S

Glucose facilitative family (SLC2)

Equilibrative nucleoside transporter family (SLC29)

Channel (pore)

Ionic channels

Figure 2 The classification of modes of solute (S) transport translocation between the extracellular (out) and intracellular (in) compartments. A few examples of transporter families are included.

primary active transport systems are coupled to an energy source that results in solar, electrical or chemical reactions, such as the hydrolysis of ATP by ion pumps (ATPases) and the ATP-binding cassette (ABC) transporters that transport the substrate in one defined direction. They are called primary transporters because no additional biochemical step is needed for solute transport. The combination of an energy-yielding reaction and transport occurs within a single catalytic cycle of the protein. The second group of transporters are the co-transporters; these use a voltage or/and ion gradient to transport both ions and solutes together. They are uniporters when only one species is transported, and symporters when both species are transported in the same direction, whereas the antiporters transport solutes and ions in opposite directions. The H + ion is the most common form of energy in prokaryotes, while Na + is more frequently

Apical membrane Basolateral membrane

Figure 3 This model shows how solutes (S) are transported in a mammalian epithelial cell having K+ channels and three types of transporters at the apical or basolateral membranes. The Na+ ,K+ -ATPase (the primary active transporter) generates a Na+ gradient, which provides the driving force for activating Na+ and H + fluxes via the Na+ ,H + antiporter (the secondary active transporter). T is the tertiary active transporter, which transports S molecules down the H + gradient in symporter mode.

Figure 3 This model shows how solutes (S) are transported in a mammalian epithelial cell having K+ channels and three types of transporters at the apical or basolateral membranes. The Na+ ,K+ -ATPase (the primary active transporter) generates a Na+ gradient, which provides the driving force for activating Na+ and H + fluxes via the Na+ ,H + antiporter (the secondary active transporter). T is the tertiary active transporter, which transports S molecules down the H + gradient in symporter mode.

encountered in eukaryotic cells.3 For example, several secondary transporters are involved in the entrance and exit of Na+ at both apical and basolateral membranes of intestinal and renal epithelial cells. Thus the rate of transport of solutes like sugars or amino acids depends on the extracellular concentration of Na+. The main role of the Na+, K+-ATPase system is to activate the cascades of multiple co-transport processes. These co-transporters are also called secondary or even tertiary transporters because the machinery of ion transport must be activated by one or two pumps before solute transport occurs (Figure 3). Thus the energy component is clearly critical and the consequence of a lack of ATP in cells, as occurs in ischemia, leads to a loss of all transport by the ABC transporters. Other sources of energy involve glutathione (GSH), HCO3, and dicarboxylate a-ketoglutarate (a-KG).

The biological significance of these proteins is highlighted by their universal distributions in both prokaryotes and eukaryotes. Transporters are involved in the biology of yeasts, plants, bacteria, and parasites, so that it is possible that these molecules in bacteria and parasites may raise resistance to drug therapy. Many of the major body functions in mammals, like gut absorption, glucose uptake by tissues, bile acid secretion into the gut from the liver, and the renal secretion or reabsorption of nutrients, are linked to the presence of protein transporters on both the plasma membranes of cells and in intracellular organelles like the endoplasmic reticulum, mitochondria, and endosomes. Therefore, the extracellular and intracellular trafficking of any solute, including exogenous compounds like drugs, depends directly on this network of protein transporters.4

5.04.3 Biology of Membrane Transport Proteins

5.04.3.1 Genes and Classification

Following the determination of the prokaryotic and eukaryotic genomes, Paulsen et al.5 determined the distribution of membrane transport proteins for all those organisms whose genomes had been completely sequenced. Paulsen predicted that 15% of the 23 000 genes in the human genome code for transport proteins; this would result in nearly 3500 transporters. Ward et al.6 assumed that 3-12% of the genes in bacterial genomes encode membrane transport proteins that can be vital for the efflux of antibiotics, the secretion of proteins or toxins, and other functions. But only about 10% of these proteins with known physiological function have been identified in mammalian cells, and only the members of two superfamilies are presently known to affect drug transport. They are the ABCs and solute carriers (SLCs). ABC proteins are widespread in all organisms, from bacteria to mammals, with about 600 referenced transporters, but only 48 genes have been identified in humans and no more than around nine ABCs have been shown to affect drug pharmacokinetics and pharmacodynamics.7 The SLC family, which may contain about 2000 members, is presently known to have 46 families, including 362 transporter genes with documented transport functions.8

