摘要:本計畫延續我們之前的生技製藥國家型計畫,其源自臨床觀察到白花苦藍盤葉萃取物對一個頑固性運動抽動患者明顯有效,我們於是篩選了白花苦藍盤葉之酒精分萃物改善甲基安非他命(MA)引起的過動症,發現KLP-1 為一有效成分。KLP-1 (i.p.)也可以改善MA、K 他命、MK-801 和PCP 在小鼠引起的前脈衝抑制(PPI)缺損。研究指出KLP 可增強GABAA受體活化,包括只表現在小腦顆粒細胞的含?6的?6-GABAAR。我們於是將KLP-1 微量注射到小腦,發現KLP-1 也可改MA、K 他命、MK-801 和PCP引起的PPI缺損,KLP-1 的這些作用可被a6-GABAAR 抑制劑逆轉,被受體促效劑仿用。KLP-1 也能有效改善PCP 在小鼠誘發的互動行為退縮情形,該作用可被D1 受體抑制劑逆轉。在92 個受體/酵素的結合篩選中,KLP-1 只結合6 個藥效標的,包括3 個benzodiazepine/ GABAA受體、COMT、MAO-A和MAO-B,並無毒性標的。本計畫中,我們擬建構化學合成KLP-1 的最適化流程 (目標1),然後申請百克級大量合成KLP-1,並申請臨床前毒理試驗 (目標2),建立妥瑞症及精神分裂症的動物模式 (目標4),觀察KLP-1 在這些動物模式的療效與對其前額葉皮質與紋狀體微透析液中多巴胺濃度的影響,並利用藥理學方法釐清GABAA/a6-GABAA受體和COMT 對KLP-1 作用的貢獻(目標5),最後化學修飾KLP-1,建構最佳分子結構,期望這個源自臨床的發現可以經由動物實驗的驗證後,轉譯回到臨床應用。
Abstract: This is a continuation project of our previous NSTPBP-funded project which was initiated bya clinical observation that a patient with intractable motor tics was responsive to the leaveextract of a local herb, Clerodendrum inerme (CI) (白花苦�盤). We have screened differentsubfractions of the ethanol extract of CI leaves in relieving methamphetamine (MA)-inducedhyperlocomotion and identified KLP-1 to be an active component. Patients with Tourettesyndrome (TS), a spectrum of motor tics, also manifest sensorimotor gating deficit, which can bereflected by a disruption in prepulse inhibition of the startle reflex (PPI) in humans and animals.PPI disruptions are also observed in patients with other neuropsychiatric disorders, especiallyschizophrenia. The CI ethanol extract and KLP-1 (i.p.) also rescued PPI disruptions in miceinduced by MA and ketamine/MK-801/PCP, based on dopamine and glutamate hypotheses ofschizophrenia, respectively. KLP-1 is a positive allosteric modulator of GABAARs, including the6 subunit consisting GABAARs (6GABAARs) which are exclusively expressed in cerebellargranule cells. Given by intracerebellar (i.c.b.) microinjection, KLP-1 also rescued PPIdisruptions in MA-, MK-801- and ketamine-treated mice. These effects were reversed by i.c.b.injection of an a6-GABAAR antagonist (furosemide) and mimicked by an a6-GABAAR agonist(Ro 15-4513). KLP-1 (i.p.) also significantly improved social withdrawal-like behaviors inchronic PCP-treated mice, an animal model mimicking the negative symptom of schizophrenia.This effect was antagonized by a D1 receptor antagonist, SCH 23390. Binding screenings on 92receptors/enzymes revealed only 6 ON targets, including 3 benzodiazepine/GABAAR bindingsites, COMT, MAO-A and MAO-B, and none of OFF target. In addition to having filed 3 patents,we will push this compound to preclinical toxicity testing. We will launch a synthetic plan forKLP-1 to optimize a feasible synthetic scheme for scale-up synthesis (Aim 1). Then, we willrequest a scale-up synthesis of KLP-1, supported from NHRI. With enough amounts of KLP-1available, we will request the preclinical toxicity tests (Aim 2). Besides, we will reinforce thetherapeutic potential of KLP-1 in TS and schizophrenia by establishing respective animal models,such as apomorphine-induced stereotypy behaviors and social withdrawal-like behaviorsinduced by MK-801 (Aim 4). The finding that KLP-1 is a COMT inhibitor is interestingsince COMT is important in controlling the dopamine level of the prefrontal cortex (PFC) and,hence, the cognitive function. We hypothesize that COMT inhibition may contribute to KLP-1’srescue of social withdrawal and PPI disruption, and a6-GABAAR activation may be involved inits improvement of hperlocomotion and PPI disruption. To validate these hypotheses, we willexamine effects of KLP-1 on apomorphine-induced stereotypy behaviors, MK-801-impairedsocial interaction, and dopamine levels in the microdialysate of the PFC and striatum infree-moving mice (Aim 5). The relative contributions of GABAARs and COMT in KLP effectswill be clarified. Two COMT inhibitors, tolcapone and OR-486 will be used as positive controlsfor COMT inhibition. Then, we will derive a structure-activity relationship study for leadoptimization (Aim3). Hopefully, this clinically-inspired project can be ultimately translated backto clinical trials, filing IND application with satisfied pre-clinical toxicity results.