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Development of tolerance to the anticonvulsant effects of clobazam
European Journal of Pharmacology, 94 (1983) 155-158
155
Elsevier
Short communication D E V E L O P M E N T OF T O L E R A N C E T O T H E A N T I C O N V U L S A N T E F F E C T S OF C L O B A Z A M J. PAUL GENT and JEREMY R.M. HAIGH
Department of Pharmacology, University of Leeds, Leeds LS2 9JT, U.K. Received 26 July 1983, accepted 24 August 1983
J.P. G E N T and J.R.M. H A I G H , Development of tolerance to the anticonvulsant effects of clobazam, European J.
Pharmacol. 94 (1983) 155-158. The anticonvulsant properties of the 1,5-benzodiazepine clobazam were studied in mice during and aftcr chronic treatment at two different dose levels. Pentylenetetrazol given by slow intravenous infusion was used as the convulsant stimulus. Tolerance to the anticonvulsant effects was observed; this was rapid in onset and could be overcome by increasing the dose of clobazam. Clobazam
Tolerance
Pentylenetetrazol
Anticonvulsant
1. Introduction Clobazam, a 1,5-benzodiazepine first marketed as an anxiolytic agent, is being used increasingly in the treatment of epilepsy (Feely et al., 1982) since animal studies (Fielding and Hoffmann, 1979) and early clinical trials (Gastaut and Low, 1979) revealed potent anticonvulsant activity. Furthermore, the side effects which limited the usefulness of conventional 1,4-benzodiazepines as antiepileptic agents, namely sedation and impairment of psychomotor performance, appeared less marked with clobazam (Hindmarch, 1979). Clobazam, like the other established anticonvulsant benzodiazepines clonazepam, diazepam and nitrazepam, is effective against a broad spectrum of seizure types in most patients. It has not yet achieved widespread use as an anticonvulsant as in more than one third of cases it has been shown to lose part or all of its efficacy after a few days or weeks of treatment (Gastaut and Low, 1979). In recent years, tolerance to the pharmacological effects of the benzodiazepine group of drugs chronic administration has become an issue of increasing importance. Whilst tolerance to the sedative actions of these compounds now seems 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
fairly well established both in man (Petursson and Lader, 1981) and in animals (File, 1981) there is more speculation over the development of tolerance to the anticonvulsant properties. Such anticonvulsant tolerance has been clearly recorded in patients receiving chronic treatment with therapeutic doses of clonazepam (Browne, 1976) and clobazam (Gastaut and Low, 1979; Feely et al., 1982) but as yet the underlying mechanism of this phenomenon has not been firmly established. We show here that tolerance to clobazam is also evident in an animal model using pentylenetetrazol (PTZ) as the convulsant stimulus.
2. Materials and methods Clobazam (Hoechst U.K.) was dissolved in a vehicle of the following composition: propylene glycol 0.4 ml; ethanol 0.1 ml; benzyl alcohol 0.015 ml; sodium benzoate 50.0 mg; benzoic acid 2.25 mg; distilled water to 1.0 ml. Adult male mice (Tuck No. 1), 25-40 g in weight, were randomly assigned to four groups. Two of these groups were injected intraperitoneally with clobazam twice daily (morning and evening) for 10 days; one at 5 m g / k g per dose and the other at 2.5 m g / k g . The
156
two remaining groups were given vehicle alone on the same schedule. All mice received the same injection volume of 2.5 m l / k g body weight. On the first day of the study and subsequently every three days, five mice were randomly chosen from each of the control and experimental groups and tested with PTZ 2 h after the morning injection. PTZ (Sigma, London; 10 m g / m l in 165 mM NaCI solution) was given by slow infusion into a tail vein of the unrestrained mouse at a constant rate of 0.3 m l / m i n until a clonic convulsion was elicited. All mice were killed after this convulsin occurred. The minimum convulsant dose of PTZ ( M C D PTZ) was thus obtained for each mouse and the mean + S.E.M. was calculated for each group. In the 5 m g / k g clobazam study, extra mice (both control and experimental) were taken through the study and on day 10 were given varying doses of clobazam before being tested with PTZ.
