Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.

To characterize the subchronic oral toxicity of resveratrol, CD rats received daily gavage doses of 0, 200, 400, or 1000 mg resveratrol/kg/day, and beagle dogs received daily capsule doses of 0, 200, 600, or 1200 mg resveratrol/kg/day for 90 days. Resveratrol induced only minimal toxicity, consisting of dose-related reductions in body weight gain in female rats and both sexes of dogs, and a statistically significant increase in bilirubin levels in rats at the 1000 mg/kg/day dose. Clinical observations, hematology, ophthalmology, neurotoxicity evaluations (functional observational batteries), organ weights, and gross pathology provided no biologically significant evidence of resveratrol toxicity in either species. In rats, the high dose of resveratrol reduced the incidence of cardiomyopathy; no other microscopic changes were seen. Histopathologic changes in dogs were limited to minimal inflammatory infiltrates in the kidney and urinary bladder, which were not considered toxicologically significant. A cardiovascular safety pharmacology (telemetry) study in dogs revealed no evidence of resveratrol toxicity. Based on body weight effects, the No Observed Adverse Effect Level (NOAEL) for resveratrol was 200mg/kg/day in rats and 600 mg/kg/day in dogs. The apparent cardioprotective activity of resveratrol in rats demonstrates that its potentially beneficial activities may extend beyond efficacy in cancer prevention.

Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs.

Standardized green tea extract was evaluated for exposure and toxicity in Beagle dogs following oral dosing by capsules. The main component (-)-epigallocatechin gallate (EGCG) accounted for 56-72% of the material. A 9-month chronic study (0, 200, 500, and 1000 mg/kg/day) was done in fasted dogs to take advantage of the reported improved catechin bioavailability with fasting. Extensive morbidity, mortality, and pathology of many major organs led to its early termination at 6.5 months and prevented identification of the toxicity mechanisms. A follow-up 13-week study examined the exposure to and toxicity of the extract. In general, toxicities were less severe than in the chronic study during the same interval. Dosing in a fed state resulted in considerably lower and less variable exposure than found under fasted conditions. Toxicity was less frequent and of lesser severity with lower exposure but limited sample size and large variability prevented reaching that definitive conclusion. Differences in mortality and morbidity between the preliminary terminated chronic and follow-up subchronic studies with the same dose of the same drug lot and similar exposure were not fully resolved as there may be other as yet unclear confounding factors.

Direct determination of selenium in rat blood plasma by Zeeman atomic absorption spectrometry.

The method was developed to be applied for direct determination of selenium in rat plasma by graphite-furnace atomic absorption spectrometry with Zeeman background correction. Blood was obtained from CD rats of both sexes 2h after dosing in weeks 7 and 13 in order to acquire data on the levels of selenium in these animals during 13-week gavage administration of l-seleno-methylselenocysteine (SeMC), a new candidate chemopreventive agent under development. Application of the commonly used method of standard addition was found to be unsuitable to calculate the selenium content in rat plasma (within-run and between-run accuracy and precision parameters were less than 85%). Therefore, a new analytical method was developed. In this method, samples of rat plasma (50 microL) were diluted 10-fold with a reducing agent containing l-ascorbic acid, a modifier solution containing palladium chloride and Triton X-100. Samples were atomized in pyrolytically coated graphite tubes and peak height signals were measured. Selenium concentrations were determined by linear least squares regression analysis based on the standard curve generated in pooled rat blank plasma. Since selenium is normally present in plasma, a three-step approach was used to calculate selenium plasma levels. Initially selenium levels were determined based on the standard curve with selenium-spiked pool plasma. In the second step, background selenium levels in the pooled plasma were determined based on the same standard curve. In the third step, background level was added to the previously derived number. The relative errors were in the range from -4.6 to 11.4% (intra-day assay) and from -0.4 to 8.8% (inter-day assay) which proved good accuracy. The relative standard deviations were in the range from 1.88 to 4.70% (intra-day precision) and from 3.28 to 5.38% (inter-day precision). In rat plasma, the following dose-dependent selenium levels (mean+/-S.D.) in males and females, respectively, were observed at 13 weeks: 655.5+/-48.8 and 595.8+/-43.9 ng/mL (control group), 927.9+/-85.3 and 859.3+/-164.3 ng/mL (0.4 mg/kg per day dose group), 1238.9+/-182.4 and 1169.9+/-112.6 ng/mL (0.8 mg/kg per day dose group), and 1476.5+/-138.1 and 1320.1+/-228.6 ng/mL (2.0mg/kg per day dose group). No significant sex differences in selenium plasma levels were seen in the SeMC-treated groups. No significant differences in selenium plasma levels were seen between mean plasma levels at 7 and 13 weeks. The described method is simple, rapid, accurate, precise and can be easily applied in other laboratories for a large number of samples.

