Biliary Clearance Research Articles

Hepatocyte Concentrations of Imaging Compounds Associated with Transporter Inhibition: Evidence in Perfused Rat Livers

In the liver, several approaches are used to investigate and predict the complex issue of drug-induced transporter inhibition. These approaches include in vitro assays and pharmacokinetic models that predict how inhibitors modify the systemic and liver concentrations of the victim drugs. Imaging is another approach that shows how inhibitors might alter liver concentrations stronger than systemic concentrations. In perfused rat livers associated with a gamma counter that measures liver concentrations continuously, we previously showed how fluxes across transporters generate the hepatocyte concentrations of two clinical imaging compounds, one with a low extraction ratio [gadobenate dimeglumine (BOPTA)] and one with a high extraction ratio [mebrofenin (MEB)]. BOPTA and MEB are transported by rat organic anion transporting polypeptide and multiple resistance-associated protein 2, which are both inhibited by rifampicin. The aim of the study is to measure how rifampicin modifies the hepatocyte concentrations and membrane clearances of BOPTA and MEB and to determine whether these compounds might be used to investigate transporter-mediated drug-drug interactions in clinical studies. We show that rifampicin coperfusion greatly decreases BOPTA hepatocyte concentrations, but increases those of MEB. Rifampicin strongly decreases BOPTA hepatic clearance. In contrast, rifampicin decreases moderately MEB hepatic clearance and blocks the biliary intrinsic clearance, increasing MEB hepatocyte concentrations. In conclusion, low concentrations prevent the quantification of BOPTA biliary intrinsic clearance, while MEB is a promising imaging probe substrate to evidence transporter-mediated drug-drug interactions when inhibitors act on influx and efflux transporters.

Laparoscopic CBD exploration, is it useful ?

Introduction: Laparoscopic CBD exploration is an alternative choice to extract stone, evaluate bilary tract or biopsy. Safe, cost-effective and reliable single stage approach are the superiority for this technique. Advancement of laparoscopic and endoscopic equipment and surgical skill need to be improved to do this procedure properly. Method: We performed LCBDE for 28 patients with CDB stone, suspected stricture or probably mass from biliary tract. (Mei 2017 - Feb 2019) Preoperatively MRCP or MSCT done for diagnosis. All CBD exploration done by video choledochoscope. Route LCBDE, duration, procedure, conversion rate, clearance rate and complication will be evaluated. Result: 28 patients LCBDE, 12 Male, 16 female Mean age 52 year, in range 30-74 years TC-LCBDE in 5 patients and C-LCBDE in 23 patients Extraction CBD stones or bypass and biopsy done. The largest CBD stone extraction 25 mm, in range 3-25 mm and 1-6 stones extract / clearance by basket or forceps. No major perioperative complication, two converted to open due to adhesion and stricture Mean operative time 210 minutes, in range 120 - 300 minutes Majority Choledochotomy done with primary closure and put drain subhepatal, only 2 cases with T-tube. All CBD clearanced, make sure with video choledochoscope. Conclusion: LCBDE is a safe procedure with low complication and higher success rate for biliary tract clearance and cholecystectomy (single stage approach) Choledochoscope is must to evaluate biliary tract clearance and biopsy. Learning curve and trained team needs to reduce time operation.

An Adaption of Human-Induced Hepatocytes to In Vitro Genetic Toxicity Tests.

Metabolic activation is essential in standard in vitro genotoxicity test systems. At present, there is a lack of suitable cell models that can express the major characteristics of liver function for predicting substance toxicity in humans. Human-induced hepatocytes (hiHeps), which have been generated from fibroblasts by lentiviral expression of liver transcription factors, can express hepatic gene programs and can be expanded in vitro and display functional characteristics of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Our purpose was to investigate whether hiHeps could be used as a more suitable model for genotoxicity evaluation of chemicals. Therefore, a direct mutagen, methylmethanesulfonate (MMS), and five promutagens [2-nitrofluorene (2-NF), benzo[a]pyrene (B[a]P), aflatoxin B1, cyclophosphamide and N-nitrosodiethylamine] were tested by the cytokinesis-block micronucleus test and the comet assay. Results from genotoxicity tests showed that the micronucleus frequencies were significantly increased by all of the six clastogens tested. Moreover, MMS, 2-NF and B[a]P induced significant increases in the % Tail DNA in the comet assay. In conclusion, our findings from the preliminary study demonstrated that hiHeps could detect the genotoxicity of indirect carcinogens, suggesting their potential to be applied as an effective tool for in vitro genotoxicity assessments.

