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This white paper examines recent progress, applications, and challenges in predicting unbound and total tissue and intra/subcellular drug concentrations using in vitro and preclinical models, imaging techniques, and physiologically based pharmacokinetic (PBPK) modeling. Published examples, regulatory submissions, and case studies illustrate the application of different types of data in drug development to support modeling and decision making for compounds with transporter-mediated disposition, and likely disconnects between tissue and systemic drug exposure. The goals of this article are to illustrate current best practices and outline practical strategies for selecting appropriate in vitro and in vivo experimental methods to estimate or predict tissue and plasma concentrations, and to use these data in the application of PBPK modeling for human pharmacokinetic (PK), efficacy, and safety assessment in drug development.

Tolvaptan, a vasopressin V2-receptor antagonist, has demonstrated efficacy in slowing kidney function decline in patients with autosomal dominant polycystic kidney disease (ADPKD). In the pivotal clinical trial, the incidence of elevated liver enzymes was higher in patients receiving tolvaptan compared with placebo. Adjudication by a panel of expert hepatologists concluded a causal link of tolvaptan to liver injury in patients with ADPKD. An ex situ isolated perfused liver (IPL) study of tolvaptan disposition was undertaken in a rodent model of ADPKD, the polycystic kidney (PCK) rat (n = 5), and compared with wild-type (WT) Sprague-Dawley rats (n = 6). Livers were perfused with tolvaptan, followed by a tolvaptan-free washout phase. Total recovery (mean ± S.D. percentage of dose; PCK vs. WT) of tolvaptan and two metabolites, DM-4103 and DM-4107, quantified by liquid chromatography-tandem mass spectroscopy, was 58.14% ± 24.72% vs. 43.40% ± 18.11% in liver, 20.10% ± 9.15% vs. 21.17% ± 12.51% in outflow perfusate, and 0.08% ± 0.01% vs. 0.39% ± 0.32% in bile. DM-4103 recovery (mean ± S.D. percentage of dose) was decreased in PCK vs. WT bile (

Drug transporter expression and function at the blood-brain barrier is altered in Alzheimer's disease (AD). However, the impact of AD on the expression of transporters and metabolizing enzymes in peripheral tissues has received little attention. The current study evaluated the expression of drug transporters and metabolizing enzymes in the small intestine and liver from 8- to 9-month-old female wild-type (WT) and APPswe/PSEN 1dE9 (APP/PS1) transgenic mice, a widely used AD model, using a quantitative targeted absolute proteomics (QTAP) approach. Furthermore, the general morphological appearance of the liver was assessed by immunohistochemistry, and lipid content was visualized using Oil Red O staining. The small intestines of APP/PS1 mice exhibited a significant 2.3-fold increase in multidrug resistance-associated protein 2 (Mrp2), a 1.9-fold decrease in monocarboxylate transporter 1 (Mct1), and a 3.6-fold increase in UDP-glucuronosyltransferase (Ugt) 2b5 relative to those from WT mice based on QTAP analysis. While the liver from APP/PS1 mice exhibited no changes in drug transporter expression, there was a 1.3-fold elevation in cytochrome P450 (Cyp) 51a1 and a 1.2-fold reduction in Cyp2c29 protein expression, and this was associated with morphological alterations including accumulation of hepatocyte lipids. These studies are the first to demonstrate that the protein expression of transporters and metabolizing enzymes important in oral drug absorption are modified in a mouse model of familial AD, which may lead to altered disposition of some orally administered drugs in AD.

Contains fulltext : 200052.pdf (Publisher’s version ) (Closed access) Drug transporters are critically important for the absorption, distribution, metabolism, and excretion (ADME) of many drugs and endogenous compounds. Therefore, disruption of these pathways by inhibition, induction, genetic polymorphisms, or disease can have profound effects on overall physiology, drug pharmacokinetics, drug efficacy, and toxicity. This white paper provides a review of changes in transporter function associated with acute and chronic disease states, describes regulatory pathways affecting transporter expression, and identifies opportunities to advance the field.

Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the "invisibility" of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases. Graphical abstract ᅟ.

Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma ). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow-up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case-by-case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.

BackgroundCW002 is an investigational nondepolarizing, neuromuscular blocking agent with a rapid onset and intermediate duration of action in animals. This is a single ascending dose, healthy subject study exploring tolerability, pharmacokinetics, and potency.MethodsPopulation pharmacokinetic and pharmacokinetic/pharmacodynamic models were developed using plasma drug concentration data from a previously published dose–response study in 28 healthy subjects receiving single doses of CW002 during sevoflurane anesthesia. Subjects included in the models were from five different dose cohorts (cohorts 3, 4, 5, 6, and 8 receiving 0.04, 0.06, 0.08, 0.10, and 0.14 mg/kg, respectively). Serial arterial plasma concentrations and muscle twitch heights were monitored.ResultsA four-compartment model was fit to the concentration–time data, whereas a transit compartment with a sigmoid Emax model was fit to the pharmacokinetic/pharmacodynamic data. The population pharmacokinetics of CW002 was linear with very low interindividual variability in clearance (10.8%). Simulations were conducted to predict the onset and offset of effect at 2×, 3×, and 4× ED95. The time to 80% block was predicted to be 1.5, 0.8, and 0.7 min for 2×, 3×, and 4× ED95 doses, respectively. The simulated 25 to 75% recovery index was independent of dose.ConclusionsCW002 has predictable pharmacokinetics and is likely to have a rapid onset with an intermediate duration of action at 3× ED95. This model provides information to inform critical decisions (e.g., dose, study design) for continued development of CW002.

