In vitro peptide degradation assay

Nerinetide (18 mg/ml) was mixed with human plasmin (1 mg/ml; Sigma-Aldrich, catalog no. P1867-150UG) for 5 min at 37°C. The reaction was stopped by transferring the tubes to 4°C. At least five aliquots of 100 μl of digest solution were collected and stored at −80°C until analysis. Samples were analyzed by LC-MS.

To determine the stability of nerinetide and d-Tat-l-2B9c in the presence of rt-PA or plasmin, they were added at 65 μg/ml to 600 μl of PBS and incubated at 37°C for 2 min for baseline time points. rt-PA (135 μg/ml) or plasmin (10 μg/ml) was added after the first sample acquisition. Experiments were performed in triplicate.

The peptide content of nerinetide in plasma was analyzed by HPLC. In brief, nerinetide or d-Tat-l-2B9c was spiked into rat or human plasma at a concentration of 65 μg/ml. Alteplase was then added after the baseline time point was collected at the specified concentrations. Alteplase administration followed the clinical dosing approach [10% bolus dose followed by 60-min infusion (90% of the dose)] using a Harvard apparatus pump.

Sample collection after nerinetide or d-Tat-l-2B9c spiking in PBS or plasma was performed at 5, 15, 30, and 45 min after dose. At each time point, about 100 μl of plasma was collected from each vial using a fresh syringe. Plasma was then collected and stored at −80°C until analyzed.