The anti-tumour effect of AQ4N and its' enhancement using a Gene Therapy Strategy

Abstract

AQ4N is a bioreductive drug, whose mechanism of activation requires hypoxia and CYP P450's. It has recently entered Phase I clinical trials in combination with radiation. The first aim of this study was to develop a method of identifying tumours that have the necessary characteristics for the metabolism of AQ4N and which could be adopted as a predictive assay for use in the clinical trial. The alkaline comet assay was used to identify DNA damage caused by AQ4, the cytotoxic metabolite of AQ4N reduction. This was carried out in whole tumours and fine needle aspirates from mice at a range of time points after AQ4N treatment. A very significant difference in DNA damage was found between treated and untreated mice using both techniques (p = 0.0001). Therefore, DNA damage measured by the comet assay can be used as a surrogate endpoint for AQ4N metabolism identifying tumours, which have both the hypoxia and CYP P450 compliment necessary for its' metabolism.

A second aim of the study was to identify which of the CYP P450's involved in AQ4N activation, was the most efficient. This was used to design and evaluate a targeted gene therapy approach to enhance AQ4N's metabolism in vitro and in vivo. This strategy was developed to benefit tumours that lacked an appropriate compliment of CYP P450's. CYP2B6 and CYPlAl were found to be the most efficient at metabolising AQ4N. CYP2B6 proved its efficacy in significantly enhancing AQ4N's metabolism in vitro demonstrated by enhanced DNA damage and poor cell survival. CYP3A4, 2B6 and lAl were all evaluated in combination with AQ4N as part of a GDEPT strategy in vivo. CYP3A4 and CYPlAl significantly enhanced anti-tumour effect in combination with 20 Gy X-rays and AQ4N compared to controls; 3 out of 9 tumours treated with CYP3A4 were locally controlled for 60 days. CYP2B6 in combination with AQ4N and 10 Gy X-rays demonstrated enhanced anti-tumour efficacy compared to controls. CYP2B6 and CYPRED in combination with cyclophosphamide and AQ4N had a significant effect on anti-tumour efficacy. A radioinducible promoter (W AF-1) was evaluated to target the expression of the therapeutic transgene CYP2B6 in vitro and in vivo. This strategy appeared ineffective and would require further investigation to evaluate its potential.

Date of Award	Apr 2003
Original language	English
Supervisor	Stephanie Mc Keown (Supervisor), Tracy Robson (Supervisor) & Ciara Hughes (Supervisor)