摘要:背景:本研究為台奧國際交流合作計畫及2016 年科技部個人型計畫之延伸。共同主持人邱教授團隊之動物模式研究已證實由奧地利團隊設計製出的嶄新化合物:化合物6 有助於治療類精神分裂和抽動症(tics)。化合物6 為一pyrazoloquinolinone compound,乃GABAA 受體之α6 次單位的專一作用子(ligand),已知α6GABAA 次單位接受器(α6GABAAR)只存在於三種神經細胞,其中之一為與偏頭痛致病機轉密切相關的三叉神經節。偏頭痛是嚴重型頭痛伴隨噁心、嘔吐、及自律神經發作,如眼睛畏光、怕吵等。約佔群體15%,因症狀嚴重,易造成生活失能,因此造成社會和家庭沉重的負擔,有必要進一步了解其疾病機轉和藥物作用方式,才能發展有效的治療。目前已知三叉神經血管系統中樞(含三叉神經核和頸椎前緣)及周邊組織(三叉神經節和大腦腦膜之神經末梢及血管)的敏感化是造成偏頭痛的主因。藥物治療主要的機轉以降低三叉頸神經之興奮性、減少腦膜神經性血管擴張(極可能經由抑制calcitonin gene related peptide (CGRP)釋出之機轉)、抑制局部腦血流及cortical spreadingdepression,然而確切之作用機轉仍不清楚。已知仍有20%偏頭痛患者對目前現有的抗偏頭痛藥物療效不佳,有必要開發新的治療偏頭痛藥物。本團隊初步實驗結果顯示,化合物6 能有效抑制中樞和周邊偏頭痛活性,然而其半生期短只有近3 小時,因此將研究半生期較長的衍生物:氘化物DK-I-56-1,並探討其藥物動力學,並與原生藥化合物6 比較。另外,將於第三年研究另一中藥萃取物hispidulin,乃非專一性α6GABAAR 正變構調節劑(positive allosteric modulators)。如此將可比較DK-I-56-1 及hispidulin 與化合物6 抗偏頭痛活性之差異,列出值得開發之藥物清單;並進一步研究氘化物藥物動力學之改變及hispidulin 抗偏頭痛之機轉,探討其抗偏頭痛機轉是否經由調節α6GABAAR。實驗方法:本研究團隊已建立以辣椒素(capsaicin)誘發的偏頭痛動物模式,以辣椒素注入枕骨大孔,檢視三叉神經血管系統之中樞神經核和周邊三叉神經節及腦膜神經末梢和血管的變化。將執行於公鼠(8-10 週),中樞部分將測量並量化c-fos 免疫活性,此乃神經元活化之標記,量化的方法則根據本團隊之前研究所得之公式。周邊部分則以免疫螢光染色呈現神經胜肽CGRP 之活化或釋出,即偏頭痛神經性發炎之表徵。並以diazepam 為陰性對照組,Ro 15-4513 和loreclezole 為陽性對照組。結論:化合物6 及其衍生物為全新合成之化合物,和hispidulin 皆為α6GABAA 次單位接受器正變構調節劑,尚未被應用在偏頭痛之研究。檢視這些α6GABAA 次單位接受器正變構調節劑之抗偏頭痛效果,一方面可以開發新的可能臨床上有效治療偏頭痛藥物,造福頑固型偏頭痛患者,尤其是有合併抽動症之病患;另一方面也有助於了解偏頭痛之致病機轉、精進臨床治療之成效,及未來發展新的抗偏頭痛藥物。
Abstract: Background: This proposal is an extension from a Taiwan-Austria international collaborating project and apersonal project in 2016 funded by MoST. In that bilateral project, the co-PI’s lab using several animalmodels mimicking schizophrenia and tics found several effective pyrazoloquinolinone compounds, such asCompound 6, which were developed by the Austrian partner’s lab and are the first-in-class high affinitypositive allosteric modulators (PAMs) selectively acting on GABAARs containing the α6 subunit(α6GABAARs). α6GABAARs are only expressed in three neuronal tissues, including the trigeminal ganglia(TG), which plays an important role in the pathogenesis of migraine. Migraine, a severe headachecharacterized with concurrent nausea, vomiting, and autonomic instability, is a prevalent disabling conditionaffecting about 15% of people with a high societal and individual burden. Trigemino-vascular activation viaboth peripheral and central sensitizations is an essential neuropathogenic mechanism of migraine. Althoughthere are several pharmacological treatments available clinically, 20% of migraineurs are refractory. Theseagents were found to reduce superior sagittal sinus-evoked firing of neurons in the trigeminocervical complex(TCC), attenuate neurogenic dural vasodilation, which is likely through inhibiting the release of calcitoningene related peptide (CGRP) from prejunctional trigeminal neurons, and inhibit propagation of corticalspreading depression in rats and cats. Our preliminary results showed Compound 6 significantly inhibit c-Fosexpression of TCC and CGRP immunoreactivity of TG as well as reversed CGRP depletion in the dura mater,which suggest the selective PAM of α6GABAARs can inhibit the TG firing activity, leading to ananti-migraine activity. Thus, α6GABAARs are novel and promising targets of antimigraine therapy. In thisresearch, we will investigate the deuterated derivative of Compound 6, DK-I-56-1, which has a longerhalf-life, and hispidulin, a flavonoid isolated from a local herb, which is a non-selective PAM ofα6GABAARs. The antimigraine activity of the three drugs will be compared as well as the pharmacokineticsof the deuterated derivative and its prodrug for generating the selectivity profile of interest in migrainetherapy. We will further explore the antimigraine mechanism of hispidulin.Methods: The migraine model induced by intra-cisternal (i.c.) capsaicin in Wistar rats (8-10 weeks) weestablished will be used. The c-fos immunoreactivity, an activated neuron marker, in the TCC (the central endof the trigeminovascular system) will be quantified by the formulas we have established. In the periphery,protein extravasation with CGRP release and expression, phenomena of neurogenic inflammation, will bemeasured by immunohistochemistry in TG and dura mater. Diazepam that is ineffective at α6GABAAR, willbe tested as negative control whereas Ro 15-4513 and loreclezole are both α6GABAAR PAMs as positivecontrols.Conclusions: Developing a novel drug is of benefit for those refractory to current migraine therapy.Furthermore, it may help understand the exact pathogenesis of migraine, improve clinical practice anddevelop new anti-migraine drugs in the future. In our experience, there are 10% of pediatric migraineurscomorbid with tics. These may be potential anti-migraine drugs for those with intractable migraine orcomorbidity with tics.