TM-2�ڴ��󡢱ȸ�Ȯ���˸�΢�����е�ø�ٷ�Ӧ����ѧ�о�

??OBJECTIVE To study the enzymatic kinetics of TM-2 in rat, Beagle dog and human liver microsomes by LC-MS/MS. METHODS TM-2 was incubated with liver microsomal incubation system. LC-MS/MS method was established for quantitative analysis of TM-2 with cabazitaxel as internal standard. The enzyme kinetics parameters V_max and K_m was calculated by the GraphPad Prism 5.0 software. RESULTS A rapid and sensitive LC-MS/MS method was developed to study the enzyme kinetics of TM-2 in rat, Beagle dog and human liver microsome. The corresponding enzymatic kinetic parameters in rat, Beagle dog and human were as follows: V_maxvalues were 16.3, 354.6 and 154.8 nmol??min^-1??mg(protein)^-1, respectively; K_m values were 25.7, 313.8 and 89.4 ??mol??L^-1, respectively; CL_int values were 0.63, 1.13 and 1.73 mL??min^-1??mg(protein)^-1, respectively. CONCLUSION The result indicates that there are species differences in the activity of metabolic enzyme and the affinity of TM-2 to the metabolic enzyme. Enzyme kinetic parameters obtained of TM-2 provide important parameters for the further study.     

替格瑞洛的体外代谢及其与他汀类药物的相互作用研究

目的 考察替格瑞洛在大鼠肝微粒体的酶动力学及其与CYP3A的底物药物辛伐他汀、洛伐他汀及阿托伐他汀的相互作用,以期为临床上替格瑞洛与他汀类药物的合理使用提供科学依据。方法 将替格瑞洛与大鼠肝微粒体进行体外共孵育,孵育一定时间后用含有内标(地西泮,10 ng·mL^-1)的甲醇终止反应,并沉淀蛋白,14 000 r·min^-1离心10 min后取上清液进行分析,采用 LC-MS/MS测定微粒体酶孵育体系中活性代谢产物AR-C124910XX的浓度,使用 Prism 5软件计算主要的酶促动力学参数K_m,V_max与CL_int。在得到 K_m后,反应体系中底物替格瑞洛的浓度选择为1/3K_m~3K_m内的3个浓度,辛伐他汀、洛伐他汀及阿托伐他汀的浓度范围为1~100 μmol·L^-1,通过体外大鼠肝微粒体代谢实验研究其与替格瑞洛代谢性相互作用。使用 SigmaPlot 12.3 软件酶动力学模块,根据 Dixon公式计算各他汀对替格瑞洛代谢的可逆性抑制常数 K_i,并根据标准差值选择最符合的模型。结果 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应动力学,其转化生成AR-C124910XX的K_m值为32.2 μmol·L^-1,V_max为149.0 pmol·min^-1·mg(pro)^-1。表观清除率CL_int为4.63 nL·min^-1·mg(protein)^-1;辛伐他汀显著抑制替格瑞洛活性代谢产物的生成,其抑制符合混合抑制模型。抑制常数K_i为0.58 μmol·L^-1,α值为7.5,β值为0.56。洛伐他汀及阿托伐他汀对替格瑞洛代谢呈现出中等强度的抑制作用,其抑制亦符合混合抑制模型,抑制常数K_i分别为2.9和7.5 μmol·L^-1,α 值分别为3和1.7,β值为0.34和0.29。 结论 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应酶动力学;辛伐他汀对替格瑞洛有显著程度的代谢性抑制作用,洛伐他汀和阿托伐他汀对替格瑞洛有中等程度的抑制作用,临床上替格瑞洛与他汀类药物合用时可能需要考虑其与辛伐他汀的相互作用。     

