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Barbiturate serum levels and protection against kindled amygdaloid seizures in the rat
Nruropharmcoloy~ Vol. 19. pp. II41 lo I144 Pergamon Press Ltd 1980. Printed in Great Bntain
BARBITURATE KINDLED
T.E.
Albertson, Departments
SERUM
LEVELS
AMYGDALOID
S.L.
Peterson,
AND PROTECTION SEIZURES
L.C.
IN THE
Stark
of Pharmacology and School of Medicine University of California Davis, California 95616 (AccepRed
13 Augwt
and
AGAINST RAT
R.C.
Baselt
Pathology
1980)
Summary The barbiturates have been reported to be very effective against ktndled the anticonvulsant effectiveness amygdaloid seizures in the rat. In this study, phenobarbital and primidone was studied in a systematic, of pentobarbital, multiple-dose evaluation. A correlation at 30 min between increased anticonvulsant protection and increased serum levels of pentobarbital and A posstble correlation at 30 min, 2 hours and 48 phenobarbital was seen. hours between summed serum levels of the metabolites phenylethylmalonamtde and phenobarbital was seen with the primidone treated animals. Serum primidone levels appeared to contribute little to the delayed protection against kindled amygdaloid seizures seen with primidone. This laboratory has been engaged in studying the effects of anttconvulsants on the kindled amygdaloid seizure (KAS) in rats (Albertson, Peterson and Stark, 1980), finding In this study, we attempted to them extremely effective in this model of epilepsy. correlate the serum levels of pentobarbital and phenobarbital with the degree of seizure a correlation was made between protection against the KAS in the rat. In additton, serum levels of primidone, its metabolites (phenylethylmalonamide and phenobarbttal) and the degree of KAS inhibition in primidone treated rats. Methods Anticonvulsant
Testing:
Male Sprague-Dawley rats weighing approximately 300 grams were implanted using standardized stereotaxic techniques with bipolar electrodes in the right amygdala (Albertson et al., 1980). The animals were allowed 7-10 days to recover from surgery. All animals used in this study were initially klndled in a variety of experiments studying kindling development utilizing a Grass S-44 stimulator which provided a one second tratn of 60 Hz square waves, each 1 mscc in duration and 400 PA of constant current to the amygdala. The same stimulation parameters were used throughout this study.
tored were
Cortical on a ranked
and Grass (O-5)
amygdaloid electroencephalograph Mpdel 78D EEG and the behavioral according to the method of Racine
(EEG) afterdtscharges were moniresponses to the induced seizures (1972).
Once kindled, the animals received a nondrug seizure test (control trtal). Animals that did not respond with a seizure rank of 5 were not used during the following drug test. The next day (drug trial), the animals were injected intraperitoneally w
Drum, Detcrminattons:
Addittonnl anticonvulsant
male testing
Sprague-Dawley closely in both
rats, age
matching and weight, 1141
the antmals were Injected
that received intraperitoneally
the
1142
Preliminary Notes
following drugs and doses with the (expressed pentobarbital (3.75, 7, and 15 mg/kg); phenobarbital primidone The animals (100 and 800 mg/kg 1. all sacrificed by decapitation. phenobarbital were collected. Animals receiving primidone were sacrificed after injection.
