Effects of membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver of drugs: A microdialysis study in rats.

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. Kpuu,liver and Kpuu,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.

The Role of P-Glycoprotein in Decreasing Cell Membranes Permeability during Oxidative Stress.

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.

Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system.

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.

Terfenadone is a strong inhibitor of CYP2J2 present in the human liver and intestinal microsomes.

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 IC50 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 IC50 values of 0.43, 0.08 and 1.07 µM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC50 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 (IC50 = 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.

Terfenadine metabolism of human cytochrome P450 2J2 containing genetic variations (G312R, P351L and P115L).

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 Kd 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 kcat and Km 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.

Sorption of citalopram, irbesartan and fexofenadine in soils: Estimation of sorption coefficients from soil properties.

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, KF (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 KF value for n = 1 was 1838 cm3 g-1) followed by fexofenadine (KF = 35.1 cm3/n µg1-1/n g-1, n = 1.19) and irbesartan (KF = 3.96 cm3/n µg1-1/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 KF values for different compounds (RIRB,FEX = 0.895, p-value<0.01; RIRB,CIT = -0.835, p-value<0.05; RFEX,CIT = -0.759, p-value<0.05) and by the reverse relationships between the KF values and soil properties in the pedotransfer functions. While the KF 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 KF 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 (R2 = 93.4), SCS and Cox for irbesartan (R2 = 96.3), and clay content and Cox for fexofenadine (R2 = 82.9).

Fexofenadine, a Putative In Vivo P-glycoprotein Probe, Fails to Predict Clearance of the Substrate Tacrolimus in Renal Recipients.

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 Cmax (-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 (R2 = 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.

CYP3A4 Activity is Markedly Lower in Patients with Crohn's Disease.

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.

Influence of P-glycoprotein on the disposition of fexofenadine and its enantiomers.

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.

D-Optimal mixture design optimization of an HPLC method for simultaneous determination of commonly used antihistaminic parent molecules and their active metabolites in human serum and urine.

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.

Binding of fexofenadine hydrochloride to bovine serum albumin: structural considerations by spectroscopic techniques and molecular docking.

The binding interaction of peripheral H1 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.

Reply to "Comment on 'In Silico Modeling of Gastrointestinal Drug Absorption: Predictive Performance of Three Physiologically Based Absorption Models'".

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.

Virtual Clinical Trial Toward Polytherapy Safety Assessment: Combination of Physiologically Based Pharmacokinetic/Pharmacodynamic-Based Modeling and Simulation Approach With Drug-Drug Interactions Involving Terfenadine as an Example.

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 Cmax 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.

Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates, astemizole and terfenadine.

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.

Prediction of area under the curve for a p-glycoprotein, a CYP3A4 and a CYP2C9 substrate using a single time point strategy: assessment using fexofenadine, itraconazole and losartan and metabolites.

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.

Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin.

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.

Imaging of drug and metabolite distribution by MS: case studies.

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.

Paroxetine decreased plasma exposure of glyburide partly via inhibiting intestinal absorption in rats.

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.

Determination of fexofenadine in Hank's balanced salt solution by high-performance liquid chromatography with ultraviolet detection: application to Caco-2 cell permeability studies.

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.

Substrate- and dose-dependent drug interactions with grapefruit juice caused by multiple binding sites on OATP2B1.

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.