Correction to: Comparative Study of the Dose-Dependence of OATP1B Inhibition by Rifampicin Using Probe Drugs and Endogenous Substrates in Healthy Volunteers.

There was a miscalculation of coproporphyrin I AUC0-24h in the published article (Volume 35, Number 7). After the correction of AUC0-24h, AUC ratio and R-square were re-calculated. Then, following corrections were made in the abstract, the body, Fig. 3, Fig. 4 and Table 2 in this article.

Evaluation of Alteration in Hepatic and Intestinal BCRP Function In Vivo from ABCG2 c.421C>A Polymorphism Based on PBPK Analysis of Rosuvastatin.

Polymorphism c.421C>A in the ABCG2 gene is thought to reduce the activity of breast cancer resistance protein (BCRP), a xenobiotic transporter, although it is not clear which organ(s) contributes to the polymorphism-associated pharmacokinetic change. The aim of the present study was to estimate quantitatively the influence of c.421C>A on intestinal and hepatic BCRP activity using a physiologically based pharmacokinetic (PBPK) model of rosuvastatin developed from clinical data and several in vitro studies. Simultaneous fitting of clinical data for orally and intravenously administered rosuvastatin, obtained in human subjects without genotype information, was first performed with the PBPK model to estimate intrinsic clearance for hepatic elementary process. The fraction of BCRP activity in 421CA and 421AA (fca and faa values, respectively) with respect to that in 421CC subjects was then estimated based on extended clearance concepts and simultaneous fitting to oral administration data for the three genotypes (421CC, 421CA, and 421AA). On the assumption that c.421C>A affects both intestinal and hepatic BCRP, clinical data in each genotype were well reproduced by the model, and the estimated terminal half-life was compatible with the observed values. The assumption that c.421C>A affects only either intestinal or hepatic BCRP gave poorer agreement with observed values. The faa values obtained on the former assumption were 0.48-0.54. Thus, PBPK model analysis enabled quantitative evaluation of alteration in BCRP activity owing to c.421C>A, and BCRP activity in 421AA was estimated as half that in 421CC.

Quantitative Analysis of Complex Drug-Drug Interactions between Cerivastatin and Metabolism/Transport Inhibitors Using Physiologically Based Pharmacokinetic Modeling.

Cerivastatin (CER) was withdrawn from the world market because of lethal rhabdomyolysis. Coadministrations of CER and cyclosporine A (CsA) or gemfibrozil (GEM) have been reported to increase the CER blood concentration. CsA is an inhibitor of organic anion transporting polypeptide (OATP)1B1 and CYP3A4, and GEM and its glucuronide (GEM-glu) inhibit OATP1B1 and CYP2C8. The purpose of this study was to describe the transporter-/enzyme-mediated drug-drug interactions (DDIs) of CER with CsA or GEM based on unified physiologically based pharmacokinetic (PBPK) models and to investigate whether the DDIs can be quantitatively analyzed by a bottom-up approach. Initially, the PBPK models for CER and GEM/GEM-glu were constructed based on the previously reported standard protocols. Next, the drug-dependent parameters were optimized by Cluster Newton Method. Thus, described concentration-time profiles for CER and GEM/GEM-glu agreed well with the clinically observed data. The DDIs were then simulated using the established PBPK models with previously obtained in vitro inhibition constants of CsA or GEM/GEM-glu against the OATP1B1 and cytochrome P450s. DDIs with the inhibitors were underestimated compared with observed data using the geometric means of reported values. To search for better described parameters within the range of in vitro values, sensitivity analyses were performed for DDIs of CER. Using the in vitro parameter sets selected by sensitivity analyses, these DDIs were well reproduced, indicating that the present PBPK models were able to describe adequately the clinical DDIs based on a bottom-up approach. The approaches in this study would be applicable to the prediction of other DDIs involving both transporters and metabolic enzymes.

Physiologically Based Pharmacokinetic Modeling of Bosentan Identifies the Saturable Hepatic Uptake As a Major Contributor to Its Nonlinear Pharmacokinetics.