The current status of transporter nomenclature and classification is in the same disarray as was that of the metabolizing enzymes in the early 1980s. However, the Human Genome Organization (HUGO) Nomenclature

Committee Database has provided a list of the ABC and SLC genes and defined families of these transporters.83 There are other classification systems, such as the Transport Classification Data Base (TCDB). This is analogous to the Enzyme Commission system for the classification of enzymes but also incorporates phylogenetic information.8b This review uses the HUGO as the primary reference for identifying genes and proteins. Human proteins (genes) are shown in capitals (e.g., ABCB1 (MDR1)), while rat and mouse proteins (genes) are indicated by an initial capital followed by small letters (proteins) and small letters (genes) (e.g., Abccl (mdrl)). Some transporter databases provide only certain properties of a specific class or group of membrane transporter proteins. But, altogether, they contain information on cDNA and amino sequences, gene family, putative membrane topology, driving force, transport direction, lists of substrates, inhibitors, inducers, transport kinetic data, tissue distribution in both humans and mice and rats, drug-drug interactions involving transporters, and altered functions caused by mutations or polymorphisms and the influence of associated diseases.9

5.04.3.2 Basic Structure

The primary and secondary structures of many transporters are known. The structures of many ABCs and SLCs remain to be determined due to the inherent difficulties in crystallizing transporters for the x-ray crystallographic analyses needed to prepare high-resolution three-dimensional (3D) structures. This needs to be addressed. Biochemical analyses of transporter proteins are more challenging than studies on soluble proteins because they are hydrophobic, high molecular weight proteins and represent a small percentage of the total cell proteins. They are also rather inaccessible because they are tightly inserted into the lipid bilayer. Nevertheless, considerable progress has been made in the past 10 years by combining several biophysical approaches to determine the structure and topology of membrane transporters in the absence of a crystal structure. For example, several homology models predicting the pharmacophore pattern for the transporter binding site(s) are now available tools for 3D structure-activity relationship (3D-QSAR) analyses (see 5.32 In Silico Models for Interactions with Transporters).

Drug transporters are integral membrane proteins that typically have 12 transmembrane domains (TMDs), although there are exceptions with 6, 8, 10, 11, 13, or even 17 TMDs. The TMDs are folded in a-helical structures within the membrane and linked at both sides by amino acid sequences floating in the internal or external cell environment. The amino acids in the external loop domains are frequently N-glycosylated, while those of the intracellular loops of both SLC and ABC proteins bear phosphorylation sites and one or two ATP binding domains in the ABCs. When observed in 3D structure, TMDs form a crown and look like a channel allowing communication between the two fluid spaces separated by the lipid bilayer. Many SLC transporters have 300-800 amino acid residues and a molecular mass of 40-90 kDa, while the ABC transporters are larger, with 1200-1500 residues and a mass of 140-190 kDa. Knowledge of the amino acid sequence can be used to classify transporters by family and subfamily according to the degree of amino acid homology. For example, a transporter protein is assigned to a specific SLC family if it has an amino acid sequence that is at least 20-25% identical to those of other members of that family. A new nomenclature system was recently proposed that is based on the classification of drug-metabolizing enzymes; the transport proteins of a superfamily are arranged in clusters of families (X40% identity) and subfamilies (X60% identity).12 Knowledge of the amino acid sequences is also extremely helpful for assessing the effect of a single mutation within the sequence that can induce a change in the conformation of the transport protein and alter its transport functions.

5.04.3.3 Transport Kinetics

Transport proteins may have one or more binding sites formed by amino acid sequences of the TDMs or the extracellular loops. Environmental factors, such as the extracellular or intracellular pH or the lipid bilayer fluidity, may influence the transport process. For example, the probability of a solute interacting with a membrane transport protein can depend on the permeability coefficient (Pe), which determines how fast a solute diffuses passively across the lipid bilayer. This condition is particularly relevant when solute molecules interact with the transport binding sites of the TDM inserted in the plasma membrane. The higher the Pe (<10 ~6cms _1) the lower the probability of the binding site interacting with the substrate because the residence time of the substrate molecules within the membrane space is too short. In contrast, a long substrate residence time within the membrane domain may facilitate the interaction between the transporter and the substrate.

Like drug-metabolizing enzymes, transporters are substrate specific and the kinetics of transport obey the capacity limit rules described by the Michaelis-Menten equation. The influx or efflux flux rate (Jin or Jex) for a single permeation process mediated by one transporter depends on the affinity of the transporter for its substrate (Km), which is the concentration at the half-maximal transport rate (Vmax):

Figure 4 Transport kinetics: relationships between the transport flux (J) and the concentration of substrate [S] in (a) influx and (b) efflux modes. Multiple components (diffusion and one or more forms of active transport) participate in the overall transport process. Kinetic parameters such as Km and Vmax (Jmax) can be derived from these plots.

Figure 4 Transport kinetics: relationships between the transport flux (J) and the concentration of substrate [S] in (a) influx and (b) efflux modes. Multiple components (diffusion and one or more forms of active transport) participate in the overall transport process. Kinetic parameters such as Km and Vmax (Jmax) can be derived from these plots.

The active process is more often coupled to a passive diffusion component, which is directly, proportional to the substrate concentration by a rate constant (kd) leading to an additional term in eqn [1]:

The permeation of a solute through biological membranes may be more complex than that described by the above equations, and results from the combination of several components associating more than one active process with the diffusion component. In this case, the net flux of the transport (Jnet) is calculated by adding all influx components and subtracting the efflux ones:

This last feature is not simply hypothetical; more and more examples of multicomponent permeation are now known. The impact of all these kinetic processes of varying complexity is illustrated in Figure 4. It can be seen that the superimposing passive diffusion and active influx and/or efflux processes leads to nonlinear relationships between Jnet and the substrate concentration. These relationships can be dissected into their diffusion and active component processes. This can be used to calculate the kinetic parameters of active transport (Km and Vmax) and to anticipate the risks of permeation saturation in situations such as exposure to a high dose of a drug or following drug-drug interactions.

5.04.4 Transporters in Drug Disposition

This section covers only those transporters that influence the A, D, or E of drugs and xenobiotics. They belong to either the ABC or the SLC superfamilies and are often classified according to the chemical nature of their substrates. Hence, they translocate organic anions or cations, peptides, or nucleosides. Most of them were first named according to their specific chemical substrate, such as the organic cation transporters (OCTs), or the organic anion transporters (OATs), before they were named using the HUGO nomenclature rules. They all have some common general properties: a broad specificity with frequent overlaps of substrate recognition, making them 'polyspecific transporters,' and they are present in several body tissues and organs. They frequently differ in the type of energy mechanism that catalyzes the transport reaction.

