Review
pubs.acs.org/molecularpharmaceutics
Passive Lipoidal Diffusion and Carrier-Mediated Cell Uptake Are Both
Important Mechanisms of Membrane Permeation in Drug
Disposition
Dennis Smith,*,† Per Artursson,‡ Alex Avdeef,§ Li Di,∥ Gerhard F. Ecker,⊥ Bernard Faller,#
J. Brian Houston,¶ Manfred Kansy,□ Edward H. Kerns,■,▽ Stefanie D. Kram
̈ er,○ Hans Lennernas̈ ,‡
●
△
▲
Han van de Waterbeemd, Kiyohiko Sugano, and Bernard Testa
†
4 The Maltings, Walmer, Kent, CT14 7AR, U.K.
Department of Pharmacy, Biomedical Centre, Uppsala University, S-752 63 Uppsala, Box 580, Sweden
§
1732 First Avenue, #102, New York, New York 10128, United States
∥
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, Connecticut 06340, United States
⊥
Department of Medicinal Chemistry, University of Vienna, Althanstrasse, 141090 Wien, Austria
#
Novartis Institutes for Biomedical Research, WSJ-350.3.04, CH-4002 Basel, Switzerland
¶
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K.
□
The Non-Clinical Safety Department, F. Hoffmann-La Roche, CH-4070 Basel, Switzerland
■
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States
○
Institute of Pharmaceutical Sciences, ETH, Zürich, Switzerland
●
14 Rue de la Rasclose, 66690 Saint André, France
△
Research Formulation, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, U.K.
▲
Department of Pharmacy, University Hospital Lausanne, CH-1011 Lausanne, Switzerland
‡
ABSTRACT: Recently, it has been proposed that drug permeation is essentially
carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes
the prevailing hypothesis of drug permeation through biological membranes,
which integrates the contribution of multiple permeation mechanisms, including
both carrier-mediated and passive lipoidal diffusion, depending on the
compound’s properties, membrane properties, and solution properties. The
prevailing hypothesis of drug permeation continues to be successful for
application and prediction in drug development. Proponents of the carriermediated only concept argue against passive lipoidal diffusion. However, the
arguments are not supported by broad pharmaceutics literature. The carriermediated only concept lacks substantial supporting evidence and successful
applications in drug development.
KEYWORDS: permeation, passive lipoidal diffusion, “carrier-mediated only” concept
■
INTRODUCTION
the data on permeation has led to a widely accepted and applied
prevailing drug permeation paradigm, which embodies the
contribution of all the mechanisms. This paradigm is routinely
applied with success in drug design to optimize efficacy and safety,
predict human PK/PD, select dose, and plan dosing regimens.
This review discusses the prevailing permeation hypothesis,
which we conclude is consistent with the experimental data.
Absorption, distribution, and elimination of drug molecules and
their metabolites from the point of dosing, throughout body
tissues, into cells, exposure to biochemical targets, and routes of
clearance involve crossing diverse tissue, cellular, and organelle
membranes. The process of membrane crossing is broadly
referred to as “permeation”, which includes mechanisms of passive
lipoidal diffusion, carrier-mediated influx, carrier-mediated efflux,
paracellular diffusion, aqueous boundary layer (mucus) diffusion,
endocytosis, and others. Owing to the importance of permeation
in drug absorption and disposition, these mechanisms have been
and continue to be an active research area. The preponderance of
© 2014 American Chemical Society
Received:
Revised:
Accepted:
Published:
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February 10, 2014
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This theory is based on results from drug disposition in
pharmaceutical research and from fundamental investigations as
discussed below.
Unifying Role of Lipoidal Permeability in Drug
Disposition. Overview. Tissues of the body are constructed
of cells. Cells are encapsulated by membranes made of
phospholipid bilayers. Proteins are embedded in the cell
membrane and function as sensors for the cell, maintain the
intracellular homeostasis, or transport nutrients and other
substances. Cells are joined, through junction proteins, in
varying degrees of tightness.
Paracellular permeation occurs when drug molecules diffuse
through the cell junctions. Examples of the role of paracellular
permeation in defining drug permeation include the following:
1. For much of the vasculature of systemic circulation, the
junctions are not tight and allow aqueous diffusion of
even large drug molecules. Thus, drug molecules in
circulation are rapidly exposed to tissue cell surfaces.
2. The capillary vasculature in the brain has very tight
junctions between cells, forming the distinct blood−brain
barrier (BBB), which greatly attenuates the diffusion of
drug molecules to the surface of the CNS tissue cells.
3. The cells of the upper gastrointestinal tract are
moderately tight and allow a portion of small drug
molecules (typically polar and less than 300 Da) to pass
through the junctions. These drugs can also reach cell
surfaces for many tissues but will have restricted access to
the CNS.
Paracellular diffusion does not account for most drug
permeation processes, such as for larger molecules, exposure
to intracellular drug targets, or penetration to CNS drug targets
through the BBB. Experimental data also support two
additional major permeation mechanisms: passive lipoidal
diffusion through the phospholipid membrane and carriermediated transport through one of the transporter proteins.
The theoretical framework and the experimental evidence for
membrane permeability of drugs have been reviewed.5,6
Additional recent data26 from fluorine NMR studies on uptake
of modified nucleosides (L-FMAU) into erythrocytes (biological systems that include transporters) provide clear
indication of two different mechanism governing uptake of
L-FMAU in erythrocytes: “facilitated transport via nucleoside
transporter and nonfacilitated diffusion”.
Removal of drug and metabolite molecules from the body is
by metabolism, renal clearance, or “biliary excretion”. Again,
transport proteins may play an important role for some
compounds. Passive lipoidal permeability has a central role in
controlling these processes, as outlined below.
Passive lipoidal permeability is correlated positively with
lipophilicity (e.g., as expressed by the log of the octanol−water
partition, log P, or the apparent value at a given pH, often
7.4, log D) and negatively with the hydrogen-bonding capacity
of a molecule (e.g., as expressed by the polar surface area, PSA,
the solvatochromic parameters α (H-bond donor) and β
(H-bond acceptor), and the molecular hydrogen-bonding
potentials, MHBPs).27−30 If a drug molecule has positive log D
and low PSA, passive lipoidal permeability is high and is a large
fraction of the total permeability, owing to the large surface area
of the cell membrane. In this case, any transporter permeability is
a low fraction of the total permeability, even if the compound has
carrier-mediated transport, because it is limited by the carrier
concentration and the Km (left diagram in Figure 1). If a drug
Importantly, this review also addresses uncertainties among
drug researchers that result from recent articles1−4 published by
a group that assert the carrier-mediated only concept (CMOC)
of drug permeation and attempt to invalidate passive lipoidal
diffusion across biological membranes into cells and across cell
layer membranes. We conclude that the CMOC lacks sufficient
evidence. The CMOC is not a sound scientific principle and
lacks experimental evidence. Carrier-mediated transport and
passive lipoidal diffusion permeation mechanisms are complementary, their relative contribution depending on many
physicochemical and biochemical factors (e.g., concentration
gradient, lipophilicity and hydrogen-bonding capacity of the
diffusing compound, transporter affinity, and Km). The
integrated system follows the laws of thermodynamics.
