Departments of
Pharmaceutics (J.L.S., A.C.C., L.L., K.K.A., T.F.K.,
D.D.S.) and
Anesthesiology (K.M.P., A.A.A.), Schools of Pharmacy and
Medicine, University of Washington
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Introduction |
VPA1
is a widely used anticonvulsant effective against a
broad spectrum of epilepsies, particularly primary generalized seizures and juvenile myoclonic epilepsy. The use of VPA in young children has
been curtailed because of the concern for rare, but potentially fatal,
VPA-induced hepatotoxicity (1-3). In addition, VPA is highly
teratogenic, which raises concern over its use in women of childbearing
age (4, 5). In view of these limitations, considerable effort has been
directed toward identifying less toxic analogs of VPA. Much attention
has focused on E-
2-VPA, a 
-oxidative
metabolite of VPA. E-2-VPA was one of the
first metabolites shown to possess anticonvulsant activity in
experimental seizure models (6). A recent series of studies in rodents
has established that E-2-VPA is much less
hepatotoxic than VPA (7, 8) and is devoid of teratogenic effects (9,
10). Single dose tolerance and pharmacokinetics of
E-2-VPA in healthy human volunteers was
reported recently (11).
Several studies in neurosurgical patients have shown that the
concentrations of VPA in the human brain are well below circulating concentrations; the respective average brain/plasma ratios for total and free drug in plasma were ~0.1 and ~0.5 (12-15). Low
brain/plasma and CSF/plasma concentration ratios of VPA and
E-2-VPA have also been observed in laboratory
animals (16, 17). These observations pointed to the existence of an
active transport system(s) mediating the clearance of VPA and
E-2-VPA from the CNS.
There have been several attempts to identify the mechanism(s)
responsible for CNS uptake and clearance of VPA and
E-2-VPA. In a recent study using the in
situ rat brain perfusion technique, Adkison and Shen (18) showed
that cerebral uptake of VPA exhibited saturation kinetics and was
inhibited by medium- and long-chain fatty acids but not by short-chain
fatty acids or 
-keto acids. They also found that uptake was
accelerated by preloading the brain with VPA and medium-chain
dicarboxylic acids. This led to the conclusion that luminal uptake of
VPA into the brain capillary endothelium occurs via a
carrier-mediated process, which operates in a manner similar to the
p-aminohippurate exchanger at the renal proximal tubule, and
a parallel nonsaturable process, presumably reflecting passive
diffusion. A follow-up study of VPA uptake by freshly isolated rat
brain microvessels (19) suggested that VPA transport at the antiluminal
domain of the brain capillary endothelium is mediated by a separate
transport system that is shared by medium-chain fatty acids and many
anionic drugs.
In the above studies (18, 19), VPA uptake into either rat brain
in vivo or isolated rat brain microvessels in
vitro was shown to be inhibited by
E-2-VPA, which suggests common CNS transport
system(s) for the two compounds. The purpose of this study is to
examine the steady-state plasma-brain-CSF distribution kinetics of
E-2-VPA in rabbits utilizing the simultaneous
iv infusion-VC perfusion technique previously employed by this
laboratory for the study of VPA kinetics (20). In this earlier study,
we found that the brain capillary endothelium rather than the choroid
epithelium was the predominant site of VPA efflux from the brain and
that the organic anion transport inhibitor, probenecid (PBD), could inhibit the endothelial transport system. Because VPA and
E-2-VPA probably use the same transporter(s),
we investigated the role of PBD-sensitive transporters in the efflux of
E-2-VPA at the brain capillary endothelium and
choroid plexus.
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Materials and Methods |
The method used in this study to investigate the steady-state,
bidirectional transport of E-2-VPA across the
brain capillary endothelium and the choroid plexus was an adaptation of
the technique described by Pollay and Davson (21) for studying the
choroidal transport of p-aminohippurate and several
inorganic anions. Briefly, this technique involves simultaneous iv
infusion of E-2-VPA and VC perfusion with mock
CSF containing the radiotracer [3H]E-2-VPA. To
determine the contribution of PBD-sensitive transporters to the efflux
of E-2-VPA from the brain, a parallel group of
rabbits was given an iv infusion of PBD in addition to
E-2-VPA.
Animals.
This study was approved by the University of Washington Animal Care
Committee. Fifteen male New Zealand White rabbits (weight range,
3.5-4.5 kg) were randomly assigned to two study groups designated as
either the control group (N = 6) or the PBD treatment group (N = 9).
Reagents.
PBD was obtained from Sigma (St. Louis, MO) and was used without
further purification. Blue dextran 2000 was obtained from Pharmacia
Biotech, Inc. (Piscataway, NJ). All analytical grade solvents and
chemicals were obtained from commercial sources without further
purification.
E-2-VPA and Tritiated
E-2-VPA.
E-2-VPA was synthesized by modifying a
procedure originally described by Rettenmeier et al. (22).
