Evaluation Of Novel Epilepsy Treatment Approaches

该项目的总体目标是通过动物模型的药理学研究和人类受试者的临床研究来探索药物治疗癫痫的策略。研究继续评估神经活性类固醇在癫痫中的作用及其在癫痫治疗中的可能用途。神经活性类固醇是内源性类固醇激素（及其合成类似物），通过直接作用于膜离子通道，包括GABA-A和NMDA受体，迅速改变神经元的兴奋性。在先前的报道中，我们证实生殖激素黄体酮具有强大的抗惊厥活性。对孕激素受体敲除（PRKO）小鼠的研究表明，孕激素对癫痫易感性的影响不是由其同源核激素受体介导的。相反，我们发现黄体酮是抗惊厥药，因为它转化为神经活性类固醇异孕酮。我们提出，在月经期（黄体酮水平下降时），许多女性癫痫患者发作频率的增加可能与停药异孕酮有关。目前，没有专门的治疗癫痫的方法。然而，我们对兔兔癫痫动物模型的研究表明，神经类固醇替代可能是有用的。此外，我们还研究了神经类固醇在应激诱导的癫痫易感性改变中的作用，特别关注脱氧皮质酮（DOC），一种肾上腺类固醇，其合成在应激时增强。我们的研究结果表明，DOC是急性应激生理效应的中介，可通过其转化为神经类固醇，对GABA-A受体（包括四氢脱氧皮质酮（THDOC））具有调节作用，从而促进应激诱导的癫痫易感性变化。我们的研究结果进一步表明，神经活性类固醇作为一种治疗压力相关癫痫发作的方法。在本报告期内，我们使用孕激素受体敲除（PRKO）小鼠进行了研究，以评估孕激素受体（PR）在孕激素抗焦虑活性中的作用。这些研究是对先前报道的研究的补充，我们使用PRKO小鼠来检查PR在黄体酮抗惊厥活性中的作用。免疫细胞化学证实PRKO脑组织中PR受体蛋白表达缺失。在PRKO小鼠及其等基因野生型（WT）幼崽中，黄体酮给药与血浆异孕酮浓度的剂量依赖性升高以及相应的升高+迷宫试验中的焦虑作用有关。PRKO小鼠表现出比WT动物更大的抗焦虑反应，尽管两种基因型的异孕醇酮水平相似。异孕酮也表现出抗焦虑作用，但两种基因型的反应程度相似。非那雄胺是一种阻断孕酮向异孕酮转化的5 α -还原酶抑制剂，对PRKO小鼠进行预处理，完全阻止了孕酮的抗焦虑活性，但对异孕酮的反应没有影响，这表明异孕酮（或其他孕酮的5 α -还原代谢物）是对母体类固醇激素的反应。我们的结果提供了直接的证据，证明黄体酮的抗焦虑作用不需要pr。然而，由于PRKO小鼠对黄体酮更敏感，孕酮激活PR可能影响抗焦虑反应。神经活性类固醇可能比黄体酮更适合用于治疗双眠性癫痫，因为神经活性类固醇不具有可导致副作用的pr介导的激素作用。目前的结果进一步支持使用神经活性类固醇而不是黄体酮治疗夜眠性癫痫，因为黄体酮可以作用于脑PR受体并可能对脑功能产生不良影响。

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. Studies with progesterone receptor knockout (PRKO) mice demonstrated that the effects of progesterone on seizure susceptibility are not mediated by its cognate nuclear hormone receptors. Instead, we found that progesterone is anticonvulsant as a result of 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). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period, we conducted studies using progesterone receptor knockout (PRKO) mice to evaluate the role of the progesterone receptor (PR) in the anxiolytic activity of progesterone. These studies are complementary to research reported previously in which we used PRKO mice to examine the role of PR in the anticonvulsant activity of progesterone. The absence of PR receptor protein expression in PRKO brain was confirmed by immunocytochemistry. In PRKO mice and their isogenic wild-type (WT) littermates, progesterone administration was associated with a dose-dependent rise in plasma allopregnanolone concentrations and corresponding anxiolytic effects in the elevated plus maze test. PRKO mice exhibited a greater anxiolytic response than WT animals although the allopregnanolone levels were similar in the two genotypes. Allopregnanolone also exhibited anxiolytic effects, but the magnitude of the response was similar in both genotypes. Pretreatment of PRKO mice with finasteride, a 5alpha-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone, completely prevented the anxiolytic activity of progesterone, but had no effect on the response to allopregnanolone, demonstrating that allopregnanolone (or other 5alpha-reduced metabolites of progesterone) accounts for the response to the parent steroid hormone. Our results provide direct evidence that the anxiolytic action of progesterone does not require PRs. However, PR activation by progesterone may influence the anxiolytic response since PRKO mice were more sensitive to progesterone. Neuroactive steroids may be preferred to progesterone in the treatment of catamenial epilepsy because neuroactive steroids do not have hormonal actions mediated by PRs which can lead to side effects. The present results provide further support for the use of neuroactive steroids rather than progesterone in the treatment of catamenial epilepsy, since progesterone can act on brain PR receptors and potentially produce undesirable effects on brain function. During this reporting period we also began studies examining the role of androgen-related steroids in the regulation of seizure susceptibility. Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone--androsterone (5alpha-androstan-3alpha-ol-17-one), a neurosteroid that acts as a positive allosteric modulator of GABA-A receptors, and its 5beta-epimer etiocholanolone (5beta-androstan-3alpha-ol-17-one)--also may be reduced in epilepsy. Androsterone has been found in adult brain, and both metabolites, which also can be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. We hypothesized that androsterone and etiocholanolone have anticonvulsant properties, so that low levels of these metabolites in men with epilepsy could lead to enhanced seizure susceptibility and poor seizure control. Supporting evidence was obtained in animal epilepsy models and in an in vitro brain slice model of epileptiform activity. Androsterone protected mice in a dose-dependent fashion from seizures in the 6-Hz electrical stimulation, pentylenetetrazol, pilocarpine, 4-AP, and maximal electroshock models. Etiocholanolone also had anticonvulsant properties, but was less potent, whereas epiandrosterone (3beta-androsterone) was inactive. Androsterone also inhibited epileptiform discharges in hippocampal slices exposed to 4-aminopyridine, whereas etiocholanolone was active but of lower potency. We conclude that androsterone and etiocholanolone have anticonvulsant properties and could represent endogenous modulators of seizure susceptibility. Reductions in the levels of these metabolites in men with epilepsy could contribute to poor seizure control. As in catamenial epilepsy, neuroactive steroid replacement is a potential therapeutic strategy.