CBO9780511781933.008.pdf

Section 1
Chapter
7
Principles of drug action
Drug transport and transporters
Roland J. Bainton and Evan D. Kharasch
Drug transporters and localized
pharmacokinetics
Xenobiotics and defining chemical space
Living organisms use biologic compartments to perform dis-
crete biochemical processes, to localize and control the natural
small molecules contained within [1]. Foreign small molecules,
called xenobiotics, share the same molecular weight range and
solubility characteristics as endogenous small molecules and are
thus able to penetrate biologic spaces. As xenobiotics also pos-
sess potent biointeraction (i.e., drug-like) properties they are the
source for nearly all clinically useful pharmaceuticals [2]. To
balance the cells’ needs of access to endogenous small molecules
and control of xenobiotic penetration, powerful biotransforma-
tion and bioelimination pathways have evolved to control
chemical space (Chapter 6). These same processes present chal-
lenges for delivering clinically useful therapies to particular
organ spaces, as xenobiotics have complex and unpredictable
relationships with the selectively expressed small molecule
transporters or specially tuned metabolic systems [2]. Thus
understanding how any one pharmaceutical interacts with bio-
logically defined chemical space requires a closer examination
of that drug’s susceptibility to highly localized pharmacokinetic
processes affecting chemical persistence at the site of action.
Lipid bilayers and drug localization
Lipid bilayers physically partition aqueous spaces [1] (Fig. 7.1),
creating cells and specialized internal subcellular compartments.
In addition, formation of cell–cell contacts in conjunction with
diffusion-tight junctional complexes divides the external cellular
milieu into different interstitial spaces [1] (Fig. 7.2). This bio-
physical boundary prevents passive diffusion of large molecules
such as proteins and nucleic acids between different compart-
ments and greatly curtails the movement of electrolytes, soluble
components of metabolism, and drugs. Drugs and xenobiotics
are often in equilibrium between charged and charge-neutral
states (Fig. 7.2). Charge-neutral molecules more readily transit
lipid bilayers and possess better penetration rates into cells by
orders of magnitude (passive permeability, Fig. 7.1). Cellular
penetration may be augmented (influx transport) or opposed
(efflux transport) by active cellular processes (see below), a fea-
ture that allows drugs to take multiple routes to a target (Fig. 7.2).
To control the ability of drugs to transit freely in cellular space
and still maintain chemical partition, cells organize molecular
systems to contain, modify, or move endogenous and foreign
lipophilic molecules from one space to another.
Small-molecule partition and transporters
Chemical partition is the sine qua non of cell biology, and cellular
transport systems play a vital role in chemical compartmental-
ization. An enormous variety of transmembrane protein trans-
porters are dedicated to selectively moving small molecules
across membranes [3]. Their role is so broad that by some
estimates 15–30% of all membrane proteins are transporters
[1]. Among the many substrates that transmembrane protein
transporters relocalize are glucose, anabolic intermediates, amino
acids, neurotransmitters, metabolic products, and lipids [3]. The
vast majority of transporters have high substrate specificity and
are organized by DNA sequence relatedness and known function.
They are referred to as solute carrier (SLC) proteins and include
46 different gene families with over 360 genes in humans [3].
Their enormous diversity speaks to the wide variety of intracel-
lular environments and their highly specific needs.
The precision, specificity, and ability of these transport
systems to act against concentration gradients allows small mol-
ecules to have discrete functions depending on their localization.
For example, plasma membrane transporters of the SLC6 class
specialize in the transport of monoamines from the interstitial
space to the cytoplasm. These transporters perform two key
functions for the cell: (1) providing amino acids to the cytoplasm
for metabolic purposes, and (2) reducing extracelluar concen-
trations of monoamines when unwanted [4]. However, when the
same molecule (or close derivative) is to be used as a
neurotransmitter, the SLC18 transporters highly concentrate
and package the molecule into synaptic vesicles. After release
into the extracelluar space for signaling at the postsynaptic
membrane, neurotransmitters are rapidly taken up by SLC6 class
Anesthetic Pharmacology, 2nd edition, ed. Alex S. Evers, Mervyn Maze, Evan D. Kharasch. Published by Cambridge University Press. # Cambridge University Press 2011.
90
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transporters. Moving small molecules across cell membranes is
entropically unfavorable, and thus transporters require energy
expenditure either directly using adenosine triphosphate (ATP),
or dissipating other existing electrochemical gradients (e.g., Na/
K) by using symport or antiport mechanisms [3]. Together,
transporter systems create the chemical preconditions for cellu-
lar function, restore chemical conditions after perturbation, and
clear chemical space of unwanted metabolites. Thus transporters
are essential actors in almost every biologic function.
Localized pharmacokinetic spaces
Acting in concert with, or after the action of, phase I and
phase II metabolism (Chapter 6), to move drugs and their
metabolites across lipid bilayers, transporters are often
referred to as phase III metabolism. Therefore transporters
are often highly expressed in organs of biotransformation such
as the liver and intestine, and metabolism and transport
together create formidable pharmacokinetic barriers. Different
organ systems tune their biodefense systems by locally
applying phase I, II, and III mechanisms [5]. Thus, for a drug
to be clinically effective, it must possess high target specificity,
favorable organismal pharmacokinetics, and complementary
properties for highly localized chemical partition processes at
the cellular level. It is in this latter context that the special role
of drug transporters and their ability to control xenobiotic
chemical space in drug action are considered.
Drug transporters
Discovery of drug efflux transporters
Xenobiotic transporters were identified as a consequence of the
study of multidrug resistance to chemical cancer treatment in
the 1970s [6,7]. Highly efficient cytotoxic resistance to a broad
spectrum of cancer drugs could occur suddenly and unexpect-
edly after initiation of chemotherapy [8]. Analysis of drug-
resistant tumor cells determined that this new-found resilience
correlated with upregulation of a few transmembrane proteins,
suggesting that chemotheraputic protection systems were quite
nucleus
hydrophobic
small/uncharged
polar
large/uncharged
polar
ions
lipid bilayer
intracellular space
extracellular space
~10–7 cm/sec ~10–11 cm/sec
~10–4 cm/sec
>10–2 cm/sec
Figure 7.1. Chemical partition across bilayers.
