Clarithromycin substantially increases steady-state bosentan exposure in healthy volunteers.

AIMS: The aim of this study was to assess the effect of the cytochrome P450 (CYP) 3A4 and organic anion-transporting polypeptide (OATP) 1B1 inhibitor clarithromycin on the pharmacokinetics of bosentan. We also aimed to evaluate the impact of CYP2C9 and SLCO1B1 (encoding for OATP1B1) genotypes and their combination. METHODS: We assessed the effect of the OATP and CYP3A inhibitor clarithromycin on bosentan pharmacokinetics at steady state and concurrently quantified changes of CYP3A activity using midazolam as a probe drug. Sixteen healthy volunteers received therapeutic doses of bosentan (125 mg twice daily) for 14 days and clarithromycin (500 mg twice daily) concomitantly for the last 4 days, and bosentan pharmacokinetics was assessed on days 1, 10 and 14. RESULTS: Clarithromycin significantly increased bosentan area under the plasma concentration-time curve of the dosing interval 3.7-fold and peak concentration 3.8-fold in all participants irrespective of the genotype. Clarithromycin also reduced CYP3A activity (midazolam clearance) in all participants; however, these changes were not correlated to the changes of bosentan clearance. CONCLUSIONS: Clarithromycin substantially increases the exposure to bosentan, suggesting that dose reductions may be necessary.

Influence of St. John's wort on the steady-state pharmacokinetics and metabolism of bosentan.

OBJECTIVE: We assessed the effect of St. John's wort (SJW) on bosentan pharmacokinetics at steady-state in different CYP2C9 genotypes in healthy volunteers. METHODS: Nine healthy extensive metabolizers of CYP2C9 and 4 poor metabolizers received therapeutic doses of bosentan (125 mg q.d. on study day 1; 62.5 mg b.i.d. on study day 2, 125 mg b.i.d. on study days 3 - 20) for 20 days and SJW (300 mg t.i.d.) concomitantly for the last 10 days. Bosentan pharmacokinetics was assessed on days 1, 10, and 20. Concurrently, we repeatedly quantified changes of CYP3A activity using low dosed midazolam (3 mg p.o.) as a probe drug. RESULTS: Due to auto-induction of its metabolism, Cl/F increased by 67%, thus significantly lowering bosentan exposure (AUC) to 60% after 10 days of bosentan administration (n = 13, p < 0.05). Concurrently, midazolam clearance (CYP3A activity) increased by 224% (n = 13, p < 0.05) and further increased after SJW by 374% compared to baseline (n = 13, p < 0.05). SJW increased midazolam clearance by 47% (n = 13, p < 0.05) but failed to alter bosentan exposure and clearance consistently. No significant differences in bosentan exposure and clearance changes were observed in CYP2C9 poor metabolizers. CONCLUSION: SJW increased CYP3A activity but had no consistent effect on bosentan clearance. However, inter-individual changes of the interaction were large, suggesting that close monitoring of bosentan effects may be advisable. The contribution of CYP2C9 to this interaction seems to be minor.

Lack of a clinically significant interaction of grapefruit juice with ambrisentan and bosentan in healthy adults.

OBJECTIVE: We assessed the effect of 1 x 300 mL/d and 3 x 300 mL/d grapefruit juice (GFJ) on ambrisentan and bosentan pharmacokinetics in healthy volunteers. METHODS: In the ambrisentan study, 12 healthy extensive metabolizers (EM) of CYP2C19 received therapeutic doses of ambrisentan (5 mg q.d. on study days 1 - 11) and GFJ (1 x 300 mL/d on study days 6 - 8 and 3 x 300 mL/d on study days 9 - 11). Ambrisentan pharmacokinetics was assessed on study days 5, 8, and 11. In the bosentan study, 16 healthy EM of CYP2C9, who were stratified into two groups (CYP3A5 expressors (n = 8) and CYP3A5 non-expressors (n = 8)), received bosentan (125 mg b.i.d. on study days 1 - 13) and GFJ (1 x 300 mL/d on study days 8 - 10 and 3 x 300 mL/d on study days 11 - 13). Bosentan pharmacokinetics was assessed on study days 7, 10, and 13. RESULTS: Whereas 1 x 300 mL/d GFJ had no effect on the pharmacokinetics of ambrisentan and its metabolite, 3 x 300 mL/d GFJ increased the exposure with ambrisentan by 8% and the molar plasma metabolic ratio by 22% (both p < 0.05). Correspondingly, the apparent oral clearance of ambrisentan decreased to 92% (p < 0.05). GFJ slightly prolonged t(max) of bosentan and increased the metabolic ratio of bosentan/hydroxy-bosentan by 19% (p < 0.05). CONCLUSION: GFJ had no clinically relevant effect on the pharmacokinetics or safety profile of ambrisentan and bosentan. Therefore, no dose adjustments are needed, and GFJ can be safely co-administered even at very high doses.