Subchronic oral toxicity studies of Se-methylselenocysteine, an organoselenium compound for breast cancer prevention.

Se-methylselenocysteine (MSC) is an organoselenium compound being developed for breast cancer chemoprevention. To characterize MSC toxicity, CD rats received daily gavage doses of 0, 0.5, 1.0, or 2.0 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day), and beagle dogs received daily gavage doses of 0, 0.15, 0.3, or 0.6 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day) for 28 days. In rats, MSC induced dose-related hepatomegaly in both sexes; mild anemia, thrombocytopenia, and elevated liver enzymes were observed in high dose females only. Microscopic pathology included hepatocellular degeneration (high dose males, all doses in females); arrested spermatogenesis (high dose males); and atrophy of corpora lutea (middle and high dose females). In dogs, MSC induced mild anemia in middle and high dose males, and in high dose females. Toxicologically significant microscopic lesions in dogs were seen only in the liver (peliosis and vacuolar degeneration in high dose males, midzonal necrosis in males in all dose groups). Based on liver pathology seen in female rats in all dose groups, the no observed adverse effect level (NOAEL) for MSC in rats is <0.5mg/kg/day. Based on alterations in hematology parameters and liver morphology in male dogs in all dose groups, the NOAEL for MSC in dogs is <0.15 mg/kg/day.

Computer-aided drug discovery and development (CADDD): in silico-chemico-biological approach.

It is generally recognized that drug discovery and development are very time and resources consuming processes. There is an ever growing effort to apply computational power to the combined chemical and biological space in order to streamline drug discovery, design, development and optimization. In biomedical arena, computer-aided or in silico design is being utilized to expedite and facilitate hit identification, hit-to-lead selection, optimize the absorption, distribution, metabolism, excretion and toxicity profile and avoid safety issues. Commonly used computational approaches include ligand-based drug design (pharmacophore, a 3D spatial arrangement of chemical features essential for biological activity), structure-based drug design (drug-target docking), and quantitative structure-activity and quantitative structure-property relationships. Regulatory agencies as well as pharmaceutical industry are actively involved in development of computational tools that will improve effectiveness and efficiency of drug discovery and development process, decrease use of animals, and increase predictability. It is expected that the power of CADDD will grow as the technology continues to evolve.

Comparing pharmacokinetic and pharmacodynamic profiles in female rats orally exposed to lovastatin by gavage versus diet.

The objective of this study was to assess how the dosing method (i.e., gavage versus diet) affects the absorption and disposition of lovastatin, as well as its effect on two biological markers of exposure, such as serum levels of cholesterol and triglycerides. In preclinical safety studies the test agent is normally administered by gavage, but in chemoprevention efficacy studies the test agent is usually administered with the diet. Therefore, extrapolation of safety and efficacy data from laboratory animals to humans should consider the influence of the method of administration on the absorption, disposition and effect of the drug. Lovastatin, a blood cholesterol-lowering drug with a short elimination half-life in humans, was used to assess the influence of two different dosing methods on the drug pharmacokinetics and pharmacodynamics. Plasma and liver concentrations of lovastatin and its active metabolite lovastatin-Na were measured in female rats at sequential times after administration. Serum concentrations of triglycerides and cholesterol were measured at similar times and used as biomarkers of effect. Significant differences in pharmacokinetics and pharmacodynamics were observed after administration of lovastatin by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of lovastatin and lovastatin-Na in plasma and liver, lower area under the concentration-time curve of lovastatin-Na in plasma and liver, and less of an effect on the serum concentrations of triglycerides and cholesterol than the corresponding diet dosing. Although no inverse linear relationship was observed between pharmacokinetic and pharmacodynamic markers, in the case of serum cholesterol a visual trend could be observed which might have proven significant had data from a larger number of dose levels been available. As in our previous study with sulindac, this study illustrates potential limitations in trying to extrapolate from data obtained using different dosing schemes to potential safety and efficacy in humans.

Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.

Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics.

