title: Methods for detection and confirmation of Hematide™/peginesatide in anti-doping samples.
abstract: Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide™) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market. To detect potential athletic abuse of this product and deter athletes who consider cheating, we initiated a collaboration to implement a detection test for anti-doping purposes. Peginesatide is a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. It is undetectable using current anti-doping tests due to its lack of sequence homology to EPO. To detect and deter potential abuse of peginesatide, we initiated an industry/antidoping laboratory collaboration to develop and validate screening and confirmation assays so that they would be available before peginesatide reaches the market. We describe a screening ELISA and a confirmation assay consisting of immune-purification followed by separation with SDS-PAGE and revelation with Western double blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in blood samples, enabling detection for several days after administration of a physiologically relevant dose. This initial report describes experimental characterization of these assays, including testing with a blinded set of samples from a clinical study conducted in healthy volunteers.
authors.affiliation: Swiss Laboratory for Doping Analyses, University Center of Legal Medecine, Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Ch. des Croisettes 22, CH-1066 Epalinges, Switzerland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Leuenberger
authors.forename: Jérémy
authors.initials: J
authors.lastname: Saugy
authors.forename: Richard B
authors.initials: RB
authors.lastname: Mortensen
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schatz
authors.forename: Sylvain
authors.initials: S
authors.lastname: Giraud
authors.forename: Martial
authors.initials: M
authors.lastname: Saugy
mesh_terms: D015153:Blotting, Western; D003299:Cooperative Behavior; D004300:Doping in Sports; D004345:Drug Industry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006397:Hematinics / Q000097:blood*; D006801:Humans; D057230:Limit of Detection; D010455:Peptides / Q000097:blood*; D058573:Performance-Enhancing Substances / Q000097:blood*; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D006397:Hematinics; D010455:Peptides; D058573:Performance-Enhancing Substances; C556270:peginesatide