Steady-state pharmacokinetics and metabolism of voriconazole in patients.

OBJECTIVES: Voriconazole exhibits non-linear pharmacokinetics in adults and is said to be mainly metabolized by CYP2C19 and CYP3A4 to voriconazole-N-oxide. The aim of this study was to obtain data on steady-state pharmacokinetics after dosing for at least 14 days in patients taking additional medication and in vivo data on metabolites other than voriconazole-N-oxide. PATIENTS AND METHODS: Thirty-one patients receiving voriconazole as regular therapeutic drug treatment during hospitalization participated in this prospective study. Pharmacokinetic profiles were obtained for the 12 h (dosing interval) after the first orally administered dose (400 mg) or (if possible and) after an orally administered maintenance dose (200 mg) following intake for at least 14 days (n = 14 after first dose; n = 23 after maintenance dose). Blood and urine samples were collected and the concentrations of voriconazole and three of its metabolites (the N-oxide, hydroxy-voriconazole and dihydroxy-voriconazole) were determined, as well as the CYP2C19 genotype of the patients. All other drugs taken by the participating patients were evaluated. RESULTS: A high variability of exposure (AUC) after the first dose was slightly reduced during steady-state dosing for voriconazole (82% to 71%) and the N-oxide (86% to 56%), remained high for hydroxy-voriconazole (79%) and even increased for dihydroxy-voriconazole (97% to 127%). In 16 of the 22 steady-state patients, trough plasma concentrations were <2 µg/mL. N-oxide plasma concentrations during steady state stayed almost constant. Hydroxylations of voriconazole seem to be quantitatively more important in its metabolism than N-oxidation. CONCLUSIONS: High variability in voriconazole exposure, as well as low steady-state trough plasma concentrations, suggest that the suggested steady-state dosage of 200 mg twice a day has to be increased to prevent disease progression. Therapeutic drug monitoring is probably necessary to optimize the voriconazole dose for individual patients.

Trimethoprim-metformin interaction and its genetic modulation by OCT2 and MATE1 transporters.

AIMS: Metformin pharmacokinetics depends on the presence and activity of membrane-bound drug transporters and may be affected by transport inhibitors. The aim of this study was to investigate the effects of trimethoprim on metformin pharmacokinetics and genetic modulation by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) polymorphisms. METHODS: Twenty-four healthy volunteers received metformin 500 mg three times daily for 10 days and trimethoprim 200 mg twice daily from day 5 to 10. Effects of trimethoprim on steady-state metformin pharmacokinetics were analysed. RESULTS: In the population as a whole, trimethoprim significantly reduced the apparent systemic metformin clearance (CL/F) from 74 to 54 l h(-1) and renal metformin clearance from 31 to 21 l h(-1) , and prolonged half-life from 2.7 to 3.6 h (all P < 0.01). This resulted in an increase in the maximal plasma concentration by 38% and in the area under the plasma concentration-time curve by 37%. In volunteers polymorphic for both OCT2 and MATE1, trimethoprim had no relevant inhibitory effects on metformin kinetics. Trimethoprim was associated with a decrease in creatinine clearance from 133 to 106 ml min(-1) (P < 0.01) and an increase in plasma lactate from 0.94 to 1.2 mmol l(-1) (P = 0.016). CONCLUSIONS: The extent of inhibition by trimethoprim was moderate, but might be clinically relevant in patients with borderline renal function or high-dose metformin.

