The Current State of Biotransformation Science 2024

To end this year of highlighted papers around the subject of biotransformation and metabolism, we feature a paper published by the “Translatability of MetID In Vitro Systems Working Group”, a subgroup of the International IQ Consortium. The paper gives a useful insight into the most commonly used in vitro MetID practices across 26 pharma companies, and crucially reveals how effective these systems are at predicting circulating human metabolites.

There are so many interesting nuggets in this paper – only a few elements are highlighted here. We recommend a read of the entire paper and look forward to future efforts to improve translatability.

Assays galore

As expected, there is wide use of in vitro biotransformation assays with 56% of respondents indicating suspension hepatocytes as the preferred primary system but many other systems are commonly employed (microsomes, S9 fractions, recombinant enzymes etc.). Notably hepatocyte co-culture seems to be relatively commonly used where tailoring of the approach is needed, particularly relevant for more metabolically resistant drugs. Interestingly 27% also utilize gut microbiota to study drug metabolism, however this increases to 62% when used more reactively to investigate unexpected circulating or fecal metabolites observed in-vivo.

However, across the systems used, a point of difference appears to be the variety of incubation conditions e.g. substrate concentration and time used, i.e. there is no consensus on optimal conditions.

Not unexpectedly, post assay work-up adaptations are often made in anticipation of the formation of particular metabolites. Most commonly this involves acidification of samples post-reaction to minimize hydrolysis and migration of acyl glucuronides.

Reporting metabolites

The most common “cutoff” for reporting metabolites at the hit-to-lead and lead optimization stages is the top 3-5 metabolites approach, although this varies a lot. This transitions into a far more in-depth investigation from candidate selection onwards with the most frequent cutoff 1-2% of total DRM (drug related material).

Generally, a major metabolite in the context of in vitro assays is considered at an abundance of around 10% or higher, but some just rank rather than use a pre-determined cut-off. It was mentioned that use of “major” may be substituted by “predominant”, “main” or “significant”, rather than the word “major” which is more generally associated with clinically relevant metabolites.

Predictability of in vitro to in vivo metabolism

Predictivity varies but the top line from the responses is that the probability of discovering a major metabolite during later stages of clinical development are low, as is complete discordance of in vitro to in vivo metabolism. Where poor prediction is observed, the top three reasons are insufficient metabolism of stable drug scaffolds, extensive metabolism to multiple metabolites (both primary and sequential), and extrahepatic metabolism.

Metabolite formation

For quantitation, before Phase 1 clinical trials, generally MS or UV/DAD are used, however beyond Phase 1 quantification is mostly performed using authentic standards or radiolabeling of the drug.

For predicted major circulating metabolites, synthesis and characterization is most commonly frontloaded (58%) with 38% waiting for confirmation in Phase 1. This is interesting to us at Hypha given we specialize in synthesizing metabolites for clients. We generally see a later action to metabolite synthesis with late Phase 1 or Phase 2 (e.g. after analysis of Single Ascending Dose (SAD) and Multiple Ascending Dose (MAD) studies) being common times these activities are actioned. Perhaps this reflects the difference in company size and resources available for such work, since the majority of respondents in the IQ working group are representatives from large pharma companies. Hypha’s clients range from small through to large pharma companies.

Characterization activities include establishment of bioanalytical assays (35%), in vitro safety studies (39%), and pharmacological activity assessment (15%).