Search

Thirteen novel hydrazone-Schiff bases (3-15) of fexofenadine were succesfully synthesized, structurally deduced and finally assessed their capability to inhibit urease enzyme (in vitro). In the series, six compounds 12 (IC 50 =10.19±0.16 μM), 11 (IC 50 =15.05±1.11 μM), 10 (IC 50 =17.01±1.23 μM), 9 (IC 50 =17.22±0.81 μM), 13 (IC 50 =19.31±0.18 μM), and 14 (IC 50 =19.62±0.21 μM) displayed strong inhibitory action better than the standard thiourea (IC 50 =21.14±0.24 μM), while the remaining compounds displayed significant to less inhibition. LUMO and HOMO showed the transferring of charges from molecules to biological transfer and MEP map showed the chemically reactive zone appropriate for drug action are calculated using DFT. AIM charges, non-bonding orbitals, and ELF are also computed. The urease protein binding analysis benefited from the docking studies., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Terfenadine is a widely prescribed non-sedating antihistamine which undergoes rapid and almost complete first pass biotransformation to an active carboxylic acid metabolite. It is unusual to find unmetabolised terfenadine in the plasma of patients taking the drug. Terfenadine in vitro is a potent blocker of the myocardial potassium channel. Overdose, hepatic compromise and the coadministration of ketoconazole and erythromycin result in the accumulation of terfenadine, which is thought to be responsible of QT prolongation and Torsades de Pointes ventricular arrhythmia in susceptible individuals. Cimetidine and ranitidine are two popular H antagonists which are often taken with terfenadine. The effects of cimetidine and ranitidine on terfenadine metabolism were studied in two cohorts of 6 normal volunteers given the recommended dose of terfenadine (60 mg every 12 h) for 1 week prior to initiation of cimetidine 600 mg every 12 h or ranitidine 150 mg every 12 h. Pharmacokinetic profiles and morning pre-dose electrocardiograms were obtained whilst the patients were on terfenadine alone and after the addition of cimetidine or rantidine. One of the subjects in each cohort had a detectable plasma level of parent compound after 1 week of terfenadine therapy alone; it did not accumulate further after addition of the H antagonist. The pharmacokinetics of the carboxylic acid metabolite of terfenadine (C, t, AUC) were not significantly changed after co-administration of either H antagonist. None of the remaining 5 subjects in either cohort demonstrated accumulation of unmetabolised terfenadine after addition of the respective H antagonist and electrocardiographic QT intervals and T-U morphology in them was not changed during the course of the study. We conclude that cimetidine and ranitidine in the dosages used in this study did not affect the metabolism of terfenadine, and that patients exposed to these drug combinations are not at increased risk of altered cardiac repolarisation. [ABSTRACT FROM AUTHOR]

The unbound concentrations of 14 commercial drugs, including five non-efflux/uptake transporter substrates-Class I, five efflux transporter substrates-class II and four influx transporter substrates-Class III, were simultaneously measured in rat liver, muscle, and blood via microanalysis. K puu,liver and K puu,muscle were calculated to evaluate the membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver. For Class I compounds, represented by antipyrine, unbound concentrations among liver, muscle and blood are symmetrically distributed when compound hepatic clearance is low. And when compound hepatic clearance is high, unbound concentrations among liver, muscle and blood are asymmetrically distributed, such as Propranolol. For Class II and III compounds, overall, the unbound concentrations among liver, muscle, and blood are asymmetrically distributed due to a combination of hepatic metabolism and efflux and/or influx transporter activity., (© 2021 Pharmaron Beijing Co Ltd. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

P-Glycoprotein (P-gp) is one of the most clinically significant representatives of the ABC transporter superfamily due to its participation in the transport of biotic components and xenobiotics across the plasma membrane. It is known that various chemicals, environmental factors, and pathological processes can affect P-gp activity and expression. In this study, we investigated the role of P-gp in limiting the cell membrane permeability during oxidative stress. Human adenocarcinoma colon cells (Caco-2) overexpressing P-gp were cultured for 72 h in the medium containing hydrogen peroxide (0.1-50 µM). The transport of the P-gp substrate fexofenadine was evaluated in a special Transwell system. The amounts of P-gp and Nrf2 transcription factor were analyzed by the enzyme-linked immunosorbent assay. The concentration of SH-groups in proteins and the contents of lipid peroxidation products and protein carbonyl derivatives were determined spectrophotometrically. Hydrogen peroxide at a concentration of 0.1-5 µM did not significantly affect the studied parameters, while incubation with 10 µM H2O2 decreased in the level of SH groups in cell lysates and increased in the amount of Nrf2 in the cell lysates. Nrf2, in its turn, mediated an increase in the content and activity of the P-gp transporter, thus limiting the increasing permeability of the cell membrane. Hydrogen peroxide at a concentration of 50 µM promoted oxidative stress, which was manifested as a decrease in the content of SH-groups, increase in the concentration of lipid peroxidation products and protein carbonyl derivatives, and decrease in the P-gp level, which led to a significantly increased permeability of the plasma membrane. These results show that the transport and protective roles of P-gp, in particular, reduction of the cell membrane permeability, are affected by the intensity of oxidative stress and can be manifested only if the extent of membrane damage is insignificant.

