Abstract 493: Potential Therapeutic Benefit of Novel DDAH Inhibitors for the Treatment of Endotoxemia
During the onset of sepsis, the induction of inducible nitric oxide synthase (iNOS) and the subsequent generation of large amounts of nitric oxide (NO) is thought to be partly responsible for the marked hypotension ultimately leading to inadequate organ perfusion and cardiovascular collapse. A potential therapeutic target in sepsis is inhibition of excess NO generation, although to date, direct NOS inhibition has proved unsuccessful. Asymmetric dimethylarginine (ADMA) is an endogenously occurring competitive inhibitor of NOS. ADMA is subject to hydrolysis catalysed by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). We therefore hypothesised that pharmacological inhibition of DDAH would raise endogenous ADMA levels and potentially inhibit the excess NO production observed during sepsis (endotoxemia). Novel inhibitors of DDAH were synthesised, co-crystallised with the enzyme and characterised using in vitro enzyme assays and in vitro and in vivo rat models of endotoxemia. Co-crystallisation revealed the binding mode of the novel inhibitor, L-291. Isolated enzyme studies demonstrated that L-291 caused a concentration dependent inhibition of DDAH activity whilst having no direct effect on NOS activity. In vitro functional studies using isolated rat aorta demonstrated that L-291 reversed the iNOS mediated dilatation produced by LPS treatment by 16% ± 4.4 at 100uM and 25%± 4.1 at 200uM. In anaesthetised rats, a bolus dose of LPS induced iNOS mediated vasodilatation resulting in a fall in blood pressure. Administration of L-291 in LPS treated rats elevated circulating ADMA levels when compared with saline control (2.48 ± 0.14uM vs. 1.47±0.21uM) and significantly attenuated the fall in blood pressure. In conclusion, we have generated a novel highly selective DDAH inhibitor, which elevates circulating ADMA levels in vivo. In a rat model of acute endotoxemia DDAH inhibition attenuates iNOS mediated hypotension and stabilises blood pressure.