Valproic acid extends Caenorhabditis elegans lifespan.

Aging is an important biological phenomenon and a major contributor to human disease and disability, but no drugs have been demonstrated to delay human aging. Caenorhabditis elegans is a valuable model for studies of animal aging, and the analysis of drugs that extend the lifespan of this animal can elucidate mechanisms of aging and might lead to treatments for age-related disease. By testing drugs that are Food and Drug Administration approved for human use, we discovered that the mood stabilizer and anticonvulsant valproic acid (VA) extended C. elegans lifespan. VA also delayed age-related declines of body movement, indicating that VA delays aging. Valproic acid is a small carboxylic acid that is the most frequently prescribed anticonvulsant drug in humans. A structure-activity analysis demonstrated that the related compound valpromide also extends lifespan. Valproic acid treatment may modulate the insulin/IGF-1 growth factor signaling pathway, because VA promoted dauer larvae formation and DAF-16 nuclear localization. To investigate the mechanism of action of VA in delaying aging, we analyzed the effects of combining VA with other compounds that extend the lifespan of C. elegans. Combined treatment of animals with VA and the heterocyclic anticonvulsant trimethadione caused a lifespan extension that was significantly greater than treatment with either of these drugs alone. These data suggest that the mechanism of action of VA is distinct from that of trimethadione, and demonstrate that lifespan-extending drugs can be combined to produce additive effects.

Prophylactic and therapeutic functions of T-type calcium blockers against noise-induced hearing loss.

Cochlear noise injury is the second most frequent cause of sensorineural hearing loss, after aging. Because calcium dysregulation is a widely recognized contributor to noise injury, we examined the potential of calcium channel blockers to reduce noise-induced hearing loss (NIHL) in mice. We focused on two T-type calcium blockers, trimethadione and ethosuximide, which are anti-epileptics approved by the Food and Drug Administration. Young C57BL/6 mice of either gender were divided into three groups: a 'prevention' group receiving the blocker via drinking water before noise exposure; a 'treatment' group receiving the blocker via drinking water after noise exposure; and controls receiving noise alone. Trimethadione significantly reduced NIHL when applied before noise exposure, as determined by auditory brainstem recording. Both ethosuximide and trimethadione were effective in reducing NIHL when applied after noise exposure. Results were influenced by gender, with males generally receiving greater benefit than females. Quantitation of hair cell and neuronal density suggested that preservation of outer hair cells could account for the observed protection. Immunocytochemistry and RT-PCR suggested that this protection involves direct action of T-type blockers on alpha1 subunits comprising one or more Ca(v)3 calcium channel types in the cochlea. Our findings provide a basis for clinical studies testing T-type calcium blockers both to prevent and treat NIHL.

Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans.

The identification and analysis of compounds that delay aging and extend lifespan is an important aspect of gerontology research; these studies can test theories of aging, lead to the discovery of endogenous systems that influence aging, and establish the foundation for treatments that might delay normal human aging. Here we review studies using the nematode Caenorhabditis elegans to identify and characterize compounds that delay aging and extend lifespan. These studies are considered in four groups: (1) Studies that address the free-radical theory of aging by analyzing candidate compounds with antioxidant activities including vitamin E, tocotrienols, coenzyme Q, and Eukarion-8/134. (2) Studies that analyze plant extracts (blueberry and Ginko biloba) that contain a mixture of compounds. (3) Studies of resveratrol, which was identified in a screen for compounds that affect the activity of the Sir2 protein that influences lifespan. (4) Studies based on screening compound libraries using C. elegans aging as a bioassay, which led to the identification of the anticonvulsant medicines ethosuximide and trimethadione. There has been exciting progress in the analysis of compounds that influence C. elegans aging, and important challenges and opportunities remain in determining the mechanisms of action of these compounds and the relevance of these observations to aging of other animals.

Effects of anticonvulsant drugs on life span.

Aging is characterized by widespread degenerative changes in tissue morphology and function and an increase in the incidence of human diseases such as cancer, stroke, and Alzheimer disease. Findings from recent genetic studies suggest that molecular mechanisms that influence life span are evolutionarily conserved, and interventions that extend the life span of model organisms such as worms and flies are likely to have similar effects on vertebrates such as humans. However, little progress has been made in identifying drugs that delay aging. We identified 3 pharmacologic compounds, ethosuximide, trimethadione, and 3,3-diethyl-2-pyrrolidinone, that extend lifespan and delay age-related degenerative changes in the nematode worm Caenorhabditis elegans. All 3 compounds are anticonvulsants that modulate neural activity in vertebrates, and ethosuximide and trimethadione are used to treat absence seizures in humans. We discuss existing evidence that these drugs might also delay vertebrate aging and suggest experiments that could test this hypothesis. Genetic and cell ablation studies conducted with model organisms have demonstrated connections between the nervous system and aging. Our studies provide additional support for the hypothesis that neural activity plays a role in lifespan determination, since ethosuximide and trimethadione regulated neuromuscular activity in nematodes. Our findings suggest that the lifespan extending activity of these compounds is related to the anticonvulsant activity, implicating neural activity in the regulation of aging. We also discuss models that explain how the nervous system influences lifespan.

