships of phenytoin for individual rats in the EEG
and CSM. F, Relationship between phenytoin concentrations and the
EEG effect measure derived after i.v. administration. The solid line
represents the best fit of the actual data to the sigmoid Emax model. l,
Relationship between phenytoin concentrations and the anticonvulsant
effect. The solid line represents the best fit to the exponential equation.
lipophilicity of phenytoin and the relatively high blood flow to
the brain. However, previous studies reported that maximum
concentrations in brain were observed 6 min after a 2-min i.v.
infusion was completed (Ramsay et al., 1979). Such a differ-
ence coincides with the delay to the onset of effect and to
maximal effect observed in both models investigated.
The temporal delay (hysteresis) between plasma concen-
trations and effect appears to be a characteristic of PK-PD
modeling of several drugs active in the central nervous sys-
tem (Mandema et al., 1991; Danhof et al., 1992a). The current
study does not permit us to establish whether distribution is
the major determinant of the observed hysteresis. However,
the rationale for this delay is easily understood if one as-
sumes that the resulting pharmacological effect or response
is preceded by drug distribution to the site of action. Thus far,
there is no consistent report demonstrating the role of other
factors in the biophase equilibration of phenytoin which are
1998
known to cause hysteresis, such as coupling mechanisms and
effectuation process that follow the drug-receptor interac-
tion. The hysteresis can be modeled mostly by the effect-
compartment approach, which postulates the existence of a
hypothetical effect compartment linked to the plasma site by
a first-order process (ke0). Our approach is based on the
assumption that distribution kinetics between plasma and
effect site is linear and that the same effect-site concentra-
tion always evokes the same response, independent of time.
This assumption may not hold when interactive metabolites
are formed or when there is development of acute tolerance.
However, it has been demonstrated that main metabolites of
phenytoin have little or no antiepileptic activity (Jones and
Wimbish, 1985). Moreover, there is no evidence of the devel-
opment of tolerance toward the EEG or anticonvulsant ef-
fects of phenytoin in these models.
The successful minimization of hysteresis under the as-
sumption of an effect compartment indicates that plasma
(central compartment) concentration can reflect the biophase
concentration properly (fig. 3). Furthermore, because the re-
sulting ke0 values for the EEG and anticonvulsant effects do
not differ significantly, one may suggest that the biophase for
both effects is pharmacokinetically indistinguishable. In fact,
this can be correlated with anatomophysiological findings.
The anatomical substrate (somatosensory cortex) for assess-
ment of both effects is the same. Furthermore, studies on the
brain regional distributions of specific [3H]phenytoin binding
have demonstrated that the cortex is a major binding site
(Wong and Teo, 1988).
Regarding the pharmacodynamics, the assessment of the
anticonvulsant action is a complex issue. Therefore, useful,
accurate measures of the anticonvulsant effect intensity for
PK-PD studies are scarce, despite the numerous animal mod-
els of epilepsy. Quantitative pharmaco-EEG on the basis of
aperiodic analysis has fulfilled most of the requirements for
PK-PD modeling. In previous studies it has been successfully
used to describe the effect of benzodiazepines (Mandema et
al., 1992; Danhof and Mandema, 1992). Application of this
technique to antiepileptic drugs has not been reported, how-
ever. For benzodiazepines a clear correlation has been estab-
lished between the EEG effect and the anticonvulsant effect
in the pentylenetetrazole model (Mandema et al., 1991).
Whether this is also the case for phenytoin remains to be
determined. To date, it is well known that phenytoin in-
creases levels of g-aminobutyric acid in rat cerebral cortex
and enhances its uptake (Wong and Teo, 1988). In addition,
phenytoin was recently shown to potentiate the increase in
the amplitude of cortical high frequency (20 30 Hz) back-
ground activity induced by N-methyl-D-aspartate antago-
nists. This EEG response is considered to be caused by the
antagonistic effect exerted by phenytoin on the release of
glutamate or on the N-methyl-D-aspartate receptor-linked
channels (Popoli et al., 1994). The existing data do not permit
the conclusion that the observed EEG effects are relevant to
the anticonvulsant action of phenytoin. Further investigation
is still required to establish a correlation between the EEG
effect and the pharmacological mechanisms involved. Not-
withstanding, the primary merit of such a parameter is to
allow PK-PD modeling of the effect according to the sigmoid
Emax model, providing meaningful pharmacodynamic esti-
mates, i.e., Emax and EC50. Moreover, it is worth noting that
PK-PD Modeling of Phenytoin 465
in vivo PK-PD modeling of a drug with nonlinear kinetics by
the sigmoid Emax model has not been reported thus far.
