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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 289:1084 –1089, 1999
Vol. 289, No. 2
Printed in U.S.A.
Active Transport of Fentanyl by the Blood-Brain Barrier1
THOMAS K. HENTHORN, YANG LIU, MRINAL MAHAPATRO, and KA-YUN NG
University of Colorado Health Sciences Center, Department of Pharmaceutical Sciences, School of Pharmacy and Departments of
Anesthesiology and Neurosurgery, School of Medicine, Denver, Colorado
Accepted for publication December 21, 1998
This paper is available online at http://www.jpet.org
Drug distribution to tissue affects pharmacokinetics, and
thus, observed pharmacodynamics. For example, drug uptake by pulmonary tissue, if extensive, markedly reduces
peak systemic arterial drug concentrations in the moments
after rapid i.v. drug administration (Roerig et al., 1994).
Thus, for drugs with rapid onsets of action, such as i.v.
anesthetics, pulmonary drug uptake could act to reduce peak
drug effect. We recently have demonstrated that the marked
pulmonary uptake of fentanyl (a lipophilic synthetic m-opiate
agonist and prototypic high pulmonary uptake drug) is generated by the pulmonary endothelium and results from both
first order passive diffusive and higher capacity saturable
processes, suggesting transporter mediation (Waters et al.,
1999).
It has been assumed that entry of lipophilic xenobiotics
into tissues occurs by passive diffusion with the equilibrium
Received for publication August 6, 1998.
1
This study was supported in part by National Institutes of Health Grant
GM47502 and was presented in part at the 1998 Annual Meeting of the
American Society of Anesthesiologists (Henthorn TK, Liu Y and Ng KY (1998)
Evidence for a fentanyl transporter at the blood-brain barrier. Anesthesiology
89:A522).
a known substrate of P-gp, previously loaded in the BBMECs
was studied in the presence or absence of either fentanyl or
verapamil, a known competitive inhibitor of P-gp. Both fentanyl
(10 mM) and verapamil (100 mM) decreased release of rhodamine 123 from BBMECs, indicating that fentanyl is a substrate
of P-gp in the BBMECs. This was further supported by the
observation that uptake of [3H]fentanyl was significantly increased in Mg21-free medium, a condition known to reduce
P-gp activity. However, release of [3H]fentanyl was significantly
increased when incubated with either unlabeled fentanyl or
verapamil. These results suggest that the active P-gp-mediated
extrusion of fentanyl in these cells is overshadowed by an
active inward transport process, mediated by an as yet unidentified transporter. In addition, verapamil was shown to be a
substrate of both P-gp and the fentanyl uptake transporter.
between plasma and tissue drug concentrations determined
by physicochemical properties such as octanol-water partitioning and protein binding (Ishizaki et al., 1997; Wood,
1997). More recently, transport proteins such as the ATPbinding cassette (ABC) transporter superfamily [e.g., P-glycoprotein (P-gp)] have been found to be extensively distributed in many endothelia (Thiebaut et al., 1987) and act to
create and maintain tissue/plasma partition gradients of lipophilic xenobiotics that would not be produced by simple
passive processes alone.
Although the increased pulmonary tissue/plasma partitioning of fentanyl may affect observed drug effects after
rapid i.v. administration, the presence of a similar uptake
phenomenon at the blood-brain barrier (BBB) would have
even more immediate pharmacodynamic implications.
Firstly, if fentanyl concentration at its site(s) of action is
controlled by an endothelial transporter, not passive diffusion, then intra- and interindividual potency variability may
not be solely dependent on m receptor differences, but on
variable plasma/brain partitioning. Secondly, if fentanyl
transport is inward at the brain endothelium, then such a
mechanism may be exploitable for reducing fentanyl effects
ABBREVIATIONS: ABC, ATP-binding cassette; BBB, blood-brain barrier; BBMEC, bovine brain microvascular endothelial cell; BPAEC, bovine
pulmonary artery endothelial cell; ECGS, endothelial cell growth supplements; EBSS, Earl’s balanced salt solution; KEQ equilibrium partition
coefficient; ko, rate constant of drug dissociation from transporter; kt, rate constant of drug association to transporter; KM, drug concentration
which leads to 50% occupancy of transporters; P-gp, P-glycoprotein; R123, rhodamine 123.
