Epilepsici, 37(4):332-335, 1996 Lippincott-Raven Publishers, Philadelphia 0 International League Against Epilepsy

Failure of Chronic Treatment with Abecarnil to Induce Contingent and Noncontingent Tolerance in

Pentylenetetrazol-Kindled Rats

M. Serra, C. A. Ghiani, E. Maciocco, M. G. Pisu, G. Tuligi, M. L. Porceddu, and G. Biggio

Department of Experimental Biology “B. Loddo, ” University of Cagliari, Italy

Summary: We examined the effect of chronic treatment with abecarnil, a selective agonist at y-aminobutyric acid, (GABA,) receptors, on the development of toler- ance to its anticonvulsant effect in pentylentetrazole (PTZ)-kindled rats. We used two different experimental protocols to differentiate between pharmacological (non- contingent) and contingent tolerance. In one group of an- imals, kindling was suspended and abecarnil (1 mg/kg in-

traperitoneally, i.p.) was administered three times daily for 15 days. In a second group of rats, PTZ-kindling was continued during chronic treatment with abecarnil. Tol- erance to the anticonvulsant effect of a subsequent chal- lenge dose of abecarnil(O.5 mg/kg i.p.) did not develop in either experimental group. Key Words: Abecarnil- Kindling-Pharmacological tolerance-Contingent toler- ance-Rats.

Among the anticonvulsant drogs currently used to treat epilepsy, benzodiazepines (BZD) have po- tent therapeutic effects. However, the clinical effi- cacy of BZD is limited by the development of tol- erance and dependence (I ) . Experimental demon- stration of tolerance to the anticonvulsant effect of BZD depends on several factors, including the treatment regimen and the type of seizures. Abe- carnil (isopropyl-6-benzyloxy-4-methoxymethyl-~- carboline-3-carboxylate), a selective agonist for specific BZD receptor subtypes ( 2 4 , does not in- duce significant pharmacological tolerance to its an- ticonvulsant effect, regardless of the degree of ex- posure and the dosage regimen (5,6).

We examined the anticonvulsant efficacy of abe- carnil after long-term treatment in pentylenetetrazol (PTZ)-kindled rats. Because repeated administra- tion of PTZ induces increased excitability of the CNS, fully kindled rats develop convulsions in re- sponse to a dose of PTZ that was initially subcon- vulsant. This PTZ-kindling model of chronic epilep- togenesis may be useful in predicting the clinical anticonvulsant efficacy and tolerance potential of

Received August 3, 1995; revision accepted December 21, 1995.

Address correspondence and reprint requests to Dr. M. Serra at Department of Experimental Biology, Via Palabanda 12,09123 Cagliari, Italy.

antiepileptic drugs (AEDs) (7) and also allows study- ing tolerance that involves a learning component. This particular kind of tolerance, originally pro- posed by Carlton and Wolgin (8), has been termed “contingent tolerance .” Contingent tolerance de- velops preferentially to the effect of a drug on re- sponses that have occurred during the period of drug exposure. Learning plays an important role in the development of tolerance to the anticonvulsant effects of BZD receptor ligands (9,lO). Loscher et al. (1 l), showed that contingent tolerance to the an- ticonvulsant effect of abecarnil developed in amygdala-kindled rats. We investigated whether contingent tolerance to the anticonvulsant effect of abecarnil develops in PTZ-kindled rats and whether the increased susceptibility to convulsions in these animals is reduced or abolished by chronic treat- ment with abecarnil.

METHODS

Animals Male Sprague-Dawley CD rats (Charles River,

Como, Italy) weighing 180-200 g at the start of treatment, were housed 4-6 per cage under con- trolled temperature (23” & 2°C) and lighting (12-h light/l2-h dark cycle) conditions with food and wa- ter freely available.

