Indian J Physiol Pharmacol 1998; 42 (4) : 491-497

INFLUENCE OF ADENOSINE AGONISTS AND ANTIEPILEPTICDRUGS ON THEOPHYLLINE-INDUCED SEIZURES IN RATS

Y. K. GUPTA* AND JATINDER MALHOTRA

Neuropharmacology Laboratory,Department of Pharmacology,All India Institute of Medical Sciences,New Delhi - 110 029

(Received on December 30, 1997)

Abstract: Seizures is a major toxicity of theophylline. The mechanism oftheophylline-induced seizures is not known, but antagonism at adenosinereceptors may be a possibility. The effect of pretreatment with differentdoses of adenosine (lOO, 500 and 1000 mg/kg, i.p.), and the adenosine Alreceptor agonist NG-cyclopentyladenosine (CPA), 1, 5 and 10 mg/kg, i.p.,was studied against seizures induced by theophylline in rats. Both thesedrugs, at all dose levels tested, failed to protect theophylline seizures.Thus adenosinergic system is unlikely to be involved in mediating theconvulsant action of theophylline. On the other hand, the conventionalanti epileptic drugs, ie. diazepam (4 mg/kg), sodium valproate (300 mg/kg)and phenobarbitone (50 mg/kg), but not carbamazepine, afforded someprotection. The modification of course of seizures, by the antiepilepticdrugs suggests the involvement of some other alternate mechanism intheophylline-induced seizures.

Key words: adenosinediazepamcarbamazepine

NG-cyclopentyladenosine (CPA)sodium valproate phenobarbitone

theophylline seizures

INTRODUCTION

Theophylline a drug of narrowtherapeutic index can potentially cause life-threatening convulsions (1), Theseconvulsions are particularly threatening asthey are not usually preceeded by otherwarning, milder, side-effects, are oftenrefractory to conventional antiepilepticdrugs (2, 3) and may be fatal (4-6).

The mechanism of convulsant andproconvulsant action of theophylline is notyet clear. It is a non-specific adenosine

'Corresponding Author

receptor blocker and some studies haverelated the epileptogenic potency ofxanthines with their adenosine receptorantagonist activity (7, 8). Adenosine hasbeen shown to exert anticonvulsanteffect in vivo and in vitro studies (9-11).Recently, we have also demonstrated ananticonvulsant effect of adenosine inpentylenetetrazole-induced seizures in ratsand that the protection is mediated byadenosine Al receptor subtype (12).

If theophylline seizures are due toadenosine At receptor antagonism, thentreatment with adenosinergic agents should

492 Gupta and Malhotra

attenuate them. In order to ascertainwhether blockade of adenosine Al receptor,is involved in the convulsant action oftheophylline, the effect of adenosine andspecific adenosine Al receptor agonist,N6-cyclopentyladenosine (CPA) was studiedin theophylline-induced seizures in rats.The effect of the established antiepilepticdrugs ego diazepam, sodium valproate,phenobarbitone and carbamazepine, wasalso evaluated for the purpose ofcomparison.

METHODS

Male 'Wistar' rats, weighing 150-200 gwere used. The animals wer group housedin plastic cages (not more than four animalsper cage) and maintained under standardlaboratory conditions with a natural light­dark cycle and controlled temperature (20­25°C) and humidity. The rats w reacclimatized to the nvironment for at leasta week prior to ~xperimenta ion. Animalswere allow d free access to food and watertill, just before the drug treatment. Eachtreatment group comprised of 8-12 animals.The animals we·re used only once in thestudy.

Theophylline-induced seizure: Theophylline(courtesy Sun Pharma, India) was dissolved

10 warm saline and administeredintraperitoneally. To establish the dose oftheophylline that produces convulsions in100% animals, it was administered in gradeddoses i e., 200, 250, 275 and 300 mg/kg.The dose of 300 mg/kg, i p, producedseizures in 100% animals and was used forexperiments with the different drugtreatments.

Indian J Physiol Pharmacol 1998; 42(4)

Observational parameters: The animals wereobserved for one hour following convulsantdose of theophylline and, the percentincidence of clonic seizures and tonic hindlimb extension as well as their latencies,were noted. The recording was donemanually and supplemented with a seizurerecording assembly previously described byus (13). The mortality during the 1 hourobservation period and also, the mortalityat 24 hours after theophylline challenge wasrecorded.

Drugs and treatment schedules: All drugswere prepared fresh and administeredintraperitoneally, in a volume not exceeding1 mIll 00 g, using a 25 gauge hypodermicneedle. Control experiments were performedwith the vehicles used for the differentdrugs i.e., 8% tween 20, 8% ethanol, 3%tween 80, saline and distilled water. Dosesselected and pretreatment times weredecided on the basis of earlier experiments(12, 14).