In cancer chemopreventive studies, test agents are typically administered via diet, while the preclinical safety studies normally employ oral gavage dosing. Correspondence in pharmacokinetic and pharmacodynamic profiles between the two dosing approaches cannot be assumed a priori. Sulindac, a non-steroidal anti-inflammatory agent with potential chemopreventive activity, was used to assess effects of the two oral dosing paradigms on its pharmacokinetics and pharmacodynamics. Time-dependent concentrations of sulindac and its sulfone metabolite were determined in plasma and potential target organ, mammary gland. Prostaglandin E(2) was used as a pharmacodynamic biomarker and measured in mammary gland. An inverse linear relationship was detected between pharmacodynamic and pharmacokinetic markers, area under the curve for prostaglandin E(2) levels and sulindac sulfone concentrations, respectively, in the mammary tissue. Marked differences in pharmacokinetics and pharmacodynamics were observed after administration of sulindac by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of sulindac in plasma and mammary tissue, higher area under concentration-time curve in plasma and mammary tissue, and greater effect on prostaglandin E(2) levels than the corresponding diet dosing. This study illustrates potential pitfalls and limitations in trying to generalize based on data obtained with different oral dosing schemes and their extrapolation to potential efficacy and health risks in humans.

Chiral high-performance liquid chromatographic analysis of enantiomers of losigamone, a new candidate antiepileptic drug.

An assay based on a single-step liquid-liquid extraction from human plasma followed by high-performance liquid chromatography on a chiral column was developed for the measurement of enantiomers of a racemic new candidate antiepileptic drug. Excellent intra- and inter-assay accuracy and precision and recovery were demonstrated in the desired concentration range of 0.031 to 5.00 microg/ml. The method is free from interferences by other anticonvulsant drugs and their metabolites. The method is being used in a clinical trial of losigamone.

Potentially reactive cyclic carbamate metabolite of the antiepileptic drug felbamate produced by human liver tissue in vitro.

Felbamate (FBM) is a novel antiepileptic drug that was approved in 1993 for treatment of several forms of epilepsy. After its introduction, toxic reactions (aplastic anemia and hepatotoxicity) associated with its use were reported. It is unknown whether FBM or one of its metabolites is responsible for these idiosyncratic adverse reactions. Although the metabolism of FBM has not been fully characterized, three primary metabolites of FBM have been identified, i.e. 2-hydroxy, p-hydroxy, and monocarbamate metabolites. In addition, the monocarbamate metabolite leads to a carboxylic acid, which is the major metabolite of FBM in humans. Formation of the hydroxylated products of FBM involves cytochrome P450 enzymes, but the enzymes involved in the formation and further metabolism of the monocarbamate have not yet been elucidated. Recently, mercapturate metabolites of FBM have been identified in human urine, and a metabolic scheme involving reactive aldehyde metabolite formation from the monocarbamate metabolite has been proposed. The present study confirmed the formation of the proposed metabolites using human liver tissue in vitro. The aldehyde intermediates were trapped as oxime derivatives, and the cyclic equilibrium product (proposed as a storage and transport form for the aldehydes) was monitored directly by HPLC or GC/MS. Formation of putative toxic aldehyde intermediates and the major carboxylic acid metabolite of FBM was differentially effected with the cofactors NADP+ and NAD+. It is possible that the cofactors may influence the relative metabolism via activation and inactivation pathways.

In vitro glucuronidation of D-23129, a new anticonvulsant, by human liver microsomes and liver slices.

1. The metabolic profile of D-23129, a new anticonvulsant agent, was studied in vitro using human liver microsomes and fresh liver slices. 2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean total radioactivity not associated with the parent compound recovered from oxidative metabolism studies from three individual liver donors was 0.7% +/- 0.6 SD and was not significantly different from [14C]-D-23129 incubated with heat inactivated microsomes, mean = 0.5% +/- 0.4 SD. 3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct N-glucuronides as the primary metabolites. These metabolites were identified by electrospray ionization LC/MS. 4. The apparent Km for one of the glucuronide metabolites was determined in human liver microsome preparations from two individual liver donors to be 131 and 264 microM respectively, Vmax determined for the same microsomal preparations yielded 48.9 and 59.9 pmol/min/mg protein.

Stereoselective metabolism of a new anticonvulsant drug candidate, losigamone, by human liver microsomes.