Altered Hepatobiliary Disposition of Tolvaptan and Selected Tolvaptan Metabolites in a Rodent Model of Polycystic Kidney Disease.

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 (<0.01% ± <0.01% vs. 0.02% ± 0.01%; P = 0.0037), and DM-4107 recovery was increased in PCK vs. WT outflow perfusate (1.60% ± 0.57% vs. 0.43% ± 0.29%; P = 0.0017). A pharmacokinetic compartmental model assuming first-order processes was developed to describe the rate vs. time profiles of tolvaptan and DM-4103 + DM-4107 in rat IPLs. The model-derived estimate of tolvaptan's biliary clearance was significantly decreased in PCK compared with WT IPLs. The model predicted greater hepatocellular concentrations of tolvaptan and DM-4103 + DM-4107 in PCK compared with WT IPLs. Increased hepatocellular exposure to tolvaptan and metabolites may contribute to the hepatotoxicity in patients with ADPKD treated with tolvaptan.

Baicalein Enhances the Oral Bioavailability and Hepatoprotective Effects of Silybin Through the Inhibition of Efflux Transporters BCRP and MRP2.

Although hepatoprotective properties of silybin are well documented, the clinical therapeutic efficacy is limited by its low bioavailability due to absorption rates, extensive phase II metabolism, and biliary excretion. As our previous study indicated that metabolic enzymes may have limited effects on the pharmacokinetic (PK) behavior of silymarin, here, we intended to increase the oral bioavailability and bio-efficacy of silybin through the inhibition of active efflux. In Caco-2 and transfected MDCKII cell models, flavone baicalein significantly inhibited the efflux of silybin as a BCRP and MRP2 inhibitor. In addition, baicalein reduced the biliary excretion index (BEI) and biliary clearance of silybin conjugates in the sandwich-cultured rat hepatocyte (SCH) model, indicating the inhibition of baicalein in biliary excretion of conjugated silybin metabolites. PK study demonstrated that baicalein significantly increased the area under the curve (AUC) and Cmax of silybin and its conjugates, suggesting enhanced absorption in vivo. Moreover, coadministration of silybin with baicalein boosted the liver protective, antioxidant, and anti-inflammatory effects of silybin in the carbon tetrachloride (CCl4)-induced liver injury model in comparison with silybin given alone. In summary, efflux transporters play a critical role in the low bioavailability of silybin, while inhibition of breast cancer resistance protein (BCRP) and multi-drug resistance protein 2 (MRP2) by baicalein can significantly increase the absorption and bio-efficacy of silybin, which provides a new combination therapeutic approach for the treatment of chronic liver diseases.

Oxidative Stress Status in Patients with Choledocholithiasis: Before and After Endoscopic Sphincterotomy and Biliary Clearance

Choledocholithiasis may cause biliary obstruction which leads to hepatocellular injury. Oxidative stress has been proposed as a possible mechanism involved in this disorder. This study evaluates the oxidative stress burden in patients with choledocholithiasis and secondary cholestasis, before and after endoscopic sphincterotomy. Experimental part: Patients diagnosed with choledocholithiasis and secondary extrahepatic cholestasis were included in the study between January 1st 2016 and October 31st 2016. In all patients oxidative stress markers were collected within 2 hours before and 48 hours after therapeutic ERCP. Selected markers were superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA). The results were compared to those from a group of 40 healthy subjects. Significantly lower concentrations of SOD (p = 0.03) and GPX (p [ 0.0001) activities, associated with an increased level of MDA level (p [ 0.0001) were shown in patients before biliary clearance compared with the healthy control group. After ERCP the only oxidative stress parameter which showed improvement was the SOD specific activity (p = 0.037). This study shows that extrahepatic cholestasis secondary to choledocholithiasis is associated with increased oxidative stress status. After biliary clearance one oxidative stress marker was significantly improved (SOD), suggesting a possible antioxidant effect of such procedure.

