- Title
- Evaluating metabolism-induced toxicity using a non-hepatic cell line
- Creator
- Weyers, Carli
- Subject
- Cytochrome P-450
- Subject
- Drugs Metabolism
- Subject
- Drugs Design
- Date Issued
- 2018
- Date
- 2018
- Type
- Master's theses
- Type
- text
- Identifier
- http://hdl.handle.net/10962/61950
- Identifier
- vital:28087
- Description
- The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data.
- Description
- Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (89 pages)
- Format
- Publisher
- Rhodes University
- Publisher
- Faculty of Pharmacy, Pharmacy
- Language
- English
- Rights
- Weyers, Carli
- Rights
- Attribution 4.0 International (CC BY 4.0)
- Rights
- Open Access
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
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