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- MATERIALS AND METHODS
Summary: Purpose: Newly designed antiepileptic drugs (AEDs) are being evaluated for their efficacy in preventing seizures and for their toxic profiles. We investigated and compared the toxic effects of two dibenz[b,f]azepine derivatives with anticonvulsant activity, 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA2-024) and (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f] azepine-5-carboxamide (BIA2-093), with the structurally related compounds carbamazepine (CBZ) and oxcarbazepine (OXC), both in current use for the treatment of epilepsy.
Methods: Primary rat hippocampal neurons were used to evaluate neuronal morphology and biochemical changes induced by the AEDs used in this study. Immunocytochemical staining against MAP-2 was used to evaluate neuronal morphology. Reactive oxygen species (ROS) and changes in mitochondrial membrane potential (Ψm) were measured by fluorescence techniques. Intracellular adenosine triphosphate (ATP) levels were quantified by high-performance liquid chromatography (HPLC).
Results: Hippocampal neurons treated for 24 h with CBZ or OXC (300 μM) showed degeneration and swelling of neurites, but this effect was not observed in neurons treated with BIA 2-024 or BIA 2-093 (300 μM). ROS production also was increased in neurons treated with OXC, but not in neurons treated with the other AEDs. ATP levels were significantly decreased only in neurons treated with OXC, although the energy charge was not altered. Furthermore, OXC led to a decrease of Ψm.
Conclusions: In all parameters assayed, OXC was more toxic than the other AEDs used. Because the new putative AEDs have previously been shown to have an efficacy in preventing seizures similar to that of CBZ and OXC, and are less toxic to neuronal cells, they may be considered as alternatives to the current available therapies for the treatment of epilepsy.
Epilepsy is a common neurologic disease estimated to affect 50 million persons worldwide. The major antiepileptic drugs (AEDs) in current clinical use [e.g., phenytoin (PHT), carbamazepine (CBZ), valproate (VPA), and phenobarbital (PB), among others] have a high efficacy in reducing seizures. An ideal AED would prevent seizures without producing side effects that adversely affect the patient's quality of life. Unfortunately, patients taking AEDs display a broad spectrum of undesirable side effects. Particularly dizziness, ataxia, drowsiness, and reduction of alertness occur in therapy with CBZ, in the beginning of treatment and with increasing doses (1). Thus, extensive research has focused on the design of new drugs with anticonvulsant activity and fewer adverse effects. New drugs have progressed in terms of longer half-lives, greatly reduced potential for drug interactions, and general lack of hepatic enzyme induction (2–5).
CBZ is a dibenz[b,f]azepine derivative (5H-dibenz[b,f]azepine-5-carboxamide (6) that, since its introduction for clinical treatment of epileptic seizures, has become the most frequently prescribed first-line drug. Besides the fact that a significant percentage of affected individuals do not respond to treatment with CBZ, this drug has the disadvantage of inducing hepatic microsomal enzymes that cause self-induction of its own metabolism. Moreover, its metabolites (particularly epoxides) have been held responsible for the general and neuronal toxicity of CBZ. Oxcarbazepine (OXC) is an analogue of CBZ, with comparable anticonvulsant efficacy, in which a keto group has been added to the position 10 of the azepine ring. This modification results in important differences in the metabolism of the two drugs. It has the advantage of a lower incidence of allergic reactions and enzyme induction. In combined therapy with other AEDs, OXC is usually better tolerated than CBZ (7).
10,11-Dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024) and (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) are representative of a new series of compounds (8,9), structurally related to CBZ and OXC, with anticonvulsant activity, as determined by maximal electroshock stimulation (9,10). As newly developed drugs should be less toxic than already existing drugs, these two compounds were specifically designed to circumvent their further degradation to toxic metabolites, such as epoxides, without losing anticonvulsant potency.
Several studies can be found in the literature regarding the toxicity of AEDs to neuronal cells. Some reports show that CBZ is able to induce apoptosis in cultured cerebellar granule cells (11–13). In a previous study, we showed that OXC and CBZ can be toxic to cultured hippocampal neurons (14), showing patterns of neuronal deterioration, mainly in cells exposed to high concentrations, and also showing increased activity of caspase-3–like enzymes, in the case of OXC. We also have seen some morphologic changes (nuclear condensation) in neurons exposed to high concentrations of either BIA 2-024 or BIA 2-093, suggesting apoptosis, although the extent of the insult was much less than that observed with CBZ or OXC. Recently, Pavone and Cardile (15) described the effects of CBZ and OXC on astrocyte cultures and showed decreased tolerance by cortical astrocytes to high concentrations of these and other AEDs.
Within this scenario, we found important to compare the toxicity profiles of the new putative AEDs, BIA 2-024 and BIA 2-093, with those of currently used and structurally similar AEDs, CBZ and OXC. We chose cultured hippocampal neurons as a model, given that the hippocampal formation is one of the most affected brain regions during status epilepticus. In this study, we further investigated the toxic effect caused by AEDs in cultured neurons. We focused on parameters related to apoptosis-like cell death and markers for mitochondrial dysfunction, which compromises the energy status of the cell and impairs neuronal survival.
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- MATERIALS AND METHODS
In this work, we show that OXC, at high concentrations, is toxic to cultured hippocampal neurons, triggering mechanisms that result in decreased intracellular ATP levels and increased ROS, compromising cell survival. We observed significant neurotoxic changes after treatment with OXC, which are not observed with treatment with the two new putative AEDs, BIA 2-024 and BIA 2-093, or with CBZ, thus sustaining the concept that this new putative AEDs are less toxic than currently used AEDs. These results are in agreement with and extend the observations in previous studies by our group (14), in which it was shown that OXC is toxic to cultured hippocampal neurons and that BIA 2-024 and BIA 2-093 are less toxic than the related compounds, CBZ and OXC, which toxicity is detected at concentrations as low as 50–100 μM.
