W. Lee

The orphan nuclear receptor HNF4α 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. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4α (HNF4α; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4α binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4α had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4α had reduced basal and inducible expression of CYP3A. These data identify HNF4α as an important regulator of coordinate nuclear-receptor–mediated response to xenobiotics.

Intestinal Drug Transporter Expression and the Impact of Grapefruit Juice in Humans

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter‐transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real‐time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver‐specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2u2009h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short‐term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Identification of nonsynonomous polymorphisms of human organic anion transporting polypeptide‐A (OATP‐A) associated with altered transport activity

Organic anion transporting polypeptides (OATP) are a growing family of uptake transporters important to drug disposition. Studies from this laboratory have shown that OATP‐A (SLC21A3) is selectively expressed in human intestine as well as kidney and brain. Accordingly, variability in the expression or function of this polyspecific drug uptake transporter may have important implications to bioavailability and tissue penetration of substrate drugs. In order to determine the extent of genetic heterogeneity in OATP‐A, PCR‐based genotyping analyses were carried out on ethnically defined genomic DNA samples (n=96 each for African‐, Chinese‐, European‐, and Hispanic‐Americans). We identified three novel nonsynonymous polymorphisms within the coding region of OATP‐A; T38C (Ile38Thr), A516C (Glu172Asp) and G559A (Ala187Thr). Genotypic frequencies of these polymorphisms were dependent on race. In vitro functional assessment revealed that the A516C (Glu172Asp) variant had markedly reduced capacity for mediating the cellular uptake of OATP‐A substrates, estrone sulfate and fexofenadine. Cell surface biotinylation experiments did not show altered plasma membrane expression of the transporter, suggesting the reduced transport activity associated with the A516C variant is likely due to altered intrinsic activity of the transporter. Our data suggest that OATP‐A polymorphisms may contribute to inter‐individual variability in intestinal absorption and CNS penetration of substrate drugs.

PIII-26Hepatic nuclear factor (HNF)1αactivates the induction of human CYP2C9 by rifampin (Rif) and interacts with HNF4α and small heterodimer partner 1 (SHP-1)

Patients with HNF1α gene mutations exhibit marked hypersensitivity to hypoglycemic drugs (CYP2C9 substrates). Rif‐induced CYP2C9 gene expression is reduced by pretreatment with chenodeoxycholic acid, which induces expression of SHP‐1. SHP‐1 can repress HNF1α gene through the suppression of HNF4α and HNF4α synergistically activates PXR/Rif induction of CYP2C9. This study examined the hypothesis that HNF1α activates Rif‐induced CYP2C9 transactivation and interacts with HNF4α and SHP‐1.

Hepatocyte nuclear factors (HNF) 4α and 1α synergistically up‐regulate car‐mediated transcriptional activity of human CYP2C9 gene

There are two CAR response elements, −1783/−1856 bp and −2899/−2883 bp, in the −3kb CYP2C9 promoter region. The proximal element is thought to mediate PXR response while the distal one may have a CAR effect. HNFs are important co‐regulators of gene expression. The aim of this study was to define the roles of HNF4α and HNF1α in CAR‐mediated transactivation of human CYP2C9 gene.

Modulation of adopted nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), by commonly prescribed chemotherapy agents

