Investigation Of Anti-post-traumatic Stress Disorder Candidate Drug And Its Possible Mechanisms

Post-traumatic stress disorder(PTSD)is a delayed and long lasting mental disorder following exposure to severe traumatic events.Patients with PTSD often show severe symptoms including re-experience,hyperarousal and avoidance of trauma-related stimuli.Comorbidity of PTSD and depression,anxiety,drug addiction and other mental disorders can as high as 50%,making it a major mental illness which aroused widespread concern in society.To date,there is still no effective treatment for PTSD due to the exact aetiology of PTSD unclear.The serotonin(5-HT)reuptake inhibitors(SSRIs)(sertraline or paroxetine)are the only medications currently approved by the Food and Drug Administration(FDA)for the treatment of PTSD,but there are problems such as serious adverse effects and low efficiency.So it is urgent to find new anti-PTSD candidate drugs and its possible mechanisms.At present,most researchers believe that the disorder of neurotransmitter/neuroregulator in the brain is the material basis for the pathogenesis of PTSD,in which the over-activation of the dopamine(DA)system and the glutamate(Glu)system plays a crucial role,the latest research suggests that endogenous cannabinoid system(ECS)may also occupy a place in the pathogenesis of PTSD.Dopamine D3 receptor is thought to have particularly relevance to neuropsychiatric diseases in the 5 DA receptor subtypes.Studies on its single nucleotide polymorphism(SNP)suggest that the D3 R SNPs may relate with impulsive disorders and PTSD.In terms of brain distribution,the D3 receptor is expressed in brain regions thought to govern emotion and emotional responses to body reward,decision making,exercise,anxiety and fear,such as the amygdala,cerebral cortex,Nucleus Accumbens(NAc)olfactory nodules and other brain regions and central nucleus.Functionally,D3 R acts as a self-receptor(presynaptic receptor)and has a significant regulatory effect on the activation of DA neurons and the concentration of DA in the synaptic cleft.Therefore,from the perspective of distribution and function,D3 receptor has potential research value in anti-PTSD.YQA14 is a patented selective D3 antagonist designed and synthesized by our institute with high affinity for D3 R.Our previous studies have indicated that YQA14 exhibited anti-schizophrenia and anti-drug addiction effects in animal models,whether it has anti-PTSD effect,has aroused great interest.Ketamine is a non-competitive glutamate N-methyl-D-aspartate(NMDA)receptor antagonist with sedative and analgesic effects at doses below the general anesthetic dose.In recent years,ketamine has been widely concerned with its rapid antidepressant effect by affecting the function of the glutamate receptor system of the central nervous system of the body,but its studies on anti-PTSD have shown contradictory results that are effective,ineffective or even aggravate the core symptoms of patients with PTSD.What’s more,anti-PTSD studies on optical isomers of ketamine have rarely been reported.From the pharmacological effects and the use of ketamine,it is of great significance to conduct preclinical studies on the anti-PTSD effects of ketamine.CBD is another major component of cannabis other than THC,with high safety and no addiction potential.Related studies suggest that CBD can effectively reduce the cardiovascular response and anxiety caused by stress stimulation,and the CBD can inhibit the acquisition and arousal of fear memory before stress stimulation.At the same time,CBD can hinder the re-consolidation of fear memory,thus reducing the experience of response to fear memory.In addition,clinical brain imaging studies have found that CBD can inhibit the amygdala function in patients with different levels of concern,and reduce the expression of c-Fos protein in mouse amygdala,suggesting that CBD may have the biological activity of down-regulating amygdala function.However,so far,there is no systematic pharmacological research report on the role of CBD in PTSD.There is no uniform report and systematic research on the timing and duration of CBD administration.ObjectiveBased on dopamine,glutamate and endocannabinoid receptor systems,the study aims to explore the anti-PTSD effects and the possible neurobiological mechanisms of D3 