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used recombinant CYP2J2 and determined whether danazol, hydroxyebastine, telmisartan, and terfenadone inhibited CYP2J2 by using four representative CYP2J2 substrates, namely albendazole, astemizole, ebastine, and terfenadine. In this study, we evaluated the inhibitory potential of these four chemicals on human liver and intestinal microsomes, which are commonly used in a reaction phenotyping study. Among the four CYP2J2 inhibitors tested, terfenadone was strongest inhibitor of CYP2J2-mediated metabolism of albendazole, astemizole, and terfenadine with IC 50 values of 0.31, 0.15, and 2.11 μM, respectively, in human liver microsomes (HLMs). In addition, terfenadone had strong inhibitory effect on the metabolism of the abovementioned drugs in human intestinal microsomes (HIMs), with IC 50 values of 0.43, 0.08 and 1.07 μM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC 50 values of 13.8 and 18.3 μM, respectively in HLMs, whereas it strongly inhibited the CYP2J2-mediated ebastine hydroxylase activity in HLMs and HIMs (IC 50  = 1.93-1.95 μM). Our data suggest that terfenadone may be used as a general CYP2J2 inhibitor in reaction phenotyping study using HLMs and HIMs regardless of the substrate used., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The sorption of 3 pharmaceuticals, which may exist in 4 different forms depending on the solution pH (irbesartan in cationic, neutral and anionic, fexofenadine in cationic, zwitter-ionic and anionic, and citalopram cationic and neutral), in seven different soils was studied. The measured sorption isotherms were described by Freundlich equations, and the sorption coefficients, K F (for the fixed n exponent for each compound), were related to the soil properties to derive relationships for estimating the sorption coefficients from the soil properties (i.e., pedotransfer rules). The largest sorption was obtained for citalopram (average K F value for n = 1 was 1838 cm 3  g -1 ) followed by fexofenadine (K F  = 35.1 cm 3/n μg 1-1/n g -1 , n = 1.19) and irbesartan (K F  = 3.96 cm 3/n μg 1-1/n g -1 , n = 1.10). The behavior of citalopram (CIT) in soils was different than the behaviors of irbesartan (IRB) and fexofenadine (FEX). Different trends were documented according to the correlation coefficients between the K F values for different compounds (R IRB,FEX  = 0.895, p-value<0.01; R IRB,CIT  = -0.835, p-value<0.05; R FEX,CIT  = -0.759, p-value<0.05) and by the reverse relationships between the K F values and soil properties in the pedotransfer functions. While the K F value for citalopram was positively related to base cation saturation (BCS) or sorption complex saturation (SCS) and negatively correlated to the organic carbon content (Cox), the K F values of irbesartan and fexofenadine were negatively related to BCS, SCS or the clay content and positively related to Cox. The best estimates were obtained by combining BCS and Cox for citalopram (R 2  = 93.4), SCS and Cox for irbesartan (R 2  = 96.3), and clay content and Cox for fexofenadine (R 2  = 82.9)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The human cytochrome P450 2J2 is involved in several metabolic reactions, including the oxidation of important therapeutics and epoxidation of endogenous arachidonic acid. At least ten genetic variations of P450 2J2 have been identified, but their effects on enzymatic activity have not been clearly characterized. Here, we evaluated the functional effects of three genetic variations of P450 2J2 (G312R, P351L, and P115L). Recombinant enzymes of wild-type and three variant P450 2J2 were heterologously expressed in Escherichia coli and purified. P450 expression levels in the wild-type and two variants (P351L and P115L) were 142-231 nmol per liter culture, while the G312R variant showed no holoenzyme peak in the CO-binding spectra. Substrate binding titrations to terfenadine showed that the wild-type and two variants displayed K d values of 0.90-2.2 μM, indicating tight substrate binding affinities. Steady-state kinetic analysis for t-butyl methyl hydroxylation of terfenadine indicated that two variant enzymes had similar k cat and K m values to wild-type P450 2J2. The locations of mutations in three-dimensional structural models indicated that the G312R is located in the I-helix region near the formal active site in P450 2J2 and its amino acid change affected the structural stability of the P450 heme environment., (Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Whether the combined use of probe drugs for CYP3A4 and P-glycoprotein can clarify the relative contribution of these proteins to pharmacokinetic variability of a dual substrate like tacrolimus has never been assessed. Seventy renal recipients underwent simultaneous 8-h pharmacokinetic profiles for tacrolimus, the CYP3A4 probe midazolam, and the putative P-glycoprotein probe fexofenadine. Patients were genotyped for polymorphisms in CYP3A5, CYP3A4, ABCB1, ABCC2 and SLCO2B1, -1B1, and 1B3. Carriers of the ABCB1 2677G>A polymorphism displayed lower fexofenadine C max (-66%; P = 0.012) and a trend toward higher clearance (+157%; P = 0.078). Predictors of tacrolimus clearance were CYP3A5 genotype, midazolam clearance, hematocrit, weight, and age (R 2 = 0.61). Fexofenadine pharmacokinetic parameters were not predictive of tacrolimus clearance. In conclusion, fexofenadine pharmacokinetics varied considerably between renal recipients but most of this variability remained unexplained, with only minor effects of genetic polymorphisms. Fexofenadine cannot be used to assess in vivo CYP3A4-P-glycoprotein interplay in tacrolimus-treated renal recipients., (© 2017 American Society for Clinical Pharmacology and Therapeutics.)

This study describes a specific, precise, sensitive and accurate method for simultaneous determination of hydroxyzine, loratadine, terfenadine, rupatadine and their main active metabolites cetirizine, desloratadine and fexofenadine, in serum and urine using meclizine as an internal standard. Solid-phase extraction method for sample clean-up and preconcentration of analytes was carried out using Phenomenex Strata-X-C and Strata X polymeric cartridges. Chromatographic analysis was performed on a Phenomenex cyano (150 × 4.6 mm i.d., 5 μm) analytical column. A D-optimal mixture design methodology was used to evaluate the effect of changes in mobile phase compositions on dependent variables and optimization of the response of interest. The mixture design experiments were performed and results were analyzed. The region of ideal mobile phase composition consisting of acetonitrile-methanol-ammonium acetate buffer (40 mm; pH 3.8 adjusted with acetic acid): 18:36:46% v/v/v was identified by a graphical optimization technique using an overlay plot. While using this optimized condition all analytes were baseline resolved in <10 min. Solvent mixtures were delivered at 1.5 mL/min flow rate and analytes peaks were detected at 222 nm. The proposed bioanalytical method was validated according to US Food and Drug Administration guidelines. The proposed method was sensitive with detection limits of 0.06-0.15 μg/mL in serum and urine samples. Relative standard deviation for inter- and intra-day precision data was found to be <7%. The proposed method may find application in the determination of selected antihistaminic drugs in biological fluids., (Copyright © 2017 John Wiley & Sons, Ltd.)

The binding interaction of peripheral H 1 receptor antagonist drug, fexofenadine hydrochloride to bovine serum albumin (BSA) is investigated by fluorescence spectroscopy in combination with UV-absorption spectroscopy under physiological conditions. The Stern-Volmer plots at different temperatures and the steady state fluorescence suggested a static type of interaction between fexofenadine and BSA. Binding constants were determined to provide a measure of the binding affinity between fexofenadine and BSA. It was found that BSA has one binding site for fexofenadine. On the basis of the competitive site marker experiments and thermodynamic results, it was considered that fexofenadine was primarily bound to the site I of BSA mainly by hydrogen bond and van der Waals force. Utilising Förster resonance energy transfer the distance, r between the donor, BSA and acceptor fexofenadine was obtained. Furthermore, the results of circular dichroism and synchronous fluorescence spectrum indicated that the secondary structure of BSA was changed in the presence of fexofenadine. Molecular docking was applied to further define the interaction of fexofenadine with BSA.

