European Journal of Pharmacology, 181(1990) 1-8
1
Elsevier EJP 51320
Development of tolerance to the anticonvulsant effect of vaiproate but not to ethosuximide in a rat model of absence epilepsy Helge W a h l e a n d H a n s - H a s s o F r e y Department of Pharmacology and Toxicology, School of Veterinary Medicine, Free University of Berlin, Berlin, F.R. G. Received 16 November 1989, revised MS received 15 January 1990, accepted 6 March 1990
Ethosuximide and valproic acid were tested for 4 and 2 weeks, respectively, in rats showing the spontaneous spike-wave syndrome. Ethosuximide suppressed the syndrome at plasma concentrations of 75-100/.tg/ml. High doses of valproate (170 mg/kg i.p., t.i.d.), resulting in plasma concentrations of about 500 t~g/ml, were necessary to suppress the syndrome, but signs of tolerance to the drug developed from day 5. Tolerance was confined to the number of spike-wave complexes, whereas the duration of the discharges was shortened to 60% of the control value, without there being signs of tolerance. It is assumed that increases in cerebral GABA, induced by the high concentration of valproate, counteracted the anti-absence effect of the drug in this model. Absence model (in rats); Ethosuximide; Valproate; Tolerance
1. Introduction
Rats with spontaneously occurring spike-wave discharges in the electrocorticogram (ECoG), associated with petit mal-like behavioural seizures, have been used as a model for absence seizures in human epilepsy (Vergnes et al., 1982). In this model, the pharmacological sensitivity is identical to that of human absences: ethosuximide, valproate, trimethadione and benzodiazepines abolish the spike-wave discharges in a dose-dependent manner, whereas carbamazepine and phenytoin are ineffective (Marescaux et al., 1984). The data available about this model indicate that these 'spontaneously epileptic rats' are a suitable model of absence seizures in human epilepsy. Since it is a chronic model, the rats can be used for chronic drug efficacy studies. Very high doses of anti-ab-
Correspondence to: H.-H. Frey, Department of Pharmacology and Toxicology, School of Veterinary Medicine, Free University of Berlin, Koserstrasse 20, D-1000 Berlin 33, F.R.G.
sence drugs are necessary to block seizures in the pentylenetetrazol seizure threshold test in mice, whereas active doses of these drugs in this rat model are lower and similar to the doses effective against absence seizures in man (Marescaux et al., 1984). Thus it seems possible to maintain active drug concentrations of valproate and ethosuximide sufficient to suppress spike-wave discharges during chronic treatment with these drugs. Although this model was first described with rats of a laboratory colony in Strasbourg, similar electrophysiological and clinical observations have been made by other authors in various strains of rats (Robinson and Gilmore, 1980; Kaplan, 1985; Van Luijtelaar and Coenen, 1986). Some of our Wistar rats, bought from a commercial breeder, also exhibit spontaneously occurring spike-wave discharges accompanied by typically twitching of the vibrissae. The purpose of the present experiments was: (1) to examine the anti-epileptic effects of valproate and ethosuximide during prolonged treatment; (2) to determine whether tolerance de-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
velops to the anti-epileptic effects of these two drugs and to determine if the alteration in the anti-epileptic effects that occurs during prolonged treatment can be related to changes in the plasma concentrations of the drugs; and (3) to assess whether there are signs of physical dependence and for anti-epileptic carry-over effects after cessation of treatment.
