This paper describes the metabolic transformation of the phosphodiesterase-4 inhibitor Roflumilast in thoroughbred horses after oral intake. Additionally, it presents in vitro experiments utilizing equine liver microsomes and Cunninghamella elegans. Plausible structures of identified metabolites were proposed through liquid chromatography-high resolution mass spectrometry. The data reported here could assist in detecting illicit uses of Roflumilast in competitive sports.

ABSTRACT

The phosphodiesterase 4 (PDE4) inhibitors constitute a relatively modern class of medications that are known for inducing bronchodilation and exhibiting anti-inflammatory properties within the body. Due to these properties, there is concern regarding their potential misuse as performance-enhancing substances in competitive sports. This study delves into the metabolic conversion of roflumilast in thoroughbred horses following oral administration and in vitro experimentation using equine liver microsomes and Cunninghamella elegans. High-performance liquid chromatography coupled with a Q Exactive Orbitrap mass spectrometer (HPLC-HRMS) was employed for analysis. The investigation identified 10 metabolites of roflumilast, including six phase I and four phase II metabolites from in vivo studies, and 11 metabolites from in vitro studies, consisting of eight phase I and three phase II metabolites. The identified biotransformation products encompassed processes such as hydroxylation, chlorine substitution, methylation, N-oxide formation, and even the dissociation of methylenecyclopropane and difluoromethane. Furthermore, the study identified three glucuronic acid and one sulfonic acid conjugated phase II metabolites of the investigated drug candidate. The aforementioned findings contribute to the detection and comprehension of the unauthorized utilization of roflumilast in equestrian sports.