Kim L. R. Brouwer

Membrane transporters in drug development

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

The Complexities of Hepatic Drug Transport: Current Knowledge and Emerging Concepts

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.

Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity

Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided.

Pharmacokinetic and Pharmacodynamic Implications of P-glycoprotein Modulation

P‐glycoprotein (P‐gp) is a cell membrane—associated protein that transports a variety of drug substrates. Although P‐gp has been studied extensively as a mediator of multidrug resistance in cancer, only recently has the role of P‐gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined. P‐glycoprotein is present in organ systems that influence drug absorption (intestine), distribution to site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues. Many marketed drugs inhibit P‐gp function, and several compounds are under development as P‐gp inhibitors. Similarly, numerous drugs can induce P‐gp expression. While P‐gp induction does not have a therapeutic role, P‐gp inhibition is an attractive therapeutic approach to reverse multidrug resistance. Clinicians should recognize that P‐gp induction or inhibition may have a substantial effect on the pharmacokinetics and pharmacodynamics of concomitantly administered drugs that are substrates for this transporter.

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca(2+)-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca(2+)-free buffer. The cumulative uptake of [(3)H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca(2+)-free buffer, due to accumulation of taurocholate in canalicular spaces. When [(3)H]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca(2+)-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from approximately 8% at day 0 to approximately 60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

Intracellular Drug Concentrations and Transporters: Measurement, Modeling, and Implications for the Liver

Intracellular concentrations of drugs and metabolites are often important determinants of efficacy, toxicity, and drug interactions. Hepatic drug distribution can be affected by many factors, including physicochemical properties, uptake/efflux transporters, protein binding, organelle sequestration, and metabolism. This white paper highlights determinants of hepatocyte drug/metabolite concentrations and provides an update on model systems, methods, and modeling/simulation approaches used to quantitatively assess hepatocellular concentrations of molecules. The critical scientific gaps and future research directions in this field are discussed.

P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide.

AbstractPurpose. 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 re-polarization, 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. Methods. 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. Results. 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. Conclusions. These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.

In vitro methods to support transporter evaluation in drug discovery and development

This white paper addresses current approaches and knowledge gaps concerning methods to assess the role of transport proteins in drug/metabolite disposition in humans. The discussion focuses on in vitro tools to address key questions in drug development, including vesicle‐ and cell‐based systems. How these methods can be used to assess the liability of compounds for transporter‐based drug–drug interactions (DDIs) in vivo is also explored. Existing challenges and approaches to examine the involvement of transporters in drug disposition are discussed.

In vitro-in vivo correlation of hepatobiliary drug clearance in humans.

The biliary clearance (Clbiliary) of three compounds was estimated using sandwich‐cultured human hepatocytes (SCHH) and compared with Clbiliary values measured in vivo. Tc‐99m sestamibi (MIBI) Clbiliary was determined in seven healthy volunteers using an oroenteric catheter to aspirate duodenal secretions, and gamma scintigraphy to determine gallbladder contraction; this technique was used previously to determine Tc‐99m mebrofenin (MEB) and piperacillin (PIP) in vivo Clbiliary. In vitro Clbiliary of MEB, MIBI, and PIP was quantified in SCHH as the ratio of mass excreted into bile canaliculi and area under the blood concentration‐time curve (AUC) in medium. MIBI Clbiliary in vivo was 5.5±1.2 mL/min/kg (mean±SD). The rank order of Clbiliary predicted from SCHH corresponded well with the in vivo Clbiliary values in mL/min/kg for MEB (7.44 vs 16.1), MIBI (1.20 vs 5.51), and PIP (0.028 vs 0.032). In conclusion, the methods developed allowed for reproducible quantification of Clbiliary of drugs in healthy humans and prediction of Clbiliary from in vitro data.

CHARACTERIZATION OF TRANSPORT PROTEIN EXPRESSION IN MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) 2-DEFICIENT RATS

Multidrug resistance-associated protein (Mrp) 2-deficient transport-deficient (TR–) rats, together with their transport-competent Wistar counterparts (wild type), have been used to examine the contribution of Mrp2 to drug disposition. However, little is known about potential variation in expression of other transport proteins between TR– and wild-type rats or whether these differences are tissue-specific. Sections of liver, kidney, brain, duodenum, jejunum, ileum, and colon were obtained from male TR– and wild-type Wistar rats. Samples were homogenized in protease inhibitor cocktail and ultracentrifuged at 100,000g for 30 min to obtain membrane fractions. Mrp2, Mrp3, Mrp4, P-glycoprotein, sodium-dependent taurocholate cotransporting polypeptide, organic anion transporting polypeptides 1a1 and 1a4, bile salt export pump, breast cancer resistance protein, ileal bile acid transporter, UDP-glucuronosyl transferase (UGT1a), glyceraldehyde-3-phosphate dehydrogenase, and β-actin protein expression were determined by Western blot. Mrp3 was significantly up-regulated in the liver (∼6-fold) and kidney (∼3.5-fold) of TR– rats compared with wild-type controls. Likewise, the expression of UGT1a enzymes was increased in the liver and kidney of TR– rats by ∼3.5- and ∼5.5-fold, respectively. Interestingly, Mrp3 expression was down-regulated in the small intestine of TR– rats, but expression was similar to wild type in the colon. Mrp4 was expressed to varying extents along the intestine. Expression of some transport proteins and UGT1a enzymes differ significantly between TR– and wild-type rats. Therefore, altered drug disposition in TR– rats must be interpreted cautiously because up- or down-regulation of other transport proteins may play compensatory roles in the presence of Mrp2 deficiency.