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??OBJECTIVE To establish a robust, fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1, and explore their kinetic features.METHODS Two selective substrates including phenacetin and dextromethorphan, which are probes of CYP1A2 and CYP2D1, were chosen for liver microsomes incubation, respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS) methods were developed for kinetic studies.RESULTS The fast and convenient UPLC-MS methods with high resolution and short running time(4~5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities;both methods showed good accuracy and precision, and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0.267 and 0.007 ??mol??L^-1, respectively. The kinetic studies showed that the Michaelis constant(K_m) for CYP1A2 and CYP2D1 were (28.4??2.7) and (13.9??1.3) ??mol??L^-1, respectively. Their activities were determined to be (1.47??0.12) and (3.98??0.09) nmol??mg^-1, respectively,when the substrate concentration was 10 ??mol??L^-1.CONCLUSION UPLC Tandem MS technique is proved to be a rapid, convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.     

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??OBJECTIVE To study the pharmacokinetics of pirfenidone in Chinese healthy volunteer after a single dose and multiple-dose administration. METHODS Twelve Chinese healthy volunteers were randomly divided into low, medium and high dose groups(200, 400, 600 mg). The multiple-dose group was administrated with pirfenidione 400 mg three times daily for 5 d. Intensive blood sampling was performed from 12 volunteers within 12 h after the single dosing and the last dose of the multiple dosing. HPLC-MS/MS was used to determine the plasma concentrations of pirfenidone. The pharmacokinetic parameters were calculated by DAS software. RESULTS The main pharmacokinetic parameters of pirfenidone after single-dose administration of 200,400,600 mg qd as follows: ??_max were(5.00??1.42),(9.43??2.74)and(14.14??3.36)mg??L^-1;t_max were(0.57??0.33),(0.60 ??0.30)and(0.60??0.38)h;t_1/2 were(2.16??0.77),(2.15??0.75)and(2.01??0.76)h; AUC_0-?? were(13.87??7.79),(29.26??12.02)and(45.85??20.25)mg??h??L^-1;AUC_0-12 were?(13.27??7.08),(27.92??10.56)and(43.98??18.14)mg??h??L^-1,respectively. The main pharmacokinetic parameters after 400 mg tid for 5 d were as follows: ??_max was(9.46??2.77)mg??L^-1,??_min was(1.14??1.11)mg??L^-1,t_max was(0.52??0.34)h,t_1/2 was(1.93??0.63)h,AUC_0-?? was(26.74??13.49)mg??h??L^-1,AUC_0-12 was (25.79 ??12.34)mg??h??L^-1,AUC_sswas(23.53??10.59)mg??h??L^-1.CONCLUSION The pharmacokinetic parameters of pirfenidone show that ??_max and AUC were linear in the dose range from 200-600 mg and the pharmacokinetic parameters were similar as reference.
     

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??OBJECTIVE To study the pharmacokinetics of hot-melt spray-dried andrographolide granules and compare it with andrographolide bulk drug. METHODS A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for determination of the concentration of andrographolide in plasma of rats which were respectively given micronized andrographolide and hot-melt spray-dried andrographolide granules, then the pharmacokinetic parameters were calculated. RESULTS The pharmacokinetic parameters of andrographolide after a single dose administration of micronized andrographolide and hot-melt andrographolide were as following: t_1/2 were (347.33??9.32) and (390.82??8.78) min, t_max were (30.00??5.94) and (60.00??3.48) min, ??_max were (1 940.14??21.21) and (1 818.22??23.64) ng??mL^-1, AUC_0-t were (427 515.71??37 350.03) and (426 406.31??20 577.75) ng??min??mL^-1, AUC_0-inf were (545 423.14??47 969.18) and (593 569.87??30 247.35) ng??min??mL^-1, Vz/F were (43.48??4.75) and (44.96??3.81) kg??L^-1, CL/F were (86.78??3.35) and (79.74??2.89) kg??L^-1??min^-1, respectively. CONCLUSION Compared with the bulk drug, the hot-melt spray-dried andrographolide granules have a longer t_1/2, lower ??_max and delayed t_max in rats.     