The modification involved the use of phenethyl, instead of ethyl, ester
intermediates, which resulted in improved yield (~50%). Phenethyl
ester of E-2-VPA was prepared by a four-step
synthesis: 1) esterification of pentanoic acid with 2-phenylethanol to
yield 2-phenethyl pentanoate; 2) condensation of phenethyl pentanoate
with propionaldehyde to form phenethyl 3-hydroxyvalproate; 3)
tosylation of phenethyl 3-hydroxyvalproate, and 4) formation of
phenethyl E-2-VPA by reaction of the tosylate
ester with diazobicycloundecene. The crude reaction product consisted
of ~80% E-isomer of phenethyl-2-valproate,
~10% Z-isomer of phenethyl-2-valproate, and
~10% phenethyl-3-hydroxy-valproate. The E-isomer was separated from
the other products by flash silica gel chromatography. Purity of the
phenethyl-(E)-2-valproate (>95%) was checked
by thin layer chromatography, proton nuclear magnetic resonance, and
gas chromatography. E-2-VPA at a purity of 92 to 95% was finally obtained by methanolic/base hydrolysis.
The same procedure was applied to the synthesis of tritium-labeled
E-2-VPA. Approximately 70 mCi of
phenethyl-3-hydroxy-2
,3-3H-valproate (specific
activity of 50 Ci/mM) was obtained by reduction of
phenethyl-2-allyl-3-hyroxypentanoate under
3H2 in the presence of 5%
palladium on charcoal in ethanol. The tritiation step was performed by
the Custom Synthesis Service at DuPont NEN. The tritiated precursor was
processed through the synthetic steps described above, yielding 19.5 mCi of 4,5-H3-E-2-VPA.
The specific activity of the final product was estimated to be 100-150
mCi/mM. The radiochemical purity was at least 96% as checked by thin
layer chromatography.
Surgical Preparation.
Each rabbit was prepared surgically for VC perfusion following the
procedure outlined by Foxworthy and Artru (23). Briefly, each rabbit
was anesthetized with halothane (0.8-1.2% inspired) and nitrous oxide
(66% inspired) in oxygen and mechanically ventilated by a small animal
respirator (Harvard Apparatus, Dover, MA) through a tracheotomy tube.
Expired CO2 was monitored by a Datex model 254 airway gas monitor (Datex Instrumentation Corp., Helsinki, Finland) and
maintained within the normal range of 32 ± 5 mm Hg.
With the animal in the supine position, the left and right femoral
veins were exposed and cannulated for separate infusions of VPA and
PBD. An ear vein was cannulated for administration of the muscle
relaxant pancuronium (0.5 mg/hr). A femoral artery was cannulated to
allow blood sampling for drug assay, blood gas, and pH determinations.
Arterial blood pressure and heart rate were determined from a cannula
placed in the left femoral artery. The electrocardiogram was monitored
using needle electrodes inserted bilaterally at the shoulders and
thighs. Rectal temperature was monitored by a thermistor probe and
maintained at 37.5-38.5°C by a servo-controlled heat lamp. Gold cup
electrodes were placed over the right frontal cortex and the
parietooccipital cortex to monitor the EEG using a Lifescan Brain
Activity System (Diatek Corp., San Diego, CA) with a band pass of
0.5-29.9 Hz. This system used aperiodic analysis to convert the analog
EEG signal into a set of digital parameters (24, 25). Computer (Zenith
Data Systems, Glenview, IL) analysis of the EEG was performed using the
Lifescan Research System program (Diatek Corp.). For each 60-sec
interval, the following values were determined: the power (calculated
as amplitude squared) and number of waves in each of the standard
frequency bins (delta, 0.5-3.0 Hz; theta, 3-8 Hz; alpha, 8-12 Hz;
and beta, 12-30 Hz), total hemispheric power, and the activity edge
(the frequency below which 95% of the hemispheric activity was
present). The EEG was monitored to ensure adequate anesthesia during
this study and to provide data on the effects of iv
E-2-VPA on EEG. Data on the EEG effects of
E-2-VPA were compared with a set of data on
the EEG effects of VPA previously collected in rabbits using the EEG
system described in this study (26, 27).
The animal was turned to the prone position, and its head was affixed
into a stereotaxic frame. The atlantooccipital membrane was exposed
surgically, and an 18-gauge iv catheter was inserted into the cisterna
magna. Correct placement of the cisterna magna catheter was indicated
by the appearance of CSF in the cannula, and then a 200-µl sample of
CSF was withdrawn. The osmolality of the CSF was measured using a
Wescor model 5100B vapor pressure osmometer (Logan, UT). Mock CSF (28)
of matching osmolality was prepared by mixing working solutions with
osmolalities of 290, 300, and 310 mOsmol/kg. Two batches of mock CSF
solution were prepared: one containing the marker blue dextran (3 mg/ml) and the other without. Mock CSF was bubbled with 5%
CO2 in 95% O2 to adjust
the pH of the solution to 7.3. A 22-gauge 3.8-cm stainless steel needle
was inserted in the left lateral ventricle through a burr hole in the
skull. The burr hole was placed 5 mm posterior and 5 mm lateral to the
intersection of the sagittal and coronal sutures. The ventricular and
cisternal catheters were connected to strain gauge transducers to
monitor inflow and outflow pressures. Entry of the catheter into the
ventricle was confirmed by a sudden drop in inflow pressure as the
catheter was advanced. Entry into the ventricle generally occurred at
4.0-4.5 mm below the surface of the dura.
Simultaneous iv Infusion-VC Perfusion.
VC perfusion was initiated by infusing clear mock CSF into the lateral
ventricle catheter at an inflow rate of 60 µl/min via a 50-ml glass
syringe that was driven by a Harvard infusion pump. The CSF perfusate
flowed through the lateral, third, and fourth ventricles and exited
through the outflow catheter in the cisterna magna. Perfusion with
clear mock CSF was continued for approximately 30 min to ensure
success. Once a stable VC perfusion was attained, the study period
began.
