乌头类生物碱组分在CYP450中的代谢指纹图谱及对CYP450活性的影响

利用超高效液相色谱-串联质谱(UPLC-MS/MS)的多反应监测(MRM)技术, 结合多探针底物方法, 对单酯型及双酯型乌头类生物碱组分对细胞色素P450(CYP450)亚型的活性影响进行了研究; 同时利用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS/MS)对单酯型和双酯型乌头类生物碱组分在CYP450中的代谢指纹图谱进行了研究. 活性影响研究结果表明, 单酯型生物碱组分对CYP2C及2D的抑制能力较强, 其IC₅₀值分别为7.44和6.74 μmol/L; 双酯型生物碱组分对CYP1A2, 3A, 2C和2D均有较弱的抑制作用, 其IC₅₀值分别为39.48, 70.44, 17.36和86.04 μmol/L. 代谢指纹图谱显示, 双酯型生物碱组分在大鼠肝微粒体中有6个特异性产物可以作为该反应的特征峰.     

4种黄酮化合物与5-脂氧合酶相互作用研究

首次采用体外抑制实验检测芦丁、异槲皮苷、紫云英苷、槲皮素4种黄酮化合物对5-脂氧合酶(5-LOX)抑制作用,4种化合物IC50值分别为44.18,94.17,172.49和468.45 μmol/L.在超滤液质联用实验中测定4种化合物与5-LOX酶的结合率,其结果与体外抑制实验结果一致,表明芦丁对5-LOX有较强的抑制作用,其次为紫云英苷,而槲皮素和异槲皮苷对5-LOX几乎无作用.     

高效液相色谱-串联质谱及特异性抑制剂探针方法研究异嗪皮啶的体外CYP450代谢产物及代谢亚型

利用高效液相色谱-串联质谱及特异性抑制剂探针方法研究了异嗪皮啶在大鼠肝微粒体CYP450中的代谢产物及代谢亚型。结果表明,异嗪皮啶在大鼠肝微粒体的代谢时间为2 h最佳,其主要代谢产物有5种。CYP 3A,CYP 2C,CYP 2E1等亚型为异嗪皮啶在大鼠肝微粒体内的主要代谢酶。本方法为进一步研究药物-药物相互作用提供了依据。     

5-苄基-4-叔丁基-2-芳氨基噻唑氢溴酸盐的合成、表征及抗肿瘤活性

以芳香胺和4,4-二甲基-1-芳基-2-溴戊-3-酮为原料, 设计合成了29种新的5-苄基-4-叔丁基-2-芳氨基噻唑氢溴酸盐化合物; 其结构经¹H NMR和元素分析等确证. 体外抗肿瘤活性测试结果表明, 化合物A4, A5, A12和A29对人非小细胞肺癌细胞A549的IC₅₀值分别为0.016, 0.019, 0.019和0.026 μmol/mL, 与阳性对照物紫杉醇(IC₅₀值为0.022 μmol/mL)相当; 化合物A5, A7, A13, A14和A19对肝癌细胞Bel7402的IC₅₀值分别为0.021, 0.021, 0.026, 0.014和0.029 μmol/mL, 与阳性对照物紫杉醇的IC₅₀值(0.030 μmol/mL)相近. 利用倒置显微镜和Hoechst33342-PI双染色法观察了细胞经化合物处理后的形态变化.     

液相色谱-质谱联用技术结合多探针底物法研究人参皂苷Rb1的体外代谢

以奥美拉唑、 苯妥英、 卡马西平和非那西丁为检测肝药酶细胞色素P450酶(CYP450)亚型的专属探针药物, 通过原型药物减少量测定法考察药物体外代谢的变化, 评价人参皂苷Rb1对CYP450不同亚型酶的作用. 结果表明, P2C9, P2C19和P3A4实验组与对照组差异不显著, P1A2实验组与对照组差异显著, 表明人参皂苷Rb1能诱导P1A2亚型酶的活性, 促进底物与酶反应, 加快底物的代谢, 而对P2C9, P2C19和P3A4三个亚型酶有弱的诱导或无诱导作用. 根据快速分离液相色谱-质谱联用(RRLC-MS/MS)检测结果推断, 人参皂苷Rb1在CYP450酶中的代谢产物可转化为人参皂苷Rb1氧化产物(Rb1+O)及人参皂苷Rd和F2.     

