Scientific background

The Role Of ABC Transporters In Drug Resistance, Metabolism And Toxicity

Mini-Review
Hristos Glavinas¹, P�ter Krajcsi^1,2, Judit Cserepes³, and Bal�zs Sarkadi⁴

¹ SOLVO Biotechnology, Szeged, Hungary
² Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
³ Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
⁴ National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Budapest, Hungary

CONTENTS

Abstract

5. Abreviations

6. Bibliography

Abstract

ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disrorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.

1. General overview of ABC transporters

1.1 The ABC Transporter Family

ABC (ATP-binding cassette) proteins form one of the largest protein families, and members of this family are found in all living organisms from microbes to humans. The wild-spread presence of these proteins with a relatively conserved structure and function suggests a fundamental role. In the present review we focus on the expression, structure and function of ABC transporters present in humans and in related model organisms. The basic structure that defines the members of this protein family is the combination of conserved ATP-binding and transmembrane domains. In mammals the functionally active ABC proteins consist of four characteristic domains, two transmembrane domains (TMDs), and two nucleotide-binding domains (NBDs). These four domains may be present within one polypeptide chain ("full transporters"), or within two separate proteins ("half transporters"). In this latter case the ABC transporters become functionally competent after specific dimerization (for reviews see 1-11). The membrane-spanning domains form the key structural background of the function of ABC transporters. The two TMDs contain polypeptide chains that span the membrane multiple times, typically forming six transmembrane α-helices per domain, a total of twelve helices in a full transporter (7, 10). The twelve transmembrane α-helices probably form a pore-like structure across the membrane, and through this path a range of different substrates can be transported by these proteins. The conformational changes within the TMD domains are believed to be responsible for the opened or closed states of these transmembrane structures. ABC proteins can utilize the energy derived from ATP hydrolysis to perform a directed transmembrane movement of their substrates (primary active transporters), open or close a specific membrane channel (e.g. ion-channels), or regulate the permeability of multi-protein channel complexes (receptors). In the ABC proteins which act as primary active transporters, the transport function depends on the hydrolysis of ATP within the NBDs. These cytoplasmic domains are attached to the intracellular regions of the TMDs, and a close interaction provides the functional connection between these two different domains. The nucleotide-binding domains bind cytoplasmic ATP and, in the active transporters, ATP hydrolysis ensures the energy for the uphill transport of a substrate. The specific, close interaction of NBDs with the TMDs provide the transmission gear of the conformational changes caused by substrate binding and the hydrolysis of ATP (8-10). During the sequencing of the human genome the full range of the human ABC genes has been described and characterized. Table I. presents the full list of the 48 human ABC genes (9), including some of their basic features. After some confusion in naming and numbering of the newly identified ABC transporters, a consistent nomenclature has been introduced, based on the sequence homology between these proteins. In this system the ABC genes are grouped into seven subfamilies, based on the similarity in the gene structure, order of the domains, and sequence homology in the NBDs and TMDs (see: http://nutrigene.4t.com/humanabc.htm, 5, 9). In the following sections we provide a general introduction for these families. We also describe examples for the expression and function of some key family members, especially for those with an already recognized medical or pharmacological importance.

Table I. Human ABC transporters and their basic features

Family	Member	Alias	Expression	Function
ABCA	ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA11 ABCA12	ABC1 ABC2 ABC3,ABCC ABCR	Ubiquitous Brain Lung Rod photoreceptors Muscle, heart, testes Liver Spleen, thymus Ovary Heart Muscle, heart Stomach Low in all tissues	Removal of cholesterol and PLs onto HDL particles Drug resistance Surfactant protection N-retinydilester-PE efflux
ABCB	ABCB1 ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11	MDR1,PGP TAP1 TAP2 PGP3,MDR3 MTABC3 ABC7 MABC1 MTABC2 SPGP,BSEP	Adrenal,kidney,brain Ubiquitous,ER Ubiquitous,ER Liver Ubiquitous Mitochondria Mitochondria Mitochondria Heart,brain Mitochondria Liver	Multidrug resistance Peptide transport into the ER Peptide transport into the ER Phosphatidylcholine transport Iron transport Heme transport Heme transport Bile salt transport
ABCC	ABCC1 ABCC2 ABCC3 ABCC4 ABCC5 ABCC6 ABCC7 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12	MRP1 MRP2, cMOAT MRP3, cMOAT-2 MRP4, MOAT-B MRP5, MOAT-C MRP6 CFTR SUR SUR2 MRP7 MRP8 MRP9	Ubiquitous Liver Lung, intestine, liver Prostate Ubiquitous Kidney, liver Exocrine tissues Pancreas Heart, muscle Low in all tissues Low in all tissues Low in all tissues	Drug resistance Organic anion transport Drug resistance Nucleoside transport Nucleoside transport Chloride ion transport Sulfonylurea receptor
ABCD	ABCD1 ABCD2 ABCD3 ABCD4	ALD ALD1,ALDR PMP70, PXMP1 PMP69, P70R	Peroxisomes Peroxisomes Peroxisomes Peroxisomes	VLCFA transport regulation
ABCE	ABCE1	OABP	Ovary, testes, spleen	Oligoadenylate-binding protein
ABCF	ABCF1 ABCF2 ABCF3	ABC50	Ubiquitous Ubiquitous Ubiquitous
ABCG	ABCG1 ABCG2 ABCG4 ABCG5 ABCG8	ABC8, Human white ABCP, MXR, BCRP White2 Steroline 1 Steroline 2	Ubiquitous Placenta, intestine Liver Liver, intestine Liver, intestine	Cholesterol transport Drug resistance Sterol transport Sterol transport

HDL: high density lipoprotein
VLCFA: very long chain fatty acid

1.1.1 The ABCA subfamily

This subfamily comprises of 12 full transporters; some of the largest ABC proteins belong to this family. This subfamily can be divided into two subgroups, based on a phylogenetic analysis (12). One subgroup comprises five proteins (ABCA5-6 and ABCA8-10), the genes encoding these transporters are clustered on chromosome 17q24. The other subgroup consists of seven proteins (ABCA1-4, A7, A12-13), encoded by genes in different chromosomes.

The ABCA1 protein is relatively well characterized, and suggested to be involved in the removal of cholesterol and phospholipids from cells onto high-density lipoproteins (HDL) particles (13). Different mutations in the ABCA1 gene cause a well defined clinical syndrome, Tangier disease. Patients with this syndrome are characterized by deficient efflux of lipids from macrophages, resulting early atherosclerosis and low level of HDL in the blood (14, 15). The ABCA1 protein is heavily glycosylated and probably resides in the plasma membrane. Recent reports described that the regulation of the expression of ABCA7 is similar to that of ABCA1, suggesting a role for ABCA7 in the lipid homeostasis as well (16).

ABCA4 is a retina-specific ABC transporter, expressed exclusively in photoreceptors, where it probably transports retinal or its conjugates from the photoreceptor outer segment disks into the cytoplasm (17). Mutations in the ABCA4 gene have been associated with distinct chorioretinal disorders (18). Complete loss of ABCA4 function leads to retinitis pigmentosa, whereas patients with moderately severe mutations have the so called Stargardt disease (STGD). Heterozygous ABCA4 mutations may play a role in increased frequency of age-related macular degeneration (AMD), or recessive retinitis pigmentosa (RP19). The pathophysiology of these disorders is poorly understood, but the abnormal accumulation of retinoids caused by ABCA4 deficiency has been postulated to be the initial mechanism in these diseases.

1.1.2 The ABCB subfamily

This is the most variable subfamily: it comprises of both full transporters and half transporters, includes transmembrane drug transporters, iron transporters and also specific peptide transporters, involved in antigen presentation. These proteins are localized in different membrane compartments, including the plasma membrane, endoplasmic reticulum, lysosomal and mitochondrial membranes.

ABCB1 (MDR1/P-glycoprotein) was the first described membrane transporter characterized by its ability to confer multidrug resistance to cancer cells (19). MDR1 is glycosylated and localized in the plasma membrane, in the apical/luminal membrane in polarized cells. It is also present in the brush border membrane of intestinal cells, the biliary canalicular membrane of hepatocytes, the luminal membrane of proximal tubule epithelial cells of the kidney, and in the endothelial cells at the blood-brain barrier (1, 3, 4, 20 - see below). ABCB1 has a crucial physiological function in these barriers, namely the protection of the cells and organs against toxic compounds and metabolites. Due to its high transport capacity and broad substrate recognition, this transporter can confer a multidrug resistance phenotype to cancer cells (see below).

ABCB2/TAP1 and ABCB3/TAP2 are half-transporters, and form heterodimers in order to function as peptide transporters involved in the MHC-I-dependent antigen presentation. They are localized in the membrane of endoplasmic reticulum, and pump degraded cytoplasmic peptides into the ER lumen where the MHC class I molecular complexes assemble (21).

ABCB4/MDR3 and ABCB11/BSEP are involved in the transport of phosphatidylcholine (ABCB4) and bile salts (ABCB11), respectively, across the hepatocyte canalicular membrane into bile. Their mutations are responsible for various forms of progressive familial intrahepatic cholestasis (PFIC); mutations in ABCB4 were found in patients with PFIC3, resulting from a defective transport of phosphatidylcholine across the canalicular membrane (22-24). Mutations in ABCB11 cause PFIC2, in this case the biliary bile salt secretion is very low (25).

ABCB6-8 are not well characterized as yet. These are ABC half-transporters, most probably localized in the mitochondrial inner membrane. They likely play a role in the mitochondrial metal homeostasis; ABCB6 probably mediates the transport of an iron complex, ABCB7 and ABCB8 are thought to be involved in the heme transport from mitochondria to the cytosol. They are also candidate genes for inherited metabolic disorders, ABCB6 for the lethal metabolic syndrome, ABCB7 for X-linked sideroblastic anemia with ataxia. ABCB9 is expressed in the lysosomes with unknown function as yet (26).

1.1.3. The ABCC subfamily

The 12 members of this family are similar in their structure, all of them being full transporters. However, their functions are fundamentally different, they act with basically diverse molecular mechanisms. This subfamily includes active, ATP-dependent transporters (ABCC1-6, see 27-30), regulated ion channel (ABCC7/CFTR), and modulators of the permeability of specific K+ channels (SUR1/ABCC8 and SUR2/ABCC9) (4-6).

The human multidrug resistance-associated protein ABCC1/MRP1 was discovered as the basis of a non-P-glycoprotein associated multidrug resistance (27). This protein is responsible for the transport of a remarkable range of drugs as it confers a cancer cell resistance to hydrophobic drugs, similarly to ABCB1/MDR1. However, further functional characterization revealed that the preferred substrates for MRP1 are organic anions, including drugs conjugated to glutathione (GSH), glucuronate, or sulfate (25-28, 30-32). MRP1 also mediates the cotransport of unconjugated amphiphilic anions, together with free GSH. The major physiological function of ABCC1 is presumably leukotriene C4 transport (28).

ABCC2/MRP2 was discovered as an organic anion transporter in the canalicular membrane of hepatocytes, and its mutations were found in patients with Dubin-Johnson syndrome (33-34). This protein is present in the liver, kidney and gut epithelium apical membranes, where it can mediate the efflux of bilirubin glucuronides and other organic anions, playing a role in the detoxification for many endogenous and xenobiotic compounds. In Dubin-Johnson syndrome the inherited mutations of ABCC2 cause a defect in the hepatobiliary secretion of amphiphilic anionic conjugates, resulting in severe conjugated hyperbilirubinemia. By now it has been documented that ABCC2 may also cause multidrug resistance in tumor cells.

ABCC3/MRP3 is also an organic anion transporter (35), and this protein prefers glucuronate conjugates as transported substrates. This protein exhibits a similar tissue distribution as MRP2, but unlike MRP2, that is present in the apical surface of cells, MRP3 is located in the basolateral membranes of polarized cells (35-36).

