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Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and Pharmacokinetics Laboratory, Mitsubishi Pharma Corporation, Chiba, Japan
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I. Introduction |
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; Venter et al., 2001), this new information about all the human genes and their functions led to a change in drug research strategies and, in particular, the processes of drug discovery and development. Following the progress in genome-based drug discovery, international competition in drug discovery has become even keener. The particular strategy adopted for drug discovery will be a critical factor in determining whether a company is successful in its research and development operations. The strategy that target proteins are identified based on genomic information, so-called "genome-based drug discovery," is likely to become very popular. However, determination of the target protein alone is insufficient to allow the development of clinically significant drugs. It is also necessary to identify the lead compounds binding the target proteins by using combinatorial chemistry synthesis and high-throughput screening, optimize these lead compounds, and then select those with clinically effective pharmacological activity and minimize any side effects. A significant number of drug candidates entering clinical development are dropped at some stage due to unacceptable pharmacokinetic properties (White, 2000; Roberts, 2001). The pharmacokinetic profile should be a primary consideration in the selection of a drug candidate, ultimately contributing to its eventual clinical success or failure. It is now recognized that selection of a "robust" candidate requires a balance among efficacy, safety, and pharmacokinetic properties, and that the screening of these characteristics should be carried out as early as possible in the discovery process. Thus, many pharmaceutical companies are now carrying out rational high-throughput drug metabolism and pharmacokinetics screening systematically and establishing pharmacokinetic selection criteria (White, 2000; Roberts, 2001). For example, the high-throughput screening for absorption using Caco-2 cells and the screening for metabolic stability and metabolic enzyme inhibition using cytochrome P450 recombinant microsomes or human liver microsomes have become extremely popular. Attention is now being focused on optimizing the pharmacokinetic profiles of drug candidates using transporter function (Ayrton and Morgan, 2001; Mizuno and Sugiyama, 2002).
Many different drug transporters are expressed in various tissues, such as the epithelial cells of the intestine and kidney, hepatocytes, and brain capillary endothelial cells (Muller and Jansen, 1997; Koepsell, 1998; Meijer et al., 1999; Suzuki and Sugiyama, 1999; Inui et al., 2000a; van Aubel et al., 2000; Gao and Meier, 2001) (Table 1). In recent years, a number of important transporters have been cloned, and considerable progress has been made in understanding the molecular characteristics of individual transporters. It has now become clear that some of these are responsible for drug transport in various tissues, and they may be key determinants of the pharmacokinetic characteristics of a drug as far as its intestinal absorption, tissue distribution, and elimination are concerned (Oude Elferink et al., 1995; Zhang et al., 1998; Kim, 2000; Dresser et al., 2001; Kusuhara and Sugiyama, 2002; Russel et al., 2002). Studies of the functional characteristics, such as substrate specificity, and of the localization of cloned drug transporters could provide important information about the mechanisms of drug disposition. Transporters have been classified as primary, secondary, or tertiary active transporters. Secondary or tertiary active transporters, such as OAT1, OATP, NTCP, OCT, OCTN, and PEPT, are driven by an exchange or cotransport of intracellular and/or extracellular ions (Burckhardt and Wolff, 2000; Dresser et al., 2001; Lee et al., 2001a). The driving force for primary active transporters like ATP-binding cassette transporters, such as MDR, MRP, and BCRP, is ATP hydrolysis (Lautier et al., 1996; Borst et al., 1999; Hooiveld et al., 2001; Lee et al., 2001a; Schinkel and Jonker, 2003). Most of the former transporters have a similar structure in that they have 12 putative membrane-spanning domains and their molecular mass is approximately 50 to 100 kDa. In contrast, the mean molecular weight of the latter transporters, involved in the cellular extrusion of xenobiotics, is comparatively high (150 -200 kDa) and they all have two ATP-binding domains, except for BCRP. Furthermore, each gene family of transporters is composed of a multiplicity of members. Owing to the increase in the number of identified transporter genes, the Human Gene Nomenclature Committee has classified transporters using standardized names, such as the solute carrier superfamily (SLC) and ATP-binding cassette (ABC) transporters (http://www/gene.ucl.ac.ul/nomenclature/genefamily.shtml). These standardized names, accompanied by the conventional names, are shown in Table 2. The tissue distribution and elimination route of some drugs is determined by the degree of expression of each transporter subtype in each tissue and its corresponding substrate affinity and transport maximum. Thus, regulating the function of transporters should allow the highly efficient development of drugs with ideal pharmacokinetic profiles. As drug discovery involving the use of transport mechanisms increases, the need for an effective in vitro screening system for transporters will also increase. Accordingly, methods allowing the rational prediction and extrapolation of in vivo drug disposition from in vitro data are urgently required. Although there has been intensive investigation of the functional analysis of the human genome, there are a large number of genes whose molecular protein function remains unknown (Venter et al., 2001). The human genome contains many genes that encode membrane transporters and related proteins (Table 3) (Venter et al., 2001). For drug discovery, development, and targeting one needs to know which transporters play a role in the disposition of a drug and its subsequent effects.
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In this article, we summarize the key role played by drug transporters in drug disposition, and the strategic use of drug transporters in drug discovery and development is discussed. We also introduce possible strategies for drug discovery using transporters, including the transporter screening systems, methods for estimating the contribution of transporters to drug disposition, and the prediction of in vivo drug disposition from in vitro data.
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II. Strategies for Drug Discovery Using Transporters |
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One of the main goals is to develop pharmaceutical agents with no adverse effects. It is also desirable to develop drugs with a wide therapeutic spectrum of activity. Drug targeting is one effective approach both to increase the pharmacological activity of drugs and to reduce their side effects by enhancing delivery to the target site. Recent research has identified many types of transporters that are expressed selectively in the liver, kidney, and other organs and which, therefore, may be a promising target for drug delivery. Some instances of drug delivery to the liver or kidney are introduced here.
