Filip Cuyckens

Human radiolabeled mass balance studies are crucial for comprehensively characterizing the absorption, distribution, metabolism, and excretion (ADME) of investigational drugs, providing essential data for drug development, regulatory evaluation, and product labeling. The IQ Consortium's Human Mass Balance Radiolabeled ADME (hADME) Working Group developed this perspective to address key considerations and highlight opportunities for these studies. This is particularly relevant as regulatory guidance, such as the 2024 US Food and Drug Administration (FDA) and China's National Medical Products Administration (NMPA), have finalized dedicated guidance documents, along with recommendations from other global regulatory bodies (including European Medicines Agency (EMA) and Pharmaceuticals and Medical Devices Agency (of Japan; PMDA)); still, there are some regional differences. This review outlines current practices and emerging trends in hADME study design. It details seven clinical designs, from conventional radiolabeled approaches to microtracer studies that employ analytical techniques, such as accelerator mass spectrometry (AMS). It further highlights alternative non‐radioactive approaches, including the use of Fluorine‐19 nuclear magnetic resonance ( ¹⁹ F‐NMR) spectroscopy. The manuscript also explores the integration of hADME studies with absolute bioavailability (ABA) assessments and highlights their impact on early drug development with case studies. This collective effort by the IQ working group highlights fit‐for‐purpose study designs to ensure comprehensive ADME characterization supporting drug registration and patient well‐being.

Radiolabeled human mass balance studies are crucial for identifying circulating metabolites and understanding drug absorption, excretion, and clearance pathways. Metabolite profiling involves quantifying all drug‐related entities, including parent drug and metabolites in plasma and excreta, using extended liquid chromatography methods coupled with detection through scintillation counting, accelerator mass spectrometry, or non‐radiolabeled approaches. Given the labor‐intensive nature of sample extraction and analysis, we propose a new paradigm that maximizes gathering information through sample pooling strategies. Our proposal introduces sample pooling strategies by integrating both individual and pooled sample schemes, simplifying decisions, and consolidating existing knowledge into a cohesive document. This aligns with the low statistical power typically associated with mass balance studies that dose six to eight subjects. In metabolite profiling, it is common practice to pool samples either from the limited number of subjects participating in a human mass balance study or from different time points of sample collection. This approach improves efficiency while preserving data integrity. Pooling reduces resource constraints and enables the concentration of samples with relatively low radioactivity levels, resulting in higher quality metabolite profiles. Nevertheless, there are situations when analyzing samples from individual subjects or time points may be preferred. This proposal presents guidance and decision trees designed to facilitate informed decisions about sample pooling to maximize data quality of metabolite profiling in human mass balance studies while efficiently managing resources. These recommendations stem from discussions within the mass balance working group of the IQ Consortium.

Metabolite identification is essential in drug metabolism and pharmacokinetics (DMPK) studies and plays a pivotal role throughout the drug development process, from informing drug design to evaluating safety and efficacy. Mass spectrometry (MS) is the analytical technique of choice for characterizing metabolites due to its selectivity and sensitivity, particularly when paired with chromatographic methods. However, MS encounters challenges in structural characterization. This study employs infrared ion spectroscopy (IRIS) to differentiate isomeric compounds and demonstrates the robustness of a newly developed IRIS platform. We showcase applications in metabolite identification by determining the site of glucuronidation and phase I oxidation in selected drug molecules. Employing density functional theory for spectral prediction, IRIS decreases reliance on reference standards and alleviates the time-consuming purification processes typically associated with metabolite analysis. The newly developed platform integrates a high-power, high-repetition-rate infrared laser and ion trap MS. This setup is very robust, as evidenced by the highly reproducible IRIS spectra recorded over a one-year period without any instrument readjustment or recalibration. Moreover, the high power and high repetition rate of the laser provide a large dynamic range that is necessary to resolve all spectral features. These results leverage IRIS toward a transformative tool in analytical chemistry, with potential applications expanding across various fields, such as impurity analysis and forensics. The introduction of a compact IRIS setup in an industrial setting not only confirms its practical applicability but also emphasizes its potential for integration into routine analytical workflows.

Exposure to atabecestat is associated with liver injury, which subsequently led to its withdrawal from development. Previous studies of patients with atabecestat induced liver injury identified T cells responsive to atabecestat and its metabolites, indicating that immune-mediated mechanisms are involved. As irreversible protein modification is suspected to drive immunogenicity, this study aimed to characterize potential atabecestat protein adducts using HSA, GSTA1, and GSTP as model proteins. We have shown that atabecestat only formed a cysteine adduct on GSTP in the presence of metabolic systems, highlighting the important role of bioactivation in adduct formation and selectivity for the binding interaction.

