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1.
Bioorg Med Chem ; 27(4): 579-588, 2019 02 15.
Article in English | MEDLINE | ID: mdl-30626555

ABSTRACT

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.


Subject(s)
Antioxidants/pharmacology , Cyclobutanes/pharmacology , Drug Discovery , Isothiocyanates/pharmacology , NF-E2-Related Factor 2/metabolism , Signal Transduction/drug effects , Animals , Antioxidants/chemical synthesis , Antioxidants/pharmacokinetics , Cell Line , Cyclobutanes/chemical synthesis , Cyclobutanes/pharmacokinetics , Gene Expression , Heme Oxygenase-1/genetics , Humans , Isothiocyanates/chemical synthesis , Isothiocyanates/pharmacokinetics , Kelch-Like ECH-Associated Protein 1/metabolism , Mice, Inbred C57BL , Molecular Structure , Oxidative Stress/drug effects , Rats , Solubility , Structure-Activity Relationship , Sulfoxides , Thiocarbamates/chemical synthesis , Thiocarbamates/pharmacokinetics , Thiocarbamates/pharmacology
2.
J Pharm Biomed Anal ; 154: 150-157, 2018 May 30.
Article in English | MEDLINE | ID: mdl-29544106

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 Adult
3.
Analyst ; 139(8): 1902-12, 2014 Apr 21.
Article in English | MEDLINE | ID: mdl-24563904

ABSTRACT

Activation of the Nrf2 stress pathway is known to play an important role in the defense mechanism against electrophilic and oxidative damage to biological macromolecules (DNA, lipids, and proteins). Chemical inducers of Nrf2 such as sulforaphane, dimethyl fumarate (Tecfidera®), CDDO-Me (bardoxolone-methyl), and 3-(dimethylamino)-4-((3-isothiocyanatopropyl)(methyl)amino)cyclobut-3-ene-1,2-dione (a synthetic sulforaphane analogue; will be referred to as ) have the ability to react with Keap1 cysteine residues, leading to activation of the Antioxidant Response Element (ARE). Due to their electrophilic nature and poor matrix stability, these compounds represent great challenges when developing bioanalytical methods to evaluate in vivo exposure. like SFN reacts rapidly with glutathione (GSH) and nucleophilic groups in proteins to form covalent adducts. In this work, three procedures were developed to estimate the exposure of in a non-GLP 7 day safety study in rats: (1) protein precipitation of blood samples with methanol containing the free thiol trapping reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to measure GSH- and N-acetylcysteine conjugated metabolites of ; (2) an Edman degradation procedure to cleave and analyze N-terminal adducts of at the valine moiety; and (3) treatment with ammonium hydroxide to measure circulating free- and all sulfhydryl bound .


Subject(s)
NF-E2-Related Factor 2/metabolism , Toxicity Tests , Animals , Area Under Curve , Chromatography, Liquid , Male , Rats , Reference Standards , Tandem Mass Spectrometry
4.
Prostaglandins Other Lipid Mediat ; 104-105: 25-31, 2013.
Article in English | MEDLINE | ID: mdl-23434473

ABSTRACT

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 effects
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(25): 2487-93, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21798825

ABSTRACT

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography-mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid-liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05-50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.


Subject(s)
8,11,14-Eicosatrienoic Acid/analogs & derivatives , Epoxide Hydrolases/metabolism , Linoleic Acids/blood , Stearic Acids/blood , 8,11,14-Eicosatrienoic Acid/blood , Biomarkers/blood , Chromatography, Liquid , Epoxide Hydrolases/blood , Female , Humans , Male , Reproducibility of Results , Sensitivity and Specificity , Tandem Mass Spectrometry
6.
J Pharm Sci ; 100(8): 3055-3061, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21484807

ABSTRACT

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/chemistry
7.
Drug Metab Dispos ; 38(7): 1015-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20382755

ABSTRACT

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 Specificity
8.
Drug Metab Dispos ; 38(5): 863-70, 2010 May.
Article in English | MEDLINE | ID: mdl-20133892