free drug I : as weight of the (7.5, 15, 30 and 60 mg/kgi; and and with pcntobarbltal treated at 30 mln and their blood was at 30 mln, 2 hours, and 48 hours
and phenylcr hylSerum samples were analyzed for primidone, phenobarbital malonamide by a high-pressure liquid chromatographic method involving direct injectIon of a protein-free plasma supernatant and ultraviolet detection at 195 nm (Kabra, 1978). Serum was analyzed for pentobarbital by flame-lonl/atlon McDonald, and Marton, gas chromatography following solvent extraction and concentration (Ream, 1974). Results The results of the antlconvulsant testing obtained with lndivldual doses of the various barbiturates and vehicles are presented in Table I. Pentobarbital produced a duration and seizure rank scores. However, dose-related decrease in afterdlscharge occurred for seizure rank only at a dose which resulted in significant protection Phenobarbital also produced a dose-related decrease in sClzure neurological deficits. rank scores and afterdischarge duration. Significant reductions of both measures were Little modification of either seen at doses not associated with neurological deficits. seizure rank scores or afterdischarge duration was seen with primldone at 30 min. When significant reduction in afterdischarge duration and the animals were tested at 2 hours, some reduction of seizure rank scores was found at the highest doses (400 and 800 The animals tested at 48 hours mg/kg) with minimal associated neurological deficits. (800 mg/kg) showed the most significant reduction in seizure rank scores .Ind afterdischarge duration without associated neurological deficits. Table I shows the increase in serum levels of pentobarhital and Additionally, phenobarbital were correlated with Increasing dose and aftcrdlschar.ec protectlen. Primidone levels were highest at 2 hours in both the high dose (800 mg/kgl and low dose (100 mg/kg) animals. In the primidone treated animals, phenobarbital levels were highest at 2 hours for the low dose group and at L8 hours for the high dose group. Phenylethylmalonamide (PEMA) levels reached their highest level at 2 hours in the low ,n the high dose dose group and were approximately the same at 2 and 48 hours animals. An apparent correlation between afterdischarge protection and summated strum levels of PEMA and phenobarbital in animals treated with prlmidone wan seen. Discussion A lack of selective anticonvulsant protection by pentobarbital against KAS was seen in this study. Only doses associated with neurological deficits provided slgniflcant reduction in both seizure rank scores and afterdischarge duration. Serum pentobarhltal levels reported in this study compare favorably with the serum pentobarhitai levels previously reported in the rat (Villa, Guanltanl, and Bartosek, 1978). A good correlation between administered dose, serum levels, and KAS protection was found with pentobarbital. The selective anticonvulsant action of phenobarbital against the KAS at dcses not associated with acute neurological deficits is consistent with previous reports iAlberlson al 1980; Babington and Wedeking, 19733. The serum levels reported here correspond -et _.’ to those reported previously of 57 mg/L one hour after a single intraperitoneal lnlectlon of 40 mg/kg of phenobarbital (Baumel, Gallagher, DiMicco, and Goico, 19731. Baumel et al. ( 1973 1 found significant protection in the rat against maximal electroshock: +tylenetetrazol and fluorothyl seizures when the serum phenobarbital level was abcve 34 mg/L. ln the KAS, we have found that serum phenobarbital levels as low as 20 mgi[, were associated with significant reduction of the seizure rank and afterdlscharge duration. When behaviorally toxic serum levels of 68 mg/L were reached, almost total inhibition of both behavioral seizure response and the EEG afterdischar,ge was spcn. Ashton and Wauquier (1979) h ave recently reported on the effects of prlmldone In controlling behavioral manifestations of the KAS in rats, using a significantly different protocol. Anticonvulsant effect was reported at 30 min with the highest dose of prlmidone tested (160 mg/kg). However, this dose of primidone was more effective in suppressing the behavioral manifestations of KAS at 24 hours than at 30 min. The EEG afterdlscharxe effects were not reported (Ashton and Wauquier, 1979). These authors proposed that this delayed effect was the result of the accumulation of the metabolites of primldone, phenylethylmalonamide and phenobarbital (Ashton and Wauquier, 1979). we did not see any effect at 30 min with primidone at much higher In our study, This may reflect a difference In doses than those tested by Ashton and Wauquier (1979). the stlmulatlon paradigms used or ihe absorption characteristics of the two vehicles, A significant reduction in Cittfrdlscharge criteria used to evaluate the seizure response. duration occurred in the high dose (400 and 800 mg/kg) prlmidone groups at 2 hours.
