Influence of sexual hormone antagonists on the anticonvulsant action of conventional antiepileptic drugs against electrically- and pentylenetetrazol-induced seizures in mice.

The present results refer to the action of three gonadal steroid antihormones, tamoxifen (TXF, an estrogen antagonist), cyproterone acetate (CYP, an antiandrogen) and mifepristone (MIF, a progesterone antagonist) on seizure phenomena in mice. TXF and CYP at their lowest protective dose in the electroconvulsive threshold test, enhanced the antiseizure efficacy of some antiepileptic drugs. TXF (20 mg/kg) potentiated the protective activity of valproate, diphenylhydantoin and clonazepam, but not that of carbamazepine or phenobarbital, against maximal electroshock-induced convulsions in female mice. CYP (40 mg/kg) enhanced the anticonvulsant action of valproate, carbamazepine, diphenylhydantoin and clonazepam, but not that of phenobarbital, against maximal electroshock in male animals. MIF failed to affect the electroconvulsive threshold or the efficacy of antiepileptic drugs in maximal electroshock. The effect of TXF or CYP upon the electroconvulsive threshold and on the action of antiepileptics was not reversed by sex steroid hormones (estradiol, testosterone, progesterone). However, the TXF-induced elevation of the electroconvulsive threshold was abolished by bicuculline, N-methyl-D-aspartic acid and kainic acid, and partially reversed by aminophylline, strychnine being ineffective in this respect. The action of CYP on the threshold for electroconvulsions was partially reversed by bicuculline and aminophylline. Both glutamatergic agonists and strychnine remained ineffective in this respect. Moreover, the action of TXF or CYP on the activity of antiepileptics was not influenced by strychnine, and reversed to various extents by the remaining convulsants. In contrast to maximal electroshock, none of the three antihormones affected the protective action of antiepileptic drugs against pentylenetetrazol-induced seizures in mice. Neither TXF nor CYP altered the free plasma levels of antiepileptic drugs, so a pharmacokinetic interaction is not probable. The combined treatment of the two antihormones with antiepileptic drugs, providing 50% protection against maximal electroshock, did not affect motor performance in mice, and did not result in significant long-term memory deficits. Our data indicate that steroid receptor-mediated events may be indirectly associated with seizure phenomena in the central nervous system and can modulate the protective activity of some conventional antiepileptic drugs.

Influence of LY 300164, an AMPA/kainate receptor antagonist upon the anticonvulsant action of antiepileptic drugs against aminophylline-induced seizures in mice.

LY 300164 [7-acetyl-3-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxazolo[4,5-h][2,3]-benzodiazepine], a novel AMPA/kainate receptor antagonist, administered intraperitoneally protected mice against aminophylline-induced seizures. At doses up to 0.5 mg/kg, which did not significantly affect the convulsant activity of aminophylline, it potentiated the protective activity of diazepam. On the other hand, LY 300164 used at the lowest protective dose of 1.0 mg/kg enhanced anticonvulsant activity of all antiepileptic drugs tested in this seizure model. However, LY 300164 neither alone nor combined with antiepileptic drugs, reduced aminophylline-induced mortality.

Influence of sex hormone antagonists on the anticonvulsant action of conventional antiepileptic drugs against amygdala-kindled seizures in male and female rats.

The effects of three gonadal steroid antihormones, tamoxifen (TXF, an estrogen antagonist), cyproterone acetate (CYP, an antiandrogen) and mifepristone (MIF, a progesterone antagonist) alone or combined with conventional antiepileptics were evaluated in amygdala-kindled seizures in male and female rats. None of the three antihormones used in this study affected any seizure parameter. TXF (50 mg/kg) and CYP (50 mg/kg), when combined with carbamazepine, or phenobarbital applied at their subprotective doses of 15 mg/kg, resulted in significant reductions of the seizure and afterdischarge durations, both in male and female rats. Additionally, the combination of carbamazepine and CYP markedly increased the afterdischarge threshold in fully-kindled rats of both genders. The interaction between antihormones and carbamazepine, or phenobarbital, was not reversed by respective sex steroid hormones (estradiol, testosterone). However, the TXF- and CYP-induced anticonvulsant effects in combinations with carbamazepine were attenuated by bicuculline, N-methyl-D-aspartate (NMDA) and aminophylline. Kainic acid and strychnine remained ineffective in this respect. The effect of a combination of TXF with phenobarbital was reversed by bicuculline and NMDA and that of CYP with phenobarbital-by bicuculline and aminophylline. Neither TXF nor CYP altered the free plasma concentrations of carbamazepine or phenobarbital, so a pharmacokinetic interaction is not probable. The combined treatment of the two antihormones with antiepileptic drugs did not affect motor performance, and did not result in significant long-term memory deficits. Our data confirm the hypothesis that sex hormone antagonist-mediated events may play some role in seizure processes in the central nervous system and can modulate the protective activity of some conventional antiepileptic drugs against kindled seizures.