Lipid bilayers (green lines) are formed by virtue of
the chemical characteristics of their constituent
phospholipids: a hydrophilic phosphate head group
and a hydrophobic carbon-chain tail. The head
groups associate with the aqueous environment,
while the tail groups self-associate and form the
internal, hydrophobic core of the bilayer. This layer
must be transited if nutrients as well as drugs are to
reach targets inside the cell, and movement across
this bilayer is also dictated by specific chemical
properties. Paracellular movement refers to passage
of molecules between cells, bypassing cell mem-
brane barriers. Transcellular movement requires pas-
sage across membrane barriers. More hydrophobic
chemicals and compounds can cross the barrier
with relative ease and have a high permeability
coefficient (expressed in the figure as cm s–1
)
whereas chemicals with low permeability coeffi-
cients (such as ions) cannot passively transit the lipid
bilayer and must cross via other means such as
carrier-mediated transport. All chemicals critical to
sustaining life fall along this continuum. Modified
with permission from Alberts et al. [1].
( )+/– ( )0
Transcellular
Paracellular
Drug
Transporters
(Ionized Form)+/– (Non-ionized Form)0
Junctional
Complexes
Interstitial Space
(vascular)
Interstitial Space
(organ)
Figure 7.2. Chemical partition across cellular
boundaries. Cells separate one interstitial space
from another. Cellular barriers rely upon two modes
of exclusion to maintain xenobiotic exclusion, as
small molecules can interact with cells in two ways.
Charged molecules (blue stars) are excluded by the
boundary function of lipid bilayers, very low rates of
endocytosis, and tight diffusion barriers provided by
special lateral-border junctional complexes (blue
ovals) of barrier epithelium. Small uncharged
molecules (red stars) pass easily through lipid
bilayers (green), but active efflux transporters (red)
move them back into the humoral space, creating
an active transport barrier. As drugs in aqueous
solution are in equilibrium between charged and
uncharged forms, a true xenobiotic barrier must
maintain both properties simultaneously to mani-
fest chemical exclusion.
Chapter 7: Drug transport and transporters
91

different from the highly specific SLC transporter systems dis-
cussed above. Ultimately these genes were identified as active
drug efflux pumps that, unlike most SLC transporters, had
surprisingly broad substrate specificity. These proteins were
found to bind and transport a wide spectrum of chemothera-
peutics back into the extracellular space, thus reducing the
effective internal concentration of cytotoxic drugs [8]. Interest-
ingly, these cells were not intrinsically more resistant to che-
motherapeutics at the target sites, but had instead coopted and
enhanced an endogenous chemical partition system to
strengthen the chemical barrier at the membrane [8].
Whole genome sequencing methods have discovered large
families of similar multidrug resistance transporter (MDR)
genes in nearly all organisms [9,10]. Most of these proteins
belong to a large and highly conserved class of transmembrane
proteins known as ATP-binding cassette (ABC) transporters
[8,11]. The archetypal ABC transporter contains two six-pass
transmembrane domains and two cytoplasmic nucleotide-
binding domains, although the proteins do vary structurally
depending on their class (Fig. 7.3). Full transporters are large
proteins with 1200–2200 amino acids, and half-transporters
have 650–800 amino acids. Half-transporters are thought to
function as dimers in the membrane, potentially generating
additional functional heterogeneity by combining different
substrate preference into one transport pocket [8].
Unfortunately, unlike other transport systems where
ligand binding is well understood, only a few specifics are
known about the mechanism of xenobiotic binding to ABC
transporters. Efficient efflux, for example, is thought to result
from conformational changes of the transporter which allow
an alternating high substrate-affinity (cell-facing)/low
substrate-affinity (extracellular-facing) organization [8]. This
permits intracellular presentation of the drug at one interface
and release (or efflux) into the extracellular milieu on the
other side. Evidence is mounting that drug binding to the
major substrate-promiscuous xenobiotic transporters occurs
in the membrane and not from the solution. Thus, these
transporters may be designed to act at a protein/lipid-mem-
brane interface [8].
The ABC gene family
The approximately 50 human ABC genes occur in seven
families (A through G), although only a subset are known
to play major roles in drug transport. The major xenobiotic
efflux transporter familes are the B, C, and G gene classes
(Table 7.1). Interestingly, while ABC gene sequence and sub-
families are highly conserved from yeast to flies to humans,
the specific roles of most gene products are unknown. ABC
transporter localization in polarized cell interfaces is essential
to chemoprotective function, as net flux of xenobiotics into or
away from various interstitial spaces is determined by the
type, quantity, efflux or influx orientation, and apical/basal
orientation at the protective interface (e.g., the vascular endo-
thelium of the blood–brain barrier, as in Figs. 7.4 and 7.5)
[8,13]. Unfortunately as the best studies of ABC transporters
in the animal are limited to a few genes (see below), it is not
known whether or how they function as a complementary
system in the biology of chemical exclusion.
Important subtypes of drug
efflux transporters
ABCB1 (P-glycoprotein)
P-glycoprotein (P-gp, ABCB1, or the multidrug resistance
transporter MDR1) was the first xenobiotic transporter
discovered in multidrug-resistant cancer cells [7] and is
Domain 2
Extracellular
Intracellular
Extracellular
Intracellular
Extracellular
Intracellular
NBD NBD
C-Terminus
N-Terminus
NBD
NBD
C-Terminus
N-Terminus
NBD
N-Terminus
Lipid Bilayer
Lipid Bilayer
Lipid Bilayer
C-Terminus
Examples
MDR1 (ABCB1)
MRP4 (ABCC4)
MRP5 (ABCC5)
MRP7 (ABCC1)
BSEP/SPGP (ABCB11)
MRP1 (ABCC1)
MRP2 (ABCC2)
MRP3 (ABCC3)
MRP6 (ABC6)
MXP/BCRP/ABC-P
(ABCG2)
Domain 1 Figure 7.3. Membrane topologies of known xeno-
biotic transporters. (Top) P-gp-like ABC transporters
have 12 transmembrane (TM) domains and two
nucleotide-binding domains (NBD). (Middle) In the
ABCC class a second membrane topology preserves
the core P-gp-like transporter with an additional five
TMs on the N-terminus. (Bottom) The half-transport-
ers of the ABCG class have six TMs and one NBD.