50
-_T--
c~ (J =E
25
10 DAYS
75
2
a u Z
25
3. Results
The results from the two chronic studies are shown in fig. 1. Of the mice given 5 m g / k g clobazam (fig. 1A) the group tested after the first dose was clearly protected ( M C D PTZ = 65.6 _+ 1.8 m g / k g ) but by day 4 the protection had been appreciably reduced ( M C D PTZ = 47.8 + 1.5 m g / k g ) and it remained at this lower level throughout the rest of the study. The initial protection afforded by 2.5 m g / k g clobazam ( M C D PTZ = 52.9 + 3.0 m g / k g ) which was lower than that with 5 m g / k g , as might be expected, was also reduced on subsequent days (fig. 1B). The significance of these results was analysed using single classification analysis of variance and the Student's t-test. In the 5 m g / k g study, the mean M C D PTZ for each group of control mice lay between 33.5 and 35.2 m g / k g ; none were significantly different from each other (F = 0.42; P > 0.05), but all were significantly different from those of the corresponding experimental groups (t >/5.31; P < 0.01). There was, however, a significant change in the mean M C D PTZ of the experimental groups throughout the study (F = 42.0;
DAYS
Fig. 1. Development of tolerance to the anti-PTZ effects of clobazam. Bars show mean and S.E.M. M C D PTZ. For each t e s t day, the left bar in the pair represents the experimental group of mice, and that on the right the corresponding group of control mice injected with vehicle alone (n ~ 5). Experimental
mice were given clobazam twice daily, at 5 mg/kg per dose in A and at 2.5 mg/kg per dose in B (for statistical analysis of significance see text). P < 0.01) and when the results were partitioned, the mean M C D PTZ on day 1 was found to be significantly different from those on all subsequent days (F = 124.7; P < 0.01). In the 2.5 m g / k g study, the mean M C D PTZ of control groups lay between 32.3 and 35.3 m g / k g ; again none were significantly different from each other (F = 0.66; P > 0.05). All were significantly different from those of the corresponding experimental groups (t >/3.24; P < 0.02) except on day 10 where the difference was not significant (t = 1.71; P > 0.05). At this dose level, although there was a significant change in the M C D PTZs of the experimental groups during the study (F = 5.46;
157 A
B
T N
50
L
25
Q U
E
ii
Fig. 2. Responses to clobazam in animals which had received chronic treatment. Bars show mean and S.E.M. M C D PTZ (n = 5). In A, the right hand bar of each pair represents the control group from day 10 of the 5 m g / k g clobazam study and this is compared in (i) with the experimental group receiving 5 m g / k g clobazam, and in (ii) with an experimental group receiving a 10 m g / k g clobazam dose, on the same day. In B, the left hand bar of each pair represents a group of animals receiving their first 5 m g / k g dose of clobazam: (i) is the experimental group from day 1 of the study, (ii) is a group given a 5 m g / k g dose after 10 days of treatment with vehicle alone; in both eases these groups are shown with the corresponding control group for that day (for statistical analysis of significance see text).
P < 0.01) partitioning revealed the reduction to be significant only by day 7 (F = 16.0; P < 0.01). Fig. 2 shows the results from the extra groups of mice tested on day 10 of the 5 m g / k g study. When the dose of clobazam given to a group of tolerant experimental animals was doubled, there was a significant increase in the mean MCD PTZ ( t = 10.4; P < 0 . 0 0 1 ; fig. 2A). The mean MCD PTZ for a group of mice which had received vehicle alone throughout the study, but were given clobazam 5 m g / k g on day 10, was not significantly different from that of the experimental group on day 1 (t = 0.47; P > 0.1; fig. 2B).
