AFB_1代谢相关的CYP450酶的表达及影响因素分析--《南京医科大学》2009年硕士论文
AFB_1代谢相关的CYP450酶的表达及影响因素分析
【摘要】：黄曲霉毒素B_1(AFB_1)属于I类强致癌物,主要污染农作物和食物。尽管肝脏一直被认为是AFB_1毒作用的重要靶器官,但也有证据显示AFB_1对人呼吸系统肿瘤的发生起重要作用。AFB_1的致癌作用和其经代谢活化形成活性中间代谢产物epoxide有关,而细胞色素P450(CYP450)酶则是其在体内代谢活化过程中最主要的代谢酶。研究显示,一些CYP450s如CYP1A2、CYP3A4以及CYP2A6与AFB_1的代谢活化有密切关系,其中CYP1A2是公认的人肝脏代谢AFB_1的优势酶。但近年来的研究发现,CYP2A13也可以高效代谢活化AFB_1,生成包括epoxides在内的多种活性代谢产物。鉴于CYP2A13在人呼吸道和肺脏中特异性的高表达,而CYP1A2几乎不在该组织中表达,提示CYP2A13可能与人呼吸系统原位代谢AFB_1并进而引起肿瘤的发生有关。但迄今,有关CYP2A13代谢活化AFB_1的证据尚显不足。因此,以体外代谢为切入点,通过酶反应学和代谢产物分析,进一步确认CYP2A13与AFB_1的关系并寻找重要代谢产物,将为AFB_1诱发人呼吸系统肿瘤提供机理上的依据。
AFB_1的体外代谢反应体系需要大量单一组分、有活性的CYP450蛋白,通过异源表达系统表达纯CYPs,是目前获得单一纯酶的主要方法。异源表达系统中,昆虫细胞的蛋白加工和修饰方式与哺乳动物细胞相似,且表达量高,可以高水平表达多种原始的CYP450蛋白。只是CYP450s活性的发挥需要NADPH-细胞色素P450还原酶(POR)、NADPH等辅酶为其提供电子,单独的表达会阻碍活性的完全发挥。有研究表明,在昆虫细胞中加入辅助因子5-ALA、柠檬酸铁(Fe3+)或Hemin,可表达有活性的CYP450s;此外,CYP450s和POR共表达可得到天然不需再修饰的微粒体膜活性蛋白,从而为体外代谢体系的建立提供较为有效的途径。但由于体外表达CYP450蛋白的复杂性,因此需要对关键影响因子进行系统研究,为体外高效表达CYP450s创造有利条件。
选择Baculovirus/Sf9系统体外表达CYP2A13及其他与AFB_1代谢相关的CYP450和POR蛋白,研究不同辅助因子Hemin、5-ALA和Fe~(3+)对CYP450酶和POR表达效率及活性的影响,并尝试CYP酶和POR的共表达,探寻稳定、高效表达高活性CYP450酶和POR的条件,为进一步构建体外代谢反应体系、系统阐明CYP2A13对AFB_1的代谢活化作用、明确关键的代谢产物提供可靠的研究平台。
使用酶切、酶连方法构建重组转移载体pFastBac1-CYPs质粒并进行酶切和PCR验证;通过转座实验,将CYPs各基因片段插入穿梭载体Bacmid DNA中,构建重组Ac-Bacmid-CYPs,进行PCR验证;使用Ac-Bacmid-CYPs DNA转染昆虫细胞Sf9,在细胞中获得完整的含CYPs基因的杆状病毒;使用含CYPs基因的杆状病毒感染Sf9细胞,加入辅助因子5-ALA和Fe~(3+),表达CYP1A2、CYP3A4、CYP2A6、CYP2A13和POR蛋白,Western blot检测各蛋白的表达;对CYP1A2、CYP3A4、CYP2A6和CYP2A13蛋白进行CO差示光谱检测;对POR蛋白进行细胞色素C分析。
在使用Baculovirus/Sf9系统表达CYP450s时,加入不同浓度、不同辅助因子5-ALA、Fe~(3+)、Hemin或其组合,使用Western blot法,细胞色素C还原法和CO差示光谱法检测并评价不同辅助因子对蛋白表达水平和活力的影响。此外,将含CYP1A2或POR基因的杆状病毒按不同比例混合联合感染Sf9细胞进行共表达,寻求最佳比例;在此基础上,初步探讨不同辅助因子5-ALA或(和) Fe~(3+)对CYP1A2和POR共表达的影响,寻找最合适的体外表达条件。
1.经过选择合适正确的酶切位点,通过酶切和连接反应,获得了pFastBac 1-CYPs(POR)质粒并经酶切反应和PCR验证无误;随后成功构建真核表达Ac-Bacmid-CYPs(POR)病毒DNA并进一步获得高滴度的重组真核表达Ac-Bacmid-CYPs(POR)病毒。
2.采用传统条件体外表达CYP1A2、CYP3A4、CYP2A6、CYP2A13和POR,Western blot检测显示上述蛋白在Baculovirus/Sf9系统中可成功表达,CYP450可出现45-55 kD的条带,而POR的条带出现在约78 kD处;CO差示光谱法检测证实各CYP450s在450nm处都有明显的吸收峰,显示有酶催化活性,每mg微粒体蛋白分别含CYP2A13、CYP2A6、CYP1A2、和CYP3A4蛋白的表达量为0.21nmol、0.057 nmol、0.027 nmol和0.023 nmol。POR蛋白对细胞色素C催化活力为1519.47 Unit/mg微粒体蛋白,且低温存放3个月后活性变化不明显(1505.01 Unit/mg微粒体蛋白)。
3.相对于单独加入5-ALA和Fe~(3+),同时加入0.1mM 5-ALA和0.1mM Fe~(3+)时,CYP1A2蛋白的表达显著提高,分别增加了40%和65%,也比单独加入Hemin时提高了32%;尽管POR的表达可能不需要5-ALA和Fe~(3+)的协助,但加入了5-ALA和/或Fe~(3+)后,POR的表达量显著提高,分别相当于不加时的2.13、1.71和1.86倍,且仅加入5-ALA时表达水平最高;同样的结果也反映在POR的活力上,加入5-ALA和/或Fe~(3+)分别提高7.1倍、4.5倍和5.4倍。不同浓度和种类辅助因子对CYP450酶的活性也有较大的影响,加入0.35mM 5-ALA和0.1mM Fe~(3+)时,CYP2A6的酶活性显著高于其它浓度组别。
4.不同比例的CYP1A2和POR病毒在Baculovirus/Sf9系统中共表达时,对各自蛋白的表达有较大的影响,在CYP1A2和POR病毒比例为2.5:1和3:1时,无论CYP1A2还是POR,其表达量都显著增加,分别比比例为2:1和4:1时提高约50%,且两者的表达比例较为适当;在CYP1A2:POR病毒比例为2.5:1的表达体系中,0.1和0.35mM 5-ALA时,CYP1A2和POR的表达量都显著高于0.6 mM;加入0.1mM Fe~(3+)时,蛋白表达水平也有较高水平;但有趣的是,当联合加入5-ALA和Fe~(3+)时,蛋白的表达量反而下降,相关机制有待进一步研究。
1.采用传统方法可获得一定量和一定活性的CYP450蛋白和POR蛋白,但不同的CYP450的活性差异较大;低温保存较长时间对POR活性影响不大;
2.不同辅助因子对CYP450和POR的表达及活性有明显影响。联合加入5-ALA和Fe~(3+)可显著增加CYP450的表达和活性,但POR略有不同,仅加入5-ALA时,POR的表达和活性明显提高;
3. CYP1A2和POR病毒比例为2.5:1和3:1时有利于两者的表达;低浓度的5-ALA或Fe~(3+)有利于共表达,但联合加入时,共表达效率反而下降。
由上可见,不同的表达条件对于在Baculovirus/Sf9系统中CYP450蛋白的表达和活性有较大的影响,应在实际工作中不断优化,确保稳定高效的表达。
【关键词】：
【学位授予单位】：南京医科大学【学位级别】：硕士【学位授予年份】：2009【分类号】：Q591【目录】：
摘要4-8ABSTRACT8-13前言13-18材料和方法18-33结果33-50讨论50-55小结55-56参考文献56-61附录61-62综述62-75 参考文献71-75主要缩略词75-76致谢76
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C Fahrmayr1, J K&nig1, D Auge1, M Mieth1, K M&nch1, J Segrestaa2, T Pfeifer2, A Treiber2 andMF Fromm1,*DOI:&10.1111/bph.12126
British Journal of Pharmacology pages 21&33, Author Information1Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universit&t Erlangen-N&rnberg, Erlangen, Germany2Actelion Pharmaceuticals Ltd, Allschwil, Switzerland*
Correspondence Martin F. Fromm, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universit&t Erlangen-N&rnberg, Emil Fischer Center, Fahrstra&e 17, 91054 Erlangen, Germany. E-mail:
Publication HistoryIssue online: 12 APR 2013Version of Record online: 12 APR 2013Accepted manuscript online: 6 FEB AM ESTManuscript Accepted: 16 DEC 2012Manuscript Revised: 5 DEC 2012Manuscript Received: 24 JUL 2012
ARTICLE TOOLS
OATP1B1;CYP3A4;UGT1A1;MRP2;quadruple-MDCKIIBackground and PurposeHepatic uptake (e.g. by OATP1B1), phase I and II metabolism (e.g. by CYP3A4, UGT1A1) and subsequent biliary excretion (e.g. by MRP2) are key determinants for the pharmacokinetics of numerous drugs. However, stably transfected cell models for the simultaneous investigation of transport and phase I and II metabolism of drugs are lacking.Experimental ApproachA newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line was used to investigate metabolism and transcellular transport of the endothelin receptor antagonist bosentan.Key ResultsIntracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) was significantly lower in all cell lines expressing MRP2 compared to cell lines lacking this transporter (P & 0.001). Accordingly, considerably higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with MRP2 expression (P & 0.001). HPLC and LC-MS measurements revealed that mainly unchanged bosentan accumulated in intracellular and apical compartments. Furthermore, the phase I metabolites Ro 48&5033 and Ro 47&8634 were detected intracellularly in cell lines expressing CYP3A4. Additionally, a direct glucuronide of bosentan could be identified intracellularly in cell lines expressing UGT1A1 and in the apical compartments of cell lines expressing