Contribution of CYP2C19 and CYP3A4 to the formation of the active nortilidine from the prodrug tilidine.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The analgesic activity of tilidine is mediated by its active metabolite, nortilidine, which easily penetrates the blood-brain barrier and binds to the µ-opioid receptor as a potent agonist. Tilidine undergoes an extensive first-pass metabolism, which has been suggested to be mediated by CYP3A4 and CYP2C19; furthermore, strong inhibition of CYP3A4 and CYP2C19 by voriconazole increased exposure of nortilidine, probably by inhibition of further metabolism. The novel CYP2C19 gene variant CYP2C19*17 causes ultrarapid drug metabolism, in contrast to the *2 and *3 variants, which result in impaired drug metabolism. WHAT THIS STUDY ADDS: Using a panel study with CYP2C19 ultrarapid and poor metabolizers, a major contribution of polymorphic CYP2C19 on tilidine metabolic elimination can be excluded. The potent CYP3A4 inhibitor ritonavir alters the sequential metabolism of tilidine, substantially reducing the partial metabolic clearances of tilidine to nortilidine and nortilidine to bisnortilidine, which increases the nortilidine exposure twofold. The lowest clearance in overall tilidine elimination is the N-demethylation of nortilidine to bisnortilidine. Inhibition of this step leads to accumulation of the active nortilidine. AIMS: To investigate in vivo the effect of the CYP2C19 genotype on the pharmacokinetics of tilidine and the contribution of CYP3A4 and CYP2C19 to the formation of nortilidine using potent CYP3A4 inhibition by ritonavir. METHODS: Fourteen healthy volunteers (seven CYP2C19 poor and seven ultrarapid metabolizers) received ritonavir orally (300 mg twice daily) for 3 days or placebo, together with a single oral dose of tilidine and naloxone (100 mg and 4 mg, respectively). Blood samples and urine were collected for 72 h. Noncompartmental analysis was performed to determine pharmacokinetic parameters of tilidine, nortilidine, bisnortilidine and ritonavir. RESULTS: Tilidine exposure increased sevenfold and terminal elimination half-life fivefold during ritonavir treatment, but no significant differences were observed between the CYP2C19 genotypes. During ritonavir treatment, nortilidine area under the concentration-time curve was on average doubled, with no differences between CYP2C19 poor metabolizers [2242 h ng ml(-1) (95% confidence interval 1811-2674) vs. 996 h ng ml(-1) (95% confidence interval 872-1119)] and ultrarapid metabolizers [2074 h ng ml(-1) (95% confidence interval 1353-2795) vs. 1059 h ng ml(-1) (95% confidence interval 789-1330)]. The plasma concentration-time curve of the secondary metabolite, bisnortilidine, showed a threefold increase of time to reach maximal observed plasma concentration; however, area under the concentration-time curve was not altered by ritonavir. CONCLUSIONS: The sequential metabolism of tilidine is inhibited by the potent CYP3A4 inhibitor, ritonavir, independent of the CYP2C19 genotype, with a twofold increase in the exposure of the active nortilidine.

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1'-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry.

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1'-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 µm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1'-hydroxymidazolam were quantified using deuterium- and (13)C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05-250 pg/mL) and 1'-hydroxymidazolam (0.25-125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1'-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1-100 µg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1'-hydroxymidazolam for at least 10 h, even after the administration of only 1 µg of midazolam.

Long-term effect of efavirenz autoinduction on plasma/peripheral blood mononuclear cell drug exposure and CD4 count is influenced by UGT2B7 and CYP2B6 genotypes among HIV patients.

OBJECTIVES: We investigated the long-term effect of efavirenz autoinduction on its plasma/peripheral blood mononuclear cell (PBMC) exposure and the CD4 count, and the importance of sex and pharmacogenetic variations. METHODS: Treatment-naive HIV patients (n = 163) received efavirenz-based antiretroviral therapy. Plasma and intracellular (PBMC) concentrations of efavirenz and 8-hydroxyefavirenz were determined at weeks 4 and 16 of antiretroviral therapy. CD4 count was determined at baseline, and at weeks 12, 24 and 48. Genotyping for CYP2B6*6, CYP3A5*3, CYP3A5*6, CYP3A5*7, ABCB1 3435C/T and UGT2B7 (-327G→A, *2) was done. RESULTS: There was a significant increase in the median plasma (32%) and intracellular (53%) 8-hydroxyefavirenz concentrations with a decrease in the efavirenz metabolic ratio (MR) (calculated by dividing the concentration of efavirenz by that of 8-hydroxyefavirenz) (20% and 5%, respectively) by week 16 compared with at week 4. While the CYP2B6 genotype significantly influenced efavirenz pharmacokinetics at weeks 4 and 16, the effect of the UGT2B7 genotype and sex was significant only at week 16. The Wilcoxon matched pairs test indicated that the change in 8-hydroxyefavirenz concentration and efavirenz MR over time was significant in females and in CYP2B6*1 and UGT2B7*1 carriers. The intracellular 8-hydroxyefavirenz level at week 16 was a negative predictor of the CD4 count at week 24 (P = 0.03) and at week 48 (P = 0.007). CYP2B6 (P = 0.02) and UGT2B7 (P = 0.05) genotypes predicted the CD4 count at week 48. Among CYP2B6*1/*1 and UGT2B7*1/*1 carriers there was no significant change in the mean CD4 count after week 24, while it continuously increased until week 48 in CYP2B6*6 and UGT2B7*2 carriers. CONCLUSIONS: The effects of long-term efavirenz autoinduction on its plasma/PBMC exposure and the CD4 count over time display wide interpatient variability, partly due to sex and CYP2B6 and UGT2B7 genetic variation. Patients with the CYP2B6*1/*1 and UGT2B7*1/*1 genotypes are at risk of suboptimal immune recovery due to pronounced long-term autoinduction.

