Concentration-dependence of multiple actions of phenytoin (PT) on mouse spinal cord neurons in primary dissociated cell culture was studied using intracellular microelectrode recording techniques. At concentrations of 2 to 50 micrograms/ml, PT did not alter resting membrane potential or input resistance. At 1 to 2 micrograms/ml, equivalent to therapeutic cerebrospinal fluid concentrations, PT limited the ability to sustain high-frequency repetitive firing of action potentials during long (500-2000 msec) depolarizing current pulses. There was a progressive reduction of maximal rate of rise (Vmax) of action potentials during the train until firing failed. Recovery of Vmax of single action potentials after repetitive firing was also prolonged. PT did not reduce Vmax of a single action potential at 1 to 2 micrograms/ml, but did so at 3 to 40 micrograms/ml in a voltage-dependent manner. Hyperpolarization partially reversed this reduction of Vmax. Thus, PT may slow recovery of sodium channels from inactivation. At concentrations above 3 micrograms/ml, PT reduced spontaneous neuronal firing with progressive increase in the number of quiescent neurons, reduced calcium-dependent action potential duration and amplitude, eradicated convulsant-induced paroxysmal bursting and augmented postsynaptic responses to iontophoretically applied gamma-aminobutyric acid. Glutamic acid responses were unaffected at PT concentrations of 10 micrograms/ml or less. These actions occurred at concentrations equivalent to toxic cerebrospinal fluid levels in patients and may be related to PT-induced toxicity. We suggest that limitation of sustained high-frequency repetitive firing may account, at least in part, for the anticonvulsant efficacy of PT.
The amino acid antiepileptic drug (AED) gabapentin (GBP) is indicated for adjunctive use in the treatment of partial seizures with or without becoming secondarily generalized in individuals older than 12 years. GBP was about as potent as phenytoin in the maximal electroshock test, but had a different profile of efficacy than standard antiepileptics in a range of animal models. Possible mechanisms of action include biochemical effects enhancing the ratio of gamma-aminobutyric acid (GABA) to glutamate, ion-channel actions (direct or indirect), and/ or enhancement of nonsynaptic GABA release. The anticonvulsant effect appears to depend on concentration of gabapentin in neurons, presumably by the L-system amino acid transporter that has been implicated in absorption from the gut. Data from studies for U.S. Food and Drug Administration (FDA) approval suggested a direct relationship of clinical response to dose and efficacy did not plateau at the doses used. The maximally effective dose, relationship of efficacy to blood level, and maximum tolerable dose are not yet known conclusively. Lack of significant binding to plasma proteins and lack of liver metabolism contribute to the absence of known limiting drug-drug interactions, particularly with other AEDs. Excretion intact in the urine affords dose adjustment on the basis of creatinine clearance. A half-life of approximately 7 h necessitates multiple doses daily for many individuals. The medication is well tolerated, in general. Side effects tend to be mild to moderate in intensity, most frequently affect the central nervous system, and resolve with time in many individuals. GBP has been prescribed for approximately 70,000 individuals worldwide without untoward incidence of severe systemic toxicity to date. Safety data continue to accumulate. GBP has been labeled category C on the basis of effects on rodent fetuses. Experience with use in pregnant women is limited and human teratogenic effects have not been reported. Data from ongoing monotherapy trials will help to clarify the range of clinical utility of gabapentin.
The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.
Effects of benzodiazepines (BDZs) and beta carbolines (beta CCs) on sustained repetitive firing at high frequency (SRF) of action potentials of mouse spinal cord neurons in cell culture were examined using intracellular recording techniques. In control medium neurons responded to depolarizing current pulses with SRF. Limitation of SRF was produced by the anticonvulsant BDZs (diazepam, clonazepam, nitrazepam and lorazepam) at low to mid nanomolar concentrations, by a convulsant BDZ which does not bind to high affinity BDZ receptors (Ro 5-4864) at high nanomolar concentrations and by a BDZ receptor weak partial agonist (Ro 15-1788) at micromolar concentrations. The limitation of SRF was accompanied by use- and voltage-dependent reduction of maximal rate of rise (Vmax) of sodium-dependent action potentials. Partial agonist and inverse agonist beta CCs did not limit SRF at concentrations up to 200 nM. The limitation of SRF by diazepam was not prevented by inverse or partial agonists at the BDZ receptor, including Ro 15-1788 and the beta CCs. These findings suggest that limitation of SRF was produced by binding of BDZs, but not beta CCs, to voltage-dependent sodium channels and not to high affinity central BDZ receptors, and that BDZs limit SRF by slowing recovery of sodium channels from inactivation. We propose that the limitation of SRF may contribute to the efficacy of BDZs against generalized tonic-clonic seizures and status epilepticus.
The actions of clinically used anticonvulsant drugs on sustained high frequency repetitive firing of action potentials and on responses to gamma aminobutyric acid (GABA) have been determined using mouse neurons in cell culture, and a classification of anticonvulsant drug actions has been developed based on these cellular actions. Actions of the anticonvulsant drugs were accepted as clinically relevant only if they occurred at concentrations achieved in cerebrospinal fluid or in plasma unbound to plasma proteins. Based on their cellular mechanisms of actions, drugs have been divided into 3 categories: (1) Phenytoin, carbamazepine and valproic acid limited sustained high frequency repetitive firing but did not alter GABA responses. (2) Phenobarbital and the benzodiazepines, clonazepam, diazepam and nitrazepam, augmented postsynaptic GABA responses. These drugs limited sustained high frequency repetitive firing only at concentrations above the therapeutic range in ambulatory patients, but equal to concentrations achieved in the acute treatment of status epilepticus. (3) Ethosuximide failed to reduce sustained high frequency repetitive firing or enhance GABA responses even at supertherapeutic concentrations. Limitation of sustained high frequency repetitive firing by anticonvulsant drugs correlated well with efficacy against generalized tonic-clonic seizures in man and against maximal electroshock seizures in experimental animals. Enhancement of postsynaptic GABA responses correlated with efficacy against generalized absence seizures in man and against pentylenetetrazol seizures in animals. Ethosuximide, however, did not alter GABA responses or sustained high frequency repetitive firing suggesting that its action against generalized absence seizures in man and pentylenetetrazol seizures in experimental animals occurs by an additional, as yet unknown, mechanism.
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