Goodman and Gilman Manual of Pharmacology and Therapeutics

Section I
General Principles

chapter 5
Membrane Transporters and Drug Response

Transporters are membrane proteins that are present in all organisms. These proteins control the influx of essential nutrients and ions and the efflux of cellular waste, environmental toxins, drugs, and other xenobiotics (Figure 5–1), consistent with their critical roles in cellular homeostasis, ~2000 genes in the human genome, ~7% of the total number of genes, code for transporters or transporter-related proteins. The functions of membrane transporters may be facilitated (equilibrative, not requiring energy) or active (requiring energy). In considering the transport of drugs, pharmacologists generally focus on transporters from 2 major superfamilies, ABC (ATP binding cassette) and SLC (solute carrier) transporters.

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Figure 5–1 Membrane transporters in pharmacokinetic pathways. Membrane transporters (T) play roles in pharmacokinetic pathways (drug absorption, distribution, metabolism, and excretion), thereby setting systemic drug levels. Drug levels often drive therapeutic and adverse drug effects.

Most ABC proteins are primary active transporters, which rely on ATP hydrolysis to actively pump their substrates across membranes. Among the best recognized transporters in the ABC superfamily are P-glycoprotein (Pgp, encoded by ABCB1, also termed MDR1) and the cystic fibrosis transmembrane regulator (CFTR, encoded by ABCC7). The SLCsuperfamily includes genes that encode facilitated transporters and ion-coupled secondary active transporters. Forty-eight SLC families with ~315 transporters have been identified in the human genome. Many SLC transporters serve as drug targets or in drug absorption and disposition. Widely recognized SLC transporters include the serotonin transporter, SERT, and the dopamine transporter, DAT, both targets for antidepressant medications.

MEMBRANE TRANSPORTERS IN THERAPEUTIC DRUG RESPONSES

PHARMACOKINETICS. Transporters important in pharmacokinetics generally are located in intestinal, renal, and hepatic epithelia, where they function in the selective absorption and elimination of endogenous substances and xenobiotics, including drugs. Transporters work in concert with drug-metabolizing enzymes to eliminate drugs and their metabolites (Figure 5–2). In addition, transporters in various cell types mediate tissue-specific drug distribution (drug targeting). Conversely, transporters also may serve as protective barriers to particular organs and cell types. For example, P-glycoprotein in the blood-brain barrier protects the central nervous system (CNS) from a variety of structurally diverse drugs through its efflux mechanisms.

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Figure 5–2 Hepatic drug transporters. Membrane transporters (red ovals with arrows) work in concert with phase 1 and phase 2 drug-metabolizing enzymes in the hepatocyte to mediate the uptake and efflux of drugs and their metabolites.

PHARMACODYNAMICS: TRANSPORTERS AS DRUG TARGETS. Membrane transporters are the targets of many clinically used drugs. SERT (SLC6A4) is a target for a major class of antidepressant drugs, the selective serotonin reuptake inhibitors (SSRIs). Other neurotransmitter reuptake transporters serve as drug targets for the tricyclic antidepressants, various amphetamines (including amphetamine-like drugs used in the treatment of attention deficit disorder in children), and anticonvulsants.

These transporters also may be involved in the pathogenesis of neuropsychiatric disorders, including Alzheimer and Parkinson diseases. Transporters that are nonneuronal also may be potential drug targets, e.g., cholesterol transporters in cardiovascular disease, nucleoside transporters in cancers, glucose transporters in metabolic syndromes, and Na+-H+ antiporters in hypertension.

DRUG RESISTANCE. Membrane transporters play critical roles in the development of resistance to anticancer drugs, antiviral agents, and anticonvulsants. Decreased uptake of drugs, such as folate antagonists, nucleoside analogs, and platinum complexes, is mediated by reduced expression of influx transporters required for these drugs to access the tumor.Enhanced efflux of hydrophobic drugs is one mechanism of antitumor resistance in cellular assays of resistance.

For example, P-glycoprotein is overexpressed in tumor cells after exposure to cytotoxic anticancer agents. P-glycoprotein pumps out the anticancer drugs, rendering cells resistant to their cytotoxic effects. The over-expression of multidrug resistance protein 4 (MRP4) is associated with resistance to antiviral nucleoside analogs.

MEMBRANE TRANSPORTERS AND ADVERSE DRUG RESPONSES

As controllers of import and export, transporters ultimately control the exposure of cells to chemical carcinogens, environmental toxins, and drugs. Thus, transporters play crucial roles in the cellular toxicities of these agents. Transporter-mediated adverse drug responses generally can be classified into 3 categories (Figure 5–3).

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Figure 5–3 Major mechanisms by which transporters mediate adverse drug responses. Three cases are given. The left panel of each case provides a representation of the mechanism; the right panel shows the resulting effect on drug levels. (Top panel) Increase in the plasma concentrations of drug due to a decrease in the uptake and/or secretion in clearance organs (e.g., liver and kidney). (Middle panel) Increase in the concentration of drug in toxicological target organs due to the enhanced uptake or reduced efflux. (Bottom panel) Increase in the plasma concentration of an endogenous compound (e.g., a bile acid) due to a drug’s inhibiting the influx of the endogenous compound in its eliminating or target organ. The diagram also may represent an increase in the concentration of the endogenous compound in the target organ owing to drug-inhibited efflux of the endogenous compound.

Transporters expressed in the liver and kidney, as well as metabolic enzymes, are key determinants of drug exposure in the circulating blood, thereby affecting exposure, and hence toxicity, in all organs (Figure 5–3top panel). For example, after oral administration of an HMG-CoA reductase inhibitor (e.g., pravastatin), the efficient first-pass hepatic uptake of the drug by the organic anion-transporting polypeptide OATP1B1 maximizes the effects of such drugs on hepatic HMG-CoA reductase. Uptake by OATP1B1 also minimizes the escape of these drugs into the systemic circulation, where they can cause adverse responses such as skeletal muscle myopathy.

