| 1. |
- Garg, Neeraj, et al.
(författare)
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Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control
- 2018
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Ingår i: Organic and biomolecular chemistry. - : ROYAL SOC CHEMISTRY. - 1477-0520 .- 1477-0539. ; 16:5, s. 698-702
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Tidskriftsartikel (refereegranskat)abstract
- Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography- mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.
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| 2. |
- Hansson, Annelie, et al.
(författare)
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Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes
- 2015
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Ingår i: Drug Testing and Analysis. - : Wiley. - 1942-7603 .- 1942-7611. ; 7:8, s. 673-683
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Tidskriftsartikel (refereegranskat)abstract
- Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.
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| 3. |
- Hansson, Annelie, et al.
(författare)
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Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
- 2018
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 1074-1075, s. 91-98
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Tidskriftsartikel (refereegranskat)abstract
- LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a not a major metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.
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| 4. |
- Hansson, Annelie, et al.
(författare)
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Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry
- 2017
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Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier BV. - 0731-7085 .- 1873-264X. ; 134, s. 228-236
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Tidskriftsartikel (refereegranskat)abstract
- FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.
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| 5. |
- Hansson, Annelie, et al.
(författare)
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Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
- 2016
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 30:7, s. 833-842
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Tidskriftsartikel (refereegranskat)abstract
- RationaleSelective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. MethodsEach SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MSE mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. ResultsThe numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. ConclusionsThe maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target.
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| 6. |
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| 7. |
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| 8. |
- Knych, Heather K., et al.
(författare)
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Pharmacokinetics and pharmacodynamics of meldonium in exercised thoroughbred horses
- 2017
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Ingår i: Drug Testing and Analysis. - : John Wiley & Sons. - 1942-7603 .- 1942-7611. ; 9:9, s. 1392-1399
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Tidskriftsartikel (refereegranskat)abstract
- Although developed as a therapeutic medication, meldonium has found widespread use in human sports and was recently added to the World Anti-Doping Agency's list of prohibited substances. Its reported abuse potential in human sports has led to concern by regulatory authorities about the possible misuse of meldonium in equine athletics. The potential abuse in equine athletes along with the limited data available regarding the pharmacokinetics and pharmacodynamics of meldonium in horses necessitates further study. Eight exercised adult thoroughbred horses received a single oral dose of 3.5, 7.1, 14.3 or 21.4 mg/kg of meldonium. Blood and urine samples were collected and analyzed using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined using non-compartmental analysis. Maximum serum concentrations ranged from 440.2 to 1147 ng/mL and the elimination half-life from 422 to 647.8 h. Serum concentrations were below the limit of quantitation by days 4, 7, 12 and 12 for doses of 3.5, 7.1, 14.3 and 21.4 mg/kg, respectively. Urine concentrations were below the limit of detection by day 44 following administration of 3.5 mg/kg and day 51 for all other dose groups. No adverse effects were observed following meldonium administration. While the group numbers were small, changes in heart rate were observed in the 3.5 mg/kg dose group (n = 1). Glucose concentrations changed significantly in all dose groups studied (n = 2 per dose group). Similar to that reported for humans, the detection time of meldonium in biological samples collected from horses is prolonged, which should allow for satisfactory regulation in performance horses. Copyright (C) 2017 John Wiley & Sons, Ltd.
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| 9. |
- Krug, Oliver, et al.
(författare)
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Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4
- 2012
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Ingår i: Journal of Equine Veterinary Science. - : Elsevier BV. - 0737-0806 .- 1542-7412. ; 32:9, s. 562-568
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Tidskriftsartikel (refereegranskat)abstract
- 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 chromatography–tandem mass spectrometry and –high-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 chromatography–tandem mass spectrometry and –high-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%.
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| 10. |
- Nilsson Broberg, Malin, et al.
(författare)
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A multivariate data analysis approach for the investigation of in vitro derived metabolites of ACP-105 in comparison with human in vivo metabolites
- 2023
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Ingår i: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 1231
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Tidskriftsartikel (refereegranskat)abstract
- Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry.The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.
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