Investigations on the synthesis and pharmacological properties of 4-alkoxy-2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl]-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones.

Synthesis of 2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl] derivatives of 4-alkoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones (8-12) is described. The chlorides used in the above synthesis can exist in two isomeric forms: chain (18-20) and cyclic (19a, 20a). The compounds 8-12 exhibited potent analgesic activity which was superior than that of acetylsalicylic acid in two different tests. Most of the investigated imides suppressed significantly spontaneous locomotor activity in mice.

Interaction of the neurosteroid alphaxalone with conventional antiepileptic drugs in different types of experimental seizures.

A number of neurosteroids exert antiseizure and/or neuroprotective properties. The aim of this study was to evaluate the effect of the neurosteroid alphaxalone on the protective action of conventional antiepileptics in four seizure tests. Alphaxalone (up to 5 mg/kg) did not exert a significant action against amygdala-kindled seizures in rats, or against pentetrazole- or aminophylline-induced convulsions in mice. The neuroactive steroid at the dose of 2.5 mg/kg significantly raised the threshold for electroconvulsions in mice. At 2.5 mg/kg, alphaxalone diminished the protective activity of valproate against maximal electroshock and at 2.5-5 mg/kg against pentetrazole-induced seizures in mice. However, alphaxalone (2.5 mg/kg) did not affect the protective activity of carbamazepine, diphenylhydantoin, phenobarbital or clonazepam against maximal electroshock and at 5 mg/kg did not affect that of phenobarbital, clonazepam and ethosuximide against pentetrazole-induced convulsions. Insignificant results were also obtained in the case of co-administration of alphaxalone with phenobarbital, valproate, clonazepam and carbamazepine against aminophylline-evoked seizures in mice. Also, in the kindling model of epilepsy, combinations of the neuroactive steroid (2.5 mg/kg) with valproate, carbamazepine, phenobarbital, diphenylhydantoin or clonazepam at their subprotective doses did not result in pro- or anticonvulsant activity. Valproate (284 mg/kg; the dose used in combination with alphaxalone) produced significant memory deficits in mice. Alphaxalone (2.5 mg/kg), valproate (at its ED(50) value of 226 mg/kg) and the combination of valproate (284 mg/kg) with alphaxalone (2.5 mg/kg) did not affect long-term memory, evaluated in the passive avoidance task with mice. Alphaxalone administered alone or in combination with valproate caused no motor impairment in experimental animals. Finally, alphaxalone (2.5 and 5 mg/kg) significantly increased the free plasma levels of valproate, strongly indicating that the neuroactive steroid-induced reduction of the protective activity of valproate is not related to pharmacokinetic phenomena. Summing up, alphaxalone does not seem to be a promising candidate for adjunctive treatment of epilepsy.

Effects of tamoxifen, mifepristone and cyproterone on the electroconvulsive threshold and pentetrazole-induced convulsions in mice.

The aim of this study was to evaluate the efficacy of three antihormones, tamoxifen (TXF, an antiestrogen), mifepristone (MIF, an antiprogesterone) and cyproterone (CYP, an antiandrogen) in two major models of experimental epilepsy, electrically and pentetrazole (PTZ)-evoked seizures in mice. TXF (20-50 mg/kg) significantly raised the threshold for electroconvulsions in female mice, whereas CYP was active in male mice. Similar effects were observed in castrated mice. Different data were obtained in sexually immature animals since both TXF and CYP exerted anticonvulsive effects in animals of both genders. MIF (5-50 mg/kg) remained without effect on electrically evoked seizures in mice. The anticonvulsive action of TXF was reversed by aminophylline, bicuculline, kainic acid and N-methyl-D-aspartic acid, but not by estradiol or strychnine. The protective action of CYP was reversed by aminophylline and bicuculline, but not by testosterone, kainic acid, N-methyl-D-aspartic acid or strychnine. All three antihormones were ineffective against PTZ-induced convulsions in mice. Our results suggest that the action of TXF and CYP might be indirectly associated with the respective hormonal receptor-mediated events, but the nature of this dependence is unclear and further investigations are needed to elucidate this phenomenon.

