Walter E. Haefeli

Nitric Oxide Is Responsible for Flow-Dependent Dilatation of Human Peripheral Conduit Arteries In Vivo

BACKGROUND Experimental evidence suggests that flow-dependent dilatation of conduit arteries is mediated by nitric oxide (NO) and/or prostacyclin. The present study was designed to assess whether NO or prostacyclin also contributes to flow-dependent dilatation of conduit arteries in humans. METHODS AND RESULTS Radial artery internal diameter (ID) was measured continuously in 16 healthy volunteers (age, 24 +/- 1 years) with a transcutaneous A-mode echo-tracking system coupled to a Doppler device for the measurement of radial blood flow. In 8 subjects, a catheter was inserted into the brachial artery for measurement of arterial pressure and infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 8 mumol/min for 7 minutes; infusion rate, 0.8 mL/min). Flow-dependent dilatation was evaluated before and after L-NMMA or aspirin as the response of the radial artery to an acute increase in flow (reactive hyperemia after a 3-minute cuff wrist occlusion). Under control conditions, release of the occlusion induced a marked increase in radial blood flow (from 24 +/- 3 to 73 +/- 11 mL/min; P < .01) followed by a delayed increase in radial diameter (flow-mediated dilatation; from 2.67 +/- 0.10 to 2.77 +/- 0.12 mm; P < .01) without any change in heart rate or arterial pressure. L-NMMA decreased basal forearm blood flow (from 24 +/- 3 to 13 +/- 3 mL/min; P < .05) without affecting basal radial artery diameter, heart rate, or arterial pressure, whereas aspirin (1 g PO) was without any hemodynamic effect. In the presence of L-NMMA, the peak flow response during hyperemia was not affected (76 +/- 12 mL/min), but the duration of the hyperemic response was markedly reduced, and the flow-dependent dilatation of the radial artery was abolished and converted to a vasoconstriction (from 2.62 +/- 0.11 to 2.55 +/- 0.11 mm; P < .01). In contrast, aspirin did not affect the hyperemic response nor the flow-dependent dilatation of the radial artery. CONCLUSIONS The present investigation demonstrates that NO, but not prostacyclin, is essential for flow-mediated dilatation of large human arteries. Hence, this response can be used as a test for the L-arginine/NO pathway in clinical studies.

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.

Voriconazole Concentration in Human Aqueous Humor and Plasma during Topical or Combined Topical and Systemic Administration for Fungal Keratitis

ABSTRACT Voriconazole (VRC) is an antifungal drug that effectively treats keratitis caused by yeasts and molds when administered orally. We retrospectively evaluated clinical outcomes and plasma and aqueous humor drug concentrations in five patients with fungal keratitis and one patient with posttraumatic endophthalmitis who were treated with VRC. VRC was administered either topically (1% eye drops every hour) or orally (400 mg twice a day). Plasma and aqueous humor samples from affected eyes were taken 12 h after oral administration or 1 h after eye drop application. The drug concentration was measured by liquid chromatography with UV or mass spectrometric detection. All six patients responded well to VRC treatment. The VRC concentration ranged from 2.93 to 3.40 mg/liter in the aqueous humor and from 3.20 to 4.20 mg/liter in the plasma after combined oral and topical treatment. Topical administration alone resulted in highly variable trough VRC concentrations of 0.61 to 3.30 mg/liter in the aqueous humor. VRC concentrations were above the MIC for Candida albicans Aspergillus fumigatus and clinical improvement was seen in all four patients with C. albicans and A. fumigatus keratitis. Combined orally and topically administered VRC resulted in aqueous humor drug concentrations of ≥2.93 mg/liter, which is above the VRC MIC for most fungi. Topical VRC treatment resulted in an aqueous humor drug concentration >0.61 mg/liter, which is above the MIC for most Candida species. The results from this small series of patients suggest that both topical and combined systemic and topical VRC therapy can be effective in treating fungal keratitis. Furthermore, the data provide preliminary support for initiation of VRC treatment with a combined topical and systemic administration until the causative fungus and its MIC are identified.

Drug Dosage in Patients with Renal Failure Optimized by Immediate Concurrent Feedback

AbstractOBJECTIVE: To examine the impact of immediate concurrent feedback on dose adjustment in patients with renal failure. DESIGN: Prospective 12-month study in patients with various degrees of renal failure, with comparison to a retrospective control group. SETTING: A 39-bed unit of a university hospital providing primary and tertiary care. PATIENTS: Patients with renal failure (estimated creatinine clearance ≤50 mL/min) receiving at least 1 pharmacologically active drug. INTERVENTIONS: Education of physicians and immediate concurrent feedback on the ward giving estimated creatinine clearance and dose recommendations for renally eliminated drugs adjusted to individual renal function. MEASUREMENTS AND MAIN RESULTS: The percentage of dosage regimens adjusted to renal function and cost assessment of drug therapy were calculated. Overall, 17% of the patients had at least 1 estimated creatinine clearance ≤50 mL/min. In the intervention group, the dose of 81% of renally eliminated drugs was adjusted to renal function, compared with 33% in the control group (P<.001). The mean difference in cost between standard and adjusted dose of renally eliminated drugs in the intervention and control groups was 5.3±12.3 and 0.75±2.8 Swiss francs (approximately US