Transporters expressed in tissues that may be targets for drug toxicity (e.g., brain) or in barriers to such tissues (e.g., the blood-brain barrier [BBB]) can tightly control local drug concentrations and thus control the exposure of these tissues to the drug (Figure 5–3middle panel). For example, endothelial cells in the BBB are linked by tight junctions, and some efflux transporters are expressed on the blood-facing (luminal) side, thereby restricting the penetration of compounds into the brain. The interactions of loperamide and quinidine are a good example of transporter control of drug exposure at this site. Loperamide is a peripheral opioid used in the treatment of diarrhea and is a substrate of P-glycoprotein. Inhibition of P-glycoprotein-mediated efflux in the BBB would cause an increase in the concentration of loperamide in the CNS and potentiate adverse effects. Indeed, coadministration of loperamide and the potent P-glycoprotein inhibitor quinidine results in significant respiratory depression, an adverse response to the loperamide.

The case of oseltamivir (the antiviral drug TAMIFLU) provides an example that dysfunction of an active barrier may cause a CNS effect. Oseltamivir and its active form, Ro64-0802, undergo active efflux across the BBB by P-glycoprotein, organic anion transporter 3 (OAT3), and multidrug resistance-associated protein 4 (MRP4). Decreased activities of these transporters at the BBB can enhance the CNS exposure to oseltamivir and Ro64-0802, contributing to an adverse effect on the CNS.

Drug-induced toxicity sometimes is caused by the concentrative tissue distribution mediated by influx transporters. For example, biguanides (e.g., metformin and phenformin), used for the treatment of type 2 diabetes mellitus, can produce lactic acidosis, a lethal side effect. Biguanides are substrates of the organic cation transporter 1 (OCT1), which is highly expressed in the liver. OCT1-mediated hepatic uptake of biguanides plays an important role in lactic acidosis. The organic anion transporter 1 (OAT1) and organic cation transporters (OCT1 and OCT2) provide other examples of transporter-related toxicity. OAT1 is expressed mainly in the kidney and is responsible for the renal tubular secretion of anionic compounds. Substrates of OAT1, such as cephaloridine (a β-lactam antibiotic), and adefovir and cidofovir (antiviral drugs), reportedly cause nephrotoxicity. In vitro experiments suggest that cephaloridine, adefovir, and cidofovir are substrates of OAT1 and that OAT1-expressing cells are more susceptible to the toxicity of these drugs than control cells. Exogenous expression of OCT1 and OCT2 enhances the sensitivities of tumor cells to the cytotoxic effect of oxaliplatin for OCT1, and cisplatin and oxaliplatin for OCT2.

Drugs may modulate transporters for endogenous ligands and thereby exert adverse effects (Figure 5–3bottom panel). For example, bile acids are taken up mainly by Na+-taurocholate cotransportingpolypeptide (NTCP) and excreted into the bile by the bile salt export pump (BSEP, ABCB11). Bilirubin is taken up by OATP1B1 and conjugated with glucuronic acid, and bilirubin glucuronide is excreted by the multidrug-resistance-associated protein (MRP2, ABCC2). Inhibition of these transporters by drugs may cause cholestasis or hyperbilirubinemia.

Uptake and efflux transporters determine the plasma and tissue concentrations of endogenous compounds and xenobiotics, thereby influencing the systemic or site-specific toxicity of drugs.

BASIC MECHANISMS OF MEMBRANE TRANSPORT

TRANSPORTERS VERSUS CHANNELS. Both channels and transporters facilitate the membrane permeation of inorganic ions and organic compounds. In general, channels have 2 primary states, open and closed, that are totally stochastic phenomena. Only in the open state do channels appear to act as pores for the selected ions. After opening, channels return to the closed state as a function of time. In contrast, a transporter forms an intermediate complex with the substrate (solute), and subsequently a conformational change in the transporter induces translocation of the substrates to the other side of the membrane.

The turnover rate constants of typical channels are 106 to 108 s–1; those of transporters are, at most, 101 to 103 s–1. Because a particular transporter forms intermediate complexes with specific compounds (referred to as substrates), transporter-mediated membrane transport is characterized by saturability and inhibition by substrate analogs, as described in “Kinetics of Transport.”

The basic mechanisms involved in solute transport across biological membranes include passive diffusion, facilitated diffusion, and active transport. Active transport can be further subdivided into primary and secondary active transport. These mechanisms are depicted in Figure 5–4.

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Figure 5–4 Classification of membrane transport mechanisms. Red circles depict the substrate. Size of the circles is proportional to the concentration of the substrate. Arrows show the direction of flux.Black squares represent the ion that supplies the driving force for transport (size is proportional to the concentration of the ion). Blue ovals depict transport proteins.

PASSIVE DIFFUSION. Simple diffusion of a solute across the plasma membrane consists of 3 processes: partition from the aqueous to the lipid phase, diffusion across the lipid bilayer, and repartition into the aqueous phase on the opposite side. Diffusion of any solute (including drugs) occurs down an electrochemical potential gradient of the solute.

FACILITATED DIFFUSION. Diffusion of ions and organic compounds across the plasma membrane may be facilitated by a membrane transporter. Facilitated diffusion is a form of transporter-mediated membrane transport that does not require energy input. Just as in passive diffusion, the transport of ionized and nonionized compounds across the plasma membrane occurs down their electrochemical potential gradient. Therefore, steady state will be achieved when the electrochemical potentials of the compound on both sides of the membrane become equal.

ACTIVE TRANSPORT. Active transport is the form of membrane transport that requires the input of energy. It is the transport of solutes against their electrochemical gradients, leading to the concentration of solutes on 1 side of the plasma membrane and the creation of potential energy in the electrochemical gradient formed. Active transport plays an important role in the uptake and efflux of drugs and other solutes. Depending on the driving force, active transport can be subdivided into primary and secondary active transport (see Figure 5–4).

