Emerging anabolic drugs - investigation of the in vitro and in vivo metabolism of selective androgen receptor modulators 

  • Anna Holderbaum

Student thesis: Doctoral ThesisDoctor of Philosophy


Selective androgen receptor modulators (SARMs) are a novel class of orally active, tissue-selective small molecule androgen receptor ligands with potential use for human therapeutic purposes. While SARMs show similar myoanabolic effects to anabolic androgenic steroids with reduced side effects, their exact underlying molecular mechanisms remain unclear. Their abuse has been reported in human and animal sports and further potential for abuse exists in livestock species. SARMs abuse therefore poses a threat to sports and food integrity as well as public health. To advance forensic testing of illicit use of SARM compounds in several relevant species and better understand their physiological impact, this thesis studies the in vitro and in vivo metabolism of key SARM compounds.

To evaluate the use of liver homogenates as an in vitro tool to rapidly produce SARM metabolites, cattle liver homogenates, S9 fractions, and microsomes were isolated, characterised (protein concentration, P450 and cytochrome b5 content, enzymatic activity), and incubated with SARM compounds ostarine, andarine, or S-1 and respective cofactors to generate phase I and II metabolites followed by ultra-high performance liquid chromatography- quadrupole time of flight mass spectrometry (UHPLC-QTOF MS) analysis. All fractions produced metabolites that have previously been described as important target analytes of the investigated SARM compounds, demonstrating that the proposed simple and low-cost liver homogenate approach holds potential for use in the rapid development of metabolite-based screening methods.

Further, the in vitro metabolism of key SARM compounds across several relevant sports (horse), livestock (cattle, pig) and laboratory (rat) species, and the correlation of in vitro to in vivo generated metabolites was investigated. After isolation and characterisation of the liver fractions, microsomes alone or in combination with S9 fractions were incubated with SARM compounds ostarine, LGD-4033, or RAD140 to generate phase I and II metabolites respectively. To characterise in vivo generated metabolites, urine samples were collected after initial and repeated administration of ostarine, LGD-4033, or RAD140 to rats. Prepared in vitro and in vivo samples were analysed by UHPLC-ion mobility-QTOF MS. Interspecies differences within determined in vitro metabolite profiles were observed, highlighting the necessity of studying the metabolism of emerging anabolic agents such as SARMs on a by-species basis. The majority of detected urinary metabolites were also produced in vitro. While RAD140 was metabolically relatively stable, ostarine and LGD-4033 were extensively metabolised in vitro in all investigated species as well as in vivo - Subsequently, cytochrome P450 (CYP) isoenzymes responsible for the formation of ostarine and LGD-4033 phase I metabolites using cattle, horse, pig, or rat liver microsomes were identified using a CYP-selective inhibitor reaction phenotyping approach followed by UHPLC-tandem mass spectrometry (UHPLC-MS/MS). Bovine, equine, murine, and porcine orthologues of human CYP2A6, CYP2B6, CYP2C8, CYP2C9 (only ostarine), and CYP3A4 were implicated in metabolism of ostarine and LGD-4033 across species.

To expand on the liver-based in vitro tools employed in this thesis, the metabolism of SARM compounds ostarine, LGD-4033, and RAD140 by multiple equine tissue microsomal and S9 fractions from liver, lung, kidney, and small intestines was investigated by UHPLC-QTOF analysis. Although it was shown that SARMs are not only metabolised by hepatic but also extrahepatic equine tissues, no additional metabolites were produced by extrahepatic tissue fractions.

Administration of SARM compounds ostarine or LGD-4033 to rats at sub-toxic levels for 17 days was shown (via two-dimensional difference gel electrophoresis (2-D DIGE) to identify differential protein levels with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis to cause a response to the hepatic proteome. Subsequent differential centrifugation of livers into subcellular fractions (nuclei, mitochondria, cytosol, and microsomes) and 2-D DIGE analysis enabled detection of more subtle changes to the protein levels in response to SARM treatment. While all investigated SARM compounds triggered changes to 12 protein spots, each compound exhibited a unique response profile. Pathway enrichment analysis revealed that identified proteins were mainly involved in metabolic processes related to amino acid, carbohydrate and energy metabolism. Several proteins involved in protein processing within the endoplasmic reticulum had elevated differential abundances suggesting a potential cellular stress response following SARM treatment.

This thesis describes in vitro strategies and in vitro/in vivo metabolite profiles of key SARM compounds in sports, livestock and laboratory species and the in vivo effects of SARMs on hepatic protein levels. It is anticipated that the results will help to inform SARM metabolite-based screening approaches in veterinary species and provide a better understanding of SARMs effects on liver as a key metabolic tissue.
Date of AwardJul 2020
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SponsorsEC-Horizon 2020
SupervisorMark Mooney (Supervisor) & Christopher Elliott (Supervisor)

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