Background: The regulatory guidances on metabolites in safety testing (MIST) by US Food
and Drug Administration (FDA) and International Conference on Harmonisation (ICH) describe the
necessity to assess exposures of major circulating metabolites in humans at steady state relative to exposures
achieved in nonclinical safety studies prior to the initiation of large scale clinical trials. This
comparison can be accomplished by measuring metabolite concentrations in animals and humans with
validated bioanalytical methods. However, bioanalysis of metabolites in multiple species and multiple
studies is resource intensive and may impact the timelines of clinical studies.
Method: A simple, reliable and accurate method has been developed for quantitative assessment of metabolite
coverage in preclinical safety species by mixing equal volume of human plasma with blank
plasma of animal species and vice versa followed by an analysis using LC-SRM or LC-HRMS. Here,
we explored the reliability and accuracy of this method in several development projects at Genentech
and compared the results to those obtained from validated bioanalytical methods.
Results: The mixed-matrix method provided comparable accuracy (within ±20%) to those obtained
from validated bioanalysis but does not require authentic standards or radiolabeled compounds, which
could translate to time and resource savings in drug development.
Conclusion: Quantitative assessment of metabolite coverage in safety species can be made using
mixed matrix method with similar accuracy and scientific rigor to those obtained from validated bioanalytical
methods. Moving forward, we are encouraging the industry and regulators to consider accepting
the mixed matrix method for assessing metabolite exposure comparisons between humans and animal
species used in toxicology studies.