The 5-HT₆ Receptor-related Mechanism For The Cognition-enhancing Properties Of Hypidone Hydrochloride（YL-0919）,a Novel Protential Antidepressant

Depression is one of the major diseases according to WHO.Statistics shows that more than 340 million patients are inflicted with depression,which has become a public medical health problem worldwide and induced critical social issues because of its high prevailing,recurring,disabling and suicide rates in addition to its low recognition,consultation and cure rates.However,Clinical first-line antidepressants including 5-HT reuptake inhibitors（SSRIs）,5-HT/NE dual reuptake inhibitors（SNRIs）and tricyclic（TCAs）have significant antidepressant and anxiolytic-like effects while they possess various deficits,including a delayed onset of action（2-6weeks）and low effective rate,and cause cognitive impairment,sexual dysfunction,suicidal tendencies,thus the development of novel multitarget antidepressant with rapid-onset and cognition-enhancing advantages has become the main direction of novel antidepressants.Hypidone hydrochloride（YL-0919）,the 5-HT_1A/6A/6 agonists and5-HT reuptake inhibitor,is a novel potent antidepressant with original chemical structure,and has been granted patents from the United States,Japan and a series of domestic and foreign countries,and received clinical approval of the national 1.1 kind of new medicine in 2015.Previous studies confirmed that YL-0919 has significant antidepressant-and anxiolytic-like effects.Compared with first-line antidepressants,YL-0919 possesses rapid-onset and cognition-enhancing advantages without causing sexual disorders.Recently,it has been found that it has high affinity with 5-HT₆receptor.Based on the close relationship between 5-HT₆ receptor and cognition,we speculate that the cognition-enhancing function of YL-0919 may be related to 5-HT₆receptor.The purpose of this study is to systematically explore the target characteristics and regulating mechanisms of 5-HT₆ receptor.Objective:To study the target characteristics of YL-0919 to 5-HT₆receptors,and to explore the relationship between the 5-HT₆ receptor and the cognition-enhancing,antidepressant/anxiolytic-like effects of YL-0919 and targeting mechanisms.Methods:The radioligand binding inhibition test and[³⁵S]-GTPγS binding assay were used to evaluate the binding affinity of YL-0919 to 5-HT₆ receptor in rat striatum,transient CHO cell line and stable Hela cell lines.Novel object recognition（NOR）,Morris water maze（MWM）and step-down test（SD）were used to evaluate the cognition-enhancing activity of YL-0919,and the selective 5-HT₆ receptor antagonist SB271046 was used to evaluate the relationship between behavioral improvement caused by YL-0919 and 5-HT₆ receptor activation.To study the 5-HT₆receptor related mechanisms of YL-0919,the competitive immunofluorescence assay were used to examine the cAMP level in h5-HT₆ receptor-expressed in the Hela cells,and the Western Blot were used to evaluate the regulating effects of YL-0919 on p-CREB,BDNF,p-ERK_1/2,Synapsin-1,PSD-95 and GluR-1 signal pathway and synaptic protein expression.Finally,the mouse tail suspension test（TST）and rat forced swimming（FST）were used to evaluate the antidepressant activity of YL-0919and its relationship with 5-HT₆ receptor;the mouse holeboard test（HB）,staircase test（ST）,elevated plus maze（EPM）,and open field test（OFT）were used to evaluate the anxiolytic activity of YL-0919 and its relationship with 5-HT₆ receptor.Results:（1）Radioligand competitive binding experiments showed that YL-0919 had high binding affinity with 5-HT₆ receptors in the rat striatum,the CHO cells transiently expressed the h5-HT₆ receptor and the Hela cells stably expressed the h5-HT₆ receptor,with K_i of 10.72,14.76 and 28.12nM respectively;[³⁵S]-GTPγS showed full agonist characteristics of YL-0919 in striatum and cells,with EC₅₀0 of71.23,64.73 and 52.92nM respectively,and the maximum efficiency(E_max)reached100%which is the same to the 5-HT₆ receptor agonist WAY208466,suggesting that YL-0919 is a full 5-HT₆ receptor agonist.（2）Cognitive-related behavioral tests showed that subchronic oral administration of YL-0919（1.25-2.5mg/kg）could significantly increase the recognition index in NOR,the entries and duration in the target quadrant,the entries crossing the platform in WMW,shortened the first time crossing the platform in MWM and the step-down latency in SD,suggesting the cognition-enhancing effects of YL-0919;compared with Vilazodone,the partial agonist of 5-HT_1AA receptor and 5-HT reuptake inhibitor,which of no such functions;Further study showed that 5-HT₆ receptor antagonist SB271046（10mg/kg）completely blocked the cognition-enhancing effects of YL-0919 without affecting the cognitive activity itself,suggesting that 5-HT₆ receptor activation might be its underlying mechanisms;（3）Mechanism study found that YL-0919 could significantly increase cAMP levels in the Hela cells stably-expressed the h5-HT₆ receptor,which could be dose-dependent blocked by SB271046,and intragastric administration of YL-0919（2.5mg/kg）significantly increase p-CREB,BDNF,p-ERK_1/2,Synapsin-1,PSD-95 and GluR-1 levels in the hippocampus,which was blocked by SB271046,suggesting that activating the 5-HT₆ receptor and enhancing the function of cAMP-CREB signaling pathway may be one of the important mechanisms of cognition-enhancing effects of YL-0919.（4）A single dose of YL-0919（2.5mg/kg）could significantly shorten the immobility time in FST and TST,and the antidepressant-like effects were blocked by selective 5-HT₆ receptor antagonist SB271046（10mg/kg）;chronic intragastric administration of YL-0919（2.5mg/kg）could significantly increase the number of head-dips and head-dipping duration in HB,decrease the number of rearings in ST,increase the time and entries into open arms in EPM,increase the entries and duration in the center in the OFT,which were blocked by SB271046（10mg/kg）treatment,suggesting that 5-HT₆ receptor mediated the antidepressant-and anxiolytic-like effects of YL-0919.Conclusion:YL-0919 is a full agonist of 5-HT₆receptor.YL-0919 showed significant cognition-enhancing effects in various kinds of animal models,and its underlying important mechanism might be activating 5-HT₆ receptor.In addition,enhancing downstream cAMP-CREB signaling pathway of 5-HT₆ receptor might at least partially mediate the above process.Moreover,5-HT₆ receptor activation might also be one of the mechanisms of antidepressant-and anxiolytic-like effects of YL-0919.In conclusion,this study confirmed the 5-HT₆ receptor-related mechanisms of YL-0919,the 1.1 types of antidepressants,laying the experimental foundation for developing novel antidepressants with cognition-enhancing effects.