Epilepsy Research, 12 (1992) 199205 0920-121 l/92/%05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved
199
EPIRES 00497
Valproic acid treatment of experimental status epilepticus
Nancy Y. Walton and David M. Treiman Neurology and Research Services, West Los Angeles Department of Veterans Affuirs Medical Center and ~epurtment
ofNearology, Universiry of CuI~orniu, Los Angeles, CA, USA
(Received 19 July 1991; revision received 30 March 1992; accepted 30 March 1992) Key worh: Valproic acid; Status epilepticus; Rat; Anticonvulsant
The efficacy of valproic acid (VPA) in control of generalized convulsive status epilepticus was tested in a rat model. Rats with cortical cobalt lesions were injected with homocysteine thiolactone to induce secondarily generalized tonic-clonic seizures (GTCS). The median effective dose (EDsa) for control of GTCS was 21 I .9 mg/kg (270 fig/ml in serum 30 mm post dose) when treatment was given intraperitoneally after the second GTCS. VPA entered both serum and brain very rapidly after injection, with little change in concentration from 5 to 30 min post dose. In earlier experiments with phenytoin, phenobarbital, diazepam and lorazepam in this model, we found that the serum concentrations produced by the EDses versus GTCS were very similar to those which have been reported to be effective in treating human status epilepticus. If this same relationship holds true for VPA, we would predict that a serum con~ntration of around 270 pg/ml VPA would be required for control of generalized convulsive status epilepticus in human patients. The safety of this high a concentration of VPA has not been tested.
Valproic acid (VPA) is a widely used anticonvulsant drug which has been FDA-approved for use in the USA since 1978. Although the current indication is only for treatment of absence seizures, considerable evidence has been generated that the drug is effective in control of other primarily generalized seizures as well as partial-onset seizures2. VPA is a branched chain carboxylic acid, a molecular structure which is very different from the heterocyclic structures of other currently available anticonvulsants. The mechanism by which VPA exerts its anticonCorrespondence to: Dr. Nancy Y. Walton, Department of Neurology, Reed Neurological Research Center (C-128), UCLA School of Medicine, Los Angeles, CA 90024, USA.
vulsant properties has not been established with certainty, although increased brain y-aminobutyric acid (GABA) levels, enhanced postsynaptic response to GABA, and changes in cellular membranes have all been suggested4’5. Clinical experience suggests that there is a temporal delay after VPA therapy is started before antiepileptic effkacy is seen3T12,26,suggesting that one or more VPA metabolites may contribute to its anticonvulsant action. Several VPA metabolites have been shown to possess anticonvulsant activity in animal The parent VPA molecule itmodels 14,16,17321*27*42. self, however, also has antiepileptic activity, because seizure protection has been reported in animal testing at times too early to be due to the effects of metabolites2’, and there are reports of efficacy when VPA was administered rectally for the treatment of status epilepticus30,34.
200
These experiments were designed to test the efficacy of VPA in a rat model of generalized convulsive status epilepticus. The model involves administration of homocysteine thiolactone epileptogenic cortical cobalt lesions. status epilepticus
includes
to rats with The resulting
a series of true secondar-
ily generalized tonic-clonic seizures (GTCS), accompanied by electroencephalographic patterns remarkably similar to those we have observed in hu-
saline. Sodium valproate by Abbott Laboratories. 500 mg VPA equivalent which was reconstituted used immediately. Measurement
of VPA
for injection was donated Each VPA vial contained as a lyophylized cake with sterile water and in blood
and
brain
was
Details of these methods have been published earlier - see references 3741. Adult, male SpragueDawley rats weighing 2OG-250 g each were used as subjects in these experiments (n = 134). Rats were housed individually after surgery in Plexiglas cages with contact bedding. Food and water were avail-
done using fluorescence polarization immunoassay (TDX, Abbott). Blood was drawn into tubes containing EDTA and immediately centrifuged to separate the serum. Samples for free valproate levels were prepared by centrifugal ultrafiltration of the fresh serum. Serum and ultrafiltrate were frozen until assayed. Brain samples were prepared for assay by sonication of tissue in phosphate buffer (pH = 10.5) followed by incubation with an alkaline protease for 60 min at 50°C. VPA was extracted from the tissue homogenate by adjusting the pH to 4.5 with 10% H3P04 and mixing with methyl-tertbutyl ether (MtBE). The sample was then centrifuged and the organic layer transferred to a conical tube containing 200 ~1 polyethylene glycol 400 (PEG). The MtBE was dried and 800 ~1 phosphate buffer (pH = 7.0) were added to the PEG to produce samples for TDX assay. Serum and ultrafiltrate samples of 50 ~1 were diluted as needed to fall within the range of the assay system. Brain homogenate derived from 0.1-1.0 g brain tissue was used for extraction of samples to be assayed. EEG was monitored daily beginning on the fifth post-operative day. Status was induced by intraper-
able ad lib. Temperature was maintained at 22°C and a 24-h diurnal lighting schedule was utilized, with lights on from 06.00 to 18.00 each day.