receptor antagonist YQA14,non-selective glutamate receptor antagonist and CBD,which may provide useful clues for the discovery of new and effective anti-PTSD drug candidates.MethodsRat single prolonged stress(SPS),time-dependent sensitization(TDS)and mice pre-shock model were used.The locomotor activity of animals was evaluated by open field test,PTSD-like behaviors were evaluated by contextual freezing test and elevated plus maze test.Results1.The anti-PTSD effects of dopamine D3 receptor antagonist YQA14 and its possible mechanism(1)The anti-PTSD effects of YQA14: In the rat SPS model,there was no significant difference between the control non-SPS rats and the post-SPS rats regarding the crossings and rears in the open field test(P> 0.05,P>0.05,n=8),suggesting that neither SPS nor drug treatment affected the locomotor activity in rats.However,SPS rats showed significant PTSD-like behaviors with enhanced freezing time in contextual fear test(P<0.001)and decreased percentage of entries into and time spent in open arms in elevated plus maze test(P<0.01,P<0.05,n=8),indicating that the SPS model was successfully developed.We also found that repeated administrations of YQA14 can either decrease the contextual freezing time(P<0.05,n=8)and increase the percentage of entries into and time spent in open arms significantly(P<0.05,P<0.05,n=8),suggesting that YQA14 has a significant inhibitory effect on fear and anxiety-like behavior in PTSD rats.(2)Possible mechanisms: In order to determine the relationship between D3 receptor and PTSD behaviors,we established a pre-shock PTSD model in mice using D3 receptor knockout mice: We found that although the freezing time significantly increased in both the wild type(WT)mice model group and the knock out(KO)mice model group(Day 5: P < 0.001,P < 0.01,n = 6-12;Day 18: P < 0.001,P < 0.05,n = 6-12),the duration of freezing was significantly reduced in the KO mice with foot-shocks compared with the WT mice model group(Day 5 and Day 18: P < 0.001,n = 6-12).In the elevated plus maze tests,the percentage of entries into the open arms and time spent in the open arms decreased significantly in the WT control mice(P < 0.05,P < 0.05,n = 6-12),while the KO mice did not change significantly and showed significant differences compared to the WT model group.There were no significant changes in the number of crossings and rears in the open field test(P > 0.05,P > 0.05,n = 6-12).The experimental results suggest that dopamine D3 receptor may be involved in the pathophysiological process of fear-like and anxiety-like behavior in PTSD mice.(3)To further determine whether the observed pharmacological effects of YQA14 on PTSD models are mediated by blockade of brain D3 receptors,we investigated and compared the effects of YQA14 in WT and KO mice.We found that the freezing time significantly increased in both the WT mice model group and the KO mice model group,compared with respective control groups(P < 0.001,P < 0.05,n = 6-9).Moreover,the duration of freezing was significantly reduced in the KO mice with foot-shocks compared with the WT mice model group(P<0.05,n=6-9).In the elevated plus maze tests,the percentage of entries into and time spent in the open arms decreased significantly in the WT control mice(P<0.05,P<0.05,n=6-9),while the KO mice did not change significantly.The results were consistent with the above results.For WT mice,daily administration of YQA14 can both reduce the freezing behaviour(P<0.05,n=6-9)and increase the percentage of entries into and time spent in the open arms(P<0.01,P<0.05,n=6-9)significantly,while the KO mice YQA14 group did not change significantly.In addition,there was no significant changes in the number of crossings and rears in the open field test(P>0.05,P>0.05,n=6-9).The results suggest that the anti-PTSD effect of YQA14 is mediated through the D3 receptor,and knocking out the D3 receptor has the effect of alleviating PTSD-like behavior.2.Anti-PTSD effects of racemate ketamine and its R,S-isomers(1)In the experiment of studying the anti-PTSD effects of ketamine,we selected the rat TDS model: in the rat TDS model,there was no significant changes in the number of crossings and rears in the open field tests(P>0.05,P>0.05,n=14),suggesting that modeling and administration of ketamine did not affect the spontaneous activity