Experiments with adenosine and specificadenosine AI receptor agonist, NG­cyclopentyladenosine (CPA): Adenosine(Sigma, St. Louis, MO, U.S.A) wassuspended in 8% tween 20 and administeredin doses of 100, 500 and 1000 mglkg, i.p., 5min before theophylline challenge. Nr._cyclopen yladenosine (CPA Sigma, St. Louis,MO, U.S.A.), an adenosine AI receptoragonist was dissolved in 8% ethanol andinjected in doses of 1, 5 and 10 mglkg, i.p.,60. mIn before convulsant dose oftheophylline.

Experiments with established antiepilepticdrugs - a) diazepam, b) sodium valproate,c) phenobarbitone, and d) carbamazepine:Diazepam (courtesy Intas Laboratories Ltd.,

Indian J Physiol Pharmacol 1998; 42(4)

India) was suspended in 3% tween 80 andadministered in a dose of 4 mg/kg, 60 minbefore theophylline challenge. Sodiumvalproate (courtesy Reckitt and ColmanLtd., India) was dissolved in distilled waterand administered in a dose of 300 mg/kg,15 min before theophylline. Phenobarbitone(courtesy Intas Laboratories Ltd., India) wasdissolved in distilled water by adding a dropof 0.1 N sodium hydroxide. It was injected60 min before convulsant challenge, in adose of 50 mg/kg. Carbamazepine (courtesyIntas Laboratories Ltd., India) was dissolvedin 40% propyleneglycol, 101Ji, ethanol and50% water. It was given 30 min before, ina dose of 20 mg/kg.

Data analysis

The results were analysed statisticallyu ing a microsoft computer programme,'microstat' copyright Ecosoft Inc., U.S.A.Fi, her's xact test was applied for incidenceof s >izures.

RESULTS

Theophylline 200 mg/kg, i.p. did notproduce seizures in any rat. The doses of

Theophylline-induced Seizures in Rats 493

250 and 275 mg/kg caused generalizedclonic seizures in 83.3% animals in both thegroups while the incidence of tonic hindlimb :.,xtension was 33.3% and 50%respectively. With theophylline 300 mg/kg,100'/', animals experienced both generalizedclonic seizures and hind limb tonicextension. The mortality during the onehour observation period was 100 percent.Typically, the seizure activity aftertheophylline 300 mg/kg, comprised ofrepeated generalized clonic seizures followedby hind limb tonic extension and death. Themean latency to first donie seizure was8.94±1.31 min while that of tonic hind limbextension was 11.44:t1.9 min.

None of the vehicles used i.e. saline, 8%tween 20, 30/1 tween 80 or 8% ethanol causedany significant change in any of the seizurecomponents studied.

Effect of adenosine and specific adenosineAI receptor agonist N'-cyc1opcntyladcn-osine(CPA) on theophylline-induced seizures:

When theophylline challenge was given5 min after adenosine, at the higher dosesi.e. 500 and 1000 mg/kg, there was no

TABLE I Effect of pretreatment with adenosine (ADO) and adenosine Al receptor agonist,N6 -cyclopentyladenosine (CPA) on latencies in seizures induced by 100%convulsant dose of theophylline (300 mg/kg). Values are mean:!: S.E.M.

Drug & Latencies (min)S.No. Dose Pretrea tmen t

(mg/kg) time (min) n Clonic seizures Hind limbextension

1. Control 12 8.94 :!: 1.31 11.44 ± 1.902. ADO 100 5 10 15.68 :!: 0.98 28.48 0.00*3 ADO 500 5 10 25.65 :!: 5.39 35.21:!: 8.844. ADO 1000 5 10 7.06:!: 0.92 8.94:!: 0.995. CPA 1 60 10 13.10 :!: 1.84 18.49:!: 2.146. CPA 5 60 10 28.47:!:6.69 11.46:!: 2.057. CPA 10 60 10 11.25 :!: 1.32 12.85:!: 2.29

*No. of animals showing hind limb toxic extension insufficient to calculate S.E.M.

494 Gupta and Malhotra Indian J Physiol Pharmacol 1998; 42(4)

Incidence

(a)100

80

20

a THEa '00 ADO 100 AOO ODD ADO 1000 CPA 1 C"'" 0 C"'" '0

Incidence

THEO 300 SV 300 DZP 4 PHB ~O

CBZ 20PHB 50SV 300THEO 300

80

(b)100

MortaIity,.

CPA 1AOO 500 ADO '000J

1 HEO 300 ADO 100

20

80

60

(b)100 -

MortaIity,.

Fig. 1 : Effect of adenosine (ADO) and specific adenosineA, receptor agonist, NG-cyclonpentyl adenosine(CPA) in theophylline-induced seizures. (a)Effect on percent incidence of generalized clonicseizures (solid columns), and tonic hind limbextension (hatched coloumns), (b) Effect onpercent mortality during one hour (solidcclumns) and in twenty four hours (hatchedcolumns).

Fig. 2 : Effect of antiepileptic drugs, diazepam (OZP),sodium valproate (SV), phenobarbitone (PHB)and carbamazepine (CSZ) against theophylline(TI-IEO)-induced seizures. (a) Effect on percentincidence of generalized clonic seizures (solidcolumns) and tonic hind limb extension(hatched columns), (b) Effect on percentmortality during one hour (solid columns) andin twenty four hours (hatched columns).

