This project is part of a long-term research effort to develop a method to accurately predict from in vitro data the biliary clearance of drugs measured in vivo in humans. This type of correlation is lacking in the current literature despite the extensive use of in vitro systems to predict disposition and metabolism of drugs in humans. One of the major obstacles encountered in studying the biliary excretion of drugs is the difficulty in accessing the gallbladder and collecting bile from healthy volunteers. This is why, for many drugs, very little is known about the actual fraction of the dose excreted in bile, and the portion of total clearance that can be attributed to the biliary route. The development and validation of a technique to determine biliary clearance in healthy human volunteers would provide novel in vivo information which could be compared to biliary clearance values generated in vitro. Several in vitro models have been used to examine mechanisms of biliary clearance. The most physiologically relevant in vitro system, which is characterized by the presence of both metabolic enzymes and transport proteins, is the sandwich-cultured human hepatocyte model. This in vitro system could be employed for moderate- to high-throughput screening of human hepatic clearance mechanisms, and perhaps more importantly, for investigating and predicting drug associated hepatotoxicity and drug-drug interactions.
In summary, the first aim of this research project was to develop a method to quantitatively collect bile in healthy volunteers after administration of probe compounds. These probes were specifically selected to exhibit various degrees of biliary excretion, in order to generate a range of in vivo biliary clearance values for these compounds. A second objective was to utilize pharmacokinetic modeling and simulations, as well as in vitro systems expressing specific transport proteins, to elucidate the mechanisms involved in the hepatobiliary
disposition of the model compound Tc-99m mebrofenin, and to explore the effects of pathophysiological changes due to disease states or drug interactions on the disposition of this compound. Additionally, the versatility of the clinical technique was demonstrated by collecting previously unknown metabolites that were excreted in bile, in addition to quantifying the biliary excretion of drugs. Finally, the sandwich-cultured human hepatocyte model was used to investigate the in vitro biliary clearance of the model compounds previously studied in vivo, in order to evaluate the accuracy of the in vitro predictions. These Aims are described briefly in the next few pages and in depth results, analyses and discussion are reported in Chapters 2-5 and in Appendices A-D.
HYPOTHESES: A method to quantitatively estimate biliary clearance of drugs in healthy human volunteers can be developed and applied to model compounds exhibiting a wide range of biliary excretion (Aim #1). Pharmacokinetic modeling and simulation studies with Tc-99m mebrofenin can explain in vivo observations in health and disease; information obtained with the aid of in vitro tools concerning the mechanisms underlying Tc-99m mebrofenin biliary excretion can be incorporated into the pharmacokinetic model (Aim #2). The clinical methodology developed in Aim #1 can be applied to the collection of labile metabolites in bile that are unique to the human species (Aim #3). The biliary clearance of the model compounds obtained in sandwich-cultured human hepatoyctes, once properly scaled, can accurately predict in vivo biliary clearance in humans (Aim #4).
Specific Aim #1 Hypothesis: It is possible to quantitatively collect bile secreted into the duodenum in healthy volunteers and obtain reliable estimates of biliary clearance for drugs that exhibit various degrees of biliary excretion.
1.a. Design an oroenteric tube and a clinical protocol to quantitatively collect duodenal secretions in humans.
1.b. Validate the oroenteric tube and clinical protocol in a study involving healthy human volunteers using Tc-99m mebrofenin.
1.c. Utilize the technique developed in 1.a and 1.b to obtain biliary clearance values in healthy volunteers for piperacillin, Tc-99m sestamibi and Tc-99m mebrofenin, compounds expected to exhibit low, intermediate and high biliary clearance, respectively.
Specific Aim #2 Hypothesis: The disposition of Tc-99m mebrofenin observed in the clinical studies can be characterized with the aid of pharmacokinetic modeling. The mechanisms of hepatobiliary transport of this probe can be clarified with the use of in vitro tools.
2.a. Use pharmacokinetic modeling as an approach to evaluate the disposition of Tc-99m mebrofenin (a model compound that undergoes high biliary clearance) in humans and simulate alterations in disposition caused by drug interactions or disease states.
2.b. Identify the human transport proteins involved in the hepatic basolateral uptake of Tc- 99m mebrofenin using Xenopus leavis oocytes transfected with OATP1B1 and OATP1B3. 2.c. Elucidate the mechanism of canalicular and basolateral excretion of Tc-99m mebrofenin using inside-out membrane vesicles prepared from HEK293 cells transfected with MRP2 or MRP3.
2.d. Evaluate the contribution of Mrp2 in the biliary excretion of Tc-99m mebrofenin in the sandwich-cultured rat hepatocyte model using cells from control (Wistar) and TR- (Mrp2- deficient) rats.
Specific Aim #3 Hypothesis: The collection of bile as soon as it is secreted into the human intestine by utilizing the oroenteric tube and clinical protocol developed in Aim #1 allows for identification of labile metabolites unique to the human species.
3.a. Use LC-MS-MS to analyze bile and urine obtained from healthy volunteers after administration of piperacillin in order to investigate the formation and excretion of Phase I and Phase II metabolites.
3.b. Demonstrate that the formation of labile metabolites identified in vivo can be generated
in vitro.
Specific Aim #4 Hypothesis: The sandwich-cultured human hepatocyte model is a useful tool for predicting biliary clearance values in humans.
4.a. Customize experimental conditions for studying the cellular accumulation of model compounds in sandwich-cultured human hepatocytes.
4.b. Determine the biliary excretion index (BEI) and in vitro biliary clearance parameters for Tc-99m mebrofenin, Tc-99m sestamibi and piperacillin in sandwich-cultured hepatocytes. 4.c. Investigate the predictive capabilities of the sandwich-cultured human hepatoycte model by comparing in vitro data with in vivo data obtained in humans.