The heteromeric steroid transporter organic solute transporter α/β (OSTα/β, SLC51A/B) was discovered over a decade ago, but its physiological significance in the liver remains uncertain. A major challenge has been the lack of suitable models expressing OSTα/β. Based on observations first reported here that hepatic OSTα/β is upregulated in nonalcoholic steatohepatitis, the aim of this research was to develop an in vitro model to evaluate OSTα/β function and interaction with drugs and bile acids. OSTα/β expression in human liver tissue was analyzed by quantitative RT-PCR, Western blotting, and immunofluorescence. Radiolabeled compounds were used to determine OSTα/β-mediated transport in the established in vitro model. The effect of bile acids and drugs, including those associated with cholestatic drug-induced liver injury, on OSTα/β-mediated transport was evaluated. Expression of OSTα/β was elevated in the liver of patients with nonalcoholic steatohepatitis and primary biliary cholangitis, whereas hepatocyte expression of OSTα/β was low in control liver tissue. Studies in the novel cell-based system showed rapid and linear OSTα/β-mediated transport for all tested compounds: dehydroepiandrosterone sulfate, digoxin, estrone sulfate, and taurocholate. The interaction study with 26 compounds revealed novel OSTα/β inhibitors: a biomarker for cholestasis, glycochenodeoxycholic acid; the major metabolite of troglitazone, troglitazone sulfate; and a macrocyclic antibiotic, fidaxomicin. Additionally, some drugs (e.g., digoxin) consistently stimulated taurocholate uptake in OSTα/β-overexpressing cells. Our findings demonstrate that OSTα/β is an important transporter in liver disease and imply a role for this transporter in bile acid-bile acid and drug-bile acid interactions, as well as cholestatic drug-induced liver injury. NEW & NOTEWORTHY The organic solute transporter OSTα/β is highly expressed in hepatocytes of liver tissue obtained from patients with nonalcoholic steatohepatitis and primary biliary cholangitis. OSTα/β substrates exhibit rapid, linear, and concentration-driven transport in an OSTα/β-overexpressing cell line. Drugs associated with hepatotoxicity modulate OSTα/β-mediated taurocholate transport. These data suggest that hepatic OSTα/β plays an essential role in patients with cholestasis and may have important clinical implications for bile acid and drug disposition.

Tolvaptan is a vasopressin V(2)-receptor antagonist that has shown promise in treating Autosomal Dominant Polycystic Kidney Disease (ADPKD). Tolvaptan was, however, associated with liver injury in some ADPKD patients. Inhibition of bile acid transporters may be contributing factors to drug-induced liver injury. In this study, the ability of tolvaptan and two metabolites, DM-4103 and DM-4107, to inhibit human hepatic transporters (NTCP, BSEP, MRP2, MRP3, and MRP4) and bile acid transport in sandwich-cultured human hepatocytes (SCHH) was explored. IC(50) values were determined for tolvaptan, DM-4103 and DM-4107 inhibition of NTCP (∼41.5, 16.3, and 95.6 μM, respectively), BSEP (31.6, 4.15, and 119 μM, respectively), MRP2 (>50, ∼51.0, and >200 μM, respectively), MRP3 (>50, ∼44.6, and 61.2 μM, respectively), and MRP4 (>50, 4.26, and 37.9 μM, respectively). At the therapeutic dose of tolvaptan (90 mg), DM-4103 exhibited a C(max)/IC(50) value >0.1 for NTCP, BSEP, MRP2, MRP3, and MRP4. Tolvaptan accumulation in SCHH was extensive and not sodium-dependent; intracellular concentrations were ∼500 μM after a 10-min incubation duration with tolvaptan (15 μM). The biliary clearance of taurocholic acid (TCA) decreased by 43% when SCHH were co-incubated with tolvaptan (15 μM) and TCA (2.5 μM). When tolvaptan (15 μM) was co-incubated with 2.5 μM of chenodeoxycholic acid, taurochenodeoxycholic acid, or glycochenodeoxycholic acid in separate studies, the cellular accumulation of these bile acids increased by 1.30-, 1.68-, and 2.16-fold, respectively. Based on these data, inhibition of hepatic bile acid transport may be one of the biological mechanisms underlying tolvaptan-associated liver injury in patients with ADPKD.

The prevalence of nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is increasing at an alarming rate. The role of bile acids in the development and progression of NAFLD to NASH and cirrhosis is poorly understood. This study aimed to quantify the bile acid metabolome in healthy subjects and patients with non-cirrhotic NASH under fasting conditions and after a standardized meal. Liquid chromatography tandem mass spectroscopy was used to quantify 30 serum and 16 urinary bile acids from 15 healthy volunteers and 7 patients with biopsy-confirmed NASH. Bile acid concentrations were measured at two fasting and four post-prandial time points following a high-fat meal to induce gallbladder contraction and bile acid reabsorption from the intestine. Patients with NASH had significantly higher total serum bile acid concentrations than healthy subjects under fasting conditions (2.2- to 2.4-fold increase in NASH; NASH 2595–3549 µM and healthy 1171–1458 µM) and at all post-prandial time points (1.7- to 2.2-fold increase in NASH; NASH 4444–5898 µM and healthy 2634–2829 µM). These changes were driven by increased taurine- and glycine-conjugated primary and secondary bile acids. Patients with NASH exhibited greater variability in their fasting and post-prandial bile acid profile. Results indicate that patients with NASH have higher fasting and post-prandial exposure to bile acids, including the more hydrophobic and cytotoxic secondary species. Increased bile acid exposure may be involved in liver injury and the pathogenesis of NAFLD and NASH.

Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of multidrug resistance protein 4 (MRP4), in addition to bile salt export pump (BSEP), might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed using 257 BSEP and 86 MRP4 inhibitors and noninhibitors in the training set. Models were externally validated and used to predict the affinity of compounds toward BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of nine well characterized MRP4 inhibitors and nine potent BSEP inhibitors. Bayesian models for BSEP and MRP4 inhibition/noninhibition were developed with cross-validated receiver operator curve values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements. Similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability.