Discussion of divergent hypotheses, based on experimental
data, is certainly desirable. Thus, recent articles5,6 have
discussed the experimental evidence that both passive lipoidal
diffusion and carrier-mediated transport permeation mechanisms affect the absorption and disposition of drugs. There is
broad consensus in the pharmaceutics field on the existence,
effects, and application of knowledge about both passive
lipoidal diffusion and carrier-mediated transport. Clearly, drug
researchers have, throughout the history of drug discovery,
utilized physicochemical properties (lipophilicity, hydrogenbonding capacity, molecular size, ionization/charge), which
have been correlated to passive lipoidal diffusion permeation
rate and not to carrier-mediated permeation rate, to enhance
the success of drug delivery.7−23 In recent years, drug discovery
researchers have also utilized knowledge about transporter
uptake to enhance drug exposure to certain tissues. For
example, liver specific transporters (OATP1B1 and 1B3)
selectively increase liver concentration of their substrates,
which minimize the exposure to peripheral tissue and reduce
toxicity.24,25
Founded on our experience with detailed experimentation on
drug permeation mechanisms, scientific literature evidence, and
application of drug permeation mechanisms in drug research,
we reiterate and support the roles of both passive lipoidal
diffusion and carrier-mediated transport in the prevailing drug
permeation theory. The purpose of this review is to provide
guidance for drug researchers, so that potential drug candidates
will not be overlooked by mistaken evaluations that ignore the
important effects of passive lipoidal diffusion.
Prevailing Drug Permeation Philosophy Restated. The
prevailing theory of drug permeation through biological
membranes is as follows:
1. There are multiple permeation mechanisms by which
drug molecules, in general, cross biological membranes
and are absorbed and distributed throughout the body,
including passive lipoidal diffusion, carrier-mediated influx,
carrier-mediated efflux, paracellular diffusion, aqueous
boundary layer (mucus) diffusion, and transcytosis.
2. The rate with which a given compound crosses specific
biological barriers (epithelium, endothelium) varies with
(a) the compound’s properties (e.g., lipophilicity, pKa,
and transporter parameters [Km and Vmax]), (b) the
membrane’s properties (e.g., composition, transporter
function, tight junction characteristics, and membrane
potential), and (c) solution properties (pH, pH gradient
across membrane, and permeant concentration gradient
across membrane).
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Figure 1. Schematic relating to drugs and their dependence on passive lipoidal diffusion and carrier-mediated transport to cross membranes. Drug
molecules with balanced lipophilicity and relatively low hydrogen-bonding capacity rapidly cross membranes by lipoidal diffusion (white double
arrows), owing to the high cell surface area, at high rates compared to minimal transporter impact (gray unidirectional arrows). Drug molecules with
greater hydrogen-bonding capacity and lower lipophilicity cross membranes by lipoidal diffusion less readily and may, therefore, be influenced by
transporters for which they have substantial affinity and transport rate.
molecule has negative log D and high PSA, passive lipoidal
permeability is low and, if the molecule has carrier-mediated
permeability, a significant portion of the total permeability may
be carrier-mediated (right diagram in Figure 1). The schematics
shown in Figure 1 illustrate the separation between drugs of
high and low lipoidal permeability.
The evidence for lipoidal diffusion is provided by many
experiments, and importantly the results are consistent for
passage across any cell type, organ, or species and are not
energy or concentration dependent.
Evidence from in Vitro Monolayer Cell Systems. Lipoidal
diffusion is well exemplified by in vitro monolayer cell systems
used to predict or rationalize drug absorption. These measure
flux in an apical to basolateral (A−B) direction and vice versa.
A highly passive lipoidally permeable drug will show the
following:
• a comparatively high rate of apical to basolateral (A−B)
flux of a group of compounds
• no effect of concentration on rate constant, up to the
limit of solubility
• near identical A−B and B−A flux regardless of
concentration
• lack of effect of inhibitors of known transport proteins
• similar rates of flux in cell lines selectively cultured for
low and high transporter expression.
There is only small variation in in vitro permeation rates of
drugs between various cell layer permeation experiments in
which large variation exists in transporters.31 The same is true
in permeation rates of drugs between Caco-2 (from human
colon carcinoma) and MDCK (from canine kidney),32,33 where
the following observations were made (Figure 2):
• There is a very close correlation between physicochemical systems (including PAMPA and liposomes) and cell
based systems.
• It does not require energy.
• It happens in all organs, species, and cell types.
Typical MCDK35 and averaged Caco-236 results are
illustrated in Table 1 for a series of beta adrenoceptor blockers.
