盐酸羟哌吡酮（YL-0919）的抗抑郁效应和机制研究

发布时间：2018-06-28 09:40

本文选题：抗抑郁药 + YL-0919　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：抑郁症是致病率和致残率最高的精神疾病之一,因其具有高发病、高自杀、高复发、高致残率,和低识别、低就诊、低治疗率成为严重的全球性公共卫生问题和突出社会问题。WHO预测,到2020年抑郁症将成为危害人类健康的第二大疾患。目前临床最为广泛的一线绝大多数抗抑郁药物,包括5-HT重摄取抑制剂(SSRIs,如氟西汀)、5-HT/NE双重重摄取抑制剂(SNRIs,如度洛西汀)等均存在起效延迟、有效率不高、导致性功能障碍和自杀倾向等较严重缺陷。近年来基于“优化的单胺策略”,5-HT1A受体部分激动和选择性的5-HT再摄取抑制双靶标抗抑郁药(serotonin partial agonist and reuptake inhibitors, SPARIs),因其具有强效、快速起效、潜在低自杀倾向等特点,以及不良反应(包括性功能障碍和体重增加等)发生率低的优势,己成为新型药物的代表方向。盐酸羟哌吡酮(YL-0919)是军事医学科学院毒物药物研究所正在研发的兼有5-HT1A部分激动和5-HT重摄取抑制双重靶标的1.1类抗抑郁专利新药,具有较强的新药研发前景,本研究旨在系统探讨其行为活性及靶标机制。目的：主要研究了兼有5-HT1A受体(5-HT1AR)激动和5-HT重摄取抑制双重活性抗抑郁新药YL-0919的活性特点及其靶标调节机制。方法：采用放射性配体竞争结合实验分别研究YL-0919与大鼠突触体蛋白中5-HT转运蛋白(SERT)和5-HT1A受体的亲合性以及受体选择性,并进一步制备含有SERT的大鼠突触体蛋白以及采用稳定表达人源SERT (hSERT)的HEK-293细胞系进行YL-0919对5-HT的重摄取抑制作用研究。分别采用小鼠悬尾和小鼠强迫游泳两个经典行为绝望模型评价新药YL-0919单次给药的抗抑郁活性,并采用相关5-HT受体阻断剂,明确YL-0919抗抑郁作用的相关受体。在小鼠获得性无助模型上评价YL-0919亚慢性给药的抗抑郁活性,在zero-迷宫、高架十字迷宫、Vogel电击冲突饮水、新奇抑制摄食行为模型中探讨单次或慢性给予大鼠YL-0919的抗焦虑活性。采用小鼠新奇抑制摄食、悬尾实验和强迫游泳实验对YL-0919和同靶标抗抑郁药维拉佐酮的抗焦虑、抗抑郁效果进行比较。在受体后机制研究中,首先在离体水平用竞争免疫荧光法检测YL-0919对正常大鼠前额皮层突触膜腺苷酸环化酶(AC)活性的影响。在整体水平,检测YL-0919长期给药后小鼠海马、前额皮层Foskolin激活的AC活性的变化。应用酶联免疫吸附法(ELISA)检测长期给药后小鼠海马PKA活性的变化,并在小鼠悬尾和小鼠强迫游泳模型上检测脑室注射选择性蛋白激酶A(PKA)抑制剂H-89对YL-0919抗抑郁活性的阻断作用。采用蛋白免疫印迹分析法(Western Blot)考察慢性给药YL-0919对小鼠海马BDNF、p-CREB、p-ERK表达的调控作用。结果：研究表明,YL-0919对SERT以及5-HT1A受体均具有高亲和力结合,Ki分别为0.72nM和0.19nM; YL-0919与NET(NE转运蛋白)和DAT (DA转运蛋白)的几乎没有亲和力。在大鼠前额皮层粗制突触体中,YL-0919强效抑制[3H]-5-HT的重摄取,ICso为1.78nM；在稳定表达人源SERT的HEK-293细胞系中,YL-0919同样对[3H]-5-HT的重摄取具有强效的抑制作用,IC50为1.93nM。并且YL-0919的5-HT重摄取抑制作用强于临床一线药物氟西汀和度洛西汀。YL-0919在浓度为10μM时对D3、D2、D4、D1、D5、5-HT1B、5-HT1D、5-HT2A、5-HT2C、5HT5A、5-HT6、β1A、α1B、α2A、M1、M2、M3、M4、M5等19种受体无亲和力。这些结果提示YL-0919对5-HT1A和SERT具有高亲和力和高选择性结合,并且是强效的5-HT重摄取抑制剂。单次给予YL-0919(0.625-2.5mg/kg, i.g)能够剂量依赖的显著缩短小鼠悬尾和强迫游泳不动时间。使用5-HT1A受体阻断剂WAY-100635预处理能够完全阻断YL-0919(2.5mg/kg,i.g)的抗抑郁活性。而5-HT2受体拮抗剂米安色林、5-HT3受体拮抗剂曲匹西隆、α2肾上腺素受体拮抗剂育亨宾对YL-0919的抗抑郁活性无阻断作用。提示YL-0919急性给药具有抗抑郁作用,并且与激动5-HT1A受体有关,这与本实验室的其他研究一致。YL-0919亚慢性给药(1.25-5mg/kg, i.g)在第2-5天显著缩短获得性无助小鼠逃避潜伏期及减少逃避失败次数,表现出了显著的抗抑郁活性。单次或慢性给予大鼠YL-0919(0.625-2.5mg/kg,i.g)显著缩短新奇抑制摄食潜伏期、增加大鼠在zero-迷宫开臂的停留时间和进入开臂的次数、增加大鼠在高架十字迷宫进入开臂次数百分比和在开臂停留时间百分比、增加电击饮水次数,提示YL-0919单次或慢性给药具有抗焦虑作用。慢性给予YL-0919(0.625-2.5mg/kg, i.g.)和维拉佐酮(1-4mg/kg, i.g.)