They are thought to dimerize to promote drug
transport function. Modified with permission from
Gottesman et al. [12].
Section 1: Principles of drug action
92

responsible for the most profound effects on localized xeno-
biotic protection of all the ABC transporter subfamily
proteins. P-gp is a twelve-pass transmembrane protein of
140 kD and is highly expressed at important chemoprotec-
tive interfaces (Table 7.2). It has the broadest substrate
specificity of the ABC genes, transporting a significant
number of drugs, including anticancer drugs, HIV-protease
inhibitors, antibiotics, antidepressants, antiepileptics, and
opioids such as morphine and loperamide (Table 7.2).
P-gp substrates are generally amphipathic and range in size
from 300 to 2000 Da.
P-gp also functions in a wide range of environments
including the GI tract, renal proximal tubule cells, the
canalicular surfaces of hepatocytes, the capillary endothelial
cells of testes, and the blood–brain barrier. Thus the poten-
tial for significant drug interactions is widespread [8]. For
example, oral bioavailability of the anti-inflammatory drug
cyclosporine A is greatly increased by amlodipine, a P-gp
substrate that doubles as a P-gp inhibitor [8]. Another
example is loperamide, an opioid antidiarrheal that pro-
duces no notable central nervous system (CNS) effects at
normal doses because intestinal P-gp-mediated efflux retards
systemic absorption [8]. However, when loperamide is given
with the P-gp inhibitor quinidine, significant systemic
absorption occurs, CNS delivery ensues, and side effects
such as respiratory depression arise [16]. Thus localized
transporter interactions can have substantial effects on drug
bioavailability. This is particularly pertinent in fentanyl
pharmacodynamics, based on the association between P-gp
polymorphisms and fentanyl respiratory depression [17].
In patients under spinal anesthesia given intravenous
fentanyl (2.5 mg kg–1
), there are differences in suppression
of ventilatory effort between three distinct P-gp genotypes
prominent in ethincally Korean populations. The presump-
tion is that P-gp transports fentanyl at CNS barriers, and
therefore P-gp polymorphisms significantly alter brain
concentration despite equivalent doses. Thus, a highly local-
ized drug partition phenomenon can affect drug responses,
but they are dependent on the relationship of the specific
interface (i.e., the blood–brain barrier) and the protected
localization of the drug target (i.e., neurons containing
opioid receptors).
ABCC transporters (MRPs)
Multidrug resistance proteins (MRPs) are the largest class
of ABC transporters relevant to drug resistance and drug
distribution and are ubiquitously expressed [18]. There are
12 proteins in this class with two distinct types of transmem-
brane protein topologies (Fig. 7.3). Although they contain
only 15% sequence identity to P-gp they are predicted to
posses a similar structure, with two nucleotide-binding
domains and similar membrane topology to ABCB transport-
ers (Fig. 7.3) [12]. Furthermore, like P-gp, they can extrude a
wide array of amphipathic anions. Interestingly, all members
of this particular subfamily, also called ABCC transporters,
are lipophilic anion transporters that are broadly involved in
cancer therapeutic resistance [18]. They are quite adept at
effluxing methotrexate, thereby lessening the effect of
an important chemotherapeutic in the brain [8]. MRP sub-
strate specificities show a preference for glutathione and
glucuronide conjugates, thus drug elimination could be
greatly facilitated when transporters are locally paired with
the appropriate phase II enzymes (e.g., glucuronyl trans-
ferases, Chapter 6). Nevertheless MRPs also efficiently trans-
port unconjugated compounds such as anthracyclines,
Table 7.1. ABC transporter genes are highly conserved. Pairwise
analysis using human ABC transporters versus Drosophila using NCBI
BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) demonstrates high
conservation of transporter number per class and high identity among
classes. Similar results are obtained from comparison with other
metazoan genomes, and thus ABC genes are highly evolutionarily
conserved and likely to play fundamental roles in cellular biology. The
major xenobiotic transporters are from the B, C, and G classes, but
A-class transporters may play roles in some cases.
Subfamily Human Drosophila Identities
(%)
Positives
(%)
A 12 10 20–37 34–56
B 11 8 43–57 59–75
C 13 14 23–50 40–67
D 4 2 56–59 71–74
E 1 1 77 88
F 3 3 41–69 56–82
G 5 (þ1) 15 22–54 41–71
Total 50 53
Vascular
Space
A
VE
P
CNS
Interstitial
Space
Figure 7.4. Organization of the blood–brain barrier (BBB). While chemical
protection is focused at discrete epithelial layers, surrounding tissue can have
essential roles in molding and assisting the localized PK potential. This is
particularly true at the BBB, where properties of the vascular endothelium (VE)
are induced and controlled by surrounding tissues. The BBB VE is thought to
receive inductive cell-fate signals from the CNS parenchyma (not shown), while
astrocytes (A) and pericytes (P) assist in and regulate localized responses to
chemical and immune stress.
93

epipodophyllotoxins, vinca alkaloids, and camptothecins
(Table 7.2) [18]. MRPs are known to localize to both apical
and basal interfaces of chemical protection interfaces and thus
can participate in moving drugs in both directions between
interstitial spaces (Fig. 7.5) [8,13].
