M1 muscarinic antagonists interact with sigma recognition sites.

The M1-selective muscarinic antagonists aprophen, caramiphen, carbetapentane, 2-DAEX, dicyclomine, hexahydrosiladifenidol, iodocaramiphen, nitrocaramiphen, oxybutynin and trihexyphenidyl potently inhibited binding to sigma sites in brain. Both basic ester and non-ester structural type compounds which exhibit affinity for the muscarinic receptor also demonstrated affinity for the sigma site, while the classical antimuscarinic agents atropine and QNB, and the tricyclic pirenzepine, were ineffective in binding to this site. We also observed a significant correlation between the Ki values for sigma compounds to inhibit [3H]pirenzepine binding and their IC50 values to inhibit carbachol-stimulated phosphoinositide turnover. These observations may aid in elucidating the relationship of sigma binding to inhibition of phosphoinositide turnover stimulated by cholinergic agonists.     

Effects of anticonvulsants and gamma-aminobutyric acid (GABA)-mimetic drugs on immunoreactive somatostatin and GABA contents in the rat brain

Immunoreactive somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) contents in the rat brain were investigated to study chronic effects of the treatment with anticonvulsants, carbamazepine (CBZ), valproic acid (VPA) and phenytoin (PHT). Decreased IR-SRIF levels were found in several brain regions after chronic treatment with VPA and CBZ. GABA concentrations were found to be increased significantly in chronic CBZ and VPA treatment in the rat brain, especially in limbic structures. PHT had no effect on both IR-SRIF and GABA contents in the rat brain. Effects of several GABA-mimetic drugs also were studied on IR-SRIF contents in the rat brain. Aminooxyacetic acid an inhibitor of GABA transaminase, induced a decrease in IR-SRIF concentration in the pyriform and entorhinal cortex, whereas ethanolamine-o-sulfate, another GABA-transaminase inhibitor and muscimol, a GABA receptor agonist had no effect on brain IR-SRIF after acute administration. The present results suggest that endogenous somatostatin has an important role for anticonvulsant properties of CBZ and VPA, but not of PHT. The relationship between the changes in IR-SRIF and the GABA transmitter system in the anticonvulsant action of CBZ and VPA remains to be clarified.     

New morphinan derivatives with negligible psychotropic effects attenuate convulsions induced by maximal electroshock in mice

Interest in dextromethorphan (DM) has been renewed because of its anticonvulsant and neuroprotective properties. However, DM at supra-antitussive doses can produce psychotomimetic effects in humans. Recently, we demonstrated that DM exerts psychotropic effects in mice [Neurosci. Lett. 288 (2000) 76, Life Sci. 69 (2001) 615]. We synthesized a series of compounds with a modified morphinan ring system, with the intention of developing compounds that retain the anticonvulsant activity with weak psychotropic effects [Bioorg. Med. Chem. Lett. 11 (2001) 1651]. In order to extend our understanding of the pharmacological intervention of these morphinans, we assessed their behavioral effects, and then examined whether they exert protective effects on maximal electroshock convulsions (MES) in mice. Repeated treatment (20 or 40 mg/kg, i.p./day x 7) with DM or dextrorphan (a major metabolite of DM; DX) significantly enhanced locomotor activity in a dose-related manner. This locomotor stimulation was accentuated more in the animals treated with DX, and might be comparable to that of phencyclidine (PCP). By contrast, treatment with a metabolite of DM [3-methoxymorphinan (3MM) or 3-hydroxymorphinan (3HM)], 3-allyloxy-17-methylmorphinan (CPK-5), or 3-cyclopropylmethoxy-17-methylmorphinan (CPK-6) did not significantly alter locomotor activity or patterns. The behavioral effects mediated by these morphinans and PCP paralleled the effects of conditioned place preference. DM, DX, CPK-5, and CPK-6 had anticonvulsant effects against MES, while 3MM and 3HM did not show any anticonvulsant effects. We found that DM, DX, CPK-5 and CPK-6 were high-affinity ligands at sigma(1) receptors, while they all had low affinity at sigma(2) receptors. DX had relatively higher affinity for the PCP sites than DM. By contrast, CPK-5 and CPK-6 had very low affinities for PCP sites, suggesting that PCP sites are not requisites for their anticonvulsant actions. Our results suggest that the new morphinan analogs are promising anticonvulsants that are devoid of PCP-like behavioral side effects, and their anticonvulsant actions may be, in part, mediated via sigma(1) receptors.     