The human MRP4, MRP5 and MRP6 have been recently characterized as primary active transporters. According to these data MRP4 can mediate the transport of several nucleoside analogs, including anti-human immunodeficiency virus drugs, such as 9-(2-phosphonylmethoxyethyl) adenine (PMEA) and azidothymidine monophosphate (AZTMP). This protein can also confer resistance to 9-(2-phosphonylmethoxyethyl) guanine (PMEG), a compound with neoplastic activity (37, 38). Similarly to MRP4, MRP5 can also catalyze the efflux of nucleoside monophosphate analogues, including nucleoside-based antiviral drugs, and drugs used in the treatment of acute lymphoblastic or myeloid leukemia (ALL/AML) (39). However, the physiological functions of MRP4 and MRP5 are unknown as yet.

Mutations affecting the human MRP6 gene were described in patients with pseudoxanthoma elasticum (PXE), a rare heritable disorder, resulting in the calcification of elastic fibers. Functional studies demonstrated that MRP6 can mediate the transport of glutathione conjugates, e.g. LTC4 and N-ethylmaleimide S-glutathione (40-41). However, the physiological function and the role of ABCC6 in the pathogenesis of this disease are still unknown. Several recently recognized ABCC proteins (ABCC10, 11 and 12) may also be involved in as yet ill-defined conjugate transport processes (42).

The gene coding for ABCC7/CFTR was identified in the late 1980-es as the gene in which mutations are responsible for the development of cystic fibrosis (CF). This is the most frequent autosomal recessive disease within Caucasian populations, with a prevalence of 1 in 2,500 births. The CF gene has been identified by positional cloning (43) and the 'cystic fibrosis transmembrane conductance regulator' protein (ABCC7/CFTR) was documented to function as a chloride ion channel, probably playing a role in exocrine secretions. The CF symptoms mostly relate to the disruption of exocrine functions: in the sweat glands (high sweat electrolyte), pancreas, bronchial glands (chronic bronchopulmonary infection), intestinal glands (meconium ileus), etc. However, additional functional role(s) of the CFTR may be involved in the development of the CF disease (44).

ABCC8/SUR1 has been documented to regulate the cell surface expression and function of ATP-sensitive potassium channels in the pancreatic islets and thus modulate insulin secretion. This protein provides a high-affinity binding site for sulfonylurea, which is widely used to treat patients with non-insulin-dependent diabetes mellitus (45).

1.1.4. The ABCD subfamily

This subfamily consists of four human proteins, all ABC half-transporters, that require a partner half-transporter to form functional homo- or heterodimeric transporters. They are localized in the peroxisomal membrane, and mutations in these genes cause different peroxisomal disorders. Adrenoleukodystrophy (ALD) is a severe X-linked disorder, the most frequent peroxisomal disorder, with a prevalence of 1 in 20,000 males, caused by mutations in the ABCD1 gene (46). This neurodegenerative disease is characterized by the accumulation of the unbranched saturated fatty acids with a chain length of 24 to 30 carbons, as well as of cholesterol esters in the brain and in adrenal cortex. The ABCD1/ALD protein is located in the peroxisomal membrane and most likely plays a role in the transport of very long chain fatty acids.

1.1.5-6. The ABCE and ABCF subfamily

The members of these subfamilies have NBDs, characteristic for ABC proteins, but have no TMDs identified, and thus not known to be involved in any membrane transport functions.

1.1.7. The ABCG subfamily

These five half-transporters, composing the ABCG subfamily, have a unique domain arrangement, with NBDs located at the N-termini and TMDs, located at the C-termini. The first described member of this subfamily was the human homolog of the Drosophila white gene, ABCG1 (47). The expression of this gene is regulated by cholesterol and lipoproteins in macrophages, suggesting that this protein is involved to the lipid metabolism in humans (48).

ABCG5 and ABCG8 are also playing a role in the lipid metabolism, and mutations of either of these genes cause a rare heritable disorder, called sitosterolemia (49-50). In these patients the increased absorption of plant and fish sterols and their decreased biliary excretion cause a high level of toxic sitosterols in the blood, resulting in premature atherosclerosis and cardiac infarct.

ABCG2, a half-transporter causing mitoxantrone resistance in carcinoma cells and multidrug resistance in breast cancer, was first found in multidrug-resistant tumor cells not expressing either MDR1 or MRP1 (51-53). ABCG2 functions as a high capacity drug transporter with a wide substrate specificity, and confers resistance to mitoxantrone, topotecan, and probably even methotrexate.

1.2 Molecular mechanism, cellular distribution, and substrate specificity of ABC transporters

Members of the ABC superfamily are associated with a broad spectrum of physiological functions, including detoxification (ABCB1/MDR1, ABCC1/MRP1), defense against xenobiotics and oxidative stress (ABCCs/MRPs), absorption and secretion processes (MDRs, MRPs), lipid metabolism (ABCA1, MDR3, ABCGs), antigen presentation (ABCB2/TAP1 and ABCB3/TAP2), etc.

Many of the ABC transporters function as active transporters, they mediate the transport of substrates against a concentration gradient. This transport activity requires the energy of ATP hydrolysis, controlled by drug interaction, and coupled to the actual substrate translocation. Most probably the substrate recognition and binding occurs in some parts of the transmembrane domains (3-6, 54). According to several reports, the hydrophobic substrates of MDR1 are recognized within the membrane bilayer or in its vicinity (55). Similarly, in the case of MRP1 it has also been suggested that the transmembrane domains are involved in the drug interactions (56). But the specific sequences responsible for the substrate recognition have not been identified as yet. Interestingly, in case of MDR1 and MRP1, where the substrate-specificities are comparable, the two proteins have only 15% amino-acid identity (57). Recent reports have revealed a few key amino acids playing a role in the recognition of certain substrates in MDR1 (58-59), but the corresponding amino acids have not been found in MRP1.

ATP binding and hydrolysis occurs in the nucleotide-binding domains, and in ABC transporter the close interaction of two NBDs results in the formation of a fully competent ATP hydrolytic site. In active transporters the specific substrate binding enhances the ATPase activity of the transporter, so the transported substrate can promote the liberation of the energy required for its translocation. Due to the molecular connection of NBDs to TMDs, the conformational changes resulted by the ATP hydrolysis can be directly transferred within the protein. Experiments analyzing the ATPase cycle of MDR1 or MRP1 revealed that the interaction of the two NBDs is an essential requirement for the catalytic reaction (60-61). These results suggest that both NBDs can bind ATP and both catalytic sites are active, and at least in the case of MDR1, the two ABC domains enter into the catalytic cycle alternatively (62-63). The ATPase activity of all ABC transporters studied so far is inhibited by vanadate. Vanadate probably replaces phosphate during ATP hydrolysis, and stabilizes a specific, transition state conformation (see ref. 64).

Thus the ATPase activity of ABC transporters reflects their catalytic, transport activity. This substrate-modulated, vanadate-sensitive ATPase activity, representing the full transport cycle, can be directly measured and thus used in various ABC transporter assay systems. In ABC transporters the catalytic reaction involves the formation of a transition state complex that contains an occluded nucleotide. This complex, when containing ADP, can be stabilized by vanadate (64), and this vanadate-dependent nucleotide trapping is significantly modulated by compounds interacting with the transporter (6, 60, 65). Again, this reaction can be used to study drug-protein interactions, as the concentration dependent modulation of nucleotide trapping correlates with the interaction of a given compound with the ABC proteins. Still, we know relatively little about the detailed catalytic mechanism of ABC transporters, although several models have already been suggested to describe this mechanism (64).

As shown in Table I, ABC transporters with multidrug transporter function (ABCB1/MDR1, ABCC1/MRP1, ABCG2) show a widespread expression profile, providing a cellular defense mechanism throughout the organism. The tissue distribution of these three major multidrug resistance proteins is overlapping but different. ABCC1 expression is high in all tissues, it is elevated in lung and the testis, and reduced in the liver. MDR1 is more restricted to tissues involved in the absorption and secretion (1-4). MDR1 is also highly expressed in tissues with pharmacological barrier function, such as the blood-brain barrier, and the choroid plexus (66-67). Recent reports have demonstrated that ABCG2 is highly expressed in the placenta, liver, intestine and in various stem cells (68-69).

In tissues where different multidrug transporters are present, the subcellular localization of these proteins can be a discriminating feature. All multidrug transporters are localized predominantly in the plasma membrane. ABCB1/MDR1 exhibits apical (luminal) expression in polarized cells, e.g. epithelial cells of the intestine and the proximal tubules of kidney, or the canalicular membranes of hepatocytes (21-22, 70). In contrast, MRP1 in polarized cells is localized solely in the basolateral membrane. The expression of ABCG2 was reported to be mostly apical (71).

The substrate specificity of MDR-ABC proteins exhibits as wide a variety as their tissue distribution and they share the ability to transport a wide range of compounds out of cells. The substrate specificities of these three proteins are again overlapping, but each transporter can handle unique compounds as well. ABCB1/MDR1 favors uncharged or positively charged hydrophobic compounds, whereas the preferred substrates for ABCC1/MRP1 are organic anions, including drugs conjugated to glutathione (GSH), glucuronate, or sulfate. Typical high affinity substrates of MRP1 are leukotriene C4 (LTC4), 17 β-glucuronosyl estradiol and bisglucuronosyl bilirubin. ABCG2 has also a remarkable range of transported substrates (mitoxantrone, anthracyclins, topotecan, flavopiridol, and methotrexate - see 11, 68, 72).

In some cases, when different transporters with overlapping substrate profiles are located in the same cells, their affinity, capacity, and the cellular drug-concentrations determine the actual fate of the substrates. In hepatocytes, where MRP2 and MRP3 are both expressed, different conjugates are transported according to the transporters affinity and capacity. MRP2 transports glucuronate conjugates with a relatively low affinity, but with high capacity, in contrast to MRP3, which is a high affinity, but low capacity transporter for these conjugates. Moreover, MRP-mediated transport is greatly increased by the presence of intracellular bile salts. Thus, under normal conditions glucuronate conjugates are extruded into the bile by MRP2, located in the apical membrane. However, at increasing cellular concentrations of glucuronate conjugates MRP3 can also remove a certain amount of these compounds into the blood stream, and defend the cells from the toxic materials (73-74).

2. ABC Transporters as Therapeutic Targets

2.1 Circumvention of Multidrug Resistance in Cancer - MDR Transporters

The multidrug resistance phenotype in tumors is associated with the overexpression of certain ABC transporters, termed MDR proteins. The P-glycoprotein (Pgp, MDR1, ABCB1) mediated multidrug resistance was the first discovered and probably still is the most widely observed mechanism in clinical multidrug resistance (1, 3, 5, 75). There are two other ABC transporters, which have been definitely demonstrated to participate in the multidrug resistance of tumors: the multidrug resistance protein 1 (MRP1/ABCC1, 27, 29, 76), and the mitoxantrone resistance protein (MXR/BCRP, ABCG2, 68). Furthermore, other human ABC proteins capable of actively transporting various compounds out of the cells, may also be players in selected cases of multidrug resistance. These include the homologues of MRP1, MRP2-MRP5. MRP2 and MRP3 seem to be key players in organic conjugate transport in various tissues, while MRP4 and MRP5, may have special functions as nucleoside transporters (36, 39, 77-78, 80).

The three major proteins involved in cancer multidrug resistance are MDR1 (P-glycoprotein, ABCB1), MRP1 (multidrug resistance protein 1, ABCC1) and the ABCG2 multidrug transporter (BCRP/ MXR). MDR1 and MRP1 can recognize and transport a large variety of hydrophobic drugs, and MRP1 can also extrude anionic drugs or drug conjugates (28-29, 81-82). The substrate specificity of ABCG2 partially overlaps with that of MDR1 and MRP1, that is the compounds transported by ABCG2 are also large, hydrophobic molecules, including mitoxantrone, topotecan, flavopiridol, methotrexate and Hoechst 33342 (81).