-_
Z(12.8) S(5) 69.5&(14) 89.2A(Z) 98.3q1.5)
100 200 400 800
0 (ml/kg)
0 (ml/kg)
6
48 .-
; 6"
48 ::
48 25.** 4.3 1.3*
5
4.4
"$,f' 28.4(15.4) 21.4(13) 62.3f$10)
17.7(11.5)
* = p 5 .05 compared to vehicle control by Mann-Whitney U-test. h = p c .05 compared to vehicle control by Students' t-test. N = Number of animals. t(hr) = The number of hours after dosing at which the animals were tested or sacrificed. x Rank = The stimulation induced behavioral score using a scale by Racine NT = Not tested.
48 hours
2 hours
(1972).
55
4 3
3
N
0
: :
1
1
.9 (.I)
7.7(3.0)
0
a.l(l.3)
5.7(1.2)
1.4(.6)
2.X.9)
P.E.M.A.
ii.1 (.6) 20.1 (.ef 54.4 (2.1) 68.4 (2.1) _-___--.--
Ph.
14.3(5.5)
.8(.6)
2.3(.3)
1.2(.5)
0
___~-
22.0
1.4
10.4
6.9
1.4
2.8
Ph.+P.E.M.A. -___--
.5(.4/
Ph.
__-.-.--____._
::: I::]
._---
Pr. = primidone P.E.H.A. = phcnylethylisdlonar;iide Ph. = phenobarbital Pent. = pentobarbital
= Each drug trial afterdischarge (AD) duration was divided by the previoils day's control AD duration and subtracted fromlOO.: to get the afterdischarge protection.
5.9(3.1) -_-~
.8(.3)
66.3(32.9)
31.0(?.6)
9.3(2.9)
-~.-Levels_JmJ~L_(>@JJ
Pent. ._-__--.-----.
serum
12.8(4.6)
Pr.
x AD Protection
3 NT NT 5
NT
3 NT NT 3
NT
NT 3 NT NT 3
5 7 5 .__.__~__~---
:: I:
0 4
0 0
ii Sedated or Ataxic --
: 0
3.4 4.1 1.8* .3* Of
66.6A 3g.3n
874; 10.6(4.3)
X AD (sEM) Protection
100 : ; 3.7 32.7(17) 200 2 5 5 32(15.7) 400 2 : 3.7 57.9A(12.83 800 2 3.7 i2.2A(l1.4) ________________________________________~~~~~~~-~~-~~~~----~~-~-------
_--
: 6 6 7
1.6' 3.3
5 5
ji Rank
TesJe$
I
0 0 0 1 1
.5 hours
Prjmidone/PGf
--
:: .5 .5 .5
7.5 D (ml/kg) 15 30 60
6 5
11 5
N
Seizure
1ARlE
0 (ml/kg) .5 7 5 11.2(3.73 100 .5 6 4.1 4.9(5.4) 200 .5 ;i 4.8 13.6(12.2) 400 .5 5 6.4(3.1) 800 .5 6 5 21.5h(8.2) ________________________________________~~-~~~_-_~-~~~~---~~-------~-~
Phenobarbital/Saline
.5
::
t(hr)
7.5 15
0 (ml/kg) 3.75
Pentobarbital/Saline
(v/kg) _--^
Dose
Drug/Solvent
_._-._.
--
E
Preliminary
Skinner,
1.
Company,
Ilii71).
from
Cailfornln,
in the
parrlai
DOVIS.
Supported
Laboratory
of
Group
CA.
Manual.
pp.
1X1-143,
W.R.
S:rund~rs
!n
Supported
the
rcqulremcnt
Ph~xrmacology L”
part
for
the
and
Toxtcnlogy
II
fcllowshlp
hy
I,octor of
the
from
of
Philosophy
Cnivcrslty ihe
of
Rank
Foundation. by
a”
Association
Supported
fulfillment
Graduate
Amertca-Gianninl
facturers
A
Ph!iadrlphla.