ABCG2 (BCRP)
Breast cancer resistance protein (BCRP), so named for its
discovery in an especially drug-resistant breast cancer cell line
[21], is the second member of the five-member
half-transporter ABCG subfamily (Fig. 7.3) [10]. BCRP is
expressed throughout the body but most notably in the
liver, intestinal epithelium, placenta, blood–brain barrier, and
a variety of stem cells. Like P-gp, BCRP is quite promiscuous
in its choice of substrate and is powered by the hydrolysis of
ATP. BCRP substrates generally include compounds that are
relatively large in molecular weight and either positively or
negatively charged and amphipathic. Specific substrates for
BCRP-mediated efflux include antineoplastic drugs and their
metabolites (e.g., methotrexate and sulfated methotrexate),
Table 7.2. Major human drug transporters P-glycoprotein (P-gp), multidrug resistance protein (MRP), breast cancer resistance protein (BRCP), organic
anion transporting polypeptide (OATP), organic cation transporter (OCT), and organic anion transporter (OAT) are the best-studied drug transporters. They can
share substrates and inhibitors. Modified with permission from Loscher and Potschka [13], Dobson and Kell [14], Zhang et al. [15].
Transporter
gene
Aliases Localization Function Selected substrates Selected inhibitors
ABC family
ABCB1 P-glycoprotein,
MDR1
BBB, BRB, intestine, liver,
kidney, placenta, adrenal,
testis
efflux digoxin, fexofenadine, morphine,
loperamide, corticoids,
cyclosporine, paclitaxel,
verapamil, methotrexate
cyclosporine,
verapamil,
quinidine, ritonavir,
ketocoanzole,
itraconazole
ABCC1 MRP1 BBB, breast, lung, heart,
kidney, blood cells, liver,
intestines, testes, placenta
efflux glutathione or glucuronide
conjugates, leukotriene C4,
etoposide, methotrexate
probenecid,
sulfinpyrazone,
cyclosporine
ABCC2 MRP2 intestine, liver, kidney,
brain, placenta
efflux bilirubin, indinavir, cisplatin cyclosporine
ABCG2 BCRP BBB, intestine, liver,
breast, placenta
efflux anthracyclines, mitoxantrone,
prazosin, topotecan, methotrexate
elacridar (GF120918),
cyclosporine,
fumitremorgin C
SLCO family
SLCO1A2 OATP1A2 Liver, kidney, brain, lung,
colon
uptake,
efflux
fexofenadine, rocuronium,
enalapril, rosuvastatin
SLCO1B1 OATP1B1 BBB, brain, kidney, liver uptake,
efflux
organic anions, bile salts, digoxin,
fexofenadine, methotrexate,
rifampin
cyclosporine,
verapamil, rifampin,
similar to P-gp
SLC family
SLC22A1 OCT1 liver uptake metformin quinidine, quinine,
verapamil, cimetidine
SLC22A2 OCT2 kidney, brain uptake metformin, amantadine,
memantine, varenicline
cimetidine
SLC22A6 OAT1 kidney, brain uptake acyclovir, adefovir, methotrexate,
zidovudine
probenecid,
cefadroxil,
cefamandole,
cefazolin, diclofenac
SLC22A8 OAT3 kidney, brain uptake cimetidine, methotrexate,
zidovudine
probenecid,
cefadroxil,
cefamandole,
cefazolin, cimetidine
BBB, blood–brain barrier; BRB, blood–retinal barrier.
94

topotecan and irinotecan (both topoisomerase I inhibitors), as
well as mitoxantrone, camptothecin and flavopiridol, xenobio-
tic toxins, food-borne carcinogens, and endogenous com-
pounds [8]. BCRP, unlike MRPs, is also adept at transporting
glutamated folates and folate antagonists [8]. Therefore, like
P-gp, BCRP is a major factor in conferring resistance to a wide
range of therapeutics and is central to pharmacokinetics and
toxicity considerations [8].
Other drug transporters
SLC21 transporters (organic anion
transporter proteins)
Despite the relative abundance of SLC transporters at protect-
ive barrier interfaces, few are known to participate directly in
drug transport. One exception is the organic anion transporter
proteins (OATPs), which are not structurally related to ABC
transporters, but belong to the solute carrier transporters class
21 (SLC21/SLCO) [3]. OATPs primarily use passive transport
mechanisms driven by electrochemical gradients or symport/
antiport mechanisms, but interestingly show other functional
similarities to P-gp. The best-characterized OATP xenobiotic
transporters are OATP1A2, OATP1B1, OATP1B3, and
OATP2B1 [14,15]. They are generally efflux transporters,
found at chemoprotective interfaces, prominently in brain
endothelial cells, and also in liver, lung, kidney, and testes.
In-situ mRNA hybridization also demonstrates broad expres-
sion in central brain tissue, suggesting a role in general cellular
protection as well as barrier function. In-vitro experiments
suggest that OATPs can transport many structurally divergent
molecules, reminiscent of P-gp. Examples include bile acids,
Intestine
MCT1 OATP-B OCTN2 MDR1 MRP2
Blood-Cerebrospinal
Fluid Barrier
Blood
Blood-Brain Barrier
others?
MCT1
MRP1/2//4/5/6
OAT3
OCTN1/2
MDR1
BCRP
PEPT1/2
OAT4
NPT1
URAT1
OCTN1/2
MDR1
MRP2/4
Kidney
others?
OCT2
OAT1/3
MRP3/6
Liver
NTCP
OATP-B/C/8
OAT2
NPT1
MRP1/3/6
others?
others?
MRP2
BCRP
MDR1
OCT3
MRP1/3
Placenta
PEPT1 OCT3 ASBT
MRP3
MCT1
OATP-A
OST α/β
others?
BCRP
?
MDR1
BSEP
BCRP
MRP2
PEPT2 OCT2
others?
