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研究癫痫的药物治疗策略。研究仍在继续，评估神经活性类固醇在癫痫中的作用以及它们在癫痫治疗中的可能用途。神经活性类固醇是一种内源性类固醇激素(及其合成类似物)，通过直接作用于包括GABA-A和NMDA受体在内的膜离子通道，迅速改变神经元的兴奋性。在以前的报告中，我们证实生殖激素孕酮具有强大的抗惊厥活性，我们证明这是由于它转化为神经活性类固醇别孕酮所致。我们提出，许多患有癫痫的女性在月经前后(孕酮水平下降时)发作频率的增加，可能部分与停用别孕酮有关。目前，月经性癫痫尚无特效治疗方法。然而，我们对月经性癫痫动物模型的研究表明，神经类固醇替代可能是有用的。我们已经开始了临床研究，以验证月经性癫痫的神经类固醇停药假说，并计划进行一项临床试验，以评估神经类固醇替代疗法的有效性。此外，我们还研究了神经类固醇在应激诱导的癫痫敏感性改变中的作用，特别是四氢脱氧皮质酮(DOC)，这是一种肾上腺类固醇，其合成在应激过程中被增强。我们的结果表明，DOC是急性应激的生理效应的中介，它可能通过转化为神经类固醇来调节GABA-A受体，包括THDOC和可能的二氢脱氧皮质酮(DHDOC)，从而导致应激诱导的癫痫敏感性的改变。我们的结果进一步表明，神经活性类固醇作为一种治疗应激相关癫痫的方法。在本报告期间，我们开发了一个体外模型来评估神经类固醇对癫痫敏感性的影响，在没有混杂因素的情况下，例如吸收、新陈代谢和大脑可及性的差异。 本实验观察了神经类固醇类化合物别孕酮及其5β-异构体孕烯醇酮和孕烯醇酮硫酸酯(PS)对印防己毒素(PTX)和4-氨基吡啶(4-AP)诱导的大鼠海马脑片CA3区癫痫样放电的影响。低浓度时，别孕酮可部分抑制PTX诱导的猝发，高浓度时可完全抑制猝发。相反，孕酮未能改变放电频率。别孕酮抑制4-AP诱导的猝发，其效力与PTX模型相似；孕酮也部分有效。在4-AP模型中，别孕酮既抑制由GABA能神经元间同步放电产生的频率较高的以正向为主的电位，也抑制频率较低的负向电位。在4-AP模型中，PS可增加PTX爆发频率，增加负电位的频率，但不改变正电位的频率。PS本身不能诱发爆裂反应。在体外癫痫模型中，类固醇的作用很大程度上与它们对GABA-A受体的活性相一致；放电的抑制可能是由于这些受体的直接激活而不是对GABA介导的突触反应的调节。我们的结论是，在消除脑生物利用度因素的情况下，PTX和4-AP诱导的海马片爆发是直接评估神经类固醇对癫痫敏感性影响的有用模型。

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, in part, 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. We have initiated clinical studies to validate the neurosteroid withdrawal hypothesis of catamenial epilepsy and we plan a clinical trial to evaluate the utility of neurosteroid replacement. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on tetrahydrodeoxycorticosterone (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 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 developed an in vitro model to assess neurosteroid effects on seizure susceptibility in the absence of confounding factors such as differences in absorption, metabolism, and brain accessibility. The neurosteroids allopregnanolone and its 5beta-epimer pregnanolone, and pregnenolone sulfate (PS) were examined for effects on spontaneous epileptiform discharges induced by picrotoxin (PTX) and 4-aminopyridine (4-AP) in the CA3 region of the rat hippocampal slice. At a low concentration, allopregnanolone partially reduced PTX-induced bursting and at higher concentrations completely suppressed bursting. In contrast, pregnanolone failed to alter the discharge frequency. Allopregnanolone depressed 4-AP-induced bursting with similar potency as in the PTX model; pregnanolone was also partially effective. In the 4-AP model, allopregnanolone inhibited both the more frequent predominantly positive-going potentials as well as the less frequent negative-going potentials that may be generated by synchronous GABAergic interneuron firing. PS enhanced the PTX bursting frequency and, in the 4-AP model, increased the frequency of negative potentials but did not alter the frequency of positive potentials. By itself, PS did not induce bursting. The effects of the steroids in the in vitro seizure models largely correspond with their activities on GABA-A receptors; suppression of discharges may occur as a result of direct activation of these receptors rather than modulation of GABA-mediated synaptic responses. We conclude that PTX and 4-AP-induced bursting in the hippocampal slice are useful models for directly assessing neurosteroid effects on seizure susceptibility under conditions that eliminate the factor of brain bioavailability.