Background: Disease-dependent changes in the activity of drug metabolizing enzymes and transporters, such as Cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp), are thought to have a major influence on the disposition of shared substrates. However, little is known regarding the in vivo relevance of these 2 proteins during drug therapy for gastrointestinal diseases. Our aim was to elucidate the activity of CYP3A4 and P-gp in subjects with Crohn's disease (CD) and to evaluate their influence on budesonide pharmacokinetics., Methods: A detailed pharmacokinetic assessment was conducted in 8 individuals diagnosed with CD on stable doses of oral budesonide, a putative shared CYP3A4, and P-gp substrate, where hepatic and intestinal CYP3A4 activity were also assessed using intravenous and oral midazolam. In addition, oral fexofenadine was used as an in vivo probe for P-gp activity., Results: Budesonide area under the curve was highly variable between subjects but similar to previously reported values in healthy subjects. The hepatic and intestinal extraction ratios for midazolam were 0.11 ± 0.06 and 0.64 ± 0.25, respectively; however, CYP3A4 activity was nearly 5-fold lower in our CD cohort compared with published data among healthy subjects. Multivariate regression revealed that only 25% budesonide clearance could be explained based on midazolam or fexofenadine clearance., Conclusions: Midazolam and fexofenadine disposition profile did not predict budesonide clearance. However, we observed a marked reduction in vivo CYP3A4 activity among individuals with CD. Therefore, changes in CYP3A4 activity in disease states such as CD may be a heretofore underappreciated determinant of variation in drug responsiveness in CD.

Objectives: P-glycoprotein (P-gp) is responsible for the efflux of a broad variety of human and veterinary drugs. Canine P-gp polymorphisms alter drug disposition and toxicity, but their impact on the disposition of enantiomeric drugs is unknown. Using fexofenadine as a model compound, we developed and validated HPLC-fluorescence methods to determine the effect of P-gp on the disposition of fexofenadine and its enantiomers., Methods: A chiral CD-Ph column was used for the separation of (R) and (S)-fexofenadine. Determination of racemic fexofenadine was achieved on an XDB-CN column. Fexofenadine and its enantiomers were detected by fluorescence at the excitation wavelength of 220 nm and emission wavelength of 300 nm. These methods were used to measure concentrations of fexofenadine and its enantiomers in Collie plasma after oral administration., Key Findings: This study demonstrates that P-gp prefers to transport (S)-fexofenadine, and P-gp deficiency causes the increase in both (R)-fexofenadine and (S)-fexofenadine in plasma. Racemic fexofenadine, (R)-fexofenadine and (S)-fexofenadine were increased in ABCB1-1Δ Collies (118.7, 72.0 and 48.3 ng/ml) compared to wild-type Collies (25.0, 16.5 and 7.7 ng/ml) at 1 h postadministration. The results demonstrate that the stereoselectivity of P-gp plays a key role in the disposition of fexofenadine enantiomers., Conclusions: The information derived from this drug model will be used to determine whether additional safety or efficacy requirements are necessary for enantiomeric drugs that would be used in dogs or humans., (© 2017 Royal Pharmaceutical Society.)

This is a reply to the comment on "In Silico Modeling of Gastrointestinal Drug Absorption: Predictive Performance of Three Physiologically Based Absorption Models" by Turner and other Simcyp associates. In the reply we address the major concerns raised by Turner et al. regarding the methodology to compare the predictive performance of the different absorption models and at the same time ensure that the systemic pharmacokinetic input was exactly the same for the different models; the selection of the human effective permeability value of fexofenadine; the adoption of model default values and settings; and how supersaturation/precipitation was handled. In addition, we also further discuss aspects related to differences in in silico models and the potential implications of such differences. Our original report should be viewed as the starting point in a thorough and transparent review of absorption prediction models with the overall aim of improving their application as validated tools for bridging studies of active pharmaceutical ingredients from various sources and origins in a regulatory context. With this reply we encourage other independent investigators to perform further model evaluations of commercial as well as other existing or recently implemented models. This will boost the overall progression of physiologically based biopharmaceutical models for predicting and simulating intestinal drug absorption both in research and development and in a regulatory context.

1. Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes. 2. Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice. 3. Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver. 4. Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes. 5. These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.

A Quantitative Systems Pharmacology approach was utilized to predict the cardiac consequences of drug-drug interaction (DDI) at the population level. The Simcyp in vitro-in vivo correlation and physiologically based pharmacokinetic platform was used to predict the pharmacokinetic profile of terfenadine following co-administration of the drug. Electrophysiological effects were simulated using the Cardiac Safety Simulator. The modulation of ion channel activity was dependent on the inhibitory potential of drugs on the main cardiac ion channels and a simulated free heart tissue concentration. ten Tusscher's human ventricular cardiomyocyte model was used to simulate the pseudo-ECG traces and further predict the pharmacodynamic consequences of DDI. Consistent with clinical observations, predicted plasma concentration profiles of terfenadine show considerable intra-subject variability with recorded C max values below 5 ng/mL for most virtual subjects. The pharmacokinetic and pharmacodynamic effects of inhibitors were predicted with reasonable accuracy. In all cases, a combination of the physiologically based pharmacokinetic and physiology-based pharmacodynamic models was able to differentiate between the terfenadine alone and terfenadine + inhibitor scenario. The range of QT prolongation was comparable in the clinical and virtual studies. The results indicate that mechanistic in vitro-in vivo correlation can be applied to predict the clinical effects of DDI even without comprehensive knowledge on all mechanisms contributing to the interaction., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The expression of carboxylesterase (CES) and the transdermal movement of an ester prodrug were studied in rat skin. Ethyl-fexofenadine (ethyl-FXD) was used as a model lipophilic prodrug that is slowly hydrolyzed to its parent drug, FXD (MW 502). Among the CES1 and CES2 isozymes, Hydrolase A is predominant in rat skin and this enzyme was involved in 65% of the cutaneous hydrolysis of ethyl-FXD. The similarity of the permeation behavior of ethyl-FXD in full thickness and stripped skin indicated that the stratum corneum was not a barrier to penetration. However, only FXD was observed in receptor fluid, not ethyl-FXD, presumably because of the high degree of binding of ethyl-FXD in viable skin. The rate of hydrolysis of ethyl-FXD was much faster than steady-state flux, such that the influx rate was the rate-limiting process for transdermal permeation. Although Hydrolase A levels gradually increased in skin taken from rats aged from 8 to 90 weeks, variations in the expression levels of the esterase hardly affected the conversion of prodrug. The present data suggest that the slow hydrolysis of the prodrug of an active ingredient in viable skin followed by slow diffusion of active drug may provide a useful approach to topical application., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: In the present age of polypharmacy, limited sampling strategy becomes important to verify if drug levels are within the prescribed threshold limits from efficacy and safety considerations. The need to establish reliable single time concentration dependent models to predict exposure becomes important from cost and time perspectives., Methods: A simple unweighted linear regression model was developed to describe the relationship between Cmax versus AUC for fexofenadine, losartan, EXP3174, itraconazole and hydroxyitraconazole. The fold difference, defined as the quotient of the observed and predicted AUC values, were evaluated along with statistical comparison of the predicted versus observed values., Results: The correlation between Cmax versus AUC was well established for all the five drugs with a correlation coefficient (r) ranging from 0.9130 to 0.9997. Majority of the predicted values for all the five drugs (77%) were contained within a narrow boundary of 0.75- to 1.5-fold difference. The r values for observed versus predicted AUC were 0.9653 (n = 145), 0.8342 (n = 76), 0.9524 (n = 88), 0.9339 (n = 89) and 0.9452 (n = 66) for fexofenadine, losartan, EXP3174, itraconazole and hydroxyitraconazole, respectively., Conclusions: Cmax versus AUC relationships were established for all drugs and were amenable for limited sampling strategy for AUC prediction. However, fexofenadine, EXP3174 and hydroxyitraconazole may be most relevant for AUC prediction by a single time concentration as judged by the various criteria applied in this study.

