title: Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy.
abstract: A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79/V79-NTR(puro)) and murine (EMT6/EMT6-NTR(puro)) cell line pairs, and were compared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 microM) precluded the determination of IC(50) values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. m.hay@auckland.ac.nz
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hay
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
authors.forename: William A
authors.initials: WA
authors.lastname: Denny
mesh_terms: D000818:Animals; D002219:Carbamates / Q000138:chemical synthesis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D004355:Drug Stability; D015316:Genetic Therapy; D006801:Humans; D009574:Nitro Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*; D011355:Prodrugs / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D002219:Carbamates; D009574:Nitro Compounds; D011355:Prodrugs; D004317:Doxorubicin; D009601:Nitroreductases