Higher clearance of micafungin in neonates compared with adults: role of age-dependent micafungin serum binding.

Micafungin, a new echinocandin antifungal agent, has been used widely for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins. Micafungin body weight-adjusted clearance is higher in neonates than in adults, but the mechanisms underlying this difference are not understood. Previous work had revealed the roles of sinusoidal uptake (Na(+) -taurocholate co-transporting peptide, NTCP; organic anion transporting polypeptide, OATP) as well as canalicular efflux (bile salt export pump, BSEP; breast cancer resistance protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 were similar in neonates and in adults. However, the micafungin fraction unbound (f(u) ) in neonatal serum was about 8-fold higher than in the adult serum (0.033±0.012 versus 0.004±0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent serum protein binding of micafungin is responsible for its higher clearance in neonates compared with adults.

In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats.

The purpose of the present study was to elucidate the transport mechanisms responsible for elimination of micafungin, a new semisynthetic echinocandin antifungal agent, which is predominantly cleared by biliary excretion in humans and rats. In vitro studies using sandwich-cultured rat and human hepatocytes were conducted. Micafungin uptake occurred primarily (∼75%) by transporter-mediated mechanisms in rat and human. Micafungin uptake into hepatocytes was inhibited by taurocholate (K(i) = 61 µM), Na(+) depletion (45-55% reduced), and 10 µM rifampin (20-25% reduced); these observations support the involvement of Na(+)-taurocholate-cotransporting polypeptide (NTCP/Ntcp) and, to a lesser extent, organic anion-transporting polypeptides in the hepatic uptake of micafungin. The in vitro biliary clearance of micafungin, as measured by the B-CLEAR technique, amounted to 14 and 19 µl/(min · mg protein) in human and rat, respectively. In vitro biliary excretion of micafungin was reduced by 80 and 75% in the presence of the bile salt export pump (BSEP) inhibitors taurocholate (100 µM) and nefazodone (25 µM), respectively. Biliary excretion of micafungin also was reduced in the presence of breast cancer resistance protein inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 µM) and fumitremorgin C (10 µM)]. In vitro biliary excretion of micafungin was not significantly altered by coincubation with P-glycoprotein or multidrug resistance-associated protein 2 inhibitors. These results suggest that NTCP/Ntcp and BSEP/Bsep are primarily responsible for hepatobiliary disposition of micafungin in human and rat. Interference with hepatic bile acid disposition could be one mechanism underlying hepatotoxicity associated with micafungin in some patients.

In vitro hepatic metabolism explains higher clearance of voriconazole in children versus adults: role of CYP2C19 and flavin-containing monooxygenase 3.

Voriconazole is a broad spectrum antifungal agent for treating life-threatening fungal infections. Its clearance is approximately 3-fold higher in children compared with adults. Voriconazole is cleared predominantly via hepatic metabolism in adults, mainly by CYP3A4, CYP2C19, and flavin-containing monooxygenase 3 (FMO3). In vitro metabolism of voriconazole by liver microsomes prepared from pediatric and adult tissues (n = 6/group) mirrored the in vivo clearance differences in children versus adults, and it showed that the oxidative metabolism was significantly faster in children compared with adults as indicated by the in vitro half-life (T(1/2)) of 33.8 + or - 15.3 versus 72.6 + or - 23.7 min, respectively. The K(m) for voriconazole metabolism to N-oxide, the major metabolite formed in humans, by liver microsomes from children and adults was similar (11 + or - 5.2 versus 9.3 + or - 3.6 microM, respectively). In contrast, apparent V(max) was approximately 3-fold higher in children compared with adults (120.5 + or - 99.9 versus 40 + or - 13.9 pmol/min/mg). The calculated in vivo clearance from in vitro data was found to be approximately 80% of the observed plasma clearance values in both populations. Metabolism studies in which CYP3A4, CYP2C19, or FMO was selectively inhibited provided evidence that contribution of CYP2C19 and FMO toward voriconazole N-oxidation was much greater in children than in adults, whereas CYP3A4 played a larger role in adults. Although expression of CYP2C19 and FMO3 is not significantly different in children versus adults, these enzymes seem to contribute to higher metabolic clearance of voriconazole in children versus adults.

Fexofenadine brain exposure and the influence of blood-brain barrier P-glycoprotein after fexofenadine and terfenadine administration.

P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administration demonstrated that fexofenadine brain penetration was 48-fold higher in mdr1a(-/-) mice than in mdr1a(+/+) mice. In contrast, the P-gp efflux ratio at the blood-brain barrier (BBB) for fexofenadine was only approximately 4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp efflux ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetration after terfenadine administration was approximately 25- to 27-fold higher than after fexofenadine administration in both mdr1a(+/+) and mdr1a(-/-) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitro incubation. The fexofenadine formation rate increased approximately 5-fold during a 2-h brain tissue homogenate incubation with hydroxyl-terfenadine, suggesting that the hydroxylation of terfenadine is the rate-limiting step in fexofenadine formation. Moreover, regional brain metabolism seems to be an important factor in terfenadine brain disposition and, consequently, fexofenadine brain exposure. Taken together, these results indicate that the fexofenadine BBB P-gp efflux ratio has been underestimated previously due to the lack of complete equilibration of fexofenadine across the blood-brain interface under typical experimental paradigms.

Role of flavin-containing monooxygenase in oxidative metabolism of voriconazole by human liver microsomes.

Voriconazole is a potent second-generation triazole antifungal agent with broad-spectrum activity against clinically important fungi. It is cleared predominantly via metabolism in all species tested including humans. N-Oxidation of the fluoropyrimidine ring, its hydroxylation, and hydroxylation of the adjacent methyl group are the known pathways of voriconazole oxidative metabolism, with the N-oxide being the major circulating metabolite in human. In vitro studies have shown that CYP2C19, CYP3A4, and to a lesser extent CYP2C9 contribute to the oxidative metabolism of voriconazole. When cytochrome P450 (P450)-specific inhibitors and antibodies were used to evaluate the oxidative metabolism of voriconazole by human liver microsomes, the results suggested that P450-mediated metabolism accounted for approximately 75% of the total oxidative metabolism. The studies presented here provide evidence that the remaining approximately 25% of the metabolic transformations are catalyzed by flavin-containing monooxygenase (FMO). This conclusion was based on the evidence that the NADPH-dependent metabolism of voriconazole was sensitive to heat (45 degrees C for 5 min), a condition known to selectively inactivate FMO without affecting P450 activity. The role of FMO in the metabolic formation of voriconazole N-oxide was confirmed by the use of recombinant FMO enzymes. Kinetic analysis of voriconazole metabolism by FMO1 and FMO3 yielded K(m) values of 3.0 and 3.4 mM and V(max) values of 0.025 and 0.044 pmol/min/pmol, respectively. FMO5 did not metabolize voriconazole effectively. This is the first report of the role of FMO in the oxidative metabolism of voriconazole.