multiple efflux pumps are involved in the transepithelial...

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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.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 9, 2009 as DOI: 10.1124/dmd.109.028282

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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:

[email protected]

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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