ABSTRACT
Information is needed on the expression of transporters in lung to inform drug development and therapeutic decisions. Much of the information currently available is from semiquantitative gene expression or immunometric densitometry studies reported in the literature. NanoLC-MS/MS (MRM mode) isotope dilution targeted quantitative proteomics was used here to quantify twelve selected transporters in fresh human lung membrane fraction samples and in the membrane fraction of selected immortalized human lung epithelial cell line samples. Fractionation was undertaken by homogenization in crude membrane lysis buffer followed by differential centrifugation of the homogenate. In lung membranes we found OATPs to be the most highly expressed transporters of those measured, followed by PEPT2 and ABCs (P-gp & BCRP). SLC22A transporters (OCTs 2 & 3 and OCTN1) were also found to be expressed. OATP2A1, also known as the prostaglandin transporter, was the most highly expressed transporter, being low in two subjects who were at least occasional smokers. One subject, a non-smoker, had an OATP2A1 concentration that was 8.4 times higher than the next nearest concentration, which itself was higher than the concentration of any other transporter. OATP2A1 is known, from gene expression and animal functional studies, to be present in lung. These results inform the understanding of xenobiotic disposition in the lung and show the distinct profile of transporters in lung compared to other tissues.
Subject(s)
Lung/metabolism , Membrane Transport Proteins/analysis , Proteomics/methods , Tandem Mass Spectrometry/methods , Adolescent , Adult , Animals , Biological Transport , Cell Line, Tumor , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Dogs , Female , Humans , Indicator Dilution Techniques , Isotopes/chemistry , Madin Darby Canine Kidney Cells , Male , Membrane Transport Proteins/metabolism , Tandem Mass Spectrometry/instrumentation , Xenobiotics/metabolism , Young AdultABSTRACT
Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclohexylamines/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Leukocytes/drug effects , Lung/drug effects , Triazines/pharmacology , 8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/metabolism , Administration, Oral , Adult , Animals , Chemokine CXCL1/biosynthesis , Dose-Response Relationship, Drug , Epoxide Hydrolases/metabolism , Exotoxins/metabolism , Female , Humans , Inflammation/enzymology , Inflammation/etiology , Inflammation/pathology , Inflammation/prevention & control , Leukocyte Count , Leukocytes/metabolism , Leukocytes/pathology , Lung/enzymology , Lung/pathology , Male , Mice , Mice, Knockout , Middle Aged , Oxidative Stress/drug effects , Rats , Stearic Acids/metabolism , Tobacco Smoke Pollution/adverse effectsABSTRACT
P-glycoprotein (P-gp)-based drug interactions are a major concern in the clinic and in preclinical drug development, especially with respect to the intestinal absorption of drugs and distribution of drugs across the blood-brain barrier. Thus, there is significant interest in developing in vitro (e.g., cell culture) and in vivo models (e.g., rodents) to predict such interactions. In order to generate accurate predictions from these models, however, an understanding of the magnitude of substrate- and species-dependent differences in P-gp inhibition is required. We have used a sensitive flow cytometry assay to measure the ability of various drugs to inhibit the initial rate of accumulation of two fluorescent drug analogs (probe substrates), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene (BODIPY)-verapamil and BODIPY-prazosin, into Lewis lung carcinoma-porcine kidney 1 (LLC-PK1) cells expressing human or rat P-gp. The inhibition of P-gp-mediated efflux of these two fluorescent substrates by several drugs, including quinidine and itraconazole, was found to be substrate- and/or species-dependent. These data suggest that to provide accurate prediction of clinically significant P-gp drug interactions, multiple P-gp substrates will need to be used in both in vitro and in vivo (including human) drug interaction studies. In addition, extrapolation of P-gp-based drug interaction in rodents to humans must be conducted with caution.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , Prazosin/pharmacokinetics , Verapamil/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Biological Transport , Boron Compounds/chemistry , Boron Compounds/pharmacokinetics , Drug Evaluation, Preclinical , Drug Interactions , Flow Cytometry , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Humans , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Prazosin/chemistry , Rats , Species Specificity , Substrate Specificity , Swine , Verapamil/chemistryABSTRACT
Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPH-cytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K(m) values of 4.8 and 2.1 microM, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 microM, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K(m) value of 150 microM, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K(m) value of 3.9 microM, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic , Animals , Baculoviridae/metabolism , Cells, Cultured , Coumarins/metabolism , Dogs , Microsomes, Liver/enzymology , NADPH-Ferrihemoprotein Reductase/biosynthesis , Phenacetin/metabolism , Spodoptera/genetics , Spodoptera/virology , Substrate SpecificityABSTRACT
The role of human UDP glucuronosyltransferase (UGT) 2B10 in the N-glucuronidation of a number of tricyclic antidepressants was investigated and compared with that of UGT1A4 in both the Sf9 expressed system and human liver microsomes. The apparent K(m) (S(50)) values for the formation of quaternary N-glucuronides of amitriptyline, imipramine, clomipramine, and trimipramine were 2.60, 16.8, 14.4, and 11.2 microM in UGT2B10 and 448, 262, 112, and 258 microM in UGT1A4, respectively. The kinetics of amitriptyline and imipramine glucuronidation in human liver microsomes exhibited a biphasic character, where the high- and low-affinity components were in good agreement with our results in expressed UGT2B10 and UGT1A4, respectively. The kinetics of clomipramine and trimipramine glucuronidation in human liver microsomes were sigmoidal in nature, and the S(50) values were similar to those found for expressed UGT1A4. The in vitro clearances (CL(int) or CL(max)) were comparable between UGT2B10 and UGT1A4 for glucuronidation of imipramine, clomipramine, and trimipramine, whereas CL(int) of amitriptyline glucuronidation by UGT2B10 was more than 10-fold higher than that by UGT1A4. Nicotine was found to selectively inhibit UGT2B10 but not UGT1A4 activity. At a low tricyclic antidepressant concentration, nicotine inhibited their glucuronidation by 33 to 50% in human liver microsomes. Our results suggest that human UGT2B10 is a high-affinity enzyme for tricyclic antidepressant glucuronidation and is likely to be a major UGT isoform responsible for the glucuronidation of these drugs at therapeutic concentrations in vivo.
Subject(s)
Amitriptyline/metabolism , Antidepressive Agents, Tricyclic/metabolism , Clomipramine/metabolism , Glucuronic Acid/metabolism , Glucuronosyltransferase/metabolism , Imipramine/metabolism , Trimipramine/metabolism , Biocatalysis/drug effects , Enzyme Inhibitors/pharmacology , Glucuronides/metabolism , Glucuronosyltransferase/antagonists & inhibitors , Glucuronosyltransferase/genetics , Humans , Kinetics , Microsomes, Liver/enzymology , Nicotine/pharmacology , Recombinant Proteins/metabolism , Sapogenins/pharmacologyABSTRACT
INTRODUCTION: P-glycoprotein is localized in numerous tissues throughout the body and plays an important role in the disposition of many xenobiotics. The contribution of P-glycoprotein-mediated drug transport is being evaluated in early drug discovery stages, particularly for compounds targeted to the central nervous system, using in vitro tools including cell lines expressing P-glycoprotein. Previous work in our laboratory suggests there are species differences in P-glycoprotein transport activity between humans and animals. The rat Abcb1a form of P-glycoprotein (formerly known as Mdr1a), the predominate isoform in the brain, has not been described in a functional cell system. Here, we describe the development and characterization of LLC-PK1 cells expressing rat Abcb1. METHODS: We cloned rat Abcb1a and generated a stable LLC-PK1 cell line. Expression and function of the cells were evaluated by immunoblot analysis, cytotoxicity analysis, cellular accumulation assays, and transcellular transport of probe substrates. The transport ratios of structurally diverse compounds obtained from parental cells or cells stably transfected with human ABCB1, mouse Abcb1a or rat Abcb1a were compared. RESULTS: Two forms of rat Abcb1a were cloned from Sprague-Dawley cDNA that differ by six amino acids and a base pair deletion. The intact form was stably transfected in LLC-PK1 cells. Immunoblot analysis demonstrated expression of the protein. The cells demonstrated P-glycoprotein-mediated function by directional transport of dexamethasone, ritonavir, and vinblastine in a transwell assay that was inhibited in the presence of cyclosporin A, verapamil, or quinidine. Likewise, the cells showed reduced cellular accumulation of Rh123 by FACS analysis that was reversed in the presence of cyclosporin A. These cells showed >or=350-fold resistance to colchicine, doxorubicin, vinblastine, and taxol and were sensitized in the presence of verapamil or cyclosporin A. Of 179 chemically diverse compounds evaluated, approximately 20% of the compounds evaluated were predicted to be substrates in one species but not in other species. DISCUSSION: Taken together, these data suggest these cells will be useful for evaluation of rat Abcb1a-mediated transport and for evaluation of species-specific P-glycoprotein-mediated transport.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , LLC-PK1 Cells/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Humans , Mice , Molecular Sequence Data , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Species SpecificityABSTRACT
The major sulfated product of 17alpha-ethynylestradiol (EE) after incubations with 3'-phosphoadenosine-5'-phosphosulfate and recombinant human sulfotransferase 2A1 (SULT2A1), or liver cytosol, is the 3-O-sulfate of EE. However, when celecoxib is also present in the incubation, sulfation is switched (in a concentration-dependent manner) from the 3-O-position to the 17beta-O-position of ethynylestradiol. In incubations with recombinant SULT2A1, increasing concentrations of celecoxib decreased the Vmax of 3-O-sulfate product formation by 3- to 4-fold, with no major change in the Km value. For 17beta-O-sulfate formation, increasing concentrations of celecoxib resulted in an 8-fold decrease in the Km and a 7-fold increase in Vmax. Celecoxib not only modulated the regioselectivity of the enzyme, but also activated the enzyme such that total sulfated product exceeded product formation by the native enzyme, 3- to 4-fold (at 250 microM celecoxib). Finally, IC50 values obtained by varying celecoxib concentrations (0-250 microM) at fixed concentrations of EE showed that 3-O-sulfation was inhibited by celecoxib to the same extent, independent of the concentration of EE. In addition, the apparent kinetic constant for celecoxib (as measured by EE 17beta-O-sulfation) decreased 2-fold in the presence of high concentrations of EE, consistent with the potential for celecoxib to bind to either the enzyme-EE complex or to free enzyme. Taken as a whole, these data suggest that celecoxib is acting as a heterotropic modulator of SULT2A1 activity, most likely involving a separate noncompetitive binding site.