盐酸丁咯地尔在人血浆和肝微粒体中代谢产物鉴定

目的研究盐酸丁咯地尔在人体的代谢情况。方法采用UPLC-QTOF/MS法发现并鉴定盐酸丁咯地尔在人血浆和肝微粒体孵化体系中的代谢产物。采用HSS T3 C18色谱柱(100 mm×2.1 mm,1.8μm),以5 mmol·L~(-1)醋酸铵水溶液(含体积分数0.05%的甲酸)(A)-甲醇(B)为流动相梯度洗脱,流速为0.4 m L·min~(-1),采用ESI离子源,正离子模式下检测。结果通过与空白生物样品进行比较,在人血浆和肝微粒体中共发现了丁咯地尔及其24种代谢产物,其中18种为首次发现的代谢产物。根据代谢产物的色谱保留时间、准分子离子及碎片离子等信息,确定并推断了丁咯地尔及其代谢产物的结构,阐明了丁咯地尔在人体的代谢途径,包括去甲基化、氧化、羟基化、硫酸和葡萄糖醛酸结合途径。结论建立了UPLC-QTOF/MS方法研究了丁咯地尔在人体的代谢情况,为其化学结构类似药物和候选化合物的代谢研究提供依据。     

UPLC-MS/MS快速鉴定乔松素在人肝微粒体中的代谢产物

目的 以超高效液相色谱质谱联用法(UPLC-MS/MS)快速鉴定乔松素在人肝微粒体中的代谢产物。 方法 以人肝微粒体体外孵育体系对乔松素进行代谢研究,以UPLC-MS/MS鉴定乔松素的体外代谢产物。 结果 建立了乔松素的体外代谢孵育体系,UPLC-MS/MS在6 min内检测到乔松素的4个代谢产物,分别鉴定为柚皮素、5,6,7-三羟基二氢黄酮、5,7,8 -三羟基二氢黄酮和乔松素-7-葡萄糖醛酸苷,这4个代谢产物均为首次发现的乔松素人肝微粒体体外代谢产物。 结论 乔松素在人肝微粒体中的主要代谢途径为羟基化和葡萄糖醛酸化,5,7,8 -三羟基二氢黄酮是其主要羟基化产物,乔松素-7-葡萄糖醛酸苷可能为乔松素的主要代谢失活形式。     

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。     

新型降糖化合物LSM-13在大鼠肝微粒体中的稳定性研究及代谢产物分析

目的:考察新型降糖化合物LSM-13在大鼠肝微粒体中的代谢稳定性,并分析其可能的代谢产物.方法:将LSM-13溶解于由还原型烟酰胺腺嘌呤二核苷酸磷酸启动的大鼠肝微粒体孵育体系中,置于37℃水浴中进行孵育,分别于孵育0、5、10、15、20、30、45、60 min时用乙腈终止反应.以吲哚美辛为内标,采用高效液相色谱法测...     

治疗黑色素瘤的新型二芳基衍生物04022a在不同种属肝微粒体中的稳定性及代谢产物分析

目的 采用SD大鼠、人、比格犬的肝微粒体酶,考察新型二芳基衍生物04022a在3个种属肝微粒体中的代谢稳定性,比较代谢的种属差异,并对产生的代谢产物进行筛选与结构表征。方法 将04022a溶解在由还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)启动的肝微粒体孵育体系中,孵育0、15、30、60、90、120 min后,加入等体积的冰甲醇终止反应。采用高效液相色谱法测定04022a的质量浓度,计算各个时间点的剩余药物百分比、体外半衰期(t_1/2)和固有清除率(CL_int)。采用超高效液相色谱串联飞行时间质谱法对04022a在肝微粒体中的代谢产物进行检测与分析。结果 孵育120 min时,04022a在大鼠、人、比格犬肝微粒体中的剩余药物百分比分别为88.30%、82.15%、98.63%。t_1/2分别为770、364、3 465 min, CL_int分别为0.001 8、0.003 8、0.000 4 mL·(min·mg)^-1。初步判断04022a在3个种属共有5个体外代谢产物。结...     

