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Multiple Efflux Pumps are Involved in the Transepithelial Transport of
Colchicine: Combined Effect of P-gp and MRP2 Leads to Decreased
Intestinal Absorption Throughout the Entire Small Intestine
Arik Dahan, Hairat Sabit and Gordon L. Amidon
The University of Michigan College of Pharmacy, Dept. of Pharmaceutical Sciences, 428
Church Street, Ann Arbor MI 48109 (A.D., H.S, G.L.A.)
DMD Fast Forward. Published on July 9, 2009 as doi:10.1124/dmd.109.028282
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: Involvement of Multiple Pumps in Colchicine Intestinal
Efflux
Corresponding Author: Prof. Gordon L. Amidon, Dept. of Pharmaceutical
Sciences, College of Pharmacy, University of Michigan,
428 Church Street, Ann Arbor, Michigan 48109-1065, Tel:
734-764-2464, Fax: 734-763-6423, E-mail:
Number of Text Pages: 30
Number of Tables: 0
Number of Figures: 10
Number of References: 42
Number of words in the Abstract: 249
Number of words in the Introduction: 607
Number of words in the Discussion: 911
Abbreviations: GIT, gastrointestinal tract; P-gp, P-glycoprotein; MRP2,
multidrug resistance-associated protein 2; BCRP, breast
cancer resistance protein; MDR, multidrug resistance; AP,
apical; BL, basolateral; ER, efflux ratio; FTC,
fumitremorgin C; BCS, biopharmaceutics classification
system.
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Abstract
The purpose of this study was to thoroughly characterize the efflux transporters involved
in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine,
and evaluate the role of these transporters in limiting its oral absorption. The effects of P-
gp, MRP2 and BCRP inhibitors on colchicine bidirectional permeability were studied
across Caco-2 cell monolayers, inhibiting one vs. multiple transporters simultaneously.
Colchicine permeability was then investigated in different regions of the rat small
intestine by in-situ single-pass perfusion. Correlation with P-gp/MRP2 expression level
throughout different intestinal segments was investigated by immunoblotting. P-gp
inhibitors (GF120918, verapamil and quinidine), and MRP2 inhibitors (MK-571,
indomethacin and p-AH) significantly increased AP-BL and decreased BL-AP Caco-2
transport, in a concentration-dependent manner. No effect was obtained by the BCRP
inhibitors FTC and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced
colchicine mucosal secretion, including complete efflux abolishment (GF120918/MK-
571). Colchicine displayed low (vs. metoprolol) and constant permeability along the rat
small-intestine. GF120918 significantly increased colchicine permeability in the ileum
with no effect in the jejunum, while MK-571 augmented jejunal permeability without
changing the ileal transport. GF120918/MK-571 combination caused similar effect to
MK-571 alone in the jejunum and to GF120918 alone in the ileum. P-gp expression
followed a gradient increasing from proximal to distal segments, while MRP2 decreased
from proximal to distal small-intestinal regions. Overall, it was revealed that combined
effect of P-gp and MRP2, but not BCRP, dominates colchicine transepithelial transport,
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leading to complete coverage of the entire small intestine, and makes the efflux transport
dominate the intestinal-permeability process.
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Introduction
ATP-dependent efflux transporters, such as P-glycoprotein (P-gp; ABCB1),
multidrug resistance-associated protein 2 (MRP2; ABCC2) and breast cancer resistance
protein (BCRP; ABCG2), have been shown to play significant roles in drug absorption,
distribution and clearance processes, as well as in drug-drug and drug-food interactions
(Dahan and Altman, 2004; Raub, 2006; Takano et al., 2006; Miller et al., 2008). These
transporters are present in many biological membranes, including the villus tip of the
apical brush border membrane of gut enterocytes, and actively cause efflux of drugs from
gut epithelial cells back into the intestinal lumen. P-gp recognizes a variety of structurally
and pharmacologically unrelated neutral and positively charged compounds (Mizuno et
al., 2003; Raub, 2006). MRP2 transports relatively hydrophilic molecules, including the
glucuronide, glutathione, and sulfate conjugates of endogenous and exogenous
compounds (Suzuki and Sugiyama, 2002). BCRP recognizes relatively hydrophilic
anticancer agents (Doyle and Ross, 2003). Due to overlapping substrate specificities,
multiple efflux pumps might be involved in limiting the membrane permeability of a drug
molecule. Drug substances that have been shown to be co-substrates for several efflux
pumps include atorvastatin (Lau et al., 2006), rosuvastatin (Kitamura et al., 2008),
cyclosporine (Mannermaa et al., 2006), sulfasalazine (Dahan and Amidon, 2009c),
saquinavir (Usansky et al., 2008), and fexofenadine (Matsushima et al., 2008). It is
necessary to determine the contribution of each transporter to the overall efflux process,
since such information could allow one to predict changes in membrane permeability
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when the functions of transporters are altered, e.g. by genetic polymorphism, disease state
or drug-drug interactions.
