The anthraquinone di-N-oxide AQ4N is a prodrug designed to be
excluded from cell nuclei until metabolised in hypoxic tumour regions to
AQ4, a DNA binder and potent inhibitor of topoisomerase II. The
antitumour effects of AQ4N in rodent neoplasms are well characterised
but the identity of enzymes responsible for the metabolism are unknown.
The aims of the present work were to identify Cytochrome P450 (CYP)
enzymes responsible for AQ4N metabolism in rat and human tissue and
to conduct a preliminary investigation into the in vivo metabolism of
AQ4N in tumour bearing rodents.
AQ4N was found to undergo a two electron reduction to the mono-Noxide
AQM followed by a subsequent two electron reduction to cytotoxic
AQ4. The process occurred in the microsomes of rat and human liver,
was cofactor dependent and was inhibited by air. In rats, CYPs 2B and
2E were found to anaerobically metabolise both AQ4N and AQM.
Kinetically, AQ4N metabolism conformed to a Michaelis-Menten model
whereas the metabolism of AQM was better described by a sigmoidal
relationship. In addition, both semi purified Cytochrome P450 reductase
(CPR) and purified Nitric oxide synthase (NOS) were both able to
anaerobically metabolise AQ4N. Both enzymes required NADPH and
CPR mediated metabolism was dependent on the presence of exogenous
haem.
In humans, the anaerobic metabolism of both AQ4N and AQM correlated
with CYP 3A activity and not with the activities of CYP 1 AI 2C and 2D.
AQM metabolism correlated also with the activity of CYP 2A. The
involvement of CYP 3A was confirmed by the use of CYP specific
inhibitors and by the use of cDNA transfected cell microsomes. Human
kidney and colonic tumours were found to anaerobically metabolise
AQ4N and tumour metabolism was inhibited by the CYP inhibitor carbon
monoxide (CO). Finally, the in vivo metabolism of AQ4N was studied in
C3H tumour bearing mice. Metabolites of AQ4N were found in all tissues
studied but the AQ4! AQ4N ratio was highest in the tumours.
Collectively, these findings have identified the enzymes responsible for
the metabolism of AQ4N and its mono-N-oxide. Differences exist
between the CYP isoforms responsible for metabolism in rodents and in
man, in humans, CYP 3A enzymes predominantly metabolise AQ4N and
this subfamily of CYP are known to be well expressed in a broad
spectrum of human cancers. With this in mind, AQ4N based therapy
should be considered as a rational treatment regime for patients bearing
solid tumour burdens