Sedative And Hypnotic Mechanisms Of Compound H057

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 GABAA 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 26% 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.