Submitted degrw
Neuroscience:
Notes
I”
Advanced
Pwdoctoral
FellowshIp
from
Foundation. part
bv
a
RIomedical
Research
Support
Grant.
the
Pharmaceutical
Manu-
of
BARBITURATE KINDLED
T.E.
Albertson, Departments
SERUM
LEVELS
AMYGDALOID
S.L.
Peterson,
AND PROTECTION SEIZURES
L.C.
IN THE
Stark
of Pharmacology and School of Medicine University of California Davis, California 95616 (AccepRed
13 Augwt
and
AGAINST RAT
R.C.
Baselt
Pathology
1980)
Summary The barbiturates have been reported to be very effective against ktndled the anticonvulsant effectiveness amygdaloid seizures in the rat. In this study, phenobarbital and primidone was studied in a systematic, of pentobarbital, multiple-dose evaluation. A correlation at 30 min between increased anticonvulsant protection and increased serum levels of pentobarbital and A posstble correlation at 30 min, 2 hours and 48 phenobarbital was seen. hours between summed serum levels of the metabolites phenylethylmalonamtde and phenobarbital was seen with the primidone treated animals. Serum primidone levels appeared to contribute little to the delayed protection against kindled amygdaloid seizures seen with primidone. This laboratory has been engaged in studying the effects of anttconvulsants on the kindled amygdaloid seizure (KAS) in rats (Albertson, Peterson and Stark, 1980), finding In this study, we attempted to them extremely effective in this model of epilepsy. correlate the serum levels of pentobarbital and phenobarbital with the degree of seizure a correlation was made between protection against the KAS in the rat. In additton, serum levels of primidone, its metabolites (phenylethylmalonamide and phenobarbttal) and the degree of KAS inhibition in primidone treated rats. Methods Anticonvulsant
Testing:
Male Sprague-Dawley rats weighing approximately 300 grams were implanted using standardized stereotaxic techniques with bipolar electrodes in the right amygdala (Albertson et al., 1980). The animals were allowed 7-10 days to recover from surgery. All animals used in this study were initially klndled in a variety of experiments studying kindling development utilizing a Grass S-44 stimulator which provided a one second tratn of 60 Hz square waves, each 1 mscc in duration and 400 PA of constant current to the amygdala. The same stimulation parameters were used throughout this study.
tored were
Cortical on a ranked
and Grass (O-5)
amygdaloid electroencephalograph Mpdel 78D EEG and the behavioral according to the method of Racine
(EEG) afterdtscharges were moniresponses to the induced seizures (1972).
Once kindled, the animals received a nondrug seizure test (control trtal). Animals that did not respond with a seizure rank of 5 were not used during the following drug test. The next day (drug trial), the animals were injected intraperitoneally w
Drum, Detcrminattons:
Addittonnl anticonvulsant
male testing
Sprague-Dawley closely in both
rats, age
matching and weight, 1141
the antmals were Injected
that received intraperitoneally
the
1142
Preliminary Notes
following drugs and doses with the (expressed pentobarbital (3.75, 7, and 15 mg/kg); phenobarbital primidone The animals (100 and 800 mg/kg 1. all sacrificed by decapitation. phenobarbital were collected. Animals receiving primidone were sacrificed after injection.