Primary Active Transport. Membrane transport that directly couples with ATP hydrolysis is called primary active transport. ABC transporters are examples of primary active transporters. In mammalian cells, ABC transporters mediate the unidirectional efflux of solutes across biological membranes.

Secondary Active Transport. In secondary active transport, the transport across a biological membrane of 1 solute S1 against its concentration gradient is energetically driven by the transport of another solute S2 in accordance with its concentration gradient. For example, an inwardly directed Na+ concentration gradient across the plasma membrane is created by Na+, K+-ATPase. Under these conditions, inward movement of Na+ produces the energy to drive the movement of a substrate S1 against its concentration gradient by a secondary active transporter, as in Na+/Ca2+ exchange. Depending on the transport direction of the solute, secondary active transporters are classified as either symporters or antiporters. Symporters, also termed cotransporters, transport S2 and S1 in the same direction, whereasantiporters, also termed exchangers, move their substrates in opposite directions (see Figure 5–4).

KINETICS OF TRANSPORT

The flux of a substrate (rate of transport) across a biological membrane via transporter-mediated processes is characterized by saturability. The relationship between the flux v and substrate concentration C in a transporter-mediated process is given by the Michaelis-Menten equation:

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where Vmax is the maximum transport rate and is proportional to the density of transporters on the plasma membrane, and Km is the Michaelis constant, which represents the substrate concentration at which the flux is half the Vmax value. Km is an approximation of the dissociation constant of the substrate from the intermediate complex.

The Km and Vmax values can be determined by examining the flux at different substrate concentrations. The Eadie-Hofstee plot provides a graphical method for determining the Vmaxand Km values (Figure 5–5).

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Figure 5–5 Eadie-Hofstee plot of transport data. The black lines show the hyperbolic concentration-dependence curve (ν vs C, left panel) and the Eadie-Hofstee transformation of the transport data (ν/C vsν, right panel) for a simple transport system. The blue lines depict transport in the presence of a competitive inhibitor (surmountable inhibition; achieves same Vmax). The red lines depict the system in the presence of a noncompetitive inhibitor that reduces the number of transporting sites by half but leaves the Km of the functional sites unchanged. Involvement of multiple transporters with different Km values gives an Eadie-Hofstee plot that is curved. Algebraically, the Eadie-Hofstee plot of kinetic data is equivalent to the Scatchard plot of equilibrium binding data.

Transporter-mediated membrane transport of a substrate is also characterized by inhibition by other compounds. The manner of inhibition can be categorized as 1 of 3 types: competitive, noncompetitive, and uncompetitive. Competitive inhibition occurs when substrates and inhibitors share a common binding site on the transporter, resulting in an increase in the apparent Km value in the presence of inhibitor. The flux of a substrate in the presence of a competitive inhibitor is

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where I is the concentration of inhibitor, and Ki is the inhibition constant.

Noncompetitive inhibition assumes that the inhibitor has an allosteric effect on the transporter, does not inhibit the formation of an intermediate complex of substrate and transporter, but does inhibit the subsequent translocation process.

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Uncompetitive inhibition assumes that inhibitors can form a complex only with an intermediate complex of the substrate and transporter and inhibit subsequent translocation.

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VECTORIAL TRANSPORT

Asymmetrical transport across a monolayer of polarized cells, such as the epithelial and endothelial cells of brain capillaries, is called vectorial transport (Figure 5–6). Vectorial transport is important for the absorption of nutrients and bile acids in the intestine. Vectorial transport plays a major role in hepatobiliary and urinary excretion of drugs from the blood to the lumen and in the intestinal absorption of drugs. In addition, efflux of drugs from the brain via brain endothelial cells and brain choroid plexus epithelial cells involves vectorial transport. The ABC transporters mediate only unidirectional efflux, whereas SLC transporters mediate either drug uptake or efflux. For lipophilic compounds that have sufficient membrane permeability, ABC transporters alone are able to achieve vectorial transport without the help of influx transporters. For relatively hydrophilic organic anions and cations, coordinated uptake and efflux transporters in the polarized plasma membranes are necessary to achieve the vectorial movement of solutes across an epithelium. Common substrates of coordinated transporters are transferred efficiently across the epithelial barrier.

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Figure 5–6 Transepithelial and transendothelial flux. Transepithelial or transendothelial flux of drugs requires distinct transporters at the 2 surfaces of the epithelial or endothelial barriers. These are depicted diagrammatically for transport across the small intestine (absorption), the kidney and liver (elimination), and the brain capillaries that comprise the blood-brain barrier.

In the liver, a number of transporters with different substrate specificities are localized on the sinusoidal membrane (facing blood). These transporters are involved in the uptake of bile acids, amphipathic organic anions, and hydrophilic organic cations into the hepatocytes. Similarly, ABC transporters on the canalicular membrane (facing bile) export such compounds into the bile. Multiple combinations of uptake (OATP1B1, OATP1B3, OATP2B1) and efflux transporters (MDR1, MRP2, and BCRP) are involved in the efficient trans-cellular transport of a wide variety of compounds in the liver by using a model cell system called “doubly transfected cells,” which express both uptake and efflux transporter on each side. In many cases, overlapping substrate specificities between the uptake transporters (OATP family) and efflux transporters (MRP family) make the vectorial transport of organic anions highly efficient. Similar transport systems also are present in the intestine, renal tubules, and endothelial cells of the brain capillaries (see Figure 5–6).

REGULATION OF TRANSPORTER EXPRESSION. Transporter expression can be regulated transcriptionally in response to drug treatment and pathophysiological conditions, resulting in induction or down-regulation of transporter mRNAs. Recent studies have described important roles of type II nuclear receptors, which form heterodimers with the 9-cis-retinoic acid receptor (RXR), in regulating drug-metabolizing enzymes and transporters (see Table 6–4 and Figure 6–8). Table 5–1 summarizes the effects of drug activation of type II nuclear receptors on expression of transporters.