4. Discussion
Tolerance to the anticonvulsant effects of benzodiazepines has been shown to occur when convulsions are evoked by bicuculline, strychnine and electroshock (for refs. see File, 1983). Recently, File (1983), using a somewhat different test procedure from that described here, has demonstrated
the occurence of tolerance to the anti-PTZ effects of diazepam and our results lend support to the previously disputed notion that tolerance can develop to the anti-PTZ effects of benzodiazepines. Two major points are evident from these resuits. Firstly, tolerance to the anticonvulsant effects of clobazam does occur in that the response to clobazam is decreased after repeated administration. Furthermore, this tolerance can be overcome by an increase in dose (fig. 2A). The reproducibility of the estimation of mean MCD PTZ in control animals throughout the course of the study indicated that neither the vehicle itself nor the techniques employed, such as handling or injection of the animals, had any marked effect on the test procedure. The protection afforded by a single dose of 5 m g / k g clobazam was no different in animals receiving their first injection from that in animals which had received chronic administration of vehicle (fig. 2B); it would therefore seem unlikely that the latter had led to changes in the pharmacokinetics and in particular the absorption of clobazam. Secondly, it would seem that the onset of this tolerance is extremely rapid, occuring within 4 days in the 5 m g / k g study. Interestingly, the initial fall in protection was followed by a level of reduced, but still significant, protection. This residual level of protection after chronic treatment supports clinical evidence from Gastaut and Low (1979) who reported that in a number of cases only a partial 'exhaustion phenomenon' occured, leaving sufficient protection to justify continuation of treatment. In the lower dose study, a similar pattern of tolerance was evident. Interpretation of these results, however, is made less clear by the smaller degree of protection afforded by 2.5 m g / k g clobazam. Clobazam has a relatively short half-life of approximately one hour in mice (Caccia et al., 1980) but its major metabolite, N-desmethylclobazam, with a half life of between 3 and 4 h, also possesses significant anticonvulsant activity (Caccia et ai., 1980). It is likely that the protection measured in our experiments results at least in part from the presence of metabolite. It is not possible from these experiments to draw conclusions on the mechanism underlying
158 the development of this tolerance. O n the one h a n d , benzodiazepines have been shown to cause enzyme i n d u c t i o n in animals, but only at very high doses. The effect of this would be difficult to predict as it is unclear whether benzodiazepines stimulate or inhibit their own m e t a b o l i s m (for refs. see Petursson a n d Lader, 1981) and also as the m a j o r metabolite of clobazam is likely to contribute to the protection seen in our experiments. O n the other hand, the tolerance might be a t t r i b u t a b l e to some event at cellular level such as down-regulation of receptors (Crawley et al., 1982). In an a t t e m p t to distinguish between these two possible m e c h a n i s m s of tolerance, further experiments must include m e a s u r e m e n t of the relative plasma levels of parent c o m p o u n d a n d metabolite throughout the course of chronic administration.
Acknowledgements We thank Hoechst U.K. for the gift of clobazam and some financial support. JRMH is an Emma and Leslie Reid scholar.
References Browne, T.R., 1976, Clonazepam: a review of a new anticonvulsant drug, Arch. Neurol. 33, 326. Caccia, S., G. Guiso, R. Samanin and S. Garattini, 1980, Species differences in clobazam metabolism and antileptazol effect, J. Pharm. Pharmacol. 32, 101. Crawley, J.N., P.J. Marangos, J. Stivers and F.K. Goodwin, ~982, Chronic clonazepam administration induces benzodiazepine receptor subsensitivity, Neuropharmacol. 21, 85. Feely, M., R. Calvert and J. Gibson, 1982, Clobazam in catamenial epilepsy, Lancet 2, 71. Fielding, S. and I. Hoffmann, 1979, Pharmacology of antianxiety drugs with special reference to clobazam, Br. J. Clin. Pharmacol. 7, 7s. File, S.E., 1981, Rapid development of tolerance to the sedative effects of lorazepam and triazolam in rats, Psychopharmacol. 73, 240. File, S.E., 1983, Tolerance to the anti-pentylenetetrazol effects of diazepam in the mouse, Psychopharmacol. 79, 284. Gastaut, H. and M.D. Low, 1979, Antiepileptic properties of clobazam, a 1-5 benzodiazepine,in man, Epilepsia 20, 437. Hindmarch, I., 1979, Some aspects of the effects of clobazam on human psychomotor performance, Br. J. Clin. Pharmacol. 7, 77s. Petursson, H. and M.H. Lader, 1981, Benzodiazepinedependence, Br. J. Addict, 76, 133.
