氯苄律定在离体大鼠简易肝微粒体的代谢动力学

目的 测试氯苄律定（CPU-86017）在离体大鼠肝简易微粒体中的代谢。方法 首先制备肝简易微粒体（含肝细胞胞浆及部分线粒体），分别加入不同尝试的CPU-86017，测定药物代谢速率，Vmax、Vm。结果 测出肝代谢t1/2为1h,Km为0.031mmol/L,Vmax为891pmol/Lmg蛋白/h。结论 肝内代谢速率较慢，可排除肝首过效应，亦未见肝本科饱和现象。     

大鼠肝微粒体中有机锡抗癌化合物DBDCT的测定与酶促动力学研究

目的建立测定大鼠肝微粒体中有机锡抗癌化合物DBDCT的方法并研究其相应的酶促动力学。方法采用HPLC测定DBDCT在体外代谢系统中的酶促动力学,用Eadie-Hofstee法分析数据,求算酶促动力学参数Km和Vmax以及肝代谢速率Clin。结果在体外代谢系统中,DBDCT代谢酶促动力学参数Km=159.07μmol·L-1,Vmax=2.813μmol·（min·mg）^-1以及肝清除率Clin=Vmax/Km=0.017L·（min·mg）^-1。结论此分析方法符合方法学验证要求且简单准确,可满足DBDCT体外代谢的研究。     

奥美拉唑在大鼠肝微粒体中的酶促反应动力学研究

目的研究奥美拉唑在大鼠肝微粒体中的酶促反应动力学。方法采用高效液相色谱法测定奥美拉唑在体外代谢系统中的产物5’-羟基奥美拉唑,并用Eadie-Hofstee作图法分析数据,计算酶促反应动力学参数最大反应速率(Vmax)、米氏常数(Km)。结果在体外代谢系统中,奥美拉唑生成5’-羟基奥美拉唑的Vmax为2010nmol/(min.mgprotein)、Km为50.3μmol/L。结论本分析方法符合方法学验证的要求,体外代谢常数准确,可满足奥美拉唑体外代谢的研究。     

异丙酚在人肝微粒体中酶促动力学研究

目的 研究异丙酚在人肝微粒体中的代谢。方法 采用高效液相方法测定孵育液中异丙酚的浓度。研究异丙酚的酶促动力学,推导出药物米氏常数(Km)和最大反应速度(Vmax);并计算体外酶对药物的清除率(CLint)。同时观察不同种类的CYP酶特异性抑制剂对异丙酚代谢的影响。结果 异丙酚在人肝微粒体中Km和Vmax分别为777.43μuM和172.413μM?h ?1?mg ?1?protein,Clint为0.22h?1?mg?1?protein。CYP2B6特异性抑制剂氯吡格雷能够显著抑制异丙酚的代谢,而CYP2C9和CYP2C19特异性抑制剂对异丙酚代谢无显著影响CYP2B6抑制剂能够减少异丙酚的代谢。结论 CYP2B6主要参与异丙酚的代谢, CYP2B6酶抑制剂可能会抑制异丙酚的代谢,造成药物药效或毒性的增加。     

罗通定在人、比格犬和大鼠肝微粒体代谢转化的体外比较研究

目的体外研究罗通定(rotundine,RTD)在大鼠、比格犬和人肝微粒体中酶代谢动力学及代谢产物差异。方法优化3个种属肝微粒体与RTD反应体系,应用LC-MS测定RTD在3种肝微粒体中的体外代谢消除,应用LC-MS/MS分析比较RTD在3种肝微粒体中代谢产物种类和生成量的差异,计算并比较相应的活性值。结果RTD在人肝微粒体中代谢转化最慢,其相应的动力学参数为Km=2.67μmol·L-1、Vmax=0.095μmol·L-1·min-1、T21=298±18.0min、CLint=14.6±0.91ml·min-1·kg-1;大鼠中相应的动力学参数为Km=3.24μmol·L-1、Vmax=0.122μmol·L-1·min-1、T12=71.0±2.30min、CLint=87.5±2.79ml·min-1·kg-1;比格犬中相应的动力学参数为Km=5.31μmol·L-1、Vmax=0.228μmol·L-1·min-1、T21=62.3±0.647min、CLint=139±1.43ml·min-1·kg-1。RTD在3个种属肝微粒体中均代谢产生4个O-去甲基后的羟基化同分异构体产物,但4个产物的相对生成百分比在不同种属肝微粒体中有一定差异。结论罗通定在体外人、大鼠和比格犬肝微粒体中主要的I相代谢途径相同,但是酶代谢动力学性质及代谢产物的生成量存在着一定的差异。     