Embryotoxicity of phenytoin in adrenalectomized CD-1 mice.

It has been proposed that the anticonvulsant drug phenytoin (PHT) and glucocorticoids induce orofacial clefting by the same mechanism. Previous work had demonstrated that PHT treatment significantly increased endogenous maternal corticosterone concentrations for approximately 48 hr after dosing in A/J mice. The purpose of the present investigation was to determine whether PHT is embryotoxic in the absence of endogenous maternal glucocorticoids. Maternal adrenal glands were removed on Day 7 of gestation, and the incidence of clefting after PHT treatment was determined. There was a high level of maternal toxicity following adrenalectomy (ADX) and PHT treatment at either 60 or 75 mg/kg. This increased toxicity did not appear to be due to altered maternal drug levels in ADX mice. There was a significant increase in the clefting incidence among offspring of ADX dams treated with PHT at 60 mg/kg. This dose of PHT did not elevate maternal corticosterone levels in ADX dams. These data suggest that PHT is capable of producing clefts in the absence of endogenous maternal corticosterone.     

Effects on toes from prenatal exposure to anticonvulsants

BACKGROUND: Changes in the distal phalanges of the fingers, including coned epiphyses and hypoplasia of the phalanges, are recognized teratogenic effects of the anticonvulsant drugs phenytoin and phenobarbital. We hypothesized that the frequency of these changes would also be increased in the toes of children exposed to these drugs in comparison to unexposed children. METHODS: We report on the findings in an analysis of radiographs of the feet of 63 children exposed in utero to either phenytoin alone, phenobarbital alone or both drugs and 56 unexposed comparison children. RESULTS: Only subtle changes were identified. The frequency of coned epiphyses and hypoplasia of phalanges of the toes was the same in both the anticonvulsant and unexposed children. Among the anticonvulsant-exposed children, however, there was a strong association between the presence of coned epiphyses in the feet and in the hands: all five children with coned epiphyses in the hands, as described previously in the same individuals by Lu et al. ([2000] Teratology 61:277-283) had coned epiphyses in their feet (P = 0.0012). Measurements showed a shortening of metatarsals in all three treatment groups, but this was significant only in the phenytoin monotherapy-exposed children. CONCLUSIONS: Subtle changes are present in the phalanges and metatarsals of the feet of anticonvulsant-exposed children, but the overall frequency is much less than occurred in the hands of the same children. We conclude that the presence of either coned epiphyses or hypoplasia of the phalanges of the toes cannot be considered a distinctive feature of the teratogenicity of the anticonvulsant drugs phenytoin and phenobarbital.     

Phenytoin teratogenicity and effects on embryonic and maternal folate metabolism [published errtum appears in Teratology 1986 Dec;34(3):487

It has been postulated that the mechanism of teratogenicity of the anticonvulsant drug phenytoin (PHT), is via a deficiency of folic acid. To test this hypothesis, Swiss Webster mice were administered PHT in the diet prior to and throughout gestation. Animals received a daily dose of approximately 75 mg/kg body weight. The maternal plasma PHT levels were within the therapeutic range for this drug. This dose increased the incidence of malformations, primarily cleft palate, in the absence of embryolethality. There was a decrease in maternal plasma folate levels on day 12 of gestation but no effect on days 10 and 18. Even in the presence of a maternal folate deficiency on day 12, PHT had no effect on total embryonic folate levels on days 10, 12, and 14. Previous experiments have demonstrated that PHT decreases activity of the enzyme 5,10-methylenetetrahydrofolate reductase in the liver of nonpregnant Swiss Webster mice. Data from the current study indicate that this enzyme activity is also decreased in hepatic tissue of pregnant mice, but it is not altered in embryos on the days examined. These data show that a teratogenic dose of PHT affects maternal folate metabolism. However embryonic folate metabolism, when measured in total embryos, was not affected.     