The most comprehensively documented case is pravastatin. Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, undergoes enterohepatic circulation, which prolongs the exposures of the liver (target organ) to the drug and minimizes adverse side effects in the peripheral tissues. This enterohepatic circulation is mediated by transporters in every process from pravastatin gastrointestinal absorption to biliary transport. Pravastatin is taken up by the liver from the portal vein by OATP family proteins located on the sinusoidal (basolateral) membrane (Hsiang et al., 1999; Nakai et al., 2001; Sasaki et al., 2002). After exhibiting its pharmacological action in the liver, pravastatin is then excreted into the bile via MRP2 with only a minimum degree of metabolic conversion (Yamazaki et al., 1996). The fraction of the drug released into the duodenum is then reabsorbed by active transport (Tamai et al., 1995). Thus, efficient hepatobiliary transport by OATP and MRP2 plays an important role in the enterohepatic circulation, which is responsible for maintaining significant concentrations of this drug in the liver. Although the mechanisms governing the pharmacokinetic properties of this drug were identified after their development, attempts to design molecules during the drug discovery process will be required in the future.
It has been found that successful targeting of anticancer drugs can be achieved using oligopeptide transporter PEPT1, expressed in tumors (Nakanishi et al., 1997, 2000). Some human cancer cell lines naturally express oligopeptide transport activity. The delivery of the peptide-mimetic anticancer drug, bestatin, a substrate of PEPT1, has been investigated. After i.v. administration of bestatin into nude mice-inoculated tumor cells, the bestatin concentration in PEPT1-transfected tumor was significantly greater than that in vector-transfected tumor (Nakanishi et al., 2000). Furthermore, repeated oral administration of bestatin specifically suppressed the growth of PEPT1-transfected tumors. It has been suggested that bestatin distributes to tumor tissues in a specific manner.
NTCP is the Na+-bile acid cotransporting protein that mediates the hepatic uptake of bile acids (Hagenbuch et al., 1991). Since NTCP is exclusively expressed on the sinusoidal membrane of the liver (Meier, 1995), this transporter may be used as a target for drug delivery to that organ. Dominguez et al. have reported that coupling of drugs to the side chains of bile acids may be a useful strategy for specifically targeting liver tumor cells (Dominguez et al., 2001). A novel cisplatin-ursodeoxycholic derivative (Bamet-UD2) is efficiently transported by NTCP (Briz et al., 2002). The concentration of Bamet-UD2 in the liver was severalfold higher than that of cisplatin, while potentially toxic drug accumulation in other tissues, such as kidney, brain, and bone marrow, was significantly reduced (Dominguez et al., 2001). Thus, in mice, Bamet-UD2 exhibited strong antitumor activity without any side effects compared with cisplatin (Dominguez et al., 2001).
The targeting strategy should focus on the differential expression of transporters between the target organ and other organs, and it is essential to design molecules that are capable of being transported by a target organ-specific transporter. In particular, the use of blood-brain barrier (BBB)-specific influx transporters is expected to be an effective approach for the brain delivery of drugs acting on the central nervous system (CNS) because drug penetration into the brain is restricted under normal conditions.
B. Role of Brain Efflux Transporters
Brain capillary endothelial cells form the BBB and act as a self-defense mechanism, preventing xenobiotics from entering the brain. However, successful penetration of the blood-brain barrier is necessary if a drug is to reach the required concentration for a desired pharmacological effect. Efflux transport systems at such barriers provide protection for the CNS by removing drugs from the brain and transferring them to the systemic circulation. This is why the brain penetration of drugs is markedly restricted (Suzuki et al., 1997; Tsuji and Tamai, 1997; Fromm, 2000; Kusuhara and Sugiyama, 2001b; Lee et al., 2001a; Schinkel, 2001; Hagenbuch et al., 2002; Sun et al., 2003). Primary active transporters, such as P-gp encoded by MDR1 or MRP transporters, are responsible for the cellular extrusion of many kinds of drugs (Cole and Deeley, 1998; Borst et al., 1999; Kool et al., 1999; Kuwano et al., 1999). P-gp transports a wide variety of lipophilic, structurally diverse drugs, such as vinca alkaloids and anthracyclines. In general, the substrate specificities of efflux transporters are remarkably broad, and their affinities for substrates are much lower (of the order of micromolar to millimolar) than the affinities for pharmacological receptors (of the order of nano-molar to picomolar). Thus, these transporters are able to recognize a large number of xenobiotics with a wide variety of structures. In normal tissue, P-gp is expressed in the liver, kidney, small and large intestine, and brain capillary endothelial cells (Troutman et al., 2001) (Table 1). Thus, the brain penetration of drugs, which are substrates of this transporter, is extremely limited (Fromm, 2000; Tamai and Tsuji, 2000; Kusuhara and Sugiyama, 2001b; Schinkel, 2001). In mice that lack P-gp encoded by the mdr1a gene, the brain distribution of P-gp substrates is significantly increased compared with that in normal mice (Table 4). Clearly, these results demonstrate that P-gp plays a key role in the BBB. The transporter gene knockout mouse is a very important tool for investigating the role of transporters in drug disposition (Schinkel et al., 1994; Wijnholds et al., 1997, 2000; Jonker et al., 2001). Efflux transport should be taken into consideration during drug development to improve brain penetration and to avoid drug-drug interactions involving these transporters and subsequent side effects. In general, BBB permeability increases with increasing lipophilicity because the passive entry of molecules into the brain increases. However, a number of highly lipophilic drugs demonstrate unexpectedly poor BBB penetration because they are effluxed by P-gp. An approach to increase lipophilicity is not always useful as far as improving brain penetration is concerned. Brain penetration of drugs that pharmacologically target the CNS could, therefore, be improved by modifying the drug so that it is not recognized by P-gp (Doan et al., 2002). Whether poor brain penetration of a drug is due to poor membrane permeability or active efflux should be investigated in detail at an early stage of drug development. The expression system of transporters is an efficient tool for screening transport activities. Human transporter gene transfected cells are especially important tools because human experimental systems are rather limited. Recent studies show that the transport activity of MDR1-transfected cells correlates well with the P-gp transport activity in vivo (Adachi et al., 2001; Yamazaki et al., 2001).
