membrane transporters and drug...

41

C H A P T E R

2

MEMBRANE TRANSPORTERS AND DRUG RESPONSE

Kathleen M. Giacomini and Yuichi Sugiyama

Transporters are membrane proteins that are present in allorganisms. These proteins control the influx of essentialnutrients and ions and the efflux of cellular waste, envi-ronmental toxins, and other xenobiotics. Consistent withtheir critical roles in cellular homeostasis, approximately2000 genes in the human genome ~7 of the total numberof genes code for transporters or transporter-related pro-teins. The functions of membrane transporters may befacilitated (equilibrative, not requiring energy) or active(requiring energy).

In considering the transport of drugs, pharmacologistsgenerally focus on transporters from two major super-families, ABC (

A

TP

b

inding

c

assette) and SLC (

s

o

l

ute

c

arrier) transporters. Most ABC proteins are primaryactive transporters, which rely on ATP hydrolysis toactively pump their substrates across membranes. Thereare 49 known genes for ABC proteins that can begrouped into seven subclasses or families (ABCA toABCG) (Borst and Elferink, 2002). Among the bestrecognized transporters in the ABC superfamily are P-glycoprotein (P-gp, encoded by

ABCB1

, also termed

MDR1

) and the cystic fibrosis transmembrane regulator(CFTR, encoded by

ABCC7

). The SLC superfamilyincludes genes that encode facilitated transporters andion-coupled secondary active transporters that reside invarious cell membranes. Forty-three SLC families withapproximately 300 transporters have been identified inthe human genome (Hediger, 2004). Many serve as drugtargets or in drug absorption and disposition. Widelyrecognized SLC transporters include the serotonin anddopamine transporters (SERT, encoded by

SLC6A4

;DAT, encoded by

SLC6A3

).Drug-transporting proteins operate in pharmacokinetic

and pharmacodynamic pathways, including pathways

involved in both therapeutic and adverse effects (Figure2–1).

MEMBRANE TRANSPORTERS IN THERAPEUTIC DRUG RESPONSES

Pharmacokinetics.

Transporters that are important inpharmacokinetics generally are located in intestinal, renal,and hepatic epithelia. They function in the selectiveabsorption and elimination of endogenous substances andxenobiotics, including drugs (Dresser

et al.

, 2001; Kim,2002). Transporters work in concert with drug-metaboliz-ing enzymes to eliminate drugs and their metabolites (Fig-ure 2–2). In addition, transporters in various cell typesmediate tissue-specific drug distribution (drug targeting);conversely, transporters also may serve as protective bar-riers to particular organs and cell types. For example, P-glycoprotein in the blood–brain barrier protects the cen-tral nervous system (CNS) from a variety of structurallydiverse compounds through its efflux mechanisms. Manyof the transporters that are relevant to drug response con-trol the tissue distribution as well as the absorption andelimination of drugs.

Pharmacodynamics: Transporters as Drug Targets.

Membrane transporters are the targets of many clinicallyused drugs. For example, neurotransmitter transporters arethe targets for drugs used in the treatment of neuropsychia-tric disorders (Amara and Sonders, 1998; Inoue

et al.

,2002). SERT (

SLC6A4

) is a target for a major class of anti-depressant drugs, the serotonin selective reuptake inhibitors(SSRIs). Other neurotransmitter reuptake transporters serve

42

Section I / General Principles

as drug targets for the tricyclic antidepressants, variousamphetamines (including amphetaminelike drugs used inthe treatment of attention deficit disorder in children), andanticonvulsants (Amara and Sonders, 1998; Jones

et al.

,1998; Elliott and Beveridge, 2005). These transporters also

may be involved in the pathogenesis of neuropsychiatricdisorders, including Alzheimer’s and Parkinson’s diseases(Shigeri

et al.

, 2004). Transporters that are nonneuronalalso may be potential drug targets,

e.g.,

cholesterol trans-porters in cardiovascular disease, nucleoside transporters in

Figure 2–1.

Roles of membrane transporters in pharmacokinetic pathways.

Membrane transporters (T) play roles in pharmaco-kinetic pathways (drug absorption, distribution, metabolism, and excretion), thereby setting systemic drug levels. Drug levels oftendrive therapeutic and adverse drug effects.

Figure 2–2.

Hepatic drug transporters.

Membrane transporters, shown as hexagons with arrows, work in concert with phase 1 andphase 2 drug-metabolizing enzymes in the hepatocyte to mediate the uptake and efflux of drugs and their metabolites.

Chapter 2 / Membrane Transporters and Drug Response

43

cancers, glucose transporters in metabolic syndromes, andNa

+

-H

+

antiporters in hypertension (Damaraju

et al.

, 2003;Pascual

et al.

, 2004; Rader, 2003; Rosskopf

et al.

, 1993).

Drug Resistance.

Membrane transporters play a criticalrole in the development of resistance to anticancer drugs,antiviral agents, and anticonvulsants. For example, P-gly-coprotein is overexpressed in tumor cells after exposure tocytotoxic anticancer agents (Gottesman

et al.

, 1996; Linand Yamazaki, 2003; Leslie

et al.

, 2005). P-glycoproteinpumps out the anticancer drugs, rendering cells resistantto their cytotoxic effects. Other transporters, includingbreast cancer resistance protein (BCRP), the organicanion transporters, and several nucleoside transporters,also have been implicated in resistance to anticancerdrugs (Clarke

et al.

, 2002; Suzuki

et al.

, 2001). The over-expression of multidrug-resistance protein 4 (MRP4) isassociated with resistance to antiviral nucleoside analogs(Schuetz

et al.

, 1999).

MEMBRANE TRANSPORTERS AND ADVERSE DRUG RESPONSES

Through import and export mechanisms, transporters ulti-mately control the exposure of cells to chemical carcino-gens, environmental toxins, and drugs. Thus, transportersplay critical roles in the cellular toxicities of these agents.Transporter-mediated adverse drug responses generally canbe classified into three categories, as shown in Figure 2–3.

Transporters in the liver and kidney affect the exposureof drugs in the toxicological target organs. Transportersexpressed in the liver and kidney, as well as metabolicenzymes, are key determinants of drug exposure in the cir-culating blood (Mizuno

et al.

, 2003) (Figure 2–3,

top panel

).For example, after oral administration of an HMG-CoAreductase inhibitor (

e.g.,

pravastatin

), the efficient first-passhepatic uptake of the drug by the organic anion–transportingpolypeptide OATP1B1 maximizes the effects of such drugson hepatic HMG-CoA reductase. Uptake by OATP1B1 alsominimizes the escape of these drugs into the systemic circu-lation, where they can cause adverse responses such as skel-etal muscle myopathy. Transporters in the liver and kidney,which control the total clearance of drugs, thus have aninfluence on the plasma concentration profiles and subse-quent exposure to the toxicological target.

Transporters in toxicological target organs or at barriersto such organs affect drug exposure by the target organs.Transporters expressed in tissues that may be targets fordrug toxicity (

e.g.,

brain) or in barriers to such tissues

[

e.g.,

the blood–brain barrier (BBB)] can tightly controllocal drug concentrations and thus control the exposure ofthese tissues to the drug (Figure 2–3,

middle panel

). Forexample, to restrict the penetration of compounds into thebrain, endothelial cells in the BBB are closely linked bytight junctions, and some efflux transporters are expressedon the blood-facing (luminal) side. The importance of theABC transporter multidrug-resistance protein (

ABCB1

,MDR1; P-glycoprotein, P-gp) in the BBB has been dem-onstrated in

mdr1a

knockout mice (Schinkel

et al.

, 1994).The brain concentrations of many P-glycoprotein sub-strates, such as

digoxin,

used in the treatment of heart fail-ure (

see

Chapters 33 and 34), and

cyclosporin A

(

see

Chapter 52), an immunosuppressant, are increased dramat-ically in

mdr1a

(–/–) mice, whereas their plasma concentra-tions are not changed significantly.

Another example of transporter control of drug exposurecan be seen in the interactions of

loperamide

and

quinidine.

Loperamide is a peripheral opioid used in the treatment ofdiarrhea and is a substrate of P-glycoprotein. Coadministra-tion of loperamide and the potent P-glycoprotein inhibitorquinidine results in significant respiratory depression, anadverse response to the loperamide (Sadeque

et al.

, 2000).Because plasma concentrations of loperamide are notchanged in the presence of quinidine, it has been suggestedthat quinidine inhibits P-glycoprotein in the BBB, resultingin an increased exposure of the CNS to loperamide andbringing about the respiratory depression. Inhibition of P-glycoprotein-mediated efflux in the BBB thus would causean increase in the concentration of substrates in the CNSand potentiate adverse effects.

Drug-induced toxicity sometimes is caused by the con-centrative tissue distribution mediated by influx transport-ers. For example, biguanides (

e.g.,

metformin

and

phen-formin

), widely used as oral hypoglycemic agents for thetreatment of type II diabetes mellitus, can produce lacticacidosis, a lethal side effect. Phenformin was withdrawnfrom the market for this reason. Biguanides are substratesof the organic cation transporter OCT1, which is highlyexpressed in the liver. After oral administration of met-formin, the distribution of the drug to the liver in

oct1

(

–/–

)mice is markedly reduced compared with the distributionin wild-type mice. Moreover, plasma lactic acid concen-trations induced by metformin are reduced in

oct1

(

–/–

)mice compared with wild-type mice, although the plasmaconcentrations of metformin are similar in the wild-typeand knockout mice. These results indicate that the OCT1-mediated hepatic uptake of biguanides plays an importantrole in lactic acidosis (Wang

et al.

, 2003). The organic anion transporter 1 (OAT1) provides another

example of transporter-related toxicity. OAT1 is expressed

44

Section I / General Principles

mainly in the kidney and is responsible for the renal tubularsecretion of anionic compounds. Some reports have indicat-ed that substrates of OAT1, such as

cephaloridine,

a

β

-lac-tam antibiotic, sometimes cause nephrotoxicity.

In vitro

experiments suggest that cephaloridine is a substrate ofOAT1 and that OAT1-expressing cells are more susceptibleto cephaloridine toxicity than control cells.

Transporters for endogenous ligands may be modulat-ed by drugs and thereby exert adverse effects (Figure 2–3,

bottom panel

). For example, bile acids are taken up

mainly by

N

a

+

-

t

aurocholate

c

otransporting

p

olypeptide(NTCP) (Hagenbuch

et al.

, 1991) and excreted into the bileby the

b

ile

s

alt

e

xport

p

ump (BSEP,

ABCB11

) (Gerloff

etal.

, 1998). Bilirubin is taken up by OATP1B1 and conjugat-ed with glucuronic acid, and bilirubin glucuronide is excret-ed by the

m

ultidrug-

r

esistance-associated

p

rotein (MRP2,

ABCC2).

Inhibition of these transporters by drugs maycause cholestasis or hyperbilirubinemia.

Troglitazone,

a thi-azolidinedione insulin-sensitizing drug used for the treat-ment of type II diabetes mellitus, was withdrawn from the

Figure 2–3.

Major mechanisms by which transporters mediate adverse drug responses.

Three cases are given. The

left panel

ofeach case provides a cartoon 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 such as the liver andkidney. (

Middle panel

) Increase in the concentration of drug in toxicological target organs due either to the enhanced uptake or to reducedefflux of the drug. (

Bottom panel

) Increase in the plasma concentration of an endogenous compound (

e.g

., a bile acid) due to a drug’sinhibiting the influx of the endogenous compound in its eliminating or target organ. The diagram also may represent an increase in theconcentration of the endogenous compound in the target organ owing to drug-inhibited efflux of the endogenous compound.

Chapter 2 / Membrane Transporters and Drug Response

45

market because it caused hepatotoxicity. The mechanism forthis troglitazone-induced hepatotoxicity remains unclear.One hypothesis is that troglitazone and its sulfate conjugateinduced cholestasis. Troglitazone sulfate potently inhibitsthe efflux of taurocholate (

K

i

= 0.2

µ

M) mediated by theABC transporter BSEP. These findings suggest that troglita-zone sulfate induces cholestasis by inhibition of BSEP func-tion. BSEP-mediated transport is also inhibited by otherdrugs, including cyclosporin A and the antibiotics

rifamycin

and

rifampicin

(Stieger

et al.

, 2000).

Thus, uptake and efflux transporters determine theplasma and tissue concentrations of endogenous com-pounds and xenobiotics and thereby can influence the sys-temic or site-specific toxicity of drugs

.

BASIC MECHANISMS OF MEMBRANE TRANSPORT

Transporters

versus

Channels.