UGT1A1 and MRP2.Conclusions and ImplicationsThese in vitro data indicate that bosentan is a substrate of UGT1A1. Moreover, the efflux transporter MRP2 mediates export of bosentan and most likely also of bosentan glucuronide in the cell system. Taken together, cell lines simultaneously expressing transport proteins and metabolizing enzymes represent additional useful tools for the investigation of the interplay of transport and metabolism of drugs.AbbreviationsCYP3A4cytochrome P450 enzyme 3A4Mrp2/MRP2rodent/human multidrug resistance protein 2OATP1B1organic anion transporting polypeptide 1B1Ro 47&86344-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-hydroxy-phenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamideRo 48&50334-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamideRo 64&10564-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-hydroxy-phenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamideRT-PCRreverse transcriptase polymerase chain reactionSLCOsolute carrier family of the OATPsUGT1A1uridine diphosphate-glucuronosyltransferase 1A1The coordinate process of drug uptake into hepatocytes, intracellular drug metabolism and the subsequent excretion of drug metabolites into bile is an important determinant for the pharmacokinetics and pharmacodynamics of orally administered drugs. Organic anion transporting polypeptides (OATPs) such as OATP1B1 (gene symbol: SLCO1B1) play an important role in the uptake process of drugs from portal venous blood into hepatocytes (K&nig, ). Following hepatic uptake, drugs often undergo phase I and/or phase II metabolism reactions [e.g. by cytochrome P450 enzyme 3A4 (CYP3A4) and/or uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1)] and drugs or their metabolites are subsequently exported into bile by efflux transporters, for example by MRP2, a member of the ABCC transporter family [gene symbol: ABCC2; (Ho and Kim, ; Funk, ; Zolk and Fromm, )].Bosentan, a dual endothelin receptor antagonist with high affinity for both endothelin A and B receptors, was approved as first oral treatment for pulmonary arterial hypertension (Clozel et&al., ; Weber et&al., ; van Giersbergen et&al., ; Treiber et&al., ; Thorin and Clozel, ). It is metabolized in vitro to a similar extent by CYP3A4 and by CYP2C9 and the known resulting metabolites are a phenol metabolite (4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-hydroxy-phenoxy)-[2,2&]bipyrimidinyl-4-yl]- Ro 47&8634), a hydroxy metabolite (4-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2&]bipyrimidinyl-4-yl]- Ro 48&5033) and a secondary metabolite containing both biochemical modifications [4-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-hydroxy-phenoxy)-[2,2&]bipyrimidinyl-4-yl]- Ro 64&1056, (Weber et&al., ; van Giersbergen et&al., ; Treiber et&al., )]. Among these metabolites, only the hydroxy metabolite is pharmacologically active. The parent compound and the hydroxy metabolite are substrates of OATP1B1 [with a Km value of 44&&M for bosentan and a Km value of 60&&M for th (Treiber et&al., )]. The formation of glucuronide conjugates of the parent compound and its phase I metabolites in primary human hepatocytes has been discussed in an abstract (Shen et&al., ), but to the best of our knowledge, it is not known which UGT isoform(s) mediate(s) these reactions. Biliary excretion of bosentan metabolites and possibly of the parent compound accounts for more than 90% of total drug elimination (Weber et&al., ; Treiber et&al., ). Fouassier et&al. () showed that bosentan alters canalicular bile formation predominantly via multidrug resistance protein 2 (Mrp2)-mediated mechanisms. However, the efflux transporter(s) mediating biliary secretion of bosentan and bosentan metabolites has/have not been identified.To test the hypothesis whether bosentan and/or its phase I metabolites are substrates of the phase II enzyme UGT1A1 and that the excretion of the parent compound and/or possibly formed conjugates is mediated by MRP2, we generated and characterized a quadruple-transfected cell line with the simultaneous expression of the uptake transporter OATP1B1, the metabolizing enzymes CYP3A4 and UGT1A1 and the hepatic efflux transporter MRP2.Cloning of the human CYP3A4 cDNACloning of the CYP3A4 coding sequence (NM_) by reverse transcription PCR using human liver cDNA synthesized from a Multiple RNA panel (Clontech, Heidelberg, Germany) was performed as described earlier (Fahrmayr et&al., ). Amplification of the full length CYP3A4 cDNA was conducted using the primer pair oCYP3A4-5&.for (5&-AGA TCT GTA AGG AAA GTA GTG ATG G-3&) and oCYP3A4-RT.rev (5&-AGC AGA TCT CCT TAG GAA AAT TCA G-3&), and the amplified fragment was cloned into the pCR2.1-TOPO vector (Invitrogen GmbH, Karlsruhe, Germany). After sequence verification (AGOWA, Berlin, Germany), the CYP3A4 cDNA was cloned into the expression vector pVITRO1-blasti-mcs (InvivoGen, San Diego, CA, USA). Five base pair exchanges resulting in amino acid exchanges compared to the reference sequence were corrected using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands). Finally, correctness and orientation of the cDNA were verified by sequencing (AGOWA).Generation of stably transfected cellsGeneration and characterization of MDCKII-Co, MDCKII-OATP1B1, MDCKII-OATP1B1-UGT1A1, MDCKII-OATP1B1-MRP2 and MDCKII-OATP1B1-UGT1A1-MRP2 cell lines have been described before (Cui et&al., ; K&nig et&al., ; Fehrenbach