Effects of denzimol on benzodiazepine receptors in the CNS: Relationship between the enhancement of diazepam activity and benzodiazepine binding sites induced by denzimol in the rat

Denzimol, a new anticonvulsant drug with a pharmacological profile similar to that of phenytoin, enhances the ataxic and antimetrazol activity of diazepam in rats without affecting its activity against picrotoxin-induced seizures. $\text{In vivo}$ and $\text{ex vivo}$ denzimol enhances the binding of ³H-flunitrazepam in cortex and in hippocampus but not in cerebellum.The possibility of this increase in the number of benzodiazepine binding sites contributing in some way to enhancement of the depressive and anticonvulsant activity of diazepam is discussed.     

Antioxidant and GSH-related enzyme response to a single teratogenic exposure to the anticonvulsant phenytoin: temporospatial evaluation

BACKGROUND: It has been proposed that the anticonvulsant drug phenytoin (PHT) requires bioactivation to reactive intermediate(s) to achieve its recognized teratogenic potential and that embryonal detoxification power may play a fundamental role in the teratogenic response. On this basis, we sought to investigate the potential effects of a teratogenic exposure to PHT on the activities of antioxidant and GSH-related detoxifying enzymes in gestational murine tissues. METHODS: Pregnant Swiss mice were injected intraperitoneally with 0 (vehicle) or 65 mg/kg of PHT on gestation day (GD) 12 (plug day = GD 1). Biochemical determinations, including activities of glutathione transferase, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, catalase, and superoxide dismutase, were carried out on maternal and embryonic/fetal livers and in placentas on GD 14 and 19. RESULTS: The major findings of this study show that (1) organogenesis-stage conceptal tissues have detectable levels of all the tested enzymes; (2) most of the embryonic liver and placental enzymes investigated undergo a significant induction within 48 hr (GD 14) after PHT administration; and (3) in the same tissues a down-regulation of enzyme activities is noted near term (GD 19). CONCLUSIONS: Overall, these findings show that teratogenic exposure to PHT is associated with a modulation of reactive-intermediates-scavenging enzyme activities, and provide further support for role of generation of reactive intermediates in PHT-induced teratogenesis.     

The effects of valproate and phenytoin on the cAMP and cGMP levels in nervous tissue

Earlier studies have demonstrated that valproic acid (VPA) and phenytoin (PHT) influence the excitability properties of crayfish axons through different mechanisms. PHT was found to antagonize the electrophysiologic effects of VPA. The purpose of the present study was to determine if the electrophysiologic effects of VPA and PHT are correlated with changes in the cellular levels of either cAMP or cGMP as these substances are known to influence membrane excitability. It was found that PHT (0.1 mM) has no effect on the levels of either cAMP or cGMP within crayfish neural tissue. VPA (4.0 mM) also has no effect on cAMP levels. However, it does significantly reduce the levels of cGMP. Pretreatment of neural tissue with PHT has been shown to eliminate the effects of VPA on membrane excitability. It was found that this pretreatment has no influence on VPA's ability to reduce cGMP levels. The effect of VPA on cGMP levels is observed in the absence of spontaneous activity. Therefore, it is concluded that the observed reduction in cGMP levels does not represent the modulation of cGMP levels that is known to accompany activity. Two experiments demonstrate that the 4-mV depolarization of membranes by VPA can not account for its effect on cGMP levels. In the first, pretreatment with PHT abolished the depolarizing effect on VPA but not its effect on cGMP. In the second, a concentration of ouabain which depolarizes crayfish neural tissue by 8-10 mV without producing spike activity had no effect on either cAMP or cGMP levels. These experiments effectively dissociate the electrophysiologic response to VPA and PHT from changes in cyclic nucleotide levels.     

Anticonvulsant Efficacy of Drugs with Cholinergic and/or Glutamatergic Antagonism Microinfused into Area Tempestas of Rats Exposed to Soman