Table 5–1

Regulation of Transporter Expression by Nuclear Receptors in Humans

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DNA methylation is one mechanism underlying the epigenetic control of gene expression. Reportedly, the tissue-selective expression of transporters is achieved by DNA methylation (silencing in the transporter-negative tissues) as well as by transactivation in the transporter-positive tissues. Transporters subjected to epigenetic control include OAT3, URAT1, OCT2, Oatp1b2, Ntcp, and PEPT2 in the SLC families; and MDR1, BCRP, BSEP, and ABCG5/ABCG8.

TRANSPORTER STRUCTURE AND MECHANISM

Predictions of secondary structure of membrane transport proteins based on hydropathy analysis indicate that membrane transporters in the SLC and ABC superfamilies are multi-membrane-spanning proteins. ABC transporters have nucleotide-binding domains (NBDs) on the cytoplasmic side that may be thought of as the motor domains with conserved motifs (e.g., Walker-A motif, ABC signature motif) that participate in binding and hydrolysis of ATP. Crystal structures of ABC transporters show 2 NBDs in contact with each other. The ABC transport mechanism appears to involve binding of ATP to the NBDs, which triggers one conformation of the transporter; dissociation of the hydrolysis products of ATP results in a conformation open toward the opposite side of the membrane. In the case of drug extrusion, when ATP binds, the transporters open to the outside, releasing their substrates to the extracellular media; when the hydrolysis products dissociate, the transporter returns to the inward-facing conformation, permitting the binding of ATP and transportable substrate. Chapter 5 of the 12th edition of the parent text provides additional details.

TRANSPORTER SUPERFAMILIES IN THE HUMAN GENOME

SLC TRANSPORTERS. The solute carrier (SLC) superfamily includes 48 families and represents ~315 genes in the human genome, some of which are associated with certain genetic diseases (Table 5–2). Transporters in the SLC superfamily transport diverse ionic and nonionic endogenous compounds and xenobiotics. SLC superfamily transporters may be facilitated transporters or secondary active symporters or antiporters.

Table 5–2

The Human Solute Carrier Superfamily

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ABC SUPERFAMILY. The 7 groups of ABC transporters are essential for many cellular processes, and mutations in at least 13 of the genes for ABC transporters cause or contribute to human genetic disorders (Table 5–3). In addition to conferring multidrug resistance, an important pharmacological aspect of these transporters is xenobiotic export from healthy tissues. In particular, MDR1/ABCB1, MRP2/ABCC2, and BCRP/ABCG2 have been shown to be involved in overall drug disposition.

Table 5–3

The ATP Binding Cassette (ABC) Superfamily in the Human Genome and Linked Genetic Diseases

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The ABC superfamily includes 49 genes, each containing 1 or 2 conserved ABC regions. The ABC region is a core catalytic domain of ATP hydrolysis and contains Walker A and B sequences and an intervening ABC transporter-specific signature C sequence. The ABC regions of these proteins bind and hydrolyze ATP, and the proteins use the energy for uphill transport of their substrates across the membrane. Although some ABC superfamily transporters contain only a single ABC motif, they form homodimers (BCRP/ABCG2) or heterodimers (ABCG5 and ABCG8) that exhibit a transport function. ABC transporters (e.g., MsbA) in prokaryotes are involved in the import of essential compounds that cannot be obtained by passive diffusion (sugars, vitamins, metals, etc.). Most ABC genes in eukaryotes transport compounds from the cytoplasm to the outside or into an intracellular compartment (endoplasmic reticulum, mitochondria, peroxisomes).

PROPERTIES OF ABC TRANSPORTERS RELATED TO DRUG ACTION

The tissue distribution of drug-related ABC transporters in humans is summarized in Table 5–4 together with information about typical substrates.

Table 5–4

ABC Transporters Involved in Drug Absorption, Distribution, and Excretion Processes

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TISSUE DISTRIBUTION OF DRUG-RELATED ABC TRANSPORTERS. MDR1 (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) are all expressed in the apical side of the intestinal epithelia, where they serve to pump out xenobiotics, including many orally administered drugs. MRP3 (ABCC3) is expressed in the basal side of the epithelial cells.

Key to the vectorial excretion of drugs into urine or bile, ABC transporters are expressed in the polarized tissues of kidney and liver: MDR1, MRP2, and MRP4 (ABCC4) on the brush-border membrane of renal epithelia; MDR1, MRP2, and BCRP on the bile canalicular membrane of hepatocytes; and MRP3 and MRP4 on the sinusoidal membrane of hepatocytes. Some ABC transporters are expressed specifically on the blood side of the endothelial or epithelial cells that form barriers to the free entrance of toxic compounds into tissues: the BBB (MDR1 and MRP4 on the luminal side of brain capillary endothelial cells), the blood-cerebrospinal fluid (CSF) barrier (MRP1 and MRP4 on the basolateral blood side of choroid plexus epithelia), the blood-testis barrier (MRP1 on the basolateral membrane of mouse Sertoli cells and MDR1 in several types of human testicular cells), and the blood-placenta barrier (MDR1, MRP2, and BCRP on the luminal maternal side and MRP1 on the antiluminal fetal side of placental trophoblasts).

