Development and Characterization of P-glycoprotein specific Multidrug Resistance Modulators

Open Access
Author:
Lee, Brian D.
Graduate Program:
Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 21, 2003
Committee Members:
  • Charles D Smith, Committee Chair
  • Melvin Lee Billingsley, Committee Member
  • Robert G Levenson, Committee Member
  • Jong Kak Yun, Committee Member
Keywords:
  • P-glycoprotein
  • multidrug resistance
  • in vivo tumor model
  • actinomycin D transport
Abstract:
Multidrug resistance (MDR) is a phenomenon by which tumor cells develop reduced sensitivity to anticancer drugs, which often leads to the failure of cancer chemotherapy. A prominent mechanism of MDR is the overexpression of the multidrug efflux pump, P-glycoprotein (P-gp), which decreases the intracellular accumulation of many anticancer drugs leading to increased tumor growth. Intensive efforts are underway to develop clinically useful MDR modulators that inhibit the function of P-gp for use in combination with established anticancer drugs. To study the in vivo systemic effects of P-gp-specific inhibitors on reversing P-gp-mediated MDR, we developed a solid tumor model utilizing immunocompetent animals. Using in vitro cytotoxicity and drug accumulation assays, two transformed murine cell lines, JC and TIB-75, were found to demonstrate the P-gp-mediated MDR phenotype. In contrast, two similar lines did not express functional P-gp. Western blot analyses confirmed the expression of P-gp and the lack of expression of the closely related drug efflux protein MRP1 in the JC and TIB-75 cell lines. The JC cell line displayed excellent tumorgenicity and consistent growth kinetics when implanted into immunocompetent Balb/c mice. Animals treated with a combination of a known MDR modulator, cyclosporin A, and a cytotoxic drug, doxorubicin, exhibited significantly reduced tumor growth compared to untreated controls or animals treated with either cyclosporin A or doxorubicin alone. Therefore, this syngeneic solid tumor model provides a new in vivo system that can be used to evaluate the efficacy of P-gp inhibitors in an immunocompetent host and should provide improved prediction of the clinical utility of these compounds. In our search for improved MDR modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-gp without modulating multidrug resistance-related protein 1 (MRP1). These compounds were evaluated for their toxicity towards drug sensitive tumor cells (i.e. MCF-7, T24) and for their ability to antagonize P-gp-mediated drug resistant cells (i.e. NCI/ADR) and MRP1-mediated resistant cells (i.e. MCF-7/VP). The in vitro cytotoxicity and drug accumulation assays demonstrated that the dihydropyrroloquinoline analogs inhibit P-gp to varying degrees without any significant inhibition of MRP1. One of the analogs, PGP-4008, showed the highest level of P-gp inhibition (P-gp antagonism score ≈ 17) in vitro and was further evaluated in vivo. PGP-4008 inhibited tumor growth in the JC murine syngeneic P-gp-mediated MDR solid tumor model when given in combination with doxorubicin. The dose of PGP-4008 (100 mg/kg) and the route of administration (intraperitoneal) used in the tumor model resulted in rapid systemic absorption with plasma concentrations exceeding the in vitro effective dose (0.8 µg/ml) for 2 h after administration. PGP-4008 did not alter the plasma distribution of concomitantly administered anticancer drugs. Furthermore, signs of systemic toxicity were not seen with the P-gp selective modulator, PGP-4008, as seen in comparison with a non-selective inhibitor, cyclosporin A. Because of their transport-selectivity, these substituted dihydropyrroloquinolines may be more effective MDR modulators than non-specific modulators in a clinical setting. We also identified a series of quinoxalinones that could reverse MDR through the inhibition of P-gp, thereby increasing the efficacy of anticancer drugs in P-gp-expressing tumor cells. One compound in this series, 2-benzyloxy-3-methyl-quinoxaline (termed BMQ), displayed a surprising result when administered with actinomycin D. Actinomycin D is a peptide-containing antitumor drug isolated from Streptomyces that inhibits RNA synthesis by intercalating into DNA. It is currently thought that actinomycin D enters cells by passive diffusion through the plasma membrane. However, studies described herein demonstrate that actinomycin D uptake by cells is saturable, temperature-dependent and energy-dependent, suggesting an active transport mechanism. BMQ markedly increased the accumulation of actinomycin D within cells overexpressing P-gp, as well as a variety of cell lines that do not express this drug efflux protein. This increased accumulation of [3H]actinomycin D by BMQ was not due exclusively to the inhibition of efflux of the drug from the cell. Treating the cells with antimycin A, 2-deoxyglucose and sodium azide, which depleted intracellular levels of ATP by at least 75%, abolished the effects of BMQ on the cellular accumulation of actinomycin D. In addition, decreasing the temperature from 37˚C to 4˚C blocked the effects of BMQ. These studies suggest that the facilitation of actinomycin D transport by BMQ is not due to inhibition of efflux, and that actinomycin D transport is energy-dependent, temperature-dependent, and saturable. Overall, these studies suggest that a membrane transporter is responsible for the passage of actinomycin D across the cellular membrane, and that BMQ can potentiate this influx mechanism.