PersPecTives

estimation of the accuracy of important

OpiniOn
pharmacokinetic parameters, such as liver
and renal clearance, can be improved.
Coexistence of passive and In the following sections, we will first
discuss the basics of membrane permeability
carrier-mediated processes and the experimental methods applied in
the measurement of passive transport and
in drug transport carrier­mediated transport. We will then give
an overview on the role of joint contribution
of passive transport and carrier­mediated
Kiyohiko Sugano, Manfred Kansy, Per Artursson, Alex Avdeef, Stefanie Bendels, transport to the total permeation of a drug,
Li Di, Gerhard F. Ecker, Bernard Faller, Holger Fischer, Grégori Gerebtzoff, focusing on in vitro and in vivo results.
Finally, some implications for drug discovery
Hans Lennernaes and Frank Senner
and development will be discussed.
Abstract | The permeability of biological membranes is one of the most important
determinants of the pharmacokinetic processes of a drug. Although it is often Basics of membrane permeation
Passive transcellular transport. Biological
accepted that many drug substances are transported across biological membranes
cellular membranes are fluidic patches com­
by passive transcellular diffusion, a recent hypothesis speculated that carrier- prising various bilayer­forming amphiphilic
mediated mechanisms might account for the majority of membrane drug transport phospholipids, cholesterol and membrane­
processes in biological systems. Based on evidence of the physicochemical anchored proteins — including transporters
characteristics and of in vitro and in vivo findings for marketed drugs, as well as — that form thin (~5 nm) hydrophobic
barriers separating aqueous environments.
results from real-life discovery and development projects, we present the view that
Consequently, lipid bilayer and carrier
both passive transcellular processes and carrier-mediated processes coexist and proteins coexist in a cellular membrane
contribute to drug transport activities across biological membranes. when the transporter is expressed in the
cells. In this article, the passive transcellular
The permeability of biological membranes permeation for many (but not all) drugs transport (for uptake, efflux, absorptive and
is a key determinant of drug pharmaco­ in many (but not all) cell types, at least in excretive directions (BOX 1)) is defined as a
kinetics. Historically, passive diffusion intestinal membrane permeation. Carrier­ concentration gradient­driven mass transport
through the lipid bilayer portion of a mediated transport is probably important of a compound (called the permeant, which
biological membrane was suggested as for drugs with low passive permeation, includes drug and drug­like molecules) from
the dominant route. However, in the past especially for brain and liver distribution, one side of the cellular membrane to the other
couple of decades, many carrier proteins and in renal excretion. However, even in through the lipid bilayer portion (FIG. 1).
(transporters) have been discovered to those three cases, passive diffusion coexists. As the fluidic lipid bilayer portion of a
transport various drugs. Consequently, Drug absorption across the intestinal membrane does not have specific binding
it would be interesting to discuss whether epithelium will be used as an example in sites, passive transcellular transport is usually
carrier-mediated transport is the ubiquitous this article because abundant drug transport not saturable, not subject to inhibition
and dominant mechanism of biological studies have been done in this tissue. and less sensitive (or not sensitive at all)
membrane permeation for most drugs in The theory for intestinal transport is to the stereospecific structure of a drug
most organs1,2 or whether it is a specialized qualitatively valid for other tissues in vivo (BOX 1). As the centre of the lipid bilayer is
mechanism for a certain drug in a certain (the quantitative contribution of each highly hydrophobic, a compound diffuses
cell type. process differs case by case, by tissues and across the lipid bilayer portion mainly as an
Based on a literature review described drugs, in accordance with the varying uncharged and largely desolvated species,
in this article, we conclude that passive levels of transporters expressed in different depending on its molecular size and affinity
transport and carrier­mediated transport tissues, and the drug concentration at the to the centre of the lipid bilayer (known as
coexist in biological membrane permeation biological membrane and the binding sites). its lipophilicity). Therefore, passive trans­
to various extents. The weight of contribu­ We are aware that there is also large interest cellular transport generally depends on the
tion from each route to the total membrane in understanding carrier­mediated trans­ uncharged fraction of a permeant (that is,
permeation should be carefully considered port in other organs, especially in the liver, it depends on its pH and pKa) and the whole
on a case by case basis (for each drug and the kidney and the brain3. Once carrier­ molecule physicochemical property of a
each cell membrane). Passive transport is mediated transport mechanisms can be permeant such as its octanol–water partition
probably the primary route of membrane properly quantified in these organs, an coefficient, its total hydrogen bond strength

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PersPectives
Box 1 | Terminology of transport routes and direction of transport
A schematic representation is shown of how compounds can permeate the intestinal membrane.
Transcellular permeation is the route passing through the epithelial cells (that consists of passive
transcellular and carrier-mediated permeation). Paracellular permeation is the route passing
between the cells. The aqueous boundary layer (ABL) is adjacent to a membrane and could be a
permeation barrier. Uptake refers to transport into a cell and efflux refers to transport out of a cell.
Apical (A) side Intestinal membrane Basolateral (B) side
Paracellular permeation (passive)
ABL permeation (passive)
Passive transcellular Passive transcellular
transport transport
Carrier-mediated Carrier-mediated
uptake efflux
Carrier-mediated Carrier-mediated
efflux uptake
Tight junction
Concentration gradient
Absorptive direction (A to B)
Excretive direction (B to A)
and its molecular size (TABLE 1), whereas Natureall
is common to almost Reviews Drug Discovery
cells. |Therefore,
stereochemically­specific interactions basic passive transcellular transport occurs
determine the carrier­mediated transport. regardless of cell type (for example between
This is called the pH partition theory4,5. in vivo organs and in vitro cells). The extent
When pKa is neglected, the correlation of passive transcellular transport could be
between the lipophilicity of a compound dependent on the lipid composition of the
and the biological membrane permeation membrane, but is usually of comparable
can become unclear for compounds that magnitude between different cell types.
undergo ionization6,7. In addition, because For instance, Caco-2 cells (derived from the
passive transcellular transport is a diffusion human colon) and madin–Darby canine
process, after normalizing for lipophilicity, kidney (mDCK) cells show similar mag­
a correlation with the molecular size of a nitudes of passive transcellular transport 12.
compound (the molecular size correlates with Therefore, in general, a lipophilic drug
the diffusion coefficient) can be observed8,9. that displays a high passive transcellular
This dependency on molecular size that is transport across the intestinal epithelial cell
observed in passive transcellular permeation membrane may also display a high passive
should not be confused with that in paracell­ transcellular transport across, for example,
ular or aqueous­pore permeation. However, the sinusoidal and canalicular cell membranes
the molecular size dependency of passive of the hepatocyte.
transcellular transport is usually less sig­
nificant than lipophilicity dependency, and Carrier-mediated transport. A membrane
often is difficult to detect for loosely packed transport process involving a protein in the
membranes such as the intestinal membrane. transcellular permeation process is called
But it could be observed for the more densely carrier­mediated in this article (the protein
packed blood–brain barrier 10,11. is called the transporter). There are more
Although there are variations in the than 400 membrane transporters, and these
lipid composition of membranes between belong to two major superfamilies of mem­
different cell types, the lipid bilayer portion brane transporters: the ATP­binding cassette
598 | AugusT 2010 | vOlume 9
© 2010 Macmillan Publishers Limited. All rights reserved
(ABC) and the solute carrier (slC) families.
About two dozen of these are of clinical rele­
vance with regard to drug disposition and/or
side effects3.
If the transport process directly or
indirectly consumes energy (ATP), it is
said to be active and may not require a
concentration gradient of a permeant.
The ABC transporter superfamily requires
binding and hydrolysis of ATP to function
and to mediate the primary active transport
process. many uptake transporters of
the slC superfamily 13,14 are driven by ion
gradients created by ATP­dependent
primary transporters, such as the na+/
K+­ATPase. These co­transporters are said
to mediate secondary active transport.
When carrier­mediated transport is not
energy driven, it is defined as a facilitated
transport process and relies on a concentra­
tion gradient of a substrate, as well as a
transporter protein. unlike passive trans­
cellular transport, the carrier­mediated
process is expected to be saturable (BOXEs 2,3,
see supplementary information s1 (table)
for examples). This occurs when the total
number of permeant molecules exceeds
the number of carrier protein binding sites
available for transport (that is, when the
concentration of a substrate is higher
than the michaelis constant (Km) (BOX 3)).
Carrier­mediated transport is also subject to
inhibition. As carrier proteins have a more
rigid structure than fluidic lipid bilayers and
as carrier proteins are made of chiral amino
acids, carrier­mediated transport is stereo­
specific and in many cases enantioselective,
whereas the molecular properties determining
passive transcellular membrane transport are
generally identical between enantiomers.
Therefore, even though the physical
forces (for example, a hydrogen bond)
governing both inter­molecular interactions
(that is, drug–carrier protein and drug–lipid
bilayer) are the same, the spatial (stereo)
patterns are different between passive and
carrier­mediated transport. Hence, for
passive transport, molecular chemical
descriptors (such as total hydrogen bond
strength, lipophilicity and dipole/polariz­
ability) are often used to investigate the
quantitative structure–permeation relation­
ship (QsPr)15. By contrast, for carrier­
mediated transport, stereostructure specific
QsPr (three­dimensional QsPr) and/
or structure­based drug design approaches
tend to be used16,17. In the literature on
carrier­mediated transport, dependence on
lipophilicity is not well documented, but can
be identified in a narrow range of chemical
structural classes. most importantly,
www.nature.com/reviews/drugdisc
 PersPectives

Octanol–water Artificial
partitioning membranes Cell culture systems

a b c d e f
Carrier-
Transcellular Paracellular mediated Cell suspension Membrane
or adherent cell vesicles