Evaluation of Drug Biliary Excretion Using Sandwich-Cultured Human Hepatocytes.

Evaluation of hepatobiliary transport of drugs is an important challenge, notably during the development of new molecular identities. In this context, sandwich-cultured human hepatocytes (SCHH) have been proposed as an interesting and integrated tool for predicting in vitro biliary excretion of drugs. The present review was therefore designed to summarize key findings about SCHH, including their establishment, their main functional features and their use for the determination of canalicular transport and the prediction of in vivo biliary clearance and hepatobiliary excretion-related drug-drug interactions. Reviewed data highlight the fact that SCHH represent an original and probably unique holistic in vitro approach to predict biliary clearance in humans, through taking into account sinusoidal drug uptake, passive drug diffusion, drug metabolism and sinusoidal and canalicular drug efflux. Limits and proposed refinements for SCHH-based analysis of drug biliary excretion, as well as putative human alternative in vitro models to SCHH are also discussed.

In Vitro Stimulation of Multidrug Resistance-Associated Protein 2 Function Is Not Reproduced In Vivo in Rats.

Previously we reported that coproporphyrin-I (CP-I) is an optimal probe substrate for multidrug resistance-associated protein 2 (MRP2), and stimulation of MRP2-mediated transport is probe substrate-dependent. In the present investigation, we assessed if the in vitro stimulation is physiologically relevant. Similar to human MRP2 transport, CP-I was transported by rat Mrp2 in a typical Michaelis-Menten kinetics with apparent Km and Vmax values of 15 ± 6 µM and 161 ± 20 pmol/min/mg protein, respectively. In vivo Mrp2 functions were monitored by biliary and renal secretion of CP-I and its isomer CP-III, in bile-duct cannulated rats before and after treatment with mitoxantrone, progesterone, and verapamil. These compounds stimulated Mrp2-mediated CP-I transport in vitro. No significant increase in biliary or renal clearances, as well as in the cumulative amount of CP-I or CP-III eliminated in bile, were detected following treatment with the in vitro stimulators, indicating an in vitro to in vivo disconnect. In presence of 10 µM bilirubin, the in vitro stimulation was suppressed. We concluded that the in vitro stimulation of CP-I transport mediated by Mrp2 is not translatable in vivo, and proposed that the presence of endogenous compounds such as bilirubin in the liver may contribute to the in vitro to in vivo disconnect.

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance.

Atovaquone, an antiprotozoal and antipneumocystic agent, is predominantly cleared by biliary excretion of unchanged parent drug. Atovaquone is ≥10,000-fold concentrated in human bile relative to unbound plasma. Even after correcting for apparent nonspecific binding and incomplete solubility in bile, atovaquone is still concentrated ≥100-fold in bile, consistent with active biliary excretion. Mechanisms of atovaquone hepatobiliary disposition were studied using a multiexperimental in vitro and in vivo approach. Atovaquone uptake was not elevated in HEK293 cells singly overexpressing OATP1B1, OATP1B3, OATP2B1, OCT1, NTCP, or OAT2. Hepatocyte uptake of atovaquone was not impaired by OATP and OCT inhibitor cocktail (rifamycin and imipramine). Atovaquone liver-to-blood ratio at distributional equilibrium was not reduced in Oatp1a/1b and Oct1/2 knockout mice. Atovaquone exhibited efflux ratios of approximately unity in P-gp and BCRP overexpressing MDCK cell monolayers and did not display enhanced uptake in MRP2 vesicles. Biliary and canalicular clearance were not decreased in P-gp, Bcrp, Mrp2, and Bsep knockout rats. In the present study, we rule out the involvement of major known basolateral uptake and bile canalicular efflux transporters in the hepatic uptake and biliary excretion of atovaquone. This is the first known example of a drug cleared by biliary excretion in humans, with extensive biliary concentration, which is not transported by the mechanisms investigated herein.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms.