et&al., ; Fahrmayr et&al., ). For generation of the MDCKII-OATP1B1-CYP3A4-UGT1A1 and the MDCKII-OATP1B1-CYP3A4-MRP2 triple-transfected and the MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 quadruple-transfected cell lines, MDCKII-OATP1B1-UGT1A1, MDCKII-OATP1B1-MRP2 and MDCKII-OATP1B1-UGT1A1-MRP2 cells were transfected with the plasmid pVITRO1-blasti-mcs-CYP3A4 using the Effectene transfection reagent kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany) respectively. To obtain single colonies of all three transfectants, cells were treated additionally with blasticidin S (7&&g&ml&1) for several weeks. Colonies grown under selection were tested for their CYP3A4 mRNA expression using reverse transcriptase (RT)-PCR and LightCycler-based quantitative RT-PCR (Roche Diagnostics-Applied Science, Mannheim, Germany) as described before (Mandery et&al., ). Primer pairs used for quantification of CYP3A4 mRNA expression were oCYP3A4-RT.for (5&-GCA AGA AGA ACA AGG ACA ACA TAG A-3&) and oCYP3A4-RT.rev (5&-AGC AGA TCT CCT TAG GAA AAT TCA G-3&) resulting in a 275 bp-fragment. Cell clones exhibiting the highest CYP3A4 expression of all three transfectants as well as the remaining cell lines used in this study (MDCKII-Co, MDCKII-OATP1B1, MDCKII-OATP1B1-MRP2, MDCKII-OATP1B1-UGT1A1-MRP2) were finally screened for their SLCO1B1 mRNA (encoding OATP1B1), their CYP3A4 mRNA (encoding CYP3A4), their UGT1A1 mRNA (encoding UGT1A1) and their ABCC2 mRNA (encoding MRP2) expression for comparative analysis. Primers used for quantitative real-time PCR have been described before (Fahrmayr et&al., ). All expression values were calculated in relation to the expression of the housekeeping gene &-actin. Cell clones showing the highest CYP3A4 mRNA expression and a SLCO1B1, UGT1A1 and ABCC2 mRNA expression comparable to the respective expression in the control cell lines (MDCKII-OATP1B1, MDCKII-OATP1B1-MRP2 and MDCKII-OATP1B1-UGT1A1-MRP2) were selected for further experiments. The expression of the respective mRNAs and proteins in the quadruple-transfected MDCKII-OATP1B1-CYP3A1-UGT1A1-MRP2 cell line and in all control cell lines has been analysed by quantitative RT-PCR and immunoblot analyses at different time points during these experiments demonstrating a stable expression over time for all transfected proteins.Immunoblot analysisGeneration of total protein homogenates and immunoblot analysis were performed as described previously (Seithel et&al., ; Mandery et&al., ). Five micrograms of cell homogenates used for detection of all four proteins (OATP1B1, CYP3A4, UGT1A1 and MRP2) were diluted with Laemmli buffer (62&mM Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromphenol blue, and 0.4&mM dithiothreitol) and incubated for 5&min at 95&C, except for MRP2 samples (Cui et&al., ). Separation of total homogenates was conducted with 7.5 (for MRP2) and 10% (for OATP1B1, CYP3A4 and UGT1A1) SDS-polyacrylamide gels. An unstained protein ladder (Protein Ladder 10&250&kDa, New England BioLabs, Frankfurt am Main, Germany) was used to visualize the protein molecular weight ranges. After separation, proteins were transferred onto a nitrocellulose membrane (Protran, Whatman GmbH, Dassel, Germany) using a tank blotting system from Bio-Rad Laboratories (Munich, Germany). Membranes were then incubated with a purified rabbit polyclonal anti-human OATP1B1 antiserum [pESL; 1:500; (K&nig et&al., )], with a CYP3A4 purified MaxPab mouse polyclonal antibody (B01P; 1:1000; Abnova, Taipei, Taiwan), with a rabbit polyclonal anti-human UGT1A1 antibody (ab6; Abcam, Cambridge, UK) and with a rabbit polyclonal anti-human MRP2 antibody [EAG5; 1:5000; kindly provided by Professor Dr. Dietrich K DKFZ, Heidelberg, G (Keppler and Kartenbeck, ; Jedlitschky et&al., )]. Secondary antibodies were a horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire, UK) used at a 1:10&000 dilution (Seithel et&al., ) and horseradish peroxidase-conjugated goat anti-mouse Fab fragments (Dianova, Hamburg, Germany) at a 1:4000 dilution. Proteins were visualized using ECL Western blotting detection reagents (GE Healthcare UK Ltd) with the ChemiDoc XRS imaging system (Bio-Rad Laboratories). To detect &-actin, membranes were thereafter stripped and reincubated with a mouse monoclonal anti-human &-actin antibody (1:10&000; Sigma-Aldrich Chemie GmbH, Munich, Germany) and developed as described above. Different amounts of homogenate of the MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 quadruple-transfected cell line further served as positive controls and for semiquatitative analysis of immunoblots. Blots were analysed using the Quantity One Software (Bio-Rad Laboratories).Determination of microsomal enzyme activitiesBefore transfection with the plasmid pVITRO1-blasti-mcs-CYP3A4, the cell lines MDCKII-OATP1B1, MDCKII-OATP1B1-MRP2 and MDCKII-OATP1B1-UGT1A1-MRP2 (Fahrmayr et&al., ) were tested for their NADPH-cytochrome P450 reductase activity. Measurement of the NADPH-cytochrome P450 reductase activity was performed as described (Gomes et&al., ). Instead of cytochrome P450, the NADPH-cytochrome P450 reductase can also reduce cytochrome c, which can be measured photometrically at 550&nm. In brief, 150&&g of total cell homogenates were equilibrated for 3&min at 25&C in 300&mM sodium phosphate buffer (pH&7.7), 1&mM KCN and 40&&M cytochrome c. Reduction of cytochrome c was started by addition of 100&&M NADPH and reduced cytochrome