Permeation rate increases with increasing lipophilicity, as
observed in many experiments,21 and clearly indicated for
Caco-2 in Table 1, which is consistent with passive diffusion
through the lipophilic core of the bilayer membrane. The high
passive lipoidally permeable compounds (lipophilic, with low
polar surface area such as bufarolol, alprenolol, and
Figure 2. Caco-2 and MDCK intrinsic permeability, log P0 (uncharged
form), values of 79 drugs thought to cross cell layers predominantly by
passive lipoidal diffusion. The expression of drug-transporting proteins
in the Caco-2 cell line differ substantially from laboratory to
laboratory.34 Furthermore, the expression of drug-transporting
proteins in Caco-2 and other cell lines differs substantially.31 In spite
of that, the two cell models indicate nearly the same intrinsic
permeability, with r2 = 0.90, slope = 1 and intercept ∼0.33
propranolol) show identical high rates of transport in either
direction across the MDR1-MDCKII cells and are not
influenced by the presence of a transporter inhibitor. Less
passive lipoidally permeable compounds, such as labetalol,
show different rates A−B and B−A (due to different heights of
the energy barrier), and are markedly affected by the presence
of the transporter inhibitor. The transport of atenolol, a low
molecular weight drug of low lipophilicity, is based on
paracellular permeation. The permeation rate is related to
ionization constant pKa and pH according to the “pH-partition
theory”37 and to intrinsic lipophilicity (log P), with permeation
rate increasing in relation to increased concentration of a
compound’s neutral species (to which log P refers), which is
the form that permeates the bilayer membrane while the
charged form has very low permeability. log D is a particularly
useful term in explaining the interplay between intrinsic
lipophilicity (log P) and pKa. In Table 1, the drugs have
similar pKa, but the degree of ionization means that their log D
at pH 7.4 is approximately 2 log units lower than their intrinsic
lipophilicity (log P). Other experiments show that permeation
rate is well correlated between a biological membrane (e.g.,
Caco-2), an artificial membrane (e.g., PAMPA), and octanol/
water partition coefficient.21,38
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Table 1. Unidirectional Permeability across MDR1-MDCKII of High Lipoidal Compounds Exemplified by Beta Adrenoceptor
Antagonists, Compared with Caco-2 Permeability35
drug
log P
log D(7.4)
PSA (Å2)
Papp,A−Ba
Papp,B−Aa
Efflux Ratio
Papp,A−Ba (plus GF120918)
efflux ratio (plus GF120918)
Caco-2 Pappa,b
propranolol
metoprolol
alprenolol
oxprenolol
bufuralol
labetalol
atenolol
3.5
2.0
3.0
2.5
3.0
1.3
0.2
1.4
−0.2
0.9
0.3
0.6
1.1
−1.9
42
51
42
51
45
96
85
40
30
46
31
64
4.1
0.3
42
36
47
42
50
36
0.3
1.0
1.2
1.0
1.4
0.8
8.8
1.0
44
37
43
31
48
7.4
0.2
1.1
1.3
0.9
1.0
0.8
1.8
0.7
494
231
99
42c
14c
31
0.7
Permeability in units of 10−6 cm/s. bAveraged measured value at pH 7.4, corrected for aqueous boundary layer and paracellular effects.36
Calculated (Avdeef, unpublished).
a
c
Figure 3 is an example of the in vitro−in vivo correlation
between the blood−brain barrier permeability and that of
Figure 4. Partitioning of (mostly basic) drugs into human red blood
cells.41 The extent and rate of partitioning of drugs into erythrocytes is
widely accepted to be a passive diffusion process at the plasma
membrane, significantly depending on lipophilicity, as represented by
the octanol−water partition coefficient, log POCT. Ke/p,u is defined as
the erythrocyte-to-plasma water partition coefficient of drug, obtained
from the ratio of the drug concentration in erythrocytes and serumprotein-free plasma water (or buffer).
Figure 3. In vivo−in vitro correlation between luminal blood−brain
barrier intrinsic permeability, as indicated by the in situ brain perfusion
model, log P0in situ,40 and the artificial membrane model, log P0PAMPA,
shows a slope near unity and an intercept near zero.39 The PAMPA
(parallel artificial membrane permeability assay) blood−brain barrier
“double-sink” model, based on 10% porcine brain lipid extract
dissolved in alkane,39 shows very high correlation of r2 = 0.84 for 85
basic drugs thought to permeate by lipoidal passive diffusion. Examples
of molecules known to be P-glycoprotein substrates, represented by
checkered circles, fall below the lower dashed line. The L-isomer of
para-F-phenylalanine is actively transported into the brain and shows
up above the upper dashed line.40 The R-isomer shows only a slightly
elevated permeability, above the “passive lipoidal diffusion” zone. Since
a purely lipoidal passive diffusion model (PAMPA) can predict in vivo
permeability, passive diffusion appears to play a significant role at the
blood−brain barrier for many drug molecules.
rate-limited in passage across a barrier that has little to do with
the transport properties of biological cell membranes. Adjacent
to the surface of any cell is a stagnant water layer, known as the
“aqueous boundary layer” (ABL). Before molecules encounter
the cell surface, they first need to traverse the ABL by passive
aqueous diffusion, which is a relatively slow process, particularly
when the assay solution is unstirred. For very lipophilic
molecules, this process is often rate limiting and can obscure
the contribution of faster carrier-mediated or lipoidal passive
diffusion processes to the measured total permeability. In Table 1,
the first five drugs (with log P ≥ 2) indicate essentially the
same MDCK Papp (43 ± 10 × 10−6 cm/s) regardless of
variation in log P or the transport directions. This should be
expected for molecules of comparable size when ABL limits the
rate of transport in both directions. Methods have been
developed (e.g., summarized in ref 36) to deconvolute the
contributions of various processes affecting transport, and it is
possible to estimate the passive lipoidal permeability due to the
cell membrane itself.
The last column in Table 1 lists Caco-2 permeability at pH
7.4 (averaged from numerous studies), after correction for
ABL, paracellular, and filter porosity contributions. Evidently,
Papp values do change substantially (14−494 × 10−6 cm/s) for
the first five drugs, suggesting that the uncorrected MDCK
permeability in the Table is ABL-limited. For ionizable drugs,
carrier-mediated effects can be recognized, but it is necessary to
perform the assay over a range of pH values (not just 7.4).
Figure 5 shows such studies performed for the β-blockers
PAMPA based on 10% porcine brain lipid extract.39 Figure 4
shows that the uptake of drugs into human red blood cells
significantly correlates with log P. Permeation rate also
decreases as the hydrogen-bond donor and/or acceptor
capacity of the molecule increases, consistent with breaking
hydrogen bonds with water to diffuse into the lipophilic core of
the membrane. Such stereoelectronic effects are expressed as
the polar surface area (PSA) and derived parameters.28,42,43
The phenomenon is illustrated by comparison of bufuralol (low
PSA, high passive lipoidal permeability compound) and
labetalol (high PSA, lower permeability compound). This
compound now shows the influence of transporters and has a
B−A flux greater than its A−B.