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ABSTRACT
Previous studies have shown that uptake of the lipophilic opioid, fentanyl, by pulmonary endothelial cells occurs by both
passive diffusion and carrier-mediated processes. To evaluate
if the latter mechanism also exists in brain endothelium, transport of [3H]fentanyl was examined in primary cultured bovine
brain microvessel endothelial cell (BBMEC) monolayers. Uptake of fentanyl appears to occur via a carrier-mediated process as uptake of [3H]fentanyl by BBMECs was significantly
inhibited in a dose-dependent manner by unlabeled fentanyl.
Fentanyl uptake was also significantly inhibited by either 4°C or
sodium azide/2-deoxyglucose, suggesting that carrier-mediated uptake of fentanyl was an active process. Fentanyl was
also tested to determine whether it might be a substrate of the
endogenous blood-brain barrier efflux transport system, Pglycoprotein (P-gp). Release of [3H]fentanyl or rhodamine 123,
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Brain Endothelial Transport of Fentanyl
or enhancing the central nervous system effects of other
drugs.
Here we report the kinetics of fentanyl transport in a
model of the BBB to determine to what extent fentanyl uptake into bovine brain microvascular endothelial cells
(BBMECs) is saturable, energy-dependent, and inhibitable.
Experimental Procedures
tively. The level of [3H]fentanyl radioactivity taken up into endothelial cells was determined using a Beckman LS6000 IC liquid scintillation counter (Beckman Instruments, Berkeley, CA) and
standardized with the amount of protein in each sample. The amount
of protein in each sample was determined by the Pierce BCA method
(Pierce Chemical, Rockford, IL). To ascertain if uptake of [3H]fentanyl by BBMEC was an active process, cellular accumulation of 50 nM
[3H]fentanyl (or 40 nM 14C-antipyrine) were also carried out at
either 4°C or in the presence of known metabolic inhibitors (5 mM
sodium azide and 50 mM 2-deoxyglucose) using a similar protocol as
described above.
Intracellular Release of [3H]fentanyl and R123. Confluent
cell cultures were washed twice with serum-free BBMEC culture
medium. Subsequently, the BBMEC monolayers were incubated
with 0.25 ml serum-free BBMEC culture medium containing either
50 nM [3H]fentanyl (0.447 mCi) or 4 mM R123 for 60 min at 37°C.
After incubation was complete, the culture media was aspirated
gently, and cells were washed three times with 1 ml of ice-cold
serum-free BBMEC culture medium to remove any extracellular
[3H]fentanyl or R123. After the washing was complete, the cells were
restored to either fresh serum-free BBMEC culture medium or serum-free BBMEC culture medium containing no additional drugs or
the P-gp inhibitor verapamil (100 mM) or nonlabeled fentanyl (10
mM) at 37°C. At the indicated time intervals for fentanyl or at 60 min
for R123 after starting the secondary postwash incubation, the culture medium was removed and the cells were washed three times
with 1.0 ml of ice-cold PBS. The cells were solubilized in 1 ml 0.2 N
NaOH and 500 ml and 25 ml aliquots of the cell lysate solution were
removed for measurement of [3H]fentanyl or R123 and protein, respectively. The intracellular concentration of R123 was determined
quantitatively by fluorescence spectrophotometry as described previously (Fontaine et al., 1996). Briefly, sample cell lysate solutions
were diluted to 1 ml with 0.5 ml 0.2 N HCl. Sample fluorescence was
then measured using a Shimadzu RF5000 Fluorescence Spectrophotometer (excitation wavelength 505 nm; emission wavelength 534
nm; Shimadzu Scientific Instruments Inc., Columbia, MD). The concentration of R123 in each sample was determined from the fluorescence measurements by the construction of a R123 standard curve
and standardized by the protein content of each sample.
Effect of Magnesium on Intracellular Accumulation of
[3H]fentanyl. Confluent cell cultures were washed twice with serum-free Earl’s balanced salt solution (EBSS; 108 mM NaCl, 26 mM
NaHCO3, 10 mM KCl, 1.8 mM CaCl2, 1 mM NaH2PO4, 5.5 mM
glucose). Subsequently, the BBMEC monolayers were preincubated
with either serum-free EBSS or serum-free magnesium-plus EBSS
(EBSS plus 5 mM MgSO4) for 30 min at 37°C. At the end of this
preincubation, the culture media was aspirated gently. The cells
were then restored to either serum-free EBSS or serum-free magnesium-plus EBSS containing 50 nM [3H]fentanyl (0.447 mCi). After
incubation with the medium containing [3H]fentanyl, the culture
medium was removed and the cells were washed three times with 1.0
ml of ice-cold PBS. The BBMEC were then solubilized in 1 ml 0.2 N
NaOH and aliquots of the cell lysate solution were removed for
measurement of [3H]fentanyl and protein as described above.