Both channels and transporters facil-itate the membrane permeation of inorganic ions and organic com-pounds (Reuss, 2000). In general, channels have two primarystates,

open

and

closed,

that are totally stochastic phenomena. Only

in the open state do channels appear to act as pores for the selectedions, allowing their permeation across the plasma membrane. Afteropening, channels return to the closed state as a function of time. Incontrast, a transporter forms an intermediate complex with the sub-strate (solute), and subsequently a conformational change in thetransporter induces translocation of the substrates to the other sideof the membrane. Therefore, there is a marked difference in turn-over rates between channels and transporters. The turnover rateconstants of typical channels are 10

6

to 10

8

s

–1

, whereas those oftransporters are, at most, 10

1

to 10

3

s

–1

. Because a particular trans-porter forms intermediate complexes with specific compounds(referred to as

substrates

), transporter-mediated membrane trans-port is characterized by saturability and inhibition by substrate ana-logs, as described below.

The basic mechanisms involved in solute transport across bio-logical membranes include passive diffusion, facilitated diffusion,and active transport. Active transport can be further subdivided intoprimary and secondary active transport. These mechanisms aredepicted in Figure 2–4 and described below.

Passive Diffusion. Simple diffusion of a solute acrossthe plasma membrane consists of three processes: parti-tion from the aqueous to the lipid phase, diffusion acrossthe lipid bilayer, and repartition into the aqueous phase onthe opposite side. Diffusion of any solute (includingdrugs) occurs down an electrochemical potential gradient∆µ of the solute, given by the equation:

Figure 2–4. Classification of membrane transport mechanisms. Light blue circles depict the substrate. Size of the circles isproportional to the concentration of the substrate. Arrows show the direction of flux. Black squares represent the ion that supplies thedriving force for transport (size is proportional to the concentration of the ion). Dark blue ovals depict transport proteins.

46 Section I / General Principles

(2–1)

where z is the charge valence of the solute, Em is themembrane voltage, F is the Faraday constant, R is the gasconstant, T is the absolute temperature, C is the concen-tration of the solute inside (i) and outside (o) of the plas-ma membrane. The first term on the right side in Eq. (2–1)represents the electrical potential, and the second repre-sents the chemical potential.

For nonionized compounds, the flux J owing to simple diffusionis given by Fick’s first law (permeability multiplied by the concen-tration difference). For ionized compounds, the difference in electri-cal potential across the plasma membrane needs to be taken intoconsideration. Assuming that the electrical field is constant, the fluxis given by the Goldman–Hodgkin–Katz equation:

(2–2)

where P represents the permeability. The lipid and water solubilityand the molecular weight and shape of the solute are determinantsof the flux in passive diffusion; they are incorporated in the per-meability constant P. The permeability constant positively corre-lates with the lipophilicity, determined by the partition betweenwater and organic solvents, such as octanol, and is also related tothe inverse of the square root of the molecular weight of the sol-ute. At steady state, the electrochemical potentials of all com-pounds become equal across the plasma membrane. In the case ofnonionized compounds, the steady-state concentrations are equalacross the plasma membrane. For ionized compounds, however,the steady-state concentration ratio across the plasma membrane isaffected by the membrane voltage and given by the Nernst equa-tion (Eq. 2–3).

(2–3)

The membrane voltage is maintained by the ion gradients across themembrane.

Facilitated Diffusion. Diffusion of ions and organiccompounds across the plasma membrane may be facili-tated by a membrane transporter. Facilitated diffusionis a form of transporter-mediated membrane transportthat does not require energy input. Just as in passivediffusion, the transport of ionized and un-ionized com-pounds across the plasma membrane occurs down theirelectrochemical potential gradient. Therefore, steadystate will be achieved when the electrochemical poten-tials of the compound on both sides of the membranebecome equal.

Active Transport. Active transport is the form of mem-brane transport that requires the input of energy. It is thetransport of solutes against their electrochemical gradi-ents, leading to the concentration of solutes on one side ofthe plasma membrane and the creation of potential energyin the electrochemical gradient formed. Active transportplays an important role in the uptake and efflux of drugsand other solutes. Depending on the driving force, activetransport can be subdivided into primary and secondaryactive transport (Figure 2–4).

Primary Active Transport. Membrane transport thatdirectly couples with ATP hydrolysis is called primaryactive transport. ABC transporters are examples of pri-mary active transporters. They contain one or two ATPbinding cassettes and a highly conserved domain in theintracellular loop region that exhibits ATPase activity.In mammalian cells, primary active transporters mediatethe unidirectional efflux of solutes across biologicalmembranes. The molecular mechanism by which ATPhydrolysis is coupled to the active transport of sub-strates by ABC transporters is a subject of currentinvestigation.

Secondary Active Transport. In secondary activetransport, the transport across a biological membraneof one solute S1 against its concentration gradient isenergetically driven by the transport of another soluteS2 in accordance with its concentration gradient. Thedriving force for this type of transport therefore isstored in the electrochemical potential created by theconcentration difference of S2 across the plasma mem-brane. For example, an inwardly directed Na+ concen-tration gradient across the plasma membrane is createdby Na+,K+-ATPase. Under these conditions, inwardmovement of Na+ produces the energy to drive themovement of a substrate S1 against its concentrationgradient by a secondary active transporter as in Na+/Ca2+ exchange.

Depending on the transport direction of the solute, secondaryactive transporters are classified as either symporters or antiport-ers. Symporters, also termed cotransporters, transport S2 and S1 inthe same direction, whereas antiporters, also termed exchangers,move their substrates in opposite directions (Figure 2–4). The freeenergy produced by one extracellular sodium ion (Na+) is given bythe difference in the electrochemical potential across the plasmamembrane:

(2–4)

The electrochemical potential of a nonionized compound ∆µsacquired from one extracellular Na+ is less than this value:

∆µ zEmF RTCi

Co------

ln+=

J PzEmF

RT--------------

Ci Co EmF RT⁄( )exp–

1 EmF RT⁄( )exp–--------------------------------------------------------–=

Ci

Co------

zEmF–

RT-----------------

exp=

∆µNa EmF RTCN a i,CN a o,---------------

ln+=

Chapter 2 / Membrane Transporters and Drug Response 47

(2–5)

Therefore, the concentration ratio of the compound is given bythe following equation:

(2–6)

Assuming that the concentration ratio of Na+ is 10 and that Em is–60 mV, ideally, symport of one nonionized organic compound withone Na+ ion can achieve a one hundredfold difference in the intra-cellular substrate concentration compared with the extracellular con-centration. When more than one Na+ ion is coupled to the move-ment of the solute, a synergistic driving force results. For the case inwhich two Na+ ions are involved,

(2–7)

In this case, the substrate ideally is concentrated intracellularlyone thousandfold relative to the extracellular space under the sameconditions. The Na+/Ca2+ antiporter shows the effect of this depen-dence in the square of the concentration ratio of Na+; Ca2+ is trans-ported from the cytosol (0.1 µM < [Ca2+] < 1 µM) to the plasma[Ca2+]free ~ 1.25 mM.

KINETICS OF TRANSPORTThe flux of a substrate (rate of transport) across a bio-logical membrane via transporter-mediated processes ischaracterized by saturability. The relationship betweenthe flux v and substrate concentration C in a transporter-mediated process is given by the Michaelis–Mentenequation:

(2–8)

where Vmax is the maximum transport rate and is propor-tional to the density of transporters on the plasma mem-brane, and Km is the Michaelis constant, which repre-sents the substrate concentration at which the flux is halfthe Vmax value. Km is an approximation of the dissocia-tion constant of the substrate from the intermediate com-plex. When C is small compared with the Km value, theflux is increased in proportion to the substrate concen-tration (roughly linear with substrate concentration).However, if C is large compared with the Km value, theflux approaches a constant value (Vmax). The Km andVmax values can be determined by examining the flux atdifferent substrate concentrations. The Eadie–Hofsteeplot often is used for graphical interpretation of satura-

tion kinetics. Plotting clearance v/C on the y axis andflux v on the x axis gives a straight line. The y interceptrepresents the ratio Vmax/Km, and the slope of the line isthe inverse of the Km value:

(2–9)

Involvement of multiple transporters with different Km

values gives an Eadie–Hofstee plot that is curved. In alge-braic terms, the Eadie–Hofstee plot of kinetic data isequivalent to the Scatchard plot of equilibrium bindingdata.

Transporter-mediated membrane transport of a sub-strate is also characterized by inhibition by other com-pounds. The manner of inhibition can be categorized asone of three types: competitive, noncompetitive, anduncompetitive.

Competitive inhibition occurs when substrates and inhibitorsshare a common binding site on the transporter, resulting in anincrease in the apparent Km value in the presence of inhibitor. Theflux of a substrate in the presence of a competitive inhibitor is

(2–10)

where I is the concentration of inhibitor, and Ki is the inhibitionconstant.

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

(2–11)

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

(2–12)

VECTORIAL TRANSPORTThe SLC type of transporter mediates either drug uptakeor efflux, whereas ABC transporters mediate only unidi-rectional efflux. Asymmetrical transport across a mono-layer of polarized cells, such as the epithelial and endo-thelial cells of brain capillaries, is called vectorialtransport (Figure 2–5). Vectorial transport is important in

∆µS ∆µNa 0≤+

Si

So-----

CN a o,CN a i,---------------

EmF–

RT--------------

exp≤

Si

So-----

CN a o,CN a i,---------------

2 2EmF–

RT------------------

exp≤

vV maxC

Km C+------------------=

vC----

V max

Km------------

CKm-------–=

vV maxC

Km 1 I Ki⁄+( ) C+---------------------------------------------=

vV max 1 I Ki⁄+( ) C⋅⁄

Km C+---------------------------------------------------=

vV max 1 I Ki⁄+( ) C⋅⁄Km 1 I Ki⁄+( )⁄ C+---------------------------------------------------=


the efficient transfer of solutes across epithelial or endo-thelial barriers. For example, vectorial transport is impor-tant for the absorption of nutrients and bile acids in theintestine. From the viewpoint of drug absorption and dis-position, vectorial transport plays a major role in hepato-biliary and urinary excretion of drugs from the blood tothe lumen and in the intestinal absorption of drugs. Inaddition, efflux of drugs from the brain via brain endothe-lial cells and brain choroid plexus epithelial cells involvesvectorial transport.

For lipophilic compounds that have sufficient mem-brane permeability, ABC transporters alone are able toachieve vectorial transport by extruding their substrates tothe outside of cells without the help of influx transporters(Horio et al., 1990). For relatively hydrophilic organicanions and cations, coordinated uptake and efflux trans-porters in the polarized plasma membranes are necessaryto achieve the vectorial movement of solutes across anepithelium. Common substrates of coordinated transport-ers are transferred efficiently across the epithelial barrier(Sasaki et al., 2002). In the liver, a number of transporterswith different substrate specificities are localized on thesinusoidal membrane (facing blood). These transportersare involved in the uptake of bile acids, amphipathicorganic anions, and hydrophilic organic cations into thehepatocytes. Similarly, ABC transporters on the canalicu-lar membrane (facing bile) export such compounds intothe bile. Overlapping substrate specificities between theuptake transporters (OATP family) and efflux transporters(MRP family) make the vectorial transport of organic

anions highly efficient. Similar transport systems also arepresent in the intestine, renal tubules, and endothelial cellsof the brain capillaries (Figure 2–5).

Regulation of Transporter Expression. Transporter expression canbe regulated transcriptionally in response to drug treatment andpathophysiological conditions, resulting in induction or down-regulation of transporter mRNAs. Recent studies have describedimportant roles of type II nuclear receptors, which form het-erodimers with the 9-cis-retinoic acid receptor (RXR), in regu-lating drug-metabolizing enzymes and transporters (Kullak-Ublick et al., 2004; Wang and LeCluyse, 2003). Such receptorsinclude pregnane X receptor (PXR/NR1I2), constitutive andros-tane receptor (CAR/NR1I3), farnesoid X receptor (FXR/NR1H4), PPARα (peroxisome proliferator-activated receptor α),and retinoic acid receptor (RAR). Except for CAR, these areligand-activated nuclear receptors that, as heterodimers withRXR, bind specific elements in the enhancer regions of targetgenes. CAR has constitutive transcriptional activity that isantagonized by inverse agonists such as androstenol andandrostanol and induced by barbiturates. PXR, also referred toas steroid X receptor (SXR) in humans, is activated by syntheticand endogenous steroids, bile acids, and drugs such as clotrim-azole, phenobarbital, rifampicin, sulfinpyrazone, ritonavir, car-bamazepine, phenytoin, sulfadimidine, taxol, and hyperforin (aconstituent of St. John’s wort). Table 2–1 summarizes the effectsof drug activation of type II nuclear receptors on expression oftransporters. The potency of activators of PXR varies amongspecies such that rodents are not necessarily a model for effectsin humans. There is an overlap of substrates between CYP3A4and P-glycoprotein, and PXR mediates coinduction of CYP3A4and P-glycoprotein, supporting their synergetic cooperation inefficient detoxification. See Table 3–4 and Figure 3–13 for infor-mation on the role of type II nuclear receptors in induction ofdrug-metabolizing enzymes.