Colchicine (Fig. 1) is an oral microtubule polymerization inhibitor, prescribed in
gout therapy (Ben-Chetrit and Levy, 1998; Terkeltaub, 2003), prevention of acute attacks
of familial Mediterranean fever (FMF) (Dinarello et al., 1974; Amital and Ben-Chetrit,
2004), and in the treatment of immune and inflammatory diseases (primary biliary
cirrhosis (Kaplan and Gershwin, 2005), systemic scleroderma (Alarcon-Segovia et al.,
1979)). Colchicine is metabolized in the liver, mainly by demethylation mediated by
cytochrome P450 3A4 (Leighton et al., 1990; Tateishi et al., 1997). In addition,
colchicine is susceptible to P-gp mediated efflux transport, and increased oral absorption,
as well as pharmacodynamic effects, due to P-gp inhibition were reported (Dintaman and
Silverman, 1999; Bittner et al., 2002). These barriers are considered to contribute to its
low and variable absorption: colchicine absolute oral bioavailability is in the range of
40% (Rochdi et al., 1994; Ferron et al., 1996). We have recently studied the
pharmacokinetic interaction of colchicine with grapefruit juice, and showed that the juice
augments colchicine intestinal absorption through P-gp inhibition mechanism (Dahan and
Amidon, 2009a). However, in all P-gp inhibition experiments, an efflux ratio (ER; PBL-
AP/PAP-BL) of 1 could not be achieved, indicating that efflux transporters other than P-gp
may be involved in colchicine intestinal absorption process, presumably contributing to
its low oral bioavailability.
The purpose of the present study was to thoroughly characterize the contribution
of efflux transporters to the intestinal permeability process of colchicine following oral
administration. The effects of various P-gp, MRP2 and BCRP inhibitors on the
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bidirectional transepithelial permeability of colchicine were studied across Caco-2 cell
monolayers, inhibiting one transporter at a time vs. multiple transporters simultaneously.
The effective permeability of colchicine was then investigated in different regions of the
rat small intestine by the in situ single-pass intestinal perfusion model, in the presence vs.
absence of P-gp (GF120918) and MRP2 (MK571) inhibitors, again, one at a time vs.
simultaneous inhibition. This was then correlated to the expression level of these
transporters throughout the different intestinal segments, measured by Western blot
analysis. Overall, this setup allowed us to confirm the role of the different efflux pumps
involved in colchicine intestinal permeability.
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Materials and Methods
Materials
Colchicine, metoprolol, phenol red, verapamil, quinidine, indomethacin, p-
aminohippuric acid (p-AH), probenecid, fumitremorgin C (FTC), lucifer yellow, MES
buffer, glucose, CaCl2, MgCl2 and trifluoroacetic acid were purchased from Sigma
Chemical Co. (St. Louis, MO). Pantoprazole, potassium chloride and NaCl were obtained
from Fisher Scientific Inc. (Pittsburgh, PA). GF120918 was generously donated by
GlaxoSmithKline Inc. (Research Triangle Park, NC). MK-571 was purchased from
Alexis Biochemicals (Lausen, Switzerland). Acetonitrile and water (Acros Organics,
Geel, Belgium) were HPLC grade. Physiological saline solution was purchased from
Hospira Inc. (Lake Forest, IL). All other chemicals were of analytical reagent grade.
Cell Culture
Caco-2 cells (passage 25-32) from American Type Culture Collection (Rockville,
MD) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum, 1% nonessential
amino acids, 1 mM sodium pyruvate, and 1% L-glutamine. Cells were grown in an
atmosphere of 5% CO2 and 90% relative humidity at 37°C. The DMEM medium was
routinely replaced by fresh medium every three days. Cells were passaged upon reaching
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approximately 80% confluence using 4 ml trypsin-EDTA (Invitrogen Corp., Carlsbad,
CA).
