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. 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-4alpha (HNF4alpha; 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 HNF4alpha binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4alpha had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4alpha had reduced basal and inducible expression of CYP3A. These data identify HNF4alpha as an important regulator of coordinate nuclear-receptor-mediated response to xenobiotics.

Increased CYP3A4 copy number in TONG/HCC cells but not in DNA from other humans.

Two recent screens for copy-number variations in the entire human genome found 12.4 gene copy number variations per person, including 2.5% of individuals with gains between 7q21.1 and 7q22.1, the chromosomal location of CYP3A4. CYP3A4 is involved in the metabolism of approximately 50% of all drugs, including many cancer chemotherapeutic agents. CYP3A4 gene copy was determined in DNA from 143 individuals: normal human livers, primary and secondary liver tumors, human hepatic cell lines, and immortalized cell lines representing eight ethnically diverse populations. CYP3A4 gene copy was normal in all but one sample, a primary human hepatocellular carcinoma cell line (TONG/HCC). Southern blots of TONG/HCC DNA revealed an approximate 10-fold increase in CYP3A and a corresponding increase in CYP3A mRNA expression and catalytic activity. Fluorescent in situ hybridization of TONG/HCC revealed specific amplification of the CYP3A4 gene on chromosome 7q21 but no amplification of the MDR1 gene that localizes 11.9 Mb upstream of CYP3A4. High resolution analysis of DNA copy number by comparative genomic hybridization confirmed amplification at 7q21.3-7q22. The amplicon spanned 1.7 Mb and contained 30 known genes, including the entire CYP3A locus. To determine whether CYP3A4 expression affected chemotherapeutic toxicity, LLC-PK1 cells were transduced with adenoviruses expressing CYP3A4 and P450 reductase. CYP3A4 conferred resistance to taxol, vinblastine and topotecan. These studies demonstrate that CYP3A4 copy number differences do not contribute to the normal variation in CYP3A4 expression. Tumors with increased CYP3A copy number (via amplification or increased chromosome 7q) would be expected to show reduced cytotoxicity to some chemotherapeutic drugs and potentially an increase in the outgrowth of drug resistant tumors.

Interaction of cytochrome P450 3A inhibitors with P-glycoprotein.

Many clinically important drug interactions occur due to inhibition of human liver cytochrome P450 3A (CYP3A) metabolism. The drug efflux pump P-glycoprotein (Pgp) can be an additional locus contributing to these drug interactions because there is overlap in drugs that are substrates for both proteins. We screened a number of CYP3A inhibitors (macrolide antibiotics, azole antifungals, and ergotpeptides) for their ability to interact with Pgp, compared with prototypical Pgp inhibitors. We used cell lines expressing human, mouse, and rat mdr1 genes. Pgp antagonism was defined by interactions of the drugs with four cell lines (LLC-PK1, L-MDR1, L-mdr1a, and L-mdr1b) using a microfluorometric calcein-AM assay and characterized for their inhibitor constant (K(i)) toward calcein-AM. The compounds were further defined for their ability to inhibit MDR1 by their effect on vinblastine accumulation into L-MDR1 cells. Representative compounds from each class of drugs were further tested as Pgp substrates, defined by the ability of human Pgp or mouse mdr1a/Pgp to transport them across a polarized kidney epithelial cell in vitro. These same compounds were administered radiolabeled in vivo to mdr1a (+/+) and (-/-) mice and the distribution of radioactivity compared. The results are summarized as follows: 1) Some drug interactions with Pgp were substrate- and/or assay-dependent. 2) Ergot alkaloids were identified as a class of MDR1/Pgp chemosensitizers. 3) The Ergot alkaloids revealed species differences in the structure-activity relationships for inhibition of Pgp. Simultaneous inhibition of Pgp by many CYP3A inhibitors contributes to human variation in the extent of drug-drug interactions.

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. Several approaches were undertaken with multiple iterations, yielding Catalyst 3D-QSAR models being able to qualitatively rank-order and predict IC(50) values for P-gp inhibitors excluded from the model in question. The success of these validations suggests that a P-gp pharmacophore for 27 inhibitors of digoxin transport in Caco-2 cells consisted of four hydrophobes and one hydrogen bond acceptor. A second pharmacophore generated with 21 inhibitors of vinblastine binding to plasma membrane vesicles derived from CEM/VLB(100) cells contained three ring aromatic features and one hydrophobic feature. A third pharmacophore generated with 17 inhibitors of vinblastine accumulation in P-gp expressing LLC-PK1 cells contained four hydrophobes and one hydrogen bond acceptor. A final pharmacophore was generated for inhibition of calcein accumulation in P-gp expressing LLC-PK1 cells and found to contain two hydrophobes, a ring aromatic feature, and a hydrogen bond donor. The similarity of features for the pharmacophores of P-gp inhibitors of digoxin transport and vinblastine binding suggest some commonality in their binding sites. Utilization of such models may prove to be of value for prediction of molecules that may modulate one or more P-gp binding sites.

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. These inhibition results were then used to generate a new pharmacophore that consisted of one hydrogen bond acceptor, one ring aromatic feature, and two hydrophobes. This model predicted the rank order of the four data sets described previously and correctly ranked the inhibitory potency of a further four verapamil metabolites identified in the literature. The degree of similarity in rank ordering prediction by these inhibitor pharmacophore models generated to date confirms a likely overlap in the sites to which the three P-gp substrates used in these studies (verapamil, vinblastine, and digoxin) bind. Alignment of the three substrate probes indicated that they are likely to bind the same or overlapping sites within P-gp. Important features on these substrates include multiple hydrophobic and hydrogen bond acceptor features, which are widely dispersed and in agreement among most of the five inhibitor pharmacophores we have described so far. These 3D-QSAR models will be useful for future prediction of likely substrates and inhibitors of P-gp.

Expression of MRP4 confers resistance to ganciclovir and compromises bystander cell killing.

The multidrug resistance protein MRP4, a member of the ATP-binding cassette superfamily, confers resistance to purine-based antiretroviral agents. However, the antiviral agent ganciclovir (GCV) has not been shown to be a substrate of MRP4. GCV is important not only in antiviral therapy, but also in the selective killing of tumor cells modified to express herpes simplex virus thymidine kinase (HSV-TK). We therefore tested the effect of MRP4 on the cytotoxicity of GCV, on the ability of GCV to kill cells genetically modified to express HSV-TK, and on the bystander effect in which unmodified target cells are killed by GCV. Cells overexpressing MRP4 had markedly increased resistance to the cytotoxicity of GCV. Although, expression of recombinant HSV-TK increased the intracellular concentration of GCV nucleotide, cells were rescued by the cytoprotective effect of MRP4. In cells that overexpressed MRP4, intracellular accumulation of GCV metabolites was reduced, efflux of these metabolites was increased, and resistance to bystander killing was increased. Therefore, MRP4 can strongly reduce the susceptibility of HSV-TK-expressing cells to GCV, and its overexpression in adjacent cells protects them from bystander cell death. These findings indicate that a nucleotide transporter, such as MRP4, modulates the cellular response to GCV and thus may influence not only the efficacy of antiviral therapy, but also prodrug-based gene therapy, which is critically dependent upon bystander cell killing.