5.04.4.1 Adenosine Triphosphate-Binding Cassette Transporter Superfamily

The transporters of the ABC superfamily have one of three types of structure, but they all have one or two nucleotide-binding domains (NBDs) (Figure 5). Two sequence motifs located 200 amino acids apart in each NBD, designated Walker A and Walker B, are conserved among all ABC transporter superfamily members. The lysine residue in the Walker A motif is involved in the binding of the b-phosphate of ATP while the aspartic acid residue in the Walker B motif interacts with Mg2 + An additional element, the signature (C) motif with a highly conserved amino acid sequence, lies just upstream of the Walker B site. The precise function of this sequence has not yet been determined although it is directly implicated in the recognition, binding, and hydrolysis of ATP.13 The two NBDs are located in the cytoplasm and transfer the energy from ATP hydrolysis to transport the substrate. The main differences between the ABC transporters is in the number of TMDs in the hydrophobic membrane-spanning domain (MSD). The MSD itself consists of several transmembrane a-helices. The ABC transporters ABCB1 (MDR1) and ABCC4, 5, 8, and 9 (multidrug resistance associated proteins (MRPs)) have a classical 12-TMD body. The structures of ABCC1, 2, 3, 6, and 7 are similar in that they possess two ATP binding sites, but they also contain an additional MSD with five TMDs at the amino-terminal end, giving them a total of 17 TMDs (see Figure 5). At the other extreme, ABBG2, also called a 'half-transporter', contains six TMDs and one ATP binding region on the amino-terminal side, but this half-transporter is thought to homodimerize to form a functional transporter. Most of the ABC transporters in eukaryotes move compounds from the cytoplasm to the outside of the cell or into intracellular organelles, such as the endoplasmic

MSD0 MSD1 MSD2

MSD0 MSD1 MSD2

Figure 5 Topological models of the best characterized ABC transporters that confer drug resistance and transport drugs and toxicants. (a) The model of MRP1 resembles MRP2, 3; (b) the model of MDR1 (P-gp) resembles MRP4, 5, 8; and (c) the model of BCRP. MSD, membrane-spanning domain; NBD, nucleotide binding domain. Transmembrane a-helical domains (TMDs) are represented by red rectangular bars; N-glycosylation sites (indicated by branches) are present on extracellular protein loops. Cytoplasmic (in) and extracellular (out) orientations are indicated.

Figure 5 Topological models of the best characterized ABC transporters that confer drug resistance and transport drugs and toxicants. (a) The model of MRP1 resembles MRP2, 3; (b) the model of MDR1 (P-gp) resembles MRP4, 5, 8; and (c) the model of BCRP. MSD, membrane-spanning domain; NBD, nucleotide binding domain. Transmembrane a-helical domains (TMDs) are represented by red rectangular bars; N-glycosylation sites (indicated by branches) are present on extracellular protein loops. Cytoplasmic (in) and extracellular (out) orientations are indicated.

reticulum, mitochondria, and peroxisomes. This means that they are frequently called efflux pumps.14'15 Phylogenetic analysis has grouped the eukaryotic ABC genes into seven subfamilies (A-G).7 Only three of these subfamilies, B, C, and G, contain transporters that influence drug disposition. Table 1 shows the main properties of the nine single ABC transporters belonging to these three subfamilies.

5.04.4.1.1 ABCB subfamily

These full transporters include ABCB1 (MDR1), ABCB4 (MDR3), and ABCB11 (sister P-gp or BSEP). ABCB1 is also called P-glycoprotein (P-gp); it is a 170-kDa protein which was the first human ABC transporter cloned and has been shown to be responsible for the multidrug resistance (MDR) phenomenom that occurs with such anticancer agents as the anthracyclines, vinca alkaloids, and taxanes.16,17 P-gp is the product of two MDR genes in humans, MDR1 and MDR2 (also called MDR3), and only the MDR1 protein is involved in the MDR phenotype; the human MDR2 gene functions as a phospholipid translocase at the canalicular membrane of hepatocytes where it is important for the secretion of phosphatidyl choline into the bile. Two genes, mdrla and lb, result in a similar MDR phenotype in rodents.

P-gp is present mainly on the apical membrane of many secretory cells, including those of the intestine, liver, kidney, choroid plexus, and adrenal gland. In the placenta, P-gp is found on the apical surface of syncytiotrophoblasts, where it can protect the fetus from toxic xenobiotics. P-gp is also abundant on hematopoietic stem cells, where it may protect the cells from toxins, and on the luminal surface of endothelial cells forming physiological barriers such as the blood-testis barrier and the BBB.18