Measurement of cell permeability can be surprisingly
complicated. For example, very lipophilic molecules are often
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Figure 5. Caco-2 permeability profiles of three β-blockers as a function
of pH, from assays employing 450 rpm stirring.44 The lower and upper
limits of directly measurable permeability are represented by the
dash−dot−dot curve (paracellular permeability, which slightly
depends on pH for ionizable molecules) and the dotted line (aqueous
boundary layer, ABL, permeability, which is pH independent),
respectively. The dashed curves represent the transcellular permeability, PC, which is pH dependent and conforms to the pH-partition
hypothesis. The maximum PC value at high pH is called the intrinsic
permeability, P0 (28840, 14125, and 46 × 10−6 cm/s for propranolol,
metoprolol, and atenolol, respectively). The solid curves represent
apparent permeability, Papp, and are the result of fitting of the
measured points to a sigmoidal function. The leveling of the solid
curves above pH 8 for the two lipophilic bases is due to the ABL
resistance (PABL = 219 × 10−6 cm/s), a rate-limiting aqueous diffusion
process. Papp measurements in alkaline pH conform to the pHpartition hypothesis and are a combined serial function of passive
lipoidal (cell bilayer membrane) and aqueous (ABL) diffusion.
Hydrophilic atenolol is not ABL-limited in transport at any pH. For
the two lipophilic bases, the solid curves start to level off in acidic pH,
very likely because of a carrier-mediated (CM) uptake process
affecting the positively charged bases,38 with PCM = 29 and 2.8 × 10−6
cm/s in the cases of propranolol and metoprolol, respectively. In the
case of atenolol, the leveling off in acidic pH is most likely due to the
paracellular pore permeability (Ppara = 0.59 × 10−6 cm/s).
Figure 6. The efflux transporters at the blood−brain barrier actively
lower the concentrations of many drugs in the brain. Kp refers to the
ratio of the total drug concentration in brain to that in plasma. An
example of steady-state brain−plasma distribution of 154 drugs is
illustrated by the plot of log Kp (sometimes called “log BB”) versus the
log of the unbound fraction of drug in plasma divided by the unbound
fraction of drug in brain homogenate. Molecules in the region between
the dashed lines (filled circles, 56% of the drugs in the example) most
probably cross the blood−brain barrier passively. Below the lower
dashed line, the efflux clearances dominate over the influx clearances.
Many of the molecules (checkered circles, 36% of the drugs) in this
lower region are known to be substrates of the P-glycoprotein efflux
pump. (Avdeef, unpublished compilation).
the brain by passive lipoidal diffusion across the blood−brain
barrier (BBB). At steady state, this is indicated by the equality
of the inward and outward clearances: CLpassive + CLinflux =
CLpassive + CLefflux.46 According to the “free drug hypothesis”,
passive lipoidal diffusion is indicated when the unbound drug
concentration is the same on both sides of a biological
membrane under steady-state equilibrium. Consequently, the
brain-to-plasma total concentration ratio, Kp, becomes equal to
the ratio of the unbound fraction of drug in plasma to the
unbound fraction of drug in brain homogenate: Kp = f u,pl/f u,br.
In Figure 6, the zone between the dashed lines defines those
molecules that permeate predominantly by passive lipoidal
diffusion. Below the lower dashed line, the efflux clearances
dominate over the influx clearances. Molecules (checkered
circles, 36% of the drugs) in this lower region, such as
doxorubicin, ivermectin, digoxin, paclitaxel, saquinavir, cetirizine, fexofenadine, methotrexate, vinblastine, etc., are known to
be substrates of the P-glycoprotein efflux pump. The BBB has
an effective efflux transporter system that attenuates the entry
of 36% of the drugs in the example.
Once inside the brain parenchyma, molecules are apparently
less subjected to transporter effects. The distribution of drugs
between the various compartments inside the brain appears to
follow the pH-partition hypothesis, that is, transport is effected
by passive lipoidal diffusion, as shown in Figure 7 for the
steady-state distribution of 88 drugs within the brain
parenchyma.47 The figure plots log f u,br (unbound fraction of
drug in brain homogenate) versus the log of the volume of
distribution of the unbound drug in brain parenchyma, Vu,br,
divided by the unbound drug partitioning in the cell, Kp,uu,CELL.
The latter partition coefficient describes the intracellular-toextracellular unbound drug concentration ratio. That is,
Kp,uu,CELL = [drug]u,CELL/[drug]u,brISF = f u,brVu,br, where brISF
= brain interstitial fluid. The estimated pH values in the
interstitial fluid, the brain cell cytosol, and the intracellular
lysosomes are 7.3, 7.06, and 5.18, respectively.47 The volumes
of the three corresponding compartments are estimated to be
propranolol, metoprolol, and atenolol.44 The intrinsic permeability
values of the uncharged forms (pH > 10) of these molecules can
reach enormous values: 28840, 14125, and 46 × 10−6 cm/s,
respectively. For very lipophilic molecules (e.g., propranolol,
metoprolol), carrier-mediated transport is generally not as fast
as passive lipoidal diffusion in alkaline solutions. According to
the pH-partition hypothesis, as the concentration of the
uncharged form of the β-blocker decreases with decreasing
pH, so does the transcellular permeability. As shown in Figure 5,
for pH < 6.5, the carrier-mediated process becomes the
predominant contribution to the observed permeability of
propranolol (29 × 10−6 cm/s) and metoprolol (2.8 × 10−6 cm/s),
as suggested by a study of the transport of verapamil in the
Caco-2 model.38 Atenolol permeability in acidic solution appears
to be due to paracellular aqueous diffusion (0.59 × 10−6 cm/s).
Evidence from in Vivo Investigations. In vivo evidence is
also fully supportive of passive lipoidal permeability being the
major influence on drug disposition. Access to intracellular and
intraorgan (CNS) targets shows, for a highly passive lipoidally
permeable drug, identical concentrations in the aqueous phase
between plasma (unbound free drug) and the intracellular or
intraorgan water.
The CNS represents an important vascular/cellular barrier
that is accessed in most cases by lipoidal diffusion and is
amenable to quantitative structure−permeation relations.45
Figure 6 considers 154 drugs, of which 56% appear to access
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The clearance of highly lipid-permeable drugs mirrors all the
above. Even if the drug is a possible substrate of a transporter,
clearance is invariably metabolic. This is evidenced by the
following:
1. A normally negligible excretion or clearance of a
lipophilic compound in urine. Even though the unbound
drug is filtered at the glomerulus and could be
concentrated in the renal tubule by transporter
involvement, passive lipoidal diffusion means the drug
is passively reabsorbed as the urine is concentrated,
resulting in negligible urinary excretion of all highly
lipoidally permeable drugs.
