止痛新標的:含a6次單元之γ-氨基丁酸-A受體

Abstract

增加脊髓當中γ-氨基丁酸的傳導性已知能夠產生止痛作用。然而，許多可以作用在γ-氨基丁酸-A (GABAA)受體的正向異位調節物(positive allosteric modulators, PAMs)，例如苯二氮平類(Benzodiazepines, BZDs)的藥物卻不能夠使用在疼痛的緩解上，其原因在於苯二氮平類藥物本身特有的副作用，包括鎮靜安眠以及肌肉無力。 根據先前的研究，我們從苦藍盤的葉子中分離出一種，名為粗毛豚草素(Hispidulin)的活性成分，能有效治療因模擬神經精神疾病而有前脈衝抑制(pre-pulse inhibition, PPI)不良的模式動物，其療效是透過正向異位調節小腦中含有次單元γ-氨基丁酸-A受體 (α6GABAAR)的傳導性；與此同時，我們也合成了一系列的pyrazoloquinolinones作為針對α6GABAAR有高度選擇性的正向異位調節物，在這些化學合成物當中，Compound 6是首要的化合物能有效治療PPI缺失，此療效也會因為小腦內注射呋塞米(Furosemide, α6GABAAR選擇性拮抗劑)而受到某種程度地抑制。α6GABAAR除了大量的表達在小腦的顆粒細胞層中，也被發現表達在脊髓中，因此，在本研究中我們想要檢視脊髓中的α6GABAAR在疼痛調控中扮演的角色為何? 我們利用hispidulin，Compound 6以及furosemide作為藥理學研究的利器，探討小鼠在熱板與福馬林實驗兩種疼痛模式下，增加脊髓中α6GABAAR的作用能否抑制疼痛產生。 從熱板實驗得到的結果顯示，分別以腹腔注射1、3、10 mg/kg的Compound 6所產生的止痛作用與劑量有著正相關(dose-dependent)。我們也測試了由化學合成Compound 6氘類衍生物的止痛效果，以利於專利的申請，這些衍生物中DK-56-1同樣以小鼠的熱板實驗，發現與劑量成正相關的止痛作用。熱板實驗中，透過腹腔注射的Compound 6 (3 mg/kg, i.p.)所產生的止痛效果，能夠媲美10 mg/kg的嗎啡(Morphine, i.p.)所產生的止痛效果，只是嗎啡的止痛的時效相較Compound 6來的稍加持久。另外，我們發現Compound 6 (3 mg/kg, i.p.)的止痛作用並不會受到納洛酮(Naloxone, 鴉片類受體拮抗劑,1 mg/kg, i.p.)所阻斷，而嗎啡的止痛效果幾乎完全消失，這意味著Compound 6的止痛效果並非源自鴉片類受體的活化。更重要的是，Compound 6與DK-56-1的止痛作用能夠被脊髓腔注射的furosemide (10 nmol)所減少，但並非完全抑制其止痛作用，由於脊髓腔注射的furosemide並沒有完全反轉Compound 6的止痛作用，我們不排除其止痛作用來自其他非專一性標的(off-target)受體的可能性。由三個可能非專一性受體中，血清素-7受體(5-HT7 receptor)是最有可能對止痛作用有貢獻的非專一性標的受體。我們利用首利安錠(Amisulpride, 血清素-7受體拮抗劑, 1 mg/kg, i.p.)來證實此假說，結果發現Compound 6 (3 mg/kg, i.p.)的止痛作用並不會受到血清素-7受體拮抗劑的影響。 另外，我們也利用熱板實驗檢視了hispidulin與其氘類衍生物的止痛效果，分別以腹腔注射3、10、30 mg/kg的hispidulin，也呈現與劑量正相關的止痛作用，然而，其氘類衍生物D-hispidulin則未在熱板實驗中見到止痛效果。 福馬林(Formalin)誘導發炎疼痛的實驗當中，Compound 6和hispidulin抑制了第二期(phase II)發炎疼痛的反應，但是在第一期(phase I)急性疼痛期卻不見療效；另外，D-hispidulin也同樣在福馬林實驗中未見其止痛效果。 最後，由免疫組織化學染色的結果，證實α6GABAARs存在於 ICR小鼠的脊髓和背根神經節中，以支持我們的論點，從這些結果推論而知，增加了脊髓中α6GABAARs所調控的傳導性，可以引發一個與活化鴉片類受體無關的止痛作用，並且不造成明顯的行為異常。因此，發展α6GABAARs的選擇性正向異位調節物，或許擁有做為發展新的疼痛緩解治療劑的潛力。

