RUTHENIUM COMPLEXES AS NITRIC-OXIDE SCAVENGERS - A POTENTIAL THERAPEUTIC APPROACH TO NITRIC OXIDE-MEDIATED DISEASES

1 Ruthenium(III) reacts with nitric oxide (NO) to form stable rutheniu m(II) mononitrosyls. Several Ru(III) complexes were synthesized and a study made of their ability to bind NO, in vitro and also in several b iological systems following expression of the inducible isoform of nit ric oxide synthase (iNOS). Here we report on the properties of two, re lated polyaminocarboxylate-ruthenium complexes: potassium ro[hydrogen( ethylenedinitrilo)tetraaceto]ruthenate (=JM1226; CAS no.14741-19-6) an d [hydrogen(ethylenedinitrilo)tetraacetato]ruthenium (=JM6245; CAS no. 15282-93-6). 2 Binding of authentic NO by aqueous solutions of JM1226 yielded a product with an infrared (IR) spectrum characteristic of an Ru(II)-NO adduct. A compound with a similar IR spectrum was obtained a fter reacting JM1226 with S-nitroso-N-acetylpenicillamine (SNAP). 3 Th e effect of JM1226 or JM6245 on nitrite (NO2-) accumulation in culture s of macrophages (RAW 264 line) 18 h after stimulating cells with lipo lysaccharide (LPS) and interferon-gamma (IFN gamma) was studied. Activ ation of RAW264 cells increased NO2- levels in the growth medium from (mean +/- s.e. mean) 4.9 +/- 0.5 mu M to 20.9 +/- 0.4 mu M. This was b locked by actinomycin D (10 mu M) or cycloheximide (5 mu M). The addit ion of JM1226 or JM6245 (both 100 mu M) to activated RAW264 cells redu ced NO2- levels to 7.6 +/- 0.2 mu M and 8.8 +/- 0.6 mu M, respectively . N-G-methyl-L-arginine (L-NMMA; 250 mu M) similarly reduced NO2- leve ls, to 6.1 +/- 0.2 mu M. 4 The effect of JM1226 or JM6245 on NO-mediat ed tumour cell killing by LPS + IFN gamma-activated macrophages (RAW 2 64) was studied in a co-culture system, using a non-adherent murine ma stocytoma (P815) line as the 'target' cell. Addition of JM1226 or JM62 45 (both 100 mu M) to the culture medium afforded some protection from macrophage-mediated cell killing: target cell viability increased fro m 54.5 +/- 3.3% to 93.2 +/- 7.1% and 80.0 +/- 4.6%, respectively (n = 6).5 Vasodilator responses of isolated, perfused, pre-contracted rat t ail arteries elicited by bolus injections (10 mu l) of SNAP were atten uated by the addition of JM1226 or JM6245 (10(-4) M) to the perfusate: the ED50 increased from 6.0 mu M (Krebs only) to 1.8 mM (Krebs + JM62 45) and from 7 mu M (Krebs only) to 132 mu M (Krebs + JM1226). Oxyhaem oglobin (5 mu M) increased the ED50 value for SNAP from 8 mu M to 200 mu M. 6 Male Wistar rats were injected with bacterial LPS (4 mg kg(-1) ; i.p.) to induce endotoxaemia. JM1226 and JM6245 (both 100 mu M) full y reversed the hyporesponsiveness to phenylephrine of tail arteries is olated from animals previously (24 h earlier) injected with LPS. Blood pressure recordings were made in conscious LPS-treated rats using a t ail cuff apparatus. A single injection of JM1226 (100 mg kg(-1), i.p.) administered 20 h after LPS (4 mg kg(-1), i.p.) reversed the hypotens ion associated with endotoxaemia. 7 The results show that JM1226 and J M6245 are able to scavenge NO in biological systems and suggest a role for these compounds in novel therapeutic strategies aimed at alleviat ing NO-mediated disease states.