itoneal (i.p.) injection of 5.5 mmol/kg HCT when one of the following criteria was met: focal motor seizures accompanied by left frontal spiking on
Epidural recording electrodes made from stainless steel machine screws were implanted under general anesthesia (ketamine/xylazine, 87/l 3 mg/kg) at the following stereotaxic coordinates (referenced to bregma): A-P + 1.5, lateral + 2.0, and A-P -2.5, lateral + 2.0. 25 mg powdered cobalt was placed onto the dura beneath the left frontal electrode. Recording electrodes were fastened into a plastic connector which was then cemented to the skull with methacrylate adhesive. Ketamine (50 mg/ml injectable) was obtained from Quad pharmaceuticals. Xylazine (20 mg/ml in normal saline) was obtained from Sigma. Homocysteine thiolactone (HCT) was obtained from Sigma and dissolved for injection in 5.0 ml/kg normal
EEG; or poly-spike bursts on EEG at least every 20 s. EEG was monitored continuously following injection of HCT. Onset of status epilepticus was defined as onset of the first GTCS and treatment injections were given i.p. immediately after the second GTCS. Rats were observed (with continuous EEG) for 30 min following treatment and all further seizure activity was noted. Experiments were videotaped to allow review of seizure activity and its classification. Rats were then anesthetized, blood samples obtained by cardiac puncture and the brain rapidly removed and frozen. Six rats were treated with each of the following VPA doses (mg/kg) in a predetermined random order: 0, 100, 140, 170,200, 300,400, 500 and 600. Six
man generalized convulsive status epilepticus’*. We found, in earlier experiments testing the efficacy of diazepam, phenytoin, phenobarbital and lorazepam in this model, that the serum concentration produced by the median effective dose (ED,,) for control of GTCS in the model closely approximates the serum concentrations for these same drugs which have been reported to be clinically effective in treating human status epilepticus37A0. We therefore believe that the model can be used successfully to predict potential clinical efficacy in new drugs for treating status epilepticus. Methods
201
TABLE
I
EDso. 9.5% confidence interval and corresponding
serum concentration for varying degrees of seizure control when status epilepticus
was
treated with VPA Seizure control
achieved
No GTCS No generalized No motor a Estimated
seizures
seizures
EDso (mg/kg
I.P.)
95% confidence
Serum concentration
interval
212
162-278
269
278
215-361
332
398
306518
597
from dose:concentration
ratios
obtained
(pg/ml)a
in these studies.
normal control rats were injected i.p. with each of the VPA doses and blood samples were obtained after a 30-min waiting period, in order to determine if status epilepticus would influence the dose:concentration ratio, protein binding or brain entry of VPA. An additional group of normal rats was used to determine the kinetics of VPA following i.p. administration. These rats were given 200 mg/kg VPA i.p., then blood samples were obtained from four rats at each of the following times (min) after injection: 5, 10, 15, 30, 60, 90, 120 and 150. EDsos and 95% confidence intervals were calculated by a computer adaptation of the method of Litchlield and Wilcoxoni3, which is based on linear regression of the probit transformation of the percentage of animals showing an effect versus the logarithm of the drug dose they received. Goodness of fit of the observed data to the linear model was confirmed by chi square testing. Statistical comparisons were made using standard parametric procedures. Results will be presented as means + SD unless noted otherwise.