均可显著缩短新奇抑制摄食潜伏期,并都显著缩短小鼠悬尾、游泳不动时间,提示YL-0919和维拉佐酮抗抑郁、抗焦虑活性相当,但YL-0919的起效剂量较维拉佐酮小1-3倍。进一步的受体后机制研究发现,在离体水平YL-0919(10-9-10-5mol/L)与正常大鼠前额皮层突触膜孵育,在1-10μM浓度时可显著激活AC活性,表现出与阳性药氟西汀和丁螺环酮一致的作用。5-HT1A受体阻断剂WAY-100635能显著拮抗YL-0919对AC的激活作用。在整体水平,长期反复给予YL-0919(1.25-2.5mg/kg,i.g.)和维拉佐酮(4mg/kg, i.g.)均能增强小鼠海马、前额皮层forskolin激活的AC活性,同时增强小鼠海马PKA活性,并显著上调小鼠海马BDNF、p-CREB、 p-ERK的表达。进一步研究证实,脑室注射选择性PKA阻断剂H-89能完全阻断单次给予YL-0919(2.5mg/kg, i.g.)在小鼠悬尾和小鼠强迫游泳模型上的抗抑郁活性。这些结果提示激活5-HT1A受体,并增强cAMP-CREB信号通路的功能是YL-0919抗抑郁作用的重要机制之结论：YL-0919与SERT蛋白和5-HT1A受体蛋白具有高亲合活性并且这种结合具高选择性；YL-0919具有强效的5-HT重摄取抑制功能,并且强于一线药物氟西汀和度洛西汀；YL-0919单次、亚慢性或慢性给药在多种动物模型上,表现出显著的抗抑郁、抗焦虑作用；YL-0919具有与维拉佐酮相当的抗抑郁、抗焦虑活性,但起效剂量更低；激活5-HT1A受体功能,并增强前额皮层、海马cAMP-CREB信号通路功能,上调海马BDNF水平从而促进神经营养是YL-0919抗抑郁、抗焦虑活性的重要机制。
[Abstract]:Depression is one of the mental diseases with the highest morbidity and disability rate. Because of its high incidence, high suicide, high relapse, high disability rate, low recognition, low treatment and low rate of treatment, it has become a serious global public health problem and a prominent social problem.WHO forecast. By 2020, depression will become the second major disease endangering human health. Most of the most widely used antidepressants in the bed, including 5-HT reuptake inhibitors (SSRIs, such as Fu Xiting), 5-HT/NE double reuptake inhibitors (SNRIs, such as duloxetine) have delayed onset and low efficiency, resulting in severe defects such as sexual dysfunction and suicidal tendencies. In recent years, the "optimized monoamine strategy", 5-HT1 A receptor partial excitation and selective 5-HT reuptake inhibits the dual target antidepressant (serotonin partial agonist and reuptake inhibitors, SPARIs), and has become a new drug because of its strong effect, rapid onset, potential low suicide, as well as the low incidence of adverse reactions (including sexual dysfunction and weight gain). YL-0919 is the 1.1 patent new antidepressant drug, which is being developed by the Toxicological Research Institute of Military Medical Science Academy of the PLA, which has both 5-HT1A partial excitation and 5-HT reuptake inhibition target. It has a strong prospect for new drug research and development. The aim of this study is to systematically explore its activity activity and target mechanism.