Modulators of very low voriconazole concentrations in routine therapeutic drug monitoring.

Very low voriconazole concentrations are commonly observed during therapeutic drug monitoring. Possible mechanisms include inappropriate dose selection, rapid metabolism (as a result of genetic polymorphisms or enzyme induction), and also nonadherence. We aimed to develop a method to distinguish between rapid metabolism of and nonadherence to voriconazole by quantification of voriconazole metabolites. In addition, the relevance of common genetic polymorphisms of CYP2C19 was assessed. In a retrospective study, samples with voriconazole concentrations 0.2 µg/mL or less in routine therapeutic drug monitoring (as quantified by high-performance liquid chromatography) were evaluated. Voriconazole and its N-oxide metabolite were quantified in residual blood using a highly sensitive liquid chromatography-tandem mass spectroscopy method (lower limit of quantitation = 0.03 µg/mL). Genetic polymorphisms of CYP2C19 were determined by real-time polymerase chain reaction using the hybridization probe format and the polymerase chain reaction-random fragment length polymorphism format. A total of 747 routine therapeutic drug monitoring plasma/blood samples of 335 patients treated with systemic voriconazole were analyzed and in 18.7% of all samples, voriconazole concentrations 0.2 µg/mL or less were found. In 32 samples (30 patients) with adequate dosage and timing of blood withdrawal, nonadherence was strongly suspected in seven patients because voriconazole-N-oxide concentrations were below 0.03 µg/mL, which was not observed in a reference group of 51 healthy volunteers with controlled drug intake. In 10 patients, of whom EDTA blood was available, the ultrarapid metabolizer genotype (CYP2C19*1\*17, CYP2C19*17\*17) was found in 80% and its prevalence was significantly higher as compared to a reference group (P = 0.02). In conclusion, quantification of voriconazole-N-oxide allowed for detection of suspected nonadherence in one of four patients with very low voriconazole concentrations. In the remaining patients, ultrarapid metabolism resulting from the CYP2C19*17 polymorphism appears to play a major role. Thus, in the case of voriconazole therapy failure, both nonadherence and genetic factors have to be considered.

Pharmacokinetics of sulfobutylether-beta-cyclodextrin and voriconazole in patients with end-stage renal failure during treatment with two hemodialysis systems and hemodiafiltration.

Sulfobutylether-beta-cyclodextrin (SBECD), a large cyclic oligosaccharide that is used to solubilize voriconazole (VRC) for intravenous administration, is eliminated mainly by renal excretion. The pharmacokinetics of SBECD and voriconazole in patients undergoing extracorporeal renal replacement therapies are not well defined. We performed a three-period randomized crossover study of 15 patients with end-stage renal failure during 6-hour treatment with Genius dialysis, standard hemodialysis, or hemodiafiltration using a high-flux polysulfone membrane. At the start of renal replacement therapy, the patients received a single 2-h infusion of voriconazole (4 mg per kg of body weight) solubilized with SBECD. SBECD, voriconazole, and voriconazole-N-oxide concentrations were quantified in plasma and dialysate samples by high-performance liquid chromatography (HPLC) and by HPLC coupled to tandem mass spectrometry (LC-MS-MS) and analyzed by noncompartmental methods. Nonparametric repeated-measures analysis of variance (ANOVA) was used to analyze differences between treatment phases. SBECD and voriconazole recoveries in dialysate samples were 67% and 10% of the administered doses. SBECD concentrations declined with a half-life ranging from 2.6 +/- 0.6 h (Genius dialysis) to 2.4 +/- 0.9 h (hemodialysis) and 2.0 +/- 0.6 h (hemodiafiltration) (P < 0.01 for Genius dialysis versus hemodiafiltration). Prediction of steady-state conditions indicated that even with daily hemodialysis, SBECD will still exceed SBECD exposure of patients with normal renal function by a factor of 6.2. SBECD was effectively eliminated during 6 h of renal replacement therapy by all methods, using high-flux polysulfone membranes, whereas elimination of voriconazole was quantitatively insignificant. The SBECD half-life during renal replacement therapy was nearly normalized, but the average SBECD exposure during repeated administration is expected to be still increased.