Drug toxicity during the development of candidate pharmaceuticals is the leading cause of discontinuation in preclinical drug discovery and development. Traditionally, the cause of the toxicity is often determined by histological examination, clinical pathology, and the detection of drugs and/or metabolites by liquid chromatography-mass spectrometry (LC-MS). While these techniques individually provide information on the pathological effects of the drug and the detection of metabolites, they cannot provide specific molecular spatial information without additional experiments. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful, label-free technique for the simultaneous detection of pharmaceuticals, metabolites, and endogenous chemical species in tissue sections, which makes it suitable for mechanistic toxicological studies to directly correlate the distribution of the drug and its metabolites with histological findings. This capability was demonstrated by the analysis of the liver from dogs dosed with discontinued drug compound B and its N-desmethyl metabolite, compound A. Histological examination showed multifocal hepatocellular necrosis, bile duct hyperplasia, periportal fibrosis, and chronic inflammation. MALDI-MSI analysis of liver tissue dosed with only compound A indicated that liver lesions were associated exclusively with the parent compound, whereas liver lesions with compound B showed the presence of the parent compound and its two metabolites (compound A and an N-oxide metabolite). Using both positive and negative ion modes, simultaneous detection and identification of endogenous molecular markers of the connective tissue, blood vessels, liver parenchyma, and bile duct epithelium was achieved, allowing optimal visualization of histological lesions by mass spectrometry imaging.

The β-amyloid precursor protein-cleaving enzyme 1 (BACE1) inhibitor JNJ-54861911, a candidate for the treatment of Alzheimer's disease, was withdrawn from clinical trials due to drug-induced liver injury (DILI). This paper describes our investigation of the metabolism of JNJ-54861911 to understand the potential contribution to the observed DILI. In human hepatocytes, JNJ-54861911 is metabolized by CYP450 3A4 to a reactive intermediate (RI), which undergoes glutathione (GSH) addition at C6 of the 2-amino-4-methyl-1,3-thiazin-4-yl moiety via glutathione S-transferase α1 (GSTA1) catalysis. Despite the preponderant role of CYP3A4 as an enabler, the adduct has the same level of oxidation as that of JNJ-54861911. The exact mechanism of RI formation might involve a sulfoxide (with further reduction) or tautomeric forms of JNJ-54861911 bearing a reactive thiazinium cation activating both the C2 and C6 positions. The cell pellet from the human hepatocyte incubated with ¹⁴C-JNJ-54861911 was analyzed via gel electrophoresis, resulting in the identification of a major protein adduct on GSTA1, a cross-link resulting from the addition of GSH and lysine 120 to JNJ-54861911, most likely on position C6 and on the nitrile, respectively. Ultimately, this major adduct might only account for 15-25% of the total covalent binding (CVB). Other important contributors to CVB might exist, like the bioactivation of the major diaminothiazine metabolite (DIAT). The level of covalent binding (CVB) burden (fraction of the dose resulting in CVB) is clearly below 1 mg/day, with a low daily dose of 25 mg. Despite this limited magnitude of CVB, it could still contribute to the liver enzyme elevations observed in approximately 20% of the patients and to the few cases of severe immune-mediated DILI. The latter could occur through a haptenization phenomenon and/or by inducing stress in hepatocytes. Such stress may activate an innate immune response, which, in turn, promotes the adaptive immune response.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit‐for‐purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

The administration of radiolabeled drug candidates is considered the gold standard in absorption, distribution, metabolism and excretion (ADME) studies for small molecule drugs, since it allows facile and accurate quantification of parent drug, metabolites and total drug related material independent of the compound structure. The choice of the position of the radiolabel, typically ¹⁴C or ³H, is critical to obtain relevant information. Sometimes a biotransformation reaction may lead to cleavage of a part of the molecule. As a result only the radiolabeled portion can be followed while information on the fate of the non-labeled metabolite may be lost. Synthesis and administration of two or more radiolabeled versions of the parent drug as a mixture or in separate studies may resolve this issue, but comes with additional challenges. In this paper we address the questions that may be considered to help make the right choice whether to use single or a multiple radiolabel approach, discuss pros and cons of different multiple labeling strategies that can be taken as well as alternative methods that allow the non-labeled part of the molecule to be followed. Significance Statement Radiolabeled studies are the gold standard in drug metabolism research, but molecules can undergo cleavage with loss of the label. This often results in discussions around potential use of multiple labels which seem to be occurring with increased frequency since an increasing proportion of the “small molecule drugs” are tending towards larger molecular weights. This review provides insight and decision criteria in considering a multiple label approach as well as pros and cons of different strategies that can be followed.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (i.v. PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as i.v. PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade.

Infrared ion spectroscopy (IRIS) can be used to identify molecular structures detected in mass spectrometry (MS) experiments and has potential applications in a wide range of analytical fields. However, MS-based approaches are often combined with orthogonal separation techniques, in many cases liquid chromatography (LC). The direct coupling of LC and IRIS is challenging due to the mismatching timescales of the two technologies: an IRIS experiment typically takes several minutes, whereas an LC fraction typically elutes in several seconds. To resolve this discrepancy, we present a heartcutting LC-IRIS approach using a setup consisting of two switching valves and two sample loops as an alternative to direct online LC-IRIS coupling. We show that this automated setup enables us to record multiple IR spectra for two LC-features from a single injection without degrading the LC-separation performance. We demonstrate the setup for application in drug metabolism research by recording six m/z-selective IR spectra for two drug metabolites from a single 2 μL sample of cell incubation extract. Additionally, we measure the IR spectra of two closely eluting diastereomeric biomarkers for the inborn error of metabolism pyridoxine-dependent epilepsy (PDE-ALDH7A1), which shows that the heartcutting LC-IRIS setup has good sensitivity (requiring ∼μL injections of ∼μM samples) and that the separation between closely eluting isomers is maintained. We envision applications in a range of research fields, where the identification of molecular structures detected by LC-MS is required.