In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse

Abstract

Hemapolin (2α,3α‐epithio‐17α‐methyl‐5α‐androstan‐17β‐ol) is a designer steroid that is an ingredient in several “dietary” and “nutritional” supplements available online. As an unusual chemical modification to the steroid A‐ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC‐MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α‐methyl‐5α‐androst‐2‐en‐17β‐ol), reduced and dihydroxylated madol (17α‐methyl‐5α‐androstane‐2β,3α,17β‐triol), and the isomeric enone metabolites 17β‐hydroxy‐17α‐methyl‐5α‐androst‐3‐en‐2‐one and 17β‐hydroxy‐17α‐methyl‐5α‐androst‐2‐en‐4‐one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell‐based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC‐MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.

Sulfated and Sulfur-Containing Steroids and Their Pharmacological Profile

“…Steroids containing an epithio group in positions 2 and 3 belong to anabolic steroids and are widely known and used in sports medicine and are of great interest for the pharmacology of sports and other aspects of medicine [ 149 , 150 , 151 , 152 , 153 , 154 ]. The most widely known are such epithio steroids that are used in sports pharmacology and medicine: epistane (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol), epitiostanol (2α,3α-epithio-5α-androstan-17β-ol, known as potent anti-estrogenic and antitumor agent), hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol), mepitiostane (epitiostanol 17β-methoxy-cyclopentyl ether), epivol (2α,3α-epithio-17α-methylethio-allo-cholanol), epivol black (2,3α-epithio-17α-methyl-5α-androstan-17β-ol), and straight epi (2,3α-epitio-17α-methyl-etioallocholane-17β-ol) [ 149 , 150 , 152 , 155 , 156 , 157 , 158 ].…”

Nontargeted detection of designer androgens: Underestimated role of in vitro bioassays

“…22,57 Notably, the metabolites may have more robust, similar, or weaker potency than their parent compound. 63 The human kidney, liver, and bone cell formats are all relevant to androgen doping interpretation, with bone cells providing some evidence of an anabolic effect and the kidney and liver cells suggesting a metabolic conversion profile. Skeletal muscle cells as host cells may also provide anabolic effect information of new designer steroids or SARMs; however, to date, there is no valid immortalized cell model for human skeletal muscle.…”

“…Hemapolin is subject to steroid metabolism, and subsequent tests showed similar strong potency of several metabolites. 63 The versatility of in vitro cell bioassays, however, is best demonstrated by studies showing their application to the detection of a wide variety of AAS 59 and designer steroids. 24 Indeed, the fact that all androgens, whether natural, synthetic, or a SARM, share a common mechanism of action underlies the underestimated value of in vitro bioassays.…”

“…These differences in metabolism capacity between mammalian cell types help explain differences between assays 22,57 . Notably, the metabolites may have more robust, similar, or weaker potency than their parent compound 63 . The human kidney, liver, and bone cell formats are all relevant to androgen doping interpretation, with bone cells providing some evidence of an anabolic effect and the kidney and liver cells suggesting a metabolic conversion profile.…”

“…In another study, this same yeast androgen bioassay demonstrated the strong androgenic potency of the designer drug, hemapolin. Hemapolin is subject to steroid metabolism, and subsequent tests showed similar strong potency of several metabolites 63 . The versatility of in vitro cell bioassays, however, is best demonstrated by studies showing their application to the detection of a wide variety of AAS 59 and designer steroids 24 …”