c was determined photometrically at 550&nM using a Bio-Rad SmartSpec&Plus spectrophotometer (Bio-Rad Laboratories) for 3&min as described previously (Vermilion and Coon, ; Tamura et&al., ; Dudka et&al., ). Enzyme activity was calculated using the extinction coefficient (&) of 21&mM&1&cm&1 (Vermilion and Coon, ; Dudka et&al., ) and expressed as pmol of cytochrome c reduced per mg of total protein homogenates per min.The activity of cytochrome P450 3A4 in the cell lines expressing this enzyme as well as in the control cell lines was assessed by measuring the amount of formed &-hydroxymidazolam after addition of the parent compound to the cell lines. Experiments were performed as described previously (Glaeser et&al., ) with some minor modifications. In brief, 100&&g of total protein homogenates were incubated either with 3 or 250&&M midazolam in 50&mM potassium hydrogen phosphate buffer (pH&7.4), 30&mM magnesium chloride and 4.8&mM NADPH in a total volume of 250&&L. Samples were equilibrated for 2&min at 37&C and the reaction was started by addition of NADPH. After 5&min, the reaction was stopped by addition of 500&&L of acetonitrile and samples were immediately stored at &80&C. The concentration of formed &-hydroxymidazolam was determined by LC-MS/MS.Quantification of &-hydroxymidazolam by LC-MS/MSConcentrations of &-hydroxymidazolam from cell lysates were measured by means of HPLC-MS-MS [Agilent 1100 HPLC System (Agilent Technologies, Waldbronn, Germany); API 4000 (Applied Biosystems, Darmstadt, Germany)]. For sample preparation, 50&&L internal standard solution [d4-&-hydroxymidazolam (50&ng&ml&1 in acetonitrile)] was added to 50&&L sample. After mixing and centrifugation, 50&&L of the supernatant was diluted with 50&&L mobile phase [12&mM ammonium acetate with acetonitrile (1:1, v/v)]. Standards and quality controls in similar matrix were prepared identically to the samples. A ZORBAX Eclipse XDB C18 (150&mm & 4.6&mm, particle size 5&&m; Agilent Technologies) with a precolumn AQ C18 (4&mm & 3& Phenomenex Ltd, Aschaffenburg, Germany) was installed as separation column. Chromatography was carried out isocratically at a flow rate of 0.6&mL&min&1. A valco valve behind the separation column was applied for cutting of salt (0.0&4.5& 4.5&6.5& 6.5&7.0&min waste). The mass transitions and collision energies were m/z 342.1 to 324.3 (31&eV) for &-hydroxymidazolam and m/z 346.0 to 328.1 (31&eV) for d4-&-hydroxymidazolam. The validated calibration range was between 0.1 and 100&ng&mL&1 and the lower limit of quantification was 0.1&ng&mL&1. The linear regression was weighted by 1/x. Correlation coefficients were at least 0.999. Intraday coefficients of variation in cell lysate ranged from 2.0 to 6.1% and the intraday accuracies ranged from 3.9 to 15.7 both at concentrations of 0.1, 0.25, 50 and 100&ng&mL&1.Vectorial transport assaysVectorial transport assays were performed as described (Cui et&al., ; Fehrenbach et&al., ; Fahrmayr et&al., ) with minor modifications. Briefly, MDCKII cells were seeded at an initial density of 4 & 105 cells&well&1 onto ThinCerts (diameter 14& pore size 0.4&&m; Greiner Bio-One GmbH, Frickenhausen, Germany) and grown for 3 days. Twenty-four hours prior to the vectorial transport experiments, cells were treated with 10&mM sodium butyrate (Merck KGaA, Darmstadt, Germany) to increase protein expression (Cui et&al., ). Radiolabelled bosentan was dissolved in uptake buffer (142&mM NaCl, 5&mM KCl, 1&mM K2HPO4, 1.2&mM MgSO4, 1.5&mM CaCl2, 5&mM glucose and 12.5&mM HEPES, pH&7.3) to a final concentration of 20&&M (4.5&kBq&ml&1) without addition of unlabelled bosentan. After washing with prewarmed (37&C) uptake buffer, 800&&L of uptake buffer were added to the apical compartment and 800&&L of uptake buffer containing bosentan were added to the basolateral compartment and cells were incubated at 37&C for 180&min. After 60 and 120&min, aliquots (100&&L) were removed from the apical compartment and plates were placed back to the incubator. After 180&min, additional 100&&L were taken from the apical compartment and radioactivity was measured by liquid scintillation counting (TriCarb 2800; PerkinElmer Life Sciences GmbH, Rodgau-J&gesheim, Germany). Afterwards, the cells were washed three times with ice-cold uptake buffer, filters were detached from the chambers and cells were lysed with 0.2% SDS. Small aliquots of lysates were both used to determine the intracellular accumulation of radioactivity by liquid scintillation counting and the protein concentrations by bicinchoninic acid assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific Inc., Rockford, IL, USA).For determination of bosentan and formed metabolites and for identification of possibly new phase II metabolites in vectorial transport assays, one assay was conducted as described above, with the exception that cells were incubated for 180&min without taking aliquots after 60 and 120&min. Samples from the intracellular and the apical compartments were measured by means of HPLC and LC-MS at Actelion Pharmaceuticals Ltd (Allschwil, Switzerland).To investigate the transcellular leakage, cells were routinely treated likewise with 50&&M [3H]inulin.Furthermore, apparent permeability coefficients (Papp) were calculated using the equation:where dQ/dt (&mol&s&1) is the initial transport rate (at 60&min), C0 (&mol/cm3) the initial concentration in the donor chamber and A (cm2) the surface area of the