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance–associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug’s inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic.less thanbr /greater thanConclusion: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity. Funding agencies: HESI; National Center for Toxicological Research (NCTR)/U.S. Food and Drug Administration (FDA) [P00800]; National Institutes of Health from the National Institute of General Medical Sciences [R01 GM041935, R35 GM122576]

The expression of hepatic transporters, including organic anion transporting polypeptides (OATPs) and multidrug resistance-associated proteins (MRPs), is altered in nonalcoholic steatohepatitis (NASH); however, functional data in humans are lacking. In this study, 99m Tc-mebrofenin (MEB) was used to evaluate OATP1B1/1B3 and MRP2 function in NASH patients. Healthy subjects (n = 14) and NASH patients (n = 7) were administered MEB (∼2.5 mCi). A population pharmacokinetic model was developed to describe systemic and hepatic MEB disposition. Study subjects were genotyped for SLCO1B1 variants. NASH increased systemic and hepatic exposure (median ± 2 SE, healthy vs. NASH) to MEB (AUC0-300,blood : 1,780 ± 242 vs. 2,440 ± 775 μCi*min/L, P = 0.006; AUC0-180,liver : 277 ± 36.9 vs. 433 ± 40.3 kcounts*min/sec, P < 0.0001) due to decreased biliary clearance (0.035 ± 0.008 vs. 0.017 ± 0.002 L/min, P = 0.0005) and decreased Vcentral (11.1 ± 0.57 vs. 6.32 ± 1.02 L, P < 0.0001). MEB hepatic CLuptake was reduced in NASH and also in healthy subjects with SLCO1B1 *15/*15 and *1A/*15 genotypes. The pharmacokinetics of drugs that are OATP1B1/1B3 and MRP2 substrates may be substantially altered in NASH.

The intracellular unbound inhibitor concentration ([I] unbound,cell ) is the most relevant concentration for predicting the inhibition of hepatic efflux transporters. However, the intracellular unbound fraction of inhibitor in hepatocytes (f u,cell,inhibitor ) is not routinely determined. Studies are needed to evaluate the benefit of measuring f u,cell,inhibitor and using [I] unbound,cell versus intracellular total inhibitor concentration ([I] total,cell ) when predicting inhibitory effects. This study examined the benefit of using [I] unbound,cell to predict hepatocellular bile acid disposition. Cellular total concentrations of taurocholate ([TCA] total,cell ), a prototypical bile acid, were simulated using pharmacokinetic parameters estimated from sandwich-cultured human hepatocytes. The effect of various theoretical inhibitors was simulated by varying ([I] total,cell / half maximal inhibitory concentration [IC 50 ]) values. In addition, the fold change was calculated as the simulated [TCA] total,cell when f u,cell,inhibitor = 1 divided by the simulated [TCA] total,cell when f u,cell,inhibitor = 0.5-0.01. The lowest ([I] total,cell /IC 50 ) value leading to a >2-fold change in [TCA] total,cell was chosen as a cutoff, and a framework was developed to categorize risk inhibitors for which the measurement of f u,cell,inhibitor is optimal. Fifteen compounds were categorized, 5 of which were compared with experimental observations. Future work is needed to evaluate this framework based on additional experimental data. In conclusion, the benefit of measuring f u,cell,inhibitor to predict hepatic efflux transporter–mediated drug-bile acid interactions can be determined a priori.

Introduction Multidrug resistance-associated protein 3 (MRP3), an efflux transporter on the hepatic basolateral membrane, may function as a compensatory mechanism to prevent the accumulation of anionic substrates (e.g., bile acids) in hepatocytes. Inhibition of MRP3 may disrupt bile acid homeostasis and is one hypothesized risk factor for the development of drug-induced liver injury (DILI). Therefore, identifying potential MRP3 inhibitors could help mitigate the occurrence of DILI. Methods Bayesian models were developed using MRP3 transporter inhibition data for 86 structurally diverse drugs. The compounds were split into training and test sets of 57 and 29 compounds, respectively, and six models were generated based on distinct inhibition thresholds and molecular fingerprint methods. The six Bayesian models were validated against the test set and the model with the highest accuracy was utilized for a virtual screen of 1470 FDA-approved drugs from DrugBank. Compounds that were predicted to be inhibitors were selected for in vitro validation. The ability of these compounds to inhibit MRP3 transport at a concentration of 100 μM was measured in membrane vesicles derived from stably transfected MRP3-over-expressing HEK-293 cells with [3H]-estradiol-17β- d -glucuronide (E217G; 10 μM; 5 min uptake) as the probe substrate. Results A predictive Bayesian model was developed with a sensitivity of 73% and specificity of 71% against the test set used to evaluate the six models. The area under the Receiver Operating Characteristic (ROC) curve was 0.710 against the test set. The final selected model was based on compounds that inhibited substrate transport by at least 50% compared to the negative control, and functional-class fingerprints (FCFP) with a circular diameter of six atoms, in addition to one-dimensional physicochemical properties. The in vitro screening of predicted inhibitors and non-inhibitors resulted in similar model performance with a sensitivity of 64% and specificity of 70%. The strongest inhibitors of MRP3-mediated E217G transport were fidaxomicin, suramin, and dronedarone. Kinetic assessment revealed that fidaxomicin was the most potent of these inhibitors (IC50 = 1.83 ± 0.46 μM). Suramin and dronedarone exhibited IC50 values of 3.33 ± 0.41 and 47.44 ± 4.41 μM, respectively. Conclusion Bayesian models are a useful screening approach to identify potential inhibitors of transport proteins. Novel MRP3 inhibitors were identified by virtual screening using the selected Bayesian model, and MRP3 inhibition was confirmed by an in vitro transporter inhibition assay. Information generated using this modeling approach may be valuable in predicting the potential for DILI and/or MRP3-mediated drug-drug interactions.

Hepatotoxicity is a key concern in the clinical translation of nanotherapeutics because preclinical studies have consistently shown that nanotherapeutics accumulate extensively in the liver. However, clinical stage nanotherapeutics have not shown increased hepatotoxicity. Factors that can contribute to the hepatotoxicity of nanotherapeutics beyond the intrinsic hepatotoxicity of nanoparticles (NPs) are poorly understood. Because of this knowledge gap, clinical translation efforts have avoided hepatotoxic molecules. Herein, by examining the hepatotoxicity of nanoformulations of known hepatotoxic compounds, we demonstrated that nanotherapeutics are associated with lower hepatotoxicity than their small molecule counterparts. We then found that the reduced hepatotoxicity is related to the uptake of nanotherapeutics by macrophages in the liver. These findings can facilitate further development and clinical translation of nanotherapeutics.

