Human metabolite profiles in early-stage drug development

You will receive the following data and information:

• Method transfer and qualification
• hrMS/MS-data collection
• Extraction recovery
• Column recovery
• Data processing
• Metabolite profile generation in 3 pooled samples (radiochromatograms)
• Relative abundances of the peaks
• Confirmation of parent and metabolites for which reference standards were available (based on retention time and/or accurate mass)
• QA approved report
• UPLC columns
• Transfer of MS files

Mass balance studies are conducted to characterize excretion routes. In a mass balance study, plasma and excreta are collected until >90% is excreted. These studies provide the routes, the rates and the quantity of drug and metabolites that are found in the excreta. Metabolite profiling separates parent drug from metabolites using UPLC. AMS then measures fractions to determine where the radiolabeled material elutes, and high-resolution MS/MS works at the same retention time to support structural identification. AMS provides the total 14C quantification signal, while hrHRMS/MS provides the structural identification. You will get insight in the % of parent drug that leaves the body unchanged and the number of metabolites that are formed in amounts that warrant further investigations (>10% of parent drug).

To enable a complete safety and efficacy assessment, it is important to know to what extent patients are exposed to the drug itself but also to drug derived metabolites. For every metabolite might have toxicological or pharmacological implications by itself. Only at the moment that we know that all metabolites are accounted for in humans, it can be assessed whether these metabolites are also present the animals that were used for safety and efficacy assessments.

Studies with AMS technology do not require microtracer radiolabels. Studies can be performed for drug products with low (microtracer) and high (conventional) radioactivity. The technology detects 14C labeled material and has the sensitivity to detect very low doses of radioactivity of 1 µCi and less in contrast to the conventional LSC method. LSC can only be used for high radioactive levels of 100 µCi and more.

In a mass balance and metabolite profiling study, typically 1 µCi of the 14C-labeled drug is dosed orally at or around therapeutic level, and plasma and excreta are collected until >90% is excreted. The low 1 µCi radioactive dose does not require a-priori animal dosimetry studies. This saves time and money at a stage where the chances of successful development are still relatively unknown.

At a later stage, to prepare for NDA or MAA filing, animal dosimetry studies may still be required by the regulatory authorities. This depends on the type of drug, the disease indication and bioavailability. By that time you are reasonably certain about the success chances of the drug.

Indeed. 6 participants is generally sufficient for regulators. Some companies have submitted NDAs using 4 participants. The minimum depends on the compound and the disease indication and is often based on company policies.

AMS is frequently used for microtracer studies which can significantly reduce overall project costs and timelines by bypassing intensive safety preparations required for traditional studies. Non-GMP-radiolabeled investigational drugs ‘Qualified material’ may be acceptable in human studies when paired with GMP-unlabeled counterparts, resulting in large cost and time benefits. Although explicit regulatory guidance on using Qualified material in hADME studies is lacking, if such material is treated as an impurity, the maximum allowed impurity level may be referenced against the qualification thresholds for degradation products in new drug products as outlined in ICH Q3B (R2) (ICH, 2006; Roffel and Hoogdalem, 2024; Zhu, S.X., 2025)

Overall, the microtracer-based mass balance, metabolite profiling and absolute bioavailability studies in early clinical developments give an enormous enrichment of the data package available at early-stage without the need for separate studies to determine human ADME data. The data richness allows better study designs for follow up clinical studies, it allows earlier assessment and risk mitigation strategies for unexpected human unique metabolites, and it eliminates the need to conduct radioactive animal mass balance/metabolite profiling studies, where many animals, and much time and money are being spent on non-relevant animal metabolites.

Incorporating radioactivity in the drug molecule is needed to ascertain that all metabolites will be found in the systemic circulation. Since virtually all drugs contain carbon, the highest scientific standard practice is to synthesize the drug with incorporation of radioactive carbon-14, and to dose this radioactive material to a small number of healthy participants or patients in a hADME study. Samples from excreta and blood or plasma can be analyzed to trace the drug and its metabolites. Analysis via AMS is up to 10,000-fold more sensitive than classical methods, for instance Liquid Scintillation Counting (LSC) and therefore very small amounts of radioactivity are sufficient.

The answer is ‘Yes’. We deliver data to our clients to support their regulatory submissions with regulatory-required or requested data such as human metabolism, routes of excretion, absolute bioavailability, and fraction absorbed. The FDA guideline on Safety Testing of Drug Metabolites Guidance for Industry (CDER) March 2020 Pharmacology/Toxicology recommends performing human in vivo metabolic evaluation as early as possible. The level of radioactivity in a microtracer study is only 0.1-1µCi. Conventional studies generally apply 100µCi. Due to the 100-fold lower radioactivity levels, ethical committees approve the use of 14C microtracers in early-stage clinical development.