2. When urinary pH is altered, compelling proof of lipoidal
permeability is shown for drugs with a positive log P
value (i.e., whose neutral form is lipophilic) and a
negative log D value (i.e., whose ionized form is
hydrophilic).50−52 Thus, the basic drug amphetamine
(log P 1.8, pKa 9.94, TPSA 26 Å2) is poorly excreted
(3−7%) in alkaline urine and markedly excreted (60%)
in acidic urine. This behavior is due to the drug
undergoing renal filtration, followed by passive reabsorption of the lipophilic neutral form, or lack of reabsorption
of the hydrophilic cationic form. Conversely, acetylsalicylic acid (log P 0.90, pKa 3.50, TPSA 64 Å2) shows
greater excretion at a urinary pH of 7 (3%) compared
to pH 5.5 (1%). Memantine, a basic drug (log P 2.1,
pKa 10.2, TPSA 26 Å2) has been examined at urinary pH
values of 5 and pH 8 and shows a 7−10-fold higher renal
clearance at acidic compared to basic urinary pH.53 At
pH 5 unbound renal clearance values (6 mL/min/kg)
exceed glomerular filtration rate (2 mL/min/kg),
indicating some active tubular secretion in addition to
glomerular filtration, illustrating how transport proteins
can exist alongside passive diffusion processes. It is
difficult to conceive of a system able to explain the effects
of pH on urinary excretion and clearance other than
passive lipoidal diffusion.
3. The rapid transfer of drugs in both directions across the
sinusoidal membrane results in intracellular hepatocyte
concentrations approximating unbound plasma concentrations. Although not direct evidence, this probably
explains the utility of the well-stirred liver model and its
robust use in in vitro to in vivo predictions of clearance
for highly lipid permeable drugs.
Thus, for an oral, high lipoidal permeability drug, only during
the absorption process will a concentration gradient exist in the
body and free drug concentrations will be identical between
plasma water, extracellular water, and total body water. The
absence of concentration gradient indicates the passive
diffusion nature of the process.
When a drug has moderate or low passive lipoidal
permeability, behavior different from that outlined above
occurs in a predictable manner. As the rate of passive lipoidal
diffusion is lower, the influence of transporter proteins can
become apparent. Transport gradients will now exist across the
aqueous compartments of the body. The evidence for the
passive diffusion of highly permeable drugs now is reflected in
almost an inversion of all the statements above for drugs of
moderate to low lipid permeability. These concepts allow the
rational understanding of drug disposition based on compound
characteristics.
0.2, 0.79, and 0.01 mL per gram of brain tissue, respectively.
For intrabrain distribution by passive lipoidal diffusion,
Kp,uu,CELL can be simply calculated by taking into account the
pKa of the drug and the pH and volumes of the three brain
compartments, as demonstrated elegantly. Examples of passive
distribution are shown in Figure 7 by filled circles (87% of the
Figure 7. Many CNS drugs distribute inside the brain by passive
lipoidal diffusion. Examples are shown by filled circles (87% of the
drugs in the example) bounded by the two dashed lines. There appear
to be net efflux processes (checkered circles, 5% of the drugs), driving
compounds from brain cells into the brain interstitial fluid. Examples
of net clearance from the interstitial fluid into the brain cells are shown
by molecules represented by unfilled circles (8% of the drugs).47
drugs in the example) bounded by the two dashed lines. There
appear to be net efflux processes for NFPS, sertraline,
sumatriptan, and trifluoperazine (checkered circles, 5% of the
drugs), driving compounds from brain cells into the brain
interstitial fluid. Examples of net clearance from the interstitial
fluid into the brain cells are shown by molecules represented by
unfilled circles (8% of the drugs), several of which bear
permanent positive charge.47 (Moxalactam is likely to be in the
passive lipoidal diffusion group, but it appears as an outlier since
its measured f u,br > 1, the physically meaningful upper limit.)
It is now widely accepted that CNS activity is determined by
the unbound brain drug concentration, Cu,br, and not the total
concentration in the brain, CbrTOT. For those molecules that are
substrates of receptors accessible from the interstitial fluid, the
unbound concentration in the interstitial fluid, Cu,brISF, is best
correlated with receptor occupancy. This was experimentally
demonstrated for 18 molecules, where more accurate prediction
of the biophase drug concentration was achieved by using Cu,br
rather than CbrTOT.48 To a lesser degree of accuracy, the
unbound plasma concentration, Cu,pl, could be used to predict
the biophase concentration in the brain.
Considerable amounts of human data are available. This is
exemplified by studying sedating (highly passive lipoidal
permeable) and nonsedating (lower passive lipoidally permeable, influenced by efflux transporters) H1 receptor antagonists
(antihistamines).49 Chorpheniramine, in contrast to the nonsedating antihistamine ebastine, when studied with PET
scanning, had a Kd calculated from CNS receptor occupancy
(7 nM) and unbound plasma concentrations matching that
measured in in vitro receptor experiments (3−4 nM). The
receptor occupancy of ebastine was much lower than its plasma
concentration would suggest. Ebastine rapidly forms the
zwitterionic (low passive lipoidal permeability) active metabolite cavebastine and is partially excluded from the brain due to
this and the activity of efflux transporters.
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Table 2. Representative Transdermal Medicines
drug
therapeutic class
major indication
formulation
MW
log P
pKa
diclofenac
17β-estradiol
fentanyl
glycol salicylate
nicotine
nitroglycerine
scopolamine
selegiline
testosterone
NSAID
estrogenic agonist
μ-opioid agonist
presumably a prodrug of salicylic acid
nicotinic agonist
vasodilator
muscarinic antagonist
MAO-B inhibitor
androgenic agonist
locomotor pain
hormone replacement therapy
narcotic analgesia
locomotor pain
nicotine withdrawal
angina pectoris
motion sickness
Parkinson’s disease
hormone replacement therapy
hydrogel, cream
gel, patch, disk
patch
hydrogel, cream
disk, patch
disk, patch
disk
patch
patch, hydrogel
296.1
272.4
336.5
182.2
162.2
227.1
303.4
187.3
288.4
4.5
4.0
4.1
2.1
1.2
1.2
0.98
2.7
3.3
4.1
10.3
8.8
Transdermal Medicines: A Case for Passive Permeation.
Skin was originally considered an essentially impermeable
barrier whose function was restricted to protecting animal
organisms from their environment. Since years, it is now known
that the skin also represents a potential portal of entry by which
many chemicals may gain access to the systemic circulation.54
This route of administration of systemically acting drugs
provides strong evidence for the determining involvement of
passive permeation processes in their transdermal permeation.
During skin absorption, drugs have to pass a complex
multilayer structure before reaching the blood.55,56 Structurally,
the skin can be viewed as a series of layers, the three major
divisions being epidermis, dermis, and subcutis or hypodermis.