Prevention of clinical MDR should significantly improve therapeutic response in a large number of tumor patients. One way to achieve this goal would be to develop anticancer agents which do not interact with any of the multidrug transporters. However, as cytotoxic drugs have to penetrate the cell membrane and the MDR proteins have an extremely wide recognition pattern, this seems to be a remote possibility. There are several suggested methods to prevent the expression or function of multidrug transporters, but pharmacological modulation seems to be the first choice at present. MDR modifying agents which inhibit the function of the MDR proteins, either competitively or non-competitively, are good candidates for such a pharmacological modulation. These compounds are expected to increase the cytotoxic action of MDR-related drugs by preventing the extrusion of anticancer drugs from the target cells.

Studies involving various in vitro assay systems (see below) led to the identification of several Pgp modulator compounds, as diverse in structure as the transported drugs. The first generation consisted of drugs that were already in clinical use, while the second generation modulators were derivatives of the above compounds. These, e.g. R-verapamil and PSC-833, had less pronounced effects on their original targets, but retained their modulatory effects. The third generation of MDR modifiers are molecules specifically devised to interact with specific MDR transporters (for recent reviews see 4, 32, 58, 81, 84-85).

As these agents are mostly in the early development phase in various research laboratories, their clinical efficiency has yet to be proven. Small hydrobhobic peptide derivatives (Reversins), interacting with P-gp/MDR1 with high affinity and selectivity, were also developed in our laboratory (86).

2.2 Lipid Disorders

ABC transporters have been documented to play a major role in lipid transport and lipid-related disorders. The association between cholesterol and atherosclerosis is thought to involve the cellular uptake and deposition of cholesterol. By removing cholesterol from the cells, the reverse cholesterol pathway provides protection for the artery wall against unwanted lipid deposition. It has been suggested that ABCA1 is the key protein in controlling the cellular apolipoprotein-mediated lipid removal pathway (87-88). Mutations in the ABCA1 gene result in Tangier disease, a genetic disorder characterized by an abnormal lipoprotein profile and the accumulation of cholesterol esters in various tissues (89-91). Cholesterol efflux from Tangier-fibroblasts to lipid-poor apolipoproteins is defective, suggesting that ABCA1 has a key role in the modulation of the reverse cholesterol transport (for reviews see 87, 89, 93).

Several other ABC transporters have also been implicated in lipid and/or cholesterol transport. The expression level of ABCG1 in human macrophages is greatly increased by cholesterol loading and by lipoproteins, suggesting that this protein is involved in the metabolism of these lipids (48). There are no published functional and localization data available as yet regarding this ABC transporter in various cell types. However, it has been documented in detail, that close relatives of ABCG1, that is ABCG5 and ABCG8, play a key role in lipid metabolism, as mutations of either of these genes was found to cause a heritable disorder altering the absorption and excretion of plant- and fish-derived steroid molecules (sitosterolemia, 49-50). In these patients high levels of blood sitosterols, and an altered cholesterol metabolism leads to atherosclerosis and cardiac diseases. ABCG5 and ABCG8 were documented to work as obligate heretodimers, and both their function and correct cellular localization depends on this dimerization process (94). ABCB4 (MDR3) is a specific phosphatidyl choline transporter residing in the bile canalicular membrane in the liver, and has an essential function in the proper bile formation (22).

Modulation of the expression and function of these lipid transporter proteins may soon become an important pharmacological target. We know very little as yet about the molecular mechanism and structure of these proteins, thus this seems to be a rapidly developing and promising area of ABC transporter research.

2.3. Other Therapeutic Targets

There are numerous ABC transporters in which specific mutations cause severe inherited diseases. These are all natural therapeutic targets in medicine and in the present review we just shortly point to some of these target proteins. In the case of the lethal inherited disease, cystic fibrosis, the most frequent mutation in the ABCC7/CFTR protein causes a misprocessing of the protein (44), thus any method helping the proper processing and localization of this mutant variant is of major interest. Major mutations in the ABCC8/SUR1 protein cause a severe hyperinsulinemic disease in early infancy, and currently the only treatment for this condition is the removal of insulin-producing pancreatic islands. Modulation of ABCC8 and the related K channel may significantly improve current treatments for various forms of diabetes. In the case of ABCA4, the large variety of eye diseases (18) caused by a malfunction of this protein call for a detailed investigation of ABCA4 and establishing methods for developing proper modifying methods and/or agents. It goes without saying, that in the case of each respective transporter, in spite of the general basic structure and mechanism, specific approaches are required for such developments.

2.4. Gene Therapy Applications

The co-expression of a human drug-resistance protein with a therapeutic gene product should allow both an enrichment of the corrected cells and an in vivo drug selection during clinical gene therapy. The use of the MDR1-P-glycoprotein as such a selectable marker has been widely investigated and advocated (95-97). However, in some experiments MDR1 expression rapidly declined in the modified cells (95, 98) In other studies, although the expression level of MDR1 remained high, this caused alterations in stem cell maturation, in some cases leading to malignant transformation (99). Also, MDR1 has a large, over 4 kbase cDNA, making the construction of therapeutic vectors difficult. Moreover, the introduction of normal human MDR1 with a naturally wide drug substrate recognition does not allow a targeted selection of the genetically modified cells.

Recent studies in our laboratory suggest that a mutant variant of the human ABCG2 multidrug resistance protein is especially applicable as a selectable marker. ABCG2, an ATP-dependent primary active transporter, extrudes a large variety of cytotoxic agents (68, 81, 100-102). The cDNA encoding this protein is approximately 2 kbase, and the active dimer is spontaneously formed within the overexpressing cells (72). ABCG2 is normally expressed in the placenta and in stem cells (69, 102, 103). It has been shown that the R482G variant of ABCG2 has a different substrate specificity than the wild-type protein (65, 104). Such a difference in drug-selectivity, caused by a point-mutation, has a special advantage in gene therapy application: the artificially introduced protein, although having the same structure and antigenicity, can be functionally distinguished from its endogenous counterpart. The in vivo applicability of such a taylor-made drug resistance ABC transporter as selectable marker still awaits a further, especially in vivo studies.

3. Role of ABC transporters in modulation of ADME/Toxicology properties of drugs

Regarding drug disposition, response or toxicity metabolism alone does not accurately account for the intersubject variability observed in clinical practice. It is increasingly recognized that transporter-mediated processes significantly modulate drug absorption, distribution, metabolism and excretion. Transporters are common sites for drug-drug interactions as well as interactions of drugs with endogenous substrates, processes leading to drug toxicity and various adverse effects.

3.1 Drug Absorption

Oral bioavailability of drugs was initially thought to be affected by drug absorption and Phase I metabolism by the liver (105). By now it has been well established that active efflux by ABC transporters in the small intestine is a major contributor to poor absorption and low bioavailability.

Four major ABC transporters have been shown to localize to the apical/luminal membrane of enterocytes, and, thus, are thought to form a barrier to intestinal absorption of substrate drugs: ABCB1/P-gp/MDR1, ABCC2/cMOAT/MRP2, ABCG2/BCRP/MXR, ABCC4/MRP4 (for review see 106). Their expression level varies between different segments of the intestine. In general, MXR, MRP2 and MDR1 are expressed at high level in the small intestine (107), considered by many in the field as the rate limiting barrier to oral drug absorption. Regarding the role in limiting intestinal absorption, MDR1 is the most thoroughly characterized ABC transporter. The most direct evidence has come from the numerous in vivo studies utilizing mdr1 knock-out mice. In a representative study Paclitaxel AUC was shown to be 6-fold greater in the mdr1a-/- mice than the wild type (mdr1+/+) mice (108). An even greater increase of paclitaxel AUC was seen in the wt mice when the Mdr1 function was blocked with PSC833, an Mdr1 inhibitor (109).

Although the expression levels of both the MRP2 and MXR are higher in the small intestine than the expression of MDR1, there are much fewer data available on their role in drug absorption. A few examples, however, have been published: MRP2 has been shown to limit absorption of a phenylimidazo[4,5-b]pyridine (PhIP) derivative, a food-derived carcinogen (110), and MXR has been shown to limit absorption of topotecan in Mdr1a/1b (-/-) mice (71).

An apparent controversy stems from the fact that some MDR1 substrate drugs have reasonable oral bioavailability. A literature search has revealed that the published KM values for MDR1-substrate drugs are relatively low (111). In fast-release formulations the intestinal concentrations for many drugs can reach the mM concentration range, when MDR1-dependent extrusion becomes saturated, and, thus, the MDR1-interaction cannot provide a coping extrusion and this function becomes quantitatively less important. On the contrary, systemic concentrations of the same drugs are usually much lower, therefore MDR1 interaction at the blood-brain-barrier, liver and kidney significantly modulate the ADME profiles of various drugs. For drugs with poor solubility, such as cyclosporin A or paclitaxel, intestinal MDR1 function is limiting their bioavailability, even at high doses.

3.2 Drug Distribution

To be effective, absorbed drugs must be transported from the site of administration to the site of action. The CNS is the tissue most frequently targeted by drugs. The blood- CNS interface is composed of the blood microvascular endothelial cells (blood-brain-barrier, BBB) and the choroid plexus (blood-cerebrospinal fluid (CSF)-barrier). It has been long known that lipophilicity is required for BBB penetration. However, many lipophilic drugs exhibit poor BBB penetration. About two decades ago these compounds were considered "outliers", without knowing the reason behind the phenomenon. Now it is known that the poor BBB permeability of these compounds is due to the efflux functions of ABC transporters.

It is well established that the MDR1 protein localized in the apical/luminal membrane of the brain capillary endothelial cells is a major barrier of CNS penetration of drugs (for a review see (112). Multiple studies have shown the pivotal importance of MDR1 in protecting the brain from xenobiotics. The most convincing studies were carried out by utilizing mdr1 knock-out animals. Each of the studies showed a dramatic increase in the brain levels and/or brain/blood ratio of drugs when MDR1 substrates were administered to Mdr1a knock-out mice (see Table II). Some MDR1 substrates, however, reached appreciable brain levels even in the wt mice. To explain the apparent controversy, it has been suggested that drugs with very high passive diffusion rate may overcome the MDR1 barrier function (113).

It is also generally accepted that MRP1 is localized in the basolateral membrane of the choroid epithelial cells, preventing CSF penetration of drugs and toxicants (for a review see 114), as clearly shown by the example of the MRP1-mediated clearance of etoposide from CSF (115).

More recently, other ABC transporters, such as MRP2 and MXR have also been implicated in protecting the brain tissue against xenobiotics. Although there are conflicting reports on the expression of the MRP2 in the brain endothelial cells, it has been shown that in MRP2 deficient TR- rats the brain levels of antiepileptic drugs were significantly higher than in the normal background strain (116), and both MRP2 and MDR1 contributes to the active barrier function against brain penetration of somatostatin analogs (117). Although no studies have been published regarding the function of MXR in the BBB, it has been recently reported, that MXR is localized in the apical/luminal membrane of the brain microvessel endothelial cells (118). Similarity between localization of MRP2, MXR and MDR1 in the brain microvessel endothelial cells and in the enteral, liver and kidney tubular epithelial cells strongly suggests a scenario where these three transporters function together to serve as physiological barriers against brain penetration of xenobiotics.

ABC transporters at the blood-CNS interface are double-edged swords. On the one hand they limit penetration of CNS-targeted drugs, on the other hand they protect against potential central toxicity of non-CNS-targeted drugs. The transporter saturation, due to high local concentration of drugs in the enterocytes, and a reasonably high passive permeability might provide a window that would allow for acceptable intestinal absorption but limited CNS penetration for ABC-transporter substrate drugs.

