Doping control in horseracing : pharmacokinetics and metabolic studies of prohibited substances/drugs in horses

Abstract

Regulatory authorities have been employing advanced technologies to tackle the doping trends observed in human and animal sports. In both equestrian sports and the horseracing industry, the fight against the misuse of prohibited substances (PS) and/or drugs is crucial, as these could distort horse performance and impact competition outcomes. Furthermore, certain medications may be misused to mask illness or injury, posing serious risks to the health and welfare of horses. To effectively manage this issue, it is essential to have a comprehensive understanding of drug metabolism and pharmacokinetics (DMPK) in horses, which is critical for identifying the proper targets for effective control. Unfortunately, the metabolic fate of many drugs in horses remains unclear, highlighting the need for extensive DMPK studies on substances with potential doping effects. Biotransformations of several potential doping agents, including estra-4,9-diene-3,17-dione, 2-hydroxyethyl salicylate and ranitidine, were studied and reported in this thesis. Anabolic androgenic steroids (AASs) are one of the prevalent classes of drug with high doping potential in equine sports. Estra-4,9-diene-3,17-dione (dienedione), an AAS marketed as a dietary supplement for bodybuilding, is prohibited in both human and equine sports due to its potential performance enhancing effect. With the rare presence of 4,9-diene configuration in endogenous steroids, dienedione has been considered as a synthetic AAS. However, the recurring detection of dienedione in entire male horse urine samples has led to the investigation of its possible endogenous nature and conjugation in entire male horses. Whilst dienedione remains exogenous in castrated horses, it could be reported with zero-tolerance once detected. To control the illicit use of dienedione in castrated horses, it is essential to study its in vivo metabolism and elimination. In chapter 2, the potential endogenous nature of dienedione in entire male horses was studied and confirmed. Dienedione-3-glucuronide was further proposed to be the major form of dienedione in entire male horse. An in-house threshold at 30 ng/mL of free and glucuronide-conjugated dienedione in entire male horse urine was established, with a risk factor of 1 in 14,269 (with a degree of freedom of 173). In chapter 3, the in vivo metabolites detected in castrated horse after dienedione administration included 17-hydroxyestra-4,9-dien-3-one (M1a and M1b), hydroxylated dienedione (M2a, M2b, M3a, M3b, M4, M5) and hydroxylated M1 (M6a, M6b, M7a, M7b, M8a and M8b), formed from hydroxylation and reduction of dienedione were presented. Metabolite M3a and M3b were identified as the appropriate target to monitor misuse of dienedione, as it gave the longest detection time. They could be detected for up to 2–5 days in urine and 0.4–4 days in plasma after the horses were given the substance. 2-Hydroxyethyl salicylate (2HES), a non-steroidal anti-inflammatory drug (NSAID), is commonly used to treat musculoskeletal injuries and inflammation in both humans and horses. Its misuse could affect the performance of horses and mask injuries. In horseracing, its use is considered an adverse finding when detected in competition. The metabolism of 2HES has not been reported in either humans or horses, leaving its metabolic fate unknown. In chapter 4, the proposed in vivo metabolites including glucuronide-conjugated 2HES (2HES-Glu) and sulfate-conjugated 2HES (2HES-SO4) resulting from phase II conjugation, likely at the hydroxyethyl group, and salicylic acid (SA) formed by the hydrolysis of 2HES were described. The parent drug, 2HES, was identified as the most suitable target for monitoring its potential misuse, as it was detectable in hydrolysed urine for up to 10 days and in plasma for up to 16 hours. Since the concentration of SA in post-administration urine and plasma samples did not exceed the corresponding international thresholds, monitoring SA levels is not an effective indicator of 2HES exposure. Ranitidine, a histamine H2-receptor antagonist, was widely used to treat gastric ulcers in horses. Therapeutic substances like ranitidine are also required to be controlled under racing regulations to ensure a level playing field. Hence, in chapter 5, the in vivo metabolism and elimination of ranitidine in horses were studied to support its effective control, with the potential to establish a screening limit that enables laboratories to report this therapeutic agent consistently. The in vivo metabolites, namely ranitidine-S-oxide (M1), ranitidine-N-oxide (M2), desmethylranitidine (M3a/b), and a furoic acid analogue of ranitidine (M4), resulting from oxidation, demethylation, and oxidative deamination, were identified. To monitor the potential misuse of ranitidine in horses, elimination profiles for urinary and plasma ranitidine were established. Free ranitidine was detectable for up to 8 days in urine and up to 72 hours in plasma. Additionally, ranitidine-S-oxide and ranitidine-N-oxide were also detectable for 8 days, making them potential screening targets alongside the parent drug to confirm that ranitidine has gone through the horse’s body. With the intended detection window, the authorities could adopt a suitable screening cut-off for its control.