DRUG DISPOSITION Clin Pharmacokinet 2003; 42 (1): 59-98

0312-5963/03/0001-0059/$30.00/0

© Adis International Limited. All rights reserved.

Role of P-Glycoprotein
in Pharmacokinetics
Clinical Implications
Jiunn H. Lin and Masayo Yamazaki
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania, USA

Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
1. Structure and Mechanism of Drug-Transporting P-Glycoprotein . . . . . . . . . . . . . . . . . . . . 61
1.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
1.2 ATP- and Substrate-Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
1.3 Substrate Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2. Polymorphisms of P-Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3. In Vitro/In Vivo Extrapolation and Species Differences . . . . . . . . . . . . . . . . . . . . . . . . . 66
4. Role of P-Glycoprotein in Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.1 Drug Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.1.1 Distribution of Intestinal P-Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.1.2 Interindividual Variability of Intestinal P-Glycoprotein . . . . . . . . . . . . . . . . . . . . 70
4.1.3 Evidence of Intestinal P-Glycoprotein Involvement in Drug Absorption . . . . . . . . . . 71
4.1.4 Saturable Efflux Transport by Intestinal P-Glycoprotein . . . . . . . . . . . . . . . . . . . 72
4.2 Drug Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.2.1 Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.2.2 Placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.3 Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.4 Drug Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.4.1 Biliary Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.4.2 Renal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5. P-Glycoprotein-Mediated Drug-Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.1 P-Glycoprotein Inhibition Does Not Follow Simple Kinetics . . . . . . . . . . . . . . . . . . . . 84
5.2 Drug Interactions Caused by P-Glycoprotein Inhibition . . . . . . . . . . . . . . . . . . . . . . 86
5.3 P-Glycoprotein Induction is a Complex Process . . . . . . . . . . . . . . . . . . . . . . . . . . 88
5.4 Drug Interactions Caused by P-Glycoprotein Induction . . . . . . . . . . . . . . . . . . . . . . 90
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Abstract P-glycoprotein, the most extensively studied ATP-binding cassette (ABC)

transporter, functions as a biological barrier by extruding toxins and xenobiotics
out of cells. In vitro and in vivo studies have demonstrated that P-glycoprotein
plays a significant role in drug absorption and disposition. Because of its localisa-
tion, P-glycoprotein appears to have a greater impact on limiting cellular uptake
of drugs from blood circulation into brain and from intestinal lumen into epithelial
cells than on enhancing the excretion of drugs out of hepatocytes and renal tubules
into the adjacent luminal space. However, the relative contribution of intestinal
60 Lin & Yamazaki

P-glycoprotein to overall drug absorption is unlikely to be quantitatively impor-

tant unless a very small oral dose is given, or the dissolution and diffusion rates
of the drug are very slow. This is because P-glycoprotein transport activity be-
comes saturated by high concentrations of drug in the intestinal lumen.
Because of its importance in pharmacokinetics, P-glycoprotein transport
screening has been incorporated into the drug discovery process, aided by the
availability of transgenic mdr knockout mice and in vitro cell systems. When
applying in vitro and in vivo screening models to study P-glycoprotein function,
there are two fundamental questions: (i) can in vitro data be accurately extrapo-
lated to the in vivo situation; and (ii) can animal data be directly scaled up to
humans? Current information from our laboratory suggests that in vivo P-glyco-
protein activity for a given drug can be extrapolated reasonably well from in vitro
data. On the other hand, there are significant species differences in P-glycoprotein
transport activity between humans and animals, and the species differences ap-
pear to be substrate-dependent.
Inhibition and induction of P-glycoprotein have been reported as the causes
of drug-drug interactions. The potential risk of P-glycoprotein-mediated drug
interactions may be greatly underestimated if only plasma concentration is mon-
itored. From animal studies, it is clear that P-glycoprotein inhibition always has
a much greater impact on tissue distribution, particularly with regard to the brain,
than on plasma concentrations. Therefore, the potential risk of P-glycoprotein-
mediated drug interactions should be assessed carefully. Because of overlapping
substrate specificity between cytochrome P450 (CYP) 3A4 and P-glycoprotein,
and because of similarities in P-glycoprotein and CYP3A4 inhibitors and induc-
ers, many drug interactions involve both P-glycoprotein and CYP3A4. Unless
the relative contribution of P-glycoprotein and CYP3A4 to drug interactions can
be quantitatively estimated, care should be taken when exploring the underlying
mechanism of such interactions.

P-glycoprotein was first identified by Juliano
and Ling as a surface phosphoglycoprotein ex-
pressed in drug-resistant Chinese hamster ovary
cells.[1] This discovery led to the finding that P-
glycoprotein is an energy-dependent efflux trans-
porter driven by ATP hydrolysis. In humans, two
members of the P-glycoprotein gene family (MDR1
and MDR3) exist, while three members of this
family (mdr1a, mdr1b and mdr2) are found in
mice.[2,3] The P-glycoprotein encoded by the hu-
man MDR1 and mouse mdr1a/1b genes functions
as a drug efflux transporter, whereas human MDR3
P-glycoprotein and mouse mdr2 P-glycoprotein
are believed to be functional in phospholipid trans-
port.[4,5] However, the involvement of human
MDR3 P-glycoprotein in drug transport has been
recently reported. An increased directional trans-
port of digoxin, paclitaxel and vinblastine across
polarised monolayers of MDR3-transfected cells
has recently been reported by Smith et al.[6] These
results suggest that MDR3 P-glycoprotein is also
able to transport a range of drugs in addition to
phospholipids.
In addition to the expression in tumour cells,
human P-glycoprotein is also highly expressed in
normal tissues. This transporter is localised on the
canalicular surface of hepatocytes in liver, the api-
cal surface of epithelial cells of proximal tubules
in kidneys, columnar epithelial cells of intestine,
epithelial cells of placenta, and the luminal sur-
face of capillary endothelial cells in brain in hu-
mans.[7,8] The anatomical localisation of P-glyco-