each (range = 6-18) during the 30 min following treatment. Cessation of further GTCS was not seen in rats given less than 170 mg/kg VPA, and 100% protection was not achieved until doses at or above 400 mg/kg VPA. EDsss, the serum concentrations they would produce 30 min after injection, and 95% confidence intervals for varying degrees of seizure control are shown in Table I. That seizure control is serum and brain concentrationdependent is evident from examination of Fig. 1, where these concentrations have been calculated for rats achieving varying degrees of seizure control, regardless of dose. We saw only a few rats in this experiment with EEG normalization. Attempts to raise the dose above 600 mg/kg resulted in fatalities during the 30 min observation window. No significant differences were seen in the VPA
Results Status was induced in these rats (n = 54) 6.7 f 1.2 days after surgery. Latency from injection of HCT to onset of status epilepticus was 28.3 + 9.4 min, and the interval from the onset of the first to onset of the second GTCS was 6.2 + 3.3 min. In addition to GTCS, brief generalized tonic seizures were also seen during the status episodes, as well as focal motor seizures of the same type as were seen prior to HCT injection. Rats treated with saline only averaged 11 GTCS
GTCS CONTINUE
NO GTCS. OTHER GEN’L CONTINUE
Fig. 1. VPA concentrations ing different after injection.
the mean
The number
above
the bars.
ALL MOTOR SZ STOP
in serum and brain from rats achiev-
degrees of seizure control
Each bar represents parentheses
NO GEN’L. SPS CONTINUE
during
status epilepticus.
+ SD of the concentration of animals Open
in each group
bars are serum
35 min shown
in
VPA while
shaded bars are brain VPA. (GTCS = generalized tonic-clonic seizure; GEN’L = generalized seizure; SPS = simple partial seizure; SZ = seizure.)
202
5 2 400*CONTROL $
2oo
ti g 0
i
0
I
I
100
200
I
I
I 300
I 500
‘loo
I 600
3
1%
l BRAIN
300 -
d 5
0 SERUM
loo
.9-r----,__
0
>
i----” 0
--
I
I 60
I 30
90
120
150
0
VPA DOSE (mglkg) MINUTES
Fig. 2. Serum VPA concentrations epilepticus is the mean
in rats treated
(open circles) and controls + SD of the concentrations
during
status
(filled circles). Each point 35 min after injection
Fig. 4. Pharmacokinetics
decline.
100
g
80
P ;;
60
I
of VPA following
i.p. Each point is the mean
a dose of 200 mg/kg
+ SD for four rats. Open circles are
serum VPA. while closed circles are brain VPA.
for six rats.
serum levels produced by the various doses of drug in the seizing versus control animals (see Fig. 2). The percentage of total serum valproate which was not bound to serum proteins increased with the serum concentration, as can be seen in Fig. 3. Brain VPA was 48.3 f 14.1% of the free VPA level, and showed no tendency to change with changes in dose or serum concentration. Results of the pharmacokinetic study, shown in Fig. 4, demonstrate that VPA entered the blood compartment rapidly and then quickly moved into brain. There was a fairly constant VPA level in both compartments from 5 to 30 min after injection, before elimination exceeded the rate of absorption, causing the concentrations to begin to
FROM INJECTION
Discussion While VPA did show efficacy in this model of status epilepticus, it was not very potent. In earlier studies of drugs with known clinical efficacy for treatment of status epilepticus, we found the serum concentrations produced by the EDses versus GTCS in this model were very similar to concentrations reported in the literature to be effective in treating human status epilepticus37-40. Table II summarizes these findings. We would predict, therefore, from the results of this experiment, that about 270 pg/ml VPA would be required for control of human status epilepticus. This concentration is far greater than the usual ‘therapeutic range’ for VPA in treatment of chronic epilepsy (S&l50 pg/ m1)2,5. The reason for the observed lack of potency in this experiment is probably also the reason that acute animal seizure model tests (maximal electroshock seizures, pentylenetetrazol seizures, etc.) have also found that concentrations above the usual hu-
0 p_
$
TABLE
40
> ii
II
Efficacious drug serum concentrations and reported in the clinical literature
20
predicted from the model
ff
Drug tested
0 -200
200250
250300
300400
400-
600
600750
750-
,900
TOTAL SERUM VPA bg/ml)
Fig. 3. Free VPA (as a percentage plotted
against
of total serum concentration)
total VPA in serum. Each bar is the mean for 9-12 rats.