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 µM OCA, 100 µM CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca2+-containing and Ca2+-free conditions (n = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CLUptake), total intrinsic basolateral efflux clearance (CLint,BL), and total intrinsic biliary clearance (CLint,Bile). Modeling results revealed that FXR agonists significantly increased CLint,BL by >6-fold and significantly increased CLint,Bile by 2-fold, with minimal effect on CLUptake Immunoblotting showed that protein levels of the basolateral transporter subunits organic solute transporter α and β (OSTα and OSTβ) in FXR agonist-treated SCHH were significantly induced by >2.5- and 10-fold, respectively. FXR agonist-mediated changes in the expression of other TCA transporters in SCHH were modest. In conclusion, this is the first report demonstrating that OCA and CDCA increased TCA efflux in SCHH, which contributed to reduced intracellular TCA concentrations. Increased basolateral efflux of TCA was consistent with increased OSTα/β protein expression in OCA- and CDCA-treated SCHH.

Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor.

Long-term in vitro liver models are now widely explored for human hepatic metabolic clearance prediction, enzyme phenotyping, cross-species metabolism, comparison of low clearance drugs, and induction studies. Here, we present studies using a long-term liver model, which show how metabolism and active transport, drug-drug interactions, and enzyme induction in healthy and diseased states, such as hepatitis B virus (HBV) infection, may be assessed in a single test system to enable effective data integration for physiologically based pharmacokinetic (PBPK) modeling. The approach is exemplified in the case of (3S)-4-[[(4R)-4-(2-Chloro-4-fluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid RO6889678, a novel inhibitor of HBV with a complex absorption, distribution, metabolism, and excretion (ADME) profile. RO6889678 showed an intracellular enrichment of 78-fold in hepatocytes, with an apparent intrinsic clearance of 5.2 µl/min per mg protein and uptake and biliary clearances of 2.6 and 1.6 µl/min per mg protein, respectively. When apparent intrinsic clearance was incorporated into a PBPK model, the simulated oral human profiles were in good agreement with observed data at low doses but were underestimated at high doses due to unexpected overproportional increases in exposure with dose. In addition, the induction potential of RO6889678 on cytochrome P450 (P450) enzymes and transporters at steady state was assessed and cotreatment with ritonavir revealed a complex drug-drug interaction with concurrent P450 inhibition and moderate UDP-glucuronosyltransferase induction. Furthermore, we report on the first evaluation of in vitro pharmacokinetics studies using HBV-infected HepatoPac cocultures. Thus, long-term liver models have great potential as translational research tools exploring pharmacokinetics of novel drugs in vitro in health and disease.

Transporter-Mediated Alterations in Patients With NASH Increase Systemic and Hepatic Exposure to an OATP and MRP2 Substrate.

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.

Organic Anion Transporting Polypeptide 1a4 is Responsible for the Hepatic Uptake of Cardiac Glycosides in Mice.