ABSTRACT

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/pharmacology
9.
Mini Rev Med Chem ; 8(3): 193-200, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18336339

ABSTRACT

P-glycoprotein (Pgp, ABCB1) is an efflux transporter for a variety of amphipathic agents that can affect the pharmacokinetics of drugs. In order to extrapolate transport and pharmacokinetic data of the drug candidates obtained from in vitro and animal models to those in humans, it is important to understand the functional differences of Pgps from various mammalian species including human, monkey, dog, rat, and mouse. Here, we review differences/similarities in the properties of Pgp from numerous mammalian species commonly used in preclinical studies and discuss their relevance to the pharmacokinetics of potential drug molecules.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Pharmaceutical Preparations/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/classification , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Animals , Antibodies/immunology , Drug Resistance/drug effects , Humans , Pharmaceutical Preparations/chemistry , Phylogeny , Structure-Activity Relationship
10.
Biochem Pharmacol ; 73(6): 880-90, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17223083

ABSTRACT

The catalytic efficiency, regioselectivity, and response to chemical inhibitors of diclofenac (DF) hydroxylation in three Old World monkey liver microsomes (rhesus, cynomolgus, and African green monkey) are different from those determined with human liver microsomes. In contrast to the high affinity-high capacity (low Km-high Vmax) characteristics of DF 4'-hydroxylation in humans, this reaction proceeded in all monkey species with catalytic efficiencies >20-fold lower. However, DF 5-hydroxylation, a negligible reaction in human liver microsomes, was kinetically favored in monkeys mainly due to the increased Vmax values. Chemical inhibitors (reversible or mechanism-based) selective to human CYP3A4 and CYP2C9 failed to differentiate monkey orthologs involved in DF hydroxylation. Immunoinhibition studies with monoclonal antibodies against human CYPs revealed the major contribution of CYP2C and CYP3A to 4'- and to 5-hydroxylation, respectively, in rhesus and cynomolgus liver microsomes. However, in African green monkeys, in addition to CYP2C, CYP3A also appeared to be involved in 4'-hydroxylation. Further studies with recombinant rhesus and African green monkey CYP2C and CYP3A enzymes (rhesus CYP2C75, 2C74, and 3A64; African green monkey CYP2C9agm and CYP3A4agm) confirmed the major role of CYP enzymes of these two subfamilies in DF 4'- and 5-hydroxylation. Clearly, while monkey CYP2C and 3A enzymes retain the same substrate selectivity towards DF hydroxylation as their human orthologs, their altered catalytic efficiency and response to chemical inhibitors may indicate different structural features of active sites as opposed to human orthologs.


Subject(s)
Diclofenac/metabolism , Enzyme Inhibitors/pharmacology , Microsomes, Liver/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Aryl Hydrocarbon Hydroxylases/physiology , Catalysis , Chlorocebus aethiops , Cytochrome P-450 CYP2C9 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/physiology , Female , Humans , Hydroxylation , Kinetics , Macaca fascicularis , Macaca mulatta , Male
11.
Drug Metab Dispos ; 34(10): 1703-12, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16815964

ABSTRACT

The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 (Komori et al., 1992). The substrate specificity of CYP3A64 for testosterone (0-250 microM), midazolam (0-200 microM), nifedipine (0-200 microM), and 7-benzoxy-4-trifluoromethylcoumarin (0-200 microM) were compared with recombinant enzymes from rat (CYP3A1, CYP3A2), dog (CYP3A12, CYP3A26), rabbit (CYP3A6), and human (CYP3A4, CYP3A5). Immunoinhibition and chemical inhibition of CYP3A64 was demonstrated using the inhibitory monoclonal antibody (MAb) 10-1-1 (anti-3A4) and ketoconazole (0-10 microM). The utility of CYP3A64 to be used as a standard in monkey induction assays was shown and the concentration of CYP3A64 protein in rhesus liver microsomes was estimated to be 72 pmol/mg protein. In summary, these results support the utilization of rhesus monkey CYP3A64 for in vitro drug metabolism studies and provide a more complete understanding of CYP3A substrate specificities and species differences in metabolic capabilities.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Macaca mulatta/genetics , Recombinant Proteins/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Base Sequence , Blotting, Western , Cell Line , Cloning, Molecular , Coumarins/metabolism , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Humans , Ketoconazole/metabolism , Kinetics , Liver/enzymology , Liver/metabolism , Macaca mulatta/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Midazolam/metabolism , Molecular Sequence Data , Nifedipine/metabolism , Recombinant Proteins/genetics , Sequence Analysis, DNA , Spodoptera , Substrate Specificity , Testosterone/metabolism
12.
Nat Rev Drug Discov ; 5(3): 219-34, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16518375