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However, reducing brain penetration by efflux transporters is expected to enable the adverse CNS side effects to be avoided in some drugs with toxic CNS effects. Some fluoroquinolone antibacterials or anticancer agents exhibit low brain distribution, despite having a high lipophilicity, because they are prevented from entering the brain by P-gp (Tamai and Tsuji, 2000). This probably explains their relative lack of CNS side effects. The brain uptake of ivermectin is markedly increased (27-fold) in mdr1a knockout mice (Table 4). Mdr1a knockout mice are much more sensitive (100-fold) than normal mice to the neurotoxic effects caused by ivermectin (Schinkel et al., 1994). Drug design, which ensures that the compounds interact with brain efflux transporter, may be a successful way to avoid the CNS toxicity of drug candidates that exhibit such an effect. However, a drug-drug interaction involving P-gp or a genetic polymorphism of MDR1 may change the brain penetration of drugs and may affect safety if the drug candidate is a P-gp substrate. Thus, it is important to select a lead compound that has no intrinsic CNS toxic potential rather than targeting the brain efflux transporter. Drug-drug interactions via brain P-gp between loperamide, a substrate for P-gp, and quinidine, an inhibitor of P-gp, have been reported (Sadeque et al., 2000). Although the antidiarrheal agent loperamide is a potent opiate, it does not produce opioid CNS effects at usual doses in patients. When a 16-mg dose of loperamide alone was administered to eight healthy volunteers, loperamide produced no respiratory depression. However, respiratory depression occurred when loperamide (16 mg) was given with quinidine at a dose of 600 mg. These changes were not explained by increased plasma loperamide concentrations. Thus, inhibition of P-gp by quinidine increases the entry of loperamide into the CNS with resulting opiate-induced respiratory depression. The lack of respiratory depression produced by loperamide, which allows it to be safely used therapeutically, can be reversed by a drug-drug interaction mediated by P-gp, resulting in serious toxic and abuse potential.
In addition, some organic anion-transporting polypeptides (OATPs) are expressed in the brain. Many members of this transporter family mediate transport of a wide spectrum of amphipathic organic anions (Hagenbuch and Meier, 2003). Rat Oatp2 (Slc21a5) is localized on both the luminal and abluminal membranes of the rat BBB (Gao et al., 1999). In humans, immunohistochemical staining of brain tissue suggested that OATP-A is expressed in the brain endothelial cells, although its localization has not been confirmed (Gao et al., 2000). Because Oatp2 can mediate bidirectional transport, involvement of Oatp2 in the efflux transport across the BBB is possible (Asaba et al., 2000; Sugiyama et al., 2001). The recently characterized human OATP-F is a high-affinity thyroxine transporter and is selectively expressed in brain (Pizzagalli et al., 2002). OATP-F is possibly involved in the uptake of thyroxin from the blood into the CNS. Moreover, OAT3, OCT3, and OCTN2 appear to be expressed in the brain (Wu et al., 1998; Kusuhara et al., 1999; Wu et al., 1999; Cha et al., 2001; Ohtsuki et al., 2002). Their localization in the brain and physiological function remain to be elucidated. It is essential to identify most of the important transporters in the brain and to characterize their function to provide a basis for developing strategies to regulate drug disposition in the brain.
C. Role of Transporters in Drug Absorption
Various transporters are expressed in the brush-border membranes of intestinal epithelial cells (Table 1). They are involved in the efficient absorption of nutrients or endogenous compounds. The use of influx transporters expressed in the gut, such as PEPT1, ASBT, OATP-B, OATP-D, OATP-E, or rat Oatp3 (Slc21a7), will help improve drug absorption (Tsuji and Tamai, 1996; Oh et al., 1999; Tamai et al., 2000a; Walters et al., 2000; Kullak-Ublick et al., 2001; Lee et al., 2001b). Rat Oatp3, but not Oatp1 (Slc21a1) or Oatp2, is expressed in the small intestine, and is localized on the apical brush-border membrane of enterocytes (Walters et al., 2000). Because Oatp3 transports taurocholate, rat oatp3 is suggested to mediate the absorption of bile acids. PEPT1 mediates the transport of peptide-like drugs such as 
-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors and the dipeptide-like anticancer drug bestatin (Hori et al., 1993; Saito and Inui, 1995; Swaan et al., 1995; Terada et al., 1997; Inui et al., 2000b). Interestingly, valacyclovir, a valyl ester prodrug of the antiviral agent acyclovir, although it does not contain a peptide bond, is transported by PEPT1 (Balimane et al., 1998; Sawada et al., 1999). From the viewpoint of drug delivery, L-valyl esterification of poorly absorbed drugs has been suggested as a useful strategy for improving their bioavailability and therapeutic efficacy.
