Detection of clostebol in sports: accidental doping?

Code: 20C15XD

Clostebol (4-chloro-testosterone; 4-chloro-4-androsten-17-ol-3-one) is an anabolic androgenic steroid (AAS) derivative from testosterone. In humans, among the legitimate therapeutic indications of anabolic steroids, clostebol acetate is approved for topical use in dermatological and ophthalmological preparations. The esterification on the 17 position also permits its oral use, protecting the compound from an extensive firstpass metabolism. Clostebol has also been used in cattle to improve the animal growth. Due to its anabolic properties the International Olympic Committee (IOC) in the past and today the World Antidoping Agency (WADA) have included clostebol and the other AAS in the yearly renewed list of prohibited substances in sports[1]. The human [2,3] and animal[4] metabolism of clostebol allowed to establish adequate methods to 
detect its illicit use. The detection of clostebol intake is traditionally based on the detection of its main metabolite (4-chloro-4-androsten-3a-ol-17-one) excreted into urine glucuronoconjugated (Figure 1.). In order to improve the detection capabilities, antidoping laboratories have developed in the last decade methods based on gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS) lowering the limits of detections or have implemented methods for the direct investigation of phase II metabolites (sulphates) using liquid chromatography coupled to mass spectrometry (LC-MS/MS) [5,6]. The presence of clostebol metabolite in an athlete’s urine sample may be due to its illicit use as anabolic, the ingestion of contaminated meat[7] and finally some case reports describe the accidental contact with cream preparations containing clostebol[8]
. 
In the last years, the improvement of the detection capabilities of the antidoping laboratories has led to a moderate increase of clostebol detection worldwide, and especially in Italy where the use of a cream containing clostebol acetate and neomicine is quite extended. Trofodermin® is a pharmaceutical preparation containing 5% clostebol acetate and 5% neomicine sulphate that can be applied by cream or spray, that can be used for the following treatments: abrasions and erosions of the skin, injuries and wounds, such as varicose ulcers, due to poor blood circulation, bedsores (due to immobility in bed) sores or trauma, fissures (cuts) on the nipple (which can occur during breastfeeding), anal fissures (small cuts around the anus), burn wounds, infected wounds, wounds that delay to form the scar, irritation, redness, and sensitization of the skin that appears after radiotherapy (radiodermatitis), dryness, cracking or peeling of the skin with ulceration. According to the Italian law, a visible symbol on the packaging indicating the presence of a substance included in the WADA list of prohibited substances must be present. Although clostebol is prohibited by all administration routes, the aim of this study is to investigate the presence of clostebol metabolite in urine after an accidental contact with the substance due to the therapeutic application of clostebol acetate in a different individual.

Main findings

The accidental contamination of an individual after getting into close contact with another individual using transdermal clostebol acetate has been demonstrated after different degrees of exposure.This project aimed to search for specific clostebol metabolic markers or concentrations thresholds that may help to distinguish an illicit use of clostebol as AAS after an oral administration from an accidental contamination after a transdermal application, or to distinguish between a transdermal or oral administration, helping to establish adequate criteria to be adopted by the antidoping community. This kind of thresholds have been already applied for other groups of substances

The metabolism of clostebol after oral and transdermal applications has been extensively described using the more common tools used by WADA accredited Laboratories, gas chromatography coupled to mass spectrometry. Ten metabolites were detected after oral administration of which 5 could not be detected after transdermal application under the assay conditions here applied. A suitable criterion to disclose an oral (late excretion) from a transdermal application was achieve. The use of concentrations of any of the metabolite is quite difficult because the variability in the individual absorption, metabolism and/or excretion. Instead, the ratios between specific metabolites M4, M3 and M2 to M1 showed plausible results to discriminated between both administrations.

The proposed metabolic ratios have a fundamental limitation for being applied worldwide by all WADA accredited laboratories. The response ratios (even among isomers) depend on the signals (transitions) selected by every laboratory. To overcome this point the metabolites considered more diagnostic need to be synthetized to allow an estimation of the concentration that would be then independent from the analytical method applied. This would allow in addition to confirm definitively the configuration of the proposed metabolites. Some metabolites are excreted in urine only after oral administrations but their response is much lower and there are more potential structure fitting with the candidates proposed here. The sulfate fraction that was in previous communications considered as a potential solution to the problem presented, were not considered here since this is a fraction not used in ITP conditions. Based on the previous considerations, we propose M2, M3 and M4 as the best targets worth to be synthetized and characterized for this purpose.