332

FAILURE OF ABECARNIL TO INDUCE TOLERANCE 333

Kindling Animals were injected intraperitoneally (i.p.)

with PTZ (30 mg/kg in 3 ml distilled water) three times a week as long as for 10 consecutive weeks. Rats were observed for 1 h after each injection, and seizures were classified according to the following scale: 0, no response; 1, ear and facial twitching; 2, myoclonic body jerks; 3, clonic forelimb convul- sions; 4, generalized clonic convulsions with epi- sodes of rearing and falling; and 5, generalized con- vulsions with tonic extension of the hindlimbs or status epilepticus. Animals that had seizure scores of 4 or 5 after three consecutive injections of PTZ were defined as kindled, and the treatment dose was reduced to 20 mg/kg. The reduction in PTZ dose from 30 to 20 was necessary because kindled rats became very sensitive to PTZ and some ani- mals died. Rats that did not kindle by the end of the tenth week of treatment were not used in the sub- sequent studies. On completion of chronic PTZ treatment, the fully kindled rats were challenged with PTZ (20 mg/kg i.p.) and abecarnil (0.5 mg/kg i.p.) before long-term treatment with abecarnil was started.

Chronic treatment with abecarnil Animals were injected intraperitoneally with abe-

carnil (1 mg/kg) three times daily (at 0800, 1400, and 2000 h) for 15 days. The drug was suspended in distilled water with 1 drop of Tween 80 per 5 ml and injected in a volume of 3 ml/kg. Control rats re- ceived an equivalent volume of vehicle. Animals were divided into four groups. Two groups received PTZ (20 mg/kg three times a week) and either abe- carnil (1 mg/kg three times a day) or vehicle. When abecarnil (or vehicle) and PTZ were administered on the same day, the animals were injected first with abecarnil and 30 min later with PTZ. The other two groups were injected with only abecarnil or ve- hicle. At the end of the chronic treatment period, animals were tested 36 h and 3 days after the last abecarnil and PTZ injection, respectively, with abe- carnil (0.5 mg/kg) and PTZ (20 mg/kg). All rats were observed for at least 1 h, during which time the onset and pattern of seizures were recorded.

Statistics Latency values are mean * SEM. Data were an-

alyzed by Fisher’s exact probability test or Stu- dent’s t test; p < 0.05 was considered statistically significant.

Chemicals Abecarnil was provided by Schering A.G. (Ber-

lin, Germany), and PTZ and Tween 80 were ob- tained from Sigma (St. Louis, MO, U.S.A.).

RESULTS

Acute treatment studies We challenged kindled rats with PTZ (20 mg/kg)

and studied the ability of abecarnil to antagonize the PTZ-induced convulsions. As expected (12), abe- carnil (0.5 mg/kg) administered 30 min before PTZ completely prevented PTZ-induced convulsions (Table 1).

Chronic treatment studies Kindled rats were divided into four groups of 15

rats each. We administered PTZ (20 mg/kg three times a week) according to the kindling protocol and either abecarnil (1 mg/kg three times a day) or vehicle for 15 days to two groups to examine the possible development of contingent tolerance to the anticonvulsant effect of abecarnil.

Abecarnil failed to induce contingent tolerance to its anticonvulsant effect (Table 2). Therefore, the challenge dose (0.5 mg/kg) of abecarnil adminis- tered 36 h and 3 days after discontinuation of either long-term abecarnil or PTZ treatment completely prevented convulsions induced by a challenge dose of PTZ. Although the animals were completely pro- tected from seizures during chronic treatment with abecarnil, they rapidly developed tolerance to the inhibitory effect of abecarnil on motor activity (3) (data not shown).