© Adis International Limited. All rights reserved. Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
protein expression suggests that the efflux trans-
porter can functionally protect the body against
toxic xenobiotics by excreting these compounds
into bile, urine and the intestinal lumen, and by
preventing their accumulation in brain. Because of
its localisation, it is believed that P-glycoprotein
may play a significant role in the processes of ab-
sorption, distribution, metabolism and excretion
of drugs in humans and animals. Indeed, the role
of P-glycoprotein in drug absorption and disposi-
tion has been demonstrated in vivo using mdr1a
and mdr1a/1b knockout mice.[9,10] A lack of either
one of the two murine P-glycoproteins (mdr1a or
mdr1b) results in significant changes in drug ab-
sorption and disposition.
The purpose of this review is to briefly summa-
rise the current knowledge regarding the struc-
ture and mechanism of drug-transporting P-glyco-
protein and its role in drug absorption, distribution,
metabolism and excretion. In addition, the poten-
tial for P-glycoprotein-mediated drug-drug inter-
actions and its clinical implications will be dis-
cussed.
1. Structure and Mechanism of
Drug-Transporting P-Glycoprotein
1.1 Structure
Since the identification of P-glycoprotein, ma-
jor efforts have been made to elucidate the struc-
ture of the protein to gain better insight into the
mechanism of its action. The cloning of genes and
structure-function analysis of the protein by ge-
netic and biochemical studies have contributed to
a better understanding of the transporter. P-glyco-
protein is composed of two homologous and sym-
metrical halves (cassettes), each of which contains
six transmembrane domains that are separated by
an intracellular flexible linker polypeptide loop
with an ATP-binding motif.[2] Interestingly, the
two halves of human P-glycoprotein are not iden-
tical, and of the amino acids aligned, only 43% are
identical,[11] suggesting that the molecules of the
two halves might have either evolved indepen-
© Adis International Limited. All rights reserved.
61
dently or have undergone major intron movement
after a duplication event.[2]
Site-directed mutagenesis and antibody map-
ping studies suggest that the two cassettes of hu-
man P-glycoprotein interact cooperatively to form
a single functional unit.[12,13] The direct evidence
that supports the hypothesis of a single functional
unit is from the study that co-expressed each cas-
sette of human P-glycoprotein. The cDNA coding
for human P-glycoprotein was divided in half and
subcloned into separate plasmids in order to ex-
press each half as a separate polypeptide and to
characterise its contribution to function. No drug-
stimulative ATPase activity was observed when
Sf9 cells were transfected separately with cDNA
coding for each cassette.[12] The hypothesis of the
single functional unit is further supported by the
mutation studies carried out by Takada et al.[14] In
this study, the key lysine and cysteine residues
in the Walker A motifs of ATP-binding domains
were substituted by methionine and alanine, re-
spectively. The results of this study clearly dem-
onstrate that if one ATP-binding domain is not
functional, there is no ATP hydrolysis even when
ATP binds to the other ATP-binding domain. In
addition to the ATP-binding domains, the intracel-
lular flexible linker loops also play a key role in
ATPase and transport activity. Deletion of the cen-
tral core of the intracellular flexible linker region
of human P-glycoprotein resulted in a protein
without functional ATPase and transport activ-
ity.[15] Collectively, these data strongly suggest
that the two cassettes of P-glycoprotein interact as
a single transporter and that the flexible linker re-
gion is important for the proper interaction of the
two cassettes.
Several mechanistic models have been pro-
posed to describe the mechanism for drug transport
activity. The initial mechanistic model hypothes-
ises that, similar to ion channel proteins, hydro-
phobic membrane-spanning regions and hydro-
philic elements of P-glycoprotein form an aqueous
transmembrane pore through which drugs are
transported from the cytosol to the extracellular
media.[16] However, another model suggests that
Clin Pharmacokinet 2003; 42 (1)
62
P-glycoprotein might extrude drugs directly from
the cell membrane even before they enter the cyto-
plasm. This second model is supported by a study
of fura-2 acetoxymethyl ester in NIH-3T3 mouse
fibroblasts.[17] Fura-2 acetoxymethyl ester, a fluo-
rescent indicator, is hydrophobic and actively ex-
truded by P-glycoprotein, whereas the hydrophilic
free acid form of the indicator, to which the ester
is rapidly hydrolysed once in the cellular cyto-
plasm, is not exported by P-glycoprotein. The
intracellular trapping of Fura-2 free acid was re-
markably reduced in P-glycoprotein-expressing
NIH-3T3 mouse fibroblasts as compared with that
in the control fibroblasts (no P-glycoprotein ex-
pression) by a factor of 10. Addition of verapamil
did not alter the intracellular concentration of
Fura-2 free acid in control cells, whereas it in-
creased the amount of trapped Fura-2 free acid in
the P-glycoprotein-expressing cells up to the level
found in the control cells. From these results, the
investigators concluded that hydrophobic mole-
cules of Fura-2 acetoxymethyl ester interacted
with the P-glycoprotein in the cell membrane be-
fore entering the cytosol, and hence cytoplasmic
esterases had no chance to ‘see’ Fura-2 acetoxy-
methyl ester. However, their conclusion is valid only
if the rate of P-glycoprotein transport is the rate-
limiting step in trapping of Fura-2 free acid, i.e. the
rate of ester hydrolysis is much faster than the rate
of transport.
More convincing evidence that supports the
concept of the interaction of substrates with P-
glycoprotein in the lipid membrane of cells came
from the study by Shapiro and Ling.[18] These in-
vestigators measured the kinetics of Hoechst
33 342 in P-glycoprotein-enriched plasma mem-
brane vesicles from Chinese hamster ovary cells.
Hoechst 33 342 is fluorescent only when bound to
the membrane, but not when in the aqueous me-
dium. Therefore, the movement of Hoechst 33 342
in and out of the membrane can be directly moni-
tored by the fluorescence intensity. Using the flu-
orometric assay, the results revealed that the initial
rate of transport was directly proportional to the
amount of dye in the lipid phase, but not to the
© Adis International Limited. All rights reserved.
Lin & Yamazaki
concentration in the aqueous phase, suggesting
that P-glycoprotein extruded Hoechst 33342 from
the lipid membrane before it entered the cytosol.
With some modifications of the second model,
a recent and more favoured model proposes that
P-glycoprotein intercepts lipophilic drugs as they
move through the lipid membrane and flips the
drugs from the inner leaflet to the outer leaflet and
into the extracellular medium.[19] This model is
consistent with the notion that the lipophilicity of
a drug is an important determinant in its interaction
with P-glycoprotein.
Recently, Rosenberg et al.[20] used high-resolu-
tion electron microscopy and image analysis to ob-
tain the first three-dimensional architecture of a
P-glycoprotein purified from Chinese hamster
ovary CHrB30 cells that retained the ability to bind
substrates and hydrolyse ATP. When viewed from
above the membrane plane, the P-glycoprotein is
toroidal, with six-fold symmetry and a diameter of
~10nm. There is a large central pore of about 5nm
in diameter, which is closed on the cytoplasmic
surface of the plasma membrane forming an aque-
ous chamber within the membrane. Overall, the ob-
served microscopic image appears to be consistent
with the proposed models.
Although in recent years there has been a great
advancement in our understanding of the structure
of P-glycoprotein through mutational and bio-
chemical studies, the precise molecular mecha-
nism of drug transport by P-glycoprotein is still not
fully understood.
1.2 ATP- and Substrate-Binding Sites
There are two ATP-binding domains of P-gly-
coprotein, located in the cytosol side. Each ATP-
binding domain contains three regions: Walker A
and B and Signature C motifs.[2] The sequences of
amino acid residues for the first ATP-binding do-
main are: from 427–435 for Walker A motif, from
531–542 for Walker B motif, and from 551–556
for Signature C motif. The corresponding amino
acid residues for the second ATP-binding domain
are from 1070–1078, from 1176–1182 and from
1196–1201, respectively. Recently, Hung et al.[21]
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
reported that a highly conserved Lys residue
within the Walker A motif of histidine permease,
an ABC transporter, is directly involved with the
binding of ATP, and a highly conserved Asp resi-
due within the Walker B motif serves to bind the
Mg2+ ion. Mutations in either one of these residues
result in nonfunctional activity of histidine perme-
ase. These results of histidine permease suggest
that the ATP-binding sites may also be restricted
to the Walker A motifs of P-glycoprotein.
It is clear now that ATP binding and subsequent
hydrolysis are essential for drug transport.[22]
Studies with photoactive analogues of ATP have
shown that these analogues bind to the ATP-bind-
ing domains.[23] Based on the data from vanadate
trapping studies, Senior and Gadsby[24] have
proposed a so-called alternate ATP-binding site
model, which explains that although both ATP-
binding sites are capable of binding ATP, only one
site participates in the catalysis at a given time, and
conformation of this catalytic site precludes the
other site from hydrolysing ATP. The stoichiome-
try of ATP hydrolysis to drug transport has been
studied, and the data indicate that, depending on
substrate, 0.6–3 molecules of ATP are hydrolysed
for every molecule of drug transported out the
cell.[25,26] The reason for the substrate-dependent
ATP stoichiometry is still unknown.
Unlike the ATP-binding sites that are restricted
to the Walker A motifs of ATP-binding domains,
many substrate-binding sites have been identified
throughout the transmembrane domains (TM) of
P-glycoprotein. Two substrate-binding sites were
found in TM6 and TM12 by using photoaffinity
probes.[27,28] After complete digestion of the P-
glycoprotein with trypsin, two major photo-
labelled fragments (5 and 4kD) were mapped by
immunological analysis. These two fragments are
located within, or immediately next to, the last
transmembrane domain of each cassette, TM6 and
TM12 of P-glycoprotein, respectively. The 5kD
fragment includes amino acid residues from 311–
456, extending a few residues beyond the Walker
A motif of the first ATP-binding site. In contrast,
the 4kD fragment includes residues from 979–
© Adis International Limited. All rights reserved.
63
1048, but not including the Walker A motif of the
second ATP-binding site. In addition to the regions
of TM6 and TM12, a region that includes TM7 and
TM8 was also reported to be photolabelled specif-
ically by an analogue of paclitaxel.[29]
Consistent with the studies of photoaffinity
probes, studies of many mutant P-glycoprotein
molecules suggest that the major drug-binding
sites reside in or near TM6 and TM12.[22] How-
ever, mutational studies also suggest that amino
acid substitutions that affect substrate specificity
are scattered throughout P-glycoprotein, including
TM1, TM4, TM6, TM10, TM11 and TM12.[30] Re-
cent studies by Taguchi et al.[31,32] suggested that
three amino acids (His61, Gly64 and Leu65) in
TM1 are involved in the formation of a binding
pocket that plays a key role in determining the suit-
able substrate sizes for P-glycoprotein. For exam-
ple, substitution of His61 by an amino acid with a
short side-chain increased resistance to vinblastine
(large molecular size; molecular weight 811),
whereas substitution of an amino acid with a long
side chain increased resistance to colchicine (small
molecular size; molecular weight 399).[31,32]
In addition to the transmembrane domains, mu-
tational analyses have suggested that the intracel-
lular linker loops of P-glycoprotein are also impor-
tant for substrate recognition.[33,34] The systematic
mutagenesis of 20 Gly residues in the cytoplasmic
loops revealed that Gly141 and Gly187 between
TM2 and TM3, Gly288 between TM4 and TM5,
and Gly812 and Gly830 between TM8 and TM9
are important in determining substrate specific-
ity.[33]
In summary, these data suggest that drug-bind-
ing sites are scattered throughout the P-glycopro-
tein molecule, including the transmembrane
domains, intracellular loops and even the ATP-
binding domains.
1.3 Substrate Recognition
One of the most intriguing aspects of P-glyco-
protein is that a single integral membrane protein
can recognise and transport so many drugs with a
wide array of chemical structures, ranging from a
Clin Pharmacokinet 2003; 42 (1)
64
molecular weight of 250 (cimetidine) to 1202
(cyclosporin).[35,36] Although most of the drugs
transported by P-glycoprotein are basic or un-
charged, there are many exceptions. The only com-
mon feature is that most of the P-glycoprotein sub-
strates are hydrophobic in nature, suggesting that
partitioning of the lipid membrane of cells is the
first step for the interaction of a substrate with the
active sites of P-glycoprotein.
Various efforts have been made to establish the
structure-activity relationship for P-glycoprotein
substrates. Originally, it was believed that a basic
nitrogen atom was a prerequisite for the interaction
of substrate and P-glycoprotein. For example,
studies of colchicine and its analogues suggested
that the nitrogen atom of the acetamido group at
the 7 position was essential for P-glycoprotein rec-
ognition.[37] However, compounds lacking a nitro-
gen atom, such as cortisol, aldosterone and dexa-
methasone, are recognised to be good substrates
for P-glycoprotein.[38] Studies by Ecker et al.[39,40]
have shown that both the lipophilicity and number
of hydrogen bonds of compounds are probably the
most important parameters in determining the af-
finity of compounds to P-glycoprotein. The higher
the lipophilicity or the larger the number of hydro-
gen bonds, the better the substrates are for the P-
glycoprotein transporter.
Similarly, Seelig and Landwojtowicz[41] have
also suggested that both lipophilicity and number
of hydrogen bonds are important determinants for
substrates and P-glycoprotein interaction. They
concluded that partitioning of the lipid membrane
is the rate-limiting step for the interaction of a sub-
strate with P-glycoprotein. Furthermore, they sug-
gested that dissociation rate of the P-glycoprotein-
substrate complex is controlled by the number of
hydrogen bonds. Based on structural analysis of
100 P-glycoprotein substrates, Seelig[42] further
proposed that in addition to lipophilicity and num-
ber of hydrogen bonds, some essential structural
elements of substrates are required for an interac-
tion with P-glycoprotein. The recognition ele-
ments of substrates are formed by two or three elec-
tron donor (hydrogen–bonding acceptor) groups
© Adis International Limited. All rights reserved.
Lin & Yamazaki
with a fixed spatial separation: 2.5 ± 0.3Å or 4.6 ±
0.6Å. Additionally, the surface area and amphiphi-
lic characteristic of the substrate also appear to
play a significant role in determining its P-glyco-
protein activity.[43]
Although a wealth of information on the rela-
tionship between physicochemical properties of
substrates and P-glycoprotein activity has been
generated in recent years, a clear structure-activity
relationship for predicting P-glycoprotein sub-
strates still cannot be established.[44] The lack of
clear structure-activity relationship for substrate
recognition is attributed mainly to the structural
complexity of P-glycoprotein.
2. Polymorphisms of P-Glycoprotein
Humans are not necessarily created equal in
terms of biological make-up. Because of evolu-
tionary and environmental factors, there is a re-
markable degree of genetic variability built into the
population. Like many cytochrome P450 (CYP)
isoenzymes,[45] genetic polymorphisms of P-gly-
coprotein in animals and humans have been re-
ported. Thus, the genetic polymorphisms of P-gly-
coprotein may also represent a major source of
individual variability in the potential toxicity and
pharmacokinetics of drugs.
The genetic polymorphism of P-glycoprotein
was first reported in CF-1 mice by Lankas and
Umbenhauer at Merck in 1997.[46,47] A subpopula-
tion of CF-1 mice, approximately 25%, was very
sensitive to neurotoxicity following exposure to
avermectin, an antiparasitic agent. The 50% lethal
dose (LD50) values were 0.3 and 120 mg/kg for the
sensitive and insensitive groups, respectively. Sub-
sequently, it is now known that this avermectin-
induced neurotoxicity is the result of a deficiency
in mdr1a P-glycoprotein that normally contributes
to a functional blood-brain barrier (BBB). In nor-
mal (wild-type) CF-1 mice the abundant P-glyco-
protein in the BBB pumps avermectin efficiently
out of the brain, but in mdr1a-deficient mice, this
protective function is absent, resulting in a more
than 80-fold higher accumulation of avermectin in
the brain. The P-glycoprotein-deficient CF-1 mice
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
are also at higher risk of birth defects caused by
avermectin.[48] When female CF-1 mice were
treated with avermectin during pregnancy, fetuses
deficient in P-glycoprotein (–/–) were 100% sus-
ceptible to cleft palate, while their heterozygote
litters (+/-) were less sensitive. The homozygous
fetuses (+/+) with abundant P-glycoprotein were
totally insensitive at the dose tested. The birth de-
fect is attributed to fetal exposure to avermectin.
The above cases of neurotoxicity and teratogenesis
demonstrate the importance of P-glycoprotein in
protecting the brain and fetus against toxic xeno-
biotics.
The molecular basis of mdr1a deficiency in
CF-1 mice was further studied at the RNA and DNA
level, using reverse transcription-polymerase chain
reaction (RT-PCR) and long PCR with oligonucle-
otides specific for mdr1a. Sequencing of the intron
between exon 22 and 23 in P-glycoprotein-defi-
cient CF-1 mice revealed an insertion of approxi-
mately 8.35kb of DNA at the exon 23 intron-exon
junction. This insertion results in the aberrant
splicing of the mRNA and loss of exon 23 during
RNA processing.[49] A subpopulation of collie dogs
is also known to be very sensitive to avermectin
and it has been speculated that the avermectin-
induced neurotoxicity in the dogs is due to P-gly-
coprotein genetic polymorphisms.[50,51] Recently,
RT-PCR studies revealed a deletion mutation of
the MDR1 gene in avermectin-sensitive collie
dogs.[52] The 4-bp deletion results in a frame shift,
generating several stop codons that prematurely
terminate P-glycoprotein synthesis.
Genetic polymorphisms of human P-glycopro-
tein were first reported from in vitro studies with
cancer cells.[53,54] However, the kinetic impact of
polymorphism on P-glycoprotein function in vivo
remained unclear until the recent report by Hoff-
meyer et al.,[55] who identified a single nucleotide
polymorphism (SNP) in exon 26 (C3435T) of
MDR1. There was a significant correlation of the
SNP and the functional activity of P-glycoprotein.
The homozygous T-allele (mutant) is associated
with more than 2-fold lower intestinal P-glycopro-
tein expression levels compared with homozygous
© Adis International Limited. All rights reserved.
65
C-allele (wild type). Individuals carrying homo-
zygous T-allele showed a lower duodenal P-glyco-
protein level and consequently higher peak plasma
concentrations (Cmax) of digoxin, a substrate of P-
glycoprotein, probably through an increase in dig-
oxin absorption as a result of decreased intestinal
P-glycoprotein. This was the first example that in-
dicated that P-glycoprotein polymorphism can di-
rectly affect drug absorption in humans. However,
a recent study by Sakaeda et al.[56] suggests that
there are no statistically significant differences in
the Cmax and 24-hour area under the curve (AUC24)
of digoxin between individuals carrying wild-type
C-allele or homozygous mutant T-allele. Simi-
larly, conflicting results of the effect of C3534T
polymorphism on the absorption of fexofenadine
have also been reported. Kim et al.[57] showed that
individuals harbouring homozygous T-allele mu-
tation tended to have lower plasma AUC24 of fexo-
fenadine. In contrast, Drescher et al.[58] claimed
that there were no significant differences between
T/T and C/T genotypes. Additionally, kinetic stud-
ies in healthy subjects and renal transplant patients
suggested that C3534T polymorphism had little
effect on the absorption of cyclosporin.[59,60]
Therefore, the impact of C3534T polymorphism
on drug absorption is still not clear.
A strong association between C3435T allele
and G2677A/G2677T alleles was observed when
MDR1 polymorphisms were investigated in 100
placentas from Japanese women.[61] Of 65 samples
with a C3435T allele, 61 (93.8%) also had a mutant
G2677T/G2677A allele. Interestingly, there ap-
peared to be a correlation between the level of P-
glycoprotein expression and G2677T/G2677A in
exon 21, and between the P-glycoprotein level and
C3435T in exon 26. The placental P-glycoprotein
expression levels for wild-type, heterozygotes and
homozygotes of G2677A/G2677T mutant allele
were 2.44, 1.97, and 1.45 (arbitrary units), respec-
tively, while the corresponding mean P-glycopro-
tein expression levels for the C/C, C/T and T/T
genotypes at position 3435 were 2.11, 1.84, and
1.51 (arbitrary units). The frequency in exon 21
occurred in 58% of the sample as heterozygosity
Clin Pharmacokinet 2003; 42 (1)
66
and 28% as homozygosity for the mutant allele,
while the frequency in exon 26 occurred in 46% of
the sample as heterozygosity and 19% as homo-
zygosity for the mutant allele. Given the high
frequency for the mutant allele G2677A/G2677T
in this study from 100 placentas, it is quite puzzling
why no G2677A/G2677T mutant allele was ob-
served from a sample population of 188 in the
aforementioned study by Hoffmeyer et al.,[55] even
though a total of 15 different SNPs, including
C3435T, were identified in their study. Recently,
Hoffmeyer’s group detected both C3435T and
G2677A/G2677T polymorphisms with high fre-
quencies in a relatively larger sample population
of 461.[62]
Similar to the ethnic variation in the polymor-
phism of CYP,[63] interethnic differences in MDR1
polymorphisms are observed. Using a polymerase
chain reaction-restriction fragment length poly-
morphism assay, 1280 subjects from 10 different
ethnic groups were evaluated for the C3435T poly-
morphism in exon 26.[64] Marked differences in
genotype and allele frequency were observed be-
tween the African and the Caucasian/Asian popu-
lations. The Ghanaian, Kenyan, African-American
and Sudanese populations have frequencies of 83,
83, 84 and 73%, respectively, for the C/C (wild
type) allele. In contrast, the British Caucasian, Por-
tuguese, Southwest Asian, Chinese, Filipino and
Saudi populations have lower frequencies of the
C/C allele compared with the African groups, rang-
ing from 34–55%. Although the level of P-glyco-
protein expression was not determined in this
study, these results suggest that P-glycoprotein
expression in African populations may be higher
than that in the Caucasian/Asian populations.
It is interesting to note that ivermectin, a potent
anthelmintic agent used for the prevention and
treatment of river blindness (onchocerciasis) in
Africa, is a very safe drug, even though this drug
causes neurotoxicity in animals with low P-glyco-
protein expression. To date, of more than 20 mil-
lion patients in Africa who had been treated with
ivermectin, not a single case of neurotoxicity has
been reported. The lack of neurotoxicity might be
© Adis International Limited. All rights reserved.
Lin & Yamazaki
attributed to the high P-glycoprotein expression in
the African population. On the other hand, the high
expression of P-glycoprotein might contribute to
the high incidence of drug resistance to cancer
treatment in individuals of African origin.[65]
In summary, the MDR1 gene is highly polymor-
phic. To date, at least 16 SNPs have been identified
in the MDR1 gene, and it is anticipated that more
SNPs will be found in the future (table I). Although
some variants lead to amino acid changes, most of
the detected polymorphisms are intronic or silent.
So far, only three SNPs (T–129C in exon 1b,
G2677A/G2677T in exon 21 and C3435T in exon
26) have been demonstrated to be associated with
variation in P-glycoprotein expression. Undoubt-
edly, variation in P-glycoprotein expression result-
ing from MDR1 polymorphism is one of the major
sources contributing to interindividual variability
in drug absorption and disposition.
3. In Vitro/In Vivo Extrapolation
and Species Differences
As will be discussed in section 4, P-glycopro-
tein plays an important role in absorption, distribu-
tion, metabolism and excretion of many drugs. Be-
cause of the importance of P-glycoprotein in
pharmacokinetics, many pharmaceutical compa-
nies have begun to incorporate P-glycoprotein
drug transport screening into the drug discovery
process. The availability of transgenic mdr knock-
out mice and in vitro cell systems has paved the
way for studies of the role of P-glycoprotein in
drug absorption and disposition. When applying in
vitro and in vivo screening models to study P-gly-
coprotein function, there are two fundamental
questions that industrial drug metabolism scien-
tists must confront daily: (i) can in vitro data be
accurately extrapolated to the in vivo situation, and
(ii) can animal data be directly scaled to humans?
To test whether in vitro P-glycoprotein activity
of drugs can be extrapolated to the in vivo situation,
we have conducted a study to measure in vitro and
in vivo P-glycoprotein activity of ten model com-
pounds.[67] The in vitro P-glycoprotein activity of
these compounds was determined using mdr1a-
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein 67

Table I. Single nucleotide polymorphisms (SNPs) in the MDR1 gene

SNPa Mutation Mutant allele frequency (%)
Hoffmeyer et al.[55] Ito et al.[66] Cascorbi et al.[62] Tanabe et al.[61] Mickley et al.[54]
5′-flanking/-41 A/G 7.3 9.4
1a/–145 C/G 1.0 1.0
1b/-129 T/C 5.9 8.3
2/–1 G/A 5.6 9.0
2/61 A/G 9.3 11.2
5/–25 G/T 16.5
5/–35 G/C 0.6
5/307 T/C 0.6 0
6/+139 C/T 40.6 37.2
6/+145 C/T 1.2
11/1199 G/A 6.5 5.5
12/1236 C/T 37.8 38.5 (T>C) 41.0 35.4 (T>C)
12/+44 C/T 5.9 4.9
17/–76 T/A 45.3 46.2
17/+137 A/G 0.6
21/2677 G 56.5 36.5 56.4
21/2677 G/T 0 41.6 41.7 43.6
21/2677 G/A 0 1.9 21.8 0
24/2956 A/G 0
24/2995 G/A 6.7
26/3220 A/C 0.2
26/3396 C/T 0.3
26/3435 C/T 48.1 53.9 49.0
28/4030 G/C 0
28/4036 A/G 25.0
a Exon/position.

transfected LLC-PK1 cells, and expressed as the
ratio of basolateral-to-apical transport to apical-to-
basolateral transport (B-to-A/A-to-B). On the
other hand, the in vivo P-glycoprotein activity was
determined using CF-1 mdr1a (+/+) and mdr1a
(–/–) mice. Following intravenous administration,
the drug concentration in brain and plasma was
measured every 15 minutes up to 60 minutes. The
ratio of brain AUC in mdr1a (–/–) mice to that in
mdr1a (+/+) mice was used as an index of in vivo
P-glycoprotein activity. There was a strong posi-
tive correlation (r2 = 0.93, p < 0.001) when the in
vitro B-to-A/A-to-B ratio was plotted against the
in vivo brain AUC ratio for these ten com-
pounds.[67] A strong correlation was also observed
when the brain concentration was normalised by
plasma concentration. These results suggest that in
vivo P-glycoprotein activity of a given drug can be
reasonably well extrapolated from in vitro data. In
our laboratory, the in vitro and in vivo correlation
was further evaluated with an additional 20 com-
pounds, and a strong correlation between in vitro
B-to-A/A-to-B ratio and the in vivo brain AUC ra-
tio was observed again (unpublished data).
The most obvious species differences in the
drug-transporting P-glycoprotein between mice
and humans is that there are two members of drug-
transporting P-glycoprotein (mdr1a and mdr1b)
for mice, and just one in humans (MDR1).[2] Al-
though the kinetics and substrate specificities are
generally similar between mouse mdr1a and mdr1b
P-glycoprotein,[68-70] species differences in func-
tional activity between human MDR1 P-glycopro-
tein and these two mouse P-glycoproteins have