monolayer (Artursson and Karlsson, ).Identification of bosentan and metabolites by HPLC/LC-MSHPLC methodThe analytical system consisted of two HPLC pumps LC-10AD VP equipped with a membrane degasser, a SCL-10AD VP system controller, a UV detector SPD-10AV VP and an autosampler model SIL-HTc (all from Shimadzu, Reinach, Switzerland). The chromatographic separation of bosentan and its metabolites was achieved using a Phenomenex Luna C18 column (250 & 4.6& particle size 5&&m; Phenomenex Ltd, Aschaffenburg, Germany) at 45&C with a flow rate of 1&mL&min&1. Mobile phases consisted of 50&mM ammonium formate adjusted to pH&4.0 with formic acid (phase A) and acetonitrile (phase B). The gradient method for the separation of bosentan and its metabolites was as follows: 0&10&min: 10% of phase B; 10&55&min: 25% of phase B; 55&60&min: 73% of phase B; 60&63&min: 95% of phase B; 63&64&min: 95% of phase B and 64&70&min: 10% of phase B. The run was stopped after 70&min. Due to the low levels of total radioactivity, post-column fractions were collected using an Agilent Technologies 1200 fraction collector (Agilent Technologies) in Deepwell LumaPlates&-96 (PerkinElmer) in intervals of 0.28&min, evaporated to dryness using an EZ-2 Evaporator (GeneVac Ltd, Ispwich, UK) and analysed offline using a TopCount-NXT& microplate luminescence counter (PerkinElmer). Data processing was performed using the RadioStar software package (version 4.6, Berthold AG, Regensdorf, Switzerland). Using these chromatographic conditions, bosentan had a retention time of 41.0&min. The variability in retention time in the different chromatograms did not exceed 1&min over a total run time of 70&min.Mass spectrometryStructure identification was performed using a LTQ Orbitrap Velo Pro mass spectrometer (ThermoFinnigan, San Jose, CA, USA) coupled to a LC-20ADXR HPLC pump (Shimadzu). The mass spectrometer was operated in positive ion mode with the following instrument settings: ESI voltage 3.0&kV, capillary temperature 300&C, source temperature 200&C, sheath gas 40&psi, auxiliary gas 5&psi, capillary voltage 13&V, S-lens RF level 45%, mass range 190&1000 and a mass resolution of 30&000. The chromatographic method was identical to that described for the HPLC method with a 1:5 flow rate split. Data processing was performed using the Xcalibur 2.0.7 software package (Thermo Electron, San Jose, CA, USA).Data and statistical analysisReal-time PCR and immunoblot analysis determining mRNA and protein expression were repeated three times. Experiments determining the CYP3A4 enzyme activity were repeated two times on separate days with a total of four samples per concentration and cell line, and experiments determining the NADPH-reductase activity were performed on two separate days with a total of four samples per cell line. Each time point in transcellular transport experiments was investigated on two separate days with three wells per day (i.e. n = 6). For measurement of bosentan and formed metabolites by means of HPLC and LC-MS one transcellular transport experiment was performed with three wells per cell line (i.e. n = 3). All data are presented as mean & SD. Multiple comparisons were analysed by anova with subsequent Tukey-Kramer multiple comparison test by using Prism 3.01 (GraphPad Software, San Diego, CA, USA). A value of P & 0.05 was considered as statistically significant.Materials[14C]Bosentan (37&MBq&ml&1), unlabelled bosentan and bosentan metabolites were from Actelion Pharmaceuticals Ltd internal sources and [3H]inulin (74&MBq&ml&1) was obtained from PerkinElmer Life Sciences GmbH (Rodgau-J&gesheim, Germany). Unlabelled inulin, poly-D-lysine hydrobromide and ammonium acetate were purchased from Sigma-Aldrich (Taufkirchen, Germany). Water-Baker analysed LC/MS reagent was from Mallinckrodt Baker B.V. (Deventer, The Netherlands). Liquid scintillation cocktail for HPLC analysis, Optiflow Safe 2, was purchased from Berthold Technologies GmbH. Sodium butyrate, cytochrome c and acetonitrile hypergrade for LC/MS were from Merck KGaA. The antibiotics zeocin, G418 (geniticin) disulfate and hygromycin were from Invitrogen (Groningen, the Netherlands) and the antibiotic blasticidin S was from InvivoGen. NADPH was obtained from AppliChem (Darmstadt, Germany) and midazolam was from Lipomed (Weil am Rhein, Germany). &-Hydroxymidazolam and the internal standard d4-&-hydroxymidazolam were purchased from LGC Standards (Wesel, Germany). All other chemicals and reagents, unless stated otherwise, were obtained from Carl Roth GmbH + Co.KG (Karlsruhe, Germany) and were of the highest grade available.Expression analysis of OATP1B1, CYP3A4, UGT1A1 and MRP2 in single-, double-, triple- and quadruple-transfected cell linesFigures& and