MRP/ABCC FAMILY. The substrates of transporters in the MRP/ABCC family are mostly organic anions. Both MRP1 and MRP2 accept glutathione and glucuronide conjugates, sulfated conjugates of bile salts, and nonconjugated organic anions of an amphipathic nature (at least 1 negative charge and some degree of hydrophobicity). They also transport neutral or cationic anticancer drugs, such as vinca alkaloids and anthracyclines, possibly by means of a cotransport or symport mechanism with reduced glutathione. MRP3 also has a substrate specificity that is similar to that of MRP2 but with a lower transport affinity for glutathione conjugates compared with MRP1 and MRP2. MRP3 is expressed on the sinusoidal side of hepatocytes and is induced under cholestatic conditions. MRP3 functions to return toxic bile salts and bilirubin glucuronides into the blood circulation. MRP4 accepts negatively charged molecules, including cytotoxic compounds (e.g., 6-mercaptopurine and methotrexate), cyclic nucleotides, antiviral drugs (e.g., adefovir and tenofovir), diuretics (e.g., furosemide and trichlormethiazide), and cephalosporins (e.g., ceftizoxime and cefazolin). Glutathione enables MRP4 to accept taurocholate and leukotriene B4. MRP5 has a narrower substrate specificity and accepts nucleotide analog and clinically important anti–human immunodeficiency virus (HIV) drugs. No substrates have been identified that explain the mechanism of the MRP6-associated disease, pseudoxanthoma.

BCRP/ABCG2. BCRP accepts both neutral and negatively charged molecules, including cytotoxic compounds (e.g., topotecan, flavopiridol, and methotrexate), sulfated conjugates of therapeutic drugs and hormones (e.g., estrogen sulfate), antibiotics (e.g., nitrofurantoin and fluoroquinolones), statins (e.g., pitavastatin and rosuvastatin), and toxic compounds found in normal food [phytoestrogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and pheophorbide A, a chlorophyll catabolite].

ABC TRANSPORTERS IN DRUG ABSORPTION AND ELIMINATION. With respect to clinical medicine, MDR1 is the most important ABC transporter yet identified. The systemic exposure to orally administered digoxin is decreased by coadministration of rifampin (an MDR1 inducer) and is negatively correlated with the MDR1 protein expression in the human intestine. MDR1 is also expressed on the brush-border membrane of renal epithelia, and its function can be monitored using digoxin (>70% excreted in the urine). MDR1 inhibitors (e.g., quinidine, verapamil, valspodar, spironolactone, clarithromycin, and ritonavir) all markedly reduce renal excretion of digoxin. Drugs with narrow therapeutic windows (e.g., digoxin, cyclosporine, tacrolimus) should be used with great care if MDR1-based drug-drug interactions are likely.

In the intestine, MRP3 can mediate the intestinal absorption in conjunction with uptake transporters. MRP3 mediates sinusoidal efflux in the liver, decreasing the efficacy of the biliary excretion from the blood, and excretion of intracellularly formed metabolites, particularly, glucuronide conjugates. Thus, dysfunction of MRP3 results in a shortening of the elimination t1/2. MRP4 substrates also can be transported by OAT1 and OAT3 on the basolateral membrane of the epithelial cells in the kidney. The rate-limiting process in renal tubular secretion is likely the uptake process at the basolateral surface. Dysfunction of MRP4 enhances the renal concentration, but has limited impact on the blood concentration.

GENETIC VARIATION IN MEMBRANE TRANSPORTERS: IMPLICATIONS FOR CLINICAL DRUG RESPONSE

There are inherited defects in SLC transporters (see Table 5–2) and ABC transporters (see Table 5–3). Polymorphisms in membrane transporters play a role in drug response and are yielding new insights in pharmacogenetics (see Chapter 7).

Clinical studies have focused on a limited number of transporters, relating genetic variation in membrane transporters to drug disposition and response. For example, 2 common single-nucleotide polymorphisms (SNPs) in SLCO1B1 (OATP1B1) have been associated with elevated plasma levels of pravastatin, a widely used drug for the treatment of hypercholesterolemia. Recent studies using genome-wide association methods have determined that genetic variants in SLCO1B1 (OATP1B1) predispose patients to risk for muscle toxicity associated with use of simvastatin. Other studies indicate that genetic variants in transporters in the SLC22A family associate with variation in renal clearance and response to various drugs including the antidiabetic drug, metformin.

TRANSPORTERS INVOLVED IN PHARMACOKINETICS

Drug transporters play a prominent role in pharmacokinetics (see Figure 5–1 and Table 5–4). Transporters in the liver and kidney have important roles in removal of drugs from the blood and hence in metabolism and excretion.

HEPATIC TRANSPORTERS

Hepatic uptake of organic anions (e.g., drugs, leukotrienes, and bilirubin), cations, and bile salts is mediated by SLC-type transporters in the basolateral (sinusoidal) membrane of hepatocytes: OATPs (SLCO) and OATs (SLC22), OCTs (SLC22), and NTCP (SLC10A1), respectively. These transporters mediate uptake by either facilitated or secondary active mechanisms.

ABC transporters such as MRP2, MDR1, BCRP, BSEP, and MDR2 in the bile canalicular membrane of hepatocytes mediate the efflux (excretion) of drugs and their metabolites, bile salts, and phospholipids against a steep concentration gradient from liver to bile. This primary active transport is driven by ATP hydrolysis.

Vectorial transport of drugs from the circulating blood to the bile using an uptake transporter (OATP family) and an efflux transporter (MRP2) is important for determining drug exposure in the circulating blood and liver. Moreover, there are many other uptake and efflux transporters in the liver (Figures 5–7 and 5–8).

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Figure 5–7 Hepatic uptake, backflux into blood, metabolism, and efflux into bile. The red circles represent parent drugs; the green triangles represent drug metabolites. PS, permeability surface product; CLmet, metabolic clearance; CLint, intrinsic clearance.

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Figure 5–8 Transporters in the hepatocyte that function in the uptake and efflux of drugs across the sinusoidal membrane and efflux of drugs into the bile across the canalicular membrane. See text for details of the transporters pictured.

The following examples illustrate the importance of vectorial transport in determining drug exposure in the circulating blood and liver and the role of transporters in drug-drug interactions.

