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.

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.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A17CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P ¼ 0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

8556 Background: The aim of this study was to profile cytochrome CYP1 family (CYP1A1/1A2, and CYP1B1) mono-oxygenase enzymes during the malignant progression of primary melanoma and metastatic disease. Methods: Tissue microarrays of primary (n = 75), and metastatic (n = 104) melanoma were constructed with the patient demographics: (1) primary melanoma; age 22 to 93 (median 59); sex M/F 36/44; Breslow thickness 0.4 to 15 mm (median 2.5 mm); ulceration 25/80, and (2) metastatic melanoma; age 26 to 92 (median 60 mm); sex M/F 54/49; ulceration 30/104; number of nodes 1 to 15 (median 2); extra-capsular spread 20/95. CYP1 protein was detected by IHC using validated selective poly- and monoclonal antibodies. Vector SG (grey) stain for CYP1 was used with nuclear fast red counterstain to aid spectral resolution from background melanin. Staining intensity was scored visually (negative 0, weak 1, moderate 2, strong 3) and using SIM at every pixel of a captured image of each melanoma core. Reference spectra of individual chromophores were used to spectrally ‘un-mix’ CYP1 staining before the mean normalised absorbance intensity was determined. Grading was by the 2002 AJCC classification system: primary stage I n = 27 (1A 8, 1B 19), and stage II n = 48 (2A 22, 2B 16, 2C 10), lymph node metastasis stage III n = 98 (3B 53, 3C 45), visceral metastasis stage IV n = 6. Normal skin (n = 27), benign naevi (n = 14), and dysplastic naevi (n = 21) were also included. Results: CYP1B1 was not in normal skin but was over-expressed in both primary and metastatic melanoma (visual: 71% & 65%, SIM: 91% & 83%). Primary melanoma (stage I & II) was significantly greater (p = 0.004) than metastasis (stage III & IV). CYP1B1 did not correlate with ulceration or Breslow thickness but did correlate with N stage lymph node metastasis (p = 0.005). CYP1B1 expression in dysplastic naevi indicated up-regulation at an early stage of melanoma progression. CYP1A1/1A2 was not expressed in normal skin nor primary/metastatic melanoma. Conclusions: CYP1B1 protein expression is maintained with advancing AJCC stage from primary through to visceral metastasis. Future work will seek to correlate protein expression with functionality with a view to exploiting CYP1B1 in the enzyme/prodrug therapy of malignant melanoma. No significant financial relationships to disclose.