CHARACTERIZATION OF NOVEL ANTHRACYCLINE PRODRUGS ACTIVATED BY HUMAN BETA-GLUCURONIDASE FOR USE IN ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY

Antibody-directed enzyme prodrug therapy (ADEPT) alms at the specific activation of a prodrug by an enzyme-immuoconjugate localized in tumor tissue. The use of an enzyme of human origin is preferable in ADEPT b ecause it might not be immunogenic when administered to patients. In t he case of human beta-glucuronidase, prodrugs should be designed that are rapidly and completely activated at a neutral pH. Four new daunoru bicin glucuronides were synthesized by coupling a glucuronide group to daunorubicin via an aliphatic (GA1 and GB1) or an aromatic (GA3, GB6) carbamate spacer, to be released by electron shift (A-type) or by rin g closure (B-type). These prodrugs were characterized in vitro for the ir usefulness in ADEPT and were compared with the previously described prodrugs epirubicin-glucuronide and doxorubicin-nitrophenyl-glucuroni de. The four new prodrugs were stable in serum, hydrophilic when compa red to the lipophilic daunorubicin, and at least 20-fold less toxic th an the parent compound. The hydrolysis rate at clinically relevant enz yme and prodrug concentrations (1 mu g/mL human beta-glucuronidase, 10 0 mu M prodrug) at pH 6.8 were similar for GA3 (T-1/2 160 min) and hig her for GB6 (T-1/2 40 min) when compared to that of doxorubicin-nitrop henyl-glucuronide (T-1/2 170 min). Epirubicin-glucuronide, GA1, and GB 1 showed a low hydrolysis rate (T-1/2 > 400 min). GA1 and GA3, but not GB1 or GB6, were activated to the parent compound. Complete activatio n was confirmed in OVCAR-3 cells pretreated with a specific antibody-h uman beta-glucuronidase conjugate, where GA3 had similar antiprolifera tive effects to those of daunorubicin.