The pharmaceutical industry has widely used in vitro metabolite identification (MetID) assays since they are simple, rapid with high throughput. The in vitro MetID assays can be used to investigate the biotransformation of test compounds for predicting in vivo metabolism and save experimental animal.
In the early stage of drug development, the in vitro MetID assays are intended to identify "soft spots" for optimizing lead compounds and screen reactive metabolites to assess the potential toxicity of a lead compound. In the preclinical development stage, the in vitro MetID focuses on comparative metabolism in human and animal, and results from the study can be used to support the selection of toxicological species. Information that identifies the major metabolites and associated metabolic pathways in in vitro can be used to design a metabolizing enzyme phenotyping experiment.
The WuXi AppTec DMPK MID team utilizes ultra-high performance liquid chromatography (UPLC) coupled with a photodiode array Detector (PDA) and high-resolution mass spectrometry (HRMS) to search and identify metabolites formed in incubation. Based on different study goals, three types of routine assay can be performed, including metabolic soft-spot analysis, reactive metabolite screening, and a metabolism comparison cross-species. The comprehensive incubation systems include but are not limited to liver microsomes, liver S9, hepatocytes, and blood/plasma. Meanwhile, some special types of assays can also be performed, such as acidified S9 and lysosomal systems used to investigate the payload-containing components released from an antibody drug conjugate (ADCs). In addition, some uncommon methods or approaches can be employed to confirm the metabolite structure, such as metabolite matching, hydrogen/deuterium (H/D) exchange, and titanium trichloride reduction.
An ADC can be delivered into the cell either by antigen receptor-mediated internalization or by nonspecific endocytosis, followed by releasing the drug in the lysosomal compartment and resulting in the tumor cell apoptosis. To determine payload-containing components released from ADCs after entering tumor cells, in vitro acidified liver S9 and lysosomes systems of human and animal species, coupled with non-targeted HRMS data mining technologies, are established and applied to identify the major payload-related component released from ADCs. Those results can support the selection of toxicological animal species and analyte(s) for PK analysis.