show the mRNA and protein expression of OATP1B1, CYP3A4, UGT1A1 and MRP2 in MDCKII-control cells (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected cells (OATP1B1-CYP3A4-UGT1A1-MRP2). As expected, only transfected cell lines showed a respective mRNA and protein expression.Figure&1. Quantitative real-time PCR (RT-PCR) analysis of SLCO1B1 (encoding OATP1B1), CYP3A4 (encoding CYP3A4), UGT1A1 (encoding UGT1A1) and ABCC2 (encoding MRP2) mRNA expression in MDCKII-control cells (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines used in this study. The RT-PCR analysis was repeated three times and data are presented as mean & SD in % &-actin mRNA expression. Statistical analyses were performed between the transfected cell lines among themselves. No expression of human SLCO1B1, CYP3A4, UGT1A1 or ABCC2 mRNA could be detected in MDCKII-control cells (Co). Multiple comparisons were analysed by ANOVA with subsequent Tukey&Kramer multiple comparison test. *P & 0.05, **P & 0.01, ***P & 0.001 versus MDCKII-OATP1B1-CYP3A4-UGT1A1 ##P & 0.01, ###P & 0.001 versus MDCKII-OATP1B1-CYP3A4-MRP2 cells.Figure&2. Immunoblot analyses of OATP1B1 (A), CYP3A4 (B), UGT1A1 (C) and MRP2 (D) expression in MDCKII-control cells (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines used in this study. A 5-&g protein homogenate of each cell line was loaded. OATP1B1 shows one glycosylated form with an apparent molecular weight of 84&kDa and one unglycosylated form (58&kDa), CYP3A4 shows one band with approximately 55&kDa, UGT1A1 one with approximately 60&kDa and MRP2 one with 190&kDa. As positive controls and for semiquantitative analysis, calibration samples (1, 2.5, 5, 7.5 and 10&&g protein) of the quadruple-transfected cell line (OATP1B1-CYP3A4-UGT1A1-MRP2) were also loaded.Determination of microsomal enzyme activitiesPrior to transfection with the plasmid pVITRO1-blasti-mcs-CYP3A4, cell lines were investigated for their NADPH-cytochrome P450 reductase activity, which is a prerequisite for CYP3A4 function. Net activity was between 11.1 and 14.5&nmol&mg protein&1&min&1 in the investigated cell lines with no significant differences.Cytochrome P450 3A4 activity was determined via formation of the metabolite &-hydroxymidazolam (Figure&) by LC-MS/MS after incubation of total homogenates of MDCKII-control cells, single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected cells (OATP1B1-CYP3A4-UGT1A1-MRP2) with 3&&M (A) or 250&&M (B) midazolam (Figure&). Only cell lines expressing the CYP3A4 enzyme showed formation of &-hydroxymidazolam, whereas no significant amounts were detectable in the other cell lines.Figure&3. Determination of CYP3A4 enzyme activity in homogenates of MDCKII-control (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines using midazolam assay. Total homogenates of cell lines were incubated with 3&&M midazolam (A) or 250&&M midazolam (B) at 37&C and the amount of the formed metabolite &-hydroxymidazolam was measured by LC-MS/MS. Statistical analyses were performed between the CYP3A4-transfected cell lines among themselves. Data are shown as mean value & SD. *P & 0.05, ***P & 0.001 versus MDCKII-OATP1B1-CYP3A4-UGT1A1; ##P & 0.01, ###P & 0.001 versus MDCKII-OATP1B1-CYP3A4-MRP2 cells.Intracellular accumulation and vectorial transport of bosentan equivalents to the apical compartment of monolayers of MDCKII-control, single-, double-, triple- and quadruple-transfected cells[14C]Bosentan was administered to the basal compartment of the cell monolayers of MDCKII-control (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected cells (OATP1B1-CYP3A4-UGT1A1-MRP2). Figures& and
show the intracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) and the transcellular transport of bosentan equivalents into the apical compartment respectively. Intracellular accumulation of bosentan equivalents was significantly lower in cell lines expressing MRP2 (Figure&; P & 0.001). Cell lines lacking MRP2 exhibited higher intracellular amounts of bosentan equivalents with the triple-transfected cell line MDCKII-OATP1B1-CYP3A4-UGT1A1 showing the highest amounts. In line with the lower intracellular values, higher amounts of bosentan equivalents were found in the apical compartment of monolayers of cell lines expressing MRP2 compared to cell lines without MRP2 expression at all investigated time points (Figure& A to C; P & 0.001). In MRP2-expressing cells apparent permeability (Papp) was more than 50% greater compared to Papp in MDCKII-Co cells (4.3 [cm&s&1]*10&6 in MDCKII-Co vs. 6.5, 7.5, 7.4 and 6.5 [cm&s&1]*10&6 in MDCKII-OATP1B1-MRP2, MDCKII-OATP1B1-CYP3A4-MRP2, MDCKII-OATP1B1-UGT1A1-MRP2 and MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 P & 0.001). Transcellular transport of bosentan equivalents to the apical compartment exhibited linear kinetics (Figure&).Figure&4. [14C]Bosentan (20&&M) was administered to the basal compartment of monolayers of MDCKII-control (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines. Intracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) in the cells after 180&min is shown. Data are shown as mean value & SD. ***P & 0.001 versus MDCKII-Co; ###P & 0.001 versus MDCKII-OATP1B1; &&&P & 0.001 versus MDCKII-OATP1B1-CYP3A4-UGT1A1 cells.Figure&5. [14C]Bosentan (20&&M) was administered to the basal compartment of monolayers of MDCKII-control (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines. Translocation of bosentan equivalents into the apical compartment after 60&min (A), 120&min (B) and 180&min (C) is shown. Data are shown as mean value & SD. ***P & 0.001 versus MDCKII-Co; ###P & 0.001 versus MDCKII-OATP1B1; +P & 0.05, +++P & 0.001 versus MDCKII-OATP1B1-MRP2; &&&P & 0.001 versus MDCKII-OATP1B1-CYP3A4-UGT1A1 cells.Figure&6. [14C]Bosentan (20&&M) was administered to the basal compartment of monolayers of MDCKII-control (Co), single- (OATP1B1), double- (OATP1B1-MRP2), triple- (OATP1B1-CYP3A4-UGT1A1, OATP1B1-CYP3A4-MRP2, OATP1B1-UGT1A1-MRP2) and quadruple-transfected (OATP1B1-CYP3A4-UGT1A1-MRP2) cell lines. Translocation of bosentan equivalents into the apical compartment after 60, 120 and 180&min is shown as