Metabolic studies of formestane in horses

Abstract

Formestane (4-hydroxyandrost-4-ene-3,17-dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4-hydroxytestosterone (M1), 3β,4α-dihydroxy-5β-androstan-17-one (M2a), 3β,4β-dihydroxy-5β-androstan-17-one (M2b), 3β,4α-dihydroxy-5α-androstan-17-one (M2c), androst-4-ene-3α,4,17β-triol (M3a), androst-4-ene-3β,4,17β-triol (M3b), and 5β-androstane-3β,4β,17β-triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post-administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post-administration urine samples. In addition, 3α,4α-dihydroxy-5α-androstan-17-one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4-hydroxytestosterone (M1) in the glucuronide-conjugated fraction. M1 could be detected for up to 34 h post-administration. In blood samples, the parent drug could be detected for up to 34 h post administration.

Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry

“…The extraction and analysis protocols used in this study for the in vitro biotransformation and the in vivo metabolic studies of ATD in urine have been established for many years and successfully applied in the authors' laboratory to two other steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) [6], and formestane [7], and a number of oral anabolic steroids. In general, the metabolites of the studied steroids were identified by GC/MS analysis as trimethylsilyl derivatives.…”

“…Furthermore, aromatase inhibitors can minimise the side effects of anabolic steroids misuse [5]. Steroidal aromatase inhibitors, such as androst-4-ene-3,6,17-trione (6-OXO) [6], formestane [7] and androsta-1,4,6-triene-3,17-dione (ATD) [8], are marketed as nutritional supplements and are readily available via the internet.…”

A review of designer anabolic steroids in equine sports

“…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.…”

“…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 .…”

Doping control analysis of anabolic steroids in equine urine by gas chromatography‐tandem mass spectrometry

“…The effective screening of anabolic steroids in doping control is critical, as according to the recently published Anti‐Doping Testing Figures Report by WADA, anabolic agents are still the most reported misused prohibited substances amongst all substance groups. The conventional method for the detection of anabolic steroids in biological samples is by the use of gas chromatography‐mass spectrometry (GC‐MS) . Liquid chromatography‐mass spectrometry (LC‐MS) has become a compatible technique for doping control testing in sports in recent years as LC‐MS has the ability to handle heat labile and large biomolecules, with faster instrument turnaround time and better sensitivity .…”