Caco-2 Permeability Studies
Transepithelial transport studies were performed using a previously described
method (Gao et al., 2001; Dahan et al., 2009a). Briefly, 5×104 cells/cm2 were seeded onto
collagen-coated membranes (12-well Transwell plate, 0.4-μm pore size, 12 mm diameter,
Corning Costar, Cambridge, MA) and were allowed to grow for 21 days. Mannitol and
Lucifer yellow permeabilities were assayed for each batch of Caco-2 monolayers (n=3),
and TEER measurements were performed on all monolayers (Millicell-ERS epithelial
Voltohmmeter, Millipore Co., Bedford, MA). Monolayers with apparent mannitol and
Lucifer yellow permeability <3×10−7 cm/sec, and TEER values >300 Ωcm2 were used for
all studies. On the day of the experiment, the DMEM was removed, and the monolayers
were rinsed and incubated for 20 min with a blank transport buffer. The transport buffer
contained 1 mM CaCl2, 0.5 mM MgCl2·6H2O, 145 mM NaCl, 3 mM KCl, 1 mM
NaH2PO4, 5 mM D-glucose, and 5 mM MES. Similar pH was used in both apical and
basolateral sides (pH 6.5) in order to maintain a constant degree of ionization in both AP-
BL and BL-AP direction experiments, and to avoid possible influence of this factor on
the permeability across the cells. Following the 20 min incubation, the drug-free transport
buffer was removed from the donor side (apical in the AP-BL direction, or basolateral in
the BL-AP direction studies), and replaced by colchicine uptake buffer solution (pH 6.5),
with or without inhibitors. Throughout the experiment, the transport plates were kept in a
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shaking incubator (50 rpm) at 37°C. Samples were taken from the receiver side at various
time points up to 120 min (100 μl from basolateral side or 70 μl from apical side), and
similar volumes of blank buffer were added following each sample withdrawal. At the
last time point (120 min), samples were taken from the donor side as well, in order to
confirm mass balance. Samples were immediately assayed for drug content. All Caco-2
monolayers were checked for confluence by measuring the TEER before and after the
transport study.
Inhibition Experiments
The concentration-dependent effects of the P-gp inhibitors GF120918 (5 and 10
μM), verapamil (10, 50 and 100 μM) and quinidine (10, 50 and 100 μM), as well as the
MRP2 inhibitors MK571 (10, 50 and 100 μM), indomethacin (10, 50 and 100 μM), p-AH
(1, 5 and 10 mM) and probenecid (10, 50 and 100 μM), and the BCRP inhibitors FTC (5,
10 and 20 μM) and pantoprazole (10, 50 and 100 μM), on the bidirectional transport of
colchicine (0.1 mM) across caco-2 cell monolayers were examined. The results of these
experiments were evaluated in comparison to the bidirectional transport of 0.1 mM
colchicine in the absence of inhibitors. The effects of the following combinations of P-gp
and MRP2 inhibitors on colchicine bidirectional transport were then investigated:
GF120918 and MK571 (10 and 100 μM, respectively), quinidine and MK571 (100 μM),
verapamil and MK571 (100 μM), quinidine and indomethacin (100 μM), and verapamil
and indomethacin (100 μM). The combination that produced the highest efflux inhibition,
GF120918 and MK571, was then selected for further analysis in rats.
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Single-Pass Intestinal Perfusion Studies (SPIP) in Rats
All animal experiments were conducted using protocols approved by the
University Committee of Use and Care of Animals (UCUCA), University of Michigan,
and the animals were housed and handled according to the University of Michigan Unit
for Laboratory Animal Medicine (ULAM) guidelines. Male albino Wistar rats (Charles
River, IN) weighing 250-280 g were used for all perfusion studies. Prior to each
experiment, the rats were fasted over night (12-18 hr) with free access to water. Animals
were randomly assigned to the different experimental groups.