HMG-CoA REDUCTASE INHIBITORS. Statins are cholesterol-lowering agents that reversibly inhibit HMG-CoA reductase, which catalyzes a rate-limiting step in cholesterol biosynthesis (see Chapter 31). Most of the statins in the acid form are substrates of uptake transporters, so they are taken up efficiently by the liver and undergo enterohepatic circulation (see Figures 5–5 and 5–8). In this process, hepatic uptake transporters such as OATP1B1 and efflux transporters such as MRP2 act cooperatively to produce vectorial trans-cellular transport of bisubstrates in the liver. The efficient first-pass hepatic uptake of these statins by OATP1B1 helps them to exert their pharmacological effect and also minimizes the systemic drug distribution, thereby minimizing adverse effects in smooth muscle. Recent studies indicate that the genetic polymorphism of OATP1B1 also affects the function of this transporter.

GEMFIBROZIL. The cholesterol-lowering agent gemfibrozil, a PPARa activator, can enhance toxicity (myopathy) to several statins by a mechanism that involves transport. Gemfibrozil inhibits the uptake of the active hydroxy forms of statins into hepatocytes by OATP1B1, resulting in an increase in the plasma concentration of the statin.

IRINOTECAN (CPT-11). Irinotecan hydrochloride (CPT-11) is a potent anticancer drug, but late-onset gastrointestinal toxic effects, such as severe diarrhea, make it difficult to use CPT-11 safely. After intravenous administration, CPT-11 is converted to SN-38, an active metabolite, by carboxylesterase. SN-38 is subsequently conjugated with glucuronic acid in the liver. SN-38 and SN-38 glucuronide are then excreted into the bile by MRP2. The inhibition of MRP2-mediated biliary excretion of SN-38 and its glucuronide by coadministration of probenecid reduces the drug-induced diarrhea, at least in rats. For additional details, see Figures 6–5 and 6–6see also Figure 6–7 in the 12th edition of the parent text.

BOSENTAN. Bosentan is an endothelin antagonist used to treat pulmonary arterial hypertension. It is taken up in the liver by OATP1B1 and OATP1B3, and subsequently metabolized by CYP2C9 and CYP3A4. Transporter-mediated hepatic uptake can be a determinant of elimination of bosentan, and the inhibition of hepatic uptake by cyclosporin A, rifampicin, and sildenafil can affect its pharmacokinetics.

The parent text contains additional examples of the contribution to clinical drug use of vectorial transport and its genetic variability.

RENAL TRANSPORTERS

ORGANIC CATION TRANSPORT. Structurally diverse organic cations are secreted in the proximal tubule. A primary function of organic cation secretion is ridding the body of xenobiotics, including many positively charged drugs and their metabolites (e.g., cimetidine, ranitidine, metformin, procainamide, and N-acetylprocainamide) and toxins from the environment (e.g., nicotine). Organic cations that are secreted by the kidney may be either hydrophobic or hydrophilic. Hydrophilic organic drug cations generally have molecular weights < 400 daltons; a current model for their secretion in the proximal tubule of the nephron is shown in Figure 5–9, involving the transporters described below.

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Figure 5–9 Organic cation secretory transporters in the proximal tubule. OC+, organic cation. See text for details of the transporters pictured.

For the transepithelial flux of a compound (e.g., secretion), the compound must traverse 2 membranes sequentially, the basolateral membrane facing the blood side and the apical membrane facing the tubular lumen. Organic cations appear to cross the basolateral membrane in human proximal tubule by 2 distinct transporters in the SLC family 22 (SCL22): OCT2 (SLC22A2) and OCT3 (SLC22A3). Organic cations are transported across this membrane down an electrochemical gradient.

Transport of organic cations from cell to tubular lumen across the apical membrane occurs through an electroneutral proton–organic cation exchange. The recent discovery of a new transporter family, SLC47A, multidrug and toxin extrusion family (MATEs), has provided the molecular identities of the previously characterized electroneutral proton–organic cation antiport mechanism. Transporters in the MATE family, assigned to the apical membrane of the proximal tubule, appear to play a key role in moving hydrophilic organic cations from tubule cell to lumen. In addition, novel organic cation transporters (OCTNs), located on the apical membrane, appear to contribute to organic cation flux across the proximal tubule. In humans, these include OCTN1 (SLC22A4) and OCTN2 (SLC22A5). These bifunctional transporters are involved not only in organic cation secretion but also in carnitine reabsorption. In the reuptake mode, the transporters function as Na+ cotransporters, relying on the inwardly driven Na+ gradient created by Na+, K+-ATPase to move carnitine from tubular lumen to cell. In the secretory mode, the transporters appear to function as proton–organic cation exchangers. That is, protons move from tubular lumen to cell interior in exchange for organic cations, which move from cytosol to tubular lumen. The inwardly directed proton gradient (from tubular lumen to cytosol) is maintained by transporters in the SLC9 family, which are Na+/H+ exchangers (NHEs, antiporters). Of the 2 steps involved in secretory transport, transport across the luminal membrane appears to be rate-limiting.

OCT2 (SLC22A2). Human, mouse, and rat orthologs of OCT2 are expressed in abundance in human kidney and to some extent in neuronal tissue such as choroid plexus. In the kidney, OCT2 is localized to the proximal tubule and to distal tubules and collecting ducts. In the proximal tubule, OCT2 is restricted to the basolateral membrane. OCT2-mediated transport of model organic cations MPP+ and TEA is electrogenic, and both OCT2 and OCT1 can support organic cation–organic cation exchange. OCT2 generally accepts a wide array of monovalent organic cations with molecular weights < 400 daltons. OCT2 is also present in neuronal tissues; however, monoamine neurotransmitters have low affinities for OCT2.

OCT3 (SLC22A3). OCT3 is located in tandem with OCT1 and OCT2 on chromosome 6. Tissue distribution studies suggest that human OCT3 is expressed in liver, kidney, intestine, and placenta, although it appears to be expressed in considerably less abundance than OCT2 in the kidney. Like OCT1 and OCT2, OCT3 appears to support electrogenic potential-sensitive organic cation transport. OCT3 may play only a limited role in renal drug elimination.

