Intestinal transport of irinotecan in Caco-2 cells and MDCK II cells overexpressing efflux transporters Pgp, cMOAT, and MRP1.

Irinotecan (CPT-11) is a water-soluble camptothecin (CPT) derivative that has been recently approved in the United States for patients as a first-line therapy in advanced colorectal cancer. Phase I clinical trials using oral CPT-11 have shown poor and variable oral bioavailability. The present study was designed to investigate the intestinal absorption and efflux mechanisms of CPT-11 using in vitro cell culture models, Caco-2 cells, and engineered Madine-Darby canine kidney (MDCK) II cells overexpressing P-glycoprotein (Pgp), canalicular multispecific organic anion transporter (cMOAT), and multidrug resistance-associated protein (MRP1). The intestinal absorptive and secretory transport of CPT-11 was investigated using Caco-2 cell monolayers. Secretory transport was concentration-dependent and saturable. The secretory efflux permeability (P(eff)) of CPT-11 decreased with decreasing temperature, with an estimated activation energy of 19.6 +/- 2.9 kcal/mol suggesting the involvement of active transporters. The involvement of potential secretory transporters was further characterized in MDCK II cells. The secretory efflux carrier permeability (P(c)) was approximately 4- and approximately 2-fold greater in MDCK II/Pgp and MDCK II/cMOAT cells than that in MDCK II/wild-type cells. Furthermore, the secretory efflux P(eff) of CPT-11 was significantly decreased by Pgp inhibitors, elacridar (GF120918) (IC50 = 0.38 +/- 0.06 microM) and verapamil (IC(50) = 234 +/- 48 microM) in MDCK II/Pgp cells and by cMOAT inhibitor 3-([(3-(2-[7-chloro-2-quinolinyl]ethyl)phenyl]-[(3-dimethylamino-3-oxoprphyl)-thio)-methyl]-thio) propanoic acid (MK571) (IC50) = 469 +/- 60 micro;M) in MDCK II/cMOAT cells. Overall, the current study suggests that Pgp and cMOAT are capable of mediating the efflux of CPT-11 in vitro. Since both Pgp and cMOAT are expressed in the intestine, liver, and kidney, it is likely that these efflux transporters play a significant role limiting the oral absorption and disposition of this important anticancer drug.     

Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells

Purpose. The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. Methods. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 M to 1000 M. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2 mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C₄(100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3°C to 37°C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. Results. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Heal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 M. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Ea of intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. Conclusions. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Kms were in the micro-molar range, and involved an energy dependent mechanism(s).     

Development of a new permeability assay using low‐efflux MDCKII cells

Permeability is an important property of drug candidates. The Madin–Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P‐glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII‐LE (low efflux), has been developed by selecting a subpopulation of low‐efflux cells from MDCKII‐WT using an iterative fluorescence‐activated cell sorting technique with calcein‐AM as a Pgp and efflux substrate. MDCKII‐LE cells are a subpopulation of MDCKII cells with over 200‐fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII‐WT. MDCKII‐LE cells showed less functional efflux activity than MDCKII‐WT based on efflux ratios. Notably, MDCKII–MDR1 showed about 1.5‐fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII‐LE cells offer clear advantages over the MDCKII‐WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII‐LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII‐LE permeability assay (4‐in‐1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost‐effective. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4974–4985, 2011     

