Johanna Weiss

Substantial pharmacokinetic interaction between digoxin and ritonavir in healthy volunteers

Ritonavir is a potent in vitro inhibitor of several cytochrome P450 isozymes and ABC transporters including the efflux pump P‐glycoprotein (P‐gp). This study assessed the effect of repetitive ritonavir administration on digoxin distribution and total and renal digoxin clearance as a marker for P‐gp activity in vivo.

Opposite effects of short‐term and long‐term St John's wort intake on voriconazole pharmacokinetics

Constituents of St Johns wort (SJW) in vivo induce the cytochrome P450 (CYP) isozymes 3A4, 2C9, and 2C19 but in vitro were shown to inhibit them. This study investigates both short‐ and long‐term effects of SJW on the antifungal voriconazole, which is metabolized by these enzymes.

CYP2C19 genotype is a major factor contributing to the highly variable pharmacokinetics of voriconazole.

In vitro data on the metabolism of the antifungal voriconazole suggest that its pharmacokinetics might be influenced by the activity of CYP2C19, CYP2C9, and CYP3A. To elucidate the genetic influence of polymorphic enzymes on voriconazole metabolism, the authors pooled the pharmacokinetic data from 2 interaction studies in which 35 participants were enrolled according to their CYP2C19 genotype to receive a single 400‐mg oral dose of voriconazole. Nine participants were homozygous for CYP2C19*1/*1, 8 heterozygous for *1/*17, 11 heterozygous for*1/*2, 2 heterozygous for *2/*17, 4 homozygous for *2/*2, and 1 with a double mutation CYP2C19*2/*2*17. Nine (heterozygous) individuals were carriers of the CYP2C9*2 or *3 variant alleles. Twenty‐five participants did not express the CYP3A5 isozyme (*3/*3), whereas in 5 individuals, the *1/*3 combination was present (active enzyme). In addition, the CYP2D6 genotype and 2 variants of the drug transporter MDR1 (C3435T and G2677T) were determined. Multiple regression analysis of voriconazole apparent oral clearance revealed that 49% of its variance can be explained solely by the CYP2C19 polymorphism (P < .0001). Including the other polymorphisms into the regression model did not show any significant contribution. The number of variant CYP2C19 alleles therefore explains a substantial part of the wide variability of voriconazole pharmacokinetics, whereas the presence of functional CYP3A5 and the CYP2C9 genotype had no significant impact on voriconazole exposure. Some minor contribution results from the MDR1 C3435T genotype.

Potent cytochrome P450 2C19 genotype-related interaction between voriconazole and the cytochrome P450 3A4 inhibitor ritonavir.

Cytochrome P450 (CYP) 2C19 and CYP3A4 are the major enzymes responsible for voriconazole elimination. Because the activity of CYP2C19 is under genetic control, the extent of inhibition with a CYP3A4 inhibitor was expected to be modulated by the CYP2C19 metabolizer status. This study thus assessed the effect of the potent CYP3A4 inhibitor ritonavir after short‐term administration on voriconazole pharmacokinetics in extensive metabolizers (EMs) and poor metabolizers (PMs) of CYP2C19.

Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by nucleoside, nucleotide, and non-nucleoside reverse transcriptase inhibitors

Many drug interactions with drugs used for the therapy of human immunodeficiency virus (HIV) occur at the level of different cytochrome P450 isozymes. Increasing evidence suggests that antiretrovirals may also modify activity and expression of active drug transport systems. Such interactions may alter drug absorption, elimination, and also drug distribution and reach clinical importance if thereby access to the target site is affected. Beyond P-glycoprotein, the family of multidrug resistance-related proteins (MRP/ABCC) substantially contributes to the elimination of numerous drugs and their metabolites. Because the interaction of MRPs with non–HIV protease inhibitor antiretrovirals has not been studied thoroughly, we investigated whether important non-nucleoside reverse transcriptase inhibitors (NNRTI) (delavirdine, efavirenz, and nevirapine), nucleoside reverse transcriptase inhibitors (NRTI) (abacavir, emtricitabine, and lamivudine), and tenofovir as a nonnucleotide reverse transcriptase inhibitor can interact with MRP1, MRP2, and MRP3 in vitro. Inhibition of these ABC transporters was quantified by confocal laser-scanning microscopy using the 5-chloromethylfluorescein diacetate assay. With the exception of abacavir, which had no effect on MRP3, all the test compounds increased intracellular 5-chloromethylfluorescein fluorescence in a concentration-dependent manner, and this effect was observed in all the overexpressing cell lines but not in the parental cell line, indicating inhibition of MRP1, MRP2, and MRP3. In conclusion, the present study provides the first evidence for a significant and concentration-dependent inhibition of MRPs by NNRTI, NRTI, and tenofovir, which was most pronounced for delavirdine, efavirenz, and emtricitabine, suggesting that this might contribute to some of the known drug interactions impairing HIV therapy and also to the superior effectiveness of combination pharmacotherapy.

