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Vol. 280, Issue 3, 1349-1356, 1997
Department of Neurology,
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of the trans-isomer of 2-en-valproate
(trans-2-en-NaVP; E-
2-en-valproate or
2-en-valproate), an unsaturated metabolite of valproic acid (VPA), on
intracellularly recorded sodium-dependent action potentials of cultured
mouse spinal cord and cortical neurons were compared with those of the
anticonvulsant sodium valproate (NaVP). The maximal rate of rise of
action potentials triggered by trains of 1-msec or 400-msec pulses
declined progressively until failure to fire in both cell types during
exposure to trans-2-en-NaVP or NaVP was observed. The limitation of
firing by both drugs was concentration, voltage, rate and time
dependent. The IC50 of trans-2-en-NaVP was 1.2 × 10
3 at 
1 hr and 4.8 × 105 M at 24 to 48 hr. Trans-2-en-NaVP did not limit sustained repetitive firing in
all cortical neurons. This may reflect slower rates of firing during
400-msec depolarizations in neurons of this type. In paired-pulse
experiments, the absolute refractory period was 7 msec in control
solution and 15 msec (P < .01 vs. control;
n = 9) in solution containing 6 × 104 M trans-2-en-NaVP. Firing was limited in all spinal
cord neurons after exposure to 0.5 mM NaVP for 24 to 48 hr; 80% were
limited by 1 mM NaVP at 1 hr. Coincubation of the spinal cord neurons with trans-2-en-NaVP and NaVP for 24 hr showed no hyperadditive effect
of these two drugs in vitro. Limitation of sustained
repetitive firing was reversed by hyperpolarization in the continuing
presence of either drug and incubation in drug-free medium. Limitation of sodium-dependent action potential firing rates could contribute, at
least in part, to the anticonvulsant effect of trans-2-en-NaVP.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Trans-2-en-NaVP is a major
metabolite of VPA in humans that results from mitochondrial
-oxidation (Baillie and Sheffels, 1995
). In amygdala-kindled rats,
VPA and trans-2-en-NaVP had approximately the same anticonvulsant
efficacy, although both drugs produced motor impairment at effective
doses (Löscher et al., 1988; Hönack et
al., 1992). Trans-2-en-NaVP was more potent than VPA in the experimental treatment of myoclonic, clonic and tonic seizures in
rodents and clonic seizures in dogs, when the two compounds were
compared at their respective times of peak effect (Löscher et al., 1991a). The anticonvulsant efficacy of
trans-2-en-NaVP occurred with less hepatotoxicity and teratogenic
potential, compared with VPA (Hönack et al., 1992;
Löscher et al., 1992, 1993). In addition,
pharmacokinetic, histopathological and clinical chemistry data obtained
from laboratory animal studies suggest that trans-2-en-NaVP might be a
valuable substitute for VPA (Löscher et al., 1991, 1992, 1993).
Among other actions (Fariello et al., 1995), VPA has been
reported to affect sodium-dependent neuronal activity. Voltage-clamp analysis in squid axons suggested that VPA (2 × 102
M) slowed sodium and potassium channel gating when applied internally (Fohlmeister et al., 1984). Voltage- and patch-clamp studies
of vertebrate neurons demonstrated blockade of both sodium and
potassium currents (Van Dongen et al., 1986; Zona and Avoli,
1990; Van den Berg et al., 1993). In addition, NaVP limited
high-frequency SRF of sodium-dependent APs in mouse central neurons in
cell culture at 37°C, at concentrations equivalent to clinically
therapeutic free (not bound to proteins) plasma levels (McLean and
Macdonald, 1986b). This led us to test the effect of trans-2-en-NaVP on
SRF in the present study.
