Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1

The effects of human immunodeficiency virus (HIV) protease inhibitors (PI) on the accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in organic anion transporting polypeptide (OATP)-1B1 and -1B3-expressing Chinese hamster ovary (CHO) cells. In addition, interaction studies in Caco-2 monolayers, known only to express the OATP2B1 isoform, were conducted using the established OATP substrate estrone 3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers. CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 microl min(-1) mg(-1) protein in OATP1B1 and OATP1B3-transfected cells, respectively. K(i)-values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and OATP1B3-expressing cells. Lopinavir was the most potent inhibitor (K(i) = 0.5-1.4 microM) of OATP1B-mediated CGamF accumulation compared with atazanavir, darunavir, ritonavir, and saquinavir (K(i) between 1.4 and 3.3 microM). Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir, and ritonavir showing substantial effects. In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver, and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine

A Physiology-Based Pharmacokinetic Framework to Support Drug Development and Dose Precision During Therapeutic Hypothermia in Neonates

Therapeutic hypothermia (TH) is standard treatment for neonates (≥36 weeks) with perinatal asphyxia (PA) and hypoxic–ischemic encephalopathy. TH reduces mortality and neurodevelopmental disability due to reduced metabolic rate and decreased neuronal apoptosis. Since both hypothermia and PA influence physiology, they are expected to alter pharmacokinetics (PK). Tools for personalized dosing in this setting are lacking. A neonatal hypothermia physiology-based PK (PBPK) framework would enable precision dosing in the clinic. In this literature review, the stepwise approach, benefits and challenges to develop such a PBPK framework are covered. It hereby contributes to explore the impact of non-maturational PK covariates. First, the current evidence as well as knowledge gaps on the impact of PA and TH on drug absorption, distribution, metabolism and excretion in neonates is summarized. While reduced renal drug elimination is well-documented in neonates with PA undergoing hypothermia, knowledge of the impact on drug metabolism is limited. Second, a multidisciplinary approach to develop a neonatal hypothermia PBPK framework is presented. Insights on the effect of hypothermia on hepatic drug elimination can partly be generated from in vitro (human/animal) profiling of hepatic drug metabolizing enzymes and transporters. Also, endogenous biomarkers may be evaluated as surrogate for metabolic activity. To distinguish the impact of PA versus hypothermia on drug metabolism, in vivo neonatal animal data are needed. The conventional pig is a well-established model for PA and the neonatal Göttingen minipig should be further explored for PA under hypothermia conditions, as it is the most commonly used pig strain in nonclinical drug development. Finally, a strategy is proposed for establishing and fine-tuning compound-specific PBPK models for this application. Besides improvement of clinical exposure predictions of drugs used during hypothermia, the developed PBPK models can be applied in drug development. Add-on pharmacotherapies to further improve outcome in neonates undergoing hypothermia are under investigation, all in need for dosing guidance. Furthermore, the hypothermia PBPK framework can be used to develop temperaturedriven PBPK models for other populations or indications. The applicability of the proposed workflow and the challenges in the development of the PBPK framework are illustrated for midazolam as model drug

Integration of Placental Transfer in a Fetal–Maternal Physiologically Based Pharmacokinetic Model to Characterize Acetaminophen Exposure and Metabolic Clearance in the Fetus

Background and Objective: Although acetaminophen is frequently used during pregnancy, little is known about fetal acetaminophen pharmacokinetics. Acetaminophen safety evaluation has typically focused on hepatotoxicity, while other events (fetal ductal closure/constriction) are also relevant. We aimed to develop a fetal–maternal physiologically based pharmacokinetic (PBPK) model (f-m PBPK) to quantitatively predict placental acetaminophen transfer, characterize fetal acetaminophen exposure, and quantify the contributions of specific clearance pathways in the term fetus. Methods: An acetaminophen pregnancy PBPK model was extended with a compartment representing the fetal liver, which included maturation of relevant enzymes. Different approaches to describe placental transfer were evaluated (ex vivo cotyledon perfusion experiments, placental transfer prediction based on Caco-2 cell permeability or physicochemical properties [MoBi®]). Predicted maternal and fetal acetaminophen profiles were compared with in vivo observations. Results: Tested approaches to predict placental t

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 CYP-specific inhibitors and antibodies were used to evaluate the oxidative metabolism of voriconazole by human liver microsomes (HLM), the results suggested that CYP-mediated metabolism accounted for ~75% of the total oxidative metabolism. The studies presented here provide evidence that the remaining ~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 °C for 5 min), a condition known to selectively inactivate FMO without affecting CYP 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 Km values of 3.0 mM and 3.4 mM and Vmax values of 0.025 pmol/min/pmol 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

Development of a pig mammary epithelial cell culture model as a non‐clinical tool for studying epithelial barrier— a contribution from the imi‐conception project

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E‐Cad and tight junctions molecules ZO‐1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Membrane protein insertion is an essential cellular process. The broad biophysical and topological range of membrane proteins necessitates multiple insertion pathways, which remain incompletely defined. Here, we have discovered a new membrane protein insertion pathway, identified the class of substrates it handles, explained why other known pathways do not work for these substrates and reconstituted the pathway using purified components

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

Micafungin, a new echinocandin antifungal agent, has been widely used for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins (>99.5%). 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, micafungin fraction unbound (fu) in neonatal serum was about 8-fold higher than in 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 to adults

ATP13A2 deficiency disrupts lysosomal polyamine export

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome—a parkinsonism with dementia1—and early-onset Parkinson’s disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson’s disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system

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 (T1/2) of 33.8 ± 15.3 versus 72.6 ± 23.7 min, respectively. The Km for voriconazole metabolism to N-oxide, the major metabolite formed in humans, by liver microsomes from children and adults was similar (11 ± 5.2 versus 9.3 ± 3.6 μM, respectively). In contrast, apparent Vmax was approximately 3-fold higher in children compared with adults (120.5 ± 99.9 versus 40 ± 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

Current knowledge, challenges and innovations in developmental pharmacology: A combined conect4children Expert Group and European Society for Developmental, Perinatal and Paediatric Pharmacology White Paper

Developmental pharmacology describes the impact of maturation on drug disposition (pharmacokinetics, PK) and drug effects (pharmacodynamics, PD) throughout the paediatric age range. This paper, written by a multidisciplinary group of experts, summarizes current knowledge, and provides suggestions to pharmaceutical companies, regulatory agencies and academicians on how to incorporate the latest knowledge regarding developmental pharmacology and innovative techniques into neonatal and paediatric drug development. Biological aspects of drug absorption, distribution, metabolism and excretion (ADME) throughout development are summarized. Although this area made enormous progress during the last two decades, remaining knowledge gaps were identified. Minimal risk and burden designs allow for optimally informative but minimally invasive PK sampling, while concomitant profiling of drug metabolites may provide additional insight in the unique PK behavior in children. Furthermore, developmental PD needs to be considered during drug development, which is illustrated by disease- and/or target organ-specific examples. Identifying and testing PD targets and effects in special populations, and application of age- and/or population-specific assessment tools are discussed. Drug development plans also need to incorporate innovative techniques like preclinical models to study therapeutic strategies, and shift from sequential enrollment of subgroups, to more rational designs. To stimulate appropriate research plans, illustrations of specific PK/PD-related as well as drug safety-related challenges during drug development are provided. The suggestions made in this joint paper of the Innovative Medicines Initiative conect4children Expert group on Developmental Pharmacology and the European Society for Developmental, Perinatal and Paediatric Pharmacology, should facilitate all those involved in drug development