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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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Lineweaver–Burk plot (a) and Eadie–Hofstee plot (b) of inhibition of 20-(S)-camptothecin efflux across Caco-2 cells at fixed etoposide concentrations of 0 μM (Δ), 100 μM (•) and 200 μM (□). Each point represents the mean (± standard deviation) for at least three observations. The inhibition constant (Ki) was determined from a secondary plot (inset) of the slopes of Lineweaver–Burk plots for various inhibitor concentrations. Peff, effective permeability; V0, initial rate.
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Figure 4: Lineweaver–Burk plot (a) and Eadie–Hofstee plot (b) of inhibition of 20-(S)-camptothecin efflux across Caco-2 cells at fixed etoposide concentrations of 0 μM (Δ), 100 μM (•) and 200 μM (□). Each point represents the mean (± standard deviation) for at least three observations. The inhibition constant (Ki) was determined from a secondary plot (inset) of the slopes of Lineweaver–Burk plots for various inhibitor concentrations. Peff, effective permeability; V0, initial rate.

Mentions: The efflux inhibition kinetics of CPT were determined by investigating the BL to AP permeability of CPT at various substrate and inhibitor concentrations. For etoposide, concentrations ranging from 100 μM to 200 μM were selected as the Km value of etoposide was estimated to be ~213 μM in our laboratory [22]. The Km values of CPT in the presence of increasing concentrations of etoposide were determined from the Lineweaver–Burk plot (Km = slope / intercept) (Fig. 4a). In the absence of etoposide, the Km value of CPT was 173.2 μM using the Lineweaver–Burk plot, which is different from that obtained using nonlinear regression (132 μM). The use of linearization techniques to analyze nonlinear data is well known to introduce error into the parameter (Km, Vmax, Jmax) estimates. The linearization techniques were therefore only used in a qualitative manner to show the kinetic behavior of the inhibitors with 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)

Lineweaver–Burk plot (a) and Eadie–Hofstee plot (b) of inhibition of 20-(S)-camptothecin efflux across Caco-2 cells at fixed etoposide concentrations of 0 μM (Δ), 100 μM (•) and 200 μM (□). Each point represents the mean (± standard deviation) for at least three observations. The inhibition constant (Ki) was determined from a secondary plot (inset) of the slopes of Lineweaver–Burk plots for various inhibitor concentrations. Peff, effective permeability; V0, initial rate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Lineweaver–Burk plot (a) and Eadie–Hofstee plot (b) of inhibition of 20-(S)-camptothecin efflux across Caco-2 cells at fixed etoposide concentrations of 0 μM (Δ), 100 μM (•) and 200 μM (□). Each point represents the mean (± standard deviation) for at least three observations. The inhibition constant (Ki) was determined from a secondary plot (inset) of the slopes of Lineweaver–Burk plots for various inhibitor concentrations. Peff, effective permeability; V0, initial rate.
Mentions: The efflux inhibition kinetics of CPT were determined by investigating the BL to AP permeability of CPT at various substrate and inhibitor concentrations. For etoposide, concentrations ranging from 100 μM to 200 μM were selected as the Km value of etoposide was estimated to be ~213 μM in our laboratory [22]. The Km values of CPT in the presence of increasing concentrations of etoposide were determined from the Lineweaver–Burk plot (Km = slope / intercept) (Fig. 4a). In the absence of etoposide, the Km value of CPT was 173.2 μM using the Lineweaver–Burk plot, which is different from that obtained using nonlinear regression (132 μM). The use of linearization techniques to analyze nonlinear data is well known to introduce error into the parameter (Km, Vmax, Jmax) estimates. The linearization techniques were therefore only used in a qualitative manner to show the kinetic behavior of the inhibitors with 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