Alterations in hepatobiliary disposition of chemotherapeutic agents by multidrug resistance modulators

摘要

The treatment of cancer with chemotherapeutic agents is complicated by the phenomenon of multidrug resistance (MDR). MDR has been a major focus of cancer research, and recent advances have led to some promising new treatment strategies to reverse MDR. One promising strategy in MDR reversal of tumor cells that overexpress P-glycoprotein (P-gp) may be to administer modulators of MDR that inhibit P-gp-mediated cellular efflux of chemotherapeutic agents. P-gp is a transmembrane glycoprotein localized on the apical membrane of numerous normal tissues and is responsible for ATP-dependent efflux of substrates. Inhibition of P-gp at sites other than cancer cells may result in pharmacologic and/or toxicologic consequences. Many chemotherapeutic agents are P-gp substrates and are eliminated primarily by hepatic processes. The objective of this research project was to determine the mechanism(s) of altered hepatobiliary disposition of chemotherapeutic agents by MDR modulators. Livers from male Sprague-Dawley rats (200-250 g) were utilized in the following model systems: isolated perfused livers (IPL), isolated hepatocyte suspensions, S9 fractions, and canalicular liver plasma membrane (cLPM) vesicles. Samples obtained from these studies were analyzed by HPLC, liquid scintillation spectrophotometry, UV/VIS spectrophotometry, or confocal microscopy. The ability to predict sites of interactions based on pharmacokinetic modeling was evaluated. The effect of MDR modulators on hepatobiliary disposition of the model P-gp substrates, daunorubicin, etoposide, or doxorubicin, was determined in each model system by comparison of vehicle controls to modulator-treated groups. Doxorubicin biliary excretion was decreased after administration to the IPL in the presence of the MDR modulators, quinidine or GF120918. The formation of the primary metabolite, doxorubicinol, was saturable in hepatic S9 fractions and was not altered significantly by GF120918 or quinidine. Quinidine decreased the apparent K$sb{rm m}$ of doxorubicinol formation slightly, indicative of competitive inhibition. The rates of sinusoidal uptake and egress of ($sp3$H) -daunorubicin in isolated hepatocytes were rapid; sinusoidal translocation was not altered by GF120918 or verapamil. The majority of the doxorubicin dose administered to the IPL was sequestered in the nucleus, as determined by confocal microscopy; fluorescence intensity of doxorubicin was not altered by GF120918 or quinidine. Biliary excretion of glutathione disulfide (GSSG), a substrate of the multidrug resistance-associated protein (MRP), was decreased only modestly by GF120918. Uptake of ($sp{14}$C) -doxorubicin in cLPM vesicles was decreased by GF120918; however, data from these studies were variable. An alternative model for assessing biliary excretion of P-gp substrates may be primary hepatocytes cultured in a collagen sandwich configuration. GF120918 increased the intracellular accumulation of ($sp{14}$C) -doxorubicin in a pilot study in this system. Pharmacokinetic modeling of data obtained from the IPL studies indicated that doxorubicin biliary excretion was decreased primarily by interactions at the canalicular membrane. The ability to predict sites of interactions based on pharmacokinetic modeling was evaluated by comparing data obtained from individual in vitro systems to results generated from the pharmacokinetic modeling.