The procedure for the in situ single-pass intestinal perfusion followed previously
published reports (Kim et al., 2006; Dahan et al., 2009b). Briefly, rats were anesthetized
with an intra-muscular injection of 1 ml/kg of ketamine-xylazine solution (9%:1%,
respectively) and placed on a heated surface maintained at 37°C (Harvard Apparatus Inc.,
Holliston, MA). The abdomen was opened by a midline incision of 3-4 cm. A proximal
jejunal segment (3 cm average distance of the inlet from the ligament of Treitz), or a
distal ileal segment (3 cm average distance of the outlet from the cecum), of
approximately 10 cm was carefully exposed and cannulated on two ends with flexible
PVC tubing (2.29 mm i.d., inlet tube 40 cm, outlet tube 20 cm, Fisher Scientific Inc.,
Pittsburgh, PA). Care was taken to avoid disturbing the circulatory system, and the
exposed segment was kept moist with 37°C normal saline solution. All solutions were
incubated in a 37°C water bath. The isolated segment was rinsed with blank perfusion
buffer, pH 6.5 at a flow rate of 0.5 ml/min in order to clean out any residual debris.
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At the start of the study, perfusion solution containing colchicine (0.1 mM), 10
mM MES buffer, pH 6.5, 135 mM NaCl, 5 mM KCl, and 0.1 mg/ml phenol red with an
osmolarity of 290 mosm/l, with or without the different inhibitors (10 μM GF120918 and
100 μM MK-571), was perfused through the intestinal segment (Watson Marlow Pumps
323S, Watson-Marlow Bredel Inc, Wilmington, MA), at a flow rate of 0.2 ml/min.
Phenol red was added to the perfusion buffer as a nonabsorbable marker for measuring
water flux. Metoprolol was co-perfused with the colchicine as well, as a compound with
known permeability that serves as a marker for the integrity of the experiment, and as a
reference standard for permeability in close proximity to the low/high permeability class
boundary (Kim et al., 2006). The perfusion buffer was first perfused for 1 hr, in order to
assure steady state conditions (as also assessed by the inlet over outlet concentration ratio
of phenol red which approaches 1 at steady state). After reaching steady-state, samples
were taken at 10 min intervals for 1 h (10, 20, 30, 40, 50, and 60 min). All samples
including perfusion samples at different time points, original drug solution, and inlet
solution taken at the exit of the syringe were immediately assayed by HPLC. Following
the termination of the experiment, the length of each perfused intestinal segment was
accurately measured.
Preparation of Rat Brush Border Homogenates for the Analysis of P-gp and MRP2
Expression Along the Small Intestine
The small intestine of male Wistar rats, starting from the ligament of Treitz and
ending at the entrance to the cecum, was removed and rinsed with ice-cold physiological
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saline solution. The intestine was cut into 5 symmetrically spaced equal segments, 10 cm
long each, and placed in PBS containing 1.5 mM EDTA, 0.5 mM dithiothreitol, pH 7.4,
and protease inhibitors cocktail (Invitrogen Corp., Carlsbad, CA). The intestinal segments
were placed on an ice-cold glass surface, and the mucosa was gently squeezed out and
placed in similar buffer containing 0.25 M sucrose. Homogenization of the intestinal
segments was achieved with a Polytron homogenizer at 25,000 rpm, 3×10 sec, on ice,
followed by 3×10 sec of ultrasonication on ice. The homogenates were centrifuged at 900
g for 10 min, and the supernatant was carefully removed and used for protein content
analysis (BioRad DC protein assay, Bio-Rad Laboratories Inc., Hercules, CA) and
immunoblotting.
Immunoblot Analysis of P-gp, MRP2 and BCRP
Immunoblot analysis of P-gp, MRP2, BCRP and villin was performed using a
previously described method with some modifications (MacLean et al., 2008). From each
sample, 100 μg of protein were resolved in 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Invitrogen Corporation, Carlsbad, CA) followed by electrophoretic
transfer onto Hybond ECL nitrocellulose membrane (Amersham, UK). Membranes were
blocked overnight in TBS-T solution containing 3% BSA at 4°C, followed by incubation
with monoclonal anti P-gp antibody (C219, 1:200 dilution in TBS-T, Zymed Laboratories
Inc., San Francisco, CA), monoclonal anti-MRP2 antibody (M2 III-6, 1:100 dilution in
TBS-T, Alexis Biochemicals, Lausen, Switzerland), monoclonal anti-BCRP antibody
(BXP-21, 1:200 dilution in TBS-T, Alexis Biochemicals, Lausen, Switzerland), or
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monoclonal anti-villin antibody (H-60, 1:400 dilution in TBS-T, Santa Cruz
Biotechnology Inc., Santa Cruz, CA) for 2 hr at room temperature. The membranes were
then incubated for 1 hr at room temperature with alkline phosphotase (AP) conjugated
goat anti-mouse IgG (1:3000 dilution, Santa Cruz Biotechnology, CA). The blots were
subsequently visualized with enhanced chemifluorescence (ECF) substrate using a
Typhoon 9200 Variable Mode Imager (GE Healthcare, NJ), and the protein bands of
interest were quantified using ImageQuant 5.2 software (GE Healthcare, NJ). P-gp and
MRP2 expression levels were first normalized to the corresponding villin expression, and
then, within each set of samples (segments 1-5 from the same rat), the lowest intensity
band was set to a value of 1, and the intensities of the other 4 segments were set relative
to that one.
