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Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4
Journal of Equine Veterinary Science 32 (2012) 562-568
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4 Oliver Krug MSc a, Andreas Thomas PhD a, Simon Beuck MSc a, Ina Schenk PhD a, Marc Machnik PhD a, Wilhelm Schänzer PhD a, Ulf Bondesson PhD b, c, Mikael Hedeland PhD b, c, Mario Thevis PhD a a b c
Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf, Cologne, Germany Department of Chemistry, Environment and Feed Hygiene, National Veterinary Institute (SVA), Uppsala, Sweden Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2011 Received in revised form 12 December 2011 Accepted 20 January 2012 Available online 14 April 2012
Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatographyetandem mass spectrometry and ehigh-resolution/ high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatographyetandem mass spectrometry and ehigh-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: SARM Equine In Vitro Metabolism LC-MS
1. Introduction A common treatment of age-related maladies and diseases in hypogonadal men (e.g., increased fat mass, frailty, decreased libido, erectile dysfunction, muscular Corresponding author at: Mario Thevis, PhD, Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf, 50933 Cologne, Germany. E-mail address: [email protected] (M. Thevis). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2012.01.005
dystrophy, and osteoporosis) is hormone replacement therapy [1,2]. When developing new drug candidates, a central aspect is the disjunction of androgenic and anabolic properties to minimize therapeutic risks such as benign prostatic hyperplasia, prostate cancer, or cardiovascular events [2,3]. In 1998, a milestone was set with the discovery of nonsteroidal agents where androgenic and anabolic effects were separated. Based on structures of the androgen receptor antagonists bicalutamide [3] and flutamide [1], which comprise an aryl-propionamide nucleus,
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to procedures published elsewhere [12-15]. Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Roche Diagnostics (Mannheim, Germany); uridine 50 -diphosphoglucoronic acid (UDPGA) and D-saccharic acid 1,4-lactone were purchased from Sigma (St. Louis, MA); and adenosine 30 -phosphate 50 -phosphosulfate (PAPS) was purchased from Calbiochem/EMD Biosciences Inc. (La Jolla, CA). All experiments were conducted using highperformance liquid chromatographyegrade solvents, and reagents of at least analytical grade were used. 2.2. Enzymes Fig. 1. Structures of aryl-propionamide-derived selective androgen receptor modulator (SARMs) S24 (A) and S4 (B).
the first selective androgen receptor modulators (SARMs) were synthesized [3]. The major benefit of SARMs as alternatives in hormone replacement therapies is due to the reported tissue selectivity [4], which leads to full anabolic activity in muscle and bone tissue and reduction of undesired side effects such as hepatic toxicity, unfavorable levels of high-density lipoproteins, gynecomastia, and a negative influence on the prostate and cardiovascular system. Other drug candidates with SARM-like properties but different core structures, for example, bicyclic hydantoins, quinolines, and tetrahydroquinolines, were also developed [5-7]. Several drug candidates are currently undergoing clinical studies; however, none has yet completed all trials. Nevertheless, the aryl-propionamide derivative andarine (S4, Fig. 1B) has been found to be available via the Internet [8], and the first adverse analytical findings have been reported [9]. The aforementioned beneficial effects may also concern other mammals, such as horses, with a similar physiology and metabolism. Hence, the community of veterinary and doping control laboratories is urged to probe for the abuse of these drugs in equine sports, and information about the metabolism of SARMs and resulting target compounds is required. In vitro experiments commonly provide rapid results and do not necessitate administration studies. However, the generated data can be considered only as a model indicating potential metabolites, which can be expected after in vivo application of the drug [10]. In case of suspected abuse, it is obligatory to confirm the presence of a substance by comparison with authentic reference material. According to International Laboratory Accreditation Cooperation-G7 guidelines, it is valid to use enzymatically synthesized reference material for this purpose [11], which is of great advantage in cases where adequate reference compounds are not commercially available. This communication reports on equine phase I and II metabolites of S24 and S4 that were synthesized by means of horse liver microsomal and S9 fraction enzymes.