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


MOLECULAR STRUCTURES OF TRANSPORTERS

Predictions of secondary structure of membrane trans-port proteins based on hydropathy analysis indicate thatmembrane transporters in the SLC and ABC superfami-lies are multi-membrane-spanning proteins. A typicalpredicted secondary structure of the ABC transporterMRP2 (ABCC2) is shown in Figure 2–6. However,understanding the secondary structure of a membranetransporter provides little information on how the trans-porter functions to translocate its substrates. For this,information on the tertiary structure of the transporter is

needed, along with complementary molecular informa-tion about the residues in the transporter that areinvolved in the recognition, association, and dissociationof its substrates.

To obtain high-resolution structures of membrane proteins, theproteins first must be crystallized, and then the crystal structuremust be deduced from analysis of x-ray diffraction patterns. Crys-tal structures generally are difficult to obtain for membrane pro-teins primarily because of their amphipathic needs for stabiliza-tion. Further, membrane proteins generally are in low abundance,so obtaining sufficient quantities for structural determination isdifficult. The few membrane transporters that have been crystal-lized are bacterial proteins that can be expressed in high abun-dance. Information on two representative membrane transportersthat have been crystallized and analyzed at relatively high resolu-

Table 2–1Regulation of Transporter Expression by Nuclear Receptors

TRANSPORTER SPECIESTRANSCRIPTION FACTOR LIGAND (DOSE) EFFECT OF LIGAND

MDR1 (P-gp) Human PXR ↑ Transcription activity (promoter assay)Rifampicin

(600 mg/day, 10 days)↑ Expression in duodenum in healthy

subjectsRifampicin

(600 mg/day, 10 days↓ Oral bioavailability of digoxin in

healthy subjectsRifampicin

(600 mg/day, 9 days)↓ AUC of talinolol after IV and oral

administration in healthy subjectsMRP2 Human PXR Rifampicin

(600 mg/day, 9 days)↑ Expression in duodenum in healthy

subjectsRifampicin/hyperforin ↑ Expression in human hepatocytes

FXR GW4064/chenodeoxy-cholate

↑ Expression in HepG2 cells

Mouse PXR PCN/dexamethasone ↑ Expression in mouse hepatocyteCAR Phenobarbital ↑ Expression in hepatocyte of PXR KO

mice (promoter assay)Rat PXR/FXR/CAR PCN/GW4064/phenobar-

bital↑ Expression in rat hepatocytes

PXR/FXR/CAR ↑ Transcription activity (promoter assay)BSEP Human FXR Chenodeoxycholate,

GW4064↑ Transcription activity (promoter assay)

Ntcp Rat SHP1 ↓ RAR mediated transcriptionOATP1B1 Human SHP1 Indirect effect on HNF1a expressionOATP1B3 Human FXR Chenodeoxycholate ↑ Expression in hepatoma cellsMDR2 Mouse PPARa Ciprofibrate (0.05% w/w

in diet)↑ Expression in the liver

See Geick et al., 2001; Greiner et al., 1999; Kok et al., 2003.


tion (<4 Å) serves to illustrate some basic structural properties ofmembrane transporters. One of the transporters, MsbA, is an ABCtransporter from E. coli with homology to multidrug-resistanceefflux pumps in mammals. The second transporter, LacY, is a pro-ton symporter, also from E. coli, that translocates lactose and otheroligosaccharides. Each of these transporters is illustrative of a dif-ferent transport mechanism.

Lipid Flippase (MsbA). MsbA is an ABC transporter in E. coli that,like other ABC transporters, hydrolyzes ATP to export its substrate.Based on an x-ray crystal structure, MsbA forms a homodimer con-sisting of two six-transmembrane units, each with a nucleotide-bind-ing domain on the cytoplasmic surface (Chang and Roth, 2001)(Figure 2–7). The hexaspanning unit consists of six α-helices. Thereis a central chamber with an asymmetrical distribution of chargedresidues. A transport mechanism that is consistent with this asym-metrical distribution of charges is a “flippase” mechanism. That is,substrates in the inner leaflet of the bilayer are recognized by MsbAand then flipped to the outer leaflet of the bilayer. This hypotheticalmechanism, although intriguing, leaves many questions unan-swered. For example, how is the energy of ATP hydrolysis coupledto the flipping process? Once in the outer leaflet, how are substratestranslocated to the extracellular space? Nevertheless, from thisstructure and other structures, we now know that transmembranedomains form α-helices, that six-unit dimers are central to the trans-port mechanism, and that there is an asymmetrical distribution ofcharged residues in a central chamber.

Lactose Permease Symporter (LacY). Lactose permease is a bacte-rial transporter that belongs to the major facilitator superfamily(MFS). This transporter is a proton-coupled symporter. A high-resolution X-ray crystal structure has been obtained for the proto-

nated form of a mutant of LacY (C154G) at a 3.5-Å α-resolution(Abramson et al., 2003) (Figure 2–8). In brief, LacY is com-prised of two units of six membrane-spanning α-helices. Thecrystal structure showed substrate located at the interface of the

Figure 2–6. Predicted secondary structure of MRP2 based on hydropathy analysis. The dark blue circles depict glycosylationsites; Walker A motif is colored light blue; black boxes represent the Walker B motif. Light gray is the middle region betweenthe two motifs. The Walker A motifs interact with α and β phosphates of di- and tri-nucleotides; the Walker B motifs help tocoordinate Mg2+.

Figure 2–7. Structure showing the backbone of MsbA fromE. coli. The structure shows a central chamber and ahomodimer formed by units of six-transmembrane α-helices.Structure was reconstructed by Libusha Kelly using the coordi-nates deposited in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ).


two units and in the middle of the membrane. This location isconsistent with an alternating-access transport mechanism inwhich the substrate recognition site is accessible to the cytosolicand then the extracellular surface but not to both simultaneously.Eight helices form the surface of the hydrophilic cavity, and eachcontains proline and glycine residues that result in kinks in thecavity. From LacY, we now know that as in the case of MsbA,six membrane-spanning α-helices are critical structural units fortransport by LacY.

TRANSPORTER SUPERFAMILIES IN THE HUMAN GENOME

Two major gene superfamilies play critical roles in the trans-port of drugs across plasma and other biological membranes:the SLC and ABC superfamilies. Web sites that have infor-mation on these families include http://nutrigene.4t.com/humanabc.htm (ABC superfamily), http://www.biopara-digms.org/slc/intro.asp (SLC superfamily), http://www.phar-maconference.org/slctable.asp (SLC superfamily), and http://www.TP_Search.jp/ (drug transporters). Information onpharmacogenetics of these transporters can be found inChapter 4 and at http://www.pharmgkb.org and http://www.pharmacogenetics.ucsf.edu.

SLC Transporters. The solute carrier (SLC) superfamilyincludes 43 families and represents approximately 300genes in the human genome. The nomenclature of thetransporters within each family is listed under the HumanGenome Organization (HUGO) Nomenclature Commit-tee database at http://www.gene.ucl.ac.uk/nomenclature/.Table 2–2 lists the families in the human SLC superfam-ily and some of the genetic diseases that are associatedwith members of selected families. The family nameprovides a description of the function(s) of each family.However, some caution should be exercised in interpre-tation of family names because individual family mem-bers may have vastly different specificities or functionalroles. All the SLC families with members in the humangenome were reviewed recently (Hediger, 2004). Inbrief, transporters in the SLC superfamily transportdiverse ionic and nonionic endogenous compounds andxenobiotics. SLC superfamily transporters may be facili-tated transporters or secondary active symporters or anti-porters. The first SLC family transporter was cloned in1987 by expression cloning in Xenopus laevis oocytes(Hediger et al., 1987). Since then, many transporters inthe SLC superfamily have been cloned and characterizedfunctionally. Predictive models defining important char-acteristics of substrate binding and knockout mousemodels defining the in vivo role of specific transporters

have been constructed for many SLC transporters(Chang et al., 2004; Ocheltree et al., 2004). In general,in this chapter we focus on SLC transporters in thehuman genome, which are designated by capital letters(SLC transporters in rodent genomes are designated bylowercase letters).

ABC Superfamily. In 1976, Juliano and Ling reportedthat overexpression of a membrane protein in colchi-cine-resistant Chinese hamster ovary cells also resultedin acquired resistance to many structurally unrelateddrugs (i.e., multidrug resistance) (Juliano and Ling,1976). Since the cDNA cloning of this first mammali-an ABC protein (P-glycoprotein/MDR1/ABCB1), theABC superfamily has continued to grow; it now con-sists of 49 genes, each containing one or two conservedABC regions (Borst and Elferink, 2002). The ABCregion is a core catalytic domain of ATP hydrolysisand contains Walker A and B sequences and an ABCtransporter-specific signature C sequence (Figure 2–6).The ABC regions of these proteins bind and hydrolyzeATP, and the proteins use the energy for uphill trans-port of their substrates across the membrane. Although

Figure 2–8. Structure of the protonated form of a mutant ofLacY. Two units of six-membrane-spanning α-helices (shownas colored ribbons) are present. Substrate (depicted as gray andblack balls) is bound to the interface of the two units and in themiddle of the membrane. Structure has been redrawn from coor-dinates in Protein Data Bank (http://www.rcsb.org/pdb/ ).


Table 2–2Families in the Human Solute Carrier Superfamily

GENE NAME FAMILY NAME

NUMBER OF FAMILY MEMBERS

SELECTED DRUG SUBSTRATES

EXAMPLES OF LINKED HUMAN DISEASES

SLC1 High-affinity glutamate and neu-tral amino acid transporter

7 Amyotrophic lateral sclerosis

SLC2 Facilitative GLUT transporter 14SLC3 Heavy subunits of the heteromeric

amino acid transporters2 Melphalin Classic cystinuria type I

SLC4 Bicarbonate transporter 10 Hemolytic anemia, blindness–auditory impairment

SLC5 Na+ glucose cotransporter 8 Glucosfamide Glucose–galactose malabsorp-tion syndrome

SLC6 Na+- and Cl–-dependent neu-rotransmitter transporter

16 Paraoxetine, fluoxetine

X-linked creatine deficiency syndrome

SLC7 Cationic amino acid transporter 14 Melphalin Lysinuric protein intoleranceSLC8 Na+/Ca2+ exchanger 3 Asymmetrical

dimethylarginineSLC9 Na+/H+ exchanger 8 Thiazide diuretics Congenital secretory diarrheaSLC10 Na+ bile salt cotransporter 6 Benzothiazepine Primary bile salt malabsorptionSLC11 H+ coupled metal ion transporter 2 Hereditary hemochromatosisSLC12 Electroneutral cation–Cl–

cotransporter family9 Gitelman’s syndrome

SLC13 Na+–sulfate/carboxylate cotransporter

5 Sulfate, cysteine conjugates

SLC14 Urea transporter 2 Kidd antigen blood groupSLC15 H+–oligopeptide cotransporter 4 ValacyclovirSLC16 Monocarboxylate transporter 14 Salicylate, atorvastatin Muscle weaknessSLC17 Vesicular glutamate transporter 8 Sialic acid storage diseaseSLC18 Vesicular amine transporter 3 Reserpine Myasthenic syndromesSLC19 Folate/thiamine transporter 3 Methotrexate Thiamine-responsive megalo-

blastic anemiaSLC20 Type III Na+–phosphate

cotransporter2

SLC21/SLC0

Organic anion transporter 11 Pravastatin

SLC22 Organic cation/anion/zwitterion transporter

18 Pravastatin, metformin Systemic carnitine deficiency syndrome

SLC23 Na+-dependent ascorbate transporter 4 Vitamin CSLC24 Na+/(Ca2+-K+) exchanger 5SLC25 Mitochondrial carrier 27 Senger’s syndromeSLC26 Multifunctional anion exchanger 10 Salicylate,

ciprofloxacinCongenital Cl–-losing diarrhea

SLC27 Fatty acid transporter protein 6

(Continued)


some ABC superfamily transporters contain only a sin-gle ABC motif, they form homodimers (BCRP/ABCG2) or heterodimers (ABCG5 and ABCG8) thatexhibit a transport function. ABC transporters (e.g.,MsbA) (Figure 2–7) also are found in prokaryotes,where they are involved predominantly in the import ofessential compounds that cannot be obtained by passivediffusion (sugars, vitamins, metals, etc.). By contrast,most ABC genes in eukaryotes transport compoundsfrom the cytoplasm to the outside or into an intracellu-lar compartment (endoplasmic reticulum, mitochon-dria, peroxisomes).

