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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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Temperature dependence of 20-(S)-camptothecin transport across Caco-2 cells for basolateral to apical transport. Plot of logarithmic effective permeability (Peff) (mean ± standard deviation) and the best-fit line versus the reciprocal of absolute temperature. Each point represents the mean (± standard deviation) for at least three observations. The activation energy (Ea), was determined based on the equation: slope = -Ea / 2.303R, where R is the gas constant (= 0.001987 kcal/deg mole).
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Figure 2: Temperature dependence of 20-(S)-camptothecin transport across Caco-2 cells for basolateral to apical transport. Plot of logarithmic effective permeability (Peff) (mean ± standard deviation) and the best-fit line versus the reciprocal of absolute temperature. Each point represents the mean (± standard deviation) for at least three observations. The activation energy (Ea), was determined based on the equation: slope = -Ea / 2.303R, where R is the gas constant (= 0.001987 kcal/deg mole).

Mentions: To determine whether CPT efflux was an active (i.e. energy dependent) process and to estimate the passive permeability component, the BL to AP transport was investigated at various temperatures ranging from 4°C to 37°C. It was observed that the efflux Peff of CPT decreased as the temperature was reduced. The energy of activation (Ea) was estimated to be ~11.3 kcal/mole from the Arrhenius plot (Fig. 2). Since activation energies greater than 4 kcal/mole are suggestive of active membrane transport [20], the secretory permeability of CPT appears to occur by an active and energy-dependent mechanism.


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)

Temperature dependence of 20-(S)-camptothecin transport across Caco-2 cells for basolateral to apical transport. Plot of logarithmic effective permeability (Peff) (mean ± standard deviation) and the best-fit line versus the reciprocal of absolute temperature. Each point represents the mean (± standard deviation) for at least three observations. The activation energy (Ea), was determined based on the equation: slope = -Ea / 2.303R, where R is the gas constant (= 0.001987 kcal/deg mole).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Temperature dependence of 20-(S)-camptothecin transport across Caco-2 cells for basolateral to apical transport. Plot of logarithmic effective permeability (Peff) (mean ± standard deviation) and the best-fit line versus the reciprocal of absolute temperature. Each point represents the mean (± standard deviation) for at least three observations. The activation energy (Ea), was determined based on the equation: slope = -Ea / 2.303R, where R is the gas constant (= 0.001987 kcal/deg mole).
Mentions: To determine whether CPT efflux was an active (i.e. energy dependent) process and to estimate the passive permeability component, the BL to AP transport was investigated at various temperatures ranging from 4°C to 37°C. It was observed that the efflux Peff of CPT decreased as the temperature was reduced. The energy of activation (Ea) was estimated to be ~11.3 kcal/mole from the Arrhenius plot (Fig. 2). Since activation energies greater than 4 kcal/mole are suggestive of active membrane transport [20], the secretory permeability of CPT appears to occur by an active and energy-dependent mechanism.

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