© Adis International Limited. All rights reserved. Clin Pharmacokinet 2003; 42 (1)
68
been reported by Tang-Wai et al.[71] Stably trans-
fected cells were developed that expressed similar
amounts of P-glycoprotein encoded by mdr1a,
mdr1b and MDR1 genes. The three transfected cell
lines were tested for their cellular resistance (cell
survival) to dactinomycin, doxorubicin, colchicine
and vinblastine. The 50% inhibitory concentra-
tions (IC50 values) for all of the drugs were much
lower (3- to 20-fold) in MDR1 transfected cells
than in mdr1a cells.[71] Likewise, with the excep-
tion of dactinomycin, the IC50 values were lower
in human MDR1 cells than in mdr1b cells. These
results suggest that there are species differences in
functional capacity between human MDR1 P-gly-
coprotein and murine P-glycoproteins.
In our laboratory, more than 640 compounds
have been evaluated for their P-glycoprotein activ-
ity in mouse mdr1a and human MDR1 transfected
LLC-PK1 cells (L-mdr1a and L-MDR1, respec-
tively). As shown in figure 1, there is a poor corre-
lation between mdr1a and MDR1 transcellular
transport activity as measured by B-to-A/A-to-B
transport ratio (r2 = 0.44). Approximately 35% of
the compounds exhibited substantial differences
(>3-fold) between mdr1a and MDR1 gene trans-
fected cells. The observed species difference in the
P-glycoprotein transport was not due to the differ-
ences in the level of the P-glycoprotein expression.
Western blotting data revealed that the P-glyco-
protein level was similar in both mdr1a and
MDR1transfected cells.
Species differences in transport activity were
also observed between human P-glycoprotein and
P-glycoprotein of other animal species. We have
recently compared the P-glycoprotein activity of
marker P-glycoprotein substrates using cell lines
expressing human, mouse, rat and canine P-glyco-
protein. Again, significant species differences in
P-glycoprotein activity were found among these
animal species (unpublished data).
Although these in vitro studies strongly suggest
the possibility of species differences in P-glyco-
protein activity, there are still no in vivo data to
support the potential of species differences. As will
be discussed in section 4, the mdr1a/1b knockout
© Adis International Limited. All rights reserved.
Lin & Yamazaki
120
100
B-to-A/A-to-B in L-MDR1 (human)
80
60
40
20
0
0 20 40 60 80 100 120
B-to-A/A-to-B in L-mdr1a (mouse)
Fig. 1. Correlation of transcellular transport ratios for 642 struc-
turally diverse compounds in monolayers of LLC-PK1 cells
transfected with mouse mdr1a or human MDR1 (L-mdr1a and
L-MDR1, respectively). A-to-B = apical to basal; B-to-A = basal
to apical.
mouse provides a very useful model for the study
of the role of P-glycoprotein in pharmacokinetics
of drugs. However, because of the potential species
differences in P-glycoprotein activity, extrapola-
tion from knockout mice to humans should be car-
ried out with discretion.
4. Role of P-Glycoprotein
in Pharmacokinetics
Although the physiological function for P-gly-
coprotein is still not fully understood, the role of
this efflux transporter in pharmacokinetics is be-
coming increasingly appreciated. In humans, P-
glycoprotein is found on the apical surface of
columnar epithelial cells of small and large intes-
tines, the biliary canalicular membrane of hepato-
cytes, the apical surface of epithelial cells of the
proximal tubules of kidney, the apical surface of
epithelial cells of placenta and the apical surface
of endothelial cells in blood capillaries of the
brain.[7,8] Because of its strategic localisation, the
P-glycoprotein transporter functionally can limit
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
cellular uptake of drugs from the blood circulation
into the brain and placenta, and from the gastroin-
testinal lumen into the enterocyte. On the other
hand, this transporter can also enhance the elimi-
nation of drugs from hepatocytes, renal tubules
and intestinal epithelial cells into the adjacent lu-
minal space. Therefore, it is very important to dis-
tinguish the localisation of P-glycoprotein in cells
in relation to drug movement – either uptake of
drugs into cells or excretion of drugs out of cells.
As will be discussed later, there is a tendency for
P-glycoprotein to have a greater impact on drug
uptake than on drug excretion.
Perhaps the most important milestone in P-gly-
coprotein research was the development of mdr-
knockout mice. Since mdr knockout mice became
available, our understanding of the role of P-gly-
coprotein in pharmacokinetics has increased expo-
nentially. As discussed above, mice have two types
of drug-transporting P-glycoprotein (mdr1a and
mdr1b), which are expressed in a tissue-specific
manner.[72] For example, only mdr1a P-glycopro-
tein is expressed in the brain and intestine of mice,
while both mdr1a and mdr1b P-glycoprotein are
expressed in the liver and kidney. Interestingly,
mdr1a and mdr1b P-glycoprotein together appear
to cover the same tissues as the single human
MDR1 P-glycoprotein, suggesting that mdr1a and
mdr1b together fulfil the same function as the sin-
gle P-glycoprotein in humans. Because of tissue-
specific expression, it is expected that genetic dis-
ruption of the mdr1a gene would have a greater
impact on drug uptake into the brain and intestine
than drug excretion from the liver and kidney.
In addition, it should be noted that genetic dis-
ruption of one or both of the mdr genes might affect
the expression and function of other transporter
systems or even drug-metabolising enzyme sys-
tems in mice. For example, it is known that the
mdr1b gene is upregulated in mdr1a knockout
mice.[8] Recently, Schuetz et al.[73] have reported
that both the protein expression and catalytic ac-
tivity of CYP is significantly increased in the
mdr1a, mdr1b and mdr1a/1b knockout mice
housed in The Netherlands. However, for the ge-
© Adis International Limited. All rights reserved.
69
netically identical mdr1a and mdr1a/1b knockout
mice housed in the US, there were no significant
changes in their protein expression and catalytic
activity of CYP. Because of the possible existence
of unrecognised factors that are associated with the
genetic disruption, data derived from mdr1a single
knockout mice or from mdr1a/1b double knockout
mice have to be interpreted with caution.
4.1 Drug Absorption
There are many factors that influence the
bioavailability of drugs, which can be broadly
categorised as physicochemical and biological
factors.[74,75] The former comprise the intrinsic
properties of the drug, such as pKa, molecular size,
lipophilicity and solubility, and the latter include
gastric and intestinal transit time, lumen pH, mem-
brane permeability, mucosa blood flow rate and
first-pass metabolism. After oral administration,
drug absorption occurs predominantly within the
small intestine, because of its large surface area
provided by epithelial folding and the villous
structures of epithelial cells. During oral absorp-
tion, drugs can be transported by either the trans-
cellular or paracellular pathway across the epithe-
lial cells, or a combination of both. The relative
contribution of the transcellular pathway to overall
absorption is highly dependent on the lipophilicity
of drugs. In an in vitro study with Caco-2 cells, the
relative contribution of the transcellular pathway
was determined to be 25, 45, 85 and 99% for chlo-
rothiazide, furosemide, cimetidine and proprano-
lol, respectively. The values correlated fairly well
with the lipophilicity of the drugs, the logP values
of which were –0.2, –0.08, 0.4 and 3.6, respec-
tively.[76] Most orally administered drugs enter the
systemic circulation by passive transcellular dif-
fusion, because of their lipophilicity. Therefore,
the intestinal absorption of a drug is often pre-
dicted on the basis of its lipophilicity.
As noted earlier, the most common physico-
chemical property for P-glycoprotein substrates
identified so far is that they are mostly lipophilic,
which implies that lipophilic drugs are likely to be
P-glycoprotein substrates. Therefore, absorption
Clin Pharmacokinet 2003; 42 (1)
70
of drugs is further complicated by the existence of
P-glycoprotein efflux transporter, which is highly
expressed on the apical surface of epithelial cells.
In the intestinal lumen, drugs that are P-glycopro-
tein substrates will be absorbed and cross the epi-
thelial cell membrane by simple diffusion. Once
inside the cells, a fraction of the drug molecules
continues to diffuse along the concentration gradi-
ent into capillary blood. However, a portion of
drug molecules will be removed by the efflux P-
glycoprotein transporter out of cells back into the
lumen and another part of the drug molecules is
subject to intestinal metabolism. Consequently, the
net amount of drug absorbed into the mesenteric
blood circulation is the difference between the
amount absorbed by the influx process and the
summation of the amount extruded by efflux trans-
port together with the amount metabolised by en-
zymes.
4.1.1 Distribution of Intestinal P-Glycoprotein
The distribution of intestinal P-glycoprotein is
not uniform among cells along the epithelial villi.
Immunohistological studies with human jejunum
and colon using MRK16 antibody revealed that
high levels of P-glycoprotein were only observed
in the apical surface of columnar epithelial cells,
but not in crypt cells.[7] Unlike hepatocytes, which
regenerate only when untimely death occurs, intes-
tinal epithelial cells have a programmed, limited
life span. The villous epithelial cells are mature
and nondividing, whereas the crypt cells continue
to mature as they ascend toward the villus and are
extruded at its tip. The time required for migration
from the crypt base to the villous tip has been esti-
mated to be 2– 6 days.[77] Whether the programmed
life span and rapid migration will have impact on
the regulation of intestinal P-glycoprotein expres-
sion is an open question, and requires further in-
vestigation.
The distribution of P-glycoprotein is also not
uniform along the length of intestine. Fojo et al.[78]
have measured the content of MDR1 mRNA ex-
pression over the total length of human gastroin-
testinal tract. The levels of mRNA appear to in-
crease progressively from the stomach to the colon
© Adis International Limited. All rights reserved.
Lin & Yamazaki
with a low level in the stomach (5 arbitrary units),
an intermediate level in the jejunum (20 arbitrary
units) and a high level in the colon (30 arbitrary
units). The uneven distribution of intestinal P-gly-
coprotein is expected to have a significant impact
on the absorption of P-glycoprotein substrates. The
influence of uneven distribution of P-glycoprotein
in intestine was demonstrated in a clinical study
with cyclosporin.[79] Cyclosporin was given to ten
healthy volunteers at different parts of the gastro-
intestinal tract (stomach, jejunum and colon). The
oral AUC of cyclosporin was in the rank order
stomach > jejunum > colon. There was a negative
correlation between MDR1 mRNA expression and
oral AUC of cyclosporin. Uneven distribution of
P-glycoprotein has also been observed in rats. Us-
ing the rat intestinal loop technique, vinblastine,
a well-known P-glycoprotein substrate, was ab-
sorbed fairly well from ileal loops, ranging from
30–60% of the dose in 30 minutes, whereas absorp-
tion of vinblastine from the jejunal loop was almost
negligible, suggesting a high level of P-glycopro-
tein expression in rat jejunum.[80]
4.1.2 Interindividual Variability
of Intestinal P-Glycoprotein
Although interindividual variability in drug-
metabolising enzymes is well documented,[45] only
a few papers deal with the issue of interindividual
variability of P-glycoprotein. In a clinical study of
25 kidney transplant recipients, expression level of
intestinal P-glycoprotein was measured using im-
munoblotting.[81] Biopsy specimens were obtained
from the second portion of the duodenum of each
patient. Because P-glycoprotein is expressed ex-
clusively in mature epithelial cells in the villous tip
of intestinal mucosa, differences in the number of
total mature cells in individual biopsies might con-
tribute to the variability. To correct this practical
problem, the investigators used villin, a constitu-
tively expressed protein in mature epithelial cells,
as an internal standard. The results from this study
indicate that there is a significant interindividual
variability in the intestinal P-glycoprotein expres-
sion. More than 8-fold differences in the P-glyco-
protein expression were observed in a small popu-
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
lation of 25 patients; the intestinal P-glycoprotein
level of duodenal biopsies ranged from 31–263
(arbitrary units).
As discussed earlier, the MDR1 gene is highly
polymorphic. At least 16 SNPs have been identi-
fied (table I). Although complete P-glycoprotein
deficiency has not been reported for these poly-
morphisms, the SNP at 3435 in exon 26 does influ-
ence the expression level of intestinal P-glycopro-
tein. The mean values of intestinal P-glycoprotein
expression for the C/C homozygotes (wild type, n
= 6), C/T heterozygotes (n = 10), and T/T homo-
zygotes (n = 5) at position 3435 were 1275, 956
and 627 (arbitrary units), respectively.[55]
Interestingly, intraindividual variability in in-
testinal P-glycoprotein expression has also been
reported. A profound intraindividual variability in
intestinal P-glycoprotein expression (mRNA) was
observed in a young patient during tacrolimus ther-
apy after small bowel transplantation.[82] Both the
mRNA expression and plasma concentration of
tacrolimus were measured periodically during the
immunosuppressant therapy. In a period of 120
days, there was a 4-fold variation in MDR1 mRNA
expression level and a 2-fold variation in trough
plasma concentration of tacrolimus. The variation
in P-glycoprotein expression inversely related
fairly well to the variation of tacrolimus concen-
trations after oral administration.
Collectively, these results suggest that interin-
dividual and intraindividual variability in intesti-
nal P-glycoprotein expression may contribute to
variability of oral absorption of drugs that are P-
glycoprotein substrates.
4.1.3 Evidence of Intestinal P-Glycoprotein
Involvement in Drug Absorption
Evidence of the involvement of intestinal P-
glycoprotein in drug absorption was first demon-
strated in vitro with Caco-2 cells in which P-gly-
coprotein was highly expressed. Using Caco-2
cells, the B-to-A transport of vinblastine and
docetaxel was 10- and 20-fold, respectively,
greater than the A-to-B transport, and the A-to-B
transport was enhanced significantly in the pres-
ence of verapamil and MRK16 by blocking the
© Adis International Limited. All rights reserved.
71
P-glycoprotein efflux function.[83,84] Caco-2 cells
have also been used to study the intestinal transport
of cyclosporin.[85] Similarly, the B-to-A transport
of cyclosporin was much greater than the A-to-B
transport. The A-to-B cyclosporin transport in-
creased and the B-to-A transport decreased after
treatment with the P-glycoprotein inhibitors pro-
gesterone and chlorpromazine. Furthermore, stud-
ies with Caco-2 cells revealed that active B-to-A
transport of peptides was inhibited by verapamil,
suggesting the involvement of P-glycoprotein in
the absorption of peptides.[86] These results from
in vitro studies clearly suggest that P-glycoprotein
plays a significant role in drug absorption by lim-
iting drug transport from intestinal lumen.
Direct evidence for the role of intestinal P-gly-
coprotein in drug absorption was derived from in
vivo studies with mdr1a (–/–) knockout mice. The
oral absorption of paclitaxel was studied in mdr1a
(–/–) and mdr1a(+/+) mice.[87] The plasma AUC
of paclitaxel was 2- and 6-fold, respectively,
higher in mdr1a (–/–) mice than mdr1a (+/+) mice
after intravenous and oral drug administration. The
increased AUC of paclitaxel after intravenous ad-
ministration in mdr1a (–/–) mice reflected a de-
crease in elimination clearance, whereas the higher
AUC after oral administration in mdr1a (–/–) mice
resulted from a combination of a decrease in the
elimination clearance and an increase in the extent
of drug absorption from intestinal lumen. Based on
the AUC values after intravenous and oral admin-
istration, the bioavailability of paclitaxel was cal-
culated to be 11 and 35% for mdr1a (+/+) and
mdr1a (–/–) mice, respectively. From this study, it
is clear that intestinal P-glycoprotein does limit
drug absorption by extruding drugs from epithelial
cells back into the intestinal lumen. Further evi-
dence for the involvement of intestinal P-glyco-
protein in drug absorption in humans is provided
by the clinical study by Hoffmeyer et al.,[55] who
showed a negative correlation between duodenal
P-glycoprotein expression and plasma level of dig-
oxin.
The efflux function of intestinal P-glycoprotein
is further supported by the observations that a sig-
Clin Pharmacokinet 2003; 42 (1)
72
nificant amount of paclitaxel was excreted directly
from blood circulation into intestinal lumen after
intravenous administration. The effect of P-glyco-
protein on intestinal excretion can be experimen-
tally determined in mice after interruption of bile
flow by gallbladder cannulation. A fraction of 11%
of the dose was excreted into the intestinal lumen
within 90 minutes after intravenous administration
of paclitaxel to mdr1a (+/+) mice with a cannulated
gallbladder, but only 2.5% of the dose in the intes-
tinal lumen of mdr1a (–/–) mice.[87] Similarly, P-
glycoprotein-mediated intestinal excretion in mice
was also reported for digoxin. Approximately 16
and 2% of the digoxin dose, respectively, was ex-
creted into the intestinal lumen of mdr1a (+/+) and
mdr1a (–/–) mice with a cannulated gallbladder
within 90 min after intravenous administration.[88]
Although the phenomenon of intestinal excretion
of drugs has been known for more than two de-
cades,[89] involvement of P-glycoprotein in intesti-
nal excretion has only been recognised recently.
From a mechanistic point of view, intestinal excre-
tion should also be considered as an additional
pathway for the elimination clearance of P-glyco-
protein substrates.
Unlike the direct evidence obtained from mdr1a
knockout mice, the involvement of MDR1 P-gly-
coprotein in drug absorption is difficult to prove
directly in humans. Thus, the role of intestinal P-
glycoprotein in drug absorption in humans is often
derived indirectly from inhibition studies. In a clini-
cal study, five patients received a safe oral dose of
paclitaxel 60 mg/m2, and nine other patients re-
ceived the same oral dose of paclitaxel combined
with one single oral dose of cyclosporin 15
mg/kg.[90] The oral bioavailability of paclitaxel
when given without cyclosporin was less than
5%, and increased to 50% when cyclosporin was
coadministered. Similar results were found in an-
other clinical study with docetaxel.[91] The bio-
availability of docetaxel in cancer patients in-
creased from 8% without cyclosporin to 88% in
combination with cyclosporin. Because cyclo-
sporin is known to be a potent P-glycoprotein in-
hibitor, these results suggest the involvement of
© Adis International Limited. All rights reserved.
Lin & Yamazaki
P-glycoprotein as an intestinal barrier in limiting
the absorption of paclitaxel and docetaxel.
Since cyclosporin is also an inhibitor of CYP-
3A4 and other CYP enzymes, it is likely that the
observed increase in oral bioavailability of these
drugs could also be partly attributed to reduced me-
tabolism by the inhibition of CYP enzymes by
cyclosporin. Moreover, cyclosporin is a potent in-
hibitor of other transporters, such as canalicular
bile salt transporter and canalicular multispecific
organic anion transporter (cMOAT). The inhibi-
tory Ki values of cyclosporin for taurocholate (bile
salt transporter), leukotriene C4 (cMOAT) and
daunorubicin (P-glycoprotein) were 0.2, 3.4 and
1.5 μmol/L, respectively.[92] Clearly, the increased
bioavailability of paclitaxel and docetaxel caused
by cyclosporin cannot be explained by P-glyco-
protein inhibition alone. Therefore, the inhibition
study can only provide a qualitative assessment
as to whether P-glycoprotein is involved in drug
absorption. Due to the lack of a specific P-glyco-
protein inhibitor, it is difficult to estimate the quan-
titative contribution of P-glycoprotein to drug ab-
sorption by the inhibition approach.
Another way by which evidence can be shown
for intestinal P-glycoprotein involvement in drug
absorption is to establish the correlation between
the absorption profile (oral AUC) of drugs and
the expression of intestinal P-glycoprotein (or
mRNA). In a clinical study, cyclosporin was given
by gavage to ten male volunteers at different parts
of the gastrointestinal tract (stomach, jejunum/-
ileum and colon) in a crossover manner, with a
washout period between the administrations of at
least 7 days.[79] There was a strong negative corre-
lation between the plasma AUC of cyclosporin af-
ter administration at different locations of the gas-
trointestinal tract and the local mRNA expression
of intestinal P-glycoprotein. Similarly, based on
the observation that a highly significant correlation
exists between enterocyte P-glycoprotein content
and cyclosporin absorption kinetics, Lown et al.[81]
concluded that intestinal P-glycoprotein plays a
significant role in the absorption of cyclosporin.
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
With several lines of supportive evidence, the
conclusion from these two correlation studies that
P-glycoprotein transport is involved in the absorp-
tion of cyclosporin appears valid and appropriate.
However, it should be noted that a good correlation
itself does not prove a causal relationship. There-
fore, it is highly desirable to have other supportive
in vitro and/or animal data when applying the cor-
relation approach.
4.1.4 Saturable Efflux Transport
by Intestinal P-Glycoprotein
Like that of drug-metabolising enzymes, the
functional activity of P-glycoprotein is saturable.
In a recent kinetic study involving Caco-2 cells,[93]
the efflux of vinblastine and digoxin by P-glyco-
protein have been demonstrated to be saturable;
the Michaelis-Menten constants (Km values) for
vinblastine and digoxin were 26 and 58 μmol/L,
respectively. A similar Km value for vinblastine
(18 μmol/L) was reported by other investigators
using Caco-2 cells.[83] By using the Ussing cham-
ber technique, the transport of digoxin was studied
in human and rat intestinal tissues. Saturable trans-
port for digoxin was also observed in both human
and rat intestinal tissues. The Km values for dig-
oxin were 81, 74, 51 and 59 μmol/L, respectively,
for rat jejunum, rat ileum, rat colon and human
colon.[86] Interestingly, the Km values for digoxin
derived from human colon tissue and Caco-2 cells
are almost identical (59 vs 58 μmol/L).[93] Using
Caco-2 cells, transport of cyclosporin was shown
to be saturable with a Km of 3.8 μmol/L.[79] This Km
value for cyclosporin was comparable to that
found in other cell lines expressing human P-gly-
coprotein (8.4 μmol/L).[94] Collectively, these re-
sults strongly suggest that the efflux function of
intestinal P-glycoprotein may be saturated when
drug concentrations in the intestinal lumen exceed
the Km values after high oral doses.
As discussed previously, for drugs that are P-
glycoprotein substrates the net amount of drug
passing through the intestinal epithelial cells is the
difference between the amount absorbed by influx
processes (passive diffusion and/or active uptake)
and the summation of the amount extruded by
© Adis International Limited. All rights reserved.
73
efflux transport together with the amount meta-
bolised by enzymes. It is evident that the P-glyco-
protein-mediated efflux and CYP-mediated meta-
bolism are saturable processes. The saturable
P-glycoprotein efflux may, at least in part, explain
the observed dose-dependent absorption of tali-
nolol (a P-glycoprotein substrate) in healthy vol-
unteers.[95] For both enantiomers, the dose-
normalised AUC increased with increasing doses
after oral administration. The dose-normalised
AUC of (S)-(–)-talinolol increased from 18 μg •
h/L at a 12.5mg dose to 36 μg • h/L at a 200mg
dose. Similar results were observed for (R)-(+)-
talinolol. Consistent with the in vivo observations,
concentration-dependent permeability across Caco-
2 cell monolayers was observed when the concen-
tration of talinolol was increased from 0.1–2
mmol/L. Similarly, saturable P-glycoprotein-
mediated efflux has also been reported for cyclo-
sporin in rats.[96] The extent of cyclosporin absorp-
tion increased with increasing doses in rats; the
bioavailability increased from 13% at an oral dose
of 6 mg/kg to 25% at 18 mg/kg. These results in-
dicate that P-glycoprotein-mediated efflux trans-
port can be saturated when higher oral doses are
given.
There is a widespread misconception that the
extent of oral absorption of a drug is always mark-
edly limited by intestinal P-glycoprotein when the
drug is a P-glycoprotein substrate. This is only true
for a few P-glycoprotein substrate drugs that are
given at low doses. Absorption of digoxin is a good
example. Digoxin, a well-known P-glycoprotein
substrate, which undergoes minimal metabolism,
is given orally at a very low oral dose of 0.5 to 1mg.
At these doses, the concentration of digoxin in in-
testinal lumen is estimated to be less than 10
μmol/L, which is well below the Km value (58
μmol/L) derived from Caco-2 cells or human co-
lon.[93] Thus, P-glycoprotein plays a quantitatively
significant role in the absorption of digoxin. The
reported low and variable absorption of digoxin
can most probably be attributed to the efflux trans-
port of intestinal P-glycoprotein.
Clin Pharmacokinet 2003; 42 (1)
74 Lin & Yamazaki