Less is known about the unsaturated metabolites of VPA, several of
which have anticonvulsant efficacy in animals. Trans-2-en-NaVP is
quantitatively predominant in adults. In snail neurons, trans-2-en-NaVP hyperpolarized more than, but lessened the frequency of depolarizing bursts less than, VPA (Altrup et al., 1992). Direct effects
on sodium currents and sodium-dependent APs have not been reported. Both trans-2-en-NaVP and VPA diffuse mainly in extracellular space of
the brain (Lucke et al., 1993) and may affect neurons in a time-dependent manner. Therefore, we examined the effects of acute (1-hr) and prolonged (24-48-hr) exposure to both trans-2-en-NaVP and
VPA (here as NaVP) on intracellularly recorded electrophysiological properties of spinal cord and cortical neurons in dissociated monolayer
cell culture.
The concentration range tested here included values from clinical
pharmacokinetic studies. For 20 patients receiving VPA as monotherapy
at an average daily dose of 1152 ± 661 ms/day (mean ± S.D.), the blood levels of VPA and trans-2-en-NaVP were 60.0 ± 22.6 µg/ml (~3.6 × 104 M) and 3.0 ± 1.4 µg/ml (~1.8 × 105 M), respectively (McLaughlin
et al., 1992). VPA in plasma (free unbound fraction) may
reflect the corresponding concentrations in tissue fluids and in brain
(Vajda et al., 1981; Löscher et al., 1992).
Vajda et al. (1981) studied nine neurosurgical patients receiving VPA orally as monotherapy, at doses between 1200 and 1600 mg/day, for at least 3 days before surgery for epilepsy. Sections of
gray and white matter were obtained during surgery. CSF and plasma
samples were taken simultaneously. VPA concentrations ranged from
approximately 1.6 × 104 to 9 × 104 M in plasma, from 1.6 × 105 to
2.3 × 104 M in CSF and from 3.4 × 105 to 1.6 × 104 M in brain. Adkison
et al. (1995) measured plasma and brain levels of VPA and
its unsaturated metabolites in 24 patients, chronically receiving 740 to 4000 mg/day VPA, who subsequently underwent surgery for epilepsy.
The mean plasma concentration of VPA was ~62.7 µg/ml (~3.8 × 104 M), the free concentration was ~10.64 µg/ml
(~6.4 × 105 M) and the brain level was ~4.6
µg/g. The trans-2-en-NaVP level in plasma was ~2.9 µg/ml
(~1.74 × 105 M). A small free concentration of
~0.02 µg/ml (~0.12 × 106 M) indicated
extensive binding to plasma proteins. However, the brain tissue level
was ~0.14 µg/g, indicating concentration in the brain over plasma.
We tested both drugs at concentrations up to 60 mM.
We found that trans-2-en-NaVP and NaVP both limited SRF. The
concentration dependence of limitation by both drugs shifted to the
left with time of exposure. VPA was slightly more potent than
trans-2-en-NaVP after prolonged exposure. Limitation by trans-2-en-NaVP occurred at concentrations that could be achieved with repeated oral
dosing. Part of this work has been presented in abstract form (Wamil
et al., 1994a).
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cell Culture
Spinal cord and neocortical cell cultures were prepared by
published methods (Wamil and McLean, 1992). Briefly, embryonic mouse
spinal cords (gestational day 13-14) and neocortices (day 14-16) were
minced and dispersed, by trituration, to single cells and small clumps.
The neurons were plated in collagen-coated dishes and maintained
in vitro for 4 to 12 weeks before experimentation. The
culture medium consisted of 80% (v/v) Eagle's minimal essential medium, 10% fetal calf serum (for the first week only), 10%
heat-inactivated horse serum, 10 ng/ml 7S nerve growth factor and 1 ml/liter Mito Serum Extender (all supplements from Collaborative
Research, Bedford, MA). Growth of non-neuronal cells was suppressed by
brief treatment with 5-fluoro-2
-deoxyuridine after 1 week. The
Eagle's minimal essential medium was supplemented with 5.5 g/liter
glucose and 1.5 g/liter sodium bicarbonate and was diluted to 300 to
325 mOsM with distilled water. After equilibration with the incubator
atmosphere containing 10% CO2, the pH of the culture
medium was 7.4. Medium was changed twice weekly.