Cell
monolayer Transport
protein

Passive transmembrane Passive transcellular and paracellular transport Passive transmembrane

transport transport

Figure 1 | The potential of a drug to pass biological membranes by
passive and carrier-mediated processes can be assessed by multiple
techniques. Dotted and solid arrows and lines indicate passive and carrier-
mediated transport routes, respectively. a | Organic solvent–water par-
titioning systems (for example, octanol, hexadecane) are often used to
model the distribution of drugs into the cell membrane, rather than their
permeation across the cell membrane. b | Artificial membranes are available
in different formats and compositions, such as black lipid membranes231,
unilamellar vesicle liposomes232,233 or parallel artificial membrane per-
meation assays. Artificial membranes allow transport studies across the lipid
bilayer through a passive transmembrane route in isolation. Membrane
composition can influence compound distribution and transport234 and
transmembrane pH gradients are important for the distributions of acids
and bases, thus supporting the importance of compound partitioning for
membrane passage234,235. c | A monolayer-forming cell line with passive
transcellular and passive paracellular pathways, and negligible carrier-
mediated transport. some cell lines originating from epithelial and
endothelial membranes differentiate into cell monolayers that form barriers
resembling the physiological barriers of, for example, the intestinal
Active/carrier-mediated transport
epithelium or the blood–brain barrier endothelium. The monolayer-forming
cells provide a paracellular transport routeNature
between the cells.
Reviews | Drug The para-
Discovery
cellular route is important for the permeation of small hydrophilic drugs in
leaky barriers, such as the upper small intestine, whereas it lacks significance
in tighter barriers, such as the colon or the blood–brain barrier. d | A mono-
layer-forming cell line with both passive drug transport and carrier-mediated
drug transport routes. A cell monolayer which expresses a carrier protein
can be used to investigate both passive transport and carrier-mediated
transport processes. To examine the contribution of passive transport, mock
transfected cell lines are usually used as controls. e | A suspension or an
adherent cell line that overexpresses either an uptake or an efflux transport
protein, for example, HeK293 cells236. cell lines that do not form monolayers
are also used for in vitro studies of transport processes. However, these
studies are limited to uptake into the cells or efflux out of the cells, rather
than transport across monolayers. Their most common application is in
studies of carrier-mediated drug transport. f | inverted membrane vesicles
from a cell line expressing an efflux transport protein. As they are inverted,
the vesicles allow efflux protein-mediated accumulation of a substrate
in the vesicles, which simplifies the experiments237.

the carrier­mediated transport of a drug
occurs through the specific cells that express
the transporter. These characteristics for
carrier­mediated transport (BOX 2) are
different from those of passive transcellular
transport. Detailed definitions and char­
acterization of carrier­mediated transport
processes are found elsewhere18,19.
There are some exceptions to these rules,
and therefore these may be classed as gen­
eral rules rather than being solely sufficient
evidence. For example, temperature and pH
dependency are now not considered criteria
for judging carrier­mediated transport, as
they are also observed in passive transcellular
membrane transport (discussed later, and
excluded from BOX 1).
investigating permeation mechanisms
Methods used to investigate passive
transport. A membrane with exactly the same
lipid composition and lipid bilayer structure
as a biological membrane, but without any
membrane proteins, would be ideal to inves­
tigate the passive transcellular transport of
a compound. However, such models are not
available. A good, but not perfect, choice for
such a reference membrane is the black lipid
membrane model20–23 and unilamellar vesicles
(liposomes)8,9. The black lipid membrane
model and liposomes consist of a single
bilayer formed from predominantly zwitter­
ionic phospholipids (for example, egg lecithin,
which is a mixture of phosphatidylcholines).
Another way of investigating passive
transport is to use a monolayer­forming cell
line that does not express functional drug
transporting proteins, such as 2/4/A1 cells24,25.
Methods used to investigate carrier-mediated
transport. monolayer­forming cell lines,
such as the Caco­2 (REFs 26–30) and mDCK
cell lines31,32, in which one, two or even four
transport proteins can be simultaneously
expressed are commonly used intestinal
barrier models. Other adherent or suspension
cell lines that can overexpress transport pro­
teins by gene transfection are also commonly
used for studies of carrier­mediated transport
(FIG. 1); for example, Hela cells, HeK293 cells
and Xenopus oocytes (see supplementary
information s1 (table)). usually when trans­
fected cells are used, non­transfected cells
(mock) are simultaneously used as a control
experiment to evaluate the contribution of
passive transcellular membrane transport.
Ex vivo and in situ rodent models such as
the Ussing chamber, everted sac and intestinal
perfusion models4,26 can also be used to inves­
tigate carrier­mediated transport. Although
extremely costly to obtain, permeation data
from human in vivo intestinal membrane
would be the most direct and the most rele­
vant evidence of the extent of carrier­mediated
transport of a drug in the human intestine33–35.
Comparison of the plasma concentration–
time profile and tissue distribution data
between normal and knockout animals36
provides direct evidence of the involvement of
a transporter in pharmacokinetics. methods to
deconvolute various components of transport
in the Caco­2 model have also been studied37.
In addition, the saturation and the inhibi­
tion of transport of a compound by a specific
inhibitor also suggests that the compound

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PersPectives

Table1 | References describing relationships between biological membrane transport and passive transport indicators
Parameter names¶ Biological membrane permeation¶ Number of compounds**,‡‡
Passive transport indicator: physicochemical properties*
Log Poct, pKa, Mr Human oral Fa 92 drugs7, 258 drugs80, 567 drugs85
Log Poct, pKa, polar surface area, hydrogen bond number Human oral Fa 170 drugs76
Polar surface area Human oral Fa 92 drugs7, 20 drugs238
Hydrogen bond strength, polarizability, dipole moment, Human oral Fa 178 drugs90
molecular volume
Polar surface area, Mr, log Poct, pKa Human Peff 22 drugs84
Log Poct, pKa, polar surface area, hydrogen bond number Human oral absorption rate 22 drugs76
Log Poct, pKa, polar surface area, hydrogen bond number rat bioavailability 1,117 in-house compounds83
Log Poct, pKa rat intestinal absorption rate 75 drugs78
Log Poct, Mr caco-2 16,227 in-house compounds75
Polar surface area caco-2 77 drugs88
Log Poct, pKa, Mr caco-2 35 drugs74
Hydrogen bond strength, polarizability, dipole moment, rat blood–brain barrier permeation 30 drugs87
molecular volume
surface activity cNs permeation 42 drugs86
Log Poct, pKa Human tissue distribution 31 drugs81
Log Poct, pKa Human volume of distribution 64 drugs77, 670 drugs79
Passive transport indicator: artificial membrane partition ‡

Liposome binding Human oral Fa 27 drugs109, 20 drugs110

immobilized artificial membrane chromatography rat oral bioavailability 12 drugs111
immobilized liposome chromatography Human oral Fa 13 drugs108

is a substrate of carrier­mediated transport.
However, being an inhibitor is not necessarily
equal to being a substrate, and many inhibi­
tors are nonspecific, as they also interact with
several transporters and drug metabolizing
enzymes38. For instance, P-glycoprotein (P­gp)
and CYP3A4 have a significant overlap in
substrate specificity 39.
Analyses of concentration dependency
data using kinetic models, such as the
michaelis–menten equation (BOX 3), are also
used to differentiate carrier­mediated trans­
port from passive transcellular membrane
transport. According to the michaelis–
menten equation, the fraction of a permeant
that crosses the membrane by passive tran­
scellular membrane transport can become
more significant when the concentration
of the permeant is higher than the Km.
practical methods in drug discovery
As permeation measurements using black
lipid membrane and liposomes are difficult,
the parallel artificial membrane permeation
assay (PAmPA)40 is an alternative used by
many pharmaceutical companies, and has
been utilized in the study of many thousands
of compounds41–43. In addition, Caco­2 cells
and mDCK cells are routinely used44. many
drugs that are reported to cross membranes
by carrier­mediated transport have been
studied in these cell lines, although they
express many of the carrier­mediated trans­
port proteins found in the human small
intestine, at levels only partly comparable to
those found in vivo45,46. The permeation of
a multitude of compound series have been
ranked in Caco­2 cells to gain an apprecia­
tion of the relative oral absorption potential
of lead analogues47–49.
However, Caco­2 cells are more labour
intensive and have a lower throughput than
artificial membranes, and thus are used as
a secondary lower throughput screen when
results from a more physiological model are
desired. Other specific models of membrane
transport such as genetically transfected
mDCK cells that express P­gp (mDCK­
MDR1 cells) can also be used to determine
how a compound may be transported across
a membrane50,51.
There are two aspects that should be
carefully considered when correlating
PAmPA, Caco­2 and mDCK data.
First, these experiments can give variable
results when not performed under stand­
ardized conditions52 and there is large
inter­laboratory and intra­laboratory
variability, partly because of missing
standardization practices53. second, carrier­
mediated and paracellular transport are
absent in standard PAmPA assays40,54.
In cases in which these aspects are neglected
(for example, compiling data from different
laboratories or including paracellular route
permeants in the data set), it could result in a
superficially insignificant correlation among
these assays55,56, possibly leading to a conclu­
sion that passive permeation cannot occur
in the permeation of biological membranes.
Separating different forms of transport
All rules have exceptions. The criteria
outlined in BOX 2 are general characteristics
of passive transcellular transport and
carrier­mediated transport and are not strict
criteria. For example, in some high capacity
transporters such as amino acid and glucose
transporters57, it may often be difficult to
obtain saturation, without which it may be
erroneously concluded that the mechanism
of permeation is passive diffusion. A strictly
linear concentration dependency is only
observed for electrically neutral molecules
or charged molecules at low concentrations.
many drugs are charged at the physiological
pH range and under these conditions

