Pharmacokinetic And Pharmacodynamic Studies Of ZCY-15,an Anti-alzheimer’s Disease Compound

Objective:Alzheimer’s disease（AD）is one of the most common types of dementia in the elderly and is characterized by a gradual loss of function of the central nervous system,with clinical manifestations of progressive memory loss,cognitive loss,and behavioral changes that eventually lead to disability and death,the pathogenesis of which is unknown.At present,there are no effective drugs and methods to treat AD.ZCY-15 is a novel compound designed and synthesized with reference to the structure of Memantine and its amide analogues,which can improve the cognition and learning ability of quinoline-induced dementia mice,and its efficacy is slightly better than that of memantine.In this study,the pharmacokinetic,tissue distribution,metabolism and excretion of ZCY-15 were systematically studied to clarify its in vivo process,providing pharmacokinetic support for the development and utilization of ZCY-15.The neuroprotective effects of ZCY-15 on AD cells and mouse models were investigated through pharmacodynamic studies,and the main drug targets and pathways of ZCY-15were explored,so as to clarify the anti-AD efficacy and provide pharmacodynamic basis for subsequent studies.Methods:1.Pharmacokinetic and tissue distribution studiesA quantitative method for determination of ZCY-15 concentration in rat plasma by LC-MS/MS was established,and its specificity,matrix effect,recovery,accuracy,precision and stability were verified.After intragastric administration at low,medium and high doses(0.75,2.25,6.75 mg·kg^-1)and tail vein administration at medium doses(2.25mg·kg^-1),plasma concentration of ZCY-15 was determined,and its pharmacokinetic behavior and absorption characteristics were analyzed.A quantitative method for determination of ZCY-15 concentration in rat tissues and organs by LC-MS/MS was established,and its specificity,matrix effect,recovery,accuracy,precision and stability were verified.The concentrations of ZCY-15 in brain,hippocampus,liver,heart,spleen,lung,kidney,fat,muscle,testis（male）or ovary（female）,large intestine,small intestine and stomach were determined at different time points after medium dose(2.25 mg·kg^-1)by gavage and tail vein.The distribution and changes of ZCY-15 in tissues and organs of rats were investigated over time.To determine the permeability of the blood-brain barrier and whether it can reach the predetermined tissues in the brain.2.Metabolic and excretory studiesA high-resolution mass spectrometric Bruker^TM Tims TOF Pro tandem series Bruker^TMELUTE UPLC was applied to identify the metabolites of ZCY-15 in rat plasma,bile,urine and feces after gavage administration at medium dose(2.25 mg·kg^-1),and to clarify the chemical structure,metabolic time and metabolic pathway characteristics of the metabolites.A quantitative method for the determination of ZCY-15 in rat excreta by LC-MS/MS was established and validated in terms of specificity,matrix effect,recovery,accuracy and precision,and stability of the method;the concentrations of ZCY-15 in rat bile,urine and feces after gavage administration at medium doses(2.25 mg·kg^-1)were determined to clarify its excretion characteristics in rats.3.Pharmacodynamic studiesThe cell survival rates of ZCY-15 induced by Aβ_1-42 and quinolinic acid were determined by CCK-8 method,and the protective effect and effective concentration range of ZCY-15were determined in vitro.Annexin V-FITC/PI double staining method was used to detect the anti-apoptotic effect of ZCY-15 on Aβ_1-42 and quinolinic acid induced AD cell model.Ion channel patch clamp method was used to detect the ion channel blocking effect of ZCY-15 on NMDA receptor.Swiss Target Prediction software was used to predict the possible targets of ZCY-15.The safety tolerance of mice to ZCY-15 was tested by maximum tolerance test.Morris water maze test was used to detect the improvement of cognitive and learning ability of AD mice with gradient concentration of ZCY-15.Immunohistochemical staining was used to detect the clearance of Aβdeposition in brain of AD mice at gradient concentration of ZCY-15.Western Blot was used to determine the regulatory effects of gradient concentration of ZCY-15 on the levels of predictive target proteins and related pathway proteins in the brain of AD mouse model,and verify the drug target.The effects of ZCY-15 on brain inflammatory cytokines in AD mice were determined by ELISA.Results:1.Pharmacokinetic and tissue distribution studiesThe LC-MS/MS analytical method established in this part can be used for ZCY-15concentration in both plasma and tissue homogenate,with linear range of 1.95～1000ng·m L^-1.The matrix effect,recovery,accuracy and precision are in accordance with the requirements of biological