Active efflux kinetics of etoposide from rabbit small intestine and colon

The aim of the present study was to investigate the directional transport kinetics of etoposide in rabbit intestinal tissues using side-by-side diffusion chambers. Etoposide is a routinely used mixed-mechanism 'efflux' inhibitor; however, its absorptive and secretory transport kinetics in rabbit intestinal tissues, a commonly used animal model, have not yet been reported. Kinetic studies revealed that the apical (AP) to basolateral (BL) (i.e. absorptive) transport of etoposide was not apparently mediated by specialized transporters, whereas secretion (i.e. BL to AP transport) by intestinal tissues was concentration dependent and saturable. Half-saturation constant values (K(m), mean+/-standard deviation (S.D.)) ranged from 53.6+/-35.8 microM to 168.7+/-127.3 microM, consistent with previous results from our group in intestinal tissues from other species and Caco-2 cell monolayers. Secretory permeability was greatest in the ileum, whereas values in the upper small intestine and colon were approximately equal, and represented only 50% of the value in the ileum. The ileal secretory transport of etoposide was temperature dependent, with the activation energy (E(a)) >4 kCal/mole at 5 microM, suggesting the involvement of the active, energy dependent mechanism. Etoposide inhibition by verapamil and saquinavir, known inhibitors of intestinal secretion, was characterized as competitive with K(i)'s equal to 193.0+/-164.4 microM and 72.6+/-53.5 microM, respectively. The current results demonstrate that the absorptive transport of etoposide in rabbit tissue was not mediated by specialized carriers, and that secretory transport was regionally dependent, mediated by a transporter or transporters, the K(m)'s were in the micromolar range, and involved the energy dependent mechanism(s). The relatively low k(m) of etoposide compared with its aqueous solubility (0.25-0.34 mM, pH 5-6.5, 25 degrees C) makes it the excellent mixed-mechanism competitive inhibitor for determining the secretory transport properties of putative drug substrates. Understanding the in vitro secretory transport kinetics of etoposide provides a mechanistic basis for ongoing studies exploring the functional role of 'efflux' in vivo.     

Potential role of ATP-binding cassette transporters in the intestinal transport of rhein

Rhein, a lipophilic anthraquinone, exhibits anti-inflammatory and anti-tumor activities; however, it is hepatotoxic. ATP-binding cassette transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2), can pump toxicants from gut epithelial cells back into the intestinal lumen to prevent poisoning. We investigated their roles in rhein transport using a rat intestinal perfusion model and Caco-2, MDCKII-MDR1 (high expression of P-gp), MDCKII-BCRP (high expression of BCRP) and MDCKII-MRP2 (high expression of MRP2) cell models. The permeability of rhein in the duodenum significantly increased with increasing perfused concentration of rhein in the rat model, suggesting that efflux transporters were involved in rhein transport. In the Caco-2 cells, the permeability of rhein from the basolateral (B) to the apical (A) was significantly higher than that from A to B. In the presence of BCRP or MRP2 inhibitor, the permeability of rhein significantly decreased from B to A direction. In the MDCKII-BCRP cells, rhein was more permeable in B to A side than that in the opposite side. However, no significant differences of rhein permeability were observed in two directions in both MDCKII-MDR1 and MDCKII-MRP2 cells. Taken together, these results suggested that only BCRP was involved in rhein transport.     

The effect of cell culture conditions on saquinavir transport through, and interactions with, MDCKII cells overexpressing hMDR1

MDCK cells are cultured using wide-ranging conditions and can produce variable results. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII-PGP) and MDCKII wild-type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 x 10(5) or 5 x 10(4) cells/cm2), substratum (polycarbonate +/- collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport. The saquinavir efflux ratio (ratio of BL --> AP/AP --> BL permeability) for MDCKII-PGP cells (6.9 x 10(5) cells/cm2) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 x 10(4) cells/cm2 on polycarbonate (78) or collagen-coated polycarbonate (126). The MDCKII/wt saquinavir efflux ratio was 9. Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen-coated membranes. Collagen coating caused increased Pgp expression and saquinavir efflux ratios correlated (r2 = 0.96) with Pgp expression levels [MDCKII-PGP (on collagen-coated polycarbonate) > MDCKII-PGP (on polycarbonate) > MDCKII/wt (on collagen-coated polycarbonate)]. These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models.     

Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein

In view of the important role of P-glycoprotein (Pgp) and other drug efflux transporters for drug distribution and resistance, the identification of compounds as substrates of Pgp-mediated transport is one of the key issues in drug discovery and development, particularly for compounds acting on the central nervous system. In vitro transport assays with Pgp-transfected kidney cell lines are widely used to evaluate the potential of compounds to act as Pgp substrates or inhibitors. Furthermore, such cell lines are also frequently utilized as a substitute for more labor-intensive in vitro or in vivo models of the blood-brain barrier (BBB). Overexpression of Pgp or members of the multidrug resistance protein (MRP) family at the BBB has been implicated in the mechanisms underlying resistance to antiepileptic drugs (AEDs) in patients with epilepsy. Therefore, it is important to know which AEDs are substrates for Pgp or MRPs. In the present study, we used monolayers of polarized MDCKII dog kidney or LLC-PK1 pig kidney cells transfected with cDNA containing either human MDR1, MRP2 or mouse mdr1a and mdr1b sequences to measure the directional transport of AEDs. Cyclosporin A (CsA) and vinblastine were used as reference standards for Pgp and MRP2, respectively. The AEDs phenytoin and levetiracetam were directionally transported by mouse but not human Pgp, whereas CsA was transported by both types of Pgp. Carbamazepine was not transported by any type of Pgp and did not inhibit the transport of CsA. In contrast to vinblastine, none of the AEDs was transported by MRP2 in transfected kidney cells. The data indicate that substrate recognition or transport efficacy by Pgp differs between human and mouse for certain AEDs. Such species differences, which are certainly not restricted to human and mouse, may explain, at least in part, the controversial data which have been previously reported for AED transport by Pgp in preparations from different species. However, because transport efficacy of efflux transporters such as Pgp or MRP2 may not only differ between species but also between tissues, the present data do not exclude that the AEDs examined are weak substrates of Pgp or MRP2 at the human BBB.     

Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells. These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1). MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay. Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay. Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability. This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity. The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line. This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay. Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial. Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies.     

Transport characteristics of 9-nitrocamptothecin in the human intestinal cell line Caco-2 and everted gut sacs

The intestinal absorptive characteristics and the efflux mechanisms of 9-nitrocamptothecin (9-NC), a novel water-insoluble camptothecin (CPT) derivative, were investigated. The Caco-2 cells and the everted gut sacs were used as models of the intestinal mucosa to assess transepithelial transport of 9-NC. The determination of 9-NC was performed by HPLC. In the Caco-2 cells, the absorptive transport of 9-NC was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of 9-NC at concentrations up to 250 microM, while secretory transport were concentration dependent and saturable process (K(m) was 49.8 +/- 1.2 microM, V(max) was 38.28 +/- 0.8 ng/cm(2)/min). In the presence of verapamil (100 microM) and CsA (10 microM), potent inhibitors of P-glyprotein (P-gp)/MRP2 (cMOAT), the P(appBL-AP)/P(appAP-BL) ratio was decreased from 3.4 to 1.4 and 1.3, respectively, and permeation of apical to basolateral was enhanced approximately two-fold. In the everted gut sacs, the absorption of 9-NC was passive diffusion and had no significant difference in different gut regions. Adding verapamil in the everted gut sacs over a concentration ranging from 10 to 100 microM, the absorption of 9-NC was significantly enhanced, especially more markedly in lower small intestine (P < 0.05). Overall, the current study suggests that pH and efflux transporters are capable of mediating the absorption and efflux of 9-NC, and they may play significant roles in limiting the oral absorption of 9-NC.     

Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides

The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug resistance-associated protein 2 (Mrp2)/Abcc2. To study the possible involvement of Mrp2, we compared intestinal uptake of quercetin-3-glucoside between control and Mrp2-deficient rats, using an in situ intestinal perfusion system. The contribution of Bcrp1 was determined using the specific inhibitor fumitremorgin C (FTC) in Mrp2-deficient rats. Furthermore, vectorial transport of quercetin was studied in Madin-Darby canine kidney (MDCK)II cells transfected with either human MRP2 or murine Bcrp1. In these MDCKII cells, we showed an efficient efflux-directed transport of quercetin by mouse Bcrp1, whereas in control and MRP2-transfected cells no vectorial transport of quercetin was observed. In Mrp2-deficient rats, intestinal uptake of quercetin from quercetin-3-glucoside, efflux of quercetin glucuronides to the gut lumen, and plasma concentration of quercetin were similar to that in control rats. When intestinal Bcrp1 was inhibited by FTC in Mrp2-deficient rats, total plasma concentrations of quercetin and its methylated metabolite isorhamnetin after 30 min of perfusion were more than twice that of controls (12.3 +/- 1.5 versus 5.6 +/- 1.3 muM; p < 0.01), whereas uptake of free quercetin from the intestinal lumen was not affected. Instead, inhibition of Bcrp1 lowered the efflux of quercetin glucuronides into the perfusion fluid by approximately 4-fold. In conclusion, Bcrp1 limits net intestinal absorption of quercetin by pumping quercetin glucuronides back into the lumen.     