Data Analysis
Permeability coefficient (Papp) across Caco-2 cell monolayers was calculated from
the linear plot of drug accumulated in the receiver side versus time, using the following
equation:
dt
dQ
ACPapp ×=
0
1
where dQ/dt is the steady-state appearance rate of the drug on the receiver (serosal in the
case of AP-BL studies, or mucosal in the case of BL-AP studies) side, C0 is the initial
concentration of the drug in the donor side, and A is the monolayer growth surface area
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(1.12 cm2). Linear regression was carried out to obtain the steady-state appearance rate of
the drug on the receiver side (R2>0.99 in all experimental groups).
The efflux ratio, ER (i.e. the net efflux of colchicine), was determined by
calculating the ratio of Papp in the secretory (BL-AP) direction divided by the absorptive
(AP-BL) direction Papp, according to the following equation:
BL-AP
AP-BL
app
app
P
PER =
The effective permeability (Peff) through the rat gut wall in the single-pass
intestinal perfusion studies was determined assuming the "plug flow" model expressed in
the following equation (Fagerholm et al., 1996):
RL
CCQP inout
eff π2
)'/'ln()cm/sec(
−=
where Q is the perfusion buffer flow rate, C’out/C’in is the ratio of the outlet concentration
and the inlet or starting concentration of the tested drug that has been adjusted for water
transport, R is the radius of the intestinal segment (set to 0.2 cm), and L is the length of
the intestinal segment.
The net water flux in the single-pass intestinal perfusion studies, resulting from
both water absorption and efflux in the intestinal segment, was determined by
measurement of phenol red, a nonabsorbed, nonmetabolized marker. The phenol red (0.1
mg/ml) was included in the perfusion buffer and co-perfused with the tested drugs. The
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measured Cout/Cin ratio was corrected for water transport according to the following
equation:
red phenol
red phenol
'
'
out
in
in
out
in
out
C
C
C
C
C
C×=
where Cin phenol red is equal to the concentration of phenol red in the inlet sample, and Cout
phenol red is equal to the concentration of phenol red in the outlet sample.
Analytical Methods
The amount of colchicine in the Caco-2 medium, and the simultaneous analysis of
colchicine, metoprolol and phenol red in the rat perfusion buffer, were assayed using a
high performance liquid chromatography (HPLC) system (Waters 2695 Separation
Module) with a photodiode array UV detector (Waters 2996). Samples were filtered
(Unifilter® 96 wells microplate 0.45 μm filters, Whatman Inc., Florham Park, NJ), and
Caco-2 medium aliquots of 50 μl, or rat perfusion aliquots of 10 μl, were injected into the
HPLC system. The HPLC conditions were as follows: XTerra, RP18, 3.5 μm, 4.6×100
mm column (Waters Co., Milford, MA); a gradient mobile phase, going from 90:10 to
50:50% v/v aqueous/organic phase respectively over 15 min; the aqueous phase was
0.1% trifluoroacetic acid in water, and the organic phase was 0.1% trifluoroacetic acid in
acetonitrile; flow rate of 1 ml/min at room temperature. The detection wavelengths were
275, 265 and 350 nm, and the retention times were 6.5, 9.5 and 12.0 min for metoprolol,
phenol red and colchicine, respectively. Separate standard curves were used for each
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experiment (R2>0.99). The inter- and intra-day coefficients of variation were <1.0 and 0.5
%, respectively.
Statistical Analysis
All Caco-2 experiments were performed in triplicate (unless stated otherwise),
and all animal experiments were n=4. The data presented as mean ± SD. To determine
statistically significant differences among the experimental groups, the non-parametric
Kruskal-Wallis test was used for multiple comparisons, and the two-tailed non-
parametric Mann-Whitney U test for two-group comparison where appropriate. A p value
of less than 0.05 was termed significant.
