Increased absorption of digoxin from the human jejunum due to inhibition of intestinal transporter-mediated efflux.

BACKGROUND AND OBJECTIVES: The contribution of transport in the small intestine by the apically located efflux pump P-glycoprotein to variable drug absorption in humans is still poorly understood. We therefore investigated whether inhibition of intestinal P-glycoprotein-mediated efflux by quinidine leads to increased absorption of the P-glycoprotein substrate digoxin. METHODS: Using a multilumen perfusion catheter, we investigated the impact of P-glycoprotein inhibition on absorption of two compounds: the P-glycoprotein substrate digoxin and the marker for passive transcellular absorption antipyrine. Two 20cm adjacent jejunal segments were isolated with the multilumen perfusion catheter in seven healthy subjects. Unlabelled and deuterated digoxin and antipyrine, respectively, were simultaneously infused into either of the intestinal segments. One of the segments was additionally perfused with the P-glycoprotein inhibitor quinidine. Intestinal perfusates were collected for 3 hours, and drug concentrations were determined in the intestinal perfusates, plasma and urine. RESULTS: Quinidine did not affect the disposition of antipyrine. In contrast, coadministration of quinidine into one jejunal segment caused a considerable increase in the amount of digoxin absorbed from this segment compared with the absorption from the other quinidine-free segment (22.3 +/- 8.9% vs 55.8 +/- 21.2% of the dose; p < 0.05). Accordingly, the area under the plasma concentration-time curve and the maximum plasma concentration of digoxin were considerably higher when luminal quinidine was coadministered (p < 0.05 and p < 0.001, respectively). Differences in digoxin absorption from the two intestinal segments were also reflected by pronounced differences in urinary digoxin elimination (5.5 +/- 3.3% vs 19.2 +/- 8.1% of the dose; p < 0.01). CONCLUSIONS: P-glycoprotein inhibition in enterocytes increases systemic exposure of orally administered drugs that are P-glycoprotein substrates. These data highlight the importance of the small intestine as an active barrier against xenobiotics.

Molecular mechanism of basal CYP3A4 regulation by hepatocyte nuclear factor 4alpha: evidence for direct regulation in the intestine.

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 4alpha (HNF4alpha) was previously shown to be associated with basal hepatic CYP3A4 expression. As how HNF4alpha 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 HNF4alpha: a far distal region at -9.0 kb and the proximal promoter region at approximately -0.2 kb. By gel shift experiments and transient transfections, we characterized direct repeat (DR) 1-type motifs in both regions as functional HNF4alpha response elements. Cooperation of the two regions was shown to be required for maximal activation by HNF4alpha. The effect of HNF4alpha 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 approximately -189 base pairs. Physiological relevance of this position for activation by HNF4alpha 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 HNF4alpha, which is probably specific for the intestine.

Functional analysis of the polymorphism -211C>T in the regulatory region of the human ABCC3 gene.

The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position -211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position -211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position -211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the -211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the -211 SNP. Taken together, we did not find any influence of the -211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

Efficacy of nelfinavir-based treatment in the central nervous system of HIV-1 infected patients.

We studied the HIV-1 load and nelfinavir (NFV) concentrations in cerebrospinal fluid (CSF) after long-term successful NFV-based therapy, using ultrasensitive methods of detection. 19 patients without virological failure in plasma, who had been treated with 2 nucleoside analogue reverse transcripaste inhibitors (NRTI) and NFV for a minimum of 18 months were included. HIV-RNA was determined in plasma and CSF using an ultrasensitive method (<2 copies/ml). Total and free concentrations of NFV were analysed using high liquid chromatography with UV-light detection. 12 out of 19 (63%) patients had <2 copies HIV-RNA/ml in CSF. Seven subjects ranged between 3 and 39 copies/ml, 2 of whom had a slightly higher viral load in CSF than in plasma. NFV was detected in CSF in 16 out of 18 patients analysed and was quantifiable in 8 patients, at concentrations ranging from 6 to 29 nM. There was no correlation between NFV concentration and HIV-RNA levels. Long-term therapy with NFV + 2 NRTI showed no increased rate of virological treatment failure within the central nervous system (CNS) in compliant patients, despite earlier reports of lack of NFV penetration to CNS. Using a highly sensitive method, NFV was detected and quantified in the CSF, although at low values, which could have contributed to the high anti-HIV-1 efficacy of the therapy seen in our subjects.

A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression.

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.

Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine.

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.