Liver microsomal and S9 fraction enzymatic proteins from one female Quarter Horse were purchased from XenoTech (Lenexa, KS). 2.3. In Vitro Metabolic Assays The in vitro syntheses were conducted according to an assay developed by Kuuranne et al. [16]. Stock solutions (1 mM) of each substrate were prepared in dimethyl sulfoxide. The stock solutions were diluted with 50-mM phosphate buffer (pH 7.4) containing 5-mM MgCl2 (incubation buffer) to obtain a substrate concentration of 100 mM in working solutions. NADPH, UDPGA, and saccharic acid lactone solutions (all 50 mM in incubation buffer) were prepared just before adding to the assay. PAPS (50 mM) and enzymes (20 mg/mL) were aliquoted and frozen at 80 C. For initial experiments, different concentrations (1, 5, 10 mM) of the substrate and incubation times (60, 90, 120 minutes) were chosen. Optimized conditions included 10mM substrate, 90 minutes of incubation time for phase I at 37 C, and mixing, followed by the addition of phase II agents, further 90 minutes of incubation at 37 C, and mixing. The total volume for phase I reactions was 50 mL, including 20 mL of incubation buffer, 10 mL of substrate working solution, 10 mL of NADPH solution, 5 mL of microsomal enzymes, and 5 mL of S9 fraction enzymes. For phase II metabolism experiments, another 50 mLdcomposed of 10 mL each of UDPGA, PAPS, NADPH, and saccharic acid lactone solutions plus 5 mL of each enzymatic fractiondwas added. After a second incubation period of 90 minutes, the metabolic reaction was stopped through the addition of 150 mL of ice-cold acetonitrile. The precipitate was removed by centrifugation (2 minutes, 17,000 g), and the supernatant was transferred to a fresh tube, dried using a vacuum centrifuge, and reconstituted in ammonium acetate buffer (10 mM, pH 3.5)/acetonitrile (99:1; v/v). Each assay included samples with reaction mixtures of (a) all agents, (b) all agents excluding substrate, and (c) all agents excluding enzymes to monitor and differentiate possible enzymatic and nonenzymatic reactions. 2.4. Liquid ChromatographyeMass Spectrometry
2. Materials and Methods 2.1. Synthesis of Aryl-Propionamide-Derived SARMs S24 and S4 The substrates S24 and S4 (see Fig.1) for the in vitro assays were prepared using in-house chemical synthesis according
LCeMS(/MS) of the in vitro-generated SARM metabolites was performed on an instrument setup consisting of an Agilent 1100 Series high-performance liquid chromatography (Agilent Technologies, Waldbronn, Germany) and an Applied Biosystems 4000 Q Trap mass spectrometer (Applied Biosystems, Darmstadt, Germany). The LC system was
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Fig. 2. Extracted ion chromatograms of diagnostic S24 metabolite fragments. High resolution/high accuracy mass spectrometry (HRMS) (M-H); electrospray ionization-; higher energy collisional dissociation (HCD) 40 eV.
equipped with a Phenomenex (Aschaffenburg, Germany) Kinetex C18 column (2.1 100 mm, particle size: 2.6 mm); the eluants were 5 mM ammonium acetate with 0.1% acetic acid (eluant A) and acetonitrile (eluant B). The used gradient started with 1% of eluant B and was ramped over 12.5 minutes to 100% of eluant B, followed by reequilibration using an isocratic step of 3.5 minutes with 1% eluant B. The flow rate was 250 mL/min, and the injection volume was 10 mL. The ionization was carried out by electrospray ionization (ESI) in negative mode with 4200 V at a source temperature of 350 C. The generated metabolites were screened in MS full-scan mode from m/z 70 to 1000 and identified by comparing the blank samples with
the incubation reaction mixture containing all relevant substances, enzymes, and cofactors. Data for further identification and characterization were subsequently obtained from MS/MS experiments by manually selecting the precursor ions, and the collision offset voltage was optimized for each analyte to retain a precursor ion signal of approximately 20% relative abundance. In all experiments, nitrogen was used as collision gas (6 103 Pa). 2.5. High-ResolutioneHigh-Accuracy Experiments For the determination of elemental compositions of generated SARM metabolites and corresponding product
Fig. 3. Extracted ion chromatograms of diagnostic S4 metabolite fragments. High resolution/high accuracy mass spectrometry (HRMS) (M-H); electrospray ionization-; higher energy collisional dissociation (HCD) 40 eV.
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Table 1 Experimental elemental composition of SARM S24 and in vitro synthesized metabolites Compound/Reaction
Precursor/Product Ions [M-H]
Elemental Composition
m/z (Exp.)
Error (ppm)
Substrate/S24
381/381 269 241 185 111 287/287 269 241 185 154 397/397 255 185 111 397/397 269 241 185 127 573/573 397 241 185 477/477 397 269 377/377
C18H13O3N2F4 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C6H4OF C12H10O3N2F3 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C7H2ONF2 C18H13O4N2F4 C11H8O2N2F3 C8H4N2F3 C6H4OF C18H13O4N2F4 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C6H4O2F C24H21O10N2F4 C18H13O4N2F4 C11H8ON2F3 C8H4N2F3 C18H13O7N2F4S C18H13O4N2F4 C12H8O2N2F3 C14H12O7N2F3
381.0872 269.0542 241.0592 185.0327 111.0248 287.0650 269.0543 241.0592 185.0328 154.0107 397.0821 255.0381 185.0328 111.0247 397.0821 269.0542 241.0590 185.0328 127.0197 573.1141 397.0812 241.0591 185.0330 477.0389 397.0810 269.0541 377.0605
1.1 0.6 0.9 2.8 3.3 0.5 0.0 1.1 2.0 2.0 1.4 2.4 2.2 4.4 1.1 0.6 1.0 2.2 2.9 0.6 1.1 1.2 1.0 0.8 1.7 2.5 0.7
201 185 281/281
C8H4ON2F3 C8H4N2F3 C8H4O4N2F3S
201.0278 185.0324 280.9849
1.6 4.5 0.0
263 201 181 297/297
C8H2O3N2F3S C8H4ON2F3 C8H2ON2F2 C8H4O5N2F3S
263.9739 201.0279 181.0215 296.9795
1.9 1.2 2.2 2.5
217
C8H4O2N2F3
217.0225
2.3
M1/dephenylation
M2a/monohydroxylation (chiral carbon atom)
M2b/monohydroxylation (B-ring)
M3/monohydroxylation and glucuronidation
M4/monohydroxylation and sulfonation
M5/monohydroxylation of 4-cyano-3-trifluoromethyl phenylamine and glucuronidation
M6/monohydroxylation of 4-cyano-3-trifluoromethyl phenylamine and sulfonation
M7/bishydroxylation of 4-cyano-3-trifluoromethyl phenylamine and sulfonation
ions, LC-ESI-MS(/MS) experiments were conducted on an Accela LC system connected to a Thermo Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The chromatographic conditions (column, gradient) were identical to those described earlier in the text for the low-resolution measurements. Also, the analyses in this study were carried out in negative mode with ESI, using an ion spray voltage of 4500 V and a source temperature of 275 C. The scan range encompassed m/z 70-1000. For identification purposes of metabolites, product ions were generated by all-ion fragmentation experiments (higher collisional energy dissociation) with collision offset voltages of 15 V and 40 V using nitrogen as collision gas. This LC and MS setup was further used for the determination of the peak areas of synthesized SARM metabolites in full-scan mode to allow for an estimation of their relative abundance under the chosen in vitro incubation conditions. Although several factors certainly influence the intensity of signals generated from these molecules in ESI, this approach was considered as the best option due to the absence of synthetic reference material that would enable accurate quantification.
