Effect of diazepam alone and in combination with chlorpromazine or propranolol in experimentally induced convulsions in mice.
Although diazepam is currently considered as an anticonvulsant of choice, it may not be adequate in controlling convulsions in severe tetanus. Previously, chlorpromazine has been reported to be of some benefit in controlling spasms in human tetanus., Recently, beta-adrenergic blocking agent propranolol has been shown to possess anticonvulsant properties in animals. Since these drugs have different mechanisms of action it is possible that a combination of these may be more useful than diazepam alone. The present work was undertaken to study the anticonvulsant properties of combination of diazepam with chlorpromazine or propranolol in experimentally induced convulsions in mice.
Experiments were carried out on male albino mice weighing between 20-30 g. obtained from the Haffkine Institute, Bombay. The animals were maintained on a diet of pellets (Hindustan Lever) with water ad lib until two hours prior to the experiment. The drugs were injected subcutaneously on the back of the animal in the region of hind limbs 30 minutes before inducing convulsions. If two injections were to be made simultaneously as in the combination drug groups they were given at two different sites. The control mice received similar volume of the solvent without the drug.
Convulsions were induced by using electroshock, pentylenetetrazol or strychnine. The method used for electroshock convulsions was similar to that described by Swinyard et al. The end point for this experiment was the current in mA required to produce tonic extension of the hind limbs. Pentylenetetrazol convulsions were induced by a method described by Toman and Everett, the end point being tonic extension of hind limbs. The dose of pentylenetetrazol employed to induce convulsions was 100 mg/kg. The mouse was considered protected when the tonic extension of hind limbs was prevented. The method used for strychnine convulsions was that described by Shah et al; the end point was tonic convulsions and/or death. Strychnine was injected in a dose of 0.6 mg/kg subcutaneously. The individual drugs were injected in different doses before giving strychnine. When the tonic convulsions were prevented the mouse was considered protected.
Effect of various drug regimen on experimental convulsions was studied in different groups, each consisting of 10 animals and compared with the control. Following drugs were studied (1) diazepam, (2) chlorpromazine, (3) propranolol, (4) diazepam + chlorpromazine and (5) diazepam + propranolol.
Diazepam powder was accurately weighed and dissolved by warming in a mixture of propylene glycol and water in a proportion of 6:4 respectively to prepare a solution for injection. The volume of the vehicle injected in all groups varied between 0.1 ml and 0.3 ml depending upon the dosage used. Chlorpromazine injection with a concentration of 10 mg/ml was diluted with distilled water so as to prepare different concentrations for injection. Propranolol powder was dissolved in distilled water.
Statistical evaluation of the results was carried out by the Chi-square test according to Bradford-Hill.
(A) Maximal Electroshock
Diazepam in doses of 10 mg/kg, 20 mg/ kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 80 mg/kg and 100 mg/kg was injected in separate groups of 10 animals each. As the dose of diazepam increased a larger current was required to obtain the end point. The maximum protection was obtained by diazepam given in the dose of approximately 30 mg/kg where the mean current required to produce convulsions was 184.2 mA. Further increase in the dose did not increase the current required to produce convulsions.
Chlorpromazine was injected in doses of 20 mg/kg, 40 mg/ /kg, 60 mg/kg, 80' mg/kg, 100 mg/kg and 120 mg//kg in groups of 10 animals each. As compared to control 20 mg/kg and 40 mg/kg of chlorpromazine showed no significant protective effect (p > 0.05). The dose of chlorpromazine that afforded maximum protection was 80 mg/kg, the mean current required to produce convulsions with this dose being 175.6 mA. Further, increase in chlorpromazine dosage did not increase the amount of protection [Fig. 1]; on the contrary, the mean current required to produce convulsions showed a significant fall (p < 0.001).
Propranolol was injected in doses of 10 mg/'kg, 20 mg/kg, 40 mg/kg, 60 mg/kg and 80 mg/kg, in groups of 10 animals each; control group received distilled water alone. Propranolol given this way did afford significant protection with all doses (p < 0.001), the maximum effect occurring with the dose of 10 mg/kg. The mean current required to produce convulsions with this dose was 168 mA.
The doses of diazepam and chlorpromazine used for the combination group were those which afforded nearly maximum protection in the above experiment. Thus, the experiments were repeated as before using combination of diazepam 28 mg/kg and chlorpromazine 75 mg/kg. Although the combination afforded protection against the electric stimulus, the mean current required to produce convulsions in 10 animals was 165.8 mA which was not significantly different from that observed from diazepam alone. (p > 0.05). Addition of propranolol to diazepam in fact showed a less protective effect. The results are summarised in [Table - 1].
(B) Pentylenetetrazol induced convulsions
Diazepam was injected 30 minutes prior to the injection of pentylenetetrazol. The doses of diazepam used were 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, and 6 mg/kg. The percentage protection increased as the dose of diazepam increased upto a dose 3 mg/kg. Thus 50% of animals were protected by prior administration of 0.5 mg/kg of diazepam while increasing the dose to 3 mg/kg gave nearly 100% protection.
