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Biomedical subjects

H J Kupferberg

Publications and source records attributed to H J Kupferberg.

At least 19 recordsLinked to original sources
PubMed1995

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.

4-Aminopyridine↗
PubMed1995

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.

4-Aminopyridine↗
PubMed1993

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.

Animals↗
PubMed1993

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.

4-Aminopyridine↗
PubMed1992

Strategies for identifying and developing new anticonvulsant drugs.

The identification of new anticonvulsant drugs depends on the use of different animal models of epilepsy. The models should be mechanism-independent, able to screen a large number of compounds, at limited cost and technical expertise. Primary screening models include genetic or reflex models of epilepsy and electrically and chemically induced seizures. Once active compounds have been identified, more advanced mechanistic and seizure-specific models are needed to refine the choice of a lead compound. These can be either in vivo or in vitro models. Models known to interact with specific receptors or the production of the putative neurotransmitters of neural excitability or inhibition are valuable in assessing possible mechanisms of action. In vitro models have evolved as important tools in correlating changes in electrical phenomena and therapeutic spectrum. The use of the hippocampal slice and the cultured neuron permits classification of anticonvulsant activity based on cellular actions of the drug. Interactions by the experimental drugs with specific subcellular fractions of the central nervous system augment information on possible mechanisms of action. The final choice of compounds for development requires synthesizing and comparing all of the pharmacodynamic information with the pharmacokinetic and toxicologic data. In the final analysis, no single animal model of epilepsy known today can assure the development of better drugs for all treatment of the epilepsies.

Animals↗
PubMed1990

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.

Amino Acids↗
PubMed1990

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.

Animals↗
PubMed1989

Antiepileptic drug development program: a cooperative effort of government and industry.

The most important step in antiepileptic drug discovery is the choice of an appropriate animal model for the initial screening as well as for the more complex procedures that elucidate mechanisms of action. The currently available models fall short in their inability to identify all drugs for all types of seizures in a mechanism-independent manner. Nevertheless, spontaneous models of epilepsy are the most commonly used, and chemically or electrically induced seizures in rodents can also identify potential anticonvulsants. In the latter models, the intensity of the seizure stimulus is of paramount importance. The Antiepileptic Drug Development Program evaluates approximately 800 compounds each year, using two models for preliminary screening. One model assesses the ability of a compound to prevent seizure spread; the other weighs the ability to raise seizure threshold. In vivo tests, featuring amygdala- and corneal-kindled seizures, and in vitro assays, employing gamma-aminobutyric acid (GABA) receptors and synaptosomal uptake of adenosine, define drug-drug interactions and elucidate the pharmacological profiles of potential anticonvulsants.

Animals↗
PubMed1988

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.

2-Amino-5-phosphonovalerate↗
PubMed1987

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.

Amino Acids↗
PubMed1986

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.

Anticonvulsants↗
PubMed1986

Enhanced bursting activity in the CA3 region of the mouse hippocampal slice without long-term potentiation in the dentate gyrus after systemic pentylenetetrazole kindling.

The repeated administration of subconvulsant doses of pentylenetetrazole (24 mg/kg, i.p.) produced chemically kindled seizures in 16 of 20 mice. Hippocampal slices prepared from the mice with kindled seizures were tested for input-output characteristics in the dentate gyrus, and for spontaneous burst discharge frequency in area CA3. The kindled slices displayed no change in the magnitude of the evoked granule cell excitatory postsynaptic potential (pEPSP) to a given stimulus intensity applied to the perforant path, nor in magnitude of the granule cell population spike for a given pEPSP. Although long-term potentiation of synaptic transmission has been proposed as the cellular mechanism of kindling, these results indicate either that long-term potentiation may not underlie kindling or that systemic pentylenetetrazole kindling and focal electrical kindling may be accomplished by different mechanisms. Hippocampal slices from kindled animals did, however, show an increased incidence of spontaneous burst discharges in area CA3 pyramidal neurons in both the absence and the presence of pentylenetetrazole in the bathing medium.

Action Potentials↗
PubMed1986

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.

Adult↗
PubMed1985

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.

Animals↗