Application of an LC‐MS/MS method to the pharmacokinetics of TM‐2, a potential antitumour agent,in rats

TM‐2 is a novel semi‐synthetic taxane derivative, selected for preclinical development based on its greater anticancer activity and lower toxicity compared with docetaxel. In this study, a rapid and sensitive analytical method based on ultra performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of TM‐2 in rat plasma. The biological samples were extracted with methyl tert‐butyl ether and separated on a C₁₈ column (50 mm × 2.1 mm, 1.7 µm) using a mobile phase consisting of acetonitrile and 2 mM ammonium acetate. The standard curves were linear over the range 5–1000 ng/mL in rat plasma. The precision (relative standard deviation, RSD, %) were within 14.5%, and the accuracy (relative error, RE, %) ranged from ?1.56 to 2.36%. Recovery and matrix effect were satisfactory in rat plasma. The validated method was successfully applied to pharmacokinetic studies after intravenous administration of TM‐2 to rats. The pharmacokinetics of TM‐2 in rats were characterized by a large volume of distribution and a long half‐life of elimination after single dose (4, 8, and 16 mg/kg), and a good correlation was observed between AUC and dose. The preclinical data will be useful for the design of subsequent trials of TM‐2. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of diammonium glycyrrhizinate on pharmacokinetics of omeprazole by regulating cytochrome P450 enzymes and plasma protein binding rate

1. In clinical practice, diammonium glycyrrhizinate is usually used with omeprazole in patients with viral hepatitis and cirrhosis accompanied by peptic ulcers. However, the drug interaction between diammonium glycyrrhizinate and omeprazole remains unclear.

2. In this study, the effects of diammonium glycyrrhizinate on the pharmacokinetics of omeprazole was investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method. Male Sprague–Dawley rats were randomly assigned to two groups: omeprazole and omeprazole?+?diammonium glycyrrhizinate, and the main pharmacokinetic parameters were estimated after oral administration. It was found that using the omeprazole along with the diammonium glycyrrhizinate increased the AUC, AUMC, C_max for omeprazole.

3. For this reason, we used the LC-MS/MS to detect the binding rate of plasma protein (BRPP) of omeprazole in rats, it was found that diammonium glycyrrhizinate could decrease the BRPP in rats. In addition, we found that diammonium glycyrrhizinate specifically inhibited the enzyme activity of the CYP2C19 and CYP3A4, which are involved in the metabolism of the omeprazole.

4. These results mean that diammonium glycyrrhizinate could inhibit the metabolism and increase the plasma concentration of the omeprazole in rats. Overall, diammonium glycyrrhizinate can influence the pharmacokinetics of omeprazole by inhibiting CYP2C19 and CYP3A4 activities and decreasing BRPP of omeprazole.     

壳三糖在大鼠体内的组织分布和排泄研究

目的 研究壳三糖在大鼠体内的组织分布及排泄。方法 建立并验证了大鼠组织、尿液和粪便中壳三糖的高效液相色谱-质谱联用（LC-MS/MS）检测方法。大鼠灌胃给药壳三糖35 mg/kg后,分别在0.5、1.5、3 h 摘取组织,0 h - 4 h、4 h - 8 h、8 h - 12 h、12 h - 24 h 、24 h - 36 h、36 – 48 h时间段收集尿液和粪便。用建立的LC-MS/MS方法测定组织、尿液和粪便中的壳三糖含量。结果 组织、尿液和粪便中壳三糖在10 - 10000 ng/mL的浓度范围内线性良好,日内、日间精密度的相对标准偏差（relative standard deviation,RSD ）≤14.20%,准确度的相对误差（relative error,RE）为?11.30%～8.33%,提取回收率为85.2% - 97.9%,没有明显的基质效应,在室温放置24 h、反复冻融以及-40℃放置30天的条件下稳定性良好,满足生物样品的检测需求。灌胃给药后,0.5 h就可以在主要组织检测到壳三糖,1.5 h肾脏中壳三糖含量明显升高,3 h未在大脑中检测到壳三糖。48 h后壳三糖在尿液、粪便中的排泄率分别为7.15%、72.80%。结论 壳三糖主要以原型的形式在大鼠体内分布和排泄,壳三糖在大鼠各组织中的分布较为广泛,以肝和肾为主,粪便是壳三糖的主要排泄途径。     

Pharmacokinetics and tissue distribution study of novel potent antiplatelet agent S007-867 in mice using HPLC-MS/MS

Abstract

1.?S007-867 is a novel antiplatelet agent that shows promising in vitro and in vivo efficacy. For further development and better pharmacological elucidation, we characterized pharmacokinetics and tissue distribution of S007-867 in a mouse model.

