Metabolite Identification (Met ID): Its Purpose & Value in Drug Discovery

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Metabolite identification (Met ID) is key to understanding the metabolic fate of a potential drug candidate. However, despite it being one of several significant activities carried out in drug discovery and development, understanding of its purpose and value can sometimes be limited.

In this metabolite identification blog our resident expert, Iain Beattie, outlines the benefits of employing Met ID at different stages in a molecule’s development. He goes on to explain how the question that actually needs answering changes depending on what stage the project is at. 

Metabolite Identification (Met ID) in Drug Discovery

Metabolic stability, alongside other properties, is a key parameter to optimize in early drug discovery when the main objective is identifying a chemical series that is worth pursuing. This can be particularly important for PROTACs, where one end of the molecule binds to a protein of interest and the other end binds to an E3 ubiquitin ligase enzyme bringing them together marking the protein of interest for degradation. If metabolism were to occur on the middle part of the molecule linking the two ends, there is potential to cleave the molecule in two. The two parts of the molecule would still be able to bind to the protein of interest and the E3 ligase but as they would no longer be connected, the protein of interest would no longer be targeted for degradation.

As the project progresses into lead optimization, metabolic stability usually becomes less of a problem and the focus shifts to determining the metabolic profile in human, usually hepatocytes, and compare this against species that may be used in the safety studies.

Although not a requirement at this stage, an in vivo study may be carried out to identify the metabolic profile, particularly in plasma, and confirm how this compares with the in vitro metabolic profile.

As it moves into development, the main requirement is to identify the main circulating metabolites ideally using pooled samples and compare with the in vitro metabolic profiles.

The same question would need to be answered once it gets into the clinic when it will be important to compare with those species used in the safety studies. 

Early Drug Discovery

In early discovery, a project may have 2-3 chemical series being explored in parallel for a particular target. As part of the Design, Make, Test cycle, the aim is to identify a potent series with good DMPK parameters, of which metabolic stability is key.

If a series is showing potential but is rapidly metabolized, the chemists will want to know whether there is anything they can change to improve stability. If there are 1 or 2 main metabolites that can be identified, then they may be able to block that route to improve stability. However, if several metabolites of similar abundance are formed, improving stability may be more complicated, and recognizing that early in the project is key.

Having this information available shortly after the stability assay has been run allows the team to be reactive and modify the next design phase accordingly. To achieve this rapid response, the intrinsic clearance samples are used.

Mid to Late Drug Discovery

As projects progress through lead optimization, metabolic stability should be less of a concern and the question becomes how the human metabolic profile compares with species that may be used for safety studies.

By the time a compound has been identified for progression, a cross species comparison of its metabolic profile, usually in hepatocytes, is carried out, and the profile obtained in human is compared with other relevant species. 

The main priority of this study would be to identify the full metabolic profile in human, highlighting any human specific, any disproportionate metabolites or any potential metabolites that may be an issue, such as, glutathione conjugates or acyl glucuronides.

In Vivo Studies

When a compound progresses into the clinic, the main two questions that need addressing are:

  1. Is the human metabolic profile similar to that of the species used in the safety studies?

  2. Does the profile obtained in vivo compare with that found in the in vitro profiles?

The primary focus would be to profile the plasma from animal PK or PD studies as well as early human clinical studies and identify the major circulating metabolites. Identifying urinary or faecal metabolites would be less of a priority but may be useful as they usually have higher levels of metabolites present and can be used to identify metabolites. This information can then be used to interrogate the plasma data.

Normally an AUC pooled sample, either per subject or pooled across subjects, would be created with the aim of identifying major circulating metabolites that are >10%.

Summary

Whilst this is a top-level explanation of how metabolite identification can be employed at different stages of the drug discovery process, it hopefully explains the immense value it can bring.

You can find out more about Charnwood Discovery’s metabolite identification service here or contact us to speak with Iain and the team directly. 

Combining Technology & Expertise

Our expert team use the Thermo ScientificTM Orbitrap Exploris 120 mass spectrometer with their Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system.

This enables us to offer metabolite identification using the high-resolution capability of the instrument to provide accurate mass data to aid the structural characterization of metabolites.

Using their combined experience of over 60 years, our team is able to provide you with analysis and insight to progress your project.

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