3. Results and Discussion The equine in vitro simulation yielded several phase I and II SARM metabolites, and no artifacts potentially generated by nonenzymatic reactions were observed. For the identification of metabolic products, product ion mass spectra were recorded after detection of newly formed compounds in full-scan analyses. Supporting evidence enabling further characterization of these compounds was obtained by high-resolution/high-accuracy MS data of precursor and diagnostic product ions (Figs. 2 and 3), the elemental compositions of which are summarized in Tables 1 and 2. In the absence of reference material for the individual metabolites, the relative quantity of these analytes was estimated from peak areas of extracted ion chromatograms with isolation windows of 0.01 Da. The observed and characterized metabolites are in close agreement with those from human in vitro and in vivo studies [16,17]. Various ions were considered as diagnostic for S24 and its hydroxylated analogs such as the aniline residue of the intact A-ring (m/z 185.03), the hydroxylated A-ring (m/z 201.02), the p-fluorophenyl residue of the intact B-ring (m/z 111.02), and its hydroxylated counterpart
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Table 2 Experimental elemental composition of SARM S4 and in vitro synthesized metabolites Compound/Reaction
Precursor/Product Ions [M-H]
Elemental Composition
m/z (Exp.)
Error (ppm)
Substrate/S4
440/440 289 261 205 150 307/307 277 205 234 398/398 289 261 205 426/426 289 261 205 456/456 289 261 166 456/456 289 261 166 410/410 259 150 616/616 440 289 205 150 632/632 440 261 205
C19H17O6N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C8H8O2N C11H10O5N2F3 C10H8O4N2F3 C7H4O2N2F3 C9H7O3NF3 C17H15O5N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C18H15O6N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C19H17O7N3F3 C11H8O4N2F3 C10H8O3N2F3 C8H8O3N C19H17O7N3F3 C11H8O4N2F3 C10H8O3N2F3 C8H8O3N C19H19O4N3F3 C11H10O2N2F3 C8H8O2N C25H25O12N3F3 C19H17O6N3F3 C11H8O4N2F3 C7H4O2N2F3 C8H8O2N C25H25O13N3F3 C19H17O6N3F3 C10H8O3N2F3 C7H4O2N2F3
440.1073 289.0440 261.0489 205.0228 150.0557 307.0544 277.0437 205.0227 234.0379 398.0968 289.0442 261.0490 205.0227 426.0291 289.0437 261.0488 205.0228 456.1024 289.0440 261.0491 166.0507 456.1022 289.0441 261.0489 166.0506 410.1335 259.0688 150.0557 616.1398 440.1070 289.0440 205.0229 150.0558 632.1347 440.1070 261.0490 205.0229
0.4 0.4 1.5 1.2 2.3 1.2 1.8 1.8 1.9 0.2 0.3 1.0 1.7 1.2 1.6 1.8 1.1 0.1 0.4 0.7 1.4 0.5 0.1 1.2 2.0 0.5 4.4 2.4 0.3 1.1 0.5 0.6 1.8 0.4 1.1 0.9 0.4
M1/dephenylation
M2/deacetylation
M3/demethylation
M4a/monohydroxylation (B-ring; rt 5.88)
M4b/monohydroxylation (B-ring; rt 6.05)
M5/nitroreduction
M6/glucuronidation
M7/monohydroxylation and glucuronidation
at m/z 127.01. The main in vitro metabolic route of the arylpropionamide derivative S24 was found to be the hydroxylation of the aniline moiety of the A-ring with subsequent glucuronidation or sulfonation (M5-M7; Fig. 4). The highest relative abundance was observed for the glucuronidated metabolite M5 with approximately 30% (Fig. 6). Further, a major metabolic route was attributed to the hydrolysis of the ether linkage, which led to the elimination of the B-ring yielding M1 (Fig. 4). A possible mechanism of this cleavage was described in earlier SARM studies as initial oxidation of the carbon atom adjacent to the O-phenyl residue with subsequent elimination of 4-hydroxy acetanilide, which gives rise to an aldehyde of the remaining propionanilide followed by reduction to the metabolic product M1 [16]. Another metabolic pathway included the hydroxylation of S24 at the methyl group attached to the carbon of the chiral center (M2a) or the B-ring (M2b). These metabolites differ in retention time as well as their fragmentation pattern (Fig. 2, Table 1). M2a elutes under the chosen chromatographic conditions approximately 0.4 minutes earlier than the B-ring hydroxylated analog. The diagnostic ions of M2a comprehend the intact A-ring (m/z 185.03) as well as the intact B-ring (m/z 111.02), whereas M2b generates a product ion at m/z 127.01 representing the monohydroxylated B-ring. In case of M2b, phase II reactions lead
to subsequent glucuronidation or sulfonation (M3 and M4, respectively), but analogous phase II conjugates are not observed for M2a. Comparable with S24, S4 and its metabolites also yielded characteristic product ions that were used for identification purposes. Ions found at m/z 205.02 (representing the aniline residue of the intact A-ring), m/z 261.04 (dephenylated and reconstituted A-ring residue [5,18]), m/z 166.05 (hydroxylated B-ring), and m/z 175.04 of the nitro-reduced aniline residue of the A-ring (Fig. 3) were used to detect and confirm structural features of the metabolic products. The main equine metabolite of S4 is the dephenylated compound (M1, Fig. 5) with a deprotonated molecule observed at m/z 307 and a relative abundance of more than 90% of all measured metabolites (Fig. 6). Additional S4 metabolites were observed in analogy to S24 with the monohydroxylated compounds M4a and M4b (presumably representing ortho- and meta-hydroxylated analogs) and the corresponding glucuronidation product M7. Moreover, the deacetylated metabolite (M2) with a relative percentage of 4.9% of the entire metabolic products, nitroreduction at the A-ring (M5), and demethylation of the acetamide group at the B-ring (M3) were recorded. It is also notable that no amide hydrolysis occurred in case of S4 and that the conducted equine in vitro metabolism of S4 showed