Drug penetration into the fetus is usually a pharmacologically undesirable consequence of drug therapy. Convincing data have been shown on the protective function of MDR1 in the placenta (fetal - maternal interface) (reviewed in 111). When mdr1a deficient female CF-1 mice were treated with ivermectin during pregnancy, 100% of the -/- mice were susceptible to cleft palate. Their heterozygotes (+/-) were much less sensitive, while the homozygous (+/+) fetuses were totally insensitive to the dose tested (119). MXR and MRP2 are co-localized with MDR1 in the placental brush-border membrane, and it is now widely accepted that MXR plays an important role in the placenta while similar information is emerging on the placental function of MRP2 (120).

Table II. Mdr1a limits brain penetration of drugs

Drug	C_brain in mdr1a(-/- ) / C_brain mdr1a(+/+)	Reference
Tacrolimus	6.0	Yokogawa K. et. al. Pharm. Res. 16, 1999, 1213-18.
Vinblastine	19.4	Schinkel, AH. et.al. Cel 77, 1994, 491-502.
Cyclosporin A	10.9	Schinkel, AH. et.al. JCI 96, 1995, 1698-1705.
Ondansetron	3.8	Schinkel, AH. et.al. JCI 97, 1996, 2517-24.
Quinidine	8.3	Kusuhara, H. et. al. JPET 283, 1997, 574-580.
Verapamil	8.3	Hendrikse, et. al. Br. J. Pharm. 124, 1998, 1413-18.
Loperamide	6.7	Schinkel, AH. et.al. JCI 97, 1996, 2517-24.

3.3. Drug Metabolism

It is getting increasingly accepted that the intestine is the site of the "first-pass" metabolism and also the site for a significant portion of non-renal elimination for many drugs (121-122). Since a fraction of the extruded molecules are reabsorbed into the enterocytes, this repetitive extrusion and reabsorption prolongs the intracellular residence time, and thus, increases the probability of intestinal metabolism. The formation of the major metabolite of indinavir (reviewed in 111), a substrate of both MDR1, and CYP3A4, the major intestinal drug metabolizing enzyme, was 6-fold greater when the drug was applied at the apical side of the Caco-2 cells than when it was applied at the basolateral side (123). These data were confirmed in vivo in a study, showing that the intestinal first-pass metabolism of indinavir increased from 6% in control rats to 34% in the group treated with dexamethasone, an inducer of the MDR1 and CYP3A4 expression (124). The 6-fold increase in intestinal metabolism of indinavir can not be explained by the 2.5-fold increase in intestinal CYP3A4 level alone, providing in vivo evidence that MDR1 enhances intestinal first-pass metabolism of indinavir. In contrast, MDR1 did not appear to influence the hepatic metabolism of indinavir. The difference might be partially explained by the fact that in the liver MDR1 is expressed at the "exit site" of the hepatocytes. Hence, MDR1 mostly interacts with compounds that have undergone uptake, intracellular distribution and metabolism.

3.4 Drug Excretion

Both the liver and the kidney play an important role in the excretion of drugs and biliary and renal excretion display similarities. Drugs in both tissues must first be taken up through the basolateral membranes of hepatocytes or tubular epithelial cells. Often biotransformation occurs before the drug or the metabolite gets to the apical membrane, where the drug traverses the membrane by passive diffusion or actively effluxed by one of the apically localized transporters. Convincing data, regarding the importance of the efflux transporters have been generated by studying digoxin metabolism in mdr1 knock-out mice (reviewed in 111). Digoxin is mainly excreted as a nonmetabolized drug in the urine and the bile. The biliary clearance of digoxin is about 3-fold greater in the mdr1a (+/+) mice than in the mdr1a (-/-) mice (125). Renal clearance of digoxin was also investigated in wt and mdr1a knock-out mice, with similar conclusions: an about 3-fold decrease in the mdr1a knock-out mice (125). These data indicate that ABCB1/Mdr1a plays a significant role in the biliary excretion of digoxin. In contrast to expectations, the renal excretion of nonmetabolized doxorubicin (126) and the excretion of three basic compounds (vecuronium, azido-procainamide and tributyltrimethylammonium) (127) in mdr 1a and mdr1b double knock-outs is significantly higher than in the wt mice. An explanation for the seemingly conflicting data would be that compensatory up-regulation of other transporter systems that transport the drugs, or enzymes that metabolize the compounds, may have occurred in the mdr1 knock-out animals.

3.5. Drug-Drug Interactions (DDI)

The multispecificity of the ABC transporters serves as a basis for drug-drug interactions. A drug interacting with a transporter might inhibit the transport of another drug either in a competitive or in an non-competitive fashion. Some of the transporters can be inhibited both from the cytoplasmic, or 'cis'-side and from the extracellular or 'trans' side (128). The inhibition of the transport usually results in an increased bioavailability, decreased clearance, hence markedly elevated AUC of the affected drug. Some data on DDI, mediated by MDR1 are shown in Table III.

Drugs, inducing ABC transporter gene expression have also been shown to modulate ADME properties of co-administered drugs. Rifampin is the classical example for an inducer drug, causing such DDI (129-130). Rifampin, an inducer of MDR1 decreases intestinal absorption, as well as the clearance of co-administered drugs (129-130). The cumulative effect is a significant decrease in AUC of the co-administered drugs. Recently components of human diet have been implicated in modulation of transporter-mediated drug-efflux. Nine out of 68 standardized food extracts showed inhibitory effect on the MDR1-mediated 3H-cyclosporin efflux in the Caco-2 monolayer efflux assay (131).

3.6 Toxicity

Interaction of drugs with ABC transporters may cause toxicity by several different mechanisms. Inhibition of the efflux function may modulate distribution of co-administered drugs, environmental toxicants, thus changing their toxicity profile. This is of particular relevance for cytotoxic drugs. Some cytotoxic drugs have tolerable CNS safety profile, as they are prevented from entering the CNS by efflux transporters. Ivermectin, a neurotoxic pesticide and an MDR1 substrate, displays CNS toxicity upon co-administration of drugs with MDR1-inhibitory potential (132), and Mdr1a knock-out mice exhibited 100-fold increased sensitivity to toxic effects of ivermectin, with markedly elevated brain levels of the drug (133).

ABC transporters serve as protective shields by preventing uptake or facilitating clearance of toxic substances. Anti-toxic effects of MRP1-3 have been studied in detail. Mrp2 is involved in hepatobiliary excretion of GSH conjugates of inorganic arsenic and likely in its chemical derivatives (134). In addition, some food-derived carcinogens and pre-carcinogens and their glucuronide conjugates are also transported by Mrp2 (110). MRP1 and MRP3 may also contribute to the toxicological defense function by eliminating a number of toxic agents and their conjugates from epithelial tissues (73). It has been observed, that MRP3 expression is strongly upregulated in the liver of the MRP2 deficient patients and animals (36, 135-136), implying that basolateral MRP1 and MRP3-mediated efflux of toxicants may become of pivotal importance when administering MRP2-interacting drugs.

ABC pumps play an important function in the homeostasis of their own endogenous substrates. Pharmacological blockade of the transport endogenous substrates may cause toxicity, and adverse effects. ABCB11/BSEP is the major liver bile-salt transporter. The endothelin antagonist bosentan inhibits BSEP in an antagonistic fashion, leading to intracellular accumulation of cytotoxic bile salts, and bile salt induced liver-damage (137). Similarly, troglitazone, an insulin sensitizer, induces cholestasis and hepatotoxicity via competitive inhibition of the BSEP-mediated bile salt transport (138). In clinical use the elevations of liver enzyme levels and in several cases a fulminant hepatic failure were observed leading to the withdrawal of this drug from the market.

4. ABC transporter assay systems in drug research and development

By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved. Oral bioavailability, entering the blood-brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins. Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics.

By using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents. We focus on such assay systems relevant for ABC transporters involved in chemoresistance of cancer, in drug metabolism, and toxicity.

4.1 Screening methods

A wide range of assay systems has been developed to study drug - ABC transporter interactions. Although knock-out mice, as well as mutant mice and rat strains, deficient in functional expression of some ABC transporter proteins, are available, this review is focusing on in vitro systems, as only in vitro techniques meet the high throughput requirements of early ADME studies. Excellent recent reviews, covering the in vivo assay systems have been published (111, 139).

The functional in vitro test systems mostly rely on the detection of the translocation of a substrate, the detection of the hydrolysis of ATP or the detection of the change in the conformation of the transporter. The interaction of the test drug with the transporter can be determined indirectly, as it modulates the transport of a reporter substrate. Alternatively, the interaction can be detected by directly measuring the translocation of the test drug. The "direct" assay or direct measurement is more suitable for substrate screening, while assays utilizing a reporter substrate are more relevant in interaction studies, and in screens to identify inhibitors.

The target protein can be included in the assays in two major forms: in whole cells expressing the transporter, or in purified membrane vesicles. For whole cell studies a plethora of selected or transfected cell lines are available. Two major drawbacks of using selected cell lines are usually noted: (i) transporter expression pattern changes with time, and (ii) the cell line overexpresses other transporters with overlapping substrate specificities. Clearly, transfected cells, if available, are the experimental system of choice, as they harbor a well-defined modification and a more stable expression.

The function of the transporters can also be studied in membrane vesicles prepared from cells or tissues that overexpress the transporter. This is a widely used technique that can be used in different high-throughput assay formats. Utilization of inside-out vesicles carry major advantages, such as (i) the test compounds and ATP have direct access to the cytoplasmic domains of the transporter, and (ii) since the direction of the transport is occurring from outside to inside, the transport can be assayed by measuring the amount of substrate trapped inside the vesicle compartment. Membrane preparations, purified from cells expressing the transporter, or reconstituted from purified check protein and lipid components have been described (86, 140-141).

Although membrane vesicles can be made from practically any kind of cells that express the transporter (e.g. selected cell lines, transfected cell lines, tissues), the most frequently used membrane preparations utilize baculovirus-infected Sf9 insect cells (141). The advantage of this expression system is the particularly high transporter protein expression level (around 5-10 % of the total membrane protein is the expressed transporter). In addition, the heterologously expressed transporter is the only mammalian protein in this system. This makes the insect membranes a powerful tool in membrane-based assays. Membrane preparations from other expression systems are usually not suitable for ATPase measurements, due to low signal-to-noise ratio, as a consequence of a low transporter expression. Some ABC-transporter overexpressing vesicular membrane preparations are by now commercially available (http://www.solvo.com). Membrane preparations reconstituted from purified transporters and lipids offer excellent signal-to-noise ratio (86). However, preparation of these membranes is laborious, and these reagents are commercially not available.

4.2 Whole cell based assays

Efflux transporter-expressing cells actively pump substrates out or the cell, which results in a lower rate of substrate accumulation, lower intracellular concentration at steady state, or a faster rate of substrate elimination from cells loaded with the substrate. If the substrate is cytotoxic, cells overexpressing the transporter are less sensitive to the toxic effect than the parental ones, and inhibitors or competing non-cytotoxic substrates restore sensitivity. These types of screens are often used in search of substrates and reversing agents of transporters involved in multidrug resistance (142).

Radioactive substrates are also used to measure the transport across the cell membrane, as the accumulation or extrusion of the radioactive signal is different for the transporter overexpressing cell lines compared to parental ones (143). As most test compounds are not available in a radioactively labeled form, the "indirect" set-up is more frequently used.

