Selection of an Optimal In Vitro Model to Assess P-gp Inhibition: Comparison of Vesicular and Bidirectional Transcellular Transport Inhibition Assays.

The multidrug resistance protein 1 (MDR1) P-glycoprotein (P-gp) is a clinically important transporter. In vitro P-gp inhibition assays have been routinely conducted to predict the potential for clinical drug-drug interactions (DDIs) mediated by P-gp. However, high interlaboratory and intersystem variability of P-gp IC data limits accurate prediction of DDIs using static models and decision criteria recommended by regulatory agencies.

In Vitro Assessment of Transporter Mediated Perpetrator DDIs for Several Hepatitis C Virus Direct-Acting Antiviral Drugs and Prediction of DDIs with Statins Using Static Models.

Inhibitory effects of asunaprevir, daclatasvir, grazoprevir, paritaprevir, simeprevir, and voxilaprevir, direct-acting antiviral (DAA) drugs for the treatment of chronic hepatitis C virus (HCV) infection, were evaluated in vitro against a range of clinically important drug transporters. In vitro inhibition studies were conducted using transporter transfected cells and membrane vesicles. The risk of clinical drug-drug interactions (DDIs) was assessed using simplified static models recommended by regulatory agencies.

Role of transporters in the disposition of a novel β-lactamase inhibitor: relebactam (MK-7655).

Objectives: To identify the transporters involved in renal elimination of relebactam, and to assess the potential of relebactam as a perpetrator or victim of drug-drug interactions (DDIs) for major drug transporters.

Methods: A series of bidirectional transport, uptake and inhibition studies were conducted in vitro using transfected cell lines and membrane vesicles. The inhibitory effects of relebactam on major drug transporters, as well as the inhibitory effects of commonly used antibiotics/antifungals on organic anion transporter (OAT) 3-mediated uptake of relebactam, were assessed.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds.

Evaluation of organic anion transporting polypeptide 1B1 and 1B3 humanized mice as a translational model to study the pharmacokinetics of statins.

Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography-tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins.

Variability in P-glycoprotein inhibitory potency (IC₅₀) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC₅₀ working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC₅₀ determinations. Each laboratory followed its in-house protocol to determine in vitro IC₅₀ values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories).

Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist.

1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner.

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers.

Introduction: P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions.

Characterization of multidrug resistance 1a/P-glycoprotein knockout rats generated by zinc finger nucleases.

The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN.

Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites.

Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein.

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (Km=162 microM), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively.

Identification of potential pharmacological and toxicological targets differentiating structural analogs by a combination of transcriptional profiling and promoter analysis in LS-180 and Caco-2 adenocarcinoma cell lines.

Detecting and understanding the potential for off-target pharmacological effects is critical in the optimization of lead compounds in drug discovery programs. Compound-mediated activation of the pregnane X receptor (PXR; NR1I2), a key regulator for drug metabolism genes, is often monitored to avoid potential drug-drug interactions. Two structural analogs, MRL-1 and MRL-2, were determined to be equivalent PXR activators in trans-activation assays.

Characterization of mice lacking the multidrug resistance protein MRP2 (ABCC2).

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains.

Toxicity of glutathione depletion in mesencephalic cultures: a role for arachidonic acid and its lipoxygenase metabolites.

The contribution of arachidonic acid (AA) release and metabolism to the toxicity that results from glutathione (GSH) depletion was studied in rat mesencephalic cultures treated with the GSH synthesis inhibitor l-buthionine sulfoximine. Our data show that GSH depletion is accompanied by increased release of AA, which is phosholipase A2 (PLA2) dependent. Exogenous AA is toxic to GSH-depleted cells.

Glutathione depletion and oxidative stress.

Oxidative stress is believed to contribute to the pathogenesis of Parkinson's disease. One of the indices of oxidative stress is the depletion of the antioxidant glutathione (GSH), which may occur early in the development of Parkinson's disease. To study the role of GSH depletion in the survival of dopamine neurons we treated mesencephalic cultures with the GSH synthesis inhibitor L-buthionine sulfoximine.

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures.

Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures.