Ⅰ.Studies On The Quality Control And Pharmacokinetics Of P91024·HBr Ⅱ.Studies On The Quality Control Of Cyclovirobuxine D

I Studies on the quality control and pharmacokinetics of P91024 HBrP91024 HBr is a novel antiplatelet drug synthesized by the New Drug Research Central of China Pharmaceutical Uruvesity.Its pharmacology test showed that it could inhibit platelet aggregation and dahension,inhibit thrombogenesis and possess protective effect on oschemic cerebral injury ..In order to corperate with its further development,we studied on its quality control method and pharmacokinetics.the main content reads as follows:Part one: Structural identification and quality control of P91024 HBr1.Structural identification of P91024 HBr . IR, UV.MS.¹H-NMR and ¹3C-NMR spectrum of P91024 HBr have been reported and all the chemical shifts of C and H have been assigned based on DEPT,COSY,HMQC and HMBC The structure conforms to that of aimed compound.2 .Analysis of physical-chemical properties of P91024 HBr .Some physical-chemical properties of P91024 HBr such as solubility,water,melting point,partition coefficent,absorptivity (E1 %, 1 cm) et al were invesgated. P91024 HBr is practically insoluble in watertight soluble in methanol and ethanol,sparingly soluble in acetone,freely in chloroform.Absorptivity(El%) in methanol isl320 in 0.1 mol L^-1 ¹HCl is 1359 ,melting point is 130.4℃, partition coefficent is 32.6 in n-butanol.3. The property comparision between P91024 and P91024 HBr. Great differences were found by means of X-ray powder differaction DSC IR and NMR. Those differences can be used to distinguished P91024 from P91024 HBr .Equilibrium solubilities at 5 temperatures in water were determined and enthalpies were calculated. The enthalpies of P91024 HBr and P91024 were 182.07 kJ mol^-1 and 21.9399 kJ mol^-1 respectively. The solubilities of P91024 HBr were greater than that of P91024 above 36℃,which suggests that P91024 added HBr may be greatly increase the bioavailability of P91024,at the same time,. P91024 HBr raise the stability of P91024 to light,both of them showed that it is necessary for P91024 to become P91024 HBr.4 .Determination of P91024 HBr by RP-HPLC. A HPLC method was established for the determination of P91024 HBr .The chromatographic conditions used are as follows:HYPERSIL C₁8 column, methanol-water(90:10) as eluents,flow rate 1mL min^-1,and wavelength for measurement at 231nm.The linear range of calibration curve was 26.7-267.10 u g mL^-1(r=0.9999),the detection limit was 2ng(S/N>3).The results of assay for 2 batches of sample were 99.0%, 100.3%,and impurities were o.5%, 0.4% respectively.The proposed method was proved simple,precise,and reproducible,the decomposed product can be separated as well,therefore,the quality of P91024 HBr can be effectively controlled.5. The preliminary stability of P91024 HBr Stress test showed P91024 HBr is stable when stored at hight temperature(40℃) and hight humidity( RH 75%),but not stable at 60℃ and exposed to light(Lx4500).Part two: Pharmacokinetics of P91024 HBr.6.Determination of P91024 HBr in rat plasma by HPLC and its pharmacokinetics study .An accurate and sensitive assay for P91024 HBr in rat plasma has been developed using RP-HPLCAfter adding methanol as protein precipitating agent and then basifying ,the plasma samples were extracted with cyclohexane.The intra-day variation on spiked samples ranged from 0.92% to 3.97%,the inter -day variation on spiked samples ranged from 2.25% to 4.30%.The lowest limit of quantitation was 5ng mL^-1 when l.0mL plasma was used.The extracted recovery ranged from 90.4% to 103.0% ,the method recovery ranged from 97.0% to 105.3%. P91024 HBr was rapidly absorbed in rats after oral administration .The plasma drug concentration -time curve conforms to a 2-compartment open model with a first order absorption The mean vaule of t_max t1/2β was 3h and 9.45h respectively .The relationship between dose and AUC of P91024 HBr was linear within the dosage range.This suggested that the disposition of P91024 HBr in rats belong to linear kinetics and the pharmacokinetic parameters ofP91024 HBr were dose independent.7.The pharmacokinetics of P91024 HBr in dogs.The main pharmacokinetic parameters of P91024 HBr in dogs after ig 20 mg kg^-1 are as follows: The mean vaule of t_max and t₁/2β was 3.33+ 1.51hand 