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Fig. 4. Discovered main equine in vitro metabolic routes of aryl-propionamide-derived SARM S24.
considerably fewer phase II reactions than S24; in particular, the lack of sulfoconjugates was remarkably recognized. Although the relative abundance of generated equine metabolites differs considerably from those resulting from human in vitro studies conducted under comparable enzyme and cofactor concentration conditions and incubation duration as well as human in vivo studies after oral application (between 10% and 200%), main metabolic routes were found to be identical, and no horse-specific analyte was identified.
4. Conclusion and Outlook The present study shows that equine phase I and phase II metabolites of aryl-propionamide-derived SARMs can be synthesized using common in vitro methodologies, and the resulting products can provide indications about metabolites potentially also generated by in vivo methods. The present data provide an informative basis for veterinary metabolism studies as well as doping control laboratories with dephenylated metabolites of S24 and S4 being potential
Fig. 5. Discovered main equine in vitro metabolic routes of aryl-propionamide-derived SARM S4.
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Fig. 6. Relative abundances of S24 and S4 in vitro metabolites.
target analytes for screening procedures. However, further experiments are required. Acknowledgment The authors thank the Center for Preventive Doping Research, Cologne, Germany, for supporting the presented work. None of the authors of this article has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the article. References [1] Chen J, Kim J, Dalton JT. Discovery and therapeutic promise of selective androgen receptor modulators. Mol Interv 2005;5:173-88. [2] Hijazi RA, Cunningham GR. Andropause: is androgen replacement therapy indicated for the aging male? Annu Rev Med 2005;56:117-37. [3] Dalton JT, Mukherjee A, Zhu Z, Kirkovsky L, Miller DD. Discovery of nonsteroidal androgens. Biochem Biophys Res Comm 1998;244:1-4. [4] Mohler ML, Bohl CE, Jones A, Coss CC, Narayanan R, He Y, et al. Nonsteroidal selective androgen receptor modulators (SARMs): dissociating the anabolic and androgenic activities of the androgen receptor for therapeutic benefit. J Med Chem 2009;52:3597-617. [5] Thevis M, Schänzer W. Mass spectrometry of selective androgen receptor modulators. J Mass Spectrom 2008;43:865-76. [6] Kamischke A, Nieschlag E. Progress towards hormonal male contraception. Trends Pharmacol Sci 2004;25:49-57. [7] Gao W, Dalton JT. Expanding the therapeutic use of androgens via selective androgen receptor modulators (SARMs). Drug Discov Today 2007;12:421-8. [8] Thevis M, Geyer H, Kamber M, Schänzer W. Detection of the arylpropionamide-derived selective androgen receptor modulator
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(SARM) S-4 (Andarine) in a black-market product. Drug Test Anal 2009;1:387-92. Grata E, Perrenoud L, Saugy M, Baume N. SARM-S4 and metabolites detection in sports drug testing: a case report. Forensic Sci Int 2011;213:104-8. Scarth JP, Spencer HA, Timbers SE, Hudson SC, Hillyer LL. The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance. Drug Test Anal 2010;2:1-10. Scarth JP, Teale P, Kuuranne T. Drug metabolism in the horse: a review. Drug Test Anal 2011;3:19-53. Tucker H, Chesterson GJ. Resolution of the nonsteroidal antiandrogen 40 -cyano-3-([fluorophenyl]sulfonyl)-2-hydroxy-2-methyl30 -(trifluoromethyl)-propionanilide and the determination of the absolute configuration of the active enantiomer. J Med Chem 1988;31:885-7. Tucker H, Crook JW, Chesterson GJ. Nonsteroidal antiandrogens. Synthesis and structur-activity relationships of 3-substituted derivatives of 2-hydroxypropionanilides. J Med Chem 1988;31:954-9. Marhefka CA, Gao W, Chung K, Kim J, He Y, Yin D, et al. Design, synthesis, and biological characterization of metabolically stable selective androgen receptor modulators. J Med Chem 2004;47:993-8. Thevis M, Kamber M, Schänzer W. Screening for metabolically stable aryl-propionamide-derived selective androgen receptor modulators for doping control purposes. Rapid Commun Mass Spectrom 2006;20:870-6. Kuuranne T, Leinonen A, Schänzer W, Kamber M, Kostiainen R, Thevis M. Aryl-propionamide-derived selective androgen receptor modulators: liquid chromatography-tandem mass spectrometry characterization of in vitro synthesized metabolites for doping control purposes. Drug Metab Dispos 2008;36:571-81. Thevis M, Thomas A, Fußhöller G, Beuck S, Geyer H, Schänzer W. Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S4) for routine doping control purposes. Rapid Commun Mass Spectrom 2010;24:2245-54. Thevis M, Thomas A, Kohler M, Beuck S, Schänzer W. Emerging drugs: mechanism of action, mass spectrometry and doping control analysis. J Mass Spectrom 2009;44:442-60.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4 Oliver Krug MSc a, Andreas Thomas PhD a, Simon Beuck MSc a, Ina Schenk PhD a, Marc Machnik PhD a, Wilhelm Schänzer PhD a, Ulf Bondesson PhD b, c, Mikael Hedeland PhD b, c, Mario Thevis PhD a a b c
Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf, Cologne, Germany Department of Chemistry, Environment and Feed Hygiene, National Veterinary Institute (SVA), Uppsala, Sweden Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2011 Received in revised form 12 December 2011 Accepted 20 January 2012 Available online 14 April 2012
Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatographyetandem mass spectrometry and ehigh-resolution/ high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatographyetandem mass spectrometry and ehigh-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: SARM Equine In Vitro Metabolism LC-MS
1. Introduction A common treatment of age-related maladies and diseases in hypogonadal men (e.g., increased fat mass, frailty, decreased libido, erectile dysfunction, muscular Corresponding author at: Mario Thevis, PhD, Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf, 50933 Cologne, Germany. E-mail address: [email protected] (M. Thevis). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2012.01.005
dystrophy, and osteoporosis) is hormone replacement therapy [1,2]. When developing new drug candidates, a central aspect is the disjunction of androgenic and anabolic properties to minimize therapeutic risks such as benign prostatic hyperplasia, prostate cancer, or cardiovascular events [2,3]. In 1998, a milestone was set with the discovery of nonsteroidal agents where androgenic and anabolic effects were separated. Based on structures of the androgen receptor antagonists bicalutamide [3] and flutamide [1], which comprise an aryl-propionamide nucleus,
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to procedures published elsewhere [12-15]. Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Roche Diagnostics (Mannheim, Germany); uridine 50 -diphosphoglucoronic acid (UDPGA) and D-saccharic acid 1,4-lactone were purchased from Sigma (St. Louis, MA); and adenosine 30 -phosphate 50 -phosphosulfate (PAPS) was purchased from Calbiochem/EMD Biosciences Inc. (La Jolla, CA). All experiments were conducted using highperformance liquid chromatographyegrade solvents, and reagents of at least analytical grade were used. 2.2. Enzymes Fig. 1. Structures of aryl-propionamide-derived selective androgen receptor modulator (SARMs) S24 (A) and S4 (B).
the first selective androgen receptor modulators (SARMs) were synthesized [3]. The major benefit of SARMs as alternatives in hormone replacement therapies is due to the reported tissue selectivity [4], which leads to full anabolic activity in muscle and bone tissue and reduction of undesired side effects such as hepatic toxicity, unfavorable levels of high-density lipoproteins, gynecomastia, and a negative influence on the prostate and cardiovascular system. Other drug candidates with SARM-like properties but different core structures, for example, bicyclic hydantoins, quinolines, and tetrahydroquinolines, were also developed [5-7]. Several drug candidates are currently undergoing clinical studies; however, none has yet completed all trials. Nevertheless, the aryl-propionamide derivative andarine (S4, Fig. 1B) has been found to be available via the Internet [8], and the first adverse analytical findings have been reported [9]. The aforementioned beneficial effects may also concern other mammals, such as horses, with a similar physiology and metabolism. Hence, the community of veterinary and doping control laboratories is urged to probe for the abuse of these drugs in equine sports, and information about the metabolism of SARMs and resulting target compounds is required. In vitro experiments commonly provide rapid results and do not necessitate administration studies. However, the generated data can be considered only as a model indicating potential metabolites, which can be expected after in vivo application of the drug [10]. In case of suspected abuse, it is obligatory to confirm the presence of a substance by comparison with authentic reference material. According to International Laboratory Accreditation Cooperation-G7 guidelines, it is valid to use enzymatically synthesized reference material for this purpose [11], which is of great advantage in cases where adequate reference compounds are not commercially available. This communication reports on equine phase I and II metabolites of S24 and S4 that were synthesized by means of horse liver microsomal and S9 fraction enzymes.