Transported substrates can also be labeled by fluorescent dyes. Furthermore, fluorescent molecules can be substrates of different transporters. As most test drugs are non-fluorescent, this assay is also usually performed in an "indirect" set-up, using a fluorescent reporter substrate, such as Rhodamine 123 (R123), which is a substrate of the MDR1 transporter (144). When cells are incubated with R123, the dye accumulates at a slower rate in cells overexpressing MDR1 than in the parental ones, or, alternatively, MDR1-expressing cells loaded with R123 extrude the dye faster than parental ones. Inhibitors or competing substrates of the MDR1 transporter modulate the rate of R123 accumulation/extrusion. The most commonly used application is the measurement of the efflux of R123. Multiple drawbacks of this assay have been reported: (i) the dye displays poor cellular retention, and it interacts with different intracellular compartments result in a spectral and intensity shift (145), (ii) the initial concentration is cell-type dependent and (iii) the concentration of R123 changes during the measurement. It has also been reported that after the initial loading, cells have to be incubated for 1-2 (146), or even 3-10 hours in clinical samples (147). This long time-frame and the extensive washing of the cells prior to the plate reader analysis, makes this assay inapplicable for high throughput screening.

Figure 1. The "vacuum cleaner model" of multidrug transporter action and the principle of the calcein assay.
Non-fluorescent, hydrophobic calcein AM, dissolved in the lipid bilayer, is picked up by the MDR1 transporter and is pumped out of the cell, thus keeping the intracellular concentration of calcein AM low. Modulators of the transporter activity reduce the rate of calcein AM extrusion, leading to increased intracellular calcein AM concentrations. Calcein AM is hydrolyzed by intracellular esterases, yielding free, fluorescent calcein, which is hydrohilic and retained inside the cells. Insert: the typical output of the calcein assay (one well). With no inhibitors added, the rate of cellular calcein accumulation is slow, and cellular fluorescence slowly increases. Addition of inhibitors of the transporter results in a faster rate of calcein accumulation.

Calcein AM has been used as a more suitable alternative for R123 transport assay (ref. 148 - see Fig. 1). Calcein AM is also a substrate for MDR1, but it is nonfluorescent. Intracellularly Calcein AM is hydrolyzed by endogenous esterases, yielding a fluorescent product, Calcein. Therefore, accumulation of the fluorescent dye can be detected without the need for washing steps, making this assay suitable for a high throughput screen format. The presence of MDR1 in the cell membrane strongly reduces Calcein accumulation, and inhibitors of MDR1 produce an increased rate of accumulation, up to the level measured in the absence of the transporter. Calcein has favorable spectral properties, such as bright fluorescence, that is insensitive to pH or to Ca2+ as well as Mg2+ concentrations, and does not show spectral changes upon accumulation in intracellular compartments or upon binding to intracellular structures (149). Both Calcein AM and free Calcein are also transported by MRP1, thus the patented calcein assay is applicable for the quantitation of both MDR1 and MRP1 activities (150). A suitable kit (MDQ kit) already exists for the specific diagnostic detection of MDR1 and MRP1 activities in clinical tumor samples (151). The Calcein-based drug screening assay is provided together with detailed application notes and specific cell lines (see http://www.solvo.com)

A similar dye transport-based assay, utilizing Hoechst 33342 has been worked out to measure the activity of ABCG2/MXR, the third MDR-ABC transporter, with broad substrate specificity (11, 72, 65, 103). Hoechst 33342 is a non-fluorescent substrate of both MDR1 and MXR, and the dye becomes fluorescent after entering the cell and binding to DNA, making a high-throughput application of the assay feasible.

In vivo, drugs have to cross pharmacological barriers in order to get absorbed (intestinal epithelial cells), distributed (blood-brain barrier endothelial cells) or excreted (hepatocytes, proximal tubule epithelial cells). This transcellular movement is modeled by the monolayer efflux assays, alternatively termed as vectorial transport assays. There are several cell lines that can be grown on a permeable support in a way that the cells form a confluent monolayer with tight junctions. The cell lines used in these assays are polarized epithelial or endothelial cells, with tight junctions. Most commonly the human intestinal epithelial line Caco-2, and transfected versions of the canine kidney cell lines MDCKI, MDCKII, or the porcine kidney epithelial cells LLC-PK1 are used. Test drugs can be applied to either side of the cell layer and the rate of transport across the monolayer is measured from the apical to basolateral (A-B) or from the basolateral to apical (B-A) direction. If an active transport process modulates the movement of the test drug across the cell membranes, the compound is transported at different rates in the two directions. Modulators, specific for efflux transporters, erase the difference in transport rate, confirming the drug - transporter interaction. Recently, a variation of the monolayer efflux assay, referred as equilibrium transport assay, has been developed (152). In this set-up, the compounds are simultaneously placed at an equal concentration to both the apical and the basolateral side. The redistribution of the compounds is monitored over up to two days.

Once again, transfected cell lines seem to be more suitable for the studies as the data can be corrected for the contribution of endogenous transporters (153), and these cells provide more stable expression of the ABC transporters. It is important to note that at least some of the endogenous ABC transporter promoters are stress-sensitive (154). Therefore, it is likely that cell lines that express ABC transporters from endogenous promoters will be more susceptible to mechanical and environmental stress (changes in temperature, acidity, oxygen and fuel supply) that may occur during cell culturing.

Binding of transported substrates or modulatory agents changes the conformation of the transporter protein, allowing the detection of drug - transporter interactions. A fluorescence-activated cell sorter (FACS) based assay has been developed that utilizes UIC2, a conformation sensitive monoclonal antibody, that recognizes a complex extracellular epitope of MDR1 (155). In the presence of substrates, modulators, or ATP-depleting agents the reactivity of MDR1 expressing cells to UIC2 markedly increases (156). A more recent method (Antibody Competition Test) utilizes two MDR1 specific antibodies. The first antibody (UIC2) only mildly effects the binding of the second one (MM12.10), unless the MDR1 expressing cells are treated with certain modulators or substrates, when UIC2 treatment completely abolishes MM12.10 (157). It is claimed by the inventors that the assay is highly suitable to screen for "strong" reversing agents.

4.3. Membrane based assays

Membranes prepared form ABC transporter overexpressing cells, or reconstituted from purified protein and lipids can be used to assay the interaction of test drugs with ABC transporters in three basic assay formats: the ATPase assay, the vesicular transport assay and the nucleotide trapping assay.

Figure 2. The principle and typical output of the MDR-ATPase assay.
During the drug transport process ATP is hydrolyzed to ADP and inorganic phosphate (Pi), which can be detected by a simple colorimetric reaction. Interacting test drugs modulate the activity of the transporter, resulting the modulation of the rate of ATP hydrolysis. Insert: typical activation and inhibition curves gained in the ATPase assay. Increasing concentrations of compound 1 result in increasing ATPase activity of the transporter (possible substrate). Increasing concentrations of compound 2 result in decreasing ATPase activity of the activated transporter (inhibitor, or slowly transported compound).

The ATPase assay is one of the most widely used assays to search for compounds that interact with different ABC transporters (141). The assay (Fig. 2) requires relatively high levels of transporter protein expression, thus mostly membrane vesicles prepared from recombinant baculovirus infected insect cells (e.g. Sf9 cells) are used for this purpose. These membrane preparations show a vanadate-sensitive ABC transporter ATPase activity that is modulated by interacting compounds. The rate of ATP hydrolysis is determined by measuring the liberation of inorganic phosphate. In the presence of transported substrates the ATPase activity of the transporter increases (activation protocol). Inhibitors that act in a non-competitive fashion, or some slowly transported compounds inhibit the ATPase activity of the stimulated transporter (inhibition protocol). The main advantage of the ATPase assay is in its simplicity and reproducibility. High throughput version of the assay has been developed (http://www.solvo.com).

Figure 3. The principle and typical output of the multidrug transporter vesicular transport assay.
In the presence of ATP, transported substrates are translocated by an ABC transporter inside the inverted membrane vesicles. Vesicles can be separated from the incubation solution by rapid filtration, and the amount of compound trapped inside the vesicles can be quantitated by a suitable analytical method.

A more direct measurement of substrate translocation is possible by quantitation of the intravesicularly trapped substrates in the vesicular transport assay (Fig 3). Successful vesicular transport studies have been reported using membranes from different sources (insect cells, transformed and selected cell lines, artificial membrane vesicle - see 86, 140-141). In a suitable membrane preparation about 10-16% of the total membranes form inside-out orientation vesicles (the rest is usually open fragments), and substrates are actively transported into the vesicles. Rapid filtration, using glass fiber filters or nitrocellulose membranes, is used to separate the vesicles from the incubation solution, and the test compound, trapped inside the vesicles, is retained on the filter. The quantity of the transported unlabelled molecules can be determined by high resolution, high sensitivity analytical methods. Alternatively, the compounds are radiolabeled or a fluorescent tag is attached, and the radioactivity or fluorescence retained on the filter is quantitated.

This assay can also be performed in an "indirect" set-up, where interacting test drugs modulate the transport rate of a labeled reporter compound. The "direct" assay clearly has an advantage, however, some "fast diffuser" compounds (e.g. verapamil) redistribute so fast that the translocation assays yield conflicting data (for review see 81). A high throughput format of the vesicular transport assay has recently been published (158).

As mentioned earlier, in ABC transporters the catalytic reaction involves the formation of a transition state complex that contains an occluded nucleotide. An occluded form of ADP can be stabilized by vanadate, which provides the basis of the screening method, called vanadate-dependent nucleotide trapping. Such a nucleotide trapping is concentration dependently modulated by the transported substrates. Experimentally the isolated cell membranes are incubated with alpha-32P labeled azido-ATP, and after washings in the presence of cold ATP, the trapped, labeled nucleotides are covalently bound to the transporter by UV photo-cross linking. Nucleotide trapping is a low throughput assay, however some subtle interactions of drugs with transporters can only be shown by this assay (40).

4.4. Screening strategy

Depending on the overall aims of the test and the transporter assayed, different methods and screening strategies should be applied. In order to screen a large library of compounds, fluorescence-based whole cell assays and the ATPase assay seem to be the methods of choice. This gives a combination of direct and indirect assays, however, even these large-scale screening methods should be used in a complementary fashion, as false positive or false negative results could be obtained if only one of these methods is used.

When screening for transported substrates of a given ABC transporter, indirect methods used alone may be misleading, as both substrates and inhibitors will show up to be positive. On the contrary, indirect assays, utilizing reporter substrates, are the methods of choice to screen for reversing agents. The above described combination of different methods may provide a wide enough range of assay systems to successfully address specific drug and ABC transporter interactions.

Acknowledgments

This work has been supported by research grants from OTKA, ETT and OM, Hungary. Bal�zs Sarkadi is a recipient of a Howard Hughes International Scholarship.

5. Abbreviations

ABC transporters	ATP-Binding Cassette transporters
ADME	Absorption-Distribution-Metabolism-Excretion
ALD	Adrenoleukodystrophy
AM	acetoxy methylester
BBB	blood-brain-barrier
CNS	central nervous system
CSF	cerebrospinal fluid
DDI	drug-drug interactions
GSH	free gluthathione
MDR1	ABCB1, human multidrug transporter (P-glycoprotein)
MRP1	ABCC1, human multidrug resistance protein
MXR	ABCG2 protein
R123	Rhodamine 123
Sf9 cells	Spodoptera frugiperda ovarian cells.

6. Bibliography

1. Endicott, J.A. and Ling, V. Annu.Rev.Biochem., 1989, 58, 137-171.

2. Higgins, C.F. Ann.Rev.Cell Biol., 1992, 8, 67-113.

3. Gottesman, M.M. and Pastan, I. Annu.Rev.Biochem., 1993, 62, 385-427.

4. Gottesman, M.M.; Fojo, T.; Bates, S.E. Nat. Rev. Cancer, 2002, 2 (1), 48-58.

5. Klein, I.; Sarkadi, B.; V�radi, A. Biochim. Biophys. Acta, 1999, 1461 (2), 237-262.

6. Ueda, K.; Taguchi, Y.; Morishima, M. Semin Cancer Biol., 1997, 8 (3), 151-159.

7. V�radi, A.; Tusn�dy, G.E.; Sarkadi, B. In ABC Proteins From Bacteria to Man, Holland, I.B.; Cole, S.P.C.; Kuchler, K.; Higgins, C.F. Ed.; Elsevier Science, 2003, pp. 37-47.

