TRANSFERRIN CONJUGATES OF DOXORUBICIN - SYNTHESIS, CHARACTERIZATION, CELLULAR UPTAKE, AND IN-VITRO EFFICACY

One strategy for improving the antitumor selectivity and toxicity prof ile of antitumor agents is to design drug carrier systems employing su itable carrier proteins. Thus, thiolated human serum transferrin was c onjugated with four maleimide derivatives of doxorubicin that differed in the stability of the chemical link between drug and spacer. Of the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzo yl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide o r 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto posi tion through a benzoyl hydrazone or phenylacetyl hydrazone bond. The a cid-sensitive transferrin conjugates prepared with the carboxylic hydr azone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line compara ble to that of the free drug (employing the BrdU (5-bromo-2'-deoxyurid ine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximate to 0.1-1 mM), whereas conjugates with t he amide derivatives showed no activity. Furthermore, antiproliferativ e activity of the most active transferrin conjugate (i.e, the conjugat e containing a benzoyl hydrazone link) was demonstrated in the LXFL 52 9 lung carcinoma cell line employing a sulforhodamine B assay. In cont rast to in vitro studies in tumor cells, cell culture experiments perf ormed with human endothelial cells (HUVEC) showed that the acid-sensit ive transferrin conjugates of doxorubicin were significantly less acti ve than free doxorubicin (IC50 values approximately 10-40 higher by th e BrdU incorporation assay), indicating selectivity of the doxorubicin -transferrin conjugates for tumor cells. Fluorescence microscopy studi es in the MDA-MB-468 breast cancer cell showed that free doxorubicin a ccumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily in the cytoplasm. The differe nces in the intracellular distribution between transferrin-doxorubicin conjugates and doxorubicin were confirmed by laser scanning confocal microscopy in LXFL 529 cells after a 24 h incubation that revealed an uptake and mode of action other than intercalation with DNA. The relat ionship between stability, cellular uptake, and cytotoxicity of the co njugates is discussed.