Endothelium-dependent production and liberation of kynurenic acid by rat aortic rings exposed to L-kynurenine.

Rat aortic slices produced and liberated the endogenous antagonist of glutamate receptors, kynurenic acid, upon exposure to L-kynurenine. Endothelium-denuded slices did not synthesize any measurable amount of kynurenic acid, indicating its endothelial origin. Aortic kynurenic acid production was diminished by modification of the ionic milieu, hypoxia and hypoglycemia, as well as by L-glutamate and L-aspartate, endogenous glutamate receptor agonists, and aminooxyacetic acid, a non-selective inhibitor of aminotransferases and mitochondrial respiration. These data pave the way for future research aimed to clarify the role of kynurenic acid in the physiology and pathology of the endothelium and vasculature.

Influence of agents affecting voltage-dependent calcium channels and dantrolene on the anticonvulsant action of the AMPA/kainate receptor antagonist LY 300164 in mice.

It was previously documented that calcium (Ca(2+)) channel inhibitors intensified the protective effects of conventional antiepileptics against electroconvulsions in mice. The aim of this study was to evaluate the effects of Ca(2+) channel inhibitors (nifedipine, nicardipine and flunarizine) on the anticonvulsant action of the new AMPA/kainate receptor antagonist, 7-acetyl-3-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxazolo[4,5-h][2,3]-benzodiazepine (LY 300164), against maximal electroshock (MES)-induced seizures in mice. Dantrolene (an inhibitor of Ca(2+)release from intracellular stores) was also included. Nifedipine (30 mg/kg) and flunarizine (15 mg/kg) raised the threshold for electroconvulsions, being ineffective at lower doses. Nicardipine (up to 30 mg/kg) and dantrolene (up to 20 mg/kg) did not affect this parameter. Flunarizine (10 mg/kg), nicardipine (20 mg/kg) and dantrolene (20 mg/kg) potentiated the efficacy of LY 300164 against MES. However, nicardipine (at 20 mg/kg) raised the free plasma concentration of LY 300164. Nifedipine (30 mg/kg), given even in a dose raising the electroconvulsive threshold, did not significantly alter the protective effect of LY 300164 against MES. Furthermore, the Ca(2+) channel agonist-BAY k-8644 (at 5 mg/kg) did not influence the protection offered by LY 300164 against MES. Finally, this Ca(2+) channel activator did not affect the enhanced efficacy of LY 300164 by Ca(2+) channel modulators. The only exception was the combination of LY 300164 with flunarizine. Combined treatment with LY 300164 and dantrolene (20 mg/kg), compared to LY 300164 alone, resulted in an impairment of motor performance in mice. Ca(2+) channel inhibitors were without effect upon this parameter evaluated in the chimney test. As shown in the passive avoidance task, LY 300164 alone (at its ED(50)) or combined with agents affecting neuronal Ca(2+) concentration did not disturb long-term memory. The present results suggest that agents preventing influx of Ca(2+) ions into neurons may enhance the protective action of LY 300164.

Molsidomine potentiates the protective activity of GYKI 52466, a non-NMDA antagonist, MK-801, a non-competitive NMDA antagonist, and riluzole against electroconvulsions in mice.