Kinetic Analysis. To evaluate whether cellular accumulation of
fentanyl is governed by processes which are first order passive,
Michaelis-Menten (active), or both, a model developed previously for
analysis of fentanyl uptake by bovine pulmonary artery endothelial
cells (BPAECs) was used (Waters et al., 1999). In this model, the
constant defining the equilibrium between the supernatant and the
endothelial cells, KEQ, is represented by the sum of two terms:
K EQ 5 H 1
R MAX
~ k o/ k t! 1 C S
(1)
a first order term, H, representing the diffusional partition coefficient and a saturable term in which RMAX is the total transport
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Materials. Dispase and collagenase/dispase were obtained from
Boehringer Mannheim (Indianapolis, IN). Type I rat tail collagen
and endothelial cell growth supplements (ECGS) were purchased
from Collaborative Biomedical (Bedford, MA). Cell culture medium
was obtained from Gibco (Grand Island, NY). [3H]fentanyl (8.89
Ci/mmol) was obtained from Research Diagnostics, Inc. (Flanders,
NJ). 3H-antipyrine (7.1 Ci/mmol) was purchased from New England
Nuclear (Boston, MA). Rhodamine 123 (R123), fentanyl citrate,
platelet poor horse serum, sodium azide, 2-deoxyglucose, dimethyl
sulfoxide, fibronectin, and verapamil hydrochloride were purchased
from Sigma Chemical Co. (St. Louis, MO). All other reagents, unless
specifically stated otherwise, were purchased from Sigma Chemical
Co.
BBMEC Isolation and Culturing. BBMEC were isolated from
the cerebral gray matter of bovine brain as described previously
(Audus et al., 1996; Ng and Schallenkemp, 1996). Briefly, brain gray
matter was collected and minced to 1- to 2-mm cubes with razor
blades before undergoing a 2.5-h dispase digestion (4 ml 12.5%
dispase solution/50 g of gray matter). The microvessels were then
separated from the cell debris by centrifugation in 13% dextran. The
isolated microvessels were further incubated on a per gram basis
with 3 ml of collagenase/dispase (at 1 mg/ml or 0.3 U collagenase and
4.12 U dispase/ml) for 4 h at 37°C. At the conclusion of this incubation, the microvessels were subjected to Percoll gradient centrifugation for final separation of microvessels from pericytes, cell debris,
and other contaminated cells. The purified microvessels were stored
frozen at 2180°C in freezing medium (36% minimum essential medium, 36% F-12 medium, 18% platelet poor horse serum, 10% dimethyl sulfoxide, 50 U/ml penicillin, 50 mg/ml streptomycin, and 125
mg/ml heparin) until used.
For cellular accumulation and release studies, purified BBMECs
were seeded at a density of 50,000 cells/cm2 onto collagen-coated and
fibronectin-treated 48-well cell culture plates in BBMEC plating
medium (45% minimum essential medium, 45% F-12 medium, 10%
platelet poor horse serum, 50 U/ml penicillin, 50 mg/ml streptomycin,
2.5 mg/ml amphotericin B, 50 mg/ml polymixin B and 25 mg/ml
ECGS). The cells were grown in a 37°C incubator with 5% CO2 and
95% humidity. The plating medium was replaced with BBMEC culture medium (plating medium less polymixin B and ECGS) on the
third day after plating and every other day thereafter. BBMECs
were allowed to grow to confluence before cellular accumulation and
release studies. Typically, formation of monolayers took about 6 to 8
days.