The experiment was divided into two phases. At the beginning of phase 1 (time zero), rabbits in the control group received a priming dose of
E-2-VPA (7.5 mg/kg) iv followed by a constant
rate iv infusion of E-2-VPA (125 µg/kg/min)
to maintain a steady-state plasma E-2-VPA
concentration of approximately 55 µg/ml. Also at time zero, the VC
perfusate was switched from clear mock CSF to mock CSF containing blue
dextran, which was added to permit determination of the CSF formation
rate. The VC perfusion was continued for 120 min, at which point phase
2 began. The VC perfusion solution was then switched to a mock CSF that
contained [3H]E-2-VPA
(0.014 µCi/ml) and blue dextran. VC perfusion with
[3H]E-2-VPA and iv
infusion of unlabeled E-2-VPA were continued
for another 105 min until the experiment ended at 225 min.
Serial perfusate samples were collected from the cisternal outflow
catheter at 5-min intervals for the first 30 min of each study phase.
Thereafter, perfusate outflow was collected at 15-min intervals. The
outflow rate was determined by gravimetric analysis of the perfusate
samples. Blood samples were obtained at the midpoint of each perfusate
collection interval during phase 1 and at the midpoint of the last
three perfusate collection intervals of phase 2. Arterial blood samples
were placed in glass tubes containing potassium EDTA (7.2 mg) and
potassium sorbate (0.010 mg) and then centrifuged immediately. The
plasma from each sample was divided into two portions, which were
frozen quickly in acetone/dry ice and stored at 
20°C for subsequent
determination of E-2-VPA protein binding and
concentration.
Animals in the PBD treatment group received identical administration of
E-2-VPA by iv infusion and
[3H]E-2-VPA by VC
perfusion. In addition, they received a priming dose of PBD (30 mg/kg)
at time zero followed by a continuous iv infusion of PBD (0.5 mg/kg/min) through the contralateral femoral vein catheter for the
duration of the experiment.
Mean arterial blood pressure, heart rate, temperature, arterial blood
gas tensions, and pH were determined just before time zero at 30 min of
phase 1, at the end of phase 1, at 30 min of phase 2, and at the end of
phase 2. EEG activity was recorded for 3-5 min just before time zero,
at 30 min of phase 1, and at the end of phase 2.
At the end of each experiment (225 min), the rabbit was sacrificed by
an iv injection of potassium chloride. The VC perfusion and iv infusion
were discontinued, and the brain was immediately removed from the
cranium, dissected into halves, rinsed with saline to remove residual
perfusate, and frozen in a dry ice bath containing acetone. Before
analysis, the left (perfused) brain hemisphere was dissected into ten
regions: frontal, parietal, and occipital cortex, hippocampus,
striatum, thalamus, hypothalamus, cerebellum, colliculi, and
pons/brainstem.
Analytical Procedures.
The concentration of blue dextran in the ventricular inflow and
cisternal outflow perfusate samples was determined by measuring the
absorbance of the samples at 620 nm on a UV-VIS spectrophotometer (Gilford Instruments, Oberlin OH). E-2-VPA
concentration in the cisternal outflow, plasma, and brain samples was
analyzed by a capillary gas chromatographic assay described by Semmes
and Shen (29). The radioactivity of
[3H]E-2-VPA in
cisternal outflow and brain homogenate was assayed by liquid
scintillation counting using a TriCarb 2000CA (Packard Instrument,
Downers Grove, IL).
The free fraction of E-2-VPA in the plasma was
determined by spiking the sample with a tracer quantity of
[3H]E-2-VPA, followed
by ultrafiltration at 38°C using the Centrifree ultrafiltration
device (Amicon, Beverley, MA). Replicate determination of plasma
fraction within a run had a coefficient of variation of <8%. There
was negligible loss of
[3H]E-2-VPA to the
filtration membrane.
Data Analysis.
CSF formation and absorption rates were calculated for each animal
using standard formulae described by Heisey et al. (30). The
cisternal outflow concentration of
[3H]E-2-VPA was
corrected for dilution by the newly formed CSF. The steady-state cisternal outflow [3H]E-2-VPA concentration
was estimated by averaging the corrected outflow concentrations after
60 min of perfusion with the radiotracer, which was then divided by the
inflow perfusate concentration to yield an estimate of steady-state VC
extraction of
[3H]E-2-VPA (21).
The brain/ventricle concentration ratio of
[3H]E-2-VPA for each
of the ten brain regions was calculated by dividing the tissue concentration of
[3H]E-2-VPA by the
logarithmic average of the inflow perfusate concentration of
[3H]E-2-VPA and
steady-state cisternal outflow concentration of
[3H]E-2-VPA, which
approximates the spatial average concentration of [3H]E-2-VPA in the
ventricular space.
The brain/cisternal outflow concentration ratio of unlabeled
E-2-VPA was calculated for each of the ten
brain regions. The steady-state cisternal outflow concentration of
unlabeled drug was calculated by averaging the outflow concentrations
of E-2-VPA from 60-120 min of phase 1 and
from 180-225 min of phase 2.
The brain/plasma concentration ratios of unlabeled
E-2-VPA were also computed for each region
using the average steady-state total or free plasma concentration of
E-2-VPA. The steady-state plasma
concentrations for each rabbit were computed by averaging the
measurements taken after 60 min of equilibration from both phases of
the experiment.
