Ind. J. Physiol. Pharmac., Volume SS, Number 4, 1989

SHORT COMMUNICATION

THE ANTIMYOCLONIC ACTION OF CLONAZEPAM THROUGH A GABA-INDEPENDENT MECHANISM

VANAJA PAUL· AND M. S. KRISHNAMOORTHY

Department ofPharmacology and Environmental Toxicology, Dr. A. L. M. Postgraduate Institute ofBasic Medical Sciences, University of Madras, Taramani, Madras - 600 113

( Received on August 28, 1989)

Abstract: The present Itudy investigatea whether clonazepam exerts itl antimyoclonic action through a GABA independent mechanism. We have Itudied the antimyoclonic effect of clona­ zepam and compared it with that of (AOAA), a GABA transaminase inhibitor, against myoclonul induced by , a GABA receptor antagonilt and aIlylglycine, a drug which inhibits synthesis and release of GABA. We have also iavestigated the effect of c10nazepam against picrotoxin-induced myodonus in rats pretreated with either AOAA or aubmyoclonic dOle of ahylgylycine.

Clonazepam pretreatment inhibited both picrotoxin and -induced myoeionul whereal AOAA was effective in inhibiting only picrotoxin-induced myoclonul. The protective effect of clonazepam against picrotoxin-induced myoclonus was potentiated by AOAA pretreatment. Moreover, cloDazepam afforded protection againlt picrotoxin-induced myoclonus in rats pretreated with a submyoc1onic GABA reducing dose of allylglycine. Thele findings indicate that a GABA independent mechanilm may also be involved in the antimyodonic action of c1onazepam.

Key worde : clonazepam picrotoxin allylglycine aminooxyacetic acid

INTRODUCTION induced by picrotoxin, a GABA receptor antagonist . (6, 7) and· that produced by allylglycine, a drug Clinical (1. 2) and experimental (3) findings which inhibits synthesis (8) and release (9) of GABA. reveal that myoclonic syndrome is causally related In another set of experiments we have investigated to an impairment of gamma aminobutyric acid the antimyoclonic potency of clonazepam against (GABA) activity in the brain. The GABA potentia­ picrotoxin-induced myoclonus in rats pretreated with ting action of clonazepam is considered to be mainly allylglycine (a GABA reducing dose with no pro­ responsible for its clinical (4, 5) and experimental vocation of myoclonus) or aminooxyacetic acid (3) antimyoclonic effect. In the present study we (AOAA) which elevates brain GABA levels by have investigated whether clonazepam exerts its inhibiting GABA-transaminase (10, 11). antimyoclonic action through a GABA-independent METHODS mechanism too. We have therefore studied the effect of clonazepam pretreatment against myoclonus Adult Wistar strain male albino rats weighing ·Corresponding Author 244 Paul and Krishnamoorthy Ind. ]. Physiol. Pharmac., Volume 33, Number 4-, 1989

150-200 g were used. Clonazepam (Roche, jerking= 1, mild intermittent jerking of head and Switzerland) was dissolved in propylene glycol. forelimbs = 2, pronounced jerking of head and Allylglycine, picrotoxin and AOAA (Sigma, U.S.A.) forelimbs=3 and a short period of myoclonic were dissolved in distilled water. All solutions were convulsions =4. Since allylglycine produced freshly prepared prior to injection (02 mljlOO g, ip). myoclonus of lesser frequency (2 or 3 bouts in 2 hr), All experiments were carried out on fresh groups of each bout was scored. The mean score of each rats at a room temperature of 30-33°C. animal was taken to compute the mean value of the group. The number of rats not responding to In the first set of experiments, groups of rats (6 picrotoxin or allylglycine (total protection during in each group) were treated with clonazepam and 30 the test p.eriod) \\as also noted. The latency data min later challenged with the myoclonic dose of were statistically analysed by the Student's t-test and picrotoxin (3 mg/kg) or allylglycine (ISO mg/kg). the scoring data by the Mann-Whitney V-test.