Experimental Protocols
General.
The methods used here have been published in detail
elsewhere (Wamil and McLean, 1992). For experiments, the culture medium was replaced with mDPBS with Mg++ concentration elevated to
suppress spontaneous synaptic activity (composition, in mM: NaCl,
143.4; KCl, 4.2; CaCl2, 0.9; MgCl2, 5.0-7.0;
glucose, 5.6; in 9.5 mM sodium-phosphate buffer at pH 7.4). The culture
dish was then placed in an aluminum block heated to 37°C, on the
stage of an inverted phase-contrast microscope. Intracellular
recordings of transmembrane potential were made during superfusion with
drug-free and drug-containing solutions. A bridge circuit in the
amplifier allowed simultaneous injection of current and recording of
potential. The first derivative of the membrane potential
(dV/dt) was obtained electronically, and the peak of the
differentiated signal was proportional to 
max. The
max indirectly reflected inward sodium current
generating the upstroke of APs (for discussion, see McLean and
Macdonald, 1983; Wamil and McLean, 1992).
Degree of repetitive firing.
A series of depolarizing
current pulses of 400-msec duration and variable amplitude were applied
through the recording electrode to elicit overshooting APs in neurons
with stable Em values more negative than or equal to 
45
mV. SRF of APs is defined as continuous high-frequency firing
throughout one or more 400-msec depolarizations. All neurons in control
medium demonstrated SRF. In drug-containing solutions, SRF could not be
obtained from a percentage of neurons, depending on the drug
concentration and the duration of drug exposure. In these neurons, a
limited train (50-250 msec) of APs was evoked with a series of
depolarizations above threshold and firing ceased for the remainder of
the pulse after progressive decline of max. With
increasing depolarization past the point of maximal firing duration,
the AP trains became shorter. This is referred to as limitation of SRF
or limited firing. Limitation was judged to be present if firing could
not be sustained throughout any of the depolarizations from the
Em or a less negative holding potential.
max of APs fired from the same Em. At
fast rates (50-150 Hz), a small number of failures occurred in control
neurons, but the max of APs that fired was not
significantly altered by high-frequency firing.
Pairs of identical 1-msec stimuli were delivered at intervals of 1 to
20 msec, in the presence of trans-2-en-NaVP or in drug-free mDPBS. The
absolute refractory period was determined as the minimum interval at
which an AP appeared in response to the second stimulus, even though
the second AP had a slower max than did the first AP. The relative refractory period was determined as the minimum interval between pulses that elicited two APs with the same
max.
Neurons damaged during impalement had low Em values,
undershooting APs that did not increase in amplitude with
hyperpolarizing current and either limited firing during depolarizing
steps or extremely fast spontaneous firing before loss of membrane
potential and cell death. Such impalements were terminated, and new
neurons were selected for study.
Drug application.
Neurons were exposed to trans-2-en-NaVP or
NaVP by superfusion with or without preincubation or by pressure
application (Wamil and McLean, 1992, 1994). Drug application was
terminated by washout of the drug with drug-free buffer. For
superfusion experiments, control recordings were obtained from at least
three neurons in each culture before drug application. If SRF was not
present in all neurons, a new culture was obtained. After control
recordings, the superfusate was changed to solution containing
trans-2-en-NaVP or NaVP.
, 1994). In several sessions, the experimenter was blinded to the
concentration of drug and the name of the drug in the culture medium
and superfusate for studies of some groups of neurons. Data from
blinded and unblinded experiments were similar and were combined for
analysis.
Drug preparation.
Trans-2-en-NaVP was supplied in aqueous
solution (100 mg/ml trans-2-en-NaVP; Desitin Arneimittel GmbH, Hamburg,
Germany). That stock solution was diluted to desired concentrations in
mDPBS. NaVP (RBI, Natick, MA) was dissolved in distilled water. Stock solutions were prepared at 102 g/ml (6 × 102 M) and then diluted to desired concentrations in
mDPBS.