Bosentan is a substrate of hepatic uptake transporter organic anion-transporting polypeptides (OATPs), and undergoes extensive hepatic metabolism by cytochrome P450 (P450), namely, CYP3A4 and CYP2C9. Several clinical investigations have reported a nonlinear relationship between bosentan doses and its systemic exposure, which likely involves the saturation of OATP-mediated uptake, P450-mediated metabolism, or both in the liver. Yet, the underlying causes for the nonlinear bosentan pharmacokinetics are not fully delineated. To address this, we performed physiologically based pharmacokinetic (PBPK) modeling analyses for bosentan after its intravenous administration at different doses. As a bottom-up approach, PBPK modeling analyses were performed using in vitro kinetic parameters, other relevant parameters, and scaling factors. As top-down approaches, three different types of PBPK models that incorporate the saturation of hepatic uptake, metabolism, or both were compared. The prediction from the bottom-up approach (models 1 and 2) yielded blood bosentan concentration-time profiles and their systemic clearance values that were not in good agreement with the clinically observed data. From top-down approaches (models 3, 4, 5-1, and 5-2), the prediction accuracy was best only with the incorporation of the saturable hepatic uptake for bosentan. Taken together, the PBPK models for bosentan were successfully established, and the comparison of different PBPK models identified the saturation of the hepatic uptake process as a major contributing factor for the nonlinear pharmacokinetics of bosentan.

Comparative Study of the Dose-Dependence of OATP1B Inhibition by Rifampicin Using Probe Drugs and Endogenous Substrates in Healthy Volunteers.

PURPOSE: To evaluate association of the dose-dependent effect of rifampicin, an OATP1B inhibitor, on the plasma concentration-time profiles among OATP1B substrates drugs and endogenous substrates. METHODS: Eight healthy volunteers received atorvastatin (1 mg), pitavastatin (0.2 mg), rosuvastatin (0.5 mg), and fluvastatin (2 mg) alone or with rifampicin (300 or 600 mg) in a crossover fashion. The plasma concentrations of these OATP1B probe drugs, total and direct bilirubin, glycochenodeoxycholate-3-sulfate (GCDCA-S), and coproporphyrin I, were determined. RESULTS: The most striking effect of 600 mg rifampicin was on atorvastatin (6.0-times increase) and GCDCA-S (10-times increase). The AUC0-24h of atorvastatin was reasonably correlated with that of pitavastatin (r2 = 0.73) and with the AUC0-4h of fluvastatin (r2 = 0.62) and sufficiently with the AUC0-24h of rosuvastatin (r2 = 0.32). The AUC0-24h of GCDCA-S was reasonably correlated with those of direct bilirubin (r2 = 0.74) and coproporphyrin I (r2 = 0.78), and sufficiently with that of total bilirubin (r2 = 0.30). The AUC0-24h of GCDCA-S, direct bilirubin, and coproporphyrin I were reasonably correlated with that of atorvastatin (r2 = 0.48-0.70) [corrected]. CONCLUSION: These results suggest that direct bilirubin, GCDCA-S, and coproporphyrin I are promising surrogate probes for the quantitative assessment of potential OATP1B-mediated DDI.

Comparison of Methods for Estimating Unbound Intracellular-to-Medium Concentration Ratios in Rat and Human Hepatocytes Using Statins.