The influence of molsidomine, a donor of nitric oxide (NO), L-arginine, a substrate for NO synthesis, and N(G)-nitro-L-arginine (NNA), an inhibitor of NO synthase, on the protective activity of CGP 40116, GYKI 52466, MK-801, and riluzole against electroconvulsions was studied in mice. Molsidomine (100 mg kg(-1); i.p.) potentiated the protective activity of GYKI 52466, MK-801, and riluzole but did not influence the protection offered by CGP 40116. In contrast to molsidomine, L-arginine (500 mg kg(-1); i.p.) did not impair the protective activity of any anticonvulsant. In a dose of 40 mg kg(-1), NNA administered i.p. did not affect the protection offered by any excitatory amino acid antagonists and riluzole. Combinations of molsidomine with either GYKI 52466 or MK-801 as well as riluzole did not cause a memory deficit in the passive avoidance task. However, the combined treatment of molsidomine with these anticonvulsants resulted in a motor impairment quantified by the chimney test. The lack of effect of L-arginine and NNA on the protective activity of excitatory amino acid antagonists suggests that molsidomine-evoked alterations in the protection provided by some excitatory amino acid antagonists against electroconvulsions are independent of the NO pathway.

Niguldipine impairs the protective activity of carbamazepine and phenobarbital in amygdala-kindled seizures in rats.

There is evidence that some calcium (Ca(2+)) channel inhibitors enhance the protective activity of antiepileptic drugs. Since clinical trials have not provided consistent data on this issue, the objective of this study was to evaluate the interaction of a dihydropyridine, niguldipine, with conventional antiepileptics in amygdala-kindled rats. Niguldipine (at 7.5 but not at 5 mg/kg) displayed a significant anticonvulsant effect, as regards seizure and afterdischarge durations in amygdala-kindled convulsions in rats, a model of complex partial seizures. No protective effect was observed when niguldipine (5 mg/kg) was combined with antiepileptics at subeffective doses, i.e. valproate (75 mg/kg), diphenylhydantoin (40 mg/kg), or clonazepam (0.003 mg/kg). Unexpectedly, the combined treatment of niguldipine (5 mg/kg) with carbamazepine (20 mg/kg) or phenobarbital (20 mg/kg) resulted in a proconvulsive action. BAY k-8644 (an L-type Ca(2+) channel activator) did not modify the protective activity of niguldipine (7.5 mg/kg) or the opposite action of this dihydropyridine (5 mg/kg) in combinations with carbamazepine or phenobarbital. A pharmacokinetic interaction is not probable since niguldipine did not affect the free plasma levels of the antiepileptics. These data indicate that the opposite actions of niguldipine alone or combined with carbamazepine (or phenobarbital) were not associated with Ca(2+) channel blockade. The present results may argue against the use of niguldipine as an adjuvant antiepileptic or for cardiovascular reasons in patients with complex partial seizures.

Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophil.

BACKGROUND: Benzodiazepine binding sites distinct from the GABA-receptor-chloride-complex in the central nervous system have been recognized in many peripheral tissues, but their physiological role remains unexplained. Our study was undertaken to examine the effects of diazepam, clonazepam, and PK 11195, a peripheral benzodiazepine receptor antagonist, on the functional and biochemical responses of platelets and neutrophils stimulated by different physiological agonists. MATERIAL/METHODS: The experiments were conducted on isolated washed rat platelets activated by arachidonic acid (AA), adenosine 5'-diphosphate (ADP), or thrombin and on isolated rat neutrophils activated by a chemotactic peptide, formyl methionyl leucyl phenylalanine (fMLP). RESULTS: The results showed that neither diazepam nor clonazepam nor PK 11195 alone augmented the response of resting platelets or modified neutrophil response, but diazepam and clonazepam in a concentration-dependent manner inhibited thrombin, ADP or AA-stimulated platelet aggregation and the thrombin-induced increase in free intracellular Ca2+. Both drugs also exerted an inhibitory effect on reactive oxygen species (ROS) produced by fMLP-stimulated neutrophils. However, diazepam was about 10 times more effective than clonazepam. PK11195 did not influence platelet and neutrophil function stimulated by agonists, but reversed the inhibitory action of both benzodiazepines on platelet activation and ROS production. CONCLUSIONS: The results indicated that in vitro diazepam, and in a much smaller degree clonazepam, may down-regulate platelet activation and release of some proinflammatory mediators by stimulated neutrophils. These effects are probably exerted by a specific benzodiazepine binding sites.

Nitric oxide and convulsions in 4-aminopyridine-treated mice.

