Transduction of
tumor cells with a
cyclophosphamide (CPA)-activating
cytochrome P-450 (P450) gene provides the capacity for localized
prodrug activation and greatly sensitizes solid
tumors to CPA treatment in vivo. The therapeutic impact of this P450-based cancer gene
therapy strategy can be substantially enhanced by cotransduction of P450
reductase, a rate-limiting component of P450-dependent intratumoral CPA activation. The present study examined the possibility of further improving P450/P450
reductase-based gene therapy by using a novel schedule of CPA administration, involving repeated CPA injection every 6 days and previously shown to have an antiangiogenic component. 9L
gliosarcoma cells transduced with the CPA-activating
enzyme couple P450 2B6/P450
reductase and grown s.c. in immunodeficient severe combined immunodeficient (scid) mice were repeatedly challenged with 140 mg/kg CPA every 6 days. Full
tumor regression leading to eradication of six of eight
tumors was observed when the
tumor size at the time of initial
drug treatment was approximately 400 mm(3) (approximately 1.5% of
body weight). Little or no overt toxicity of the repeated CPA treatment regimen was observed. The same CPA schedule was much less effective in inducing regression of 9L
tumors that were not transduced with P450/P450
reductase. Repeated CPA treatment of mice bearing large, late-stage P450/P450
reductase-transduced
tumors (approximately 9-16% of
body weight) resulted in major (> or =95%) regression in 15 of 16
tumors, with
tumor eradication observed in 2 cases. Although CPA resistance was found to emerge in the population of P450/P450
reductase-transduced
tumors, this resistance primarily involved a loss of expression of the transduced P450 and/or P450
reductase gene, rather than development of intrinsic cellular resistance to the activated form of CPA. These findings demonstrate that repeated CPA treatment on a 6 day schedule can be highly effective when combined with P450/P450
reductase gene therapy and suggest that repeated transduction of
tumors with
prodrug-activation genes may be necessary to achieve
tumor eradication and a sustained therapeutic response.