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Vol. 29, Issue 7, 954-956, July 2001
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
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This study investigated the effects of racemic methadone (MET) on P-glycoprotein (P-gp) activity in cell culture. MET showed no differential rates of passage between the basolateral to apical (B to A) and apical to basolateral (A to B) direction across Caco-2 cell monolayers in a transwell system. MET transport in either direction was not importantly influenced by the P-gp inhibitor verapamil. However, MET was a potent inhibitor (IC50 = 7.5 µM) of rhodamine123 B to A transport across Caco-2 cell monolayers, causing a reduction to 25% of control at 100 µM MET. In this model of Caco-2 monolayers, rates of MET passage between B to A and A to B directions could not be distinguished. However, MET can inhibit P-gp activity at intraluminal concentrations that might be achieved clinically. This may lead to increased bioavailability of coadministered compounds.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Methadone
(MET1) is a synthetic opioid used for maintenance
therapy of opiate dependence. MET is metabolized by hepatic and intestinal CYP3A4 (Moody et al., 1997
; Foster et al., 1999) and has
been shown to inhibit hepatic CYP2D6 and CYP3A4 (Wu et al., 1993;
Iribarne et al., 1997). In addition, interactions of MET with
P-glycoprotein (P-gp), the product of the human MDR1 gene (Fardel et al., 1996), have been suggested (Callaghan and Riordan, 1993; Bouer et al., 1999; Thompson et al., 2000). P-gp is an
ATP-dependent drug efflux pump constitutively expressed in several
human tissues, including epithelia of the small intestine and blood
brain barrier, liver, and kidney (Sugawara et al., 1988; Cordon-Cardo
et al., 1989), as well as in cancer cells (Roninson, 1992). The human colon adenocarcinoma cell line Caco-2 resembles small intestinal epithelial cells (Pinto et al., 1983; Quaroni and Hochman, 1996) and is
frequently used for studies modeling intestinal drug transport. This
study was designed to investigate the effects of MET on P-gp activity
in Caco-2 cells in order to anticipate P-gp related drug-drug interactions.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Chemicals. Drugs and chemicals were purchased from commercial sources or were kindly provided by their pharmaceutical manufacturers.
Cell Lines. The human colon adenocarcinoma cell line Caco-2 was kindly provided by Douglas Jefferson, Ph.D. (Tufts University School of Medicine and the New England Medical Center, Boston, MA) and used at passages 30 to 40. Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Rockville, MD) supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids (Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.
Transport Experiments. Caco-2 cells were seeded at 2 × 104/cm2 in polycarbonate membrane transwell plates (3-µm pore size) (Corning Costar Corp., Cambridge, MA) and grown in a humidified chamber (37°C, 5% CO2) with media changes every 3 to 5 days. Experiments were conducted on day 16 to 20 after seeding. Drug solutions were prepared in Hanks' balanced salt solution (Invitrogen) containing 10 mM HEPES, 4.2 mM NaHCO3, and 0.5% dimethyl sulfoxide.
P-gp inhibition. Hanks' balanced salt solution containing Rh123 (5 µM) was added to the apical (A) or basolateral (B) chamber, with the inhibitor (100 µM verapamil or 1-100 µM MET) present in both chambers. Cells were incubated for 90 min, and Rh123 concentrations in the chamber initially not containing Rh123 were determined by fluorescence spectrophotometry at 500/550 nm (excitation/emission). Rh123 B to A transport across Caco-2 monolayers was linear between 0.3 and 100 µM Rh123, indicating that the 5 µM concentration used in the transport study was within the linear range. The duration of incubation (90 min) also was within the linear range.
P-gp substrates. Racemic MET (10 µM) was added to the A or B chamber, in the presence and absence of verapamil (100 µM) in both chambers. Cells were incubated for 90 and 180 min to assure measurable concentrations in the receiving chamber. MET concentrations were determined by high-performance liquid chromatography using a 3.9- × 300-mm µBondapak (C18) column (Waters Associates, Milford, MA) and a mobile phase consisting of 45% acetonitrile and 55% 0.05 M KH2PO4 (pH 5.5) delivered at 2.4 ml/min, with UV detection at 220 nm. Retention times were 6.9 min for verapamil, 8.3 min for nefazodone (internal standard), and 12.8 min for methadone. The average CV for triplicate samples was 5.9% at 1.5 µM MET. The detection limit was 5 ng of MET.
Data Analysis. Statistical Analysis was performed using analysis of variance followed by Bonferroni t tests against the control group with a significance level of p < 0.05.
Since the baseline diffusion of Rh123 from A to B is consistently low in our cell line (approximately 1-2% of the corresponding B to A transport), it has not been considered in calculating the percentage of inhibition of B to A Rh123 transport. IC50 values for Rh123 transport inhibition were determined using nonlinear regression (SigmaPlot 4.0, SPSS Inc., Chicago, IL) based on eqs. 1 and 2.