sample detection.After gavage administration of gradient concentrations of ZCY-15,T_max was less than 1.4 h in both male and female rats;T_1/2 was greater than 4 h in both female and male rats;C_max and AUC_0-∞in male rats were smaller than those in the corresponding female rats;CL was significantly lower in female rats than in male rats;the absolute oral bioavailability was（4.59±1.36）%and（5.81±0.86）%in male and female rats,respectively.The highest concentrations of ZCY-15 were found in the large intestine,small intestine and stomach of male and female rats after gavage administration,followed by adipose tissue.The presence of ZCY-15 was detected in both brain tissue and hippocampus.The plasma concentrations increased proportionally with increasing dose administration.2.Metabolic and excretory studiesA total of 10 possible metabolites were detected in plasma,bile,urine and feces of rats after gavage administration of ZCY-15 at medium doses(2.25 mg·kg^-1),of which MP2F2was a plasma and fecal co-metabolite,MP3U1 and MP4U2 were plasma and urine co-metabolites,MP1 was a plasma-exclusive metabolite,MU3 was a urine-exclusive metabolite,MF1,MF3 and MF4 are unique metabolites of feces,and MB1 and MB2 are unique metabolites of bile.All the above metabolites are produced by oxidation reaction in phase I metabolic reaction.The LC-MS/MS analytical method developed in this part was used for the determination of ZCY-15 concentrations in bile,urine and feces,with the linear range of 1.00～200.0 ng·m L^-1.The matrix effect,recovery,accuracy and precision were all in accordance with the requirements for the determination of biological samples.The excretion rate of ZCY-15 in bile of rats after gavage administration was only（0.0002±0.00009）%;ZCY-15 was not detected in urine samples;the excretion rate of ZCY-15 in feces was only（0.99±0.57）%;the drug may be excreted mainly as metabolites.3.Pharmacodynamic studiesZCY-15 exhibited significant neuroprotective and anti-apoptotic effects in Aβ_1-42 and quinolinic acid-induced AD cell models,respectively,with effective concentrations ranging from 1.56 to 12.50μM and 3.13 to 12.50μM,respectively,and calculated EC₅₀s of 4.04±0.20μM and 7.34±0.45μM,respectively.ZCY-15 did not block NMDA receptor ion channels,indicating that it is not an NMDA receptor antagonist.The reverse target finding software calculated that EPHX2 may be the main target of action of ZCY-15.ZCY-15 has a good safety profile in mice in the dose range of 0～5 g·kg^-1.Morris water maze experiments showed that gradient concentrations(1.5,3.0,4.5mg·kg^-1)of ZCY-15 significantly reduced the plateau crossing latency and increased the number of plateau crossings in the AD mouse model.Immunohistochemical staining showed that gradient concentrations(1.5,3.0,4.5 mg·kg^-1)of ZCY-15 significantly reduced the number of cells deposited with Aβin the brain of the AD mouse model.WB experiments showed that gradient concentrations(1.5,3.0,4.5 mg·kg^-1)of ZCY-15significantly downregulated the expression of EPHX2,i NOS,GFAP and other proteins in the brain of the AD mouse model.ELISA results showed that gradient concentrations(1.5,3.0,4.5 mg·kg^-1)of ZCY-15 significantly downregulated the levels of TNF-α,IL-1β,IL-6 inflammatory cytokines in the brains of AD mice,and up-regulated IL-3 levels,which played a good protective role against inflammatory injury triggered by Aβdeposition.Conclusions:1.Pharmacokinetic and tissue distribution studiesThe LC-MS/MS method was established for the first time to detect the concentration of ZCY-15 in plasma and tissue homogenates.ZCY-15 can be quickly absorbed into the blood through the small intestine,and the blood concentration reaches the peak in a short time.The plasma concentration increased in proportion to the dose,and the pharmacokinetic characteristics were linear.The absolute bioavailability of oral administration was low and there was gender difference.The kidneys may be the main excretory organ.ZCY-15 can target hippocampus selectively through BBB.2.Metabolic and excretory studiesTen metabolites were detected in plasma,urine,bile and feces of rats,all of which were produced by oxidation reaction in phaseⅠmetabolism.The LC-MS/MS method was established for the first time to detect the concentration of ZCY-15 in bile,urine and feces homogenates.The amount of ZCY-15 in bile and feces was very small in rats,and it was mainly excreted in the form of metabolites.3.Pharmacodynamic studiesZCY-15 is a novel epoxide hydrolase inhibitor with good safety and tolerability,showing a good anti-apoptotic effect on AD cell models.By regulating EPHX2 and other proteins and pathways,ZCY-15 can reduce Aβdeposition in brain,reduce inflammatory injury in brain,improve cognition and learning ability of AD mouse model,and show good anti-AD efficacy.