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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.

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.

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.

Purpose. The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions.

Partial purification of ribonucleoside diphosphate reductase from rabbit bone marrow was achieved by size exclusion HPLC of the crude homogenate. This step, requiring15 min, led to 9- to 13-fold purification of the reductase and removal of 64% of the contaminating kinase/phosphatase activities, which in the crude extract degrade95% of substrate CDP when reductase is assayed. A systematic study was conducted to evaluate the influence of contaminating kinase/phosphatase activities on CDP concentration during the reductase-catalyzed reaction with either ATP or its kinase-inhibiting analog, 5'-adenylylimidodiphosphate (AMP-PNP), as the allosteric effector. Our studies demonstrated that in the presence of ATP, CDP levels fell instantly to24% but thereafter remained fairly constant due to recycling via CTP. In contrast, in the presence of AMP-PNP, CDP levels decreased continuously. The Km values of the reductase for CDP determined in the presence of ATP were significantly higher than those in the presence of AMP-PNP. Furthermore, we also found that the concentration of the ultimate electron donor dithiothreitol (DTT) required for optimum activity of the reductase varies significantly with the level of purity of the reductase preparation. Interestingly, DTT is an inhibitor of the reductase above the optimum concentration. This purification method and the optimized assay together with the understanding of the fate of CDP in partially purified preparations should find application in studies with reductases from other eukaryotic sources.

Human development of an individual from a fertilized ovum to maturity alters the body anatomy and physiology. Changes of size and function, from birth onwards, cause significant alterations in the pharmacokinetics (PK) of drugs and subsequently their response pharmacodynamics (PD) in infants and children from those in adults. During the last three decades, hundreds of mechanistic and clinical pharmacology studies have been conducted to investigate the age-mediated changes of absorption, distribution, metabolism and excretion processes of drugs, which subsequently affect the pharmacology response and the safety in pediatric patients compared to adults. The practice of determining pediatric dose based on simplistic scaling of an adult dose assuming linear relationship between postnatal age and body weight or surface area that may lead to under prediction of therapeutic dose or over prediction of the dose is now under scrutiny. By understanding the disposition mechanism of therapeutic agents thoroughly, their potential drug interactions and their PK/PD relationships can be better determined in pediatric populations. As such, dosing regimens can be estimated based on actual clearance and exposure and not just by simplistic scaling of an adult dose. Accurate prediction of clearance in pediatrics is so critical that extensive translational research is warranted to improve our ability to estimate safe and efficacious doses in different pediatric populations from retrospective clinical studies in adults. Biotherapeutics, proteins and peptides-based drugs, generally depend on absorption (A), distribution (D), metabolism (M), and excretion (E) in their disposition as small molecules, but the underlying mechanisms and potential drug interaction propensity can be very different. In this article, the factors that alter pediatric and adult PK parameters are compared, and pediatric and adult PK parameters and potential drug interactions for selected biotherapeutics are summarized. Moreover, challenges of studying therapeutic proteins and peptides in pediatrics are discussed.

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.

Over the past 20 years, there has been a growing recognition that the discovery of effective therapeutic agents involves designing compounds that possess appropriate “pharmaceutical” or “drug-like” properties in addition to high affinity for their biological targets. The pharmaceutical or drug-like properties include solubility, permeation across barriers such as the intestinal epithelium or blood-brain barrier, and metabolic and excretory clearance. Appropriate balance of these properties enables drug molecules to attain and maintain sufficient systemic and/or target concentrations to exert therapeutic effects through optimum absorption, distribution, metabolism, and excretion (ADME) processes. The ADME processes, in conjunction with the biological properties, define therapeutic profiles of drug molecules (Thakker, 2006). A drug that is poorly absorbed, rapidly metabolized, or rapidly excreted via the renal or hepatic route will not attain its full therapeutic potential. Such a drug will require higher doses to achieve sufficiently high systemic or target concentrations for efficacy, which may not be practical in some cases or may cause adverse effects in others. Thus, good pharmaceutical or drug-like properties are often defined as physicochemical properties of drug candidates that enable a drug candidate to navigate through the physical, biochemical, and physiological barriers posed by the ADME processes. The pharmaceutical properties of a drug candidate are optimized by de novo designing appropriate physicochemical attributes into the molecule or via formulation of the drug candidate with agents that can improve certain aspects of the physicochemical properties.

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.

Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 μmol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by >50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 μmol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 μmol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 μmol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 μmol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.