It is essential to estimate concentrations of unbound drugs inside the hepatocytes to predict hepatic clearance, efficacy, and toxicity of the drugs. The present study was undertaken to compare predictability of the unbound hepatocyte-to-medium concentration ratios (Kp,uu) by two methods based on the steady-state cell-to-medium total concentration ratios at 37°C and on ice (Kp,uu,ss) and based on their initial uptake rates (Kp,uu,V0). Poorly metabolized statins were used as test drugs because of their concentrative uptake via organic anion-transporting polypeptides. Kp,uu,ss values of these statins provided less interexperimental variation than the Kp,uu,V0 values, because only data at longer time are required for Kp,uu,ss Kp,uu,V0 values for pitavastatin, rosuvastatin, and pravastatin were 1.2- to 5.1-fold Kp,uu,ss in rat hepatocytes; Kp,uu,V0 values in human hepatocytes also tended to be larger than corresponding Kp,uu,ss To explain these discrepancies, theoretical values of Kp,uu,ss and Kp,uu,V0 were compared with true Kp,uu (Kp,uu,true), considering the inside-negative membrane potential and ionization of the drugs in hepatocytes and medium. Membrane potentials were approximately -30 mV in human hepatocytes at 37°C and almost abolished on ice. Theoretical equations considering the membrane potentials indicate that Kp,uu,ss values for the statins are 0.85- to 1.2-fold Kp,uu,true, whereas Kp,uu,V0 values are 2.2- to 3.1-fold Kp,uu,true, depending on the ratio of the passive permeability of the ionized to nonionized forms. In conclusion, Kp,uu,ss values of anions are similar to Kp,uu,true when the inside-negative membrane potential is considered. This suggests that Kp,uu,ss is preferable for estimating the concentration of unbound drugs inside the hepatocytes.

A Clinical Cassette Dosing Study for Evaluating the Contribution of Hepatic OATPs and CYP3A to Drug-Drug Interactions.

PURPOSE: To demonstrate the relative importance of organic anion-transporting polypeptides (OATPs) and cytochrome P450 3A (CYP3A) in the hepatic elimination of substrate drugs. METHODS: A cocktail of subtherapeutic doses of bosentan, repaglinide, clarithromycin, darunavir, simeprevir, and midazolam (CYP3A probe) was administered orally to eight healthy volunteers. Rifampicin (OATP inhibitor; 600 mg, p.o.) and itraconazole (CYP3A inhibitor; 200 mg, i.v.) were coadministered with the cocktail in the second and third phases, respectively. Based on the extended clearance concept, in vivo ß values (fraction of metabolism plus biliary excretion among all the intracellular fates of drugs including basolateral efflux) and Rdif values (ratio of diffusional uptake to active uptake) were estimated. RESULTS: Rifampicin increased plasma AUCs of bosentan (×3.2), repaglinide (×1.9), clarithromycin (×1.9) and simeprevir (×7.2). Itraconazole increased those of clarithromycin (×2.3), simeprevir (×2.2) and midazolam (×3.7), which had relatively small ß values. The plasma AUC of bosentan (with relatively large ß and small Rdif) was dominated by OATP-mediated uptake. The AUC of simeprevir was also dominated by OATP-mediated uptake because of its small Rdif value. CONCLUSIONS: The DDI study clarified the rate-determining processes of OATP/CYP3A substrates. Our analyses provide valuable information for predicting complex drug-drug interactions involving multiple processes.

Investigation of Glycochenodeoxycholate Sulfate and Chenodeoxycholate Glucuronide as Surrogate Endogenous Probes for Drug Interaction Studies of OATP1B1 and OATP1B3 in Healthy Japanese Volunteers.

PURPOSE: To assess the use of glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate 3- or 24-glucuronide (CDCA-3G or -24G) as surrogate endogenous substrates in the investigation of drug interactions involving OATP1B1 and OATP1B3. METHODS: Uptake of GCDCA-S and CDCA-24G was examined in HEK293 cells transfected with cDNA for OATP1B1, OATP1B3, and NTCP and in cryopreserved human hepatocytes. Plasma concentrations of bile acids and their metabolites (GCDCA-S, CDCA-3G, and CDCA-24G) were determined by LC-MS/MS in eight healthy volunteers with or without administration of rifampicin (600 mg, po). RESULTS: GCDCA-S and CDCA-24G were substrates for OATP1B1, OATP1B3, and NTCP. The uptake of [3H]atorvastatin, GCDCA-S, and CDCA-24G by human hepatocytes was significantly inhibited by both rifampicin and pioglitazone, whereas that of taurocholate was inhibited only by pioglitazone. Rifampicin elevated plasma concentrations of GCDCA-S more than those of other bile acids. The area under the plasma concentration-time curve for GCDCA-S was 20.3 times higher in rifampicin-treated samples. CDCA-24G could be detected only in plasma from the rifampicin-treatment phase, and CDCA-3G was undetectable in both phases. CONCLUSIONS: We identified GCDCA-S and CDCA-24G as substrates of NTCP, OATP1B1, and OATP1B3. GCDCA-S is a surrogate endogenous probe for the assessment of drug interactions involving hepatic OATP1B1 and OATP1B3.

