Greer, Mhairi Louise; (2005) Validation of cytochrome p450 1b1 as a target for the enzyme prodrug therapy of cancer. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study is comprised of three parts including: (1) clinical validation of cytochrome P450 CYP1B1 in clinical head and neck squamous cell carcinoma (HNSCC) and associated pre-malignant tissue, (2) the identification of a human tumour xenograft model which exhibits either constitutive or inducible expression of CYP1B1 for the evaluation of novel prodrug therapies in vivo, and (3) the identification of stable prodrugs targeted at CYPl-family enzymes designed to deliver nitric oxide to tumours. CYP1B1 protein was detected in HNSCC by immunohistochemistry (IHC) with a selective monoclonal antibody and was over-expressed to a high frequency in carcinoma (94 %, n = 70) and associated pre-malignant, hyperplastic (100 %, n = 14), and dysplastic tissues (100 %, n = 22). Quantitative analysis of CYP1B1 staining intensity by spectral imaging microscopy facilitated correlations with other clinico-pathological parameters including tumour grade, stage and anatomical site. CYP1B1 is mainly localised in the cancer tissue and is not present in adjacent stromal tissue, suggesting it may be a possible target for the enzyme prodrug therapy of cancer. A primary human tumour xenograft model was identified that constitutively expresses CYP1B1. Target enzyme protein expression was also inducible in a time- and dose-dependent manner in vivo as detected by western blotting, IHC and spectral imaging microscopy. Parallel studies also demonstrated CYPlA1 induction in the MCF-7 breast tumour xenograft model. This work has helped validate two potential human tumour xenograft models for the in vivo evaluation of both CYP1A1 - and CYP1B1 -activated prodrugs for cancer therapy. In order to exploit the cytotoxic and hypoxic cell radiosensitising properties of nitric oxide (NO•), 10 novel benzo- or pyrido-fused indole oximes designed as prodrugs for NO• were screened for their selectivity for CYPl-family enzymes. GCI 510 was identified as a selective prodrug for CYP1Al, which generated NO• efficiently. This study has contributed to an understanding of the structural features of these prodrugs which confer selectivity for individual CYPl-family enzymes. Future work will seek to identify CYP1B1-selective prodrugs for NO• based on data from this study.

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