Potentiation of methoxymorpholinyl doxorubicin antitumor activity by P450 3A4 gene transfer
1Department of Biology, Division of Cell and Molecular Biology, Boston University, Boston, MA 02215, USA.
Abstract:
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.
Insights
This study explores using the highly expressed human CYP3A4 enzyme for cancer prodrug therapy with methoxymorpholinyl doxorubicin (MMDX). Gene transfer of CYP3A4 significantly enhanced MMDX effectiveness in tumor cells and reduced tumor growth in mice.
Area of Science:
- Biochemistry
- Pharmacology
- Gene Therapy
Background:
- Previous gene-directed enzyme prodrug therapy focused on low-expressing CYP2B enzymes.
- The potential of highly expressed human liver CYP3A enzymes for this therapy is unexplored.
- CYP3A enzymes are abundant in the human liver, offering a potential therapeutic target.
Purpose of the Study:
- To investigate the gene therapeutic potential of CYP3A enzymes for anticancer prodrug activation.
- To evaluate methoxymorpholinyl doxorubicin (MMDX), a novel CYP3A-activated prodrug.
- To assess the efficacy of MMDX in combination with CYP3A4 gene transfer in preclinical cancer models.
Main Methods:
- Retroviral and adenoviral gene transfer of CYP3A4 into cancer cells (gliosarcoma, lung, brain).
- Assessment of chemosensitivity to MMDX and ifosfamide in cells with varying CYP3A4 and P450 reductase expression.
- In vivo studies using immunodeficient mice with 9L gliosarcoma tumors treated with MMDX via intravenous, intratumoral, or intraperitoneal administration.
Main Results:
- CYP3A4 gene transfer increased MMDX chemosensitivity in gliosarcoma cells by 120-fold.
- Co-expression of P450 reductase with CYP3A4 further enhanced MMDX and ifosfamide sensitivity.
- Intratumoral MMDX administration significantly delayed tumor growth in mice with CYP3A4-expressing tumors, with lower toxicity compared to other routes.
- Hepatic first-pass metabolism of MMDX limited systemic efficacy.
Conclusions:
- Human CYP3A4 demonstrates significant potential for MMDX prodrug activation therapy.
- Endogenous CYP3A4 expression within tumor cells is a critical determinant of MMDX responsiveness.
- Targeting tumor cell-specific CYP3A4 expression may overcome limitations of hepatic first-pass metabolism.
Related Concept Videos
01:11Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase
01:27Pharmacogenetics of Phase II Enzymes: N-acetyltransferase, Thiopurine S-methyltransferase, UDP-glucuronosyltransferase
01:28Pharmacogenetics of Phase I Enzymes: Cytochrome P450 Isozymes