Detection and in vitro metabolism of AOD9604

Abstract

AOD9604 is a peptide consisting of the C-terminal fragment of human growth hormone from amino acids 177-191 with an additional tyrosine residue at the N-terminus of the peptide. It is reported to mimic the lipolytic properties of growth hormone without the diabetogenic side effects. Therefore, AOD9604 may be used as a performance enhancing drug and is banned by the World Anti-doping Agency (WADA). The peptide is available on several Internet websites and was recently identified in confiscated vials in the USA. To detect abuse of the peptide in athletes, a solid-phase extraction method was validated in urine with a limit of detection of 50 pg/mL. The method has good linearity, precision (<20%), specificity and recovery (62%). Six potential metabolites of the peptide were identified after incubation of AOD9604 in serum and urine. Quantification of the metabolites in serum identified a single metabolite, consisting of amino acids CRSVEGSCG, which is significantly more stable than the other metabolites or the parent compound. Screening for AOD9604 and the stable metabolite may potentially allow an increased window of detection.

Naturally derived cytokine peptides limit virus replication and severe disease during influenza A virus infection

“… 8 , 9 , 10 , 11 LAT8881 can undergo further N‐terminal cleavage in vivo to a peptide CRSVEGSCGF (LAT9991F) that retains its disulphide bond and ostensibly its cyclic 3D structure. 9 , 34 , 35 Proteolytic processing of GH at sites of tissue damage and inflammation (e.g. in viral lung infection or osteoarthritis) to release C‐terminal fragments may represent an endogenous pathway to help limit inflammatory damage and promote recovery.…”

“…LAT8881 is a 16‐amino‐acid synthetic form of the C‐terminal fragment of human GH (H‐YLRIVQCRSVEGSCGF‐OH), which contains an additional N‐terminal tyrosine residue and two cysteine residues linked by a disulphide bond. LAT9991F is a 10‐amino‐acid synthetic peptide (H‐CRSVEGSCGF‐OH), which is a truncated form and a known metabolite of LAT8881 34 . LAT7771 (H‐CRIIHNNNC‐OH) is the 9‐amino‐acid structural homologue of LAT9991F, derived from prolactin and was included as a control peptide.…”

“…LAT9991F is a 10‐amino‐acid synthetic peptide (H‐CRSVEGSCGF‐OH), which is a truncated form and a known metabolite of LAT8881. 34 LAT7771 (H‐CRIIHNNNC‐OH) is the 9‐amino‐acid structural homologue of LAT9991F, derived from prolactin and was included as a control peptide. The cysteines in both LAT9991F and LAT7771 were also disulphide linked.…”

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

“…The C-terminal fragment of human growth hormone comprising of the amino acids 177-191 plus an additional tyrosine residue at the N-terminus of the peptide (referred to as AOD9604) has been the subject of extensive discussions in the past. [33] Its metabolism was studied by Cox et al, who incubated the analyte in human serum and urine, [34] and, in the absence of in vivo data, suggested the implementation of a comparably stable metabolite into routine doping controls supported by in vitro simulation of the peptide's degradation. The additional target analyte was composed of nine amino acid residues (182)(183)(184)(185)(186)(187)(188)(189)(190) and added to a test method consisting of solid-phase extraction (SPE) and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, enabling an limit of detection (LOD) for AOD9604 of 50 pg/mL in human urine.…”

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry

“…Nearly every performance-enhancing biological axis in the human body can be targeted with peptide hormones: the growth hormone (GH)/insulin-like-growth factor-I levels can be manipulated by usage of Growth Hormone Releasing Peptides (GHRPs) or Growth Hormone Releasing Hormones (GHRHs), the steroid profile can be influenced by the use of Gonadorelins (LH-RH, Buserelin, Leuprolide), and recently, with ARA-290 a new non-hematopoietic erythropoetin (EPO) analogue has been added to the Prohibited List [3][4][5][6][7][8][9][10][11][12][13][14][15] [https://www.wada-ama.org/en/resources/science-medicine /prohibited-list]. In addition to that, different effects were described for miscellaneous low molecular mass peptides such as Desmopressin (masking agent), TB-500 (active fragment of Thymosin ␤4) and AOD-9604 (C-terminal fragment of GH) [15][16][17][18][19]. Altogether they share similar properties in terms of size (from 300 to 1800 Da), polarity, functional groups, ionization efficiency (using electrospray ionization), administration-dosage and route (oral, intranasal, i.v.…”