Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.
Abstract: The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH (M1), SCHFGPLTWVCKPQGG-NH (M2), WVCKPQGG-NH (M3) and VCKPQGG-NH (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.
© 2023 John Wiley & Sons Ltd.
Publication Date: 2023-05-29 PubMed ID: 37248680DOI: 10.1002/dta.3522Google Scholar: Lookup
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Summary
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This study has discovered a substance known as erythropoietin mimetic peptide 1 linear form (EMP1-linear), a performance-enhancing prodrug, in a substance suspected to be used for doping in horse racing. The researchers have also outlined a method for detecting the prodrug and its metabolites in equine biological samples, suggesting a new path for detecting doping in horse sports.
Identification of EMP1-linear
- The researchers identified erythropoietin mimetic peptide 1 linear form (EMP1-linear) in a white crystalline powder contained in sealed vials.
- The identification was carried out using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).
Role of EMP1-linear in Doping
- The researchers found that EMP1-linear may be used for doping in horse racing, with the substance making up about 2% of the analyzed powder.
- EMP1-linear can be converted into EMP1 in equine plasma, a well-known erythropoiesis stimulating agent capable of enhancing performance by binding to and activating the EPO cytokine receptor.
Metabolic Study of EMP1-linear
- In an in vitro study using horse liver S9 fraction, EMP1-linear was found to remain predominantly in its cyclic form as EMP1 after incubation.
- Four truncated in vitro metabolites were also identified: TYSCHFGPLTWVCKPQGG-NH (M1), SCHFGPLTWVCKPQGG-NH (M2), WVCKPQGG-NH (M3), and VCKPQGG-NH (M4).
Administration Study and Detection of EMP1-linear
- An intravenous administration study was performed using the white crystalline powder on three retired thoroughbred geldings.
- EMP1 was only detected in postadministration plasma samples, while the four identified in vitro metabolites were found in both postadministration plasma and urine samples.
- Metabolite M3 showed the longest detection time in both plasma and urine, remaining detectable for up to 4 and 27 hours postadministration, respectively.
Implications for Doping Control
- The study provides a new target for doping detection in equine sports, looking at the use of EMP1-linear and its metabolic pathways.
- The detection of metabolite M3 for extended periods post-administration suggests a pinnacle target for doping control tests.
Cite This Article
APA
So YM, Wong JKY, Wong ASY, Tse ATL, Wan TSM, Ho ENM.
(2023).
Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.
Drug Test Anal.
https://doi.org/10.1002/dta.3522 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, Hong Kong, China.
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- The signal responses of the cyclic disulfide-bonded EMP1, M1 and M2 were found to be lower than their corresponding linear form. Therefore, in order to maximise detection sensitivity, all samples were treated with TCEP to break the disulfide bonds in EMP1, M1 and M2. For identifying the presence of cyclic disulfide-bonded EMP1, M1 and M2 in post-administration samples, the step treated with TCEP was skipped.
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