All data were expressed as means ± SE. Mean values for
physiologic variables, EEG activity, E-2-VPA
plasma and brain concentrations and their ratios, and the VC extraction
and brain/ventricle concentration ratio of
[3H]E-2-VPA were
compared within and between groups using a two-way repeated measures
analysis of variance with repeated measures on one factor (31).
Student-Newman-Keul's test was used to make post hoc
comparisons where indicated. Statistical significance was defined as
p 
0.05.
Compartmental Modeling.
As a further effort to elucidate the similarities and differences in
the observed CNS kinetics between E-2-VPA and
VPA and the effects of PBD cotreatment on the kinetics of the
respective drugs, pharmacokinetic modeling of the steady-state distribution of unlabeled and tritium-labeled drug between plasma, total brain tissue, i.e. composed of extracellular fluid and
intracellular space, and the CSF was undertaken. Figure
1 shows the compartmental model that was
used to describe E-2-VPA distribution within
the brain. Accordingly, the following set of rate equations represents
the concentrations of systemically administered, unlabeled
E-2-VPA and perfused
[3H]E-2-VPA in the
extracellular (Ce), intracellular
(Ci) and ventricular (Cv) spaces within the rabbit brain. A
glossary of terms is presented in table
1. The following equations represent
unlabeled E-2-VPA.
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(1)
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(2)
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(3)
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Note that the K constants have the dimension of
volume/time. In the absence of a direct measurement of unlabeled
E-2-VPA concentration in the ventricles, it
was assumed that Cv can be approximated by
the cisternal outflow concentration
(Cperf,out).
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Fig. 1.
A compartmental model for the distribution
of E-2-VPA or VPA within the central nervous system (see
table 1 for glossary of terms).
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Assuming linear kinetics, a parallel set of equations representing
radiolabeled
[3H]E-2-VPA were
written. The superscript asterisk (*) denotes the tritium-labeled drug.
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(4)
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(5)
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(6)
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Note that in eq. 4, plasma concentration of
[H3]E-2-VPA
(Cp*) was assumed to be negligible because
of dilution of radiolabeled drug in the systemic tissues.
At steady state, drug concentrations in all compartments become
constant. Therefore, the rate eq. 1 through eq. 6 can be set equal to
zero and rearranged to yield the following equations for the
steady-state concentrations in all three brain compartments. Eqs. 7
through 9 are for unlabeled E-2-VPA.
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(7)
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(8)
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(9)
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Eqs. 10 through 12 are for radiolabeled
[3H]E-2-VPA.
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(10)
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(11)
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(12)
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The concentration of drug in total brain tissue
(Cb) can be related to the concentrations
in the extracellular fluid and the intracellular space by the following
equation,
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(13)
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where fe and
fi are the respective volume fraction of
extracellular fluid and intracellular space. Substituting for
Ci from eq. 8 for unlabeled drug or from
eq. 11 for radiolabeled drug yields eq. 14 and eq. 15,
respectively.
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(14)
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(15)
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Eqs. 7 through 15 were used to generate eq. 16 for the brain
tissue/plasma ratio for unlabeled drug, and eq. 17 and eq. 18 were the
brain/ventricle concentration ratio for unlabeled drug and radiolabeled, respectively.
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(16)
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(17)
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(18)
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It was assumed that
KveCv/KepCp
is sufficiently small compared with
Kpe/Kep and
that Kev/Kep
1, allowing the expression for the brain tissue/plasma ratio (eq.
16) to be simplified to eq. 19.
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(19)
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Results |
There were no significant differences between the control and PBD
groups in terms of mean body weight, CSF formation rate, or CSF
absorption rate. Moreover, blood gases, blood pH, bicarbonate required
to maintain proper blood pH, mean arterial blood pressure, heart rate,
temperature, or ventricular inflow pressure did not differ between the
control and PBD groups.
Table 2 presents a comparison of the
aperiodic analysis of EEG activity between the control rabbits in this
study (i.e. rabbits given iv
E-2-VPA and
[3H]E-2-VPA through VC
perfusion) and the control rabbits from two previous studies with VPA
(i.e. rabbits given iv VPA and
[3H]VPA through VC perfusion) (26, 27). In
comparison with the mean of the pooled baseline EEG activity recorded
in the present study and those gathered in our previous studies with
VPA, E-2-VPA selectively decreased EEG power
(an index of waveform amplitude) in the theta frequency range
(p < 0.05). In contrast, at a similar plasma
level, VPA did not cause any change in EEG activity.
E-2-VPA also increased the number of waveforms
in the beta frequencies as compared with earlier data from
the VPA studies (p < 0.05).
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TABLE 2
Aperiodic analysis of electroencephalographic activity in the control
groups from the present study and from the previous VPA
studya
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Inhalation anesthetics, such as halothane that was used in this study,
typically increase the amplitude of the lower EEG frequencies (delta
and theta), and decrease activity in the higher frequencies (alpha and
beta) as compared with the awake state. The ability of
E-2 -VPA to decrease the amplitude of theta
frequencies and increase activity in the beta frequencies indicates a
partial reversal of the EEG effects of the anesthetic. Moreover, this
effect was not observed previously with VPA, suggesting some subtle
differences in CNS pharmacology between the unsaturated and saturated
fatty acids.
Equilibration Kinetics in Plasma and Cisternal Perfusate.