The latter lies below the dermis and the vascular system, and is
therefore not relevant to percutaneous penetration. The dermis
(2 mm) is essentially an acellular collagen-based connective
tissue which supports the many blood vessels, lymphatic
channels, and nerves of the skin. Hair follicles and sweat glands
originate in this layer and open directly onto the skin surface.
The avascular epidermis consists of stacked layers of cells
whose thickness varies between about 0.1 mm (eyelids) and
0.8 mm (palms, soles).57 The outermost layer of the epidermis,
namely, the stratum corneum, is composed of 10−15 layers of
flat keratin-filled cells, closely packed in a nonpolar lipid matrix
composed of ceramides, cholesterol, and fatty acids, but largely
devoid of phospholipids.58,59 In contrast and to the best of our
knowledge, there is no influx transporter in the stratum
corneum.
The transdermal administration of systemically acting topical
medications relies on specific formulations (e.g., hydrogels,
creams, ointments, patches, or disks), from which drugs exit
down a concentration gradient. Given the absence of
transporters, the main rate-determining step in skin penetration
is then diffusion of the solute across the stratum corneum,
which represents the principal penetration barrier of the
skin.57,60 Under conditions of normal passive diffusion, it
appears that the intercellular route predominates over the
transcellular route.61−63 Moreover, an important property of
the stratum corneum is its ability to become heavily hydrated,
modifying also the permeation profile of chemicals.57,64
A direct in vivo proof that transcutaneous medicines are
indeed absorbed through the stratum corneum and able to
reach their target tissues or organs is found in the mere fact that
they were cleared for marketing. Table 2 (data taken mainly
from the Canadian Drug Bank65) offers a short list of
transdermally active medicines to illustrate the variety of
tissues and organs they can target (muscles and joints, sex
hormone receptors, central receptors, etc.). Also noteworthy is
the fact that basic, acidic, and neutral compounds coexist in this
list, as is the wide range covered by log P (1−4.1) and pKa
8.8
6.9
8.7
values (4−10). Indirect evidence obtained from the statistical
analysis of in vivo and in vitro skin permeation studies have
revealed excellent correlations with molecular descriptors such
as molecular weight and volume, log P, log D, and H-bonding
parameters.66−71
Why Debate the Role of Passive Lipoidal Permeability? There are now authors who doubt the role of passive
lipoidal diffusion in drug permeation. It is possible their view is
colored by a growing interest in transporters as drugs become
less lipid-permeable to conform to target active site characteristics (see above). Some of these authors state that carrier
mediation is the only existing form of permeation, at the
exclusion of lipoidal permeation (CMO concept). While
healthy debate is warranted, the group most advocating the
CMOC has made statements to discredit passive lipoidal
diffusion and its foundational evidence. This is contrary to the
conclusion of many, including the authors. Passive lipoidal
diffusion exists alongside carrier-mediated transport, and the
pathways are defined by physicochemical characteristics. The
proponents of the CMOC state that “...evidence and reasoning
we describe may be seen as circumstantial...”.1 The statements
against passive lipoidal diffusion have been strongly refuted,5,6
and a brief recounting follows.
A Debate on the CMOC or an Integrated System Defined
by Lipoidal Diffusion. The major points made in support of the
CMOC are followed by a response. Readers will find greater
detail in the articles.5,6 Arguments 1−13 are from Dobson and
Kell;1 argument 14 is from Dobson et al.;2 argument 15 is from
Kell et al.:3
1. Lipophilic cations are charged and cannot cross
membranes owing to Born charging. Response: Drug
molecule ions are in equilibrium with neutral nonionized
drug molecules, which have much higher lipophilicity
and much higher passive diffusion permeation rate.
According to the pH-partition theory, permeation rate
varies with solution pH and a compound’s pKa such that
an increasing ratio of nonionized/ionized forms
correlates with increasing permeation rate. The term
log D is a descriptor incorporating the effects of
lipophilicity and ionization.6
2. The mass ratio of protein:lipid in vivo (1/1 to 3/1)
affects the transport properties of lipids. Artificial
membranes do not model biological membranes, owing
to the high protein content in vivo. Response: These
ratios include the cytoplasmic and exoplasmic portions of
membrane protein mass, not just the relevant transmembrane fraction. The lipid:protein molar ratio is
estimated as 40:1, making lipid an important portion of
the membrane exposed to drug molecules.6 A further
refined consideration would take into account the
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relative cross-sectional area at the membrane surface of the
40 phospholipid molecules to one typical protein. The
lipid surface area would still be significantly greater than
that of the transporter protein.
3. Correlations of drug uptake with log P and Caco-2
permeation can be weak. Response: For drugs
permeating predominantly by passive lipoidal diffusion,
the apparent Caco-2 permeability coefficient, Papp, can be
(and often is) affected by the aqueous boundary layer,
filter, paracellular, and lipoidal (transcellular) permeability, as well as the solution pH, as illustrated by the
examples in Figure 5. log P cannot be directly compared
to log Papp. The Caco-2 intrinsic permeability, log P0, is
the rational term to compare to log P. P0 is easy to
deduce from Papp,36 but this is seldom done, which often
leads to “weak” correlation, as “apple seeds are compared
to whole watermelons.” Caco-2 cells from 10 different
laboratories were compared in terms of transporter
mRNA levels of 72 drug and nutrient transporters, and
17 other targets. It was concluded that “Caco-2 cells from
different laboratories produce different results even when
using standard protocols for transport studies. The
differences may be due to transporter expression as
shown for e.g. PepT1 and MDR1 which in turn is
determined by the culture conditions. Although the
majority of the laboratories used similar culture
conditions, absolute expression of genes was variable
indicating that even small differences in culture
conditions have a significant impact on gene expression,
although the overall expression patterns were similar”.34
Therefore, it is not astonishing that results of Caco-2
cell based permeabilities, when correlated with octanol
log P/D values, sometimes show differences in
correlations. This is mainly due to the origin and
composition of the analyzed data set (ratio actively
versus passively [lipoidal diffusion] transported compounds) and use of partition coefficients (log P) or pHdependent distribution coefficients (log D). Interestingly,
correlations of transport studies performed with different
cell lines (e.g., Caco-2/MDCK) commonly used in
absorption prediction, with presumably different transporterexpression levels, often give excellent correlations,32
further supporting the coexistence of active and passive
transport in biological systems.