大鼠肝微粒体中有机锡抗癌化合物DBDCT的测定与酶促动力学研究

目的 建立测定大鼠肝微粒体中有机锡抗癌化合物DBDCT的方法并研究其相应的酶促动力学。方法 采用HPLC测定DBDCT在体外代谢系统中的酶促动力学,用Eadie-Hofstee法分析数据,求算酶促动力学参数Km和Vmax以及肝代谢速率Clin。结果 在体外代谢系统中,DBDCT代谢酶促动力学参数Km=159.07 μmol·L^-1,Vmax=2.813 μmol·(min·mg)^-1以及肝清除率Clin=V_max/Km=0.017 L·(min·mg)^-1。结论 此分析方法符合方法学验证要求且简单准确,可满足DBDCT体外代谢的研究。     

抗癫痫新药氯桂丁胺在大鼠肝微粒体中的酶促反应动力学研究北大核心CSCD

目的观察肝微粒体药物代谢酶系对抗癫痫新药氯桂丁胺生物转化的影响及酶促动力学特征。方法差速离心法制备大鼠肝微粒体,高效液相色谱法检测氯桂丁胺及其主要代谢物的浓度,双倒数作图法计算酶促动力学参数。结果原形药的Km为13.46μg.mL-1,Vmax为14.88 nmol.min-1.mg-1蛋白,t1/2为48.53 min。代谢物M1,M2和M3的Km分别为8.82,0.26和43.37μg.mL-1,Vmax分别为0.17,4.44×10-2和3.24 nmol.min-1.mg-1蛋白。结论氯桂丁胺经肝微粒体代谢是其主要的消除形式,并呈反应时间、酶蛋白浓度和底物依赖性酶促动力学特征。     

大黄素在大鼠肝微粒体酶动力学及胡椒碱对其代谢的影响

目的分别研究大黄素在雌性和雄性大鼠肝微粒体中酶促动力学及胡椒碱对其代谢的影响。方法将系列浓度的大黄素分别与雌性或雄性大鼠肝微粒体进行体外共孵育,采用HPLC法测定孵育液中大黄素的浓度,并计算酶动力学参数。另将不同浓度的胡椒碱与大黄素在大鼠肝微粒体中进行共孵育,计算胡椒碱对大黄素代谢的抑制常数(Ki),进一步探讨其代谢抑制类型,并推测其对大黄素体内代谢的影响。结果大黄素在雌性和雄性大鼠肝微粒体中代谢的最大反应速率(Vmax)分别为(84.49±9.62)和(32.97±7.37)μmol·min~(-1)·mg protein~(-1),米氏常数(Km)分别为(60.50±8.71)和(34.48±4.96)μmol·L~(-1),内在清除速率(CLint)分别为(1 406±112)和(965±73.7)μL·min~(-1)·mg protein~(-1),雌性和雄性大鼠的Ki值分别为0.35和0.31μmol·L~(-1)。结论大鼠肝微粒体中胡椒碱抑制大黄素的葡萄糖醛酸代谢呈浓度依赖性,根据图形推断其抑制类型为混合性抑制,并推测合用胡椒碱后,大黄素在大鼠体内的血药浓度显著增加,为其进一步开发研究提供了参考依据。     