UPLC-TSQ-Endure质谱分析Rg_3对2型糖尿病大鼠P450酶活性的影响

应用超高效液相色谱-质谱联用法(UPLC-MS)结合多探针底物法建立了同时定量测定5个细胞色素P450酶探针底物的方法,同时考察正常大鼠和2型糖尿病大鼠CYP亚型酶活性的变化,分析人参皂苷Rg_3对CYP亚型酶的调控作用。结果表明,与正常组比较,2型糖尿病大鼠肝微粒体酶中P3A4和P2E1活性被诱导,P2D6,P1A2和P2C9活性被抑制。人参皂苷Rg_3诱导2型糖尿病大鼠P3A4和P1A2活性,抑制P2E1和P2C9活性,对P2D6活性无明显影响。人参皂苷Rg_3能够使糖尿病大鼠CYP2E1和CYP1A2亚型酶活性趋于恢复到正常生理状态,其在CYP450酶中的代谢产物为人参皂苷Rh2。方法适用于评价P450酶诱导或抑制的酶活性测定,在临床上指导2型糖尿病患者合理用药。     

毛秔子梢中天然高活性的神经氨酸酶抑制成分的研究

从天然产物中筛选高效低毒的神经氨酸酶抑制剂(NAI), 以期发现可抗流感病毒(H1N1)的化合物. 方法: 采用经典的NA活性检测实验(FL-MH-NANA method), 对从豆科植物毛秔子梢中分离得到10个化合物进行筛选, 检测这些化合物对NA的抑制活性. 结论: 化合物1, 2, 3, 5对流感病毒株的神经氨酸酶(NA)均具有抑制作用, 其中化合物1的抑制活性最强, 其IC₅₀值为16.76 μmol/L. 化合物1～4为新化合物, 化合物5～10为该植物中首次分得.     

新型-3-O-乙酰-11-酮基-β-乳香酸衍生物的合成及其体外抗肿瘤活性

以3-O-乙酰基-11-酮-β-乳香酸（AKBA）为原料，经过氯化、取代反应，合成了8个新型的AKBA酰胺类衍生物（S137、S151、S179、S184、S443、S634、S667和S673），其结构经¹H NMR和MS（ESI）表征。采用CCK8法测试了目标化合物对人原髓细胞白血病细胞（HL-60）、人结肠癌细胞（HCT116）和非小细胞肺癌细胞（A549）的体外抗肿瘤活性。结果表明：除S184和S443外，S137、S151、S179、S634、S667、S673对3种细胞株的活性均有一定的抑制作用，其中S137对HL-60和A549的抑制效果较好，IC₅₀分别为1.781和1.763 μM，优于AKBA（IC₅₀=16.160和10.900 μM）；S151对HL-60抑制效果最好，IC₅₀为1.255 μM，优于AKBA（IC₅₀=16.160 μM）；S634对HCT116的抑制效果最好，IC₅₀为1.193 μM，优于AKBA（IC₅₀=13.020 μM）。      

常春藤皂苷元-半酯-AZT衍生物的合成及抗病毒蛋白酶活性

以常春藤皂苷元为原料,合成了5个常春藤皂苷元-半酯-AZT复合物,其结构经¹HNMR、¹³CNMR和ESI-MS表征。并对所有目标分子体外抗HIV-1和HCV NS3/4A蛋白酶的活性进行了初步评价,化合物5显示较强的抗HCV NS/4A蛋白酶的活性,IC₅₀=5.8 μM,其余化合物IC₅₀为10.6～42.3 μM。所有衍生物无抗HIV蛋白酶活性,揭示该类衍生物选择性抑制HCV NS3/4A蛋白酶。      

新型异吲哚酮衍生物的合成与活性研究

为了寻找结构新颖,活性较好的抗肿瘤化合物,本文设计并合成了15个未见报道的异吲哚酮衍生物(I₁~I₅, II₁~II₅和III₁~III₅),其结构经¹H NMR和HR-MS(ESI)表征。使用四甲基偶氮唑盐(MTT)法,初步的生物活性测试结果表明：部分化合物对人肺癌细胞A549(II₄, IC₅₀=15.49μM)、人结肠癌细胞Colo205(II₄, IC₅₀=15.54μM)、人胰腺癌细胞PNAC-1(II₄, IC₅₀=37.07μM)和人骨髓瘤细胞U266(II₅, IC₅₀=75.27μM)表现出明显的抑制作用。      

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.     