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.

Inhibition of bile acid transport by troglitazone (TGZ) and its major metabolite, TGZ sulfate (TS), may lead to hepatocellular accumulation of toxic bile acids; TS accumulation and hepatotoxicity may be associated with impaired TS biliary excretion. This study evaluated the impact of impaired transport of breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein 2 (Mrp2) on the hepatobiliary disposition of generated metabolites, TS and TGZ glucuronide (TG). Sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR(-)) rats in combination with Bcrp knockdown using RNA interference were employed. The biliary excretion index (BEI) of generated TS was not significantly altered by impaired Bcrp (20.9 to 21.1%) and/or Mrp2 function (24.4% and 17.5% in WT and TR(-) rat SCH, respectively). Thus, loss-of-function of Mrp2 and/or Bcrp do not appear to be risk factors for increased hepatocellular TS accumulation in rats, potentially because of a compensatory transporter(s) that excretes TS into bile. Further investigations revealed that the compensatory TS biliary transporter was not the bile salt export pump (Bsep) or P-glycoprotein (P-gp). Interestingly, TGZ sulfation was significantly decreased in TR(-) compared with WT rat SCH (total recovery: 2.8 versus 5.0% of TGZ dose), resulting in decreased hepatocellular TS accumulation, even though sulfotransferase activity in TR(-) rat hepatocyte S9 fraction was similar. Hepatocellular TG accumulation was significantly increased in TR(-) compared with WT rat SCH due to increased glucuronidation and negligible TG biliary excretion. These data emphasize that the interplay between metabolite formation and excretion determines hepatocellular exposure to generated metabolites such as TS and TG.

Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 μM) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e.g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI.

Basolateral efflux clearance (CLBL) contributes significantly to rosuvastatin (RSV) elimination in sandwich-cultured hepatocytes (SCH). The contribution of CLBL to RSV hepatic elimination was determined in single-pass isolated perfused livers (IPLs) from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor, elacridar (GF120918); clearance values were compared with SCH. RSV biliary clearance (CLBile) was ablated almost completely by GF120918 in TR(-) IPLs, confirming that Mrp2 and Bcrp primarily are responsible for RSV CLBile. RSV appearance in outflow perfusate was attributed primarily to CLBL, which was impaired in TR(-) IPLs. CLBL was ≈ 6-fold greater than CLBile in the linear range in WT IPLs in the absence of GF120918. Recovery of unchanged RSV in liver tissue increased in TR(-) compared with WT (≈ 25 versus 6% of the administered dose) due to impaired CLBL and CLBile. RSV pentanoic acid, identified by high-resolution liquid chromatography-tandem mass spectroscopy, comprised ≈ 40% of total liver content and ≈ 16% of the administered dose in TR(-) livers at the end of perfusion, compared with ≈ 30 and 3% in WT livers, consistent with impaired RSV excretion and "shunting" to the metabolic pathway. In vitro-ex vivo extrapolation between WT SCH and IPLs (without GF120918) revealed that uptake clearance and CLBL were 4.2- and 6.4-fold lower, respectively, in rat SCH compared with IPLs; CLBile translated almost directly (1.1-fold). The present IPL data confirmed the significant role of CLBL in RSV hepatic elimination, and demonstrated that both CLBL and CLBile influence RSV hepatic and systemic exposure.

Transporters responsible for hepatic uptake and biliary clearance (CLBile) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CLBL) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CLBile versus CLBL remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CLBL in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CLBile was nearly ablated by GF120918 in TR(-) SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CLBile. Pharmacokinetic modeling revealed that CLBL and CLBile represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (Km = 21 ± 7 µM, Vmax = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CLBL due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication.

Leslie Z. Benet is Professor and Former Chair at the University of California San Francisco (UCSF), Department of Bioengineering and Therapeutic Sciences, and Department of Pharmaceutical Chemistry. Professor Benet has had a profound impact on the development of fundamental concepts in pharmacokinetics, and the application of these principles in patient care and drug development. This theme issue is dedicated to Professor Benet in tribute to his significant contributions to the pharmaceutical sciences. An outstanding commentary that details Professor Benet’s career path and scientific achievements over the past 50 years was published recently in Pharm Res.1 We chose to provide a different perspective for the reader by focusing this commentary on those intangible factors that make Professor Benet truly a legend in the pharmaceutical sciences. In gathering information for this “C versus T” commentary, we interviewed Professor Benet and contacted scientific colleagues, professional mentees, and family. We asked each to share a brief perspective from their vantage point on this giant in the field. Many great “Les Benet” attributes emerged including energy, pas

Drug-induced cholestasis is an important form of acquired liver disease and is associated with significant morbidity and mortality. Bile acids are key signaling molecules, but they can exert toxic responses when they accumulate in hepatocytes. This review focuses on the physiological mechanisms of drug-induced cholestasis associated with altered bile acid homeostasis due to direct (e.g., bile acid transporter inhibition) or indirect (e.g., activation of nuclear receptors, altered function/expression of bile acid transporters) processes. Mechanistic information about the effects of a drug on bile acid homeostasis is important when evaluating the cholestatic potential of a compound, but experimental data often are not available. The relationship between physicochemical properties, pharmacokinetic parameters, and inhibition of the bile salt export pump among 77 cholestatic drugs with different pathophysiological mechanisms of cholestasis (i.e., impaired formation of bile vs. physical obstruction of bile flow) was investigated. The utility of in silico models to obtain mechanistic information about the impact of compounds on bile acid homeostasis to aid in predicting the cholestatic potential of drugs is highlighted. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3037–3057, 2013

