Indian J Physiol Pharmacol 2002; 46(4): 393 – 395

Benzodiazepines (BZDs) have been reported to possess significant sedative, anxiolytic, anticonvulsant and muscle relaxant properties. Rapid development of tolerance to these pharmacological effects has been shown (1). Although BZDs are effective anticonvulsants, the development of tolerance limits their use. Gallager et al (2) reported that periodic administration of R015-1788 (Flumazenil) prevented the development of BZD withdrawal symptoms in primates. Further, coadministration of phenytoin with diazepam has also been shown to prevent the development of tolerance to its anticonvulsant effect (3). Hence administration of an agent which can prevent the development of tolerance with a BZD could be an effective addition to the armamentarium of anticonvulsant therapy. The aim of the present study was investigate whether intermittent administration of flumazenil alongwith diazepam at an interval longer than the duration of significant competitive antagonism, prevents the development of tolerance to the anticonvulsant effect of diazepam.

The study was conducted in Swiss albino mice of either sex weighing 20-25 g. The animals were procured from Central Animal House of University College of Medical Sciences and kept in natural light/dark cycle and controlled temperature (22 ± 2^oC ) conditions. Pellet diet and water were allowed ad libitum. The care of animals was as per the ‘Guidelines for the Care and Use of Laboratory Animals’ made by the Indian National Science Academy, New Delhi (4).

To titrate the dose of PTZ which produced convulsive score of sufficient intensity with no or minimal mortality (<20%), pilot studies were conducted with different doses (400-100 mg, ip). Since PTZ in doses 70-100 mg/kg produced more than 68-80% mortality, a dose of 60 mg/kg which produced a seizure score of upto 4 with no mortality was selected for the study. The seizures were scored as follows: 0 – no convulsive response: normal locomotor and exploratory behaviour; 1-Stunning with splaying of hind limbs; 2-Facial twitching, myoclonic head jerks; 3-Myoclonic body jerks with forelimb clonic convulsions; 4-Generalized clonic convulsions (single or repetitive attacks) with rearing and loss of posture (Kangaroo posture); 5-Generalized clonic convulsions with tonic hind limb extension/mortality. Flumazenil was dispersed in 1% Tween 80 and diluted with saline. Diazepam injections containing 1.5% w/v benzyl alcohol were used. In acute studies four groups, each comprising eight mice wee made. Animals of Group I, Ii, III and IV were treated with vehicle, diazepam (5 mg/kg, ip), Flumazenil (5 mg/kg, ip) and diazepam (5 mg/kg, ip) plus flumazenil (5 mg/kg, ip), respectively. Diazepam and flumazenil were administered on either side of the mid line 30 min before PTZ (60 mg/kg, ip) injection. In chronic studies mice were divided into three groups of eight animals each and were administered either vehicle, diazepam (5 mg/kg, ip/day) for diazepam (5 mg/kg, ip/day) + flumazenil (5 mg/kg, ip) every third day for 21 days. After 48 h of drug free period animals of all the groups were treated with diazepam (5 mg/kg, ip) followed by PTZ (60 mg/kg, ip) after 30 min and seizures were scored.

The results are expressed as mean ± SEM. The data were analyzed using Student’s ‘t’ test and P values < 0.05 were considered significant. Administration of PTZ in the vehicle treated groups, produced convulsions in all the animals (100%), 3.75 ± 0.15 and 3.25 ± 0.29 in acute and chronic studies, respectively. In acute experiment animals treated with diazepam showed significant (P < 0.001) decrease in mean seizure score. Flumazenil, per se failed to modify the seizure score. However, pretreating the animals with flumazenil attenuated the anticonvulsant effect of diazepam, the mean seizure score being 2.75 ± 0.15 as compared to 0.25 ± 0.15 in the diazepam alone treated group (Table I). Animals chronically treated with diazepam for 21 days exhibited significant development of tolerance to anticonvulsant effect. When diazepam was injected after 48 h of withdrawal followed by PTZ, there was no protection as seen in acute experiment, the mean seizure score was 5 ± 0 as compared to 0.25 ± 0.15 in the acute diazepam treated group (Table I). Administration of flumazenil on every third day along with diazepam prevented the development of tolerance. The seizures score after diazepam administration was 0.25 ± 0.15 in the flumazenil + diazepam treated group as compared to 5 ± 0 in the group treated with diazepam alone (Table I).

TABLE I : Effect of acute and chronic (21 days) treatment with diazepam on pentylenetetrazol (PTZ)-induced convulsions.

