Frank Klont

The extent of pharmacogenetic (PGx) drug dispensing among Dutch adults receiving medications for cardiovascular disease (CVD) is unknown. Using the University of Groningen IADB.nl pharmacy database, we performed a serial cross‐sectional study (2019–2023) to estimate the annual prevalence of PGx drug dispensing and annual rates of initiation. We also identified the most dispensed PGx drug classes, most frequently associated genes, and the proportion of individuals with potential opportunities for reuse of PGx testing results. Adults on CVD medication treatment were defined as those with ≥2 prescriptions for the same CVD medication (ATC classes B01, C01, C03, C07–C10) within 180 days in a calendar year. PGx drugs were defined as drugs with actionable drug–gene interactions according to international CPIC or national DPWG guidelines. The annual cohort size ranged from 47,602 in 2019 to 41,846 in 2023 (mean age ~70 years; ~48% male). The prevalence of ≥1 PGx drug dispensing (~90%) and multiple (≥2) PGx drug dispensing (~68%) remained stable across 5 years, both increasing significantly with age. CVD‐related PGx drugs were most common (75%), followed by proton pump inhibitors (55%). In 2023, CYP2C19, CYP2D6, and SLCO1B1‐associated drugs had most users (60%, 53%, and 51%). Among those receiving drugs associated with CYP2C19, CYP2D6, and SLCO1B1, 24%, 23%, and 4% would have opportunities for reuse of testing results with single‐gene testing, and 66%, 65%, and 83% with panel testing. In conclusion, PGx drug dispensing was highly prevalent among people receiving CVD medications, warranting further research into the (cost‐)effectiveness of PGx testing.

Purpose: Protein kinase inhibitor (PKI) therapy increasingly relies on personalized approaches such as therapeutic drug monitoring and pharmacogenetic testing. Understanding PKI metabolism is essential for these methods to be effective, notably for determining the substance to be quantified and the enzyme for which the genotypic metabolizer status needs to be predicted. For most drugs, however, human in vivo metabolite profiles are derived from the mass balance studies conducted by drug developers, typically in small and homogeneous populations. Methods: We studied mass balance study characteristics for PKIs, assessing number of participants, sex, and health state, as well as dosing regimen, study location, study duration, and consideration of genetic variation in metabolic enzymes and transporters. Data were primarily extracted from European Public Assessment Reports, supplemented with information from the Drugs@FDA database, ClinicalTrials.gov, and peer-reviewed mass balance study publications. Results: Mass balance studies were conducted for 68 out of the 69 PKIs listed as “authorized” human drugs in the European Medicines Agency database on July 7, 2025 (updated April 18, 2026; +3 PKIs). The studies included 2–12 participants (median = 6), with 90% enrolling only healthy volunteers, 88% enrolling only males, 97% administering a single dose, and four studies mentioning consideration of genetic variation in metabolic enzymes during participant inclusion. Conclusion: PKI mass balance studies exhibit similar, rather homogeneous design characteristics, potentially limiting the generalizability of their insights into drug metabolism across diverse patient populations. Still, most align with recent FDA guidelines on mass balance studies, despite being issued after study conduct.

Environmental scientists are increasingly monitoring therapeutic drugs and their metabolites in water systems, requiring knowledge of human drug metabolism and excretion. Many published studies, however, rely on data from small-scale human metabolism trials, typically involving around six (healthy, young, male) volunteers. Their generalizability to real-world drug users may be limited, potentially biasing environmental monitoring efforts. Here, we leveraged untargeted LC-SWATH/MS pharmacometabolomics data from 283 potential living kidney donors and 688 kidney transplant recipients to characterize the 24 h urinary excretion profiles of two widely used diuretics frequently monitored in wastewater, hydrochlorothiazide and furosemide. Both are expected to be excreted largely unchanged, which our analyses confirmed. For hydrochlorothiazide, however, we also identified (using reference standards) the previously underreported transformation products chlorothiazide and salamide. These findings highlight the relevance and capability of using untargeted metabolomics data from human excreta to provide insights from large, real-world cohorts into which chemicals enter wastewater systems, with both drugs serving as exemplary case studies for analogous analyses of other drugs. In particular, the qualitative information obtained (e.g., accurate mass, retention time, fragment spectra) may inform targeted biomonitoring and highlight cases where consensus-based estimates of excreted drug or metabolite fractions are overestimated.

