Cephalosporin Derivatives of Doxorubicin as Prodrugs for Activation by Monoclonal Antibody-.beta.-Lactamase Conjugates

Abstract

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.

High-Yield Synthesis of Monomeric LMWP(CPP)-siRNA Covalent Conjugate for Effective Cytosolic Delivery of siRNA

“…The IC 50 values (i.e. the dose that causes 50% cell death) for siRNA are normally in the pica-molar range 12, comparing to the micro-molar range for conventional small anti-tumor agents such as doxorubicin 13, thus allowing these drugs to eradicate a tumor at significantly lower, by 6-8 orders of magnitudes, bioavailable concentrations at the target tissues. However, siRNAs are highly negatively charged (about -40 charges) large molecules (~13,300 Da), they cannot successfully diffuse across cell membrane to enter the cytosolic compartment where the RNA-induced silencing complex (RISC) machineries locate; because cell membranes are permeable to only small (<700 Da) and hydrophobic compounds 14-16.…”

A β-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy

“…This allows the design of prodrugs that are not converted by any human enzyme. Of particular interest is h-lactamase, which has been reported to activate a variety of prodrugs to release commonly used cancer drugs, such as doxorubicin (13), vinblastine (14), Taxol (15), paclitaxel (16), and melphalan (17). Lactamases are produced by bacteria in response to treatment with lactam antibiotics.…”

Developing bifunctional β‐lactamase molecules with built‐in target‐recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage‐display selection

“…TEM-1 β -lactamase efficiently cleaves the penicillins and some cephalosporins; however, extended-spectrum cephalosporins are poor substrates for this enzyme (Cantu et al ., 1997). P99 β -lactamase has been reported to show versatility and very high activity towards broad- and extended-spectrum cephalosporins and their prodrug conjugates, some of which have been used in preclinical ADEPT studies (Meyer et al ., 1993; Vrudhula et al ., 1993; Kerr et al ., 1995; Svensson et al ., 1995; Vrudhula et al ., 1995). The development of catalytically active P99 β -lactamase molecules that have a built-in target-recognizing site should be a very effective alternative to the antibody- β -lactamase conjugates in the enzyme prodrug therapy.…”