Metabolite identification studies are an essential element in drug discovery, drug development and clinical development programs. As the compound being studied progresses through the discovery - development – clinical continuum the goals of the metabolite analysis may alter as will the detail with which the experiment is conducted.
The graphic below highlights the metabolite studies that are frequently conducted at the different stages of the discovery - development – clinical continuum.
The goals and approaches taken in each of these experiments are described below.
Early in drug discovery the compounds being synthesized frequently have poor PK characteristics; thus in-vitro studies are performed using liver microsomes or hepatocytes as a model to assess the metabolic stability of the compound. The choice of the most appropriate model (microsomes or hepatocytes) is best made knowing the routes of excretion (Phase I or Phase II metabolites) exhibited by that structural scaffold. Accurate Mass instrumentation is sufficiently analytically specific to enable the analysis of bile and urine without the use of a radiolabel. Thus the combination of accurate mass metabolite profiling with samples from a bile duct cannulated study will provide an understanding of the major excretion routes.
The resultant data will enable the drug discovery team to choose the best in-vitro model for their metabolic stability studies.
Since RMI utilizes modern high resolution mass spectrometers, we are able to provide assays where both quantitative and qualitative data are obtained. We provide metabolic stability and metabolite softspot data from the same incubation samples, additionally we provide PK data and an in-vivo metabolite snapshot from plasma samples. Learn more >>.
Early in drug discovery the compounds being synthesized are subject to a metabolic stability assay to aid in generating compounds more resistant to metabolic turn-over. The medicinal chemist needs to determine which part of the compound is subject to metabolism so that structural modification can be made to reduce it. Metabolite identification studies that provide definitive data on which structural moiety is the site of metabolism greatly enhance the effectiveness of the metabolic stability assay. These metabolite profiling data need to be provided in a synthetically meaningful time-frame, so that they assist in the decisions made as to the next series of structures to be synthesized.
RMI Laboratories offers a rapid metabolite profiling service providing these data in a 3 – 5 day time frame.
Sometimes compounds on being metabolized generate reactive chemical species which may covalently bind to proteins, and these have been implicated in idiosyncratic toxicities. Examining the propensity for a compound to form such reactive species provides information that will be included in risk assessments and in determining the probability of success. The way such experiments are conducted is to incubate the compound with a trapping agent, such as reduced glutathione, that will form conjugates that are identified. These experiments can be conducted at any stage in the drug discovery-development continuum and are often part of the decision process for advancing a drug candidate.
At the later stages of drug discovery a determination needs to be made as to which pre-clinical safety species should be used to provide the best toxicological coverage for the human clinical studies. In-vitro metabolite identification studies are typically conducted with human rat, dog and monkey incubates to demonstrate that the safety species chosen generate the same metabolites as those observed in the human samples.
Regulatory agencies require that mass balance studies are performed to determine the fate of any dosed zenobiotic; so that information on which routes of excretion are most important in the elimination of a drug from the body are available. These data may have implications for drug-drug interactions and for defining individuals that are to be excluded from a target clinical population. Such studies are performed using radio labeled drugs so that a quantitative assessment can be made as to the major elimination routes (i.e. renal or hepatic). Metabolite identification studies are also carried-out on these samples so that there is an understanding of which metabolites are the most important in defining elimination routes. Ideally an LC-FSA-MS (liquid chromatography-flow scintillation analyzer-mass spectrometry) assay should be performed to ensure good correlation between the radio-chromatographic and metabolite identification data.
The metabolite profile obtained from in-vivo studies, (either with or without a radio-tracer) are compared with the metabolite profile obtained from in-vitro experiments of the same species to confirm that the in-vitro data are an adequate representation of the metabolism that is observed in-vivo. This comparison is used to justify the safety species selection made based on the cross-species in-vitro metabolite profiling data.
Both the FDA and ICH have issued guidelines requiring as assessment of the abundance of circulating human metabolites and proof that these are also observed circulating in the safety species at an equivalent or greater abundance. These data are used to determine the need for additional toxicity studies of specific metabolites. Modern high resolution mass spectrometers enable these experiments to be performed without the use to a radio-tracer. Thus routine PK samples can also be used to generate the needed metabolite coverage data, when compared with data from PK samples derived from pre-clinical safety studies.
Data on the routes of excretion and the structure of observed metabolites are required for final drug approval. These data are obtained by dosing a radio-tracer to human subjects and determining which metabolites are eliminated vial the urine or feces. An LC-FSA-MS (liquid chromatography-flow scintillation analyzer-mass spectrometry) experiment is conducted to afford a quantitative measure of which metabolites are observed in the urine or feces providing a human mass-balance profile.