Statistics.
Parametric data are given as means ± 1 S.E.M. The significance of differences between treatment groups was
tested with the Student t test and Wilcoxon signed rank
test. The IC50 values for limitation of SRF and the
significance of differences between values obtained at different times
of exposure to trans-2-en-NaVP or NaVP were determined using
computer-assisted linear regression analysis based on the method of
Litchfield and Wilcoxon (1949).
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Passive membrane properties.
During control or superfusion
experiments with trans-2-en-NaVP and/or NaVP, the resting
Em in cortical or spinal cord neurons ranged from 40 to
80 mV. Spinal cord neurons in control mDPBS had a mean Em
of 60.7 ± 4.4 mV (n = 82). In a range of
concentrations from 6 × 109 to 6 × 104 M (n = 121), the Em
averaged 58.4 ± 2.7 mV (n = 65). The difference was insignificant. Rin was 46.7 ± 2.9 M
in control
solution and 50.2 ± 4.1 M in trans-2-en-NaVP-containing
solution (n = 29). In neocortical neurons,
Em was 53.1 ± 2.3 mV (n = 14) and
did not change more than 2 mV during experiments with given cells; Rin was 47.8 ± 3.8 M (n = 10) and
did not change substantially during exposure to either drug.
Concentration-dependent limitation of SRF by trans-2-en-NaVP and
VPA.
SRF was observed in spinal cord and cortical neurons in
control solution. Acute (up to 1 hr) superfusion with trans-2-en-NaVP led to limitation of SRF, in a concentration-dependent manner. Limitation of high-frequency firing of APs occurred in spinal cord and
cortical neurons (n = 36 and n = 10, respectively) with progressive reduction of max
before cessation of firing for the remainder of 400-msec
depolarizations during superfusion with 6 × 104 M
trans-2-en-NaVP for 1 hr (fig. 1). The effect was
reversible by hyperpolarization (fig. 1, A, 70 mV, and B, 72 mV)
during exposure to the drug and by washout with drug-free mDPBS (fig. 1, POST).
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3 M (total
n = 66) and 4.8 × 105 M (total
n = 101), respectively.
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4 M (~100 µg/ml) (fig. 2B). The IC50
for limitation of firing in neurons exposed chronically was 1.2 × 105 M (total n = 93). These findings
suggested that limitation of SRF by prolonged exposure of
trans-2-en-NaVP and NaVP to spinal cord neurons was time dependent.
Time-dependent limitation of SRF.
Figure 3A
shows the percentage of neurons with sustained firing as a function of
time of exposure to a submaximal concentration of trans-2-en-NaVP. The
percentage of spinal cord neurons with limited firing increased with
time of exposure to trans-2-en-NaVP. About 50% of neurons had limited
firing after exposure to 6 × 107 M for 48 hr (fig.
3A). Fewer neurons were limited at different time intervals between 3 min and 48 hr (n = 36) (fig. 3A). With 6 × 104 M trans-2-en-NaVP, 9 of 10 neurons were limited in
48 hr (fig. 3A).
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7 M and six of eight treated with 6 × 104 M trans-2-en-NaVP (fig. 3A, WASH).
Different extents of limitation of SRF in cortical and spinal cord
neurons by trans-2-en-NaVP.
Sustained firing was observed in 10 cortical neurons incubated for 24 hr with 6 × 108 M
trans-2-en-NaVP. Firing was limited in 8 of 14 cortical neurons at
6 × 107 M and in 5 of 10 cortical neurons at 6 × 106 and 6 × 105 M trans-2-en-NaVP
(fig. 3B). A maximum of 70% limitation (7 of 10 neurons) was observed
in cortical neurons exposed to 6 × 102 M
trans-2-en-NaVP. All spinal cord neurons were limited at 6 × 103 M trans-2-en-NaVP. In spinal cord neurons, under the
same conditions, limitation was observed in 90% (9 of 10 neurons) at
6 × 104 M (fig. 3B). Thus, cortical neurons
appeared to be less sensitive to trans-2-en-NaVP, probably due to a
lower frequency of AP firing (fig. 1) (see "Frequency Dependence").
Effect of the combination of trans-2-en-NaVP and NaVP on SRF.
Figure 4 shows results obtained from neurons incubated
for 24 hr with trans-2-en-NaVP, NaVP or both, at concentrations that limited SRF in a small percentage of neurons (fig. 2). Low
concentrations of both drugs were selected to test for additive and
hyperadditive effects of drug combinations. No hyperadditive effect was
found using the three combinations shown in figure 4.
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Frequency dependence.
The effect of 6 × 104 M trans-2-en-NaVP on trains of APs depended on
stimulation frequency. In control solution (fig. 5,
Control, n = 8), stimulation at 1 and 100 Hz with
1-msec depolarizing current pulses elicited APs with nearly constant
max, despite some failures at 100 Hz. In neurons
with limited firing during 400-msec pulses after overnight incubation
with 6 × 104 M trans-2-en-NaVP, many failures
occurred even at 10 Hz (n = 10) (fig.
6A). The max of APs that fired
declined progressively, in a frequency-dependent manner (fig. 5). As
shown in figure 6A, 82% of stimuli at 100 Hz fired APs
in control solution; only 38% of stimuli fired APs at 100 Hz in
solution containing 6 × 104 M trans-2-en-NaVP
(P < .05 vs. control by Wilcoxon signed rank test; for
17 neurons, Em was 59.4 ± 4.7 mV). In four neurons with persistent unlimited SRF after overnight incubation with 6 × 104 M trans-2-en-NaVP, max declined
slightly if at all at high frequencies of stimulation with trains of
brief pulses. Stimulation of these neurons at 100 Hz resulted in few
failures.
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Effect of trans-2-en-NaVP on refractoriness.
Refractoriness
and recovery from inactivation were prolonged during exposure to NaVP
(McLean and Macdonald, 1986b). To test the effect of trans-2-en-NaVP on
these parameters, pairs of 1-msec depolarizing current pulses,
identical in intensity and duration, were applied at variable intervals
in neurons superfused with drug-free mDPBS or preincubated (12-24 hr)
and superfused with 6 × 104 M trans-2-en-NaVP.
Figure 7 shows recordings from a spinal cord neuron
(Em = 60 mV) stimulated by paired pulses delivered every 1 sec at 1 Hz (maximum interval, 40 msec). In control solution, the
absolute refractory period was 7 msec. During exposure to 6 × 104 M trans-2-en-NaVP, this neuron had a 15-msec
refractory period. Figure 6B compares refractoriness of
trans-2-en-NaVP-treated neurons in which pairs of pulses were delivered
at different intervals (0.2, 0.5 and 1 Hz) to each of nine neurons
(Em = 52.8 ± 4.5 mV). At the three intervals
between delivery of paired pulses, the absolute refractory periods were
1.3 ± 0.2 (n = 5), 1.8 ± 0.4 (n = 7) and 3.2 ± 1.0 (n = 6)
msec, respectively, in control solution. In trans-2-en-NaVP-containing
solution, absolute refractoriness at the three intervals was 4.4 ± 0.8 (n = 5), 5.4 ± 1.0 (n = 10) and 7.3 ± 1.8 (n = 7) msec, respectively
(fig. 6B). Absolute refractoriness in neurons treated with
trans-2-en-NaVP was significantly different from that in control
neurons at all three intervals (P < .01 at 0.2 Hz; P < .05 at 0.5 and 1 Hz; unpaired t test).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Trans-2-en-NaVP has been shown to have anticonvulsant activity in
animal models of partial and generalized seizures. The models include
1) amygdala kindling, a model of partial seizures secondarily generalized; 2) electroshock-induced seizures, a pharmacological model
of tonic-clonic seizures; 3) generalized tonic-clonic seizures in
genetically seizure-prone gerbils; 4) generalized absence-like activity
in genetically seizure-prone rats; and 5) myoclonic and clonic seizures
induced by pentylenetetrazole (Löscher et al., 1991;
Semmes and Shen, 1991; Hönack et al., 1992). VPA, a
broad-spectrum, clinically used, antiepilepsy medication, is effective
in these models also. On this basis, trans-2-en-NaVP might be predicted to have a broad spectrum of clinical utility if adequate brain concentrations could be reached. The practical reason to determine this
is that trans-2-en-NaVP is less toxic than VPA and could potentially
become a substitute for the parent compound as a clinical antiepilepsy
drug (Hönack et al., 1992).
To gain insight into the cellular mechanisms of action of
trans-2-en-NaVP, we studied its effect on SRF of sodium-dependent APs
by mouse central neurons in cell culture, an in vitro action that parallels efficacy against maximal electroshock-induced seizures in animals and partial seizures, with or without secondary
generalization, clinically (for review, see Macdonald, 1983).
Phenytoin, carbamazepine and VPA protect almost equally against these
seizure types (Mattson et al., 1985, 1992). All three
antiepilepsy drugs also limit SRF at concentrations equivalent to free
(not bound to albumin) plasma levels that are therapeutically relevant
(McLean and Macdonald, 1983, 1986a,b). High-frequency firing has been
detected along subcortical pathways from a penicillin-induced cortical
epileptogenic focus (Sypert and Reynolds, 1974). Limitation of such
firing may be important in preventing the spread of seizures.
Limitation by trans-2-en-NaVP was concentration, use and voltage
dependent, as were the effects of the clinically used drugs.
Limitation of SRF by both trans-2-en-NaVP and NaVP was also
time-dependent here. NaVP was slightly (about 4-fold) more potent in
limiting SRF of spinal cord neurons after prolonged exposure. However,
NaVP limited firing in only 75 to 80% of spinal cord neurons at the
highest concentrations tested acutely. In previous experiments,
inability of NaVP to limit firing in all neurons and inefficacy of 120 µM 2-en-VPA (not the trans-conformer) were observbed
during exposures of <2 hr (McLean and Macdonald, 1986, fig. 4). Here,
during superfusion for up to 60 min, a high concentration (60 mM) of
trans-2-en-NaVP, but not NaVP, limited all exposed neurons. Both NaVP
and trans-2-en-NaVP were more potent after prolonged exposure than they
were acutely. Also, with time, NaVP became more potent than
trans-2-en-NaVP in limiting SRF. A time-dependent shift in the
concentration dependence of limitation of SRF was observed previously
with gabapentin (Wamil and McLean, 1994), oxcarbazepine (Wamil et
al., 1994), remacemide (Wamil et al., 1996) and
phenytoin (M. J. McLean, unpublished observations). Exposure to 5 µg/ml gabapentin (equivalent to a therapeutic plasma level in
clinical practice) for <5 min limited firing in only ~10% of
neurons. Firing was limited in 40% of neurons exposed to this
concentration for 1 hr, 60% exposed for 5 hr and ~80% exposed for
24 to 48 hr. These and the present findings suggest that prolonged
exposure to investigational antiepilepsy compounds should be tested
before the potential clinical significance of in vitro
actions of the drugs is judged.
Mechanisms that could account for the delayed effects on SRF include
slow uptake of trans-2-en-NaVP into neurons, slow access to a binding
site or sites (possibly within or on the membrane and associated with
the sodium channel), intracellular modification of the drug or its
target site (e.g., phosphorylation of sodium channels)
and/or modification of biochemical pathways. Perlman and Goldstein
(1984) reported VPA to be a potent membrane-disordering agent. Because
of the lipophilicity of VPA and trans-2-en-NaVP, this action should be
fast and is less likely to account for the observed delay. The methods
used here cannot distinguish among these possibilities, and we found no
published data testing them critically in relation to efficacy. The
delay parallels the delay to peak anticonvulsant activity of
trans-2-en-NaVP (Löscher et al., 1993) and VPA (Wilder
and Karas, 1982) in animals after i.v. administration. This is
consistent with the interpretation that transport into neurons, an
effect on enzymes (Löscher and Frey, 1977; Van der Laan et
al., 1979; Löscher et al., 1991) or slow binding
to a membrane site must occur before the anticonvulsant effect.
