DDAH inhibition - A novel approach to targeting hypotension in septic shock

Manasi Nandi, Belen Torondel, Neil McDonald, Sharon Rossiter, Judith Murray-Rust, Patrick Vallance, James Leiper
1University College, London, United Kingdom, 2Birkbeck College, London, United Kingdom

The leading cause of mortality in intensive care units is septic shock (hyperdynamic, hypotensive state), and primarily arises as a result of an infection. The induction of inducible nitric oxide synthase (iNOS) in response to infection results in the generation of large amounts of nitric oxide (NO) and is believed to contribute towards disease pathology. However, direct NOS inhibition has proved unsuccessful in clinical trials possibly as a result of inhibition of beneficial as well as detrimental NO. Asymmetric dimethylarginine (ADMA) is an endogenously occurring competitive inhibitor of NOS. ADMA is metabolised by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) of which there are two isoforms (DDAH1 and DDAH2) which have different tissue distributions. We hypothesized that targeted inhibition of DDAH could raise local levels of ADMA and thereby regulate NOS in sepsis without affecting beneficial NO production.

Novel inhibitors of DDAH were synthesised and characterised using in vitro and ex vivo enzyme assays and in vivo (anesthetised and instrumented for blood pressure recordings) rat models of acute endotoxemia using lipopolysaccharide (LPS).

The novel DDAH inhibitor, L-291 was co-crystalised with mammalian DDAH1 and shown to bind to the active site. Isolated enzyme studies revealed that L-291 resulted in a concentration dependent inhibition of DDAH activity whilst having no direct effect on NOS activity. L-291 (100μM) fully inhibited the activity of recombinant DDAH1 whilst having no effect on recombinant DDAH2. L-291 (100μM) reversed iNOS mediated NO production in LPS (5μg/ml) stimulated mouse aortic rings (which express both DDAH1 and 2) whilst having no effect on LPS stimulated murine peritoneal macrophages (which solely express DDAH2).

In anaesthetised rats, a bolus dose of LPS (40mg/kg i.v) induces iNOS mediated vasodilatation resulting in systemic hypotension. Administration of L-291 in LPS treated rats elevated circulating ADMA levels when compared with saline control (2.42 ± 0.12uM vs. 1.17±0.09uM) and significantly attenuated the fall in blood pressure (85±4.2mmHg vs 62.2±8.54mmHg).

We have generated a novel and selective DDAH1 inhibitor, which elevates circulating ADMA levels in vivo and stabilises blood pressure in endotoxemic rats. DDAH1 inhibition could therefore provide an alternative means to regulate the detrimental NO generation. We believe this compound works by resetting the arginine:ADMA ratio in activated cells in which iNOS is expressed. The lack of DDAH1 expression in macrophages suggests that the beneficial antimicrobial effect of NOS will remain unaffected.