Fig. 2 shows a typical time course for
unlabeled E-2-VPA concentrations in the plasma
and cisternal outflow and for
[3H]E-2-VPA
radioactivity in the cisternal outflow during a simultaneous iv
infusion-VC perfusion experiment in a control rabbit. Steady-state plasma concentrations of E-2-VPA were achieved
within 5-10 min and maintained throughout the experiment.
E-2-VPA concentration in cisternal outflow
increased gradually and achieved a plateau by approximately 60 min. The
gradual increase of cisternal E-2-VPA
concentration reflected the time required for systemically administered
E-2-VPA to cross the blood-brain barrier
(BBB), diffuse through the brain parenchyma to reach the ventricular
fluid, and transit through the ventricular space and outflow tubing.
Cisternal outflow concentrations of
[3H]E-2-VPA also
reached steady-state concentrations within 60 min after switching from
plain mock CSF to mock CSF containing
[3H]E-2-VPA. The time
required for cisternal
[3H]E-2-VPA to reach
steady-state concentrations was governed by the transit time of
[3H]E-2-VPA through
the ventricular space and outflow tubing and the time required for
equilibration of
[3H]E-2-VPA between
CSF, brain tissue, and blood.
VC Extraction and Brain-Perfusate Distribution of
[3H]E-2-VPA.
No significant difference in the mean steady-state VC extraction ratio
of [3H]E-2-VPA was
found between the control and PBD groups; 64 ± 3.4% of the
inflow perfusate [3H]E-2-VPA was extracted
during transit through the ventricles for the control rabbits, compared
with 61 ± 3.2% for the PBD-treated animals.
Distribution of
[3H]E-2-VPA from the
ventricular space into the brain tissue was examined by comparing the
steady-state concentration of
[3H]E-2-VPA present in
the ten different brain regions with the logarithmic average of the
inflow and outflow perfusate concentrations. Table 3 lists the mean
[3H]E-2-VPA
tissue/perfusate concentration ratios for the brain regions studied.
Those structures furthest from the ventricles, such as the frontal
cortex, had the lowest tissue/perfusate concentration ratios, whereas
those closest to the ventricles, such as the hippocampus and striatum,
had the highest concentration ratios. Intravenous administration of PBD
tended to increase the mean
[3H]E-2-VPA
concentration ratios in all ten brain regions. However, the differences
between the groups did not reach statistical significance in any of the
brain regions studied. In the brain region with the highest
steady-state concentration of
[3H]E-2-VPA,
i.e. the hippocampus in the PBD group, the tissue
concentration reached only 29% of the average ventricular
concentration. Hence, accumulation in the brain tissue cannot fully
account for the extensive loss of
[3H]E-2-VPA upon
passage through the ventricles.
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TABLE 3
Brain tissue-to-perfusate concentration ratio of
[3H]E-2-VPA in ten brain regions during
steady-state VC perfusiona
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Blood-Brain-Cisternal Perfusate Distribution of Unlabeled
E-2-VPA.
The mean steady-state plasma concentration of
E-2-VPA reached the target level and did not
differ between control and PBD-treated animals, as shown in table
4. Coadministration of PBD did cause a
small increase in the plasma-free fraction of
E-2-VPA. However, the difference in mean free
plasma concentration between the two groups did not reach statistical
significance. The mean concentration of
E-2-VPA in the cisternal outflow for both
groups was much lower than either total plasma or free plasma
concentration (~10% by comparison). The steady-state cisternal
outflow concentration of E-2-VPA in the
PBD-treated animals tended to be higher than the corresponding values
for the control animals; however, the increase was not statistically
significant.
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TABLE 4
Comparison of the mean steady-state concentrations of
E-2-VPA in plasma and cisternal outflow in control and
PBD treatment groupsa
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A comparison of the mean steady-state E-2-VPA
concentrations in the ten brain regions between control and PBD
treatment groups is presented in table 5.
The brain concentrations of E-2-VPA were much
lower than either total or free plasma concentrations in both groups.
Cotreatment with PBD led to a 3- to 5-fold increase in brain tissue
E-2-VPA concentration for all the dissected
brain regions (p < 0.05).
The mean steady-state brain/total plasma concentration ratios of
E-2-VPA in the ten brain regions (fig.
3) were very low. The ratios ranging from
0.014 to 0.023 in the control animals increased to 0.066-0.096 in the
PBD-treated animals, i.e. a 4- to 6-fold gain. The occipital
cortex was the only brain region where the difference in the mean
brain/total plasma concentration ratio between the control and PBD
groups did not reach statistical significance.
When corrected for plasma protein binding, the distribution ratios,
i.e. steady-state brain/free plasma concentration ratios, of
E-2-VPA were still lower than unity for all
ten brain regions in both study groups (see fig.
4). Brain-to-free plasma concentration ratios ranged from 0.085 to 0.138 for control animals and from 0.358 to
0.545 for those treated with PBD. Coadministration of PBD caused a 3- to 5-fold increase in the brain/free plasma concentration ratio in all
ten brain regions; the PBD-induced increases were statistically
significant in all regions (p < 0.05) except
the parietal and occipital areas of the cortex.
The brain tissue/cisternal outflow concentration ratio of
E-2-VPA (fig. 5)
was examined to assess the steady-state partitioning of unlabeled
E-2-VPA between the brain parenchyma and the
ventricular fluid space. The ratios in control animals approached
unity. However, coinfusion of PBD caused a 3- to 4-fold increase over
control values, i.e. mean brain/cisternal outflow
concentration ratios ranging from 2.91 to 4.33. The increases were
statistically significant in all brain regions except the occipital
cortex.
