Transferrin Conjugates of Doxorubicin: Synthesis, Characterization, Cellular Uptake, and in Vitro Efficacy||

Abstract

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing suitable carrier proteins. Thus, thiolated human serum transferrin was conjugated 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 benzoyl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive transferrin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable to that of the free drug (employing the BrdU (5-bromo-2'-deoxyuridine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximately 0.1-1 mM), whereas conjugates with the amide derivatives showed no activity. Furthermore, antiproliferative activity of the most active transferrin conjugate (i.e. the conjugate containing a benzoyl hydrazone link) was demonstrated in the LXFL 529 lung carcinoma cell line employing a sulforhodamine B assay. In contrast to in vitro studies in tumor cells, cell culture experiments performed with human endothelial cells (HUVEC) showed that the acid-sensitive transferrin conjugates of doxorubicin were significantly less active than free doxorubicin (IC50 values approximately 10-40 higher by the BrdU incorporation assay), indicating selectivity of the doxorubicin-transferrin conjugates for tumor cells. Fluorescence microscopy studies in the MDA-MB-468 breast cancer cell showed that free doxorubicin accumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily in the cytoplasm. The differences 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 relationship between stability, cellular uptake, and cytotoxicity of the conjugates is discussed.

Receptor Mediated Endocytosis and Cytotoxicity of Transferrin-Mitomycin C Conjugate in the HepG2 Cell and Primary Cultured Rat Hepatocyte.

“…17) Transferrin receptors were found to be more widely distributed on the tumor tissues than the normal tissues. 18) Transferrin can be exploited as a carrier of drugs, such as neocarzinostatin, 19) adriamycin, 20,21) and cisplatin. 22,23) We have previously described the covalent attachment of MMC to give TF-MMC, which aided the delivery of MMC to the tumor cells.…”

Schiff bases: An emerging potent class of pharmaceuticals

“…Hydrazones play an important role in improving the antitumor selectivity and toxicity profile of antitumor agents by forming drug carrier systems employing suitable carrier proteins (Kratz et al, 1998). They also have anti-inflammatory and analgesic activity equal or close to that of aspirin (Sondhi et al, 2006).…”

Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells

“…Transferrin has several adventages over other drug carriers. It is not an immunogenic, but is a commercially available protein [6]. The transferrin receptor (known as CD71), a type II transmembrane glycoprotein found on the surface of cells, is a protein involved in iron uptake and the regulation of cell growth [7].…”

“…The transferrin receptor (known as CD71), a type II transmembrane glycoprotein found on the surface of cells, is a protein involved in iron uptake and the regulation of cell growth [7]. The expression of transferrin receptors is significantly upregulated in a variety of malignant cells [7], so the conjugation of anticancer drugs with transferrin might aid in delivering the therapeutics directly to neoplastic cells (via receptor-mediated endocytosis) with reduced injury to normal cells [4][5][6]. A number of studies have indicated that the conjugation of doxorubicin with the serum protein transferrin significantly increased the cytotoxic effect against tumour cells both in vitro [6,8,9] and in vivo [10].…”

“…The expression of transferrin receptors is significantly upregulated in a variety of malignant cells [7], so the conjugation of anticancer drugs with transferrin might aid in delivering the therapeutics directly to neoplastic cells (via receptor-mediated endocytosis) with reduced injury to normal cells [4][5][6]. A number of studies have indicated that the conjugation of doxorubicin with the serum protein transferrin significantly increased the cytotoxic effect against tumour cells both in vitro [6,8,9] and in vivo [10]. DOX-TRF is toxic against a variety of human cell lines including HL60-promyelocytic leukemia [11], K562-erythroleukemia [11], Hep2-liver carcinoma [10], HeLa-cervical adenocarcinoma [12], SBC-3-small cell lung cancer [13] and cell lines resistant to DOX: KB-oral carcinoma [14], MCF-7-breast cancer [9], and SBC-3/ADM [13].…”