Losigamone (LSG) is a new candidate anticonvulsant drug under going preclinical and clinical development. Metabolism of racemic (+/-)-LSG and its two enantiomers, AO-242 [(+)-LSG] and AO-294 [(-)-LSG], was studied using human liver microsomes and recombinant cytochrome P450 isozymes. HPLC with both UV and electrochemical detection was used for analysis of the incubation media. Five metabolites (M1, M2, M3, M4, and M5) were generated from racemic (+/-)-LSG by both human liver microsomes and recombinant enzymes. Stereoselective metabolism was observed when each enantiomer was incubated separately with human liver microsomes. M1 was the major metabolite produced from (+)-LSG, whereas M3, M4, and M5 were primarily produced from (-)-LSG. The production of M1 from (+)-LSG was markedly inhibited by (-)-LSG, indicating a metabolic enantiomer/enantiomer interaction. (+/-)-LSG enantiomers were selectively metabolized by recombinant cytochrome P450 2A6, and the metabolism of (+)-LSG and (-)-LSG by human liver microsomes was preferentially inhibited by coumarin, a cytochrome P450 2A6-selective compound.

The effects of D-23129, a new experimental anticonvulsant drug, on neurotransmitter amino acids in the rat hippocampus in vitro.

D-23129 [N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester] and D-20443 (dihydrochloride of D-23129) are promising anticonvulsant compounds with a broad spectrum activity in animal models of epilepsy. Their effects on de novo synthesis of excitatory (glutamate and aspartate) and inhibitory (GABA) amino acids were studied in rat hippocampal slices. Like phenytoin, carbamazepine, lamotrigine, losigamone, U54494A, and flupirtine, D-23129 and D-20443 were effective in preventing the effects of a chemoconvulsant, 4-aminopyridine, on de novo synthesis of the three amino acids. However, unlike the other compounds, D-23129 and D-20443 also preferentially increased the concentrations of newly synthesized GABA. Their effect on the neosynthesis of GABA was unique, dose dependent, and not tetrodotoxin sensitive. A total of 15 compounds (including standard, new and candidate anticonvulsants) either had no effect on new GABA or decreased it. Therefore, D-23129 and D-20443 exhibited two different effects on de novo synthesis of neurotransmitter amino acids, both of which could potentially be anticonvulsant in nature.

The effects of anticonvulsant compounds on 4-aminopyridine-induced de novo synthesis of neurotransmitter amino acids in rat hippocampus in vitro.

4-Aminopyridine, a voltage-dependent potassium channel blocker, causes tonic-clonic and electrographic seizures in vivo and evokes epileptiform activity and release of glutamate, aspartate and GABA in vitro. This study examined the effects of 4-aminopyridine (4AP) on de novo synthesis of neuroactive amino acids and a subsequent response to various anticonvulsant compounds (phenytoin, carbamazepine, phenobarbital, valproate, ethosuximide, diazepam, lamotrigine, felbamate, losigamone, U54494A, CPP, MK801 and CNQX) using a hippocampal slice preparation. 4-Aminopyridine had a minimal effect on total tissue concentrations of glutamate, aspartate, and GABA, but caused a significant increase in their de novo synthesis. Phenytoin, carbamazepine, lamotrigine, losigamone and U54494A were the only compounds which were effective in blocking the 4AP-induced increase in all newly synthesized amino acids. It appears that these compounds inhibit 4AP effects in this paradigm by blocking depolarization, probably at use-dependent voltage-sensitive sodium channels. Therefore, this paradigm may be useful in selectively identifying anticonvulsants which act by blocking depolarization.

The effects of anticonvulsant agents on 4-aminopyridine induced epileptiform activity in rat hippocampus in vitro.

Six anticonvulsant drugs, phenytoin (PHT), carbamazepine (CBZ), valproate (VPA), U-54494A, losigamone (LOS), and D-20443, were studied using rat hippocampal slices and standard electrophysiological techniques. The K+ channel blocker, 4-aminopyridine (4-AP), was used as neuronal stimulant. The extracellular parameters evaluated in areas CA3 and CA1 were: (1) interictal-type bursting, (2) evoked population spike (PS) amplitude, (3) latency to PS onset, and (4) duration of the excitatory postsynaptic potential (EPSP). VPA was ineffective in altering any of the parameters. PHT and CBZ partially reversed the increase in EPSP duration produced by 4-AP in area CA3, while the spontaneous bursting was not affected. The experimental drugs, U-54494A, LOS, and D-20443 (dihydrochloride salt of D-23129 from Asta Medica), tended to reverse to varying degrees the 4-AP effects, especially the increase in the EPSP duration. U-54494A tended to depress responses even under control conditions. LOS partially reversed the 4-AP excitation, but abolished bursting in only one of five slices. D-20443 abolished bursting in all slices. It also partially reversed the 4-AP induced increase in the EPSP duration without depressing the normal evoked potential. The results show that 4-AP induced changes in vitro can help differentiate drugs with similar in vivo spectrums of anticonvulsant activity. While the drug induced changes may not truly define the mechanisms of action of these promising new agents, these experimental anticonvulsants can be differentiated from standard agents using the experimental paradigm in this study.

