Evaluation Of Novel Epilepsy Treatment Approaches

Neurological Disorders And Stroke

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Abstract

The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity and we demonstrated that this results from its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on deoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. Our results demonstrated that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including tetrahydrodeoxycorticosterone (THDOC) and possibly also dihydrodeoxycorticosterone (DHDOC). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period, we demonstrated that the 6 Hz seizure model is useful in the evaluation of neuroactive steroids and other GABA-A receptor modulators. Low-frequency (6 Hz), long-duration (3 s) electrical stimulation in mice produces seizures characterized by immobility, focal clonus, and automatic behaviors reminiscent of human limbic epilepsy. Renewed interest has been expressed in this seizure model with the recognition that it is sensitive to a broad spectrum of anticonvulsants (AEDs) and may have distinct pharmacologic responsiveness from other in vivo tests of AED efficacy. We sought to determine whether allopregnanolone and several structural analogues with varying degrees of activity as positive allosteric modulators of GABA-A receptors are protective in this model. Mice were pretreated with neuroactive steroids or clonazepam and then subjected to 6-Hz corneal stimulation (32 mA). Animals that failed to exhibit immobility were considered protected. The neuroactive steroids prevented 6-Hz seizures with rank order of potencies that corresponded with their potencies as modulators of GABA-A receptors. Clonazepam also was protective. The potencies of the neuroactive steroids and clonazepam were similar to their previously reported activities in the pentylenetetrazol (PTZ) seizure model. We conclude that neuroactive steroids have comparable potencies in the 6-Hz and PTZ models. The structural specificity of neuroactive steroids in both models corresponds with their activities as positive allosteric modulators of GABA-A receptors. The 6-Hz model may be a valuable tool in drug development for the identification of GABAergic AEDs. During this reporting period we also examined the question of whether the anticonvulsant actions of progesterone occur through interactions with progesterone receptors (PR), nuclear transcription factors that mediate many of the biological actions of progesterone. As noted, we have observed that progesterone protects against seizures in animal models. To examine the role of PRs, we used PR knnockout [PRKO(-/-)] mice bearing a targeted deletion of the PR gene that eliminates both isoforms of the PR. The protective activity of progesterone was examined in female and male homozygous PRKO mice and isogenic wild-type controls in the pentylenetetrazol (PTZ), maximal electroshock, and amygdala-kindling seizure models. In all three models, the anticonvulsant potency of progesterone was undiminished in PRKO mice compared with control mice. On the contrary, there was a substantial increase in the anticonvulsant potency of progesterone in the PTZ and kindling models. The antiseizure activity of progesterone in PRKO mice was reversed by pretreatment with finasteride, a 5alpha-reductase inhibitor that blocks the metabolism of progesterone to allopregnanolone. Unlike progesterone, the neurosteroids allopregnanolone and THDOC exhibited comparable anticonvulsant potency in PRKO and wild-type mice. The basis for the heightened progesterone responsiveness of PRKO mice is not attributable to pharmacokinetic factors, because the plasma allopregnanolone levels achieved after progesterone administration were not greater in the PRKO mice. These studies provide strong evidence that PRs are not required for the antiseizure effects of progesterone, which mainly occurs through its conversion to the neurosteroid allopregnanolone.