Therapeutic Mechanism Of 7,8-Dihydroxyflavone Derivatives For Treating Alzheimer’s Disease

BDNF/TrkB signaling cascades are essential for synaptic plasticity,learning and memory.BDNF is reduced in Alzheimer’s disease(AD).Accumulative evidence shows that BDNF delivery is a good strategy for treating AD.Nevertheless,BDNF cannot pass Blood-brain-barrier(BBB)and possesses insurmaountable pharmacokinetic obstacles.A potent small molecular TrkB agonist,7,8-dihydroxyflavone(7,8-DHF),mimics BDNF physiological actions in a variety of animal models.However,7,8-DHF possesses modest oral bioavailability.In the current study,we employed a prodrug strategy to improve its druggability by improving its PK profiles.In 5XFAD mouse model,we demonstrated that the optimal prodrug R13 increased the half-life,oral bioavailability and brain exposure of 7,8-DHF.Most importantly,R13 robustly exhibited promising therapeutic efficacy by strongly activating TrkB and repressing AEP,a newly identified delta-secretase that played a crucial role in AD pathogenesis,leading to eliminating the senile plaques in the AD mouse brain.The main research contents and results are listed as bellows:(1)Organic Synthesis of 7,8-DHF derivatives via ester or carbamate groups and identification of the optimal prodrug with appropriate in vitro pharmacokinetic profiles.Chemical structure of side groups modified on the catechol group in the parent compound 7,8-DHF.To identify the optimal prodrug,we tested the aqueous and room temperature stability,intestine microsome,liver microsome and plasma stability,and Caco-2 permeability in vitro.Among 20 derivatives,only R5,R7,R13,R16,R17 and R18 were relatively stable in intestinal microsomes and hydrolyzed in both liver microsomes and plasma.However,R7 barely yielded free 7,8-DHF in human liver microsomes,R16 and R17 failed to release detectable 7,8-DHF.Meanwhile,R18 showed poor Caco-2 permeability regardless its stability and hydrolyzability into 7,8-DHF.Taken together,the in vitro screening assays supported R13 as the only appropriate candidate for further investigation.(2)In vivo PK/BBB(Pharmakenetic/Blood-brain-barrier)profiles of R13 was tested through dosing mice with both 7,8-DHF and R13.In vivo pharmakinetic study showed that following a single oral administration of R13 at 75 mg/kg.bw,the mean Cmax and Tmax values for 7,8-DHF were 129 ng/mL and 30 min,and the mean area under curve(AUC)value was 14777.5 min ng/mL.The mean value of oral bioavailability for R13 was 10.5%.The T1/2 release of 7,8-DHF from R13 was 219.6 min versus 134 min by parent compound.BBB study showed that following a single oral administration of R13 at a dose of 72.5 mg/kg.bw,the mean Cmax and Tmax values for 7,8-DHF were 56 ng/ml and 2 h in plasma and 46 ng/ml and 4 h in the brain,which is higher than those from parent compound on oral administration.Overall,R13 possessed the favourable druggable properties.(3)Chronic oral administration of R13 activated TrkB and its downstream signaling pathways in mouse brain and prevents synaptic loss in 5XFAD mice.As expected,TrkB receptors were more prominently phosphorylated in 5XFAD mice treated with R13 than in those treated with vehicle control,so were the downstream AKT and ERK/MAPK pathways,reversing the loss of synaptic density in a dose-dependent manner.(4)Chronic oral administration of R13 inhibited AEP activation and APP and Tau proteolytic cleavage in 5XFAD mice.As compared with vehicle group,oral administration of R13 repressed AEP and blocked APP and Tau pathological fragmentation,and alleviated inflammation in the brain in a dose-dependent manner.(5)Chronic oral administration of R13 alleviated Aβ deposition and rescueed memory deficits in 5XFAD mice.Results showed that oral administration of R13 alleviated A(3 deposition in both hippocampus and cortex and reduced the concentration of total Aβ,therefore,rescued the memory deficits in 5XFAD mice.(6)Toxicity evaluation of long-term oral administration of R13.Oral intervention trials were performed on 2-month-old AD model mice 5XFAD with an intragastric dose of 21.8 mg/kg.bw/d.The results showed that long-term oral administration of R13 had no significant effect on the body weight of mice.As compared with vehicle group,the morphology of major organs and bone marrow cells of R13 intervention mice did not change after intervention.Chronic oral administration of R13 barely changed the biochemical profiles of the blood,indicating that R13 had low toxicity and long-term oral administration of R13 was safe.