外消旋普罗帕酮在经诱导的大鼠肝微粒体中N-去丙基化的立体选择性

:采用对照及 β-萘黄酮 (β- NF)或地塞米松(Dex)诱导的大鼠肝微粒体 ,应用 GITC柱前衍生化 ,反相高效液相色谱法研究了消旋普罗帕酮〔(R/S) - PPF〕体外代谢的立体选择性 .实验结果表明 ,在 Dex,β- NF诱导的微粒体中有 N-去丙基普罗帕酮生成。在 β- NF,Dex预处理组 ,(R/S) - PPF低浓度时的经肝微粒体代谢具有立体选择性 ,R(- )对映体的清除大于 S(+)对映体 ,高浓度时的代谢无立体选择性 .R(- )对映体的 Km 值显著低于 S(+)对映体 ,而 Vmax值无显著性差异 .在 Dex预处理组中的立体选择性大于 β- NF组 .在对照组中代谢无立体选择性 ,且 Km,Vmax值均小于β- NF,Dex预处理组。结果提示 ,CYP1 A,CYP3A4亚族对普罗帕酮 (PPF)的 N-去丙基化有贡献 .(R/S) - PPF的N-去丙基化具有浓度依赖性的立体选择性     

黄芩素在人及不同种属肝微粒体中的UDP-glucuronosyltransferase代谢差异（摘要）

目的：研究黄芩素在不同种属肝微粒体中的UDP-葡萄糖醛酸转移酶（UDP-glucuronosyltransferase, UDPGA）代谢差异特性。方法使用肝微粒体体外代谢孵育法、HPLC-UV分析方法,选用不同种属的肝微粒体进行黄芩素UDPGA体外代谢研究。结果黄芩素在人肝微粒体及不同种属的肝微粒中,加入UDPGA进行37℃恒温孵育,孵育结束后离心,取上清液,经HPLC-UV分离检测得到3个代谢产物,分别是：黄芩素-7-O-β-葡萄糖醛酸结合物、黄芩素-6-O-β-葡萄糖醛酸结合物和黄芩素-6-O-葡萄糖醛酸结合物-7-O-β-葡萄糖醛酸结合物;通过与标准品对照确定黄芩素的三个代谢产物都是葡萄糖醛酸化的代谢产物。同时,不同种属间UGT代谢物的活性表现出较大差异,黄芩素-7-O-β-葡萄糖醛酸结合物在人肝微粒体中的代谢活性最强,Km=1.61,Vmax=0.77（BG在人肝微粒体中的代谢活性是SD雌鼠的25.2倍）;黄芩素-6-O-β-葡萄糖醛酸结合物在比格犬肝微粒体中代谢活性最强,Km=3.05,Vmax=3.51（雄性比格犬肝微粒体的活性是雄性恒河猴肝微粒体的2.6倍）;黄芩素-6-O-葡萄糖醛酸-7-O-β-葡萄糖醛酸结合物在猪肝微粒体中代谢活性最强,Km=5.38, Vmax=0.17（猪肝微粒体的活性是人肝微粒体的13.6倍）,其他依次是犬、恒河猴、鼠和人。结论黄芩素在人及不同种属肝微粒体UGT代谢中均生成上述三种葡萄糖醛酸化代谢产物,但是不同种属间的代谢表现出酶动力学的差异。     

美托洛尔在经左氧氟沙星诱导的大鼠肝微粒体P450系统中的代谢特征

采用正常及经不同剂量 ( 80、160、2 40mg/kg ,ip ,7d)左氧氟沙星诱导的大鼠肝微粒体 ,研究美托洛尔代谢特征及酶动力学参数。HPLC法用于美托洛尔浓度的测定。研究结果显示 ,经不同剂量的左氧氟沙星诱导后 ,大鼠的肝重及P45 0含量均明显降低 ,中剂量组及高剂量组分别与对照组及低剂量组比较有极显著性差异 (P <0 0 1)。对美托洛尔的代谢率随诱导剂剂量的增加而降低 ,低剂量组与对照组比较无差异 ,中剂量组及高剂量组分别与对照组及低剂量组比较有统计学意义。酶动力学参数测定结果显示 ,经左氧氟沙星诱导的细胞色素P45 0酶系统对美托洛尔代谢的Km 和Vmax值均增加 ,与对照组比较有极显著性差异 (P <0 0 1)。结论 :左氧氟沙星可抑制大鼠肝微粒体细胞色素P45 0系统对美托洛尔的代谢。     