OCTN1 (SLC22A4). OCTN1 seems to operate as an organic cation–proton exchanger. OCTN1-mediated influx of model organic cations is enhanced at alkaline pH, whereas efflux is increased by an inwardly directed proton gradient. OCTN1 contains a nucleotide-binding sequence motif, and transport of its substrates appears to be stimulated by cellular ATP. OCTN1 also can function as an organic cation–organic cation exchanger. OCTN1 functions as a bidirectional pH- and ATP-dependent transporter at the apical membrane in renal tubular epithelial cells.

OCTN2 (SLC22A5). OCTN2 is a bifunctional transporter, it functions as both a Na+-dependent carnitine transporter and a Na+-independent organic cation transporter. OCTN2 transport of organic cations is sensitive to pH, suggesting that OCTN2 may function as an organic cation exchanger. The transport of L-carnitine by OCTN2 is a Na+-dependent electrogenic process, and mutations in OCTN2 appear to be the cause of primary systemic carnitine deficiency.

MATE1 AND MATE2-K (SLC47A1 AND SLC47A2). Multidrug and toxin extrusion family members MATE1 and MATE2-K interact with structurally diverse hydrophilic organic cations including the antidiabetic drug metformin, the H2 antagonist cimetidine, and the anticancer drug, topotecan. In addition to cationic compounds, the transporters also recognize some anions, including the antiviral agents acyclovir and ganciclovir. The zwitterions cephalexin and cephradine are specific substrates of MATE1. The herbicide paraquat, a bis-quaternary ammonium compound, which is nephrotoxic in humans, is a potent substrate of MATE1. Both MATE1 and MATE2-K have been localized to the apical membrane of the proximal tubule. MATE1, but not MATE2-K, is also expressed on the canalicular membrane of the hepatocyte. These transporters appear to be the long-searched-for organic cation–proton antiporters on the apical membrane of the proximal tubule, that is, an oppositely directed proton gradient can drive the movement of organic cations via MATE1 or MATE2-K. The antibiotics, levofloxacin and ciprofloxacin, though potent inhibitors, are not translocated by either MATE1 or MATE2-K.

Polymorphisms of OCTs. OCT1 exhibits the greatest number of amino acid polymorphisms, followed by OCT2 and then OCT3. Recent studies suggest that genetic variants of OCT1 and OCT2 are associated with alterations in the renal elimination and response to the anti-diabetic drug, metformin.

ORGANIC ANION TRANSPORT. The primary function of organic anion secretion appears to be the removal from the body of xenobiotics, including many weakly acidic drugs (e.g., pravastatin, captopril, p-aminohippurate [PAH], and penicillins) and toxins (e.g., ochratoxin). Organic anion transporters move both hydrophobic and hydrophilic anions but also may interact with cations and neutral compounds. A current model for the transepithelial flux of organic anions in the proximal tubule is shown in Figure 5–10.

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Figure 5–10 Organic anion secretory transporters in the proximal tubule. Two primary transporters on the basolateral membrane mediate the flux of organic anions from interstitial fluid to tubule cell: OAT1 (SLC22A6) and OAT3 (SLC22A8). Hydrophilic organic anions are transported across the basolateral membrane against an electrochemical gradient in exchange with intracellular α-ketoglutarate, which moves down its concentration gradient from cytosol to blood. The outwardly directed gradient of α-ketoglutarate is maintained at least in part by a basolateral Na+-dicarboxylate uptake transporter (NaDC3). The Na+ gradient that drives NaDC3 is maintained by Na+, K+-ATPase. OA, organic anion; α-KG, α-ketoglutarate.

OAT1 (SLC22A6). Mammalian isoforms of OAT1 are expressed primarily in the kidney, with some expression in brain and skeletal muscle. OAT1 generally transports small-molecular-weight organic anions that may be endogenous (e.g., PGE2 and urate) or ingested drugs and toxins.

OAT2 (SLC22A7). OAT2 is present in both kidney and liver. In the kidney, the transporter is localized to the basolateral membrane of the proximal tubule, and appears to function as a transporter for nucleotides, particularly guanine nucleotides such as cyclic GMP.

OAT3 (SLC22A8). OAT3 transports a wide variety of organic anions, including model compounds, PAH and estrone sulfate, as well as many drug products (e.g., pravastatin, cimetidine, 6-mercaptopurine, and methotrexate).

OAT4 (SLC22A9). In humans, OAT4 is expressed in placenta and kidney (on the luminal membrane of the proximal tubule). Organic anion transport by OAT4 can be stimulated by transgradients of α-ketoglutarate, suggesting that OAT4 may be involved in the reabsorption of organic anions from tubular lumen into cell.

OTHER ANION TRANSPORTERS. URAT1 (SLC22A12) is a kidney-specific transporter confined to the apical membrane of the proximal tubule. URAT1 is primarily responsible for urate reabsorption, mediating electroneutral urate transport that can be trans-stimulated by Cl gradients. NPT1 (SLC17A1) is expressed on the luminal membrane of the proximal tubule as well as in the brain. NPT1 transports PAH, probenecid, and penicillin G. It appears to be part of the system involved in organic anion efflux from tubule cell to lumen.

MRP2 (ABCC2) is considered to be the primary transporter involved in efflux of many drug conjugates such as glutathione conjugates across the canalicular membrane of the hepatocyte. However, MRP2 is also found on the apical membrane of the proximal tubule, where it is thought to play a role in the efflux of organic anions into the tubular lumen. In general, MRP2 transports larger, bulkier compounds than do most of the organic anion transporters in the SLC22 family. MRP4 (ABCC4), localized on the apical membrane of the proximal tubule, transports a wide array of conjugated anions, including glucuronide and glutathione conjugates. MRP4 appears to interact with drugs, including methotrexate, cyclic nucleotide analogs, and antiviral nucleoside analogs.