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Discussion |
We have previously shown that VPA and
E-2-VPA may utilize the same carrier system(s)
at the brain capillary endothelium and/or the choroid plexus in the rat
(18, 19). The present data on the CNS distribution kinetics of
E-2-VPA in the rabbit generally seem to be
similar to those previously reported for VPA. However, important
quantitative differences do exist between
E-2-VPA and its saturated precursor. The
following discussion will focus on 1) differences in distribution
kinetics between E-2-VPA and VPA in control
animals, i.e. without PBD cotreatment, and 2) the respective
effects of PBD on the distribution of E-2-VPA
and VPA.
Comparison of E-2-VPA and VPA.
The mean steady-state VC extraction of
[3H]E-2-VPA is
slightly higher (64 ± 3.4%) than that previously reported for
VPA (57 ± 7%) (20). These extractions are quite extensive in
comparison with the previously reported VC extraction of other organic
anions in the rabbit, such as p-aminohippurate (17%),
thiocyanate (37%), penicillin (40%) (21, 32), and PBD (26%) (20).
The high VC extraction ratio of
[3H]E-2-VPA could be
explained by rapid efflux at the choroid plexus, extensive
sequestration in the brain parenchyma, or an efficient transport system
at the brain capillary endothelium. The findings from this study and
those of the parallel study with VPA suggest that the last explanation
is most likely. First, coadministration of the organic anion transport
inhibitor PBD had no effect on VC extraction of
[3H]E-2-VPA. A similar
finding was observed with [3H]VPA in the
earlier study (20). The VPA study also showed that a sufficient
concentration of PBD is reached in the ventricular fluid during iv
infusion. We surmise that there is not a significant efflux of
E-2-VPA from the CNS via a PBD-sensitive
transport system at the choroid plexus. Second, there was little tissue
localization of E-2-VPA, as evidenced by the
fact that [3H]E-2-VPA
steady-state concentrations in the brain tissue reached at most 29% of
the average ventricular concentration (table 3). In the previous VPA
study, the mean steady-state brain tissue concentration of
[3H]VPA was only 4% of the average ventricular
concentration of [3H]VPA (20). These
observations point to the presence of a transporter at the brain
capillary endothelium that facilitates transport of
E-2-VPA from the brain to the blood upon entry
of the drug into the brain parenchyma. Furthermore, it is generally
recognized that the brain capillaries rather than the choroid plexus
are the predominant sites of elimination for drugs that gain access to
the brain parenchyma because the surface area of the brain capillaries
is approximately 5000 times greater than that of the choroidal
epithelium (33, 34). The observed differences in VC extraction and
steady-state tissue concentrations between
E-2-VPA and VPA may reflect differences in
their efficiency of transport by the carrier(s) at the brain capillary
endothelium.
Table 6 presents a comparison of the mean
tissue-to-ventricular fluid or -plasma distribution ratios in control
rabbits from the present E-2-VPA study with
those from the earlier VPA study. To facilitate our analysis, a set of
equations (see eq. 17, eq. 18, and eq. 19 under Materials and
Methods and reproduced in table 6) was derived for the
steady-state partitioning of labeled and unlabeled drug between plasma,
brain tissue, and ventricular perfusate using the compartmental model
shown in fig. 1.
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TABLE 6
Summary of compartmental model analysis of the plasma-brain-ventricle
distribution data of E-2-VPA as compared with those of
VPA
|
|
The mean brain/cisternal outflow concentration ratios
(Cb/Cv) for
unlabeled E-2-VPA were similar to those of
VPA. In contrast, tissue accumulation of tritiated drug derived from
the ventricular perfusate differed between
E-2-VPA and VPA. The brain tissue/average
ventricular concentration ratio
(Cb*/Cv*) was
significantly higher for
[3H]E-2-VPA than for
[3H]VPA in both control and PBD-treated
animals. There were also notably lower brain/total plasma and
brain/free plasma concentration ratios
(Cb/Cp) for
E-2-VPA than for VPA.
Equation 17 (see Materials and Methods or table 6) shows the
factors governing the brain/cisternal outflow concentration ratio (Cb/Cv) for
systemically administered unlabeled drug. The rate constants
Kve and Kev
refer to the rate of diffusion of drug across the leaky ependymal layer
from the ventricles to the brain extracellular fluid and from the brain
extracellular fluid to the ventricles, respectively. Because these
parameters reflect bulk flow or diffusional rates, they should be
similar for E-2-VPA and VPA. The parameter
Q is the perfusion flow rate of mock CSF through the
ventricles and was the same in the two studies at 60 µl/min. Hence,
any differences in brain/CSF concentration ratios between
E-2-VPA and VPA are governed largely by the
ratio Kei/Kie,
which describes the steady-state partitioning of drug between the
intracellular and extracellular compartments, i.e. drug
exchange across the neural cell membranes. It is useful to note that
the brain/cisternal ratio is not affected by the exchange kinetics at
the BBB. Because the mean brain/cisternal concentration ratios for
E-2-VPA and VPA did not differ, it means that
Kei/Kie for
E-2-VPA is nearly equal to that of VPA. In
other words, there is little difference in the rate of exchange of
E-2-VPA and VPA across the neural cell
membranes.
Equation 18 (see Materials and Methods or table 6) is an
expression for the brain/average ventricular concentration ratio for
perfused radiolabeled drug
(Cb*/Cv*).
