Targeted metabolism and non-targeted plasma profiling in equine model

Abstract

Previous in-vitro studies on anabolic androgenic steroids (AAS) were performed by other horseracing clubs around the world. The studies demonstrated the extensive metabolism of hydrophobic designer steroids in the horse liver suggesting the fast elimination of the parent drug in-vivo. Hence, there is a potential challenge in detecting selected parent drug during targeted screening and further raises the pertinent question of whether some of the metabolites of AAS are pharmacologically active. It is therefore important to determine the appropriate analytes to monitor, like parent drug, metabolites or both. Due to ethical issues related to animal research, alternative approaches have to be used to study the metabolic stability and metabolite identification of AAS. There is also the constant challenge of detecting new AAS abuse in equine sports.

Our present study was designed to investigate both the non-targeted and targeted metabolic profiling approaches to bridge the gaps in the detection of doping agents. In-vitro experiments were carried out using horse liver microsomes to understand the metabolic stability and identify the metabolites of ethylestrenol, metribolone, ecdysone and 20-hydroxy ecdysone. Global profiling of equine plasma was carried out to differentiate the different gender of horses.

Ethylestrenol is a short acting AAS with progesterone-like activity and little androgenic activity. Ethylestrenol has been studied in-vivo in equine studies. In addition, in-vitro models in cattle and rats have also been investigated. From the small-scale synthesis experiment, it is clear that the unknown metabolite generated in horse liver microsomes is possibly associated with 17a-ethyl-5a-estrane-3?,17? diol. With the possible structure of the metabolite, large-scale synthesis experiments could be conducted and the product can be characterized. This metabolite could be explored as an analyte to be monitored in ethylestrenol abuse.

Metribolone is a potent synthetic AAS reported since the late 1960s. Androgen binding assays were done on metribolone and it was found to have high affinity for androgen receptors. However it was never marketed as an AAS due to its high liver toxicity. Information on metribolone metabolism is limited. Our data showed that metribolone metabolizes in horse liver microsomes to produce multiple metabolites. The major metabolite was elucidated to be 16-hydroxy metribolone. 16-hydroxy metribolone could be explored as an analyte to be monitored in metribolone abuse.

Ecdysone and 20-hydroxyecdysone are ecdysteroids which are insect-molting hormones present in various arthropods and plants. Their structures are related to cholesterol. They are known to increase protein synthesis upon administration. In addition, studies on 20-hydroxyecdysone have shown that it is capable of enhancing muscle growth without the negative effects of synthetic AAS. Ecdysteroid metabolism was previously studied in humans. Our data showed that there is no significant metabolism of ecdysone and 20-hydroxy ecdysone in horse liver microsomes and suggested that it might be more appropriate to monitor the parent drug in dope testing.

Global profiling of equine plasma obtained from horses of different genders was performed in the current study. As the chemometric models were not sufficiently robust, the results were not conclusive in differentiating the different genders of the horses. Nevertheless, the broad classification trends suggested that there might be unique metabotype associated with each gender. Further studies need to be performed to determine the baseline metabotype of each gender of the horses before the metabonomic approach can be tested in dope testing.