- 0. Overview
The action of a drug is affected by its distribution and disposition in the body. Taking anticancer drugs as an example, it is well known that those which can kill cancer cells in the test-tube do not necessarily exhibit satisfactory antitumor effects when given to patients. This is because the anticancer drugs also distribute to normal cells, producing a variety of adverse effects before the tumor cells are completely destroyed. Consequently, major advances in drug therapy can be expected if drug delivery systems (DDS) can be developed to control disposition in the body and selectively attack target tissues. The development of ideal DDS is the ultimate goal of our research and, at present, work is in progress to examine the major factors which govern drug disposition in the body and to clarify the mechanisms involved inf membrane transport in the liver, brain, kidney, intestine and the tumor itself.
In the life sciences, research normally starts at the body (in vivo) level and progresses to the organ, cell, protein and gene level in order to understand the principles involved in the functions of the human body. Such an approach has met with a great deal of success. However, "the prediction and control of drug effects and safety" will never be fully achieved unless we develop methodology to reconstruct quantitatively in vivo phenomena from in vitro data. Before drugs can exert their ultimate effects they have to undergo a variety of different processes: (1) absorption from the site of administration to reach the circulating blood, (2) detoxification via metabolism and excretion, (3) distribution to the tissue(s) involved in their pharmacological (or adverse) effects and (4) binding to pharmacological receptors, followed by signal transduction etc. It is possible to describe these processes using physiological and anatomical parameters such as blood flow, tissue volume, pH and the membrane potential difference between the outside and inside of the cells as well as biochemical parameters representing drug binding to blood and tissue macromolecules, membrane permeability, interaction with metabolic enzymes, transport carriers and pharmacological and toxicological receptors and acceptors. Indeed, by incorporating such physiological and biochemical parameters into appropriate mathematical models, we have been attempting to predict the output "exertion of pharmacological and adverse effects" from the input "dose of drug, route of administration and pathophysiological condition of the patients". The research project now underway can be divided into seven areas as described left.
- 1. Prediction of drug disposition from in vitro data using physiologically-based pharmacokinetic (PBPK) models
In order to predict both the pharmacological and adverse effects of drugs, It is essential to be able to predict their blood concentration-time profiles. The blood concentration-time profile of a drug can be described from a knowledge of the absolute values of 1) hepatic clearance for metabolism and biliary excretion 2) renal clearance and 3) volume of distribution (extent of distribution to tissues). We have developed a method for predicting the human disposition of a drug from in vitro data with the aid of a physiologically-based pharmacokinetic model. In this, the in vitro data on metabolism, transport and binding, obtained in isolated membrane vesicles, isolated cells and perfused tissues, were successfully used to predict the in vivo disposition. For many drugs, renal clearance in humans has been successfully predicted by extrapolating from animal data based on an allometric equation (the animal scale-up method). The prediction of human hepatic metabolism from animal data, however, is difficult due to the large interspecies difference. We are currently trying to predict human in vivo hepatic clearance based on in vitro data obtained using human hepatocytes, microsomes and recombinant P-450 isozymes.
The adverse effects of drugs resulting from drug-drug interactions has long been a serious problem but has recently begun to attract increasing attention. Pharmacokinetic factors which can be altered to produce drug-drug interactions include plasma-protein binding, carrier-mediated drug transfer across biological membranes, and metabolism. We are establishing a method for accurately predicting the occurrence of such drug-drug interactions are, in particular, we are focusing on interactions involving metabolism and transport processes. These series of studies are closely linked to the development of novel drugs with fewer adverse effects and to the more efficient and safer used of drugs.
Fig 1. Prediction of drug disposition from in vitro data using PBPK models
- 2. Clarification of transport systems in the blood-brain and blood-cerebrospinal fluid barries, and prediction of drug disposition in the central nervous system
- Investigation of the efflux transport mechanism across the blood-brain and blood-cerebrospinal fluid barriers, and establishment of a method for predicting disposition of a drug in the central nervous system
It is necessary to have information about the permeability of a drug across the key barriers, i.e. the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) in order to predict its disposition in the central nervous system. An experimental system using isolated choroid plexus was established for that purpose. Using this technique, it was found that many drugs are actively eliminated from the cerebrospinal fluid through the BCSFB into the blood, and the driving force for this has been identified. At present, the development of a method for predicting the intracerebral distribution in vivo has been carried out using a series of drugs as model compounds including an anti-AIDS agent, a number of quinolone antibiotics and antidementia agents. Such analyses, have recently allowed us to collect a great deal of interesting data. Although the BBB has been regarded as a "static wall" consisting of anatomical features such as the tight junction which connects endothelial cells to each other, recent advances in kinetic and molecular biological research have changed the way we look at this dynamic barrier. It is now known that efflux transporters, such as P-glycoprotein, also provide a barrier function by transporting xenobiotics including drugs from inside the brain capillary endothelial cells to the blood resulting in an apparently low BBB permeabilitiy. It is suggested that transporters other than P-glycoprotein also play a role in the detoxification of xenobiotics in the BBB based on our recent studies using the brain efflux index method. This method enables us to measure efflux transport across the BBB in vivo, using membrane vesicles and a number of molecular biological approaches.
Fig 2. Transporters expressed in the BBB and BCSFB
- 3. 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.
Fig 3. Transporters expressed in the human liver
Fig 4. Prediction of hepatic clearances using in vitro experimental systems
- 4. Kinetic and molecular analysis of drug-drug interactions
In normal clinical drug therapy, some drugs are often concomitantly prescribed rather than used as monotherapy. Consequently, an unexpected reduction in pharmacological effects and/or side effects is sometimes observed in some of patients due to drug-drug interactions. In particular, we have focused on the pharmacokinetic interaction, which occurs in determinant molecules (metabolic enzymes and transporters) involving the time-profile of drug concentrations at the target sites (receptors) and we have established experimental systems to quantitatively predict in vivo drug-drug interactions from in vitro experiments (Fig 5).
Among the drug interactions caused by metabolic enzymes, competitive and non-competitive inhibition of CYP (cytochrome P450) enzymes, which mainly catalyze phase-I oxidative reactions, have been extensively studied. Moreover, we have also investigated other cases where drugs, such as rifampicin and phenobarbital, which can induce the expression of metabolic enzymes, increase the metabolism of co-administered drugs and some metabolic products covalently bind to metabolic enzymes and irreversibly inhibit their metabolism (mechanism-based inhibition).
To explain several forms of drug interactions quantitatively, we have proposed various kinds of mathematical models and demonstrated that we can predict a change in the in vivo plasma concentration profiles by using kinetic parameters obtained from in vitro experiments. Our prediction methods for drug interactions that avoid false negative predictions have been adopted in the guideline from the Japanese Ministry of Health, Labour and Welfare and are now widely used in the field of drug discovery and development.
Since the history of transporter research is shorter than that of metabolic enzymes, we are carrying out detailed investigations of drug interactions mediated by transporters. In this laboratory, we were the first to demonstrate that the drug interaction between cyclosporin A and cerivastatin can be quantitatively explained by the action of a hepatic uptake transporter (OATP2) from the results of both in vitro and in vivo experiments. This finding, showing that we must pay attention to transporter-mediated drug interactions, has had a major impact in the field of the pharmaceutical sciences. Now we are carrying out research integrating molecular biological approaches and kinetic considerations to propose a prediction method for preventing the severe side effects during the drug development stage and subsequent clinical use.
Fig 5. Mechanisms of drug-drug interactions involving drug transporters