Pharmacological Characterization of Novel High Affinity CGRP Receptor Antagonists that are Selective for Human CGRP Receptors

Abstract

Human-α-calcitonin gene-related peptide (CGRP) is a 37 amino acid residue peptide produced from alternative splicing of the calcitonin gene. CGRP mediates vascular relaxation and other effects through CGRP receptors, which activate adenylyl cyclase. We found that benzoylation of the N-terminus and benzylation of the His10 side-chain of the antagonist CGRP(8-37) produced a high affinity antagonist with selectivity for human CGRP receptors. CGRP(8-37) was synthesized on a p-MBHA resin by rapid assembly methods using Boc-chemistry and in situ neutralization solid phase peptide synthesis. His10 benzylation was accomplished by reaction with benzyl bromide and N-terminal acylation was accomplished by reaction with benzoic anhydride. The peptide was cleaved form the resin using TFMSA, purified by reversed-phase HPLC to > 98% and characterized by amino acid analysis and mass spectrometry. CGRP(8-37), N-benzoyl-CGRP(8-37) and N-benzoyl-(His10-benzyl)-CGRP(8-37) produced concentration dependent inhibition of CGRP-mediated vasodilation of mouse thoracic aortas studied in vitro, with KB values of 1000.3, 88.9 and 22.9 nM, respectively. These antagonists also produced concentration dependent inhibition of CGRP-mediated cAMP production in human SK-MC cells with KB values of 797.0, 15.4 and 0.65 nM, respectively. N-terminus benzoylation combined with His10 benzylation significantly increased the affinity and selectivity for human SK-N-MC cells. Thus, this antagonist inhibited CGRP-mediated responses in vitro and may be a useful tool to investigate CGRP-mediated functional responses in human tissue.