However, primary active efflux transporters, such as P-gp, MRP2, or BCRP, are expressed on the brush-border membrane of enterocytes (Table 1) and excrete their substrates into the lumen, resulting in a potential limitation of net absorption (Gotoh et al., 2000; Hirohashi et al., 2000a; Jonker et al., 2000; Taipalensuu et al., 2001). Active secretion of absorbed drug is now becoming recognized as a significant factor in oral drug bioavailability (Wacher et al., 2001; Zhang and Benet, 2001). P-gp contributes to the absorption of many drugs because of its broad substrate specificity (Borst et al., 1999; Fromm, 2000; Troutman et al., 2001). The intestinal P-gp content correlates with the AUC after oral administration of digoxin, a P-gp substrate, in humans (Greiner et al., 1999). This result suggests that P-gp in the epithelium of the gut wall determines the plasma concentration of orally administered digoxin. Another report involving a patient undergoing a small bowel transplant has also demonstrated that the plasma concentration of orally administered tacrolimus, a substrate of both P-gp and CYP3A4, correlated well with the mRNA expression of intestinal MDR1, but not CYP3A4 (Masuda et al., 2000). These results suggest that intestinal P-gp, rather than CYP3A4, is a good probe to predict intraindividual variations in tacrolimus pharmacokinetics. Furthermore, high levels of MDR1 are strongly associated with reductions in survival rates after living-donor liver transplantation and subsequent immunosuppressive therapy with tacrolimus (Hashida et al., 2001). Intestinal MDR1 is also a powerful prognostic indicator of living-donor liver transplantation outcomes.
BCRP is a multidrug-resistance protein that is a new member of the ATP-binding cassette transporter family (Doyle et al., 1998). BCRP has only one ATP-binding cassette and six putative transmembrane domains (Rocchi et al., 2000), suggesting that BCRP is a half-transporter, which may function as a homo- or heterodimer. BCRP plays a role in the secretion of clinically important drugs such as topotecan (Jonker et al., 2000). When both topotecan, a substrate of BCRP, and GF120918, an inhibitor of both BCRP and P-gp, were administered orally, the bioavailability of topotecan was increased in P-gp-deficient mice (over 6-fold) compared with mice given vehicle alone (Jonker et al., 2000) (Table 5). Thus, BCRP appears to be a major determinant of the bioavailability of topotecan following oral administration. Because GF120918 inhibits both P-gp and BCRP, P-gp-deficient mice have been used to exclude any confounding effects of P-gp inhibition. Topotecan is a weak-to-moderate substrate of P-gp, thus P-gp also appears to play a role in the bioavailability of topotecan. BCRP is expressed not only in the intestine, but also in the bile canalicular membrane and placenta (Maliepaard et al., 2001). Thus, treatment with GF120918 reduced the plasma clearance and hepatobiliary excretion of topotecan (Table 5). Furthermore, in pregnant GF120918-treated, P-gp-deficient mice, the fetal penetration of topotecan was 2-fold higher than that in pregnant mice given vehicle alone (Maliepaard et al., 2001). These results indicate that BCRP plays an important role in protecting the fetus from topotecan. The bioavailability of topotecan in humans is moderate, with a high interpatient variability (30 ± 8%) (Schellens et al., 1996). By combining oral topotecan with an effective BCRP inhibitor, the bioavailability of topotecan might be markedly improved and the interindividual variability might be reduced (de Bruin et al., 1999). Although GF120918 inhibits not only BCRP but also P-gp, fumitremorgin C, an extract of Aspergillus fumigatus, has been shown to potently inhibit BCRP but not P-gp or MRP, suggesting that fumitremorgin C is a highly selective inhibitor of BCRP (Rabindran et al., 1998; Ozvegy et al., 2001). So, the strategic application of BCRP inhibitors may lead to more effective oral chemotherapy with topotecan or other drugs that are BCRP substrates.
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In principle, the inhibition of intestinal efflux transporters is a useful way to improve the oral bioavailability of a coadministered drug (Sikic et al., 2000). It has been shown that treatment with water-soluble vitamin E (d-
-tocopheryl polyethylene glycol 1000 succinate [TPGS]) enhances the absorption of cyclosporine in healthy volunteers or liver transplant recipients (Sokol et al., 1991; Chang et al., 1996). Another report has demonstrated that TPGS also increased the solubility of amprenavir, an HIV protease inhibitor, and inhibited the efflux transport systems and enhanced the permeability of amprenavir through Caco-2 cell monolayers (Yu et al., 1999). Overall, TPGS enhances the absorption flux of amprenavir by increasing its solubility and permeability. This improvement is very significant since the bioavailability of amprenavir in conventional capsule formulations is almost zero, and the softgel formulation containing vitamin E-TPGS is 69% bioavailable for dogs (Yu et al., 1999). Surfactants, such as Cremophor EL or Tween 80, have been found to be potent inhibitors of P-gp (Lo et al., 1998; van Zuylen et al., 2001). Both are used as formulation vehicles for a variety of poorly water-soluble drugs, including the anticancer agents paclitaxel and docetaxel. The use of these surfactants may increase the intestinal absorption of some drugs through P-gp inhibition and, thus, improve the drug bioavailability of P-gp substrates.
