Detection and quantification of the metabolite Ac‐Tβ_1–14 in in vitro experiments and urine of rats treated with Ac‐Tβ4: A potential biomarker of Ac‐Tβ4 for doping tests

Abstract

Thymosin β4 (Tβ4) was reported to exert various beneficial bioactivities such as tissue repair, anti‐inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti‐Doping Agency. However, no metabolism studies of Tβ4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac‐Tβ31–43, Ac‐Tβ17–43, Ac‐Tβ1–11, Ac‐Tβ1–14, Ac‐Tβ1–15, and Ac‐Tβ1–17) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tβ4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tβ4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac‐Tβ1–14 was developed and validated using Q‐Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac‐Tβ1–14 were 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r2 = 0.9998). As a result, among the six metabolites above, Ac‐Tβ1–14, as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tβ4, and the urine of rats intraperitoneally treated with 20‐mg/kg Tβ4. And the metabolite Ac‐Tβ1–14 was quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac‐Tβ1–14 was not detected in non‐treated control groups, including human blank urine. These results suggest that Ac‐Tβ1–14 in urine is a potential biomarker for screening the parent Tβ4 in doping tests.

The 41st Manfred Donike workshop on doping analysis

“…Conversely, the optimized sensitivity of analytical approaches regarding the growth hormone secretagogue capromorelin proved capable of detecting trace amounts of the drug in a doping control urine sample, which was attributable to a contamination scenario that an athlete was exposed to when administering the drug to a dog, 18 thus presenting another facet of the athlete's exposome. 19 Advancing the knowledge concerning peptide-based drug metabolites for sports drug testing purposes was conducted using in vitro and animal in vivo experiments, contributing new information for thymosin-β4, 20 the insulin-mimetic peptides S519 and S597, 21 and myostatin inhibitory peptides (e.g., DF-3 and DF-25). 22 Finally, and particularly relevant for result management and in avoiding unintentional anti-doping rule violations, higenamine excretion profiles in urine after ingestion of higenamine-containing fruit as well as enriched dietary supplement were investigated, suggesting that concomitantly observed markers of benzylisoquinoline alkaloid biosynthetic pathway in distinct abundance ratios are particularly valuable for a differentiation of diet-related exposure and doping scenarios.…”

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…The metabolic fate of thymosin β4 was studied further by Rahaman et al employing both in vitro and rat in vivo experiments, aiming at providing best‐possible target analytes for human urine analyses 80 . The peptidic drug candidate that currently undergoes a variety of clinical trials was exposed to leucyl aminopeptidase, human kidney microsomes, and human umbilical vein endothelial cells, and was also injected to rats at 20 mg/kg body weight as a single dose.…”