Abstract
CYP3A4 is one of the most abundant P450s in human liver, accounting for 10 to 60% of total liver P450 (Guengerich, 1990; Krivoruk et al., 1994; Shimada et al., 1994) and has been estimated to metabolise 60% of pharmaceuticals currently in use, for example cyclosporin, nifedipine and terfenadine (Cholerton et al., 1992; Jounaidi et al., 1994). The levels of CYP3A4 can be up-regulated in vivo by a number of structurally diverse compounds, for example the glucocorticoid dexamethasone, and the antibiotic rifampicin (Watkins, 1992). Due to the potential for clinically significant drug interactions it is important to determine whether a new chemical entity is an inducer of CYP3A4 early in the drug developmental process. Previously in our laboratory (Williams, 1997), a plasmid construct containing approximately 1kb of the 5'-regulatory region of the human CYP3A4 gene, coupled to a reporter gene for human secreted placental alkaline phosphatase (SPAP), was engineered and used to develop an in vitro transfection system for the assessment of the induction of human CYP3A4 by xenobiotics. This system was used to demonstrate the induction of CYP3A4-dependent reporter gene expression by dexamethasone, PCN and phenobarbitone. The initial aim of the research presented in this thesis was to further develop this system in an attempt to improve the reproducibility and robustness. The ultimate aim of this approach would be to modify the system to render it useful for the high throughput screening of xenobiotics. Another goal of the project was to attempt to generate a stable cell line expressing the alkaline phosphatase reporter gene under the direct control of the CYP3A4 regulatory region. Success in this approach would dramatically reduce problems associated with poor reproducibility and would greatly increase the potential for the use of this system as a high throughput screen. The final aim of this project was to use the system to attempt to gain information concerning the molecular mechanisms underlying induction of CYP3A4 by xenobiotics. Specifically, the major advances made during this research have been: 1) The system originally developed in this laboratory has been extensively modified, rendering it more reproducible, more robust, and suitable for use in high-throughput screening. 2) Induction of CYP3A4-dependent reporter gene expression by carbamazepine, dexamethasone, metyrapone, PCN, phenobarbitone, phenylbutazone, phenytoin, rifampicin, spironolactone and sulfinpyrazone has been demonstrated. 3) Clotrimazole, erythromycin and triacetyloleandomycin did not induce CYP3A4-dependent reporter gene expression, indicating that these compounds may act at a post-transcriptional level to induce CYP3A4. 4) The anti-glucocorticoid RU-486 was found to block dexamethasone-dependent induction of CYP3A4-dependent reporter gene expression. 5) A role for the human glucocorticoid receptor in CYP3A4 induction by a number of agents was also demonstrated; the precise molecular mechanisms of this role were not elucidated, but possibilities have been discussed. These results demonstrate that this assay provides a sensitive, specific and high throughput screen for the assessment of xenobiotic induction of human CYP3A4 in vitro.