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MIST evaluation toolbox – what is in there? Part 4

25 February 2022

The overall aim of the safety metabolism studies is to better understand human metabolism. How is the drug cleared, what metabolites are formed, what is the relative exposure to these and can they be considered safety tested through adequate exposures in toxicology species. The 4th part of this blog series concerns the MIST analysis, i.e. the metabolite exposure comparison performed utilising samples from repeated dose studies in man and toxicology species.

Metabolite exposure comparison, why and when?

MIST analysis – what is that?

The ultimate tool in MIST (Metabolites in safety testing) evaluation is the so-called MIST analysis, which comprises the exposure comparison of human metabolites between human and toxicology species. MIST analysis can be conducted when samples from repeated dosing are available. It is an analytical approach that allows exposure comparison of metabolites between species, without absolute quantification. MIST analysis identifies potential safety testing issues, their magnitudes and allows mitigation well in time.

Which samples are used in the MIST analysis?

In vivo samples from repeated dose studies in toxicology species and human are used in this work. For toxicology species these samples are most often the GLP-tox TK (toxicokinetic) samples and for human the MAD (multiple ascending dose) samples. At the time of analysis, typically when MAD samples are available, the most recent tox samples are preferred. Samples from the last day of dosing in each study are chosen, where steady state of parent compound has been reached. It is notable that knowledge about metabolites and steady state levels may not be available at this stage. Still “the last day of dosing samples” can be considered to present the worst-case scenario in terms of accumulated metabolite levels after repeated dosing.

Which doses are of interest?

Adequate exposure to human metabolites must be confirmed in toxicology species at doses with no adverse effects. Therefore, the doses assigned as no observed adverse effect level (NOAEL) are preferred. For human samples, the most practical is to choose all three dose cohorts from the MAD study since the therapeutic dose normally is not fully confirmed yet. Including all doses allows revisiting the data as therapeutic doses are evaluated.

How can exposures be compared without absolute quantification?

The PK/TK samples over a dose interval are pooled in a time proportionate manner to generate one sample per species and dose group/cohort. Samples are matrix matched to address matrix effects in LC/MS analysis. Samples are prepared and analysed at same occasion and LC/MS peak areas of the metabolites correspond to their respective exposure and can be compared between human and the toxicology species without knowing their absolute levels.

Which in vivo metabolites are of interest?

The main focus is on the human metabolites. Therefore, the work starts with human metabolite profiling and characterisation in the AUC0-xh pools. In the MIST guideline1, human metabolites of concern, requiring adequate exposure, are those constituting >10% of the total drug-related material/exposure. Due to MS response differences, it is difficult to draw final conclusions about relative levels using non-radiolabeled compounds, and therefore the exposure comparison includes all human metabolites detected in the AUC pool. If data concludes that all human metabolites are adequately exposed in the animal species, their relative levels are irrelevant. If a metabolite demonstrates non-adequate exposure, its relative concentration level needs to be understood through additional work.

Outcome

The acquired data from MIST analysis will reveal if there are any disproportionate or unique human metabolites in terms of exposure, regardless of their relative level. Relative level of any identified disproportionate or unique human metabolite at this point is concluded assuming equal LC/MS response. The risk for the metabolite being >10 % of total drug related material needs to be further investigated with the relevant additional tools and analytical approaches. It must at some point be considered if it is necessary to synthetize a metabolite of a potential concern for a) confirmation of relative level, b) confirmation of disproportionality by GLP bioanalysis and c) in the end for administration to toxicology species.

Next step

The final step in MIST evaluation will be the human ADME study where radiolabelled test compound is administered to healthy volunteers. Despite only single dose is administered, the samples allow quantitative profiling of metabolites in plasma. If necessary, also response factors can be derived to understand which of the metabolites are of concern. The MIST analysis data demonstrates its exposure in toxicology species in relation to human.

Written by Johanna Haglund

P.S. The previous parts of the safety metabolism blog post series you will find from here.

1st part: In vitro cross species comparison, why and when?
2nd part: In vivo Met ID in toxicology species, why and when?
3rd: Human Met ID, why and when?

References:
1) Safety testing of Drug Metabolites, FDA 2016

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