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(1) |
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(2) |
) and can be used for an empirical
determination of IC50 (Venkatakrishnan et al.,
1998).
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Results and Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Substrate. In a transwell system, transport of MET across a Caco-2 cell monolayer was evaluated in both directions, B to A (indicative of active transport) and A to B (indicative of passive diffusion). MET showed no differential transport compared with a >20-fold difference observed for the positive control Rh123, a known P-gp substrate (Table 1). The P-gp inhibitor verapamil (100 µM) reduced Rh123 B to A transport to 5% of control. Verapamil had no effect on the B to A transport of MET at either time point. A to B passage of MET appeared to be affected by verapamil, but in opposite directions at the 90- and 180-min points. Therefore, the findings, although statistically significant, do not reflect a consistent biological effect.
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Inhibition. MET (100 µM) reduced B to A Rh123 transport across Caco-2 monolayers to 25% of control, while a reduction to 5% was achieved by the P-gp inhibitor verapamil (100 µM) used as a positive control (Fig. 1a). The corresponding A to B Rh123 transport was <10% of the B to A transport and was not affected by MET or verapamil (Fig. 1a). The inhibition was concentration-dependent, with an IC50 of 7.5 µM (Fig. 1b).
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). However, intraluminal
concentrations can be expected to transiently exceed plasma
concentrations after oral administration of usual therapeutic doses.
This finding is consistent with a previous study showing increased
vinblastine accumulation in drug-resistant cells in the presence of MET
(Callaghan and Riordan, 1993). Although the
Km of Rh123 transport in this system is not
established, transport linearity was demonstrated up to at least 100 µM Rh123. This suggests that the Rh123 concentration used in the
present study is well below the transport
Km and that IC50
values generated using Rh123 as the index substrate are likely to apply
to other substrates for transport.
In Caco-2 cells, no differential transport of MET was observed, and
transport from B to A was not affected by verapamil. In contrast, Bouer
et al. (1999) reported a 60% increase in MET transport across everted
rat intestine in the presence of verapamil, and a study by Thompson et
al. (2000) demonstrated an increased analgesic effect of MET in
mdr1 knockout mice. The discrepancy could be attributable to
interspecies differences in substrate specificity of P-gp similar to
that previously shown for the related opioid morphine. LLC-PK1 cells
transfected with the mouse mdr1a gene showed differential
transport of morphine, while cells transfected with human
MDR1 did not (Schinkel et al., 1995). In a clinical study,
coadministration of the P-gp inhibitor PSC833 did not affect morphine
pharmacokinetics or cause adverse events in healthy volunteers (Drewe
et al., 2000), while in mdr1 knockout mice an increased analgesic effect of morphine was reported (Thompson et al., 2000). Also, regional differences in P-gp expression depending on section of
the small intestine used may confound studies using rat intestine (Makhey et al., 1998). Finally, note that the density of P-gp in
nonselected Caco-2 cells is considerably lower than in other cell lines
such as NIH3T3/MDR1 cells (Ibrahim et al., 2000). Thus P-gp-mediated transport of a lipophilic substance such as methadone, which may be quantitatively important in other models or in vivo, may
not be evident in the present model due to the greater quantitative importance of passive diffusion.
Caco-2 cells are an accepted in vitro model for intestinal transport
(Artursson and Karlsson, 1991; Artursson et al., 1996; Makhey et al.,
1998). However, expression of transporters other than P-gp (multidrug
resistance protein, lung resistance protein, and dipeptide
transporters) in that cell line (Bailey et al., 1996; Makhey et al.,
1998) require the use of index compounds that are relatively specific
for the transporter studied. Rh123 is frequently used as a P-gp index
substrate. A good correlation of Rh123 transport with expression of
P-gp, but not multidrug resistance-associated protein, was found
in a drug screen in 58 different cell lines (Lee et al., 1994).
Verapamil has been shown to inhibit P-gp in
MDR-1-transfected Madin-Darby canine kidney cells
that constitutively express only low levels of transport proteins
(Braun et al., 2000); however, verapamil may also inhibit transporters
other than P-gp (Aszalos et al., 1999).
Our data suggest a potential for MET to cause drug interactions by
inhibition of drug transport, which may increase bioavailability and
central nervous system penetration or reduce renal or biliary clearance of coadministered drugs that are substrates for P-gp or other transporters.
Elke Störmer
Michael D. Perloff
Lisa L. von
Moltke
David J. Greenblatt
Department of Pharmacology
and Experimental Therapeutics,
Tufts University School of Medicine,
Boston, Massachusetts
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Footnotes |
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Received November 17, 2000; accepted April 11, 2001.
David J. Greenblatt, M.D., Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: dj.greenblatt{at}tufts.edu
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Abbreviations |
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Abbreviations used are: MET, methadone; P-gp, P-glycoprotein; Rh123, rhodamine123; A and B chambers, apical and basolateral chambers, respectively.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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