Identification of novel enzyme–prodrug combinations for use in cytochrome P450-based gene therapy for cancer

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Abstract

Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene–CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated ∼250-fold by liver microsomes from dexamethasone-induced rats (IC50 (MMDX) ∼0.1 nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug–CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.

Section snippets

Chemicals

CPA (Cat#C0768), dacarbazine (Cat#D2390), NADPH (Cat#N6505), and troleandomycin (TAO) (Cat#T6514) were purchased from Sigma–Aldrich (St. Louis, MO). IFA was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). MMDX-HCl (PNU-152243) was a gift from Pharmacia & Upjohn (Milan, Italy). Procarbazine (Cat#P6858) and tamoxifen (Cat#T0250) were purchased from LKT Laboratories (St. Paul, MN). Ketoconazole (Cat#30.152.82) was obtained from Research Diagnostics

Evaluation of microsome–prodrug activation using a growth inhibition assay

Evaluation of microsomal CYP–prodrug combinations for potential use in prodrug-activation-based cancer gene therapy was carried out using 9L rat gliosarcoma cells grown in 96-well tissue culture plates. In an initial study, 9L cells were seeded at 0, 250, 450, and 1000 cells/well and allowed to grow for 3, 4, 5, or 6 days, followed by staining with crystal violet. Cell growth was essentially linear over all time points for all cell densities (Fig. 2). The A595 of the stained 1000-cell/well

Discussion

A new generation of treatment strategies is currently being developed to improve the efficacy and reduce the side effects of cancer chemotherapy. One such strategy is cytochrome P450-based prodrug activation gene therapy for cancer treatment [8]. By the delivery of a prodrug-activating CYP gene directly to a tumor, the tumor acquires the capacity for prodrug activation, enabling the localized production of cytotoxic CYP-activated prodrug metabolites. This approach may decrease systemic exposure

Acknowledgements

Supported in part by N.I.H. Grant CA49248 (to D.J.W.). This article is dedicated to Dr. Ronald W. Estabrook in appreciation of the generous support and encouragement that he has extended to young investigators in the field of cytochrome P450 for the past four decades.

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    Present address: GlaxoSmithKline, Research Triangle Park, NC 27502, USA.

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