Administration study of black market myostatin propeptide

Code: 20C17CR

Chapter S4 of WADA’s Prohibited List 2020 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter 4 (“Agents preventing activing receptor IIB activation, Myostatin inhibitors”) as prohibited substance. So far, no approved myostatin propeptide pharmaceuticals are available. On the other hand, myostatin propeptide is sold on the black market (labelled “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”). But the administration of black market myostatin propeptide to human test persons will be ethically not justifiable. For that reason we plan a study with rats. The test animals will receive black market myostatin propeptide at a dosage, which can be clearly detected in serum (10 mg/kg BW). After 24, 48 and 168 hours, serum and urine will be collected and tested for myostatin propeptide by electrophoresis and Western blotting. The study will help to clarify (1) how long black market myostatin propeptide is detectable in blood, and (2) if it can also be observed in urine. We have already shown that black market myostatin propeptide can be differentiated from endogenous myostatin propeptide by electrophoresis (SDS-PAGE) and Western blotting.

Main findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter S4.3. (“Agents preventing activin receptor IIB activation, Myostatin inhibitors such as myostatin-binding proteins (e.g. follistatin, myostatin propeptide)”) as prohibited substance. Currently, myostatin propeptide is only available on the black market. Since administration of black market products to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- an animal study (administration of black market myostatin propeptide (“GDF-8”) and recombinant myostation propeptide standard to rats followed by collection of serum and urine)

- an investigation of the electrophoretic detectability of black market GDF-8 in rat serum and urine after circulation in blood for 24, 48, and 168 hours. Additionally, the detectability of recombinant myostation propeptide standard after 24 hours was also investigated

- an in vitro metabolism study of myostation propeptide using human and rat liver microsomes

Results:

After a single dose administration of black market GDF-8 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. However, it was no longer traceable in the samples taken after 48 h and 168 h. No GDF-8 was found in the urine samples at all three time-points. Recombinant myostation propeptide was neither detectable in serum nor urine 24 hours post administration.

In order to reveal possible differences in the metabolism of myostation propeptide between humans and rats, it was incubated with human and rat liver microsomes (5, 60, 120, 300 min, 24 hours). The protein proved quite stable - only after 24 hours degradation of the main band was observed.

Conclusions:

Black market myostation propeptide can be detected in rat serum samples by SDS-PAGE and immunoblotting with a monoclonal myostation propeptide-specific antibody. For the extraction from rat serum and urine, polyclonal myostation propeptide-antibodies captured by magnetic beads or coated on ELISA-wells were used. After single dose administration, the protein remained detectable for only 24 hours in rat serum samples. No signals were obtained on Western blots in serum after 48 and 168 hours and at all three time-points in urine samples.