Metabolism and detection of ghrelin and new ghrelin mimetics

Code: 18A03MT 

Ghrelin and Ghrelin mimetics belong to the prohibited substances according to the current WADA Prohibited List. After administration, they induce the secretion of growth hormone into the circulation and are considered as performance enhancing accordingly. While Ghrelin itself is an endogenous peptide hormone, Ghrelin mimetics (such as Capromorelin, Macimorelin and Tabimorelin) are low molecular mass synthetic peptides (or peptide similars) which act as agonists at the ghrelin receptor. Although prohibited, efficient detection methods for these drugs are scarce. Thus, it is planned to develop analytical methods to determine the potential misuse of the new hGhrelin mimetics (Capromorelin, Macimorelin and Tabimorelin) in doping control samples. This will include the investigation of efficient extraction procedures, metabolism studies (in-vitro/ in-vivo) and identification of reliable target analytes in the respective matrix. Results will be validated and published.

Main Findings: 

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572), and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a ‘dilute-and-inject’ approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs’ biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin, and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency (WADA)-compliant initial testing (LOD: 0.02-0.60 ng/mL) and confirmation procedures (LOI: 0.18-0.89 ng/mL) for human urine and blood matrices. The obtained results allow extending the test spectrum of doping agents in multi-target screening assays for growth hormone-releasing factors from human urine.

For ghrelin and its desacylated analog, a quantitative test method with an LOD of 20 pg/mL and an LOQ of 100 pg/mL was developed and subsequently, a representative population of athletes’ blood samples was analyzed. The intact (non-digested) ghrelin exhibited poor chromatographic properties due to the presence of 7 basic amino acids in the sequence and, therefore, the approach was designed to include an enzymatic hydrolysis step which yielded the tryptic peptide t1 with 11 amino acids and only one Arg residue at the C-terminus. In accordance with literature data, the analyzed plasma samples from professional athletes yielded concentrations between 26 and 177 pg/mL, which is significantly lower than concentrations reported for ghrelin post-administration plasma samples that reached peak levels of 17500 pg/mL in clinical studies.