+ SD
Predicted
from
Reported
from human
model
clinical trials
Phenytoin
19.4 pg/m137,39
24.6’
Diazepam
40 ng/m13’
50 ng/ml”
Phenobarbital
15 pg/m138 196 ng/m14’
60-330
900
Lorazepam
18.3 pg/mlz9 ng/m13’.3h
203
man ‘therapeutic’ range were required for seizure contro15, namely, that the full anticonvulsant effect of VPA in chronic therapy depends not only on the initial anticonvulsant action of the VPA, but also on either the action of a metabolite or else some secondary action of VPA. One cannot wait for such secondary effects when treating human status epilepticus. VPA has only rarely been studied in humans given large i.v. bolus doses, and none of these studies has achieved serum concentrations greater than 120 pg/ml, less than half of what we anticipate would be needed to treat human status Dupuis et al.9 have reportepilepticus ‘,‘8*19,21-23*43. ed survival of a child who had greater than 900 pg/ ml VPA in serum following an accidental overdose. A dose of about 2-2.5 g VPA would be required in humans to produce 270 pug/ml in serum. This high VPA serum concentration would, of course, pose little problem clinically unless it produced potentially dangerous systemic toxicity. The usual dosedependent side effects of sedation, gastrointestinal upset, tremor and hair loss, which have been reported with chronic VPA therapy, would not be clinically significant if the drug controlled status epilepticus. In addition, such side effects would need to be tolerated only briefly. VPA, however, has been reported to cause hyperammonemia after i.v. administration; the presence of barbiturates worsens this problem19,42. The increasing VPA free fraction as total serum levels increased over the range from less than 200 to more than 900 fig/ml confirms reports by others at the lower end of this range and shows that binding behaves as predicted as levels increase6*‘5,24Y25*28,43 Cramer et al.6 reported that human free fractions increased from 7% to 30% as total serum VPA increased from 50 to 150 pg/ml. VPA free fractions in our rats increased from 40% to 80% as total serum VPA rose from less than 200 to greater than 900 pg/ml. Brain VPA concentrations were slightly less than half of the free concentration, with no tendency for this percentage to rise as the free concentration rose. Similar brain:serum VPA ratios have been reported in other species, as well as some limited data from humans’ 1,17,20,24Y33 In addition to the very high VPA serum concentrations needed to control GTCS in this model.
further concern is raised by the inability of this compound to completely control epileptiform EEG activity, even when serum levels exceeded 1000 pg/ml. A similar failure of VPA to control focal epileptiform activity has been reported by others, using cobalt or alumina gel to produce the foci’“,35. We have some concern that this failure to control epileptiform EEG activity in the model may mean that i.v. VPA will not be capable of successfully treating the more refractory cases of status epilepticus, which do not respond to the usual drugs at the usual doses. Drugs which have been reported to have some success in treating refractory status in humans, lorazepam and phenobarbital, are able to suppress even focal epileptiform activity in this model when doses are pushed high enough38*40. It is, of course, possible or even likely that i.v. VPA may be the treatment of choice for those patients with primarily generalized epilepsies who develop status epilepticus. However, such patients respond readily to the benzodiazepines available now. The more commonly seen types of status epilepticus involve secondarily generalized convulsions, or patients for whom no information is available as to any pre-existing epilepsy. Many physicians may question the validity of making predictions about the response of human patients to drugs based on data from animal models. It is important to keep two points in mind, however. First, the validity of the prediction is not based on the similarity of rats or what is done to them to people or how they are treated, but rather to the similarity of the serum concentrations effective in the model to those which control human status. Second, initial tests of potential anti-status efficacy of any new drugs cannot be done in humans, due to ethical and legal constraints. This model, and the experimental work we have done with it thus far, is an attempt to discover a method by which probable efficacious serum concentrations of new drugs might be estimated. Such information will be essential if new drugs for treating status epilepticus are to be developed within the current medico-legal system.
204
Acknowledgements This research
Kang in the conduct knowledged. Portions
was supported
by grants
from Ab-
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2-oique,
C.R. Sot. Biol. (Paris),
43 Yu, H.-Y.,
Clinical
epileptic children
implications
during
du
172 (1978) 7077712. of serum protein
sodium valproate
apy, Ther. Drug Monit., 6 (1984) 414423.
31 (1981) 486487.