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??OBJECTIVE To prepare compound aspirin and esomeprazole magnesium enteric-coated pellet capsules and evaluate the drug release in vitro/in vivo. METHODS The aspirin pellet cores were prepared by using extrusion-spheronization method, and the esomeprazole magnesium-containing drug pellets were prepared with fluidized bed. By using fluidized bed coating method, the two kinds of drug-containing pellets were respectively coated with enteric layer to obtain enteric-coated pellets. After determining the loading capacity by measuring drug content, the two kinds of drug-containing pellets were filled into No.1 capsules. In vitro release was evaluated by measuring release percentage. The in vivo release behavior was evaluated by determination of pharmacokinetic parameters in rats. RESULTS The cumulative release percentage of the two drugs was less than 5% in 2 h in 0.1 mol??L^-1 hydrochloric acid solution. The cumulative release percentage of aspirin was more than 70% in 45 min in pH 6.8 PBS and it was more than 80% in 30 min for esomeprazole magnesium. Aspirin was metabolized to salicylic acid in plasma and its main pharmacokinetic parameters were as follows:t_1/2=9.47 h, MRT_0-??=14.43 h, t_max=3.00 h, ??_max=51.34 mg??L^-1, AUC _0-24=703.39 mg??h??L^-1, AUC _0-??=860.52 mg??h??L^-1. The pharmacokinetic parameters for esomeprazole magnesium were as follows:t_1/2=3.72 h, MRT_0-??=7.44 h, t_max=1.50 h, ??_max=2.71 mg??L^-1, AUC_0-24=11.89 mg??h??L^-1, AUC_0-??=13.79 mg??h??L^-1. CONCLUSION The formulation of compound enteric-coated pellet capsules is reasonable, and the preparation technology has good reproducibility. The drug release is located in the intestinal tract, thus esomeprazole magnesium can antagonize the gastrointestinal side effects of aspirin and aspirin can produce better antithrombotic effect .     

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??OBJECTIVE To investigate the pharmacokinetics of matrine injection by different routes of administration. METHODS Twenty healthy SD rats were enrolled in this study. They were randomly divided into two groups and received intraperitoneal and intravenous administration of matrine injection at dose of 15 mg??kg^-1 respectively. Blood samples (0.3-0.4 mL) were immediately collected into heparinized tubes before injection and at 0.033, 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12 h after injection. Plasma sample concentrations were determined by a validated LC-MS/MS method. The pharmacokinetic parameters including AUC_0-12, AUC_0-??, MRT_0-12, MRT_0-??, t_1/2, Vd, CL and ??_max were calculated. RESULTS The main pharmacokinetic parameters for matrine after intraperitoneal and intravenous administration at dose of 15 mg??kg^-1 were as follows:AUC_0-12 (10 166??2 426), (12 217??2 968) ng??mL^-1??h;AUC_0-?? (10 230??2 432), (12 300??3 031)- ng??mL^-1??h;MRT_0-12 (1.91??0.41), (2.14??0.54) h;MRT_0-?? (2.01??0.41), (2.26??0.64) h; t_1/2(2.26??0.89), (2.60??1.25) h;Vd(4 998??2 010), (6 175??2 540) mL;CL (1 531??315.0), (1 727??475.6) mL??h^-1??kg^-1; ??_max (5 246??1 187), (8 503??1 101) ng??mL^-1, respectively. The bioavailability of intraperitoneal administration is 83.21%. CONCLUSION No significant differences were observed in AUC, MRT, t_1/2 and CL values of matrine between different administrations except for ??_max and Vd.     

