Development of Nano-Liposomal Formulations of Epidermal Growth Factor Receptor Inhibitors and their Pharmacological Interactions on Drug-Sensitive and Drug-Resistant Cancer Cell Lines
Trummer, Brian J.
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A rapidly expanding understanding of molecular derangements in cancer cell function has led to the development of selective, targeted chemotherapeutic agents. Growth factor signal transduction networks are frequently activated in an aberrant fashion, particularly through the activity of receptor tyrosine kinases (RTK). This has spurred an intensive effort to develop receptor tyrosine kinase inhibitors (RTKI) that are targeted to specific receptors, or receptor subfamilies. Chapter 1 reviews the pharmacology, preclinical, and clinical aspects of RTKIs that target the epidermal growth factor receptor (EGFR). EGFR inhibitors demonstrate significant success at inhibiting phosphorylation-based signaling pathways that promote cancer cell proliferation. Additionally RTKIs have physicochemical and structural characteristics that enable them to function as inhibitors of multi-drug resistance transport proteins. Thus EGFR inhibitors and other RTKIs have both on-target and off-target activities that could be beneficial in cancer therapy. However, these agents exert a number of side effects, some of which arise from their hydrophobic nature and large in vivo volume of distribution. Side effects of the EGFR inhibitor gefitinib include skin rash, severe myelotoxicity when combined with certain chemotherapeutic agents, and impairment of the blood brain barrier to xenobiotics. Weighing the preclinical and clinical observations with the EGFR inhibitors, we developed the primary overall hypothesis of this research: that drug-carrier formulations of RTKIs such as the EGFR inhibitors could be developed based on nanoparticulate liposomal carriers. Theoretically, this carrier strategy would ameliorate toxicity and improve the biodistribution and tumor selectivity of these agents. We hypothesized specifically that liposomal formulations could shift the biodistribution of EGFR inhibitors such as gefitinib away from skin, bone marrow, and the blood brain barrier, and toward solid tumors, due to leaky tumor vasculature and the resulting Enhanced Permeability and Retention (EPR) phenomenon. In Chapter 2 we report that both gefitinib and the structurally similar EGFR inhibitor erlotinib display environment-dependent fluorescence properties. Peak excitation was 345 nm, and the emission peak ranged from 365 to 476 nm, depending upon the polarity of the environment and physical state of the drug. The fluorescence was negligible in aqueous solution, but intense in organic solvents or membrane bilayers. The environment-sensitive fluorescence properties of these drugs enabled rapid evaluation of numerous parameters affecting liposomal drug incorporation and performance. Up to 4-6 mol% of gefitinib could be incorporated in the liposome bilayer, based upon hydrophobic interactions with membrane bilayers. In contrast, 40-60 mol% could be loaded into the aqueous core of pre-formed liposomes at high efficiency, using a remote loading procedure. A stable formulation consisting of distearoylphosphatidylcholine: polyethylene glycol-distereoylphosphatidylethanolamine: cholesterol (DSPC:PEGDSPE:Chol, 9:1:5 mol:mol:mol) and containing drug at 50-60 mol% gefitinib (L-GEF) showed minimal leakage in serum-containing medium over 24 h at 37°C, which should be sufficient to improve biodistribution in vivo. Chapter 3 investigated the pharmacological activity of liposome-encapsulated gefitinib, alone and in combination with several prevalent anticancer agents. Experiments with MCF7 breast cancer cell lines demonstrated that liposome encapsulated gefitinib formulation (L-GEF) had a 2-fold higher IC 50 (concentration of drug resulting in half-maximal growth inhibition) than free gefitinib. Lower in vitro potency would be consistent with delayed drug release from the carrier. Therapeutic effects were investigated in combination with the cytotoxic agents paclitaxel and doxorubicin. The drug-resistant MCF7R cell line was 23-fold more resistant to paclitaxel than the parental, drug-sensitive MCF7S cell line, and MCF7R was 12-fold more resistant than MCF7S to doxorubicin. A concentration of 3 μM gefitinib, which had no discernible effect upon MCF7R cell proliferation, reduced resistance to paclitaxel to 6-fold, relative to the parental MCF7S cell line, and reduced resistance to doxorubicin to 8-fold, compared to MCF7S. The cytostatic effect of a wider range of gefitinib:paclitaxel ratios was evaluated in order to permit quantitative analysis of the mechanisms of drug interaction, using response surface models. LGEF in combination with paclitaxel was found to be synergistic in MCF7R multidrug-resistant cells, with a psi value of 0.29. Free GEF combined with paclitaxel was also synergistic, with a psi value of 0.40. The confidence intervals for both psi values were below 1, indicating statistical significance for synergy. The fluorescence of gefitinib and doxorubicin permitted visualization of cellular uptake and intracellular distribution of these drugs in multicellular spheroids of rat 9L gliosarcoma cells, which recapitulate some of the barrier functions to drug distribution within tumors. Gefitinib altered the intracellular distribution of doxorubicin, and overcame the nuclear exclusion of doxorubicin in these drug-resistant cells. Chapter 4 discusses the significance of the findings relating both to the formulations produced and to their pharmacology, and concludes that nanoparticulate formulations of gefitinib have the properties necessary to alter drug biodistribution and pharmacokinetics. Such formulations have the potential to provide an effective means to enhance EGFR-inhibitor efficacy in combination chemotherapy.