Pharmacokinetics, metabolism and bioavailability of the triazole antifungal agent voriconazole in relation to CYP2C19 genotype

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Pharmacokinetic variability of voriconazole is largely caused by CYP3A4- and CYP2C19-mediated metabolism. * Oral bioavailability of voriconazole has been claimed to be almost 100%, thus facilitating a change from intravenous to oral application without dose adjustment. WHAT THIS STUDY ADDS * For the first time voriconazole exposure after intravenous and oral administration in relation to CYP2C19 activity is reported. * In addition, the predominant metabolic pathway is the hydroxylation that seems to be influenced by the CYP2C19 genotype. * Enterohepatic circulation of both hydroxylated metabolites must be anticipated. AIMS The aim was to determine the pharmacokinetics of voriconazole after a single oral dose in comparison with intravenous (i.v.) administration in healthy individuals stratified according to the cytochrome P450 (CYP) 2C19 genotype. In addition, the possible metabolic pathways and their modulation according to CYP2C19 genotype were investigated after oral and i.v. administration of voriconazole. METHODS In a single-centre, open-label, two-period crossover study 20 participants received single doses of 400 mg voriconazole orally and 400 mg voriconazole intravenously in randomized order. Blood and urine samples were collected up to 96 h post dose and the voriconazole and three major metabolites were quantified by high-performance liquid chromatography coupled to mass spectroscopy. RESULTS Absolute oral bioavailability of voriconazole was 82.6% (74.1, 91.0). It ranged from 94.4% (78.8, 109.9) in CYP2C19 poor metabolizers to 75.2% (62.9, 87.4) in extensive metabolizers. In contrast to voriconazole and its N-oxide, the plasma concentrations of both hydroxylated metabolites showed a large second peak after 24 h. Independent of the route of administration, voriconazole partial metabolic hydroxylation after i.v. administration was eightfold higher compared with N-oxidation [48.8 ml min(-1) (30.5, 67.1) vs. 6.1 ml min(-1) (4.1, 8.0)]. The formation of the metabolites was related to CYP2C19 activity. CONCLUSIONS Independent of the route of administration, voriconazole exposure was three times higher in CYP2C19 poor metabolizers compared with extensive metabolizers. Voriconazole has a high bioavailability with no large differences between the CYP2C19 genotypes. The hydroxylation pathway of voriconazole elimination exceeded the N-oxidation, both influenced by the CYP2C19 genotype.

Localization of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) in Lipid Rafts/Caveolae and Modulation of Its Activity by Cholesterol in Vitro

Breast cancer resistance protein (BCRP/ABCG2) is an active efflux pump that belongs to the ATP-binding cassette (ABC) transporter family. It is located in various tissues involved in drug absorption, distribution, and elimination and plays an important role in multidrug resistance. For P-glycoprotein, another member of the ABC transporter family, it is well established that it is at least partly located in cholesterol and sphingolipid-enriched domains of the plasma membrane called “lipid rafts” and that the composition of the membrane lipids may modulate its efflux activity. This study addressed the compartmentalization of BCRP in the plasma membrane and the influence of membrane cholesterol on the efflux activity of BCRP. As a cell model, we used the canine kidney epithelial cell line MDCKII-BCRP transfected with the cDNA encoding human BCRP and the corresponding parental cell line MDCKII. Cholesterol depletion with methyl-β-cyclodextrin (MβCD) provoked a 40% decrease in BCRP activity (p < 0.01) assessed with flow cytometry (pheophorbide A efflux assay). Cholesterol repletion with MβCD/cholesterol-inclusion complexes restored BCRP function, and cholesterol saturation of native cells did not further enhance BCRP activity. Coimmunoprecipitation experiments indicated a physical interaction between BCRP and caveolin-1, and Western blot analysis after density gradient ultracentrifugation demonstrated that BCRP is located in detergent-resistant membranes that also contain caveolin-1. In conclusion, our results demonstrate for the first time that BCRP is located in membrane rafts and that cholesterol has impact on its efflux activity.