Background/Objectives: Clopidogrel is a widely prescribed antiplatelet prodrug that requires bioactivation, primarily by the polymorphic CYP2C19 enzyme. Genetic variation in this enzyme leads to differences in active metabolite formation and has prompted the development of pharmacogenetics-guided prescribing. However, current pharmacogenetic strategies are grounded in drug metabolism knowledge derived from mass balance studies conducted in small groups of healthy volunteers. This narrow evidence base may limit the data’s applicability to real-world settings, where factors like polypharmacy or altered organ function may influence drug response. Methods: Pharmacogenetics could benefit from real-world drug metabolism and excretion studies, which we conducted for clopidogrel in 38 kidney and 16 liver transplant recipients from the TransplantLines Biobank and Cohort Study (NCT03272841), utilizing existing LC-SWATH/MS pharmacometabolomic data. Clopidogrel-associated metabolic signals were identified using xenobiotic metabolism knowledge and literature-reported pathways. Results: Across both transplant groups, 26 clopidogrel-associated features were prioritized, of which some matched previously reported urinary metabolites, had previously been observed in plasma, or represented previously unreported metabolites. Clopidogrel carboxylic acid predominated in kidney transplant recipients, whereas its glucuronide form was most abundant in liver transplant recipients. Notably, unmetabolized clopidogrel was consistently detected across all patients. Moreover, our data support a thiol desulfurization route, aligning with emerging evidence of clopidogrel’s role as a hydrogen sulfide-releasing drug. Conclusions: More (putative) clopidogrel metabolites were detected than previously reported, demonstrating the value of pharmacometabolomics in expanding our understanding of drug metabolism. This approach provides novel data that may complement pharmacogenetics research to understand clopidogrel response variability among treated patients.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease predominantly of the small airways and parenchyma. COPD lungs exhibit an influx of circulating innate immune cells, which, when isolated, display impaired functions, including imbalanced protease secretion. In addition to immune cells, the extracellular matrix (ECM) plays a crucial role in COPD pathology. Remodeling of the ECM can generate ECM fragments, which can be released into circulation and subsequently induce pro-inflammatory responses. COPD is a heterogeneous disease, and serological biomarkers can be used to sub-categorize COPD patients for targeted treatments and optimal recruitment in clinical trials. This study evaluated fragments of calprotectin, collagen type VI, and versican, generated by neutrophil elastase and matrix metalloproteinases (MMP-) 2 and 12, respectively, as potential biomarkers of COPD disease, severity, and endotypes. Lower plasma levels of a neoepitope marker of calprotectin, indicative of activated neutrophils (nordicCPa9-HNETM), were detected in COPD donors compared to controls. CPa9-HNE was associated with milder disease, higher degree of air-trapping, and higher serum levels of MMP-2. Deposition of CPa9-HNE levels in lung tissue revealed no differences between groups. Taken together, CPa9-HNE was found to be a potential marker of mild COPD, but further studies are warranted to validate our findings.

Background/Objectives: The increased use of antibiotics is raising concerns about environmental contamination and antibiotic resistance, exemplified by the case of cotrimoxazole, a widely prescribed combination of sulfamethoxazole and trimethoprim. After oral administration and absorption, both drugs are excreted in their parent and metabolized forms, which is a factor that is commonly considered in environmental studies. Many studies, however, rely on pharmacokinetic data from drug developers, who mostly investigate drug metabolism in healthy male volunteers rather than in actual patient populations. Methods: We investigated the real-world metabolism and urinary excretion of cotrimoxazole in an LC-SWATH/MS-based pharmacometabolomics study of 149 kidney transplant recipients who took part in the TransplantLines Biobank and Cohort Study (NCT0327284). Results: Our study confirmed (as “putatively characterized compound classes”) the presence of all the expected metabolites, and we (putatively) identified several previously unreported metabolites, including glucuronide conjugates of both drugs and two isoxazole ring-opened variants of sulfamethoxazole. The relative metabolite profiles furthermore indicated that the active drug trimethoprim accounted for 75% of the total signal intensity. For sulfamethoxazole, its acetylated metabolite was the main metabolite (59%), followed by the active parent drug (17%) and its glucuronide (7%). Alongside trimethoprim, these substances could serve as analytical targets for environmental cotrimoxazole monitoring, given their abundance (all three substances), activity (parent drug), and/or back-transformation potential (both conjugated metabolites). The isoxazole ring-opened variants (2–3%) may also warrant attention, considering their (presumed) absolute excreted quantities and potential pharmacological activity. Conclusions: This study underscores the value of pharmacometabolomics in elucidating real-world metabolite profiles, and it provides novel insights into cotrimoxazole metabolism and excretion, with implications for environmental and clinical monitoring.

