Metabolic stability and metabolite identification using hepatocytes

Abstract

Introduction: This project is about in vitro assays of metabolic stability and metabolite identification during early drug discovery.

Background: Predicting metabolic stability is as essential part of drug discovery. Several in vitro systems exist including microsomes, S9 fractions and hepatocytes. Currently, hepatocytes are not used at the company, but there is demand from clients and projects. For low clearance compounds, the in vitro testing is challenging as it requires longer incubation time to be able to measure metabolism. Identifying metabolites formed can assist in designing novel compounds.

Aim(s): The aim of the project was to design and compare a few different protocols to assess metabolic stability and metabolite profiling using hepatocytes in order to identify the assay that best meets the projects’ needs throughout the development phase for new drugs.

Methods: Protocols for hepatocytes in suspension and plated hepatocytes were designed and tested on a set of known drugs. After analysis with LC-MS/MS the in vitro intrinsic clearance was determined and used to predict an in vivo clearance, which was compared to published in vivo studies. Metabolites were evaluated for two known drugs and one project compound using data from LC-MS/QTOF and the software Molecule Profiler (Sciex) and prevalence was compared to published data.

Results: The hepatocyte suspension assay was able to predict in vivo clearance within 2-fold for 75-80% of the drugs, while the plated hepatocyte assay was not able to do so for any of the drugs. The identified metabolites were similar between the assays, and nearly all identified metabolites have been published before. For indomethacin, the expected phase II metabolites were not observed in this study.

Conclusion: The hepatocyte suspension assay was able to accurately predict in vivo clearance and most major metabolites could be found by analysing samples from the metabolic stability assay. It is therefore a suitable assay for the company.

Popular Abstract

Predicting how drugs will behave in the body

In the body, the liver is the organ responsible for breaking down drugs to eliminate them from the body. When developing new drugs, a large amount of candidate molecules goes through rigorous testing in a lab before being tested on an animal or human. The purpose is to predict how the molecule will behave in the body of a living being. Using that information, some molecules are discarded and some are selected for further development and testing. Examples of things that can be tested in the lab are how soluble the molecule is in water, how much it binds to proteins in the blood, how well it binds to the intended cellular target that creates the medicinal effect, and how fast it is broken down by liver proteins or liver cells.

This project was performed at a company that has not use liver cells to measure the breaking down of drug before, so the main goal was to find a method that could accurately predict the behavior of the drug in a living being and identify what the drug is mainly broken down to. Known drugs were tested so that the results could be compared to published results from previous studies on living rats and humans.

Two methods were tested, one with short incubation time, and one with long incubation time. In the short method, the cells were floating free in suspension in a solution containing the drug for 60 minutes. Samples were taken regularly to be able to follow the disappearance of the drug over time. The method with long incubation was made for compounds that are broken down very slowly, so that no change can be seen in only 60 minutes. In this method the cells were allowed to attach to the bottom of a container, which enables them to stay alive for days instead of hours. Then the drug was added and the incubation lasted for 24 hours. Samples were taken regularly. Using factors specific for each species, for example number of cells per gram liver, the results were scaled up to how fast it would disappear in a rat or human. In this study I found that the short method was able to predict the disappearance found in earlier studies very well, but the long method did not produce acceptable results.

To investigate what the drug is broken down to, I analysed the samples using a method that scans everything present in the sample and used a software to identify which molecules increased over the incubation time. I essentially found the same major metabolites as previous research, but some were not present in the samples.

In conclusion, the method with the short incubation time could accurately predict behavior in living beings, so it is a good method to implement at the company.

• author: Dahlqvist, Sofia
• supervisor: Jessica Larsson
• organization: Department of Chemistry
• course: KEMR30 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Analytical chemistry, Drug discovery, Drug metabolites, Hepatocytes, Metabolic stability
• language: English
• id: 9128847
• date added to LUP: 2023-06-22 11:15:17
• date last changed: 2023-06-22 11:15:17