摘要：背景：本研究為台奧國際交流合作計畫及2016-2017年科技部個人型計畫之延伸。已證實由奧團隊開發的嶄新化合物:化合物6有助於治療類精神分裂、抽動症(tics)及偏頭痛之動物模式。化合物6為一pyrazoloquinolinone，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(TG)。偏頭痛是嚴重型頭痛，易造成生活失能。目前已知三叉神經血管系統中樞及周邊組織(TG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之一是降低三叉神經血管系統興奮性、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)，然而確切之作用機轉仍不清楚。已知20%偏頭痛患者對現有藥物療效不佳，有必要開發新的治療藥物。flumazenil和allopregnanolone為臨床使用的老藥，前者用於拮抗benzodiazepines，從未被報告應用於偏頭痛的治療;後者為黃體素之神經活性代謝物，類黃體素已用以治療偏頭痛女性經期症狀。二藥皆α6GABAAR正變構調節劑(positive allosteric modulators, PAM)。延續本團隊的研究顯示，α6GABAAR PAM能有效抑制中樞和周邊偏頭痛活性，因此本研究將檢視flumazenil是否也有抗偏頭痛之效及其作用機轉是否經由α6GABAAR?而類黃體素雖已被用於治療偏頭痛，然而療效不一。將重新檢視allopregnanolone抗偏頭痛的效果及其機轉。並與化合物6比較列出值得開發之藥物清單。實驗方法：將辣椒素(capsaicin)注入公鼠(8-10週)枕骨大孔，檢視三叉神經血管系統之中樞神經核和周邊TG及腦膜神經末梢和血管的變化。中樞部分將測量並量化c-fos免疫活性(神經元活化標記)。周邊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。且以diazepam為陰性對照組，Ro 15-4513和loreclezole為陽性對照組。結論：檢視α6GABAAR PAM之抗偏頭痛效果，有助於了解偏頭痛之致病機轉，並可開發臨床用藥新方向，造福頑固型偏頭痛患者，尤其是有合併抽動症之病患。
Abstract: Background: This proposal is an extension from a Taiwan-Austria international collaborating project and a personal project in 2016-2017 funded by MoST. In that bilateral project, the co-PI’s lab using several animal models mimicking schizophrenia and tics found several effective pyrazoloquinolinone compounds, such as Compound 6, which were developed by the Austrian partner’s lab and are the first-in-class high affinity positive allosteric modulators (PAMs) selectively acting on GABAARs containing the α6 subunit (α6GABAARs). α6GABAARs are expressed in limited neuronal tissues, including the trigeminal ganglia (TG), which plays an important role in the pathogenesis of migraine. Migraine is a prevalent disabling condition affecting about 15% of people with a high societal and individual burden. Trigemino-vascular activation via both peripheral and central sensitizations is an essential neuropathogenic mechanism of migraine. Around 20% of migraineurs are refractory to current pharmacological treatments. These agents were found to reduce superior sagittal sinus-evoked firing of neurons in the trigeminocervical complex (TCC), attenuate neurogenic dural vasodilation, likely through inhibiting the release of calcitonin gene related peptide (CGRP) from prejunctional trigeminal neurons, and inhibit propagation of cortical spreading depression in rats and cats. We found Compound 6 significantly inhibit c-Fos expression of TCC and CGRP immunoreactivity of TG as well as reversed CGRP depletion in the dura mater, suggesting the selective α6GABAARs PAM can inhibit the TG firing activity, leading to an anti-migraine activity. Thus, α6GABAARs are novel and promising targets of antimigraine therapy. In this research, we will investigate flumazenil and allopregnanolone, both clinically available drugs and non-selective PAMs of α6GABAARs. Flumazenil, the benzodiazepine antagonist, used to treat benzodiazepine overdose or dependence, exerts both agonist and antagonist actions on different GABA subunits. The antimigraine activity of flumazenil has not been studied yet. Hormonal interventions have been explored as a prophylactic treatment of menses-related migraine or migraine experienced during the pill-free period. Steroids have also been used for acute therapy of migraine clinically. However, the mechanism remains unclear. We will investigate the antimigraine activity of flumazenil and allopregnanolone and further explore their antimigraine mechanism and site of action by using the blood brain barrier impermeable selective α6GABAARs antagonist, furosemide.Methods: The migraine model induced by intra-cisternal (i.c.) capsaicin in Wistar rats (8-10 weeks) we established will be used. The c-fos immunoreactivity, an activated neuron marker, in the TCC (the central end of 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 be measured by immunohistochemistry in TG and dura mater. Diazepam that is ineffective at α6GABAAR will be tested as negative control whereas Ro 15-4513 and loreclezole are both α6GABAAR PAMs as positive controls. Conclusions: Repurposing a clinically available drug may benefit those refractory to current migraine therapy. Furthermore, it may help understand the exact pathogenesis of migraine, improve treatment outcome and develop new anti-migraine drugs in the future. In our experience, 10% of pediatric migraineurs are comorbid with tics, who may benefit from these potential anti-migraine drugs.
