Mechanistic Studies on AMD6221: A Ruthenium-Based Nitric Oxide Scavenger

Abstract

Nitric oxide is a mediator of many disease states. Previous studies have demonstrated that ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. Using RAW264 cells it was confirmed that [Ru(H3dtpa)(Cl)] (AMD6221) was neither acting at the level of iNOS induction, nor as an inhibitor of iNOS by measuring iNOS mRNA by RT-PCR and protein by Western blot and enzyme activity. Using HPLC, the nitrosyl adduct of reaction of AMD6221, [Ru(H2dtpa)NO], was identified in the medium of stimulated RAW264 cells co-incubated with AMD6221, concomitant with a stoichiometric reduction in nitrite/nitrate levels, thus confirming that the ruthenium(III) polyaminocarboxylates exert their pharmacological effect by scavenging NO.