Identification and Characterization of Trimethylamine-N-oxide Uptake and Efflux Transporters.

Trimethylamine-N-oxide (TMAO) is a recently identified predictor of cardiovascular and chronic kidney disease. TMAO is primarily generated through gut-microbiome mediated conversion of dietary choline and carnitine to TMA, which is converted to TMAO by hepatic flavin monooxygenase 3 (FMO3) and subsequently undergoes renal elimination. We investigated the role of uptake and efflux drug transporters in TMAO disposition in vitro and in vivo.

Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance.

The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters.

Contribution of hepatic organic anion-transporting polypeptides to docetaxel uptake and clearance.

The antimicrotubular agent docetaxel is a widely used chemotherapeutic drug for the treatment of multiple solid tumors and is predominantly dependent on hepatic disposition. In this study, we evaluated drug uptake transporters capable of transporting radiolabeled docetaxel. By screening an array of drug uptake transporters in HeLa cells using a recombinant vaccinia-based method, five organic anion-transporting polypeptides (OATP) capable of docetaxel uptake were identified: OATP1A2, OATP1B1, OATP1B3, OATP1C1, and Oatp1b2.

An allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1.

The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac potassium channel KCNQ1.

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E.

Functional characterization of genetic variants in the apical sodium-dependent bile acid transporter (ASBT; SLC10A2).

Background And Aim: The major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity.

Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity.

Objective: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance.

Methods: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis.

Modulation of drug block of the cardiac potassium channel KCNA5 by the drug transporters OCTN1 and MDR1.

Background And Purpose: A common site for drug binding on voltage-gated ion channels is at the interior face of the channel pore. In this study, we tested the hypothesis that the extent of drug block of the human cardiac KCNA5 (K(v) 1.5) channel underlying the atrial-specific, ultra-rapidly activating, delayed K(+) current (I(Kur) ) is modulated by the drug uptake and efflux transporters encoded by organic cation transporter 1 (OCTN1) and multiple drug-resistant gene 1 (MDR1) and expressed in human heart.

Polymorphic variants in the human bile salt export pump (BSEP; ABCB11): functional characterization and interindividual variability.

Objectives: Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11), and to assess interindividual variability in BSEP expression.

Methods: We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method.

Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

Rationale: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins.

Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe.

Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe.

A common polymorphism in the bile acid receptor farnesoid X receptor is associated with decreased hepatic target gene expression.

The farnesoid X receptor (FXR or NR1H4) is an important bile-acid-activated, transcriptional regulator of genes involved in bile acid, lipid, and glucose homeostasis. Accordingly, interindividual variations in FXR expression and function could manifest as variable susceptibility to conditions such as cholesterol gallstone disease, atherosclerosis, and diabetes. We performed an FXR polymorphism discovery analysis of European-, African-, Chinese-, and Hispanic-Americans and identified two rare gain-of-function variants and a common single nucleotide polymorphism resulting in a G-1T substitution in the nucleotide adjacent to the translation initiation site (FXR*1B) with population allelic frequencies ranging from 2.

Ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibit bile acid transport in human and rat hepatocytes.

Human immunodeficiency virus-infected patients on antiretroviral drug therapy frequently experience hepatotoxicity, the underlying mechanism of which is poorly understood. Hepatotoxicity from other compounds such as bosentan and troglitazone has been attributed, in part, to inhibition of hepatocyte bile acid excretion. This work tested the hypothesis that antiretroviral drugs modulate hepatic bile acid transport.

Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics.

Background & Aims: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, target liver HMG-CoA and are of proven benefit in the prevention of coronary heart disease. Rosuvastatin is an effective statin notable for liver selectivity and lack of significant metabolism. We assessed the extent and relevance of hepatic transporters to rosuvastatin uptake.

Development and characterization of LLC-PK1 cells containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to human and mouse.

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.

Hepatic uptake of the novel antifungal agent caspofungin.

Caspofungin (CANCIDAS, a registered trademark of Merck & Co., Inc.) is a novel echinocandin antifungal agent used in the treatment of esophageal and invasive candidiases, invasive aspergillosis, and neutropenia.

Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry.

Organic anion-transporting polypeptide 1A2 (OATP1A2) is a drug uptake transporter known for broad substrate specificity, including many drugs in clinical use. Therefore, genetic variation in SLCO1A2 may have important implications to the disposition and tissue penetration of substrate drugs. In the present study, we demonstrate OATP1A2 protein expression in human brain capillary and renal distal nephron using immunohistochemistry.

Identification of amino acids in rat pregnane X receptor that determine species-specific activation.

The pregnane X receptor (PXR) is a nuclear receptor significantly involved in the transcriptional regulation of drug-metabolizing enzymes and transporters. Interestingly, certain PXR ligands such as rifampin have been shown to readily induce human and rabbit but not rodent members of the cytochrome P450 3A. Because drugs of divergent chemical structures seem to be similarly affected, we hypothesized that specific amino acid residue(s) or domains in rat PXR affect receptor activation by certain human PXR ligands.

Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition.

The key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, functionally relevant polymorphisms in this transporter would be predicted to have an important impact on bile acid homeostasis/liver function.

The orphan nuclear receptor HNF4alpha determines PXR- and CAR-mediated xenobiotic induction of CYP3A4.

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression.

Human organic anion transporting polypeptide-C (SLC21A6) is a major determinant of rifampin-mediated pregnane X receptor activation.

Rifampin, a member of the rifamycin class of antibiotics, is well known for its ability to induce drug-metabolizing enzymes and transporters, through activation of the pregnane X receptor. Available data suggest rifampin entry into hepatocytes may be transporter-mediated. Accordingly, it is therefore plausible that modulation of the achievable intracellular concentration of rifampin by drug uptake transporters would influence the degree of induction.

Application of three-dimensional quantitative structure-activity relationships of P-glycoprotein inhibitors and substrates.

Using in vitro data, we previously built Catalyst 3-dimensional quantitative structure activity relationship (3D-QSAR) models that qualitatively rank and predict IC(50) values for P-glycoprotein (P-gp) inhibitors. These models were derived and tested with data for inhibition of digoxin transport, calcein accumulation, vinblastine accumulation, and vinblastine binding. In the present study, 16 inhibitors of verapamil binding to P-gp were predicted using these models.

Three-dimensional quantitative structure-activity relationships of inhibitors of P-glycoprotein.

P-glycoprotein (P-gp) is an efflux transporter involved in limiting the oral bioavailability and tissue penetration of a variety of structurally divergent molecules. A better understanding of the structural requirements of modulators of P-gp function will aid in the design of therapeutic agents. Toward this goal, three-dimensional quantitative structure-activity relationship (3D-QSAR) models were generated using in vitro data associated with inhibition of P-gp function.

Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine.

Objectives: Our objective was to examine the effect of different fruits and their constituents on P-glycoprotein and organic anion transporting polypeptide (OATP) activities in vitro and on drug disposition in humans.

Methods: P-glycoprotein-mediated digoxin or vinblastine efflux was determined in polarized epithelial cell monolayers. OATP-mediated fexofenadine uptake was measured in a transfected cell line.