Development of a competition ELISA to assess stability and measure ShK‐186 in plasma samples

ShK-186, a potent (IC50 70 pM) and selective peptide blocker of Kv1.3, is scheduled to begin human phase 1 trials in 2011 as a therapeutic for autoimmune disease. Kv1.3 selectivity is due to a negatively charged phosphotyrosine (pTyr) at the N-terminus. Here, we describe the development of a competition ELISA (cELISA) to measure ShK-186 concentrations in plasma samples. Six monoclonal antibodies (mAbs) were raised and specificities were determined against a panel of ShK analogs. Four mAbs reacted with the pTyr moiety (e.g. P2F12), and two reacted only to the peptide-body. A cELISA was developed with P2F12 to detect the intact fully phosphorylated form. Standard curves generated with varying concentrations of ShK-186 in plasma were reproducible (n=9), and QC samples were measured within 9% precision. Alpha-1 acid glycoprotein, a plasma protein known to bind cationic peptides, sequestered ShK-186 in a time-dependent manner. High salt reversed this effect, suggesting a charge-charge interaction. Dephosphorylation, detected by loss of P2F12 reactivity and confirmed by LC-MS, was evident in 1 hour in rat and human plasma, and was anti-coagulant dependent. In patch-clamp experiments, the dephosphorylated peptide (ShK-198) blocked Kv1.3 (IC50 4.3 pM) and Kv1.1 (IC50 3 pM) with greater potency but lower selectivity than ShK-186. PK studies in rats are underway. Supported by NIH (RO1NS48252) and SBIR (1R43A1085691-01)