Preclinical and clinical studies of CYP gene-directed
enzyme prodrug therapy have been focused on anticancer
prodrugs activated by CYP2B
enzymes, which have low endogenous expression in human liver; however, the gene therapeutic potential of
CYP3A enzymes, which are highly expressed in human liver, remains unknown. This study investigated
methoxymorpholinyl doxorubicin (
MMDX;
nemorubicin), a novel CYP3A-activated anticancer
prodrug. Retroviral transfer of
CYP3A4 increased 9L
gliosarcoma cell chemosensitivity to
MMDX 120-fold (IC(50)=0.2 nM in 9L/3A4 cells). In CHO cells, overexpression of P450
reductase in combination with
CYP3A4 enhanced chemosensitivity to
MMDX, and to
ifosfamide, another
CYP3A4 prodrug, 11- to 23-fold compared with
CYP3A4 expression alone.
CYP3A4 expression and
MMDX chemosensitivity were increased in human lung (A549) and brain (U251)
tumor cells infected with replication-defective adenovirus encoding
CYP3A4.
Coinfection with Onyx-017, a replication-conditional adenovirus that coamplifies and coreplicates the Adeno-3A4 virus, led to large increases in
CYP3A4 RNA but only modest increases in
CYP3A4 protein and activity.
MMDX induced remarkable growth delay of 9L/3A4
tumors, but not the P450-deficient parental 9L
tumors, in immunodeficient mice administered low-dose
MMDX either intravenous or by direct intratumoral (i.t.) injection (60 microg kg(-1), every 7 days x 3). Notably, the i.t. route was substantially less toxic to the mouse host. No antitumor activity was observed with intraperitoneal
MMDX treatment, suggesting a substantial hepatic first pass effect, with activated
MMDX metabolites formed in the liver having poor access to the
tumor site. These studies demonstrate that human
CYP3A4 has strong potential for
MMDX prodrug-activation
therapy and suggest that endogenous
tumor cell expression of
CYP3A4, and not hepatic
CYP3A4 activity, is a key determinant of responsiveness to
MMDX therapy in
cancer patients in vivo.