Investigation of Equine In Vivo and In Vitro Derived Metabolites of the Selective Androgen Receptor Modulator (SARM) ACP-105 for Improved Doping Control

Abstract

Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collected over a time period of 96 h. The in vivo samples were compared with five in vitro incubation models encompassing Cunninghamella elegans, microsomes and S9 fractions of both human and equine origin. The analyses were performed using ultra-high performance liquid chromatography coupled to high resolution Q Exactive^TM Orbitrap^TM mass spectrometry (UHPLC-HRMS). A total of 21 metabolites were tentatively identified from the in vivo experiments, of which several novel glucuronides were detected in plasma and urine. In hydrolyzed urine, hydroxylated metabolites dominated. The in vitro models yielded several biotransformation products, including a number of monohydroxylated metabolites matching the in vivo results. The suggested analytical target for equine doping control in plasma is a dihydroxylated metabolite with a net loss of two hydrogens. In urine, the suggested targets are two monohydroxylated metabolites after hydrolysis with β-glucuronidase, selected both due to prolongation of the detection time and the availability of reference material from the in vitro models.

Keywords: ACP-105; Cunninghamella elegans; SARM; Selective Androgen Receptor Modulator; doping; horse; mass spectrometry; metabolites; microsomes.

Figure 1

The structure of ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile).

Figure 2

MS/MS spectrum and suggested cleavage sites for the product ions of ACP-105 with the pre-cursor ion [C₁₆H₂₀ClN₂O]⁺ with m/z 291.1262. The suggested cleavage sites do not represent the final hydrogen distribution between the detected fragment and the neutral loss for all fragments, full information available in Table A1 in Appendix A.

Figure 3

Combined extracted ion chromatograms of the metabolites of ACP-105 fulfilling the set criteria from the UHPLC-HRMS analysis. A full description of the metabolites and their denotations can be found in Table A1 in Appendix A. (A) metabolites in plasma 12 h after administration; (B) metabolites in urine 24 h after administration and; (C) metabolites in hydrolyzed urine 24 h after administration. The metabolite labels represent the following metabolic transformations: monohydroxylation (M1), dihydroxylation (M2), loss of 2H (M3), loss of 2H and monohydroxylation (M4), loss of 2H and dihydroxylation (M5), loss of 2H and trihydroxylation (M6), glucuronidation (M7), monohydroxylation and glucuronidation (M8) and dihydroxylation and glucuronidation (M9).

Figure 4

Time profile of the metabolites from ACP-105 present in hydrolyzed urine. The metabolites M1a, M1c and M5b were detected up to 96 h. The figure shows the time from administration of ACP-105 (x-axis) and the chromatographic peak area (y-axis).

Figure 5

Suggested fragmentation pattern of the major metabolites in comparison with ACP-105. (a) Parent ACP-105—precursor ion [C₁₆H₂₀ClN₂O]⁺ at m/z 291.1262; (b) monohydroxylated metabolite M1a—precursor ion [C₁₆H₂₀ClN₂O₂]⁺ at m/z 307.1209; (c) monohydroxylated metabolite M1c—precursor ion [C₁₆H₂₀ClN₂O₂]⁺ at m/z 307.1209; (d) dihydroxylated metabolite with formed double bond M5b—precursor ion [C₁₆H₁₈ClN₂O₃]⁺ at m/z 321.1003. The exact position of the formed double bond within the aliphatic ring structure is not known, but it will change the previous chair formation; (e) monohydroxylated and glucuronidated metabolite M8a—precursor ion [C₂₂H₂₈ClN₂O₈]⁺ at m/z 483.1532; (f) monohydroxylated and glucuronidated metabolite M8d—precursor ion [C₂₂H₂₈ClN₂O₈]⁺ at m/z 483.1530.

Figure 6

Extracted ion chromatograms of monohydroxylated ACP-105 [C₁₆H₂₀ClN₂O₂]⁺ that present the metabolites detected in hydrolyzed urine, C. elegans incubates and incubates of equine and human microsomes and S9 fractions. In hydrolyzed urine, the peak at 8.65 min represents metabolite M1a, the peak at 9.01 min represents the metabolite M1b and the peak at 9.14 min represents the metabolite M1c. The figure shows the retention time in minutes (x-axis) and the relative mass spectrometric intensity (y-axis).

Figure 7

The proposed structures and location of metabolic transformations of the major metabolites from ACP-105. The information within the brackets indicates if they are present in plasma (P) urine (U), hydrolyzed urine (H.U) and incubates with C. elegans (C.E), equine microsomes (E.M), equine S9 fractions (E.S), human microsomes (H.M) and human S9 fractions (H.S). For metabolite M5b, the exact position of the double bond within the aliphatic ring structure is not known.