2. Materials and methods
2.1. Animals Female Wistar rats (Winkelmann Versuchstierzuchtanstalt, Borchen, F.R.G.), weighing 260270 g, were used in this study. They were housed singly in plastic cages at constant temperature (24-26 o C) and humidity (50%) under a 12-h light cycle beginning at 7 : 0 0 a.m. and were supplied with food (Altromin 1324 standard diet; Altromin, Lage, F.R.G.) and water ad libitum. 2.2. Surgery The rats were chronically provided with ECoG electrodes under chloral hydrate anesthesia (400 m g / k g i.p.). The animals were fixed with earbars in a stereotaxic instrument. The skull was exposed and holes (diameter 0.7 mm) were drilled with a dental bore to place four electrodes bilaterally above the cortex. The following stereotaxic coordinates, with the bregma as reference, were used: 3.0 anterior, 3.0 lateral and 5.0 posterior, 4.0 lateral. Stainless steel screws served as electrodes and were connected to a teflon-coated stainless steel wire (Medwire Corp., Mt. Vernon, NY, U.S.A.) and a female connector. The whole assembly was held in place by dental acrylic cement applied to the exposed skull. The animals were allowed to recover for at least 7 days after surgery before the first ECoG recordings were carried out. 2.3. Recordings For ECoG recordings, freely moving animals were placed singly into glass boxes and connected
to an electroencephalograph (San-Ei rectilinear thermal-writing-oscillograph 8K 22; San-Ei Instrument Co., Tokyo, Japan). The glass boxes were placed in a grounded Faraday cage. The ECoG was derived between two ipsilateral electrodes from either the right or left hemisphere. 2.4. Experimental procedures Animals displaying reliable spike-wave discharges when untreated were used for the experiment. Table 1 shows the number of spike-wave discharges/30 min under control conditions. In both experiments, a 30-min reference ECoG was recorded after habituation to the experimental conditions for 15 min on 5 successive days. During the recording periods, the freely moving rats were monitored continuously and were prevented from falling asleep by gentle sensory stimulation. The number of discharges occurring during the 30-min period were counted and the duration of each discharge was measured from original ECoG tracings. The cumulative discharge duration and mean duration of each discharge during this period were calculated. The arithmetic means of these three variables (number of discharges, cumulative discharge duration, mean duration of each discharge) for 5 days were taken as control values for each rat and were set to 1.0. Discharges lasted at least 1 s and had an amplitude at least twice the background activity. When the interval between two discharges was less than 1 s, the discharge complexes were regarded as a single discharge. The efficacy of treatment was expressed in relation to the three control values (set to 1.0). 2.5. Treatment schedule 2.5.1. Ethosuximide treatment A group of nine rats with spike-and-wave discharges were orally treated b.i.d. (at 08 : 00 h and 20:00 h) with 45 m g / k g ethosuximide for 26 days. The treatment was started with an initial dose of 125 mg/kg. ECoGs were recorded 1 h after the morning dose (at 09 : 00 h) on day 1 (i.e. after the first dose), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24 and on day 26 (i.e. after the last application), as
well as 1, 2, 3, 4, 5, 6, 7 and 10 days after cessation of treatment. The animals were habituated for 15 min to the recording conditions before ECoG recordings were made. 2.5.2. Determination of plasma levels of ethosuximide To determine the plasma levels of ethosuximide, six rats were treated as the experimental group, but no ECoG was recorded. Blood was sampled by puncture of the retro-orbital plexus. Blood samples were always drawn 75 min after drug administration (corresponding to the middle of ECoG recordings in the experimental group) from three animals. 2.5.3. Valproate treatment A group of six rats was treated t.i.d. (at 07 : 30, 15:30 and 23:30 h) with 170 m g / k g valproate i.p. for 14 days. ECoG recordings were made 15 min after the morning dose (at 07:45 h) for 30 min on every day of treatment, as well as 1, 2, 3, 4, 5, 8, 10 and 12 days after cessation of the treatment. The animals were put in the recording cage immediately after drug administration to allow them to become habituated to their environment. 2.5.4. Determination of plasma levels of valproate To determine plasma levels of valproate, six rats were treated as described above. Blood samples were always drawn 30 min after drug administration (corresponding to the middle of ECoG recordings in the experimental group) from three animals.