155
Elsevier
Short communication D E V E L O P M E N T OF T O L E R A N C E T O T H E A N T I C O N V U L S A N T E F F E C T S OF C L O B A Z A M J. PAUL GENT and JEREMY R.M. HAIGH
Department of Pharmacology, University of Leeds, Leeds LS2 9JT, U.K. Received 26 July 1983, accepted 24 August 1983
J.P. G E N T and J.R.M. H A I G H , Development of tolerance to the anticonvulsant effects of clobazam, European J.
Pharmacol. 94 (1983) 155-158. The anticonvulsant properties of the 1,5-benzodiazepine clobazam were studied in mice during and aftcr chronic treatment at two different dose levels. Pentylenetetrazol given by slow intravenous infusion was used as the convulsant stimulus. Tolerance to the anticonvulsant effects was observed; this was rapid in onset and could be overcome by increasing the dose of clobazam. Clobazam
Tolerance
Pentylenetetrazol
Anticonvulsant
1. Introduction Clobazam, a 1,5-benzodiazepine first marketed as an anxiolytic agent, is being used increasingly in the treatment of epilepsy (Feely et al., 1982) since animal studies (Fielding and Hoffmann, 1979) and early clinical trials (Gastaut and Low, 1979) revealed potent anticonvulsant activity. Furthermore, the side effects which limited the usefulness of conventional 1,4-benzodiazepines as antiepileptic agents, namely sedation and impairment of psychomotor performance, appeared less marked with clobazam (Hindmarch, 1979). Clobazam, like the other established anticonvulsant benzodiazepines clonazepam, diazepam and nitrazepam, is effective against a broad spectrum of seizure types in most patients. It has not yet achieved widespread use as an anticonvulsant as in more than one third of cases it has been shown to lose part or all of its efficacy after a few days or weeks of treatment (Gastaut and Low, 1979). In recent years, tolerance to the pharmacological effects of the benzodiazepine group of drugs chronic administration has become an issue of increasing importance. Whilst tolerance to the sedative actions of these compounds now seems 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
fairly well established both in man (Petursson and Lader, 1981) and in animals (File, 1981) there is more speculation over the development of tolerance to the anticonvulsant properties. Such anticonvulsant tolerance has been clearly recorded in patients receiving chronic treatment with therapeutic doses of clonazepam (Browne, 1976) and clobazam (Gastaut and Low, 1979; Feely et al., 1982) but as yet the underlying mechanism of this phenomenon has not been firmly established. We show here that tolerance to clobazam is also evident in an animal model using pentylenetetrazol (PTZ) as the convulsant stimulus.
2. Materials and methods Clobazam (Hoechst U.K.) was dissolved in a vehicle of the following composition: propylene glycol 0.4 ml; ethanol 0.1 ml; benzyl alcohol 0.015 ml; sodium benzoate 50.0 mg; benzoic acid 2.25 mg; distilled water to 1.0 ml. Adult male mice (Tuck No. 1), 25-40 g in weight, were randomly assigned to four groups. Two of these groups were injected intraperitoneally with clobazam twice daily (morning and evening) for 10 days; one at 5 m g / k g per dose and the other at 2.5 m g / k g . The
156
two remaining groups were given vehicle alone on the same schedule. All mice received the same injection volume of 2.5 m l / k g body weight. On the first day of the study and subsequently every three days, five mice were randomly chosen from each of the control and experimental groups and tested with PTZ 2 h after the morning injection. PTZ (Sigma, London; 10 m g / m l in 165 mM NaCI solution) was given by slow infusion into a tail vein of the unrestrained mouse at a constant rate of 0.3 m l / m i n until a clonic convulsion was elicited. All mice were killed after this convulsin occurred. The minimum convulsant dose of PTZ ( M C D PTZ) was thus obtained for each mouse and the mean + S.E.M. was calculated for each group. In the 5 m g / k g clobazam study, extra mice (both control and experimental) were taken through the study and on day 10 were given varying doses of clobazam before being tested with PTZ.