Accumulating evidences have shown that diabetes is often accompanied with depression, thus it is possible that oral antidiabetic agent glyburide and antidepressive agent paroxetine are co-administered in diabetic patients. The aim of this study was to assess interactions between glyburide and paroxetine in rats. Effect of paroxetine on pharmacokinetics of orally administered glyburide was investigated. Effect of naringin (NAR), an inhibitor of rat intestinal organic anion transporting polypeptides 1a5 (Oatp1a5), on pharmacokinetics of glyburide was also studied. The results showed that co-administration of paroxetine markedly reduced plasma exposure and prolonged Tmax of glyburide, accompanied by significant increase in fecal excretion of glyburide. Co-administration of naringin also significantly decreased plasma exposure of glyburide. Data from intestinal perfusion experiments showed that both paroxetine and naringin significantly inhibited intestinal absorption of glyburide. Caco-2 cells were used to investigate whether paroxetine and naringin affected intestinal transport of glyburide and fexofenadine (a substrate of Oatp1a5). The results showed that both paroxetine and naringin greatly inhibited absorption of glyburide and fexofenadine. All results gave a conclusion that co-administration of paroxetine decreased plasma exposure of glyburide in rats via inhibiting intestinal absorption of glyburide, which may partly be attributed to the inhibition of intestinal Oatp1a5 activity., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The drug-transporting proteins can affect the pharmacokinetics and pharmacodymanics of many drugs, resulting in an erratic and unpredictable pharmacological response. The Caco-2 monolayer is routinely applied to investigate the carrier-mediated transport of drugs. Therefore, the selection of a marker compound able to characterize the activity of such transporters is crucial. Fexofenadine (FEX), a P-gp/OATP substrate, can be considered a suitable probe. However, in order to use be used as a marker compound, it is mandatory to develop an analytical method able to quantify this drug during the in vitro permeability assay. An HPLC method with ultraviolet detection was developed; the mobile phase consisted of phosphate buffer (pH 3.2) containing 10 m m of sodium octanosulphonate and acetonitrile (60:40) and the flow rate was set at 1.2 mL/min. Fexofenadine was eluted at 40°C, the retention time was about 4.6 min. The LOD and LOQ values were 1.9 and 6.2 ng/mL, respectively. Verapamil and ketoconazole, the most common P-gp inhibitors, were eluted as distinct peaks of that corresponding to fexofenadine The method was successfully applied to quantify the amount of FEX transported across the Caco-2 monolayer and could be an additional tool for those investigating the role of membrane transporters on drug absorption., (Copyright © 2014 John Wiley & Sons, Ltd.)

Analysis of drug and metabolite distribution is essential for understanding of the mechanisms underlying the pharmacological or toxicological effects. MS imaging (MSI) can visualize the distribution of drugs or biological molecules in tissue sections without radiolabeling, and distinguish between the distribution of a drug and that of its metabolites in tissue sections. Therefore, it is expected to be a potent imaging technique for drug distribution studies. This article includes cases in which MSI was used to analyze drug and metabolite distribution, and discusses the impact of data obtained by MSI in drug discovery and development.

Purpose: OATP2B1-mediated grapefruit juice (GFJ)-drug interactions are substrate-dependent; for example, GFJ ingestion significantly reduces bioavailability of fexofenadine, but not pravastatin. In the present study, we aimed to establish whether this observation can be explained by the presence of multiple binding sites (MBS) on OATP2B1., Methods: OATP2B1-mediated drug uptake was evaluated using a Xenopus oocyte expression system. Drug concentration was quantified by LC/MS/MS analysis., Results: OATP2B1-mediated uptake of pravastatin and fexofenadine exhibited biphasic saturation kinetics, indicating the presence of MBS on OATP2B1. GFJ strongly inhibited pravastatin uptake mediated by the high-affinity site on OATP2B1, while no significant inhibition of the low-affinity site was observed. In contrast, high-affinity transport of fexofenadine was only modestly inhibited by GFJ, while significant inhibition of the low-affinity site was observed. Contribution analysis indicated that both drugs are transported via the low-affinity site on OATP2B1 at therapeutically relevant concentrations. These findings indicate that only fexofenadine is expected to interact with GFJ on OATP2B1 at therapeutic concentrations, in accordance with the clinical observations., Conclusion: Substrate- and dose-dependent GFJ-drug interactions mediated by OATP2B1 might be explained in terms of the presence of MBS: interaction occurs only when drug and GFJ components share the same binding site on OATP2B1.

The research consists in the investigation of the sex differences of P-glycoprotein functional activity and expression in Chinchilla rabbits. P-glycoprotein functional activity was assessed by the pharmacokinetics of its probe substrate--fexofenadine after its single oral administration. P-glycoprotein expression was investigated by immunohistochemistry method. It is shown that male's maximal concentration of fexofenadine, its areas under concentration-time curves, half-life and retention time were higher and its clearance was lower than female's. The efficient differences in pharmacokinetic parameters of fexofenadine confirm more intensive excretion and less intensive absorption in gastro-intestinal tract of fexofenadine. This data indicate that P-glycoprotein activity is more active in female than in male. Immunohistochemistry analysis shows that total liver and intestine P-glycoprotein expression is more intensive in females, than in males that correlates with its more active functioning.

