Administration study of black market ACE-031 (Ramatercept)

Code: 21C11CR

Chapter S4 of WADA’s Prohibited List 2021 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter 3 (“Agents preventing activing receptor IIB activation, Activin receptor IIB competitors – Decoy activing receptors”) as prohibited substance. ACE-031 (Ramatercept) is a soluble fusion protein, which blocks activin receptor type II B (ActRIIB) ligands including myostatin. So far, no approved ACE-031 pharmaceuticals are available. On the other hand, ACE-031 is sold on the black market. However, the administration of black market ACE-031 to human test persons will be ethically not justifiable. For that reason we plan a study with rats. Page 2/7 The test animals will receive black market ACE-031 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 ACE-031 by electrophoresis and Western blotting. In addition, the metabolism of black market ACE-031 will be investigated in human microsomes as the metabolites in rat may be different from those in humans. The study will help to clarify (1) how long black market ACE-031 is detectable in blood, (2) if it can also be observed in urine, and (3) if metabolites are different in rats and humans. We have already developed a method for the detection of black market ACE-031 after electrophoretic separation and Western blotting (SDS-, SAR-PAGE).

Main findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter S4.3 (“Agents preventing activing receptor IIB activation, Activin receptor IIB competitors such as decoy activin receptors (e.g. ACE-031)”) as prohibited substance. Currently, ACE-031 is only available on the black market. Since administration of black market ACE-031 to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- the characterization of 14 black market ACE-031 products by SDS-PAGE followed by Coomassie staining or Western blotting using ACVR2B-, follistatin- and His-tag-specific antibodies

- an investigation of the electrophoretic detectability of black market ACE-031 in rat serum and urine after circulation in blood for 24, 48, and 168 hours

- an in vitro metabolism study of ACE-031 using human and rat liver microsomes

Results:

Of the 14 tested ACE-031 products, 13 contained Coomassie-stainable proteins. Western blotting revealed that 12 products were indeed ACVR2B-related proteins. One product was mislabelled and contained black market follistatin instead. It could be further shown that on all 12 ACE-031 products His-tags were present as already demonstrated for black market follistatin.

After a single dose administration of black market ACE-031 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. While it remained traceable in the majority of the 48 h samples, it was undetectable after 7 days (168 h). No ACE-031 was found in the urine samples at all three time-points.

In order to reveal possible differences in the metabolism of ACE-031 between humans and rats, ACE-031 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 ACE-031 can be detected by SDS-PAGE and immunoblotting with a monoclonal ACVR2B-specific antibody. Additionally, it contains immunoreactive His-tags. For extraction of ACE-031 from rat serum and urine samples, a polyclonal ACVR2B-antibody linked to magnetic beads was used. After single dose administration, the protein remained detectable for at least 48 hours in most rat serum samples. No signals were obtained on Western blots after 168 hours in serum and in all urine samples.