However, the oral dose for most drugs is high
(>50mg) and drugs in the intestinal lumen can eas-
ily reach the mmol/L concentration range. A de-
tailed literature survey revealed that all of the re-
ported Km values for P-glycoprotein drugs are
relatively low, ranging from 4–213 μmol/L (table
II). Given the low Km values for P-glycoprotein
drugs, P-glycoprotein activity can readily be satu-
rated when drugs are administered at high doses,
and hence the role of intestinal P-glycoprotein in
drug absorption becomes quantitatively less signif-
icant. Indinavir, an HIV protease inhibitor, is a P-
glycoprotein substrate and is given orally at a dose
of 800mg. At this high dose, the indinavir concen-
tration in the intestinal lumen is expected to be
greater than 1 mmol/L, which is much higher than
the Km value for P-glycoprotein transport (140
μmol/L) derived from Caco-2 cells.[97] Therefore,
at high doses, the effect of intestinal P-glycopro-
tein on indinavir absorption becomes quantita-
tively less important. This can explain why in-
dinavir has a reasonably good bioavailability
(>60%) in patients, even though it is a good P-gly-
coprotein substrate.[98] Interestingly, in a recent lit-
erature survey, Chiou et al.[99] have concluded that
the in vivo oral absorption of 13 drugs is not sig-
nificantly impeded by efflux transport, in spite of
being good P-glycoprotein substrates.
However, there are exceptions that intestinal P-
glycoprotein still plays a significant role in absorp-
tion for some drugs, even when they are given at
high doses. For example, the clinical oral dose is
200–700mg for cyclosporin and 100–200mg for
paclitaxel, but clinical studies clearly indicate
that P-glycoprotein does play a significant role in
limiting their oral absorption.[79,82,87,101] This can
be explained by the fact that both cyclosporin and
paclitaxel have very poor water solubility, slow
dissolution rate and large molecular weight (1202
for cyclosporin and 854 for paclitaxel). The poor
water solubility and slow dissolution rate can result
in low drug concentration in the intestinal lumen
in relation to their Km value for P-glycoprotein
transport, and the large molecular size can impede
the rate of passive diffusion across the cell mem-
branes. The notion of slow dissolution rate and/or
slow membrane diffusion rate of cyclosporin in in-
testine is supported by the fact that peak concen-
trations of the drug occur slowly at 3–4 hours
after administration to patients.[101]

Table II. Apparent values of the Michaelis-Menten constant (Km) for P-glycoprotein substrates
Compound Material (flux evaluated) Apparent Km (μmol/L) References
Cyclosporin Caco-2 (net B-to-A) 3.8 79
Digoxin Caco-2 (net B-to-A) 58 93
Stripped rat jejunum (net B-to-A) 81 93
Stripped rat ileum (net B-to-A) 74 93
Stripped rat colon (net B-to-A) 51 93
Stripped human colon (net B-to-A) 59 93
Etoposide Caco-2 (B-to-A) 213 100
Stripped rat jejunum (B-to-A) 94 100
Stripped rat colon (B-to-A) 119 100
Indinavir Caco-2a (net B-to-A) 140 97
Verapamil Stripped rat jejunum (B-to-A) 31 100
Stripped rat ileum (B-to-A) 29 100
Stripped rat colon (B-to-A) 4.4 100
Vinblastine Caco-2 (net B-to-A) 19 83,84
Caco-2 (net B-to-A) 27 93
Stripped rat ileum (net B-to-A) 48 93
Stripped rat colon (net B-to-A) ~100 93
a Treated with calcitriol.
B-to-A = basal to apical.