Background: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time., Results: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations., Conclusion: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

The purpose of this study was to determine the impact of CYP3A5 expression on inhibitory potency (Ki or IC50 values) of CYP3A inhibitors, using recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) and CYP3A5 genotyped human liver microsomes (HLMs). IC50 ratios between rCYP3A4 and rCYP3A5 (rCYP3A5/rCYP3A4) of ketoconazole (KTZ) and itraconazole (ITZ) were 8.5 and 8.8 for midazolam (MDZ), 4.7 and 9.1 for testosterone (TST), 1.3 and 2.8 for terfenadine, and 0.6 and 1.7 for vincristine, respectively, suggesting substrate- and inhibitor-dependent selectivity of the two azoles. Due to the difference in the IC50 values for CYP3A4 and CYP3A5, nonconcordant expression of CYP3A4 and CYP3A5 protein can significantly affect the observed magnitude of CYP3A-mediated drug-drug interactions in humans. Indeed, the IC50 values of KTZ and ITZ for CYP3A-catalyzed MDZ and TST metabolism were significantly higher in HLMs with CYP3A5*1/*1 and CYP3A5*1/*3 genotypes than in HLMs with the CYP3A5*3/*3 genotype, showing CYP3A5 expression-dependent IC50 values. Moreover, when IC50 values of KTZ and ITZ for different HLMs were kinetically simulated based on CYP3A5 expression level and enzyme-specific IC50 values, a good correlation between the simulated and the experimentally measured IC50 values was observed. Further simulation analysis revealed that both the Ki ratio (for inhibitors) and Vmax/Km ratio (for substrates) between CYP3A4 and CYP3A5 were major factors for CYP3A5 expression-dependent IC50 values. In conclusion, the present study demonstrates that CYP3A5 genotype and expression level have a significant impact on inhibitory potency for CYP3A-catalyzed drug metabolism, but that the magnitude of its effect is inhibitor-substrate pair specific.

Enzyme-based grapefruit juice (GFJ)-drug interactions are mainly due to mechanism-based irreversible inhibition of metabolizing enzyme CYP3A4 by GFJ components, but the transporter organic anion transporting polypeptide (OATP)2B1 is also a putative site of interaction between drugs and fruit juices (FJ) in the absorption process. Here we aimed to investigate the effect of preincubation with FJ on OATP2B1-mediated transport of drugs in vitro. When OATP2B1-expressing Xenopus oocytes were preincubated with GFJ, orange juice (OJ), or apple juice (AJ), AJ induced a remarkable decrease in OATP2B1-mediated estrone-3-sulfate uptake in a concentration-dependent manner (IC(50) = 1.5%). A similar but less potent effect was observed with OJ (IC(50) = 21%), whereas GFJ had no effect. Similar results were obtained in preincubation studies using fexofenadine. Preincubation with OJ and AJ resulted in time-dependent inhibition of OATP2B1. Again, AJ had the more potent effect; its action lasted for at least 240 minutes, suggesting that AJ irreversibly inhibits OATP2B1-mediated drug uptake. Kinetic analysis revealed that coincubation and preincubation with AJ reduced OATP2B1-mediated estrone-3-sulfate uptake via competitive and noncompetitive mechanisms, respectively. Thus, OATP2B1 is functionally impaired through both competitive and long-lasting inhibition mechanisms by AJ and OJ, but not GFJ. Interestingly, although GFJ but not AJ is able to irreversibly inhibit CYP3A4, in the case of OATP2B1, AJ but not GFJ has a long-lasting inhibitory effect. Accordingly, complex FJ-drug interactions may occur in vivo, and their clinical significance should be examined.

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

Objectives: Fexofenadine contains a chiral carbon in its chemical structure and is orally administered as a racemic mixture. This study evaluated the selective uptake of fexofenadine enantiomers by Caco-2 cells as a model of intestinal epithelial cells., Methods: R(+)-fexofenadine or S(-)-fexofenadine was applied to Caco-2 cells, followed by incubation. After incubation, the amounts of fexofenadine enantiomers in cells were determined. The kinetic parameters for the uptake of fexofenadine enantiomers by Caco-2 cells were estimated using the Michaelis-Menten equation., Key Findings: The transporter-mediated uptake rate of R(+)-fexofenadine was 1.7-fold higher than that of S(-)-fexofenadine. The difference in transporter-mediated R(+)-fexofenadine and S(-)-fexofenadine uptake was completely diminished under ATP-depleted conditions and in the presence of organic anion transporter peptide (OATP) inhibitors. Also, a Dixon plot showed that each fexofenadine enantiomer was competitively inhibited by the other enantiomer. The ratio of R(+)-fexofenadine uptake to S(-)-fexofenadine uptake in the case of a racemic mixture was higher than that in the case of a single enantiomer., Conclusion: This study suggested that the selective absorption of fexofenadine enantiomers by intestinal epithelial cells might have been due to the selective uptake mediated by OATPs and that the difference in intestinal absorption was enhanced with a racemic mixture., (© 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.)

Background and Purpose: Terfenadine has been reported to cause cardiac death. Hence, we investigated its pro-arrhythmic potential in various in vitro models., Experimental Approach: Pro-arrhythmic effects of terfenadine were investigated in rabbit isolated hearts and left ventricular wedge preparations. Also, using whole-cell patch-clamp recording, we examined its effect on the human ether-à-go-go-related gene (hERG) current in HEK293 cells transfected with hERG and on the I(Na) current in rabbit ventricular cells and human atrial myocytes., Key Results: Terfenadine concentration- and use-dependently inhibited I(Na) in rabbit myocytes and in human atrial myocytes and also inhibited the hERG. In both the rabbit left ventricular wedge and heart preparations, terfenadine at 1 µM only slightly prolonged the QT- and JT-intervals but at 10 µM, it caused a marked widening of the QRS complex, cardiac wavelength shortening, incidences of in-excitability and non-TdP-like ventricular tachycardia/fibrillation (VT/VF) without prolongation of the QT/JT-interval. At 10 µM terfenadine elicited a lower incidence of early afterdepolarizations versus non- Torsades de Pointes (TdP)-like VT/VF (100% incidence), and did not induce TdPs. Although the concentration of terfenadine in the tissue-bath was low, it accumulated within the heart tissue., Conclusion and Implications: Our data suggest that: (i) the induction of non-TdP-like VT/VF, which is caused by slowing of conduction via blockade of I(Na) (like Class Ic flecainide), may constitute a more important risk for terfenadine-induced cardiac death; (ii) although terfenadine is a potent hERG blocker, the risk for non-TdP-like VT/VF exceeds the risk for TdPs; and (iii) cardiac wavelength (λ) could serve as a biomarker to predict terfenadine-induced VT/VF., (© 2012 Janssen Pharmaceutica NV. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues.

