Introduction. Our previous in vitro (1-3) studies demonstrated a higher anticancer activity of HPMA copolymer-bound doxorubicin (P(GFLG)-DOX) when compared to free DOX, especially in DOX resistant human ovarian carcinoma cells. Being internalized in membrane-limited organelles, HPMA copolymer bound DOX was protected from the cellular drug efflux and detoxification mechanisms, and preserved its activity during intracellular trafficking. As the result it activated apoptosis and necrosis signaling pathways more significantly than free DOX, and simultaneously inhibited cellular defensive systems. In contrast, free DOX activated the defense mechanisms (3). The differences in the cytotoxic mechanisms of free DOX and P(GFLG)-DOX were also confirmed in an animal model of solid tumor (4). However, the observed in vivo antitumor activity of HPMA copolymer-bound DOX, especially in case of the DOX resistant tumors was significantly higher than it could be predicted from in vitro experiments. We hypothesize that this phenomenon was caused by the amplification of the EPR effect for macromolecules carrying cytotoxic drugs. The aim of the present study was to verify this hypothesis and investigate the peculiarities and mechanisms of the EPR effect for macromolecules coupled either with a cytotoxic drug (DOX) or with non-toxic drug models (Texas Red, FITC) in an animal model of solid tumor resistant to doxorubicin (subcutaneous A2780/AD human ovarian carcinoma xenografts).
Results and Discussion. The EPR effect was significantly different for macromolecules, which contained cytotoxic drug when compared to macromolecules without drug. The cytotoxicity of macromolecular therapeutics amplified the EPR effect, lead to a more homogenous distribution of the drug within the tumor, increased the average drug concentration in the tumor and, consequently, augmented the cytotoxicity of the conjugate. The accumulation of free DOX lead to the over-expression of the VEGF gene and increase in the vascular permeability. This enhanced the drug accumulation in the same location resulting in a highly inhomogeneous drug distribution within the tumor and ultimately in a decreased antitumor effect. In contrast, the accumulation of a significant amount of P(GFLG)-DOX in tumor down-regulated the VEGF gene and decreased vascular permeability at the accumulation site. This prevented additional drug accumulation in already dead necrotic tissue, resulting in a more uniform distribution and enhanced antitumor activity of HPMA copolymer-bound DOX.
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2. T. Minko, P. Kopecková, and J. Kopecek. Chronic exposure to HPMA copolymer-bound adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell line. J. Controlled Rel. 59, 133-148 (1999).
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Acknowledgements. We thank Dr. A Suarato (Pharmacia-Upjohn, Milano, Italy) for the generous gift of doxorubicin. This research was supported in part by NIH grant CA 51578 from the National Cancer Institute.
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