Objective: To study the activity characteristics and target regulation mechanism of the double active antidepressant antidepressant YL-0919, which has both 5-HT1A receptor (5-HT1AR) and 5-HT reuptake inhibition.
Methods: the affinity and acceptor selectivity of 5-HT transporter (SERT) and 5-HT1A receptor in YL-0919 and rat synaptosomes were studied by radioactivity ligand competition assay, and the synaptosomes in rats with SERT and HEK-293 cell lines with stable expression of human SERT (hSERT) were further prepared and YL-0919 pairs of 5-HT were carried out. The antidepressant activity of the new drug YL-0919 single dose was evaluated by two classical behavioral despair models of mouse hanging tail and forced swimming in mice. The related 5-HT receptor blockers were used to identify the related receptors of the antidepressant effect of YL-0919. The subchronic YL-0919 administration was evaluated in the mouse acquired helplessness model. Antidepressant activity in the zero- labyrinth, the elevated cross maze, the Vogel electric shock drinking water, the novel inhibition feeding behavior model to explore the single or chronic anti anxiety activity of YL-0919 in rats. The antidepressant and antidepressant of YL-0919 and the same target antidepressant, vistadone, were used in the novel inhibition feeding, tail suspension experiment and forced swimming test. The effects were compared. In the post receptor mechanism study, the effects of YL-0919 on the activity of synaptic adenylate cyclase (AC) in the prefrontal cortex of normal rats were first detected by competitive immunofluorescence. At the overall level, the changes in the AC activity of the hippocampus in mice and the Foskolin activated in the frontal cortex after the long-term administration of YL-0919 were detected. The changes of PKA activity in the hippocampus of mice after long-term administration were detected by ELISA. The blocking effect of the selective protein kinase A (PKA) inhibitor H-89 on the antidepressant activity of YL-0919 was detected on the mouse suspension tail and the mice forced swimming model. The chronic administration of YL-0919 to mice was examined by the protein immunoblotting analysis (Western Blot). The regulation of the expression of BDNF, p-CREB and p-ERK in the hippocampus.