© Adis International Limited. All rights reserved. Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
In conclusion, it is clear that the effect of intes-
tinal P-glycoprotein on drug absorption is unlikely
to be quantitatively important unless a very small
oral dose is given, or the dissolution and/or mem-
brane diffusion rates of the drug are very slow.
4.2 Drug Distribution
Drugs are often administered at a location dis-
tant from their intended site of action. To be effec-
tive, the drug must be absorbed and transported
from the site of administration across several
biomembranes to reach the target tissue and the
site of action. Penetrating cell membranes is a
complex process that is highly dependent on the
nature of the membrane and the physicochemical
properties of the drug. The physical and biochem-
ical properties of membranes, such as lipid bilayer
structure and dynamics, play an important role in
drug penetration. In addition, the physicochemical
properties of drugs, such as hydrophobicity, ioni-
sation profile, molecular size and number of hy-
drogen bonds, also play a significant role in mem-
brane penetration.[102]
Although extensive efforts have been made to
study the molecular mechanisms of the processes
of drug absorption, metabolism and excretion,
drug distribution has historically received much
less attention than the other processes, in spite of
its importance as a key factor in determining drug
response. Because of this lack of attention, drug
distribution has been regarded as a forgotten rela-
tive in clinical pharmacokinetics.[103] The lack of
attention stems partly from a lack of useful exper-
imental tools in studying drug distribution. How-
ever, the importance of drug distribution is becom-
ing increasingly recognised. For example,
Schinkel et al.[8,9] demonstrated large differences
in drug distribution into the brain and other tissues
between mdr1a (–/–) and mdr1a (+/+) mice. In ad-
dition, with recent advances in the molecular biol-
ogy and biochemistry of transporter systems, sev-
eral in vitro systems have now been developed for
studying drug distribution.[104] These newly devel-
oped tools and experimental methodologies will
© Adis International Limited. All rights reserved.
75
certainly provide important insights into the mech-
anisms of drug distribution in the very near future.
4.2.1 Blood-Brain Barrier
Lipophilicity and Brain Penetration
The brain is different from other organs of the
body in many aspects. One of the most important
features is that the brain is anatomically separated
from the blood circulation by the BBB. All other
organs are perfused by capillaries lined with endo-
thelial cells that have small pores to allow for
movement of drugs into the organ interstitial fluid
from the circulation. However, the endothelial
cells in brain capillary blood vessels are closely
joined to each other, leaving no space between
cells. Consequently, only lipophilic drugs can
cross endothelial cells and enter the BBB by way
of passive diffusion. A strong positive correlation
between lipophilicity and brain penetration of
drugs has been reported by many investiga-
tors.[105,106] In addition, factors other than lipophil-
icity may also play an important role in the trans-
port of drugs across the BBB. For example, a
negative correlation was found between the BBB
permeability of lipophilic compounds (steroid hor-
mones and peptides) and the total number of hy-
drogen bonds; the greater the total number of hy-
drogen bonds, the lower the permeability.[105,107]
In addition, the molecular size of drugs is also an
important determinant for brain penetration.[108]
Although lipophilicity is an important factor in
determining the BBB penetration of drugs, many
lipophilic drugs have exhibited poor BBB penetra-
tion. In a rat study, Levin[108] reported a good cor-
relation between the in vivo BBB permeability co-
efficient of 22 compounds and their lipophilicity.
However, they found that vincristine and epipo-
dophylotoxin displayed poor BBB permeability,
despite relatively high lipophilicity (logP value of
2.8). Additionally, many other lipophilic com-
pounds also exhibit poor BBB penetration. For ex-
ample, a potent CCKB receptor antagonist candi-
date is a lipophilic compound with a logP value of
3.6, and it has poor brain penetration.[109] The poor
BBB penetration of these drugs cannot be ex-
plained by the number of hydrogen bonds and
Clin Pharmacokinet 2003; 42 (1)
76
molecular weight. Twenty years ago, these com-
pounds were regarded as ‘outlier compounds’ for
brain penetration without knowing the exact cause.
The possible efflux function of P-glycoprotein
in the BBB was not connected with the observed
poor BBB permeability of lipophilic drugs until the
findings of P-glycoprotein in brain capillaries by
Thiebaut et al.[110] and Cordon-Cardo et al.[111] Us-
ing monoclonal antibodies, they demonstrated that
P-glycoprotein is highly expressed on the apical
surface of the endothelial cells of the brain capil-
laries. With these findings, it is now clear that the
observed poor BBB permeability of those lipo-
philic drugs is due mainly to the efflux function of
P-glycoprotein.
Localisation of P-Glycoprotein in Brain
Localisation of P-glycoprotein in the brain is a
widely disputed issue that has become the centre
of much controversy in recent years. Beaulieu et
al.[112] provided very convincing evidence for P-
glycoprotein being predominantly localised in the
luminal membrane of endothelial cells of rat brain
capillaries facing blood circulation. Using a novel
technique with cationic colloidal silica, the inves-
tigators were able to selectively isolate the luminal
membrane of endothelial cells of rat brain capillar-
ies. The isolation procedures resulted in a mem-
brane preparation with a 9.9-fold enrichment of the
endothelial membrane marker protein GLUT1
(glucose transporter 1) and a 17-fold enrichment of
P-glycoprotein relative to isolated brain capillar-
ies. Enrichment of GLUT1 and P-glycoprotein rel-
ative to whole brain membrane preparations was
280- and 500-fold, respectively. However, glial
fibrillary acidic protein (GFAP), a specific marker
for astrocytes, was enriched only 1.4-fold relative
to brain capillary, suggesting a very minor contam-
ination of astrocytes in the luminal membrane
preparations. The co-enrichment of P-glycoprotein
(500-fold) and GLUT1 (280-fold) in brain capil-
lary luminal membranes compared with whole
brain membrane preparations strongly suggests
that P-glycoprotein is expressed predominantly in
the luminal membrane of brain endothelial cells.
© Adis International Limited. All rights reserved.
Lin & Yamazaki
Consistent with Beaulieu’s results, Barrand et
al.,[113] using a dual immunostaining approach in
combination with confocal microscopy, concluded
that endothelial marker C219 staining did not co-
localise with astrocyte marker GFAP staining in rat
brain microvessels. Similarly, Matsuoka et al.[114]
have demonstrated that P-glycoprotein is localised
in the brain capillaries of rats, using P-glycoprotein
and GFAP double-immunolabelling technique.
Furthermore, a recent study by Decleves et al.[115]
showed that P-glycoprotein is expressed in both
cultured rat endothelial cells and astrocytes. In
this study, RT-PCR analysis showed that mdr1b
mRNA was preferentially expressed in astrocytes,
whereas both mdr1a and mdr1b mRNA were de-
tected in endothelial cells. In this study, Western
blotting analysis revealed much higher expression
level of P-glycoprotein in endothelial cells com-
pared with astrocytes. Collectively, these results
consistently suggest that brain P-glycoprotein is
predominantly expressed on the apical surface of
endothelial cells of capillaries.
Although most immunohistochemical studies
indicate that P-glycoprotein is predominantly
localised on the surface of endothelial cells facing
the luminal side, Pardridge et al.[116] claim that P-
glycoprotein is localised mainly to astrocyte foot
processes. They conducted an immunochemical
study with human brain microvessels using (a)
the MRK16 antibody to human P-glycoprotein, (b)
an antiserum to GFAP and (c) an antiserum to
GLUT1. They observed that the P-glycoprotein-
specific antibody MRK16 bound to microvessels
with a similar, discontinuous staining pattern as an
antiserum directed against GFAP. The apparently
discontinuous and abluminal localisation of MRK-
16 staining in isolated brain capillaries and the
similarity in immunostaining patterns by MRK-
16 and anti-GFAP antibodies led the investigators
to conclude that P-glycoprotein is expressed in as-
trocyte foot processes, rather than in endothelial
cells. The conclusion was further supported by
their subsequent findings that staining of the endo-
thelial membrane marker protein GLUT1 was con-
tinuous and showed only minimal overlap with
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
MRK16 staining. With their observations, Golden
and Pardridge[117] have proposed a revised kinetic
model of P-glycoprotein in the brain in contrast to
the classic kinetic model.
According to the revised kinetic model of Gold-
en and Pardridge,[117] a deficiency (or inhibition)
of P-glycoprotein would have no effect on BBB
permeability, but would cause a decrease in the
drug concentration in the interstitial fluid (ISF) of
brain. On the other hand, according to the classic
model, a deficiency (or inhibition) of P-glycopro-
tein would result in an increase in BBB permeabil-
ity as well as an increase in drug concentration in
the ISF of brain. Therefore, comparison of brain
uptake of P-glycoprotein substrates in normal and
P-glycoprotein-deficient mice can be used to ad-
dress the question of whether the P-glycoprotein
transporter is localised in the capillary endothe-
lial cells or in the astrocytes. Using an intracereb-
ral microdialysis technique, de Lange et al.[118]
showed that a deficiency of P-glycoprotein in mice
resulted in the same degree of increase in drug con-
centrations of rhodamine-123 (a P-glycoprotein
substrate) in the brain as well as ISF. After an in-
travenous infusion of rhodamine-123, the total
brain concentrations were about four times higher
in the mdr1a (–/–) mice compared with wild-type
mice. Similarly, the rhodamine-123 concentra-
tions in ISF were also about four times higher in
mdr1a (–/–) mice than in mdr1a (+/+) mice. These
results appear to be consistent with the classic con-
cept that in mice the P-glycoprotein transporter is
expressed in the brain capillary endothelial cells.
Recently, Lee et al.[119] have demonstrated that
P-glycoprotein is expressed and functional in brain
microglia. Using a continuous rat brain microglia
cell line (MLS-9), immunocytochemistry studies
revealed the location of P-glycoprotein along the
nuclear envelope and plasma membrane of mi-
croglia. Furthermore, the accumulation of digoxin
by microglia was significantly enhanced by
valspodar (PSC-833), a potent P-glycoprotein in-
hibitor. Consistent with these findings, P-glyco-
protein has also been detected in mixed glial cells,
but not in primary cultured neurons, by other in-
© Adis International Limited. All rights reserved.
77
vestigators.[114] The expression of P-glycoprotein
in glial cells may partly explain the intriguing
pharmacokinetics and pharmacodynamics of [D-
penicillamine2,5]enkephalin (DPDPE) in mdr1a
(–/–) and mdr1a (+/+) mice demonstrated by Chen
and Pollack.[120] Although the brain concentrations
of DPDPE were 2- to 4-fold higher in mdr1a (–/–)
mice than in mdr1a (+/+) mice after intravenous
administration, the dose required to elicit compa-
rable antinociception was more than 30-fold lower
in mdr1a (–/–) mice compared with mdr1a (+/+)
mice. Based on brain concentrations, the EC50
(concentration producing half-maximal antinoci-
ception effect) of DPDPE was 13 times lower in
mdr1a (–/–) mice compared with wild-type mice
(12 versus 160 ng/g). These results suggest either
differences in DPDPE distribution within the brain
or differences in the intrinsic activity of δ-opioid
receptors between mdr1a (–/–) mice and mdr1a
(+/+) mice. Subsequent pharmacokinetic and phar-
macodynamic modelling suggested that the differ-
ence in antinociception between mdr1a (–/–) and
mdr1a (+/+) mice was due to the distribution of
DPDPE within the brain as well as between the
blood and brain, but not due to differences in in-
trinsic response. These results are consistent with
the notion that P-glycoprotein is expressed in other
type of brain cells in addition to the endothelial
cells of capillaries.
Evidence of P-Glycoprotein
Involvement in Brain Uptake
The first experimental evidence that P-glyco-
protein is involved in drug transport in the BBB
came from Tsuji and coworkers.[121] Immunostain-
ing with a P-glycoprotein antibody (MRK16) dem-
onstrated an exclusively apical localisation of
P-glycoprotein in the cultured bovine brain endo-
thelial cells. Kinetic studies showed that efflux
transport of vincristine from the bovine brain en-
dothelial cells was inhibited by verapamil, result-
ing in a significant increase in intracellular drug
concentration. Similar results were also reported
by Tsuruo and colleagues[122] with mouse brain
capillary endothelial cells. They showed by im-
munochemical studies that P-glycoprotein was
Clin Pharmacokinet 2003; 42 (1)
78
localised on the apical surface of endothelial cells
of mouse brain. The unidirectional transport of vin-
cristine from basolateral side to apical side was
demonstrated in the polarised monolayer of mouse
endothelial cells. Similarly, efflux transport of
cyclosporin was also observed in cultured endothe-
lial cells of bovine and mouse brain capillar-
ies.[123,124] Immunostaining with P-glycoprotein
antibody also demonstrated an exclusively apical
localisation of P-glycoprotein in human brain en-
dothelial cells.[125] These in vitro studies provide
evidence of functional involvement of P-glycopro-
tein in the BBB penetration of drugs.
Although, in vitro studies have shown that ATP
is essential for P-glycoprotein transport func-
tion,[126,127] the involvement of ATP in P-glyco-
protein-mediated transport was first demonstrated
in vivo by Tsuji and colleagues.[128] They demon-
strated that the BBB permeability coefficient of
doxorubicin increased from 14 μl/min/g brain in
control rats to 243 μl/min/g brain in rats ATP-
depleted by occlusion of vertebral and common
carotid arteries. The BBB permeability coefficient
of doxorubicin was determined by using in situ
brain perfusion technique. Under the experimental
conditions, the ATP content in the rats with tran-
sient brain ischaemia was only 3% of that in normal
rats (0.04 versus 1.43 μmol/g brain). Similarly, an
increase in the BBB permeability coefficient of
cyclosporin in ATP-depleted rats was also reported
by Tsuji’s group.[129] These results from the ATP-
depleted rat studies are consistent with the classic
concept that the P-glycoprotein is expressed in the
brain capillary endothelial cells, because the BBB
permeability decreases when P-glycoprotein func-
tion is impaired.
The knockout animal model of mdr1a(–/–) mice
also provides a powerful tool for studying brain
uptake of drugs. Oral administration of [3H]iver-
mectin in mdr1a (–/–) and mdr1a (+/+) mice re-
sulted in 87-fold higher levels of radioactivity in
the brain of mdr1a (–/–) mice as compared with
wild-type mice, whereas the levels in liver, kidney,
small intestine and plasma were increased by less
than 4-fold.[9] In another study, when [3H]digoxin
© Adis International Limited. All rights reserved.
Lin & Yamazaki
and [3H]cyclosporin were given intravenously,
markedly higher brain levels of radioactivity (17-
and 55-fold, respectively) were observed in mdr1a
(–/–) mice than in mdr1a (+/+) mice.[130] Again,
only a moderate increase in radioactivity levels (2-
to 3-fold) of these two drugs was observed for
liver, kidney, small intestine and plasma of mdr1a
(–/–) mice. The large differences in brain concen-
tration were also observed between mdr1a/1b (–/–)
double knockout and normal mice.[10] A 27-fold
increase in the brain concentration of digoxin was
observed in mdr1a/1b (–/–) double knockout mice
compared with the wild-type mice, with only a 2.5-
fold increase in digoxin concentration in the liver,
kidney and plasma of double knockout mice.
It is puzzling why the most marked increase in
drug concentration of P-glycoprotein substrates in
mdr1a (–/–) knockout mice is always observed in
the brain, while the increases in drug concentration
in other tissues in which P-glycoprotein is also
highly expressed, such as liver and kidney, are rel-
atively modest. As mentioned earlier, the mdr gene
in mice is expressed in a tissue-specific manner.
Both mdr1a and mdr1b genes are expressed in the
liver and kidney, while only mdr1a gene is ex-
pressed in the brain of mice.[131] At first glance, one
might assume that the less profound increases in
drug concentration in the liver and kidney in mdr1a
(–/–) mice are due to the extra protective functions
of mdr1b P-glycoprotein in these tissues. How-
ever, even in mdr1a/1b (–/–) double knockout
mice, increases in drug concentrations in the liver
and kidney are also much lower than that in the
brain.[10] The marked increases in drug concentra-
tion in the brain of P-glycoprotein-deficient mice
also cannot be explained by the loss of BBB integ-
rity. Experiments performed using fluorescein and
fluorescein-dextran-4000 as integrity markers
showed that there were no differences in
brain/plasma concentration ratio of these com-
pounds between mdr1a (–/–) and wild-type mice,
being 2.3 and 1.4%, respectively, indicating main-
tenance of BBB integrity in the absence of P-gly-
coprotein.[118] The underlying mechanism for the
marked increases in brain concentration remains
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
unknown. Regardless of the underlying mecha-
nisms, the data from the mdr1a and mdr1a/1b-
knockout mice studies suggest that the brain is
more sensitive to changes in P-glycoprotein func-
tion than other tissues. Therefore, P-glycoprotein
inhibitors should be used with caution to avoid po-
tential neurotoxicity.
Another important observation from these
mdr1a and mdr1a/1b knockout mice studies is that
for certain P-glycoprotein substrates, an apprecia-
ble amount of drug is still observed in the brain of
wild-type mdr1a (+/+) mice. For example, the
brain concentrations of tacrolimus were about 2.7-
fold higher than the corresponding plasma concen-
trations at the same time point in mdr1a(+/+) mice
after intravenous administration.[132] Similarly,
appreciable brain concentrations of cyclosporin,
ranging from 30–70% of the corresponding plasma
concentration, were observed in mdr1a (+/+)
mice.[130] Kinetically, the drug concentration in the
brain is determined by the difference between the
amount of drug transported by influx processes
(passive diffusion and active uptake) and the
amount of drug extruded by the P-glycoprotein-
mediated efflux process. A fraction of drug mole-
cules can reach the brain tissue if the influx diffu-
sion rate is greater than the P-glycoprotein efflux
rate. Nonspecific binding to brain tissue may also
be a contributing factor in determining drug con-