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 PersPectives

Table1 (cont.) | References describing relationships between biological membrane transport and passive transport indicators
Parameter names¶ Biological membrane permeation¶ Number of compounds**,‡‡
Passive transport indicator: artificial membrane permeation§
Octanol membrane caco-2 16 drugs74
egg lecithin PAMPA# Human oral Fa 92 drugs7, 25 drugs40
Hexadecane PAMPA Human oral Fa 32 drugs106
Biomimetic PAMPA Human oral Fa 80 drugs54,101,104
Phospholipid–octanol membrane Human oral Fa 21 drugs6
Liposome PAMPA Human oral Fa 21 drugs96
Trilayer PAMPA Human oral Fa 35 drugs239
Biomimetic PAMPA Human Peff 18 drugs105
Double-sink PAMPA Human Peff 8 drugs91
Double-sink PAMPA rat Peff 17 drugs26
Double-sink PAMPA caco-2 18 drugs37
egg lecithin PAMPA caco-2 92 drugs7
Biomimetic PAMPA caco-2 20 drugs103
Trilayer PAMPA caco-2 35 drugs239
PAMPA MDcK-MDR1 40 drugs and 72 in-house compounds97
Blood–brain barrier PAMPA Brain to plasma ratio 30 drugs and 14 in-house compounds95
Blood–brain barrier PAMPA Mouse blood–brain barrier permeation 130 drugs93
silicon PAMPA Human skin Kp 19 drugs102
Double-sink PAMPA Ki/ic90 34 in-house compounds99
silicon PAMPA Uptake in fish 20 chemicals98
Passive transport indicator: cells without functioning transporters||
2/4/A1 cells Human oral Fa 30 drugs100
Fa, fraction of a dose absorbed; ic90, concentration that produces 90% inhibition in a cell-based assay; Kp, human skin permeability coefficient value; Ki, inhibition
dissociation constant or binding affinity of the inhibitor; log Poct, octanol–water partition coefficient of a compound; Mr, molecular mass; Peff, effective intestinal
membrane permeation and is the sum of passive transport and carrier-mediated transport; pKa, dissociation constant. *Partition coefficients in different solvent
systems and additional physicochemical properties can be used to describe biological membrane permeation and can suggest that passive transport is dominant
for these drugs and compounds. ‡Partition into artificial membranes can be used to describe biological membrane permeation and can suggest that passive
transport is dominant for these drugs and compounds. §Permeations derived by passive permeation measurements using artificial membranes are predictive for
biological membrane permeation and can suggest that passive transport is dominant for these drugs and compounds. ||Permeations derived by measurements
using cells that do not express a functioning transporter are predictive for biological membrane permeation and can suggest that passive transport is dominant for
these drugs and compounds. ¶For details of each parameter please refer to the references. #There are various parallel artificial membrane permeation assay
(PAMPA) technologies to improve predictability for biological membrane permeation. For details of each PAMPA please refer to the references. **Drugs refer to a
compound that has been used clinically. in-house compounds refers to a compound in the drug discovery process. Both are cited as these two could have different
drug-like properties. ‡‡Typical drugs used for these investigations (the number in the parentheses is oral Fa in humans): acebutolol (80–90), acetaminophen (80),
acetylsalicylic acid (84–100), acyclovir (20–23), allopurinol (90), alprenolol (93–96), amiloride (50), ampicillin (62), antipyrine (97), atenolol (50–54), aztreonam (1),
barbital (90), bromocriptine (28), bupropion (87), caffeine (100), carbamazepine (70–100), ceftriaxone (1), cefuroxime (5), cephalexin (0), chloramphenicol (90),
chlorothiazide (13), chlorpromazine (100), cidofovir (3), cimetidine (64–95), ciprofloxacin (69), cloxacillin (37–60), clozapine (100), corticosterone (100), coumarin
(100), creatinine (80), cymarin (47), cytarabine (20), desferrioxamine (2), desipramine (100), dexamethasone (80–100), diazepam (100), diclofenac (100), dicloxacillin
(35–76), dilthiazem (80), diltiazem (92), dipyridamole (66), doxycycline (90–100), enalapril (66), erythritol (90), ethambutol (80), ethionamide (80), etoposide (50),
famotidine (38–45), fenoterol (60), flecainide (81), flucytosin (75–90), foscarnet (17), furosemide (50–61), ganciclovir (3), gentamycin (0), guanabenz (75–80),
HBeD (5), hydrocortisone (55–91), imipramine (99–100), indomethacin (100), isoniazid (80), ketoprofen (100), labetalol (90), lactulose (0.6), lansoprazole (85),
lincomycin (28), mannitol (16–26), metaproterenol (44), metformin (86), methotrexate (20), methylprednisolone (82), metolazone (64), metoprolol (95),
nadolol (35–57), naltrexone (96), naproxen (99–100), nicotinic acid (88), norfloxacin (71), olsalazine (2), oxacillin (30–35), oxprenolol (97), oxybutynin (6),
oxytetracycline (60), phenobarbital (100), phenytoin (90), pindolol (87–92), piroxicam (100), practolol (100), pravastatin (34), prazosin (77–95), prednisolone
(99), procainamide (75–95), progesterone (91), propranolol (90–100), propylthiouracil (76), quinidine (81), quinine (90), raffinose (0.3), ranitidine (50–64),
ribavirin (33), salicylic (acid) (100), sotalol (60), streptomycin (1), sulphasalazine (12–59), sulindac (90), sulpiride (35–44), sumatriptan (57), terbutaline (62–73),
testosterone (98–100), tetracycline (75–80), theophylline (98–100), tiacrilast (99), timolol (72–95), tolbutamide (85), tranexamic (acid) (55), valsartan (55),
verapamil (95–100), warfarin (93–98), zidovudine (100).

membrane binding and passive diffusion
could be nonlinear (owing to a change in
the surface potential of the membrane
on drug partitioning 10) and could closely
resemble carrier­mediated transport (BOX 3).
In some cases of passive transcellular
transport (such as transport of ionized
molecules by ion pairing with lipid
components or by electric potential differ­
ences across membranes), saturation and
inhibition of passive permeation were also
observed58,59. several efflux transporters show
a high degree of promiscuity in their sub­
strate recognition, but the promiscuity is still
narrower than passive transport. Although
partitioning processes of compounds into
membranes (or octanol as a surrogate) can be
regarded as the precondition for membrane
passage, partition processes are not sufficient
to describe membrane permeation for all
compound classes correctly.
For instance, highly charged or polar
amphiphilic compounds have the potential to
accumulate in the polar membrane region,

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PersPectives
Box 2 | General features of passive and carrier-mediated transports
carrier-mediated transport
• Concentration dependent (saturable)
• Subject to inhibition
• More structure specific than passive transport, but dependence on lipophilicity could be
identified in a narrow chemical series
• Cell type specific; requires expression of the transporter
Passive transport
• Not concentration dependent (non-saturable)
• Not subject to inhibition
• Less structure specific than carrier-mediated transport; there is a general dependence on
lipophilicity for structurally diverse compounds
• Less cell type specific than carrier mediated transport
mimicking compounds that have a high
permeation potential but without passing
this barrier 60. lipophilic basic amidine­
containing inhibitors of the coagulation
pathway can belong to this class of com­
pounds61,62. The influence of charge
distribution on the permeation of cationic
amphiphilic compounds has been described,
showing that permanently charged lipophilic
molecules can pass membranes provided
that the charge can be spread over several
aromatic ring systems or neutralized by
making an ion pair complex with a counter
anion63,64. Therefore, a thorough study of
carrier­mediated transport should include
multiple indicators of carrier­mediated
transport 65.
Why not temperature and pH dependency?
Two experimental observations that were
used to support carrier­mediated transport
— temperature and pH dependency — can
be commonly observed for passive diffusion.
energy consumption and active trans­
port processes are minimal at 0–4 °C,
whereas energy consumption is normal and
active transport processes are functioning
at 37 °C. Consequently, it has often been
assumed that when a compound is taken up
at 37 °C but not at 0–4 °C, the transport is
carrier­mediated, but when the compound
is taken up at both temperatures then
transport is thought to be passive. However,
passive transport is also highly temperature
dependent, because partition/distribution
coefficients and passive transcellular
(and paracellular) permeation can be
strongly influenced by temperature66,67,
as exemplified in FIG. 2. For example,
the activation energy values for passive
transport and active transport across cell
monolayers were indistinguishable for
indomethacin and salicylic acid27.
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many cephalosporins have oral
bioavailability higher than expected from
their lipophilicity properties, which is due in
part to peptide transporter 1 (PePT1; also
known as sCl15A1)­mediated transport 68.
As PePT1­mediated transport is proton­
dependent, pH dependency was used to
support the involvement of PePT1­mediated
transport. However, several cephalosporins
have an acidic functional group69, which
could explain the apparent pH gradient
effect as being solely due to the consequence
of the pH partition theory of passive trans­
cellular permeation. As both passive
transport and PePT1­mediated transport
are pH dependent, it is difficult to quantify
the contribution from each mechanism
by the pH dependency of the permeation.
The pH­dependent in situ intestinal mem­
brane permeation of benzoic acid in the rat
is a good example showing the importance
of thorough consideration of the effect of
pH4 (FIG. 3), which shows that the acid micro­
climate is the most important factor that
determines the effective intestinal membrane
permeation (Peff) of benzoic acid70.
Theoretical aspects
In the following sections we first discuss
the existence or non­existence of both pas­
sive transcellular membrane transport and
carrier­mediated transport, and then discuss
the relative contribution of these transport
processes on the pharmacokinetics of drugs.
Rationale for the existence of passive trans-
cellular membrane transport. molecules
diffuse across the membrane in proportion
to their concentration gradient across the
membrane. In black lipid membranes and
liposomal membranes, numerous reports
suggest that compounds with mid to high
lipophilicity (for example, a log Doct > 0)23,71
rapidly permeated, whereas compounds
with low lipophilicity slowly permeated
(for example, glycerol (log Doct = –1.76) and
urea (log Doct = –1.66)72). These studies indi­
cate that many drug­like compounds can
pass through the lipid bilayer in proportion
to their lipophilicity 73. Correlation between
indicators of biological membrane permea­
tion and passive permeation (the whole
molecule physicochemical property and
artificial membrane permeation) for struc­
turally diverse compounds also suggests that
passive transcellular membrane transport of
drugs exists (TABLE 1).
Rationale for the existence of carrier-
mediated transport. given the difficulty in
identifying carrier­mediated transport as
discussed above, we revisited 55 publica­
tions (published later than 1996) that have
been cited to provide evidence for a more
prominent role of carrier­mediated trans­
port1 (total 100 drug–carrier protein com­
binations (see supplementary information
s1 (table)). These reports tended to use a
thorough investigation of the criteria listed
in BOX 2, especially uptake into transfected
cells overexpressing the transporter of
interest (for example, PePT1, PePT2 (also
known as sCl15A2), P­gp, breast cancer
resistance protein (BCrP; also known as
ABCg2), multidrug resistance­associated
protein 1 (mrP1; also known as ABCC1),
organic anion transporting polypeptides
(OATPs), organic cation transporters
(OCTs)), as demonstrating the involvement
of carrier­mediated transport. It is clear
that carrier­mediated transport exists for
drugs permeating biological membranes,
but it is interesting that in most of these
publications, passive transport was also
reported, usually in control experiments in
non­transfected cells.
Clear indications of the involvement of
passive transport were presented in 81 cases.
In 46 cases, passive transcellular transport
contributed to more than 30% of total
uptake and/or permeance. This suggests
that even among these publications focusing
on carrier­mediated transport, passive
transcellular transport coexisted. Therefore,
it is common practice to subtract the base­
line passive transcellular transport from the
total permeation in order to separate the
contribution of carrier­mediated transport.
However, these experiments cannot suggest
the quantitative contribution of passive trans­
cellular transport and of carrier­mediated
transport in the total (net) permeation
in vivo, as there are differences between
in vitro, in vivo and clinical conditions.
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 PersPectives

