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Hepatic cell lines serve as economical and reproducible alternatives for primary human hepatocytes. However, the utility of hepatic cell lines to examine bile acid homeostasis and cholestatic toxicity is limited due to abnormal expression and function of bile acid-metabolizing enzymes, transporters, and the absence of canalicular formation. We discovered that culturing HuH-7 human hepatoma cells with dexamethasone (DEX) and 0.5% dimethyl sulfoxide (DMSO) for two weeks, with Matrigel overlay after one week, resulted in a shorter and improved differentiation process. These culture conditions increased the expression and function of the major bile acid uptake and efflux transporters, sodium taurocholate co-transporting polypeptide (NTCP) and the bile salt export pump (BSEP), respectively, in two-week cultures of HuH-7 cells. This in vitro model was further characterized for expression and function of bile acid-metabolizing enzymes, transporters, and cellular bile acids. Differentiated HuH-7 cells displayed a marked shift in bile acid composition and induction of cytochrome P450 (CYP) 7A1, CYP8B1, CYP3A4, and bile acid-CoA: amino acid N-acyltransferase (BAAT) mRNAs compared to control. Inhibition of taurocholate uptake and excretion after a 24-h treatment with prototypical cholestatic drugs suggests that differentiated HuH-7 cells are a suitable model to examine cholestatic hepatotoxicity.

Objective: Pharmacokinetic (PK) data to guide cefazolin dosing in premature infants is virtually non-existent. Therefore, we aimed to characterize cefazolin PK in infants aged ≤32 weeks of gestation at birth. Study Design: We conducted a prospective, open-label PK and safety study of cefazolin in infants ≤32 weeks gestation from a University Medical Center. We administered intravenous cefazolin and collected both timed and scavenged blood samples. We analyzed data using non-linear mixed effect modeling and simulated several dosage regimens to achieve target concentrations against methicillin-susceptible Staphylococcus aureus. Results: We analyzed 40 samples from 9 infants and observed that premature infants had lower clearance and greater volume of distribution for cefazolin compared to older children. The median (range) individual Bayesian estimates were 0.03 L/h/kg (0.01-0.08) for clearance and 0.39 L/kg (0.31-0.52) for volume. Conclusion: Simulations suggested reduced cefazolin dosing based on postmenstrual age achieve target concentrations and potentially reduce unnecessary exposure.

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.

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.

The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion. These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes.Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively. Transporter function was evaluated by measuring [D-penicillamine2,5]enkephalin (3H-DPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes.P-gp and the canalicular marker protein dipeptidyl peptidase IV (DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy. Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture. Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes. Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased. OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes. OATP1B3 expression was constant in SC human hepatocytes. In vitro biliary excretion of the opioid peptide 3H-DPDPE correlated with the proper localization of canalicular proteins in both species. Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function.These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.

Recently, hepatic transport processes have been recognized as important determinants of drug disposition. Therefore, it is not surprising that characterization of the hepatic transport and biliary excretion properties of potential drug candidates is an important part of the drug development process. Such information also is useful in understanding alterations in the hepatobiliary disposition of compounds due to drug interactions or disease states. Basolateral transport systems are responsible for translocating molecules across the sinusoidal membrane, whereas active canalicular transport systems are responsible for the biliary excretion of drugs and metabolites. Several transport proteins involved in basolateral transport have been identified including the Na(+)-taurocholate co-transporting polypeptide [NTCP (SLC10A1)], organic anion transporting polypeptides [OATPs (SLCO family)], multidrug resistance-associated proteins [MRPs (ABCC family)], and organic anion and cation transporters [OATs, OCTs (SLC22A family)]. Canalicular transport is mediated predominantly via P-glycoprotein (ABCB1), MRP2 (ABCC2), the bile salt export pump [BSEP (ABCB11)], and the breast cancer resistance protein [BCRP (ABCG2)]. This review summarizes current knowledge regarding these hepatic basolateral and apical transport proteins in terms of substrate specificity, regulation by nuclear hormone receptors and intracellular signaling pathways, genetic differences, and role in drug interactions. Transport knockout models and other systems available for hepatobiliary transport studies also are discussed. This overview of hepatobiliary drug transport summarizes knowledge to date in this rapidly growing field and emphasizes the importance of understanding these fundamental processes in hepatic drug disposition.

This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagen-coated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM) + 1.0 microM dexamethasone (DEX) + 5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM + 0.1 microM DEX, DMEM +/- 0.1 microM DEX + 5% FBS, Williams' medium E + 0.1 microM DEX + 1% ITS+, DMEM + 1.0 microM DEX, DMEM + 1.0 microM DEX + 5% FBS, or modified Chee's medium (MCM) + 0.1 microM DEX + 1% ITS+. [3H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca2+-free HBSS was measured, and the biliary-exeretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI = 0.649 [0.1 ml collagen]; BEI = 0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI = 0.538 [0.1 ml collagen]; BEI = 0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI = 0.396). Differing DEX concentrations (0.1 microM versus 1.0 microM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.

Purpose. This study characterized the gastrointestinal (GI) absorptionof zafirlukast after oral and colonic administration in humans.