ABC transporters can be divided into seven groupsbased on their sequence homology: ABCA (12 members),ABCB (11 members), ABCC (13 members), ABCD (4members), ABCE (1 member), ABCF (3 members), andABCG (5 members). ABC genes are essential for many

cellular processes, and mutations in at least 13 of thesegenes cause or contribute to human genetic disorders(Table 2–3).

In addition to conferring multidrug resistance (Sadeeet al., 1995), an important pharmacological aspect ofthese transporters is xenobiotic export from healthy tis-sues. In particular, MDR1/ABCB1, MRP2/ABCC2, andBCRP/ABCG2 have been shown to be involved in over-all drug disposition (Leslie et al., 2005).

Properties of ABC Transporters Related to Drug Action

The tissue distribution of drug-related ABC transporters in the bodyis summarized in Table 2–4 together with information about typicalsubstrates.

Tissue Distribution of Drug-Related ABC Transporters. MDR1(ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) are all expressedin the apical side of the intestinal epithelia, where they serve to

SLC28 Na+-coupled nucleoside transport 3 Gemcitabine, cladribine

SLC29 Facilitative nucleoside transporter 4 Dipyridamole, gemcitabine

SLC30 Zinc efflux 9SLC31 Copper transporter 2 CisplatinSLC32 Vesicular inhibitory amino acid

transporter1 Vigabatrin

SLC33 Acetyl-CoA transporter 1SLC34 Type II Na+–phosphate

cotransporter3 Autosomal-dominant hypo-

phosphatemic ricketsSLC35 Nucleoside-sugar transporter 17 Leukocyte adhesion deficiency

type IISLC36 H+-coupled amino acid transporter 4 D-Serine,

D-cycloserineSLC37 Sugar-phosphate/phosphate

exchanger4 Glycogen storage disease non-1a

SLC38 System A and N, Na+-coupled neutral amino acid transporter

6

SLC39 Metal ion transporter 14 Acrodermatitis enteropathicaSLC40 Basolateral iron transporter 1 Type IV hemochromatosisSLC41 MgtE-like magnesium transporter 3SLC42 Rh ammonium transporter (pending) 3 Rh-null regulatorSLC43 Na+-independent system-L-like

amino acid transporter2

Table 2–2Families in the Human Solute Carrier Superfamily (Continued)


NUMBER OF FAMILY MEMBERS

SELECTED DRUG SUBSTRATES

EXAMPLES OF LINKED HUMAN DISEASES


pump out xenobiotics, including many clinically relevant drugs.The kidney and liver are major organs for overall systemic drugelimination from the body. The liver also plays a role in presys-temic drug elimination. Key to the vectorial excretion of drugsinto urine or bile, ABC transporters are expressed in the polarizedtissues of kidney and liver: MDR1, MRP2, and MRP4 (ABCC4)on the brush-border membrane of renal epithelia, and MDR1,MRP2, and BCRP on the bile canalicular membrane of hepato-cytes. Some ABC transporters are expressed specifically on theblood side of the endothelial or epithelial cells that form barriersto the free entrance of toxic compounds into naive tissues: theBBB (MDR1 and MRP4 on the luminal side of brain capillaryendothelial cells), the blood–cerebrospinal fluid (CSF) barrier(MRP1 and MRP4 on the basolateral blood side of choroid plexusepithelia), the blood–testis barrier (MRP1 on the basolateral mem-brane of mouse Sertoli cells and MDR1 in several types of humantesticular cells), and the blood–placenta barrier (MDR1, MRP2,and BCRP on the luminal maternal side and MRP1 on the antilu-minal fetal side of placental trophoblasts).

Substrate Specificity of ABC Transporters. MDR1/ABCB1 sub-strates tend to share a hydrophobic planar structure with positive-ly charged or neutral moieties as described in Table 2–4 (see alsoAmbudkar et al., 1999). These include structurally and pharmaco-logically unrelated compounds, many of which are also substrates

of CYP3A4, a major drug-metabolizing enzyme in the humanliver and GI tract. Such overlapping substrate specificity impliesa synergistic role for MDR1 and CYP3A4 in protecting the bodyby reducing the intestinal absorption of xenobiotics (Zhang andBenet, 2001). After being taken up by enterocytes, some drugmolecules are metabolized by CYP3A4. Drug molecules thatescape metabolic conversion are eliminated from the cells viaMDR1 and then reenter the enterocytes. The intestinal residencetime of the drug is prolonged with the aid of MDR1, therebyincreasing the chance of local metabolic conversion by theCYP3A4 (see Chapter 3).

MRP/ABCC Family. The substrates of transporters in the MRP/ABCC family are mostly organic anions. The substrate specificitiesof MRP1 and MRP2 are similar: Both accept glutathione and gluc-uronide conjugates, sulfated conjugates of bile salts, and nonconju-gated organic anions of an amphipathic nature (at least one negativecharge and some degree of hydrophobicity). They also transportneutral or cationic anticancer drugs, such as vinca alkaloids andanthracyclines, possibly via a cotransport or symport mechanismwith reduced glutathione (GSH).

MRP3 also has a substrate specificity that is similar to that ofMRP2 but with a lower transport affinity for glutathione conju-gates compared with MRP1 and MRP2. Most characteristicMRP3 substrates are monovalent bile salts, which are never trans-ported by MRP1 and MRP2. Because MRP3 is expressed on the

Table 2–3The ATP Binding Cassette (ABC) Superfamily in the Human Genome and Linked Genetic Diseases


NUMBER OF FAMILY MEMBERS EXAMPLES OF LINKED HUMAN DISEASES

ABCA ABC A 12 Tangier disease (defect in cholesterol transport; ABCA1), Stargardt syndrome (defect in retinal metabolism; ABCA4)

ABCB ABC B 11 Bare lymphocyte syndrome type I (defect in antigen-presenting; ABCB3 and ABCB4), progressive familial intrahepatic cholestasis type 3 (defect in biliary lipid secretion; MDR3/ABCB4), X-linked sideroblas-tic anemia with ataxia (a possible defect in iron homeostasis in mito-chondria; ABCB7), progressive familial intrahepatic cholestasis type 2 (defect in biliary bile acid excretion; BSEP/ABCB11)

ABCC ABC C 13 Dubin–Johnson syndrome (defect in biliary bilirubin glururonide excre-tion; MRP2/ABCC2), pseudoxanthoma (unknown mechanism; ABCC6), cystic fibrosis (defect in chloride channel regulation; ABCC7), persistent hyperinsulinemic hypoglycemia of infancy (defect in inwardly rectifying potassium conductance regulation in pancreatic B cells; SUR1)

ABCD ABC D 4 Adrenoleukodystrophy (a possible defect in peroxisomal transport or catabolism of very long-chain fatty acids; ABCD1)

ABCE ABC E 1ABCF ABC F 3ABCG ABC G 5 Sitosterolemia (defect in biliary and intestinal excretion of plant sterols;

ABCG5 and ABCG8)


Table 2–4ABC Transporters Involved in Drug Absorption, Distribution, and Excretion

TRANSPORTER NAME

TISSUE DISTRIBUTION

PHYSIOLOGICAL FUNCTION SUBSTRATES

MDR1 Liver Detoxification of xenobiotics?

Characteristics: Neutral or cationic compounds with bulky structure(ABCB1) Kidney

Intestine Anticancer drugs: etoposide, doxorubicin, vincristineBBB Ca2+ channel blockers: diltiazem, verapamilBTB HIV protease inhibitors: indinavir, ritonavirBPB Antibiotics/antifungals: erythromycin, ketoconazole

Hormones: testosterone, progesterone Immunosuppressants: cyclosporine, FK506 (tacrolimus)Others: digoxin, quinidine

MRP1 Ubiquitous (kidney, BCSFB, BTB)

Leukotriene (LTC4) secre-tion from leu-kocyte

Characteristics: Amphiphilic with at least one negative net charge(ABCC1)

Anticancer drugs: vincristine (with GSH), methotrexate Glutathione conjugates: LTC4, glutathione conjugate of

ethacrynic acid Glucuronide conjugates: estradiol-17-D-glucuronide,

bilirubin mono(or bis)glucuronide Sulfated conjugates: estrone-3-sulfate (with GSH) HIV protease inhibitors: saquinavir Antifungals: grepafloxacinOthers: folate, GSH, oxidized glutathione

MRP2 Liver Excretion of bilirubin glu-curonide and GSH into bile

Characteristics: Amphiphilic with at least one negative net charge (similar to MRP1)(ABCC2) Kidney

Intestine Anticancer drugs: methotrexate, vincristineBPB Glutathione conjugates: LTC4, GSH conjugate of

ethacrynic acid Glucuronide conjugates: estradiol-17-D-glucuronide,

bilirubin mono(or bis)glucuronideSulfate conjugate of bile salts: taurolithocholate sulfateHIV protease inhibitors: indinavir, ritonavirOthers: pravastatin, GSH, oxidized glutathione

MRP3 Liver ? Characteristics: Amphiphilic with at least one negative net charge (Glucuronide conjugates are better sub-strates than glutathione conjugates.)

(ABCC3) KidneyIntestine

Anticancer drugs: etoposide, methotrexateGlutathione conjugates: LTC4, glutathione conjugate of

15-deoxy-delta prostaglandin J2Glucuronide conjugates: estradiol-17-D-glucuronide,

etoposide glucuronideSulfate conjugates of bile salts: taurolithocholate sulfateBile salts: glycocholate, taurocholateOthers: folate, leucovorin

(Continued)


sinusoidal side of hepatocytes and is induced under cholestaticconditions, backflux of toxic bile salts and bilirubin glucuronidesinto the blood circulation is considered to be its physiologicalfunction.

MRP4 and MRP5 have narrower substrate specificities. Theyaccept nucleotide analogues and clinically important anti–human immunodeficiency virus (HIV) drugs. Although sometransport substrates have been identified for MRP6, no physio-logically important endogenous substrates have been identifiedthat explain the mechanism of the MRP6-associated diseasepseudoxanthoma.

BCRP/ABCG2. BCRP accepts both neutral and negatively chargedmolecules, including cytotoxic compounds (e.g., mitoxantrone, topote-can, flavopiridol, and methotrexate), sulfated conjugates of therapeuticdrugs and hormones (e.g., estrogen sulfate), and toxic compoundsfound in normal food [2-amino-1-methyl-6-phenylimidazo[4,5-b]pyri-dine (PhIP) and pheophorbide A, a chlorophyll catabolite].

Physiological Roles of ABC Transporters. The physiological sig-nificance of the ABC transporters is illustrated by studies involv-ing knockout animals or patients with genetic defects in thesetransporters. Mice deficient in MDR1 function are viable and fer-

MRP4 Ubiquitous (kid-ney, prostate, lung, muscle, pancreas, testis, ovary, bladder, gallbladder, BBB, BCSFB)

? Characteristics: Nucleotide analogues(ABCC4) Anticancer drugs: 6-mercaptopurine, methotrexate

Glucuronide conjugates: estradiol-17-D-glucuronideCyclic nucleotides: cyclic AMP, cyclic GMPHIV protease inhibitors: adefovirOthers: folate, leucovorin, taurocholate (with GSH)

MRP5 Ubiquitous ? Characteristics: Nucleotide analogues(ABCC5) Anticancer drugs: 6-mercaptopurine

Cyclic nucleotides: cyclic AMP, cyclic GMPHIV protease inhibitors: adefovir

MRP6 Liver ? Anticancer drugs: doxorubicin*, etoposide*

(ABCC6) Kidney Glutathione conjugate of: LTC4

Other: BQ-123 (cyclic peptide ET-1 antagonist)BCRP Liver Normal heme

transport dur-ing maturation of erythrocytes

Anticancer drugs: methotrexate, mitoxantrone, camptothecin analogs (SN-38, etc.), topotecan(MXR) Intestine

(ABCG2) BBB Glucuronide conjugates: 4-methylumbelliferone glucu-ronide, estradiol-17-D-glucuronide

Sulfate conjugates: dehydroepiandrosterone sulfate, estrone-3-sulfate

Others: cholesterol, estradiolMDR3 Liver Excretion of

phospholipids into bile

Characteristics: Phospholipids(ABCB4)

BSEP Liver Excretion of bile salts into bile

Characteristics: Bile salts(ABCB11)ABCG5 and

ABCG8Liver Excretion of plant

sterols into bile and intestinal lumen

Characteristics: Plant sterolsIntestine

NOTE: Representative substrates and cytotoxic drugs with increased resistance (*) are included in this table (cytotoxicity with increased resistance isusually caused by the decreased accumulation of the drugs). Although MDR3 (ABCB4), BSEP (ABCB11), ABCG5, and ABCG8 are not directlyinvolved in drug disposition, inhibition of these physiologically important ABC transporters will lead to unfavorable side effects.