A group of antiparkinson drugs (benactyzine, biperiden, caramiphen, procyclidine, and trihexyphenidyl) has been shown to possess both anticholinergic and antiglutamatergic properties, making these agents very well suited as anticonvulsants against nerve agents. The first purpose of this study was to make a comparative assessment of the anticonvulsant potencies of the antiparkinson agents when microinfused (1 μl) into the seizure controlling area tempestas (AT) of rats 20 min before subcutaneous injection of soman (100 μg/kg). The second purpose was to determine whether cholinergic and/or glutamatergic antagonism was the effective property. The results showed that only procyclidine (6 μg) and caramiphen (10 μg) antagonized soman-induced seizures. Cholinergic, and not glutamatergic, antagonism was likely the active property, since atropine (100 μg), and scopolamine (1 μg) caused anticonvulsant effects, whereas MK-801 (1 μg), and ketamine (50 μg) did not. Soman (11 nmol) injected into AT resulted more frequently in clonic convulsions than full tonic–clonic convulsions. AT may serve as both a trigger site for soman-evoked seizures and a site for screening anticonvulsant potencies of future countermeasures. Special issue article in honor of Dr. Frode Fonnum.     

Ropizine concurrently enhances and inhibits [3H]dextromethorphan binding to different structures of the guinea pig brain: autoradiographic evidence for multiple binding sites

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.     

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.     

Anticonvulsant action of di-N-propylacetate combined with benzodiazepines, phenobarbital and phenytoin

The combined use of di-n-propylacetate with phenazepam, diazepam, phenobarbital or phenytoin was shown to be followed by reciprocal potentiation of the anticonvulsant activity of the drugs in a variety of experimental epileptic seizures in mice according to the tests of shock and antagonism with corasole and thiosemicarbazide. The potentiating effect of the subthreshold dose of di-n-propylacetate on anticonvulsant effects of benzodiazepines, phenobarbital and phenytoin was more pronounced than the effect of the drugs administered in the subthreshold doses on the anticonvulsant activity of di-n-propylacetate. Of both combinations, di-n-propylacetate plus benzodiazepines proved to be most efficacious one. The unidirectional effect of the combined drugs on the different stages of the development of GABA-ergic system inhibitory function in the CNS activity is assumed to be of importance in the mechanism of reciprocal potentiation.     

Phenytoin-induced stress protein synthesis in mouse embryonic tissue

Several proteins have been shown to be synthesized in response to various environmental stimuli, including treatment with teratogens. The role of these proteins in the teratogenic process is unknown. Pregnant A/J mice were treated with either a teratogenic or a non-teratogenic dose of the anticonvulsant drug, phenytoin (PHT). Protein synthesis in embryonic craniofacial (target) tissue or forelimb buds (non-target) was determined by incorporation of radiolabeled leucine and analysis by two-dimensional polyacrylamide gel electrophoresis. Synthesis of three proteins in target tissue and one protein in non-target tissue was stimulated by drug treatment. These results suggest that synthesis of specific stress proteins may serve as biomarkers of drug-target tissue interaction.     

Benzodiazepine Receptors on Primary Cultures of Mouse Astrocytes

Benzodiazepines bind to glial membranes on a single type of site, with a high affinity (KD = 5 x 10(-9) M) on about 100 fmol of sites per mg protein. The number of binding sites is increased when the membranes are treated with Triton X-100. Antiepileptic drugs such as clonazepam and phenobarbital and hypnotic drugs such as Ro-11-3128 and Ro-11-6896 are able in pharmacological concentrations to displace [3H]flunitrazepam from its glial binding sites.     

Effects of different classes of antiepileptic drugs on brain-stem pathways

Antiepileptic drugs probably act by preventing the spread of the abnormal paroxysmal activity from the epileptogenic focus to surrounding normal neurons. An investigation of the mechanism of action of established anticonvulsant drugs on normal neuronal systems may therefore offer useful insights into the pathogenesis of the seizure disorders that these drugs serve to control. Antiabsence drugs (ethosuximide, valproate) depress reticular inhibitory pathways. Drugs effective against generalized tonic-clonic seizures (phenytoin, carbamazepine, valproate) depress reticular excitatory pathways. Drugs that are also effective against trigeminal neuralgia (phenytoin, carbamazepine) also depress afferent excitation and facilitate segmental inhibition in the trigeminal complex. Drugs that depress afferent excitation and facilitate segmental inhibition but do not depress the reticular system (baclofen) are effective against trigeminal neuralgia but do not have clinical antiepileptic properties. These observations indicate that the ability to depress the reticular core is an important characteristic of antiepileptic drugs, and suggest that the reticular core is involved in the spread and generalization of clinical seizures.     

