In vitro metabolic profiling of weight-loss-inducing amylin receptor agonists in the context of preventive doping research.
This study investigated the metabolic profiles of three amylin receptor agonists — pramlintide, cagrilintide, and KBP-066 — in the context of sports anti-doping research. Motivated by growing concerns about misuse of weight-loss peptide hormones in athletic disciplines where weight management is performance-relevant, researchers used comprehensive in vitro models (human skin and kidney S9 fractions, biological fluids) to characterize how these compounds are broken down. High-resolution tandem mass spectrometry (HRMS/MS) was used to identify metabolites, and authentic post-administration rat plasma samples were analyzed for cagrilintide to assess in vivo relevance. The study found that all three peptides underwent N-terminal and C-terminal degradation, producing multiple stable metabolites considered suitable as analytical detection targets. Metabolites predicted from in vitro experiments for cagrilintide were confirmed in rat plasma samples. The researchers developed and validated an LC-MS/MS-based detection method applicable to anti-doping screening. Limitations include the primary reliance on in vitro models; the only in vivo data came from rat samples, not humans. This represents the first systematic metabolic characterization of these three compounds in an anti-doping context and lays groundwork for future human monitoring programs.
Why this grade: The study relies predominantly on in vitro human tissue models with no human in vivo administration; the only biological in vivo data come from rat plasma samples, precluding direct evidence about human metabolism or effects.
Peptide hormone-based weight-loss therapeutics have gained increasing attention, driven amongst other reasons, by the clinical and commercial success of semaglutide. Their increasing accessibility raises concerns about their potential misuse in sports, especially in disciplines where weight management is decisive for athletic performance. Semaglutide has been included in the World Anti-Doping Agency's monitoring program since 2024. Given that amylin signalling is a key therapeutic target for next-generation weight-loss drugs, amylin receptor agonists such as pramlintide, cagrilintide and KBP-066 also warrant consideration as to metabolism and detection strategies in sports drug testing programs. This study aimed to characterize the metabolic profiles of pramlintide, cagrilintide and KBP-066, identify analytically suitable metabolites and develop and validate a LC-MS/MS-based detection approach. Comprehensive in vitro models, including human skin S9 fraction, kidney S9 fraction and biological fluids, were used to investigate metabolic pathways. HRMS/MS was employed to characterize metabolites and evaluate their suitability as analytical targets. For comparison, authentic post-administration rat samples were analysed for cagrilintide and respective biotransformation products. All three peptides underwent N-terminal and C-terminal degradation, yielding multiple stable metabolic products suitable as detection targets. Cagrilintide metabolites predicted from in vitro experiments were observed in authentic post administration rat plasma samples, confirming in vivo relevance. Finally, suitable preparation and detection methods were established and validated. This study provides the first systematic metabolic characterization of pramlintide, cagrilintide and KBP-066. The identified metabolites and LC-MS/MS detection approach offer a foundation for future monitoring of emerging weight-loss peptide hormone analogues in anti-doping contexts.
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