percentage of administered radioactivity. All cell lines show linear transport kinetics.Qualitative measurements of samples from transcellular transport experiments by means of HPLC and LC-MS identified the phase I metabolites Ro 48&5033 and Ro 47&8634 in the intracellular compartments of cell lines expressing the phase I enzyme CYP3A4 (Table&). The majority of radioactivity accumulating in the intracellular compartments consisted of bosentan itself. In addition to the phase I metabolites Ro 48&5033 and Ro 47&8634, bosentan glucuronide could be identified in the intracellular compartments of all cell lines expressing the phase II enzyme UGT1A1 (Table&). Glucuronides of the phase I metabolites were not detected. Results of these measurements are summarized in Table&. HPLC- and LC-MS-measurements revealed that mainly bosentan was translocated into the apical compartments of investigated cell lines confirming that bosentan itself is a substrate of MRP2. Furthermore, small amounts of bosentan glucuronide were detected in the apical compartments of cell lines expressing UGT1A1 and MRP2 indicating that also bosentan glucuronide is a substrate of MRP2.Table&1.&Qualitative determination of bosentan and metabolites in the intracellular compartments of the investigated cell lines after transcellular transport experiments using [14C]bosentanIdentified compoundsCell linesCoOATP1B1OATP1B1-MRP2OATP1B1-CYP3A4-UGT1A1OATP1B1-CYP3A4-MRP2OATP1B1-UGT1A1-MRP2OATP1B1-CYP3A4-UGT1A1-MRP2
Bosentan+++++++
Ro 48&5033&&&++&+
Ro 47&8634&&&++&+
Ro 64&1056n.d.n.d.n.d.n.d.n.d.n.d.n.d.
Bosentan glucuronide&&&+&++This study using a newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line had the following major findings: (i) transporter-mediated uptake, phase I and II metabolism and efflux transport can be investigated using this new cellular system. (ii) UGT1A1 is involved in formation of bosentan glucuronide. (iii) MRP2 mediates secretion of bosentan and most likely also of bosentan glucuronide.In order to gain more insights into the hepatobiliary transport of drugs, their directed transport through monolayers of cell lines stably expressing uptake as well as efflux transporters using in vitro cell models has intensively been studied (Cui et&al., ; Kopplow et&al., ; Ishiguro et&al., ; Nies et&al., ; Hirouchi et&al., ; K&nig et&al., ). However, most drugs undergo phase I and/or phase II metabolism so that the coordinate process of drug transport and metabolism cannot be investigated using models expressing only transport proteins. Previously, experiments using an OATP1B1-UGT1A1-MRP2 triple-transfected cell line demonstrated that cell lines stably simultaneously expressing transport proteins and metabolizing enzymes provide useful tools to study the interplay of drug transport and drug metabolism (Fahrmayr et&al., ).Based on the OATP1B1-UGT1A1-MRP2 triple-transfected cell line, we established and characterized a cell line with the additional expression of the phase I enzyme CYP3A4. For the characterization of this cell line, we used the dual endothelin receptor antagonist bosentan. In vitro and in vivo studies clearly revealed that the hepatic uptake transporters OATP1B1 and OATP1B3 as well as the cytochrome P450 enzymes CYP3A4 and CYP2C9 are important determinants of the disposition of bosentan (Treiber et&al., ; van Giersbergen et&al., ). In the present study, however, we could not detect a significant difference in the amount of radioactivity accumulating in the intracellular compartments of the OATP1B1 single-transfected cell line in comparison to the control cell line. This could be due to the much longer incubation time in this study compared to uptake experiments leading to an increased contribution of additional transport processes (e.g. basolateral efflux).Transcellular transport experiments using radiolabelled bosentan and subsequent liquid scintillation counting of intracellular samples revealed lower amounts of bosentan equivalents (i.e. bosentan and possible metabolites) in cell lines expressing MRP2. Accordingly, significantly higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with an expression of MRP2 which demonstrated a contribution of MRP2 to the export of bosentan. To investigate this hypothesis, a further transcellular transport experiment was conducted with the subsequent measurement of samples by means of HPLC and LC-MS. In fact, these data revealed that mainly bosentan was transported into the apical compartments of the tested cell lines. Moreover, bosentan glucuronide was detected in the apical compartments of cell lines expressing UGT1A1 and MRP2 indicating a contribution of MRP2 to the translocation of the metabolite. It should be noted that significant amounts of bosentan equivalents were also detectable in MDCKII-control cells indicating that endogenous canine efflux transporters (e.g. canine Mrp2) also contribute to apical accumulation.Several studies investigated the interaction of bosentan with transporters expressed in the canalicular membrane. However, none of them directly identified the transporter mediating biliary excretion of bosentan (Weber et&al., ; Fattinger et&al., ; Fouassier et&al., ; Treiber et&al., ; Hartman et&al., ). In vitro studies conducted with primary human hepatocytes showed a decrease in the biliary elimination of the MRP2 substrate [2-D-Penicillamin, 5-D-penicillamine] enkephalin during simultaneous incubation with bosentan