2.?A sensitive, selective and robust LC-MS/MS method was developed and validated in the mouse plasma and tissue for quantification of S007-867. The chromatographic separation was performed on Waters Symmetry Shield C18 column (150?×?4.6?mm, 5?µm) using methanol and ammonium acetate buffer.

3.?S007-867 was rapidly absorbed and distributed to various tissues. Following single oral administration of S007-867 in the mouse, the concentration was in the order of C intestine?>?C liver?>?C kidney?>?C heart?>?C spleen?>?C lungs?>?C brain. Tissue to plasma area under the plasma curve ratio suggested that the maximum amount of drug was found in the intestine and liver. Half life of S007-867 was found longer in the heart (8.08?h), spleen (～?7.94?h) and kidney (～?15.41?h) as compared with other tissues.

4.?The preclinical pharmacokinetics and tissue distribution data obtained using this LC-MS/MS method are expected to assist the future clinical investigations of S007-867 as a promising antiplatelet agent.     

LC-MS/MS同时测定5种探针底物代谢产物和快速评价细胞色素P450同工酶的活性

目的建立同时测定奥美拉唑（CYP2C19）、右美沙芬（CYP2D6）、睾酮（CYP3A4）、氯唑沙宗（CYP2E1）和甲苯磺丁脲（CYP2C9）5种探针底物代谢产物的Cocktail体外方法,快速评价药物对人肝微粒体细胞色素P450同工酶活性的影响。方法通过人肝微粒体与5种探针底物在还原系统NADPH的作用下,于37℃水浴条件下共同孵育,采用液质联用（LC-MS/MS）分析方法,测定探针底物的代谢产物生成量来评价各P450同工酶的活性。结果建立了同时测定5种P450同工酶活性的酶反应条件和LC-MS/MS分析方法,数据显示该方法具有良好的线性和分析灵敏度,以及具有良好的准确度、精密度、重现性,可用于药物间相互作用的体外快速评价。结论可用于体外快速评价药物对相应的CYP450亚型酶活性的影响,以预测潜在的药物相互作用。     

The in vivo pharmacokinetics,tissue distribution and excretion investigation of mesaconine in rats and its in vitro intestinal absorption study using UPLC-MS/MS

1. Mesaconine, an ingredient from Aconitum carmichaelii Debx., has been proven to have cardiac effect. For further development and better pharmacological elucidation, the in vivo process and intestinal absorptive behavior of mesaconine should be investigated comprehensively.

2. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of mesaconine in rat plasma, tissue homogenates, urine and feces to investigate the in vivo pharmacokinetic profiles, tissue distribution and excretion. The intestinal absorptive behavior of mesaconine was investigated using in vitro everted rat gut sac model.

3. Mesaconine was well distributed in tissues and a mass of unchanged form was detected in feces. It was difficultly absorbed into blood circulatory system after oral administration. The insufficient oral bioavailability of mesaconine may be mainly attributed to its low intestinal permeability due to a lack of lipophilicity. The absorption of mesaconine in rat’s intestine is a first-order process with the passive diffusion mechanism.     

LC-MS/MS法测定氢溴酸东莨菪碱在大鼠体内的组织分布

目的:研究氢溴酸东莨菪碱在大鼠体内各组织的分布特点。方法:液相色谱分离采用C18(3.0 mm×100 mm,3.5μm)色谱柱,甲醇-2 mmol.L-1甲酸铵溶液(40∶60,甲酸调pH至3.52)为流动相,流速0.4 mL.min-1;质谱检测采用ESI离子源,正离子MRM方式检测。采取大鼠灌胃给药,运用液相色谱-质谱/质谱法测定氢溴酸东莨菪碱在大鼠各个组织中的药物浓度。结果:氢溴酸东莨菪碱在各个组织中的一定浓度范围内线性关系良好,各个组织中低、中、高浓度氢溴酸东莨菪碱的回收率及精密度均符合方法学要求。氢溴酸东莨菪碱在体内分布广泛,以脾、肝、肺中分布较高。结论:本方法简便、准确,灵敏度高,可用于氢溴酸东莨菪碱的体内分析研究。     