Purpose: To conduct a mechanistic investigation of the interaction between aliskiren and grapefruit juice in healthy subjects., Methods: Twenty-eight subjects received an oral dose of aliskiren 300 mg (highest recommended clinical dose) with 300 mL of either water or grapefruit juice in a two-way crossover design. Safety and pharmacokinetic analyses were performed. In vitro studies were performed in HEK293 cells to investigate the role of organic anion transporting polypeptide (OATP) transporter-mediated uptake of aliskiren., Results: Co-administration of a single dose of aliskiren with grapefruit juice decreased the plasma concentration of aliskiren, with mean decreases in the AUC(inf), AUC(last), and C(max) of 38, 37, and 61%, respectively. The uptake of [¹⁴C]aliskiren into OATP2B1-expressing cells was essentially the same as that into control cells, and the inhibitor combination atorvastatin and rifamycin had no effect on [¹⁴C]aliskiren accumulation in either cell type. The uptake of [¹⁴C]aliskiren and [³H]fexofenadine was linear in OATP1A2-expressing cells and was reduced by naringin, with IC₅₀ values of 75.5 ± 11.6 and 24.2 ± 2.0 μM, respectively., Conclusions: Grapefruit juice decreases exposure of aliskiren partially via inhibition of intestinal OATP1A2.

The purpose of this study was to establish an in vitro system that evaluates the effects of P-glycoprotein (P-gp)-mediated efflux on the oral absorption of P-gp substrates. An in vitro system (dissolution/permeation system, D/P system) was developed that consisted of apical and basal chambers and a Caco-2 cell monolayer mounted between the chambers. Both sides of the monolayer were filled with physiological solution and were stirred at 200rpm. The dissolution in the apical medium and permeation to the basal medium were monitored for 2h after P-gp substrates were applied to the apical side of the system. When erythromycin existed in the apical medium, the permeations of fexofenadine and talinolol were significantly enhanced without change in their dissolution. The prediction of oral absorptions of fexofenadine and talinolol from in vitro data indicated that co-administration of erythromycin results in 2.1- and 1.9-fold higher oral absorptions, respectively. Moreover, the D/P system could estimate the effect of cremophor EL on the oral absorption of saquinavir. These estimations corresponded well to in vivo human observations. Our in vitro system is useful in assessment of the effect of P-gp-mediated efflux on in vivo oral absorption of P-gp substrates., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration.

The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans.

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450(terf), was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV-Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450-substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with k(m) values at the μM level, and k(cat) values around 30min(-1). Sequencing of the p450(terf) gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56-65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (V(max)/K(m)) that were lowered considerably (by 84-60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased K(m) with or without decreased V(max) values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, K(m) values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but V(max) values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34-52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.

After the dosing of an extended-release (ER) formulation, compounds may exist in solutions at various concentrations in the colon because the drugs are released at various speeds from the ER dosage form. The aim of this study was to investigate the relationship between the drug concentration profiles in plasma and the drug doses in the colon. Several drug solutions of different concentrations were directly administered into the ascending colon of dogs using a lubricated endoscope, and the effects of the drug dose on colonic absorption were estimated. As a result, dose-dependency of colonic absorption varied from compound to compound. Although the relative bioavailability of colonic administration of diclofenac, metformin and cevimeline compared to oral administration was similar regardless of the drug doses in the colon, colonic absorption of diltiazem varied according to the doses. From the results of the co-administration of verapamil and fexofenadine, it was clear that diltiazem underwent extensive hepatic and gastrointestinal first-pass metabolism, resulting in a low area under the curves (AUC) at a low drug dose. During the design of oral ER delivery systems, a colonic absorption study of candidate compounds should be carried out at several solutions of different drug concentrations and assessed carefully.

P-Glycoprotein accounts for multidrug resistance in chemotherapy patients and contributes to reduced oral bioavailability and distribution of drugs in the brain. The aim of this study was to establish the qualitative and quantitative structure-activity relationships (QSAR) of flavonoid modulation effects on P-glycoprotein (P-gp)'s function. Using human colorectal adenocarcinoma (HCT15) cells as an in vitro model and fexofenadine as a P-gp substrate, the modulation effects on P-gp at three concentration levels of 22 representative compounds from four flavonoid families were evaluated. Results showed that the modulation (enhanced or inhibitory) effects could be divided into three ranges designated as enhanced (<100%) as compared to the control, low inhibitory (100-217%) and high inhibitory (>217%) as compared to verapamil. An optimal QSAR was constructed for Y1 (adjusted R(2)=0.4798), Y2 (adjusted R(2)=0.6809), and Y3 (adjusted R(2)=0.5902), respectively. This was further confirmed by a highly correlated plot of the predicted percent inhibition against observed values from a respective QSAR equation.

Hyaluronic acid (HA)-based microspheres containing PEG 6000 and/or sodium taurocholate (NaTC) were prepared by the spray-drying method for nasal delivery of fexofenadine hydrochloride (HCl). Their physicochemical properties including particle size and drug contents were determined, while their morphology examined by a scanning electron microscope (SEM). The effects of the solubilizer (PEG 6000) and the permeation enhancer (NaTC) on the in vitro release characteristics of fexofenadine x HCl were observed. Moreover, the in vitro permeation of fexofenadine x HCl was determined using the human nasal cell (HNE) monolayers cultured on Transwell inserts. After nasal administration, fexofenadine x HCl in rabbit plasma was determined by the LC-MS/MS system. Fexofenadine x HCl-loaded microspheres were of spherical shape with 20-30 microm mean diameter. The loading efficiency was about 95%. In vitro release of fexofenadine x HCl from the microspheres was significantly increased with the addition of PEG 6000. Although NaTC did not alter the in vitro release of drug from the microspheres, its addition further increased the in vitro permeation of fexofenadine x HCl across the HNE cell monolayers. Moreover, the bioavailability of fexofenadine x HCl after nasal administration of the microsphere formulation to rabbits was increased up to about 48% while that of the control solution was only about 3%. These results indicated that the HA microsphere formulation could further be developed into a clinically useful nasal delivery system of fexofenadine x HCl., (2010 Elsevier B.V. All rights reserved.)