Inhibition studies using P-gp inhibitors in Caco-2 cell monolayers are simple to perform and are widely used to evaluate the contribution of P-gp to the absorption of a drug candidate. However, there are few studies describing any quantitative investigations or the theoretical aspects involved. Table 6 shows the effect of P-gp inhibitors on the apical-to-basal or basal-to-apical flux of P-gp substrates across Caco-2 cell monolayers. As a result, the changes in the flux of P-gp substrates can be classified into three types (Fig. 1). In the first type, the basal-to-apical flux scarcely changes and the apical-to-basal flux increases markedly in the presence of a P-gp inhibitor (Fig. 1A). In the second case, both fluxes are changed but the degree of change in the apical-to-basal flux is greater than that in the basal-to-apical flux in the presence of a P-gp inhibitor (Fig. 1B). In the third case, both fluxes are changed but the degree of change in the basal-to-apical flux is greater than that in the apical-to-basal flux in the presence of a P-gp inhibitor (Fig. 1C). An example of the first case is grepafloxacin, the second case is illustrated by saquinavir and indinavir, while examples of the third case include Rhodamine 123, cyclosporine, vinblastine, and digoxin (Table 6). Figure 2 shows a schematic diagram illustrating the transcellular transport of P-gp substrates in Caco-2 cell monolayers. PS1 and PS2 represent the permeability-surface area (PS) products for the influx and non-P-gp-mediated efflux across the apical membrane of Caco-2 cell monolayers, respectively; PS3 and PS4 represent the PS products for the efflux and influx across the basal membrane of Caco-2 cell monolayers, respectively; and PSP-gp represents the PS product for P-gp-mediated efflux across the apical membrane. CLA-B and CLB-A represent the steady-state transport clearances in the apical-to-basal direction and the basal-to-apical direction, respectively. Supposing a steady-state flux (constant velocity of transcellular transport) and "sink" conditions (constant concentration gradients), CLA-B and CLB-A are given by eqs. 1 and 2 of Fig. 2, respectively. The flux ratio across the monolayer (Rcaco), defined as the ratio of CLA-B to CLB-A, is given by eq. 3 of Fig. 2. The degree of change in CLA-B and CLB-A, when P-gp in completely inhibited by P-gp inhibitor, is given by eqs. 4 and 5 of Fig. 2, respectively. CLA-B(+I) and CLB-A(+I) represent the clearances when P-gp is completely inhibited by a P-gp inhibitor. The experimental data in Table 6 can be interpreted using eqs. 3 to 5 of Fig. 2, where it has been estimated that PS2 >> PS3 in the first case (Fig. 1A), PS2 
PS3 in the second case (Fig. 1B), and PS2 << PS3 in the third case (Fig. 1C). Furthermore, it has been estimated that the value of PSP-gp is 2-fold greater in the case of grepafloxacin, 6- to 8-fold greater in the case of saquinavir and indinavir, and 6- to 21-fold greater in case of Rhodamine 123, cyclosporine, vinblastine, and digoxin compared with the non-P-gp-mediated efflux clearance (PS2). In some cases, it has been found that the basal-to-apical flux is still greater than the apical-to-basal flux with P-gp inhibitors. The reason for this phenomenon may be not only that the inhibitor concentration is insufficient, but also that the efflux transporters on the apical membrane, other than P-gp, also play a role in the efflux of these drugs.
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D. Control of Elimination by Drug Transporters (Uptake and Efflux Transporters in the Liver and Kidney)
Multispecific transporters are expressed in the liver and kidney and play an important role in the elimination of many xenobiotics, acting as a detoxification system. Many drugs are excreted into the urine via organic anion and cation transport systems, expressed on brush-border and basolateral membranes of renal tubular cells (Table 1) (Burckhardt and Wolff, 2000; Inui et al., 2000a; Sekine et al., 2000; van Aubel et al., 2000; Dresser et al., 2001; Masereeuw and Russel, 2001; Russel et al., 2002). As far as the liver is concerned, a wide variety of transporter families are known to be present at the sinusoidal and canalicular membranes and play a significant role in hepatobiliary excretion (Table 1) (Oude Elferink et al., 1995; Yamazaki et al., 1996; Muller and Jansen, 1997; Keppler and Konig, 2000; Kullak-Ublick et al., 2000; Faber et al., 2003). Secondary active transporters expressed on the sinusoidal membrane are responsible for the uptake of drugs from the blood into hepatocytes (Meier et al., 1997). Primary active transporters expressed on the canalicular membrane are involved in the biliary excretion of both parent drugs and their metabolites (Kusuhara et al., 1998; Hooiveld et al., 2001). Since some transporters are specifically expressed on hepatocytes or renal tubular cells, they can be used as a target for drug delivery to the liver or kidney, possibly resulting in direct control of the elimination process. The transporters expressed in the liver and kidney are introduced here.
1. Organic Anion Transporting Polypeptide (SLC21A) Family.
Organic anion transporting polypeptides (OATPs) have been isolated from rats, at first as candidates for the sodium-independent uptake system in the liver (Meier et al., 1997; Muller and Jansen, 1997). OATPs form a growing gene superfamily and mediate transport of a wide spectrum of amphipathic organic anions such as bromosulfophthalein, estradiol-17-glucuronide (E2-17G), bile acids, thyroid hormones, and drugs such as pravastatin, temocaprilat, and BQ-123 (Meier et al., 1997; Muller and Jansen, 1997; Ishizuka et al., 1998; Kakyo et al., 1999; Reichel et al., 1999; Abu-Zahra et al., 2000). Although some important members of this transporter superfamily are selectively expressed in rodent and human livers, most OATPs are expressed in multiple tissues including the BBB, choroid plexus, lung, heart, intestine, kidney, placenta, and testes (Hagenbuch and Meier, 2003). A human OATP-C (also referred to as OATP2 and LST-1) is predominantly expressed in the liver (Abe et al., 1999; Hsiang et al., 1999; Konig et al., 2000a; Tamai et al., 2000a). Due to its broad substrate specificity, OATP-C is considered to play a major role in the hepatic uptake of organic anions. Recently, Cui et al. demonstrated that OATP-C transports bilirubin and its mono- and diglucuronide, suggesting that OATP-C is important from a physiological point of view (Cui et al., 2001b). In addition to OATP-C, OATP-B and OATP8 are also localized on the sinusoidal membrane of hepatocytes (Table 1) (Konig et al., 2000b; Kullak-Ublick et al., 2001). The tissue distribution of OATP-B is much broader than that of liver specific OATP-C. Although the expression of OATP-B is most abundant in human liver, it is also present in the pancreas, lung, gut, ovary, testes, and spleen. (Tamai et al., 2000a; Kullak-Ublick et al., 2001; St-Pierre et al., 2002). OATP-B transports sulfate conjugates of steroids, but not glucuronide conjugates and bile salts, whereas OATP-C transports both types of steroid conjugates (Kullak-Ublick et al., 2001; Tamai et al., 2001). OATP8 is exclusively expressed on the basolateral membrane of hepatocytes (Konig et al., 2000b). Although OATP-C and OATP8 exhibit broad overlapping substrate specificities, OATP8 is unique in transporting digoxin and exhibits an especially high transport activity for anionic peptides [D-penicillamine(2,5)] enkephalin (opioid-receptor agonist), BQ-123 (endothelin-receptor antagonist), and cholecystokinin-8 (gastrointestinal peptide hormone 8) (Ismair et al., 2001; Kullak-Ublick et al., 2001). The bile salts, substrates for OATP-C, are reportedly not transported by OATP8 (Konig et al., 2000b). Because OATP-C, OATP-B, and OATP8 are localized on the same membrane domain with overlapping substrate specificity, the contribution of OATP-C, OATP-B, and OATP8 to the total hepatic uptake of each ligand needs to be clarified.
