基于高效液相色谱-串级质谱法研究肝微粒体中T-2毒素代谢的种属差异性

目的比较T-2毒素在不同种属动物肝微粒体中代谢的差异性。方法将T-2毒素与小鼠、大鼠、比格犬、猴和人肝微粒体37℃孵育不同时间,孵育液经蛋白沉淀后采用高效液相色谱-质谱法检测,比较T-2毒素在不同种属动物中代谢动力学参数及代谢产物生成量的差异。结果 T-2毒素在人肝微粒体中半衰期(t_(1/2))<1 mim,在小鼠和猴肝微粒体中为2~4 min,在比格犬肝微粒体中为13 min,在大鼠肝微粒体中为39 min。5种动物对T-2毒素的肝清除能力可分为3组,即人、比格犬和大鼠为1组;猴和小鼠各为1组其中小鼠组对T-2毒素的肝清除率是人、比格犬和大鼠组的3~4倍不同种属的肝微粒体对T-2毒素的亲和力存在显著差异,其中T-2毒素在小鼠肝微粒体中的亲和力最高,其余依次为人、比格犬、大鼠和猴。酶的转化速率以在猴肝微粒体中最大,大鼠和比格犬中略小,而人和小鼠肝微粒体中酶转化速率仅为猴肝微粒体中转化速率的1/10~6。T-2毒素在猴肝微粒体中主要代谢产物为3'-OH-T-2和新茄病镰刀菌烯醇,在人和大鼠肝微粒体中为T-2三醇和HT-2毒素,在比格犬肝微粒体中以HT-2毒素和3'-OH-T-2毒素为主,在小鼠中则以T-2三醇和3'-OH-T-2毒素为主。T-2毒素在小鼠、大鼠、比格犬和人肝微粒体中主要以水解代谢转化为主,而在猴肝微粒体中则以羟基化代谢为主。结论 T-2毒素的代谢参数、代谢产物及其生成量、代谢途径均存在种属差异性。     

UPLC-MS/MS方法研究pongachin在肝微粒体中的代谢特征

目的:采用不同种属肝微粒体,研究黄酮类化合物pongachin的体外代谢特性,明确参与pongachin代谢的CYP酶亚型。方法:选择UPLC-MS/MS测定方法,通过测定pongachin的剩余浓度,考察pongachin在人、大鼠和猴3个种属肝微粒体中的代谢稳定性,以及在大鼠肝微粒体中的代谢表型。结果:pongachin在人、大鼠和猴肝微粒体中t1/2分别为36.86、30.13和20.50 min;其清除率为人大鼠猴。在大鼠肝微粒体中,CYP2E1、CYP2C、CYP1A2对pongachin的代谢有较强抑制作用。结论:pongachin在人和大鼠肝微粒体中代谢稳定性较好(30 min),且在人和大鼠肝微粒体中代谢相似,在临床上与其他抑制CYP2E1、CYP2C、CYP1A2代谢的药物合用时应注意药物间的相互作用。     

雌二醇在人、比格犬和大鼠的肝微粒体中体外代谢比较研究

采用体外肝微粒体孵育体系, 研究雌二醇在大鼠、比格犬和人肝微粒体中酶代谢动力学及代谢产物差异。通过对雌二醇浓度、肝微粒体蛋白含量和孵育时间等条件的考察, 优化雌二醇与肝微粒体的反应体系; 应用LC-MS/MS定量检测孵育体系中的雌二醇及代谢产物, 分析比较雌二醇在3个种属、不同性别肝微粒体中代谢产物种类和生成量的差异, 计算并比较相应的动力学参数。在3个种属肝微粒体中均发现9个I相代谢产物, 且百分比有种属差异。结果表明, 3个种属的肝微粒体对雌二醇I相代谢途径基本相同, 但是代谢产物的生成量及雌二醇的药物代谢动力学性质存在一定的差异。     

