To assess the association of CYP2B6 allelic diversity with efavirenz (EFV) pharmacokinetics, we performed extensive genotyping of 15 relevant single nucleotide polymorphism in 169 study participants, and full resequencing of CYP2B6 in individuals with abnormal EFV plasma levels. Seventy-seven (45.5%) individuals carried a known (CYP2B6*6, *11, *15, or *18) or new loss/diminished-function alleles. Resequencing defined two new loss-of-function alleles: allele *27 (marked by 593T>C [M198T]), that results in 85% decrease in enzyme activity and allele *28 (marked by 1132C>T), that results in protein truncation at arginine 378. Median AUC levels were 188.5 microg h/ml for individuals homozygous for a loss/diminished-function allele, 58.6 microg h/ml for carriers, and 43.7 microg h/ml for noncarriers (P<0.0001). Individuals with a poor metabolizer genotype had a likelihood ratio of 35 (95% CI, 11-110) of presenting very high EFV plasma levels. CYP2B6 poor metabolizer genotypes explain to a large extent EFV pharmacokinetics and identify individuals at risk of extremely elevated EFV plasma levels.

Human CYP3A enzymes play a pivotal role in the metabolism of many drugs, and the variability of their expression among individuals may have a strong impact on the efficacy of drug treatment. However, the individual contributions of the four CYP3A genes to total CYP3A activity remain unclear. To elucidate the role of CYP3A7, we have studied its expression in human liver and intestine. In both organs, expression of CYP3A7 mRNA was polymorphic. The recently identified CYP3A7*1C allele was a consistent marker of increased CYP3A7 expression both in liver and intestine, whereas the CYP3A7*1B allele was associated with increased CYP3A7 expression only in liver. Because of the replacement of part of the CYP3A7 promoter by the corresponding region of CYP3A4, the CYP3A7*1C allele contains the proximal ER6 motif of CYP3A4. The pregnane X and constitutively activated receptors were shown to bind with higher affinity to CYP3A4-ER6 than to CYP3A7-ER6 motifs and transactivated only promoter constructs containing CYP3A4-ER6. Furthermore, we identified mutations in CYP3A7*1C in addition to the ER6 motif that were necessary only for activation by the constitutively activated receptor. We conclude that the presence of the ER6 motif of CYP3A4 mediates the high expression of CYP3A7 in subjects carrying CYP3A7*1C.Cytochrome P450 enzymes play a pivotal role in the oxidative, peroxidative, and reductive metabolism of many endogenous compounds, procarcinogens, and drugs. The CYP3A subfamily composed of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 in humans is of special importance because it accounts for as much as 30% of total liver cytochrome P450 content (1). At least 50% of all medicines are metabolized by enzymes of the CYP3A subfamily (2). The most abundant CYP3A isoform in liver and intestine is CYP3A4. Its interindividual hepatic expression varies 60-fold (3), and the in vivo function as assessed by clearance displays at least a 20-fold difference (4). Induction by xenobiotics (e.g. rifampin) and endogenous compounds (e.g. steroid hormones) further modulates the variability of CYP3A4 expression among individuals. The induction of CYP3A4 and most likely that of other CYP3A genes is mediated by the nuclear receptor NR1I2 (pregnane X receptor (PXR) 1 ) (reviewed in Ref. 5). CYP3A4-inducing compounds bind to PXR and stimulate the transcriptional activity of the receptor. Additional nuclear receptors such as NR1I3 (constitutively activated receptor (CAR)) and NR1I1 (vitamin D receptor) have also been implicated in the transcriptional regulation of CYP3A4 (6, 7). Although the substrate specificity of CYP3A5 is similar to that of CYP3A4, CYP3A5 has been regarded to be less important for drug elimination because it is expressed at much lower levels than CYP3A4 in most livers of Caucasian origin (8). CYP3A43 is expressed at very low levels in adult human livers, accounting for only 0.1-0.2% of CYP3A4 transcripts (9, 10). Therefore, its contribution to the elimination of CYP3A substrates is regarded to be negligible (10). This variability in CYP3A...

MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1 . The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.

The multidrug resistance protein 4 (MRP4) is an efflux transporter involved in the transport of endogenous substrates and xenobiotics. We measured MRP4 mRNA and protein expression in human livers and found a 38-and 45-fold variability, respectively. We sequenced 2 kb of the 5 0 -flanking region, all exons and intron/exon boundaries of the MRP4 gene in 95 patients and identified 74 genetic variants including 10 non-synonymous variations, seven of them being located in highly conserved regions. None of the detected polymorphisms was significantly associated with changes in the MRP4 mRNA or protein expression. Immunofluorescence microscopy indicated that none of the non-synonymous variations affected the cellular localization of MRP4. However, in cholestatic patients the MRP4 mRNA and protein expression both were significantly upregulated compared to non-cholestatic livers (protein: 2997138 vs 100760a.u., Po0.001). Taken together, human hepatic MRP4 expression is highly variable. Genetic variations were not sufficient to explain this variability. In contrast, cholestasis is one major determinant of human hepatic MRP4 expression.

ABSTRACT:Cytochrome P450 3A4 plays an outstanding role in the metabolism of clinically used drugs and shows a marked interindividual variability in expression even in the absence of inducing agents. Thus, regulation of basal expression contributes considerably to variability. The nuclear receptor hepatocyte nuclear factor 4␣ (HNF4␣) was previously shown to be associated with basal hepatic CYP3A4 expression. As how HNF4␣ regulates basal expression of CYP3A4 still remains elusive, we systematically screened 12.5 kilobase pairs (kb) of the CYP3A4 5 upstream region for activation by the receptor in the human intestinal cell line LS174T. In this study, we newly identified two widely separated regions mediating the activation by HNF4␣: a far distal region at ؊9.0 kb and the proximal promoter region at ϳ؊0.2 kb. By gel shift experiments and transient transfections, we characterized direct repeat (DR) 1-type motifs in both regions as functional HNF4␣ response elements. Cooperation of the two regions was shown to be required for maximal activation by HNF4␣. The effect of HNF4␣ was antagonized by chicken ovalbumin upstream promoter transcription factor II, which was shown to bind to one of the DR1 motifs. Furthermore, activation of CYP3A4 via the DR1 element in the proximal promoter depends on an additional, yet unknown, factor, which is binding at ϳ؊189 base pairs. Physiological relevance of this position for activation by HNF4␣ in vivo is suggested by the presence of a binding activity in small intestine similar to that in LS174T cells. In summary, we here have elucidated a molecular mechanism of direct regulation of CYP3A4 by HNF4␣, which is probably specific for the intestine.