Overlap of the concentration dependence of limitation of SRF with
clinically attainable concentrations suggests that limitation of SRF
might be a clinically important mechanism of the protective efficacy of
trans-2-en-NaVP. Plasma levels of trans-2-en-NaVP ranged between 370 and 600 µg/ml (~2.2-3.6 mM) in amygdala-kindled rats given 150 mg
i.p. three times daily (Hönack et al., 1992). In
epileptic patients, trans-2-en-NaVP derived from orally administered VPA actually accumulated in brain, relative to plasma concentrations, albeit at low levels (0.143 ± 0.026 µg/g vs.
0.022 ± 0.004 µg/ml) (Adkison et al., 1995). These
low concentrations make it unlikely that accumulation of
trans-2-en-NaVP derived from VPA is sufficient to enhance the effect of
the parent compound. However, assuming accumulation after large
systemic doses of trans-2-en-NaVP, brain concentrations as high as 14 to 25 mM might result at steady state. Such levels could make
trans-2-en-NaVP a monotherapeutic antiepilepsy drug, if tolerable.
Prolonged incubation with trans-2-en-NaVP led to limitation of SRF in
all spinal cord neurons tested at 1 mM, with an IC50 value
of 4.8 × 105 M. The concentration-response curve
stretched over 6 log units, suggesting that the desired concentration
could be finely adjusted over time with large or small changes in oral
dose. Acutely, exposure to 10 mM trans-2-en-NaVP limited firing in all
neurons. The IC50 for limitation was 1.3 × 103 M (~216 µg/ml) acutely, but it was 4.8 × 105 M (~8 µg/ml) after prolonged exposure, simulating
chronic, repeated, clinical dosing. A maximum of 70% of cortical
neurons had limited firing after prolonged exposure to 60 mM
trans-2-en-NaVP, but firing of 50% of cortical neurons was limited at
6 × 106 and 6 × 105 M
trans-2-en-NaVP (~1 and 10 µg/ml, respectively). Thus, systemic dosing could produce adequate CSF concentrations to limit firing rates
in a large percentage of neurons. Presumably, as with phenytoin and
carbamazepine, this could underlie anticonvulsant and antiepileptic efficacy, at least in part.
No hyperadditive effect was seen when trans-2-en-NaVP was combined with
NaVP. Simple additive effects on SRF suggest a shared mechanism or
mechanisms of antiepileptic action (Löscher et al., 1988; Hönack et al., 1992). The ability of
trans-2-en-NaVP to limit SRF in vitro is shared by
therapeutically relevant concentrations of several clinically used
antiepilepsy compounds effective against partial seizures, with or
without secondary generalization, including phenytoin (McLean and
Macdonald, 1983), carbamazepine (McLean and Macdonald, 1986a), VPA
(McLean and Macdonald, 1986b), felbamate (White et al.,
1992), lamotrigine (Cheung et al., 1992), gabapentin (Wamil
and McLean, 1994b) and oxcarbazepine (Wamil et al., 1994). Benzodiazepines (McLean and Macdonald, 1988) and barbiturates (Macdonald and McLean, 1986) limited SRF at concentrations encountered in treating status epilepticus. Phenobarbital protects many
patients against partial seizures with or without secondary
generalization. Thus, limitation of SRF is not the sole mechanism for
efficacy against these seizure types. MK-801 (Wamil and McLean, 1992)
and remacemide (Wamil et al., 1996) limited SRF at
concentrations that are nontoxic in patients and that overlap the upper
end of the range of concentrations required for each drug to block
N-methyl-D-aspartate responses in
vitro. Ethosuximide did not limit firing at 5 times the upper
value in the clinically therapeutic range of concentrations (McLean and
Macdonald, 1986b). Thus, limitation of SRF is unlikely to account for
the broad spectrum of efficacy of trans-2-en-NaVP in animals and of
felbamate and lamotrigine clinically.