When dealing with a disease such as epilepsy (21), which is a life-long condition and requires continuous treatment after diagnosis, bioaccumulation of AEDs cannot be excluded, and the plasma levels of the drug can surpass the therapeutic dose; hence the interest of studying the effect in neuronal survival of high concentrations of AEDs. Pavone and Cardile (15) characterized the effect of various concentrations of CBZ and OXC (ranging from 1 to a 100 μg/ml), as well as other AEDs, on the viability of cultured cortical astrocytes, showing increased cellular stress. To our knowledge, little is known about the toxicity triggered by AEDs in neurons. High concentrations of CBZ were shown to be neurotoxic in cultured cerebellar neurons (11–13) and in cultured hippocampal neurons (14). OXC also was described to be toxic to hippocampal neurons (14). It is very important to assess the neurotoxic profile of AEDs because metabolic failure due to drug interaction and/or hepatic dysfunction or renal failure might lead to an increase in plasma levels of the drug, and thus the interest in choosing a therapy with as few toxic effects as possible to neuronal cells.
We found that OXC had a strong deleterious effect in the morphology of cultured hippocampal neurons: the neurites became swollen and shorter, and the network of fine processes that is observed in control cultures was greatly reduced. The CBZ effect was not as dramatic as that of OXC, although the damage in neuronal cultures was clear. Treatment of cultures with OXC (100 μM) did not cause an apparent retraction and swelling of neurites (data not shown), although a decrease of MTT reduction and increase in the number of apoptotic-like nuclei were already observed at this concentration (14). Apparently, morphologic changes are secondary to biochemical detection of neurodegeneration, as when the MTT reduction is significantly decreased or the appearance of apoptotic-like nuclei is clearly higher, but still no morphologic changes at the neurite network level are yet visible. The hippocampal cultures treated with BIA 2-024 and BIA 2-093 did not show evident morphologic markers of neurodegeneration, suggesting that these drugs are not as toxic as OXC or CBZ. Previous work with BIA 2-024 and BIA 2-093 showed that at the same concentration used in the present study (300 μM), BIA 2-093 significantly decreases MTT reduction by hippocampal neurons in culture, but BIA 2-024 did not (14). However, in the same study, the new putative AEDs did not cause an increase in caspase-3–like enzymes activity, as OXC or CBZ did, and neuronal morphology appears to be intact, as we observed.
Mitochondria are central players in the metabolic processes of eukaryotic cells, having a critical role in ATP synthesis. Additionally, mitochondria are highly involved in other cellular processes besides energy production, such as intracellular Ca2+ buffering and cell death. We screened for the effect of AEDs in intracellular ATP levels and the EC of cultured hippocampal neurons exposed to these drugs. Only treatment with OXC but not with the other AEDs caused a decrease in intracellular ATP levels. Although a consistent and significant decrease in intracellular ATP levels is caused by OXC exposure, the decrease observed is not massive, and thus this is not traduced by a significant decrease in energy charge, which might be explained by the fact that apoptosis rather than necrosis is occurring. Our current data and previous work point to a situation of apoptosis. ATP is required for apoptotic cell death, and depletion of the intracellular ATP levels and a dramatic decrease of the cell energy charge would cause a shift from apoptosis to necrotic cell death (22), which does not seem to be the case. Likewise, an increase in ROS production as well as a decrease in Ψm, observed only in OXC-treated neurons, suggests that the toxic effects of OXC might be mediated by a mitochondrial pathway, because the majority of ROS generated in the cells are of mitochondrial origin.
OXC rapidly undergoes reduction of the carbonyl group in the liver, to form the active agent 10,11-dihydro-10-hydroxycarbamazepine, and is thought to be a good alternative to CBZ in the treatment of epilepsy, because its metabolism does not involve formation of an epoxide metabolite, thus being less toxic. Compared with the parent compound (CBZ), hepatic microsomal enzyme induction and autoinduction are greatly reduced, and the clinical efficacy of OXC compares favorably with that CBZ in clinical trials (5). OXC has fewer side effects and reduced potential for drug interaction in patients that did not respond well to CBZ therapy. However, of all the drugs tested in this study, OXC was the more toxic drug in all the parameters assayed. In vitro, OXC was not converted to other metabolites in cultured hippocampal neurons (data not shown), suggesting that the toxic effect of treatment with OXC in vitro is due to OXC itself and not to its metabolites. Being so, because in vivo OXC is rapidly metabolized to 10,11-dihydro-10-hydroxycarbamazepine, OXC will not reach the brain unless metabolic impairment occurs, and hepatic dysfunction is not uncommon in patients with epilepsy, which can often result from the AED therapy itself as an undesirable side effect. Thus care should be taken when administering OXC, because it proved to be toxic to cultured hippocampal neurons at concentrations not far from the therapeutic levels (14).
Within this scenario, because the two new putative AEDs have been shown to have efficacy in preventing seizures similar to that of CBZ and OXC (8,9), being less toxic to neuronal cells, there are advantages in considering these new drugs as possible alternatives to the current therapies available for the treatment of epilepsy. BIA 2-093 proved to be safe in a double-blind, placebo-controlled clinical trial in healthy male volunteers (23).