Purpose. The purpose of this study was to evaluate the extent of ranitidine absorption from an externally activated drug-delivery system in two distinct regions of the intestine (jejunum and ileum) in healthy human volunteers. This investigation also was designed to evaluate the utility of the InteliSite® capsule for studying regional intestinal drug absorption in humans.

Purpose. This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions.

Purpose. Ranitidine plasma concentration vs. time profiles and the extent of ranitidine absorption were examined in the presence and absence of pancreatico-biliary secretions in order to elucidate factors which may contribute to secondary peaks after oral ranitidine administration.

Purpose. The objective of this study was to evaluate the effect of a potent P-gp inhibitor, GF120918, on the systemic pharmacokinetics and antinociceptive pharmacodynamics of a single intravenous dose of morphine in rats.

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides.

Purpose. Ranitidine gastrointestinal distribution was examined in the rat small intestine after oral administration to determine whether intestinal transit or secretion (exsorption) may influence the appearance of secondary peaks in ranitidine serum concentration-time profiles.

Purpose. Ranitidine absorption from isolated segments of rat small intestine (duodenum, midgut, and terminal ileum) was investigated to examine the influence of pH and 50% bile, and to determine if ranitidine is absorbed preferentially from a specific region. Methods. Ranitidine (50 mg/kg) was administered into each segment in pH 5 or pH 7 buffer, or in 50% bile. Venous blood was collected at various times for 40 min from the right jugular vein. Results. When ranitidine was administered in pH 7 buffer or in 50% bile, C max and AUC0–40 were significantly greater after administration into the terminal ileum compared to the duodenum and midgut. AUC0–40 was significantly greater when ranitidine was administered in pH 5 buffer or in 50% bile into the duodenum compared to the midgut. C max was significantly different between administration into the duodenum and midgut only when ranitidine was administered in 50% bile. Ranitidine administration in pH 5 buffer significantly decreased AUC0– 40 and C max after administration into the midgut, and AUC0–40 after administration into the terminal ileum compared to administration with pH 7 buffer or in 50% bile. Bile had no significant effect on AUC0–40 after ranitidine administration into the duodenum and midgut compared to administration in pH 7 buffer. However, bile significantly increased AUC0–40and C max after ranitidine administration into the terminal ileum compared to administration with pH 7 and pH 5 buffer. Conclusions. Results suggest that ranitidine is absorbed from the entire small intestine. However, the terminal ileum is the optimal site of gastrointestinal absorption. Furthermore, bile enhances ranitidine absorption from the terminal ileum.

Purpose. The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work.

Conventional analysis of initial uptake and egress rates in isolated hepatocytes is limited in the ability to distinguish between rates of metabolite formation and egress, and to separate basolateral and canalicular transport processes. The present study examined the applicability of kinetic modeling in describing acetaminophen glucuronide (AG) and acetaminophen sulfate (AS) formation and egress in hepatocytes after acute exposure to phenobarbital or p-hydroxyphenobarbital glucuronide (p-OHPBG) in vitro, or in vivo phenobarbital pretreatment. A significant pretreatment effect on AG and AS disposition was seen based on initial rates of egress. In vivo phenobarbital pretreatment decreased the initial egress rate of AG compared to vehicle pretreatment, and the initial egress rate of AS compared to all other treatments. A pharmacokinetic model incorporating AG and AS formation in hepatocytes as well as egress processes (including diffusional and active transport components) was fit to the data. Parameter estimates derived from model fits to the data showed the expected increase in acetaminophen glucuronidation and decrease in sulfation after phenobarbital pretreatment; in addition, an increase in the AG diffusional rate constant and a decrease in the AS diffusional rate constant was apparent. The excretion Vmax for AG was decreased statistically after acute phenobarbital exposure in vitro, and in vivo phenobarbital pretreatment, with a concomitant statistical increase in the Km for AG excretion. In vitro acute p-OHPBG exposure also decreased significantly the excretion Vmax for AG. These data are consistent with the hypothesis that phenobarbital-impaired biliary excretion of AG is a function of impaired canalicular transport due to the presence of p-OHPBG. They further suggest that the mechanism may not be simple competitive inhibition. This work demonstrates the utility of a kinetic modeling approach to differentiate metabolic and transport processes when analyzing data from isolated hepatocyte studies. Additional information may be gained that would not be apparent by conventional methods of analysis.

Double peaks in the plasma concentration-time profile following oral administration have been reported for several compounds. A pharmacokinetic model incorporating discontinuous absorption was developed to simulate concentration-time profiles with double peaks. The gastrointestinal (GI) tract was divided into N compartments, with absorption occurring only from the second and Nth compartments. A two-compartment model was used to describe systemic drug disposition. The effect of gastric emptying and GI transit rate constants (Kl and K1, respectively), number of hypothetical gut compartments, and absorption rate constant at each site (Ka1, Ka2) on the time of occurrence of each peak (Tp1, Tp2), the theoretical fraction of the dose absorbed at each site (phi 1, phi 2), and the contribution of the second site to systemic drug exposure (expressed as phi 2rel) were examined. Simulated concentration-time profiles demonstrated that Tp2 was determined by Kt and N, while Tp1 was determined by K1 and Kt. Changes in Ka1 and Ka2 had no effect on Tp1 or Tp2. phi 1, phi 2, and phi 2rel were determined by Ka1, Ka2, and Kt, and simulations indicated that a secondary peak in the concentration-time profile will be evident only when phi 2rel is substantial. In addition, concentration-time data for ranitidine and cimetidine, which displayed double peaks, were fit with the model. The present model described both data sets well, and realistic pharmacokinetic and physiologic parameters (absorption rate constants, systemic bioavailabilities, GI residence times) were obtained.

