Harold H. Wolf

Topiramate enhances GABA-mediated chloride flux and GABA-evoked chloride currents in murine brain neurons and increases seizure threshold

The anticonvulsant topiramate is effective in laboratory animals against maximal electroshock seizures, amygdala kindling, and spike-wave discharges and has demonstrated efficacy in humans for the treatment of complex partial seizures. However, its mechanism of action has yet to be clearly elucidated. When the chloride-sensitive fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used as a tool for estimating the effect of anticonvulsant drugs on GABA receptor function, topiramate was observed to enhance GABA-stimulated chloride (Cl-) flux. At a therapeutic concentration, topiramate (10 microM) enhanced GABA-stimulated (10 microM) Cl- influx into cerebellar granule neurons but did not significantly increase Cl- influx alone. Phenytoin (10 microM) and acetazolamide (300 microM) did not enhance GABA-stimulated Cl- influx. In patch-clamp electrophysiological studies, topiramate also enhanced GABA-evoked whole cell Cl- currents in mouse cerebral cortical neurons in culture. In vivo anticonvulsant studies confirmed that topiramate, like phenytoin, is primarily effective against tonic extension seizures induced by maximal electroshock and is ineffective against clonic seizures induced by the subcutaneously administered chemoconvulsants pentylenetetrazol (PTZ), bicuculline (Bic), and picrotoxin (Pic). In contrast to phenytoin, topiramate, at a dose equivalent to the MES median effective dose (ED50), was found to elevate seizure threshold as estimated by the intravenous PTZ seizure threshold test. Taken together these results support the conclusion that enhancement of GABA-mediated Cl- flux may represent one mechanism that contributes to the anticonvulsant activity of topiramate.

Topiramate Modulates GABA-Evoked Currents in Murine Cortical Neurons by a Nonbenzodiazepine Mechanism

Purpose: These studies further investigate the ability of topiramate (TPM) to enhance γ‐aminobutyric acid (GABA)‐mediated inhibition through a benzodiazepine‐insensitive pathway.

D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures

The anticonvulsant activity of the novel drug D-23129 (N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester) was evaluated in animal models of epileptic seizures. D-23129 was active after oral and intraperitoneal administration in rats and mice in a range of anticonvulsant tests at nontoxic doses. The compound was active against electrically induced seizures (MES, ED50 rat p.o. = 2.87 mg/kg), against seizures induced chemically by pentylenetetrazole (s.c. PTZ, ED50 mouse p.o. = 13.5 mg/kg), picrotoxin and N-methyl-D-aspartate (NMDA) and in a genetic animal model, the DBA/2 mouse. It was not active against seizures induced by bicuculline and strychnine. Motor impairment, evaluated with the rotarod test and by observation in the open field, was minimal at doses showing anticonvulsant activity. D-23129 was very effective in elevating the threshold for electrically and chemically induced seizures. Considering the dose increasing the MES threshold by 50% (TID50 mouse i.p. = 1.6 mg/kg; TID50 rat i.p. = 0.72 mg/kg) and the TD50 obtained in the rotarod test, the protective index of D-23129 is better than that of valproate and phenytoin. During 14 days chronic oral treatment with 15 mg/kg, no development of tolerance was observed. D-23129 thus presents an orally active, safe, broad spectrum anticonvulsant agent, which is structurally unrelated to anticonvulsants currently used. We expect that D-23129 will improve the treatment of refractory seizures in humans.

A Neuropharmacological Evaluation of Felbamate as a Novel Anticonvulsant

Summary: Felbamate (2‐phenyl‐1,3‐propanediol dicarbamate, FBM) was subjected to a series of carefully selected in vivo and in vitro tests to provide additional insight into mechanism of action, margin of safety, and clinical potential. FBM was effective against intracere broventricular (i.c.v.) N‐methyl‐D‐aspartate (NMDA)‐induced clonus and i.c.v. NMDA‐ and quisqualic acid (quis)‐induced forelimb tonic extension in mice and ineffective against i.c.v. quis‐induced clonus in mice. FBM was also effective in preventing the expression of Stage 5 kindled seizures in corneal‐kindled rats. The calculated protective indices (rotorod median toxic dose divided by anticonvulsant median effective dose) ranged from 28 to 146 for those tests in which FBM displayed activity. With the in vitro tests, FBM did not significantly displace [3H]MK‐801 from its binding site. In contrast, FBM was effective in blocking sustained repetitive firing in mouse spinal cord neurons grown in tissue culture (median inhibitory concentration 67 μg/ml). This effect on repetitive firing suggests indirectly that FBM modulates sodium channel conductance. The results, when compared to similar data for phenytoin, carbamazepine, valproate, and ethosuximide, support the concept that FBM is a relatively nontoxic agent with a unique profile of anticonvulsant action, a broad margin of safety, and a clinical potential that includes at least generalized tonic‐clonic and complex partial seizures.