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。     

吖啶酮衍生物H64在5种种属肝微粒体中的代谢产物分析

目的：应用肝微粒体体外孵育体系,对吖啶酮衍生物H64在人、SD大鼠、小鼠、恒河猴和比格犬的肝微粒体中的代谢产物进行鉴定,并提出H64的主要体外代谢途径。方法：生物样品经过前处理,乙腈和0.1%甲酸水溶液为流动相,通过UHPLC-Q-Exactive Plus液质进行分析,使用Xcalibur 4.2以及Compound Discoverer^TM 2.0软件对数据进行处理,及后期的人工筛选,鉴定H64在5种种属肝微粒体中可能的代谢产物。结果：共检测到11个代谢产物,其中9个为Ⅰ相代谢产物,包括氧化以及脱氧还原的代谢物;其余2个为Ⅱ相代谢产物,包括甲基化、乙酰化的代谢物。在人、SD大鼠、小鼠、恒河猴和比格犬的肝微粒体中分别检测出了9、6、7、7、7种代谢产物。结论：H64在各种属肝微粒体中,最主要的代谢途径为氧化。本研究能反映H64的体内代谢产物的大致情况。小鼠和人肝微粒体中的代谢产物种类最为接近,为H64早期代谢研究的实验动物的选择上提供了参考。     

乌头碱在豚鼠和小鼠肝微粒体细胞色素氧化酶中的代谢作用研究

目的:研究乌头碱(AC)在豚鼠和小鼠体外肝微粒体中代谢产物的差异。方法:优化2个种属肝微粒体与AC的P450反应体系,应用高效液相色谱-高分辨质谱联用(HPLC-HRMS)和超高相液相色谱-多级质谱(UPLC-MS/MS)法测定AC在2种肝微粒体中的细胞色素氧化酶(CYP)代谢产物。结果:AC在2种肝微粒体中均代谢产生8种代谢产物,其中6种为CYP代谢产物;脱甲基、脱乙基、脱氢和氧化反应是其主要代谢特征。结论:乌头碱在豚鼠和小鼠体外肝微粒体中的CYP主要代谢途径相同。     

DHDK肝微粒体代谢动力学研究

目的 研究抗肿瘤候选药物DHDK在人和大鼠肝微粒体的代谢动力学特征,比较种属差异,为临床前研究提供依据。方法 建立HPLC法测定DHDK在肝微粒体孵育体系中的含量,用于考察DHDK体外代谢稳定性和酶动力学,进而推算其在体内的肝清除率(CL_h)与肝提取率(ER)。结果 肝微粒体孵育试验表明DHDK在两个种属之间的体外代谢稳定性和酶动力学行为无显著性差异(P>0.05),但在人体内的肝清除率显著低于大鼠(P<0.05)。结论 DHDK体外代谢主要依赖于烟酰胺腺嘌呤二核苷酸磷酸和肝微粒体酶,属于中等程度代谢消除。     

氮烯乙茶在大鼠、犬、猴肝亚细胞成分中的生物转化

目的:利用肝亚细胞成分研究I类抗癌新药氮烯乙茶的生物转化及其种属差异。方法:以高效液相色谱法检测肝9 000 ×g 上清液(S-9) 和微粒体中氮烯乙茶及其代谢产物的浓度。结果:大鼠、犬、猴肝S-9中氮烯乙茶的固有清除率(CL_int) 分别为14-9,10-4,30-4 μL·min^-1·mg^-1 protein,代谢产物为7-乙基-8-氨基茶碱(7-ethyl-8-aminotheophylline,EAT),氮烯乙茶在肝的生物转化部位在微粒体。结论:预计氮烯乙茶在生物转化途径上的种属差异较小;肝亚细胞成分是研究药物代谢及种属差异的较好体外模型。     