[3H]Fentanyl Cellular Accumulation Studies. After the establishment of a confluent BBMEC monolayer was confirmed (by
phase contrast microscopy examination), cellular accumulation of
[3H]fentanyl was measured. Briefly, confluent cell cultures were
washed twice with serum-free BBMEC culture medium. Subsequently, the cells were incubated at 37°C with 0.25 ml serum-free
BBMEC culture medium containing 50 nM [3H]fentanyl (0.447 mCi/
ml) and various concentrations of nonlabeled fentanyl for 60 min
(three incubations at each of nine nonlabeled fentanyl concentrations). After incubation, the cellular accumulation studies were terminated by removing the assay solutions and washing the BBMEC
monolayers three times with 1.0 ml of ice-cold PBS. The BBMEC
were then solubilized by incubation with 1 ml of 0.2 N NaOH overnight. Aliquots (500 ml and 25 ml) of the cell lysate solution were
removed for analysis of [3H]fentanyl and protein content, respec-
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Vol. 289
Results
Inhibition of Fentanyl Endothelial Uptake and Release. [3H]fentanyl uptake into BBMECs was found to occur
by both a first order (passive) and saturable (carrier-mediated) processes. The evidence for this is in Fig. 1, which shows
that KEQ is significantly higher at low doses of unlabeled
fentanyl and lower at high doses. The line in Fig. 1 is the best
Fig. 1. The ratio of cell-associated [3H]fentanyl concentrations (nmol/mg
protein) to supernatant fentanyl concentrations (nmol/ml) versus supernatant fentanyl concentrations (nmol/ml, micromolar). Observed values
are solid circles and the line represents the best fit of these data to eq. 1.
Note the sigmoid shape of this line with the upper plateau delineating the
combined effects of diffusional equilibrium (H) and active transport, the
lower plateau representing H alone, and the KM at the midpoint of the
vertical portion.
fit of eq. 1 to our data; a sigmoid relationship that was well
predicted by our model (three parameters, adjusted r2 5
0.662). From this fit we determined H to be 0.040 ml/mg
protein, RMAX to be 0.11 pmol/mg protein and KM to be 3.19
mM. This fit was significantly better than a linear concentration-dependent relationship (two parameters, adjusted r2 5
0.352) or one that assumed fentanyl uptake occurred only by
a simple diffusion mechanism (one parameter, i.e., a constant
KEQ, adjusted r2 , 0.001).
To corroborate these findings, a study was done to determine the rates of release of [3H]fentanyl from cells preloaded
with [3H]fentanyl under control conditions, and one was done
in which the supernatant contained 10 mM cold fentanyl
(EC75 for saturating the inward transport mechanism). Figure 2 shows that [3H]fentanyl was released from BBMECs at
a faster rate when 10 mM cold fentanyl was present, consistent with its inhibition of a transporter directed inwardly.
The ratio of the rate constants for passive diffusion (unsaturable) to that for the transporter (saturable) was 0.38,
which is identical with the ratio of H to RMAX from the uptake
model (eq. 1) of 0.38. A simpler model was also fit to the data.
In this model there was no inhibitable rate constant directed
inward (i.e., all data in Fig. 2 is fit to a single line). The full
model in which there is an additional parameter for the
inward transporter (under control conditions) was supported
by the Akaike information criterion. The percent coefficients
of variation for the diffusive rate constant and the transporter rate constant were 7.0 and 8.5%, respectively.
Energy Depletion and Fentanyl Endothelial Uptake.
Pretreatment of the BBMECs with the metabolic inhibitors
sodium azide and 2-deoxyglucose reduced [3H]fentanyl uptake into BBMECs to that of diffusion alone (Fig. 3) as
predicted by the saturation model (eq. 1 and Fig. 1). These
data indicate that the saturable component of the uptake
process for fentanyl is energy-dependent. In contrast, pretreatment of the BBMECs with the same metabolic inhibitors had no effect on the uptake of antipyrine, a prototypic
lipophilic diffusion tracer (data not shown). Fentanyl uptake
was similarly reduced when the experiments were conducted
at 4°C, confirming the energy dependence of this process.
P-gp Inhibition. Because fentanyl is very lipophilic
(Stanski and Hug, 1982) and metabolized by CYP3A4 (Labroo et al., 1997), it is a candidate substrate for the endogenous BBB efflux system, P-gp (Zhang et al., 1998), which
Fig. 2. Results of the pulse-trace experiment showing the labeled cellular
fentanyl concentrations versus time for the BBMECs preloaded with
[3H]fentanyl. At time t 5 0 the culture medium containing [3H]fentanyl
was removed and after washing, replaced with either culture medium (E,
n 5 3 at each point) or culture medium plus 10 mM unlabeled fentanyl (‚,
n 5 3 at each point). The lines represent the best fit of these data to the
respective models (see Materials and Methods).