In the second set the animals received either RESULTS AOAA, 6 hr prior to, or allylglycine (l00 mg/kg, submyoclonic dose) 100 min prior to c1onazepam. The myoclonic latency of picrotoxin was signi­ Thirty min after c1ona~epam treatment they were ficantly prolonged in c10nazepam pretreated animals. challenged with the myoclonic dose of picrotoxin In these rats a dose-dependent inhibition of the (3 mg/kg). intensity of myoclonic movements was found. Clonazepam, at 0.1 and 02 mg/kg' dose levels In the third set, the animals were treated with produced total protection in I and 3 animals, AOAA and challenged 6.5 hr or 6 hr later with the respectively (Table I A). Though c10nazepam myoclonic dose of picrotoxin (3 mg/kg) or pretreatment did not alter the myoclonic latency of allylglycine (150 mg/kg) respectively. Control allylglycine (I50 mg/kg) it however, did reduce the groups received requisite volume of the vehicle myoclonic score in a dose-dependent manner (ip). (Table II A). AOAA pretreatment, in a dose­ dependent manner, inhibited picrotoxin (Table I B) Picrotoxin: and allylglycine. challenged animals but not allylglycine (Table II B)-induced myoclonus. were caged singly and observed for I hr and 2 hr, A potentiation of the effect of clonazepam against respectively. The onset .of the first jerky movement picrotoxin-induced myoclonus \\as found in AOAA (myoclonic lat~ncy) was recorded in each rat. pretreated animals (Table I B). PreHeatment)with Picrotoxin produced intermittent JIlyoclonu8 every 8ubmyoclonic dose (100 mg/kg)' of allylglycine 5-10 min. Each animal was observed over a 10 min resulted in a shortening of the myoclonic latency of period at 10 min intervals (10-20, 30-40 and 50-60 picrotoxin. In these animals' clonaxepam was min) and the intensity of the jerking movement was . effective in signifieantly prolonging the myoclonic assessed according to the scoring method of Slater latency of picrotoxin and in reducing the myoclonic LInd Dickinson (12) : no jerking=O, weak, occasional score (Table I C). Ind. J. Physio!. Pharmac., Volume 33, Number 4, 1989 Antimyodonic Action of Clonazepam 245

TABLE I: Effect of clonazepam alone (A) and in rats pretreated with AOAA (B) or submyoclonic dose of allylglycine (C) against picrotoxin-induced myoclonus. Clonazepam was injected 6 hr after AOAA or 100 min after allylglycine. Picrotoxin (3 mg/kg) was injected 30 min after clonazepam. Values given are means±SEM (n=6). Figures in parenthesis indicate the number of rats in the group not responding to picrotoxin.

Myoclonic Score Pretreatment Dose ofclonazepam Myoclonic (Mean±SEM) (milkg) (mglkg) Latency (min) (Mean±SEM) Min after picrotoxin 10·20 30-40 50·60

A. Control 9.6 3.0 3.2 2.4 ±O.S ±0.3 ±O.I ±0.2 0.05 15.3+ 2.4 2.2* 1.5* ±1.I ±O.S ±02 ±04 0.1 22.2++ (I) 1.4* 1.2* 0.0** ±1.6 ±0.3 ±0.2 0.2 27.6++ (3) 0.0** 0.3** 0.0** ±1.3 ±0.2 B Control 9.8 3.2 3.0 2.4 ±0.8 ±0.4 ±0.3 ±0.2 AOAA 2.0 11.3 2.3* 2.3* 1.1* ±1.0 ±O.I ±O.I ±O.I 4.0 13.1+ (I) 1.5* 1.6* 0.7* ±1.1 ±05 ±O.S ±0.3 8.0 21.3++ (3) 0.5** 0.9** 0.0" ±I.O ±0.2 ±0.3 2.0 0.05 28.0t (3) 0.0## 0.8# 0.0## I ±1.0 ±0.3 C. Control 9.6 3.U 2.8 2.0 ±0.8 ±0.2 ±0.3 ±0.4 Allylglycine 100 5.6+ 3.4 3.2 28 ±0.2· ±0.4 ±0.3 ±0.2 100 0.1 15.3+'0 2.2.'* 2.0.'* 0.8.'* ±I.l ±0.2 ±0.2 ±0.2 + P

T ABLE II : Effect of clonazepam (A) and AOAA (B) against DISCUSSION allylglycine-induced myoclonus. Allylglycine (150 mg/kg) was injected 30 min after clonazepam and 6 hr after AOAA. Values given are means Pi~rotoxin, which is known to produce myoclonus ±SEM In=6). Figures in parenthesis indicate the number of rats in each group not responding by antagonizing GABA receptor activity (3), is to allylglycine.. unlikely to produce this effect if GABA-ergic activity