抗肿瘤药LS-177在大鼠和人肝微粒体中的代谢产物研究

目的：推测LS-177在大鼠和人肝微粒体中代谢产物的结构及其可能的体外代谢途径。方法：体外孵育大鼠和人肝微粒体代谢模型,采用超高效液相质谱联用（LC-MSⁿ）法,推测LS-177的体外代谢产物的结构。结果：通过质谱数据、保留时间和碎片离子,在肝微粒体中共检测到5个代谢产物,初步推测LS-1177在肝微粒体中可能的代谢途径。结论：建立LC-MSⁿ方法,初步推测LS-177在大鼠和人肝微粒体中代谢产物的结构,为其体内外代谢的进一步研究以及化学结构类似物的体外代谢研究提供一定的参考依据。     

诃子酸在不同种属肝微粒体中的代谢稳定性和代谢酶表型研究

目的 建立测定肝微粒体孵育体系中诃子酸浓度的方法,并比较其在人、犬、猴、小鼠、大鼠肝微粒体中的Ⅰ相、Ⅱ相代谢稳定性及种属差异,确定其在人肝微粒中的代谢表型。方法 将诃子酸与不同种属肝微粒体共同孵育,应用UPLCMS/MS检测孵育液中诃子酸的含量,考察其代谢稳定性及体外动力学参数。采用化学抑制剂法确定其在人肝微粒中的代谢表型。将诃子酸与各CYP450同工酶CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1和CYP3A4的特异性抑制剂（α-萘黄酮、香豆素、磺胺苯吡唑、噻氯匹定、奎尼丁、二乙基二硫代氨基甲酸钠、酮康唑）共同孵育,确定其代谢酶表型。结果 诃子酸在Ⅰ相、Ⅱ相孵育体系中均可代谢,在Ⅰ相代谢中,犬肝微粒体孵育与人最为相似,半衰期（t_1/2）分别为115.50 min和121.58 min;Ⅱ相代谢中5个种属代谢稳定性均中等,其中猴与人肝微粒体代谢趋势最为相近。诃子酸在人肝微粒中的代谢是由多种CYP酶共同介导的,其中CYP2C9、CYP2E1和CYP3A4是主要的同工酶。结论 建立的UPLCMS/MS方法简便、快速、专属性强、灵敏性高,可用于肝微粒体孵育体系中诃子酸浓度的测定及体外代谢的研究。诃子酸在人、犬、猴、小鼠、大鼠肝微粒体中代谢存在一定种属差异,且其代谢过程与多种CYP酶相关。     

Role of glutathione in reduction of arsenate and of gamma-glutamyltranspeptidase in disposition of arsenite in rats

Arsenate (AsV), the environmentally prevalent form of arsenic, is converted sequentially in the body to arsenite (AsIII), monomethylarsonic acid (MMAsV), monomethylarsonous acid (MMAsIII), and dimethylarsinic acid (DMAsV) and some trimethylated metabolites. Although the biliary excretion of arsenic in rats is known to be glutathione (GSH)-dependent, involving transport of arsenic-GSH conjugates, the role of GSH in the reduction of AsV to the more toxic AsIII in vivo has not been defined. Therefore, we studied how the fate of AsV is influenced by buthionine sulfoximine (BSO), which depletes GSH in tissues. Control and BSO-treated rats were given AsV (50 micromol/kg, i.v.) and arsenic metabolites in bile, urine, blood and tissues were analysed by HPLC-HG-AFS. BSO increased retention of AsV in blood and tissues and decreased appearance of AsIII in blood, bile (by 96%) and urine (by 63%). The biliary excretion of MMAsIII was also nearly abolished, the appearance of MMAsIII and MMAsV in the blood was delayed and the renal concentrations of these monomethylated arsenicals were decreased by BSO. Interestingly, appearance of DMAsV in blood and urine remained unchanged and the concentrations of this metabolite in the kidneys and muscle were even increased in response to BSO. To test the role of gamma-glutamyltranspeptidase (GGT) in arsenic disposition, the effect of the of the GGT inhibitor acivicin was investigated in rats injected with AsIII (50 micromol/kg, i.v.). Acivicin lowered the hepatic and renal GGT activities and increased the biliary as well as urinary excretion of GSH, but failed to alter the disposition (i.e. blood and tissue concentrations, biliary and urinary excretion) of AsIII and its metabolites. In conclusion, shortage of GSH decreases not only the hepatobiliary transport of arsenic, but also reduction of AsV and the formation of monomethylated arsenic, while not hindering the production of dimethylated arsenic. While GSH plays an important role in the disposition and toxicity of arsenic, GGT, which hydrolyses GSH and GSH conjugates, apparently does not influence the fate of the GSH-reactive trivalent arsenicals in rats.     