Treatment (mg / kg, ip)	PTZ (60 mg / kg, ip) seizure score (Mean ± SEM)
Acute
Vehicle	3.75±0.15
Diazepam (5)	0.25±0.15
Flumazenil (5) + Diazepam (5)	2.75±0.15
Chronic
Vehicle	3.25±0.29
[Diazepam (5) x 21 days] + Diazepam (5)	5.00 ± 0.00
[“Flumazenil (5) + Diazepam (5) x 21 days] + Diazepam (5)	0.25±0.15

‘’Diazepam – administered continuously; Flumazenil-

administered every third day

*P<0.001

^a Compated to vehicle treated group

^b Compared to diazepam treated group

^c Compared to acute diazepam treated group

^d Compared to chronic diazepam treated group.

Results of current study show that diazepam provides nearly complete protection against PTZ-induced seizures. However, administration of diazepam for a period of 21 days resulted in development of tolerance, i.e. no protection was observed. This effect may be because of progressive development of sub sensitivity of GABA-A receptors (5), reduced coupling between the benzodiazepine agonist site and the chloride channel 6) or change in the expression of genes encoding for GABA-A receptor subunits (7). In chronic study the animals rather showed exaggerated response to PTZ when administered 30 minutes after diazepam. It is reported that convulsions result from an imbalance between inhibitory and excitatory neurotransmission. Administration of flumazenil, a competitive antagonist of diazepam prevented the development of tolerance to anticonvulsant effect of diazepam when administered along with it. The short elimination half life of flumazenil (0.7 – 1.3 h) as compared to long half life of diazepam (20 – 28 h) (1) and administration of flumazenil once in 3 days, i.e. at an interval much longer than the duration of competitive antagonism, is not likely to reverse the beneficial anticonvulsant effect of diazepam. Further, it has been observed that concomitant administration of flumazenil with diazepam does not decrease the antiepileptic effect of the latter (9). This contention is strengthened by the observations of the present study where diazepam when given to animals chronically treated with flumazenil + diazepam afforded as much protection as when diazepam is given in acute experiment showing thereby that chronic administration of flumazenil has not attenuated the anticonvulsant effect of diazepam. The prevention of development of tolerance to diazepam by flumazenil could be attributed to prevention of development of sub sensitivity of GABA-A receptor by the presence of an antagonist (5, 10) or restoration of functioning of the GABA-A chloride channel complex to control levels (6). The study, thus, provides an idea for development of drugs which can be used as adjunct along with an effective anticonvulsive BZD agents to prevent the development of tolerance to their therapeutic (anticonvulsive) effects.

REFERENCES

1.                  Trevor AJ, Way WL. Sedative-hypnotic drugs. In: Katzung BG, editor. Basic and Clinical Pharmacology 9^th ed. New York: McGraw-Hill; 2001: 64-381.

2.                  Gallager DW, Heninger K, Heninger G. Periodic benzodiazepine antagonist administration prevents benzodiazepine withdrawal symptoms in primates. Eur J Pharmacol 1986; 12: 31-38.

3.                  Sharma KK, Sangraula H, Jain A. Attenuation of tolerance to anti-pentylenetetrazole (PTZ) effect of diazepam by chronic treatment with phenytoin. Indian J Pharmacol 2001; 3:17.

4.                  Anonymous. Guidelines for Care and Use of Animals in Scientific Research, Revised ed. New Delhi: Indian National Science Academy, 2000.

5.                  Gonsalves SF, Gallager DW. Spontaneous and Ro 15-1788 – induced subsensitivity to GABA following chronic benzodiazepine. Eur J Pharmacol 1985; 110:163.

6.                  Allan AM, Baier LD, Zhang X. Effects of lorazepam tolerance and withdrawal on GABA-A receptor operated chloride channels. J Pharmacol Exp Ther 1992; 261:395-422.

7.                  Impagnatiello F, Persold C, Longone P, Caruncho H, Fritschy JM, Costa E, Guidott A. Modification of gamma-amino butyric acid-A receptor subunit expression in rat neocortex during tolerance to diazepam. Mol Pharmacol 1966; 49:822-831.

8.                  Johnston MV. Neurotransmitters and epilepsy. In: Wyllie E, editor. The Treatment of Epilepsy. Principles an0ad Practice, 2^nd ed. Baltimore: Williams and Wilkins, 1997: 122-138.

9.                  Reisner-Keller LA, Pham Z. Oral flumazenil in the treatment of epilepsy. Ann Pharmacother 1995; 29:530-531.

10.              File SE. Recovery from lorazepam tolerance and the effect of benzodiazepine antagonist (Ro 15-1788) on the development of tolerance. Psychopharmacology (Berl.) 1982; 77: 284-288.