MRP1
Figure 7.5. Membrane localization of drug transporters at the blood–brain barrier. Organ spaces subject to specific chemical constraints usually organize the
tightest control of chemical space at one cellular layer. These chemical barriers use tight junctions and transporter content, type, and apical/basal localization to
modulate the protected interstitial space to the purpose and susceptibilities of the underlying tissue [19,20]. The vascular endothelium of the BBB is one of the
most potent localized pharmacokinetic interfaces and has a vast array of drug efflux and influx transporters at both vascular and CNS interfaces. Similar
organization is repeated at the blood–cerebrospinal fluid barrier, renal epithelium of the kidney, intestinal epithelium, hepatocyte/bile-duct interfaces, and
placenta; thus drug action at an individual organ can depend greatly on the chemoprotective efficiency of a particular exposure interface. For transcellular
transport, drugs need to cross two different membranes on the basal and apical sides. In the intestine, drugs are absorbed from the luminal side (brush border
membrane) and excreted into the portal blood across the basolateral membrane. In the liver, drugs are taken up into hepatocytes across the sinusoidal membrane
and excreted into the bile. In the kidney, drugs undergo secretion (urinary excretion) or reabsorption. In the case of secretion, drugs are taken up from the
basolateral side and excreted in the urine across the apical side. However, in the case of reabsorption, drugs are taken up from the urine and excreted in the
circulating blood. Reproduced with permission from Tsuji [19].
95

steroids, estrogen, many anionic compounds, and glutathione
or glucuronide conjugates [22]. Certain drug–food inter-
actions (e.g., grapefruit juice) [23] may be attributable to
inhibition of OATPs, but further characterization of the in-
vivo role of OATPs in drug interactions is needed. Interest-
ingly, unlike ABC transporters, some OATPs can also function
as drug influx transporters (Fig. 7.4). Bidirectional transport
and/or the counteracting of efflux transport occurs at numer-
ous tissue interfaces including the gut, liver, kidney, and
blood–brain barrier [19,20]. How these competing transporter
processes are regulated, coordinated, and potentially circum-
vented or controlled will require a better understanding of
drug–transporter interactions at cellular interfaces and
methods for analyzing net localized pharmacokinetic dispos-
ition of drugs.
Specially protected anatomic
spaces
Specialized cellular boundary systems have evolved to limit
access to sensitive biologic spaces [12,24]. Transporters are
critical to the processes of drug uptake, distribution, metabol-
ism, and excretion and may play multiple roles simultaneously
depending on their localization, expression levels, and activity.
Transporters are especially important in highly sensitive
organs such as the CNS, where they are important determin-
ants of drug pharmacodynamics [25].
CNS drug barriers
Blood–brain barrier
The blood–brain barrier (BBB) is the largest pharmacokinetic
interfacewithintheCNS(about20m2
)[26],andthustheprimary
focusofthe manyefforts to understand and controldrugaccess to
the brain [27]. The BBB evolved due to the brain’s unique chem-
ical sensitivity and requirement for the highest level of chemical
protection. BBB drug transporters can profoundly influence the
clinical effectiveness of drugs acting in the brain [13].
The BBB has a complex cellular makeup that is functionally
integrated with surrounding brain tissue as part of the neuro-
vascular unit [11,28]. The complete neurovascular unit com-
prises the capillary vascular endothelium, pericytes, a basement
membrane, closely associated astrocytic glia, and nearby
neurons [29] (Fig. 7.4). The highly polarized vascular endothe-
lium is the primary site of chemical exclusion. It possesses very
tight lateral border junctions [30] and a diverse array of apically
(i.e., vascular) facing efflux drug transporters, including P-gp,
MRP1, and BCRP (Fig. 7.5). The functional importance of
these transporters to drug partition in the brain has been
studied using gene-specific genetic null mice (knockouts)
[31–35]. In single transporter loss-of-function experiments,
two- to ten-fold increases in CNS penetration of specific ABC
transporter substrates including chemotherapeutics can result
after IV administration [36]. That ABC transporters can so
profoundly affect brain drug concentration by their presence
or absence in a single endothelial layer suggests a plausible
mechanism for increased drug sensitivity of the CNS in trans-
porter-associated pharmacogenomic studies [17] (see P-gp,
above). Furthermore these studies promote the idea of specif-
ically targeting xenobiotic transporter function to more readily
concentrate drugs in the brain for treating CNS illnesses such as
brain cancer, multiple sclerosis, and Alzheimer’s disease [27].
Astrocytic glia form a continuous circumferential and close
association with the basement membrane, further isolating the
vascular and CNS interstitial spaces, but their role in chemical
exclusion is unclear. The astroglial cell layer is less than a
micron thick and lacks very tight junctions at cell–cell contacts,
and xenobiotic transporters should therefore not directly affect
CNS glial cell chemopartition. Indeed, genomic data show a
much lower level of xenobiotic transporter and junctional pro-
tein expression in the astroglia compared to vascular endothe-
lium (R. Daneman and B. Barres, personal communication and
[37]). Thus, while astrocytes are essential for BBB maintenance
and function, their role in chemical protection may be to sense
or integrate chemical, metabolic, and inflammatory stresses
which must be coordinated to maintain proper physiologic
balance for neurons [11]. Substantial efforts are under way to
develop model systems and new methods to identify regulatory
controls and chemoresponsive physiologies in the hope of
specifically modulating drug barrier function at the BBB and
ultimately improving clinical treatment options [27].
Blood-retinal barrier (BRB)
Transport of molecules between the vitreous/retina and sys-
temic circulation is restricted by the blood-retinal barrier
(BRB, also called the retinal BBB or RBBB), composed of
retinal pigment epithelium (RPE) and endothelial cells of the
retinal blood vessels. The RPE maintains a tight barrier to
diffusion to the interstitial fluid of the surrounding connective
tissue of the eye. Vascular access to the retina is primarily
through the retinal blood vessels, which penetrate with the
optic nerve and run parallel to the retinal surface. The capillary
bed of the retinal vascular endothelium maintains tight junc-
tions and apically facing transporters morphologically similar
to the BBB in the central brain. A unique feature of the eye
is the ability to bypass the barrier by topical application or
injection into the vitreous humor. However, while drug
delivery to the CNS is uniquely possible in the eye, transport
processes resident at the RBBB promote very rapid removal of
small molecules into the systemic circulation. For example,
common topical ophthalmic drugs such as erythromycin, dex-
amethasone, and cyclosporine are substrates for prominent
RBBB transporters and can be found in the systemic circula-
tion after ocular administration [38]. These transporters
include MDR1, MRP1, BRCP, and numerous OATPs [38].