The NK1 receptor antagonist aprepitant does not alter the pharmacokinetics of high-dose melphalan chemotherapy in patients with multiple myeloma.

AIMS: The objective of this investigation was to assess the effect of aprepitant on the pharmacokinetics of high-dose melphalan used as conditioning therapy before blood stem cell transplantation in multiple myeloma. METHODS: Aprepitant (125 mg) or placebo was administered 1 h before melphalan therapy (1 h infusion of 100 mg m⁻²). Eleven plasma samples were obtained over 8 h and melphalan was quantified using an LC/MS/MS method. Standard pharmacokinetic parameters were calculated and nonparametric testing was applied to assess the differences between aprepitant and placebo treatment. RESULTS: Twenty patients received placebo and 10 patients aprepitant treatment. There were no differences observed for C(max) at the end of melphalan infusion (placebo 3431 ± 608 ng ml⁻¹ vs. aprepitant 3269 ± 660 ng ml⁻¹). In addition, AUC and terminal elimination half-life were not changed by aprepitant. Total clearance of melphalan was 304 ± 58 ml min⁻¹ m⁻² (placebo) which was not influenced by aprepitant (288 ± 78 ml min⁻¹ m⁻²). CONCLUSIONS: The administration of the NK1 receptor antagonist aprepitant 1 h before a high-dose chemotherapy does not influence the exposure and the elimination of melphalan. Therefore, oral administration of 125 mg aprepitant 1 h before melphalan infusion does not alter the disposition of intravenously administered melphalan.

Inhibition of the active principle of the weak opioid tilidine by the triazole antifungal voriconazole.

AIMS: To investigate in vivo the influence of the potent CYP2C19 and CYP3A4 inhibitor voriconazole on the pharmacokinetics and analgesic effects of tilidine. METHODS: Sixteen healthy volunteers received voriconazole (400 mg) or placebo together with a single oral dose of tilidine (100 mg). Blood samples and urine were collected for 24 h and experimental pain was determined by using the cold pressor test. Noncompartimental analysis was performed to determine pharmacokinetic parameters of tilidine, nortilidine and voriconazole, whereas pharmacodynamic parameters were analysed by nonparametric repeated measures ANOVA (Friedman). RESULTS: Voriconazole caused a 20-fold increase in exposition of tilidine in serum [AUC 1250.8 h*ng ml(-1), 95% confidence interval (CI) 1076.8, 1424.9 vs. 61 h*ng ml(-1), 95% CI 42.6, 80.9; P < 0.0001], whereas the AUC of nortilidine also increased 2.5-fold. After voriconazole much lower serum concentrations of bisnortilidine were observed. The onset of analgesic activity occurred later with voriconazole, which is in agreement with the prolonged t(max) of nortilidine (0.78 h, 95% CI 0.63, 0.93 vs. 2.5 h, 95% CI 1.85, 3.18; P < 0.0001) due to the additional inhibition of nortilidine metabolism to bisnortilidine. After voriconazole the AUC under the pain withdrawal-time curve was reduced compared with placebo (149 s h(-1), 95% CI 112, 185 vs. 175 s h(-1), 95% CI 138, 213; P < 0.016), mainly due to the shorter withdrawal time 0.75 h after tilidine administration. CONCLUSIONS: Voriconazole significantly inhibited the sequential metabolism of tilidine with increased exposure of the active nortilidine. Furthermore, the incidence of adverse events was almost doubled after voriconazole and tilidine.

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.

In vitro metabolism of the opioid tilidine and interaction of tilidine and nortilidine with CYP3A4, CYP2C19, and CYP2D6.