Kinetic factors involved in the metabolism of benzene in mouse lung and liver

Benzene is an occupational and environmental toxicant. The main human health concern associated with benzene exposure is acute myelogenous leukemia. Benzene produces lung tumors in mice, while its effects on human lung are not clear. The adverse effects of benzene are dependent on its metabolism by the cytochrome P-450 enzyme system. The isozymes CYP2E1 and CYP2F2 play roles in the metabolism of benzene at low, environmentally relevant concentrations. Previous studies indicate that the mouse lung readily metabolizes benzene and that CYP2F2 plays a role in this biotransformation. The significance of CYP2E1 and CYP2F2 in benzene metabolism was determined by measuring their apparent kinetic parameters K(m) and V(max). Use of wild-type and CYP2E1 knockout mice and selective inhibitors allowed the determination of the individual importance of both CYP2E1 and CYP2F2 in mouse liver and lung. A simple Michaelis-Menten relationship involving Lineweaver-Burk plots for the microsomal metabolism of benzene shows the apparent kinetic factors are different between the wild-type (K(m): 30.4 microM, V(max): 25.3 pmol/mg protein/min) and knockout (K(m): 1.9 microM, V(max): 0.5 pmol/mg protein/min) mouse livers. Wild-type lung has a K(m) of 2.3 microM and V(max) of 0.9 pmol/mg protein/min. CYP2E1 knockout lung has similar affinity and metabolic activity with a K(m) of 3.7 microM and V(max) of 1.2 pmol/mg protein/min. These data suggest CYP2E1 is less important in the lung than liver, and that it has a lower affinity for benzene but higher rate of hydroxylated metabolite production than does CYP2F2, which plays the predominant role in metabolizing benzene in mouse lung.     

CPU86017, a berberine derivative, attenuates cardiac failure through normalizing calcium leakage and downregulated phospholamban and exerting antioxidant activity

Aim:

To investigate whether {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017, a berberine derivative, attenuates heart failure by blocking calcium influx and exerting its antioxidant activity.

Methods:

Myocardial infarction was induced in male Sprague-Dawley rats for 17 d followed by isoproterenol (ISO) (5 mg/kg, sc) treatment for 5 d to reduce cardiac function. The rats were divided into 5 groups: sham operation, myocardial infarction (MI), MI plus ISO, and co-treated (in mg/kg, po) with either propranolol (PRO, 10) or {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017 (80). Hemodynamic measurements were conducted, and measurements of the redox system, calcium handling proteins and endothelin (ET) system in vivo were done. Furthermore, calcium flux studies and PLB immunocytochemistry were conducted in vitro.

Results:

Compared to sham operation, HF was evident following MI and further worsened by ISO treatment. This occurred in parallel with downregulated mRNA and protein production of SERCA2a, PLB, and FKBP12.6, and was associated with upregulation of preproET-1, endothelin converting enzyme, and PKA mRNA production in the myocardium in vivo. Calcium leakage was induced by ISO treatment of isolated beating myocytes in vitro. These changes were attenuated by treatment with either PRO or {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017. PLB fluorescence in myocytes was downregulated by ISO treatment, and was relieved significantly by treatment with antioxidant aminoguanidine, ascorbic acid or {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017 in vitro.

Conclusion:

HF, calcium leakage, downregulated PLB, FKBP12.6, SERCA2a production, and upregulated PKA were caused by ISO treatment, and were abolished by {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017 treatment. The beneficial effects of {"type":"entrez-protein","attrs":{"text":"CPU86017","term_id":"899077019","term_text":"CPU86017"}}CPU86017 are attributable to its antioxidant and calcium influx blocking effects.     