2. 7. Analytical methods Ethosuximide and valproate were determined by gas chromatography as described by L/Sscher and G~3bel (1978). Analysis was performed with a Varian 3740 gas chromatograph. 3. Results
3.1. Spontaneous discharges in Wistar rats About 15% of the operated Wistar rats exhibited spontaneous, bilaterally synchronous, paroxysmally occurring spike-and-wave discharges (fig. 1) on the first recording (age 3-4 months) after electrode implantation. These discharges lasted from 1 to 40 s and occurred when the rats were immobile. Spike frequency in bursts varied between 8-9/s, spike amplitude varied between 400-1000 /~V and was 2-7 times greater than the background activity. The rats did not have motor convulsions during discharges, but rhythmic twitching of the vibrissae was observed. Studies with older animals (age 7-10 months) showed that the response increased with age. 3.2. Pretreatment control values Control values for ethosuximide- and valproate-treated rats, determined from ECoG recordings on 5 successive days, are shown in table 1. There were considerable interindividual differences, but the incidence of discharges for each animal was relatively stable. 3.3. Chronic treatment with ethosuximide
2.6. Drugs Valproate (sodium salt), kindly provided by Desitin Arzneimittel G m b H (Hamburg, F.R.G.), was dissolved in 0.9% NaC1 and injected in a volume of 3 ml/kg. All doses refer to the free acid. A commercial preparation of ethosuximide was used (Petnidan Saft, Desitin), which was diluted with distilled water and administered in a volume of 2.5 ml/kg. The half-lives of the drugs have been determined previously: ethosuximide 10 h (El Sayed et al., 1978); valproic acid 4.6 h (L/Sscher, 1978).
3.3.1. ECoG parameters during chronic treatment with ethosuximide The effect of chronic administration of etho! 1
I,,
~ J
Fig. 1. Bilateral spike-wave discharge in the ECoG of a rat. Upper tracing left, lower tracing right hemisphere; calibration 1 s (horizontal) and 0.2 mV (vertical).
TABLE 1 Pretreatment control values of ethosuximide- (group 1) and valproate- (group 2) treated rats. The data represent m e a n s + S.D. of five 30-min ECoG recording periods on 5 successive days. Rat. No.
Number of discharges S.D.
Mean duration in s
Total duration in s/30 min
~
S.D.
~
S.D.
Group 1 1 2 3 4 5 6 7 8 9
51 64 37 29 41 39 49 51 28
9.2 15 7.0 5.4 2.0 5.9 12.0 8.7 8.9
8.1 6.3 11.0 5.0 7.0 9.3 7.4 3.8 14.0
0.59 0.51 1.70 0.25 1.3 0.69 0.79 0.13 2.6
414 399 405 145 286 386 362 195 364
76 87 126 27 58 70 81 39 80
63 81 59 42 33 31
14.0 11.0 14.0 4.3 10.0 7.0
34.6 6.7 4.4 10 3.3 7.7
0.76 0.95 0.44 1.2 0.34 1.0
287 540 260 438 107 239
54 60 70 76 30 63
slightly, but not significantly during treatment. After the first administration of 125 mg/kg, the plasma concentrations varied between 82 and 104 (mean 94) /~g/ml and on day 3 of treatment between 104 and 136 (mean 115)/lg/ml. A steady state concentration was reached on day 5 of treatment (table 3). The plasma concentrations fell to below the level of detection within 3 days of withdrawal. 3.4. Subchronic treatment with valproate 3.4.1. ECoG parameters during subchronic treatment with valproate The effect of subchronic administration of valproate, injected t.i.d, at 170 m g / k g for 2 weeks,
Group 2 10 11 12 13 14 15
suximide on the ECoG parameters measured is shown in table 2. The drug suppressed the number of spontaneous discharges during treatment with a median efficacy of 70-80%. After the first dosing with 125 m g / k g ethosuximide, as well as on day 3 of treatment, ethosuximide abolished the spikeand-wave discharges almost completely. The drug only had a slight effect on the mean duration of the discharges, and this effect lasted for the duration of the treatment. The cumulative discharge duration (a function of the number of discharges and mean duration of discharges) was also suppressed with a median efficacy of 70-80%. 3.3.2. ECoG parameters after cessation of chronic treatment with ethosuximide All three ECoG parameters reached pretreatment control values one day after the cessation of treatment (table 2). 3.3.3. Concentration of ethosuximide in plasma during and after treatment The plasma levels of ethosuximide determined 75 min after drug administration decreased