In contrast to the EEG response, the pharmacodynamic
endpoint in the CSM is a direct measure of the anticonvul-
sant action of phenytoin. Particularly important is the fact
that the CSM is one of the few models that allow a graded,
reproducible and clinically relevant measure of the antiepi-
leptic effect in an individual animal (Hoogerkamp et al.,
1994). In addition, the pharmacological meaning of each
threshold has been investigated in previous studies (Voskuyl
et al., 1992). Analogous to the difference in efficacy in the
pentylenetetrazole and maximal electroshock screening mod-
els, the threshold for localized seizure activity has been
shown to reflect the capacity of a drug to block the onset or
triggering of seizure activity but the threshold for general-
ized seizure activity to encompass the capacity of a drug to
prevent propagation of the seizure activity. Thus, the eleva-
tion of the TGS without alteration in the TLS agrees with the
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postulated mechanisms of action of phenytoin, which involve
inhibition of the propagation of seizure activity.
The PK-PD relationship presented a nonlinear pattern
with a steep increase of the effect at the concentration range
of 15 to 25 mg z ml21 (fig. 5). As in previous studies Emax could
not be measured. This seems to be a common feature of the
method for all the antiepileptic drugs (Hoogerkamp et al.,
1994). Such a phenomenon might be explained by the impos-
sibility of more frequent measurement of the effect at the
early distribution phase. Furthermore, because the current
that was applied to the cortex does not discriminate between
inhibitory or excitatory systems, another hypothesis to con-
sider is the triggering and overlapping of different mecha-
nisms which distort the actual effect profile at higher current
intensity. This has been partially investigated in a compari-
son between the concentration-anticonvulsant effect profiles
of antiepileptic drugs in the pentylenetetrazole model and in
the CSM. The results indicated that the mechanisms by
which seizure activity is evoked in these models are respon-
sible for the differences in effect pattern (Dingemanse et al.,
1990). This does not diminish the quality and accuracy of the
information obtained from the thresholds, however. In terms
of modeling, such a nonlinear profile can be fitted by either
an exponential function, as the one we have applied here, or
by the log-linear model, which is a simplification of the sig-
moid Emax model. However, the log-linear model does not
explain the effect in the absence of the drug and requires the
assumption of a threshold for concentrations to produce a
significant effect (Holford and Sheiner, 1981).
In fact, the parameters derived from this approach seem to
provide a realistic insight into the anticonvulsant effect of
phenytoin. Despite the practical constraints mentioned
above, both EC50 values in the EEG model and the concen-
tration range of effective anticonvulsant action in the CSM
are of the same magnitude as the therapeutic levels in hu-
mans. Correcting for protein binding, the EEG effect oc-
curred in the range of 1.8 to 6.0 mg z ml21 whereas a signifi-
cant elevation of the TGS was observed between 1.75 and 10
mg z ml21. This is consistent with plasma concentrations
yielding effective therapeutic responses in humans (Wolf,
1993; Yoshida et al., 1993).
In conclusion, our results show that the use of concomitant
PK-PD modeling provided independent, accurate informa-
tion about the transport of phenytoin to the site of action,
466 Paschoa et al.
understanding about the nature of the observed effects and
the underlying concentration-effect relationship. Application
of this methodology represents an important step forward in
the comprehension of the pharmacodynamics of drugs with
nonlinear pharmacokinetics.
Acknowledgments
The authors thank K. B. Postel-Westra and M. Langemeijer for
their technical assistance.
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