Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N.

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.

A cytochrome P450 2B6 meditated gene therapy strategy to enhance the effects of radiation or cyclophosphamide when combined with the bioreductive drug AQ4N.

BACKGROUND: AQ4N is metabolised in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Most solid tumours are known to contain regions of hypoxia whereas levels of CYPs have been found to vary considerably. Enhancement of CYP levels may be obtained using gene-directed enzyme prodrug therapy (GDEPT). We have therefore examined the potential of a CYP2B6-mediated GDEPT strategy to enhance the anti-tumour effect of the combination of AQ4N with radiation or cyclophosphamide (CPA). METHODS: In vitro and in vivo transient transfection of human CYP2B6 +/- CYP reductase (CYPRED) was investigated in RIF-1 mouse tumours. Efficacy in vitro was assessed using the alkaline comet assay (ACA). In vivo, the time to reach 4x the treatment volume (quadrupling time; VQT) was used as the end point. RESULTS: When CYP2B6 was transfected into RIF-1 cells and treated with AQ4N under hypoxic conditions there was a significant increase in DNA damage (measured by the ACA) compared with non-transfected cells. In vivo, a single intra-tumoural injection of a CYP2B6 vector construct significantly enhanced tumour growth delay in combination with AQ4N (100 mg/kg) and 10 Gy X-rays. AQ4N (100 mg/kg) and CPA (100 mg/kg) with CYP2B6 and CYPRED also enhanced tumour growth delay; this effect became significant when the schedule was repeated 14 days later (p = 0.0197). CONCLUSIONS: The results show the efficacy of a CYP2B6-mediated GDEPT strategy for bioreduction of AQ4N; this may offer an additional approach to target radiation- and chemo-resistant hypoxic tumours that should enhance overall tumour control.