PURPOSE. The bioavailability of a crushed tolvaptan tablet suspended in water and administered by nasogastric (NG) tube was compared to the bioavailability from the tablet administered whole. METHODS. In a randomized crossover study, 28 healthy adults received a single 15-mg dose of tolvaptan on two occasions (one dose given as an intact tablet swallowed whole and the other as a crushed tablet in suspension given by NG tube), with a washout interval of ≥7 days. During each administration period, blood samples were collected at 15 time points over 36 hours. A validated liquid chromatography–tandem mass spectrometry assay was used to obtain plasma tolvaptan concentrations. Plasma tolvaptan time–concentration data were analyzed using noncompartmental methods, and pharmacokinetic data including maximum concentration (C(max)), time to C(max) (t(max)), area under the concentration–time curve (AUC) from time zero to the time of the last measurable concentration (AUC(t)), and AUC extrapolated to infinity (AUC(∞)) resulting from oral and NG tube tolvaptan delivery were compared via repeated-measures, mixed-effects analysis of variance. Due to differences in total drug exposure seen, an in vitro experiment was conducted on three dose levels to quantify drug sequestration. RESULTS. The ratios of geometric mean C(max), AUC(t), and AUC(∞) values (expressed as a percentage) with NG tube versus oral tolvaptan administration were 88.9%, 74.3%, and 74.2%, respectively; the latter two values were not within the specified bioequivalence tolerance limits (80–125%). In vitro analysis showed that approximately 11% of all tolvaptan doses evaluated was sequestered by the NG tube. CONCLUSION. In healthy adults, a single 15-mg dose of tolvaptan administered as a crushed tablet suspended in water by NG tube resulted in AUC(t) and AUC(∞) values that were approximately 25% lower than those observed after oral administration of a 15-mg tolvaptan tablet swallowed intact.

Tolvaptan is a selective vasopressin V2 receptor antagonist, approved in several countries for the treatment of hyponatremia and autosomal dominant polycystic kidney disease (ADPKD). No liver injury has been observed with tolvaptan treatment in healthy subjects and in non-ADPKD indications, but ADPKD clinical trials showed evidence of drug-induced liver injury (DILI). Although all DILI events resolved, additional monitoring in tolvaptan-treated ADPKD patients is required. In vitro assays identified alterations in bile acid disposition and inhibition of mitochondrial respiration as potential mechanisms underlying tolvaptan hepatotoxicity. This report details the application of DILIsym software to determine whether these mechanisms could account for the liver safety profile of tolvaptan observed in ADPKD clinical trials. DILIsym simulations included physiologically based pharmacokinetic estimates of hepatic exposure for tolvaptan and2 metabolites, and their effects on hepatocyte bile acid transporters and mitochondrial respiration. The frequency of predicted alanine aminotransferase (ALT) elevations, following simulated 90/30 mg split daily dosing, was 7.9% compared with clinical observations of 4.4% in ADPKD trials. Toxicity was multifactorial as inhibition of bile acid transporters and mitochondrial respiration contributed to the simulated DILI. Furthermore, simulation analysis identified both pre-treatment risk factors and on-treatment biomarkers predictive of simulated DILI. The simulations demonstrated that in vivo hepatic exposure to tolvaptan and the DM-4103 metabolite, combined with these 2 mechanisms of toxicity, were sufficient to account for the initiation of tolvaptan-mediated DILI. Identification of putative risk-factors and potential novel biomarkers provided insight for the development of mechanism-based tolvaptan risk-mitigation strategies.

Transporter-mediated alterations in bile acid disposition may have significant toxicological implications. Current methods to predict interactions are limited by the interplay of multiple transporters, absence of protein in the experimental system, and inaccurate estimates of inhibitor concentrations. An integrated approach was developed to predict altered bile acid disposition due to inhibition of multiple transporters using the model bile acid taurocholate (TCA). TCA pharmacokinetic parameters were estimated by mechanistic modeling using sandwich-cultured human hepatocyte data with protein in the medium. Uptake, basolateral efflux, and biliary clearance estimates were 0.63, 0.034, and 0.074 mL/min/g liver, respectively. Cellular total TCA concentrations (Ct,Cells) were selected as the model output based on sensitivity analysis. Monte Carlo simulations of TCA Ct,Cells in the presence of model inhibitors (telmisartan and bosentan) were performed using inhibition constants for TCA transporters and inhibitor concentrations, including cellular total inhibitor concentrations ([I]t,cell) or unbound concentrations, and cytosolic total or unbound concentrations. For telmisartan, the model prediction was accurate with an average fold error (AFE) of 0.99–1.0 when unbound inhibitor concentration ([I]u) was used; accuracy dropped when total inhibitor concentration ([I]t) was used. For bosentan, AFE was 1.2–1.3 using either [I]u or [I]t. This difference was evaluated by sensitivity analysis of the cellular unbound fraction of inhibitor (fu,cell,inhibitor), which revealed higher sensitivity of fu,cell,inhibitor for predicting TCA Ct,Cells when inhibitors exhibited larger ([I]t,cell/IC50) values. In conclusion, this study demonstrated the applicability of a framework to predict hepatocellular bile acid concentrations due to drug-mediated inhibition of transporters using mechanistic modeling and cytosolic or cellular unbound concentrations.

Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH.

Extracorporeal membrane oxygenation (ECMO) is a cardiopulmonary bypass device used in the pediatric critical care setting that is life-saving for infants with refractory cardiorespiratory failure. Blood is drained from the central venous system via a surgically placed cannula, pumped through an artificial lung (oxygenator) where oxygen is added and carbon dioxide is removed, and then oxygenated blood is returned to either the venous or arterial circulation. ECMO has been used successfully in multiple disease states including meconium aspiration syndrome, persistent pulmonary hypertension of the newborn, fulminant myocarditis, and sepsis.1 Despite these successes, infants supported with ECMO are at high risk for ECMO-related complications, including nosocomial infections.2 Invasive candidiasis is the second most common nosocomial infection in infants on ECMO and is often fatal.2,3 The incidence of fungal infections in patients on ECMO varies by center, with reports ranging from 0.7–10%.2,3.Standard treatment for invasive candidiasis in many patient populations consists of antifungal agents (e.g., fluconazole) and removal or replacement of intravascular catheters due to the organism’s ability to adhere to indwelling catheters.4 Catheter removal for infants on ECMO is impossible if the infant cannot be disconnected from the ECMO circuit; therefore, successful treatment of invasive candidiasis in infants on ECMO relies upon optimal antifungal drug therapy. Optimal antifungal management for patients on ECMO depends on dosing that has not been previously described and can differ greatly from other populations due to the pharmacokinetic (PK) changes induced by the ECMO circuit. PK changes attributed to the ECMO circuit include increased volume of distribution and decreased clearance, but these vary by drug.5–8 This study describes the ECMO-related PK changes of fluconazole and their impact on pharmacodynamic (PD) targets for fungal prophylaxis and treatment of invasive candidiasis.