P-gp transports not only antineoplastic agents, but also a wide variety of substrates that are not structurally closely related: they are mostly hydrophobic compounds that are either neutral or positively charged, and are probably presented to the transporter binding sites directly from the lipid bilayer. The range of substrates that P-gp recognizes overlaps with those of the main drug-metabolizing enzymes CYP3A4/5. These enzymes are known to metabolize about 50% of the drugs currently on the market. Immunosuppressive agents (cyclosporin A and its analog PSC833), cardiac glycosides (digoxin), protease inhibitors (saquinavir and indinavir), antibiotics (rifampicin), calcium channel blockers (verapamil), and quinoline (quinidine) have been found to interact with P-gp as both substrates and inhibitors.19 It is now well accepted that P-gp has several drug-binding domains lying within the TMDs (see 5.32 In Silico Models for Interactions with Transporters). This helps to clarify why so many substrates are transported by P-gp. A complete catalytic cycle includes the hydrolysis of two ATP molecules, but the two events do not occur simultaneously, although both are needed to transport one substrate molecule. The substrate becomes bound to P-gp and the first ATP hydrolysis causes a change in the conformation of P-gp that releases the substrate to either the outer leaflet of membrane or the extracellular space. The second ATP hydrolysis allows the 're-set' of P-gp so that it can bind another molecule of substrate.20

The use of P-gp inhibitors to improve bioavailability of P-gp substrate drugs taken orally and their brain penetration has many clinical interests and is not limited to anticancer therapy. Extensive pharmacotherapy programs have tested three generations of chemical inhibitors. The first-generation inhibitors of P-gp were drugs like verapamil and cyclosporin A that act as competitive inhibitors. They failed in clinical application because they were bound poorly by P-gp; this meant that high doses had to be used, and that led to the concomitant inhibition of the main drug-metabolizing enzyme, CYP3A4, and hence pharmacokinetic interactions. The second-generation P-gp inhibitors, like the cyclosporin A analogue valspodar (PSC 833), are more potent but they also inhibit CYP3A4, again limiting their clinical application. The third-generation inhibitors, such as elacridar (GF120918), zosuquidar (LY335979), and tariquidar (XR9576), are more clinically useful as they are potent inhibitors of P-gp and do not interact with CYP3A4.21 The combination of P-gp on the apical or luminal membranes of epithelial or endothelial cells with its capacity to extrude compounds from the cell cytosol or to impede their entry into cells makes this transporter a strategic pharmacokinetic effector that limits absorption of a drug by the intestine, its distribution to the brain, testis, placenta, and stem cells, and enhances its elimination in the bile and urine. The pharmacological and toxicological effects of active drug transport by P-gp have been demonstrated in knockout mice that lack one or both of the murine homologs of the genes encoding human MDR1, Mdr1a, and Mdr1b.22 The two murine isoforms have approximately 30% amino acid identity with MDR1. Murine P-gps confer resistance to a similar spectrum of anticancer agents as does MDR1. Nevertheless, the two rodent isoforms differ in their tissue distribution. Mdr1a is relatively abundant in the BBB, placenta, liver, and kidney, while Mdr1b is found in the adrenal gland, uterus, ovary, placenta, liver, and kidney. Studies using the Mdr1a or 1b or Mdr1a/1b knockout-mice, which are viable and fertile but more susceptible to developing colitis than wild-type mice, have made a marked contribution to elucidation of the function of P-gp in pharmacology and toxicology.21

The discovery in recent years of MDR1 polymorphisms has amplified the question of the importance of P-gp in the response to drugs and their toxicity in humans. A total of 29 single nucleotide polymorphisms (SNPs) have been reported in the MDR1 gene.23 SNPs in exons 26 (C3435T), 21 (G2677T/A), and 12 (C1236T) are the most frequent genotypic combinations and the allele frequencies of the three variant sites differ markedly between populations of

Table 1 Current human ABC protein, gene nomenclature and properties

Human gene name Protein name

Common name (aliases)

Predominant substrates

Transport mode energy source

Tissue distribution (cellular expression)

Predominant transport impact

ABCB1 (MDR1) ABCB1 (MDR1)

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