| Alzheimer's disease(AD),also calledsenile dementia,was a kind of irreversible and anamorphosis brain functional disturbance,also was one of the most severe health problems of the aged.Clinical manifestations were dysmnesy,intelligence decrescence,and any other cognition functional lesion.Origin,prevention and cure of AD were always extremely complicate problems.Acetylcholinesterase(AChE)inhibitors were the first and the most developed group of drugs approved for AD symptomatic treatment.BYZX,[(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinde n-one],was one of a series of 2-phenoxy-indan-1-one derivatives tested as acetyl cholinestera -se inhibitors,synthesized by structural modification based on Donepezil,the second filial generation AChE inhibition.It exhibited high AChE inhibitory activity(IC50=50nmol·L-1), and the molecular docking study indicated that it was nicely accommodated by AChE.In the progress of new drug development,it was important to understand the interaction to P-gp,the in vitro metabolism of NCE and the drug-drug interactions.Since the use of BYZX will be frequently accompanied by the use of multiple drugs,which may cause adverse effect by drug-drug interactions.Hence It was important to consider of the in vitro metabolism and the risk for drug interaction for further clinical research.Simultaneously,due to P-gp had a considerable influence on the drug absorption,so elucidate whether BYZX was the substrate or inhibitor of P-gp was also very important.Our studies focused on elucidate the interactions between BYZX and P-gp,the in vitro metabolism of BYZX in HLM and the drug-drug interactions of BYZX to provide a principle for clinical rational administration, and evaluate the security and rationality of co-administration of BYZX with other drugs. Meanwhile,also provide a theory evidence for the further development of BYZX.1.The research of interactions between BYZX and P-gpAIM:A reversed-phase high-performance liquid chromatography method was developed to simultaneously detemine BYZX uptaken by Bcap37 and Bcap37/MDR1 cells.It was authenticated that whether BYZX was the substrate of P-gp by the different accumulation of BYZX in the two cells.Meanwhile,distinguished the inhibitive effects of BYZX on the P-gp by the rhodamine 123.METHODS:A Diamonsil?C18column(250mm×4.6mm i.d.,5μm) was used for the reversed-phase high-performance liquid chromatography(RP-HPLC) separations of BYZX.The mobile phases were acetonitrile-10mmol·L-1KH2PO4(0.8%TEA, pH=4.0)(25:75,v/v),and the flow rate was 1.0 ml·min-1.UV detection wavelength set at 325 nm.Rhodamine 123 was determined by the 96 wells plate-scanning readers at Ex=485nm, Em=538nm.RESULTS:The calibration curves of BYZX in the concentration range from 0.1μmol·L-1to 20μmol·L-1ranges were linearity.The relative standard deviations of within-day were 1.16%～5.09%(n=5),and those of between-day were 2.38%～6.81%(n= 5).The limit of detection(LOD)was 0.026μmol·L-1(S/N=3)and the limit of quantification (LOQ)was 0.1μmol·L-1(RSD＜5%,n=5).The determination recoveries of BYZX were in the range of 97.10%～117.10%(n=5).The calibration curves of rhodamine123 were linear over the concentration range from 0.01μmol·L-1to 0.4μmol·L-1.The relative standard deviations of within day were 2.47%～5.27%(n=5),and between days were 5.63%～6.92%(n=5).The average method recoveries of rhodamine123 were 103.4%,the average absolute recoveries of rhodamine123 were 98.6%.Bcap37/MDR1 and Bcap37 cells were treated with BYZX and rhodamine 123.The concentrations of intracellular BYZX in Bcap37/MDR1 cells were as many as those in Bcap37 cells,when incubated at the same concentratin and time.And the concentrations of intracellular BYZX in the Bcap37/MDR1 cells did not increase when incubated with P-gp inhibitor verapamil.Furthermore,different concentrations of BYZX also had no any inhibitive effects on the efflux of rhodamine 123.CONCLUSION:The results indicated that there were no interactions between BYZX and P-gp.BYZX will not be pumped out of the cells,which was better for its absorption.2.In vitro metabolism of BYZX in human liver