Liver microsomal and S9 fraction enzymatic proteins from one female Quarter Horse were purchased from XenoTech (Lenexa, KS). 2.3. In Vitro Metabolic Assays The in vitro syntheses were conducted according to an assay developed by Kuuranne et al. [16]. Stock solutions (1 mM) of each substrate were prepared in dimethyl sulfoxide. The stock solutions were diluted with 50-mM phosphate buffer (pH 7.4) containing 5-mM MgCl2 (incubation buffer) to obtain a substrate concentration of 100 mM in working solutions. NADPH, UDPGA, and saccharic acid lactone solutions (all 50 mM in incubation buffer) were prepared just before adding to the assay. PAPS (50 mM) and enzymes (20 mg/mL) were aliquoted and frozen at 80 C. For initial experiments, different concentrations (1, 5, 10 mM) of the substrate and incubation times (60, 90, 120 minutes) were chosen. Optimized conditions included 10mM substrate, 90 minutes of incubation time for phase I at 37 C, and mixing, followed by the addition of phase II agents, further 90 minutes of incubation at 37 C, and mixing. The total volume for phase I reactions was 50 mL, including 20 mL of incubation buffer, 10 mL of substrate working solution, 10 mL of NADPH solution, 5 mL of microsomal enzymes, and 5 mL of S9 fraction enzymes. For phase II metabolism experiments, another 50 mLdcomposed of 10 mL each of UDPGA, PAPS, NADPH, and saccharic acid lactone solutions plus 5 mL of each enzymatic fractiondwas added. After a second incubation period of 90 minutes, the metabolic reaction was stopped through the addition of 150 mL of ice-cold acetonitrile. The precipitate was removed by centrifugation (2 minutes, 17,000 g), and the supernatant was transferred to a fresh tube, dried using a vacuum centrifuge, and reconstituted in ammonium acetate buffer (10 mM, pH 3.5)/acetonitrile (99:1; v/v). Each assay included samples with reaction mixtures of (a) all agents, (b) all agents excluding substrate, and (c) all agents excluding enzymes to monitor and differentiate possible enzymatic and nonenzymatic reactions. 2.4. Liquid ChromatographyeMass Spectrometry
2. Materials and Methods 2.1. Synthesis of Aryl-Propionamide-Derived SARMs S24 and S4 The substrates S24 and S4 (see Fig.1) for the in vitro assays were prepared using in-house chemical synthesis according
LCeMS(/MS) of the in vitro-generated SARM metabolites was performed on an instrument setup consisting of an Agilent 1100 Series high-performance liquid chromatography (Agilent Technologies, Waldbronn, Germany) and an Applied Biosystems 4000 Q Trap mass spectrometer (Applied Biosystems, Darmstadt, Germany). The LC system was
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Fig. 2. Extracted ion chromatograms of diagnostic S24 metabolite fragments. High resolution/high accuracy mass spectrometry (HRMS) (M-H); electrospray ionization-; higher energy collisional dissociation (HCD) 40 eV.
equipped with a Phenomenex (Aschaffenburg, Germany) Kinetex C18 column (2.1 100 mm, particle size: 2.6 mm); the eluants were 5 mM ammonium acetate with 0.1% acetic acid (eluant A) and acetonitrile (eluant B). The used gradient started with 1% of eluant B and was ramped over 12.5 minutes to 100% of eluant B, followed by reequilibration using an isocratic step of 3.5 minutes with 1% eluant B. The flow rate was 250 mL/min, and the injection volume was 10 mL. The ionization was carried out by electrospray ionization (ESI) in negative mode with 4200 V at a source temperature of 350 C. The generated metabolites were screened in MS full-scan mode from m/z 70 to 1000 and identified by comparing the blank samples with
the incubation reaction mixture containing all relevant substances, enzymes, and cofactors. Data for further identification and characterization were subsequently obtained from MS/MS experiments by manually selecting the precursor ions, and the collision offset voltage was optimized for each analyte to retain a precursor ion signal of approximately 20% relative abundance. In all experiments, nitrogen was used as collision gas (6 103 Pa). 2.5. High-ResolutioneHigh-Accuracy Experiments For the determination of elemental compositions of generated SARM metabolites and corresponding product
Fig. 3. Extracted ion chromatograms of diagnostic S4 metabolite fragments. High resolution/high accuracy mass spectrometry (HRMS) (M-H); electrospray ionization-; higher energy collisional dissociation (HCD) 40 eV.
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Table 1 Experimental elemental composition of SARM S24 and in vitro synthesized metabolites Compound/Reaction
Precursor/Product Ions [M-H]
Elemental Composition
m/z (Exp.)