Involvement of P-glycoprotein,Multidrug Resistance Protein 2 and Breast Cancer Resistance Protein in the Transport of Belotecan and Topotecan in Caco-2 and MDCKII Cells

Purpose  To investigate the underlying mechanism of low bioavailabilities of the water-soluble camptothecin derivatives, belotecan and topotecan. Methods  The bioavailability of belotecan and topotecan in rats was determined following oral administration of each drug at a dose of 5 mg/kg body weight. The vectorial transport of each drug was measured in Caco-2 and engineered MDCK II cells. Results  The bioavailability of belotecan (11.4%) and topotecan (32.0%) in rats was increased to 61.5% and 40.8%, respectively, by the preadministration of CsA at a dose of 40 mg/kg. Contrary to the absorptive transport, the secretory transport of these drugs across the Caco-2 cell monolayer was concentration-dependent, saturable, and significantly inhibited by the cis presence of verapamil (a P-gp substrate), MK-571 (an MRP inhibitor), bromosulfophthalein (BSP, an MRP2 inhibitor), fumitremorgin C (FTC, a BCRP inhibitor) and cyclosporine A (CsA, an inhibitor of P-gp and BCRP, and a substrate of P-gp) suggesting the involvement of these transporters, which could be further confirmed in MDCKII/P-gp, MDCKII/MRP2 and MDCKII/BCRP cells. Conclusion  The involvement of secretory transporters P-gp, MRP2 and BCRP, particularly for belotecan, as well as a low passive permeability, appears to be responsible for the low bioavailability of belotecan and topotecan. Hong Li and Hyo-Eon Jin have contributed equally to this work.     

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.     

Functional characterization of peptide transporters in MDCKII-MDR1 cell line as a model for oral absorption studies

MDCKII-MDR1 cell line has been extensively selected as a model to study P-gp-mediated drug efflux. Recently, investigators have employed this cell line for studying influx of peptide prodrug derivatives of parent compounds, which are P-gp substrates. Therefore, the objective of this study is to functionally characterize the peptide mediated uptake and transport of [(3)H] Glycylsarcosine ([(3)H] Gly-Sar), a model peptide substrate across MDCKII-MDR1 cells. [(3)H] Gly-Sar uptake from apical (AP) and basolateral (BL) membranes was found to be time-dependent and saturable. Michaelis-Menten (K(m)) constants of [(3)H] Gly-Sar uptake across the AP and BL directions in MDCKII-MDR1 cell line were found to be 457+/-37 and 464+/-85microM, respectively. V(max) values in AP and BL directions for the peptide transporters in MDCKII-MDR1 cell line were calculated to be 0.035+/-0.001 and 0.35+/-0.034pmol/minmg protein, respectively. Uptake of [(3)H] Gly-Sar was significantly inhibited in the presence of aminocephalosporins and ACE-Inhibitors, known substrates for peptide transporters in both the AP and BL directions. Permeability of [(3)H] Gly-Sar in the BL direction was maximal at pH 4 as compared to pH 5, 6 and 7.4 whereas such permeability in the AP direction was optimal at pH 7.4. Transepithelial transport of [(3)H] Gly-Sar in the AP-BL direction was significantly lower than from BL-AP direction at all observed pHs. No statistical difference was observed in the transepithelial permeability of [(3)H] Gly-Sar across both AP and BL directions over 4-10 days of growth period. The present study indicates that peptide transporters are effectively involved in the bidirectional transport of Gly-Sar across MDCKII-MDR1 cell line; the BL peptide transporter can transport Gly-Sar at a greater rate as compared to the AP peptide transporter. Results from these studies suggest the application of MDCKII-MDR1 cell line as a rapid effective tool to study peptide mediated influx of compounds that may be substrates for both P-gp and peptide transporters.     