Table 2–4ABC Transporters Involved in Drug Absorption, Distribution, and Excretion (Continued)

TRANSPORTER NAME

TISSUE DISTRIBUTION

PHYSIOLOGICAL FUNCTION SUBSTRATES


tile and do not display obvious phenotypic abnormalities otherthan hypersensitivity to toxic drugs, including the neurotoxic pes-ticide ivermectin (one hundredfold) and the carcinostatic drugvinblastine (threefold) (Schinkel et al., 1994). mrp1 (–/–) miceare also viable and fertile without any obvious difference in littersize. However, these mice are hypersensitive to the anticancerdrug etoposide. Damage is especially severe in the testis, kidney,and oropharyngeal mucosa, where MRP1 is expressed on thebasolateral membrane. Moreover, these mice have an impairedresponse to an arachidonic acid–induced inflammatory stimulus,which is likely due to a reduced secretion of leukotriene C4 frommast cells, macrophages, and granulocytes. MRP2-deficient rats(TR– and EHBR) and Dubin–Johnson syndrome patients are nor-mal in appearance except for mild jaundice owing to impaired bil-iary excretion of bilirubin glucuronide (Ito et al., 1997; Paulusmaet al., 1996).

BCRP knockout mice are viable but highly sensitive to thedietary chlorophyll catabolite phenophorbide, which induces pho-totoxicity. These mice also exhibit protoporphyria, with a tenfoldincrease in protoporphyrin IX accumulation in erythrocytes,resulting in photosensitivity. This protoporphyria is caused by theimpaired function of BCRP in bone marrow: Knockout mice trans-planted with bone marrow from wild-type mice become normalwith respect to protoporphyrin IX level in the erythrocytes andphotosensitivity.

As described earlier, complete absence of these drug-relatedABC transporters is not lethal and even can remain unrecognizedwithout exogenous perturbation owing to food, drugs, or toxins.Inhibition of physiologically important ABC transporters (especiallythose related directly to the genetic diseases described in Table 2–3)by drugs should be avoided to reduce the incidence of drug-inducedside effects.

ABC Transporters in Drug Absorption and Elimination. With respectto clinical medicine, MDR1 is the most important ABC trans-porter yet identified, and digoxin is one of the most widely stud-ied of its substrates. The systemic exposure to orally adminis-tered digoxin (as assessed by the area under the plasma digoxinconcentration–time curve) is increased by coadministration ofrifampin (an MDR1 inducer) and is negatively correlated withthe MDR1 protein expression in the human intestine. MDR1 isalso expressed on the brush-border membrane of renal epithelia,and its function can be monitored using digoxin as a probe drug.Digoxin undergoes very little degradation in the liver, and renalexcretion is the major elimination pathway (>70%) in humans.Several studies in healthy subjects have been performed withMDR1 inhibitors (e.g., quinidine, verapamil, vaspodar, spirono-lactone, clarythromycin, and ritonavir) with digoxin as a probedrug, and all resulted in a marked reduction in the renal excre-tion of digoxin. Similarly, the intestinal absorption of cyclospor-ine is also related mainly to the MDR1 level rather than to theCYP3A4 level, although cyclosporine is a substrate of bothCYP3A4 and MDR1.

Alteration of MDR1 activity by inhibitors (drug–drug interac-tions) affects oral absorption and renal clearance. Drugs with nar-row therapeutic windows (such as the cardiac glycoside digoxinand the immunosuppressants cyclosporine and tacrolimus) shouldbe used with great care if MDR1-based drug–drug interactions arelikely.

Despite the broad substrate specificity and distinct localizationof MRP2 and BCRP in drug-handling tissues (both expressed on the

canalicular membrane of hepatocytes and the brush-border mem-brane of enterocytes), there has been very little integration of clini-cally relevant information. Part of the problem lies in distinguishingthe biliary transport activities of MRP2 and BCRP from the contri-bution of the hepatic uptake transporters of the OATP family. MostMRP2 or BCRP substrates also can be transported by the OATPfamily transporters on the sinusoidal membrane. The rate-limitingprocess for systemic elimination is uptake in most cases. Under suchconditions, the effect of drug–drug interactions (or genetic variants)in these biliary transporters may be difficult to identify. Despitesuch practical difficulties, there is a steady increase in the informa-tion about genetic variants and their effects on transporter expres-sion and activity in vitro. Variants of BCRP with high allele fre-quencies (0.184 for V12M and 0.239 for Q141K) have been foundto alter the substrate specificity in cellular assays. The clinicalimpact of these variants and drug–drug interactions needs to bestudied in more detail in humans and under in vivo conditions usingappropriate probe drugs.

GENETIC VARIATION IN MEMBRANE TRANSPORTERS: IMPLICATIONS FOR CLINICAL DRUG RESPONSE

Inherited defects in membrane transport have been knownfor many years, and the genes associated with severalinherited disorders of membrane transport have beenidentified [Table 2–2 (SLC) and Table 2–3 (ABC)].Reports of polymorphisms in membrane transporters thatplay a role in drug response have appeared only recently,but the field is growing rapidly. Cellular studies havefocused on genetic variation in only a few drug transport-ers, but progress has been made in characterizing thefunctional impact of variants in these transporters. Fur-ther, large-scale studies in the area of single-nucleotidepolymorphisms (SNPs) in membrane transporters and cel-lular characterization of transporter variants have beenperformed (Burman et al., 2004; Gray et al., 2004; Leab-man et al., 2003; Osato et al., 2003; Shu et al., 2003) (seeChapter 4). The clinical impact of membrane transportervariants on drug response has been studied only recently.Like the cellular studies, the clinical studies have focusedon a limited number of transporters.

The most widely studied drug transporter is P-glyco-protein (MDR1, ABCB1), and results from clinical studieshave been controversial. Associations of the ABCB1 gen-otype with responses to anticancer drugs, antiviral agents,immunosuppressants, antihistamines, cardiac glycosides,and anticonvulsants have been described (Anglicheau etal., 2003; Drescher et al., 2002; Fellay et al., 2002;Hoffmeyer et al., 2000; Illmer et al., 2002; Johne et al.,2002; Macphee et al., 2002; Pauli-Magnus et al., 2003;Sai et al., 2003; Sakaeda et al., 2003; Siddiqui et al.,


2003; Verstuyft et al., 2003). ABCB1 SNPs also havebeen associated with tacrolimus and nortriptyline neuro-toxicity (Roberts et al., 2002; Yamauchi et al., 2002) andsusceptibility for developing ulcerative colitis, renal cellcarcinoma, and Parkinson’s disease (Drozdzik et al.,2003; Schwab et al., 2003; Siegsmund et al., 2002).

Recently, two common SNPs in SLCO1B1 (OATP1B1)have been associated with elevated plasma levels of prav-astatin, a widely used drug for the treatment of hypercho-lesterolemia (Mwinyi et al., 2004; Niemi et al., 2004) (seeChapter 35).

TRANSPORTERS INVOLVED IN PHARMACOKINETICS

Hepatic Transporters

Drug transporters play an important role in pharmacokine-tics (Koepsell, 1998; Zamek-Gliszczynski and Brouwer,2004) (Figure 2–1). Hepatic uptake of organic anions (e.g.,drugs, leukotrienes, and bilirubin), cations, and bile salts ismediated by SLC-type transporters in the basolateral (sinu-soidal) membrane of hepatocytes: OATPs (SLCO) (Abe etal., 1999; Konig et al., 2000) and OATs (SLC22) (Sekineet al., 1998), OCTs (SLC22) (Koepsell, 1998) and NTCP(SLC10A1) (Hagenbuch et al., 1991), respectively. Thesetransporters mediate uptake by either facilitated or secon-dary active mechanisms.

ABC transporters such as MRP2, MDR1, BCRP,BSEP, and MDR2 in the bile canalicular membrane ofhepatocytes mediate the efflux (excretion) of drugs andtheir metabolites, bile salts, and phospholipids against asteep concentration gradient from liver to bile. This pri-mary active transport is driven by ATP hydrolysis. SomeABC transporters are also present in the basolateralmembrane of hepatocytes and may play a role in theefflux of drugs back into the blood, although their physi-ological role remains to be elucidated. Drug uptake fol-lowed by metabolism and excretion in the liver is amajor determinant of the systemic clearance of manydrugs. Since clearance ultimately determines systemicblood levels, transporters in the liver play key roles insetting drug levels.

Vectorial transport of drugs from the circulating bloodto the bile using an uptake transporter (OATP family) andan efflux transporter (MRP2) is important for determiningdrug exposure in the circulating blood and liver. More-over, there are many other uptake and efflux transportersin the liver (Figure 2–9). Two examples illustrate the

importance of vectorial transport in determining drugexposure in the circulating blood and liver: HMG-CoAreductase inhibitors and angiotensin-converting enzyme(ACE) inhibitors.

HMG-CoA Reductase Inhibitors. Statins are cholesterol-loweringagents that reversibly inhibit HMG-CoA reductase, which cata-lyzes a rate-limiting step in cholesterol biosynthesis (see Chapter35). Statins affect serum cholesterol by inhibiting cholesterol bio-synthesis in the liver, and this organ is their main target. On theother hand, exposure of extrahepatic cells in smooth muscle tothese drugs may cause adverse effects. Among the statins, prava-statin, fluvastatin, cerivastatin, atorvastatin, rosuvastatin, andpitavastatin are given in a biologically active open-acid form,whereas simvastatin and lovastatin are administered as inactiveprodrugs with lactone rings. The open-acid statins are relativelyhydrophilic and have low membrane permeabilities. However,most of the statins in the acid form are substrates of uptake trans-porters, so they are taken up efficiently by the liver and undergoenterohepatic circulation (Figures 2–5 and 2–9). In this process,hepatic uptake transporters such as OATP1B1 and efflux trans-porters 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 after their oraladministration helps to exert the pharmacological effect and alsominimizes the escape of drug molecules into the circulating blood,thereby minimizing the exposure in a target of adverse response,smooth muscle. Recent studies indicate that the genetic polymor-phism of OATP1B1 also affects the function of this transporter(Tirona et al., 2001).

Temocapril. Temocapril is an ACE inhibitor (see Chapter 30). Itsactive metabolite, temocaprilat, is excreted both in the bile and inthe urine via the liver and kidney, respectively, whereas otherACE inhibitors are excreted mainly via the kidney. The specialfeature of temocapril among ACE inhibitors is that the plasmaconcentration of temocaprilat remains relatively unchanged evenin patients with renal failure. However, the plasma area under thecurve AUC of enalaprilat and other ACE inhibitors is markedlyincreased in patients with renal disorders. Temocaprilat is a bisub-strate of the OATP family and MRP2, whereas other ACE inhibi-tors are not good substrates of MRP2 (although they are taken upinto the liver by the OATP family). Taking these findings intoconsideration, the affinity for MRP2 may dominate in determiningthe biliary excretion of any series of ACE inhibitors. Drugs thatare excreted into both the bile and urine to the same degree thusare expected to exhibit minimum interindividual differences intheir pharmacokinetics.

Irinotecan (CPT-11). Irinotecan hydrochloride (CPT-11) is a potentanticancer drug, but late-onset gastrointestinal toxic effects, such assevere diarrhea, make it difficult to use CPT-11 safely. After intra-venous administration, CPT-11 is converted to SN-38, an activemetabolite, by carboxy esterase. SN-38 is subsequently conjugatedwith glucuronic acid in the liver. SN-38 and SN-38 glucuronide arethen excreted into the bile by MRP2. Some studies have shown thatthe inhibition of MRP2-mediated biliary excretion of SN-38 and itsglucuronide by coadministration of probenecid reduces the drug-induced diarrhea, at least in rats. For additional details, see Figures3–5 and 3–7.


Drug–Drug Interactions Involving Transporter-Medi-ated Hepatic Uptake. Since drug transporters are deter-minants of the elimination rate of drugs from the body, trans-porter-mediated hepatic uptake can be the cause of drug–drug interactions involving drugs that are actively taken upinto the liver and metabolized and/or excreted in the bile.

Cerivastatin (currently withdrawn), an HMG-CoA reductaseinhibitor, is taken up into the liver via transporters (especiallyOATP1B1) and subsequently metabolized by CYP2C8 and CYP3A4.Its plasma concentration is increased four- to fivefold when coadmin-istered with cyclosporin A. Transport studies using cryopreservedhuman hepatocytes and OATP1B1-expressing cells suggest that thisclinically relevant drug–drug interaction is caused by inhibition ofOATP1B1-mediated hepatic uptake (Shitara et al., 2003). However,cyclosporin A inhibits the metabolism of cerivastatin only to a limitedextent, suggesting a low possibility of serious drug–drug interactionsinvolving the inhibition of metabolism. Cyclosporin A also increasesthe plasma concentrations of other HMG-CoA reductase inhibitors. Itmarkedly increases the plasma AUC of pravastatin, pitavastatin, androsuvastatin, which are minimally metabolized and eliminated fromthe body by transporter-mediated mechanisms. Therefore, these phar-macokinetic interactions also may be due to transporter-mediatedhepatic uptake. However, the interactions of cyclosporin A with pro-drug-like statins (lactone form) such as simvastatin and lovastatin aremediated by CYP3A4.