Again, Kve and
Kev should not differ between
E-2-VPA and VPA. The equation then is governed
by Kep, which is the rate constant for
translocation of drug from the brain extracellular space to the plasma
across the BBB, and the ratio
Kei/Kie. The mean brain/average ventricular concentration ratios were higher for
[3H]E-2-VPA than they
were for [3H]VPA in most brain regions. This is
the case when either the rate of efflux of
[3H]E-2-VPA from the
brain into the plasma is lower than that of
[3H]VPA, i.e.
Kep [E-2-VPA] < Kep [VPA], or the ratio
Kei/Kie is
higher for [3H]E-2-VPA
than for [3H]VPA. In the preceding discussion
on the brain/cisternal ratio for the unlabeled drug, we argued that the
ratio Kei/Kie
does not differ between the two drugs. Accordingly, the higher
brain/ventricle concentration ratio for
[3H]E-2-VPA compared
with [3H]VPA is probably because of a slower
efflux of [3H]E-2-VPA
than [3H]VPA across the BBB.
Equation 19 (see Materials and Methods or table 6) is an
approximate derivation for the brain/plasma concentration ratio of iv-administered unlabeled drug
(Cb/Cp). This
ratio is governed primarily by the term
Kpe/Kep for the
exchange of drug across the BBB, and the term
Kei/Kie. For
all ten brain regions, the mean tissue/plasma concentration ratios were
lower for E-2-VPA than for VPA. This situation
can arise in three ways: the ratio
Kei/Kie for
E-2-VPA is lower than that for VPA, the ratio
Kpe/Kep for
E-2-VPA is lower than that for VPA, or both.
We already argued that there is no difference in
Kei/Kie between
E-2-VPA and VPA. Therefore, the lower
brain/plasma concentration ratios for E-2-VPA
must be due to differences in exchange rates of the two drugs at the
BBB, reflected by a lower
Kpe/Kep ratio
for E-2-VPA than for VPA. Furthermore, there
are two mechanisms that can contribute to a lower
Kpe/Kep for
E-2-VPA: transport from plasma to the brain
extracellular fluid is less efficient for
E-2-VPA, i.e.
Kpe [E-2-VPA] < Kpe [VPA], and/or the rate of efflux from
the brain extracellular fluid to the plasma is higher for
E-2-VPA, i.e.
Kep [E-2-VPA] > Kep [VPA]. Based on the data for
Cb*/Cv* (eq.
18), we deduced that the rate of efflux of
E-2-VPA across the BBB is lower than that of
VPA, which rules out the possibility that
Kep [E-2-VPA] > Kep [VPA]. Therefore, the lower
brain-to-plasma concentration ratios observed for
E-2-VPA compared with VPA are most likely
because of a much slower transport of E-2-VPA
from plasma water into the brain extracellular fluid across the BBB.
In summary, the main difference in the steady-state CNS distribution
between E-2-VPA and VPA lies in their
bidirectional transport across the BBB. The
intracelluar-to-extracellular partitioning does not seem to differ
between the two congeners.
Effects of Probenecid.
We now turn our attention to the effects of PBD on the distribution
kinetics of E-2-VPA. The cotreatment of
rabbits with PBD caused a remarkable 3- to 5-fold increase in
steady-state brain tissue concentrations of unlabeled
E-2-VPA; this compares with a 1.5- to 2-fold
increase in steady-state VPA brain tissue concentrations in our
previous VPA study. Coadministration of PBD did result in a modest
elevation in the mean plasma-free fraction of
E-2-VPA and a slight but statistically
insignificant increase in free plasma concentration of
E-2-VPA. This was likely because of
competition for plasma protein binding sites between PBD and
E-2-VPA. The increase in free plasma
concentration was small (1.4-fold) in comparison with the increase in
steady-state brain tissue concentration of
E-2-VPA. Therefore, the increase in
steady-state brain tissue concentration of
E-2-VPA could be explained to a minor extent
by the increase in free plasma concentration if brain uptake is
restricted to free drug in plasma. A more reasonable explanation for
the observed elevation in brain E-2-VPA
concentration upon treatment with PBD is a preferential inhibition of
mechanisms that move E-2-VPA from the brain
into the blood. The transport of E-2-VPA out
of the brain seemed to be more sensitive to PBD treatment than did that
of VPA; indeed, PBD induced only 2-fold increases in brain tissue
concentrations of VPA.
PBD cotreatment elevated brain tissue concentrations of
E-2-VPA to a level three to four times higher
than the cisternal outflow concentration of
E-2-VPA (fig. 5 and table
7). PBD had a similar, yet less dramatic, effect on VPA brain tissue/cisternal outflow concentration ratios in
our earlier study (20). Brain tissue concentrations of VPA were
elevated 1.5-fold over the cisternal outflow VPA concentration. It
is generally accepted that there is rapid and free exchange of drugs
between the CSF and brain interstitium across the ependyma (35).
Accordingly, the cisternal outflow concentration of
E-2-VPA was expected to have been increased by
PBD to nearly the same extent as the brain tissue concentration,
i.e. no change in brain/cisternal outflow concentration
ratio. One explanation for the unexpected increase in the
brain/cisternal ratio may be the presence of a PBD-sensitive transport
system for the exchange of E-2-VPA across the
neural cell membranes, in which case PBD could inhibit the
translocation of E-2-VPA from the
intracellular compartment to the extracellular space, resulting in
intracellular retention of E-2-VPA and
elevation in brain tissue E-2-VPA
concentration relative to cisternal E-2-VPA
concentration. In fact, by means of microdialysis, we recently showed
that in rabbits the steady-state concentration of VPA in the brain
extracellular space is lower than VPA concentrations inside the neural
cells for both control and PBD-treated animals, with the latter group
exhibiting significantly higher intracellular/extracellular VPA
concentration ratios than the controls (36). Further support for this
hypothesis comes from a study by Nilson et al. (37), which
showed that VPA was transported into cultured astroglial cells by a
saturable, carrier-mediated process.