Results: the study showed that YL-0919 had high affinity for both SERT and 5-HT1A receptors, and Ki was 0.72nM and 0.19nM, respectively, and there was almost no affinity between YL-0919 and NET (NE transporter) and DAT (DA transporter). In the HEK-293 cell line of human SERT, YL-0919 also has a strong inhibitory effect on [3H]-5-HT, IC50 is 1.93nM. and the 5-HT reuptake inhibition of YL-0919 is stronger than the clinical frontline drug fluoxetine and duloxetine.YL-0919 at the concentration of 10 mu M. 19 receptors, such as 2A, M1, M2, M3, M4, M5, have no affinity. These results suggest that YL-0919 has a high affinity and high selectivity for 5-HT1A and SERT, and is a potent inhibitor for 5-HT reuptake.
A single dose of YL-0919 (0.625-2.5mg/kg, i.g) could significantly shorten the dose dependent mice suspension and forced swimming time. The 5-HT1A receptor blocker WAY-100635 pretreatment could completely block the antidepressant activity of YL-0919 (2.5mg/kg, i.g). The 5-HT2 receptor antagonist, michalin, the 5-HT3 receptor antagonist, traepimacon, and the alpha 2 adrenaline The anti depressant activity of YL-0919 was not blocked by a prime receptor antagonist, suggesting that YL-0919 acute administration has antidepressant effect and is associated with the activation of 5-HT1A receptor. This is consistent with other studies in the laboratory, which is consistent with the other studies in the laboratory (1.25-5mg/kg, i.g), which significantly shortened the escape latency and decrease of the Acquired Helplessness mice on the 2-5 day. The single or chronic YL-0919 (0.625-2.5mg/kg, i.g) significantly shortened the incubation period of new inhibition of feeding, increased the duration of the opening of the arm in the zero- maze and the number of times to enter the arm, and increased the percentage of the opening arm of the rat in the high cross maze and the open arm in the open arm. The percentage of retention time increased the frequency of electric shock and drinking water, suggesting that the single or chronic administration of YL-0919 had anti anxiety effect. Chronic YL-0919 (0.625-2.5mg/kg, i.g.) and Vilazzo ketone (1-4mg/kg, i.g.) could significantly shorten the incubation period of new inhibition of feeding, and shorten the tail of mice and swim time, suggesting YL-0919 and Vera R. Antidepressant and anti anxiety activity were equivalent, but the dose of YL-0919 was 1-3 times smaller than that of group.
Further postreceptor mechanism study found that the activity of AC was significantly activated at the concentration of YL-0919 (10-9-10-5mol/L) in the prefrontal cortex of the normal rat and at the concentration of 1-10 micron M, showing that the.5-HT1A receptor blocker WAY-100635, in accordance with the positive drug fluoxetine and buspirone, could significantly antagonize the activation of YL-0919 to AC. At the overall level, YL-0919 (1.25-2.5mg/kg, i.g.) and Vilazzo ketone (4mg/kg, i.g.) can enhance the activity of forskolin activated in the hippocampus and the prefrontal cortex, and enhance the activity of PKA in the hippocampus of mice, and increase the expression of BDNF, p-CREB, p-ERK in the hippocampus of mice. Further studies have proved that selective PKA blockers are injected into the ventricle. -89 can completely block the antidepressant activity of a single dose of YL-0919 (2.5mg/kg, i.g.) on mouse tail and mouse forced swimming. These results suggest that activating the 5-HT1A receptor and enhancing the function of the cAMP-CREB signaling pathway is an important mechanism for the antidepressant effect of YL-0919.
Conclusion: YL-0919 has high affinity to SERT protein and 5-HT1A receptor protein and this binding has high selectivity; YL-0919 has strong inhibitory function of 5-HT reuptake, and is stronger than fluoxetine and duloxetine; YL-0919 single, subchronic or chronic administration in multiple animal models, showing significant antidepressant. Anti anxiety effect; YL-0919 has antidepressant, anti anxiety activity with Vera R, but lower starting dose; activating 5-HT1A receptor function, enhancing the function of the prefrontal cortex, hippocampal cAMP-CREB signal pathway, up regulating the BDNF level of the hippocampus and promoting neuronutrition is an important mechanism of YL-0919 antidepressant and anti anxiety activity.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【共引文献】