Among organic anion transporting polypeptide (Oatp) family transporters expressed in the rodent liver, such as Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1, Oatp1a4 has a unique character to recognize neutral cardiac glycosides as a substrate in addition to organic anions. The relative contribution of Oatp1a4 to the substrate uptake into hepatocytes has not been clarified. In this study, we investigated the importance of Oatp1a4 in the hepatic uptake of its substrate drugs using Slco1a4-/- mice. The hepatic mRNA expression of Slco1a4 was decreased significantly in Slco1a4-/- mice, whereas no differences were seen in other hepatic transporters between wild-type and Slco1a4-/- mice. We determined the plasma concentrations and liver-to-plasma concentration ratios (Kp,liver) of Oatp1a4 substrates, including ouabain, digoxin, BQ-123, fexofenadine, rosuvastatin, pravastatin, nafcillin, and telmisartan, after continuous intravenous infusion. The plasma concentrations of ouabain and rosuvastatin were 2.1-fold and 1.7-fold higher in Slco1a4-/- mice, and Kp,liver of ouabain and digoxin were 13.4-fold and 4.3-fold lower in Slco1a4-/- mice, respectively. Furthermore, the biliary clearance of ouabain and digoxin with regard to plasma concentration were 21.9-fold and 4.1-fold lower in Slco1a4-/- mice, respectively, accompanied with a marked reduction in their Kp,liver, whereas the systemic clearance of ouabain, but not digoxin, was reduced significantly in Slco1a4-/- mice. These results suggest that Oatp1a4 plays a major role in the hepatic accumulation of cardiac glycosides in mice.

Systemic clearance and brain distribution of carbazole-based cyanine compounds as Alzheimer\u2019s disease drug candidates

SLM and SLOH, two analogues of carbazole-based cyanine compounds, have been shown to inhibit β-amyloid peptide aggregation in vitro and in Alzheimer’s disease model mice, which could be potentially developed into drugs for disease treatment. To pave the way for further pharmacokinetics-pharmacodynamics study, we set to investigate these compounds’ systemic clearance pathways and their brain exposure. We found that they generally exhibited relatively low plasma clearance which comprised of hepatic clearance and biliary clearance. Phase I oxidative metabolites for SLM and for SLOH upon microsomes incubation were identified, and the metabolism by CYP3A4 were found to be the major (>70%) hepatic clearance pathway, while the efflux by P-gp and BCRP located in the canalicular membrane of hepatocytes led to high biliary clearance. The permeation of SLM and SLOH through the brain endothelium was affected by the efflux transporters (P-gp and BCRP) and influx transporter (OATP2B1). The unbound interstitial fluid to plasma ratio (Kpuu,brain) was 8.10 for SLOH and 11.0 for SLM, which favored brain entry and were several folds higher than that in wild-type mice. Taken together, these carbazole compounds displayed low plasma clearance and high brain permeability, which entitle further development.

Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin Alfa).

GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.

Role of tangeretin as a potential bioavailability enhancer for silybin: Pharmacokinetic and pharmacological studies

Biological responses of a variety of naturally occurring compounds in vivo were restrained by their poor oral bioavailability. Silybin, as one of the active ingredients of silymarin, has presented promising bioactivity for the treatment of chronic liver diseases and cancer. However, its exposure in body was limited. In this study, silybin was demonstrated to be substrates of both BCRP and MRP2 by utilizing monolayer Caco-2 cell model and confirmed in MDCK cells overexpressing specific efflux transporter. Of all compounds screened, tangeretin, a potent inhibitor of efflux transporters of BCRP, MRP2 and P-gp, was able to enhance exposure of silybin by inhibiting functions of the barriers mediating transcellular transport. Moreover, study carried out in sandwich-cultured rat hepatocyte (SCH) model showed that the biliary excretion index (BEI) and in vitro biliary clearance of silybin decreased as levels of tangeretin increased, indicating efflux transporters mediating biliary excretion of silybin might be involved. Pharmacokinetic behaviors of silybin in rats were altered by co-administration of tangeretin, in terms of increased AUC and Cmax of silybin by comparing with that of silybin given alone. In addition, results coming from CCl4-induced acute liver injury rat model revealed that protection effect of silybin against liver damage in the presence of tangeretin was significantly enhanced. All these data were evident that efflux transporters play a critical role in transcellular transport of silybin and account for its low bioavailability. Enhanced bioavailability of silybin with co-administration of tangeretin by significantly inhibiting the efflux transporters further boost its bioactivity which is of particular importance in clinical use.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2