Error (ppm)
Substrate/S24
381/381 269 241 185 111 287/287 269 241 185 154 397/397 255 185 111 397/397 269 241 185 127 573/573 397 241 185 477/477 397 269 377/377
C18H13O3N2F4 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C6H4OF C12H10O3N2F3 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C7H2ONF2 C18H13O4N2F4 C11H8O2N2F3 C8H4N2F3 C6H4OF C18H13O4N2F4 C12H8O2N2F3 C11H8ON2F3 C8H4N2F3 C6H4O2F C24H21O10N2F4 C18H13O4N2F4 C11H8ON2F3 C8H4N2F3 C18H13O7N2F4S C18H13O4N2F4 C12H8O2N2F3 C14H12O7N2F3
381.0872 269.0542 241.0592 185.0327 111.0248 287.0650 269.0543 241.0592 185.0328 154.0107 397.0821 255.0381 185.0328 111.0247 397.0821 269.0542 241.0590 185.0328 127.0197 573.1141 397.0812 241.0591 185.0330 477.0389 397.0810 269.0541 377.0605
1.1 0.6 0.9 2.8 3.3 0.5 0.0 1.1 2.0 2.0 1.4 2.4 2.2 4.4 1.1 0.6 1.0 2.2 2.9 0.6 1.1 1.2 1.0 0.8 1.7 2.5 0.7
201 185 281/281
C8H4ON2F3 C8H4N2F3 C8H4O4N2F3S
201.0278 185.0324 280.9849
1.6 4.5 0.0
263 201 181 297/297
C8H2O3N2F3S C8H4ON2F3 C8H2ON2F2 C8H4O5N2F3S
263.9739 201.0279 181.0215 296.9795
1.9 1.2 2.2 2.5
217
C8H4O2N2F3
217.0225
2.3
M1/dephenylation
M2a/monohydroxylation (chiral carbon atom)
M2b/monohydroxylation (B-ring)
M3/monohydroxylation and glucuronidation
M4/monohydroxylation and sulfonation
M5/monohydroxylation of 4-cyano-3-trifluoromethyl phenylamine and glucuronidation
M6/monohydroxylation of 4-cyano-3-trifluoromethyl phenylamine and sulfonation
M7/bishydroxylation of 4-cyano-3-trifluoromethyl phenylamine and sulfonation
ions, LC-ESI-MS(/MS) experiments were conducted on an Accela LC system connected to a Thermo Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The chromatographic conditions (column, gradient) were identical to those described earlier in the text for the low-resolution measurements. Also, the analyses in this study were carried out in negative mode with ESI, using an ion spray voltage of 4500 V and a source temperature of 275 C. The scan range encompassed m/z 70-1000. For identification purposes of metabolites, product ions were generated by all-ion fragmentation experiments (higher collisional energy dissociation) with collision offset voltages of 15 V and 40 V using nitrogen as collision gas. This LC and MS setup was further used for the determination of the peak areas of synthesized SARM metabolites in full-scan mode to allow for an estimation of their relative abundance under the chosen in vitro incubation conditions. Although several factors certainly influence the intensity of signals generated from these molecules in ESI, this approach was considered as the best option due to the absence of synthetic reference material that would enable accurate quantification.
3. Results and Discussion The equine in vitro simulation yielded several phase I and II SARM metabolites, and no artifacts potentially generated by nonenzymatic reactions were observed. For the identification of metabolic products, product ion mass spectra were recorded after detection of newly formed compounds in full-scan analyses. Supporting evidence enabling further characterization of these compounds was obtained by high-resolution/high-accuracy MS data of precursor and diagnostic product ions (Figs. 2 and 3), the elemental compositions of which are summarized in Tables 1 and 2. In the absence of reference material for the individual metabolites, the relative quantity of these analytes was estimated from peak areas of extracted ion chromatograms with isolation windows of 0.01 Da. The observed and characterized metabolites are in close agreement with those from human in vitro and in vivo studies [16,17]. Various ions were considered as diagnostic for S24 and its hydroxylated analogs such as the aniline residue of the intact A-ring (m/z 185.03), the hydroxylated A-ring (m/z 201.02), the p-fluorophenyl residue of the intact B-ring (m/z 111.02), and its hydroxylated counterpart
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Table 2 Experimental elemental composition of SARM S4 and in vitro synthesized metabolites Compound/Reaction
Precursor/Product Ions [M-H]
Elemental Composition
m/z (Exp.)
Error (ppm)
Substrate/S4
440/440 289 261 205 150 307/307 277 205 234 398/398 289 261 205 426/426 289 261 205 456/456 289 261 166 456/456 289 261 166 410/410 259 150 616/616 440 289 205 150 632/632 440 261 205
C19H17O6N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C8H8O2N C11H10O5N2F3 C10H8O4N2F3 C7H4O2N2F3 C9H7O3NF3 C17H15O5N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C18H15O6N3F3 C11H8O4N2F3 C10H8O3N2F3 C7H4O2N2F3 C19H17O7N3F3 C11H8O4N2F3 C10H8O3N2F3 C8H8O3N C19H17O7N3F3 C11H8O4N2F3 C10H8O3N2F3 C8H8O3N C19H19O4N3F3 C11H10O2N2F3 C8H8O2N C25H25O12N3F3 C19H17O6N3F3 C11H8O4N2F3 C7H4O2N2F3 C8H8O2N C25H25O13N3F3 C19H17O6N3F3 C10H8O3N2F3 C7H4O2N2F3
440.1073 289.0440 261.0489 205.0228 150.0557 307.0544 277.0437 205.0227 234.0379 398.0968 289.0442 261.0490 205.0227 426.0291 289.0437 261.0488 205.0228 456.1024 289.0440 261.0491 166.0507 456.1022 289.0441 261.0489 166.0506 410.1335 259.0688 150.0557 616.1398 440.1070 289.0440 205.0229 150.0558 632.1347 440.1070 261.0490 205.0229
0.4 0.4 1.5 1.2 2.3 1.2 1.8 1.8 1.9 0.2 0.3 1.0 1.7 1.2 1.6 1.8 1.1 0.1 0.4 0.7 1.4 0.5 0.1 1.2 2.0 0.5 4.4 2.4 0.3 1.1 0.5 0.6 1.8 0.4 1.1 0.9 0.4
M1/dephenylation
M2/deacetylation
M3/demethylation
M4a/monohydroxylation (B-ring; rt 5.88)
M4b/monohydroxylation (B-ring; rt 6.05)
M5/nitroreduction
M6/glucuronidation
M7/monohydroxylation and glucuronidation
