Aerobic 2- and 4-nitroreduction of CB 1954 by human liver.
5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is an anti-tumour prodrug which recently entered clinical trials in combination with Escherichia coli nitroreductase in a gene-directed enzyme prodrug therapy (GDEPT) context. A Phase I trial of the prodrug, however, revealed dose-limiting hepatotoxicity (transaminitis). The aim of this study was to find out whether the prodrug undergoes reductive metabolism in human liver to cytotoxic metabolites which may contribute to this clinical toxicity. CB 1954 (2.5-250 microM) was incubated with human liver preparations (2-8 mg/mL of S9, cytosolic or microsomal proteins) in the presence of NAD(P)H (1 mM). The NADH- and NADPH-dependent formation of both 2- and 4-nitroreduction products was demonstrated, with NADPH being the preferred cofactor, by HPLC and mass spectrometry. The major metabolite formed in all three human liver preparations was the 4-hydroxylamine, a potent DNA cross-linking cytotoxin. The 2-hydroxylamine and 2-amine metabolites were also detected, both of which have also been demonstrated to be highly cytotoxic. 2-Nitroreduction was far greater in S9 compared with cytosol and was not detected in microsomal preparations. Although 2- and 4-nitroreduction of CB 1954 was inhibited under hyperoxic conditions, substantial metabolism was observed under atmospheric oxygen levels. These studies demonstrate that human liver is capable of aerobic reductive bioactivation of CB 1954 to cytotoxic metabolites in vitro, possibly involving multiple enzymes, which may account for the clinical hepatotoxicity observed.
Tang
Magdalene Huen Yin
MH
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand. m.tang@auckland.ac.nz
Helsby
Nuala A
NA
Wilson
William R
WR
Tingle
Malcolm D
MD
D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors / Q000378:metabolism; D001388:Aziridines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002248:Carbon Monoxide / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D017321:Clinical Trials, Phase I as Topic; D003600:Cytosol / Q000201:enzymology; D003603:Cytotoxins / Q000378:metabolism; D001728:Dicumarol / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006898:Hydroxylamines / Q000009:adverse effects / Q000737:chemistry / Q000378:metabolism; D000860:Hypoxia; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D009243:NAD / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000187:drug effects / Q000378:metabolism; D009601:Nitroreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism
D000970:Antineoplastic Agents; D001388:Aziridines; D003603:Cytotoxins; D006898:Hydroxylamines; D011355:Prodrugs; D009243:NAD; D009249:NADP; C100099:tretazicar; D001728:Dicumarol; D002248:Carbon Monoxide; D009601:Nitroreductases