Tilidine is one of the most widely used narcotics in Germany and Belgium. The compound's active metabolite nortilidine easily penetrates the blood-brain barrier and activates the mu-opioid receptor. Thus far, the enzymes involved in tilidine metabolism are unknown. Therefore, the aim of our study was to identify the cytochrome P450 isozymes (CYPs) involved in N-demethylation of tilidine in vitro. We used human liver microsomes as well as recombinant CYPs to investigate the demethylation of tilidine to nortilidine and quantified nortilidine by liquid chromatography-tandem mass spectrometry. Inhibition of CYPs was quantified with commercial kits. Moreover, inhibition of ABCB1 and ABCG2 was investigated. Our results demonstrated that N-demethylation of tilidine to nortilidine followed a Michaelis-Menten kinetic with a K(m) value of 36 +/- 13 microM and a v(max) value of 85 +/- 18 nmol/mg/h. This metabolic step was inhibited by CYP3A4 and CYP2C19 inhibitors. Investigations with recombinant CYP3A4 and CYP2C19 confirmed that the demethylation of tilidine occurs via these two CYPs. Inhibition assays demonstrated that tilidine and nortilidine can also inhibit CYP3A4, CYP2C19, CYP2D6, ABCB1, but not ABCG2, whereas inhibition of CYP2D6 and possibly also of CYP3A4 might be clinically relevant. By calculating the metabolic clearance based on the in vitro and published in vivo data, CYP3A4 and CYP2C19 were identified as the main elimination routes of tilidine. In vivo, drug-drug interactions of tilidine with CYP3A4 or CYP2C19 inhibitors are to be anticipated, whereas substrates of CYP2C19, ABCB1, or ABCG2 will presumably not be influenced by tilidine or nortilidine.

Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry.

Selective and sensitive methods for the determination of the cationic dye and anti-malarial methylene blue in human liquid whole blood, dried whole blood (paper spot), and plasma depending on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) have been developed, validated according to FDA standards, and applied to samples of healthy individuals and malaria patients within clinical studies. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for liquid whole blood and plasma. For the extraction of methylene blue from paper spots aqueous acetonitrile was used. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as internal standard. Depending on the sample volume (whole blood and plasma 250 microL, and 100 microL on paper spots) the method was linear at least within 75 and 10,000 ng/mL and the limit of quantification in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood, plasma, and paper spot methods varied between -4.5 and +6.6%, -3.7 and +7.5%, and -5.8 and +11.1%, respectively, with corresponding precision ranging from 3.8 to 11.8% CV. After a single oral dose (500 mg) methylene blue concentrations were detectable for 72 h in plasma. The methods were applied within clinical studies to samples from healthy individuals and malaria patients from Burkina Faso.

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

OBJECTIVES: 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. METHODS: In a randomized, placebo-controlled crossover study, 20 healthy participants who were stratified according to CYP2C19 genotype received oral ritonavir (300 mg twice daily) or placebo for 2 days. Together with the first ritonavir or placebo dose, a single oral dose of 400 mg voriconazole was administered. Voriconazole was determined in plasma and urine by liquid chromatography-mass spectrometry, and pharmacokinetic parameters were estimated by noncompartmental analysis. RESULTS: When given alone, the apparent oral clearance of voriconazole after single oral dosing was 26%+/-16% (P > .05) lower in CYP2C19*1/*2 individuals and 66%+/-14% (P < .01) lower in CYP2C19 PMs. The addition of ritonavir caused a major reduction in voriconazole apparent oral clearance (354+/-173 mL/min versus 202+/-139 mL/min, P = .0001). This reduction occurred in all CYP2C19 genotypes (463+/-168 mL/min versus 305+/-112 mL/min [P = .023] for *1/*1, 343+/-127 mL/min versus 190+/-93 mL/min [P = .008] for *1/*2, and 158+/-54 mL/min versus 22+/-11 mL/min for *2/*2) and is probably caused by inhibition of CYP3A4-mediated voriconazole metabolism. CONCLUSIONS: Coadministration of a potent CYP3A4 inhibitor leads to a higher and prolonged exposure with voriconazole that might increase the risk of the development of adverse drug reactions on a short-term basis, particularly in CYP2C19 PM patients.

Voriconazole brain tissue levels in rhinocerebral aspergillosis in a successfully treated young woman.

Invasive aspergillosis of the central nervous system has a mortality rate exceeding 90%. We describe a 29-year-old woman with a medical history of chronic polyarthritis who developed a proven rhinocerebral Aspergillus fumigatus infection refractory to first-line treatment with liposomal amphotericin B. The patient responded successfully to salvage combination treatment with voriconazole and caspofungin. Furthermore, for the first time, voriconazole levels in an intracerebral abscess were measured in this patient undergoing voriconazole oral therapy.

Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine.

The development of safe, effective and affordable drug combinations against malaria in Africa is a public health priority. Methylene blue (MB) has a similar mode of action as chloroquine (CQ) and has moreover been shown to selectively inhibit the Plasmodium falciparum glutathione reductase. In 2004, an uncontrolled dose-finding study on the combination MB-CQ was performed in 435 young children with uncomplicated falciparum malaria in Burkina Faso (CQ monotherapy had a > 50% clinical failure rate in this area in 2003). Three serious adverse events (SAE) occurred of which one was probably attributable to the study medication. In the per protocol safety analysis, there were no dose specific effects. The overall clinical and parasitological failure rates by day 14 were 10% [95% CI (7.5%, 14.0%)] and 24% [95% CI (19.4%, 28.3%)], respectively. MB appears to have efficacy against malaria, but the combination of CQ-MB is clearly not effective in the treatment of malaria in Africa.

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

OBJECTIVES: Constituents of St John's 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. METHODS: In a controlled, open-label study, single oral doses of 400 mg voriconazole were administered to 16 healthy men stratified for CYP2C19 genotype before and on day 1 and day 15 of concomitant SJW intake (300 mg LI 160 3 times daily). Plasma and urine concentrations of voriconazole were determined by liquid chromatography with mass-spectrometric detection. RESULTS: During the initial 10 hours of the first day of SJW administration, the area under the voriconazole plasma concentration-time curve was increased by 22% compared with control (15.5 +/- 6.84 h . microg/mL versus 12.7 +/- 4.16 h . microg/mL, P = .02). After 15 days of SJW intake, the area under the plasma concentration-time curve from hour 0 to infinity was reduced by 59% compared with control (9.63 +/- 6.03 h . microg/mL versus 23.5 +/- 15.6 h . microg/mL, P = .0004), with a corresponding increase in oral voriconazole clearance (CL/F) from 390 +/- 192 to 952 +/- 524 mL/min (P = .0004). The baseline CL/F of voriconazole and the absolute increase in CL/F were smaller in carriers of 1 or 2 deficient CYP2C19*2 alleles compared with wild-type individuals (P < .03). CONCLUSIONS: Coadministration of SJW leads to a short-term but clinically irrelevant increase followed by a prolonged extensive reduction in voriconazole exposure. SJW might put CYP2C19 wild-type individuals at highest risk for potential voriconazole treatment failure.

Safety of the methylene blue plus chloroquine combination in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso [ISRCTN27290841].

BACKGROUND: Safe, effective and affordable drug combinations against falciparum malaria are urgently needed for the poor populations in malaria endemic countries. Methylene blue (MB) combined with chloroquine (CQ) has been considered as one promising new regimen. OBJECTIVES: The primary objective of this study was to evaluate the safety of CQ-MB in African children with uncomplicated falciparum malaria. Secondary objectives were to assess the efficacy and the acceptance of CQ-MB in a rural population of West Africa. METHODS: In this hospital-based randomized controlled trial, 226 children (6-59 months) with uncomplicated falciparum malaria were treated in Burkina Faso. The children were 4:1 randomized to CQ-MB (n = 181; 25 mg/kg CQ and 12 mg/kg MB over three days) or CQ (n = 45; 25 mg/kg over three days) respectively. The primary outcome was the incidence of severe haemolysis or other serious adverse events (SAEs). Efficacy outcomes were defined according to the WHO 2003 classification system. Patients were hospitalized for four days and followed up until day 14. RESULTS: No differences in the incidence of SAEs and other adverse events were observed between children treated with CQ-MB (including 24 cases of G6PD deficiency) compared to children treated with CQ. There was no case of severe haemolysis and also no significant difference in mean haemoglobin between study groups. Treatment failure rates were 53.7% (95% CI [37.4%; 69.3%]) in the CQ group compared to 44.0% (95% CI [36.3%; 51.9%]) in the CQ-MB group. CONCLUSION: MB is safe for the treatment of uncomplicated falciparum malaria, even in G6PD deficient African children. However, the efficacy of the CQ-MB combination has not been sufficient at the MB dose used in this study. Future studies need to assess the efficacy of MB at higher doses and in combination with appropriate partner drugs.