微透析取样技术在中药体内分析中的应用研究进展

本文综述了微透析取样技术在中药体内分析中的应用,介绍微透析取样技术的原理、组成、探针类型、特点,重点阐述了微透析取样技术在测定脑、血液、皮肤等组织器官中中药有效成分浓度的应用实例。表明微透析取样技术在中药药效研究中具有广阔的前景。     

应用PASS系统分析我院2010年住院医嘱

目的:了解我院2010年住院患者的合理用药情况,探讨如何利用合理用药监测系统( PASS)提高合理用药水平.方法:利用PASS对我院2010年15 966例住院患者的1 184 997条用药医嘱进行监测,以黑色警示医嘱为依据,收集不合理用药信息,并对监测结果进行统计、分析.结果:不合理用药医嘱50 261条,发生率为4.24％.绝对禁止黑色医嘱5441条,主要为药物相互作用(66.54％)、注射液体外配伍(17.86％)、用法用量(15.46％)、儿童警告(1.14％).结论:应用PASS系统能有效监测医嘱中的不合理用药情况,有利于提高临床合理用药水平,但PASS系统尚存在局限性,有待进一步完善.     

Changes in levels of dependency and predictors of mortality in elderly people in institutional care in Dunedin

The 1983 study of dependency of subjects in institutional care in Dunedin was repeated two years later. A significant increase in levels of dependency in residential homes, particularly in the Religious and Welfare sector was found. In 1983 there were 29 high dependency residents and 73 medium dependency residents in residential homes. In 1985 these numbers had increased to 55 and 86 respectively. There was no change in the number of low dependency residents. In 1983, 6 high dependency residents had been admitted to residential home care in the year prior to the study. In 1985 the number of high dependency residents recently admitted had increased to 23. There had also been a significant increase in the dependency of patients in Religious and Welfare continuing care hospitals. Of the 933 subjects in institutional care in 1983 who were able to be followed, 354 (37.9%) died in the following 2 years. Mortality rate was higher for those in hospital care (48.1%) than for those in residential home care (29.6%). Mortality rates were higher in more dependent subjects and this was evident for each measure of dependency.     

应用PASS系统对我院2008年住院医嘱的分析

目的监测分析2008年我院住院患者用药情况。方法将PASS系统嵌入医生工作站、临床药学工作站等子系统,构建合理用药计算机网络系统,对住院医嘱进行及时监测,将监测结果向医生反馈,并对其进行统计、分析。结果2008年共监测医嘱3 620 241条,不合理医嘱908条,占0.02%。不合理医嘱中,配伍禁忌(381条)占41.96%,用法用量(381条)占41.96%,药物相互作用(108条)占11.89%,儿童用药(38条)占4.19%。经与医生沟通后,更改不合理医嘱856条,占94.27%。结论PASS系统可有效监测医嘱中的不合理用药,通过与医生交流,大大减少药物不良事件的发生,值得临床推广应用,也为临床药师开展工作带来了极大的便利。但PASS系统尚存在局限性,有待进一步完善。     

Role of desacetylation in the detoxification of cephalothin in renal cells in culture