F.J.E.,
R.W.,
N.Y. and Treiman,
experimental
(1985) 323-325. 32 Treiman,
cus, Ann. Neural., 37 Walton,
39 Walton,
and phenytoin
phenytoin,
J.E., Homan,
Bell, R.D. and Tassker,
homocysteine
Drug Monit., 12 (1990) 117-123.
34 Vajda,
Rectal
Epilepsia, 16 (1975) 83-90.
6168.
tion
P.F.,
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binding
maintenance
in
ther-
199
EPIRES 00497
Valproic acid treatment of experimental status epilepticus
Nancy Y. Walton and David M. Treiman Neurology and Research Services, West Los Angeles Department of Veterans Affuirs Medical Center and ~epurtment
ofNearology, Universiry of CuI~orniu, Los Angeles, CA, USA
(Received 19 July 1991; revision received 30 March 1992; accepted 30 March 1992) Key worh: Valproic acid; Status epilepticus; Rat; Anticonvulsant
The efficacy of valproic acid (VPA) in control of generalized convulsive status epilepticus was tested in a rat model. Rats with cortical cobalt lesions were injected with homocysteine thiolactone to induce secondarily generalized tonic-clonic seizures (GTCS). The median effective dose (EDsa) for control of GTCS was 21 I .9 mg/kg (270 fig/ml in serum 30 mm post dose) when treatment was given intraperitoneally after the second GTCS. VPA entered both serum and brain very rapidly after injection, with little change in concentration from 5 to 30 min post dose. In earlier experiments with phenytoin, phenobarbital, diazepam and lorazepam in this model, we found that the serum concentrations produced by the EDses versus GTCS were very similar to those which have been reported to be effective in treating human status epilepticus. If this same relationship holds true for VPA, we would predict that a serum con~ntration of around 270 pg/ml VPA would be required for control of generalized convulsive status epilepticus in human patients. The safety of this high a concentration of VPA has not been tested.
Valproic acid (VPA) is a widely used anticonvulsant drug which has been FDA-approved for use in the USA since 1978. Although the current indication is only for treatment of absence seizures, considerable evidence has been generated that the drug is effective in control of other primarily generalized seizures as well as partial-onset seizures2. VPA is a branched chain carboxylic acid, a molecular structure which is very different from the heterocyclic structures of other currently available anticonvulsants. The mechanism by which VPA exerts its anticonCorrespondence to: Dr. Nancy Y. Walton, Department of Neurology, Reed Neurological Research Center (C-128), UCLA School of Medicine, Los Angeles, CA 90024, USA.
vulsant properties has not been established with certainty, although increased brain y-aminobutyric acid (GABA) levels, enhanced postsynaptic response to GABA, and changes in cellular membranes have all been suggested4’5. Clinical experience suggests that there is a temporal delay after VPA therapy is started before antiepileptic effkacy is seen3T12,26,suggesting that one or more VPA metabolites may contribute to its anticonvulsant action. Several VPA metabolites have been shown to possess anticonvulsant activity in animal The parent VPA molecule itmodels 14,16,17321*27*42. self, however, also has antiepileptic activity, because seizure protection has been reported in animal testing at times too early to be due to the effects of metabolites2’, and there are reports of efficacy when VPA was administered rectally for the treatment of status epilepticus30,34.