only in one of three donors (Hartman et&al., ). Furthermore, co-incubation experiments in primary human hepatocytes with bosentan and probenecid, a known inhibitor for MRP2, showed a reduced uptake and an increased elimination of bosentan (Hartman et&al., ). Moreover, Mano et&al. () reported that bosentan stimulated MRP2-mediated ATP-dependent vesicular transport of [3H]estradiol. Thus, our study is the first one identifying unconjugated bosentan as substrate of MRP2. Although MRP2 is known as transporter of glutathione and glucuronide conjugates, MRP2-mediated, glutathione-dependent transport of the unconjugated anticancer agents etoposide, vincristine and vinblastine has been shown (Cui et&al., ; van Aubel et&al., ; Evers et&al., ) suggesting a cotransport of these agents with glutathione (Jedlitschky et&al., ). Furthermore, a cotransport of drugs or endogenous substances with glutathione by other MRP transporters (MRP1 and MRP4) has also been shown (Rappa et&al., ; Cole and Deeley, ; Loe et&al., ; Rius et&al., ). Pilot experiments indicate that there is a trend towards higher total glutathione concentrations in the apical compartments of MDCKII-OATP1B1-MRP2 monolayers compared to the concentrations in the apical compartments of MDCKII-control cells after addition of bosentan (20&&M) to the respective basal compartments (data not shown).The metabolism of bosentan by the cytochrome P450 enzymes CYP3A4 and CYP2C9 to three phase I metabolites (Ro 48&5033, Ro 47&8634 and Ro 64&1056) has been elucidated (van Giersbergen et&al., ; Treiber et&al., ). However, the glucuronidation of bosentan and its phase I metabolites by uridine diphosphate-glucuronosyltransferases has not been clarified in detail yet (Shen et&al., ). Transcellular transport experiments with labelled bosentan and measurement of samples by HPLC and LC-MS revealed the phase I metabolites Ro 48&5033 and Ro 47&8634 in the intracellular compartments of cell lines expressing the enzyme CYP3A4. However, the majority of radioactivity accumulating in the intracellular compartment of all cell lines consisted of bosentan itself. This could be due to the fact that bosentan is a rather poor substrate of CYP3A4 than the model substrate midazolam used to test the functionality of the enzyme in the cell lines. Shen et&al. () characterized the metabolism of bosentan to the phase I metabolites Ro 48&5033 and Ro 47&8634 in human liver microsomes and determined respective Km values of 65&&M (Ro 48&5033) and 73&&M (Ro 47&8634). In contrast, Km values for the metabolism of midazolam to &-hydroxymidazolam are 3.9&&M in human liver microsomes and 0.8&&M for recombinant CYP3A4 (Patki et&al., ).In addition to the phase I metabolites, bosentan glucuronide could be detected in cells with an expression of UGT1A1. Thus, a further metabolic reaction in the metabolic pathways of bosentan (i.e. direct glucuronidation of bosentan by UGT1A1) could be identified. A schematic representation of the extended metabolic pathway of bosentan is shown in Figure&. Glucuronides of the phase I metabolites, although possible on the basis of their chemical structure, could not be detected in the current study. This could be due to the fact that intracellular amounts of phase I metabolites were quite low, thus rendering the quantification of glucuronides of these metabolites was difficult. In accordance with our findings, Shen et&al. () mentioned in their abstract minor amounts of conjugates derived from glucuronidation of bosentan itself and of phase I metabolites using human hepatocytes. However, it should be noted that so far, no phase II metabolites of bosentan and its phase I metabolites have been detected in humans.Figure&7. Metabolic pathways of bosentan.Currently, we see the primary benefit of the recently generated triple-transfected cell line [expressing OATP1B1, UGT1A1 and MRP2; (Fahrmayr et&al., )] and the quadruple-transfected cell line described here in the qualitative analysis of the interplay of uptake, metabolism and efflux. For example, phase I or phase II metabolites formed in these cell lines can be studied as MRP2 substrates, whereas this might not be possible using inside-out vesicles of recombinant cells expressing MRP2 due to unavailability of the respective pure compounds. A limitation of these cell lines is that they express with some variability transporters and drug-metabolizing enzymes of particular importance for drug disposition, but certainly not all hepatic proteins affecting hepatic drug handling. Thus, quantitative in vitro-in vivo extrapolations should be investigated using other systems such as sandwich-cultured hepatocytes albeit this system has also certain limitations (for review see, e.g. Swift et&al., ).Taken together, we could demonstrate that it is technically possible to establish a recombinant cell system with the expression of all components necessary for hepatobiliary elimination of drugs. Furthermore, the present study shows that these newly established cell models in addition to primary hepatocytes, animal models and clinical studies represent useful tools to gain more insights into the hepatic disposition of drugs.This work was supported by the DOKTOR ROBERT PFLEGER-STIFTUNG B and in part by the Deutsche Forschungsgemeinschaft (Fr ).Treiber, Pfeifer and Segrestaa are employees of Actelion Pharmaceuticals Ltd (Allschwil, Switzerland).
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