2. Organic Anion Transporter (SLC22A) Family.
OAT1 and OAT3 are mainly expressed in the kidney and localized on the basolateral membrane of the proximal tubules (Table 1) (Sekine et al., 1997; Hosoyamada et al., 1999; Kusuhara et al., 1999; Sekine et al., 2000; van Aubel et al., 2000; Dresser et al., 2001; Kojima et al., 2002). Their substrates include relatively small and hydrophilic organic anions, such as p-aminohippurate (PAH), methotrexate, -lactam antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and antiviral nucleoside analogs (Uwai et al., 1998; Apiwattanakul et al., 1999; Cihlar et al., 1999; Jariyawat et al., 1999; Wada et al., 2000). Recently, Oat3 knockout mice have been developed, and Oat3-/- mice exhibit impaired organic anion transport function in renal and choroid plexus epithelia but not in the liver (Sweet et al., 2002). This indicates that Oat3 plays an essential role in renal, but not hepatic, organic anion uptake.
Generally, amphipathic organic anions with a relatively high molecular weight, such as OATP substrates, are eliminated from the liver by metabolism and/or biliary excretion, while small and hydrophilic organic anions, such as OAT substrates, are excreted into the urine. The tissue distribution and elimination pathways of drugs can be explained by similarities and differences in the substrate recognition by these transporters expressed in the liver and kidney. Thus, modifying the drug so that it is recognized by OATP or OAT may lead to liver or kidney organotropism. Although, in general, OAT families are mainly expressed in the kidney, OAT2 is abundantly expressed in the liver and, to a lesser extent, in the kidney, and localized to the basolateral membrane of the liver (Simonson et al., 1994; Sekine et al., 1998). OAT2 transports relatively small and hydrophilic organic anions, such as indomethacin and salicylate, and may be involved in the hepatic uptake of these drugs (Morita et al., 2001). However, the OATP family is supposed to be responsible for the hepatic uptake of amphipathic organic anions.
The reported toxicity of some drugs is occasionally caused by concentrative tissue distribution due to active transport. The OAT1-mediated transport of ochratoxin A, a potent nephrotoxin, has been reported, suggesting that accumulation of the toxin via OAT1 in proximal tubules may be the primary event in the development of ochratoxin A-induced nephrotoxicity (Tsuda et al., 1999; Jung et al., 2001b). A similar effect has been proposed for adefovir, cephalosporin antibiotics, and -lactam antibiotics, which accumulate extensively in the tubules (Cihlar et al., 1999; Jariyawat et al., 1999; Takeda et al., 1999, 2002a). Active transport processes may increase the intracellular concentration and appear to be directly related the development of tubular injury. Thus, designing a drug that is not transported by OAT1 or coadministering OAT1 inhibitors may be an effective way of reducing the nephrotoxicity of these compounds (Cihlar et al., 2001). A fluorescence assay to screen for novel human OAT1 inhibitors has been developed (Cihlar and Ho, 2000) and it has been suggested that NSAIDs may reduce adefovir nephrotoxicity since they efficiently inhibit the human OAT1-specific transport of adefovir at clinically relevant concentrations (Apiwattanakul et al., 1999; Mulato et al., 2000).
3. Organic Cation Transporter (SLC22A) Family.
The OCT family of proteins is involved in the uptake of organic cations into the liver or kidney from blood. OCT1 and OCT2 are expressed in epithelial cells of the kidney, liver, and intestine, and appear to be localized to the basolateral membranes of the cells (Table 1) (Meyer-Wentrup et al., 1998; Urakami et al., 1998). These transporters mediate the uptake of a variety of organic cations, such as dopamine, choline, 1-methyl-4-phenylpyridinium (MPP+), N1-methylnicotinamide, TEA, and cimetidine (Martel et al., 1996; Zhang et al., 1997; Breidert et al., 1998; Koepsell, 1998; Urakami et al., 1998; Zhang et al., 1998). Rat Oct1 is expressed in both the liver and kidney, although its human counterpart is expressed predominantly in the liver, while human and rat Oct2 are present mainly in kidney and brain (Grundemann et al., 1994; Gorboulev et al., 1997). Rat Oct3 mRNA has been found to be most abundant in the placenta, with a moderate presence in the intestine, heart, and brain (Kekuda et al., 1998).
Recently, the pharmacological and physiologic role of Oct1 has been investigated using Oct1 knockout (Oct1-/-) mice (Jonker et al., 2001). The distribution and excretion of the model substrate TEA after intravenous administration has been compared in wild-type and Oct1-/- mice. In Oct1-/- mice, accumulation of TEA in liver was 4- to 6-fold lower than in wild-type mice, indicating that for TEA, Oct1 is the main uptake system in the liver (Table 7). In addition, direct intestinal excretion of TEA was reduced about 2-fold, showing that Oct1 also mediates basolateral uptake of TEA into enterocytes (Table 7). Excretion of TEA into urine over 1 h accounted for 53% of the dose in wild-type mice compared with 80% in knockout mice, probably because in Oct1-/- mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney (Table 7).
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Similarly, the distribution of metformin, a biguanide, to the liver and intestine in Oct1-/- mice was significantly lower than that in wild-type mice, whereas distribution to the kidney and the urinary excretion profile showed only minimal differences (Wang et al., 2002). Oct1 is responsible for hepatic uptake as well as playing a role in the intestinal uptake (via basolateral membrane) of metformin, while the renal distribution and excretion are mainly governed by other transport mechanisms. Biguanides are oral antihyperglycemic agents used for the treatment of type 2 diabetes mellitus, but they are associated with lactic acidosis, a potentially fatal side effect. Following the administration of metformin, the blood lactate concentration significantly increased in wild-type mice, whereas only a slight increase was observed in Oct1-/- mice (Wang et al., 2003). The hepatic concentration of metformin in Oct1-/- mice was markedly reduced, whereas its plasma concentration time profile was similar in wild-type and Oct1-/- mice. These results indicate that the Oct1-mediated hepatic uptake of biguanides plays an important role in lactic acidosis.
4. Multidrug Resistance-Associated Protein 2 (ABCC2).
MRP2, located on the bile canalicular membrane, is involved in the biliary excretion of clinically important anionic drugs as well as the intracellularly formed glucuronide- and glutathione-conjugates of many drugs (Paulusma et al., 1996; Ito et al., 1997; Keppler et al., 1997; Ito et al., 1998b; Konig et al., 1999a). In the liver, xenobiotics are metabolized by the so-called phase I and II enzymes, which are mainly cytochrome P450 and conjugating enzymes, respectively. After these enzymatic reactions, the conjugated metabolites produced are pumped out from hepatocytes into the bile. This ATP-dependent efflux transporter plays a physiologically important role as the "phase III" xenobiotic detoxification system (Ishikawa, 1992). In addition, MRP2 mediates the biliary excretion of not only conjugated metabolites, but also unchanged organic anions, such as grepafloxacin (a new fluoroquinolone antibiotics) or cefodizime and ceftriaxone (-lactam antibiotics) (Sathirakul et al., 1993; Kusuhara et al., 1998; Sasabe et al., 1998). These antibiotics have been shown to be effective in the treatment of inflammatory conditions affecting the biliary tract because they are efficiently excreted into the bile (Suzuki and Sugiyama, 1999). The biliary excretion of these antibiotics gives these drugs a pharmacological advantage due to the target organ.
However, in some cases there is a major accumulation of drugs in the bile duct via MRP2 expressed on the bile canalicular membrane, which results in toxic effects on bile epithelial cells or gastrointestinal cells. It is supposed that the reactive glucuronide of the NSAID diclofenac is selectively transported into bile via MRP2, where it exhibits toxic effects on the bile canalicular membrane (Seitz et al., 1998). Similarly, methotrexate is concentrated in bile compared with plasma and undergoes enterohepatic circulation, resulting in adverse effects in the intestine. It has been reported that the biliary excretion of methotrexate is mediated by MRP2 (Masuda et al., 1997). A structural modification of such drugs to reduce their biliary excretion would be useful.
Although CPT-11 is an effective anticancer drug, its clinical use is frequently limited by a form of gastrointestinal toxicity, severe diarrhea (Rowinsky et al., 1994). Such severe diarrhea exhibits a large degree of interpatient variability. The action of its active metabolite, SN-38, on gastrointestinal cells is believed to be responsible for this toxicity (Araki et al., 1993). The biliary excretion of SN-38 and SN-38 glucuronide and subsequent uptake by gastrointestinal epithelial cells may be associated with this diarrhea (Kaneda and Yokokura, 1990). It has been shown that MRP2 is involved in the biliary excretion of SN-38 and SN-38 glucuronide (Chu et al., 1997a,b), and there is a large degree of interindividual variability in the transport activity of SN-38 via MRP2, as shown by a study using human bile canalicular membrane vesicles (CMVs) (Chu et al., 1998). Thus, the biliary excretion of its metabolites mediated by MRP2 has been proposed to be linked to its unpredictable gastrointestinal toxicity. An attempt to prevent this toxicity using potent MRP2 inhibitors has been investigated. Probenecid is a potential candidate, which can be used clinically to inhibit the biliary excretion of CPT-11 metabolites (Horikawa et al., 2002b). In actual fact, it has been shown that coadministration of probenecid markedly reduces both SN-38 exposure and CPT-11-induced late-onset toxicity in the gastrointestinal tissues of rats, possibly by inhibiting the biliary excretion of CPT-11 and/or its metabolites (Horikawa et al., 2002a). It is expected that this agent will be used clinically to prevent toxicity. Approaches using intentional drug-drug interactions (positive drug interactions) like this case may become more important in the future.
Control of the elimination route, such as biliary or urinary excretion, is also one of the strategies used to avoid potentially toxic effects. In some cases, transporters expressed in the liver or kidney may determine the elimination route affecting the systemic plasma concentrations of drugs. Many ACE inhibitors are actually administered as prodrugs and are metabolized to their active forms, such as enalaprilat, captoprilat, cilazaprilat, ramiprilat, and spirapprilat. They are excreted predominantly into the urine. In contrast, temocaprilat is excreted via both bile and urine (Oguchi et al., 1993). The presence of an excretion route other than the urinary one confers a pharmacokinetic advantage, particularly in the treatment of patients with renal failure. In such patients, the AUC and Cmax values of captopril and enalapril are markedly increased because these ACE inhibitors are eliminated primarily via renal excretion (Oguchi et al., 1993). In contrast, alterations in these pharmacokinetic parameters are minimal for temocaprilat because of the presence of the biliary excretion pathway (Oguchi et al., 1993). A multiple elimination pathway will result in a relatively stable pharmacokinetic profile compared with only a single elimination pathway. Although the biliary excretion of temocaprilat is governed by MRP2 at the canalicular membrane, it has been suggested that other ACE inhibitors are not good substrates of MRP2 (Ishizuka et al., 1997, 1999). The affinity for MRP2 is expected to be the predominant factor in determining the biliary excretion of any series of ACE inhibitors. Drugs that are excreted into both the bile and urine to the same degree may be expected to exhibit minimal interindividual differences in their pharmacokinetics.
Mrp2-deficient rats, such as transport-deficient rats and Eisai hyperbilirubinemic rats, exhibit hyperbilirubinemia such as the Dubin-Johnson syndrome due to a defect in the biliary excretion of bilirubin glucuronides. Mrp3 is induced on the hepatic basolateral membrane of Mrp2-deficient animals (Hirohashi et al., 1998; Donner and Keppler, 2001; Soroka et al., 2001) and is able to excrete glucuronide conjugates of xenobiotics (Hirohashi et al., 1999, 2000b). Thus, these results are consistent with the hypothesis that Mrp3 may be involved in the sinusoidal efflux of glucuronide conjugates in these mutants. It is plausible that in the cholestatic liver, bilirubin glucuronides are effluxed from the liver into the blood via sinusoidal Mrp3, resulting in jaundice. Moreover, immunohistochemical studies have indicated that MRP3 is induced in the sinusoidal membrane of patients suffering from Dubin-Johnson syndrome (Konig et al., 1999b).
5. Bile Salt Export Pump (ABCB11).
Intrahepatic cholestasis can be induced by interference with the secretion of biliary constituents resulting in an intracellular accumulation of bile salts and other toxic bile constituents within hepatocytes. BSEP, located on the canalicular membrane, mediates the transport of bile acids such as taurocholic acid (Gerloff et al., 1998; Kullak-Ublick et al., 2000). Cholestasis induced by some drugs is mediated, at least in part, by inhibition of BSEP, resulting in intracellular accumulation of cytotoxic bile salts. The immunosuppressant, cyclosporine, has been shown to produce cis-inhibition of BSEP-mediated bile salt transport (Stieger et al., 2000). A similar mechanism has been postulated for rifampicin and glibenclamide (Stieger et al., 2000). In contrast, the cholestatic estrogen metabolite, E2-17G, causes trans-inhibition of BSEP-mediated bile salt transport and, therefore, exerts its cholestatic action only after its excretion by MRP2 into the canalicular lumen (Stieger et al., 2000). In addition, some other MRP2 substrates cause trans- inhibition of the BSEP-mediated transport of bile acids (Akita et al., 2001). Horikawa et al. have reported the inhibition potential of a series of therapeutic drugs, producing clinical cholestasis, on BSEP and MRP2 (Horikawa et al., 2003). Although most of the drugs have only a minimal inhibitory effect on Bsep- and Mrp2-mediated transport in rat CMVs, cloxacillin inhibited BSEP-mediated transport in both rat and human CMVs. Since the inhibitory effect on BSEP-mediated transport by cloxacillin was more marked in human CMVs than in rat CMVs, species differences in inhibitory potential need to be considered (Horikawa et al., 2003). Troglitazone is a thiazolidinedione insulin-sensitizing agent for the treatment of noninsulin-dependent diabetes mellitus, but it was withdrawn from the market because of liver toxicity (Funk et al., 2001a). Although the mechanism underlying this troglitazone-associated hepatotoxicity is at present unclear, it has been suggested that a cholestatic mechanism is involved (Funk et al., 2001a). Troglitazone and, to a much greater extent troglitazone sulfate, the main troglitazone metabolite eliminated into bile, competitively inhibit ATP-dependent taurocholate transport via BSEP (Funk et al., 2001a,b). This inhibition of the hepatobiliary export of bile salts by troglitazone and troglitazone sulfate may lead to a drug-induced intrahepatic cholestasis in humans, possibly contributing to the hepatotoxicity of troglitazone. One should consider the possibility that drugs which inhibit BSEP may cause cholestasis. The evaluation of BSEP inhibition will play an important role in the identification of compounds that could be a potential cause of cholestasis.
Obtaining more data on the substrate specificities and expression level of each human transporter will be of great help in improving drug design by targeting specific transporters and controlling their elimination. The route of elimination may be controlled by using transporters that are expressed selectively in either the liver or kidney.
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III. Clinical Implications of Transporter-Mediated Drug Interactions |
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Drug-drug interactions involving membrane transport can be classified into two categories: one caused by competition for the substrate binding sites of the transporters, and the other caused by a change in the expression level of the transporters. Due to the broad substrate specificity of P-gp, drug-drug interactions involving P-gp are very likely (Lin, 2003). Table 8 gives an overview of the known drug interactions that involve, at least in part, P-gp. This gives an indication of the interactions that one may expect during combination therapy. P-gp inhibitors, such as quinidine, valspodar, and verapamil, are known to increase plasma concentrations of digoxin, a cardiac glycoside, because they block its biliary and/or urinary excretion via P-gp (Table 8) (Hedman et al., 1990, 1991; Kovarik et al., 1999). Since the therapeutic range of digoxin is small, changes in its plasma