200
These experiments were designed to test the efficacy of VPA in a rat model of generalized convulsive status epilepticus. The model involves administration of homocysteine thiolactone epileptogenic cortical cobalt lesions. status epilepticus
includes
to rats with The resulting
a series of true secondar-
ily generalized tonic-clonic seizures (GTCS), accompanied by electroencephalographic patterns remarkably similar to those we have observed in hu-
saline. Sodium valproate by Abbott Laboratories. 500 mg VPA equivalent which was reconstituted used immediately. Measurement
of VPA
for injection was donated Each VPA vial contained as a lyophylized cake with sterile water and in blood
and
brain
was
Details of these methods have been published earlier - see references 3741. Adult, male SpragueDawley rats weighing 2OG-250 g each were used as subjects in these experiments (n = 134). Rats were housed individually after surgery in Plexiglas cages with contact bedding. Food and water were avail-
done using fluorescence polarization immunoassay (TDX, Abbott). Blood was drawn into tubes containing EDTA and immediately centrifuged to separate the serum. Samples for free valproate levels were prepared by centrifugal ultrafiltration of the fresh serum. Serum and ultrafiltrate were frozen until assayed. Brain samples were prepared for assay by sonication of tissue in phosphate buffer (pH = 10.5) followed by incubation with an alkaline protease for 60 min at 50°C. VPA was extracted from the tissue homogenate by adjusting the pH to 4.5 with 10% H3P04 and mixing with methyl-tertbutyl ether (MtBE). The sample was then centrifuged and the organic layer transferred to a conical tube containing 200 ~1 polyethylene glycol 400 (PEG). The MtBE was dried and 800 ~1 phosphate buffer (pH = 7.0) were added to the PEG to produce samples for TDX assay. Serum and ultrafiltrate samples of 50 ~1 were diluted as needed to fall within the range of the assay system. Brain homogenate derived from 0.1-1.0 g brain tissue was used for extraction of samples to be assayed. EEG was monitored daily beginning on the fifth post-operative day. Status was induced by intraper-
able ad lib. Temperature was maintained at 22°C and a 24-h diurnal lighting schedule was utilized, with lights on from 06.00 to 18.00 each day.
itoneal (i.p.) injection of 5.5 mmol/kg HCT when one of the following criteria was met: focal motor seizures accompanied by left frontal spiking on
Epidural recording electrodes made from stainless steel machine screws were implanted under general anesthesia (ketamine/xylazine, 87/l 3 mg/kg) at the following stereotaxic coordinates (referenced to bregma): A-P + 1.5, lateral + 2.0, and A-P -2.5, lateral + 2.0. 25 mg powdered cobalt was placed onto the dura beneath the left frontal electrode. Recording electrodes were fastened into a plastic connector which was then cemented to the skull with methacrylate adhesive. Ketamine (50 mg/ml injectable) was obtained from Quad pharmaceuticals. Xylazine (20 mg/ml in normal saline) was obtained from Sigma. Homocysteine thiolactone (HCT) was obtained from Sigma and dissolved for injection in 5.0 ml/kg normal
EEG; or poly-spike bursts on EEG at least every 20 s. EEG was monitored continuously following injection of HCT. Onset of status epilepticus was defined as onset of the first GTCS and treatment injections were given i.p. immediately after the second GTCS. Rats were observed (with continuous EEG) for 30 min following treatment and all further seizure activity was noted. Experiments were videotaped to allow review of seizure activity and its classification. Rats were then anesthetized, blood samples obtained by cardiac puncture and the brain rapidly removed and frozen. Six rats were treated with each of the following VPA doses (mg/kg) in a predetermined random order: 0, 100, 140, 170,200, 300,400, 500 and 600. Six
man generalized convulsive status epilepticus’*. We found, in earlier experiments testing the efficacy of diazepam, phenytoin, phenobarbital and lorazepam in this model, that the serum concentration produced by the median effective dose (ED,,) for control of GTCS in the model closely approximates the serum concentrations for these same drugs which have been reported to be clinically effective in treating human status epilepticus37A0. We therefore believe that the model can be used successfully to predict potential clinical efficacy in new drugs for treating status epilepticus. Methods
201
TABLE
I
EDso. 9.5% confidence interval and corresponding
serum concentration for varying degrees of seizure control when status epilepticus
was
treated with VPA Seizure control
achieved
No GTCS No generalized No motor a Estimated
seizures
seizures
EDso (mg/kg
I.P.)
95% confidence
Serum concentration
interval
212
162-278
269
278
215-361
332
398
306518
597
from dose:concentration
ratios
obtained
(pg/ml)a
in these studies.
normal control rats were injected i.p. with each of the VPA doses and blood samples were obtained after a 30-min waiting period, in order to determine if status epilepticus would influence the dose:concentration ratio, protein binding or brain entry of VPA. An additional group of normal rats was used to determine the kinetics of VPA following i.p. administration. These rats were given 200 mg/kg VPA i.p., then blood samples were obtained from four rats at each of the following times (min) after injection: 5, 10, 15, 30, 60, 90, 120 and 150. EDsos and 95% confidence intervals were calculated by a computer adaptation of the method of Litchlield and Wilcoxoni3, which is based on linear regression of the probit transformation of the percentage of animals showing an effect versus the logarithm of the drug dose they received. Goodness of fit of the observed data to the linear model was confirmed by chi square testing. Statistical comparisons were made using standard parametric procedures. Results will be presented as means + SD unless noted otherwise.