were obtained when high quality data

Box 3 | Michaelis–Menten equation for membrane permeation
were used (however, again with some
The rate of total mass transport across a cellular membrane (dM/dt) is the sum of passive transport outliers)7,26,37,43,95,99,118–121 (FIG. 5a). In a recent
(PT) and carrier-mediated (CM) transport, and can be described by using the Michaelis–Menton study that compared a Caco­2 cell­based
equation (see equation and figure below). Passive transcellular transport is in proportion to the assay with PAmPA, 85% concordance
concentration gradient across the membrane (in this equation the concentration on the right side between both assays was obtained for struc­
of the membrane is C and that in the right side is assumed to be zero).
turally diverse compounds122, suggesting
F/ #×
2  2 ×%#×2 ×% 8OCZ×% that these compounds permeated across
26 %/ 26
FV -O % Caco­2 and mDCK cells mainly by passive
Where A is the surface area of a membrane (length2); P is the permeability (length/time); C is
transcellular transport.
the concentration of a permeant (amount/length3); Km is the Michaelis constant (amount/length3); The permeation coefficients of 30 drugs
Vmaxis the maximum carrier-mediated transport (amount/time) in 2/4/A1 cells showed a good correla­
tion with the human Fa of these drugs123.
In these studies, the relative importance
of passive transport and carrier­mediated
Total (net) transport transport was investigated using drugs
(carrier-mediated
+ passive) that are more hydrophilic (therefore have
Carrier- Carrier- lower passive transcellular transport) than
mediated
lipophilic (which would have high passive
dM/dt

mediated
Vmax transport
Passive
transport
Km Concentration
so we conclude that both passive trans­
cellular transport and carrier­mediated
transport exist in the biological membrane
permeation of drugs.
practical aspects
In this section, we discuss the relative con­
tribution of passive transcellular transport
and carrier­mediated transport in vivo,
using intestinal membrane permeation as
an example. As there are more than 1,000
marketed drugs and much larger numbers
of drug­like compounds in discovery, it is
difficult to have an exact percentage but we
attempt to draw a conclusion.
Contribution of passive transport in
intestinal membrane permeation. Although
there are large differences between octanol
and lipid bilayers, octanol–water partition
coefficients or distribution coefficients
(log Poct, log Doct) can still be regarded as the
gold standard lipophilicity scale that is used
to describe distribution processes of drugs
into membranes. In a study of more than 550
structurally diverse drugs, log Doct correlated
with the oral absorption rate in rats, the frac­
tion of a dose absorbed (Fa) in humans and
Caco­2 membrane permeation7,74–85 (TABLE 1).
In addition, various other physicochemical
properties also correlated with Fa and
human Peff (REFs 7,86–90) (TABLE 1).
transport
Passive
transcellular
transport
PAmPA6,7,26,37,40,43,54,91–107
Nature Reviews and other
| Drugartificial
Discovery
membrane systems83,108–111 showed good
correlations with clinical Fa for more than
100 structurally diverse drugs (TABLE 1)
albeit with some outliers40,97,112. most regis­
tered oral drugs (for example, propranolol,
desipramine and naproxen), as well as
typical drug candidates, have mid to high
PAmPA permeations43, suggesting that
these compounds are absorbed mainly by
passive transcellular transport. A bilinear
relationship between PAmPA permeations
and log Poct/Doct (FIG. 4) have been described
for structurally diverse drugs113,114. Those
results supported the principles described
by Kubinyi115,116, which state that the rate
constants of transport of a drug from an
aqueous phase into an organic phase (k1) and
in the reverse direction (k2) can be described
by nonlinear relationships. As lipophilicity
increases, k1 increases whereas k2 decreases,
resulting in the bilinear relationship.
Interestingly, the most frequent lipophilicity
range for structurally diverse registered oral
drugs (70% of oral drugs have log Doct values
of +0.5 to +3) overlapped with the range that
gives a high membrane permeance, which
depends on the method applied83,117 (FIG. 4) .
Despite the restrictions described
earlier in the direct comparison of Caco­2,
mDCK and PAmPA measurements, good
correlations for structurally diverse drugs
transcellular transport). Therefore,
carrier­mediated transport should be more
readily observed than passive transcellular
transport. These drugs are usually incom­
pletely absorbed in the human intestine100
(Biopharmaceutics classification system (BCs)
class III). The data set was selected so that
half of the drugs were known to be at least
partly transported by different carrier
proteins of the human intestine, whereas
for the remaining part of the data set, no
clear indications of carrier­mediated trans­
port had been published. The permeation
of these compounds was tested in three
in vitro models: artificial membranes,
Caco­2 cells and 2/4/A1 cells. The best
correlation between the human Fa and
in vitro permeance was obtained for 2/4/A1
cells, which do not express any functioning
carrier proteins. Importantly, 3 out of the
approximately 15 actively transported com­
pounds were outliers. Indeed, two of these,
a model substrate for PePT1 (gly­sar) and
methotrexate (a substrate for the reduced
hydrofolate transporter) are known to be
highly dependent on active transport for
intestinal permeation.
Correlations between indicators of
passive permeation (that is, log Doct, artificial
membrane permeabilities and 2/4/A1 cell
permeation) and in vivo intestinal mem­
brane permeations (oral absorption rates,
Peff and Fa) for structurally diverse drugs and
compounds were calculated. These correla­
tions provided the evidence supporting the
premise that passive transcellular membrane
transport is the primary transport route for
most drugs in in vivo intestinal membrane
permeation (TABLE 1). At the same time, the
existence of outliers suggested a possibility
that carrier­mediated transport could be
present for these drugs.

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PersPectives

20 concentration is larger than Km, passive

4 °C
transcellular transport contributes signifi­
18 cantly to the total permeance, or is even
37 °C
the dominating mechanism.
16
This aspect was recently proposed for
angiotensin­converting enzyme (ACe)
inhibitors132,133, some of which are his­
14 torically claimed to be a substrate for the
PAMPA permeation (1 × 10–6 cm s–1)

proton­dependent oligopeptide transporters

12 PePT1 and PePT2. Of the 14 investigated
ACe inhibitors, the majority displayed weak
10 or no carrier­mediated transport. low trans­
port currents were observed for only four of
the inhibitors, including enalapril and lisi­
8
nopril, which have previously been cited as
drugs that are transported by PePT1. When
6 the authors considered the low affinities,
low transport activities, relatively moderate
4 to high lipophilicity (log Doct > 0 in 10 out
of 14 drugs) and daily dose, they thought it
2 highly probable that passive diffusion mainly
controls the in vivo intestinal absorption of
many of the ACe inhibitors. Although this
0
controversial conclusion is supported by
rbu e
e
e

n
Pir ran

asa lol

Va ne

rap n
il
ofe ole

Pit icam
Pro tatin
ole ntip e

oth atin
Fur azide

er fen
Me llow
ide
kin e

Na rolo
Da 8

am
in
din
De pson
pin

Na roxe
n

a
in-

other publications118,134,135, further in vivo

t
o

i
Dip rami
yri

laz
tal
gat
em
Fex dam

r
Lu opro
t

Su pran

lsa
ye
na
ze

top

s
ch vas

ox
i

ava
p
psa
os

studies are needed to confirm the findings.

