Abstract P320: Novel Alpha-calcitonin Gene Related Peptide Agonist Analogs In The Treatment Of Cardiovascular Disease

Abstract

Alpha-calcitonin gene related peptide (α-CGRP) is a potent vasodilator and protects against heart failure and hypertension in various animal models; however, rapid clearance of the peptide from the circulation makes it difficult to use as a therapeutic agent. Recently, we have synthesized two α-CGRP agonist analogs linked with two n-methoxyethylglycine (NMEG) peptoid molecules at either end: 1)- α-CGRP with NMEG molecules at the N-terminal end (N-ter NMEG), and 2)- α-CGRP with NMEG molecules at the C-terminal end (C-ter NMEG). Bioactivity and toxicity of modified peptides were evaluated in in vitro and in vivo conditions. Peptide-peptoid hybrids, N-ter NMEG and C-ter NMEG, were synthesized using a solid-phase peptide synthesis method with a >98% purity. To test the in vitro cell viability of the analogs, rat H9C2 cardiac myocyte cells were treated with 1 μM, 3 μM, and 10 μM concentrations of N-ter NMEG and C-ter NMEG peptides for 4 days followed by an MTT assay. Our in vitro results demonstrated that neither α-CGRP analogs were toxic to H9C2 cells. To test the bioactivity of these peptides, blood pressure (BP) was measured in wild-type C57BL6 mice using a tail-cuff BP analysis system. A bolus dose of 1.2, 3.6, and 12 (in mg/kg b.wt./mouse) of either peptide was injected subcutaneously (n= 4 mice/dose) followed by BP measurement at increasing time points. Our results demonstrate that there is a variance in the bioactivity of the two peptides. Subcutaneous delivery of N-ter NMEG reduced BP similar to the native peptide. After ten minutes of N-ter NMEG delivery, the BP (in mmHg ±SD) at a dose of 1.2 mg/kg was 76 ±5 (baseline BP= 118 ±15), at a dose of 3.6 mg/kg was 79 ±7 (baseline BP= 113 ±9), and at a dose of 12 mg/mg was 83 ±12 (baseline BP= 104 ±6). In contrast, C-ter NMEG administration did not reduce BP at any peptide concentration tested. In summary, in this study we determined that the α-CGRP analog N-ter NMEG, but not C-ter NMEG, is bioactive. Furthermore, N-ter NMEG showed no toxic effects on H9C2 cardiac myocytes in an in vitro viability assay. These results demonstrate that the analog N-ter NMEG is an effective α-CGRP agonist and a promising candidate molecule to treat cardiac diseases.