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??OBJECTIVE To develop a highly sensitive and specific LC-MS/MS method to explore the pharmacokinetic properties of araloside A. METHODS Araloside A was administered in a dose of 50 mg??kg^-1 via gastric in fusion and 5 mg??kg^-1 by intravenous injection in rats.Araloside A was analyzed by a validated LC-MS/MS method in plasma after intravenous and intragastric administration. The pharmacokinetic parameters were evaluated by software DAS 3.0. RESULTS The RESULTS of pharmacokinetic study showed that the linear range of araloside A was good in 1.0-10 000.0 ??g??L^-1(r>0.994 8). The specificity, precision and accuracy, matrix effect and extraction recovery rate and stability all meet the requirements. The main pharmacokinetic parameters for intragastric administration with araloside A 50 mg??kg^-1 and intravenous injection of araloside A 5 mg??kg^-1 were as follows:t_1/2 was(8.65??3.22) and(2.00??0.21)h, AUC_0-t was(277.14??101.00) and (21 194.59??4 385.13)ng??h??L^-1, MRT_0-t was (7.88??0.64) and (1.21??0.11)h, Vd/F was (2 229.99??1 013.97) and (0.71??0.20)L??kg^-1, CL/F was(149.11??62.28) and (0.24??0.05) L??h^-1??kg^-1, respectively; ??_max was (32.68??10.74) ??g??L^-1 for intragastric administration and t_max reached(1.21??0.70) h, oral bioavailability of araloside A was about 0.14%. CONCLUSION The LC-MS/MS method established is specific and sensitive, and can be successfully applied in basic pharmacokinetic study of araloside A in rat plasma.     

黏质沙雷氏菌发酵制备舍雷肽酶及基本酶学性质研究

目的 开发微生物发酵生产舍雷肽酶的工艺,并了解基本酶学特性。方法 从家蚕蛹的肠道中分离产酶菌株,单因素实验和响应面实验优化发酵培养基,采用(NH₄)₂SO₄沉淀、超滤和DEAE离子交换层析3步纯化舍雷肽酶,电泳法测定酶的相对分子质量和等电点,考察pH和温度对酶活力和稳定性的影响,并测算酶的反应动力学常数。结果 分离获得一株产舍雷肽酶的黏质沙雷氏菌LL-413菌株,摇瓶发酵产酶活力达到1 126 U·mL^-1,10-L发酵罐中发酵产酶活性达到1 505 U·mL^-1;舍雷肽酶相对分子质量和等电点分别约为52×10³和7.2;酶活最适pH为8.0,在pH 5.0~10.0之间稳定;酶活最适温度为40 ℃,在45 ℃以下稳定。以酪蛋白为底物时,米氏常数(K_m)和最大反应速度(V_max)分别为22.3 mg·mL^-1和1 355 mg·L·min^-1。结论 利用黏质沙雷氏菌LL-413菌株生产舍雷肽酶发酵单位高,酶的蛋白水解能力强、pH和热稳定性好,研究结果为该酶的生产和应用奠定了基础。     

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??OBJECTIVE To study the pharmacokinetics and bioequivalence of hydroxysafflor yellow A (HSYA) and hydroxysafflor yellow A nanoemulsion (HYAN) in rats.METHODS Twelve male rats were randomly divided into two groups. The rats were administered intragastrically with HSYA or HYAN, respectively, and then blood was collected from the venous plexus at different time points. HPLC method was used for the determination of HSYA blood concentration.RESULTS The main pharmacokinetic parameters of HYAN were as follows: the area under curve (AUC_{0-24 h}), peak concentration (??_max), peak time (t_max) and clearance (CL) were (31.56??4.58) mg??L??h^-1, (12.75??2.64) mg??L^-1, (0.83??0.54) h and (1.89??0.93) L??h^-1??kg^-1, respectively. The AUC_{0-24 h}, ??_max and t_max of HYAN increased by 5.49, 10.22 and 2.50 times, respectively, and the CL of HYAN was only 1/4 of that of HSYA. The 90% confidence intervals for AUC_{0-24 h} and ??_max were not within the prescribed range of bioequivalence criteria.CONCLUSION Relative to HSYA, the high plasma concentration and prolonged peak time of HYAN in vivo can significantly improve the oral bioavailability of HSYA. HSYA solution and HYAN are not bioequivalent.