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Results
The Effect of Different Efflux Transporters on Colchicine Transcellular
Permeability Across Caco-2 Cell Monolayers
The flux of colchicine across Caco-2 monolayers in the AP-BL and BL-AP
directions, in the presence vs. absence of different concentrations of P-gp or MRP2
inhibitors, is presented in Figures 2 and 3, respectively. All P-gp inhibitors (GF120918,
verapamil and quinidine) significantly increased AP-BL and decreased BL-AP direction
transport, in a concentration-dependent manner. Among these inhibitors, GF120918
showed the most potent effect, however, none of these inhibitors produced an efflux ratio
of 1, i.e. complete inhibition of the drug efflux. Similarly, the MRP2 inhibitors MK-571,
indomethacin and p-AH, displayed a concentration-dependent decrease in colchicine
mucosal secretion, in both AP-BL and BL-AP directions. Again, an efflux ratio of 1
could not be achieved. Among these inhibitors, MK-571 showed the most potent effect.
Probenecid, a MRP2 inhibitor, showed some inhibition of colchicine BL-AP transport,
but no effect on the AP-BL direction.
The effects of the BCRP inhibitors FTC and pantoprazole on colchicine
bidirectional transport across Caco-2 cell monolayers is presented in Fig. 4. It can be seen
that colchicine transepithelial permeability was not affected by the inhibition of BCRP.
The Caco-2 expression of P-gp, MRP2 and BCRP was validated using Western blot
analysis (Fig. 5), confirming that these efflux transporters were indeed present in the cell
culture experiments in the protein level.
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The effects of different combinations of P-gp and MRP2 inhibitors, on the
bidirectional Caco-2 transport of colchicine is presented in Fig. 6, and the resulting efflux
ratios of colchicine from the different inhibition experiments are summarized in Fig. 7.
All P-gp and MRP2 inhibitors combinations were able to greatly reduce colchicine
mucosal secretion, and the combination of the two most potent inhibitors, GF120918 and
MK-571, abolished colchicine efflux transport completely. Hence, this combination was
chosen for further evaluation in rats.
The Effect of P-gp and MRP2 on Colchicine Intestinal Permeability Along the Rat
Small Intestine
Colchicine permeability coefficients (Peff) obtained following in situ perfusion to
the rat proximal jejunum or distal ileum, in the presence vs. absence of the P-gp inhibitor
GF120918 and the MRP2 inhibitor MK-571, are presented in Fig. 8. Without inhibitors,
colchicine displayed low (relative to metoprolol) and constant permeability in the
proximal and distal segments of the rat small intestine. Segmental-dependent colchicine
permeability was obtained in the presence of the efflux transporters inhibitors; while
inhibition of P-gp significantly increased the permeability in the ileum with no effect on
the jejunal transport, MRP2 inhibition augmented colchicine permeability in the jejunum,
with no change in the ileal Peff. When both inhibitors were simultaneously co-perfused
with colchicine, comparable permeability in the proximal and distal segments of the rat
small intestine was obtained; GF120918/MK-571 combination caused similar effect to
MK-571 alone in the jejunum and to GF120918 alone in the ileum.
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P-gp and MRP2 Protein Expression Levels Along the Rat Small Intestine
Fig. 9 shows representative Western blots of rat mucosal samples from different
segments along the small intestine, obtained with monoclonal antibodies against P-gp,
MRP2 and villin. A semi-quantitative analysis of the efflux protein bands, normalized to
villin, is presented in Fig. 10. Significant regional dependent P-gp and MRP2 expression
levels were found throughout the rat small intestine, with the two transporters exhibiting
gradients of opposing trends; while P-gp expression levels increased from the proximal to
the distal segments, MRP2 levels decreased from the proximal to the distal small
intestinal regions.
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Discussion
Segmental-dependent expression level of different transporters, both influx and
efflux, has significant implications for oral drug delivery. With regard to efflux
transporters, we recently showed that the low expression of P-gp in the proximal regions
of the small intestine leads to a minimal role for this transporter in the absorption of P-gp
substrates from these segments, enabling a window for such drug compounds to be
relatively well absorbed following oral administration (Dahan and Amidon, 2009b). The
same phenomenon, in the opposite direction, may be obtained for MRP2 substrate drugs.