Gemfibrozil is another cholesterol-lowering agent that acts by a dif-ferent mechanism and also causes a severe pharmacokinetic interactionwith cerivastatin. Gemfibrozil glucuronide inhibits the CYP2C8-medi-ated metabolism and OATP1B1-mediated uptake of cerivastatin more

potently than does gemfibrozil. Laboratory data show that the glucu-ronide is highly concentrated in the liver versus plasma probablyowing to transporter-mediated active uptake and intracellular forma-tion of the conjugate. Therefore, it may be that gemfibrozil glucu-ronide, concentrated in the hepatocytes, inhibits the CYP2C8-mediatedmetabolism of cerivastatin. Gemfibrozil markedly (four- to fivefold)increases the plasma concentration of cerivastatin but does not greatlyincrease (1.3 to 2 times) that of unmetabolized statins pravastatin,pitavastatin, and rosuvastatin, a result that also suggests that this inter-action is caused by inhibition of metabolism. Thus, when an inhibitorof drug-metabolizing enzymes is highly concentrated in hepatocytesby active transport, extensive inhibition of the drug-metabolizingenzymes may be observed because of the high concentration of theinhibitor in the vicinity of the drug-metabolizing enzymes.

The Contribution of Specific Transporters to theHepatic Uptake of Drugs. Estimating the contributionof transporters to the total hepatic uptake is necessary forunderstanding their importance in drug disposition. Thisestimate can help to predict the extent to which a drug–drug interaction or a genetic polymorphism of a transport-er may affect drug concentrations in plasma and liver. Thecontribution to hepatic uptake has been estimated success-fully for CYP-mediated metabolism by using neutralizingantibody and specific chemical inhibitors. Unfortunately,specific inhibitors or antibodies for important transportershave not been identified yet, although some relativelyspecific inhibitors have been discovered.

Figure 2–9. Transporters in the hepatocyte that function in the uptake and efflux of drugs across the sinusoidal membrane andefflux of drugs into the bile across the canalicular membrane. See text for details of the transporters pictured.


The contribution of transporters to hepatic uptake can be estimat-ed from in vitro studies. Injection of cRNA results in transporterexpression on the plasma membrane of Xenopus laevis oocytes(Hagenbuch et al., 1996). Subsequent hybridization of the cRNA withits antisense oligonucleotide specifically reduces its expression. Com-parison of the drug uptake into cRNA-injected oocytes in the presenceand absence of antisense oligonucleotides clarifies the contribution ofa specific transporter. Second, a method using reference compoundsfor specific transporters has been proposed. The reference compoundsshould be specific substrates for a particular transporter. The contribu-tion of a specific transporter can be calculated from the uptake of testcompounds and reference compounds into hepatocytes and transport-er-expressing systems (Hirano et al., 2004):

(2–13)

where CLhep,ref and CLexp,ref represent the uptake of reference com-pounds into hepatocytes and transporter-expressing cells, respective-ly, and CLhep,test and CLexp,test represent the uptake of test compoundsinto the corresponding systems. For example, the contributions ofOATP1B1 and OATP1B3 to the hepatic uptake of pitavastatin havebeen estimated using estrone 3-sulfate and cholecystokinine octapep-tide (CCK8) as reference compounds for OATP1B1 and OATP1B3,respectively. However, for many transporters, reference compoundsspecific to the transporter are not available.

Renal Transporters

Secretion in the kidney of structurally diverse mole-cules including many drugs, environmental toxins andcarcinogens is critical in the body’s defense againstforeign substances. The specificity of secretory path-ways in the nephron for two distinct classes of sub-strates, organic anions and cations, was first describeddecades ago, and these pathways were well character-ized using a variety of physiological techniques includ-ing isolated perfused nephrons and kidneys, micro-puncture techniques, cell culture methods, and isolatedrenal plasma membrane vesicles. However, not untilthe mid-1990s were the molecular identities of theorganic anion and cation transporters revealed. Duringthe past decade, molecular studies have identified andcharacterized the renal transporters that play a role indrug elimination, toxicity, and response. Thus, we nowcan describe the overall secretory pathways for organiccations and their molecular and functional characteris-tics. Although the pharmacological focus is often onthe kidney, there is useful information on the tissuedistribution of these transporters. Molecular studies usingsite-directed mutagenesis have identified substrate-rec-ognition and other functional domains of the transport-ers, and genetic studies of knockout mouse modelshave been used to characterize the physiological rolesof individual transporters. Recently, studies have iden-

tified and functionally analyzed genetic polymorphismsand haplotypes of the relevant transporters in humans.Our understanding of organic anion transport has pro-gressed in a similar fashion. In some cases, transportersthat are considered organic anion or organic cationtransporters have dual specificity for anions and cat-ions. The following section summarizes recent work onhuman transporters and includes some information ontransporters in other mammals. An excellent review ofrenal organic anion and cation transport has been pub-lished recently (Wright and Dantzler, 2004).

Organic Cation Transport. Structurally diverse organ-ic cations are secreted in the proximal tubule (Dresser etal., 2001; Koepsell and Endou, 2004; Wright and Dantz-ler, 2004). Many secreted organic cations are endoge-nous compounds (e.g., choline, N-methylnicotinamide,and dopamine), and renal secretion appears to be impor-tant in eliminating excess concentrations of these sub-stances. However, a primary function of organic cationsecretion is ridding the body of xenobiotics, includingmany positively charged drugs and their metabolites(e.g., cimetidine, ranitidine, metformin, procainamide,and N-acetylprocainamide), and toxins from the envi-ronment (e.g., nicotine). Organic cations that are secret-ed by the kidney may be either hydrophobic or hydro-philic. Hydrophilic organic drug cations generally havemolecular weights of less than 400 daltons; a currentmodel for their secretion in the proximal tubule of thenephron is shown in Figure 2–10.

For the transepithelial flux of a compound (e.g., secretion), it isessential for the compound to traverse two membranes sequentially,the basolateral membrane facing the blood side and the apical mem-brane facing the tubular lumen. Distinct transporters on each mem-brane mediate each step of transport. Organic cations appear tocross the basolateral membrane by three distinct transporters in theSLC family 22 (SCL22): OCT1 (SLC22A1), OCT2 (SLC22A2), andOCT3 (SLC22A3). Organic cations are transported across this mem-brane down their electrochemical gradient (–70 mV). Previous stud-ies in isolated basolateral membrane vesicles demonstrate the pres-ence of a potential-sensitive mechanism for organic cations. Thecloned transporters OCT1, OCT2, and OCT3 are all potential sensi-tive and mechanistically coincide with previous studies of isolatedbasolateral membrane vesicles.

Transport of organic cations from cell to tubular lumen acrossthe apical membrane occurs via an electroneutral proton–organiccation exchange mechanism in a variety of species, includinghuman, dog, rabbit, and cat. Transporters assigned to the apicalmembrane are in the SLC22 family and termed novel organiccation transporters (OCTNs). In humans, these include OCNT1(SLC22A4) and OCTN2 (SLC22A5). These bifunctional transportersare involved not only in organic cation secretion but also in car-nitine reabsorption. In the reuptake mode, the transporters functionas Na+ cotransporters, relying on the inwardly driven Na+ gradient

ContributionCLhep,ref CLexp,ref⁄CLhep,test CLexp,test⁄------------------------------------------------=


created by Na+,K+-ATPase to move carnitine from tubular lumen tocell. In the secretory mode, the transporters appear to function asproton–organic cation exchangers. That is, protons move from tubu-lar lumen to cell interior in exchange for organic cations, whichmove from cytosol to tubular lumen. The inwardly directed protongradient (from tubular lumen to cytosol) is maintained by transport-ers in the SLC9 family (NHEs), which are Na+/H+ exchangers (anti-porters). The bifunctional mechanism of OCTN1 and OCTN2 maynot totally explain the organic cation–proton exchange mechanismthat has been described in many studies in isolated plasma mem-brane vesicles. Of the two steps involved in secretory transport,transport across the luminal membrane appears to be rate-limiting.

OCT1 (SLC22A1). OCT1 (SLC22A1) was first cloned from arat cDNA library (Koepsell and Endou, 2004). Subsequently,orthologs were cloned from mouse, rabbit, and humans. Mammali-an isoforms of OCT1, which vary in length from 554 to 556 aminoacids, have 12 putative transmembrane domains (Figure 2–11) andinclude several N-linked glycosylation sites. A long extracellularloop between transmembrane domains 1 and 2 is characteristic ofthe OCTs. The gene for the human OCT1 is mapped to chromo-some 6 (6q26). There are four splice variants in human tissues,one of which is functionally active, OCT1G/L554 (Hayer et al.,1999). In humans, OCT1 is expressed primarily in the liver, withsome expression in heart, intestine, and skeletal muscle. In mouse

and rat, OCT1 is also abundant in the kidney, whereas in humans,very modest levels of OCT1 mRNA transcripts are detected in kid-ney. The transport mechanism of OCT1 is electrogenic and satura-ble for transport of model small-molecular-weight organic cationsincluding tetraethylammonium (TEA) and dopamine. Interesting-ly, OCT1 also can operate as an exchanger, mediating organic cat-ion–organic cation exchange. That is, loading cells with organiccations such as unlabeled TEA can trans-stimulate the inward fluxof organic cations such as MPP+. It also should be noted thatorganic cations can transinhibit OCT1. In particular, the hydro-phobic organic cations quinine and quinidine, which are poor sub-strates of OCT1, when present on the cytosolic side of a mem-brane, can inhibit (transinhibit) influx of organic cations viaOCT1.

The human OCT1 generally accepts a wide array of monovalentorganic cations with molecular weights of less than 400 daltons,including many drugs (e.g., procainamide, metformin, and pindolol)(Dresser et al., 2001). Species differences in the substrate specificityof OCT1 mammalian orthologs have been described. Inhibitors ofOCT1 are generally more hydrophobic. Detailed structure–activityrelationships have established that the pharmacophore of OCT1consists of three hydrophobic arms and a single cationic recognitionsite. The kinetics of uptake and inhibition of model compounds withhuman OCT1 differ among studies and may be related to experi-

Figure 2–10. Model of organic cation secretory transporters in the proximal tubule. Hexagons depict transporters in the SLC22family, SLC22A1 (OCT1), SLC22A2 (OCT2), and SLC22A3 (OCT3). Circles show transporters in the same family, SLC22A4(OCTN1) and SLC22A5 (OCTN2). MDR1 (ABCB1) is depicted as a dark blue oval. Carn, carnitine; OC+, organic cation.


mental techniques, including a range of heterologous expressionsystems. Key residues that contribute to the charge specificity ofOCT1 have been identified by site-directed mutagenesis studies andinclude a highly conserved aspartate residue (corresponding to posi-tion 475 in the rat ortholog of OCT1) that appears to be part of themonoamine recognition site. Since OCT1 mammalian orthologshave greater than 80% amino acid identity, evolutionarily noncon-served residues among mammalian species clearly are involved inspecificity differences (Wright and Dantzler, 2004).

OCT2 (SLC22A2). OCT2 (SLC22A2) was first cloned from a ratkidney cDNA library in 1996 (Okuda et al., 1996). Human, rabbit,mouse, and pig orthologs all have been cloned. Mammalianorthologs range in length from 553 through 555 amino acids. Simi-lar to OCT, OCT2 is predicted to have 12 transmembrane domains,including one N-linked glycosylation site. OCT2 is located adjacentto OCT1 on chromosome 6 (6q26). A single splice variant of humanOCT2, termed OCT2-A, has been identified in human kidney.OCT2-A, which is a truncated form of OCT2, appears to have alower Km (or greater affinity) for substrates than OCT2, although alower affinity has been observed for some inhibitors (Urakami etal., 2002). Human, mouse, and rat orthologs of OCT2 are expressedin abundance in human kidney and to some extent in neuronal tissuesuch as choroid plexus. In the kidney, OCT2 is localized to theproximal tubule and to distal tubules and collecting ducts. In theproximal tubule, OCT2 is restricted to the basolateral membrane.OCT2 mammalian species orthologs are greater than 80% identical,whereas OCT1 and OCT2 paralogs are approximately 70% identi-cal. The transport mechanism of OCT2 is similar to that of OCT1.In particular, OCT2-mediated transport of model organic cationsMPP+ and TEA is electrogenic, but like OCT1, OCT2 can supportorganic cation–organic cation exchange (Koepsell et al., 2003).Some studies show modest proton–organic cation exchange. Morehydrophobic organic cations may inhibit OCT2 but may not betranslocated by it.

