Synthesis and in Vitro Efficacy of Transferrin Conjugates of the Anticancer Drug Chlorambucil

Beyer, Ulrich; Roth, Thomas; Schumacher, Peter; Maier, Gerhard; Unold, Anuschka; Frahm, A. W.; Fiebig, Heinz H.; Unger, Clemens; Kratz, Felix

doi:10.1021/jm9704661

Cited by 82 publications

(53 citation statements)

References 17 publications

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“…Our time-dependent stability studies with the active acid-sensitive conjugates Tf-Hyd 1 and Tf-Hyd 2 in 10% FCS and "cell-conditioned" cell culture medium rule out the possibility that their antiproliferative activity is due to a rapid hydrolysis of the hydrazone bond realized in the conjugates as only very small amounts of doxorubicin are released under these conditions during 24 h. The importance of the acidsensitivity of the linker between doxorubicin and the carrier protein for anticancer activity has been noted by Greenfield et al 32 and by our group for acid-sensitive daunorubicin and chlorambucil conjugates. 33, 34 Faulk has prepared transferrin conjugates in which doxorubicin is bound to transferrin through glutaraldehyde coupling, 15 the chemical link between transferrin and the amino sugar position of doxorubicin presumably being realized by an imine bond. From a chemical point of view this bond exhibits acid-sensitivity.…”

Section: Discussionmentioning

confidence: 99%

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

Kratz¹,

Beyer²,

Roth³

et al. 1998

Journal of Pharmaceutical Sciences

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149

123

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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.

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Section: Discussionmentioning

confidence: 99%

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

Kratz¹,

Beyer²,

Roth³

et al. 1998

Journal of Pharmaceutical Sciences

Self Cite

149

123

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“…And preliminary toxicity studies in mice showed that this conjugate can be administrated at higher doses compared with unbound chlorambucil. 186 Transferrin-mitomycin C (MMC), the chemotherapeutic DNA crosslinking agent, has been demonstrated to be a useful hybrid as a receptor-mediated targeting system. [187][188][189] The Tf-MMC conjugate bound and was internalized into the human hepatoma cell line HepG2 cell, normal cultures rat hepatocyte, human leukemia cell line HL60 cells and Sarcoma 180 cells.…”

Section: A Transferrin Conjugates In Drug Deliverymentioning

confidence: 99%

Transferrin/transferrin receptor‐mediated drug delivery

Qian

2002

Medicinal Research Reviews

410

247

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Since transferrin was discovered more than half a century ago, a considerable effort has been made towards understanding tranferrin-mediated iron uptake. However, it was not until recently with the identification and characterization of several new genes related to iron homeostasis, such as the hemochromatosis protein HFE and the iron transporter DMT1, that our knowledge has been advanced dramatically. A major pathway for cellular iron uptake is through internalization of the complex of iron-bound transferrin and the transferrin receptor, which is negatively modulated by HFE, a protein related to hereditary hemochromatosis. Iron is released from transferrin as the result of the acidic pH in endosome and then is transported to the cytosol by DMT1. The iron is then utilized as a cofactor by heme and ribonucleotide reductase or stored in ferritin. Apart from iron, many other metal ions of therapeutic and diagnostic interests can also bind to transferrin at the iron sites and their transferrin complexes can be recognized by many cells. Therefore, transferrin has been thought as a "delivery system" for many beneficial and harmful metal ions into the cells. Transferrin has also be widely applied as a targeting ligand in the active targeting of anticancer agents, proteins, and genes to primary proliferating malignant cells that overexpress transferrin receptors. This is achieved by conjugation of transferrin with drugs, proteins, hybride systems with marcomolecules and as liposomal-coated systems. Conjugates of anticancer drugs with transferrin can significantly improve the selectivity and toxicity and overcome drug resistance, thereby leading to a better treatment. The coupling of DNA to transferrin via a polycation such as polylysine or via cationic liposomes can target and transfer of the extrogenous DNA particularly into proliferating cells through receptor-mediated endocytosis. These kinds of non-viral vectors are potential alternatives to viral vectors for gene therapy, if the transfection efficiency can be improved. Moreover, transferrin receptors have shown potentials in delivery of therapeutic drugs or genes into the brain across blood-brain barrier.

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“…The Kratz group has also used transferrin [124] and albumin [125] as carriers for the targeting of the anticancer drug chlorambucil (Fig. 45).…”

Section: -Hydrazone Linkagesmentioning

confidence: 99%

Reversible Covalent Chemistry in Drug Delivery

West

Otto

2005

CDDT

150

109

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The targeting of drugs specifically to their sites of action is an important strategy for increasing drug efficacy. Chemists have come up with many elegant schemes that aim to convert drugs into magic bullets. This review focuses on the chemistry that underlies these schemes, with particular emphasis on two types of cleavable covalent bonds that are frequently used to link drugs to their various carriers: disulfide bonds and hydrazone bonds. These linkages have been used to release drugs under specific conditions; in the case of disulfides, cleavage is triggered by the mildly reducing environment found in intracellular fluids, and in the case of hydrazones, the acidic conditions that prevail in endosomes cause release of the drug. The applications of these chemistries in drug delivery are reviewed.

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Synthesis and in Vitro Efficacy of Transferrin Conjugates of the Anticancer Drug Chlorambucil

Cited by 82 publications

References 17 publications

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

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

Transferrin/transferrin receptor‐mediated drug delivery

Reversible Covalent Chemistry in Drug Delivery

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