ma

sip

Ve
sto

yri

dro Flu

lph

Te
Ke
A

cif
lor
rba

cy

In addition, many drugs that were identi­

Ca

fied as a P­gp substrate in an in vitro study

Ch

Hy

were later shown to be completely absorbed

Figure 2 | Temperature dependency (4 °c and 37 °c) of passive transport through an artificial
membrane system of various drugs. Permeation experiments for numerous
under conditions as previously described but at two different temperatures63,65,114, 4 °c and 37 °c.
PAMPA, parallel artificial membrane permeation assay.
Contribution of carrier-mediated transport
to intestinal membrane permeation — results
from knockout mice experiments. Knockout
mice124 and transgenic mice with organ­
specific expression of human transporters125
have been used to evaluate the contribution
of carrier­mediated transport in vivo. The
effect of P­gp on the area under the plasma
concentration–time curve (AuC) (or plasma
concentration at a specific time point such as
tmax) was determined by oral or intravenous
administration of well­established P­gp sub­
strates in P­gp­deficient mice (P­gp knockout)
and wild­type animals36. even though
bioavailability also includes the effect of gut wall
and liver metabolism, and excretion (so that
there is overestimation of the role of P­gp in
intestinal membrane permeation126–128), these
data contain one of the most relevant pieces
of information for the contribution of carrier­
mediated transport on intestinal membrane
permeation36,126,128. For several compounds,
the bioavailability ratio of drugs after oral
administration was >2. However, as shown in
TABLE 2, P­gp affects brain distribution more
significantly than it affects oral absorption.
drugs were
Nature Reviews | Drugperformed
Discovery
On the other hand, the contribution of
PePT1 to in vivo oral absorption is ~60% for
cefixime (comparing wild­type and knock­
out mice)129 and is ~50% for cephalexin
(from rat in situ permeation studies)130.
In addition, several clinical studies show
that genetic variance of transporters affected
the AuC after oral administration of several
drugs (reviewed in REF. 131). This evidence
suggests that, at least for several propor­
tions of drugs, carrier­mediated transport
determines more than 50% of membrane
permeation in a certain organ.
Cases when carrier-mediated transport
does or does not display a significant effect
on oral absorption. For several substrates
of P­gp, oral absorption was not affected
by P­gp knockout (TABLE 2). As shown in
BOX 3, being a transporter substrate does
not necessarily mean that carrier­mediated
transport dominates the total (net) permea­
tion of the substrate in vivo. According to
the michaelis–menten equation for mem­
brane permeation (BOX 3), when passive
transcellular permeation is high and/or drug
in vivo136,137, for example, verapamil. One
reason suggested for this discrepancy
was the difference of the concentrations
between in vitro assays and in vivo intestinal
fluid137,138. usually, in vitro cellular assays
use a low concentration to identify a P­gp
substrate, whereas a higher concentration
can be achieved in vivo and clinically (for
example, when 100 mg dose of a compound
of molecular mass 400 daltons is dosed
in humans, the concentration could be
1,000 μm (intestinal fluid volume is assumed
to be 250 ml))139. The apparent Km of P­gp is
<200 μm for most cases140. Therefore, results
obtained solely from cell culture studies
using a low concentration might have led to
an overestimation of the importance of P­gp
transport on the understanding of the main
intestinal absorption process.
Another reason suggested for the discrep­
ancy between in vitro and in vivo findings is
that passive transcellular transport could be
rapid enough to overcome the efflux carrier­
mediated transport 141–144. Direct comparison
of mDCK­MDR1 cells and PAmPA permea­
tions highlighted the importance of the
balance of passive and carrier­mediated
efflux transport in total permeance145 (FIG. 5b).
As shown using a large set of compounds,
active efflux from the basolateral to the
apical side of the cells can mainly be expected
for those compounds that exhibit low passive

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 PersPectives

Luminal pH permeation144. similar findings are described

2 3 4 5 7 9 10 11 for bidirectional transport in Caco­2 cells146.
a usually the fraction of compounds with
a large efflux ratio (efr >5) increases with
decreasing permeation. This experimental
–2 observation was further confirmed in
pKa (3.98) Caco­2 cells147 (FIG. 6). These data suggest
pH partition that in order for the efflux P­gp transport
theory to be significant, it has to surmount passive
O
transcellular transport in the absorptive
Air-segmented perfusion OH direction148,149. For instance, digoxin,
–3
hABL 103 µm (estimated) a typical P­gp substrate150, had absorption of
>80%151–153. verapamil is another example154.
Log Peff (cm s–1)

Benzoic acid
For intestinal membrane permeation,
Normal perfusion (0.5 ml min–1) (5.35) the contribution of the paracellular pathway
hABL 531 µm can also be significant54,155. For example, in
–4 (6.05)
the case of ampicillin (a PePT1 substrate),
a significant contribution of the paracellular
route on the total permeance was suggested
Ionic in rats156 in addition to PePT1. moreover,
the total permeance of a paracellular pathway
compound, for example, ranitidine, is less
3 4 5 6 7 8
affected by P­gp efflux 143. As cellular uptake
Microclimate pH assays cannot assess the paracellular route
and as in vitro monolayer cells (for example,
Caco­2) can have a tighter paracellular route,
b pH 5.0 pH 6.5 pH 7.4 these assays would underestimate the contri­
bution of the paracellular pathway. notably,
2/4/A1 cells have paracellular permeance
most comparable to that of the human small
intestine and show good predictability for Fa
of both passive transcellular and paracellular
transport157.
Therefore, carrier­mediated transport
processes in the intestine can be expected to
be more important for low dose or low solu­
bility compounds (hence low concentration
in the intestinal fluid) with low passive
Passive (neutral) Passive (ion) Aqueous boundary layer Paracellular
permeance (in both passive transcellular
membrane transport and paracellular trans­
Figure 3 | pH-dependent permation of benzoic acid in the rat intestine. a | rat in situ intes-
tinal membrane perfusion of benzoic acid as a function of the microclimate pH on the surface of
port). Indeed, this aspect has been repeat­
enterocytes based on published absorption–pH rate data4, which were transformed edly cited in the literature3,136,142–144,154,158–162.
Nature Reviews | into
Drugpermea-
Discovery
tion values. The pH partition theory curve without the interference by the aqueous boundary layer
(ABL) is indicated by the green solid line. The blue dashed curve corresponds to the theoretical Human intestinal membrane permeability.
curve under the air-segmented flow perfusion conditions (efficient stirring in the perfusate, less even though it can be expensive to obtain,
ABL effect) and the red dashed curve corresponds to that in the normal flow perfusion conditions the human Peff is the most authentic method
(large ABL effect). The red circles represent experimental data in normal flow perfusion conditions to investigate intestinal membrane permeation
and the green circles represent experimental data in air-segmented flow perfusion conditions. The relevant to clinical pharmacokinetics. Among
numbers in parentheses are apparent pKa values under air-segmented flow (5.35) and normal flow more than 40 drugs investigated for human
(6.05) conditions, indicating the extent of the ABL-induced ‘artefact shift’ in absorption curves Peff (REFs 34,35), amoxicillin, cephalexin, enal­
from the intrinsic pKa value (3.98). The departure of red and blue dashed curves from the solid green april, lisinopril and valacyclovir are PePT1
curve for pH > 6.5 indicates the minor contribution from the transport of the ionic form of benzoic
substrates, and cyclosporine, cimetidine, fex­
acid. At pH 6, under normal flow perfusion conditions, 44% of the transport follows the original
ofenadine and verapamil are P­gp substrates.
prediction of the pH partition theory that is passive diffusion. Under air-segmented flow condi-
tions, the number increases to 78% of the transport, as a result of the reduction of the ABL resist- Fexofenadine and l­3,4­dihydroxyphenyla­
ance (54% to 18%). When the luminal pH was used as it is (without converting to the microclimate lanine (l­DOPA) are absorbed through an
pH), the pH–permeation curve did not follow the pH partition theory. Therefore, it could be mis- OATP and an amino acid transporter, respec­
leading to conclude that the pH partition theory is inapplicable. The most important factor for tively. In addition, cimetidine is an OCT1 and
rationalizing the observed curve shape is the acid microclimate. b | estimated factors controlling an OCT2 substrate.
transport in the rat in situ intestinal membrane perfusion experiment described in part a, at micro- Amoxicillin (log Doct = –1.7), cephalexin
climates of pH 5.0, pH 6.5 and pH 7.4. Part a is reproduced, with permission from, REF. 41 © (2008) (log Doct= –1.1), valacyclovir (log Doct= –1.4)
elsevier science. and l­DOPA (log Doct < –2) showed Peff values