Treatnunt Dose MyOClonic latency Myoclonic score is enhanced in the brain The GABA-elevating (mg/kg) (min, mean±SFM) (Mean±SEM) action of AOAA (10,11) and the GABA potentiating A. Control 106± 4.8 3.5±0.2 action of clonazepam (3) have been proposed for Clonazepam 0.05 109±13.1 2.8±0.2* 0.1 117±132(1, 1.5±0.3** their protective effect against picrotoxin-induced 0.2 121 ± 16.2 (2) 0.8±0.3** myoclonus. A synergism between these two agents B. Control 105± 55 3.4±0.3 AOAA 2.0 109± 5.0 3.2±0.2 is likely and the same has been demonstrated in this 40 107± 50 3.0±0.2 8.0 108± 7.0 2.S±0.3 study. The present study also shows the failure of AOAA to suppress myoclonus that resulted from an * P<0.05, ** P

release indicating that AOAA produces antimyoclonic compounds have been shown to produce myorelaxant action solely through a GABA-ergic mechanism. action without the involvement of GABA (13). Interestingly, clonazepam, unlike AOAA, has Electrophysiological evidence (14, 15) has further inhibited allylglycine-induced myoclonus. Moreover, indicated that these compounds are able to produce clonazepam afforded protection against picrotoxin­ neuronal inhibition without the participation of induced myoclonus in rata pretreated with a sub­ GABA. myoclonic GABA reducing dose of allylglycine. Taken together, these findings suggest that ACKNOWLEDGEMENTS clonazepam, in addition to its GABA potentiating action, might also be acting through a GABA­ The authors thank Mr. C. Azhaganambi for independent mechanism for its antimyoclonic effect. assistance. In support uf this proposal, the

REFERENCES

1. Enna SJ. Stern LZ, Wastek GJ, Yamamura HI. ultrastructural changes by drug allylglycine. Cerebrospinal fluid GABA variations in neurological Bioch,m PMrmacol 1969; 18 : 137-43. disorders. Arch N,urol 1977; 34 : 683-85. 9. Tapia R, Sandoval ME, Contreras P. Evidence for a 2. Airaksinen E, Leino EM. Decrease in the GABA in the activity as a regulatory cerebrospinal fluid of patients with progressive myoclonus mechanism of cerebral excitability. J N,urochlm 1975: epilepsy and its correlation with the decrease of 5-HIAA 24: 1283-5. and HVA. Acta N,urol Scandinav 1982; 66: 666-72. 10. Loscher W. Effect of inhibition of GABA-T on the 3. Tarsy D, Pycock CJ, Meldrum RS, Marsden CD. Focal of GABA in brain tissues and synaptosomal contralateral myoclonus produced by inhibition of GABA fractions. J Neuroch,m 1981: 36: 1521-7. in the caudate nuclens ofrats. Brain 1978; 101 : 143-62. 11. Gale K, Iadarola MJ. protection and increased 4. Goldberg MA, Dorman JD. Intention myoclonus : nerveterminal GABA : Delayed effects of GABA tran.­ successful treatment with clynazepam. N,urology 1976: aminase inhibition. Sci,nc, 1980; 208: 288-91. 26: 24-26. 12. Slater P, Dickinson SL. Role of acetylcholine and 5. Chadwick D, Hallett M, Jenner P, Reynolds FH, dopamine in myoclonus induced by intrastriatal Marsden CD, Harries R. Clinical and biochemical picrotoxin. N,urosci utt 1982: 28 : 253-7. factors distinguishing myoclonus responsive to 5-HTP 13. Lloyd KG, Morel E, Perrault G, Zivkovic B. plus a MAOI and clonazepam. Brain 1977; 100: 455-87. GABA-dependent and GABA-independent effects of 6. Ticku MK, Ban M, Olsen R W. Binding of (8H)-L­ and . Br J Pharmacol 1986: , a gamma aminobutyric acid 87: 38P. synaptic antagonist, to rat brain membranes. Mol 14. MacDanald JF, Barker JL. Multiple actions of pico­ Pharmacol 1978; 14: 391 402. molar concentrations of flurazepam on the excitability 7. Olsen RW. The GABA-postsynaptic membrane of cultured mouse spinal neurons. Brain R,s 1982; 246: receptor-ionophore complex: site of action of convulsant 257-64. and drugs. Mol Cell Bioc",m 1981; 39 : 15. Carlen PL, Gurevich N, Pole P. Low-dose benzodiaze­ 261-79. pine neuronal inhibition : enhanced CaH -mediated 8. Alberci M, Rodriguez de Lores Arnaiv G, DeRobertis E. K+ -conductance. Brain Res 1983; 271 : 358-64. and decarboxylase inhibition and