Like OCT1, OCT2 generally accepts a wide array of monovalentorganic cations with molecular weights of less than 400 daltons. Theapparent affinities of the human OCT1 and OCT2 paralogs for someorganic cation substrates and inhibitors have been shown to be dif-ferent in side-by-side comparison studies. Isoform-specific inhibi-

tors of the OCTs are needed to determine the relative importance ofOCT1 and OCT2 in the renal clearance of compounds in rodents, inwhich both isoforms are present in kidney. OCT2 is also present inneuronal tissues. However, studies with monoamine neurotransmit-ters demonstrate that dopamine, serotonin, histamine, and norepi-nephrine have low affinities for OCT2. These studies suggest thatOCT2 may play a housekeeping role in neurons, taking up onlyexcess concentrations of neurotransmitters. OCT2 also may beinvolved in recycling of neurotransmitters by taking up breakdownproducts, which in turn enter monoamine synthetic pathways.

OCT3 (SLC22A3). OCT3 (SLC22A3) was cloned initially from ratplacenta (Kekuda et al., 1998). Human and mouse orthologs have alsobeen cloned. OCT3 consists of 551 amino acids and is predicted tohave 12 transmembrane domains, including three N-linked glycosyla-tion sites. hOCT3 is located in tandem with OCT1 and OCT2 onchromosome 6. Tissue distribution studies suggest that human OCT3is expressed in liver, kidney, intestine, and placenta, although itappears to be expressed in considerably less abundance than OCT2 inthe kidney. Like OCT1 and OCT2, OCT3 appears to support electro-genic potential-sensitive organic cation transport. Although the speci-ficity of OCT3 is similar to that of OCT1 and OCT2, it appears tohave quantitative differences in its affinities for many organic cations.Some studies have suggested that OCT3 is the extraneuronal mono-amine transporter based on its substrate specificity and potency ofinteraction with monoamine neurotransmitters. Because of its rela-tively low abundance in the kidney, OCT3 may play only a limitedrole in renal drug elimination.

OCTN1 (SLC22A4). OCTN1, cloned originally from humanfetal liver, is expressed in the adult kidney, trachea, and bone mar-row (Tamai et al., 1997). The functional characteristics of OCTN1suggest that it operates as an organic cation–proton exchanger.OCTN1-mediated influx of model organic cations is enhanced atalkaline pH, whereas efflux is increased by an inwardly directedproton gradient. OCTN1 contains a nucleotide-binding sequencemotif, and transport of its substrates appears to be stimulated by cel-lular ATP content. OCTN1 also can function as an organic cation–organic cation exchanger. Although the subcellular localization ofOCTN1 has not been demonstrated clearly, available data collec-tively suggest that OCTN1 functions as a bidirectional pH- and

Figure 2–11. Secondary structure of OCT1 (SLC22A1) constructed from hydropathy analysis. The transmembrane topologydiagram was rendered using transmembrane protein display software available at the UCSF Sequence Analysis Consulting Group Website, http://www.sacs.ucsf.edu/TOPO/topo.html. The blue circles show putative N-glycosylation sites.


ATP-dependent transporter at the apical membrane in renal tubularepithelial cells. Its physiological role is not yet known because stud-ies in octn1 knockout mice are not available.

OCTN2 (SLC22A5). OCTN2 was first cloned from human kidneyand determined to be the transporter responsible for systemic carnitinedeficiency (Tamai et al., 1998). Rat OCTN2 mRNA is expressed pre-dominantly in the cortex, with very little expression in the medulla,and is localized to the apical membrane of the proximal tubule.

OCTN2 is a bifunctional transporter. That is, it transports L-car-nitine with high affinity in an Na+-dependent manner, whereas, Na+

does not influence OCTN2-mediated transport of organic cationssuch as TEA. Thus, OCTN2 is thought to function as both an Na+-dependent carnitine transporter and an Na+-independent organic cat-ion transporter. Similar to OCTN1, OCTN2 transport of organic cat-ions is sensitive to pH, suggesting that it may function as an organiccation exchanger. Studies in mice containing a missense mutation inSlc22a5 suggest that organic cations are transported in a secretorydirection by OCTN2, whereas carnitine is transported in a reabsorp-tive direction (Ohashi et al., 2001). Therefore, transport of L-car-nitine by OCTN2 is an Na+-dependent electrogenic process. Muta-tions in OCTN2 have been found to be the cause of primarysystemic carnitine deficiency (OMIM 212140) (Nezu et al., 1999).

Polymorphisms of OCTs. Polymorphisms of OCTs have been identi-fied in large post–human genome SNP discovery projects (Kerb et al.,2002; Leabman et al., 2003; Shu et al., 2003). OCT1 exhibits thegreatest number of amino acid polymorphisms, followed by OCT2and then OCT3. Furthermore, allele frequencies of OCT1 amino acidvariants in human populations generally are greater than those ofOCT2 and OCT3 amino acid variants. Functional studies of OCT1and OCT2 polymorphisms have been performed. OCT1 exhibits fivevariants with reduced function. These variants may have importantimplications clinically in terms of hepatic drug disposition and target-ing of OCT1 substrates. In particular, individuals with OCT1 variantsmay have reduced liver uptake of OCT1 substrates and thereforereduced metabolism. Clinical studies need to be performed to ascer-tain the implications of OCT1 variants to drug disposition andresponse. For OCT2, several polymorphisms exhibited altered kineticproperties when expressed in Xenopus laevis oocytes. These variantsmay lead to alterations in renal secretion of OCT2 substrates.

Organic Anion Transport. A wide variety of structural-ly diverse organic anions are secreted in the proximaltubule (Burckhardt and Burckhardt, 2003; Dresser et al.,2001; Wright and Dantzler, 2004). As with organic cationtransport, the primary function of organic anion secretionappears to be the removal from the body of xenobiotics,including many weakly acidic drugs [e.g., pravastatin,captopril, p-aminohippurate (PAH), and penicillins] andtoxins (e.g., ochratoxin). Organic anion transporters moveboth hydrophobic and hydrophilic anions but also mayinteract with cations and neutral compounds.

A current model for the transepithelial flux of organicanions in the proximal tubule is shown in Figure 2–12.Two primary transporters on the basolateral membranemediate the flux of organic anions from interstitial fluid totubule cell: OAT1 (SLC22A6) and OAT3 (SLC22A8).Energetically, hydrophilic organic anions are transported

across the basolateral membrane against an electrochemi-cal gradient in exchange with intracellular α-ketoglut-arate, which moves down its concentration gradient fromcytosol to blood. The outwardly directed gradient of α-ketoglutarate is maintained at least in part by a basolateralNa+-dicarboxylate transporter (NaDC3). The Na+ gradientthat drives NaDC3 is maintained by Na+,K+-ATPase.Transport of small-molecular-weight organic anions bythe cloned transporters OAT1 and OAT3 can be driven byα-ketoglutarate. Coupled transport of α-ketoglutarate andsmall-molecular-weight organic anions (e.g., p-aminohip-purate) has been demonstrated in many studies in isolatedbasolateral membrane vesicles. The molecular pharmacol-ogy and molecular biology of OATs have recently beenreviewed (Eraly et al., 2004).

The mechanism responsible for the apical membrane transport oforganic anions from tubule cell cytosol to tubular lumen remainscontroversial. Some studies suggest that OAT4 may serve as theluminal membrane transporter for organic anions. However, recentstudies show that the movement of substrates via this transportercan be driven by exchange with α-ketoglutarate, suggesting thatOAT4 may function in the reabsorptive, rather than secretory, fluxof organic anions. Other studies have suggested that in the pig kid-ney, OATV1 serves as an electrogenic facilitated transporter on theapical membrane (Jutabha et al., 2003). The human ortholog ofOATV1 is NPT1, or NaPi-1, originally cloned as a phosphate trans-porter. NPT1 can support the low-affinity transport of hydrophilicorganic anions such as PAH. Other transporters that may play a rolein transport across the apical membrane include MRP2 and MRP4,multidrug-resistance transporters in the ATP binding cassette familyC (ABCC). Both transporters interact with some organic anions andmay actively pump their substrates from tubule cell cytosol to tubu-lar lumen.

OAT1 (SLC22A6). OAT1 was cloned from rat kidney (Sekine etal., 1997; Sweet et al., 1997). This transporter is greater than 30%identical to OCTs in the SLC22 family. Mouse, human, pig, andrabbit orthologs have been cloned and are approximately 80% iden-tical to human OAT1. Mammalian isoforms of OAT1 vary in lengthfrom 545 to 551 amino acids, with features similar to those shownin Figure 2–11. The gene for the human OAT1 is mapped to chro-mosome 11 and is found in an SLC22 cluster that includes OAT3and OAT4. There are four splice variants in human tissues, termedOAT1-1, OAT1-2, OAT1-3, and OAT1-4. OAT1-2, which includes a13-amino-acid deletion, transports PAH at a rate comparable withOAT1-1. These two splice variants use the alternative 5′-splice sitesin exon 9. OAT1-3 and OAT1-4, which result from a 132-bp (44-amino-acid) deletion near the carboxyl terminus of OAT1, do nottransport PAH. In humans, rat, and mouse, OAT1 is expressed pri-marily in the kidney, with some expression in brain and skeletalmuscle.

Immunohistochemical studies suggest that OAT1 is expressedon the basolateral membrane of the proximal tubule in human andrat, with highest expression in the middle segment, S2. Based on aquantitative polymerase chain reaction (PCR), OAT1 is expressed ata third of the level of OAT3, the other major basolateral membraneorganic anion transporter. OAT1 exhibits saturable transport oforganic anions such as PAH. This transport is trans-stimulated by


other organic anions, including α-ketoglutarate. Thus, the insidenegative-potential difference drives the efflux of the dicarboxylateα-ketoglutarate, which, in turn, supports the influx of monocarboxy-lates such as PAH. Regulation of expression levels of OAT1 in thekidney appears to be controlled by sex steroids.

OAT1 generally transports small-molecular-weight organic anionsthat may be endogenous (e.g., PGE2 and urate) or ingested drugs andtoxins. Some neutral compounds are also transported by OAT1 at alower affinity (e.g., cimetidine). Key residues that contribute to trans-port by OAT1 include the conserved K394 and R478, which areinvolved in the PAH–glutarate exchange mechanism.

OAT2 (SLC22A7). OAT2 was cloned first from rat liver (andnamed NLT at the time) (Sekine et al., 1998; Simonson et al.,1994). This transporter has a gender-based tissue distributionbetween the liver and the kidney in rodents but not in humans,OAT2 is present in both kidney and liver. In the kidney, the trans-porter is localized to the basolateral membrane of the proximaltubule. Efforts to stimulate organic anion–organic anion exchangevia OAT2 have not been successful, leading to the speculation thatOAT2 is a basolateral membrane transporter that serves in thereabsorptive flux of organic anions from tubule cell cytosol tointerstitial fluids. OAT2 transports many organic anions, includingPAH, methotrexate, ochratoxin A, and glutarate. Human, mouse,and rat orthologs of OAT2 have high affinities for the endogenousprostaglandin, PGE2.

OAT3 (SLC22A8). OAT3 (SLC22A8) was cloned originally fromrat kidney (Kusuhara et al., 1999). Human OAT3 consists of twovariants, one of which transports a wide variety of organic anions,including PAH and estrone sulfate. The longer OAT3 in humans,a 568-amino-acid protein, does not support transport. It is likelythat the two OAT3 variants are splice variants. Northern blottingsuggests that the human ortholog of OAT3 is primarily in the kid-ney. Mouse and rat orthologs show some expression in the brainand liver. OAT3 mRNA levels are higher than those of OAT1,which in turn are higher than those of OAT2 or OAT4. HumanOAT3 is confined to the basolateral membrane of the proximaltubule.

OAT3 clearly has overlapping specificities with OAT1,although kinetic parameters differ. For example, estrone sulfate istransported by both OAT1 and OAT3, but OAT3 has a much high-er affinity in comparison with OAT1. The weak base cimetidine(an H2-receptor antagonist) is transported with high affinity byOAT1, whereas the cation TEA is not transported. Domains andresidues involved in the charge specificity of OAT3 have beenidentified in several studies. Interestingly, changing two basicamino acid residues in OAT3 (R454D and K370A) shifts thecharge specificity of OAT3 from anionic to cationic. Like OAT1,OAT3 appears to be an exchanger that couples the outward flux ofα-ketoglutarate to the inward flux of organic anions: The insidenegative-potential difference repels α-ketoglutarate from the cells

Figure 2–12. Model of organic anion secretory transporters in the proximal tubule. Rectangles depict transporters in the SLC22family, OAT1 (SLC22A6) and OAT3 (SLC22A8), and hexagons depict transporters in the ABC superfamily, MRP2 (ABCC2) andMRP4 (ABCC4). NPT1 (SLC17A1) is depicted as a circle. OA–, organic anion; α-KG, α-ketoglutarate.


via OAT3, which in turn transports its substrates against a concen-tration gradient into the tubule cell cytosol.