1和3—硝基苯并[a]芘混合物在大鼠肝微粒体中代谢产物的探讨

本文研究用3-甲基胆蒽诱导大鼠肝微粒体与等量1-和3-硝基苯并[a]芘混合物在体外有氧条件下进行培培养,分析代谢产物和代谢速率,并同单纯的1-硝基苯并[a]芘和3-硝基苯[a]芘的代谢产物进行比较。结果表明,1-和3-硝基苯并[a]芘混合物的代谢产物与单纯的1-硝基苯并[a]和3-硝基苯并[a]芘的代谢产物是相同的,两者按摩尔比等量混合后的代谢速度也是一致的;需要把高压液相色谱仪的反相柱和正相柱结合起来使用,才能把1-和3-硝基苯并[a]芘混合物的代谢产物完全分开。     

双环醇在大鼠和人肝微粒体的代谢

目的研究参与双环醇代谢的主要药物代谢酶及代谢动力学参数，分离鉴定双环醇代谢产物。方法双环醇与大鼠和人肝微粒体进行温孵，以高效液相色谱、质谱、核磁共振技术检测并分离鉴定双环醇及其代谢产物。结果双环醇在地塞米松诱导大鼠肝微粒体中的代谢速率显著高于正常大鼠肝微粒体，酮康唑可显著抑制双环醇的代谢。双环醇主要代谢产物为:4-羟基-4′-甲氧基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯和4-甲氧基-4′-羟基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯。结论双环醇在大鼠和人肝微粒体的主要代谢产物为4-羟基-4′-甲氧基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯和4-甲氧基-4′-羟基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯，细胞色素P450 3A主要参与双环醇代谢。     

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。     

枸橼酸爱地那非人肝微粒体代谢产物鉴定及性别差异

目的研究枸橼酸爱地那非在不同性别来源的人肝微粒体体外代谢孵育体系中的代谢差异。方法将枸橼酸爱地那非与不同性别来源的人肝微粒体进行体外共孵育,采用超高效液相色谱-四极杆-飞行时间质谱仪,鉴定并推测爱地那非在不同性别来源的人肝微粒体中的代谢产物。色谱柱为Acquity UPLC BEH C₁₈(2.1 mm×100 mm×1.7μm),流动相为乙腈与0.4%甲酸溶液,梯度洗脱,流速0.3 mL·min^-1。采用电喷雾离子源,正离子模式检测。结果结合色谱保留时间和质谱碎片离子信息,在体外肝微粒体孵育体系中鉴定了原型分子爱地那非,以及爱地那非经Ⅰ相代谢反应产生的8种代谢产物。所有代谢产物均为水溶性分子,无明显毒性结构。不同性别肝微粒体孵育体系中检测到的代谢产物种类相同。结论建立了爱地那非的体外肝微粒体孵育体系模型,初步研究推测不同性别人群对爱地那非的肝脏微粒体代谢过程一致。     

米氮平在鼠肝微粒体中的体外代谢研究

目的:通过不同诱导剂预处理鼠肝,研究米氮平在肝微粒体中的代谢主要受何种酶影响,同时研究米氮平对介导其自身代谢的P450酶亚型是否有影响,为米氮平的临床合理应用提供科学依据。方法:将米氮平与不同诱导剂诱导的鼠肝微粒体进行体外孵育代谢,以乙腈中止反应,样品用25%氨水碱化后以环己烷提取。用RPHPLC测定剩余的米氮平含量,流动相为甲醇水(含10mmol·L-1NH4AC,5mmol·L-1SDS,pH3.5)6238(VV),检测波长为307nm。结果:本文测定方法回收率高,日内和日间精密度均良好,符合生物样本检测要求。苯巴比妥诱导的鼠肝微粒体对米氮平的代谢具有明显的催化作用,利福平也有一定的催化能力,米氮平诱导的鼠肝微粒体与对照组对米氮平的代谢无明显差异。结论:由苯巴比妥诱导的P450酶亚型(主要为细胞色素P4503A4)和利福平诱导的P450酶亚型(主要为细胞色素P4502C92C19,同时也对P4503A4有一定的诱导作用)在米氮平的体外代谢中起着重要作用;而米氮平诱导组对于米氮平代谢无明显影响,预示米氮平对介导其自身代谢的P450酶亚型无明显的诱导或抑制作用。     

