Preliminary Preclinical Study Of New Acridone Beta-secretase Inhibitors

Aβ oligomers formed by the abnormal accumulation ofβ-amyloid protein（Aβ）and the eventually formed amyloid plaques（commonly known as senile plaques）are prominent pathological hallmarks in the pathogenesis of Alzheimer’s disease（AD）except neurofibrillary tangles.The formation of many senile plaques is a late manifestation of AD.At this time,the damage to neurons is very serious,which cannot be reversed,and it is too late to intervene in this case.Therefore,the early treatment by targeting the production of Aβmay effectively delay the occurrence and development of AD based on the early diagnosis.Based on biochemical and molecular biology study,β-secretase（BACE1）is one of the important enzyme for Aβproduction.If this enzyme is inhibited,the development of AD might be halted.In the previous study,collaborated with the relevant pharmaceutical chemistry lab,we obtained a high score of new acridone compounds by virtual of screening compound libraries.We further structurally modified this new c lass of compounds,and totally synthesized 12 potential compounds that may inhibit the activity ofβ-secretase（BACE1）.And these new compounds were screened for further drug development study,which laid the foundation for the preclinical research of new compounds as new drugs.Molecularly,we evaluated toxicity of the compounds,the effect of each compound on the expression of Aβ40 and Aβ42 protein,and the effect on the activity ofβ-secretase in the APP/PS1 transgenic Chinese hamster ovarian cancer cells（CHO）.Taken together,079 showed better results than other compounds.This compound had no significant effect on the viability of CHO,effectively reduced the level of Aβ40 and Aβ42 protein secreted by transgenic CHO cells,and the EC₅₀ values are 0.08±0.01μΜand 0.05±0.01μΜrespectively.In addition,the result of ELISA showed that 079 had specific selectivity for BACE1.The results of western blotting showed that079 had no significant effect on the expression of APP protein,but increased the expression of enkephalinase.These results indicated that 079 not only effectively inhibits BACE1 activity in APP/PS1 transgenic CHO cells to reduce the production of Aβprotein,but also promots the expression of enkephalinase to accelerate the degradation of Aβprotein.At the same time,we used Caco-2 cells to simulate intestinal mucosal cells in order to detect the intestinal absorption capacity of 079.The results showed that 079 had a good permeability in Caco-2 enterocytes.When verapamil,the P-glycoprotein inhibitor,was added,the detected concentration of 079 on the basolateral side was significantly increased.In the in vivo experiments,we firstly used SD rats to initially examine the pharmacokinetics of 079 on the whole animals.The compound（50mg/kg）was administered via gavage and vein injection separately.The results showed that the plasma concentration of 079 reached a maximum after 1.4 h,and its half-life was 5.3 h.It had the ability to penetrate the blood-brain barrier and enter the brain,and the relative bioavailability of which was 19.8%.Secondly,we used Kunming species mice to study the acute toxicity of 079 on the whole animals.When the mice were orally administered with 5 g/kg of 079,no mice died.Therefore,the MTD of 079was more than 5 g/kg,indicating that 079 had low toxic ity.Finally,we utilized APP/PS1 transgenic mice to test the pharmacodynamics of 079initially on whole animals.The results showed that oral administration with079（80 mg/kg）for 2 months increased the ability of spontaneous activity,reduced the anxiety,and improved the cognitive impairment o f the transgenic mice.In addition,results of immunofluorescence and ELISA showed that 079 significantly reduced the level of Aβprotein in the cortex and hippocampus of the APP/PS1 transgenic mice.In summary,compound 079 can specifically inhibit the activity of BACE1 and promote the expression of enkephalinase to reduce the level and deposition of Aβprotein,which ultimately improves the cognitive function of the APP/PS1 transgenic mice.These results suggest that this compound is of great potential to be developed as an AD therapeutic agent.