大黄素对大鼠肝脏CYP450酶的诱导研究

目的 探讨大黄素对大鼠肝脏细胞色素P450酶(CYP450)及其主要亚型的影响。方法 20只雄性SD大鼠, 随机分成4组, 每组5只, 分别为溶剂对照组, 170、500和1 500 mg/kg大黄素染毒组, 大黄素蒸馏水混悬后连续经口给药16 d, 结束后次日取大鼠肝脏组织制作微粒体, 分别采用CO还原差示光谱法、分光光度法及化学发光法检测大鼠肝脏微粒体总CYP450水平, 红霉素脱甲基酶(CYP3A)、氨基比啉-N-脱甲基酶, CYP1A、CYP2B和CYP2E1酶活性变化。结果 大黄素连续经口给药16 d, 能够引起大鼠肝脏微粒体总CYP450显著升高、可轻度诱导CYP3A、CYP1A、CYP2E1和CYP2B酶, 500 mg/kg剂量组最明显。结论 大黄素对大鼠肝脏中CYP3A、CYP1A、CYP2B和CYP2E1酶均有诱导作用。     

The utility of cold-preserved human hepatocytes in studies on cytochrome P450 induction and hepatic drug transport

Abstract

1. Human hepatocytes that had been cold-preserved in SureTran^TM matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012).

2. After 5?d of cold preservation, the viability was still more than 70%, and after 8?d it was around 60%. In hepatocytes that had been cold-preserved for 3?d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8?µM rifampicin for 72?h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3?d and thereafter treated with 1?mM phenobarbital for 72?h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3?d, while the activity increased in cells treated with 0.3–25?µM β-naphthoflavone for 72?h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers.

3. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2?d.

4. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.     

Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers

AIMS: To assess the age-associated changes over time of plasma paraxanthine/caffeine (PAX/CAF) ratios used as a probe for CYP1A2 activity. METHODS: Intraindividual and interindividual variabilities in PAX/CAF ratio were compared by phenotyping with caffeine, 16 young and 16 elderly healthy subjects on five occasions. RESULTS: PAX/CAF ratio variability was comparable regardless of age (intraindividual CV: 17.6 +/- 6% and 16.2 +/- 5.9%, interindividual CV: 48.1 +/- 2.9% and 42.7 +/- 3.6% in young and elderly, respectively). The PAX/CAF ratio was lower in elderly than in young subjects (95% CI for the difference: 0.004, 0.32) but the difference was not significant in nonsmokers compared separately. CONCLUSIONS: The variability over time of the PAX/CAF ratio is not influenced by age.     

毛蕊花糖苷在大鼠体内吸收、分布及排泄研究

目的:采用液相色谱-质谱联用(LC-MS/MS)法测定大鼠血浆及组织样品中毛蕊花糖苷的浓度,并探讨其在大鼠体内吸收、分布及排泄研究。方法:SD大鼠灌胃给予毛蕊花糖苷20,40,80,160 mg·kg^-1后,于不同时间点采血,给予40 mg·kg^-1剂量进行分布和排泄试验,测定血浆、组织和排泄物中的毛蕊花糖苷浓度,并用DAS 2.0软件拟合并计算药动学参数。结果:大鼠给药20,40,80,160 mg·kg^-1的毛蕊花糖苷后,药时曲线呈二室开放模型,主要药动学参数t_max,C_max,t_1/2α,AUC_0-t,AUC_0-∞,CL/F,V/F分别为(17.50±10.37)min、(0.313±0.04)mg·L^-1、(6.79±12.10)min、(21.39±4.03)mg·L^-1·min^-1、(22.39±3.89)mg·L^-1·min^-1、(1.83±0.30)L·min^-1·kg^-1、(179.10±52.77)L·kg^-1。大鼠给予40 mg·kg^-1的毛蕊花糖苷后,毛蕊花糖苷在尿液和粪便中36 h内的累积排泄百分数分别为(0.037±0.005)%、(0.004 2±0.000 8)%,胆汁中12 h内的累积排泄率基本为零。结论:毛蕊花糖苷在大鼠体内吸收符合一级动力学,分布在小肠和肺浓度最高,其次为胃和肌肉,其他组织都有少量的分布;且通过尿液、粪便和胆汁排泄量较少,其可能主要通过代谢过程进行消除。     

细胞色素氧化酶CYP 1A2基因多态性与物质代谢和癌症发生相关性的研究进展

细胞色素氧化酶CYP 1A2亚家族是近年来药物代谢研究领域较受关注的热点之一。该酶具有高度的个体间差异,并参与多种临床药物以及环境致癌物质的代谢,与癌症、炎症、心肌梗塞等疾病的发病易感性相关。CYP 1A2具有抗氧化作用;CYP 1A2基因多态性和表型差异的研究,可用于评价临床药物治疗效果;探针药物的应用是研究CYP 1A2活性的主要方法;人源化CYP 1A2转基因动物模型,是癌症发生研究中、新的研究手段。     