Localized pharmacokinetic methods for measuring efflux
across the BRB are limited; therefore reliable quantitative
96

estimation of the roles of inner and outer BRB has not been
well established in humans. In rabbits, however, MDR1 pre-
sent at the RBBB can be selectively modulated using traditional
inhibitors such as quinidine and cyclosporine A [39]. Further-
more, diffusion barrier studies have been carried out in mice,
where labeled mannitol is used as a biomarker for retinal
diffusion barrier integrity [40] and has shown diffusion-tight
function of the BRB. Thus experimental models identify the
BRB as an effective barrier similar to the BBB.
Blood–cerebrospinal fluid barrier and choroid plexus
The blood–cerebrospinal fluid barrier (B-CSFB) is derived
from a special confluence of the pia mater and a single layer
of CSF-facing glial cells (the ependyma). This meeting of two
barrier layers is known as the choroid plexus and is the site of
CSF production. Throughout the rest of the CNS, ependymal
cells become diffusion-tight, utilizing the same modes of sep-
aration discussed at the capillary–brain interface above. Here,
therefore, chemical exclusivity of the CNS is maintained by an
anatomically simpler system than at the BBB. The CSF pro-
duction cycle is completed by rapid uptake at arachnoid granu-
lations. Together, the CSF production and brain barrier system
is thought to perform cleansing functions, further augmenting
the chemical protection provided by the BBB [41].
Because the diffusion of polar substances at the B-CSFB is
less hindered than at the BBB [41], drug administration into
CSF has been proposed to circumvent the BBB and gain access
to the CNS. However, potent chemical exclusion properties are
present at the B-CSFB (i.e., ependymal cells), as at the choroid
plexus [42]. Specifically, transporters OAT, OATP1, OATP2,
MRP1, and MDR1 are present in high levels, suggesting an
explanation for why the CSF has not proven to be a depend-
able access point for the administration of chemotherapy or
other drugs into the CNS. Nevertheless, certain drugs can
overcome these barrier processes by a combination of favor-
able penetration chemistry and poor transportability. For
example, sodium-channel blockers such as bupivicaine are
used for subarachnoid block.
Gastrointestinal tract
Intestinal drug uptake usually occurs by mass action and
passive permeability when high concentrations are presented
to the microvillus absorption surface. An empirical set of rules,
known as Lipinski’s Rule of Fives, describes how small mol-
ecules passively cross the lipid bilayer. Oral drugs can violate
only one of the following criteria: (1) not more than five
hydrogen bond donors, (2) not more than 10 hydrogen bond
acceptors, (3) molecular weight under 500 Da, and (4) an
octanol/water partition coefficient log P less than 5. Pharma-
ceuticals that possess these properties are better at passing the
chemoprotective interface of the gut, but these constraints do
not allow small molecules to bypass the action of transporters.
Xenobiotic transporters are abundantly represented
throughout the enteral space (Fig. 7.5) [43,44]. P-gp is apically
localized in microvilli throughout the gut epithelium, found in
highest amounts in the ileum and jejunum, and counteracts
the passive or active absorption of xenobiotics [25]. For
instance, mouse P-gp knockouts demonstrate increased
absorption and bioavailability of many oral xenobiotics [45],
and uptake of some drugs is markedly increased by small
molecules that inhibit P-gp function [16]. BCRP is the trans-
porter expressed in highest abundance throughout the human
small intestine [46]. In addition to countering the absorption
of some orally administered drugs, certain transporter systems,
particularly ABCC proteins, counter the absorption of drug
glucuronides that are excreted in bile. Intestinal efflux trans-
porters act in concert with intestinal phase I metabolism to
limit drug bioavailability.
Liver
Hepatic drug elimination depends on passive or active hepatic
uptake, intracellular metabolism, excretion of metabolites or
parent drug into bile, and/or excretion of metabolites or parent
drug into sinusoidal blood. Transporters are involved in
uptake, biliary excretion, and sinusoidal efflux (Fig. 7.5) [23].
SLC-type transporters in the basolateral (sinusoidal) mem-
brane (OATPs, OATs, OCTs) mediate the uptake of organic
cations, anions, bilirubin, and bile salts. ABC transporters
(P-gp, BCRP, MRPs) at the canalicular membrane mediate
the efflux into bile of drugs, metabolites, and bile salts. MRPs
may also mediate drug and metabolite efflux back into the
blood. The balance of vectorial uptake and efflux drug trans-
port together with intracellular metabolism, determines drug
elimination. Hepatic drug transporters are subject to pharma-
cogenetic variability, and drug–drug interactions.
In murine models, transporter disruption can effect the
distribution of important anesthetic drugs such as opiates.
Morphine-3-glucuronide (M3G), the most abundant metab-
olite of morphine, is transported in vitro by human MRP2
(ABCC2) that is present in the apical membrane of hepato-
cytes. Loss of biliary M3G secretion in MRP2(-/-) mice results
in its increased sinusoidal transport, which can be attributed
to MRP3. Combined loss of MRP2 and MRP3 leads to a
substantial accumulation of M3G in the liver, resulting in
low rates of sinusoidal membrane transport and the pro-
longed presence of M3G in plasma [47]. These results suggest
that in vertebrates MRP2 and MRP3 transporters provide
alternative routes for the excretion of a glucuronidated sub-
strate from the liver in vivo. This illustrates the potential
complexity of interactions between transporters and modify-
ing enzymes intrinsic to particular chemoprotective
environments.