In vitro metabolic stability of moisture-sensitive rabeprazole in human liver microsomes and its modulation by pharmaceutical excipients

A reliable method to assess in vitro metabolic stability of rabeprazole and its modulation by Generally Recognized As Safe (GRAS)-listed pharmaceutical excipients was established in human liver microsomes. The metabolic stability of rabeprazole decreased as a function of incubation time, resulting in the formation of thioether rabeprazole via nonenzymatic degradation and enzymatic metabolism. Buffer type was also a determining factor for the degree of both nonenzymatic degradation and enzymatic metabolism. The net extent of enzymatic drug metabolism, obtained by calculating the difference in drug degradation between a microsome-present reaction system and a microsome-free solution, was about 9.20 ± 0.67% in phosphate buffer and 2.27 ± 1.76% in Tris buffer, respectively. Rabeprazole exhibited first-order kinetics in microsome-free solution but showed non-linear kinetics in the microsome-present reaction system. The maximal velocity, V_max, in phosphate buffer was 5.07 μg mL⁻¹ h⁻¹ and the Michaelis-Menten constant, K_m, was 10.39 μg mL⁻¹ by computer-fitting to the classical Michaelis-Menten equation for pattern of time-dependent change in the substrate concentration. The intact drug and its thioether form were well resolved and successfully identified by HPLC chromatography and liquid chromatography mass spectroscopy (LC/MS). The metabolic stability of rabeprazole was also modulated by the presence of pharmaceutical excipients. Among the five pharmaceutical excipients tested, poloxamer 188 and Gelucire 44/14 had potentially inhibitory effects on rabeprazole metabolism in human liver microsomes (p < 0.05). A greater understanding of metabolic stability and its modulation by pharmaceutical excipients would be useful for optimizing the bioavailability of rabeprazole at the early formulation stages.     

Characterization of fimasartan metabolites in human liver microsomes and human plasma

1.?The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects.

2.?We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode.

3.?The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards.

4.?In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3?min in the presence of NADPH, UDPGA and NADPH?+?UDPGA, respectively.

5.?The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation.

6.?In humans orally administered with 120?mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0–8-h pooled plasma sample of each subject.

7.?This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.     

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes,liver cytosol and plasma preparations

1. The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days and from adult humans.

2. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes.

3. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.

4. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.

5. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes.

6. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90?day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

     

Effects of some commonly used Saudi folk herbal medications on the metabolic activity of CYP2C9 in human liver microsomes

Objective

To investigate the potential effects of eleven of the most commonly used Saudi folk herbal medications on the metabolic activity of CYP2C9 in human liver microsomes.

Method

CYP2C9-mediated 4′-hydroxylation of tolbutamide (TB) to 4′-hydroxytolbutamide (4-OH-TB) was utilized to assess the metabolic activity of CYP2C9. In the present study, an initial screening of the eleven herbs was carried out by incubating TB with microsomes and NADPH in absence or presence of a fixed concentration (25 μg/ml) of alcoholic extracts of different herbs and the metabolite formed was measured by HPLC. Herbs that showed significant effects were further investigated at a lower range of concentration.

Results

Among the investigated herbal extracts, only aniseed and curcuma showed statistically significant effects on the formation of 4-OH-TB in human liver microsomes. Curcuma produced a potent inhibition on the metabolite formation and its maximum (about 45% inhibition) was observed at the highest extract concentrations (10 and 25 μg/ml). On the other hand, aniseed significantly activates the formation of 4-OH-TB and the maximum activation (about 55%) was observed at 2.5 μg/ml of aniseed extract.

Conclusion

The results of this study have shown that alcoholic extracts of curcuma and aniseed were capable of inhibiting and activating; respectively, the CYP2C9-mediated 4-OH-TB formation in human liver microsomes, suggesting that these herbs have the potential to interact with CYP2C9 drug substrates. None of the other nine investigated herbs was able to produce any statistically significant effect.     

