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In Vitro metabolism

In vitro metabolic screening offers a cost-effective and efficient way to evaluate compound metabolism during the hit-to-lead and lead optimisation stages of discovery.

We can provide metabolic stability assessments in both microsomes and hepatocytes and offer two levels of analysis:

  • A discovery level metabolite ID study where the top 3 metabolites are identified to give an overview of metabolic hotspots is typically appropriate for early stage compounds and helps to narrow down the number of clinically relevant compounds;
  • Comprehensive metabolic profiling of all significant metabolites (typically >1% of total compound-related material) is recommended for later stage lead compounds.

For in vitro metabolite ID analysis, we require 50 µL of a 10 mM solution in DMSO or 1 mg of solid compound.

We also offer a cross-species metabolic profiling comparison in human and preclinical hepatocytes. This has two important benefits. First, it increases confidence around the choice of toxicology species when moving to early development. And second, it can flag up in vitro human specific metabolites (HSMs) or any disproportionate human metabolites (DHMs) that might prove problematic as a molecule moves towards candidate nomination and development. Gaining maximum value from these data is important when de-risking a development candidate. Being aware of potential difficulties well ahead of time will greatly assist in mitigating risks, as dictated by industrial guidelines such as ICH M3(R2) and FDA Metabolites in Safety Testing (MIST).

In Vivo metabolism

If the metabolites that are formed in preclinical species are identified, they can give crucial insights to inform the later stages of drug discovery. They can provide critical information about how a molecule might be metabolised, and the routes by which it might be eliminated. These data can be particularly useful in elucidating PK/PD disconnects. They also provide valuable information to drive discussions around efficacy.

By combining relevant analytical approaches, including high-resolution mass spectrometry with highly skilled manual data interpretation and chemically intelligent software algorithms, we are able to enhance metabolic coverage and provide greater confidence in the characterisation of metabolites in plasma, urine, blood and bile samples.

Reactive metabolite screening

Reactive metabolites have been linked to idiosyncratic toxicity which is a major concern for patient safety and has led to drug withdrawals and black box warnings. Early assessment of the likelihood of reactive metabolite formation will allow mitigation of associated risks.

Sygnature offers in vitro reactive metabolite trapping assays, the Glutathione (GSH) Trapping assay and the Cyanide Trapping assay for early assessment of drug candidates. These assays do not require radiolabelled compound.

Metabolism of some drug compounds can generate chemically reactive metabolites which can react with cellular molecules such as proteins or DNA, leading to adverse reactions. This risk can be assessed with tailored microsomal incubations using glutathione or cyanide to covalently bond with electrophilic species, thereby ‘trapping’ them. Any detected glutathione or cyanide adducts can provide a flag for potential liabilities around toxicity. The samples are acquired with non-targeted high-resolution mass spectrometry and adducts are detected with high selectivity of characteristic fragment ions or isotope patterns, enabling detection down to trace levels. This approach gives a wide coverage and provides an accurate assessment of overall liabilities around potential reactivity in a time-efficient manner. Subsequent structural characterisation of key metabolites can be performed where relevant and feasible, which can help direct a program to eliminate chemical liability by structural modification.

The reactive metabolites identification assays can be tailored to answer specific questions relevant to your project. We have a collaborative and flexible approach to maximise the impact of your data. If you would like to discuss these assays in more detail or have particular requirements, please do not hesitate to contact us using the contact form or by email:

Definitive structural identification

Where characterisation of a metabolite is required, either unequivocally or in more detail than high-resolution mass spectrometric analysis can provide, further analysis by nuclear magnetic resonance (NMR) spectroscopy can be instigated to give greater insights into the site of biotransformation, where feasible. These in-depth studies can provide early, unequivocal data around metabolite structure that can better inform PK/PD relationships, and inform early assessments around metabolite safety.

We have a Bruker 500 MHz spectrometer in-house as well as access to several Bruker spectrometers with cryoprobes. Our in-house expertise to analyse the NMR data and scientific leadership in the field enables us to suggest confident, data-led outcomes in the context of the questions being asked.

Bespoke context driven analysis

We offer a tailored service to meet our customers’ needs. It is very much a conversation – we will listen to what they require and suggest how we can use all the techniques and tools at our disposal to achieve that. The process is always led by high-quality science, and it is important to focus on the question we are trying to answer. The starting point is a discussion about what is feasible and aligning this with realistic expectations.

Please contact us to discuss your needs, and how we can help you meet them.

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