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Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2).

Lalloo AK, Luo FR, Guo A, Paranjpe PV, Lee SH, Vyas V, Rubin E, Sinko PJ - BMC Med (2004)

Bottom Line: The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable.The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA. lalloo@eden.rutgers.edu <lalloo@eden.rutgers.edu>

ABSTRACT

Background: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition.

Methods: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.

Results: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 microM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family.

Conclusions: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

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Inhibition of 20-(S)-camptothecin (10 μM) efflux transport across Caco-2 cells. Each point represents the mean (± standard deviation) for at least three observations. * Efflux permeability (with inhibitor) is significantly different from the control (without inhibitor), P < 0.05. Peff, effective permeability.
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Figure 3: Inhibition of 20-(S)-camptothecin (10 μM) efflux transport across Caco-2 cells. Each point represents the mean (± standard deviation) for at least three observations. * Efflux permeability (with inhibitor) is significantly different from the control (without inhibitor), P < 0.05. Peff, effective permeability.

Mentions: The concentration and temperature dependency studies suggest that active transporters possibly mediate the efflux of CPT. To confirm this hypothesis, BL to AP transport of CPT was investigated in the presence of etoposide, a mixed mechanism efflux inhibitor, and GF120918, a specific PGP and BCRP inhibitor (Fig. 3). Co-incubation of etoposide with CPT reduced the efflux permeability by 1.3-fold (200 μM), while in the presence of GF120918 (0.4 μM) the CPT efflux was reduced by 2.1-fold. It has been reported that GF120918 fully reverses PGP-mediated multidrug resistance at concentrations ranging from 0.05 μM to 0.1 μM [21]. However, in the presence of increasing concentrations of GF120918 (0.8–1.2 μM, data not shown), where PGP activity is expected to be fully reversed, the efflux of CPT was not further reduced. This suggests that other transporters may also be involved in the transport of CPT.


Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2).

Lalloo AK, Luo FR, Guo A, Paranjpe PV, Lee SH, Vyas V, Rubin E, Sinko PJ - BMC Med (2004)

Inhibition of 20-(S)-camptothecin (10 μM) efflux transport across Caco-2 cells. Each point represents the mean (± standard deviation) for at least three observations. * Efflux permeability (with inhibitor) is significantly different from the control (without inhibitor), P < 0.05. Peff, effective permeability.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC411064&req=5

Figure 3: Inhibition of 20-(S)-camptothecin (10 μM) efflux transport across Caco-2 cells. Each point represents the mean (± standard deviation) for at least three observations. * Efflux permeability (with inhibitor) is significantly different from the control (without inhibitor), P < 0.05. Peff, effective permeability.
Mentions: The concentration and temperature dependency studies suggest that active transporters possibly mediate the efflux of CPT. To confirm this hypothesis, BL to AP transport of CPT was investigated in the presence of etoposide, a mixed mechanism efflux inhibitor, and GF120918, a specific PGP and BCRP inhibitor (Fig. 3). Co-incubation of etoposide with CPT reduced the efflux permeability by 1.3-fold (200 μM), while in the presence of GF120918 (0.4 μM) the CPT efflux was reduced by 2.1-fold. It has been reported that GF120918 fully reverses PGP-mediated multidrug resistance at concentrations ranging from 0.05 μM to 0.1 μM [21]. However, in the presence of increasing concentrations of GF120918 (0.8–1.2 μM, data not shown), where PGP activity is expected to be fully reversed, the efflux of CPT was not further reduced. This suggests that other transporters may also be involved in the transport of CPT.

Bottom Line: The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable.The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA. lalloo@eden.rutgers.edu <lalloo@eden.rutgers.edu>

ABSTRACT

Background: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition.

Methods: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.

Results: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 microM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family.

Conclusions: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

Show MeSH
Related in: MedlinePlus