本科室与北京协和医学院药物研究所药化室张纯贞研究员课题组合作，从一系列哌嗪类化合物中定向筛选出化合物H057，其结构新颖且具有较强的镇静催眠作用。本论文旨在对H057的镇静催眠作用进行药效学评价，并对其机制进行初步探索。

腹腔注射H057（≥5 mg/kg）5 min后，即可观察到小鼠出现明显的镇静现象。给药15 min后，利用开场实验测定小鼠自主活动。结果表明，H057 (5和25 mg/kg，i.p.) 能显著抑制小鼠的自主活动，抑制率分别为25%和66%。腹腔注射H057 (≥60 mg/kg) 能直接诱导100%小鼠翻正反射消失，60 mg/kg和80 mg/kg组小鼠睡眠潜伏期分别为 (24±11) min和 (20±7) min，睡眠时间分别为 (96±15) min和 (124±64) min。协同阈下剂量戊巴比妥钠实验结果显示，H057能显著增加阈下剂量戊巴比妥钠诱导小鼠的入睡率，5 mg/kg和25 mg/kg组小鼠的入睡率分别增加为87.5% 和100%。同时，直接经侧脑室给予H057 (20 μg, 1 min/只)，也能引起小鼠翻正反射消失，显示H057能够以原型起效。以上实验结果表明H057具有显著的镇静催眠作用。通过悬挂实验测试H057对小鼠肌张力的影响，结果发现给药40 min后，溶剂对照组小鼠全部能悬挂于绳上超过1 min，10 mg/kg和20 mg/kg组小鼠只有1/2能悬挂于绳上超过1 min。测定给予H057前后不同时间点小鼠的肛温，结果显示H057 (5 和10 mg/kg, i.p.) 能分别引起小鼠体温下降0.9℃和1.8℃。摄食实验结果显示，H057 (25 mg/kg, i.p.) 给药16小时内对小鼠摄食的抑制率为26%。

通过合并给予GABA_A受体拮抗剂荷包牡丹碱，测定小鼠自主活动以检测荷包牡丹碱对H057镇静作用的影响。实验结果表明，荷包牡丹碱不能拮抗H057对小鼠自主活动的抑制作用。利用印防己毒素、戊四唑和NMDA诱导小鼠致惊厥模型，来评价H057对小鼠惊厥潜伏期的影响。实验结果显示，H057 (20 mg/kg，i.p.) 对印防己毒素和NMDA诱导小鼠惊厥的潜伏期有显著延长作用，惊厥潜伏期分别延长51%和500%。采用高效液相-电化学检测法测定H057对小鼠大脑皮层、纹状体、下丘脑和海马脑匀浆中抑制性氨基酸GABA含量的影响。结果表明，H057对不同脑区脑匀浆中GABA含量无显著差异。应用微透析技术在体动态监测H057对大鼠大脑皮层细胞外液GABA含量的影响。实验结果显示H057能显著升高大脑皮层细胞外液GABA水平，给药后10 min升高26%。采用离体孵育脑片的方法测定H057对小鼠大脑皮层和下丘脑细胞外液GABA含量的影响。结果显示H057（10^-5 moL/L）能显著升高下丘脑和大脑皮层细胞外液GABA水平，分别升高了160%和130%。

前期研究发现H057能显著抑制小鼠的摄食，因此，我们推测H057发挥镇静催眠作用可能跟Orexin能神经系统相关。通过评价侧脑室注射Orexin受体激动剂Orexin A对H057 (80 mg/kg，i.p.) 诱导小鼠睡眠时间的影响，我们发现侧脑室注射Orexin A (0.10、0.15和0.20 nmol，i.c.v..) 能剂量依赖性的缩短H057诱导小鼠的睡眠持续时间，各剂量组的缩短率为37%、55%和95%。同时，我们发现H057（80 mg/kg，i.p.）不能引起预先侧脑室注射Orexin A的小鼠翻正反射消失。以上两个实验结果提示H057可能是Orexin受体的拮抗剂。Orexin主要通过激活单胺能神经系统来促进觉醒。采用高效液相-电化学检测法测定H057对小鼠大脑皮层、纹状体、下丘脑和海马脑匀浆中单胺神经递质含量的影响。结果表明，H057能显著降低小鼠纹状体内DA和下丘脑内NE的含量，分别降低了43%和28%。应用微透析技术在体实时动态监测H057对大鼠大脑皮层细胞外液单胺神经递质含量的影响。结果显示H057能显著降低大鼠大脑皮层细胞外液的NE和5-HT水平，分别降低38%和50%。采用离体孵育脑片的方法测定H057对小鼠大脑皮层和下丘脑细胞外液单胺神经递质含量的影响。实验结果表明H057（10^-5 moL/L）能降低两个脑区的DA和5-HT水平，大脑皮层分别降低80%和64%，下丘脑分别降低79%和66%。我们利用荧光假神经递质FFN102标记DA神经元来测定H057是否对单胺囊泡转运体2（VMAT2）有抑制作用。结果发现H057能显著性降低FFN102的荧光强度，推测H057对VAMT2可能有抑制作用。

以上结果提示，化合物H057发挥镇静催眠作用的可能通路为：1）H057能

够拮抗Orexin受体，阻断Orexin神经元对单胺神经的激活，降低单胺神经递质水平；2）Orexin能神经系统对单胺能神经系统的抑制使GABA能神经系统间接激活，增加了细胞外液的GABA含量；3）H057能够抑制VMAT2的活性，破坏DA的储存，降低DA的水平。

We collaborated with Professor Zhang Chunzhen in the department of pharmaceutical chemistry in institute of Materia Medica and found a novel compound H057 with the potential effect of sedative hypnotic from a series of compounds.This article is to study the sedative and hypnotic mechanisms of a novel compound H057.