centration in the brain. Therefore, one should take
the rate of influx diffusion, rate of P-glycoprotein
efflux transport and nonspecific binding of com-
pounds into consideration when predicting brain
penetration.
4.2.2 Placenta
As noted earlier, Lankas et al.[48] have clearly
demonstrated the protective role of placental P-
glycoprotein in reducing fetal exposure to
xenobiotics in CF-1 mice. The role of placental
P-glycoprotein in protection of the fetus has been
further evaluated using mdr1a/1b (–/–) double
knockout mice.[133] Heterozygous mdr1a/1b (+/–)
female mice were mated with heterozygous male
mice to produce fetuses of three genotypes:
mdr1a/1b (–/–), mdr1a/1b (+/–) and mdr1a/1b (+/+).
© Adis International Limited. All rights reserved.
79
Following intravenous administration of digoxin,
saquinavir and paclitaxel to pregnant heterozy-
gous dams, fetal drug exposure was much higher
in the mdr1a/1b (–/–) fetus than the wild-type
mdr1a/1b (+/+) fetus. The ratio of drug concentra-
tion in the mdr1a/1b (–/–) fetus to that in the wild-
type fetus was 2.5, 5 and 16, respectively, for dig-
oxin, saquinavir and paclitaxel. On the other hand,
the drug concentrations in the heterozygous
mdr1a/1b (+/–) fetus were similar to those in the
wild-type fetus, suggesting that the P-glycoprotein
level in the placenta of the heterozygous fetus is
still sufficient to protect the fetus. In this study, the
investigators further demonstrated that the P-gly-
coprotein inhibitors valspodar and FG-120918
were able to completely block the placental P-
glycoprotein function. The fetal drug concentrations
in the wild-type fetus were increased and were
comparable to those in the mdr1a/1b (–/–) fetus
after oral administration of the P-glycoprotein in-
hibitors to heterozygous mothers. As in the brain,
these results clearly demonstrate that placenta is
very sensitive to changes in P-glycoprotein func-
tion. Because of potential function blockade, it
is recommended that P-glycoprotein inhibitors
should not be used in women during pregnancy to
avoid excessive fetal exposure to xenobiotics.
P-glycoprotein is also highly expressed in hu-
man placenta. Using monoclonal antibody C219,
immunostaining showed a high expression of P-
glycoprotein in trophoblasts of human placen-
ta.[134] Functional P-glycoprotein activity has
been demonstrated in cultured human placenta
choriocarcinoma epithelial cells (BeWo cells).[135]
Western blotting studies with monoclonal anti-
body C219 or JSB-1 indicated that P-glycoprotein
is highly expressed in BeWo cells. In the BeWo
monolayer, the B-to-A transport of vinblastine,
vincristine and digoxin was significantly greater
than the A-to-B transport. Addition of cyclosporin
resulted in an increase in the A-to-B transport of
the drugs and a decrease in the B-to-A transport.
These results suggest that the placental P-glyco-
protein acts as an efflux transporter by removing
xenobiotics from cells. Since P-glycoprotein is ex-
Clin Pharmacokinet 2003; 42 (1)
80
pressed in human placental trophoblasts, it is likely
that placental P-glycoprotein also protects fetuses
from xenobiotics in humans as well.
Genetic variation in the expression level of pla-
cental P-glycoprotein was studied by Western blot-
ting in 100 placentas obtained from Japanese
women.[61] In this study, nine SNPs were identified
with an allelic frequency of 0.005–0.42 (table I).
Of these SNPs, G2677A (allelic frequency 0.18)
and G2677T (0.39) in exon 21 were associated
with an amino acid conversion from Ala to Thr and
to Ser, respectively. Comparison of the MDR1-
genotyping and corresponding placental P-glyco-
protein level revealed a correlation between the P-
glycoprotein expression level and SNPs in exon 1b
(T–129C) and exon 21 (G2677A/G2677T). Indi-
viduals with heterozygous T–129C (T/C) had sig-
nificantly lower levels of P-glycoprotein than the
wild-type (T/T) individuals (1.07 vs 1.99, arbitrary
units). The expression levels of placental P-glyco-
protein in homozygotes for wild-type allele, het-
erozygotes and homozygotes of mutant allele in
exon 21 were 2.44, 1.97 and 1.45 (arbitrary units),
respectively. Although the clinical implications of
interindividual variability in placental P-glycopro-
tein remains to be investigated, high placental lev-
els of P-glycoprotein may provide a better protec-
tion for the fetus against xenobiotics.
4.3 Drug Metabolism
It has been widely accepted that the liver is the
major site of drug metabolism because of its size
and high content of drug-metabolising enzymes. In
addition to the liver, the small intestine and kidney
may contribute significantly to overall metabolism
in the body.[136] In humans, CYP3A4 is the princi-
pal enzyme involved in the hepatic and intestinal
metabolism of drugs. Studies in rats by Debri et
al.[137] suggest that the CYP3A enzymes in the liver
are evenly distributed. The data of Watkins et
al.,[138] who investigated the CYP3A content at ten
different locations in a human liver, also indicate
that hepatic CYP3A is homogeneously distributed.
Unlike the liver, the distribution of CYP3A4 is not
uniform along the length of the small intestine, or
© Adis International Limited. All rights reserved.
Lin & Yamazaki
along the villi within a cross-section of mucosa.
Using a monoclonal antibody to CYP3A, the co-
lumnar absorptive epithelial cells of the villi exhib-
ited the strongest immunoreactivity, whereas no
immunostaining was detectable in the goblet and
crypt cells.[139] It has also been shown that CYP-
3A4 expression varied along the length of small
intestine: median values of 31, 23 and 17 pmol/mg
microsomal protein were found in human duode-
num, distal jejunum, and distal ileum.[140] In con-
trast to CYP3A4, the expression of P-glycoprotein
appears to increase progressively along the length
of intestine.[78]
There is a striking overlap between CYP3A4
substrates and P-glycoprotein substrates. Because
of overlapping substrate specificity, and because
of coexpression of CYP3A enzymes and P-glyco-
protein in the intestine, kidney and liver, it is
conceivable that P-glycoprotein may play an im-
portant role in drug metabolism. However, the
magnitude of the effect of P-glycoprotein on me-
tabolism appears to be dependent on the spatial
relationship between P-glycoprotein and CYP3A
enzymes. In the liver and kidney, P-glycoprotein is
localised on the luminal membrane of hepatic can-
aliculi facing the bile duct lumen or on the luminal
brush-border membrane of renal proximal tubular
cells facing the renal tubule lumen. This means that
P-glycoprotein is localised at the exit site of hepa-
tocytes and renal epithelial cells. Therefore, P-gly-
coprotein only ‘sees’ drug molecules after cellular
uptake, intracellular distribution and metabolism
in both the liver and kidney.
In contrast to the situation in the liver and kid-
ney, P-glycoprotein is localised at the entrance site
of epithelial cells of intestines. Drug molecules are
exposed to P-glycoprotein prior to intracellular
distribution and metabolism. A large fraction of
drug molecules is extruded by intestinal P-glyco-
protein from the inside of the epithelial cells back
into the intestinal lumen after the drug molecules
gain access across the luminal surface of the epi-
thelial cells; however, a portion of the extruded
drugs then can be reabsorbed into the epithelial
cells. Through the repetitive processes of extrusion
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
and reabsorption, P-glycoprotein prolongs the in-
tracellular residence time of drug molecules and
increases the probability of exposure to drug-me-
tabolising enzymes. Consequently, P-glycoprotein
may enhance intestinal metabolism of drugs,
whereas it has less of an effect on drug metabolism
in liver and kidney.
The effect of P-glycoprotein on CYP3A4-
mediated intestinal metabolism of indinavir, a
substrate for both CYP3A4 and P-glycoprotein,
has been carefully evaluated in our laboratory us-
ing calcitriol-treated Caco-2 cells expressing both
CYP3A4 and P-glycoprotein.[97,141] The formation
of the major metabolite (M6), expressed as the ra-
tio of the amount of the metabolite formed to the
amount of the parent drug transported across the
monolayer, was more than 6-fold greater when the
drug was applied at the apical side than when the
drug was applied at the basolateral side. Similarly,
the metabolism of cyclosporin in Caco-2 cells
was higher from the apical side than from the baso-
lateral side.[142] These results strongly suggest a
role of P-glycoprotein in enhancement of CYP3A4-
mediated intestinal metabolism of drugs.
The effect of P-glycoprotein on intestinal meta-
bolism was also shown in vivo. The intestinal first-
pass metabolism of indinavir increased from 6%
in control rats compared with 34% in dexametha-
sone-treated rats.[143] Pretreatment of rats with dex-
amethasone (40 mg/kg orally for 3 days) resulted
in a 2.5-fold increase in both CYP3A and P-glyco-
protein levels in the intestine. The 6-fold increase
in intestinal first-pass metabolism of indinavir can-
not be explained by the 2.5-fold increase in intes-
tinal CYP3A level alone. The increased intestinal
first-pass metabolism is most probably due to a
combination of increased intestinal CYP3A and
P-glycoprotein levels, providing in vivo evidence
that P-glycoprotein enhances the intestinal first-
pass metabolism of indinavir.
In this study, the effect of P-glycoprotein on
hepatic metabolism was also investigated. Pre-
treatment of rats with dexamethasone also induced
hepatic first-pass metabolism of indinavir, and the
increased hepatic CYP3A enzyme activity alone
© Adis International Limited. All rights reserved.
81
appeared to be able to explain the increased hepatic
first-pass metabolism, suggesting that P-glycopro-
tein plays a less significant role in hepatic metabo-
lism as compared with intestinal metabolism.[143]
These results clearly suggest that P-glycopro-
tein may play an important role in intestinal
metabolism of drugs. However, it should be re-
emphasised that the effect of P-glycoprotein on
drug metabolism becomes quantitatively less sig-
nificant when high doses are given.[144]
4.4 Drug Excretion
Drugs are generally eliminated from the body
by metabolism and/or excretion. Both the liver and
kidney play an important role in the excretion of
unchanged drugs and their metabolites. In princi-
ple, biliary excretion and renal tubular excretion
share certain characteristics. For biliary excretion,
a drug must first traverse the sinusoidal (basolat-
eral) membrane of the hepatocytes by passive dif-
fusion and/or hepatic uptake transporters. The si-
nusoidal membrane of the hepatocyte contains a
number of active transporters responsible for the
uptake of cations, anions and endogenous sub-
stances into hepatocytes from the circulation.[145]
Once in the hepatocytes, the drug molecules con-
tinue to diffuse and reach the canalicular mem-
brane, where P-glycoprotein and other efflux
transporter systems will pump the drug molecules
into bile. Often, biotransformation occurs when
the drug molecules are passing through the hepa-
tocytes. Therefore, hepatic uptake, intracellular
diffusion and metabolism, as well as other efflux
transporter systems, have to be taken into consid-
eration when biliary excretion of drugs is evalu-
ated.
Similarly, uptake of drugs across the basolateral
membrane of renal epithelial cells is the first step
in renal excretion, and biotransformation may oc-
cur. The basolateral membrane contains a number
of active transporters responsible for drug uptake.
The luminal brush-border membrane also contains
numerous active transporters, including P-glyco-
protein, which is responsible for the last step of
Clin Pharmacokinet 2003; 42 (1)
82
excretion of P-glycoprotein substrates into the
urine.[146]
4.4.1 Biliary Excretion
The involvement of P-glycoprotein in the bil-
iary excretion of drugs was first suggested by im-
munohistochemical studies showing that P-glyco-
protein is highly expressed on the canalicular
membrane of hepatocytes.[6] Earlier experimental
evidence of the potential involvement of P-glyco-
protein in biliary excretion had come from in vitro
studies with highly purified canalicular membrane
vesicles and isolated perfused rat liver. Kamimoto
et al.[127] demonstrated ATP-dependent transport
of daunomycin in canalicular membrane vesicles.
Furthermore, they showed that the transport of
daunomycin was inhibited by verapamil, a potent
P-glycoprotein inhibitor. These results suggest the
involvement of P-glycoprotein in the biliary excre-
tion of daunomycin. The involvement of P-glyco-
protein in the biliary excretion of vincristine has
also been demonstrated by using isolated per-
fused rat liver.[147] Vincristine is eliminated in rats
mainly by biliary excretion; approximately 50% of
the dose is excreted as unchanged drug into the
bile. The biliary excretion of vincristine has been
shown to be saturable and inhibited by verapamil.
Similarly, studies in isolated perfused rat liver also
suggest that P-glycoprotein plays a significant role
in the biliary excretion of doxorubicin.[148]
More direct evidence for the involvement of P-
glycoprotein in biliary excretion of drugs has come
from studies with mdr1a knockout mice. Digoxin
is mainly excreted as unchanged drug in the bile
and urine of mice; only a minor fraction of digoxin
(<3%) is metabolised. The biliary clearance of dig-
oxin is substantially greater in mdr1a (+/+) mice
(2.3 ml/min/kg) than in mdr1a (–/–) mice (0.84
ml/min/kg).[149] Approximately 45% of the dose is
excreted as unchanged digoxin in the bile of mdr1a
(+/+) mice. These results clearly indicate that P-
glycoprotein plays a significant role in the biliary
excretion of digoxin in mice. Similarly, the role of
P-glycoprotein in biliary excretion has also been
reported for doxorubicin and vinblastine.[150] The
excretion of unchanged doxorubicin and vinblas-
© Adis International Limited. All rights reserved.
Lin & Yamazaki
tine into bile is 3- to 5-fold greater in mdr1a (+/+)
mice than in mdr1a (–/–) mice. However, the ab-
solute biliary recovery of unchanged drugs in
mdr1a (+/+) mice is only 5 and 13% of the dose for
vinblastine and doxorubicin, respectively. The low
biliary excretion of doxorubicin and vinblastine in
mice is partly attributed to their high hepatic me-
tabolism. From these two studies, it is important to
note that not all P-glycoprotein substrates are sub-
ject to significant biliary excretion.
Because both mdr1a and mdr1b genes are ex-
pressed on the canalicular membrane, biliary ex-
cretion of drugs was further investigated in a dou-
ble knockout mouse model in which both mdr1a
and mdr1b genes were disrupted. It was found that
the complete absence of both mdr1a and mdr1b
P-glycoprotein at the canalicular membrane results
in a greater decrease in biliary excretion of a num-
ber of basic drugs compared with mdr1a (–/–) sin-
gle knockout and wild-type mice.[151,152] Vecuron-
ium, a neuromuscular blocking agent, is eliminated
predominantly by biliary excretion in mice, and the
biliary excretion of vecuronium is profoundly re-
duced in mdr1a/1b(–/–) double knockout mice
compared with mdr1a (–/–) single knockout and
wild-type mice. The biliary clearance of vecuron-
ium in mdr1a/1b (–/–) double knockout mice was
found to be about six times lower than in wild-type
mice, whereas the biliary clearance in mdr1a (–/–)
mice was only 2.5 times smaller than in the wild-
type mice.[149,150] Interestingly, appreciable resid-
ual biliary excretion of vecuronium was still ob-
served in mdr1a/1b (–/–) double knockout mice,
suggesting that additional carrier systems are in-
volved in the biliary excretion of cationic drugs.
Collectively, the results from both in vitro and
in vivo studies clearly demonstrate that P-glyco-
protein plays a significant role in biliary excretion
of P-glycoprotein substrates. However, additional
factors, such as hepatic uptake, intracellular distri-
bution and metabolism have to be taken into con-
sideration when quantitatively assessing the role of
P-glycoprotein in biliary excretion.
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
4.4.2 Renal Excretion
Renal excretion of drugs usually involves three
processes: glomerular filtration, renal tubular se-
cretion, and reabsorption from the renal tubular lu-
men. The relationship between renal clearance
(CLR) and these processes can be expressed as
equation 1:
CLR = fu • GFR + CLS – CLRA
Rearrangement of equation 1 yields equation 2:
CLR/(fu • GFR) = 1 + (CLS – CLRA)/(fu • GFR)
where GFR, CLS, and CLRA are glomerular filtra-
tion rate, secretion clearance and reabsorption
clearance, respectively, and fu is the unbound frac-
tion of drug in plasma. GFR is a passive process
by which only unbound drugs can be filtered, while
tubular secretion and reabsorption often involve
active transporters. Non-filtered drugs must first
cross the basolateral membrane and then the apical
membrane of epithelial cells of the renal tubule,
either by passive diffusion or carrier-mediated pro-
cesses.
Immunohistochemical studies[6] reveal that P-
glycoprotein is localised at the apical brush-border
membrane of the proximal renal tubule, which is
the major site of renal secretion. The finding of the
localisation of renal P-glycoprotein has led to rec-
ognition of the importance of this transporter in
tubular secretion of drugs. In vitro systems have
proven to be very useful tools for the study of the
P-glycoprotein role in tubular secretion. Human
MDR1 gene-transfected Madin-Darby canine kid-
ney (MDCK) and porcine kidney (LLC-PK1) epi-
thelial cell lines are the two most widely used mod-
els for the study of renal P-glycoprotein function.
The B-to-A transepithelial transport of digoxin
across LLC-PK1 monolayers expressing human P-
glycoprotein is much greater than the A-to-B trans-
port by a factor of 7. Addition of cyclosporin re-
sults in a marked decrease in the B-to-A transport
and an increase in the A-to-B transport.[153] Simi-
larly, LLC-PK1 cells expressing human P-glyco-
protein exhibit greater B-to-A transport of vinblas-
tine than A-to-B transport.[154] Transepithelial
transport of vinblastine has also been observed in
© Adis International Limited. All rights reserved.
83
MDCK monolayers expressing human P-glyco-
protein.[155] The B-to-A transport of vinblastine
has been observed to be about six times higher than
A-to-B transport. These results suggest that the P-
glycoprotein functions as an efflux transporter at
the apical membrane of epithelial cells of the renal
tubule.
The isolated perfused kidney technique has also
been used to investigate the role of P-glycoprotein
in tubular secretion. As shown in equation 2, there
are three processes involved in renal excretion. Al-
though quantification of each process of renal ex-
cretion is difficult due to practical limitations, the
CLR/(fu • GFR) ratio can be used as a simple way
to assess the relative contribution of each process
to overall renal excretion. When the CLR/(fu • GFR)
ratio of a drug is greater than unity, this means
that tubular secretion of the drug occurs. Con-
versely, when the ratio is less than unity, reabsorp-
