Abstract Body: Introduction: Cardiac arrest (CA) affects about 650,000 people annually. The overall survival rate for OHCA is less than 10% and 25% for IHCA. Early intervention after CA during CPR is essential to the likelihood of survival. We have demonstrated that a novel peptide TAT-PHLPP9c, when administered during CPR, improves survival following asystole, ventricular fibrillation, and pulseless electrical activity arrest in mouse and swine models. To further optimize the use of this peptide for resuscitation, we investigated two clinically relevant factors that could affect TAT-PHLPP9c effectiveness, degradation and storage stability.
Hypothesis: TAT-PHLPP9c plasma concentrations could be affected by proteases and its activity by changes in storage temperature.
Methods: TAT-PHLPP9c (7.5 mg/kg) was administered to mice via retro-orbital injection. Plasma concentrations were measured at 5, 10 min, up to 1 h by ELISA assay. An in vitro study was also performed by adding 100 nM of TAT-PHLPP9c to the plasma of mice, pigs and humans. The concentrations at baseline, 30, 60, and 120 min were measured. To study the stability, the lyophilized powder of TAT-PHLPP9c was stored at -80, -20, 4, and 20 °C for 6 months. The peptide bioactivity was Akt activation measured by Western blot. In C57BL/6 retired female mice (n=20), a 12-min asystolic arrest was induced with KCl followed by CPR and blinded randomized administration of TAT-PHLPP9c or saline control during CPR. Survival (4h) was assessed.
Results: The baseline concentration of TAT-PHLPP9c was about 94 µg/ml. Its concentration was 3 µg/ml at 5 min, 0.5 µg/ml at 10 min, and disappeared by 30 min post-injection. In vitro studies demonstrated that its plasma concentration at baseline was 25 nM in mice, 50 nM in pigs, and 60 nM in humans, respectively. The concentration was decreased by 50% in mice and about 20% in pigs and humans, which was abrogated by the protease inhibitor complex III. Despite its rapid degradation by proteases, TAT-PHLPP9c administered during CPR in the mouse CA model improved CA outcomes after 12 min arrest. For the stability study, Akt phosphorylation was significantly reduced after 3 months for all the storage temperatures except for -80 °C.
Conclusions: TAT-PHLPP9c degradation in plasma is primarily mediated by proteases, but despite degradation can retain significant activity. Formulation modifications will be needed to improve stability during storage conditions common to the ambulance environment.