The synthesis of a series of cephalosporin doxorubicin derivatives that differ with respect to the substituent at position 7 of the cephem nucleus is described. These compounds are designed as prodrugs of doxorubicin for activation by monoclonal antibody-beta-lactamase conjugates. The key step in the synthesis of this series of compounds involves the use of the phenylacetamido group as an enzymatically removable protecting group for the 7-amino group on the cephem. In vitro cytotoxicity assays with H2981 lung adenocarcinoma cells revealed that cephalosporin doxorubicin derivatives were all less toxic than the released drug. Prodrugs containing negatively charged groups in the side chain, such as the delta-carboxybutanamido derivative 4 and the alpha-sulfophenylacetyl derivative 5, displayed the least cytotoxic activity and were 46- and 26-fold less toxic than doxorubicin, respectively. The efficiency of activation of all the prodrugs was evaluated in cytotoxicity assays on H2981 cells with the beta-lactamases from Enterobacter cloacae P99, Escherichia coli TEM-1, and Bacillus cereus (type II). In general, the E. cloacae enzyme was found to most rapidly activate the majority of these prodrugs. Phenylacetamido prodrug 2 and delta-carboxybutanamido prodrug 4 were both activated in an immunospecific manner by L6-E. cloacae beta-lactamase, a monoclonal antibody conjugate that binds to receptors on H2981 lung adenocarcinoma cells.
7-Aminocephalosporin doxorubicin (AC-Dox) was condensed with monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (5 kDa) or with a branched form of poly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (10 kDa), forming M-PEG-AC-Dox and B-PEG-AC-Dox, respectively. These polymer drug derivatives were designed such that doxorubicin would be released upon Enterobacter cloacae beta-lactamase (bL)-catalyzed hydrolysis. Both M-PEG-AC-Dox (IC50 = 80 microM) and B-PEG-AC-Dox (IC50 = 8 microM) were less toxic to H2981 human lung adenocarcinoma cells than doxorubicin (IC50 = 0.1-0.2 microM) and could be activated in an immunologically specific manner by L6-bL, a monoclonal antibody-bL conjugate that bound to H2981 cell surface antigens. In addition, the polymers were relatively stable in mouse plasma (< 26% hydrolysis after 24 h at 37 degrees C) and were less toxic to mice (maximum tolerated dose > 52 mumol/kg) than doxorubicin (maximum tolerated dose = 13.8 mumol/kg). Pharmacokientic studies were performed in mice bearing subcutaneous 3677 melanoma tumors. B-PEG-AC-Dox cleared from the blood more slowly than M-PEG-AC-Dox and was retained to a 2.1-fold greater extent in human 3677 melanoma tumor xenografts over a 4 h period. The intratumoral concentrations of both polymers far exceeded that of doxorubicin. Thus, the PEG-AC-Dox polymers offer the possibility of generating large intratumoral doxorubicin concentrations owing to their reduced toxicities, the amounts that accumulate in tumors, and the fact that doxorubicin is released upon beta-lactam ring hydrolysis.
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