Validation of novel HPLC methods to analyse metabolic reaction products catalysed by CYP450 enzymes and 'in vitro' measurement of Drug-Drug Interactions

Background: The inhibition of hepatic cytochrome P450 activity is one of the most significant mechanisms of a drug interaction which can result in a change in the pharmacokinetic behaviour of the drug. Severe adverse events have been associated with drug interactions caused during coadministration. COVID-19 infection has rapidly grown into a worldwide pandemic in 2020, and this has had a significant impact on human health. Dexamethasone, aspirin, ibuprofen, remdesivir, and omeprazole are some of the drugs being used in the treatment of COVID-19 during the pandemic. Aim: This research aims to determine the inhibitory effects of aspirin, ibuprofen, remdesivir, and omeprazole on dexamethasone metabolism (CYP3A2 activity) and the effect of testosterone on Vitamin D metabolism (CYP2C11 activity) in rat liver microsomes using High-Performance Liquid chromatography. Methods: A Dexamethasone and 6β-hydroxydexamethasone assay was developed and validated, and inhibition parameters were calculated using Lineweaver and Michaelis-Menten plots in Chapter 2. Using the previously developed HPLC assay, inhibition parameters were calculated using Lineweaver and Michaelis-Menten plots in Chapter 3. In chapter 4, a Vitamin D3 and Vitamin D2 and metabolites (25(OH)D3 and 25(OH)D2) assay was developed and validated on HPLC using the isocratic mode of elution. Results: The linearity (r2 > 0.99), intra and interday precision (<15%), accuracy and recovery (80-120%), and stability study values of the newly developed methods for CYP3A2 and CYP2C11 substrates and metabolites were following International Conference on Harmonization (ICH) guidelines. Our inhibition study data showed that aspirin is a competitive inhibitor (weak) with the Ki = 95.46 ± 4.25 µM and IC50 = 190.92 ± 8.50 µM for the CYP3A2 assay. The results also showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. Moreover, a study of inhibition parameters was not conducted due to the presence of interferences and extremely low concentrations of Vitamin D and metabolites. Conclusion: The findings of this PhD research represent that there is a minimal risk of toxicity when dexamethasone is co-administrated with aspirin, ibuprofen, remdesivir, and omeprazole and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.