each (range = 6-18) during the 30 min following treatment. Cessation of further GTCS was not seen in rats given less than 170 mg/kg VPA, and 100% protection was not achieved until doses at or above 400 mg/kg VPA. EDsss, the serum concentrations they would produce 30 min after injection, and 95% confidence intervals for varying degrees of seizure control are shown in Table I. That seizure control is serum and brain concentrationdependent is evident from examination of Fig. 1, where these concentrations have been calculated for rats achieving varying degrees of seizure control, regardless of dose. We saw only a few rats in this experiment with EEG normalization. Attempts to raise the dose above 600 mg/kg resulted in fatalities during the 30 min observation window. No significant differences were seen in the VPA
Results Status was induced in these rats (n = 54) 6.7 f 1.2 days after surgery. Latency from injection of HCT to onset of status epilepticus was 28.3 + 9.4 min, and the interval from the onset of the first to onset of the second GTCS was 6.2 + 3.3 min. In addition to GTCS, brief generalized tonic seizures were also seen during the status episodes, as well as focal motor seizures of the same type as were seen prior to HCT injection. Rats treated with saline only averaged 11 GTCS
GTCS CONTINUE
NO GTCS. OTHER GEN’L CONTINUE
Fig. 1. VPA concentrations ing different after injection.
the mean
The number
above
the bars.
ALL MOTOR SZ STOP
in serum and brain from rats achiev-
degrees of seizure control
Each bar represents parentheses
NO GEN’L. SPS CONTINUE
during
status epilepticus.
+ SD of the concentration of animals Open
in each group
bars are serum
35 min shown
in
VPA while
shaded bars are brain VPA. (GTCS = generalized tonic-clonic seizure; GEN’L = generalized seizure; SPS = simple partial seizure; SZ = seizure.)
202
5 2 400*CONTROL $
2oo
ti g 0
i
0
I
I
100
200
I
I
I 300
I 500
‘loo
I 600
3
1%
l BRAIN
300 -
d 5
0 SERUM
loo
.9-r----,__
0
>
i----” 0
--
I
I 60
I 30
90
120
150
0
VPA DOSE (mglkg) MINUTES
Fig. 2. Serum VPA concentrations epilepticus is the mean
in rats treated
(open circles) and controls + SD of the concentrations
during
status
(filled circles). Each point 35 min after injection
Fig. 4. Pharmacokinetics
decline.
100
g
80
P ;;
60
I
of VPA following
i.p. Each point is the mean
a dose of 200 mg/kg
+ SD for four rats. Open circles are
serum VPA. while closed circles are brain VPA.
for six rats.
serum levels produced by the various doses of drug in the seizing versus control animals (see Fig. 2). The percentage of total serum valproate which was not bound to serum proteins increased with the serum concentration, as can be seen in Fig. 3. Brain VPA was 48.3 f 14.1% of the free VPA level, and showed no tendency to change with changes in dose or serum concentration. Results of the pharmacokinetic study, shown in Fig. 4, demonstrate that VPA entered the blood compartment rapidly and then quickly moved into brain. There was a fairly constant VPA level in both compartments from 5 to 30 min after injection, before elimination exceeded the rate of absorption, causing the concentrations to begin to
FROM INJECTION
Discussion While VPA did show efficacy in this model of status epilepticus, it was not very potent. In earlier studies of drugs with known clinical efficacy for treatment of status epilepticus, we found the serum concentrations produced by the EDses versus GTCS in this model were very similar to concentrations reported in the literature to be effective in treating human status epilepticus37-40. Table II summarizes these findings. We would predict, therefore, from the results of this experiment, that about 270 pg/ml VPA would be required for control of human status epilepticus. This concentration is far greater than the usual ‘therapeutic range’ for VPA in treatment of chronic epilepsy (S&l50 pg/ m1)2,5. The reason for the observed lack of potency in this experiment is probably also the reason that acute animal seizure model tests (maximal electroshock seizures, pentylenetetrazol seizures, etc.) have also found that concentrations above the usual hu-
0 p_
$
TABLE
40
> ii
II
Efficacious drug serum concentrations and reported in the clinical literature
20
predicted from the model
ff
Drug tested
0 -200
200250
250300
300400
400-
600
600750
750-
,900
TOTAL SERUM VPA bg/ml)
Fig. 3. Free VPA (as a percentage plotted
against
of total serum concentration)
total VPA in serum. Each bar is the mean for 9-12 rats.