N-Glucuronidation of some 4-arylalkyl-1H-imidazoles by rat, dog, and human liver microsomes.

N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.     

Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.     

CPU86017: a novel Class III antiarrhythmic agent with multiple actions at ion channels

CPU86017 is a novel Class III antiarrhythmic agent derived from berberine and with an improved pharmacological profile, solubility and bioavailability. It is active in suppressing arrhythmias in several animal models. The ED(50) of CPU86017 for suppressing ischemia/reperfusion arrhythmias in rats was 0.22 mg/kg against 2.23 mg/kg for lidocaine. CPU86017 is about 10-fold more potent than lidocaine. It blocks I(K(R.tail)), I(K(S)), and I(Ca(L)) currents with IC(50) values of 25, 14.4, and 11.5 microM, respectively. The plasma t(1/2) of CPU86017, i.v. bolus, in rabbits and dogs is approximately 90 min. The effective plasma levels of CPU86017 in rabbits required to delay the appearance of oubain-induced ventricular arrhythmias is in the range of 0.13-0.31 microg/mL. Higher levels of the drug are required to eliminate ventricular arrhythmias produced by two-stage ligation of the coronary artery in anesthetized dogs. Drug levels in myocardium are much higher than in plasma. CPU80617 has an antioxidant effect that is likely to contribute to its antiarrhythmic activity. The abnormal expression of the ryanodine receptor type 2 (RyR2) and of FKBP12.6 is reduced by CPU80617 during its ventricular tachyarrhythmia-suppressing action. CPU86017 appears to be a promising antiarrhythmic agent with a cardioprotective action. It can be expected to protect from malignant arrhythmias and sudden cardiac death by suppressing molecular events caused by channelopathies.     

CPU 86017 suppression of arrhythmias induced by ischemia/reperfusion,ouabain, aconitine,and elevation of ventricular fibrillatory threshold

Protection by CPU 86017 against reperfusion-induced arrhythmias was studied in Langendorff perfused hearts and anesthetized rats. Inhibition of arrhythmias induced by ouabain and aconitine was observed, and the influence of CPU 86017 on ventricular fibrillation threshold (VFT) was determined. CPU 86017 exerted a dose-dependent antiarrhythmic effect in animal models. In Langendorff's perfused hearts, CPU 86017 significantly reduced the incidence and duration of ventricular fibrillation and ventricular tachycardia, as well as arrhythmic scores in a dose-dependent manner. The approximate ED₅₀ of CPU 86017 against ventricular fibrillation was 2.10 μM. The antiarrhythmic effect of CPU 86017 against arrhythmia persisted for more than 6 h. In anaesthetized rats, CPU 86017 provided potent antiarrhythmic and antifibrillatory effects by po or iv routes. CPU 86017 was similar in potency to propafenone by the po route, and 10 times more potent than lidocaine by the iv route. CPU 86017 significantly attenuated the exaggerated arrhythmia and ventricular fibrillation in hypertrophic hearts induced by 1-thyroxine. Against ouabain-induced arrhythmias CPU 86017 exerted a dose-dependent inhibitory effect, which was 6.5-fold more potent than berberine. CPU 86017 significantly reduced ventricular fibrillation incidence and prevented the further deterioration of ventricular arrhythmias induced by aconitine. In conclusion, CPU 86017 is a potent antiarrhythmic agent with multiple mechanisms of action. Drug Dev. Res. 39:184–190. © 1997 Wiley-Liss, Inc.     

Modelling the loss of metabolic capacities of cultured hepatocytes: application to measurement of Michaelis-Menten kinetic parameters in in vitro systems

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V(max) and K(m) from extended experiments, by modelling V(max) as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V(max) and K(m) were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V(max) decrease with culture time from estimates of time parameters, t(1/2) or t(50). In our system, this V(max) decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments.