In vivo and in vitro metabolism of the designer anabolic steroid furazadrol in thoroughbred racehorses

The artificial increase of the physical capability of horses using drugs is well known in racing and other equine sports. Both illicit and therapeutic substances are regarded as prohibited substances in competition in most countries. Some countries make distinctions for a few, specific drugs which are, however, allowed for use in other countries. The primary objective in the case of doping control is the detection of any trace of drug exposure, either parent drug or any of its metabolites, using the most powerful analytical methods which are generally based on chromatographic/mass spectrometric techniques. Of major concern in horseracing is the absence of a single organization regulating the anti-doping framework; instead of this, individual racing authorities provide rules and regulations often resulting in variations in the applied doping control programmes of different countries. The aim of this paper is to review the recent literature (approximately from 2012 to mid-2016) to highlight the numerous and diverse challenges faced in doping control of racing and equestrian sports, including the detection of designer drugs (anabolic steroids or stimulants) and of other emerging prohibited substances, such as peptides and noble gases in horse urine and plasma. Moreover, the application of 'omics' techniques (especially of metabolomics) deserves attention for establishing possible fingerprints of drug abuse as well as the evolution of instrumental analysis resulting a powerful ally in the fight against doping in equine sports.

Section: Nickelmentioning

confidence: 99%

Challenges in detecting substances for equine anti‐doping

Fragkaki

Kioukia‐Fougia

Kiousi

et al. 2017

“…To model the liver, which is the principle organ involved in detoxification and metabolism of exogenous substances, preparations involving liver metabolic enzymes are amongst the most commonly used in vitro methods . Equine liver microsomes and S9 fraction are most commonly used to model equine metabolism due their ease of use and commercial availability.…”

Section: In Vitro Equine Steroid Metabolismmentioning

confidence: 99%

“…Typically, the reaction of glucose‐6‐phosphate with a glucose‐6‐phosphate dehydrogenase (G6PDH) enzyme is used, which regenerates NADH/NADPH as a by‐product of oxidation. This allows for the use of a catalytic amount of these co‐factors in the metabolism reaction . Recent reports have also demonstrated the practicality of using homogenized whole liver tissue to perform in vitro studies, in an effort to closely replicate in vivo metabolism .…”

Section: In Vitro Equine Steroid Metabolismmentioning

confidence: 99%

“…In contrast, LC‐MS analysis has the advantage of not requiring these preparatory steps allowing much higher throughput in sample analysis. Additionally, LC‐MS allows the direct detection of intact phase II conjugates, which may offer advantages in the detection of some steroid compounds which are primarily excreted as these conjugates and that may otherwise only be detected indirectly by GC‐MS methods after hydrolysis and derivatization . LC‐MS is not a complete replacement for GC‐MS however, as the analysis of some compounds is difficult with LC‐MS, including the study of saturated steroids and steroid diols, which ionize poorly under the electrospray ionization (ESI) and atmospheric‐pressure chemical ionization (APCI) conditions common to LC‐MS systems .…”

Section: Anti‐doping Screening For Designer Anabolic Steroidsmentioning

confidence: 99%

“…Due to ethical concerns over the potential detrimental effects to the health and welfare of animal subjects, in vitro techniques are rapidly gaining attention as tools to study the metabolism of these compounds. These techniques, as highlighted by multiple examples throughout this review, are rapidly improving, and can more faithfully generate a metabolic profile representative of in vivo systems which will greatly assist anti‐doping analysis . Currently, AORC criteria allow for the use of in vitro ‐derived materials as standards for confirmatory analysis, and improvements to in vitro techniques may allow for the direct detection and confirmation of in vivo phase II metabolites by LC‐MS/MS methods …”

Section: Future Directionsmentioning

confidence: 99%

See 2 more Smart Citations

A review of designer anabolic steroids in equine sports

Waller

McLeod

2016

Self Cite

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in 'dietary' or 'nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols.

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate

Weththasinghe

Waller

Fam

et al. 2017

Self Cite

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na SO providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17β-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.