Metabolic characteristics of Tanshinone I in human liver microsomes and S9 subcellular fractions

1. Tanshinone I (TSI) is a lipophilic diterpene in Salvia miltiorrhiza with versatile pharmacological activities. However, metabolic pathway of TSI in human is unknown.

2. In this study, we determined major metabolites of TSI using a preparation of human liver microsomes (HLMs) by HPLC-UV and Q-Trap mass spectrometer. A total of 6 metabolites were detected, which indicated the presence of hydroxylation, reduction as well as glucuronidation.

3. Selective chemical inhibition and purified cytochrome P450 (CYP450) isoform screening experiments revealed that CYP2A6 was primarily responsible for TSI Phase I metabolism. Part of generated hydroxylated TSI was glucuronidated via several glucuronosyltransferase (UGT) isoforms including UGT1A1, UGT1A3, UGT1A7, UGT1A9, as well as extrahepatic expressed isoforms UGT1A8 and UGT1A10. TSI could be reduced to a relatively unstable hydroquinone intermediate by NAD(P)H: quinone oxidoreductase 1 (NQO1), and then immediately conjugated with glucuronic acid by a panel of UGTs, especially UGT1A9, UGT1A1 and UGT1A8. Additionally, NQO1 could also reduce hydroxylated TSI to a hydroquinone intermediate, which was immediately glucuronidated by UGT1A1.

4. The study demonstrated that hydroxylation, reduction as well as glucuronidation were the major pathways for TSI biotransformation, and six metabolites generated by CYPs, NQO1 and UGTs were found in HLMs and S9 subcellular fractions.

     

High-resolution mass spectrometry-based approach for the identification and profiling of the metabolites of taletrectinib formed in liver microsomes

Taletrectinib is a potent, orally active, and selective ROS1/NTRK kinase inhibitor. The aim of this study was to study the metabolism of taletrectinib in rat, dog, and human liver microsomes. The biotransformation of taletrectinib was carried out using rat, dog, and human liver microsomes supplemented with nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) and uridine diphosphate glucuronic acid (UDPGA). The microsomal incubations were conducted at 37°C for 60 min. The formed metabolites were identified by ultrahigh performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS) using electrospray ionization in the positive ion mode. They were identified by accurate masses and MS/MS spectra and based on their fragmentation pathways. With UHPLC-HRMS, a total of 10 metabolites including one glucuronide conjugate (M7) were structurally identified. M9 and M10 were unambiguously identified as taletrectinib alcohol and taletrectinib ketone, respectively, using reference standards. The phase I metabolic pathways of taletrectinib involved N-dealkylation, O-dealkylation, oxidative deamination, and oxygenation; the phase II metabolic pathways referred to glucuronidation. The current study investigated the in vitro metabolic fate of taletrectinib in animals and human species, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.     

Update on metabolism of abemaciclib: In silico,in vitro,and in vivo metabolite identification and characterization using high resolution mass spectrometry

Abemaciclib was approved by the US Food and Drug Administration in 2015 as an advanced treatment for metastatic breast cancer. Identification and characterization of limited numbers of abemaciclib metabolites have been reported in the literature. Therefore, the current study focused on the investigation of the in vitro and in vivo metabolic fate of abemaciclib using high resolution mass spectrometry. Initially, a vulnerable site of metabolism was predicted by the Xenosite web predictor tool. Later, in vitro metabolites were identified from pooled rat liver microsomes, rat S9 fractions, and human liver microsomes. Finally, in vivo metabolites have been detected in plasma, urine, and feces matrix of male Sprague–Dawley rats. A total of 12 putative metabolites (11 phase I and 1 phase II) of abemaciclib and their metabolic pathways were proposed by considering accurate mass, mass fragmentation pattern, nitrogen rule, and ring double bonds of the detected metabolites. Abemaciclib was metabolized via hydroxylation, N‐oxidation, N‐dealkylation, oxidative deamination followed by reduction and sulfate conjugation. In the human liver microsomes, maximum numbers of metabolites (11 metabolites) were observed, from which M7, M8, M9, and M11 were human specific.     

Selective inhibition of CYP2C8 by fisetin and its methylated metabolite,geraldol, in human liver microsomes

Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb–drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC₅₀ shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs.