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Putative Agmatinase Inhibitor for Hypoxic-Ischemic New Born Brain Damage

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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 3 h. Upon reoxygenation, the slices were treated with piperazine-1-carboxamidine or agmatine (50 μg/ml agents), or saline, and 15 h 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 2.5 h. Piperazine-1-carboxamidine at 50 mg/kg i.p. was given 15 min post-reoxygenation and continued twice daily for 3 days. 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 22 days 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.

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Acknowledgments

This research was supported by a grant from the National Institutes of Health, R21 NS054862-1-3. For the analytical chemistry of newly-synthesized PZC, we gratefully acknowledge the Mississippi College Diffraction Facility supported by National Science Foundation grant MRI 0618148, as well as the W. M. Keck Foundation for crystallographic resources. For the brain slice procedure we thank the advice of Dr. Robert Mitchell, and for the slice hypoxia protocol we thank the advice of Dr. Rubin Mestril (both of Loyola University Medical Center, Chicago). For immunohistochemical procedures we gratefully acknowledge the advice of Dr. Rita Cowell of the University of Alabama and of Dr. Julie Fudge of the University of Rochester, NY.

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Correspondence to John E. Piletz or Michael A. Collins.

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Piletz, J.E., Klenotich, S., Lee, K.S. et al. Putative Agmatinase Inhibitor for Hypoxic-Ischemic New Born Brain Damage. Neurotox Res 24, 176–190 (2013). https://doi.org/10.1007/s12640-013-9376-5

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