of the rats.Compared with the control group,the freezing time of the rats in the model group in the contextual freezing tests was significantly prolonged(P< 0.001,n=14),the percentage of entries into and time spent in the open arms in the elevated plus maze were significantly reduced(P<0.001,P<0.001,n=14),suggesting that the model was established successfully with significant fear-like behavior and anxiety-like behavior.After continuous administration of ketamine,the ketamine group had a reduced freezing time compared with the model group,but there was no significant difference(P>0.05,n=14).In the elevated plus maze tests,the percentage of the entries into and time spent in the open arm were significantly higher than those of the model group(P<0.05,P<0.05,n=14),suggesting that ketamine has improved PTSD anxiety-like behavior in rats.(2)Anti-PTSD effects of R,S-ketamine on rat TDS model: There was no significant change in the number of crossings and rears in the open field test(P>0.05,P>0.05,n= 8),suggesting that modeling and administration of the dose of ketamine did not affect the spontaneous activity of rats.Moreover,compared with the model group,S-ketamine significantly reduced the freezing time in rats(P<0.05,n=8),and the percentage of time spent in the open arms in the elevated plus maze test was significantly increased(P<0.05,n=8),while the effect of R-ketamine was not obvious.The results suggested that S-ketamine had a significant effect on improving PTSD-like behaviors under the conditions of this experiment,and R-ketamine had no such effect.3.Study on the anti-PTSD effects of cannabidiol(1)In the mice pre-shock model,after continuous administration of 10 mg/kg or 30 mg/kg(CBD)for 5 days after model establishment(preventive effect),the number of crossings and rears of mice in the open field test were not significant(P>0.05,P>0.05,n=12),which suggested that this dose of CBD did not affect the spontaneous activity of mice.In the contextual freezing test and elevated plus maze test,10mg/kg of CBD can significantly reduce the freezing time of mice on the 8th day(P<0.01,n=12)and significantly increase the percentage of time spent in the open arms(P < 0.05,n = 12).The results suggested that continuous administration of CBD in the acute phase of PTSD can significantly improve the fear and anxiety-like behaviors of PTSD mice.(2)In the acute therapeutic effect tests,10 mg/kg or 30 mg/kg CBD had no significant effect on the number of crossings and rears in the open field test(P>0.05,P>0.05,n=12),which suggested that this dose of CBD did not affect the spontaneous activity of mice.In the contextual freezing test,pre-administration of 10 mg/kg CBD significantly reduced the freezing time of the mice(P<0.05,n=10),and significantly reversed the decreased entries into and time spent in open arms in the elevated plus maze test(P<0.05,P<0.05,n=10).The experimental results suggested that acute administration of CBD during the test period can significantly improve the fear and anxiety-like behavior of PTSD mice.Conclusions1.The administration of the dopamine D3 receptor antagonist YQA14 has a significant inhibitory effect on PTSD-like behaviors,which is achieved by blocking the D3 receptor,and knocking out the D3 receptor can significantly alleviate PTSD-like behavior in mice.The study demonstrated for the first time that D3 receptors are likely to participate in the pathophysiological processes of fear and anxiety-like behavior in mice PTSD-like behavior.2.Repeated administration of ketamine can improve the anxiety-like behavior in PTSD rats.Repeated administration of S-ketamine significantly counteracts PTSD-like behavior in rats at a dose lower than that of racemic ketamine,whereas R-ketamine have no such effect.3.In the pre-shock mice model,acute administration of CBD during the phase-concomitant administration or test period can significantly reduce the fear-like and anxiety-like behavior of PTSD mice.In summary,we found that dopamine D3 receptor blocker YQA14,ketamine and CBD have different degrees of anti-PTSD effect,suggesting that DA,Glu and endogenous cannabinoid systems may be involved in the pathophysiology of PTSD.In-depth understanding of the pathogenesis of PTSD,exploring new therapeutic targets and developing new anti-PTSD drug mechanisms provide useful clues.