Clarification of the Mechanism of Clopidogrel-Mediated Drug-Drug Interaction in a Clinical Cassette Small-dose Study and Its Prediction Based on In Vitro Information.

Clopidogrel is reported to be associated with cerivastatin-induced rhabdomyolysis, and clopidogrel and its metabolites are capable of inhibiting CYP2C8 and OATP 1B1 in vitro. The objective of the present study was to identify the mechanism of clopidogrel-mediated drug-drug interactions (DDIs) on the pharmacokinetics of OATP1B1 and/or CYP2C8 substrates in vivo. A clinical cassette small-dose study using OATPs, CYP2C8, and OATP1B1/CYP2C8 probe drugs (pitavastatin, pioglitazone, and repaglinide, respectively) with or without the coadministration of either 600 mg rifampicin (an inhibitor for OATPs), 200 mg trimethoprim (an inhibitor for CYP2C8), or 300 mg clopidogrel was performed, and the area under the concentration-time curve (AUC) ratios (AUCRs) for probe substrates were predicted using a static model. Clopidogrel increased the AUC of pioglitazone (2.0-fold) and repaglinide (3.1-fold) but did not significantly change the AUC of pitavastatin (1.1-fold). In addition, the AUC of pioglitazone M4, a CYP2C8-mediated metabolite of pioglitazone, was reduced to 70% of the control by coadministration of clopidogrel. The predicted AUCRs using the mechanism-based inhibition of CYP2C8 by clopidogrel acyl-ß-glucuronide were similar to the observed AUCRs, and the predicted AUCR (1.1) of repaglinide using only the inhibition of OATP1B1 did not reach the observed AUCR (3.1). In conclusion, a single 300 mg of clopidogrel mainly inhibits CYP2C8-mediated metabolism by clopidogrel acyl-ß-glucuronide, but its effect on the pharmacokinetics of OATP1B1 substrates is negligible. Clopidogrel is expected to have an effect not only on CYP2C8 substrates, but also dual CYP2C8/OATP1B1 substrates as seen in the case of repaglinide.

Ticlopidine, a cholestatic liver injury-inducible drug, causes dysfunction of bile formation via diminished biliary secretion of phospholipids: involvement of biliary-excreted glutathione-conjugated ticlopidine metabolites.

The antiplatelet drug, ticlopidine (TIC), reportedly causes cholestatic liver injuries. The present study analyzed the effect of TIC on bile formation, revealing that the biliary secretion of phospholipids was significantly decreased in TIC-administered Sprague Dawley (SD) rats. However, the effect of TIC on biliary phospholipids was not observed in SD rats pretreated with diethylaminoethyl diphenylpropylacetate that inhibits cytochrome P450s (P450), or in Eisai hyperbilirubinemic rats (EHBR) lacking functional multidrug resistance-associated protein 2 (MRP2/ABCC2). These results suggest that glutathione-conjugated TIC metabolites (TIC-SGs), which were formed in the liver after P450s-mediated metabolism and were excreted extensively into bile by MRP2, mediated the observed alterations of the bile composition. Administration of TIC caused significant liver injuries in SD rats, with decreased biliary phospholipids, but not in EHBR, consistent with the in vitro observation that phospholipid-bile acid-mixed micelles moderated the cytotoxic effects of bile acids. Further analyses revealed that TIC-SGs did not directly inhibit multidrug resistance 3 P-glycoprotein (MDR3/ABCB4)-mediated phosphatidylcholine efflux in vitro. Because the diminished biliary secretion of phospholipids with TIC administration was restored by taurocholate infusion in SD rats, the decreased biliary concentration of bile acids, due to the stimulation of bile acid-independent bile flow driven by TIC-SGs, might have indirectly attenuated phospholipid secretion. In conclusion, extensive biliary excretion of TIC-SGs decreased the biliary secretion of phospholipids, which might have increased the risk of TIC-induced cholestatic liver injury.