The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil.

The purpose of the present study was to explore the analgesic effects of the low voltage-activated T-type Ca2+ channel blockers ethosuximide, trimethadione, and mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects of the compounds were also determined in the intravenous pentylenetetrazol seizure model. Following intraperitoneal administration, ethosuximide and trimethadione dose-dependently reversed capsaicin-induced mechanical hyperalgesia. Similarly, the highest dose of mibefradil tested (30 microg, intracisternal) reversed capsaicin-induced mechanical hyperalgesia. Ethosuximide and mibefradil produced statistically significant analgesic effects in both early and late phase formalin-induced behaviors and trimethadione reduced late phase behaviors. Additionally, ethosuximide and trimethadione produced antinociceptive effects in the rat-tail flick reflex test. In contrast, following intracisternal administration, mibefradil had no effect in the tail flick reflex test. In addition, the anticonvulsants ethosuximide and trimethadione increased the doses of pentylenetetrazol required to produce both first twitch and clonic seizures. In contrast however, mibefradil had no anticonvulsant effect. The present results demonstrate that the clinically used anticonvulsants ethosuximide and trimethadione provide analgesic effects at doses, which are anticonvulsant. Furthermore, the data further supports the idea that T-type Ca2+ channels may be important targets for treating persistent pain syndromes.

Anticonvulsant medications extend worm life-span.

Genetic studies have elucidated mechanisms that regulate aging, but there has been little progress in identifying drugs that delay aging. Here, we report that ethosuximide, trimethadione, and 3,3-diethyl-2-pyrrolidinone increase mean and maximum life-span of Caenorhabditis elegans and delay age-related declines of physiological processes, indicating that these compounds retard the aging process. These compounds, two of which are approved for human use, are anticonvulsants that modulate neural activity. These compounds also regulated neuromuscular activity in nematodes. These findings suggest that the life-span-extending activity of these compounds is related to the anticonvulsant activity and implicate neural activity in the regulation of aging.

Embryonic arrhythmia by inhibition of HERG channels: a common hypoxia-related teratogenic mechanism for antiepileptic drugs?

PURPOSE: There is evidence that drug-induced embryonic arrhythmia initiates phenytoin (PHT) teratogenicity. The arrhythmia, which links to the potential of PHT to inhibit a specific potassium channel (Ikr), may result in episodes of embryonic ischemia and generation of reactive oxygen species (ROS) at reperfusion. This study sought to determine whether the proposed mechanism might be relevant for the teratogenic antiepileptic drug trimethadione (TMO). METHODS: Effects on embryonic heart rhythm during various stages of organogenesis were examined in CD-1 mice after maternal administration (125-1,000 mg/kg) of dimethadione (DMO), the pharmacologically active metabolite of TMO. Palatal development was examined after administration of a teratogenic dose of DMO and after simultaneous treatment with DMO and a ROS-capturing agent (alpha-phenyl-N-tert-butyl-nitrone; PBN). The Ikr blocking potentials of TMO and DMO were investigated in HERG-transfected cells by using voltage patch-clamping tests. RESULTS: DMO caused stage-specific (gestation days 9-13 only) and dose-dependent embryonic bradycardia and arrhythmia at clinically relevant maternal plasma concentrations (3-11 mM). Hemorrhage in the nasopharyngeal part of the embryonic palate (within 24 h) preceded cleft palate in fetuses at term. Simultaneous treatment with PBN significantly reduced the incidence of DMO-induced cleft palate, from 40 to 13%. Voltage patch-clamping studies showed that particularly DMO (70% inhibition), but also TMO, had Ikr blocking potential at clinically relevant concentrations. CONCLUSIONS: TMO teratogenicity, in the same way as previously shown for PHT, was associated with Ikr-mediated episodes of embryonic cardiac arrhythmia and hypoxia/reoxygenation damage.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre- and postsynaptic calcium fluxes in rat hippocampal slices.