TABLE 2 Effect of chronic oral treatment with ethosuximide on the number of discharges, mean duration of each discharge and cumulative discharge duration during a 30-min ECoG recording period on the days indicated. The rats were treated 26 days b.i.d, with 45 m g / k g of ethosuximide after an initial dose of 125 mg/kg. During treatment, the ECoG was recorded 1 h after the morning dose of the drug. After the cessation of treatment, the ECoG was recorded at the same times as during treatment. The data represent the median and range expressed as percentage of control values for a group of nine rats. Number of discharges %
Day of treatment 1 0 (0- 18) 3 5 8 10 12 15 17 19 22 24 26
7 (020 (414 (025 (329 (1023 (024 (1027 (820 (320 (1231 (10-
15) 68) 39) 37) 57) 41) 40) 46) 46) 48) 51)
Mean duration %
Total duration %
78 65 77 75 73 86 71 73 75 80 94 87
(60- 98) (47- 84) (53-122) (50-119) (60-114) (57-151) (27- 87) (55-151) (56-127) (59-130) (56-161) (69-127)
0 4 18 11 20 32 18 17 18 14 19 34
87 90 99 95 99 87 101 85
(70-124) (72-132) (76-130) (83-113) (67-116) (76-114) (82-119) (69-122)
132 115 114 116 78 78 111 110
(0(0(2(0(2(6(0(9(6(2(10(12-
17) 11) 68) 39) 31) 57) 31) 38) 46) 51) 56) 41)
Day after withdrawal 1 2 3 4 5 6 7 10
107 (75-168) 117 (58-144) 114 (72-168) 112 (43-154) 77 (51-151) 84 (54-192) 123 (77-150) 118 (81-147)
(65-162) (67-157) (75-181) (61-154) (37-166) (54-165) (67-167) (58-148)
TABLE 3 Plasma levels of ethosuximide in female rats during chronic treatment (45 m i n / k g p.o., b.i.d., after an initial dose of 125 m g / k g p.o.) for 26 days and after the cessation of treatment. Data are means and S.D. of three rats, determined 75 roan after the morning dose at the days indicated and at day 1, 2 and 3 after the cessation of treatment. /zg/ml
S.D.
Day ofadministration 1
3 5 8 10 12 15 17 19 22 24 26
94 115 93 91 98 89 83 83 79 81 76 76
11 18 17 15 9 15 22 6 12 8 6 5
Day after withdrawal 1 2 3
24 4
1
Elsevier EJP 51320
Development of tolerance to the anticonvulsant effect of vaiproate but not to ethosuximide in a rat model of absence epilepsy Helge W a h l e a n d H a n s - H a s s o F r e y Department of Pharmacology and Toxicology, School of Veterinary Medicine, Free University of Berlin, Berlin, F.R. G. Received 16 November 1989, revised MS received 15 January 1990, accepted 6 March 1990
Ethosuximide and valproic acid were tested for 4 and 2 weeks, respectively, in rats showing the spontaneous spike-wave syndrome. Ethosuximide suppressed the syndrome at plasma concentrations of 75-100/.tg/ml. High doses of valproate (170 mg/kg i.p., t.i.d.), resulting in plasma concentrations of about 500 t~g/ml, were necessary to suppress the syndrome, but signs of tolerance to the drug developed from day 5. Tolerance was confined to the number of spike-wave complexes, whereas the duration of the discharges was shortened to 60% of the control value, without there being signs of tolerance. It is assumed that increases in cerebral GABA, induced by the high concentration of valproate, counteracted the anti-absence effect of the drug in this model. Absence model (in rats); Ethosuximide; Valproate; Tolerance
1. Introduction
Rats with spontaneously occurring spike-wave discharges in the electrocorticogram (ECoG), associated with petit mal-like behavioural seizures, have been used as a model for absence seizures in human epilepsy (Vergnes et al., 1982). In this model, the pharmacological sensitivity is identical to that of human absences: ethosuximide, valproate, trimethadione and benzodiazepines abolish the spike-wave discharges in a dose-dependent manner, whereas carbamazepine and phenytoin are ineffective (Marescaux et al., 1984). The data available about this model indicate that these 'spontaneously epileptic rats' are a suitable model of absence seizures in human epilepsy. Since it is a chronic model, the rats can be used for chronic drug efficacy studies. Very high doses of anti-ab-
Correspondence to: H.-H. Frey, Department of Pharmacology and Toxicology, School of Veterinary Medicine, Free University of Berlin, Koserstrasse 20, D-1000 Berlin 33, F.R.G.
