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Abstract P2033: Rationale For A Novel Approach To Hypertension Treatment Based On Immuno-neutralization Of Angiotensin-(1-12) Catalytic Sites With A Polyclonal Antibody


 The biochemical pathways for the formation of biological active angiotensins continues to undergo significant revision with the demonstration of angiotensin-(1-12) as an endogenous substrate forming angiotensin II (Ang II) by a non-renin dependent mechanism. Research done in our laboratory established that Ang II generation from Ang-(1-12) is primarily mediated by ACE in the circulation and by chymase in cardiac tissues. In this study, we have utilized an antigen-antibody binding approach to block the cleavage sites of human Ang-(1-12) peptide with an anti-Ang-(1-12) polyclonal antibody (pAb) as a potential therapeutic strategy to prevent the catalytic action of chymase and ACE enzymes to generate Ang II. The pAb was generated against the C-terminus of the human Ang-(1-12) peptide sequence and its specificity for human Ang-(1-12) and cross-reactivity with other angiotensin peptides were determined in competitive binding assays. To block the cleavage sites, the radiolabeled human Ang-(1-12) [
 125
 I-Ang-(1-12)] substrate was first neutralized by pre-incubating with or without anti-Ang-(1-12) pAb and then re-incubated with human recombinant chymase (hrChymase, 0.325 μg/mL) or 10 μL of rat serum (for ACE) in 50 mM Tris-HCl buffer solution containing 150 mM NaCl (pH 8.0) for 30 min at 37
 o
 C. The reaction was stopped by adding an equal volume of 1% phosphoric acid and
 125
 I-Ang metabolic products were quantified by HPLC connected to an in-line flow-through gamma detector. Anti-Ang-(1-12) pAb showed high specificity for the human Ang-(1-12) sequence (EC
 50
 = 29.6 ng/mL) and negligible cross-reactivity with closely related angiotensin peptides. The non-neutralized human
 125
 I-Ang-(1-12) substrate was rapidly metabolized by both hrChymase and rat serum ACE to
 125
 I-Ang II (81% and 35%, respectively). In contrast, generation of
 125
 I-Ang II from neutralized
 125
 I-Ang-(1-12) substrate by either hrChymase or ACE was only 17% and 7%, respectively. The effective and potent blockade of Ang II generation from either chymase or ACE by combining Ang-(1-12) with an anti-Ang-(1-12) pAb now paves the way to develop new therapeutic approaches to hypertension treatment using Ang-(1-12) antibodies directed against the human sequence of this Ang II-forming substrate.