free drug I : as weight of the (7.5, 15, 30 and 60 mg/kgi; and and with pcntobarbltal treated at 30 mln and their blood was at 30 mln, 2 hours, and 48 hours
and phenylcr hylSerum samples were analyzed for primidone, phenobarbital malonamide by a high-pressure liquid chromatographic method involving direct injectIon of a protein-free plasma supernatant and ultraviolet detection at 195 nm (Kabra, 1978). Serum was analyzed for pentobarbital by flame-lonl/atlon McDonald, and Marton, gas chromatography following solvent extraction and concentration (Ream, 1974). Results The results of the antlconvulsant testing obtained with lndivldual doses of the various barbiturates and vehicles are presented in Table I. Pentobarbital produced a duration and seizure rank scores. However, dose-related decrease in afterdlscharge occurred for seizure rank only at a dose which resulted in significant protection Phenobarbital also produced a dose-related decrease in sClzure neurological deficits. rank scores and afterdischarge duration. Significant reductions of both measures were Little modification of either seen at doses not associated with neurological deficits. seizure rank scores or afterdischarge duration was seen with primldone at 30 min. When significant reduction in afterdischarge duration and the animals were tested at 2 hours, some reduction of seizure rank scores was found at the highest doses (400 and 800 The animals tested at 48 hours mg/kg) with minimal associated neurological deficits. (800 mg/kg) showed the most significant reduction in seizure rank scores .Ind afterdischarge duration without associated neurological deficits. Table I shows the increase in serum levels of pentobarhital and Additionally, phenobarbital were correlated with Increasing dose and aftcrdlschar.ec protectlen. Primidone levels were highest at 2 hours in both the high dose (800 mg/kgl and low dose (100 mg/kg) animals. In the primidone treated animals, phenobarbital levels were highest at 2 hours for the low dose group and at L8 hours for the high dose group. Phenylethylmalonamide (PEMA) levels reached their highest level at 2 hours in the low ,n the high dose dose group and were approximately the same at 2 and 48 hours animals. An apparent correlation between afterdischarge protection and summated strum levels of PEMA and phenobarbital in animals treated with prlmidone wan seen. Discussion A lack of selective anticonvulsant protection by pentobarbital against KAS was seen in this study. Only doses associated with neurological deficits provided slgniflcant reduction in both seizure rank scores and afterdischarge duration. Serum pentobarhltal levels reported in this study compare favorably with the serum pentobarhitai levels previously reported in the rat (Villa, Guanltanl, and Bartosek, 1978). A good correlation between administered dose, serum levels, and KAS protection was found with pentobarbital. The selective anticonvulsant action of phenobarbital against the KAS at dcses not associated with acute neurological deficits is consistent with previous reports iAlberlson al 1980; Babington and Wedeking, 19733. The serum levels reported here correspond -et _.’ to those reported previously of 57 mg/L one hour after a single intraperitoneal lnlectlon of 40 mg/kg of phenobarbital (Baumel, Gallagher, DiMicco, and Goico, 19731. Baumel et al. ( 1973 1 found significant protection in the rat against maximal electroshock: +tylenetetrazol and fluorothyl seizures when the serum phenobarbital level was abcve 34 mg/L. ln the KAS, we have found that serum phenobarbital levels as low as 20 mgi[, were associated with significant reduction of the seizure rank and afterdlscharge duration. When behaviorally toxic serum levels of 68 mg/L were reached, almost total inhibition of both behavioral seizure response and the EEG afterdischar,ge was spcn. Ashton and Wauquier (1979) h ave recently reported on the effects of prlmldone In controlling behavioral manifestations of the KAS in rats, using a significantly different protocol. Anticonvulsant effect was reported at 30 min with the highest dose of prlmidone tested (160 mg/kg). However, this dose of primidone was more effective in suppressing the behavioral manifestations of KAS at 24 hours than at 30 min. The EEG afterdlscharxe effects were not reported (Ashton and Wauquier, 1979). These authors proposed that this delayed effect was the result of the accumulation of the metabolites of primldone, phenylethylmalonamide and phenobarbital (Ashton and Wauquier, 1979). we did not see any effect at 30 min with primidone at much higher In our study, This may reflect a difference In doses than those tested by Ashton and Wauquier (1979). the stlmulatlon paradigms used or ihe absorption characteristics of the two vehicles, A significant reduction in Cittfrdlscharge criteria used to evaluate the seizure response. duration occurred in the high dose (400 and 800 mg/kg) prlmidone groups at 2 hours.