Background and Objective Current immunosuppressive treatment to prevent graft rejection in organ transplant recipients commonly includes mycophenolate mofetil (MMF) and a calcineurin inhibitor. After absorption, MMF is activated to mycophenolate (MPA) by the carboxylesterase (CES) enzymes, which is considered to occur rapidly and completely. Recent research utilizing pharmacometabolomics (PMx), however, identified an unknown/unreported MMF glucuronide metabolite in several kidney transplant recipients (KTR). This finding indicates incomplete MMF prodrug activation by CES, thereby suggesting enzyme saturation and/or inhibition, which warrants further study. In this work, we aimed to identify clinical factors that could (partially) explain incomplete MMF activation as observed in KTR. Methods We analyzed untargeted urinary PMx data to determine MMF prodrug activation in 321 KTR from the TransplantLines Biobank and Cohort Study (NCT03272841) and 403 KTR from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT02811835). Beta regression was used to associate incomplete MMF activation with clinical parameters. Subsequently, in vitro experiments using human S9 liver extracts were performed to compare the influence of potential CES inhibitors on MMF activation. Results Beta regression linked an impaired MMF activation with increasing MMF dose and kidney function as well as with cyclosporine (CsA) use. Regarding the latter, in vitro experiments revealed a decreased MMF activation caused by the pharmaceutical excipient Kolliphor^® EL, which is present in CsA capsules, rather than by CsA itself. Conclusion Substantially reduced MMF prodrug activation was observed in large numbers of KTR, indicating relevant attenuation of the MMF-converting CES enzymes, which may be due to enzyme saturation and inhibition. However, there may be other factors affecting MMF activation, which require elucidation to improve immunosuppression therapy.

Background and Objective Metoprolol is primarily metabolized via the polymorphic cytochrome P450-2D6 (CYP2D6) enzyme, which underlies interindividual variation in conversion rates and may benefit from pharmacogenetics-driven therapy personalization. However, the field relies heavily on knowledge of a drug’s metabolism, often originating from early-phase clinical trials with single-dose administration in small samples of healthy volunteers. Pharmacogenetics could thus benefit from real-world drug metabolism studies. Methods We conducted a real-world drug metabolism study for metoprolol in 18 (potential) living kidney donors and 374 kidney transplant recipients from the Transplant Lines Food and Nutrition Biobank and Cohort Study (NCT02811835) using existing liquid chromatography–high resolution mass spectrometry pharmacometabolomic data. Results In both groups, we confirmed the presence of seven expected metabolites, including the high-abundance substances metoprolol acid and hydroxymetoprolol. We were unable to detect deisopropylmetoprolol and a metabolite known as “H 119/68”. However, we did find putative further oxidized forms, namely the expected variant of deisopropylmetoprolol in which the primary amine is removed and the leftover methyl group is oxidized into a carboxylic acid (“H 104/83”) and an unknown/unreported metoprolol metabolite that we refer to as “metoprolol benzoic acid”. Moreover, we found nine other previously unknown/unreported metabolites, putatively reflecting N-glucuronidated metoprolol, four glucuronidated versions of hydroxymetoprolol, and a formylated, a glucuronidated, and two hydroxylated versions of metoprolol acid. Interestingly, the same metabolites were detected in potential living kidney donors and kidney transplant recipients, and metabolite profiles did not differ between both groups in principal component analysis. Conclusion We found more metoprolol metabolites than previously reported, calling for replication studies and evaluation of pharmacogenetic testing approaches to realize safer, more effective metoprolol therapy.