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PersPectives
a b 100
Partition Hydrocortisone Corticosterone
90 Naproxen
Aqueous phase Coumarin Testosterone
Cocaine
Flux: mass tranfer via artificial
80
C1
P = C1 /C2
70 Papaverine
membrane (%)
C2 60
Proquazone
Lipid phase 50
Pindolol
40
Permeation Metergoline
30 Procainamide
k1 Tetracycline
20 Chlorprothixen
10
Acyclovir
0 Saccharin
k2 –1 0 1 2 3
Log D pH 7.4
Figure 4 | Partitioning versus permeation. a | Principal difference between the partitioning and
distribution, and membrane permeation of drug-like compounds. b | relationship between permea-
Nature Reviews | Drug Discovery
tion through a parallel artificial membrane permeation assay (PAMPA) and octanol–water partitioning
(expressed as the percentage flux: mass transfer via artificial membrane) at physiological pH (log D pH
7.4). A rough nonlinear relationship between lipophilicity and permeation is observed. Bilinear rela-
tionships in compound mass transfer as described by Kubinyi115,116, depend on the rate constants of
transfer of the drug through aqueous and organic compartments. in simple in vitro systems the rate
constant k1 of transport of a drug from an aqueous phase into an organic phase and the rate constant
k2 of the reverse process can be described as functions of the partition coefficient P, where c
represents concentration. Data in panel b plotted from REF. 114.
that are higher than that expected from their to apical direction being 28­fold to 85­fold
lipophilicity. This is in good agreement with higher than the Papp (apical to basolateral)
the notion that carrier­mediated transport in the concentration range 10–1,000 μm163.
has a significant role in oral absorption. Indeed, Papp from the basolateral to the apical
enalapril (log Doct = 0.08) and lisinopril (log direction decreased with increasing con­
Doct = –1.32) were already discussed above. centration (Vmax = 5.21 nmol cm per second
Cyclosporine and verapamil showed a good and Km = 150 μm), suggesting the saturation
permeation (1.6 × 10–4 and 6.80 × 10–4 cm of an apical efflux transporter by this drug.
per second, respectively), although they are In addition, data obtained using Caco­2
P­gp substrates, suggesting that high passive cells suggest that the in vitro permeation
transcellular transport overcame P­gp efflux. was increased in the apical to basolateral
Other drugs (more than 30) were thought to direction by approximately 2–3 fold in the
be absorbed by passive diffusion35,84. presence of various P­gp inhibitors, such
To further investigate the joint contribu­ as verapamil, ketoconazole and gF 120918
tion of passive transcellular transport and (REFs 163,164,166). However, the Papp (apical
carrier­mediated transport, fexofenadine to basolateral) was independent of the con­
and cimetidine are discussed in detail below centration applied, suggesting that the effect
as two examples of BCs class III drugs, of carrier­mediated transport in the apical
which are probably influenced by carrier­ to the basolateral direction is minimal or the
mediated transport and because in vitro, apparent Km in the apical to the basolateral
in vivo and clinical permeation data were direction is higher than 1 mm.
available in the literature. In a clinical investigation, the plasma
exposure of fexofenadine was discovered to
Fexofenadine. Fexofenadine is a well­ be linear over a wide dose range of 40–800
established substrate for P­gp (efflux mg 167. In addition fexofenadine had similar
direction basolateral to apical) and OATP bioavailability when given orally as a micro­
(absorptive direction apical to basola­ dose (<100 μg) and at a higher dose of
teral)163,164. The Peff in humans was low 120 mg (41% versus 30%, respectively)168.
(0.1–0.2 × 10–4 cm per second) and variable, This is in agreement with the linear in vitro
which classifies it as a compound with low permeance in the apical to basolateral direc­
membrane permeation (BCs class III)165. tion as described above in the Caco­2 model.
Fexofenadine displays polarized transport This linear increase of exposure after oral
in Caco­2 cells, with the apparent perme­ administration suggests that passive diffu­
ability coefficient (Papp) from the basolateral sion would be the main route in intestinal
606 | AugusT 2010 | vOlume 9
© 2010 Macmillan Publishers Limited. All rights reserved
membrane permeation, although, as we
discussed above, a sole experimental find­
ing should not be used as the main evidence
to support this transport mechanism. As
the substrate concentration in the cytosol
is lower than that on the apical side169, the
apparent Km value could be higher than that
of the intrinsic Km value, depending on the
intrinsic passive permeation and the mem­
brane surface area170 of both apical and baso­
lateral membranes. The apparent Km of P­gp
value in the apical to the basolateral direction
was seen to be higher than that in the baso­
4
lateral to the apical direction and can reach >
1,000 μm172,173 in specific cases. In addition,
the clinical interaction between fexofenadine
and nagrignin suggests that about 50–70%
of absorptive permeation in the intestine
might be mediated by passive transcellular
membrane transport and 30–50% mediated
by OATP174. Therefore, the possibility of a
cancelling out effect between P­gp and OATP
that results in a linear increase of exposure
cannot be entirely excluded.
most reported clinical drug–drug inter­
actions of fexofenadine, including those for
verapamil and ketoconazole, which partly
result in an increase of plasma levels (AuC)
of fexofenadine, were explained by the inhi­
bition of intestinal efflux by P­gp. The clini­
cal fexofenadine and verapamil interaction
suggests that verapamil increased the AuC
of fexofenadine by up to 2–3­fold in total,
although it was difficult to identify which
transporter is responsible for this increase175.
However, there was little or no acute effect
by either of these P­gp inhibitors, verapamil
and ketoconazole, on the Peff of fexofenadine
in humans and rats165,176. An in vivo per­
fusion study with simultaneous assessment
of intestinal transport and plasma pharma­
cokinetics suggests that liver uptake of fexo­
fenadine is mediated by OATP1B1 and/or
OATP1B3, which could also be inhibited by
verapamil and ketoconazole177. In addition,
by using double transfected cells expressing
OATP1B1/mrP2 or OATP1B3/mrP2, it
was shown that OATP1B1 and OATP1B3 are
involved in the hepatic uptake of fexofena­
dine178. Therefore, this evidence suggests
that the drug–drug interaction could occur
in the liver uptake process rather than at the
intestinal membrane permeation process,
which is also supported by a physiologically
based pharmacokinetic model179.
In conclusion, for fexofenadine, passive
transcellular transport is suggested as one
of the main intestinal membrane transport
mechanisms in humans, but several other
transporters may also be involved, especially
in the liver and the kidney 180. The intrinsic
www.nature.com/reviews/drugdisc
 PersPectives

a –3 b 30 inhibitors used in these studies (for example,

Correlation = 0.84
cyclosporine) could be both inhibitors of the
Log Papp MDCK (1 × 10–6 cm s–1)

–4 25 transporter P­gp and the enzyme CYP3A4

RGF: PappGF120918a–b/Pappa–b
(REF. 39). In addition, it is suggested that P­gp
20
–5 and CYP3A4 are synergistically operating to
15
avoid absorption of xenobiotics in the intes­
–6
tine196,197. P­gp can reduce the permeance of
10 the drug, thereby increasing the chance of
the drug to be metabolized by CYP3A4 in
–7
5 the gut wall.
An interesting example of this phenom­
–8 0
–8 -7 -6 -5 -4 -3 0.01 0.1 1 10 100
enon is paclitaxel, which is reported to have
Log Papp HDM-PAMPA (1 × 10–6 cm s–1) Papp HDM-PAMPA (1 × 10–6 cm s–1) a low bioavailability following oral admin­
istration. The reason for its low bioavail­
Figure 5 | competition between passive and active transport. a | correlation between the
Nature Reviews | Drug Discovery
ability was first thought to be mainly due to
apparent passive permeation (Papp) for 37 drugs in MDcK cells and in hexadecane membrane (HDM)-
parallel artificial membrane permeation assay (PAMPA). compounds studied were acyclovir, alprenolol, intestinal efflux 193,198,199 but it could be due
amiloride, antipyrine, atenolol, ceftriaxon, chloramphenicol, chloroquine, cimetidine, cyclosporine, to a combination of its low solubility and its
desipramine, digoxin, doxorubicin, fluvastatin, furosemide, guanabenz, imipramine, lansoprazole, extensive first pass metabolism198,200. Paclitaxel
methotrexate, metoprolol, midazolam, mitoxantrone, naproxen, omeprazole, pantoprazole, prazosine, has a relatively high in vitro permeance in
propranolol, pumafentrine, quinidine, ranitidine, ritonavir, sulphasalazine, sulpiride, terbutaline, Caco­2 cells (4.4 × 10–6 cm per second, apical
testosterone, tolafentrine, topotecan and verapamil. A good correlation (r = 0.84) was observed to basolateral)201, which suggests that the
between the PAMPA and MDcK cell permeations of the drugs, suggesting that passive transcellular drug will be effectively absorbed in the intes­
transport is dominant in the permeation of these compounds in MDcK cells. b | correlation between tinal tract and that presystemic metabolism
P-glycoprotein (P-gp) (efflux) function determined as the ratio of apparent permeation across caco-2 is the major reason for incomplete bioavaila­
cells in the absence and presence of the P-gp inhibitor GF120918 (REF. 145) and passive permeation
bility. Increased solubility and dissolution rate
determined by HDM-PAMPA. All compounds analysed are known to show P-gp-ATPase activity. The
dashed lines indicate the threshold ratios of 0.5 and 1.25, which discriminate between compounds provided higher luminal concentration that
that are actively transported (above the lines). A compound between the threshold ratios of 0.5 and could more efficiently saturate the intestinal
1.25 indicates the marginal involvement of P-gp. Only compounds with low passive permeation show efflux and/or metabolism for paclitaxel200,202.
significant active transport expressed by the rGF ratio (apical (a) to basolateral (b) transport rate ratio It is worth noting that extensive investi­
(inhibited/non inhibited)). These data confirm findings that transporter-related efflux has to surmount gations are currently being undertaken to
passive permeation in order to be significant. Data plotted from REF. 145. identify a specific inhibitor for each trans­
porter 38, which will allow us to quantify
the contribution of each transport process
in vivo and in clinical pharmacokinetics.
low passive membrane permeance of fexo­ permeation mechanism in intestinal mem­
fenadine, together with it being a transporter brane permeation of cimetidine in humans. intestinal membrane transport: summary
substrate, determines the low permeance If the organic cation transporters, OCT1 For many drugs that are substrates of
in the small intestine and the liability of the and OCT2, were the primary mechanism it carrier­mediated transport (for example,
AuC for fexofenadine to be largely affected by would be expected that the human intestinal ACe inhibitors, cimetidine, fexofenadine
hepatic uptake and/or other transporters. in vivo permeance would have been signifi­ and verapamil), passive transport con­
cantly higher than the Caco­2 permeation, tributes significantly to the total intesti­
Cimetidine. Cimetidine is a relatively as the expression of OCT1 and OCT2 is nal membrane permeation, especially at
hydrophilic compound (log Poct = 0.48). higher in the human small intestine than it clinical doses.
The rate and the extent of intestinal absorp­ is in the Caco­2 model34,185,187. Therefore, the Conversely, some antibiotics (for example,
tion of cimetidine has been extensively main determinant of the intestinal membrane cefixime)129, folic acid derivatives (for
investigated, with the Fa after oral admin­ permeation of cimetidine is considered to be example methotrexate)203,204 and amino
istration estimated to be 75%181–183 (BCs passive diffusion188. acid derivatives (for example, l­DOPA)205,
class III). Cimetidine is a substrate for P­gp the structures of which are closely related
and/or OCT1 and OCT2 (REFs 184–187). interplay of mechanisms and properties to those of nutritional compounds206, are
The human Peff (in vivo) is 0.22 ± 0.13 × In the above discussion, only passive trans­ mainly absorbed in the intestine by carrier­
10–4 cm per second and 0.32 ± 0.18 × 10–4 cellular transport and carrier­mediated mediated transport. These are hydrophilic
cm per second at luminal concentrations of transport were considered. However, the drugs with little expected passive transport
1.0 mm and 2.7 mm (that is, clinical dose situation surrounding the bioavailability but good oral absorption. In addition, from
200–400 mg (3–6 mm, 250 ml intestinal of a drug may be more complex. the pharmacokinetic data of P­gp knockout
fluid volume)), respectively 34. several clinical studies have claimed that animals and clinical genetic variants of P­gp,
The lack of difference between perme­ an inhibition of intestinal efflux (especially oral absorption of several P­gp substrates
ance at the two concentrations, together with by inhibiting P­gp) is the major cause behind have been proven to be limited by P­gp
the observation that the human intestinal increased bioavailability when certain in vivo36,126,128 (TABLE 2).
membrane permeance quantitatively cor­ drugs are coadministered189–195. However, These results collectively suggest that pas­
relates well to that of the Caco­2 model, in many of these reports, involvement of sive transport and carrier­mediated transport
suggests that passive transport is the primary CYP3A4 cannot be excluded51,138,184–187, as the coexist in intestinal membrane permeation.

