Drug metabolite identification: Bridging nonclinical to clinical drug development

    Drug metabolism research runs through the entire process of drug development, and understanding the metabolic pathway and metabolite structure of compounds is very important for the design and optimization of lead compounds, the selection of clinical drug candidates, the support of clinical studies in the development phase and the evaluation of drug interactions and safety. The ICH and various regulatory agencies (e.g. USFDA, EMA and NMPA) emphasize that when the assessed exposure exceeds 10% of the total drug-related exposure, and the exposure of the metabolite in humans significantly exceeds the maximum exposure in toxicological test animals (≥ 2x), the risk of exposure is increased. Corresponding non-clinical characterization of the metabolite is required prior to the commencement of large-scale clinical trials (usually phase III).

    During the screening and optimization of lead compounds, metabolite identification can screen compounds for potential safety risks by forming reactive metabolites. Studies based on in vitro metabolite identification can quickly identify metabolic soft points to optimize the structure of lead compounds for better efficacy.

    In the pre-clinical research stage, in vitro metabolite identification studies of different species can obtain in vitro metabolic differences among different genera. Although these in vitro methods have limited ability to predict circulating metabolites in vivo, based on the data from these in vitro metabolic comparative studies, toxicological tests can be carried out in the early stage of development to select relevant animal species to cover human metabolites as much as possible and improve the ability to predict safety risks related to human metabolites. At this stage, qualitative and quantitative analysis of metabolites in animals can also be carried out, and comparative analysis can be conducted with the results of in vitro metabolic studies to better understand the predictive ability of these results of in vitro metabolic tests on in vivo metabolism.

    Conduct human metabolite studies as early as possible in the Phase I clinical phase to identify differences in the quality and/or quantity of metabolites between humans and animals as early as possible so that adequate non-clinical assessment of human-specific or non-proportional metabolites that may raise safety concerns can be conducted before large-scale clinical trials begin. Anticipate and reduce unintended safety risks for subjects.

    By comparing the metabolites in animal and human plasma samples, it is possible to preliminarily determine whether the metabolites in animals are sufficient to cover the metabolites in humans. According to the preliminary comparison results, the researcher can make a decision on whether to carry out further research. If the metabolites in animals are not enough to cover the metabolites in humans, the researcher can choose to carry out further research, such as identifying the structure of the metabolites and using standard substances to quantify the metabolites.

    Radioisotope tracer technology can effectively track the metabolic distribution and clearance of drugs in animals. During Phase I clinical trials, material balance studies of radiolabelling in rodents are usually conducted, and the researchers may also quantify human metabolites using reaction factors of radiolabelling reactions shared by animals and humans. Human absorption, metabolism and excretion experiments with radioisotope tracer can more clearly and intuitively obtain the distribution of metabolites in plasma, which can be used to determine the exposure of drugs and their metabolites in the systemic circulation, and analyze the metabolism and clearance pathways of drugs.

    Prior to the completion of Phase II clinical trials and/or Phase III clinical trials, all necessary metabolite studies should have been completed in accordance with the relevant technical guidelines, including human ADME studies using radiolabelated drugs to fully elucidate the pharmacokinetic processes of drug-related substances in vivo. A comprehensive spectrum and quantitative information of metabolites in human plasma were obtained. For specific metabolites or non-proportional metabolites found in humans (i.e., 10% of the total drug-related exposure, and the corresponding metabolite exposure in the animal subject to toxicology testing does not cover the human metabolite exposure), the corresponding non-clinical studies should be completed prior to large-scale clinical trials (usually Phase III clinical trials).

    With the development of high resolution mass spectrometry technology, the detection of trace metabolites has gradually become a routine, and time-flight mass spectrometry and Orbitrap mass spectrometry have become powerful analytical equipment in the study of metabolites. However, absolute quantification of metabolites is not possible based on mass spectrometry data. In some cases, UV data can provide the relative abundance of the metabolite to the prototype drug. Radioisotopically labeled drugs can be quantified relatively accurately between metabolites and prototype drugs by on-line flow detection after liquid chromatography separation or scintillation after microplate enrichment.

   At present, Hongren Pharmaceutical has accumulated more than 70 clinical and non-clinical metabolic product identification projects. Many of these items are challenging to identify metabolites, such as: polypeptides, PDC, oligonucleotides, low exposure, endogenous interference and endogenous lysate metabolites.