In this paper, we demonstrate the combined effect of P-gp and MRP2 on a drug substance
which is a substrate for both P-gp and MRP2 mediated efflux. This powerful
‘collaboration’ leads to complete coverage of the entire small intestinal length, and makes
the efflux transport dominate the intestinal permeability process. The data presented in
this paper can explain colchicine low and variable oral absorption, as well as reveal
potential drug interactions.
Colchicine P-gp mediated efflux has been demonstrated before, both by us
(Dahan and Amidon, 2009a) and others (Dintaman and Silverman, 1999; Troutman and
Thakker, 2003; Niel and Scherrmann, 2006). However, an efflux ratio of 1 could not be
achieved under inhibition of P-gp alone, indicating the involvement of efflux transporters
other than P-gp in its intestinal absorption process. Hence, we investigated the role of
MRP2 and BCRP in colchicine bidirectional permeability. The MRP2 inhibitor MK-571
exhibited a strong concentration-dependent increase in AP-BL transport, accompanied by
a decrease in BL-AP, illustrating, for the first time, that colchicine is susceptible to
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MRP2 mediated efflux transport (Fig. 3). Surprisingly, probenecid, which is a non-
specific inhibitor of MRP2, had little effect on the BL-AP transport, and no effect on the
AP-BL direction. A similar phenomenon was reported before for fluvastatin, where
although the involvement of MRP2 in the intestinal absorption and biliary secretion of
this HMG-CoA reductase inhibitor was evident, probenecid showed neither in vitro nor
in vivo effects on its transport (Lindahl et al., 2000; Lindahl et al., 2004). This may be
explained by a relatively poor affinity of probenecid for MRP2 compared to the
investigated drug. Indeed, probenecid was reported to have higher affinity for MRP1 than
for MRP2, and to be less effective as an MRP2-mediated efflux inhibitor than
indomethacin (Bakos et al., 2000). The non-specific MRP2 inhibitors indomethacin and
p-AH displayed a concentration-dependent increase in AP-BL and a decrease in BL-AP
transport, further supporting the involvement of this transporter in colchicine efflux
process. The specific BCRP inhibitors FTC and pantoprazole had no effect on colchicine
bidirectional transport across Caco-2 monolayers (Fig. 4), indicating that this efflux
pump is not involved in colchicine intestinal permeability process. Overall, it was
revealed that the combined effect of P-gp and MRP2, but not BCRP, mediates the
transepithelial efflux transport of colchicine. This was made further evident by the
experiments involving combination of P-gp and MRP2 inhibitors (Fig. 6); All P-gp and
MRP2 inhibitors combinations were able to greatly reduce colchicine mucosal secretion,
and the combination of the two most potent inhibitors, GF120918 and MK-571,
completely abolished colchicine efflux transport, as was evident by an efflux ratio of 1
(Fig. 7). Hence, a thorough characterization of colchicine transepithelial efflux process
was achieved.
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We previously showed that according to the biopharmaceutics classification
system (BCS) (Amidon et al., 1995), colchicine is a class III drug, i.e. high-solubility
low-permeability compound (Kasim et al., 2004; Dahan and Amidon, 2009a). As a class
III P-gp/MRP2 substrate, colchicine is more susceptible to show efflux dependent in vivo
intestinal absorption; the intrinsic low gut wall permeability of this class of drugs
essentially leads to limited amounts of drug inside the enterocyte, with potentially sub-
saturated P-gp/MRP2 levels (Dahan and Amidon, 2009b). This was made further evident
by the significantly increased intestinal permeability observed in the in-situ rat perfusion
studies in the presence of P-gp/MRP2 inhibitors (Fig. 8), since this experimental model
has been reported to provide a precise method to predict in vivo intestinal absorption (i.e.