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© 2010 Macmillan Publishers Limited. All rights reserved

PersPectives

Table 2 | Data from knockout mice for bioavailability and brain to plasma ratio*
Drug Plasma Auc or concentration ratio Bioavailability ratio Brain to plasma ratio
intravenous orally
Amprenavir NA 1.3 <1.3 21
Asimadoline 1.0 1.1 1.1 9.1
Benzo(a)pyrene 0.8 0.8 1.0 1.6
cyclosporine 1.1 0.6–0.9 0.5–0.8, 1.6 11–29
Digoxin NA 2.4 2 4–28
Dihydroergocryptine 1.8 1.8 1.0 1.1
erythromycin 1.5 3.4 2.3 1.2
Fexofenadine 1.0, 4.6 4.6, 6.5 1.0, 6.5 1.9
Fluconazone 1.2 1.2 1.0 0.9
indinavir 0.7 2.0 2.9 3–10
ivermectin NA 1.9–3.7 <1.9–3.7 17–27
Loperamide 2.0 2.0 1.0 6.7
Nelfinavir 1.3 4.8 3.7 31
Paclitaxel 1.1–2 5.0–6.0 2.5–5.5, 3.18–6.71 7.9
resperine 1.2 1.2 1.0 2.4
retinoic acid 1.0 1.1 1.1 1.0
rifampicin NA 3.5 NA NA
ritonavir 1.0 1.0 1.0 6.9
s 09788 NA 2.4 3.4 NA
salinomycin NA NA 1.5 NA
saquinavir 0.7–1.1 6.5 1.55, 5.9–9.3 2.2–6.8
Tacrolimus 2.3 8.2 3.5, 3.6 6
Talinolol NA 2.9 NA NA
Topotecan NA 2.3 <2.3 2.0
UK-224671 1.1 >40 >36 NA
verapamil NA 1 1 8.3
vinorelbine NA NA 1.5 NA
AUc, area under the plasma concentration–time curve; NA, not available. For details see reFs 36, 125, 126, 128. *Brain to plasma ratio refers to the concentration
ratio of a drug in the brain compared with that in the plasma.

For most drugs, the intestinal membrane
permeation is primarily determined by pas­
sive transport207, and for a proportion of
drugs it is affected by carrier­mediated trans­
port84,208. As exemplified by fexofenadine, the
contribution of carrier­mediated transport
could become more significant in post­
absorption processes, for example, for liver
clearance, than in the intestinal membrane
permeation. even when carrier­mediated
transport is involved, the contribution of pas­
sive diffusion to the overall permeance could
become significant when the concentration
at the permeation site is high and/or passive
transcellular permeation is rapid. In vitro
permeation studies at a low concentra­
tion could overestimate the contribution of
carrier­mediated transport in vivo. Therefore,
the weight of contribution of passive
transcellular transport and carrier­mediated
transport should be carefully discussed in
a case by case manner.
Other drug disposition processes
We have already highlighted that there is
evidence showing that carrier­mediated
transport has a more important role in
blood–brain barrier permeation126,209
(TABLE 2), renal210 and hepatic211 secretions
than it does in the intestine. recent progress
in the understanding of carrier­mediated
transport showed that this could be more
important than usually thought1,3. For exam­
ple, 7 out of 50 compounds that had been
progressed to clinical studies were catego­
rized as being eliminated by carrier­mediated
transport (biliary and/or renal excretion)212.
This is probably due to the fact that the
concentration of a drug that is not bound to
plasma proteins is lower than the concentra­
tion in the intestinal fluid. In addition, the
transporters in the excretive direction, such
as P­gp, tend to have wide substrate specifi­
city 213,214. The in vivo and clinical effect of
carrier­mediated transport in the excretive
direction has been shown for many drugs by
using knockout animals and by investigating
humans with genetic variants124.
Distribution: blood–brain barrier. The
blood–brain barrier expresses various trans­
porters such as P­gp, mrPs and OATPs. P­gp
affects brain distribution more significantly
compared to oral absorption (TABLE 2). At
the same time, passive transcellular trans­
port also has an important role93–95,159,215.
The effect of the efflux transporter on the

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© 2010 Macmillan Publishers Limited. All rights reserved