TABLE II Effect of preteatment with diazepam (DZP), sodium valproate (SV), carbamazepine(CBZ) and phenobarbitone (PHS) on latencies in seizures induced by 100%convulsant dose of theophylline (300 mg/kg). Values are mean ± S.E.M.

Drug &S.No. Dose

{mg/ kg}

Pretreatmenttime (min)

n

Latencies {min}

Clonic seizures Hind limbextension

1. Con trol2. DZP 43. SV 3004. PHB 505. CSZ 20

60156030

1212121212

8.94±1.3113.32 ± 3.0419.00 ± 1.860.00**

1l1l±0.75

11.44 ± 1.9026.13 *

0.00**0.00* *0.00**

*No. of animals showing hind limb extension insufficient to calculate S.E.M.uNone of the animals showed seizures.

Indian J Physiol Pharmacol 1998; 42(4)

change either in the incidence of seizuresor the associated mortality. With adenosine100 mg/kg, an increase in latencies ofdifferent components and decrease inincidence of hind limb tonic extension wasobserved. Though there was no mortalityduring the one hour observation periodhowever, all animals died during thenext 24 hours. With the specificadenosine Al receptor agonist, CPA, therewas no significant change in theincidence of different components oftheophylline-induced seizures, at the dosestested i.e. 1, 5 and 10 mglkg. The latenciesand perc en t mortality as compared totheophylline control animala also remainedunchanged (Fig. 1 and Table 1).

Effect of diazepam, sodium ualproate,phenobarbitone and carbamazepine, ontheophylline-induced seizures:

Fig. 2 shows the change In percentincidence and mortality with theantiepileptic drugs administered beforetheophylline challenge. The correspondingchanges in latencies are shown in Table II.Diazepam 4 mg/kg, afforded only partialprotection. The incidence of tonic hind limbextension was decreased to 16.6%. Sodiumvalproate 300 mg/kg decreased the incidenceof clonic seizures and in the animalsshowing clonic activity, the latency wasincreased. In addition, none of the valproatetreated animal showed tonic hind limbextension. In phenobarbitone 50 mg/kgtreated rats, none of the animal experiencedeither clonic or tonic seizures.Carbamazepine 20 mg/kg had no effect ontheophylline-induced seizures except forabolishing the hind limb tonic extension.Notably, In sodium valproate and

Theophylline-induced Seizures in Rats 495

phenobarbitone treated animals, there wasno mortality during the observation period.However, all animals were found dead at 24hours.

DISCUSSION

Theophylline has been used to treatacute bronchial asthma and the acuteexacerbation of chronic obstructivepulmonary disease (COPD), usually as theethylenediamine complex, aminophylline. Ithowever, has a narrow therapeutic windowand life threatening toxicities are welldocumented. One of the serious toxicity oftheophylline, in humans is seizures whichare frequently resistant to treatment andoften cause death (4-6).

In experimental animals, non-convulsivedoses of methylxanthines have beenshown to potentiate the convulsanteffect of many chemoconvulsants e.g.pentyleneterazole, kainic acid or pilocarpine(15, 16). Concurrent administration ofmethylxanthines (in subconvulsant doses)also impairs the antiepileptic activity of theantiepileptic drugs like diazepam, sodiumvalproate and carbamazepine in vivo (14,17, 18). While, ECT induced seizures areenhanced in patients on theophylline (19).

Multitude of mechanisms have beenimplicated in the action of xanthines. Theseare phosphodiesterase (PDE) inhibition andthereby increase in cAMP levels, calciummobilization and adenosine receptorantagonism 0, 20). More recently,methylxanthines have also been shown tointeract at GABA-benzodiazepine receptorsite (21), dose-dependently effect theregional release of catecholamines,

496 Gupta and Malhotra Indian J Physiol Pharmacol 1998; 42(4)

These observations however appearcontrary to the notion that theophyllineseizures are mostly refractory toantiepileptic drugs in humans. Perhapshigher doses are required to elicit theprotection or the observed protection maybe due to species variation.

with cyclohexyl adenosine, an adenosine AIreceptor agonist and dipyridamole anadenosine uptake blocker failed to inhibitthe ability of theophylline to cause tonicseizures. Morgan and Durcan, (30) haverather shown a proconvulsant effect of bothperipherally and centrally administered N­ethylcarboxamidoadenosine (NECA), anadenosine agonist, in caffeine-inducedseizures, in mice.

Interestingly, in the present study, whileboth adenosine and CPA were ineffective,diazepam, sodium val pro ate andphenobarbitone, but not carbamazepineshowed protection against theophyllineseizures. Such a protection has also beendocumented by other workers (31). But themortality at 24 hours remained unaffected.This mortality may perhaps be attributableto other side effects of theophylline e.g.cardiovascular.

To conclude, it is apparent from thisstudy that adenosinergic mechanisms areunlikely to be responsible for the convulsantaction of theophylline and alternatemechanisms need to be explored.

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