Both mRNA and protein levels of the carboxylesterase (CES) isozymes, hCE1 and hCE2, in Caco-2 cells increase in a time-dependent manner, but hCE1 levels are always higher than those of hCE2. In human small intestine, however, the picture is reversed, with hCE2 being the predominant isozyme. Drugs hydrolyzed by hCE1 but not by hCE2 can be hydrolyzed in Caco-2 cells, but they are barely hydrolyzed in human small intestine. The results in Caco-2 cells can be misleading as a predictor of what will happen in human small intestine. In the present study, we proposed a novel method for predicting the absorption of prodrugs in the absence of CES-mediated hydrolysis in Caco-2 cells. The specific inhibition against CES was achieved using bis-p-nitrophenyl phosphate (BNPP). The optimal concentration of BNPP was determined at 200 microM by measuring the transport and hydrolysis of O-butyryl-propranolol (butyryl-PL) as a probe. BNPP concentrations of more than 200 microM inhibited 86% of hydrolysis of butyryl-PL, resulting in an increase in its apparent permeability. Treatment with 200 microM BNPP did not affect paracellular transport, passive diffusion, or carrier-mediated transport. Furthermore, the proposed evaluation system was tested for ethyl fexofenadine (ethyl-FXD), which is a superior substrate for hCE1 but a poor one for hCE2. CES-mediated hydrolysis of ethyl-FXD was 94% inhibited by 200 microM BNPP, and ethyl-FXD was passively transported as an intact prodrug. From the above observations, the novel evaluation system is effective for the prediction of human intestinal absorption of ester-type prodrugs.

A mechanistic model was applied to quantitatively derive the kinetic parameters from in vitro hepatic uptake transport data. These parameters were used as input to simulate in vivo elimination using a fully mechanistic physiologically based pharmacokinetic (PBPK) model. Fexofenadine and napsagatran, both BDDCS class 3 drugs, were chosen as model compounds. In rat, both compounds are hardly metabolized and are eliminated unchanged mostly through biliary excretion. Uptake was estimated in this study based on plated rat hepatocytes, and a mechanistic model was used to derive the active and passive transport parameters, namely Michaelis-Menten uptake parameters (V(maxI) and K(mI,u)) together with passive diffusion (P(dif)) and nonspecific binding. Maximum transport velocity and passive diffusion were scaled to in vivo parameters (J(maxI) and PS(TC)) using hepatocellularity. Biliary excretion, through passive and active transport, was assessed from in vivo studies. These transport parameters were then used as input in a whole body physiologically based model in which the liver compartment was parametrized for the different passive and active transport processes. Each of the processes was linked to the free concentration in the relevant compartment. For napsagatran hepatic uptake, no passive diffusion and no binding were detected in vitro besides the active transport (K(mI,u) = 88.4 +/- 8.1 microM, V(maxI) = 384 +/- 19 pmol/mg/min). Fexofenadine was rapidly taken up into rat hepatocytes (K(mI,u) = 271 +/- 35 microM, V(maxI) = 3162 +/- 274 pmol/mg/min), and some contribution of passive diffusion to the uptake (P(dif) = 2.08 +/- 0.67 microL/mg/min) was observed. For fexofenadine, the biliary export rate was found to be slower than the uptake, leading to drug accumulation in liver. No accumulation was observed for napsagatran where excretion was faster than hepatic uptake. Observed plasma, liver and bile concentration time profiles were compared to PBPK simulations based on scaled in vitro transport kinetic parameters. An uncertainty analysis indicated that for both compounds the scaled in vitro uptake clearance had to be adjusted with an additional empirical scaling factor of 10 to match the plasma and liver concentrations and biliary excretion profiles. Applying this model, plasma clearance (CL(P)) and half-life (t(1/2)), maximum liver concentration (C(maxL)) and fraction excreted in bile (f(bile)) were predicted within 2-fold. In vitro uptake data had most impact on the simulated plasma and biliary excretion profiles, while accurate simulations of liver concentrations required also quantitative estimates of biliary excretion transport. This study indicated that the mechanistic model allowed for accurate evaluation of in vitro experiments; and the scaled kinetic parameters of hepatic uptake transport enabled the prediction of in vivo PK profiles and plasma clearances, using PBPK modeling.

We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug-drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with K(i) values of 3.9 and 4.1 micromol/l, respectively. The [I](in,max,u)/K(i) ratios were 0.004 and 0.003, respectively. The K(i) values were about 300-fold greater than the [I](in,max,u), therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.

To characterize drug binding to the human ether-a-go-go related gene (hERG) channel, a synergic approach interplaying patch-clamp experiments and a docking study was developed. Mutations were introduced into concatenated dimers of the hERG channel that were assembled into a heterotetramer with mutated diagonal subunits. The binding affinities of three drugs (cisapride, terfenadine, and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride (E-4031, 1)) to a set of mutant channels were examined electrophysiologically to assess the involved residues, their number, and relative positions. Cisapride and 1 interacted with Tyr652 residues on adjacent subunits, while terfenadine interacted with Tyr652 residues on diagonal, but not on adjacent, subunits. Phe656 was involved in the binding of all three drugs, and Ser624 was found to be only involved in cisapride and 1. The docking models demonstrated that pi-pi and CH-pi interactions rather than cation-pi interaction play a key role in drug binding to the hERG channel.

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V(max) and K(m), as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P(dif)). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V(max) and K(m) were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5-6-fold higher permeability at 37 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.

Block of cardiac hERG K+ channels by the antihistamine terfenadine and the prokinetic agent cisapride is associated with prolonged ventricular repolarization and an increased risk of ventricular arrhythmia. Here, we used a site-directed mutagenesis approach to determine the molecular determinants of hERG block by terfenadine and cisapride. Wild-type and mutant hERG channels were heterologously expressed in Xenopus laevis oocytes and characterized by measuring whole cell currents with two-microelectrode voltage clamp techniques. Mutation of T623, S624, Y652, or F656 to Ala reduced channel sensitivity to block by terfenadine. The same mutations reduced sensitivity to cisapride. These data confirm our previous findings that polar residues (T623, S624) located near the base of the pore helix and aromatic residues (Y652, F656) located in the S6 domain are key molecular determinants of the hERG drug binding site. Unlike methanesulfonanilides (dofetilide, MK-499, E-4031, ibutilide) or clofilium, mutation of V625, G648, or V659 did not alter the sensitivity of hERG channels to terfenadine or cisapride. As previously proposed by molecular modeling studies (Farid R, et al. Bioorg Med Chem. 2006;14:3160-3173), our findings suggest that different drugs can adopt distinct modes of binding to the central cavity of hERG.