The toxicity of three cephalosporin antibiotics to rabbit kidney cells in culture was compared to their known nephrotoxic potential in vivo (cephaloridine greater than cefazolin greater than cephalothin). While cephalothin is considered to be a relatively nonnephrotoxic cephalosporin when administered to many species including humans and rabbits, in several in vitro systems involving rabbit renal tissue, cephalothin was comparatively more toxic than anticipated based on in vivo data. Cephalothin is extensively desacetylated in rabbits to a less microbiologically active metabolite, desacetylcephalothin. When a microsomal S9 fraction from rabbit kidney was added to the in vitro assay in cultured rabbit renal cells, cephalothin was desacetylated and its toxicity to kidney cells was reduced. The addition of S9 in vitro provided a toxicity ranking of the cephalosporins that correlated with their known in vivo nephrotoxic potentials (cephaloridine greater than cefazolin greater than cephalothin). The in vitro detoxification of cephalothin by S9 was blocked by the coadministration of the esterase inhibitor, aminocarb. Desacetylcephalothin was relatively nontoxic to rabbit renal tissue in vitro. These results suggest that the desacetylation of cephalothin in vivo represents a previously unrecognized mechanism of detoxification of this cephalosporin antibiotic. Furthermore, this mechanism of detoxification may be applicable to other acetylated cephalosporins.     

2009年某院住院患者抗真菌药用药调查

目的:分析讨论某院抗真菌药使用的合理性,为临床安全有效地使用抗真菌药提供参考。方法:回顾性统计分析某院2009年住院患者抗真菌药用药信息。结果:2009年某院住院患者抗真菌药DDDs排名前3名分别为:氟康唑、制霉菌素和伊曲康唑;使用金额排名前3名分别为:氟康唑、米卡芬净及卡泊芬净;更换一种抗真菌药进行治疗的患者数为176人,在全部患者中占13.4%。结论:应进一步强化用药指征的意识,提高标本送检率,同时改善某些抗真菌用药不合理更换的现象,以避免耐药性发生,从而更好更长远地体现抗真菌药的治疗价值。     

Kinetics of in vitro metabolism of methoxyphenamine in rats

1. Methoxyphenamine (MP) was metabolized in vitro by rat liver preparations to O-desmethylmethoxyphenamine (O-desmethyl-MP), N-desmethylmethoxyphenamine (N-desmethyl-MP) and 5-hydroxymethoxyphenamine (5-hydroxy-MP). These metabolic pathways were inhibited by SKF 525-A and carbon monoxide, which indicates that these reactions were mediated at least partly by an NADPH-dependent cytochrome P-450 system. 2. Strain differences in the metabolism of this drug in vitro were observed in female Lewis and Dark Agouti (DA) rats, which are proposed models for human debrisoquine phenotypes. Methoxyphenamine O-demethylase and 5-hydroxylase activity in DA rats were lower than those in Lewis rats. 3. The metabolic transformation of methoxyphenamine in vitro to O-desmethyl-MP was inhibited competitively by debrisoquine and sparteine. This indicates that the cytochrome P-450 isoenzyme mediating the metabolism of MP to O-desmethyl-MP is similar to that mediating metabolism of debrisoquine and sparteine. However, no inhibition was observed with methenytoin.     

Comparison of in vitro cell models in predicting in vivo brain entry of drugs

Although several in vitro models have been reported to predict the ability of drug candidates to cross the blood-brain barrier, their real in vivo relevance has rarely been evaluated. The present study demonstrates the in vivo relevance of simple unidirectional permeability coefficient (P(app)) determined in three in vitro cell models (BBMEC, Caco-2 and MDCKII-MDR1) for nine model drugs (alprenolol, atenolol, metoprolol, pindolol, entacapone, tolcapone, baclofen, midazolam and ondansetron) by using dual probe microdialysis in the rat brain and blood as an in vivo measure. There was a clear correlation between the P(app) and the unbound brain/blood ratios determined by in vivo microdialysis (BBMEC r=0.99, Caco-2 r=0.91 and MDCKII-MDR1 r=0.85). Despite of the substantial differences in the absolute in vitro P(app) values and regardless of the method used (side-by-side vs. filter insert system), the capability of the in vitro models to rank order drugs was similar. By this approach, thus, the additional value offered by the true endothelial cell model (BBMEC) remains obscure. The present results also highlight the need of both in vitro as well as in vivo methods in characterization of blood-brain barrier passage of new drug candidates.