The contribution of T-, L-, N-, P-, and Q-type Ca2+ channels to pre- and postsynaptic Ca2+ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca2+ concentration ([Ca2+]o). [Ca2+]o was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca2+ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca2+]o decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca2+ entry (Capre), while [Ca2+]o decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca2+ entry (Capost). Ethosuximide and trimethadione were ineffective in concentrations up to 1 mM. At 10 mM, they reduced Capost and, much less, also Capre. Nimodipine (25 microM) reduced Capost and, to a minor extent, Capre. omega-Agatoxin IVA (0.4-1 microM) and omega-contoxin MVIIC (1 microM) also reduced both Capre and Capost, but with a stronger action on Capre. omega-Conotoxin GVIA (3-8 microM) reduced Capost without effect on Capre. We conclude that during stimulus-induced, high-frequency neuronal activity Capost is carried by P/Q-, N-, and L- type channels and probably a further channel type different from these channels. Capre includes at least P/Q- and possibly L-type channels. N-type channels did not contribute to Capre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca2+ entry in this situation.

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: ethosuximide, trimethadione, and dimethadione.

Spontaneous generalized epileptiform discharges were elicited in rodent thalamocortical slices by perfusion with a medium containing no added Mg2+. In multiple-channel extracellular field potential recordings in thalamus and cortex, several distinct types of discharges were recorded, with two principal variants bearing marked similarity to spike-wave and generalized tonic-clonic seizure discharges recorded in patients with generalized seizure disorders. These discharges were termed sTBCs and cTBCs, respectively, for simple and complex thalamocortical burst complexes. The sensitivity of these discharges to the generalized absence anticonvulsants ethosuximide, trimethadione and dimethadione (the active metabolite of trimethadione) was studied. sTBCs were reduced or blocked by ethosuximide and dimethadione, when these drugs were applied in clinically relevant concentrations. The order of effectiveness of these agents was dimethadione > or = ethosuximide >> trimethadione. This paralleled the relative efficacy of these drugs in blocking T current in thalamic neurons. cTBCs were unaffected or exacerbated by these drugs. Structural control drugs including succinimide, the behaviorally inactive ring base of ethosuximide, and alpha, alpha-dimethyl-beta-methylsuccinimide, a convulsant succinimide, were inactive or exacerbated either sTBCs or cTBCs, respectively. These spontaneous generalized thalamocortical discharges in rodent thalamocortical slices may represent a potentially valuable in vitro model of generalized seizure discharges, with marked pharmacological and physiological similarities to various forms of clinical epileptic seizure activity.

Antiepileptic drug mechanisms of action.

Established antiepileptic drugs (AEDs) decrease membrane excitability by interacting with neurotransmitter receptors or ion channels. AEDs developed before 1980 appear to act on sodium channels, gamma-aminobutyric acid type A (GABAA) receptors, or calcium channels. Benzodiazepines and barbiturates enhance GABAA receptor-mediated inhibition. Phenytoin (PHT), carbamazepine (CBZ), and possibly valproate (VPA) decrease high-frequency repetitive firing of action potentials by enhancing sodium-channel inactivation. Ethosuximide (ESM) and VPA reduce a low threshold (T-type) calcium-channel current. The mechanisms of action of the new AEDs are not fully established. Gabapentin (GBP) binds to a high-affinity site on neuronal membranes in a restricted regional distribution of the central nervous system. This binding site may be related to a possible active transport process of GBP into neurons; however, this has not been proven, and the mechanism of action of GBP remains uncertain. Lamotrigine (LTG) decreases sustained high-frequency repetitive firing of voltage-dependent sodium action potentials that may result in a preferential decreased release of presynaptic glutamate. The mechanism of action of oxcarbazepine (OCBZ) is not known; however, its similarity in structure and clinical efficacy to CBZ suggests that its mechanism of action may involve inhibition of sustained high-frequency repetitive firing of voltage-dependent sodium action potentials. Vigabatrin (VGB) irreversibly inhibits GABA transaminase, the enzyme that degrades GABA, thereby producing greater available pools of presynaptic GABA for release in central synapses. Increased activity of GABA at postsynaptic receptors may underline the clinical efficacy of VGB.

Tolerance to the anticonvulsant effects of phenobarbital, trimethadione, and clonazepam in kindled rats: cross tolerance to carbamazepine.

