Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3).

Background: Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult.

Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation.

The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important.

Advantages of cell-based high-volume screening assays to assess nuclear receptor activation during drug discovery.

Introduction: Adverse drug effects and drug-drug interactions (DDIs) can be elicited by the activation of several nuclear receptors (NRs). Of the NRs that regulate expression of drug metabolizing enzymes and transporters and alter cellular processes, the most important are pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor. Screening for the activation of these receptors can be achieved during drug discovery by using various high-throughput analyses including ligand binding and transactivation assays.

Cell-based systems to assess nuclear receptor activation and their use in drug development.

The evolution of scientific information relating to the regulation of xenobiotic disposition has extended to the discovery of an intricate group of receptor systems now recognized as master regulators. These ligand-activated transcription factors are commonly designated as "nuclear receptors", and include CAR (NR1I3), PXR (NR1I2), PPAR (NR1C1, NR1C2, and NR1C3) and AhR (HLHE76). As regulators of gene expression, activation of these receptors can elicit a plethora of drug-drug interactions.

Utility of DPX2 cells for predicting CYP3A induction-mediated drug-drug interactions and associated structure-activity relationships.

The increase in cytochrome P450 (P450) enzyme activity noted upon exposure to therapeutics can elicit marked drug-drug interactions (DDIs) that may ultimately result in poor clinical outcome or adverse drug effects. As such, in vitro model systems that can rapidly and accurately determine whether potential therapeutics activate the human pregnane X receptor (PXR) and thus induce CYP3A P450 levels are highly sought after tools for drug discovery. To that end, we assessed whether DPX2 cells, a HepG2-derived cell line stably integrated with a PXR expression vector plus a luciferase reporter, could detect agents that not only cause PXR activation/CYP3A induction but also elicit clinical DDIs.

Current in vitro high throughput screening approaches to assess nuclear receptor activation.

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors.

Nanosilver particle effects on drug metabolism in vitro.

Nanosilver particles are present in consumer and health care products. Their effects on human microsomal cytochrome P450 (P450) activities and induction in luciferase reporter-engineered Caco-2 (MDR1.C) and HepG2 (DPX2 and 1A2DRE) cells have been investigated.

Conversion of the HIV protease inhibitor nelfinavir to a bioactive metabolite by human liver CYP2C19.

Antiretroviral therapy for human immunodeficiency virus (HIV) infection includes treatment with both reverse transcriptase inhibitors and protease inhibitors, which markedly suppress viral replication and circulating HIV RNA levels. Cytochrome P450 (P450) enzymes in human liver, chiefly CYP3A4, play a pivotal role in protease inhibitor biotransformation, converting these agents to largely inactive metabolites. However, the protease inhibitor nelfinavir (Viracept) is metabolized mainly to nelfinavir hydroxy-t-butylamide (M8), which exhibits potent antiviral activity, and to other minor products (termed M1 and M3) that are inactive.

Regulation of CYP2E1 by ethanol and palmitic acid and CYP4A11 by clofibrate in primary cultures of human hepatocytes.

CYP2E1 and CYP4A11 are cytochrome P450 enzymes that are regulated by physiological conditions including diabetes and fasting. In addition, the xenochemical clofibrate has been reported to induce both rodent CYP2E1 and CYP4A. These findings suggest similar modes of regulation.

Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products.

Human CYP3A4 metabolizes a majority of clinically important substrates at variable rates. Accounting for these unpredictable rates is the wide variation noted in expression of this enzyme that is due, in part, to xenobiotic exposure. We used primary cultures of human hepatocytes from 17 individuals to assess the inducibility of CYP3A4 mRNA by prototypical inducers, dietary flavonoids, and botanicals.

Genetic polymorphisms of flavin-containing monooxygenase (FMO).

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU).

Expression and induction of CYP2C P450 enzymes in primary cultures of human hepatocytes.

Although CYP2C8, CYP2C9, and CYP2C19 play an important role in drug biotransformation, factors influencing the expression and activity of these CYP2C P450s in human liver remain largely undefined. We used primary cultures of human hepatocytes from 15 subjects to assess the inducibility of CYP2C enzyme expression by prototypical inducer agents, including rifampicin, dexamethasone, and phenobarbital. After culture for 72 h in serum-free medium on collagen, Western blotting revealed that CYP2C9 was the only CYP2C enzyme expressed at appreciable levels in untreated hepatocytes.