Felbamate modulates the strychnine-insensitive glycine receptor

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a novel anticonvulsant substance whose mechanism of action is not clearly understood. The present investigation examined its ability to modulate the strychnine-insensitive glycine receptor associated with the N-methyl-D-aspartate (NMDA) receptor. Felbamate decreased the magnitude of glycine (100 microM)-enhanced NMDA (100 microM)-induced intracellular calcium ([Ca2+]i) transients in mouse cerebellar granule cells which had been loaded with the Ca(2+)-sensitive fluorescent probe indo-1 acetoxymethyl ester (indo-1/AM). This effect of felbamate was concentration dependent, with a maximal effect observed at 300 microM (65 +/- 4% of control). In the Frings audiogenic seizure-susceptible mouse model of reflex epilepsy, the glycine agonist D-serine (150 nmol, i.c.v.) completely blocked the anticonvulsant activity of a maximally effective dose of felbamate (19 mg/kg, i.p.). This effect of D-serine could be reversed by increasing the administered dose of felbamate to 29 mg/kg. Furthermore, administration of D-serine (300 nmol, i.c.v.) to felbamate-treated Frings mice produced a parallel right shift in felbamates anticonvulsant dose-response curve (ED50s: 9.4 mg/kg for felbamate vs. 17.7 mg/kg for felbamate + D-serine). The results obtained in this investigation suggest that the ability of felbamate to modulate the strychnine-insensitive glycine receptor may be physiologically and behaviorally relevant to its anticonvulsant mechanism of action.

The anticonvulsant profile of rufinamide (CGP 33101) in rodent seizure models

Purpose: To evaluate the anticonvulsant profile and behavioral toxicity of rufinamide in animal seizure models compared to the established antiepileptic drugs (AEDs): phenytoin, phenobarbital, valproate, and ethosuximide, or vehicle.

A Self‐Complementary, Self‐Assembling Microsphere System: Application for Intravenous Delivery of the Antiepileptic and Neuroprotectant Compound Felbamate

Felbamate (FBM) is a novel antiepileptic drug (AED) and neuroprotectant (NP) compound that interacts with strychnine-insensitive (SI) glycine receptors in brain (IC(50) = 374 microM). FBM concentrations required to interact with SI glycine receptors are consistent with brain levels following oral and intraperitoneal administration of AED and NP doses. Because of the solubility limits of FBM, an intravenous (iv) form has not been developed. Nevertheless, an iv form could be important for the treatment of disorders such as status epilepticus and neuronal damage due to hypoxic/ischemic events. Substituted diketopiperazines precipitate in acid to form microspherical particles of uniform size ( approximately 2 microm). The microsphere system entraps drugs on precipitation and dissolves near physiological pH to release the drug cargo. Therefore, microspheres were used to produce an iv formulation of FBM. Mice were administered the FBM/microsphere (20-60 mg/kg FBM) and tested for protection against tonic extension seizures using maximal electroshock. The FBM/microsphere was effective in a time- and dose-dependent manner following iv administration. The median effective dose (ED(50)) for protection against MES seizures at 30 min was 27.2 mg/kg [95% confidence interval (CI) = 20.8-33.4, slope = 6.5]. The ED(50) for minimal motor impairment at 30 min was 167 mg/kg (95% CI = 155-177, slope = 28.1). Thus, the feasibility of encapsulating FBM or similar aqueous insoluble compounds in a microsphere system with delivery by the iv route for treatment of epilepsy and various central nervous system disorders has been clearly demonstrated. Studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Pentylenetetrazol May Discriminate Between Different Types of Benzodiazepine Receptors

Abstract: The effect of various concentrations of pentylenetetrazol (PTZ) on [3H]flunitrazepam binding to benzodiazepine receptors was investigated by Hofstee and Hill plot analyses. These analyses indicate the presence of two PTZ binding sites in forebrain, whereas a single PTZ binding site is present in cerebellum. The relative proportions of the two PTZ binding sites in forebrain are close to those of benzodiazepine Type II and Type I receptors, respectively. These results suggest that PTZ may actually discriminate between different types of benzodiazepine receptors.