The kindled-convulsion model was used to assess the development of tolerance and cross tolerance to the anticonvulsant effects of antiepileptic drugs. In Experiment 1, tolerance developed to the anticonvulsant effects of bidaily (one every 48 h) IP injections of phenobarbital, trimethadione, and clonazepam on convulsions elicited 1 h after each injection in kindled rats by amygdala stimulation. In Experiment 2, kindled rats that were tolerant to the anticonvulsant effects of phenobarbital, trimethadione, or clonazepam received bidaily IP injections of carbamazepine, each followed 1 h later by a convulsive amygdala stimulation. There was a statistically significant transfer of tolerance from phenobarbital to carbamazepine, but not from either trimethadione or clonazepam to carbamazepine. Apparently, tolerance to anticonvulsant drugs is most likely to transfer between drugs that are effective against similar kinds of clinical and experimental seizures and have similar putative mechanisms of action.

Substantia nigra regulates action of antiepileptic drugs.

The cholinergic agonist pilocarpine triggers sustained limbic seizures in rodents. Pilocarpine seizures were blocked by systemic administration of benzodiazepines, barbiturates, valproate and trimethadione, while diphenylhydantoin did not affect, and ethosuximide increased the susceptibility of rats to such seizures. This pattern of action of antiepileptic drugs is characteristic for pilocarpine seizures and different from other rodent models of epilepsy. Although the anatomical substrates in the forebrain involved in the expression of anticonvulsant activity are unknown, the basal ganglia are believed to be essential for the motor expression of pilocarpine seizures. Bilateral microinjections into the substantia nigra, a major output station of the basal ganglia, of midazolam (ED50 38.5 nmol; range 29-52 nmol), phenobarbital (ED50 16 nmol; range 7-39 nmol) and trimethadione (ED50 30 nmol; range 16-56 nmol) protected rats against pilocarpine seizures (380 mg/kg i.p.) Diphenylhydantoin (up to 100 nmol) remained inactive, while ethosuximide (ED50 38 nmol; range 22-65.5 nmol) reduced the threshold for pilocarpine seizures, converting subconvulsant doses of pilocarpine (200 mg/kg i.p.) into convulsant ones. The profiles of action of antiepileptic drugs on pilocarpine seizures were similar following intranigral and systemic administration. These observations suggest that the substantia nigra may mediate some actions of antiepileptic drugs.

Gamma-hydroxybutyrate stimulation of the formation of cyclic GMP and inositol phosphates in rat hippocampal slices.

The presence of gamma-hydroxybutyrate (GHB) (300-600 microM) in the incubation medium of rat hippocampal slices led to an increase of intracellular cyclic GMP and inositol phosphates. This phenomenon is dependent on the time and the dose of GHB used and might be the result of the stimulation of GHB receptor sites which are abundant in rat hippocampus. The increase of cyclic GMP and inositol phosphates is blocked by some anticonvulsants and opiate antagonists. These results seems to indicate that, like many substances inducing epileptic phenomena, GHB provokes neuronal depolarization in hippocampus which is accompanied by formation of cyclic GMP and inositol phosphates. The effect of opiate antagonists can be explained by the possible implication of an opiate synapse which mediates GHB effects in rat hippocampus.

Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat.

Electroencephalographic (EEG) studies were performed to examine the effects of several antiepileptic drugs (AEDS) on absence-like and tonic seizures in the spontaneously epileptic rat (SER: zi(zi), tm/tm,), a double mutant rat, which was obtained by mating the tremor heterozygous animals (tm/ +) with the zitter homozygous animals (zi/zi), and to determine whether the seizures in the SER correspond to human absence and tonic seizures. Spontaneous EEG was continuously recorded from the frontal cortex and hippocampus using implanted electrodes. The SER showed paroxysmal and synchronized 5-7-Hz spike-wave-like complexes in both cortical and hippocampal EEG during the absence-like state, which was characterized by immobility and staring. The animal also exhibited tonic convulsion without external stimulation concomitant with low-voltage fast waves on cortical and hippocampal EEG. In some animals, sporadic low-amplitude spikes appeared in the low-voltage fast waves during tonic convulsion. the absence-like seizures were inhibited by trimethadione (100 mg/kg intraperitoneally, i.p.) and ethosuximide 100 mg/kg i.p.), whereas the tonic convulsion was not affected by these drugs. In contrast, phenytoin (20 mg/kg i.p.) inhibited the tonic seizures without affecting the absence-like seizures. Phenobarbital (10 mg/kg i.p.) and valproate (200 mg/kg i.p.) inhibited both seizures to a similar degree. These results suggest that the SER, with both absence-like and tonic seizures, is a useful model for evaluation of AEDS.