Chlorpromazine given in doses of 5 mg/ kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg and 60 mg/kg gave significant protection against pentylenetetrazol induced convulsions. Thus 50% of animals were protected with a dose of 10 mg/kg and 80% with 20 mg/kg of chlorpromazine. The results are given in [Table - 2]. However, unlike diazepam further increase in doses of chlorpromazine had the opposite effect [Fig. 2]. Propranolol was injected in doses of 4 mg,/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, 16 mg/kg, 18 mg/kg, and 20 mg/ kg. The percentage of mice protected against pentylenetetrazol increased as the dose of propranolol increased. Thus, 507o of animals were protected with a dose of 12 mg/kg of propranolol while 18 mg/kg gave 90'% protection. Further increase in the dose did not increase the protection.
In order to study the effect of diazepam + chlorpromazine combination, animals received ED50 of diazepam with the ED50 of chlorpromazine (diazepam 0.5 mg/kg and chlorpromazine 10 mg/kg) prior to pentylenetetrazol. The combination protected 8 out of 10 animals (80%). As compared to this, the combination of diazepam 0.5 mg/kg with propranolol 12 mg/kg protected 7 out of 10 animals (70%). The difference between these two groups is not statistically significant (p > 0.05). Further, the results obtained following diazepam + chlorpromazine and diazepam + propranolol combinations were not different from maximally effective dose of diazepam, chlorpromazine or propranolol, given alone (p > 0.05) [Table - 2].
(C) Strychnine induced convulsions
in this study the mice were divided into four groups of ten each depending on the drug regimen given to the animals The methodology used was similar to the previous experiment with pentylenetetrazol.
Diazepam given in increasing doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg and 100 mg/kg caused an increase in percentage protection upto a dose of 100 mg/ kg. Thus 50% animals were protected against strychnine induced convulsions by prior administration of 20 mg/kg of diazepam while 100 mg/kg gave nearly 100% protection.
Chlorpromazine used in the dose ranging from 20 mg/kg to 120 mg/kg did not afford any protection against strychnine and hence, its combination with diazepam was not evaluated.
Propranolol was given in doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, 60 mg/kg, 80 mg/kg and 100 mg/kg. Fifty per cent of the animals were protected with a dose of 40 mg/kg of propranolol. The maximum protection obtained was 80% with a dose of 60, mg/kg. Further increase in the dose did not increase the protection afforded. The combination of ED50 of diazepam + ED50 of propranolol gave a protection of 70% which though higher than that afforded by diazepam alone was not significantly different (p X0.05). Results are given in [Table - 3].
Experimental methods used for the evaluation of anticonvulsant drugs commonly involve electroshock, pentylenetetrazol and strychnine induced convulsions in mice. Diazepam is known to modify maximal electroshock seizure pattern and pentylenetetrazol seizures.  It also possesses antistrychnine properties. Clinically, diazepam is useful in the treatment of status epilepticus, tetanus, strychnine poisoning and other convulsive disorders., ,  The present study confirmed that diazepam affords protection against convulsions induced by all the three methods. The results are in confirmity with those reported earlier. The exact mechanism of action of diazepam as an anticonvulsant is not known. It has been suggested that the anticonvulsant properties of diazepam may result from their mimicking the effects of putative inhibitory neurotransmitter glycine at strychnine sensitive synapses. There is also considerable evidence that diazepam enhances synaptic inhibitions in many CNS regions likely to be mediated by GABA.
Chlorpromazine showed anticonvulsant actions on maximal electroshock convulsions, pentylenetetrazol convulsions, and experimental tetanus in different species of animals., , ,  In the present study chlorpromazine afforded significant protection and thus it raised the threshold for maximal electroshock convulsions in mice. But it was observed that after the maximal dose was reached subsequent increase in doses caused reduction in the threshold. Chlorpromazine did not protect against strychnine induced convulsions. This is in confirmity with the earlier observation that chlorpromazine increases the strychnine induced toxicity in mice.
The anticonvulsant properties of chlorpromazine could be due to its depressant action of the reticular system. The drug also depresses spinal activity and in particular the gamma motor neurons by an action on the brainstem. Chlorpromazine is also known to depress monosynaptic as well as polysnaptic reflexes. The combination of diazepam + chlorpromazine, however, showed no advantage over diazepam given alone in mice.
Propranolol, an adrenergic beta blocking agent, is recommended in patients with tetanus for blocking the sympathetic overactivity. Propranolol which readily penetrates the blood brain barrier and freely permeates the brain tissue, has been shown to suppress or modify amphetamine induced behaviour and to antagonise seizures. Further, propranolol and some other beta blockers have been shown to possess protective action against maximal electroshock induced convulsions, pentylenetetrazol induced convulsions and strychnine convulsions in mice.,  In the present study, propranolol given alone raised the threshold of maximal electroshock convulsions and afforded protection against pentylenetetrazol seizures and strychnine induced seizures. Further, combination of diazepam and propranolol appeared to give more protection in this respect. Because of this property, in addition to its well known beta adrenergic blocking action, propranolol may prove useful in the management of convulsions in tetanus.