Antagonism of the anticonvulsant action of phenytoin,phenobarbital and acetazolamide by 6-hydroxydopamine

The ability of phenytoin, phenobarbital and acetazolamide to prevent the tonic extensor component of the maximal electroshock seizure was evaluated 30–50 days after treatment with 6-hydroxydopamine (6-OHDA). In the case of all 3 drugs, protection of rats from tonic extension was markedly reduced in the catecholamine amine deficient animals. However, the 6-OHDA-induced antagonism of anticonvulsant action was in all cases surmountable by increasing the dose of the anticonvulsant. These findings suggest a nonspecific antagonism of anticonvulsant action in 6-OHDA treated rats probably resulting from the increase in seizure susceptibility associated with catecholamine depletion.     

Anticonvulsant drugs and the genetically epilepsy-prone rat

Anticonvulsant drugs were evaluated in members of two colonies of genetically epilepsy-prone rats (GEPR). Virtually all of the animals in the first colony experience a wild running fit that terminates in a generalized clonic convulsion when they are stimulated by sound. According to our convulsion intensity scoring system, these animals have an audiogenic response score (ARS) of 3 and the colony is designated the GEPR-3 colony. In the second colony, more than 95% of the animals experience a wild running phase terminating in a tonic extensor convulsion when they are stimulated by sound. That is, they have an ARS of 9 and the colony is designated the GEPR-9 colony. All of the established antiepileptic drugs that were tested produced anticonvulsant effects in the GEPR. Three tricyclic antidepressant agents acted as anticonvulsants in doses substantially lower than the toxic doses that produced spontaneous convulsions. Two of the established anticonvulsants, phenobarbital and ethosuximide, produced anticonvulsant effects in very similar doses in members of GEPR-3 and GEPR-9 colonies. Valproic acid produced an anticonvulsant effect in GEPR-3 in significantly lower doses than in GEPR-9. Carbamazepine, phenytoin, imipramine, amitriptyline, and desipramine produced anticonvulsant effects in essentially equimolar doses and in each case the protective dose was significantly lower in GEPR-9 than in GEPR-3 colonies. GEPR did not experience the convulsive effects of imipramine, amitriptyline, and desipramine at lower doses than did control animals. Thus, these epilepsy-prone animals are no more likely to experience convulsions in response to overdose of one of these three drugs than are nonepileptic subjects.     

Pharmacology of GABA-mediated inhibition of spinal cord neurons in vivo and in primary dissociated cell culture

Summary In this paper it is shown that the postsynaptic GABA-receptor chloride ion channel complex is composed of several functional subunits. There are probably at least two stereospecific locations on the receptor for GABA-binding and both must be occupied to obtain an increase in chloride conductance. The interaction between these sites is uncertain but there could be either positive cooperativity between the sites or only a requirement that both sites are occupied without occupation of either site affecting the affinity for GABA of the other site. There is a chloride conductance channel coupled to the GABA receptor which opens for an average of 20 msec and has an average conductance of 18 pS. The GABA-coupled chloride channel may or may not have the same composition as the glycine coupled chloride channel.In addition to the GABA-recognition site and the chloride ion channel, GABA-receptors must have additional binding sites or modulator sites where drugs can bind to modify GABA activation of the GABA-receptor. The convulsant PICRO binds to a site which is independent of the GABA-recognition site and PICRO reduces GABA responses. Barbiturates and benzodiazepines augment GABA-responses without reducing GABA-binding and thus they must bind to a modulator site independent of the GABA recognition site. Whether or not this is the same site as the PICRO binding site is uncertain. Thus, the GABA-receptorchloride ion channel complex is composed of at least: 1) two GABA-binding sites; 2) a chloride ion channel; 3) a convulsant binding site (PICRO-binding site) and 4) an anticonvulsant binding site. This organization serves several obvious purposes. First, since two GABA-molecules are required to activate GABA-coupled chloride ion channels, the dose-response relationship for GABA is sigmoidal and steep. Thus minor shifts in GABA affinity will produce large alterations in GABA-responses and the GABA receptor can be easily modulated. Second, since the receptor has binding sites for convulsant and anticonvulsant compounds which decrease and increase GABA-responses, GABAergic inhibition can easily be modulated.