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capacity and ko/kt is the KM or the supernatant drug concentration
CS, which leads to 50% occupancy (inhibition) of transporters.
Cell-associated [3H]fentanyl data, after being normalized for their
protein content, were all fit simultaneously to eq. 1 using a constant
weight and TableCurve2D (SPSS, Chicago, IL). Simpler models in
which the transport component was linearly related to CS or in which
there was no transport component at all were also tested and the
best model selected based on the adjusted r2 (r2 penalized for additional parameters).
Evaluation of the kinetics of release of [3H]fentanyl from BBMECs
was based on a two-compartment model in which a dose of 3Hfentanyl is injected into the cellular compartment at time t 5 0 and
equilibration between the supernatant and cellular compartments is
defined by rate constants from the cell-to-supernatant (diffusional
rate constant) and by the supernatant-to-cell (sum of the diffusional
and saturable transporter rate constants). Note that in this experiment the transporter was assumed to be at either the RMAX state (no
added fentanyl) or at a fully inhibited state (10 mM fentanyl added),
thus reducing the model to two rate constants. In addition, a simpler
model in which there only is only a bidirectional diffusional rate
constant (i.e., there are no differences in the [3H]fentanyl concentrations at the various times for the 0 mM versus the 10 mM fentanyl
conditions) was tested. All data were fit simultaneously using reciprocal weighting and the compartmental module of SAAM II (SAAM
Institute, Seattle, WA). Model selection was based on the Akaike
information criterion (Ludden et al., 1994).
Statistical Analysis. All other data were compared to control
with a one-way ANOVA. If statistically significant differences were
detected, post hoc analysis consisted of a Tukey test. P was set to p ,
.05. The statistical analyses were performed with SigmaStat (SPSS,
Inc., Chicago, IL).
1999
functions to actively pump out lipophilic drugs that enter the
brain endothelial cells (Schinkel et al., 1996). To determine
whether fentanyl may also be a substrate for P-gp, pulsetrace experiments were performed to study the effects of both
unlabeled fentanyl and the competitive P-gp inhibitor verapamil on the intracellular release (trace-step) of [3H]fentanyl
or the P-gp substrate R123 (Fontaine et al., 1996; Rose et al.,
1998). If fentanyl is also a competitive inhibitor of P-gp, both
fentanyl and verapamil should decrease the egress of R123
and [3H]fentanyl from BBMECs. Figure 4 shows that both
verapamil (100 mM) and fentanyl (10 mM) significantly decreased the egress of R123 from BBMECs. However, the
reverse was true for [3H]fentanyl for which both verapamil
(100 mM) and unlabeled fentanyl (10 mM) significantly increased the egress of [3H]fentanyl from BBMECs (Fig. 5).
These inhibition findings suggest that verapamil is a substrate for the inwardly directed fentanyl transporter (in addition to its being a substrate for P-gp) and that fentanyl is a
substrate for the outwardly directed P-gp (in addition to the
evidence for an inward transporter shown in Figs. 1 and 2).
To determine whether P-gp alone was transporting bidirectionally (i.e., fentanyl in and R123 out) we performed an
[3H]fentanyl uptake study in which the incubation media did
or did not contain Mg21, an essential ion for P-gp ATPase
Fig. 4. Cell-associated R123 from pulse-trace studies at t 5 60 min after
removal of culture medium containing R123 and replacing it with culture
medium alone or culture medium containing either verapamil (100 mM)
or fentanyl (10 mM); n 5 4 for each condition. * indicates differences from
control (P , .05). Note that R123 release from BBMECs decreased when
verapamil or fentanyl was present in the supernatant. These are the
reverse of the findings with [3H]fentanyl in Fig. 5.
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Fig. 5. Cell-associated [3H]fentanyl from pulse-trace studies at t 5 60
min after removal of culture medium containing [3H]fentanyl and replacing it with culture medium alone or culture medium containing either
verapamil (100 mM) or fentanyl (10 mM); n 5 3 for each condition. *
indicates differences from control (P , .05). Note that fentanyl release
from BBMECs increased when verapamil or fentanyl was present in the
supernatant.
function (Awasthi et al., 1994). In the Mg21-depleted condition
there was more fentanyl uptake by BBMECs (Fig. 6), consistent
with the loss of outward transport of fentanyl by P-gp.