Polymorphisms of OATs. Polymorphisms in OAT1 and OAT3 have been identified in ethnically diverse human populations.

TRANSPORTERS AND PHARMACODYNAMICS: DRUG ACTION IN THE BRAIN

Neurotransmitters are packaged in vesicles in presynpatic neurons, released in the synapse by fusion of the vesicles with the plasma membrane, and, excepting acetylcholine, are then taken back into the presynaptic neurons or postsynaptic cells (see Chapter 8). Transporters involved in the neuronal reuptake of the neurotransmitters and the regulation of their levels in the synaptic cleft belong to 2 major superfamilies, SLC1 and SLC6. Transporters in both families play roles in reuptake of γ-aminobutyric acid (GABA), glutamate, and the monoamine neurotransmitters norepinephrine (NE), serotonin (5HT), and dopamine (DA). These transporters may serve as pharmacologic targets for neuropsychiatric drugs. SLC6 family members localized in the brain and involved in the reuptake of neurotransmitters into presynaptic neurons include the NE transporter (NET, SLC6A2), the DA transporter (DAT, SLC6A3), the serotonin transporter (SERT, SLC6A4), and several GABA reuptake transporters (GAT1, GAT2, and GAT3).

SLC6 family members depend on the Na+ gradient to actively transport their substrates into cells. Cl is also required, although to a variable extent depending on the family member. Through reuptake mechanisms, the neurotransmitter transporters in the SLC6A family regulate the concentrations and dwell times of neurotransmitters in the synaptic cleft; the extent of transmitter uptake also influences subsequent vesicular storage of transmitters. Many of these transporters are present in other tissues (e.g., kidney and platelets) and may serve other roles. Further, the transporters can function in the reverse direction. That is, the transporters can export neurotransmitters in a Na+-independent fashion.

SLC6A1 (GAT1), SLC6A11 (GAT3), AND SLC6A13 (GAT2). GAT1 is the most important GABA transporter in the brain, expressed in GABAergic neurons and found largely on presynaptic neurons. GAT1 is found in abundance in the neocortex, cerebellum, basal ganglia, brainstem, spinal cord, retina, and olfactory bulb. GAT3 is found only in the brain, largely in glial cells. GAT2 is found in peripheral tissues, including the kidney and liver, and within the CNS in the choroid plexus and meninges. Physiologically, GAT1 appears to be responsible for regulating the interaction of GABA at receptors. The presence of GAT2 in the choroid plexus and its absence in presynaptic neurons suggest that this transporter may play a primary role in maintaining the homeostasis of GABA in the CSF. GAT1 is the target of the antiepileptic drug tiagabine, which presumably acts to increase GABA levels in the synaptic cleft of GABAergic neurons by inhibiting the reuptake of GABA. GAT3 is the target for the nipecotic acid derivatives that are anticonvulsants.

SLC6A2 (NET). NET is found in central and peripheral nervous tissues as well as in adrenal chromaffin tissue. NET colocalizes with neuronal markers, consistent with a role in reuptake of monoamine neurotransmitters. The transporter functions in the Na+-dependent reuptake of NE and DA. NET limits the synaptic dwell time of NE and terminate its actions, salvaging NE for subsequent repackaging. NET participates in the regulation of many neurological functions, including memory and mood. NET serves as a drug target for the antidepressant desipramine, other tricyclic antidepressants, and cocaine. Orthostatic intolerance, a rare familial disorder characterized by an abnormal blood pressure and heart rate response to changes in posture, has been associated with a mutation in NET.

SLC6A3 (DAT). DAT is located primarily in the brain in dopaminergic neurons. The primary function of DAT is the reuptake of DA, terminating its actions. Although present on presynaptic neurons at the neurosynaptic junction, DAT is also present in abundance along the neurons, away from the synaptic cleft, suggesting that DAT may play a role in clearance of excess DA in the vicinity of neurons. Physiologically, DAT is involved in functions attributed to the dopaminergic system, including mood, behavior, reward, and cognition. Drugs that interact with DAT include cocaine and its analogs, amphetamines, and the neurotoxin MPTP.

SLC6A4 (SERT). SERT plays a role in the reuptake and clearance of serotonin in the brain. Like the other SLC6A family members, SERT transports its substrates in a Na+-dependent fashion and is dependent on Cl and possibly on the countertransport of K+. Substrates of SERT include 5HT, various tryptamine derivatives, and neurotoxins such as 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) and fenfluramine. SERT is the specific target of the selective serotonin reuptake inhibitor antidepressants (e.g., fluoxetine and paroxetine) and 1 of several targets of tricyclic antidepressants (e.g., amitriptyline). Genetic variants of SERT have been associated with an array of behavioral and neurological disorders. The precise mechanism by which a reduced activity of SERT, caused by either a genetic variant or an antidepressant, ultimately affects behavior, including depression, is not known.

BLOOD-BRAIN BARRIER AND BLOOD-CEREBROSPINAL FLUID BARRIER

Drugs acting in the CNS have to cross the BBB or blood-CSF barrier, which are formed by brain capillary endothelial cells and epithelial cells of the choroid plexus, respectively. These are not static anatomical barriers but dynamic ones in which efflux transporters play a role. P-glycoprotein extrudes its substrate drugs on the luminal membrane of the brain capillary endothelial cells into the blood, thereby, limiting the brain penetration. Thus, recognition by P-glycoprotein as a substrate is a major disadvantage for drugs used to treat CNS diseases. Other transporters involved in the efflux of organic anions from the CNS are being identified in the BBB and the blood-CSF barrier and include the members of OATP1A4 and OATP1A5 and OAT3 families. They mediate the uptake of organic compounds such as β-lactam antibiotics, statins, p-aminohippurate, H2 receptor antagonists, and bile acids on the plasma membrane facing the brain-CSF. Further clarification of influx and efflux transporters in the barriers will enable delivery of CNS drugs efficiently into the brain while avoiding undesirable CNS side effects.