microsomesAIM:Studied the metabolism of BYZX in vitro,a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease.Elucidated the metabolite of BYZX and investigated the metabolite formation pathways.METHODS.BYZX and human liver microsomes were co-incubation in vitro at 37℃.The reaction was terminated by add ice acetonitrile(contained internal standard donepezil).Precipitated protein,supernatant was injected into the RP-HPLC system for determining the residual BYZX and the formation of metabolite,A Finnigan LCQ Deca XPplusion trap mass spectrometer,coupled with an Agilent 1100 series HPLC instrument via an ESI interface was used to elucidate the metabolite of BYZX.A Diamonsil?C18column(250mm×4.6mm i.d.,5μm)was used for the reversed-phase high-performance liquid chromatography(RP-HPLC)separations.The mobile phases were acetonitrile-10mmol·L-1KH2PO4(0.8%TEA,pH=4.0)(28:72,v/v),and the flow rate was 0.8 ml·min-1.UV detection wavelength set at 325 nm.RESULTS:The calibration curves of BYZX were linear over the concentration range from 5.07μmol·L-1to 101.37μmol·L-1.The relative standard deviations of within-day and between-day were less than 5%(n=5).The limit of detection(LOD)was 0.73μmol·L-1(S/N=3)and the limit of quantification(LOQ)was 2.49μmol·L-1(RSD＜7%,n=5).The determination recoveries of BYZX were in the range of 98.2%～104.8%.The apparent Km of BYZX in HLM was 53.25±17.2μmol·L-1,the Vmax was 0.94±0.77μmol·L-1·min-1·mg-1protein,and the intrinsic clearance value(Clint)was 0.018±0.02ml·min-1·mg-1protein.BYZX was catalyzed by CYP3A4.The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC-MS-MS. CONCLUTION:CYP3A4 was the major isozyme responsible for BYZX metabolism. N-dealkylation was the major metabolic pathway of BYZX.N-des-ethyl-BYZX was the predominant metabolite of BYZX,detected in vitro phase I metabolism in HLM.3.The metabolic interactions between BYZX and some drugsAIM:Study the metabolic drug interactions of-BYZX with some medicine and the inhibition of BYZX to CYP enzymes,to provide some useful informations for further research. METHODS:BYZX was co-incubated with several kinds of drugs,respectively.These drugs were 5 kinds of antipyretic analgesic drugs such as Buprofen etc,12 kinds of cardiovascular drugs such as Dobutamine etc,4 kinds of antibacterial drugs such as Azlocillin sodium etc,4 kinds of antidepressant drugs such as Chlorpromazine etc,4 kinds of uropoietic system drugs such as Tolterodine tartrate etc,5 kinds of antiallergic drugs such as Chlorphenamine Maleate etc,7 kinds of diping drugs such as Felodipine etc.The interactions were evaluated by IC50or Ki of the co-incubated drugs on the metabolism of BYZX.Then the substrates of CYP3A4, CYP1A2,CYP2C9,CYP2D6,CYP2C19,CYP2E1 were co-incubated with BYZX,and the inhibitive effects of BYZX were observed.RESULTS:Diping drugs showed fairly strong inhibitive effects on the metabolism of BYZX,with Ki value between 11.44～33.25μmol·L-1. Among these drugs,Cilnidipine had the strongest inhibition,with Ki value 11.44μmol·L-1,the inhibition of Felodipine was significantly weakened compared with the other dipings.Among the antidepressant drugs,Fluvoxamine,Chlorpromazine,Sertraline hydrochloride and Fluoxertine hydrochloride all had more or less inhibitive effects on the metabolisn of BYZX, with the IC5055.55μmol·L-1,74.81μmol·L-1,75.12μmol·L-1,77.03μmol·L-1,respectively. Among the carrdiovascular drugs,only Quinidine sulfate had small effect on the metabolism of BYZX,with the IC50value 80.4μmol·L-1.Uropoietic system drugs,antipyretic analgesic, antiallergic drugs,antibacterial agent all had no inhibitive effects on the metaboliam of BYZX.Otherwise,BYZX also did not inhibite the metabolism of substrates of CYP3A4, CYP1A2,CYP2C9,CYP2D6,CYP2C19,CYP2E1(Testosterone,Nifedipine,Caffeine, Phenacetin,Flurbiprofen,Diclofenac potassium,Dextromethorphan,Omeprazole,Diazepam, chlorzoxazone).CONCLUTION:The results indicated that diping drugs had strong inhibitive effects on the in vitro metabolism of BYZX.But BYZX had no any inhibitive effects on the CYP450s.
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