The in vitro cytotoxicity of the prodrugs was assessed
The in vitro cytotoxicity of the prodrugs was assessed in the LS174T human colon cell line (). The results were found to reflect the stability of the prodrugs, with the unstable urea compounds ( and ) giving relatively poor cytotoxicity differentials between prodrugs and parents (i.e., approximately 2:1 for both after 1h exposure). However, the carbamate prodrugs ( and ) gave much better cytotoxicity differentials between the parent PBDs and prodrugs, with differentials of >7.1 and >15.4, respectively, for 1h exposure rising to >14.3 and >44.7 for continuous exposure. All of the compounds examined were between 5- and 27-times more cytotoxic on continuous exposure compared to 1h incubation, presumably due to both increased exposure time and hydrolysis of the prodrugs.
Finally, the carbamate monomer and dimer prodrugs and were incubated with 1U of CPG2 and both compounds were shown to be good substrates (). The monomer prodrug was completely converted into the cytotoxic parent PBD within 50min with no apparent chemical degradation observed in the assay buffer (100mM Tris–HCl/260μM ZnCl, pH 7.3) in the absence of CPG2 over the same time period. Similarly, the dimer prodrug was completely converted into the cytotoxic PBD dimer in 75min with no chemical degradation in the absence of CPG2. Interestingly, in the latter case, it was possible to observe the formation of an intermediate (○, Right-hand panel, ) thought to be the mono-protected PBD dimer with only one glutamic mitoxantrone residue cleaved. As anticipated, this intermediate converted into the fully de-protected PBD dimer during the course of the experiment. To confirm the potential value of these prodrugs in ADEPT therapy, the monomer () and dimer () prodrugs were incubated with CPG2 in the presence of LS174T cells for 1h. This reduced the IC values for and by 7.6- and 9.0-fold, respectively, thus confirming their transformation into cytotoxic species.
In conclusion, these results demonstrate that it is possible to synthesize N10-protected PBD prodrugs suitable for CPG2-based ADEPT therapy. The prodrugs are, in the case of the more stable carbamate derivatives and , significantly less cytotoxic than the parent compounds, and are good substrates for CPG2, being rapidly converted into the cytotoxic monomer and dimer parent PBDs upon exposure to enzyme. As the released PBD dimer () and related analogues (e.g., SJG-136, ) are known to produce DNA interstrand cross-links that are difficult for cancer cells to repair,, the potential exists to develop second-generation ADEPT prodrugs that are less prone to the development of resistance compared to mustard-based agents. Further work is underway to study the behaviour of the prodrugs in human tumour xenografts and to improve the stability of the prodrugs and the cytotoxicity of the released PBDs.
Systemic cancer therapy is limited by a lack of tumor selectivity, which leads to harmful side effects in normal tissues, and by drug resistance. Antibody-directed enzyme prodrug therapy (ADEPT) is designed to overcome both problems., , , ADEPT is a two step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at the tumor site or its metastases. In the first step, a tumor selective antibody chemically linked to an enzyme (antibody–enzyme conjugate) is administered and allowed to fix itself on the tumor site. In the second step after a suitable time, to allow the clearance of the antibody–enzyme conjugate from other tissues, the process is followed by the administration of a relatively nontoxic prodrug. This prodrug will be enzymatically converted into the active drug only at the tumor site and the usually nonspecific cytotoxicity will be minimized. The active drug is generated extracellularly with a higher concentration and diffuses itself throughout the tumor mass, killing not only cells expressing tumor antigen but also neighboring antigen-negative tumor cells. Thus selectivity is achieved by the tumor specificity of the antibody and by delaying prodrug administration until there is a large differential between tumor and normal tissue enzyme levels. Drug resistance can be overcome by generating high levels of an alkylating agent in the tumor, and this is achieved through the capacity of each enzyme to convert many molecules of prodrug into drug. Such a complex system has already been applied in clinical trials and in the last few years, a number of new monoclonal antibodies-based drugs have been clinically approved (Rituxan, Herceptin, Erbitux, Avastin and Mylotarg), and numerous other mAb-based agents have also progressed to phase II/phase III clinical trials in cancer.,