In the other two groups of rats, the kindling stim- ulus was discontinued 24 h before the onset of long- term treatment with abecarnil or vehicle. Again abecarnil did not induce pharmacological tolerance to its anticonvulsant effect (Table 3). A challenge dose of abecarnil(O.5 mg/kg) administered 36 h after drug discontinuation completely antagonized con- vulsions elicited by a challenge dose of PTZ in rats chronically treated with abecarnil as well in those chronically treated with vehicle. Moreover, re- peated administration of abecarnil in animals in which PTZ treatment was suspended failed to re- duce the increased excitability of the CNS. There- fore, the same low dose of PTZ (20 mglkg) induced

TABLE 1. Effect of acute abecarnil treatment on PTZ-induced convulsions in kindled rats

Convulsions

Treatment Latency (rnin) No. of animals

Vehicle 6.5 2 1.5 20/30 (67%) Abecarnil >60“ 0/30 (O%)b

PTZ, pentylenetetrazol. Kindled rats were challenged with PTZ (20 mg/kg intraperito-

neally, i.p.) 30 min after administration of abecarnil (0.5 mg/kg i.p.) or vehicle. Animals were observed for 1 h after challenge.

p < 0.01 versus vehicle (Fisher’s exact probability test). a p < 0.01 versus vehicle (Student’s t test).

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334 M . SERRA ET AL.

TABLE 2. Effect of chronic treatment with abecarnil on the development of contingent tolerance in

PTZ-kindled rats

Convulsions

Experimental group Latency (treatment, mg/kg) (min) No. of animals

Chronic vehicle PTZ (20) 4.5 * 0.6 13/15 (86%) PTZ + abecarnil (0.5) >60“ 0/15 (O%)b

Chronic abecarnil PTZ (20) 4.8 & 1.7 11/15 (73%) PTZ + abecarnil (0.5) >60” 0115 (O%)b

PTZ, pentylenetetrazol. Kindled animals were injected with abecarnil ( I mg/kg) or ve-

hicle three times daily and with PTZ (20 mg/kg) three times a week for 15 days. Thirty-six hours and 3 days after the last injections of abecarnil and PTZ, respectively, rats were chal- lenged with abecarnil(O.5 mg/kg) or vehicle followed 30 min later by the injection of PTZ (20 mg/kg). Animals were observed for I h after PTZ administration.

a p < 0.01 versus PTZ-treated rats (Student’s t test). p < 0.01 versus PTZ-treated rats (Fisher’s exact probability

test).

similar patterns of generalized clonic seizures in rats chronically treated with abecarnil and in those treated with vehicle (Table 3).

DISCUSSION

Several studies have shown that. abecarnil effec- tively antagonizes convulsions induced by various drugs (FTZ, isoniazid, pilocarpine, 3-mercaptopro- pionic acid, kainic acid) or by electrical stimulation of the amygdala or cornea in mice, rats, gerbils, dogs, rats, or baboons (2,3,5,12).

Our results also support a possible role for abe- carnil as an effective AED. We showed that abe- carnil not only antagonized PTZ-induced convul-

TABLE 3. Effect of chronic treatment with abecarnil on the development of pharmacological tolerance in

PTZ-kindled rats

Convulsions

Experimental group Latency (treatment, mg/kg) (min) No. of animals

Chronic vehicle PTZ (20) 3 +- 0.4 15/15 (100%) PTZ + abecarnil (0.5) >60” O h 5 (O%)b

PTZ (20) 2.8 +- 0.7 15/15 (100%) PTZ + abecarnil (0.5) >60b 0/15 (O%)b

Chronic abecarnil

PTZ, pentylenetetrazol. Kindled animals were injected with abecarnil (1 mg/kg) or ve-

hicle three times daily for 15 days. Thirty-six hours after the last injections with abecarnil, rats were challenged with abecarnil (0.5 mg/kg) or vehicle and, 30 min later with PTZ (20 mg/kg). Animals were observed for 1 h after PTZ administration.

p < 0.01 versus PTZ-treated rats (Student’s t test). p < 0.01 versus PTZ-treated rats (Fisher’s exact probability

test).