tion of the drug from the tubular lumen occurs.
Using the CLR/(fu • GFR) ratio approach, Hori et
al.[156] showed that digoxin was actively secreted
in the isolated perfused rat kidney with a CLR/(fu
• GFR) ratio of 2.5. The P-glycoprotein inhibitors
quinidine and verapamil inhibited tubular secre-
tion and decreased the ratio. In the presence of
quinidine and verapamil at a concentration of 8
μmol/L, the CLR/(fu • GFR) ratio became unity,
suggesting minimal reabsorption process of dig-
oxin. Similar results were also observed for dig-
oxin when the drug was studied in dog isolated
perfused kidney using the single-pass multiple in-
dicator dilution method.[157] These results provide
further evidence that digoxin is actively secreted
into the renal tubular lumen by P-glycoprotein.
Although transgenic mice have also been used
to study the role of P-glycoprotein in renal excre-
tion, the results from these mouse studies have re-
mained controversial. Renal clearance of digoxin
was compared in mdr1a (+/+) and mdr1a (–/–)
mice following intravenous administration. As ex-
pected, the renal clearance of digoxin in mdr1a
(+/+) mice was three times greater than that in
mdr1a (–/–) mice, at 3.15 and 0.99 ml/min/kg, re-
spectively.[149] Because doxorubicin is a P-glyco-
Clin Pharmacokinet 2003; 42 (1)
84
protein substrate, it is expected that the absence of
P-glycoprotein in renal tubule will result in a de-
crease in renal excretion of doxorubicin. However,
in contrast with the expectation, the renal excretion
of unchanged doxorubicin was found to be higher
in mdr1a (–/–) mice (15% of the dose) than in
mdr1a(+/+) mice (10%) after intravenous admin-
istration.[158] Likewise, the renal excretion of three
basic compounds (tributylmethylammonium, azido-
procainamide and vecuronium) in mdr1a/1b (–/–)
double knockout mice was significantly higher
than that in mdr1a/1b (+/+) mice.[152] Since these
three basic compounds are P-glycoprotein sub-
strates, lower renal excretion in mdr1a/1b (+/+)
mice was not expected. The reason for the conflict-
ing results is presently not known. One possible
explanation is that other transporter systems may
be involved in the renal excretion of doxorubicin
and these basic compounds, and disruption of
mdr1a and mdr1b genes may increase the expres-
sion of other transporter systems.
5. P-Glycoprotein-Mediated
Drug-Drug Interactions
Inhibition and induction of CYP enzymes, par-
ticularly CYP3A4, are probably the most common
causes for documented drug interactions.[159,160]
Several prominent drugs have been withdrawn
from the market because of serious adverse effects
as a result of CYP-mediated interactions.[161,162]
Therefore, CYP-mediated drug interactions have
always been a major concern for clinicians and pa-
tients. Like CYP-mediated drug interactions, P-
glycoprotein-mediated drug interactions may be
anticipated when P-glycoprotein substrates and
P-glycoprotein inhibitors (or inducers) are co-
administered. Inhibition and induction of P-glyco-
protein in animals and humans have been reported,
although less frequently than for CYP enzymes,
and their pharmacokinetic consequences are sim-
ilar to those observed for inhibition and induction
of CYP enzymes.
© Adis International Limited. All rights reserved.
Lin & Yamazaki
5.1 P-Glycoprotein Inhibition Does
Not Follow Simple Kinetics
As discussed in section 1, P-glycoprotein has
more than one drug-binding site, even though the
exact number of binding sites is not yet known. In
addition, two ATP-binding domains are also in-
volved in the P-glycoprotein function of drug
transport. All of the drug-binding sites and ATP-
binding domains interact cooperatively as a func-
tional unit. Thus, inhibition of P-glycoprotein
transport of a drug by other drugs could potentially
result from either competition for drug-binding
sites or from blockage of the ATP hydrolysis pro-
cess. For example, verapamil inhibits the transport
function in a competitive manner without inter-
rupting the cyclic activity (ATP hydrolysis) of
P-glycoprotein, vanadate interacts with the ATP-
binding domains of P-glycoprotein without inter-
acting with the substrate-binding sites, and cyclo-
sporin inhibits transport function by interfering
with both substrate recognition and ATP hydroly-
sis.[163-166] These results clearly indicate that mul-
tiple mechanisms are responsible for P-glycopro-
tein inhibition.
Because of the complexity, it is difficult to as-
sess the mechanism and type of P-glycoprotein in-
hibition when P-glycoprotein substrate drugs and
P-glycoprotein inhibitors are given simultaneous-
ly. As shown in the following examples, the pattern
of P-glycoprotein inhibition appears to be sub-
strate-dependent. For instance, the P-glycoprotein-
mediated transepithelial transport of digoxin in
LLC-PK1 cells was inhibited by cyclosporin, but
digoxin surprisingly did not inhibit the P-glyco-
protein-mediated transepithelial transport of cyclo-
sporin.[153] Furthermore, in vitro studies with multi-
drug-resistant P388 leukaemia cells reveal that
verapamil competitively inhibits P-glycoprotein-
mediated daunomycin uptake, whereas vinblastine
shows noncompetitive inhibition with daunomy-
cin.[167] On the other hand, P-glycoprotein-medi-
ated transepithelial transport of vinblastine in
Caco-2 cells was inhibited by verapamil in a com-
petitive manner, and by dideoxyforskolin in a non-
competitive manner.[83] Similarly, the P-glycopro-
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
tein-mediated uptake of azidopine into plasma
membrane vesicles obtained from P-glycoprotein-
expressing multidrug-resistant cells was non-
competitively inhibited by vinblastine or by cyclo-
sporin, whereas vinblastine uptake into plasma
membrane vesicles was competitively inhibited by
cyclosporin.[164,168]
P-glycoprotein-mediated interactions have also
been investigated by measuring the displacement
of reversible binding to P-glycoprotein using
membrane vesicles in the absence of ATP. The
displacement of morphine binding by verapamil
was only partial, although vinblastine binding was
completely displaced by verapamil.[169] The ATPase
activity assay has also been used as a convenient
tool for determining the type of interaction be-
tween P-glycoprotein substrates. A systematic
analysis of inhibition patterns of verapamil-
induced P-glycoprotein ATPase activity revealed
that noncompetitive inhibition of verapamil-
stimulated ATPase activity was found with vana-
date, whereas competitive inhibition was found
with cyclosporin.[170] Similarly, in another ATPase
activity study, nicardipine displayed competitive
interaction with vinblastine but noncompetitive in-
teraction with verapamil.[171]
Although the competition of two substrates for
the same P-glycoprotein usually results in an in-
hibitory effect on the P-glycoprotein-mediated
transport of the substrates, activation of P-glyco-
protein-mediated efflux transport has been re-
ported in some cases. The P-glycoprotein-medi-
ated doxorubicin efflux out of multidrug-resistant
HCT-15 colon cells was significantly increased by
some flavonoids.[172] Similarly, rhodamine 123
and Hoechst 33 342 stimulated the rate of P-glyco-
protein-mediated transport of each other in P-gly-
coprotein-enriched plasma membrane vesicles iso-
lated from Chinese hamster ovary CHrB30 cells.[173]
Furthermore, the transport of Hoechst 33 342 was
stimulated by daunorubicin and doxorubicin, but
the transport of rhodamine 123 was inhibited by
daunorubicin and doxorubicin.[173] Stimulation
was also observed in ATPase activity studies. For
example, verapamil-induced ATPase activity was
© Adis International Limited. All rights reserved.
85
stimulated by progesterone, diltiazem, amitripty-
line and propranolol.[170] Collectively, these re-
sults suggest that two substrates are able to bind
simultaneously to P-glycoprotein at different sites
that may interact allosterically.
It is of interest to note that activation is not a
phenomenon limited to P-glycoprotein interac-
tion; activation of CYP3A4 has also been re-
ported.[174] Similarly, the concept of multiple bind-
ing sites has also been proposed to explain many
unusual enzyme kinetics observed for CYP3A4.[174]
The interaction between substrates and inhibitors
of CYP3A4 does not always follow simple enzyme
kinetics. Wang et al.[175] have demonstrated that
in vitro interaction patterns of CYP 3A4 substrates
are substrate-dependent. Mutual inhibition, partial
inhibition and activation have been observed in the
interactions of testosterone with terfenadine, tes-
tosterone with midazolam and terfenadine with
midazolam, respectively. To test the hypothesis of
multiple binding sites, Shou et al.[176] have suc-
cessfully described the enzyme kinetics of CYP-
3A4-mediated metabolism of diazepam and its de-
rivatives with a kinetic model which consists of
two substrate-binding sites (apoprotein) and one
catalytic site (prosthetic haem).
In summary, the interaction between P-glyco-
protein substrates does not always follow simple
kinetics. The pattern of P-glycoprotein interaction
can be classified into at least three major catego-
ries: competitive inhibition, noncompetitive inhi-
bition and co-operative stimulation. Competitive
inhibition suggests that two substrates act on the
same sites of P-glycoprotein and that only one or
the other can bind at any one time. Noncompetitive
inhibition means that two substrates are able to
bind simultaneously to P-glycoprotein molecule at
distinct sites that are functionally independent.
The situation can be even more complicated if al-
losteric effects are involved in interaction between
substrate and inhibitor. The complexity of the mo-
lecular mechanism for P-glycoprotein inhibition
prevents our ability to predict the potential of P-
glycoprotein-mediated drug-drug interactions,
either quantitatively or qualitatively.
Clin Pharmacokinet 2003; 42 (1)
86
5.2 Drug Interactions Caused by
P-Glycoprotein Inhibition
Because of overlapping substrate specificities
and inhibitors between CYP3A4 and P-glycopro-
tein, many drug interactions may involve both
CYP 3A4 and P-glycoprotein. Therefore, it is im-
portant to distinguish CYP3A4-mediated inhibi-
tion from P-glycoprotein-mediated inhibition in
order to make appropriate interpretation of drug
interaction data. For this reason, Wandel et al.[177]
have proposed to use the ratio of IC50 for CYP3A4
to IC50 for P-glycoprotein as an index of the rela-
tive selectivity of a drug for P-glycoprotein-medi-
ated inhibition versus CYP3A4-mediated inhibi-
tion. If the ratio is much greater than unity, it means
that the relative contribution by P-glycoprotein-
mediated inhibition is quantitatively more signifi-
cant.
Relative selectivity is best exemplified by the
following study by Choo et al.[178] The IC50 values
for ketoconazole to inhibit digoxin transport
and nifedipine metabolism were 1.2 and 0.15
μmol/L, respectively, and the corresponding val-
ues for LY-335979 were 0.024 and 5 μmol/L.[178]
In mdr1a (+/+) mice, pretreatment with LY-
335979 (25 mg/kg intravenously) resulted in a 15-
fold increase in brain concentrations of nelfinavir,
but had little effect on plasma concentrations. On
the other hand, coadministration of ketoconazole
(50 mg/kg intravenously) caused an 8.5-fold in-
crease in brain concentrations of nelfinavir and a
3.5-fold increase in plasma concentrations. From
the ratio of IC50 values (208 for LY-335979 and
0.13 for ketoconazole), it is clear that the increased
brain concentrations of nelfinavir by LY-335979
are caused mainly by P-glycoprotein inhibition. In
contrast, the inhibitory effect of ketoconazole on
the brain and plasma concentrations of nelfinavir
is attributed mainly to CYP3A-mediated inhibi-
tion, and to a lesser extent to P-glycoprotein inhi-
bition.
As shown in the above study, it is clear that
coadministration of a P-glycoprotein inhibitor
causes a much greater increase in drug concentra-
tion in brain than in plasma. Extensive inhibition
© Adis International Limited. All rights reserved.
Lin & Yamazaki
of BBB P-glycoprotein function was also observed
when valspodar or GF-120918 (potent P-glycopro-
tein inhibitors) were coadministered with other
drugs in mice. Pretreatment with GF-120918 (250
mg/kg/day for 4 days, orally) led to a 13-fold in-
crease in brain concentrations, but only a modest
increase (2-fold) in plasma concentrations, of
amprenavir in mdr1a (+/+) mice.[179] In another
study, a 10-fold increase in brain concentrations of
digoxin was also observed when valspodar 50
mg/kg was given orally to mdr1a (+/+) mice.[180]
Similarly, pretreatment with intravenous valspo-
dar 25 mg/kg resulted in an 80-fold increase in
brain concentrations of nelfinavir in mdr1a (+/+)
mice.[178]
These experimental findings present a major
problem that may confound attempts to use P-gly-
coprotein modulators in the clinical setting.
Although the use of effective P-glycoprotein mod-
ulators (P-glycoprotein inhibitors), such as LY-
335979, valspodar and FG-120918, may improve
the treatment of cancers, these P-glycoprotein
modulators also inhibit P-glycoprotein function in
normal cells, resulting in increased toxicity. This
is particularly true for the brain; the profound in-
crease of drug concentration in the brain by these
P-glycoprotein modulators increases the risk of
neurotoxicity.
The increased neurotoxicity caused by P-glyco-
protein modulators is best exemplified by a clinical
study with loperamide, an antidiarrhoeal agent.[181]
Loperamide 16mg was administered to healthy
male volunteers with or without coadministration
of quinidine 600mg. Loperamide produced no re-
spiratory depression when administered alone, but
respiratory depression occurred when loperamide
was given with quinidine. Since loperamide is a
P-glycoprotein substrate, under normal conditions
the brain penetration of this drug is limited as a
result of P-glycoprotein extrusion. However, in the
presence of quinidine (a potent P-glycoprotein in-
hibitor), the deliver of loperamide to the brain in-
creases, resulting in serious neurotoxicity.
Inhibition of hepatic and intestinal P-glycopro-
tein has also been reported. The inhibitory effect
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
of valspodar on hepatic and intestinal P-glycopro-
tein was compared in wild-type mice and mdr1a/1b
(–/–) double knockout mice, using digoxin as a
model compound.[180] As expected, oral pretreat-
ment with valspodar 50 mg/kg led to a marked de-
crease in both intestinal and biliary excretion of
digoxin in wild-type mice. However, appreciable
residual biliary excretion of digoxin (14% of an
intravenous dose) was still observed in mdr1a/1b
(–/–) double knockout mice, and pretreatment with
valspodar caused an unexpected decrease in biliary
excretion of digoxin (<5%) in these animals. These
results suggested that other unidentified transport
systems, rather than P-glycoprotein, may also be
involved in the biliary excretion of digoxin in
mice, and that these unidentified transporters can
be inhibited by valspodar. An important lesson to
be learned from these studies is that the under-
lying mechanisms for drug interactions can be
very complicated, and care should be exercised to
make appropriate interpretations.
P-glycoprotein inhibition as a cause of drug
interaction has also been reported in humans. Per-
haps the most compelling clinical evidence of
P-glycoprotein-mediated drug interactions in hu-
mans is the interaction of digoxin with other car-
diac drugs, such as verapamil and quinidine.[182-185]
A daily dosage of verapamil 160mg caused a 40%
increase in digoxin plasma concentration, whereas
a daily dosage of 240mg caused a 60–80% in-
crease, suggesting dose-dependent P-glycoprotein
inhibition. Because digoxin is exclusively elimi-
nated in humans by renal excretion as unchanged
drug,[186] it is highly likely that the observed drug
interaction between digoxin and verapamil (or
quinidine) is due to inhibition of P-glycoprotein
activity, resulting in increased absorption and de-
creased elimination of digoxin. Consistent with the
clinical data, increased absorption of digoxin by
verapamil and quinidine has been shown in
rats,[187,188] and decreased renal excretion of dig-
oxin by verapamil and quinidine has been demon-
strated in in vitro studies using isolated perfused
rat and dog kidney.[156,157]
© Adis International Limited. All rights reserved.
87
An important issue related to P-glycoprotein in-
hibition is the concept of P-glycoprotein modula-
tion in cancer research. Resistance of tumour cells
to chemotherapeutic agents is a major problem in
the treatment of human cancers. Tumour cells that
exhibit multidrug resistance are often associated
with overexpression of P-glycoprotein. In 1981,
Tsuruo et al.[189] reported that verapamil, a potent
P-glycoprotein inhibitor, is able to restore the in
vitro sensitivity to vincristine in multidrug-resis-
tant cell lines by inhibiting P-glycoprotein-medi-
ated drug transport. This finding implies that clin-
ical drug resistance can be circumvented through
the concomitant administration of P-glycoprotein
inhibitors and anticancer drugs. The idea of revers-
ing P-glycoprotein-mediated drug resistance has
led to intensive efforts to develop potent and spe-
cific P-glycoprotein modulators, such as valspo-
dar, FG-120918 and LY-335979. As expected, P-
glycoprotein-mediated interactions were observed
when anticancer drugs were given with these po-
tent P-glycoprotein modulators in cancer patients.
For example, both valspodar and FG-120918
caused an increase in systemic exposure to doxo-
rubicin associated with a decrease in doxorubicin
clearance in patients.[190,191] However, the phar-
macodynamic results from clinical modulation
studies are less than encouraging.[192]
One of the main reasons for the clinical disap-
pointment is that the coadministration of P-glyco-
protein modulators with anticancer drugs fails to
improve the toxicity profiles of the chemothera-
peutic agents. Although the P-glycoprotein modu-
lators might completely inhibit P-glycoprotein
function in tumour cells and restore drug sensitiv-
ity, the modulators could also inhibit the P-glyco-
protein protective function of normal cells, leading
to cytotoxicity. As shown in the mouse studies dis-
cussed above, treatment with P-glycoprotein mod-
ulators resulted in a much greater increase in drug
concentration in tissues than in plasma. Therefore,
the lack of improvement of toxicity profiles might
result from the increased tissue distribution of
drugs caused by P-glycoprotein modulators. The
digoxin intoxication by quinidine and verapamil
Clin Pharmacokinet 2003; 42 (1)
88
can also be explained by the increased drug con-
centration in tissues. During combination therapy
with digoxin and verapamil (or quinidine), both
verapamil and quinidine enhanced digoxin toxicity
even when digoxin plasma concentrations were in
the therapeutic range observed in the absence of
the P-glycoprotein inhibitors.[184,193,194] Unless a
specific P-glycoprotein modulator that selectively
inhibits tumour cell P-glycoprotein can be devel-
oped, manipulation of the balance between tumour
cell kill and toxicity to normal tissue will be very