at m/z 127.01. The main in vitro metabolic route of the arylpropionamide derivative S24 was found to be the hydroxylation of the aniline moiety of the A-ring with subsequent glucuronidation or sulfonation (M5-M7; Fig. 4). The highest relative abundance was observed for the glucuronidated metabolite M5 with approximately 30% (Fig. 6). Further, a major metabolic route was attributed to the hydrolysis of the ether linkage, which led to the elimination of the B-ring yielding M1 (Fig. 4). A possible mechanism of this cleavage was described in earlier SARM studies as initial oxidation of the carbon atom adjacent to the O-phenyl residue with subsequent elimination of 4-hydroxy acetanilide, which gives rise to an aldehyde of the remaining propionanilide followed by reduction to the metabolic product M1 [16]. Another metabolic pathway included the hydroxylation of S24 at the methyl group attached to the carbon of the chiral center (M2a) or the B-ring (M2b). These metabolites differ in retention time as well as their fragmentation pattern (Fig. 2, Table 1). M2a elutes under the chosen chromatographic conditions approximately 0.4 minutes earlier than the B-ring hydroxylated analog. The diagnostic ions of M2a comprehend the intact A-ring (m/z 185.03) as well as the intact B-ring (m/z 111.02), whereas M2b generates a product ion at m/z 127.01 representing the monohydroxylated B-ring. In case of M2b, phase II reactions lead
to subsequent glucuronidation or sulfonation (M3 and M4, respectively), but analogous phase II conjugates are not observed for M2a. Comparable with S24, S4 and its metabolites also yielded characteristic product ions that were used for identification purposes. Ions found at m/z 205.02 (representing the aniline residue of the intact A-ring), m/z 261.04 (dephenylated and reconstituted A-ring residue [5,18]), m/z 166.05 (hydroxylated B-ring), and m/z 175.04 of the nitro-reduced aniline residue of the A-ring (Fig. 3) were used to detect and confirm structural features of the metabolic products. The main equine metabolite of S4 is the dephenylated compound (M1, Fig. 5) with a deprotonated molecule observed at m/z 307 and a relative abundance of more than 90% of all measured metabolites (Fig. 6). Additional S4 metabolites were observed in analogy to S24 with the monohydroxylated compounds M4a and M4b (presumably representing ortho- and meta-hydroxylated analogs) and the corresponding glucuronidation product M7. Moreover, the deacetylated metabolite (M2) with a relative percentage of 4.9% of the entire metabolic products, nitroreduction at the A-ring (M5), and demethylation of the acetamide group at the B-ring (M3) were recorded. It is also notable that no amide hydrolysis occurred in case of S4 and that the conducted equine in vitro metabolism of S4 showed
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Fig. 4. Discovered main equine in vitro metabolic routes of aryl-propionamide-derived SARM S24.
considerably fewer phase II reactions than S24; in particular, the lack of sulfoconjugates was remarkably recognized. Although the relative abundance of generated equine metabolites differs considerably from those resulting from human in vitro studies conducted under comparable enzyme and cofactor concentration conditions and incubation duration as well as human in vivo studies after oral application (between 10% and 200%), main metabolic routes were found to be identical, and no horse-specific analyte was identified.
4. Conclusion and Outlook The present study shows that equine phase I and phase II metabolites of aryl-propionamide-derived SARMs can be synthesized using common in vitro methodologies, and the resulting products can provide indications about metabolites potentially also generated by in vivo methods. The present data provide an informative basis for veterinary metabolism studies as well as doping control laboratories with dephenylated metabolites of S24 and S4 being potential
Fig. 5. Discovered main equine in vitro metabolic routes of aryl-propionamide-derived SARM S4.
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Fig. 6. Relative abundances of S24 and S4 in vitro metabolites.
target analytes for screening procedures. However, further experiments are required. Acknowledgment The authors thank the Center for Preventive Doping Research, Cologne, Germany, for supporting the presented work. None of the authors of this article has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the article. References [1] Chen J, Kim J, Dalton JT. Discovery and therapeutic promise of selective androgen receptor modulators. Mol Interv 2005;5:173-88. [2] Hijazi RA, Cunningham GR. Andropause: is androgen replacement therapy indicated for the aging male? Annu Rev Med 2005;56:117-37. [3] Dalton JT, Mukherjee A, Zhu Z, Kirkovsky L, Miller DD. Discovery of nonsteroidal androgens. Biochem Biophys Res Comm 1998;244:1-4. [4] Mohler ML, Bohl CE, Jones A, Coss CC, Narayanan R, He Y, et al. Nonsteroidal selective androgen receptor modulators (SARMs): dissociating the anabolic and androgenic activities of the androgen receptor for therapeutic benefit. J Med Chem 2009;52:3597-617. [5] Thevis M, Schänzer W. Mass spectrometry of selective androgen receptor modulators. J Mass Spectrom 2008;43:865-76. [6] Kamischke A, Nieschlag E. Progress towards hormonal male contraception. Trends Pharmacol Sci 2004;25:49-57. [7] Gao W, Dalton JT. Expanding the therapeutic use of androgens via selective androgen receptor modulators (SARMs). Drug Discov Today 2007;12:421-8. [8] Thevis M, Geyer H, Kamber M, Schänzer W. Detection of the arylpropionamide-derived selective androgen receptor modulator
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