Clarification of the mechanism of hepatic uptake and biliary excretion of drugs

Because the liver is one of the major organs for detoxification of xenobiotics, it is important to clarify the mechanism of hepatic uptake and biliary excretion of drugs. We have already used a variety of kinetic approaches to show that several anionic and cationic compounds are taken up by active transport systems.
Recently, many transporters have been cloned in rodents and humans. For example, OATP (organic anion transporting polypeptide) family transporters, NTCP (Na+-taurocholate cotransporting polypeptide), and OCTs (organic cation transporters) have been shown to be involved in the transport of organic anions, bile acids and organic cations, respectively (Fig. 3). These transporters generally accept many kinds of endogenous compounds (e.g. bile acids and conjugated steroids) and drugs (e.g. pravastatin and methotrexate). We are now studying the functional analyses of several transporters by using transporter-expressed mammalian cell lines and have established a method for evaluating the contribution of each transporter to the overall pharmacokinetics. We have recently been able to evaluate human liver uptake using human cryopreserved hepatocytes. To evaluate the quantitative contribution of each transporter, we are adopting several approaches not only by using transporter-specific inhibitors and substrates, but also by using knockout mice and gene specific knockdown by RNAi (RNA interference).
We have also used a number of experimental techniques to show that primary active transporters driven by ATP hydrolysis are responsible for the biliary excretion of organic anions. Initially, we showed that the hyperbilirubinemia that appeared naturally in one colony of SD rats (presently named EHBR (Eisai hyperbilirubinemic rats)) is caused by a nonsense mutation in the Mrp2 (multidrug resistance associated protein 2) gene and we have succeeded in cloning the cDNA of rat Mrp2. We have also investigated many aspects of Mrp2, such as its transport properties, by using membrane vesicles and important amino acids for substrate recognition and transport. The homologous MRP2 gene in humans has been identified as a causal gene of Dubin-Johnson syndrome and we are now carrying out functional analyses of human MRP2. MRP2 can recognize many kinds of organic anions as in the case of uptake transporters and is responsible for the biliary excretion of many endogenous compounds and drugs. Also expressed in the apical membrane are P-gp (P-glycoprotein) for the transport of organic cationic and neutral compounds, BSEP (bile salt export pump) for bile acids and BCRP (breast cancer resistance protein) (Fig. 3).
In particular, we were the first to show that BCRP can preferentially accept many kinds of sulfate conjugates and this is the first candidate transport system for the biliary excretion of sulfate conjugates. We are now studying the importance of BCRP for in vivo pharmacokinetics using knockout mice.
Biliary excretion is mediated by both uptake and efflux transporters. In our laboratory, we have constructed double transfected MDCKII cells which express uptake transporter (OATP2) on the basal side and efflux transporter (MRP2) on the apical side and have succeeded in observing the vectorial transcellular transport of bisubstrates of uptake and efflux transporters from the basal to apical compartment. Moreover, we have also demonstrated that the clearance of transcellular transport of each compound in an Oatp4/Mrp2 double transfectant correlates well with the in vivo biliary clearance in rats, which suggests that this experimental system can be used as a model of biliary excretion in hepatocytes. We are now proceeding to construct of several kinds of double transfectants which express important uptake and efflux transporters and are carrying out a detailed kinetic analyses to predict in vivo hepatic transport from in vitro experiments (Fig. 4).
It has been shown that transporters exhibit multiplicity and genetic polymorphisms like metabolic enzymes, causing some major problems in predicting the pharmacological and toxicological effects (incl. drug-drug interactions) when determining the optimum dose regimen for each patient and developing new drugs. In our laboratory, we have identified genetic polymorphisms of some transporters and we are now investigating whether mutated transporters alter their intracellular localization and transport function or not. Regarding the inter-individual variability of their expression levels, we are also trying to clarify the molecular mechanism(s) governing regulation and induction of the level of expression.