[Development of Prediction Methods for Drug-Drug Interactions Based on the Construction of Physiologically Based Pharmacokinetic Models for Hepatic OATP Substrate Drugs and Endogenous Substrates].

Quantitative prediction of the potential for drug-drug interaction (DDI) is essential to guarantee the safety and efficacy of drugs. DDI screening, modeling, and prediction is standard practice in the pharmaceutical industry. This review describes our work on (1) the establishment of a standard framework for determining physiologically based pharmacokinetic (PBPK) model structures and parameters useful for quantitatively analyzing DDIs via hepatic organic anion transporting polypeptides (OATPs). By analyzing clinically observed DDIs involving several statins as substrates, and cyclosporin A and rifampicin as inhibitors, similar in vivo inhibition constants for OATPs by each inhibitor were obtained, regardless of the substrate. (2) We took a PBPK modeling-based approach to define rate-determining processes in hepatic elimination of several OATPs and CYP3A dual substrates using our clinical DDI data with specific inhibitors for OATPs and CYP3A. Essential in vivo parameters (the passive diffusion/active transport ratio in the uptake, and the fraction of intrinsic clearance in the total drug elimination from the hepatocytes) dominating the rate-determining process in hepatic elimination were estimated quantitatively. (3) Finally, using our clinical DDI data with rifampicin, we established a PBPK model for coproporphyrin I (CP-I), which is expected to act as an endogenous substrate (biomarker) supporting the prediction of DDI involving hepatic OATPs. Our PBPK modeling-based approach with several in vitro experiments using CP-I and OATP probe substrates (statins) demonstrated the usefulness of the translation of the effect of an OATP inhibitor on CP-I pharmacokinetics into that on OATP probe substrates in drug discovery and development.

NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport.

BACKGROUND & AIMS: Biliary cholesterol secretion helps maintain cholesterol homeostasis; it is regulated by the cholesterol exporter adenosine triphosphate-binding cassettes G5 and G8 (ABCG5/G8) and the cholesterol importer Niemann-Pick C1-like 1 (NPC1L1). We studied another putative regulator of cholesterol secretion into bile, Niemann-Pick C2 (NPC2)--a cholesterol-binding protein secreted by the biliary system--and determined its effects on transporter-mediated biliary secretion of cholesterol. METHODS: Mice with hepatic knockdown of Npc2 or that overexpressed NPC2 were created using adenovirus-mediated gene transfer; biliary lipids were characterized. The effects of secreted NPC2 on cholesterol transporter activity were examined in vitro using cells that overexpressed ABCG5/G8 or NPC1L1. RESULTS: Studies of mice with altered hepatic expression of NPC2 revealed that this expression positively regulates the biliary secretion of cholesterol, supported by the correlation between levels of NPC2 protein and cholesterol in human bile. In vitro analysis showed that secreted NPC2 stimulated ABCG5/G8-mediated cholesterol efflux but not NPC1L1-mediated cholesterol uptake. Consistent with these observations, no significant changes in biliary cholesterol secretion were observed on hepatic overexpression of NPC2 in ABCG5/G8-null mice, indicating that NPC2 requires ABCG5/G8 to stimulate cholesterol secretion. Analyses of NPC2 mutants showed that the stimulatory effect of biliary NPC2 was independent of the function of lysosomal NPC2 as a regulator of intracellular cholesterol trafficking. CONCLUSIONS: NPC2 is a positive regulator of biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport.

Cluster Gauss-Newton method analyses of PBPK model parameter combinations of coproporphyrin-I based on OATP1B-mediated rifampicin interaction studies.

