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.

Genistein, resveratrol, and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside induce cytochrome P450 4F2 expression through an AMP-activated protein kinase-dependent pathway.

Activators of AMP-activated protein kinase (AMPK) increase the expression of the human microsomal fatty acid ω-hydroxylase CYP4F2. A 24-h treatment of either primary human hepatocytes or the human hepatoma cell line HepG2 with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which is converted to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate, an activator of AMPK, caused an average 2.5- or 7-fold increase, respectively, of CYP4F2 mRNA expression but not of CYP4A11 or CYP4F3, CYP4F11, and CYP4F12 mRNA.

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.

Opposing roles of peroxisome proliferator-activated receptor alpha and growth hormone in the regulation of CYP4A11 expression in a transgenic mouse model.

CYP4A11 transgenic mice (CYP4A11 Tg) were generated to examine in vivo regulation of the human CYP4A11 gene. Expression of CYP4A11 in mice yields liver and kidney P450 4A11 levels similar to those found in the corresponding human tissues and leads to an increased microsomal capacity for omega-hydroxylation of lauric acid. Fasted CYP4A11 Tg mice exhibit 2-3-fold increases in hepatic CYP4A11 mRNA and protein, and this response is absent in peroxisome proliferator-activated receptor alpha (PPARalpha) null mice.

Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies.

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined.

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11.

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS.

Kidney dysfunction and hypertension: role for cadmium, p450 and heme oxygenases?

Cadmium (Cd) is a metal toxin of continuing worldwide concern. Daily intake of Cd, albeit in small quantities, is associated with a number of adverse health effects which are attributable to distinct pathological changes in a variety of tissues and organs. In the present review, we focus on its renal tubular effects in people who have been exposed environmentally to Cd at levels below the provisional tolerable intake level set for the toxin.

Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1.

The present study examined accumulation of the metal toxins cadmium (Cd) and lead (Pb) in relation to the abundance of cytochrome P450 4F2 (CYP4F2), CYP2E1 and concentrations of zinc and copper in liver and kidney samples using immunoblotting coupled with metal analysis. The post mortem liver and kidney cortex samples were from 23 males and 8 females aged 3-89 years. All were Caucasians who had not been exposed to metals in the workplace.

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.

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.