ABSTRACT

Effective treatment of metastatic cancers usually requires the use of toxic chemotherapy. In most cases, multiple drugs are used, as resistance to single agents occurs almost universally. For this reason, elucidation of mechanisms that confer simultaneous resistance to different drugs with different targets and chemical structures - multidrug resistance - has been a major goal of cancer biologists during the past 35 years. Here, we review the most common of these mechanisms, one that relies on drug efflux from cancer cells mediated by ATP-binding cassette (ABC) transporters. We describe various approaches to combating multidrug-resistant cancer, including the development of drugs that engage, evade or exploit efflux by ABC transporters.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Neoplasms/metabolism , Animals , Humans , Neoplasms/drug therapy
13.
J Pharmacol Toxicol Methods ; 54(1): 78-89, 2006.
Article in English | MEDLINE | ID: mdl-16545584

ABSTRACT

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 Specificity
14.
Drug Metab Dispos ; 32(11): 1260-4, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15483193

ABSTRACT

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.


Subject(s)
Ethinyl Estradiol/metabolism , Pyrazoles/pharmacology , Sulfonamides/pharmacology , Sulfotransferases/metabolism , Celecoxib , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Humans , Sulfates/metabolism
15.
J Pharmacol Exp Ther ; 311(1): 307-14, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15163679

ABSTRACT

Stimulation by quinidine of warfarin metabolism in vitro was first demonstrated with liver microsomal preparations. We report herein that this drug interaction is reproducible in an animal model but that it exhibits profound species differences. Thus, using rabbit liver microsomes and a kinetic model incorporating two binding sites, the hepatic intrinsic clearance of R-warfarin via the 10-hydroxylation pathway (CL(int)(W)) was projected to be 6 +/- 1 and 128 +/- 51 microl/min/g liver, respectively, in the absence and presence of 21 microM unbound quinidine. These estimates were consistent with the results from studies in which rabbit livers (n = 5) were perfused in situ with R-warfarin or R-warfarin plus quinidine. The CL(int)(W) increased from 7 +/- 3 to 156 +/- 106 microl/min/g liver after increasing the hepatic exposure of unbound quinidine from 0 to 21 microM. In contrast, when liver microsomes or intact livers from rats were examined, R-warfarin metabolism was inhibited by quinidine, the CL(int)(W) decreasing to 26% of the control value after exposure of perfused rat livers (n = 5) to 22 microM unbound quinidine. The third example involved monkey liver microsomes, in which the rate of 10-hydroxylation of R-warfarin was little affected in the presence of quinidine (<2-fold increase). In all three species, the 10-hydroxylation of R-warfarin was catalyzed primarily by members of CYP3A, based on immuno- and chemical inhibition analyses. These findings not only highlight the variability of drug interactions among different species but also suggest that changes in hepatic clearance resulting from stimulation of cytochrome P450 activity may be projected based on estimates generated from corresponding liver microsomal preparations.


Subject(s)
Microsomes, Liver/metabolism , Quinidine/pharmacology , Warfarin/metabolism , Animals , Drug Interactions , Hydroxylation/drug effects , Metabolic Clearance Rate , Muscarinic Antagonists/pharmacology , Perfusion , Rabbits , Rats , Species Specificity
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