fraction of drug absorbed) in humans (Kim et al., 2006; Lennernas, 2007). In light of the
report that colchicine follows linear pharmacokinetics following oral administration in
the dose range of 0.5-1.5 mg, with AUC values proportional to the dose (Girre et al.,
1989), colchicine plasma levels following oral administration are expected to be
proportional to the fraction of drug absorbed, and hence changes in colchicine plasma
levels may be the result of altered P-gp/MRP2 function. This analysis highlights that the
significance of the data presented in this paper goes beyond contributing to a better
understanding of the mechanisms behind colchicine low oral bioavailability, but may also
be relevant to the prediction of different clinical scenarios, e.g. side effects or drug-drug
interactions. Although the focus of this work is the intestinal absorption process
following oral administration, our data may be relevant to other colchicine
pharmacokinetic processes, e.g. distribution and clearance. Indeed, decreased colchicine
biliary clearance from 0.122 to 0.024 ml/min/kg by cyclosporine through P-gp inhibition
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has been reported (Speeg et al., 1992), leading to increased colchicine plasma levels, and
to a clinically relevant interaction. Since MRP2 is expressed on the canalicular membrane
and plays an important role in the biliary excretion of various drugs, the fact that
colchicine is a MRP2 substrate revealed in this paper may point out new mechanism-
based drug-drug interactions. Being a drug with a narrow therapeutic index and severely
toxic side effects (effective steady state plasma concentrations range from 0.5 to 3 ng/ml
with toxic effects appearing at approximately 3 ng/ml) (Ferron et al., 1996), this may be a
most relevant issue.
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Legends for Figures
Fig. 1. Colchicine molecular structure.
Fig. 2. The concentration-dependent effects of various P-gp inhibitors on colchicine
flux (0.1 mM) across Caco-2 cell monolayers in the absorptive (AP-BL; top)
and secretory (BL-AP; bottom) directions. The investigated inhibitors are (left
to right): GF120918, verapamil and quinidine. Data presented as mean ± SD;
n=3 in each experimental group.
Fig. 3. The concentration-dependent effects of various MRP2 inhibitors on colchicine
flux (0.1 mM) across Caco-2 cell monolayers in the absorptive (AP-BL; top)
and secretory (BL-AP; bottom) directions. The investigated inhibitors are (right
to left): MK-571, indomethacin, p-AH and probenecid. Data presented as mean
± SD; n=3 in each experimental group.
Fig. 4. The concentration-dependent effects of the BCRP inhibitors FTC (top) and
pantoprazole (bottom) on colchicine flux (0.1 mM) across Caco-2 cell
monolayers in the absorptive (AP-BL; right) and secretory (BL-AP; left)
directions. Data presented as mean ± SD; n=3 in each experimental group.
Fig. 5. Analysis of P-gp, MRP2 and BCRP expression in the Caco-2 cells used in this
paper by Western immunoblotting. P-gp was probed with the monoclonal
antibody C219, MRP2 was probed with the monoclonal antibody M2 III-6, and
BCRP was probed with the monoclonal antibody BXP-21.
Fig. 6. The effect of different P-gp/MRP2 inhibitors combinations on colchicine flux
(0.1 mM) across Caco-2 cell monolayers in the absorptive (AP-BL) and
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secretory (BL-AP) directions. Data presented as mean ± SD; n=3 in each
experimental group.
Fig. 7. The efflux ratios (ER; PBL-AP/PAP-BL) obtained for colchicine transport across
Caco-2 cell monolayers in the different inhibition experiments (highest inhibitor
concentration). Data presented as mean ± SD; n=3 in each experimental group.
Fig. 8. The permeability coefficients (Peff; cm/sec) obtained for colchicine following
in-situ single-pass intestinal perfusion to the proximal jejunum and to the distal
ileum of the rat, without inhibitors, in the presence of GF120918 or MK-571,
and in the presence of both GF120918 and MK-571 simultaneously. Data
presented as mean ± SD; n=4 in each group; ** p<0.01. Metoprolol
permeability was similar in all experimental groups (2.4E-5 ± 0.4E-5; n=12).
Fig. 9. Analysis of P-gp, MRP2 and villin levels in different segments along the rat
small intestine. Segments 1-5 correspond to equal, 10 cm long, symmetrically
spaced segments, starting from the ligament of Treitz (segment 1) and ending at
the entrance to the cecum (segment 5). P-gp was probed with the monoclonal
antibody C219, MRP2 was probed with the monoclonal antibody M2 III-6, and
villin was probed with the monoclonal antibody H-60. Data shown are
representative preparations from three animals.
Fig. 10. Semi-quantitative analysis of P-gp and MRP2 protein bands, normalized to
villin, in the different segments 1-5 along the rat small intestine. In each set of
samples (segments 1-5 from the same rat), the lowest intensity band was set to
1, and the other 4 segments were set relative to it. Data presented as mean ± SD;
n=3 in each experimental group.
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