There has been a growing interest in circadian regulation of the expression and function of drug transporters. In this study, we investigated circadian rhythm in the expression and function of multidrug resistance-associated protein 2 (Mrp2) in mouse liver and involvement of circadian clock in their regulations by using the circadian clock genes (period 1 and period 2) knockout mice. The mRNA and protein expression of Mrp2, P-glycoprotein, and breast cancer resistance protein was measured in the mouse liver at different times of the day. Circadian variation of hepatobiliary excretion of phenolsulfonphthalein, a model substrate of Mrp2, was also investigated in mice. Circadian oscillation of Mrp2 protein expression was clearly observed in the mouse liver with levels down at the light phase and up at the dark phase. The cumulative biliary excretion and biliary clearance of phenolsulfonphthalein from the liver to the bile was 2.37- and 1.74-fold greater in mice administered during the dark phase than in those administered during the light phase, respectively. The circadian oscillation in mRNA expression of Mrp2 disappeared in period 1 and period 2 double knockout mice. These results suggest that the expression and function of Mrp2 show the circadian rhythm, controlled by circadian clock genes.

Understanding the Interplay Between Uptake and Efflux Transporters Within In Vitro Systems in Defining Hepatocellular Drug Concentrations

One of the most holistic in vitro systems for prediction of intracellular drug concentrations is sandwich-cultured hepatocytes (SCH); however, a comprehensive evaluation of the utility of SCH to estimate uptake and biliary clearances and the need for additional kinetic parameters has yet to be carried out. Toward this end, we have selected 9 compounds (rosuvastatin, valsartan, fexofenadine, pravastatin, repaglinide, telmisartan, atorvastatin, saquinavir, and quinidine) to provide a range of physicochemical and hepatic disposition properties. Uptake clearances were determined in SCH and compared with conventional monolayer and suspension hepatocyte systems, previously reported by our laboratory. CLuptake ranged from 1 to 41 μL/min/106 cells in SCH which were significantly lower (1%-10%) compared with the other hepatocyte models. The hepatocyte-to-media unbound concentration ratio (Kpu) has been assessed and ranged 0.7-59, lower compared with other hepatocyte systems (8-280). Estimates of in vitro biliary clearance (CLbile) for 4 drugs were determined and were scaled to predict in vivo values using both intracellular concentration and media drug concentrations. These studies demonstrate that reduced uptake in rat SCH may limit drug access to canalicular efflux transport proteins and highlight the importance of elucidating the interplay between these proteins for accurate prediction of hepatic clearance.

Hepatobiliary Clearance Prediction: Species Scaling From Monkey, Dog, and Rat, and In Vitro–In Vivo Extrapolation of Sandwich-Cultured Human Hepatocytes Using 17 Drugs

Hepatobiliary elimination can be a major clearance pathway dictating the pharmacokinetics of drugs. Here, we first compared the dose eliminated in bile in preclinical species (monkey, dog, and rat) with that in human and further evaluated single-species scaling (SSS) to predict human hepatobiliary clearance. Six compounds dosed in bile duct–cannulated (BDC) monkeys showed biliary excretion comparable to human; and the SSS of hepatobiliary clearance with plasma fraction unbound correction yielded reasonable predictions (within 3-fold). Although dog SSS also showed reasonable predictions, rat overpredicted hepatobiliary clearance for 13 of 24 compounds. Second, we evaluated the translatability of in vitro sandwich-cultured human hepatocytes (SCHHs) to predict human hepatobiliary clearance for 17 drugs. For drugs with no significant active uptake in SCHH studies (i.e., with or without rifamycin SV), measured intrinsic biliary clearance was directly scalable with good predictability (absolute average fold error [AAFE] = 1.6). Drugs showing significant active uptake in SCHH, however, showed improved predictability when scaled based on extended clearance term (AAFE = 2.0), which incorporated sinusoidal uptake along with a global scaling factor for active uptake and the canalicular efflux clearance. In conclusion, SCHH is a useful tool to predict human hepatobiliary clearance, whereas BDC monkey model may provide further confidence in the prospective predictions.