Increasing GABAergic transmission in the spinal cord is known to be anti-nociceptive. However, benzodiazepines (BDZs), acting as positive allosteric modulators (PAMs) of GABAA receptors, are not used for pain relief due to their unwanted side effects, such as sedation and muscle weakness. Previously, we have identified an active constituent from a local herb, hispidulin that was effective in animal models mimicking neuropsychiatric disorders with impaired pre-pulse inhibition (PPI) via acting as a PAM of the alpha 6 subunit-containing GABAA receptors (α6GABAARs) in the cerebellum. Meanwhile, we have found a series of synthetic pyrazoloquinolinones that are PAMs highly-selective to α6GABAARs also effective in PPI-impaired animal models. Among these, Compound 6 is the lead compound that effectively rescued PPI impairment in a manner prevented by intra-cerebellar furosemide, a selective α6GABAAR antagonist. In addition to being abundant in cerebellar granule cells, α6GABAARs are also expressed in the spinal cord. We, therefore, examined whether spinal α6GABAARs play a role in pain control using hispidulin, Compound 6 and furosemide as pharmacological tools in two pain models in mice, hot-plate and formalin tests. In the hot-plate test, Compound 6 induced antinociception at 1, 3 and 10 mg/kg (i.p.) dose-dependently. We have also tested effects of deuterated derivative of Compound 6 that were synthesized for the consideration of patent applications. DK-56-1, a deuterated derivative of Compound 6, also dose-dependently inhibited the nociceptive response in the mouse hot-plate test. The antinociceptive effect of Compound 6 at 3 mg/kg was comparable to that produced by 10 mg/kg morphine (i.p.) while having a slightly shorter duration. The effect of Compound 6 was not affected by naloxone, suggesting it is opioid-independent. Importantly, the antinociceptive effects of either Compound 6 or DK 56-1, were significantly, but not completely, reduced by intrathecal injection of furosemide (10 nmol). This incomplete reversal of Compound 6-induced antinociception induced by intrathecal injection of furosemide prompted us to examine the possible contribution of off-targets. Among three off-target receptors, the serotonin 5-HT7 receptor was a most likely candidate. We therefore have verified that 5-HT7 receptors might not the action targets of Compound 6 since a 5-HT7-selective antagonist, amisulpride (1 mg/kg, i.p.), did not affect the antinociceptive effect of Compound 6. We also examined effects of hispidulin and its deuterated derivative on the hot-plate test. Hispidulin at doses of 3, 10 and 30 mg/kg (i.p.) also reduced the nociceptive response dose-dependently in the mouse hot-plate test. However, deuterated-hispidulin had no effect in the hot-plate test. In the formalin test, both Compound 6 and hispidulin (i.p.) inhibited the nociceptive response in phase II, but not phase I. However, deuterated hispidulin had no effect in the formalin test. Finally, we have also performed an immunofluorescence experiment to support notion that α6GABAARs are expressed and functional in the spinal cord and dorsal root ganglion of ICR mice. These results suggest that enhancing α6GABAAR-mediated transmission in the spinal dorsal horn can initiate an opioid-independent antinociceptive effect without apparent behavioral changes. Therefore, α6GABAAR-selective PAMs may have the potential to be developed as novel therapeutic agents for pain relief.