PROJECT SUMMARY During an asthma exacerbation, the debilitating symptoms of breathlessness is largely driven by airway smooth muscle (ASM) contraction. To relax the ASM, current therapies are directed either at antagonizing pro-contractile ASM receptors (e.g. muscarinic antagonists, cysteinyl leukotriene receptor antagonists), or activating pro- relaxant ASM receptors (e.g. 2-agonists). Despite their widespread use, these therapies remain inadequate in controlling symptoms. Rather than targeting receptor-mediated pathways that are complex, indirect, and susceptible to desensitization, our key premise is that a more robust strategy for treating asthma is to directly target the ASM contractile apparatus. To disrupt the ASM contractile apparatus, we have discovered a compelling action for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors, ‘statins’, in the mevalonate (MA) pathway. First, using human ASM cells and human precision cut lung slices (PCLS), we observed that statins inhibit basal-, histamine-, and methacholine (MCh)-induced ASM contraction according to their physiochemical properties (i.e. lipophilic versus hydrophilic statins), and the most pronounced effect is conferred by the lipophilic statin, pitavastatin. Second, we observed that the ASM-relaxing effects of pitavastatin occurs by inhibiting Rho kinase (ROCK)-1 activity, myosin light chain (MLC)-2 phosphorylation, and F-actin stress fibers, by a MA- and geranylgeranylpyrophosphate (GGPP)-dependent mechanism. Third, pitavastatins’ effect is additive to 2−agonists. Fourth, independent of its ASM-relaxing effect, pitavastatin also inhibits ASM proliferation, and IL13-induced eotaxin-3 and IL17/TNF-induced IL6 and IL8 production by a MA/GGPP- dependent mechanism. Finally, in a non-inflammatory mouse model of MCh-induced airway hyper- responsiveness (AHR), intratracheal (i.t.) instillation of pitavastatin inhibited bronchoconstriction by 48%. Empowered by these findings, we hypothesize that pitavastatin when delivered intratracheally can provide optimal therapy for bronchoconstriction by ameliorating ASM contraction and ASM inflammation. AIM 1: Establish the potential of pitavastatin to inhibit ASM contraction and bronchoconstriction. AIM 2: Determine pitavastatin’s mechanisms for inhibiting ASM contraction and inflammation. AIM 3: Examine the effects of asthma pathobiology on the efficacy of pitavastatin therapy. Long-Term Impact: By inhibiting two key hallmark features of ASM dysfunction in asthma – contraction and inflammation – inhaled pitavastatin may be superior to or enhance current inhaled bronchodilator therapies for the treatment of asthma.

PROJECT NARRATIVE We have discovered that the statin drugs (‘statins’) induce airway smooth muscle (ASM) relaxation in disease models of airway hypercontractility. We will investigate the mechanism(s) and therapeutic efficacy of this key observation, and establish that delivering statins by inhalation is effective and safe for the treatment of asthma.