The sedative and hypnotic effects of H057 were evaluated by open field test (OFT) and the experiment of the latency and duration of loss of righting reflex (LORR). In open field test, the spontaneous locomotor activity was decreased by 25% and 66% in H057 (5 and 25 mg/kg，i.p.) treated mice, respectively. In the experiment of LORR, H057 (≥60 mg/kg, i.p.) can compeletely induce LORR in mice. The latency of LORR at dose of 60 and 80 mg/kg were (24.2±11.1) min and (19.6±6.9) min, the duration of LORR were (96.4±14.9) min and (124±63.6) min.We found that H057 (5 and 25 mg/kg，i.p.) increased sleep onset rate induced by subthreshold dosage of sodium pentobarbital to 87.5% and 100% in mice. The loss of righting reflex can be found in H057 (20 μg/mouse, i.c.v.) treated mice. The effect of H057 on the muscle strength was observed in the traction test and half of mice dropped from the rope within 1 min after administration of H057 (10 and 20 mg/kg, i.p.), while none of mice dropped from the rope within 1 min in vehicle group. Moreover, H057 (10 and 20 mg/kg, i.p.) can decrease body temperature in mice by 0.9℃and 1.8℃, respectively. These results showed that H057 may have potent sedative-hypnotic effect.

The interaction between H057 and GABA_A receptor was revealed by investigating the antagonism effect of bicuculline on the spontaneous locomotor activity in mice injected with H057. Bicuculline has no effect on the spontaneous locomotor activity in H057 (20mg/kg, i.p.) treated mice. The anti-convulsion effect of H057 was revealed by picrotoxin, pentylenetetrazol and NMDA-induce convulsion in mice. H057 (25 mg/kg，i.p.) significantly increased the latency of convulsion induced by picrotoxin and NMDA by 51% and 500% in mice. The contents of GABA in different brain regions in mice were measured by high performance liquid chromatograghy-electrochemistry (HPLC-EC). H057 (50 mg/kg，i.p.) had no significantly effect on the contents of GABA in different brain regions in mice. The brain GABA levels in the extracellular fluid in cerebral cortex and hypothalamus were measured by microdialysis in vivo and incubating brain slices in vitro combined with HPLC-EC. H057 (25 mg/kg, i.p.) could significantly increase the GABA level by 126% in the dialysate in cerebral cortex in rats. The results from incubating brain slices revealed that H057 (25 mg/kg, i.p.) increased sharply GABA level by 130% and 160% in the extracellular fluid in the hypothalamus and cerebral cortex in mice.

H057 (25 mg/kg, i.p.) could also significantly inhibit the food intake in mice. So the interaction between H057 and Orexin receptor was revealed by investigating the effect of Orexin A (an orexin receptor agonist) on LORR induced by H057 in mice. Orexin A (0.10、0.15 and 0.20 nmol, i.c.v.) shortened significantly the latency of LORR induced by H057 (80 mg/kg, i.p.) by 37%、55% and 95%. Orexins stabilize wakeness mainly by activating the monoamine neurons. The brain monoamine neurotransmitters levels in different brain region in mice were measured by high performance liquid chromatograghy-electrochemistry. The results showed that H057 (50mg/kg, i.p.) significantly decreased DA level by 43% in the striatum and decreased NE level by 28% in the hypothalamus in mice. The brain monoamine neurotransmitters levels in the extracellular fluid in cerebral cortex and hypothalamus were measured by microdialysis in vivo and incubating brain slices in vitro. In vivo microdialysis, H057 (25mg/kg, i.p.) can decrease the NE and 5-HT levels by 38% and 50% in the extracellular fluid in cerebral cortex in mice.The results from incubating brain slices revealed that H057 (25 mg/kg, i.p.) decreased the DA and 5-HT levels by 79% and 66% in the hypothalamus, decreased by 80% and 64% significantly in the cerebral cortex in mice.Then we used a fluorescent false neurotransmitter 102 (FFN102) as optical tracer selectively labels dopamine neuron to reveal whether H057 can affect dopamine storage. The result shows that H057 can weaken the fluorescence intensity of FFN102.

In summary, these results demonstrated that H057 produces the potent sedative and hypnotics effects maybe following the sereral pathways: 1) H057, as a novel orexin antagonist, can block the orexin receptor to inhibit the activities of monoamine neurons and decrease the content of monoamine neurotransmitters in the extracellular fluid; 2) The GABAergic system is activated because of the inhibition of orexinergic system and nomoaminergic system; 3) H057 can inhibit the activity of VMAT2 and destroy the dopamine storage.