Kidney
Transporters mediate active tubular secretion and reabsorp-
tion in the kidney, which, together with glomerular filtration,
determine drug elimination (Fig. 7.5). The primary focus of
renal excretion is the proximal tubule, which actively takes up
organic anions and cations at the basolateral membrane and
97

extrudes them across the apical brush border membrane into
the urine; hence transepithelial flux requires two distinct sets
of transporters. Many drugs (weak acids) and their phase II
conjugates are organic anions. OATs 1 and 3 are the primary
organic anion uptake transporters at the basolateral mem-
brane (although they may also interact with neutral and
cationic compounds), while efflux into the tubular lumen is
mediated primarily by the ABC subfamily C transporters
MRP2, MRP4, and BCRP [48]. Organic cations are typically
transported into renal tubular cells at the basolateral mem-
brane by OCTs 1 and 2, and effluxed into urine at the apical
membrane by P-gp, OCT3, and novel organic cation trans-
porters OCTN1 and OCTN2. Selective expression and spe-
cific localization of transporters at different apical and basal
interfaces in the tubule, pharmacogenetic variability in their
activity, and drug interactions play a determinative role in
drug elimination.
Transporter-based drug interactions can occur in the
kidney by competing for elimination. For example, probencid
inhibits OATP-mediated excretion of antibiotics [49]. In add-
ition, methotrexate has strong potential interactions with non-
steroidal anti-inflammatory drugs (NSAIDs), particularly after
prolonged high-dose chemotheraputic use [49].
Placenta
The placenta is the sole medium of metabolic exchange
between fetus and mother; hence it protects the fetus by
providing a major obstacle to chemical penetration. The
important chemical properties determining placental transfer
by passive diffusion are similar to other organs: molecular
weight, pKa of drug, lipid solubility, and plasma protein
binding. The transport barrier in placenta is found in the
syncytiotrophoblast, a multinucleated single cell layer that
performs nutrient and waste exchange between mother and
fetus [50]. This layer is further elaborated into two distinct
interfaces: a maternal-facing brush border and a fetal-facing
basal membrane [50].
It is in these two interfaces that a variety of membrane
transporters perform directional fluxing of metabolites and
drugs. Specifically, the brush border membrane contains all
three major classes of ABC transporters (i.e., P-gp, MRP1–3,
and BCRP) as well as specialized transporters such as the
serotonin, norepinephrine, and carnitine transporters (SERT,
NET, OCTN2) [50]. On the other side of the divide, the fetal-
facing basal membrane contains transporters such as the
organic anion transporters OAT4 and OATP-B in addition
to the reduced folate transporter RFT-1 [50]. Combinations
of genetic loss of P-gp function with specific inhibitors of
BCRP markedly increase drug penetration of topetecan to the
fetus, which is not seen in the genetically sensitized back-
ground alone. Thus the placenta is an essential chemical filter
for the fetal protected space and exemplifies the potential for
overlapping and redundant function of chemoprotective
mechanisms [51].
Transporter biology: special topics
Drug transporter methods
The field of transporter biology is relatively new, and a brief
description of experimental methods used to identify the role
of transporters in drug disposition may be helpful. Accurate
physical measurement of localized drug concentration can be
difficult in very compact biologic spaces like the BBB/CNS
interface, and consequently methods of measurement require
some explanation [52].
In-vitro methods are most commonly used to study human
transporter activity and identify candidate transporters for a
given substrate. Cell culture systems amenable to transfection
of influx or efflux transporter genes, or direct injection of
transporter mRNA into Xenopus oocytes, express and localize
transporter proteins to the cell surface. Isolated membrane
vesicles can also be prepared. Exposure to drug on one side
of a cell layer, then measurement on both sides, to determine
the ratio of substrate penetration to negative controls or by
using transporter inhibitors, allows individual assessment of
luminal and abluminal transporters and their mechanisms.
Unfortunately, heterologous systems have several problems
when attributing a specific physiologic function to a trans-
porter, because it is specifically overexpressed. Studies using
membranes of confluent primary human cells can provide
more information, particularly on the contribution of relevant
transporters for a specific drug. Ex-vivo assays, however, lack
functional context since organ-specific gross anatomy and
transporter interface conditions do not mimic the animal. In
addition, the transporter must function away from its normal
molecular constituents, and thus potential modulators of sub-
strate preference and functional compensations are lost.
In animals (and occasionally in humans) ex-vivo or in-situ
organ perfusion allows direct measurement of drug influx and
efflux concentrations. Advantages of this method are a close
control of the time course of drug administration and concen-
tration. Furthermore, drug goes directly from perfusate to the
brain (or other organ studied), bypassing systemic biotrans-
formation. Although this experimental methodology is more
precise, it still requires bulk tissue concentration measurement,
and hence does not provide data about discrete localization in
the brain (or other organ). Calculation of the brain efflux
index can be used as an indirect measure of chemical partition
at the BBB. A drug is injected directly into the brain with an
impermanent tracer. The ratio of test solute to the reference
tracer determines the brain efflux activity. An improved
method is tissue (often intracerebral) microdialysis. This
method directly samples small-molecule solutes in brain inter-
stitial fluid through a semiporous membrane. Microdialysis is
difficult and costly to perform, however, and may have its own
artifacts. Transgenic rodent models using knockouts or knock-
ins of transporters are a common approach to identifying
transporter function with respect to drug disposition and
98

effect. However, there is still a great deal of argument sur-
rounding the similarity of physiologic function even between
highly homologous vertebrate systems.
In humans, pharmacogenomic approaches often attempt to
identify transporter polymorphisms which may alter pharma-
cokinetics or pharmacodynamics. The use of drug probes to
selectively inhibit a transporter’s function, and assess the effect
on pharmacokinetics or pharmacodynamics, is limited by the
lack of transporter-selective inhibitor probes. This is unlike the
methods used to profile cytochrome P450s in drug metabol-
ism, where P450-selective inhibitor drugs are available.