A system for testing the development and reversal of anticonvulsant tolerance to benzodiazepines in mice.

Tolerance to the anticonvulsant effects of benzodiazepines limits their use in epilepsy treatment. Animal models producing tolerance have been developed, but they require repetitive injections over several days or use silastic capsules which must be made for each drug and do not provide a constant infusion rate. Alzet 2001 osmotic pumps deliver at a constant rate (1 microliter/h) and dosage can be easily adjusted. Various solvents, PEG 400, propylene glycol, 2% Tween, 50% DMSO, saline, Molecusol, and 0.5% methyl cellulose, were tried and found unsuitable because benzodiazepines were not maintained in solution or proconvulsant activity was seen. Tetraglycol was chosen as it did not demonstrate these shortcomings. Anticonvulsant activity was evaluated by PTZ i.v. tail infusion using forelimb clonus as the endpoint. This study describes a simple method for testing the development of tolerance and its reversal with flumazenil or ZK 93426. At 72 h of pump infusion with diazepam or flunitrazepam, tolerance to anticonvulsant activity was evident. Acute treatment with flumazenil or ZK 93426 reversed this tolerance. When flumazenil or ZK 93426 was given to diazepam tolerant mice, this reversal was complete. In flunitrazepam tolerant mice reversal with flumazenil was partial, but significant. When flumazenil was chronically coinfused with diazepam or flunitrazepam, anticonvulsant activity was antagonized. Similarly, when ZK 93426 was coinfused with diazepam, anticonvulsant activity was antagonized. The method described is suitable for screening putative anticonvulsant drugs for development of tolerance and the reversal of tolerance by other compounds.

Time-related loss of glutamine from hippocampal slices and concomitant changes in neurotransmitter amino acids.

A dramatic, time-dependent loss of L-glutamine was observed in mouse and rat hippocampal slices equilibrated in normal artificial CSF under static (no-flow) and superfused (constant-flow) conditions. Concomitant with the decline in L-glutamine, there was a significant, but less pronounced, decrease in levels of the neurotransmitter amino acids, gamma-aminobutyric acid, L-aspartate, and L-glutamate. The disappearance of L-glutamine was a result of diffusion from the tissue to the artificial CSF rather than chemical or biochemical transformation. The loss of amino acids from the hippocampal slices was prevented to different degrees by the addition of 0.5 mM exogenous L-glutamine to the artificial CSF. The levels of newly synthesized amino acids were also determined, because they may be more indicative of the neuronal activity than the total tissue levels of amino acids. The effects of perturbations in glutamine (length of the equilibration time and addition of exogenous glutamine) on newly synthesized glutamate were more pronounced under 4-aminopyridine-stimulated than control (unstimulated) conditions. Therefore, a loss of L-glutamine from the hippocampal slices may have neurophysiological effects and warrants further investigation.

GABA-mediated synaptic interaction between the visual and vestibular pathways of Hermissenda.

The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual-vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CNS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 microM), the cholinergic antagonist atropine (250 microM), and the serotonergic antagonist imipramine (50 microM). In contrast, the GABAA antagonist bicuculline (250 microM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 microM) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 microM) onto the terminal branches of the B cell resulted in a concentration-dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABAB agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from -70 to -80 mV and were consistent with mixed Cl- and K+ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual-vestibular interactions in this animal, and suggest a possible role of GABA in visual-vestibular associative learning.

Analysis of antiepileptic drugs.

This review discussed various analytical methods for the determination of antiepileptic drugs and their metabolites in biological tissues. The emphasis was on the reports published since their last review [J. T. Burke and J. P. Thenot, J. Chromatogr., 340 (1985) 199]. Both chromatographic and immunological procedure were cited and compared. Methods for individual and simultaneous quantitation of standard antiepileptic drugs and their metabolites were considered. In addition, a discussion of free drug determination and procedures for new candidate antiepileptic drugs were included.

Antagonism between intracerebroventricularly administered N-methyl-D-aspartate and bicuculline methiodide in induction of clonic seizures in mice.

N-Methyl-D-aspartate and bicuculline were administered alone or as a combination by intracerebroventricular injection to mice, and their convulsant activity was monitored. Both of these compounds elicited clonic seizures, though by different mechanisms. However, their simultaneous administration resulted in less than additive induction of clonic activity.