50
-_T--
c~ (J =E
25
10 DAYS
75
2
a u Z
25
3. Results
The results from the two chronic studies are shown in fig. 1. Of the mice given 5 m g / k g clobazam (fig. 1A) the group tested after the first dose was clearly protected ( M C D PTZ = 65.6 _+ 1.8 m g / k g ) but by day 4 the protection had been appreciably reduced ( M C D PTZ = 47.8 + 1.5 m g / k g ) and it remained at this lower level throughout the rest of the study. The initial protection afforded by 2.5 m g / k g clobazam ( M C D PTZ = 52.9 + 3.0 m g / k g ) which was lower than that with 5 m g / k g , as might be expected, was also reduced on subsequent days (fig. 1B). The significance of these results was analysed using single classification analysis of variance and the Student's t-test. In the 5 m g / k g study, the mean M C D PTZ for each group of control mice lay between 33.5 and 35.2 m g / k g ; none were significantly different from each other (F = 0.42; P > 0.05), but all were significantly different from those of the corresponding experimental groups (t >/5.31; P < 0.01). There was, however, a significant change in the mean M C D PTZ of the experimental groups throughout the study (F = 42.0;
DAYS
Fig. 1. Development of tolerance to the anti-PTZ effects of clobazam. Bars show mean and S.E.M. M C D PTZ. For each t e s t day, the left bar in the pair represents the experimental group of mice, and that on the right the corresponding group of control mice injected with vehicle alone (n ~ 5). Experimental
mice were given clobazam twice daily, at 5 mg/kg per dose in A and at 2.5 mg/kg per dose in B (for statistical analysis of significance see text). P < 0.01) and when the results were partitioned, the mean M C D PTZ on day 1 was found to be significantly different from those on all subsequent days (F = 124.7; P < 0.01). In the 2.5 m g / k g study, the mean M C D PTZ of control groups lay between 32.3 and 35.3 m g / k g ; again none were significantly different from each other (F = 0.66; P > 0.05). All were significantly different from those of the corresponding experimental groups (t >/3.24; P < 0.02) except on day 10 where the difference was not significant (t = 1.71; P > 0.05). At this dose level, although there was a significant change in the M C D PTZs of the experimental groups during the study (F = 5.46;
157 A
B
T N
50
L
25
Q U
E
ii
Fig. 2. Responses to clobazam in animals which had received chronic treatment. Bars show mean and S.E.M. M C D PTZ (n = 5). In A, the right hand bar of each pair represents the control group from day 10 of the 5 m g / k g clobazam study and this is compared in (i) with the experimental group receiving 5 m g / k g clobazam, and in (ii) with an experimental group receiving a 10 m g / k g clobazam dose, on the same day. In B, the left hand bar of each pair represents a group of animals receiving their first 5 m g / k g dose of clobazam: (i) is the experimental group from day 1 of the study, (ii) is a group given a 5 m g / k g dose after 10 days of treatment with vehicle alone; in both eases these groups are shown with the corresponding control group for that day (for statistical analysis of significance see text).
P < 0.01) partitioning revealed the reduction to be significant only by day 7 (F = 16.0; P < 0.01). Fig. 2 shows the results from the extra groups of mice tested on day 10 of the 5 m g / k g study. When the dose of clobazam given to a group of tolerant experimental animals was doubled, there was a significant increase in the mean MCD PTZ ( t = 10.4; P < 0 . 0 0 1 ; fig. 2A). The mean MCD PTZ for a group of mice which had received vehicle alone throughout the study, but were given clobazam 5 m g / k g on day 10, was not significantly different from that of the experimental group on day 1 (t = 0.47; P > 0.1; fig. 2B).