difficult to achieve.
5.3 P-Glycoprotein Induction
is a Complex Process
Like some of the CYP isoenzymes, the expres-
sion of P-glycoprotein is inducible. Because CYP
enzymes and P-glycoprotein function together to
protect the body from accumulation of toxins and
xenobiotics, it is not surprising, from an evolution-
ary point of view, that both defensive systems pos-
sess adaptive manoeuvres to reinforce their protec-
tive role.
Regulation of P-glycoprotein expression in re-
sponse to inducers has been extensively studied in
vitro using cell lines derived from animals and hu-
mans. Fardel et al.[195] reported that 3-methylchol-
anthrene strongly induced functional P-glycopro-
tein levels in a dose-dependent manner through an
increased expression of the mdr gene in rat liver
epithelial cells. In another study, mdr RNA levels
were found to increase substantially in rat and
mouse cell lines following acute exposure to cyto-
toxic drugs, such as doxorubicin, daunomycin and
mitoxantrone.[196] Interestingly, although these
cytotoxic drugs induced mdr RNA in all rat and
mouse cell lines, they had no effects in human cell
lines. These results suggest species differences in
inductive response to P-glycoprotein inducers.
Species differences are well known for the induc-
tion of CYP enzymes,[159] and recently it has been
demonstrated that the species differences in
CYP3A induction are due mainly to sequence dif-
ferences in the ligand-binding domain of pregnane
X receptor (PXR).[197] As will be discussed below,
© Adis International Limited. All rights reserved.
Lin & Yamazaki
P-glycoprotein is also likely to be regulated by
PXR, and therefore it is possible that the observed
species differences in CYP3A induction and P-gly-
coprotein induction have a similar molecular basis.
Dexamethasone has been shown to induce the
mdr1a and mdr1b P-glycoproteins in a mouse cell
line.[198] Similarly, the inductive effect of dexa-
methasone on P-glycoprotein expression in cul-
tured rat hepatocytes has been reported.[199] Con-
sistent with in vitro observations, pretreatment of
rats with dexamethasone (40 mg/kg/day orally for
3 days) resulted in significant increases in both in-
testinal and hepatic P-glycoprotein expression by
approximately 2- to 3-fold.[143] In another study, a
5-fold increase of rat hepatic P-glycoprotein level
was reported after intraperitoneal administration
of dexamethasone at 100 mg/kg/day for 4 days.[200]
These results suggest that the induction of P-gly-
coprotein expression is a dose-dependent process.
Similarly, dose-dependent induction of P-glyco-
protein expression was observed in rats receiving
cyclosporin orally at 2, 10 or 30 mg/kg/day, or
subcutaneously at 1, 5 or 15 mg/kg/day, for 28
days.[201] Western blot analysis showed that cy-
closporin induced renal P-glycoprotein in a dose-
dependent manner. In another study, the induction
of P-glycoprotein expression by cyclosporin was
demonstrated to be not only dose-dependent, but
also time-dependent. When cyclosporin was given
subcutaneously at 10 mg/kg/day for 5, 10 and 15
days, the increase of the P-glycoprotein levels in
liver, intestine, kidney and lungs was maximal after
10 days of treatment in most tissues of rats.[202]
Interestingly, the inductive response to P-gly-
coprotein inducers also appears to be tissue-
dependent. After administration of cyclosporin 10
mg/kg/day for 15 days, an increase in the expres-
sion of P-glycoprotein was detected in many, but
not all, tissues of rats.[202] In kidneys, intestine,
stomach, liver and lungs, the amount of P-glyco-
protein detected was increased by 256, 239, 161,
144 and 69% compared with control groups, re-
spectively. In all of these tissues, the increase was
maximal after 10 days of treatment. In heart, testis,
and spleen, the increase in P-glycoprotein expres-
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
sion was 82, 74 and 50% compared with control
groups, respectively, and the induction was maxi-
mal after 5 days of treatment in heart and after 15
days of treatment in testis and spleen. However, no
change in the level of P-glycoprotein expression
was found in the brain during treatment. The lack
of P-glycoprotein induction in the brain could be
due to the high basal level of P-glycoprotein in the
brain capillaries. The high level of P-glycoprotein
in this tissue could cause a rapid extrusion of
cyclosporin from the capillary endothelial cells,
leaving no opportunity for this compound to in-
duce P-glycoprotein. Similarly, tissue differences
in P-glycoprotein induction were also observed af-
ter administration of cycloheximide to rats.[203]
Cycloheximide caused an 8-fold increase in mRNA
of mdr1a P-glycoprotein in lung, but only a 1.5-
fold increase was observed for small intestine. Col-
lectively, these results clearly point to a tissue-
dependent inductive response to P-glycoprotein
inducers. However, the mechanisms underlying
tissue differences in the inductive response require
further investigation.
Recently, Durr et al.[204] have reported that St
John’s Wort induces P-glycoprotein and CYP3A
enzymes in rats and humans. Administration of St
John’s Wort extract to rats at an oral dose of 1000
mg/kg/day for 14 days resulted in a 3.8-fold in-
crease of intestinal P-glycoprotein and a 2.5-fold
increase of hepatic CYP3A2 expression level. Oral
administration of St John’s Wort extract to eight
healthy male volunteers for 14 days at a dose of
300mg three times daily resulted in a 1.4- and 1.5-
fold increase of duodenal P-glycoprotein and
CYP3A4, respectively. These results suggest the
coordinated regulation of P-glycoprotein and
CYP3A enzymes in both rats and humans. The in-
ducing effect of St John’s Wort on intestinal P-gly-
coprotein was considerably more pronounced in
rats than in humans. These differences most prob-
ably reflect a dose-dependent effect, because the
rats received a dose of St John’s Wort that was
approximately 50 times higher per kilogram body-
weight than that received by the volunteers. An
alternative explanation could be that species dif-
© Adis International Limited. All rights reserved.
89
ferences exist in the susceptibility to P-glycopro-
tein induction. Similarly, co-induction of P-glyco-
protein and CYP3A enzymes by dexamethasone
was also observed in rats.[143]
Consistent with in vivo observations, coordi-
nated regulation between CYP3A and P-glycopro-
tein has been reported in vitro in animal and human
cell lines. In a cell line derived from human colon
adenocarcinoma LS 180/WT and its doxorubicin-
resistant subline (LS 180/AD 50), both P-glyco-
protein and CYP3A4 were induced after treatment
with many known inducers, including phenobar-
bital, rifampicin (rifampin), clotrimazole and re-
serpine.[205] Similarly, dexamethasone, a potent
CYP3A4 inducer, has been shown to induce P-
glycoprotein in human hepatoma.[199,206] Collec-
tively, these in vitro and in vivo observations have
led to the speculation that regulation of CYP3A4
and MDR1 gene expression is coordinated through
a similar mechanism. The possible coordinate reg-
ulation of P-glycoprotein and CYP3A4 gene ex-
pression was further hypothesised by Wacher et
al.[207] based on the proximity of the chromosomal
loci of these two genes. The human MDR gene has
been mapped to chromosome locus 7q21.1,[208]
whereas the CYP3A4 gene is located at 7q22.1.[209]
Recent in vitro studies have demonstrated that
the PXR (also known as SXR) plays a central role
in regulating CYP3A4 transcription.[210] A diverse
array of drugs, including rifampicin, phenobarbi-
tal, clotrimazole and hyperforin (one of the constit-
uents of St John’s Wort) are known to activate the
nuclear receptor PXR and induce CYP3A4 expres-
sion.[211-213] It has been speculated that induction
of P-glycoprotein is also attributed to the same
PXR activation. However, direct evidence was
only made available recently, when it was shown
that the PXR activates the expression of the MDR1
gene.[214,215] Using pharmacological and genetic
approaches, Synold et al.[214] showed that the PXR
is activated by paclitaxel and rifampicin, and this
activation results in an increase in MDR1 mRNA
and P-glycoprotein expression in primary human
hepatocytes and LS 180 colon cancer cells. Pacli-
taxel and rifampicin also induce CYP3A4 and
Clin Pharmacokinet 2003; 42 (1)
90
CYP2C8 mRNA and enzyme protein expressions
in primary human hepatocytes through PXR-
mediated transcriptional effects. These findings
strongly suggest that PXR plays an important role
in regulating drug-metabolising enzymes and the
P-glycoprotein transporter.
The molecular mechanism of PXR-mediated P-
glycoprotein induction by rifampicin has been
studied in detail by Geick et al.[215] using the hu-
man colon carcinoma cell line LS174T. By using
DNA binding assay and transfections, it was
shown that the induction of MDR1 is mediated by
a DR4 nuclear response element at about –8
kilobase pairs of the MDR1 upstream region to
which PXR binds. The above two studies provide
direct evidence that a similar mechanism is respon-
sible for induction of CYP3A4 and MDR1 by
xenobiotics. Interestingly, in addition to the liver
and intestine, the nuclear receptor PXR and P-gly-
coprotein are co-expressed in a number of other
tissues, including the kidney and placenta.[211,216]
The question as to whether the tissue-dependent
differences in P-glycoprotein induction are attrib-
uted to the tissue differences in the expression level
of PXR, or to different mechanisms in different
tissues, remains to be explored.
Although it is evident that the nuclear receptor
PXR plays a central role in P-glycoprotein induc-
tion, the inductive effects of a single drug may be
mediated by multiple mechanisms. Recently,
Lee[203] reported that the induction of rat mdr1a
and mdr1b mRNA transcripts by cycloheximide is
mediated by different mechanisms. The induction
of mdr1a P-glycoprotein is transcriptionally reg-
ulated, whereas the induction of mdr1b P-glyco-
protein is post-transcriptionally regulated. The
post-transcription is probably due to mRNA stabi-
lisation, although it is currently not known how the
mRNA is stabilised. Clearly, the regulatory pro-
cesses of P-glycoprotein induction are very com-
plex and every inducer has its own pattern of in-
duction. Therefore, a detailed knowledge of the
inductive processes for each inducer is required in
order to understand its implications and conse-
quences.
© Adis International Limited. All rights reserved.
Lin & Yamazaki
5.4 Drug Interactions Caused by
P-Glycoprotein Induction
The most compelling evidence to date for P-gly-
coprotein induction as a cause of drug interactions
was provided by Greiner et al.[217] in a clinical
study comparing the pharmacokinetics of digoxin
before and during coadministration of rifampicin
600 mg/day for 10 days in eight healthy volunteers.
The plasma Cmax and AUC of digoxin decreased
from 5.4 μg/L and 55 μg • h/L before rifampicin
pretreatment to 2.6 μg/L and 38 μg • h/L, respec-
tively, during rifampicin pretreatment when the
volunteers received a single oral dose of digoxin
1mg. However, pretreatment with rifampicin had
little effect on the AUC and renal clearance of dig-
oxin after intravenous administration. Duodenal
biopsies were obtained from each volunteer before
and after administration of rifampicin. Rifampicin
treatment increased intestinal P-glycoprotein con-
tent 3.5-fold, which correlated inversely with the
oral AUC of digoxin. Since digoxin is eliminated
exclusively by renal excretion, and not by metabo-
lism, and since digoxin is given orally at a very low
dose, these results strongly suggest that the dig-
oxin-rifampicin interaction mainly occurs at the
level of the intestine through P-glycoprotein-
mediated induction. This means that the decreased
plasma concentration of digoxin during rifampi-
cin treatment is caused by reduced bioavailability
of digoxin as a result of induction of intestinal P-
glycoprotein. Similarly, administration of St John’s
Wort extract (three doses of 300mg per day for 14
days) resulted in an 18% decrease of plasma AUC
after a single digoxin dose of 0.5mg in healthy vol-
unteers.[204] Treatment with the extract resulted
in a 40% increase in the expression of duodenal
P-glycoprotein. The decreased plasma AUC corre-
lated reasonably well with the increased intestinal
P-glycoprotein expression.
Pretreatment with rifampicin also significantly
decreased systemic exposure to fexofenadine in
healthy volunteers.[218] Twenty volunteers received
a 60mg oral dose of fexofenadine before and after
treatment with oral rifampicin 600mg for 6 days.
The Cmax and AUC of fexofenadine decreased by
Clin Pharmacokinet 2003; 42 (1)
P-Glycoprotein
2- and 3-fold, respectively, in volunteers after ri-
fampicin treatment. With the assumption that
fexofenadine is predominantly eliminated by bil-
iary excretion, but not by metabolism, the investi-
gators concluded that the decreased plasma pro-
files are the result of a reduced bioavailability
caused by induction of intestinal P-glycoprotein.
However, the assumption of lack of metabolism
may not be valid. From a human mass balance
study, it was shown that approximately 80 and
11% of an oral dose of [14C]fexofenadine were re-
covered in the faeces and urine, respectively.[219]
MDL-4,829, a metabolite of fexofenadine, ac-
counted for about 20% of the radioactivity in urine.
However, it is unknown whether the faecal com-
ponent represents unabsorbed drug or the result of
biliary excretion, and information on the metabo-
lites in faeces is not available.[219] If based on the
urinary recovery, metabolism of fexofenadine is
not insignificant. Therefore, it is possible that the
observed interaction between fexofenadine and
rifampicin is due to a combination of both CYP-
mediated and P-glycoprotein-mediated induction.
Furthermore, it has recently been demonstrated that
organic anion transporting polypeptide (OATP)
is involved in the hepatic uptake of fexofenad-
ine.[220] It is possible that rifampicin is able to in-
duce OATP. Thus, the involvement of OATP in
the hepatobiliary excretion of fexofenadine may
further complicate the interpretation of the ob-
served interaction between fexofenadine and ri-
fampicin.
The observed interaction between cyclosporin
and rifampicin in healthy volunteers is also prob-
ably due to a combination of both CYP3A4 and
P-glycoprotein induction.[221] The pharmacokinet-
ics of cyclosporin were studied in six healthy vol-
unteers after administration of cyclosporin orally
(10 mg/kg) and intravenously (3 mg/kg) with and
without rifampicin pretreatment (600 mg/day for
11 days). Blood clearance of cyclosporin increased
from 0.3 L/h/kg (5 ml/min/kg) before rifampicin
treatment to 0.42 L/h/kg (7 ml/min/kg) during ri-
fampicin treatment, and bioavailability decreased
from 27% without rifampicin to 10% with rifam-
© Adis International Limited. All rights reserved.
91
picin. Rifampicin not only increased elimination
clearance of cyclosporin, but also decreased its
bioavailability to a greater extent than would have
been predicted by the increased clearance. Since
cyclosporin is a substrate for both CYP3A4 and
P-glycoprotein, and since rifampicin can induce
both CYP3A4 and P-glycoprotein, the increased
clearance and decreased bioavailability of cyclo-
sporin during rifampicin treatment is most proba-
bly due to a combination of CYP3A4 and P-glyco-
protein induction.
Because of overlapping substrate specificity
between CYP3A4 and P-glycoprotein, and be-
cause of the similarities in the inhibitors and induc-
ers between these two proteins, many drug inter-
actions may involve both P-glycoprotein and
CYP3A4. Unless the relative contribution of P-
glycoprotein and CYP3A4 to overall drug interac-
tions can be quantitatively differentiated, care
should be taken in interpreting data for drug inter-
actions, particularly in terms of the underlying
mechanisms. Although attempts have been made
to quantify the relative contribution of CYP3A4
and P-glycoprotein to overall interaction,[222] there
is still no simple way by which the relative contri-
bution of these two systems can be quantified be-
cause of the complexity of the interplay involved
between intestinal and hepatic CYP3A4 and P-gly-
coprotein.
6. Conclusions
Although the physiological function of P-gly-
coprotein is still not fully understood, the role of
this efflux transporter in drug absorption and dis-
position is becoming increasingly defined. P-gly-
coprotein is highly expressed in various tissues,
and the anatomical localisation of P-glycoprotein
in relation to the sequences of drug movement (cel-
lular uptake, intracellular distribution, metabolism
and excretion) is a very important factor in deter-
mining P-glycoprotein function. From transgenic
mouse studies, it appears that P-glycoprotein has a
greater impact on limiting cellular uptake of drugs
from the blood circulation into the brain and pla-
centa, and from intestinal lumen into epithelial
Clin Pharmacokinet 2003; 42 (1)
92
cells, than on enhancing the excretion of drugs in
the liver and kidney into the adjacent luminal
space. Although the transgenic mdr1a (–/–) and
mdr1a/1b (–/–) mice provide a powerful tool for
studying drug absorption and disposition, the inter-
pretation of the data derived from the transgenic
mouse studies are not always straightforward, and
therefore should be viewed carefully.
Inhibition and induction of P-glycoprotein have
been reported as the cause of drug-drug interac-
tions. Although the magnitude of the reported P-
glycoprotein-mediated drug interactions, based
on plasma AUC, appears to be quantitatively less
important compared with CYP-mediated drug in-
teractions, the potential risk of P-glycoprotein-
mediated drug interactions might be underesti-
mated if only the plasma concentration is monitored.
From animal studies, it is clear that P-glycoprotein
inhibition has a much greater impact on tissue dis-
tribution, particularly with regard to the brain and
placenta, than on plasma concentration of drugs.
Therefore, one should carefully assess the poten-
tial risk of P-glycoprotein-mediated drug interac-
tions when potent P-glycoprotein inhibitors and P-
glycoprotein substrates are administered together.
Another important lesson to be learned from the
literature is that pharmacokinetic drug interactions
often involve multiple mechanisms. Because of
overlapping substrate specificity between CYP-
3A4 and P-glycoprotein, and because of similari-
ties in P-glycoprotein and CYP3A4 inhibitors and
inducers, many drug interactions involve both P-
glycoprotein and CYP3A4. Unless the relative
contribution of P-glycoprotein and CYP3A4 to
overall drug interactions can be quantitatively es-
timated, care should be taken when exploring the
underlying mechanism of drug interactions.
Acknowledgements
The authors have provided no information on sources of
funding or on conflicts of interest directly relevant to the
content of this review.
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Correspondence and offprints: Dr Jiunn H. Lin, Drug Meta-
bolism, Merck Research Laboratories, West Point, WP75A-
203, PA 19486, USA.
E-mail: jiunn_lin@merck.com
Clin Pharmacokinet 2003; 42 (1)