Putative Agmatinase Inhibitor for Hypoxic-Ischemic New Born Brain Damage

Abstract

Agmatine is an endogenous brain metabolite, decarboxylated arginine, which has neuroprotective properties when injected intraperitoneally (i.p.) into rat pups following hypoxic-ischemia. A previous screen for compounds based on rat brain lysates containing agmatinase with assistance from computational chemistry, led to piperazine-1-carboxamidine as a putative agmatinase inhibitor. Herein, the neuroprotective properties of piperazine-1-carboxamidine are described both in vitro and in vivo. Organotypic entorhinal-hippocampal slices were firstly prepared from 7-day-old rat pups and exposed in vitro to atmospheric oxygen depletion for 3h. Upon reoxygenation, the slices were treated with piperazine-1-carboxamidine or agmatine (50μg/ml agents), or saline, and 15h later propidium iodine was used to stain. Piperazine-1-carboxamidine or agmatine produced substantial in vitro protection compared to post-reoxygenated saline-treated controls. An in vivo model involved surgical right carotid ligation followed by exposure to hypoxic-ischemia (8% oxygen) for 2.5h. Piperazine-1-carboxamidine at 50mg/kg i.p. was given 15min post-reoxygenation and continued twice daily for 3days. Cortical agmatine levels were elevated (+28.5%) following piperazine-1-carboxamidine treatment with no change in arginine or its other major metabolites. Histologic staining with anti-Neun monoclonal antibody also revealed neuroprotection of CA1-3 layers of the hippocampus. Until endpoint at 22days of age, no adverse events were observed in treated pups' body weights, rectal temperatures, or prompted ambulation. Piperazine-1-carboxamidine therefore appears to be a neuroprotective agent of a new category, agmatinase inhibitor.