The potential influence of alterations in several physiologic processes (hepatocellular egress, biliary excretion, gastrointestinal transit) and biotransformation steps (oxidative metabolism, glucuronidation) on the disposition of agents subject to significant enterohepatic recirculation (ER) via the glucuronide conjugate was examined in a series of simulation experiments. The model of ER developed was based upon the disposition of valproic acid (VPA) and valproate glucuronide (VPA-G) in the rat. The systemic disposition of VPA was simulated following changes in several processes contributing to (or competing with) ER: hepatic oxidative metabolism, hepatic glucuronidation, sinusoidal egress of glucuronide conjugate, canalicular egress of glucuronide conjugate, and gastrointestinal transit. Changes in the formation clearance of VPA-G resulted in a less than proportional change in systemic clearance of VPA, whereas changes in oxidative metabolism led to a greater than proportional change in systemic clearance. Furthermore, alterations in hepatocellular egress of VPA-G affected the disposition of the parent compound, suggesting that drug interactions or disease state effects on metabolite transport may be misinterpreted as effects at the level of metabolite formation. Analytical methods are proposed to recover the intrinsic kinetic parameters (formation clearances of metabolites, renal clearance of parent, volume of distribution) in the presence of ER from the systemic disposition of the parent alone.

The influence of severe thermal injury (full-thickness burns involving 50% of the body surface area) on hepatic blood flow in the rat was assessed using the tricarbocyanine dye indocyanine green (ICG). In a randomized crossover fashion, rats received sequential infusions of ICG through both the femoral vein and the portal vein, allowing the estimation of total hepatic plasma clearance and trans-hepatic extraction of the dye. These two parameters, along with the hematocrit, were used to calculate intrinsic hepatic clearance of ICG and hepatic blood flow. Animals were examined at 0 (control), 0.5, 12, or 24 hr following infliction of scald burns. Hepatic blood flow was decreased significantly by 0.5 hr postburn and remained approximately 20% below normal throughout the remainder of the study. The intrinsic efficiency of the liver in removing ICG from the systemic circulation was also decreased by thermal injury. The potential mechanisms involved in these two physiologic perturbations are discussed.

The protein binding and pharmacokinetics of diazepam, ACC-9653 (a phenytoin prodrug), and phenytoin were evaluated in nine healthy male volunteers following administration of diazepam and ACC-9653, alone or concomitantly, in a randomized crossover design. No significant differences were observed in the fraction unbound or pharmacokinetic parameters of ACC-9653, phenytoin, or diazepam when ACC-9653 was administered alone compared to concomitant administration with diazepam. The phenytoin fraction unbound increased significantly with increased concentrations of ACC-9653, indicating displacement of phenytoin from its binding sites by ACC-9653. ACC-9653 also demonstrated concentration dependent binding. The lack of a significant pharmacokinetic drug interaction between ACC-9653 and diazepam suggests that these drugs may be safely administered together, although this conclusion should be confirmed in the intended patient population.

The biliary tract is an important route of elimination and potential site of toxicity for many xenobiotics. Quantification of biliary excretion in healthy volunteers is logistically challenging and is rarely defined during drug development. The current study uses a novel oroenteric tube to examine the pharmacokinetics of mebrofenin, a compound that undergoes rapid hepatic uptake and extensive biliary excretion. A custom-made multi-lumen oroenteric tube was positioned in the duodenum of four healthy volunteers (3 male, 1 female), subjects were positioned under a gamma camera and 2.5 mCi of 99mTc-mebrofenin was administered intravenously. Duodenal aspirates, blood samples, and urine were collected periodically for 3 h. Sincalide (cholecystokinin-8) was administered intravenously 2 h after mebrofenin to stimulate contraction of the gallbladder, and gamma scintigraphy was used to determine gallbladder ejection fraction. Total body clearance of mebrofenin approximated liver blood flow, and the oroenteric tube efficiently recovered the majority of excreted bile and mebrofenin. When corrected for gallbladder ejection fraction, 77–101% of the dose was recovered. This novel oroenteric tube and clinical protocol provide a useful method to quantify biliary excretion in healthy volunteers. (See Table) Clinical Pharmacology & Therapeutics (2004) 75, P35–P35; doi: 10.1016/j.clpt.2003.11.132 Table 1. Subject 1 2 3 4 Ejection fraction 0.86 0.82 0.12 0.86 Dose collected in bile (%) 84 66 35 84 Clearance (ml/min/kg) 16.58 17.02 19.88 17.33 Biliary clearance (ml/min/kg) 15.02 11.47 7.81 15.61