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Use of A New HPLC Method in Rat Liver Microsomal Testosterone Monooxygenation and Its Application to Study the Sex Dependent Expression of Several Hydroxylases

Abstract

An HPLC method described by Mancilla and Gil [Analytical Letters 17, (B9), 1984, 873-886] has been applied to study the sex dependent expression of several rat liver testosterone hydroxylases. A sample clean up procedure has been developed using SEP-PACK C- 18 cartridges which retained testosterone and its microsomal oxidative products. Undesired components were not retained or selectivly eluted with organic solvents. The clean steroid sample was eluted with a mixture of n-hexane and 2-propanol. HPLC of testosterone microsomal oxidation products was performed by normal phase In a Lichrosorb diol column using an Isocratic mixture of n-hexane and 2-propanol. The main five testosterone metabolites produced by male and female rat liver microsomes were determined In only 24 min. The turnover rates for testosterone oxidation were similar in male and female microsomes, but significant differences were observed in the rate of production of different metabolites. Male microsomes catalized mainly oxidation at positions 2 α, and 7 α; whereas female microsomes produced mainly 7 α OHT and androstenedione. These results might be explained by the different contribution of some cytochrome P-450 isozymes in microsomes from the different sexes. This method provides a useful tool to study the P-450 isozymic contributions to microsomal activities in different tissues and might facilitate the comparison of P-450 isozymes purified in different laboratories.     

Efficient quantification of the phenolic profiles of Zanthoxylum bungeanum leaves and correlation between chromatographic fingerprint and antioxidant activity

Sixteen subsequent fractions were prepared from the ethyl acetate fraction of Zanthoxylum bungeanum leaves after bio-guided chromatographic separation. The HPLC profiles and antioxidant activity of the various fractions indicated that the content of eight phenolic compounds (chlorogenic acid, epicatechin, rutin, hyperoside, trifolin, quercitrin, afzelin and quercetin) and antioxidant activity vary significantly, and high concentrations of a combination of eight phenolic compounds would result in an increase of the antioxidant activity. These results suggested that the eight compounds could be used as chemical markers for quality assessment of Z. bungeanum leaves. Correlation between chromatographic fingerprint and antioxidant activity of the fractions showed that quercitrin and hyperoside play crucial roles in the antioxidant activity, and they can be seen as the milestone for quality control. The findings also suggested that five obtained fractions (E-3-3, E-2-4, E-7, E-5 and E-4) could become useful supplements for functional food ingredients and health-related products.     

In vitro metabolism of a new cardioprotective agent, KR-32570, in human liver microsomes

KR-32570 (5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new reversible Na+/H+ exchanger inhibitor for preventing ischemia-reperfusion injury. This study was performed to identify the metabolic pathway of KR-32570 in human liver microsomes. Human liver microsomal incubation of KR-32570 in the presence of NADPH and UDPGA resulted in the formation of six metabolites, M1-M6. M1 was identified as O-desmethyl-KR-32570, on the basis of liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis with the synthesized authentic standard. M2 and M3 were suggested to be hydroxy-KR-32570 and hydroxy-O-desmethyl-KR-32570, respectively. M1, M2, and M3 were further metabolized to their glucuronide conjugates, M4, M5, and M6, respectively. In addition, the specific P450 isoforms responsible for KR-32570 oxidation to two major metabolites, O-desmethyl-KR-32570 and hydroxy-KR-32570, were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 isoforms. The inhibitory potency of KR-32570 on clinically major P450s was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-32570 to hydroxy-KR-32570, and CYP1A2 play the predominant role in O-demethylation of KR-32570. KR-32570 was found to inhibit moderately the metabolism of CYP2C8 substrates.     

Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug–drug interaction

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti‐inflammation and anticancer activities. In this study, we utilized recombinant human UDP‐glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 μm , and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 μm , respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin‐containing herbs.     

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1'-hydroxylation kinetics in human liver microsomes

The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.