8. Karpowich, N.; Martsinkevich, O.; Millen, L.; Yuan, Y.R.; Dai, P.L.; MacVey, K.; Thomas, P.J.; Hunt, J.F. Structure, 2001, 9 (7), 571-86.

9. Dean, M.; Rzhetsky, A.; Allikmets, R. Genome Res., 2001, 11 (7), 1156-1166.

10. Linton, K.J.; Rosenberg, MF.; Kerr, I.D.; Higgins, C.F. In ABC Proteins From Bacteria to Man, Holland, I.B.; Cole, S.P.C.; Kuchler, K.; Higgins, C.F. Ed.; Elsevier Science, 2003, pp. 65-80

11. Bates, S. In ABC Proteins From Bacteria to Man, Holland, I.B.; Cole, S.P.C.; Kuchler, K.; Higgins, C.F. Ed.; Elsevier Science, 2003, pp. 359-391.

12. Arnould, I.; Schriml, L.; Prades, C.; Lachtermacher-Triunfol, M.; Schneider, T.; Maintoux, C. GeneScreen, 2001, 1, 157-164.

13. Young, S.G. and Fielding, C.J. Nat. Genet, 1999, 22 (4), 316-318.

14. Bodzioch, M.; Orso, E.; Klucken, J.; Langmann, T.; Bottcher, A.; Diederich, W.; Drobnik, W.; Barlage, S.; Buchler, C.; Porsch-Ozcurumez, M.; Kaminski, W.E.; Hahmann, H.W.; Oette, K.; Rothe, G.; Aslanidis, C.; Lackner, K.J.; Schmitz, G. Nat Genet. 1999, 22 (4), 347-51.

15. Rust, S.; Rosier, M.; Funke, H.; Real, J.; Amoura, Z.; Piette, J.C.; Deleuze, J.F.; Brewer, H.B.; Duverger, N.; Denefle, P.; Assmann, G. Nat Genet., 1999, 22 (4), 352-5.

16. Kaminski, W.E.; Orso, E.; Diederich, W.; Klucken, J.; Drobnik, W.; Schmitz, G. Biochem Biophys Res Commun., 2000, 273 (2), 532-8.

17. Allikmets, R.; Singh, N.; Sun, H.; Shroyer, N.F.; Hutchinson, A.; Chidambaram, A.; Gerrard, B.; Baird, L.; Stauffer, D.; Peiffer, A.; Rattner, A.; Smallwood, P.; Li, Y.; Anderson, K.L.; Lewis, R.A.; Nathans, J.; Leppert, M.; Dean, M.; Lupski, J.R. Nat. Genet, 1997, 15 (3), 236-246.

18. Allikmets, R. Am. J. Hum. Genet., 2000, 67 (4), 793-799.

19. Juliano, R.L. and Ling, V.A. Biochim. Biophys. Acta, 1976, 455 (1), 152-162.

20. Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M.M.; Pastan, I.; Willingham, M.C. Proc. Natl. Acad. Sci. U S A. 1987, 84 (21) 7735-7738.

21. Abele, R.; Tampe, R. Biochim Biophys Acta. 1999 1461 (2), 405-19.

22. van Helvoort, A.; Smith, A.J.; Sprong, H.; Fritzsche, I.; Schinkel, A.H.; Borst, P.; van Meer, G. Cell, 1996, 87 (3), 507-517.

23. Deleuze, J.F.; Jacquemin, E.; Dubuisson, C.; Cresteil, D.; Dumont, M.; Erlinger, S.; Bernard, O.; Hadchouel, M. Hepatology, 1996, 23 (4) 904-8.

24. de Vree, J.M.; Jacquemin, E.; Sturm, E.; Cresteil, D.; Bosma, P.J.; Aten, J.; Deleuze, J.F.; Desrochers, M.; Burdelski, M.; Bernard, O.; Oude Elferink, R.P.; Hadchouel, M. Proc Natl Acad Sci U S A., 1998, 6, 95 (1) 282-7.

25. Strautnieks, S.S.; Bull, L.N.; Knisely, A.S.; Kocoshis, S.A.; Dahl, N.; Arnell, H.; Sokal, E.; Dahan, K.; Childs, S.; Ling, V.; Tanner, M.S.; Kagalwalla, A.F.; Nemeth, A.; Pawlowska, J.; Baker, A.; Mieli-Vergani, G.; Freimer, N.B.; Gardiner, R.M.; Thompson, R.J. Nat. Genet. 1998, 20 (3), 233-238.

26. Zhang, F.; Zhang, W.; Liu, L.; Fisher, C.L.; Hui, D.; Childs, S.; Dorovini-Zis, K.; Ling, V. J Biol Chem. 2000, 275 (30), 23287-94.

27. Cole, S.P.; Bhardwaj, G.; Gerlach, J.H.; Mackie, J.E.; Grant, C.E.; Almquist, K.C.; Stewart, A.J.; Kurz, E.U.; Duncan, A.M.; Deeley, R.G. Science, 1992, 258 (5088), 1650-1654.

28. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. J. Natl. Cancer. Inst., 2000, 92 (16), 1295-1302.

29. Deeley, R.G. and Cole, S.P.C. Semin.Cancer Biol., 1997, 8 (3), 193-204.

30. Paulusma, C.C.; Bosma, P.J.; Zaman, G.J.; Bakker, C.T.; Otter, M.; Scheffer, G.L.; Scheper, R.J.; Borst, P.; Oude Elferink, R.P. Science, 1996, 271 (5252), 1126-1128.

31. Cole, S.P.; Sparks, K.E.; Fraser, K.; Loe, D.W.; Grant, C.E.; Wilson, G.M.; Deeley, R.G. Cancer Res., 1994, 54 (22), 5902-10.

32. Leslie, E.M.; Deeley, R.G.; Cole, S.P. Toxicology. 2001, 167, (1), 3-23.

33. Kartenbeck, J.; Leuschner, U.; Mayer, R.; Keppler, D. Hepatology, 1996, 23 (5),1061-6.

34. Paulusma, C.C.; Kool, M.; Bosma, P.J.; Scheffer, G.L.; ter Borg, F.; Scheper, R.J.; Tytgat, G.N.; Borst, P.; Baas, F.; Oude Elferink, R.P. Hepatology, 1997, 25 (6), 1539-1542.

35. Kool, M.; van der Linden, M.; de Haas, M.; Scheffer, G.L.; de Vree, J.M.; Smith, A.J.; Jansen, G.; Peters, G.J.; Ponne, N.; Scheper, R.J.; Elferink, R.P.; Baas, F.; Borst, P. Proc Natl Acad Sci USA, 1999, 96 (12), 6914-9.

36. Konig, J.; Rost, D.; Cui, Y.; Keppler, D. Hepatology, 1999, 29 (4),1156-63

37. Schuetz, J.D.; Connelly, M.C.; Sun, D.; Paibir, S.G.; Flynn, P.M.; Srinivas, R.V.; Kumar. A.; Fridland, A. Nat. Med., 1999, 5, (9) 1048-51.

38. Rius, M.; Nies, A.T.; Hummel-Eisenbeiss, J.; Jedlitschky, G.; Keppler, D. Hepatology. 2003, 38 (2), 374-84.

39. Wijnholds, J.; Mol, C.A.; van Deemter, L.; de Haas, M.; Scheffer, G.L.; Baas, F.; Beijnen, J.H.; Scheper, R.J.; Hatse, S.; De Clercq, E.; Balzarini, J.; Borst, P. Proc. Natl. Acad. Sci. U S A., 2000, 97, (13) 7476-81.

40. Ilias, A.; Urban, Z.; Seidl, T.L.; Le Saux, O.; Sinko, E.; Boyd, C.D.; Sarkadi, B.; Varadi, A. J. Biol. Chem., 2002, 277 (19), 16860-7.

41. Belinsky, M.G.; Chen, Z.S.; Shchaveleva, I.; Zeng, H.; Kruh, G.D. Cancer Research, 2002, 62 (21), 6172-7.

42. Tammur, J.; Prades, C.; Arnould, I.; Rzhetsky, A.; Hutchinson, A.; Adachi, M.; Schuetz, J.D.; Swoboda, K.J.; Ptacek, L.J.; Rosier, M. Dean, M.; Allikmets, R. Gene. 2001, 273 (1):89-96.

43. Rommens, J.M.; Iannuzzi, M.C.; Kerem, B.; Drumm, M.L.; Melmer, G.; Dean, M.; Rozmahel, R.; Cole, J.L.; Kennedy, D.; Hidaka, N.; et al. Science, 1989, 245 (4922), 1059-65.

44. Hanrahan, J.W.; Gentzsch, M.; Riordan, J.R. In ABC Proteins From Bacteria to Man, Holland, I.B.; Cole, S.P.C.; Kuchler, K.; Higgins, C.F. Ed.; Elsevier Science, 2003, pp. 589-618.

45. Aguilar-Bryan, L.; Nichols, C.G.; Wechsler, S.W.; Clement, J.P.; Boyd, A.E.; Gonzalez, G.; Herrera-Sosa, H.; Nguy, K.; Bryan, J.; Nelson, D.A. Science. 1995, 268 (5209), 423-6.

46. Mosser, J.; Douar, A.M.; Sarde, C.O.; Kioschis, P.; Feil, R.; Moser, H.; Poustka, A.M.; Mandel, J.L.; Aubourg, P. Nature, 1993, 361 (6414), 726-730.

47. Chen, H.; Rossier, C.; Lalioti, M.D.; Lynn, A.; Chakravarti, A.; Perrin, G.; Antonarakis, S.E. Am. J. Hum. Genet., 1996, 59, (1), 66-75.

48. Klucken, J.; Buchler, C.; Orso, E.; Kaminski, W.E.; Porsch-Ozcurumez, M.; Liebisch, G.; Kapinsky, M.; Diederich, W.; Drobnik, W.; Dean, M.; Allikmets, R.; Schmitz, G. Proc Natl Acad Sci USA, 2000, 97, (2), 817-822.

49. Berge, K.E.; Tian, H.; Graf, G.A.; Yu, L.; Grishin, N.V.; Schultz, J.; Kwiterovich, P.; Shan, B.; Barnes, R.; Hobbs, H.H. Science, 2000, 290 (5497),1771-1775.

50. Shulenin, S.; Schriml, L.M.; Remaley, A.T.; Fojo, S.; Brewer, B.; Allikmets, R.; Dean, M. Cytogenet. Cell Genet., 2001, 92 (3-4), 204-208.

51. Allikmets, R.; Schriml, L.M.; Hutchinson, A.; Romano-Spica, V.; Dean, M. Cancer Res., 1998, 58 (23), 5337-5339.

52. Doyle, L.A.; Yang, W.; Abruzzo, L.V.; Krogmann, T.; Gao, Y.; Rishi, A.K.; Ross, D.D. Proc Natl Acad Sci USA, 1998, 95 (26) 15665-15670.

53. Miyake, H.; Tolcher, A.; Gleave, M.E. Cancer Res., 1999, 59 (16), 4030-4.

54. Loo, T.W.; Clarke, D.M. J. Biol. Chem., 2001, 276 (40), 36877-36880.

55. Higgins, C.F. and Gottesman, M.M. Trends. Biochem. Sci., 1992, 17 (1), 18-21.

56. Qian, Y.M.; Qiu, W.; Gao, M.; Westlake, C.J.; Cole, S.P.; Deeley, R.G. J. Biol. Chem., 2001, 276 (42), 38636-38644.

57. Homolya, L.; Varadi, A.; Sarkadi, B. Biofactors, 2003, 17 (1-4), 103-14.

58. Ambudkar, S.V.; Dey, S.; Hrycyna, C.A.; Ramachandra, M.; Pastan, I.; Gottesman, M.M. Annu Rev Pharmacol Toxicol. 1999, 39, 361-98.

59. Loo, T.W.; Clarke, D.M. J Biol Chem., 2001, 276 (18), 14972-9.

60. Szab�, K.; Welker, E.; Bakos, �.; M�ller, M.; Roninson, I.; Varadi, A.; Sarkadi, B. J. Biol. Chem. 1998, 273 (17), 10132-10138.

61. Hrycyna, C.A.; Ramachandra, M.; Ambudkar, S.V.; Ko, Y.H.; Pedersen, P.L.; Pastan, I.; Gottesman, M.M. J. Biol. Chem., 1998, 273 (27), 16631-16634.

62. Hrycyna, C.A.; Airan, L.E.; Germann, U.A.; Ambudkar, S.V.; Pastan, I.; Gottesman, M.M. Biochemistry, 1998, 37 (39), 13660-13673.

63. Sauna, Z.E.; Ambudkar, S.V. J. Biol. Chem., 2001, 276 (15), 11653-11661.

64. Senior, A.E.; Gadsby, D.C. Semin Cancer Biol. 1997, 8 (3), 143-50.

65. Ozvegy, C.; Varadi, A.; Sarkadi, B. J Biol Chem, 2002, 277 (50), 47980-90.

66. Rao, V.V.; Dahlheimer, J.L.; Bardgett, M.E.; Snyder, A.Z.; Finch, R.A.; Sartorelli, A.C.; Piwnica-Worms, D. Proc. Natl. Acad. Sci., 1999, 96 (7), 3900-3905.

67. Bart, J.; Groen, H.J.; Hendrikse, N.H.; van der Graaf, W.T.; Vaalburg, W.; de Vries, E.G. Cancer Treat. Rev., 2000, 26 (6), 449-462.

68. Bates, S.E.; Robey, R.; Miyake, K.; Rao, K.; Ross, D.D.; Litman, T. J. Bioenerg. Biomembr., 2001, 33 (6) 503-511.

69. Zhou, S.; Schuetz, J.D.; Bunting, K.D.; Colapietro, A.M.; Sampath, J.; Morris, J.J.; Lagutina, I.; Grosveld, G.C.; Osawa, M.; Nakauchi, H.; Sorrentino, B.P. Nat. Med., 2001, 7 (9), 1028-1034.

70. Kipp, H.; Arias, I.M. Semin. Liver Dis., 2000, 20 (3), 339-351.

71. Jonker, J.W.; Smit, J.W.; Brinkhuis, R.F.; Maliepaard, M.; Beijnen, J.H.; Schellens, J.H.; Schinkel, A.H. J. Natl. Cancer Inst., 2000, 92 (20), 1651-1656.

72. Ozvegy, C.; Litman, T.; Szakacs, G.; Nagy, Z.; Bates, S.; Varadi, A.; Sarkadi, B. Biochem Biophys Res Commun., 2001, 285 (1), 111-7.

73. Bodo, A., Bakos, E., Szeri, F., Varadi, A., Sarkadi, B.; J Biol Chem. 2003, 278, (26), 23529-37

74. Zelcer, N.; Huisman, M.T.; Reid, G.; Wielinga, P.; Breedveld, P.; Kuil, A.; Knipscheer, P.; Schellens, J.H.; Schinkel, A.H.; Borst, P. J Biol Chem. 2003, 278, (26), 23538-44. Epub 2003 Apr 17

75. Goldstein, L.J.; Pastan, I.; Gottesman, M.M. Crit Rev Oncol Hematol. 1992, 12 (3), 243-53.

76. Borst, P.; Elferink, R.O. Annu Rev Biochem., 2002, 71, 537-92. Epub 2001 Nov 09.

77. Borst, P.; Kool, M.; Evers, R. Semin Cancer Biol., 1997, 8 (3) 205-13.

78. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J. Biochim Biophys Acta., 1999, 1461 (2) 347-57.

79. Hipfner, D.R.; Deeley, R.G.; Cole, S.P. Biochim Biophys Acta. 1999, 1461 (2), 359-76.

80. Hirohashi, T.; Suzuki, H.; Sugiyama, Y. J Biol Chem., 1999, 274 (21), 15181-5.

81. Litman, T.; Druley, T.E.; Stein, W.D.; Bates, S.E. Cell Mol Life Sci, 2001, 58 (7), 931-59.

82. Lecureur, V.; Courtois, A.; Payen, L.; Verhnet, L.; Guillouzo, A.; Fardel, O. Toxicology., 2000, 153 (1-3), 203-19.

83. Smith, A.J.; van Helvoort, A.; van Meer, G.; Szabo, K.; Welker, E.; Szakacs, G,; Varadi, A.; Sarkadi, B.; Borst, P. J Biol Chem., 2000, 275 (31), 23530-9.

84. List, A.F.; Kopecky, K.J.; Willman, C.L.; Head, D.R.; Persons, D.L.; Slovak, M.L.; Dorr, R.; Karanes, C.; Hynes, H.E.; Doroshow, J.H.; Shurafa, M.; Appelbaum, F.R. Blood, 2001, 98 (12), 3212-20.

85. Krishna, R.; Mayer, L.D. Eur J Pharm Sci., 2000, 11 (4), 265-83.

86. Sharom, F.J.; Yu, X.; Lu, P.; Liu, R.; Chu, J.W.; Szabo, K.; Muller, M.; Hose, C.D.; Monks, A.; Varadi, A.; Seprodi, J.; Sarkadi, B. Biochem Pharmacol. 1999, 58 (4), 571-86.

87. Oram, J.F.; Lawn, R.M. J Lipid Res., 2001, 42 (8), 1173-9.

88. Srivastava, N. Mol Cell Biochem., 2002, 237 (1-2), 155-64.

89. Schmitz, G.; Langmann, T. Curr Opin Lipidol., 2001, 12 (2), 129-40.

90. Oram, J.F. Trends Mol Med., 2002, 8 (4), 168-73.

91. Oram, J.F. Curr Opin Lipidol., 2002, 13 (4), 373-81.

92. Schmitz, G.; Kaminski, W.E.; Front Biosci., 2001, 6, D505-14.

93. Santamarina-Fojo, S.; Remaley, A.T.; Neufeld, E.B.; Brewer, H.B. Jr. J Lipid Res., 2001, 42 (9), 1339-45.

94. Graf, G.A.; Li, W.P.; Gerard, R.D.; Gelissen, I.; White, A.; Cohen, J.C.; Hobbs, H.H. J Clin Invest., 2002, 110 (5), 659-69.

95. Bunting, K.D.; Zhou, S.; Lu, T.; Sorrentino, B.P. Blood., 2000, 96 (3), 902-9.

96. Hafkemeyer, P.; Licht, T.; Pastan, I.; Gottesman, M.M. Hum Gene Ther., 2000, 11, (4), 555-65.

97. Schiedlmeier, B.; Schilz, A.J.; Kuhlcke, K.; Laufs, S.; Baum, C.; Zeller, W.J.; Eckert, H.G.; Fruehauf, S. Hum Gene Ther., 2002, 13 (2), 233-42.

98. Sellers, S.E.; Tisdale, J.F.; Agricola, B.A.; Metzger, M.E.; Donahue, R.E.; Dunbar, C.E.; Sorrentino, B.P. Blood., 2001, 97 (6), 1888-91.

99. Smeets, M.E.; Raymakers, R.A.; Vierwinden, G.; Pennings, A.H.; Wessels, H.; de Witte, T. Blood., 1999, 94 (7), 2414-23.

100. Ross, D.D.; Yang, W.; Abruzzo, L.V.; Dalton, W.S.; Schneider, E.; Lage, H.; Dietel, M.; Greenberger, L.; Cole, S.P.; Doyle, L.A. J Natl Cancer Inst. 1999, 91 (5), 429-33.

101. Litman, T.; Brangi, M.; Hudson, E.; Fetsch, P.; Abati, A.; Ross, D.D.; Miyake, K.; Resau, J.H.; Bates, S.E. J Cell Sci., 2000, 3 ( Pt 11), 2011-21.

102. Bunting, K.D. Stem Cells., 2002, 20 (1), 11-20.

103. Kim, M.; Turnquist, H.; Jackson, J.; Sgagias, M.; Yan, Y.; Gong, M.; Dean, M.; Sharp, J.G.; Cowan, K. Clin Cancer Res, 2002, 8 (1), 22-28.

104. Honjo, Y.; Hrycyna, C.A.; Yan, Q.W.; Medina-Perez, W.Y.; Robey, R.W.; van de Laar, A.; Litman, T.; Dean, M.; Bates, S.E. Cancer Res., 2001, 61 (18), 6635-9.

105. Aungst, B.J.; J Pharm Sci, 1993, 82 (10), 979-987.

106. Kruijtzer, C.M.; Beijnen, J.H.; Schellens, J.H. The Oncologist, 2002, 7 (6), 516-30.

107. Taipalensuu, J.; Tornblom, H.; Lindberg, G.; Einarsson, C.; Sjoqvist, F.; Melhus, H.; Garberg, P.; Sjostrom, B.; Lundgren, B.; Artursson, P. J Pharm Exp Ther, 2001, 299 (1), 164.

108. Sparreboom, A.; van Asperen, J.; Mayer, U.; Schinkel, A.H.; Smit, J.W.; Meijer, D.K.; Borst, P.; Nooijen, W.J.; Beijnen, J.H.; van Tellingen, O. Proc Nat Acad Sci USA, 1997, 94 (5), 2031-5.

109. van Asperen, J.; van Tellingen, O.; Sparreboom, A.; Schinkel, A.H.; Borst, P.; Nooijen, W.J.; Beijnen, J.H. Br J Cancer, 1997, 76 (9), 1181-3.

110. Dietrich, C.G.; de Waart, D.R.; Ottenhoff, R.; Schoots, I.G.; Elferink, R.P. Mol Pharmacol, 2001, 59 (5), 974-80.

111. Lin H.J. and Yamazaki M, Clin Pharmacokinet, 2003, 42, (1), 59-98

112. Sugiyama et al. (1999) J Cont Release 62: 179-186

113. Mahar Doan, K.M.; Humphreys, J.E.; Webster, L.O.; Wring, S.A.; Shampine, L.J.; Serabjit-Singh, C.J.; Adkison, K.K.; Polli, J.W. J Pharm Exp Therapeutics, 2002, 303 (3), 1029-1037.

114. Ghersi-Egea, J.F.; Strazielle, N. Microsc Res Techn, 2001, 52 (1), 83-88.

115. Wijnholds, J.; deLange, E.C.; Scheffer, G.L.; van den Berg, D.J.; Mol, C.A.; van der Valk, M.; Schinkel, A.H.; Scheper, R.J.; Breimer, D.D.; Borst, P. J Clin Invest, 2000, 105 (3), 279-85.

116. Potschka, H.; Fedrowitz, M.; Loscher, W. J Pharmacol Exp Ther, 2003, 306 (1), 124-31.

117. Fricker, G.; Nobmann, S.; Miller, D.S. Br J Pharmacol, 2002, 135 (5), 1308-14.

118. Cooray, H.C.; Blackmore, C.G.; Maskell, L.; Barrand, M.A. Neuroreport, 2002, 13 (16), 2059-63.

119. Lankas, G.R.; Wise, L.D.; Cartwright, M.E.; Pippert, T.; Umbenhauer, D.R. Reprod Toxicol, 1998, 12 (4), 457-463.

120. St-Pierre, M.V.; Serrano, M.A.; Macias, R.I.; Dubs, U.; Hoechli, M.; Lauper, U.; Meier, P.J.; Marin, J.J. Am J Physiol Regul Integr Comp Physiol., 2000, 279 (4), R1495-503.

