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.

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.

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.

Use of stable isotopes and gas chromatography-mass spectrometry in the study of different pools of neurotransmitter amino acids in brain slices.

A method was developed for simultaneous determination of endogenous and newly synthesized neurotransmitter amino acids (4-aminobutyric acid, glutamate and aspartate) and glutamine in brain in vitro. Brain slices were incubated in artificial cerebrospinal fluid in the presence of 13C-labeled precursors (glucose, pyruvate or acetate). After the incubation, the slices were homogenized in cold 80% ethanol and the supernatants were evaporated to dryness. The resultant residues were derivatized with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide and analyzed by capillary gas chromatography-mass spectrometry in the electron-impact mode. N(O)-tert.-Butyldimethylsilyl derivatives of the naturally occurring amino acids, their 13C-enriched counterparts and deuterated internal standards were detected as their [M-57]+ fragments using selected-ion monitoring. The method was shown applicable to studying compartmentation of neurotransmitter amino acids.

Effects of pharmacological manipulations on basal and newly synthesized levels of GABA, glutamate, aspartate and glutamine in mouse brain cortex.

Concentrations of basal and newly synthesized inhibitory (gamma-aminobutyric acid, GABA) and excitatory (glutamate and aspartate) neurotransmitter amino acids and glutamine were determined in mouse brain cortex. Isotopic enrichment following an intravenous infusion of a stable-labeled precursor, [13C6]D-glucose, was used to estimate the newly synthesized amino acid content. Effects of various pharmacological agents (valproate, aminooxyacetic acid, 3-mercaptopropionic acid, N-methyl-D-aspartate, and 2-amino-7-phosphonohepatanoic acid) were evaluated. The effects of 3-mercaptopropionic acid (an inhibitor of glutamate decarboxylase, a GABA-synthesizing enzyme) were restricted to the GABAergic system. On the other hand, N-methyl-D-aspartate (an agonist of a glutamate receptor subtype) was selective for the glutamate-glutamine system, and its effects were prevented by its selective antagonist, 2-amino-7-phosphonoheptanoic acid. In some cases, divergent effects were observed on basal and new amino acids. This suggested that basal and new amino acids may represent different compartments. The anticonvulsant drug valproate caused an increase in basal but a decrease in newly synthesized GABA. Aminooxyacetic acid caused a dramatic increase in basal GABA without affecting the newly synthesized GABA. This approach may be useful in studying compartmentation and fluxes of neurotransmitters.

Determination of 4-aminobutyric acid, aspartate, glutamate and glutamine and their 13C stable-isotopic enrichment in brain tissue by gas chromatography-mass spectrometry.

A selected-ion monitoring method was developed for measuring 4-aminobutyric acid, aspartate, glutamate, and glutamine in brain tissue. Natural isotopes of these amino acids and their stable-isotopic enrichment following intravenous infusion of a precursor, [13C]glucose, were quantitated. Frozen mouse brain tissue was homogenized in cold 80% ethanol, and the supernatant, equivalent to 1 mg of wet weight brain tissue, was extracted using solid-phase bonded silica ion-exchange columns. Aspartate and glutamate (dicarboxylic acids) were isolated from strong anion-exchange columns, whereas 4-aminobutyric acid and glutamine (neutral amino acids) were isolated from strong-cation exchange columns. n-Butyl ester pentafluoropropionyl amide derivatives of these amino acids were analyzed by gas chromatography-mass spectrometry using a methane positive chemical ionization mode after gas chromatographic separation on a wide-bore, fused-silica capillary column. The method is applicable to determination of brain concentrations of these amino acids as well as their fluxes following administration of a stable-isotopic tracer.

Determination in plasma of a new antiepileptic drug, dl-(5 alpha,9 alpha,11S*)-5,6,9,10-tetrahydro-N,N-dimethyl-5,9-methanobenzocycloo cten-11-amine hydrochloride, and its N-desmethyl metabolite by liquid-solid extraction and capillary gas chromatography.

We have developed a sensitive and accurate method for the determination in plasma of the antiepileptic drug dl-(5 alpha,9 alpha,11S*)-5,6,9,10- tetrahydro-N,N-dimethyl-5,9-methanobenzocycloocten-11-amine hydrochloride and its N-desmethyl metabolite. The extraction procedure utilizes base-treated disposable C2 solid-phase columns, with the analyte eluted with organic solvent. Nitrogen-selective gas chromatography is used for detection. Linear regression analysis showed that the method is linear between 4 and 1500 ng/ml for the parent drug and between 8 and 3000 ng/ml for the N-desmethyl metabolite. Intra- and inter-day variability, as shown by the coefficient of variation, is less than 8% for both compounds. The method is applicable to routine plasma determination of both these compounds in clinical pharmacokinetic studies.