We studied whether N(G)-nitro-L-arginine (NNA), an inhibitor of nitric oxide (NO) synthase as well as L-arginine and molsidomine, two agents elevating NO, influenced convulsions caused by 4-aminopyridine, a K+ channel blocker in mice. NNA, in a dose known to decrease level of NO (40 mg x kg(-1)), enhanced the seizure susceptibility to intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) 4-aminopyridine. L-arginine (500 mg x kg(-1)) and molsidomine (20 mg x kg(-1)) alone did not influence 4-aminopyridine-induced seizure activity. Surprisingly, the proconvulsant effect of NNA upon clonic and tonic seizures was potentiated by molsidomine (20 mg x kg(-1)). No influence of L-arginine on the proconvulsant effect of NNA was found. Taking into account the proconvulsant effect of NNA, an involvement of NO-mediated events in the mechanism of convulsive activity of 4-aminopyridine might be postulated. However, the ineffectiveness of L-arginine and molsidomine to suppress the convulsive activity of 4-aminopyridine as well as a paradoxical potentiation of the proconvulsant effect of NNA by molsidomine seem to exclude the impact of NO pathway on 4-aminopyridine-induced convulsions in mice. Our data suggest that the proconvulsant effect of NNA in this seizure model is caused by other, not related to NO, mechanisms.

Propranolol and metoprolol enhance the anticonvulsant action of valproate and diazepam against maximal electroshock.

The anticonvulsive potential of classical antiepileptics co-administered with beta-adrenergic receptor antagonists against generalized tonic-clonic seizures was evaluated in the model of maximal electroshock (MES)-induced convulsions. Propranolol, acebutolol, metoprolol and atenolol were tested in the doses not affecting the electroconvulsive threshold. Propranolol and metoprolol lowered the ED(50) of valproate and diazepam. Acebutolol reduced valproate's but not diazepam's ED(50) value. In contrast, hydrophilic atenolol, not penetrating via blood-brain barrier, affected neither the action of valproate nor diazepam. None of the studied drugs changed the protective activity of carbamazepine and phenytoin against MES. beta-blockers per se did not alter the motor performance of mice. Moreover, propranolol and metoprolol did not influence diazepam-evoked impairment of locomotor activity. The free plasma and brain levels of antiepileptic drugs were not affected by beta-blockers. In conclusion, the use of certain beta-adrenoceptor antagonists, such as propranolol and metoprolol, might improve the antiepileptic potential of valproate and diazepam.

The influence of interferon alpha on the rat liver injured by chronic administration of carbon tetrachloride.

Due to their complex and not fully known etiopathogenesis as well as difficulties in treatment, chronic hepatitis and cirrhosis still remain one of the main problems of hepatologists. Nowadays, the use of IFN alpha is considered the most effective method of treatment in chronic hepatitis. Recently, a new property of IFN, i.e. its effects on the reduction of fibrosis, has been discovered. The aim of the paper was to examine the effects of IFN alpha on biochemical parameters (AlAt and AspAt activities), on the metabolic function of the liver and its morphologic picture observed under the light and electron microscope after the 3- and 6-week CCl4-induced damage. The experiments were carried out in Wistar male rats. To evaluate the liver function, the test of aminophenazone elimination in the isolated perfused rat livers was used according to Miller modified by Hafte. Additionally, AspAt and AlAt activities were determined. The liver specimens were analysed under the light and electron microscope and using immunohistochemical methods. The findings show that after the 3-week CCl4-induced liver damage, IFN alpha does not significantly affect AlAt and AspAt activities, irrespective of the dose used. IFN alpha administered after the 6-week damage significantly changes those activities when the doses used are high. It was found that carbon tetrachloride does not result in evident cirrhotic changes, however it activates Ito cells, causes focal retraction of the stroma and fibrosis. The increased number of Ito cells in Disse's space observed in immunohistochemical and ultrastructural examinations is indicative of the activation of liver fibrotic processes following CCl4 administration in both variants used. IFN alpha substantially weakens fibrogenesis of the CCl4-damaged liver which is visible in the decreased number of Ito cells and weaker expression of the stroma retraction. Moreover, IFN alpha administered to the experimental animals after the CCl4-induced injury of the liver increases aminophenazone clearance, especially when used in higher doses. Positive effects of IFN confirmed in the studies suggest that the drug may be used in patients with chronic hepatitis and early cirrhosis since it is likely not only to eliminate the virus but also to improve the liver function and reduce fibrosis.