4. Transport across model artificial membranes is stated to
occur via pore defects or dissolution in the lipid mixture
that are not seen in vivo. Response: The studies cited
are computational simulations (so-called molecular
dynamics) of Na+ and Cl− ion (non-drug-like) transport
under unusual conditions. No convincing experimental
evidence for the relevance of pores has been reported.
Other experiments indicate the unimportance of pores.
Membrane resistance excluding pore diffusion is usually
determined by conductivity measurements. Otherwise
function of, e.g., ion channels could not be determined.6
5. A dominant role for carrier-mediated transport (and
against passive diffusion) is inferred from the hundreds of
publications on drug transporters. Response: A large
number of papers have been published in recent years on
transporters. These result from the recent intense
research on transporters. However, it is a logical fallacy
and a sleight of hand to state that this is evidence of the
rate and extent of dominance of carrier-mediated
6.
7.
8.
9.
10.
11.
1734
permeation over passive lipoidal diffusion. (An analogy
would be to state that newspapers contain a predominance of articles about bad events (e.g., fires, wars,
violence, accidents), therefore, bad events dominate good
events in the world.) Thus, the large number of citations
of publications on transporter research is misleading,
because the research they report or review was not
undertaken nor concluded by the publication authors as
evidence that supports CMOC, as is implied (“There is
considerable and increasing evidence that drugs get into
cells more or less solely by hitchhiking on carriers
normally used for the transport of nutrients and
intermediary metabolites”.3
Selected small molecules, urea and glycerol, which cross
BLM, permeate to some extent in vivo via transporters,
except in yeast because glycerol is an osmolyte.
Response: Urea and glycerol are more hydrophilic
than typical drugs that permeate membranes, thus, they
are not good models of permeants on which to support
theories.
In liposomes the rate of transfer of nonelectrolytes
depends on MW rather than log P. Response: Liposomes
correlate well with the permeation behaviors usually
observed in artificial and biological membranes.18,36,72−74
Molecular weight is partly correlated with lipophilicity
and hydrogen-bonding capacity, and as molecular weight
increases in drugs normally so does hydrogen bonding.
Molecular weight is therefore a hybrid term expected to
show a relationship to lipoidal membrane permeation.
In vitro models of diffusion rates across membranes are
not based on large sample numbers and validated with
compounds not used in the method development.
Response: This is out of date information.36
The flux across in vitro PAMPA membranes can be poor
even when human absorption is good (e.g., cephalexin,
tiacrilast). Response: This is out of date information and
also is misleading. PAMPA membranes serve to model
passive diffusion, whereas cephalexin and a number of
other molecules are carrier-mediation transported, as
extensively compiled.36 The present authors claim that
passive and active transport processes coexist. PAMPA
has been described to only account for passive
membrane permeation processes. Therefore, it is not
astonishing that actively transported compounds like,
e.g., cephalexin cannot be correctly predicted regarding
human absorption by methods exclusively focusing on
passive transport.
Activities of anesthetics were previously thought to be
controlled by passive diffusion and correlated to log P,
but are now thought to be protein-binding related.
Response: There is common agreement that drug
molecules and anesthetics might interact with proteins,
but this is misleading in the context of the discussion,
which is around drug transport and not mechanism of
action. A recent publication on anesthetics has
summarized thus: “The molecular mechanism of general
anesthesia is still a controversial issue. Direct effect by
linking of anesthetics to proteins and indirect action on
the lipid membrane properties are the two hypotheses in
conflict.”75
Many molecules (e.g., ethanol) have relatively specific
receptors, so they may have similar protein binding
(unidentified) that effects membrane permeation.
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Permeability can also affect in vitro cell-based assays in drug
discovery because molecules must cross cell membranes to
reach intracellular targets.
Many of the rates for the preceding permeability processes
are measured using in vitro and in vivo protocols during drug
discovery and development to observe the rates of permeation
of an experimental compound in compound screening for lead
optimization and in detailed characterization for preclinical
studies predicting human pharmacokinetics and safety. The in
silico, in vitro, and in vivo tools that have been developed using
the prevailing drug permeation hypothesis provide reliable
predictions of drug disposition in vitro, in preclinical species and
in human. Drug researchers regularly apply these tools and have
achieved notable progress and success in drug design and
optimization.81
Major New Evidence Is Needed To Support the
CMOC. There is insufficient evidence supporting CMOC to
lead to its significant application in drug development, and the
prevailing permeation hypothesis continues to be widely
applied. A fresh collection of experiments initiated based on
the unique claims of CMOC has not been reported. Commentaries on CMOC have mainly focused on reinterpretation
of data from earlier experiments, not excluding elements of
confirmation bias.82 Only one research paper83 with results
from experiments that are based on CMOC found previously
unknown linkage of carriers to permeability of selected
compounds that are toxic to yeast. These carriers have not,
to our knowledge, been validated and characterized with the
type of rigor expected of any new target or transporter in drug
research or for publication.
Since its first description in 2008, the CMOC has not been
the basis for new advances in drug delivery and disposition.
There have been no original research articles, attributed to a
foundation of CMOC, in which carriers were demonstrated to
completely explain the permeation of clinical drugs that were
previously thought to permeate mainly by passive lipoidal
diffusion. Of the many in silico tools used widely in drug
disposition prediction, CMOC has not been embraced for
incorporation. Considerable experimental evidence and peer
review concurrence would be needed to gain acceptance for
CMOC, in accordance with the usual practices for permeation
research. These include the following:
Response: This is an assumption and generalization
awaiting to be proven by experimental data, but which
currently does not rule out transport by passive (lipoidal
diffusion) mechanism.
12. Carrier-mediated drug uptake is observed where it has
been studied. (Presumably this circumstantially indicates
that transporters will be found for all drugs.) Response:
Carrier-mediated drug uptake may be observed, but it
may not account for 100% of the transport. In
Michaelis−Menten analysis, the nonsaturable term
usually is related to the passive diffusion contribution.
13. Drugs can concentrate in specific tissues beyond the
stoichiometry of internal binding sites. This phenomenon absolutely requires an active uptake process.