细胞色素P450酶诱导和抑制效应高通量筛选系统研究进展*

细胞色素P450酶（CYP450）活性的诱导或抑制，是引起临床药物代谢性相互作用的主要作用机制。目前确定候选药物出现此类相互作用可能性的主要方法是通过体外CYP450诱导和抑制潜能的快速筛选。现从CYP450诱导机制、目前发展的综合活性筛选系统和诱导筛选系统、抑制效应筛选系统以及硅上虚拟筛选系统等几个方面，对CYP450诱导和抑制效应高通量筛选系统的研究进展作一综述。     

Evaluation of the pharmacokinetics,tissue distribution and excretion studies of YMR-65, a tubulin polymerization inhibitor with potential anticancer activity,in rats using UPLC-MS/MS

1.YMR-65, 5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4, 5-dihydro-1H-pyrazole-1-carboxamide, is a new tubulin polymerization inhibitor with encouraging anticancer activity.

2.The validated ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method was successfully applied to the pharmacokinetics, tissue distribution and excretion study of YMR-65 after oral and intravenous administration. The area under concentration-time curve (AUC_0-∞) for YMR-65 were 151.67?±?54.48 and 459.45?±?49.23?ng/ml*h for oral and intravenous administration at the dosage of 1.5?mg/kg, respectively and the oral bioavailability was about 33.01%. Moreover, YMR-65 was extensively distributed in heart, liver, spleen, lung, kidney, stomach, intestine and testis and the highest were detected in heart, followed by stomach, intestine and liver. The majority of YMR-65 was excreted via feces and its accumulative excretion ratio during the period of 96?h was 19.83?±?3.01%, but only 1.54?±?0.37 and 0.215?±?0.026% for urine within 96?h and bile within 10?h after intravenous administration, respectively, though the fecal and urine excretion were incomplete within 96?h.

3.In summary, this study defined the pharmacokinetic characteristics of YMR-65 in vivo and the important data can be a useful resource for further research and development.     

丹参注射液对人肝微粒体酶CYP2C9,CYP2C19,CYP2D6体外抑制作用的研究

目的 评价丹参注射液在体外对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6活性的影响。方法 将丹参注射液与CYP450酶3种亚型(CYP2C9、CYP2C19、CYP2D6)的特异性探针底物甲苯磺丁脲、奥美拉唑、右美沙芬与大鼠肝微粒体进行孵育,采用LC-MS/MS法测定对应3种代谢产物4-羟基甲苯磺丁脲、5-羟基奥美拉唑、右啡烷的浓度,求算出IC₅₀。结果 丹参注射液对CYP2C9、CYP2C19和CYP2D6的IC₅₀值均>50 μg/mL。结论 丹参注射液对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6没有抑制作用。     

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase,glutathione S-transferase,and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.     

Functional significance of a C-->A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine

AIMS: The cytochrome P450 enzyme CYP1A2 metabolises several drugs and carcinogens. We wanted to determine how much of the variability of CYP1A2 activity is explained by a newly discovered gene polymorphism in intron 1. METHODS: A single nucleotide polymorphism in intron 1 of the CYP1A2 gene at position 734 downstream of the first transcribed nucleotide was identified by DNA sequence analysis. The functional significance of this C/A polymorphism was assessed in 185 healthy Caucasian non-smokers and in 51 smokers by genotyping and phenotyping using caffeine (100 mg oral dose). RESULTS: Out of the total sample, 46% were homozygous for the variant A, 44% were heterozygous, and 10% were homozygous for the variant C. The ratio of 1,7-dimethylxanthine (17X) plus 1,7-dimethyluric acid divided by caffeine in 0-5 h urine samples from 185 non-smokers did not differ significantly between the three CYP1A2 genotypes. In the 51 smokers, analysis of variance revealed significant differences in the 5 h plasma 17X/caffeine ratios between the genotypes (P=0.008, F-test). The mean ratio was 1.37 in carriers of the A/A genotype, 0.88 in heterozygotes and 0.82 in carriers of C/C. The mean difference between the A/A and C/A groups was 0.48 (95% confidence interval 0. 15-0.81; P=0.01). CONCLUSIONS: The A/A genotype, which may represent a CYP1A2 high inducibility genotype, may either be a direct cause of increased CYP1A2 activity, or be genetically linked to polymorphisms conferring high inducibility. Further studies are needed to define the role of this polymorphism on the pharmacokinetics of drugs metabolised by CYP1A2 and in the activation of carcinogens.