11.68 ± 4.12h respectively The mean AUC was 8758+2901 ng h mL^-1.8.Distribution of P91024 HBr in ratsAfter ig P91024 HBr 20 mg kg^-1 to rats, the drug can be found in all determinated tissues and most of tissues have higher concentration than that of plasma The parent drug can be found in brain,it suggests that P91024 HBr can pass blood-brain barrier.the concentrations of all the tissues are higher than that of the plasma except brain at 12h,it shows that P91024 HBr has strong affinity for tissues,it may be have something to do with the strong fat-solubility of P91024 HBr.9. Excretion of P91024 HBr in rats. After ig P91024 HBr 20 mg kg^-1 to rats no unchanged drug be found in urine,the biliary excretion of the parent drug amounted to only 0.011% of the dosage within 24h.and in faeces to 35% within 48h after dosing, the excretion in faeces amounted to 34% within original 12h..10. Plasma protein binding of P91024 HBr in rats.Equilibrium dialysis was employed to determine protein binding ,the mean protein binding was 82.7% with RSD 5.37% when P91024 HBr ranged from 50 to 400 ng mL^-1 in buffer solution (as dialysis solution).Part three: How to establish an analytical method for pharmacokinetics study(review).The pharmacokinetics study has become an important part and base for new drug evaluation,but whether the result of the pharmacokinetics study can be used in clinical practice and the explanation of the result of drug research,depends on the design of the research protocol,especially the design ofbiopharmaceutical analysis method..This paper discusses several problems involving in the establishing a biopharmaceutical analysis method,so as to increase the level and quality of established method.II Study on the quality control of cyclovirobixune DCyclovirobixune D is extracted from Buxus microphylla sieb ET Zucc. Var.Sinica Rehd .et wils ,it contains a proportionally similar alkaloids, At present ,the exsisted analysis methods can not control impurities in it owing to the lack of specificity,therefore,we estalished chromatographic method with good specificity. The main content is:l.Seletion of TLC condition for cyclovirobixune DAfter many times tests,the chosen chromatographic condition is:silica-gel plate,acetic ether-methanol-ammonia water(l 7:2:1) as developping agent,improved potassium iodohydrargyrate as picture agent,.Under this condition,the product passed column chromatography appeared two impurities spots,,the colour of impurities are lighter than that of 5% RS solution. We found that the item of testing other alkaloids in present china pharmacopiea is unreasonable,because the reproducibility is poor,the amount of loaded sample is too small,.the standard cannot control related substances and should be improved..2. Determination of cyclovirobuxine D by RP-HPLC with precolumn ultraviolet derivatization. The effect of several factors including the reaction medium,temperature,time and amount of phenyl-isocyanate on the yield of the derivatization was investigated systematically .The reaction condition was optimized and the method was also certificated according to the requirement of the china pharmacopoeia.lt showed the developped method was accurate,reproducible,and simple,and can be used in routine quality control.3. Determination of cyclovirobuxine D by RP-HPLC with precolumn flurorescence derivatition. An RP-HPLC method for determination of cyclovirobuxine D was developped . Cyclovirobuxine D reacted with a derivative reagent 1-naphthyl isocyanate in chloroform to form fluorescence derivatives, stopped the reaction by adding the mobile phase and then directly injected the solution into the chromatograph to seperate it by RP-HPLC.The analysis was carried out on C\g column,the mobile phase is methanol-water (85:15), the excitation wavelength was set at 305nm,emission at wavelength 385nm,and the flow rate is 1mL min^-1.The effect of several factors including the reaction medium,temperature,time and amount of 1-naphthyl isocyanate on the yield of the derivatization was also investigated systematically. The absence of interference between the derivative peak responses of cyclovirobuxine D and its related substances were verified by UV diode array detecter and MS.The linearity was obtained from 0.75 g mL^-1 to 2.5 g mL^-1 of cyclovirobuxine D derivatives with a...