Response: This can be due to pH gradients between
intracellular and extracellular compartments as described
for, e.g., basic amines and safety relevant lysosome
accumulation (phospholipidosis).76
14. Biophysical forces in drug−lipid membrane interactions
(e.g., lipophilicity, hydrogen bonding) are no different
from drug−protein interaction. Thus, physicochemical
properties and the rule of 5 need not be evidence of
passive diffusion.77 Response: Of course biophysical
forces apply to both carriers and bilayers. However, the
physical property differences between the rate limiting
barriers for a particular drug in carriers and bilayers can
dictate the predominant route of transport.19 A second
argument is that ligand−protein recognition is dominated by highly selective stereoelectronic features far
more than by global (molecular) physicochemical
properties.78−80
15. The notion of passive lipoidal permeation is traced back
to artificial membrane systems, which are not successful
predictors of membrane permeation. Response: On the
contrary, artificial membrane models have been successful predictors of passive lipoidal permeation.11,14,17−20,36
Accurate Understanding of Drug Permeation Mechanisms is Important for Drug Development Success. A
major value of the prevailing permeation hypothesis is that it
guides drug research and development, providing a reliable
foundation for lead selection, candidate optimization, preclinical studies, and product development. Therefore, it is
imperative that accurate permeation theories are implemented.
Translation of a discovery therapeutic “hit” to a clinical
candidate, in part, requires accurate prediction of drug exposure
to the target, which is enabled, along with models of other drug
properties (e.g., solubility, metabolism), by an accurate
permeation model. The prevailing permeation hypothesis has
facilitated success in drug design, interpretation of pharmacokinetics, and planning clinical dose and dosing regimens.
Permeability is a component of many in vivo pharmacokinetic
processes in drug absorption and disposition, including the
following examples in which permeation rate can be ratedetermining:
• absorption: permeation across the gastrointestinal
epithelial membranes to reach blood capillaries
• hepatic clearance: permeation across the sinusoidal and
canalicular hepatocyte cell membrane
• renal clearance: permeation across the nephron tubules
• CNS exposure: permeation across blood−brain barrier
• efficacy: permeation across the cell membrane for
intracellular targets
1. For the large number of drugs that are currently
concluded to primarily permeate via passive diffusion,
the CMOC should provide data that
a. Identify the specific transporter(s), whether known
or new, that transport each. Note that the CMOC
postulates the existence of a large number of
unidentified transporters to explain transport
across any lipoidal barrier. Specific evidence,
however, is still missing.
b. Account quantitatively for the known permeation
rates and in vivo disposition effects. Indeed, the
CMOC proponents note that many approved
drugs interact with a transporter,4 but no data are
provided to characterize the rate and extent of
these interactions and effects on in vivo disposition.
For each tissue to which the drug is known to have
exposure, proof is needed that the purported
transporter(s) are in sufficient concentration to
produce the observed level of drug exposure.
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has contained some unusual elements. First, we agree with the
significant role of carrier-mediated transport in drug permeation, however, we disagree with the attempt to invalidate the
well-established scientific process of passive lipoidal diffusion,
which is done, seemingly, to support the new CMO concept.
The arguments against passive lipoidal diffusion are frequently
illogical, and they cherry-pick the data and overlook the large
body of data that support the major contribution of passive
lipoidal diffusion in drug permeation. Second, insufficient
experiments have been planned, conducted, and reported that
are based on the necessary outcomes of CMOC, which would
differentiate it from the prevailing permeation theory. Third, we
do not think that CMOC is widely accepted and supported, as
has been implied by the statement, “There is burgeoning
evidence for the carrier-mediated view of drug uptake...”.4 The
references that are listed in support of this view are reports of
transporter research or literature reviews by authors who do not
conclude that their research supports CMOC. Until significant
direct evidence is provided that supports CMOC, drug
researchers should withhold decisions based on CMOC that
could affect drug discovery and development.
Expansion of investigation on drug permeation is greatly
encouraged. The prevailing drug permeation hypothesis, which
incorporates several parallel active and passive permeation
mechanisms (i.e., passive lipoidal diffusion, carrier-mediated
influx, carrier-mediated efflux, paracellular diffusion, mucus
resistance, endocytosis, transcytosis), whose rates are determined by the properties of the compound and the membrane,
is consistent with the experimental evidence and provides
successful predictions for drug discovery and development.
CMOC has not produced new evidence and predictions that
warrant changing drug permeation theories, nor their
application to drug research.
c. For cases where the drug is known to permeate
transcellularly (i.e., across both membranes, such
as the gastrointestinal epithelium, blood−brain
barrier, Caco-2), CMOC-based investigations
should provide evidence for both the apical and
basolateral transporters for each drug and
metabolites. Furthermore, where the drug is
known to permeate transcellularly in both
directions (i.e., apical-to-basolateral and basolateral-to-apical, such as in Caco-2), CMOC
proponents should identify the transporters in
both apical and basolateral membranes that allow
transport in both directions for that membrane.
2. Discover major new influx transporters whose substrates
are lipophilic compounds.
a. These carriers should recognize and transport
compounds having a physicochemical profile
(lipophilicity, MW, hydrogen-bonding capacity)
currently linked to passive lipoidal diffusion. This
demand is in line with the argument of CMOC
proponents that new, yet unidentified, transporters
will transport lipophilic compounds that satisfy the
rule of 5, rules 1 to 4.
b. There is a clear need to clone the genes for the
purported new transporters, express them, and
study their characteristics in an in vitro cell line.
These transporters should demonstrate the following common transporter characteristics, except in
limited cases:
i. Display saturation of permeation rate with
increasing drug concentration, since it is far
from sufficient to speculate, on behalf of
CMOC, that there are so many transporters
for every compound that permeation rate
does not saturate. Note however that a few
transporters (e.g., PEPT1) that have very
high capacity are limited cases that do not
readily saturate.
ii. Show inhibition of permeation rates by an
inhibitor.
iii. Show stereospecific differences in permeation rates.
c. Exhibit variations of permeation rate with pH that
are consistent with the pH-partition theory.
d. Show that the transporters’ permeation rates follow
the observed concentration gradient dependence.
3. Discover and characterize new high-flux transporters in
Caco-2, MDCK, 2/4/1A, and other cell lines commonly
used for in vitro cell layer permeation experiments. This
demand is warranted by the speculations of CMOC
proponents that many new significant transporters will
be discovered in these highly studied cell lines.
4. Discover a mechanism that stops or slows down passive
lipoidal diffusion in biological membranes. This demand
is justified by the fact that passive lipoidal diffusion has
been demonstrated in liposomes in vitro, and these are a
model of the observed passive lipoidal permeation of
biological membranes, as discussed above.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
Present Address
▽
21705 Mobley Farm Drive, Laytonsville, MD 20882, United
States.
Notes
The authors declare no competing financial interest.
■
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