Pharmacokinetic and pharmaceutic interaction between digoxin and Cremophor RH40

The pharmacokinetics of digoxin is modulated by the efflux pump P‐glycoprotein. Cremophor EL (BASF Aktiengesellschaft, Ludwigshafen, Germany) (polyoxyl 35 castor oil), a castor oil derivative used to improve the solubilization of drugs and vitamins, has been shown to inhibit this membrane transporter in vitro and in vivo. So far, no study in humans has evaluated the effect of Cremophor RH40 (BASF Aktiengesellschaft) (polyoxyl 40 hydrogenated castor oil) on P‐glycoprotein.

Impact of drug transporters on cellular resistance towards saquinavir and darunavir

OBJECTIVES Highly active antiretroviral therapy is complicated by drug-drug interactions and the development of viral resistance. Drug interactions involve transporters that may critically affect the pharmacokinetics of many antiretroviral drugs and contribute to the formation of functional sanctuary sites. We therefore investigated the effect of saquinavir and darunavir on drug transporter expression and functional consequences for cellular resistance towards these compounds. METHODS Induction of transporters was investigated in LS180 cells over a period of 4 weeks by means of RT-PCR, and for some transporters also at the protein and functional levels. Cellular resistance was measured by growth inhibition assays. RESULTS Incubation with 10 µM darunavir for 1 week significantly increased mRNA expression of P-glycoprotein (P-gp/MDR1/ABCB1) 3.8-fold and of organic anion-transporting polypeptide 2B1 (SLCO2B1) 1.9-fold. In contrast, 10 µM saquinavir significantly increased mRNA expression of P-gp 5.7-fold, multidrug resistance-associated protein 1 (MRP1/ABCC1) 2.3-fold, MRP2/ABCC2 4.5-fold, MRP3/ABCC3 2.0-fold, MRP4/ABCC4 1.8-fold, MRP5/ABCC5 3.8-fold, breast cancer resistance protein (BCRP/ABCG2) 4.1-fold, SLCO1B1 4.6-fold, SLCO2B1 1.8-fold and SLCO3A1 1.8-fold. P-gp induction was also confirmed at the protein and functional levels. Induction by darunavir caused an increase in cellular resistance towards this compound, as measured in growth inhibition assays; however, saquinavir treatment did not cause reduced sensitivity of cells, indicating unchanged intracellular concentration. Hence, induction by darunavir increased drug efflux and might therefore lead to a suboptimal intracellular concentration of darunavir. CONCLUSIONS The study revealed substantial induction of several drug transporters by saquinavir and darunavir, possibly leading to decreased efficacy of antiretrovirals and drugs used to treat co-morbidity.

Potential of novel antiretrovirals to modulate expression and function of drug transporters in vitro

OBJECTIVES The chemokine receptor antagonists maraviroc and vicriviroc and the integrase inhibitors elvitegravir and raltegravir are novel antiretroviral agents for the treatment of HIV-1 infections. ATP-binding cassette (ABC) transporters as modulators of the effectiveness and safety of therapy can mediate viral resistance and drug-drug interactions. To expand knowledge on drug-drug interactions of these antiretrovirals we investigated whether these compounds are substrates, inhibitors or inducers of important ABC transporters. METHODS We evaluated P-glycoprotein (P-gp/ABCB1) inhibition by the calcein assay in P388/dx and L-MDR1 cells, breast cancer resistance protein (BCRP/ABCG2) inhibition in MDCKII-BCRP cells by pheophorbide A efflux, and inhibition of the multidrug resistance-associated protein 2 (MRP2/ABCC2) by using the MRP2 PREDIVEZ™ Vesicular Transport Kit. Substrate characteristics were evaluated by growth inhibition assays in MDCKII cells overexpressing particular ABC transporters. Induction of transporters was quantified by real-time RT-PCR in LS180 cells and for ABCB1 also at the functional level. RESULTS Elvitegravir and vicriviroc inhibited ABCB1 in P388/dx and L-MDR1 cells (f2 values 1.9±0.2 µmol/L and 8.5±3.6 µmol/L, respectively). The IC50 for ABCG2 inhibition was 15.7±5.7 µmol/L for elvitegravir and 236.7±93.3 µmol/L for vicriviroc. Raltegravir and maraviroc showed no evidence of ABCB1 or ABCG2 inhibition. Maraviroc and vicriviroc stimulated ABCC2 transport function. Growth inhibition assays suggest that elvitegravir, raltegravir and vicriviroc are substrates of ABCB1. Induction assays demonstrate that mRNA expression of several ABC transporters is induced by these antiretrovirals in LS180 cells. CONCLUSIONS The new antiretrovirals bear the potential to modulate expression and function of several ABC transporters, with elvitegravir revealing the highest interaction potential.