+ SD
Predicted
from
Reported
from human
model
clinical trials
Phenytoin
19.4 pg/m137,39
24.6’
Diazepam
40 ng/m13’
50 ng/ml”
Phenobarbital
15 pg/m138 196 ng/m14’
60-330
900
Lorazepam
18.3 pg/mlz9 ng/m13’.3h
203
man ‘therapeutic’ range were required for seizure contro15, namely, that the full anticonvulsant effect of VPA in chronic therapy depends not only on the initial anticonvulsant action of the VPA, but also on either the action of a metabolite or else some secondary action of VPA. One cannot wait for such secondary effects when treating human status epilepticus. VPA has only rarely been studied in humans given large i.v. bolus doses, and none of these studies has achieved serum concentrations greater than 120 pg/ml, less than half of what we anticipate would be needed to treat human status Dupuis et al.9 have reportepilepticus ‘,‘8*19,21-23*43. ed survival of a child who had greater than 900 pg/ ml VPA in serum following an accidental overdose. A dose of about 2-2.5 g VPA would be required in humans to produce 270 pug/ml in serum. This high VPA serum concentration would, of course, pose little problem clinically unless it produced potentially dangerous systemic toxicity. The usual dosedependent side effects of sedation, gastrointestinal upset, tremor and hair loss, which have been reported with chronic VPA therapy, would not be clinically significant if the drug controlled status epilepticus. In addition, such side effects would need to be tolerated only briefly. VPA, however, has been reported to cause hyperammonemia after i.v. administration; the presence of barbiturates worsens this problem19,42. The increasing VPA free fraction as total serum levels increased over the range from less than 200 to more than 900 fig/ml confirms reports by others at the lower end of this range and shows that binding behaves as predicted as levels increase6*‘5,24Y25*28,43 Cramer et al.6 reported that human free fractions increased from 7% to 30% as total serum VPA increased from 50 to 150 pg/ml. VPA free fractions in our rats increased from 40% to 80% as total serum VPA rose from less than 200 to greater than 900 pg/ml. Brain VPA concentrations were slightly less than half of the free concentration, with no tendency for this percentage to rise as the free concentration rose. Similar brain:serum VPA ratios have been reported in other species, as well as some limited data from humans’ 1,17,20,24Y33 In addition to the very high VPA serum concentrations needed to control GTCS in this model.
further concern is raised by the inability of this compound to completely control epileptiform EEG activity, even when serum levels exceeded 1000 pg/ml. A similar failure of VPA to control focal epileptiform activity has been reported by others, using cobalt or alumina gel to produce the foci’“,35. We have some concern that this failure to control epileptiform EEG activity in the model may mean that i.v. VPA will not be capable of successfully treating the more refractory cases of status epilepticus, which do not respond to the usual drugs at the usual doses. Drugs which have been reported to have some success in treating refractory status in humans, lorazepam and phenobarbital, are able to suppress even focal epileptiform activity in this model when doses are pushed high enough38*40. It is, of course, possible or even likely that i.v. VPA may be the treatment of choice for those patients with primarily generalized epilepsies who develop status epilepticus. However, such patients respond readily to the benzodiazepines available now. The more commonly seen types of status epilepticus involve secondarily generalized convulsions, or patients for whom no information is available as to any pre-existing epilepsy. Many physicians may question the validity of making predictions about the response of human patients to drugs based on data from animal models. It is important to keep two points in mind, however. First, the validity of the prediction is not based on the similarity of rats or what is done to them to people or how they are treated, but rather to the similarity of the serum concentrations effective in the model to those which control human status. Second, initial tests of potential anti-status efficacy of any new drugs cannot be done in humans, due to ethical and legal constraints. This model, and the experimental work we have done with it thus far, is an attempt to discover a method by which probable efficacious serum concentrations of new drugs might be estimated. Such information will be essential if new drugs for treating status epilepticus are to be developed within the current medico-legal system.
204
Acknowledgements This research
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was supported
by grants
from Ab-
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