brain distribution of a drug (Kpp,b, the brain
concentration of a drug in the presence and
the absence of an efflux transporter) can be
expressed as Kpp,b = 1 + Clcarrier­mediated, efflux/
Clpassive transcellular membrane (REF. 216), showing that
the balance of passive transcellular transport
and carrier­mediated transport affect the
distribution of a drug in the brain.
Distribution to other organs. Passive trans­
cellular transport is essential in describ­
ing in vivo distribution properties of drug
candidates (TABLE 1). The tissue distribution
coefficients were successfully predicted by
log Poct and pKa in physiologically based
pharmacokinetic modelling. For an overview
of this technique, see REFs 82,217.
At the same time, for some specific drugs
in certain organs, carrier­mediated transport
could be crucial. For example, genetically
modified mice showed increased sensitivity
to a neurotoxic drug and a carcinostatic
drug218, suggesting that efflux carrier­mediated
transport significantly contributes to the
permeation of a drug into these tissues.
Excretion and metabolism: hepatic uptake
and biliary excretion. About a dozen drug
transporters are known to be expressed in
hepatocytes211,219. It is established that OATPs
have a significant role in the hepatic uptake
of some hydrophilic statins (for example,
pravastatin, rosuvastatin and cerivasta­
tin)3,220. At the same time, passive transcell­
ular transport also has an important role in
hepatic uptake221. It has been suggested that
carrier­mediated transport becomes impor­
tant when passive transcellular transport is
low, whereas passive transcellular transport
has a dominant role when passive transcell­
ular transport is high210,211 (for example, Papp,
passive transcellular membrane > 5 × 10–6
cm per second)159,222.
Excretion and metabolism: renal excretion.
renal excretion is determined by the balance
of glomerular filtration, tubular secretion
and reabsorption. The glomerular filtration
is the ultrafiltration of unbound drugs and
no carrier­mediated transport is involved. In
addition, many drugs are reabsorbed by pas­
sive transcellular transport depending on the
lipophilicity of the drug. However, carrier­
mediated transport has a significant role in
tubular secretion210. OCTs, OATPs and mrPs
are expressed in the renal epithelium. When
Papp, passive transcellular membrane is > 10 × 10–6 cm per
second, the passive transcellular membrane
reabsorption becomes predominant and little
renal excretion is anticipated. By contrast,
when Papp, passive transcellular membrane is < 1 × 10–6 cm
nATure revIeWs | Drug Discovery
12
> 10
< 10
<8
10 <6
<4
<2
8
6
Efflux ratio
4
2
1.0
0.8
0.6
0.4
Apical efflux by
0.2
transporters
(CLint,sec, ml min–1)
Figure 6 | influence of passive permeation and carrier-mediated efflux on the efflux ratio in
caco-2 cells, described by a theoretical model. The efflux ratio depends on a saturable apical
efflux (cLint,sec) as well as on passive diffusion (cLd = A × Ppassive (BOX 3)).Nature Reviews
with high passive permeation. Data plotted from REF. 147.
per second, the renal clearance often exceeds
the glomerular filtration rate159,223, suggesting
a significant contribution of carrier­mediated
transport in proximal tubular secretion.
Excretion and metabolism: drug–drug
interactions. Drug–drug interactions that
occur through carrier­mediated transport
processes can be a safety concern3, for
example drug–drug interactions between
statins and other drugs by OATP transport
in hepatic uptake220. even when mem­
brane permeation of a drug is determined
by passive diffusion, the drug could be an
inhibitor for carrier­mediated transport of a
co­administered drug.
implications for practical drug discovery
most drug candidates in development are
in all probability highly permeable and
low solubility compounds (BCs class II)224.
This is as a result of a large number of per­
meation and solubility screening processes.
excluding drugs for specific indications (for
© 2010 Macmillan Publishers Limited. All rights reserved
PersPectives
0.2
n –1
)
0.4
mi
d,ml
0.6
(CL
on
usi
0.8
iff
d
ve
ssi
Pa
1.0
0.0
The efflux ratio| Drug Discovery
approaches unity
example, antibiotics) and non­oral delivery
routes, about 80% (based on experience at
F. Hoffmann­la roche) of compounds cur­
rently in lead identification or lead optimiza­
tion and preclinical or clinical development
have medium to high passive permeance
(m.K. and F.s., unpublished observations).
usually such compounds with high passive
permeance have adequate in vivo exposure
when not modified by metabolic degrada­
tion or restricted by low intestinal solubility.
PAmPA experiments can be used to identify
high or low passive transcellular transport
compounds. In addition, low passive tran­
scellular transport compounds are further
characterized in cell­based systems to inves­
tigate carrier­mediated transport 225.
Being a substrate of carrier­mediated
transport can add value to a drug. For exam­
ple, an acyclovir prodrug (valacyclovir)
was designed to be transported by PePT1
(REFs 226,227). Being a P­gp substrate would
also decrease the central nervous system side
effects of a drug, for example, ivermectin228.
vOlume 9 | AugusT 2010 | 609
PersPectives
glossary
Active transport
An energy dependent, carrier protein-mediated transport
process that can be against a concentration gradient.
Area under the plasma concentration–time curve
(AUC). A measure of how much of an administered drug
reaches the bloodstream in a defined period of time.
Bioavailability
In this article, bioavailability means absolute
bioavailability. Bioavailability describes the fraction of an
administered dose (unchanged drug) that reaches the
systemic circulation. Intravenously administered drugs
by definition have a bioavailability of 100%. Other routes
of administration may lead to lower bioavailability,
usually expressed by a ratio or percentage of the
maximum value.
Biopharmaceutics classification system
(BCs). A classification system for drug molecules that
considers solubility and permeation. There are four
classes: BCs class I (molecules have high permeation/high
solubility), BCs class II (molecules have high permeation/
low solubility), BCs class III (molecules have low
permeation/high solubility) and BCs class IV (molecules
have low permeation/low solubility). secondary factors
considered in BCs include the rate of dissolution and
the pH.
Black lipid membrane
A single bilayer phospholipid membrane and optically
black in appearance. It slowly forms when a small quantity
of an egg lecithin dissolved in n-decane is placed over a
small hole in a thin sheet of Teflon suspended in an
aqueous buffer solution. such membranes have been
viewed as useful models of the more complex natural
membranes.
Caco-2 cells
An immortalized line of heterogeneous human epithelial
colorectal adenocarcinoma cells used as a drug transport
model for assessing intestinal absorption. Transport
measurements can be performed in two directions:
apical to basolateral or basolateral to apical.
Carrier-mediated transport
This refers to active or facilitated transport that has a
limited capacity and thus is saturable and subject to
inhibition.
CYP3A4
(Cytochrome P450 3A4). The most important member
of the cytochrome P450 mixed-function oxidase
system. It is involved in the metabolism of xenobiotics.
However, currently, it is difficult to inten­
tionally design a compound that is a dual
substrate for both carrier­mediated transport
(especially for transport in the absorptive
direction) and a pharmacological target.
Inevitably, when designing an orally available
drug, it is the passive transport across mem­
branes in the intestine that offers the highest
probability of success. For instance, most of
the prodrug approaches to increase the intes­
tinal membrane permeation were designed
to increase passive transcellular transport229.
610 | AugusT 2010 | vOlume 9
CYP3A4 metabolism can occur in the gut wall and in
the liver.
Efflux ratio
(EfR). The ratio of the apparent permeation of a
compound in the absorptive (apical to basolateral)
direction to that in the secretory (basolateral to apical)
direction, as determined in cell-based experiments.
The EfR is used to determine possible active transport.
Fa
The fraction of a dose of drug that is absorbed after oral
administration. Fa depends on membrane permeance,
solubility and the dissolution rate of a compound. It is
expressed as a percentage.
First pass metabolism
After intestinal absorption, drug molecules first pass
through the liver before entering the systemic circulation.
During this process, the drug can be metabolized in the
liver and the systemic bioavailability can be reduced.
Lipophilicity
The affinity of a compound for a lipid environment, for
example, the hydrocarbon core of a phospholipid bilayer.
It can also be described as the inter-molecular interaction
between a compound and the solvent environment, such
as the total hydrogen bond strength and dipole/
polarizability.
Log Doct
The octanol–water apparent partition coefficient at a
certain pH. Both dissociated and undissociated
(uncharged) molecular species are taken into account. In
this article, when log Doct is less than zero, it is referred to
as having low lipophilicity, when log Doct is greater than
zero but less than 2, it is referred to as having moderate
lipophilicity and when log Doct is greater than 2 it is
referred to as having high lipophilicity.
Log Poct
The octanol–water partition coefficient of a compound
(neutral form). This parameter is most widely used as a
whole molecule lipophilicity parameter.
Oral bioavailability
The fraction or percentage of a drug that reaches the
systemic circulation following oral administration. It is
determined by the fraction absorbed and the fraction not
metabolized in the gut wall and in the liver.
Papp
(Permeability coefficient). The apparent permeation
for example, in Caco-2 cell-based permeation assays.
At the same time, carrier­mediated transport
could be a reason for drug–drug inter­
actions and other side effects. Therefore, it
is important to survey the involvement of
carrier­mediated transport for individual
drugs, especially when passive transcellular
transport of the drug is low to moderate.
Concluding thoughts
We conclude that passive transport and
carrier­mediated transport coexists, and that
passive permeation remains a significant
© 2010 Macmillan Publishers Limited. All rights reserved
Papp is the sum of passive transport and carrier-
mediated transport when the effect of the aqueous
boundary layer and the paracellular pathway is
neglected.
Passive transport
This refers to the movement of a permeant across a
membrane from a region of high concentration to
that of low concentration by a process of diffusion.
The rate of passive transport is proportional to the
concentration gradient of the permeant across the
membrane.
Peff
(Effective intestinal membrane permeation). Peff is
measured by an in vivo experiment, for example, the
in situ perfusion method. Peff is the sum of passive
transport and carrier-mediated transport.
P-glycoprotein
(P-gp). Also called ABCB1, P-gp is a protein involved in
active efflux transport processes.
pH partition theory
A theory that describes passive transport based on the
pH of the aqueous phase, the dissociation constant (pKa)
and the lipophilicity of a permeant. It states that an
ionizable drug moves across a membrane by passive
diffusion as its uncharged form, depending on its
lipophilicity.
pKa
The dissociation constant pKa for an ionizable molecule is
the pH at which it would be ionized by 50%. The degree
(%) of ionization is pH dependent. For an acid it
decreases by lowering pH. For a base it increases by
lowering pH.
Tissue distribution coefficient
This represents the degree of drug molecule distributing
into a tissue. This value depends on both passive
transport and carrier-mediated transport across the
cellular membrane of the tissue.
Total permeation
This refers to the combination of passive transport and
carrier-mediated transport processes.
Ussing chamber
An instrument used to measure transport (for example,
of drugs) across epithelial barriers. A sheet of epithelia
(for example, intestinal mucosa) is clamped between
two chambers and drug transport across the epithelia is
measured.
(and often primary) factor of membrane
permeation of drug­like molecules. At the
same time, a significant proportion of drugs
were proved to be carrier­mediated trans­
port substrates in specific organs, potentially
leading to significant drug–drug interactions
and side effects. Therefore, further studies of
the balance of how passive transcellular and
carrier­mediated transport influences tissue
and organ distribution, as well as safety rele­
vant aspects, are highly desirable. Direct cor­
relation between in vitro transport and the
www.nature.com/reviews/drugdisc

accurate in vivo pharmacokinetic variables,
and development of in vitro–in vivo extrapo­
lation models are crucial for further progress
in the field230. Therefore, efforts should be
increased to improve our understanding of
the interplay between passive transport and
carrier­mediated transport processes, and to
apply the gained knowledge in the discovery
and development of successful drugs.
Kiyohiko Sugano is at Pfizer, Research Formulation,
Sandwich Laboratories, Ramsgate Road,
Sandwich, Kent CT13 9NJ, UK.
Manfred Kansy, Stefanie Bendels, Holger Fischer,
Grégori Gerebtzoff and Frank Senner are at
The Non-Clinical Safety Department, F. Hoffmann-La
Roche, CH-4070 Basel, Switzerland.
Per Artursson and Hans Lennernaes are at the
Department of Pharmacy, Biomedical Centre, Uppsala
University, S-752 63 Uppsala, BOX 580, Sweden.
Alex Avdeef is at pION, 5 Constitution Way,
Woburn, Massachussetts 01801, USA.
Li Di is at the Pharmacokinetics,
Dynamics and Metabolism Department,
Pfizer, Groton, Connecticut 06340, USA.
Gerhard F. Ecker is at the Department
of Medicinal Chemistry, University of Vienna,
Althanstrasse 141090 Wien, Austria.
Bernard Faller is at the Novartis Institutes
for Biomedical Research, WSJ-350.3.04,
CH-4002 Basel, Switzerland.
Correspondence to M.K. and K.S.
e-mails: manfred.kansy@roche.com;
kiyohiko.sugano@pfizer.com
doi:10.1038/nrd3187
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Acknowledgements
This paper is the joint effort of several colleagues from
academia and the pharmaceutical industry working on a
topic of common interest. We have to thank all involved
colleagues for rapid feedback to support this task. We have
to thank H. Kubinyi for comments at the initial phase of this
activity.
Competing interests statement
The authors declare no competing financial interests.
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