Single and multiple dose kinetics of a new antiepileptic drug, Org 6370, and its desmethyl metabolite, Org 6363.

The pharmacokinetic parameters of the new antiepileptic drug, Org 6370, and its desmethyl metabolite, Org 6363, were studied in healthy male volunteers. Plasma concentrations of the compounds were determined by a new method using liquid-solid extraction and capillary gas-chromatographic separation with a nitrogen-selective detector. The kinetic parameters obtained after a single oral dose of Org 6370 were not good predictors of multiple-dose parameters. With long-term treatment, there was unanticipated accumulation of the parent drug and especially the metabolite. The clinical implication of these findings is that caution must be exercised in clinical trials of Org 6370.

Disposition of mephenytoin and its metabolite, nirvanol, in epileptic patients.

We investigated the conversion of mephenytoin to nirvanol in five patients with uncontrolled complex partial seizures. After a 50-mg single oral dose, mean peak mephenytoin level was 0.48 microgram/ml and nirvanol 0.37 microgram/ml. After 400 mg, peak mephenytoin level was 3.9 micrograms/ml and nirvanol 2.5 micrograms/ml. On 400 mg daily, mephenytoin reached a mean steady-state level of 1.5 micrograms/ml. Nirvanol mean steady-state level was 18 micrograms/ml. Mean plasma half-life was 17 hours for mephenytoin and 114 hours for nirvanol. Two patients had reduced seizures during mephenytoin therapy and one a transient increase during drug withdrawal. No toxicity was seen, but mephenytoin was not more effective than phenytoin.

Relationship between pentylenetetrazol-induced seizures and brain pentylenetetrazol levels in mice.

Pentylenetetrazol (PTZ) is often used in experimental models of epilepsy. The relationship of the PTZ-induced seizure sequence of myoclonus, clonus and hindlimb extension (TE) to brain PTZ levels has not been reported. This study examined this relationship and determined how different routes of PTZ administration affected brain PTZ uptake and seizure development. The critical brain PTZ level for onset of clonus ranged from 20 to 50 microg/g. Brain PTZ uptake was rapid after I.P. injection of PTZ convulsant dose (CD55) for clonus/and clonus onset occured at 4.0+/- 1.6 min. uptake was slower after S.C. administration; clonus onset occurred at 9.9 +/- 3.7 min. at a CD for TE (CD40), clonus onset occured at 5.1 +/- 3.0 and 2.4 +/- 2.4 min for S.C. and I.P. routes of administration, respectively. TE onset did not appear to depend solely on brain PTZ levels were falling . Factors that could modulate the appearance of TE are discussed.

Gas chromatographic-mass spectrometric studies on the metabolic fate of ethotoin in man.

Unchanged ethotoin and 11 metabolic products of ethotoin were detected in the urine of subjects (2 men and 1 woman) receiving ethotoin. Nine of these products were identified by comparison of their retention times and mass spectra with those of authentic synthetic samples. Hydroxylation of the hydantoin ring at the 5-position produced 5-hydroxyethotoin and 5-hydroxy-5-phenylhydantoin. Aryl hydroxylation resulted in the formation of p-hydroxyethotoin, o-hydroxyethotoin, m-hydroxyethotoin, 3-methoxy-4-hydroxyethotoin, and 3,4-dihydroxyethotoin. Most of these were excreted as the glucuronide conjugate. A dihydrodiol of ethotoin and 3-ethyl-5-hydroxy-5-(4-hydroxyphenyl)hydantoin were isolated along with unchanged ethotoin and 5-phenylhydantoin. 2-Phenylhydantoic acid was also isolated and was shown to have the (R)(-)-configuration.

A single-dose study of mexiletine (Kö 1173).

Serum concentrations of mexiletine after a single dose were determined in 8 adult Caucasian males with complex partial seizures who were continuing to receive other antiepileptic drugs. Two patients each received a single dose of either 100, 200, 300, or 400 mg mexiletine. Serum concentrations were determined by two gas chromatographic methods. Serum concentrations ranged up to 795 ng/ml. Peak concentrations occurred 1 to 3 hr after administration of the drug and were significantly different between the 100- and 300-mg, 100- and 400-mg, 200- and 300-mg, and 200- and 400-mg doses. Differences between the other doses were not significant. Serum concentrations declined monoexponentially. Half-life ranged from 2.7 to 7.2 hr. Numerous papers have appeared in the European literature on the use of mexiletine to treat cardiac arrhythmias. Preliminary studies in the United States suggest the use of mexiletine as an adjunct for therapy of epilepsy.