sence drugs are necessary to block seizures in the pentylenetetrazol seizure threshold test in mice, whereas active doses of these drugs in this rat model are lower and similar to the doses effective against absence seizures in man (Marescaux et al., 1984). Thus it seems possible to maintain active drug concentrations of valproate and ethosuximide sufficient to suppress spike-wave discharges during chronic treatment with these drugs. Although this model was first described with rats of a laboratory colony in Strasbourg, similar electrophysiological and clinical observations have been made by other authors in various strains of rats (Robinson and Gilmore, 1980; Kaplan, 1985; Van Luijtelaar and Coenen, 1986). Some of our Wistar rats, bought from a commercial breeder, also exhibit spontaneously occurring spike-wave discharges accompanied by typically twitching of the vibrissae. The purpose of the present experiments was: (1) to examine the anti-epileptic effects of valproate and ethosuximide during prolonged treatment; (2) to determine whether tolerance de-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
velops to the anti-epileptic effects of these two drugs and to determine if the alteration in the anti-epileptic effects that occurs during prolonged treatment can be related to changes in the plasma concentrations of the drugs; and (3) to assess whether there are signs of physical dependence and for anti-epileptic carry-over effects after cessation of treatment.
2. Materials and methods
2.1. Animals Female Wistar rats (Winkelmann Versuchstierzuchtanstalt, Borchen, F.R.G.), weighing 260270 g, were used in this study. They were housed singly in plastic cages at constant temperature (24-26 o C) and humidity (50%) under a 12-h light cycle beginning at 7 : 0 0 a.m. and were supplied with food (Altromin 1324 standard diet; Altromin, Lage, F.R.G.) and water ad libitum. 2.2. Surgery The rats were chronically provided with ECoG electrodes under chloral hydrate anesthesia (400 m g / k g i.p.). The animals were fixed with earbars in a stereotaxic instrument. The skull was exposed and holes (diameter 0.7 mm) were drilled with a dental bore to place four electrodes bilaterally above the cortex. The following stereotaxic coordinates, with the bregma as reference, were used: 3.0 anterior, 3.0 lateral and 5.0 posterior, 4.0 lateral. Stainless steel screws served as electrodes and were connected to a teflon-coated stainless steel wire (Medwire Corp., Mt. Vernon, NY, U.S.A.) and a female connector. The whole assembly was held in place by dental acrylic cement applied to the exposed skull. The animals were allowed to recover for at least 7 days after surgery before the first ECoG recordings were carried out. 2.3. Recordings For ECoG recordings, freely moving animals were placed singly into glass boxes and connected
to an electroencephalograph (San-Ei rectilinear thermal-writing-oscillograph 8K 22; San-Ei Instrument Co., Tokyo, Japan). The glass boxes were placed in a grounded Faraday cage. The ECoG was derived between two ipsilateral electrodes from either the right or left hemisphere. 2.4. Experimental procedures Animals displaying reliable spike-wave discharges when untreated were used for the experiment. Table 1 shows the number of spike-wave discharges/30 min under control conditions. In both experiments, a 30-min reference ECoG was recorded after habituation to the experimental conditions for 15 min on 5 successive days. During the recording periods, the freely moving rats were monitored continuously and were prevented from falling asleep by gentle sensory stimulation. The number of discharges occurring during the 30-min period were counted and the duration of each discharge was measured from original ECoG tracings. The cumulative discharge duration and mean duration of each discharge during this period were calculated. The arithmetic means of these three variables (number of discharges, cumulative discharge duration, mean duration of each discharge) for 5 days were taken as control values for each rat and were set to 1.0. Discharges lasted at least 1 s and had an amplitude at least twice the background activity. When the interval between two discharges was less than 1 s, the discharge complexes were regarded as a single discharge. The efficacy of treatment was expressed in relation to the three control values (set to 1.0). 2.5. Treatment schedule 2.5.1. Ethosuximide treatment A group of nine rats with spike-and-wave discharges were orally treated b.i.d. (at 08 : 00 h and 20:00 h) with 45 m g / k g ethosuximide for 26 days. The treatment was started with an initial dose of 125 mg/kg. ECoGs were recorded 1 h after the morning dose (at 09 : 00 h) on day 1 (i.e. after the first dose), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24 and on day 26 (i.e. after the last application), as
