Metabolism studies are integral parts of the development process that aid in safety and efficacy assessments of drug candidates. Differences in metabolic profiles between humans and animals used for toxicological testing should be investigated as early as possible to identify unique and/or disproportionate human metabolites.
The incubation of drug candidates with primary hepatocyte suspensions from human and animals (e.g., minipig, dog, monkey, rabbit, rat, and mouse) aims to identify and quantify hepatic metabolites in order to enable or confirm the selection of species for toxicological studies. Occurrence of highly abundant metabolites may also trigger further profiling for pharmacodynamic effects or drug-drug interaction (DDI) potential. Structural elucidation of identified metabolites is supported by orthogonal parameters e.g., accurate mass and MSn experiments.
Our cross-species metabolism assay is generally conducted with radiolabeled test compounds in which parent drug and metabolites are quantified by radioactive measurement applying off-line scintillation counting. The use of unlabeled test compounds is feasible, but allows only semi-quantitative assessments based on mass spectrometric responses.
The assay can be supplemented by the determination of covalent binding of radiolabeled test compounds to hepatocyte or microsomal proteins to obtain indications for the formation of reactive metabolites and to assess potential risks for idiosyncratic toxicity caused by the drug candidate.
In vivo metabolite profiling and structural elucidation can be offered in virtually all matrices collected from preclinical species in pharmacokinetic and toxicokinetic studies, as well as from humans in clinical trials, including human mass balance (hADME).
We highly recommend to already invest in the identification of circulating human metabolites in individual or volume- and time-weighted Hamilton plasma pool samples even within the first in human (FiH, SAD) trial. Quantitation of metabolites can be accomplished by using reference standards (if available) or radiolabeled calibration samples generated with relevant in vitro test systems (e.g., hepatocytes) and applying high resolution mass spectrometry (HR-MS) combined with off-line radio detection.
The evaluation whether human metabolites are formed at sufficient levels by the animal toxicity species can be performed by analyzing matrix matched plasma samples, not even requiring reference standards. Resulting relative metabolite exposures (human vs. preclinical species) will support the safety assessment of your drug candidate.