Active Transport of Fentanyl by the Blood-Brain Barrier1

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 [H]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 [H]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 [H]fentanyl or rhodamine 123, 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 [H]fentanyl was significantly increased in Mg-free medium, a condition known to reduce P-gp activity. However, release of [H]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. 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 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 intraand 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 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). 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. 0022-3565/99/2892-1084$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289:1084–1089, 1999 1084 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from 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 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). [H]fentanyl (8.89 Ci/mmol) was obtained from Research Diagnostics, Inc. (Flanders, NJ). H-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 1to 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/cm 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. [H]Fentanyl Cellular Accumulation Studies. After the establishment of a confluent BBMEC monolayer was confirmed (by phase contrast microscopy examination), cellular accumulation of [H]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 [H]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 [H]fentanyl and protein content, respectively. The level of [H]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 [H]fentanyl by BBMEC was an active process, cellular accumulation of 50 nM [H]fentanyl (or 40 nM C-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 [H]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 [H]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 [H]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 [H]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 [H]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 [H]fentanyl (0.447 mCi). After incubation with the medium containing [H]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 [H]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: KEQ 5 H 1 RMAX ~ko/kt! 1 CS (1) a first order term, H, representing the diffusional partition coefficient and a saturable term in which RMAX is the total transport 1999 Brain Endothelial Transport of Fentanyl 1085 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from capacity and ko/kt is the KM or the supernatant drug concentration CS, which leads to 50% occupancy (inhibition) of transporters. Cell-associated [H]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 r (r penalized for additional parameters). Evaluation of the kinetics of release of [H]fentanyl from BBMECs was based on a two-compartment model in which a dose of Hfentanyl 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 [H]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).