well as 1, 2, 3, 4, 5, 6, 7 and 10 days after cessation of treatment. The animals were habituated for 15 min to the recording conditions before ECoG recordings were made. 2.5.2. Determination of plasma levels of ethosuximide To determine the plasma levels of ethosuximide, six rats were treated as the experimental group, but no ECoG was recorded. Blood was sampled by puncture of the retro-orbital plexus. Blood samples were always drawn 75 min after drug administration (corresponding to the middle of ECoG recordings in the experimental group) from three animals. 2.5.3. Valproate treatment A group of six rats was treated t.i.d. (at 07 : 30, 15:30 and 23:30 h) with 170 m g / k g valproate i.p. for 14 days. ECoG recordings were made 15 min after the morning dose (at 07:45 h) for 30 min on every day of treatment, as well as 1, 2, 3, 4, 5, 8, 10 and 12 days after cessation of the treatment. The animals were put in the recording cage immediately after drug administration to allow them to become habituated to their environment. 2.5.4. Determination of plasma levels of valproate To determine plasma levels of valproate, six rats were treated as described above. Blood samples were always drawn 30 min after drug administration (corresponding to the middle of ECoG recordings in the experimental group) from three animals.
2. 7. Analytical methods Ethosuximide and valproate were determined by gas chromatography as described by L/Sscher and G~3bel (1978). Analysis was performed with a Varian 3740 gas chromatograph. 3. Results
3.1. Spontaneous discharges in Wistar rats About 15% of the operated Wistar rats exhibited spontaneous, bilaterally synchronous, paroxysmally occurring spike-and-wave discharges (fig. 1) on the first recording (age 3-4 months) after electrode implantation. These discharges lasted from 1 to 40 s and occurred when the rats were immobile. Spike frequency in bursts varied between 8-9/s, spike amplitude varied between 400-1000 /~V and was 2-7 times greater than the background activity. The rats did not have motor convulsions during discharges, but rhythmic twitching of the vibrissae was observed. Studies with older animals (age 7-10 months) showed that the response increased with age. 3.2. Pretreatment control values Control values for ethosuximide- and valproate-treated rats, determined from ECoG recordings on 5 successive days, are shown in table 1. There were considerable interindividual differences, but the incidence of discharges for each animal was relatively stable. 3.3. Chronic treatment with ethosuximide
2.6. Drugs Valproate (sodium salt), kindly provided by Desitin Arzneimittel G m b H (Hamburg, F.R.G.), was dissolved in 0.9% NaC1 and injected in a volume of 3 ml/kg. All doses refer to the free acid. A commercial preparation of ethosuximide was used (Petnidan Saft, Desitin), which was diluted with distilled water and administered in a volume of 2.5 ml/kg. The half-lives of the drugs have been determined previously: ethosuximide 10 h (El Sayed et al., 1978); valproic acid 4.6 h (L/Sscher, 1978).
3.3.1. ECoG parameters during chronic treatment with ethosuximide The effect of chronic administration of etho! 1
I,,
~ J
Fig. 1. Bilateral spike-wave discharge in the ECoG of a rat. Upper tracing left, lower tracing right hemisphere; calibration 1 s (horizontal) and 0.2 mV (vertical).
TABLE 1 Pretreatment control values of ethosuximide- (group 1) and valproate- (group 2) treated rats. The data represent m e a n s + S.D. of five 30-min ECoG recording periods on 5 successive days. Rat. No.
Number of discharges S.D.
Mean duration in s
Total duration in s/30 min
~
S.D.
~
S.D.