Inhibition by antiepileptics of carbachol but not lithium- or 5-methoxytryptamine-induced wet dog shakes in rats.

The influence of antiepileptic drugs on the wet dog shakes (WDS) induced by intracerebroventricular injections of carbachol (30 micrograms icv) was investigated in rats. Diphenylhydantoin (DPH, 8 and 4 mg/kg), diazepam (0.4, 0.2 and 0.1 mg/kg), phenobarbital (12.5, 6.25 and 3.12 mg/kg), sodium valproate (Depakine, 200, 100 and 50 mg/kg) and trimethadione (200, 100 and 50 mg/kg) given ip inhibited the WDS in a dose-dependent manner. These drugs at the same doses did not change the intensity of shaking behavior induced by lithium chloride or 5-hydroxytryptamine. As the antiepileptic drugs tested in these experiments did not have anticholinergic activity and at used doses were not able to prevent electrical convulsions or pentetrazol-induced seizures, it appears that carbachol-induced WDS could be connected with convulsive activity and could be the initial stage of seizures.

Evidence for involvement of the astrocytic benzodiazepine receptor in the mechanism of action of convulsant and anticonvulsant drugs.

The anticonvulsant drugs carbamazepine, phenobarbital, trimethadione, valproic acid and ethosuximide at pharmacologically relevant concentrations inhibit [3H]diazepam binding to astrocytes in primary cultures but have much less effect on a corresponding preparation of neurons. Phenytoin as well as pentobarbital (which is not used chronically as an anticonvulsant) are equipotent in the two cell types. The convulsants picrotoxinin and pentylenetetrazol, the convulsant benzodiazepine RO 5-3663 and the two convulsant barbiturates DMBB and CHEB similarly inhibit diazepam binding to astrocytes but have little effect on neurons. On the basis of these findings it is suggested that these convulsants and anticonvulsants owe at least part of their effect to an interaction with the astrocytic benzodiazepine receptor, perhaps by interference with a calcium channel.

Effect of anticonvulsant drugs on gamma-hydroxybutyrate release from hippocampal slices: inhibition by valproate and ethosuximide.

The effects of some anticonvulsant drugs have been investigated on gamma-hydroxybutyrate release from rat hippocampal and striatal slices. Sodium valproate and ethosuximide inhibited the depolarization-evoked release of gamma-hydroxybutyrate induced by 40 mM K+. The IC50 values for these two drugs are in the concentration range of valproate and ethosuximide that exists in rat brain after administration of anticonvulsant doses to the animals. Trimethadione and pentobarbital are without significant effects. It can be concluded that the inhibition of gamma-hydroxybutyrate release, particularly that observed for hippocampus, might explain the protective effect of valproate and ethosuximide on gamma-hydroxybutyrate-induced seizures and perhaps on other kinds of epileptoid phenomenon.

Effects of anticonvulsant drugs on substantia nigra pars reticulata neurons.

Enhancement of gamma-aminobutyric acid transmission within the substantia nigra has been shown to prevent the motor manifestations of chemically induced and kindled seizures. These findings raise the possibility that the substantia nigra might constitute a site of anticonvulsant drug action if these drugs share an ability to suppress propagation of seizure activity from the nigra to motor effector sites. The current studies monitored effects of a diverse group of anticonvulsant drugs on the extracellular, single unit activity of nondopaminergic neurons of the substantia nigra pars reticulata in awake, paralyzed and locally anesthetized male rats. Intravenous phenytoin (1.25-160 mg/kg) and carbamazepine (1.25-40 mg/kg) did not alter neuronal firing at any dose. Conversely, both diazepam (31.25-8,000 micrograms/kg) and clonazepam (2-500 micrograms/kg) partially inhibited firing (to 46 +/- 11% and 59 +/- 6% of base-line rates, respectively), although clonazepam was approximately 16 times more potent in eliciting equivalent degrees of inhibition. Valproic acid (5-640 mg/kg) and phenobarbital (1.25-80 mg/kg) also slowed firing to 65 to 70% of base-line rates, but did so only at the highest doses administered. However, the anesthetic barbiturate pentobarbital (0.3125-80 mg/kg) completely suppressed firing by the highest dose tested. Of those drugs used exclusively for treatment of absence seizures, trimethadione (12.5-800 mg/kg) caused dose-related inhibitions to 37.6 +/- 9.8% of base-line, whereas ethosuximide (12.5-800 mg/kg) markedly stimulated firing, nearly doubling firing rates after the 200 mg/kg dose.(ABSTRACT TRUNCATED AT 250 WORDS)