Detailed knowledge regarding the influence of hepatic transport proteins on drug disposition has advanced at a rapid pace over the past decade. Efflux transport proteins located in the basolateral and apical (canalicular) membranes of hepatocytes play an important role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites. This review focuses on the role of these efflux transporters in hepatic drug excretion. The impact of these proteins as underlying factors for disease is highlighted, and the importance of hepatic efflux proteins in the efficacy and toxicity of drugs is discussed. In addition, a brief overview of methodology to evaluate the function of hepatic efflux transport proteins is provided. Current challenges in predicting the impact of altered efflux protein function on systemic, intestinal, and hepatocyte exposure to drugs and metabolites are highlighted.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in cancer patients have been well described. The current study was conducted to document the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with glomerulonephritis. The study was necessary due to the numerous clinical abnormalities that are present in glomerulonephritis (altered glomerular filtration rate, proteinuria, hypoalbuminaemia) which can alter the disposition of drugs. WHAT THIS STUDY ADDS • This study documents the differences in pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis vs. the cancer population. The current results demonstrate that clinical and pharmacogenetic covariates can both alter the disposition of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis. AIMS Cyclophosphamide, the precursor to the active 4-hydroxycyclophosphamide, is used in active glomerulonephritis despite limited pharmacokinetics data. The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide were evaluated. The influence of laboratory and pharmacogenomic covariates on pharmacokinetics was evaluated as a secondary aim. METHODS Glomerulonephritis patients (n = 23) participated in a pharmacokinetic evaluation. Blood was serially collected and assayed for cyclophosphamide and 4-hydroxycyclophosphamide by LC/MS methods. Kidney function, serum albumin and polymorphisms in drug metabolism or transport genes were evaluated. Analyses included non-compartmental pharmacokinetics and parametric and non-parametric statistics. RESULTS The mean area under the plasma concentration–time curve (AUC(0,∞)) data were 110 100 ± 42 900 ng ml−1 h and 5388 ± 2841 ng ml−1 h for cyclophosphamide and 4-hydroxycyclophosphamide, respectively. The mean metabolic ratio was 0.06 ± 0.04. A statistically significant relationship was found between increased serum albumin and increased half-life (0.584, P = 0.007, 95% CI 0.176, 0.820) and a borderline relationship with AUC(0,∞) (0.402, P = 0.079, 95% CI –0.064, 0.724) for 4-hydroxycyclophosphamide. Covariate relationships that trended toward significance for cyclophosphamide included decreased serum albumin and increased elimination rate constant (–0.427, P = 0.061, 95% CI 0.738, 0.034), increased urinary protein excretion and increased AUC(0,∞) (–0.392, P = 0.064, 95% CI –0.699 to 0.037), decreased Cmax (0.367, P = 0.085, 95% CI –0.067, 0.684) and decreased plasma clearance (–0.392, P = 0.064, 95% CI –0.699, 0.037). CYP2B6*9 variants vs. wildtype were found to have decreased elimination rate constant (P = 0.0005, 95% CI 0.033, 0.103), increased Vd (P = 0.0271, 95% CI −57.5, −4.2) and decreased Cmax (P = 0.0176, 95% CI 0.696, 6179) for cyclophosphamide. ABCB1 C3435T variants had a borderline decrease in cyclophosphamide elimination rate constant (P = 0.0858; 95% CI –0.005, 0.102). CONCLUSIONS Pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with lupus nephritis and small vessel vasculitis are similar. Clinical and pharmacogenetic covariates alter disposition of cyclophosphamide and 4-hydroxycyclophosphamide. Clinical findings of worsened glomerulonephritis lead to increased exposure to cyclophosphamide vs. the active 4-hydroxycyclophosphamide, which could have relevance in terms of clinical efficacy. The CYP2B6*9 and ABCB1 C3435T polymorphisms alter the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis.

Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR® technology, BAs were measured in cells+bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells+bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.85 μM in CTL rat and 183 ± 55.6 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.210 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na+-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account.

Multidrug resistance-associated protein (Mrp) 2-deficient (TR(-)) Wistar rats have been used to elucidate the role of Mrp2 in drug disposition. Decreased breast cancer resistance protein (Bcrp) levels were reported in sandwich-cultured hepatocytes (SCH) from TR(-) rats compared with those from wild-type (WT) rats. This study was designed to characterize hepatic Bcrp expression and function in TR(-) rats, using nitrofurantoin and pitavastatin as substrates. Bcrp was knocked down by RNA interference in rat SCH. Antibody BXP53, but not BXP21, specifically detected Bcrp knockdown in SCH. Bcrp protein levels were decreased markedly in TR(-) but not Mrp2-deficient Sprague-Dawley [Eisai hyperbilirubinemic rats (EHBR)] rats. Bcrp mRNA levels were decreased significantly in TR(-) livers as determined by TaqMan real-time reverse transcriptase-polymerase chain reaction. Biliary excretion of nitrofurantoin, a specific Bcrp substrate, was decreased significantly in SCH and isolated perfused livers from TR(-) rats compared with those from WT controls, indicating that hepatic Bcrp function is decreased in TR(-) rats. In Bcrp knockdown SCH, the biliary excretion index and in vitro biliary clearance of pitavastatin were decreased significantly to ∼ 58 and ∼ 52% of control, respectively, indicating that Bcrp plays a role in pitavastatin biliary excretion. Pitavastatin biliary excretion was decreased significantly in perfused livers from TR(-) compared with those from WT rats. In conclusion, expression and function of hepatic Bcrp are decreased significantly in TR(-) rats. The potential role of both Bcrp and Mrp2 should be considered when data generated in TR(-) rats are interpreted. TR(-) and EHBR rats in combination may be useful in differentiating the role of Mrp2 and Bcrp in drug/metabolite disposition.