Discussion
The distribution of lipophilic drugs to tissue has traditionally been attributed to the processes of tissue blood flow and
passive diffusion with the ultimate partitioning of drug between blood and tissue being dependent on the physicochemical properties of drug as it relates to the composition of
adjacent media (Ishizaki et al., 1997). For instance, drugs
with high octanol/water partition ratios tend to have high
tissue/blood partition ratios, especially in tissues such as
adipose, which have high lipid content (Barton et al., 1997).
This simplistic view of drug distribution has been challenged
recently by the discovery of transporters for lipophilic drugs
at the blood/tissue interface (Wood, 1997). There is evidence
for both inward and outward vectors of transport relative to
the microvascular lumen. The ABC transporter P-gp has
been found to be extensively distributed in many endothelia
(Thiebaut et al., 1987; Schinkel et al., 1995), where it transports lipophilic xenobiotics away from tissue back into the
microvascular lumen. At the BBB, P-gp actively pumps a
variety of lipophilic drugs away from brain tissue, thus reducing the tissue/blood partition ratio that would exist by
passive diffusion alone (Schinkel et al., 1996).
In contrast to the outward vector produced by P-gp, the
processes mediating the extensive first pass pulmonary uptake of the lipophilic basic amine fentanyl (Roerig et al.,
1987) include a saturable mechanism, suggesting a trans-
Fig. 6. Cell-associated [3H]fentanyl from uptake studies under control
conditions or when BBMECs were treated with a culture medium without
Mg21 (n 5 3 for each condition). * indicates differences from control (P ,
.05). Note that uptake of fentanyl by BBMECs was increased by this
condition that directly inhibits P-gp and other ABC-type transporters.
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Fig. 3. Cell-associated [3H]fentanyl from uptake studies under control
conditions (culture medium at 37°C) or when the cells were treated with
the combination of sodium azide and 2-deoxyglucose added to the culture
medium (37°C) or with reduced temperature (4°C); n 5 6 for each condition. * indicates differences from control (P , .05). Note that uptake of
fentanyl by BBMECs was reduced by conditions that inhibit active processes.
Brain Endothelial Transport of Fentanyl
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Henthorn et al.
inhibition) of an inwardly-directed transporter (Fig. 5). Verapamil (100 mM) was an equally effective inhibitor of this
process. These results suggest that verapamil is a substrate
of the fentanyl uptake transporter and, because verapamil is
a classic P-gp substrate/competitive inhibitor, fentanyl uptake may be mediated by P-gp. In contrast to these results,
verapamil greatly reduced the egress of R123 from BBMECs
(Fig. 4), confirming the findings of others that verapamil and
R123 are substrates of the outwardly directed P-gp. Fentanyl
was equally effective to verapamil at reducing R123 transport by P-gp, suggesting that fentanyl too is a substrate of
this outwardly directed transporter. This finding is consistent with the increased sensitivity to fentanyl of mice treated
with PCS 833 and patients treated with cyclosporin A, but is
inconsistent with the current findings of a net inward transport of this drug at the BBB under control conditions.
To clarify this issue we inhibited P-gp by means other than
competitive inhibition, i.e., removal of Mg21 from the supernatant. Under these conditions, fentanyl uptake was increased, suggesting that fentanyl is indeed actively transported out of BBMECs by P-gp and consistent with both the
current R123 results and increased fentanyl sensitivity in
subjects treated with P-gp inhibitors other than verapamil.
The other major implication of these findings is that there is
an active transporter for fentanyl in addition to P-gp— one
that transports fentanyl and verapamil inward and has a
higher capacity than P-gp at the BBB. The lower transporter
contribution to the inward transport of fentanyl in BBMECs
in this study (2.6 times greater than diffusion alone) compared with the previous results in BPAECs (3.8 times greater
than diffusion alone) may owe to the fact that P-gp content is
higher in the BBB, thus negating a portion of the inward
transport in BBMECs. It is not entirely surprising that verapamil is a substrate, like fentanyl, of a transporter directed
inward across the endothelium. Both drugs are lipophilic
basic amines, a fact that may underlie a shared substrate
specificity, and both are classic examples of drugs demonstrating high pulmonary uptake (Roerig et al., 1987, 1989).
