n-(4-glucuronyl-3-nitrobenzyloxycarbonyl)doxorubicin and Fibrosarcoma

When acted on by beta-glucuronidase (BG), HMR1826 is metabolized to doxorubicin. Use of this prodrug with adenoviral transfer of beta-glucuronidase (AdBG) is limited by the drug's inability to enter cells and intracellular retention of BG after transduction. We evaluated a system combining AdBG, transfer of the proapoptotic gene bax (AdBax) at a low multiplicity of infection, and HMR1826 administration.. Fibrosarcoma cells were treated with AdBG alone, AdBG plus HMR1826, AdBG followed by beta-galactosidase (AdLacZ) plus HMR1826, and AdBG followed by AdBax with no prodrug. In the experimental group, cells were transfected with AdBG, followed by AdBax plus HMR1826. Viability was measured 24 h after transfection and prodrug administration. Western blots for BG were performed on cell lysates and supernatants.. Minimal cellular killing was noted in the AdBG alone, AdBG plus HMR 1826, or AdBG:AdLacZ plus HMR 1826 groups, and Western blot did not demonstrate BG in the supernatant even though all AdBG-transfected cell lysates were positive. Cell killing was noted in the AdBG:AdBax group, but less than in the AdBG:AdBax plus HMR 1826 group (without prodrug versus with prodrug: 1:1 to 55.5% versus 75.9%, 5:1 to 10.0% versus 75.9%, 10:1 7.6% versus 49.0%, 20:1 4.6% versus 24.9%, P = 0.037). Western blot demonstrated BG in the supernatant of the AdBG:AdBax groups.. We have devised a novel enzyme prodrug method of killing tumor cells and engendering a bystander effect. AdBax leads to BG release from dying cells after AdBG transduction and conversion of HMR1826 to an active anthracycline.

Topics: Adenoviridae; Animals; Anthracyclines; Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Combined Modality Therapy; Doxorubicin; Drug Screening Assays, Antitumor; Fibrosarcoma; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glucuronates; Glucuronidase; Intracellular Membranes; Prodrugs; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Inbred F344