121. von Richter, O.; Greiner, B.; Fromm, M.F.; Fraser, R.; Omari, T.; Barclay, M.L.; Dent, J.; Somogyi, A.A.; Eichelbaum, M. Clin Pharmacol Ther, 2001, 70 (3), 217-227.

122. Mayer, U.; Wagenaar, E.; Dorobek, B.; Beijnen, J.H.; Borst, P.; Schinkel, A.H. J Clin Invest, 1997, 100 (10), 2430-2436.

123. Hochman, J.H.; Chiba, M.; Yamazaki, M.; Tang, C.; Lin, J.H. J Pharmacol Exp Ther, 2001, 298 (1), 323-330.

124. Lin, J.H.; Chiba, M.; Chen, I.W.; Nishime, J.A.; deLuna, F.A.; Yamazaki, M.; Lin, Y.J. Drug Metab Dispos, 1999, 27 (10), 1187-1193.

125. Kawahara, M.; Sakata, A.; Miyashita, T.; Tamai, I.; Tsuji, A. J Pharm Sci, 1999, 88 (12), 1281-1287.

126. van Asperen, J.; van Tellingen, O.; Tijssen, F.; Schinkel, A.H.; Beijnen, J.H. Br J Cancer, 1999, 79 (1), 108-13.

127. Smit, J.W.; Schinkel, A.H.; Weert, B.; Meijer, D.K. Br J Pharmacol, 1998, 124 (2), 416-424.

128. Stieger, B.; Fattinger, K.; Madon, J.; Kullak-Ublick, G.A.; Meier, P.J. Gastroenterology, 2000, 118 (2), 422-430.

129. Greiner, B.; Eichelbaum, M.; Fritz, P.; Kreichgauer, H.P.; von Richter, O.; Zundler, J.; Kroemer, H.K J Clin Invest, 1999, 104 (2), 147-153.

130. Hamman, M.A.; Bruce, M.A.; Haehner-Daniels, B.D.; Hall, S.D. Clin Pharmacol Ther, 2001, 69 (3), 114-121.

131. Deferme, S.; Van Gelder, J.; Augustijns, P. J Pharm Pharmacol, 2002, 54 (9), 1213-1219.

132. Marques-Santos, L.F.; Bernardo, R.R.; de Paula, E.F.; Rumjanek, V.M. Pharmacol Toxicol, 1999, 84 (3), 125-129.

133. Schinkel, A.H.; Smit, J.J.; van Tellingen, O.; Beijnen, J.H.; Wagenaar, E.; van Deemter, L.; Mol, C.A.; van der Valk, M.A.; Robanus-Maandag, E.C.; te Riele, H.P. et al. Cell, 1994, 77 (4), 491-502.

134. Kala, S.V.; Neely, M.W.; Kala, G.; Prater, C.I.; Atwood, D.W.; Rice, J.S.; Lieberman, M.W. J Biol Chem, 2000, 275 (43), 33404-33408.

135. Scheffer, G.L.; Kool, M.; de Haas, M.; de Vree, J.M.; Pijnenborg, A.C.; Bosman, D.K.; Elferink, R.P.; van der Valk, P.; Borst, P.; Scheper, R.J. Lab Invest, 2002, 82 (2), 193-201.

136. Hirohashi, T.; Suzuki, H.; Ito, K.; Ogawa, K.; Kume, K.; Shimizu, T.; Sugiyama, Y. Mol Pharmacol, 1998, 53 (6), 1068-1075.

137. Fattinger, K.; Funk, C.; Pantze, M.; Weber, C.; Reichen, J.; Stieger, B.; Meier, P.J. Clin Pharmacol Ther, 2001, 69 (4), 223-231.

138. Funk, C.; Ponelle, C.; Scheuermann, G.; Pantze, M. Mol Pharmacol, 2001, 59 (3), 627-635.

139. Zhang, Y.; Bachmeier, C.; Miller, D.W. Adv Drug Deliv Rev, 2003, 55 (1), 31-51.

140. Zeng, H.; Liu, G.; Rea, P.A.; Kruh, G.D.; Cancer Res. 2000, 60 (17), 4779-84.

141. Sarkadi, B.; Price, E.M.; Boucher, R.C.; Germann, U.A.; Scarborough, G.A. J Biol Chem. 1992, 267 (7), 4854-8.

142. Breuninger, L.M.; Paul, S.; Gaughan, K.; Miki, T.; Chan, A.; Aaronson, S.A.; Kruh, G.D. Cancer Res, 1995, 55 (22), 5342-7.

143. Reid, G.; Wielinga, P.; Zelcer, N.; De Haas, M.; Van Deemter, L.; Wijnholds, J.; Balzarini, J.; Borst, P. Mol Pharmacol. 2003, 63 (5), 1094-103.

144. Feller, N.; Kuiper, C.M.; Lankelma, J.; Ruhdal, J.K.; Scheper, R.J.; Pinedo, H.M.; Broxterman, H.J. Br J Cancer, 1995, 72 (3), 543-9. Erratum in: Br J Cancer 1996, 74, 2042.

145. Weaver, J.L.; Pine, P.S.; Aszalos, A.; Schoenlein, P.V.; Currier, S.J.; Padmanabhan, R.; Gottesman, M.M. Exp Cell Res 1991, 196 (2), 323-329.

146. Lee, J.S.; Paull, K.; Alvarez, M.; Hose, C.; Monks, A.; Grever, M.; Fojo, A.T.; Bates, S.E. Mol Pharmacol 1994, 46 (4), 627-638.

147. Chaudhury, P.M. and Roninson, I.B. Cell, 1991, 66 (1), 85-94.

148. Homolya, L.; Hollo, Z.; Germann, U.A.; Pastan, I.; Gottesman, M.M.; Sarkadi, B. J Biol Chem. 1993, 268 (29), 21493-6.

149. Haugland, R.P. and Larison, K.D. In Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, OR, 1992-94, pp 163, 172-173

150. Hollo, Z.; Homolya, L.; Hegedus, T.; Sarkadi, B. FEBS Lett 1996, 383 (1-2), 99-104.

151. Karaszi, E.; Jakab, K.; Homolya, L.; Szakacs, G.; Hollo, Z.; Telek, B.; Kiss, A.; Rejto, L.; Nahajevszky, S.; Sarkadi, B.; Kappelmayer, J. Br J Hematol 2001, 112 (2), 308-314.

152. Gaillard, P.J.; van der Sandt, I.C.; Voorwinden, L.H.; Vu, D.; Nielsen, J.L.; de Boer, A.G.; Breimer, D.D. Pharm Res, 2000, 17 (10), 1198-1205.

153. Adachi, Y.; Suzuki, H.; Sugiyama, Y. Pharm. Res, 2001, 18 (12), 1660-1668.

154. Sukhai, M. and Piquette-Miller, M. J Pharm Pharmaceut Sci, 2000<, 3 (2), 268-280.

155. Mechetner, E.B.; Roninson, I.B. Proc Nat Acad Sci USA, 1992, 89 (13), 5824-8.

156. Mechetner, E.B.; Schott, B.; Morse, B.S.; Stein, W.D.; Druley, T.; Davis, K.A.; Tsuruo, T.; Roninson, I.B. Proc Nat Acad Sci USA, 1997, 94 (24), 12908-13.

157. Nagy, H.; Goda, K.; Arceci, R.; Cianfriglia, M.; Mechetner, E.; Szabo, G. Jr. Eur J Biochem, 2001, 268 (8), 2416-20.

158. Tabas, L.B. and Dantzig, A.H. Anal Biochem, 2002, 310 (1), 61-66.

159. Bakos �., Evers R., Sink� E., V�radi A., Borst P., Sarkadi B.: Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol, 2000; 57:760-768

160. Holl� Zs., Homolya L., Davis C.W., Sarkadi B.: Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta 1994; 1191: 384-388

161. Homolya L., Holl� Z., M�ller M., Mechetner E.B., Sarkadi B.: A new method for quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells. Br J Cancer 1996; 73: 849-855

162. Sarkadi B., Muller M., Homolya L. Hollo Zs., Seprodi J., Germann U.A., Gottesman M.M., Price E.M., Boucher R.C.: Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J. 1994, 8: 766-770

163. Szabo K., Welker E., Bakos E., Muller M., Roninson I., Varadi A., Sarkadi B.: Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains. J Biol Chem 1998; 273: 10132-10138

164. Taguchi Y., Yoshida A., Takada Y., Komano T., Ueda K.: Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP). FEBS Lett. 1997; 401: 11-14

165. Bod� A., Bakos �., Szeri F., V�radi A., Sarkadi B.: The role of multidrug transporters in drug availability, metabolism and toxicity. Toxicology Letters Toxicol Lett. 2003 Apr 11;140-141:133-43.

166. Madon J, Eckhardt U, Gerloff T, Stieger B, Meier PJ.: Functional expression of the rat liver canalicular isoform of the multidrug resistance-associated protein. FEBS Lett. 1997 Apr 7;406(1-2):75-8.

167. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, Meier PJ.: The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem. 1998 Apr 17;273(16):10046-50

168. Byrne JA, Strautnieks SS, Mieli-Vergani G, Higgins CF, Linton KJ, Thompson RJ.: The human bile salt export pump: characterization of substrate specificity and identification of inhibitors. Gastroenterology. 2002 Nov;123(5):1649-58.

169. No� J, Stieger B, Meier PJ.: Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology. 2002 Nov;123(5):1659-66.

170. Funk C, Pantze M, Jehle L, Ponelle C, Scheuermann G, Lazendic M, Gasser R.: Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology. 2001 Oct 5;167(1):83-98.

171. Stieger B, Fattinger K, Madon J, Kullak-Ublick GA, Meier PJ.: Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology. 2000 Feb;118(2):422-30.

172. Kullak-Ublick GA, Stieger B, Meier PJ.: Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology. 2004 Jan;126(1):322-42

173. Faber KN, Muller M, Jansen PL.: Drug transport proteins in the liver. Adv Drug Deliv Rev. 2003 Jan 21;55(1):107-24.

174. Kaplowitz N, DeLeve LD, Drug-induced liver disease. Eds Part II: Jansen Peter LM, M�ller M.: The role of membrane transport in drug-induced hepatotoxicity and cholestasis; Marcel Dekker. 2003

175. Cserepes J, Szentpetery Z, Seres L, Ozvegy-Laczka C, Langmann T, Schmitz G, Glavinas H, Klein I, Homolya L, Varadi A, Sarkadi B, Elkind NB.: Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization. Biochem Biophys Res Commun. 2004 Jul 30;320(3):860-7.

176. Van Aubel RA, Smeets PH, van den Heuvel JJ, and Russel FG. Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites. Am J Physiol Renal Physiol 288: F327-F333, 2004

177. Van Aubel RA, Smeets PH, van den Heuvel JJ, Russel FG. Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites. Am J Physiol Renal Physiol. 2005 Feb;288(2):F327-33. Epub 2004 Sep 28. PMID: 15454390 [PubMed - indexed for MEDLINE]

178. Homolya L, Varadi A, Sarkadi B. Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. Biofactors. 2003;17(1-4):103-14. Review. PMID: 12897433 [PubMed - indexed for MEDLINE]

179. Van Aubel RA, Smeets PH, Peters JG, Bindels RJ, and Russel FG. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 13: 595-603, 2002

Scientific background

The Role Of ABC Transporters In Drug Resistance, Metabolism And Toxicity

Abstract

1. General overview of ABC transporters

2. ABC Transporters as Therapeutic Targets

3. Role of ABC Transporters in ADME/Toxicity properties of drugs

4. ABC transporter assay systems in drug research and development

5. Abreviations

6. Bibliography

Abstract

1. General overview of ABC transporters

2. ABC Transporters as Therapeutic Targets

3. Role of ABC transporters in modulation of ADME/Toxicology properties of drugs

4. ABC transporter assay systems in drug research and development

5. Abbreviations

6. Bibliography