Group 1 1 2 3 4 5 6 7 8 9
51 64 37 29 41 39 49 51 28
9.2 15 7.0 5.4 2.0 5.9 12.0 8.7 8.9
8.1 6.3 11.0 5.0 7.0 9.3 7.4 3.8 14.0
0.59 0.51 1.70 0.25 1.3 0.69 0.79 0.13 2.6
414 399 405 145 286 386 362 195 364
76 87 126 27 58 70 81 39 80
63 81 59 42 33 31
14.0 11.0 14.0 4.3 10.0 7.0
34.6 6.7 4.4 10 3.3 7.7
0.76 0.95 0.44 1.2 0.34 1.0
287 540 260 438 107 239
54 60 70 76 30 63
slightly, but not significantly during treatment. After the first administration of 125 mg/kg, the plasma concentrations varied between 82 and 104 (mean 94) /~g/ml and on day 3 of treatment between 104 and 136 (mean 115)/lg/ml. A steady state concentration was reached on day 5 of treatment (table 3). The plasma concentrations fell to below the level of detection within 3 days of withdrawal. 3.4. Subchronic treatment with valproate 3.4.1. ECoG parameters during subchronic treatment with valproate The effect of subchronic administration of valproate, injected t.i.d, at 170 m g / k g for 2 weeks,
Group 2 10 11 12 13 14 15
suximide on the ECoG parameters measured is shown in table 2. The drug suppressed the number of spontaneous discharges during treatment with a median efficacy of 70-80%. After the first dosing with 125 m g / k g ethosuximide, as well as on day 3 of treatment, ethosuximide abolished the spikeand-wave discharges almost completely. The drug only had a slight effect on the mean duration of the discharges, and this effect lasted for the duration of the treatment. The cumulative discharge duration (a function of the number of discharges and mean duration of discharges) was also suppressed with a median efficacy of 70-80%. 3.3.2. ECoG parameters after cessation of chronic treatment with ethosuximide All three ECoG parameters reached pretreatment control values one day after the cessation of treatment (table 2). 3.3.3. Concentration of ethosuximide in plasma during and after treatment The plasma levels of ethosuximide determined 75 min after drug administration decreased
TABLE 2 Effect of chronic oral treatment with ethosuximide on the number of discharges, mean duration of each discharge and cumulative discharge duration during a 30-min ECoG recording period on the days indicated. The rats were treated 26 days b.i.d, with 45 m g / k g of ethosuximide after an initial dose of 125 mg/kg. During treatment, the ECoG was recorded 1 h after the morning dose of the drug. After the cessation of treatment, the ECoG was recorded at the same times as during treatment. The data represent the median and range expressed as percentage of control values for a group of nine rats. Number of discharges %
Day of treatment 1 0 (0- 18) 3 5 8 10 12 15 17 19 22 24 26
7 (020 (414 (025 (329 (1023 (024 (1027 (820 (320 (1231 (10-
15) 68) 39) 37) 57) 41) 40) 46) 46) 48) 51)
Mean duration %
Total duration %
78 65 77 75 73 86 71 73 75 80 94 87
(60- 98) (47- 84) (53-122) (50-119) (60-114) (57-151) (27- 87) (55-151) (56-127) (59-130) (56-161) (69-127)
0 4 18 11 20 32 18 17 18 14 19 34
87 90 99 95 99 87 101 85
(70-124) (72-132) (76-130) (83-113) (67-116) (76-114) (82-119) (69-122)
132 115 114 116 78 78 111 110
(0(0(2(0(2(6(0(9(6(2(10(12-
17) 11) 68) 39) 31) 57) 31) 38) 46) 51) 56) 41)
Day after withdrawal 1 2 3 4 5 6 7 10
107 (75-168) 117 (58-144) 114 (72-168) 112 (43-154) 77 (51-151) 84 (54-192) 123 (77-150) 118 (81-147)
(65-162) (67-157) (75-181) (61-154) (37-166) (54-165) (67-167) (58-148)
TABLE 3 Plasma levels of ethosuximide in female rats during chronic treatment (45 m i n / k g p.o., b.i.d., after an initial dose of 125 m g / k g p.o.) for 26 days and after the cessation of treatment. Data are means and S.D. of three rats, determined 75 roan after the morning dose at the days indicated and at day 1, 2 and 3 after the cessation of treatment. /zg/ml
S.D.
Day ofadministration 1
3 5 8 10 12 15 17 19 22 24 26
94 115 93 91 98 89 83 83 79 81 76 76
11 18 17 15 9 15 22 6 12 8 6 5
Day after withdrawal 1 2 3
24 4