-_
Z(12.8) S(5) 69.5&(14) 89.2A(Z) 98.3q1.5)
100 200 400 800
0 (ml/kg)
0 (ml/kg)
6
48 .-
; 6"
48 ::
48 25.** 4.3 1.3*
5
4.4
"$,f' 28.4(15.4) 21.4(13) 62.3f$10)
17.7(11.5)
* = p 5 .05 compared to vehicle control by Mann-Whitney U-test. h = p c .05 compared to vehicle control by Students' t-test. N = Number of animals. t(hr) = The number of hours after dosing at which the animals were tested or sacrificed. x Rank = The stimulation induced behavioral score using a scale by Racine NT = Not tested.
48 hours
2 hours
(1972).
55
4 3
3
N
0
: :
1
1
.9 (.I)
7.7(3.0)
0
a.l(l.3)
5.7(1.2)
1.4(.6)
2.X.9)
P.E.M.A.
ii.1 (.6) 20.1 (.ef 54.4 (2.1) 68.4 (2.1) _-___--.--
Ph.
14.3(5.5)
.8(.6)
2.3(.3)
1.2(.5)
0
___~-
22.0
1.4
10.4
6.9
1.4
2.8
Ph.+P.E.M.A. -___--
.5(.4/
Ph.
__-.-.--____._
::: I::]
._---
Pr. = primidone P.E.H.A. = phcnylethylisdlonar;iide Ph. = phenobarbital Pent. = pentobarbital
= Each drug trial afterdischarge (AD) duration was divided by the previoils day's control AD duration and subtracted fromlOO.: to get the afterdischarge protection.
5.9(3.1) -_-~
.8(.3)
66.3(32.9)
31.0(?.6)
9.3(2.9)
-~.-Levels_JmJ~L_(>@JJ
Pent. ._-__--.-----.
serum
12.8(4.6)
Pr.
x AD Protection
3 NT NT 5
NT
3 NT NT 3
NT
NT 3 NT NT 3
5 7 5 .__.__~__~---
:: I:
0 4
0 0
ii Sedated or Ataxic --
: 0
3.4 4.1 1.8* .3* Of
66.6A 3g.3n
874; 10.6(4.3)
X AD (sEM) Protection
100 : ; 3.7 32.7(17) 200 2 5 5 32(15.7) 400 2 : 3.7 57.9A(12.83 800 2 3.7 i2.2A(l1.4) ________________________________________~~~~~~~-~~-~~~~----~~-~-------
_--
: 6 6 7
1.6' 3.3
5 5
ji Rank
TesJe$
I
0 0 0 1 1
.5 hours
Prjmidone/PGf
--
:: .5 .5 .5
7.5 D (ml/kg) 15 30 60
6 5
11 5
N
Seizure
1ARlE
0 (ml/kg) .5 7 5 11.2(3.73 100 .5 6 4.1 4.9(5.4) 200 .5 ;i 4.8 13.6(12.2) 400 .5 5 6.4(3.1) 800 .5 6 5 21.5h(8.2) ________________________________________~~-~~~_-_~-~~~~---~~-------~-~
Phenobarbital/Saline
.5
::
t(hr)
7.5 15
0 (ml/kg) 3.75
Pentobarbital/Saline
(v/kg) _--^
Dose
Drug/Solvent
_._-._.
--
E
Preliminary
Skinner,
1.
Company,
Ilii71).
from
Cailfornln,
in the
parrlai
DOVIS.
Supported
Laboratory
of
Group
CA.
Manual.
pp.
1X1-143,
W.R.
S:rund~rs
!n
Supported
the
rcqulremcnt
Ph~xrmacology L”
part
for
the
and
Toxtcnlogy
II
fcllowshlp
hy
I,octor of
the
from
of
Philosophy
Cnivcrslty ihe
of
Rank
Foundation. by
a”
Association
Supported
fulfillment
Graduate
Amertca-Gianninl
facturers
A
Ph!iadrlphla.
Submitted degrw
Neuroscience:
Notes
I”
Advanced
Pwdoctoral
FellowshIp
from
Foundation. part
bv
a
RIomedical
Research
Support
Grant.
the
Pharmaceutical
Manu-
of