Anticonvulsant Profiles of the Potent and Orally Active GABA Uptake Inhibitors SK&F 89976‐A and SK&F 100330‐A and Four Prototype Antiepileptic Drugs in Mice and Rats

Summary: The anticonvulsant profiles of two potent and orally active 7‐aminobutyric acid (GABA) uptake inhibitors, l‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid hydrochloride (SK&F 89976‐A) and 1‐(4,4‐diphenyl‐3‐butenyl)‐l,2,5,6‐tetrahydro‐3‐pyridine‐carboxylic acid hydrochloride (SK&F 100330‐A), were determined with a battery of well‐standardized tests in mice and rats and compared with the profiles of pheny‐toin (PHT), carbamazepine (CBZ), valproate (VPA) and clonazepam (CZP) when subjected to the same tests. ED50 values were calculated and compared with TD50 values for minimal motor impairment to provide protective indexes (PI = TD50/ED50). The anticonvulsant profiles of SK&F 89976‐A and SK&F 100330‐A were similar and suggest that these compounds raise the threshold for seizure initiation rather than inhibit seizure spread. Like intraperitoneal (i.p.) PHT, CBZ, VPA, and CZP, SK&F 89976‐A and SK&F 100330‐A inhibited seizures in corne‐ally kindled rats. The profiles of SK&F 89976‐A and SK&F 100330‐A were most similar to that of CZP and virtually opposite to that of PHT. Intraperitoneal SK&F 100330‐A provided complete protection against pentyl‐enetetrazol‐induced seizures [subcutaneous (s.c.) PTZ] in mice but was ineffective against seizures induced by maximal electroshock (MES) at doses slightly greater than its TD50. SK&F 100330‐A provided complete protection against picrotoxin‐induced seizures (s.c. Pic) and against both clonus and forelimb tonic extension induced by NMDA Af‐methyl‐D‐aspartate [intracerebralventricular (i.c.v.) NMDA] in mice; however, SK&F 100330‐A was ineffective against seizures induced by bicuculline (s.c. Bic) and strychnine (s.c. Strych) at doses slightly greater than its TD50. SK&F 89976‐A was similar but provided partial protection against NMDA‐induced clonus. SK&F(R)‐89976‐A, which is more potent than the (5> enantiomer as a GABA uptake inhibitor in vitro, was also more potent in the PTZ test. SK&F 89976‐A and SK&F 100330‐A also inhibited PTZ‐induced seizures in mice and rats after oral (p.o.) administration but were more potent in mice. Both substances were adequately absorbed after p.o. administration. Neither compound inhibited MES in mice or rats after p.o. administration. The data support the concept that SK&F 89976‐A and SK&F 100330‐A may be useful in treatment of partial seizures.

Effect of GABA agonists on the neurotoxicity and anticonvulsant activity of benzodiazepines

Progabide (50 mg/kg, i.p.), a GABA receptor agonist, significantly decreases the median minimal neurotoxic dose (TD50) of clobazam, chlordiazepoxide, and diazepam; the receptor binding of these substances is highly enhanced by muscimol. Progabide has no significant effect on the TD50 of clonazepam and triazolam; the receptor bindings of these substances is either only slightly enhanced or not altered by muscimol. Progabide also significantly decreases the median antimaximal electroshock dose (MES ED50) of all the benzodiazepines tested. However, progabide has no effect on the median antipentylenetetrazol dose (PTZ ED50) of the benzodiazepines. Likewise, THIP (2.5 mg/kg, i.p.) significantly decreases the TD50 of chlordiazepoxide but not that of triazolam. THIP significantly decreases the MES ED50 of chlordiazepoxide and triazolam but has no effect on the PTZ ED50 of these two substances. The above data suggest that benzodiazepine receptors linked to GABA receptors contribute to the minimal neurotoxicity and anti-MES activity but not to the anti-PTZ activity of benzodiazepines.