sions in kindled rats (12) but also maintained its ability to block seizures in kindled animals after chronic administration. These data are consistent with results of several previous studies showing that pharmacological tolerance to the anticonvul- sant effect of abecarnil fails to develop after re- peated administration in dogs, rats, and mice (5 ,6 , 11). The failure of chronic abecarnil administration to induce pharmacological tolerance is not limited to the anticonvulsant effect. Indeed, the anticonflict effect of abecarnil was not attenuated after repeated treatment (13). On the contrary, abecarnil-induced decreased motor function (3) disappeared soon after the beginning of treatment, a clear demonstration of pharmacological tolerance. In our experimental paradigm, repeated administration of abecarnil failed to influence the pattern of seizures when kin- dling was fully developed. Indeed, in contrast to abecarnil’s marked efficacy in preventing seizures elicited by a challenge dose of PTZ in kindled rats, chronic administration of abecarnil failed to abolish or reduce the expression or severity of kindling in rats in which kindling was suspended during chronic administration of abecarnil or in which PTZ administration was continued simultaneously with long-term abecarnil treatment. Moreover, in con- trast to evidence indicating that many GABAergic and nonGABAergic drugs prevent the development of kindling when they are administered concomi- tantly with repeated doses of FG 7142, an inverse agonist for BZD receptors with a proconvulsant ac- tivity (14), the simultaneous administration of PTZ and abecarnil failed to prevent the development of kindling (M. Serra et al., unpublished observa- tions).

In the present study, abecarnil did not induce contingent tolerance to its anticonvulsant effect. The drug maintained its pharmacological efficacy after chronic administration in the presence of re- peated convulsant stimuli. Abecarnil appears to dif- fer from other anticonvulsant drugs in that convul- sant stimulation facilitates the development of tol- erance to carbamazepine, diazepam, valproate, phenobarbital, and ethanol (9,15,16). Because abe- carnil was administered at a dose (0.5 mg/kg) that completely prevented PTZ-induced convulsions in PTZ-kindled rats, a partial loss of efficacy might have been concealed. Therefore, the anticonvulsant efficacy of abecarnil may decrease; the data of Lo- scher et al. ( 1 1) showing that contingent tolerance to the anticonvulsant effect of abecarnil developed in amygdala-kindled rats support this hypothesis. However, this apparent discrepancy between the data of Loscher et al. and ours may also be attrib- utable to differences in the kindling procedures.

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FAILURE OF ABECARNIL TO INDUCE TOLERANCE 335

Molecular biological studies have indicated that mRNAs for several subunits of the GABAA recep- tor are differentially affected in different types of kindling. Kokaia et al. (17) showed that electrical kindling of the left hippocampus induced a decrease in the abundance of p3 and y2 subunit rnRNAs in the dentate gyrus. In contrast, an increase in the abun- dance of a,, a3, p2, and yZL subunit mRNAs was apparent after 31 days of FG 7142 kindling (18). To our knowledge, GABA, receptor subunit mRNA abundance after PTZ or amygdala kindling has not been examined in molecular studies. These two kin- dling procedures may induce different long-lasting changes in the expression of genes encoding spe- cific GABAA receptor subunits, resulting in the as- sembly of receptors with different sensitivities to abecarnil. Knoflach et al. (4) showed that the intrin- sic activity of abecarnil at recombinant GABAA re- ceptors depends on receptor subunit composition: The drug acts as a full agonist at receptors contain- ing ctl and aj subunits and as a partial agonist at receptors containing at and a5 subunits.

PTZ test and amygdala kindling procedures have been suggested to reflect different clinical condi- tions, with the PTZ model representing generalized absence or myoclonic seizures (19) and the amygdala model representing refractory partial sei- zures (20). Therefore, the evidence that abecarnil induces contingent tolerance in amygdala-kindled rats but not in PTZ-kindled rats may be predictive in terms of the clinical efficacy of the drug for the treatment of different types of epileptic seizures. The most widely used AEDs, such as carbarnaze- pine, phenytoin, phenobarbital, and valproate, are ineffective against many types of generalized sei- zures (absence, myoclonic, tonic, and atonic) in hu- mans but are very effective in amygdala kindling (7). If our results can be reproduced clinically, abe- Farnil may represent a therapeutic alternative to classic AEDs in the treatment of drug-resistant ep- ilepsy.

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