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

Hansson, Annelie; Thevis, Mario; Cox, Holly D.; Miller, Geoffrey D.; Eichner, Daniel; Bondesson, Ulf; Hedeland, Mikael

doi:10.1016/j.jpba.2016.11.041

Cited by 21 publications

(22 citation statements)

References 16 publications

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“…This finding agrees with the study on the synthetic cannabinoid AM2201 that glucosidation and sulfation are the only phase II reactions observed for C. elegans metabolism while glucuronidation is the major phase II transformation in humans [33]. Similarly, for some other drugs, glucosidation and sulfation are reported to be the phase II reactions of C. elegans while the same drugs are glucuronidated and sulfated in humans [38,48]. The latter experiments did not involve extraction step as a sample preparation, supporting that poor recovery of glucuronides in dichloromethane is not likely a reason for the absence of glucuronides in this study.…”

Section: Comparison Of Fungal Metabolites With Reported Human Metabolsupporting

confidence: 91%

“…There is no literature on specific enzymes responsible for the metabolism of 8-quinolinyl 1-(5-fluoropentyl)-1H-indole-3-carboxylate (5F-PB-22) and 8-quinolinyl 1-pentyl-1H-indole-3-carboxylate (PB-22), the other two drugs investigated. Some of the advantages of C. elegans include low cost, no ethical concern involved, simple and cheap maintenance of stock fungal cultures that can be subcultured, high reproducibility, diverse metabolic profile and larger amounts of metabolite produced, which might result in more fragment ions in mass spectrometric analysis, facilitating structural elucidation [30,38]. More importantly, production of large amounts of metabolites have allowed for the structural characterisation of metabolites by NMR spectroscopy [39,40].…”

Section: Introductionmentioning

confidence: 99%

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Metabolic Profile of Synthetic Cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans

Watanabe

Kuzhiumparambil

Nguyen

et al. 2017

AAPS J

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The knowledge of metabolic profile of synthetic cannabinoids is important for the detection of the drugs in urinalysis due to the typical absence or low abundance of parent cannabinoids in human urine. The fungus Cunninghamella elegans has been reported to be a useful tool for metabolism study and thus applicability to synthetic cannabinoids metabolism was examined. In this study, 8-quinolinyl 1-(5-fluoropentyl)-1H-indole-3-carboxylate (5F-PB-22), 8-quinolinyl 1-pentyl-1H-indole-3-carboxylate (PB-22), [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11) and (1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) were incubated with Cunninghamella elegans and the metabolites were identified using liquid chromatographyquadrupole time-of-flight mass spectrometry. The obtained metabolites were compared with reported human metabolites to assess the suitability of the fungus to extrapolate human metabolism. 5F-PB-22 underwent, dihydroxylation, dihydrodiol formation, oxidative defluorination, oxidative defluorination to carboxylic acid, ester hydrolysis and glucosidation, alone and/or in combination. The metabolites of PB-22 were generated by hydroxylation, dihydroxylation, trihydroxylation, dihydrodiol formation, ketone formation, carboxylation, ester hydrolysis and glucosidation, alone and/or in combination. XLR-11 was transformed through hydroxylation, dihydroxylation, aldehyde formation, carboxylation, oxidative defluorination, oxidative defluorination to carboxylic acid and glucosidation, alone and/or in combination. UR-144 was metabolised by hydroxylation, dihydroxylation, trihydroxylation, aldehyde formation, ketone formation, carboxylation, N-dealkylation and combinations. These findings were consistent with previously reported human metabolism except for the small extent of ester hydrolysis observed and absence of glucuronidation. Despite the limitations, Cunninghamella elegans demonstrated the capacity to produce a wide variety of metabolites including some major human metabolites of XLR-11 and UR-144 at high abundance, showing the potential for metabolism of newly emerging synthetic cannabinoids.

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Section: Comparison Of Fungal Metabolites With Reported Human Metabolsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Metabolic Profile of Synthetic Cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans

Watanabe

Kuzhiumparambil

Nguyen

et al. 2017

AAPS J

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“…[1][2][3] The use of HIF prolyl hydroxylase inhibitors (PHIs) has been shown to allow for correcting and maintaining target haemoglobin levels in chronic kidney disease (CKD) patients, [4][5][6] and although full clinical approval has not been issued for any representative of this emerging class of therapeutics, concerns regarding illicit use, especially in professional and amateur sport, arose approximately a decade ago. Consequently, pilot studies aimed at testing model substances and in vitro derived metabolites were initiated [7][8][9] before 2011 the World Anti-Doping Agency (WADA) first mentioned HIF PHI explicitly in the annually issued Prohibited List. [10] Banned at all times, HIF PHI have since been on the radar of doping control laboratories, [11][12][13] and anti-doping efforts have lately been substantially facilitated by collaborative studies with the pharmaceutical industry.…”

Section: Introductionmentioning

confidence: 99%

lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG‐4592) and its main metabolites into routine doping controls

Eichner¹,

Wagoner²,

Brenner

et al. 2017

Drug Testing and Analysis

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The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma-and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-forpurpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass.

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“…Efficiently targeting new, non‐approved substances is particularly challenging when (urinary) metabolites are unknown. For the HIF stabilizer FG‐4592 (Figure , 4 ), different in vitro models were employed to comprehensively generate and characterize phase I metabolites of the drug candidate which were subsequently compared to those metabolites observed in a doping control sample that tested positive for FG‐4592 . The extent of in vitro metabolism was found to be modest in human and equine liver microsomes, with 1 identical hydroxylated metabolite being predominant in both media.…”

Section: Peptide Hormones Growth Factors Related Substances and MImentioning

confidence: 99%

Annual banned‐substance review: Analytical approaches in human sports drug testing

Thevis

Kuuranne

Geyer

2017

Drug Testing and Analysis

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Several high-profile revelations concerning anti-doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti-doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti-Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug-testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug-testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned-substance review evaluates literature published between October 2016 and September 2017 offering an in-depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List.

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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

Cited by 21 publications

References 16 publications

Metabolic Profile of Synthetic Cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans

Metabolic Profile of Synthetic Cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans

lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG‐4592) and its main metabolites into routine doping controls

Annual banned‐substance review: Analytical approaches in human sports drug testing

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