The disposition of fexofenadine, a commonly used antihistamine drug, is governed primarily by active transport. Biliary excretion of the parent compound is the major route of systemic clearance. Previous studies demonstrated that fexofenadine hepatic uptake is mediated by organic anion transporting polypeptides. Recently, we showed that in mice fexofenadine is excreted into bile primarily by multidrug resistance-associated protein (Mrp) 2 (Abcc2). In the present study, the roles of Mrp3 (Abcc3) and Mrp4 (Abcc4) in the hepatobiliary disposition of fexofenadine were examined in knockout mice using in situ liver perfusion. Compared with that in wild-type mice, basolateral excretion of fexofenadine was impaired, resulting in a approximately 50% decrease in perfusate recovery in Abcc3(-/-) mice; in contrast, fexofenadine hepatobiliary disposition was unaltered in Abcc4(-/-) mice. As expected, in Abcc2(-/-) mice, fexofenadine was redirected from the canalicular to the basolateral membrane for excretion. In Abcc2(-/-)/Abcc3(-/-) double-knockout mice, fexofenadine biliary excretion was impaired, but perfusate recovery was similar to that in wild-type mice and more than 2-fold higher than that in Abcc3(-/-) mice, presumably due to compensatory basolateral transport mechanism(s). These results demonstrate that multiple transport proteins are involved in the hepatobiliary disposition of fexofenadine. In addition to Mrp2 and Mrp3, other transport proteins play an important role in the biliary and hepatic basolateral excretion of this zwitterionic drug.

Fexofenadine (FEX), an H(1)-receptor antagonist, is eliminated from the liver mainly in an unchanged form. Our previous study suggested that organic anion-transporting polypeptide (OATP) 1B3 contributes mainly to the hepatic uptake of FEX. On the other hand, a clinical report demonstrated that a T521C mutation of OATP1B1 increased its plasma area under the plasma concentration-time curve. Several compounds are reported to have a drug interaction with FEX, and some of this may be caused by the inhibition of its hepatic uptake. We determined which transporters are involved in the hepatobiliary transport of FEX by using double transfectants and examined whether clinically reported drug interactions with FEX could be explained by the inhibition of its hepatic uptake. Vectorial basal-to-apical transport of FEX was observed in double transfectants expressing OATP1B1/multidrug resistance-associated protein 2 (MRP2) and OATP1B3/MRP2, suggesting that OATP1B1 as well as OATP1B3 is involved in the hepatic uptake of FEX and that MRP2 can recognize FEX as a substrate. The inhibitory effects of compounds on FEX uptake in OATP1B3-expressing HEK293 cells were investigated, and the maximal degree of increase in plasma AUC of FEX by drug interaction in clinical situations was estimated. As a result, cyclosporin A and rifampicin were found to have the potential to interact with OATP1B3-mediated uptake at clinical concentrations. From these results, most of the reported drug interaction cannot be explained by the inhibition of hepatic uptake of FEX, and different mechanisms such as the inhibition of intestinal efflux should be considered.

The aim of this study is to evaluate the potential use of first-generation (G1) polyamidoamine (PAMAM) dendrimers as drug carriers to enhance the permeability, hence oral absorption, of drugs that are substrates for P-glycoprotein (P-gp) efflux transporters. G1 PAMAM dendrimer-based prodrugs of the water-insoluble P-gp substrate terfenadine (Ter) were synthesized using succinic acid (suc) or succinyl-diethylene glycol (suc-deg) as a linker/spacer (to yield G1-suc-Ter and G1-suc-deg-Ter, respectively). In addition, the permeability of G1-suc-deg-Ter was enhanced by attaching two lauroyl chains (L) to the dendrimer surface (L2-G1-suc-deg-Ter). All of the G1 dendrimer-terfenadine prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer at pH 7.4 (log K(app)). The influence of the dendrimer prodrugs on the integrity and viability of human Caucasian colon adenocarcinoma cells (Caco-2) was determined by measuring the transepithelial electrical resistance (TEER) and leakage of lactate dehydrogenase (LDH) enzyme, respectively. The LDH assay indicated that the dendrimer prodrugs had no impact on the viability of Caco-2 cells up to a concentration of 1 mM. However, the IC(50) of the prodrugs was lower than that of G1 PAMAM dendrimer because of the high toxicity of terfenadine. Measurements of the transport of dendrimer prodrugs across monolayers of Caco-2 cells showed an increase of the apparent permeability coefficient (P(app)) of terfenadine in both apical-to-basolateral (A --> B) and basolateral-to-apical (B --> A) directions after its conjugation to G1 PAMAM dendrimer. The A --> B P(app) of the dendrimer prodrugs was significantly greater than B --> A P(app). The surface-modified dendrimer prodrug L2-G1-suc-deg-Ter showed the highest A --> B permeability among the conjugates.

1. The aim of the present study was to examine the effect of bacterial lipopolysaccharide (LPS) on the disposition of an organic anion transporting polypeptide and P-glycoprotein substrate in the rat isolated perfused liver. 2. Male Sprague-Dawley rats were divided into four groups. Three of the groups received 1, 2.5 or 5 mg/kg, i.p., Escherichia coli LPS in sterile saline. The fourth group received an equivalent volume of sterile saline i.p. Twenty-four hours after treatment, rats were anaesthetized and the liver isolated and perfused with fexofenadine at an initial concentration of 2000 ng/mL in a recirculating system. Perfusate and bile samples were collected for 60 min and the liver was collected at the end of the perfusion. Fexofenadine concentrations were determined by HPLC. Fexofenadine pharmacokinetic parameters, the final liver : perfusate (L : P) and bile : liver (B : L) concentration ratios were determined. 3. Injection of LPS changed the hepatic disposition of fexofenadine. The changes were most marked in the 5 mg/kg LPS group. Notably, clearance from the perfusate (CL) and into the bile (CLB; 5.9 +/- 0.6 and 1.24 +/- 0.20 mL/min, respectively), L : P (44 +/- 11) and B : L (17 +/- 2) were all reduced (P < 0.05) in this group compared with control (CL 10.0 +/- 1.1 mL/min; CLB 2.7 +/- 0.5 mL/min; L : P 87 +/- 14; and B : L 30 +/- 4). 4. In conclusion CL and CLB were reduced following treatment with LPS in a manner consistent with downregulation of both canalicular and sinusoidal transport.