Coproporphyrin I (CP-I) is an endogenous biomarker supporting the prediction of drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptide 1B (OATP1B). We previously constructed a physiologically-based pharmacokinetic (PBPK) model for CP-I using clinical DDI data with an OATP1B inhibitor, rifampicin (RIF). In this study, PBPK model parameters for CP-I were estimated using the cluster Gauss-Newton method (CGNM), an algorithm used to find multiple approximate solutions for nonlinear least-squares problems. Eight unknown parameters including the hepatic overall intrinsic clearance (CLint,all ), the rate of biosynthesis (vsyn ), and the OATP1B inhibition constant of RIF(Ki,u,OATP ) were estimated by fitting to the observed CP-I blood concentrations in two different clinical studies involving changing the RIF dose. Multiple parameter combinations were obtained by CGNM that could well capture the clinical data. Among those, CLint,all , Ki,u,OATP , and vsyn were sensitive parameters. The obtained Ki,u,OATP for CP-I was 5.0- and 2.8-fold lower than that obtained for statins, confirming our previous findings describing substrate-dependent Ki,u,OATP values. In conclusion, CGNM analyses of PBPK model parameter combinations enables estimation of the three essential parameters for CP-I to capture the DDI profiles, even if the other parameters remain unidentified. The CGNM also clarified the importance of appropriate combinations of other unidentified parameters to enable capture of the CP-I concentration time course under the influence of RIF. The described CGNM approach may also support the construction of robust PBPK models for additional transporter biomarkers beyond CP-I.

Physiologically-based pharmacokinetic model-based translation of OATP1B-mediated drug-drug interactions from coproporphyrin I to probe drugs.

The accurate prediction of OATP1B-mediated drug-drug interactions (DDIs) is challenging for drug development. Here, we report a physiologically-based pharmacokinetic (PBPK) model analysis for clinical DDI data generated in heathy subjects who received oral doses of cyclosporin A (CysA; 20 and 75 mg) as an OATP1B inhibitor, and the probe drugs (pitavastatin, rosuvastatin, and valsartan). PBPK models of CysA and probe compounds were combined assuming inhibition of hepatic uptake of endogenous coproporphyrin I (CP-I) by CysA. In vivo Ki of unbound CysA for OATP1B (Ki,OATP1B ), and the overall intrinsic hepatic clearance per body weight of CP-I (CLint,all,unit ) were optimized to account for the CP-I data (Ki,OATP1B , 0.536 ± 0.041 nM; CLint,all,unit , 41.9 ± 4.3 L/h/kg). DDI simulation using Ki,OATP1B reproduced the dose-dependent effect of CysA (20 and 75 mg) and the dosing interval (1 and 3 h) on the time profiles of blood concentrations of pitavastatin and rosuvastatin, but DDI simulation using in vitro Ki,OATP1B failed. The Cluster Gauss-Newton method was used to conduct parameter optimization using 1000 initial parameter sets for the seven pharmacokinetic parameters of CP-I (ß, CLint, all , Fa Fg , Rdif , fbile , fsyn , and vsyn ), and Ki,OATP1B and Ki,MRP2 of CysA. Based on the accepted 546 parameter sets, the range of CLint, all and Ki,OATP1B was narrowed, with coefficients of variation of 12.4% and 11.5%, respectively, indicating that these parameters were practically identifiable. These results suggest that PBPK model analysis of CP-I is a promising translational approach to predict OATP1B-mediated DDIs in drug development.

Itraconazole-induced cholestasis: involvement of the inhibition of bile canalicular phospholipid translocator MDR3/ABCB4.

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [¹4C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [³H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS): synthesis of derivatization reagents for carboxylic acids.

The derivatization reagents for carboxylic acids, N-(Pyridin-3-yl)hydrazinecarbothioamide, N-[4-(dimethylamino)phenyl]hydrazinecarbothioamide, 1-(2-aminoethyl)-3-(pyridin-3-yl)thiourea, 1-(2-aminoethyl)-3-[4-(dimethylamino)phenyl]thiourea and 4-(2-aminoethyl)-N-phenylpiperazine-1-carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed-phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision-induced dissociation, and thus they were suitable for MS/MS detection.