Comparison of furosemide and vinblastine secretion from cell lines overexpressing multidrug resistance protein (P-glycoprotein) and multidrug resistance-associated proteins (MRP1 and MRP2)

Recent studies in our laboratory have shown that the loop diuretic, furosemide, is actively secreted by Caco-2 cells and rat jejunal tissue. This active secretion could be the result of efflux transporters such as P-gp, MRP1 or MRP2 (cMOAT). To determine if any of these transporters is responsible for the secretion of furosemide, we compared directional permeability in the wild-type cell lines, MDCK strains I and II, and LLC-PK1, vs. cell lines that overexpress a single transporter, in both the presence and absence of various inhibitors, for furosemide as compared to vinblastine. Sulfinpyrazone significantly inhibited the transport of vinblastine in MRP2 expressing cells, but not the wild-type controls. Vinblastine could not be confirmed as a substrate of MRP1. We were also unable to demonstrate that any particular transporter affected furosemide in excess of the background effects of endogenous transporters in the parental cell lines. Furosemide secretion from these kidney-derived cell lines is probably not the primary result of any of the well characterized efflux transporters (P-gp, MRP1 or MRP2), although they may still play a role in the observed Caco-2 secretion. This equivocal result acknowledges the difficulty in trying to determine the effect of a single protein in a complicated expression system.     

Intestinal ciprofloxacin efflux: the role of breast cancer resistance protein (ABCG2)

Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum.     

Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor

Polarized epithelial non-human (canine) cell lines stably transfected with human or murine complementary DNA (cDNA) encoding for various efflux transporters (P-gp/MDR1, MRP1, MRP2, and Bcrp1) were used to study transepithelial transport of Lopinavir (LVR) and compare results with the MDCKII-wild type cells. These transmembrane proteins cause multidrug resistance by decreasing the total intracellular accumulation of drugs. Lopinavir efflux was directional and was completely inhibited by MK-571, a selective MRP family inhibitor in the MDCKII-MRP2 cell line. Similarly, LVR efflux was also inhibited by P-gp inhibitors P-gp-4008 and GF120918 in the MDCKII-MDR1 cell line. The efflux ratios of LVR in the absence of any efflux inhibitors in the MDCK-wild type, MDCKII-MDR1, MDCKII-MRP1 and MDCKII-MRP2 cell monolayers were 1.32, 4.91, 1.26 and 2.89 respectively. The MDCKII-MDR1 and MDCKII-MRP2 cells have significantly increased LVR efflux ratio relative to the parental cells due to the apically directed transport by MDR1 and MRP2 respectively. The efflux ratios in MRP2 and MDR1 transfected cell lines were close to unity in the presence of MK-571 and P-gp-4008, respectively, indicating that LVR efflux by MRP2 and P-gp was completely inhibited by their selective inhibitors. MDCKII-MRP1 cells did not exhibit a significant reduction in the LVR efflux relative to the parental cells, indicating that LVR is not a good substrate for MRP1. Transport studies across MDCKII-Bcrp1 cells indicated that LVR is not transported by Bcrp1 and is not a substrate for this efflux protein. In conclusion, this study presents direct evidence that LVR is effluxed by both P-gp and MRP2 which may contribute to its poor oral bioavailability and limited penetration into the CNS.     

Peptide prodrugs: improved oral absorption of lopinavir, a HIV protease inhibitor

Lopinavir (LVR) is extensively metabolized by CYP3A4 and is prevented from entering the cells by membrane efflux pumps such as P-gp and MRP2. In an approach to evade the first-pass metabolism and efflux of LVR, peptide prodrugs of LVR [valine-valine-lopinavir (VVL) and glycine-valine-lopinavir (GVL)] were synthesized. Prodrugs were identified with 1H and 13C NMR spectra and LC/MS/MS was employed to evaluate their mass and purity. Solubility studies indicated that the prodrugs have enhanced aqueous solubilities relative to parent LVR. Accumulation and transport data of VVL and GVL across MDCKII-MDR1 and MDCKII-MRP2 cells indicated evasion of prodrugs' efflux by P-gp and MRP2 significantly. Permeability studies across Caco-2 cells indicated that the prodrugs are transported by peptide transporters and have increased permeability as compared with LVR. VVL and GVL exhibited significantly better degradation rate constants as compared with LVR in rat liver microsomes. Enzymatic stability studies in Caco-2 cell homogenate indicated that the peptide prodrugs are first converted to the ester intermediate (amino acid prodrug VL) and then finally to the parent drug. Overall, the advantages of utilizing peptide prodrugs include chemical modification of the compound to achieve targeted delivery via peptide transporters present across the intestinal epithelium, significant evasion of efflux and CYP3A4 mediated metabolism and significantly better solubility profiles. Therefore, in vitro studies demonstrated that peptide prodrug derivatization of LVR may be an effective strategy for evading its efflux and enhancing its systemic concentrations.     

Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 microM doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin resistance points to the fragility of Pgp1-mediated MXR defence mechanism in fish.     

Analysis of xenobiotic detoxification system mediated by efflux transporters

The excretion of drugs mediated by transporters plays an important role in the detoxification of xenobiotics. In this article, I will summarize recent progress we have made in this field, particularly focusing on the roles of transporters responsible for exporting drugs. As far as the biliary excretion of xenobiotics is concerned, it has been suggested that canalicular multispecific organic anion transporter/multidrug resistance associated protein 2 (cMOAT/MRP2) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. By comparing the transport across this membrane between normal rats and Eisai hyperbilirubinemic rats whose cMOAT/MRP2 function is hereditarily defective, we were able to demonstrate the substrate specificity of cMOAT/MRP2. This includes non-conjugated anionic drugs, and glutathione- and glucuronide-conjugates of xenobiotics. The role of cMOAT/MRP2 in drug disposition has also been clarified. Moreover, the cDNA of cMOAT/MRP2 has been cloned and its functional analysis has been completed. Thus, it may be possible to predict in vivo transport across the bile canalicular membrane from in vitro data using the recombinant transporter. We also cloned MRP3 as an inducible transporter in the liver under the cholestatic conditions. Although MRP3 mediates the cellular export of non-conjugated organic anions and glucuronide-conjugates, the substrate specificity of MRP3 is different from that of cMOAT/MRP2 in that glutathione-conjugates are poor substrates for MRP3. It is possible that MRP3 plays an important role under certain pathological conditions in the liver. Since it has been shown that cMOAT/MRP2 and MRP 3 are expressed in the small intestine under physiological conditions, it seems reasonable that these transporters are responsible for the previously reported cellular extrusion of organic anions. We also found that there was MRP activity in the blood-brain and blood-cerebrospinal fluid barriers. RT-PCR resulted in the amplification of MRP1, 5 and 6 from freshly isolated rat cerebral endothelial cells. It has been suggested that there is basolateral localization of MRP1 in the choroid plexus. In conjunction with the P-glycoprotein located on the luminal membrane of cerebral endothelial cells, these transporters play significant roles in restricting the entry of xenobiotics from the circulating blood into the central nervous system. Regulation of the activity of these efflux transporters allows the disposition of drugs to be altered.     

Multiple pathways for fluoroquinolone secretion by human intestinal epithelial (Caco-2) cells

1. Human intestinal epithelial Caco-2 cells, T84 cells, and MDCKII cells transfected with human MDR1, were used to investigate the mechanistic basis of transintestinal fluoroquinolone secretion. 2. The fluoroquinolone grepafloxacin was secreted across Caco-2 monolayers by a saturable process (V(max)=16.9 +/- 3.4 nmol.cm(-2).h(-1)). Net secretion was reduced by 2-deoxyglucose/azide treatment to reduce intracellular ATP. 3. Grepafloxacin inhibited [(14)C]-ciprofloxacin (100 microM) secretion across Caco-2 monolayers (K(0.5)=0.8 mM), and concurrently increased the cellular accumulation of ciprofloxacin from the basal medium, indicating inhibition of export across the apical membrane. 4. The unconjugated bile acid, cholic acid, was secreted across Caco-2 monolayers, and this secretion was sensitive to inhibition by the MRP-selective inhibitor MK-571, suggesting MRP2 involvement. Secretion of cholic acid (10 microM) across the apical membrane was also inhibited by grepafloxacin (K(0.5)=0.3 mM), but not by ciprofloxacin. 5. In MDCKII-MDR1 monolayers, net secretion of grepafloxacin was increased by 3.5 fold compared with untransfected controls. Neither ciprofloxacin nor cholic acid showed net secretion in either MDCKII or MDCKII-MDR1 monolayers, showing that in contrast to grepafloxacin, neither are substrates for MDR1. 6. In T84 monolayers, which express MDR1 but not MRP2, neither ciprofloxacin nor cholic acid was secreted, whilst the V(max) for grepafloxacin secretion was lower than in Caco-2 cells, which express both MDR1 and MRP2. 7. In conclusion, the transepithelial secretion of grepafloxacin is mediated by both MRP2 and MDR1, whereas ciprofloxacin is a substrate for neither. Grepafloxacin also competes for the ciprofloxacin-sensitive pathway, which remains to be elucidated.