Received for publication July 27, 2004.
Revised August 30, 2004.
Accepted for publication September 29, 2004.

Anti-tumor Activity of Methoxymorpholinyl Doxorubicin: Potentiation by Cytochrome P450 3A Metabolism

Abstract

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (CYP)-activated anti-cancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, MMDX was activated by a panel of human liver microsomes with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC₅₀ values in growth inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells) and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that MMDX was activated by rat CYP3A1 and human CYP3A4 more efficiently than by rat CYP3A2, and that human CYPs 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in CHO cells following stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that over-expresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t_1/2 of ~5.5 hr at 37°C. MMDX was rapidly activated by CYP3A at low (~1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2 hr exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived and cell-permeable cytotoxic metabolite, and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.

Key words: Cytochrome P450, Mechanisms of cell killing/apoptosis, Pharmacokinetics, metabolism & activation

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