Evaluation of Hepatic Uptake of OATP1B Substrates by Short Term-Cultured Plated Human Hepatocytes: Comparison With Isolated Suspended Hepatocytes.

Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PSinf) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PSinf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (Kp,uu). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and Kp,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding.

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS).

Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopentylamino)-2,1,3-benzoxadiazole (DAABD-AP) and 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminobutylamino)-2,1,3-benzoxadiazole (DAABD-AB), for short-chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60 degrees C for 60 min. The generated derivatives were separated on the reversed-phase column and detected by ESI-MS/MS with the detection limits of 0.1-0.12 pmol on column. Upon collision-induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD-AP and DAABD-AB are suitable as the derivatization reagents in LC/ESI-MS/MS analysis.

Effect of OATP1B1 genotypes on plasma concentrations of endogenous OATP1B1 substrates and drugs, and their association in healthy volunteers.

This study aimed to elucidate the impact of OATP1B1 genotype (*1b/*1b, *1b/*15, and *15/*15) on plasma concentrations of endogenous OATP1B1 substrates. Healthy volunteers with OATP1B1 *1b/*1b (n = 10), *1b/*15 (n = 7), or *15/*15 (n = 2) received oral administration of a cocktail of statins (atorvastatin, pitavastatin, rosuvastatin, and fluvastatin). Mean area under the plasma concentration of atorvastatin, pitavastatin, and rosuvastatin in OATP1B1 *15/*15 were 2.2, 1.7 and 1.58-times greater than the corresponding values in OATP1B1 *1b/*1b, respectively, whereas that of fluvastatin was identical to those in other OATP1B1 genotypes. OATP1B1 *15/*15 also showed higher mean plasma concentrations of OATP1B1 endogenous substrates compared with the other OATP1B1 genotypes, such as coproporphyrin I, glycochenodeoxycholate sulfate (GCDCA-S), lithocholate sulfate (LCA-S), glycolithocholate sulfate (GLCA-S) and taurolithocholate sulfate (TLCA-S), but not total or direct bilirubin, chenodeoxycholate-24-glucuronide, or ω-dicarboxylic long-chain fatty acids. Area under the plasma concentration-time curves of plasma coproporphyrin I and GLCA-S discriminated OATP1B1 genotype *15/*15 from the other genotypes. In combination with previously published clinical studies, these results support the notion that coproporphyrin I, and GLCA-S and GCDCA-S could be a surrogate probe for assessing human in vivo OATP1B1 activities.

Total hepatocellular disposition profiling of rosuvastatin and pitavastatin in sandwich-cultured human hepatocytes.

This study describes the total disposition profiling of rosuvastatin (RSV) and pitavastatin (PTV) using a single systematic procedure called D-PREX (Disposition Profile Exploration) in sandwich-cultured human hepatocytes (SCHH). The biliary excretion fractions of both statins were clearly observed, which were significantly decreased dependent on the concentration of Ko143, an inhibitor for breast cancer resistance protein (BCRP). Ko143 also decreased the basolateral efflux fraction of RSV, whereas that of PTV was not significantly affected. To understand these phenomena, effects of Ko143 on biliary excretion (BCRP and multidrug resistance-associated protein (MRP) 2) and basolateral efflux (MRP3 and MRP4) transporters were examined using transporter-expressing membrane vesicles. BCRP, MRP3 and MRP4-mediated transport of RSV was observed, and Ko143 inhibited these transporters except MRP3. BCRP and MRP4 also mediated the transport of PTV, but the Ko143-mediated inhibition was only clear for BCRP. These results might explain the Ko143-mediated complete and partial inhibition of the biliary excretion and the basolateral efflux of RSV, respectively, in SCHH. In conclusion, D-PREX with sequential sampling of supernatants prior to cell lysis enables the evaluation of total drug disposition profiles resulting from complex interplays of intracellular pathways, which would provide high-throughput evaluation of drug disposition during drug discovery.