Sulindac is a commonly used nonsteroidal anti-inflammatory drug. This study tested the hypothesis that sulindac-mediated drug-drug interactions and/or hepatotoxicity may be caused, in part, by inhibition of proteins responsible for the hepatic transport of drugs and/or bile acids by sulindac and/or sulindac metabolites [sulindac sulfone (S-sulfone) and sulindac sulfide (S-sulfide)]. The uptake and excretion of model substrates, [(3)H]taurocholate (TC), [(3)H]estradiol 17-beta-glucuronide (E217G), and nitrofurantoin (NF), were investigated in rat and human suspended and sandwich-cultured hepatocytes (SCH). In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 24.9 +/- 6.4 and 12.5 +/- 1.8 microM, respectively) and Na(+)-independent E217G initial uptake (IC(50) of 12.1 +/- 1.6 and 6.3 +/- 0.3 microM, respectively). In rat SCH, sulindac metabolites (100 microM) decreased the in vitro biliary clearance (Cl(biliary)) of TC, E217G, and NF by 38 to 83%, 81 to 97%, and 33 to 57%, respectively; S-sulfone and S-sulfide also decreased the TC and NF biliary excretion index by 39 to 55%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 42.2 and 3.1 microM, respectively); S-sulfide also inhibited the TC Cl(biliary) in human SCH. Sulindac/metabolites markedly inhibited hepatic uptake and biliary excretion of E217G by 51 to 100% in human SCH. In conclusion, sulindac and metabolites are potent inhibitors of the uptake and biliary clearance of bile acids in rat and human hepatocytes and also inhibit substrates of rat breast cancer resistance protein, rat and human organic anion-transporting polypeptides, and human multidrug resistance-associated protein 2. Inhibition of multiple hepatic transport proteins by sulindac/metabolites may play an important role in clinically significant sulindac-mediated drug-drug interactions and/or liver injury.

Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na(+)-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [(3)H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Cl(biliary)) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Cl(biliary) by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n=2-4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI.

Breast cancer resistance protein (BCRP, ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sex-dependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP) and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg–/– (Bcrp-deficient) mice. Pharmacokinetic modeling was used to estimate parameters governing the hepatobiliary disposition of APAP, AG, and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males than in females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice, and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female mouse livers compared with that in male mouse livers. It is interesting to note that multidrug resistance-associated protein 4 was higher in female than in male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers.

BCRP transports numerous drugs/derived metabolites and toxins, and exhibits overlapping substrate specificity with P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). Assessing the contribution of BCRP to drug/metabolite biliary excretion in the intact hepatocytes remains a challenge. Current studies were designed to develop a novel in vitro tool to specifically assess the contribution of Bcrp to drug biliary excretion. Adenoviral vectors expressing short hairpin (sh) RNA targeting Bcrp (Ad-si01Bcrp) or a non-target control (Ad-siNT) were packaged and infected into sandwich-cultured rat hepatocytes (SCRH). Protein levels were detected by immunoblot. Biliary excretion index (BEI) and in vitro biliary clearance (Clbiliary) of nitrofurantoin (BCRP substrate) and digoxin (P-gp substrate) were compared among non-infected, Ad-siNT- and Ad-si01Bcrp-infected cells in SCRH. shRNA targeting Bcrp efficiently knocked down Bcrp in SCRH, while levels of other transport proteins (P-gp, Mrp2, Bsep, Mrp4 and Oatp1a1) were unaffected. In SCRH exhibiting Bcrp knockdown, cellular accumulation of nitrofurantoin was increased markedly and nitrofurantoin BEI and in vitro Clbiliary were decreased to 11% and 14% of control, respectively. Digoxin values were unaffected by knockdown of Bcrp. Results indicated that Bcrp contributed predominantly to nitrofurantoin biliary excretion, but played a negligible role in digoxin biliary excretion. In summary, Bcrp knockdown in SCRH is the first in vitro model utilizing intact hepatocytes to assess the contribution of Bcrp to the biliary excretion of drugs. This approach may be useful in predicting drug-drug interactions in biliary excretion and the consequence of impaired BCRP function on the hepatic exposure of drugs/derived metabolites.

Silymarin, an extract from seeds of Silybum marianum, is used by 8 to 33% of patients to self-treat chronic viral hepatitis C in the United States. Studies in humans and rodents suggest that biliary excretion of glucuronide and sulfate conjugates is the major route for silymarin's elimination. To determine the role of multidrug resistance-associated protein 2 (Mrp2) (Abcc2) in the biliary excretion of silymarin, the hepatobiliary disposition of the six major silymarin flavonolignans was studied using isolated perfused livers (IPRLs) from wild-type (WT) and Mrp2-deficient (TR(-)) Wistar rats. For all the flavonolignans, approximately 96% of the dose was cleared from perfusate within 30 min in both WT and TR(-) livers, and

The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction (fu), biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Cl’biliary) were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Cl’biliary of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Cl’biliary for digoxin and pravastatin, which represent low and moderately-bound compounds, respectively. The BEI and intrinsic Cl’biliary of taurocholate, a highly-bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks, based on carboxydichlorofluorescein (CDF) retention. The addition of physiological concentrations of calcium, or the addition of fatty acids to BSA-FAF, was unable to restore the BEI or intrinsic Cl’biliary of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.