4. Discussion
Tolerance to the anticonvulsant effects of benzodiazepines has been shown to occur when convulsions are evoked by bicuculline, strychnine and electroshock (for refs. see File, 1983). Recently, File (1983), using a somewhat different test procedure from that described here, has demonstrated
the occurence of tolerance to the anti-PTZ effects of diazepam and our results lend support to the previously disputed notion that tolerance can develop to the anti-PTZ effects of benzodiazepines. Two major points are evident from these resuits. Firstly, tolerance to the anticonvulsant effects of clobazam does occur in that the response to clobazam is decreased after repeated administration. Furthermore, this tolerance can be overcome by an increase in dose (fig. 2A). The reproducibility of the estimation of mean MCD PTZ in control animals throughout the course of the study indicated that neither the vehicle itself nor the techniques employed, such as handling or injection of the animals, had any marked effect on the test procedure. The protection afforded by a single dose of 5 m g / k g clobazam was no different in animals receiving their first injection from that in animals which had received chronic administration of vehicle (fig. 2B); it would therefore seem unlikely that the latter had led to changes in the pharmacokinetics and in particular the absorption of clobazam. Secondly, it would seem that the onset of this tolerance is extremely rapid, occuring within 4 days in the 5 m g / k g study. Interestingly, the initial fall in protection was followed by a level of reduced, but still significant, protection. This residual level of protection after chronic treatment supports clinical evidence from Gastaut and Low (1979) who reported that in a number of cases only a partial 'exhaustion phenomenon' occured, leaving sufficient protection to justify continuation of treatment. In the lower dose study, a similar pattern of tolerance was evident. Interpretation of these results, however, is made less clear by the smaller degree of protection afforded by 2.5 m g / k g clobazam. Clobazam has a relatively short half-life of approximately one hour in mice (Caccia et al., 1980) but its major metabolite, N-desmethylclobazam, with a half life of between 3 and 4 h, also possesses significant anticonvulsant activity (Caccia et ai., 1980). It is likely that the protection measured in our experiments results at least in part from the presence of metabolite. It is not possible from these experiments to draw conclusions on the mechanism underlying
158 the development of this tolerance. O n the one h a n d , benzodiazepines have been shown to cause enzyme i n d u c t i o n in animals, but only at very high doses. The effect of this would be difficult to predict as it is unclear whether benzodiazepines stimulate or inhibit their own m e t a b o l i s m (for refs. see Petursson a n d Lader, 1981) and also as the m a j o r metabolite of clobazam is likely to contribute to the protection seen in our experiments. O n the other hand, the tolerance might be a t t r i b u t a b l e to some event at cellular level such as down-regulation of receptors (Crawley et al., 1982). In an a t t e m p t to distinguish between these two possible m e c h a n i s m s of tolerance, further experiments must include m e a s u r e m e n t of the relative plasma levels of parent c o m p o u n d a n d metabolite throughout the course of chronic administration.
Acknowledgements We thank Hoechst U.K. for the gift of clobazam and some financial support. JRMH is an Emma and Leslie Reid scholar.
References Browne, T.R., 1976, Clonazepam: a review of a new anticonvulsant drug, Arch. Neurol. 33, 326. Caccia, S., G. Guiso, R. Samanin and S. Garattini, 1980, Species differences in clobazam metabolism and antileptazol effect, J. Pharm. Pharmacol. 32, 101. Crawley, J.N., P.J. Marangos, J. Stivers and F.K. Goodwin, ~982, Chronic clonazepam administration induces benzodiazepine receptor subsensitivity, Neuropharmacol. 21, 85. Feely, M., R. Calvert and J. Gibson, 1982, Clobazam in catamenial epilepsy, Lancet 2, 71. Fielding, S. and I. Hoffmann, 1979, Pharmacology of antianxiety drugs with special reference to clobazam, Br. J. Clin. Pharmacol. 7, 7s. File, S.E., 1981, Rapid development of tolerance to the sedative effects of lorazepam and triazolam in rats, Psychopharmacol. 73, 240. File, S.E., 1983, Tolerance to the anti-pentylenetetrazol effects of diazepam in the mouse, Psychopharmacol. 79, 284. Gastaut, H. and M.D. Low, 1979, Antiepileptic properties of clobazam, a 1-5 benzodiazepine,in man, Epilepsia 20, 437. Hindmarch, I., 1979, Some aspects of the effects of clobazam on human psychomotor performance, Br. J. Clin. Pharmacol. 7, 77s. Petursson, H. and M.H. Lader, 1981, Benzodiazepinedependence, Br. J. Addict, 76, 133.