Currently, works are being undertaken to identify and
characterize this transporter. It can be expected that this
information should greatly enhance our understanding of the
contribution of the BBB to the pharmacokinetics and pharmacodynamics of fentanyl. It is further anticipated that this
transporter may be exploitable for either reducing fentanyl
effects or enhancing the central nervous system effects of
other drugs.
In conclusion, these results demonstrate that the major
factor governing the uptake of fentanyl into BBMECs is
active carrier-mediated transport, not passive diffusion. The
results also indicate that fentanyl is a substrate of P-gp in
BBMECs, but the active P-gp-mediated extrusion of fentanyl
in these cells is overshadowed by an active inward transport
process, mediated by an as yet unidentified transporter. In
addition, verapamil was shown to be a substrate of both P-gp
and the fentanyl uptake transporter.
References
Audi SH, Linehan JH, Krenz GS, Dawson CA, Ahlf SB and Roerig DL (1995)
Estimation of the pulmonary capillary transport function in isolated rabbit lungs.
J Appl Physiol 78:1004 –1014.
Audus KL, Ng L, Wang W and Borchardt RT (1996) Brain microvessel endothelial
cell culture systems. Pharm Biotechnol 8:239 –258.
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porter directed toward lung tissue, thus increasing the tissue/blood partition ratio (Waters et al., 1999). Because fentanyl is an opioid with full m-agonist properties, a lungdirected drug transporter has pharmacokinetic implications
after rapid i.v. administration (i.e., the initial arterial fentanyl concentrations delivered to the brain depend on the extent of pulmonary uptake). However, a tissue-directed transporter at fentanyl’s central nervous system site of action
would affect its pharmacodynamics (i.e., onset time and plasma-apparent EC50).
This report demonstrates that BBMECs, like BPAECs,
possess a high-capacity fentanyl uptake process that is saturable (KM 5 3.19 mM). The results in Fig. 1 indicate that at
the low clinical concentrations of fentanyl (2–10 nM; Shafer
and Varvel, 1991), the saturable mechanism increased the
uptake of fentanyl into brain endothelium by more than
2.6-fold over that produced by simple diffusion. These results
are similar to findings in BPAECs where the saturable process (KM 5 2.8 mM) increased endothelial uptake 3.8-fold
over that by simple diffusion alone (Waters et al., 1999). In
addition, BBMEC uptake of fentanyl was reduced by ATP
depletion and 4°C (Fig. 3) to a similar degree, confirming that
the saturable uptake process is energy-dependent and suggesting that an active transport process is involved.
In uptake studies, whether in vivo or in vitro, cellular
equilibration with the supernatant (or blood) is assumed to
occur very quickly (Audi et al., 1995). In in vitro studies,
equilibrium between cells and the supernatant is assumed to
be complete within seconds. Indeed, in pilot studies we found
that fentanyl uptake into endothelial cells was indistinguishable at incubation times between 5 and 120 min. Therefore,
in uptake experiments, partition coefficients rather than
transfer rate constants are estimated. In pulse-trace studies,
changes in cellular drug content can be measured over time
because of the large dimensions and capacity of the supernatant relative to the cellular monolayer, allowing estimation of
transfer rate constants. In theory, the ratio of the diffusional
equilibrium constant to the total transporter capacity from
an uptake equilibrium model ought to mirror the ratio of the
diffusional rate constant to the transporter rate constant of
the pulse-trace kinetic model. Indeed, we found these ratios
to be 0.38 in both instances, confirming that the inward
transporter capacity is more than 2.6-fold greater than diffusional transport as well as confirming that direct comparisons between results from uptake and pulse-trace paradigms can be made.
Previous findings suggest that fentanyl may be a substrate
of the outwardly-directed P-gp in BBMECs. First, fentanyl is
a lipophilic drug and a substrate of CYP3A4 (Labroo et al.,
1997), characteristics shared by other P-gp substrates
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substrate/competitive inhibitor verapamil (100 mM; Maia et
al., 1998) as potential competitive inhibitors. As reported
above, 10 mM fentanyl greatly enhanced the egress of fentanyl from BBMECs, consistent with saturation (competitive
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Send reprint requests to: Dr. Thomas K. Henthorn, M.D., Department of
Anesthesiology, University of Colorado Health Sciences Center, Campus Box
B113 4200 E. 9th Ave., Denver, CO 80262. E-mail: tkhenthorn@ski.
uhcolorado.edu
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