OAT4 (SLC22A9). OAT4 (SLC22A9) was cloned from a humankidney cDNA library (Cha et al., 2000). Quantitative PCR indicatesthat the expression level of OAT4 in human kidneys is approximate-ly 5% to 10% of the level of OAT1 and OAT3 and is comparablewith OAT2. OAT4 is expressed in human kidney and placenta; inhuman kidney, OAT4 is present on the luminal membrane of the prox-imal tubule. At first, OAT4 was thought to be involved in the secondstep of secretion of organic anions, i.e., transport across the apicalmembrane from cell to tubular lumen. However, recent studies dem-onstrate that organic anion transport by OAT4 can be stimulated bytransgradients of α-ketoglutarate (Ekaratanawong et al., 2004), sug-gesting that OAT4 may be involved in the reabsorption of organicanions from tubular lumen into cell. The specificity of OAT4 is nar-row but includes estrone sulfate and PAH. Interestingly, the affinityfor PAH is low (>1 mM). Collectively, emerging studies suggestthat OAT4 may be involved not in secretory flux of organic anionsbut in reabsorption instead.

Other Anion Transporters. URAT1 (SLC22A12), first cloned fromhuman kidney, is a kidney-specific transporter confined to the api-cal membrane of the proximal tubule (Enomoto et al., 2002). Datasuggest that URAT1 is primarily responsible for urate reabsorp-tion, mediating electroneutral urate transport that can be trans-stimulated by Cl– gradients. The mouse ortholog of URAT1 isinvolved in the renal secretory flux of organic anions includingbenzylpenicillin and urate.

NPT1 (SLC17A1), cloned originally as a phosphate transporterin humans, is expressed in abundance on the luminal membrane ofthe proximal tubule as well as in the brain (Werner et al., 1991).NPT1 transports PAH, probenecid, and penicillin G. It appears to bepart of the system involved in organic anion efflux from tubule cellto lumen.

MRP2 (ABCC2), an ABC transporter, initially called the GS-Xpump (Ishikawa et al., 1990), has been considered to be the primarytransporter involved in efflux of many drug conjugates such as glu-tathione conjugates across the canalicular membrane of the hepato-cyte. However, MRP2 is also found on the apical membrane of theproximal tubule, where it is thought to play a role in the efflux oforganic anions into the tubular lumen. Its role in the kidney may beto secrete glutathione conjugates of drugs, but it also may supportthe translocation (with glutathione) of various nonconjugated sub-strates. In general, MRP2 transports larger, bulkier compounds thando most of the organic anion transporters in the SLC22 family.

MRP4 (ABCC4) is found on the apical membrane of the proxi-mal tubule and transports a wide array of conjugated anions, includ-ing glucuronide and glutathione conjugates. However, unlikeMRP2, MRP4 appears to interact with various drugs, includingmethotrexate, cyclic nucleotide analogs, and antiviral nucleosideanalogs. It is possible that MRP4 is involved in the apical flux ofmany drugs from cell to tubule lumen. Other MRP efflux transport-ers also have been identified in human kidney, including MRP3 andMRP6, both on the basolateral membrane. Their roles in the kidneyare not yet known.

Polymorphisms of OATs. Polymorphisms in OAT1 and OAT3 havebeen identified in ethnically diverse human populations. Two aminoacid polymorphisms (allele frequencies greater than 1%) in OAT1have been identified in African-American populations (OAT1-R50H). Three amino acid polymorphisms and seven rare amino acid

variants in OAT3 have been identified in ethnically diverse U.S.populations (see www.pharmgkb.org).

TRANSPORTERS INVOLVED IN PHARMACODYNAMICS: DRUG ACTION IN THE BRAIN

Neurotransmitters are packaged in vesicles in presynpaticneurons, released in the synapse by fusion of the vesicleswith the plasma membrane, and, excepting acetylcholine,are then taken back into the presynaptic neurons orpostsynaptic cells (see Chapter 6). Transporters involvedin the neuronal reuptake of the neurotransmitters and theregulation of their levels in the synaptic cleft belong totwo major superfamilies, SLC1 and SLC6. Transportersin both families play roles in reuptake of γ-aminobutyricacid (GABA), glutamate, and the monoamine neurotrans-mitters norepinephrine, serotonin, and dopamine. Thesetransporters may serve as pharmacologic targets for neu-ropsychiatric drugs.

SLC6 family members localized in the brain andinvolved in the reuptake of neurotransmitters into presynap-tic neurons include the norepinephrine transporters (NET,SLC6A2), the dopamine transporter (DAT, SLC6A3), theserotonin transporter (SERT, SLC6A4), and several GABAreuptake transporters (GAT1, GAT2, and GAT3) (Chen etal., 2004; Hediger, 2004; Elliott and Beveridge, 2005).Each of these transporters appears to have 12 transmem-brane secondary structures and a large extracellular loopwith glycosylation sites between transmembrane domains 3and 4. These proteins are typically approximately 600amino acids in length. SLC6 family members depend on theNa+ gradient to actively transport their substrates into cells.Cl– is also required, although to a variable extent dependingon the family member. Residues and domains that form thesubstrate recognition and permeation pathways are current-ly being identified.

Through reuptake mechanisms, the neurotransmittertransporters in the SLC6A family regulate the concentra-tions and dwell times of neurotransmitters in the synapticcleft; the extent of transmitter uptake also influences sub-sequent vesicular storage of transmitters. It is important tonote that many of these transporters are present in othertissues (e.g., kidney and platelets) and may serve otherroles. Further, the transporters can function in the reversedirection. That is, the transporters can export neurotrans-mitters in an Na+-independent fashion. The characteristicsof each member of the SLC6A family of transporters thatplay a role in reuptake of monoamine neurotransmittersand GABA merit a brief description.


SLC6A1 (GAT1), SLC6A11 (GAT3), and SLC6A13(GAT2). GAT1 (599 amino acids) is the most importantGABA transporter in the brain, expressed in GABAergicneurons and found largely on presynaptic neurons (Chenet al., 2004). GAT1 is found in abundance in the neocor-tex, cerebellum, basal ganglia, brainstem, spinal cord, ret-ina, 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 thechoroid plexus and meninges.

GAT1, GAT2, and GAT3 are approximately 50% identical inamino acid sequence. Functional analysis indicates that GAT1 trans-ports GABA with a 2:1 Na+:GABA– stoichiometry. Cl– is required.Residues and domains responsible for the recognition of GABA andsubsequent translocation have been identified: Tyr140 appears to becrucial for binding GABA. Physiologically, GAT1 appears to beresponsible for regulating the interaction of GABA at receptors. Thepresence of GAT2 in the choroid plexus and its absence in presynapticneurons suggest that this transporter may play a primary role in main-taining the homeostasis of GABA in the CSF. GAT1 and GAT3 aredrug targets. GAT1 is the target of the antiepileptic drug tiagabine,which presumably acts to increase GABA levels in the synaptic cleftof GABAergic neurons by inhibiting the reuptake of GABA. GAT3 isthe target for the nipecotic acid derivatives that are anticonvulsants.

SLC6A2 (NET). NET (617 amino acids) is found in cen-tral and peripheral nervous tissues as well as in adrenalchromaffin tissue (Chen et al., 2004). In the brain, NETcolocalizes with neuronal markers, consistent with a rolein reuptake of monoamine neurotransmitters. The trans-porter functions in the Na+-dependent reuptake of norepi-nephrine and dopamine and as a higher-capacity norepi-nephrine channel. A major role of NET is to limit thesynaptic dwell time of norepinephrine and to terminate itsactions, salvaging norepinephrine for subsequent repack-aging. NET knockout mice exhibit a prolonged synaptichalf-life of norepinephrine (Xu et al., 2000). Ultimately,through its reuptake function, NET participates in the reg-ulation of many neurological functions, including memo-ry and mood. NET serves as a drug target; the antidepres-sant desipramine is considered a selective inhibitor ofNET. Other drugs that interact with NET include other tri-cyclic antidepressants and cocaine. Orthostatic intoler-ance, a rare familial disorder characterized by an abnor-mal blood pressure and heart rate response to changes inposture, has been associated with a mutation in NET.

SLC6A3 (DAT). DAT is located primarily in the brain indopaminergic neurons. Although present on presynapticneurons at the neurosynapatic junction, DAT is also presentin abundance along the neurons, away from the synapticcleft. This distribution suggests that DAT may play a role

in clearance of excess dopamine in the vicinity of neurons.The primary function of DAT is the reuptake dopamine,terminating its actions, although DAT also weakly interactswith norepinephrine. Physiologically, DAT is involved inthe various functions that are attributed to the dopaminergicsystem, including mood, behavior, reward, and cognition.The half-life of dopamine in the extracellular spaces of thebrain is prolonged considerably in DAT knockout mice(Uhl, 2003), which are hyperactive and have sleep disor-ders. Drugs that interact with DAT include cocaine and itsanalogs, amphetamines, and the neurotoxin MPTP.

SLC6A4 (SERT). SERT is located in peripheral tissuesand in the brain along extrasynaptic axonal membranes(Chen et al., 2004; Olivier et al., 2000). SERT clearlyplays a role in the reuptake and clearance of serotonin inthe brain. Like the other SLC6A family members, SERTtransports its substrates in an Na+-dependent fashion andis dependent on Cl– and possibly on the countertransportof K+. Substrates of SERT include serotonin (5-HT), vari-ous tryptamine derivatives, and neurotoxins such as 3,4-methylene-dioxymethamphetamine (MDMA; ecstasy) andfenfluramine. The serotonin transporter has been one ofthe most widely studied proteins in the human genome.First, it is the specific target of the antidepressants in theselective serotonin reuptake inhibitor class (e.g., fluoxe-tine and paroxetine) and one of several targets of tricyclicantidepressants (e.g., amitriptyline). Further, because ofthe important role of serotonin in neurological functionand behavior, genetic variants of SERT have been associ-ated with an array of behavioral and neurological disor-ders. In particular, a common promoter region variant thatalters the length of the upstream region of SLC6A4 hasbeen the subject of many studies. The short form of thevariant results in a reduced rate of transcription of SERTin comparison with the long form. These differences inthe rates of transcription alter the quantity of mRNA and,ultimately, the expression and activity of SERT. The shortform has been associated with a variety of neuropsychia-tric disorders (Lesch et al., 1996). The precise mechanismby which a reduced activity of SERT, caused by either agenetic variant or an antidepressant, ultimately affectsbehavior, including depression, is not known.

BLOOD–BRAIN BARRIER AND BLOOD–CSF BARRIER

Drugs acting in the CNS have to cross the BBB orblood–CSF barrier. These two barriers are formed by


brain capillary endothelial cells and epithelial cells of thechoroid plexus, respectively. Recent studies have shownthat this is not only a static anatomical barrier but also adynamic one in which efflux transporters play a role(Begley and Brightman, 2003; Sun et al., 2003). P-glyco-protein was identified initially as an efflux transporter,and it extrudes its substrate drugs on the luminal mem-brane of the brain capillary endothelial cells into theblood. Thus, recognition by P-glycoprotein as a substrateis a major disadvantage for drugs used to treat CNS dis-eases. In addition to P-glycoprotein, there is accumulat-ing evidence for the presence of efflux transport systemsfor anionic drugs. The transporters involved in the effluxtransport of organic anions from the CNS are being iden-tified in the BBB and the blood–CSF barrier and includethe members of organic anion transporting polypeptide(OATP1A4 and OATP1A5) and organic anion transport-er (OAT3) families (Kikuchi et al., 2004; Mori et al.,2003). They facilitate the uptake process of organic com-pounds such as β-lactam antibiotics, statins, p-aminohip-purate, H2 antagonists, and bile acids on the plasma mem-brane facing the brain–CSF. The transporters involved inthe efflux on the membranes that face the blood stillremain to be identified, although several candidate primaryactive transporters, such as MRP and BCRP, already havebeen proposed. Members of the organic anion transportingpolypeptide family also mediate uptake from the blood onthe plasma membrane facing blood. Further clarification ofinflux and efflux transporters in the barriers will enabledelivery of CNS drugs efficiently into the brain whileavoiding undesirable CNS side effects and help to definethe mechanisms of drug–drug interactions and interindi-vidual differences in the therapeutic CNS effects.

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