AP firing frequency can be modulated by potassium and calcium currents.
However, several of the present findings suggest an effect of
trans-2-en-NaVP on sodium channels. These include 1) progressive
reduction of max until cessation of firing of APs, during both 400-msec depolarizing pulses and trains of 1-msec depolarizing steps at high frequency from a constant Em; 2)
prolongation of the absolute refractory period; and 3) prolongation of
recovery from blockade. In addition, there was no effect on
Em and Rin. We did not observe the
hyperpolarization observed by others in snail neurons (Altrup et
al., 1992), perhaps because different conductances were present in
the neurons studied here. A small percentage of cortical neurons showed
no limitation of SRF when preincubated with trans-2-en-NaVP for 24 hr,
perhaps due to the frequency dependence of block. Cortical neurons
fired more slowly than did spinal cord neurons during 400-msec current
pulses (fig. 1). Slow firing could result from activation of a
calcium-dependent potassium conductance present in cortical, but not
spinal cord, neurons. Nonetheless, cortical and hippocampal pyramidal
neurons are capable of sustained high-frequency firing when this
conductance is blocked, e.g., by biogenic amines (for
review, see Nicoll, 1990). Thus, limitation of firing rates could occur
in neurons in several different regions of the central nervous system.
Proving the molecular mechanism of the effect on sodium-dependent APs will require further investigation with voltage (patch)-clamp techniques.
In conclusion, it appears that limitation of high-frequency AP firing could contribute to the anticonvulsant efficacy of trans-2-en-NaVP in animal models in which concentrations in the range of those required to limit SRF were achieved. Such concentrations are not achieved in patients when trans-2-en-NaVP is derived from VPA. Proof that effective levels of trans-2-en-NaVP could be achieved by oral dosing would require pharmacokinetic and efficacy testing in epileptic patients. The lower incidences of organ toxicity and teratogenic effects in animals with trans-2-en-NaVP than with VPA and the efficacy of trans-2-en-NaVP against a broad range of seizures in experimental models provide an impetus for such studies.
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Acknowledgments |
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The authors thank Ron Thomas for expert tissue culture assistance.
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Footnotes |
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Accepted for publication November 8, 1996.
Received for publication March 26, 1996.
1 Current address: Department of Anesthesiology, VUMC, 1161 21st Avenue South, T-4216 Medical Center North, Nashville, TN 37232-2125.
2 Supported by a Merit Review Award from the Department of Veterans Affairs and funds received in collaboration with the Holcomb Medical Research Institute.
Send reprint requests to: Michael J. McLean, M.D., Ph.D., Department of Neurology, Vanderbilt University Medical Center, 2100 Pierce Avenue, 351 MCS, Nashville, TN 37212.
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Abbreviations |
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AP, action potential;
CSF, cerebrospinal fluid;
Em, resting membrane potential;
mDPBS, modified Dulbecco's
phosphate-buffered saline;
NaVP, sodium valproate;
Rin, input resistance;
SRF, sustained repetitive firing;
trans-2-en-NaVP, trans-isomer of 2-en-valproate;
max, maximal rate of rise of action potential;
VPA, valproic acid.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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-aminobutyric acid in mouse brain.
Naunyn-Schmiedeberg's Arch. Pharmacol.
296: 263-269, 1977[Medline].-carbolines, limit high frequency repetitive firing of action potentials of spinal cord neurons in cell culture.
J. Pharmacol. Exp. Ther.
244: 789-795, 1988This article has been cited by other articles:
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