Drug–drug interactions and transporters
Drugs themselves can act to modulate xenobotic transport, and
any new pharmaceutical is a potential effector of chemical
partition. While the number of current examples is small, as
new therapeutics are developed the opportunity for drug–drug
interactions through transporter inhibition becomes increas-
ingly problematic. This issue is further exacerbated by the wide
tissue distribution of transporters like P-gp, which makes it
difficult to predict the potential for affecting drug distribution
and efficacy in a site-dependent fashion. As noted above, P-gp
loss of function can affect both brain and gut uptake
depending on the drug in question [36,45]. This is not just a
theoretical problem but occurs with many commonly used
medications. For instance, verapamil and the cyclosporine
analog PSC833 lead to increased brain concentration of anti-
epileptics such as carbamazapine, phenytoin, and phenobarbi-
tol [13,53]. The combination of quercetin, a P-gp substrate,
and digoxin also has demonstrated toxicity [53]. Lastly,
b-blockers such as talinolol exhibit increased oral bioavailability
when intestinal P-gp activity is reduced in both animal models
and human observations [53]. Unfortunately there is no set of
chemoprotective rules that can warn clinicians of potential inter-
actions. Currently all drug–drug interactions are determined
empirically, and hence the need for a more complete analysis of
the organization of chemical protection in the animal.
Modulating transporter function
in vivo for clinical purposes
The BBB is a dynamic structure regulated by many competing
inputs [11]. In vertebrates, transcellular diffusion can be modi-
fied by adjusting either the level or the activity of transporter
proteins [54]. Transcription of a number of efflux transporters
including P-gp, MRP2, MRP3, and OATP2 is regulated in part
by the pregnane X receptor (PXR), a nuclear receptor that is
activated by naturally occurring steroids as well as a wide
variety of xenobiotics [55–57]. Higher levels of transporter
expression result in more efficient xenobiotic efflux and a
tighter BBB [58]. The activity of some transporters can also
be influenced by cell-signaling pathways. For example, binding
of endothelin 1 to the ETB receptor rapidly and reversibly
inhibits transport activity of P-gp via nitric oxide synthase
and protein kinase-C mediated pathways [59].
ABC transporter chemopartition function can also be
modulated by drugs. In this case one drug acts as competitive
substrate antagonist against more favored transport molecules
(Table 7.1). This explains why many drugs are listed as both
substrate and antagonist in substrate/inhibitor tables. Several
clinical scenarios have attempted to introduce drugs like
“selective” transport inhibitors at the BBB interface to improve
penetration of chemotherapeutics, but no clinical benefit has
yet been obtained [13,60]. Nevertheless the search for more
selective competitive inhibitors or allosteric modulators of
ABC transporters is quite active in both academic and indus-
trial research [8].
Future directions in the study
of chemical protection
The ability of an organism to protect itself against environ-
mental threats, whether predation or chemical insult, is as
essential as the ability to convert food into energy. Chemical
transporters of the variety discussed in this chapter are present
across evolutionary time, with the essential structure of pro-
teins such as members of the ABC superfamily changing very
little across phyla. For example, there is increasing evidence
that species as distant as fruit flies and humans use very similar
means to exclude xenobiotics from privileged compartments
such as the CNS [61]. The decoding of the fruit fly Drosophila
melanogaster genome has enabled investigators to identify
genes and proteins that are conserved across species, including
analogs of many of the ABC transporters relevant to human
physiology and pathology (Table 7.1) [10]. While the compon-
ent parts of chemoprotective systems are just coming to light
(i.e., transporters and metabolic enzymes that are chemically
organized), there is little understanding of the regulation and
adaptations such a system requires. Model systems using
human cell culture, animal models, and the tools of pharma-
cogenomics will play a large role in furthering the understanding
of chemical protection. Once there are better understandings of
localized cellular or subcellular drug concentrations and identifi-
cation of novel modulators of chemical partition, better methods
of therapeutic drug delivery can be developed to improve clinical
outcomes.
Summary
Biophysical boundaries prevent passive diffusion of large mol-
ecules, such as proteins and nucleic acids, between different
compartments and greatly curtail the movement of electro-
lytes, soluble components of metabolism, and drugs. An enor-
mous variety of transmembrane protein transporters are
dedicated to selectively moving small molecules across mem-
branes. Transporters are essential actors in almost every bio-
logic function, as transporter systems create the chemical
preconditions for cellular function, restore chemical condi-
tions after perturbation, and clear chemical space of unwanted
metabolites. The precision, specificity, and ability of these
99

transport systems to act against concentration gradients allow
small molecules to have discrete functions depending on their
localization. Drugs share the same molecular weight range and
solubility characteristics as many endogenous small molecules
and are subject to the same physiologic processes, and the
same transporters, as endogenous molecules.
Drug (xenobiotic) transporters were initially identified
during studies of multidrug cancer resistance. These proteins
were efflux pumps with broad substrate specificity, capable of
binding and transporting a wide spectrum of chemotherapeu-
tics back into the extracellular space, thereby reducing the
effective intracellular concentration of cytotoxic drugs. Cancer
cells had coopted and enhanced an endogenous chemical par-
tition system to strengthen the cell-membrane chemical barrier
and confer drug resistance.
Cell-surface transporters are now known to be ubiquitous,
present across evolutionary time with relatively conserved
functions across species, and capable of influx and/or efflux
transport of numerous drugs. P-glycoprotein (abbreviated
P-gp, and also termed ABCB1 or the multidrug resistance
transporter, MDR1) was the first xenobiotic transporter dis-
covered in multidrug-resistant cancer cells. P-gp also functions
in a wide range of normal tissues such as the gastrointestinal
tract, renal proximal tubule cells, the canalicular surfaces of
hepatocytes, the capillary endothelial cells of testes, and the
blood–brain barrier. Transporters may influence drug absorp-
tion, distribution, tissue penetration, metabolism, and excre-
tion. There is also the potential for significant drug
interactions.
How these competing transporter processes are regulated,
coordinated, and potentially circumvented or controlled will
require a better understanding of drug–transporter inter-
actions at cellular interfaces and methods for analyzing net
localized pharmacokinetic disposition of drugs. For a drug to
be clinically effective, it must possess high target specificity,
favorable organismal pharmacokinetics, and complementary
properties for highly localized chemical partition processes at
the cellular level. Drugs themselves can act to modulate xeno-
botic transport, and any new pharmaceutical is a potential
effector of chemical partition.
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