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TABLE 7
Summary of compartmental model analysis of the effect of probenecid
cotreatment on plasma-brain-ventricle distribution of E-2-VPA
|
|
The above observations and intuitive deductions on the effects of PBD
are supported by our compartmental analysis. With respect to the
PBD-induced increase in the brain/cisternal concentration ratio of
unlabeled E-2-VPA, compartmental analysis (see
eq. 17 and table 7) confirms that
Kei/Kie or
intracellular-to-extracellular partitioning is the key factor. The
postulated increase in
Kei/Kie could
arise in one of two ways: either PBD increases the rate of transport of
drug into the neural cell compartment from the extracellular space,
i.e. Kei [PBD] > Kei [control], or PBD blocks efflux of the drug from the intracellular compartment into the extracellular space, i.e. Kie [PBD] < Kie [control]. It seems unlikely that PBD
would stimulate the transport of drug from the extracellular space into
the intracellular compartment. Therefore, the PBD-induced elevation of
the brain/cisternal concentration ratio of unlabeled drug is best
explained by a blockade of drug efflux from the neural cell
compartment.
PBD cotreatment caused a modest (< 2-fold) and statistically
insignificant increase in brain/average ventricular concentration ratio
(Cb*/Cv*) of
perfused tritiated E-2-VPA. According to eq.
18 (see table 7), the increase most likely reflects the increase in
Kei/Kie.
Because Cb*/Cv*
did not increase to the same extent as
Cb/Cv, we
surmise that if any change occurs in Kep,
it would have to be an increase in the rate constant to offset the
large increase in
Kei/Kie,
implying that PBD stimulated E-2-VPA efflux
transport across the BBB. However, such an effect of probenecid is not
likely.
The effect of PBD on the brain/plasma concentration ratio for unlabeled
drug can be explained using eq. 19 (see table 7). Again, the equation
is governed by the ratios
Kpe/Kep and
Kei/Kie. The
brain/plasma concentration ratios were substantially higher for
PBD-treated rabbits than for control animals. This can occur in the
following two ways: the ratio
Kpe/Kep could
be higher for the PBD group than the control group or the ratio
Kei/Kie could be higher for the PBD group than the control group. Considering first
the ratio
Kpe/Kep, an
increase could result from a higher steady-state flux of drug from the
plasma to the brain extracellular fluid for PBD-treated animals as
compared with controls, i.e. Kpe
[PBD] > Kpe [control] or conversely a
lower steady-state efflux of drug from the brain extracellular fluid to
the plasma for the PBD treatment group, i.e.
Kep [PBD] < Kep [control]. It is unlikely that PBD
stimulated transport of E-2-VPA or VPA from
the blood into the brain. We have also ruled out a significant
inhibition of E-2-VPA efflux from brain
extracellular fluid to plasma in our preceding consideration of
Cb*/Cv*. Hence,
the increase in brain-to-plasma partitioning probably does not reflect
changes in BBB transport. This leaves the increase in ratio
Kei/Kie as the
most likely explanation, which, as argued previously, most likely
reflects a blockade of PBD-sensitive efflux of
E-2-VPA and VPA at the neural cell membranes.
In conclusion, simultaneous iv infusion-VC perfusion of
E-2-VPA in rabbits showed that the VC
extraction of
[3H]E-2-VPA was
extensive and was not subject to inhibition by PBD. Brain tissue
localization of
[3H]E-2-VPA could not
account for the extensive loss of
[3H]E-2-VPA from the
ventricular perfusate. This leads to the conclusion that
[3H]E-2-VPA was being
cleared from the CSF by an efficient efflux system at the brain
capillary endothelium. Cotreatment with PBD elevated brain tissue
concentrations of unlabeled E-2-VPA by as much
as 3- to 5-fold compared with control values. In addition,
E-2-VPA tissue concentrations became higher
than E-2-VPA cisternal outflow concentrations
in PBD-treated animals, which indicates the presence of a
probenecid-inhibitable transport of E-2-VPA
across the neural cell membrane aside from the previously recognized
probenecid-sensitive transport process at the BBB. In fact,
pharmacokinetic modeling of the data suggests that the major effect of
PBD is an inhibition in the translocation of
E-2-VPA from the intracellular sites to the
extracellular space, resulting in intracellular trapping and greater
tissue retention of E-2-VPA. Finally,
simultaneous iv infusion-VC perfusion technique and pharmacokinetic
modeling of the steady-state distribution data prove to be a powerful
approach in characterizing the multi-step transport processes governing
the uptake, retention, and efflux of drugs in the CNS.
This research was supported by National Institutes of Health
Grant NS-30738.
Abbreviations used are:
VPA, valproic acid;
CSF, cerebrospinal fluid;
CNS, central nervous system;
VC, ventriculocisternal;
PBD, probenecid;
BBB, blood-brain barrier.
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2-Valproic Acid
in Rabbits
Top
Abstract
Introduction