Previous reports have indicated that in vitro biliary clearance (Cl(biliary)) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl(biliary) for limited sets of compounds. This study was designed to estimate the in vitro Cl(biliary) in sandwich-cultured rat hepatocytes (SCRHs) of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism, to compare the estimated Cl(biliary) values with published in vivo Cl(biliary) data in rats, and to characterize the mechanism(s) of basolateral uptake and canalicular excretion of these drugs in rats. The average biliary excretion index (BEI) and in vitro Cl(biliary) values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin were 15, 19, 43, 45, and 20%, respectively, and 1.7, 3.2, 4.4, 46.1, and 34.6 ml/min/kg, respectively. Cl(biliary) predicted from SCRHs, accounting for plasma unbound fraction, correlated with reported in vivo Cl(biliary) for these drugs. The rank order of Cl(biliary) values predicted from SCRHs was consistent with in vivo Cl(biliary) values. Bromosulfophthalein inhibited the uptake of all drugs. BEI and Cl(biliary) values of olmesartan, valsartan, pravastatin, and rosuvastatin, known multidrug resistance-associated protein (Mrp) 2 substrates, were reduced in SCRHs from Mrp2-deficient (TR(-)) compared with wild-type (WT) rats. Although Mrp2 plays a minor role in pitavastatin biliary excretion, pitavastatin BEI and Cl(biliary) were reduced in TR(-) compared with WT SCRHs; Bcrp expression in SCRHs from TR(-) rats was decreased. In conclusion, in vitro Cl(biliary) determined in SCRHs can be used to estimate and compare in vivo Cl(biliary) of compounds in rats and to characterize transport proteins responsible for their hepatic uptake and excretion.

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.

Host-gut microbial interactions contribute to human health and disease states and an important manifestation resulting from this co-metabolism is a vast diversity of bile acids (BAs). There is increasing interest in using BAs as biomarkers to assess the health status of individuals, and therefore, an increased need for their accurate separation and identification. In this study, the negative ion fragmentation behaviors of C24 BAs were investigated by UPLC-ESI-QTOF-MS. The step-by-step fragmentation analysis revealed a distinct fragmentation mechanism for the unconjugated BAs containing a 12-hydroxyl group. The unconjugated BAs lacking 12-hydroxylation fragmented via dehydration and dehydrogenation. In contrast, the 12-hydroxylated ones, such as deoxycholic acid (DCA) and cholic acid (CA), employed dissociation routes including dehydration, loss of carbon monoxide or carbon dioxide, and dehydrogenation. All fragmentations of the 12-hydroxylated unconjugated BAs, characterized by means of stable isotope labeled standards, were associated with the rotation of the carboxylate side-chain and the subsequent rearrangements accompanied with proton transfer between 12-hydroxyl and 24-carboxyl groups. Compared to DCA, CA underwent further cleavages of the steroid skeleton. Accordingly, the effects of stereochemistry on the fragmentation pattern of CA were investigated using its stereoisomers. Based on the knowledge gained from the fragmentation analysis, a novel BA, 3β, 7β, 12α-trihydroxy-5β-cholanic acid, was identified in the postprandial urine samples of patients with nonalcoholic steatohepatitis. The analyses used in this study may contribute to a better understanding of the chemical diversity of BAs and the molecular basis of human liver diseases that involve BA synthesis, transport, and metabolism.

Background Candida is a leading cause of infection in infants on extracorporeal membrane oxygenation (ECMO). Optimal micafungin dosing is unknown in this population because ECMO can alter drug pharmacokinetics (PK). Methods To characterize micafungin pharmacokinetics and safety in infants on ECMO, we conducted an open-label pharmacokinetics trial. Infants on ECMO either received intravenous micafungin 4 mg/kg every 24 h for invasive candidiasis prophylaxis or 8 mg/kg every 24 h when a fungal infection was suspected or confirmed. We collected plasma samples after single and multiple micafungin doses. We defined the therapeutic target as the adult exposure associated with efficacy in phase III trials and the prophylactic target as one-half of the therapeutic target. Results We enrolled 12 infants (124 samples) with a median age of 59 days. Using a 1-compartment model, median weight-normalized volume of distribution and clearance were 0.64 L/kg and 0.041 L/kg/h, respectively. Dose-exposure simulations revealed that doses of 2.5 and 5 mg/kg every 24 h matched exposure targets for prophylaxis and treatment of invasive candidiasis, respectively. We did not observe any drug-related adverse events. Conclusions In infants on ECMO, micafungin volume of distribution was higher and clearance was in the upper range of previously published values for infants not on ECMO. Based on these data, we recommend dosing of 2.5 and 5 mg/kg every 24 h for prophylaxis and treatment of invasive candidiasis, respectively, to match adult exposure proven effective against Candida spp.

Freshly isolated hepatocytes are widely accepted as the “gold standard” for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and “legacy” network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.

Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp- or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR(-)) rats were perfused in a single-pass manner with 0.5 muM FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 muM) was employed to inhibit P-gp and Bcrp. The biliary excretion rate of FEX was decreased 85% in Mrp2KO relative to wild-type mice (18.4 +/- 2.2 versus 122 +/- 34 pmol/min/g liver). In mice, more than 50% of FEX unbound intrinsic biliary clearance (CL(bile, int)(') = 3.0 ml/h/g liver) could be attributed to Mrp2 (Mrp2-dependent CL(bile, int)(') approximately 1.7 ml/h/g liver), with P-gp and Bcrp playing a minor role (P-gp- and Bcrp-dependent CL(bile, int)(') approximately 0.3 ml/h/g liver). Approximately one third of FEX CL(bile, int)(') was attributed to unidentified mechanisms in mice. In contrast to mice, FEX biliary excretion rate (245 +/- 38 and 250 +/- 25 pmol/min/g liver) and CL(bile, int)(') (9.72 +/- 2.47 and 6.49 +/- 0.68 ml/h/g liver) were comparable between TR(-) and control Wistar rats, respectively, suggesting that unidentified transport mechanism(s) can completely compensate for the loss of Mrp2 function in rats. Mrp2 clearly plays a major role in FEX biliary excretion in mice. In conclusion, remarkable species differences exist in FEX hepatobiliary transport mechanisms.