The adopted nuclear receptors (NRs), CAR and PXR are key regulators of genes involved in drug disposition and may contribute to variable drug response and toxicity. Currently, little is known regarding the contribution of the NRs to chemotherapy‐associated drug interactions or toxicities. We utilized a cell‐based reporter assay to identify potential novel interactions between PXR or CAR and commonly prescribed chemotherapy drugs. In single agent studies, CAR‐dependent activation of the CYP3A4 promoter was enhanced by vinblastine (EC50=28.6 nM) and CPT‐11 (EC50=16.9 μM). Conversely, camptothecin (CPT) appeared to inhibit CAR activation (IC50=0.34 μM). CPT and cisplatin did not exert any effects as single agents; however both agents appeared to attenuate rifampin‐mediated PXR activation in a dose‐dependent manner, suggesting those agents may be antagonists of PXR. Interestingly, rifampin‐mediated PXR activation was further enhanced by the co‐incubation with etoposide or CPT‐11. In primary human hepatocytes, rifampin‐mediated CYP3A induction was similarly attenuated by the co‐incubation with CPT and cisplatin, suggesting that NR‐based reporter assays may be a useful tool for predicting conditions in vivo. Overall, our studies demonstrate that NRs interact with a number of commonly prescribed chemotherapy agents and that certain combination regimens may modulate NRs in an unexpected manner and contribute to the observed variability in efficacy and toxicity.

Incorporating Pharmacogenomics into Cancer Therapy

Heritable variations in genes associated with drug disposition and effects (i.e., drug metabolism, transport, and therapeutic target) contribute to individual heterogeneity in drug treatment response and tolerance. The terms pharmacogenetics or pharmacogenomics, which are often used interchangeably, refer to the study of how an individual’s genetic inheritance affects the body’s response to drugs and the use of this genetic information to predict the safety, toxicity, and/or efficacy of drugs in individual patients or groups of patients. In some cases, the term “pharmacogenetics” is used to refer to the study of differing phenotypes in association with a single gene or set of candidate genes and the term “pharmacogenomics” is used to refer to genome-wide approaches for identification or discovery of the genetic factors determining differing phenotypes. Throughout this chapter, the term “pharmacogenomics” will be used in reference to this scientific discipline. Pharmacogenomics, as it relates to cancer treatment, presents particular difficulties in comparison to other therapeutic areas, in that acquired somatic mutations in the tumors may also alter treatment outcomes and, that the narrow therapeutic window of chemotherapeutic agents makes titrations in drug dose to affect response or toxicity be a suboptimal approach. The application of pharmacogenomics into the treatment of cancer could have a considerable impact on optimizing cancer treatment for individual patients and groups of patients and also developing new chemotherapeutic agents.

OII-B-1The role of NA+-taurocholate cotransporting polypeptide (NTCP) in the hepatic uptake of rosuvastatin

Rosuvastatin is an HMG‐CoA reductase inhibitor (statin) used in the treatment of dyslipidemia. Rosuvastatin is not subject to significant metabolism and we have previously shown a role of OATP transporters for rosuvastatin uptake.

Evaluation of ethnicity, gender, and OATP-C genotypes as determinants of pravastatin disposition

Pravastatin is an HMG‐CoA reductase inhibitor (statin), widely used in the treatment of hypercholesterolemia. Previous studies have revealed that drug transporters are critical to the cellular uptake and elimination of pravastatin. The rate‐limiting step in the hepatic clearance of pravastatin appears to be cellular uptake, mediated by organic anion transporting polypeptide C (OATP‐C; SLC21A6), a liver‐specific uptake transporter with broad substrate specificities. Accordingly, pravastatin may be a useful in vivo probe of OATP‐C activity in humans. We have conducted a clinical study of pravastatin pharmacokinetics in a population of healthy Caucasian and African‐American volunteers after single dose (40 mg) administration of pravastatin. Preliminary (n=27) results show no significant differences in the area under the concentration‐time curve (AUC) from 0 to 3 hr between Caucasian and African‐American subjects (103.9 vs 106.4 ng*hr/ml). However, AUC values suggest gender‐dependent differences in pravastatin pharmacokinetics may exist. In addition, several single‐nucleotide polymorphisms (SNPs) previously identified by this laboratory to be important to OATP‐C function are being assessed for potential genotype:phenotype correlations. (see Table 1 )

Stable and tightly regulated expression of human NA+ taurocholate cotransporting polypeptide (NTCP) using a tetracycline‐inducible system

Clinical Pharmacology & Therapeutics (2003) 73, P13–P13; doi: