- Epigenetic silencing by promoter methylation and chromatin remodelling affects hundreds of genes and is a causal event for lung cancer. Treatment of patients with low doses of the demethylating agent 5-azacytidine in combination with the histone deacetylase inhibitor entinostat has yielded clinical responses. The subcutaneous dosing route for consecutive days and reduced bioavailability of 5-azacytidine because of inactivation by cytidine deaminase may limit the expansion of epigenetic therapy into Phase III trials. To mitigate these barriers, an aerosol of 5-azacytidine was generated and characterised.The effect of aerosol vs systemic delivery of 5-azacytidine on tumour burden and molecular response of engrafted lung tumours in the nude rat was compared.Pharmacokinetics revealed major improvement in the half-life of 5-azacytidine in lung tissue with aerosol delivery. Aerosolised 5-azacytidine significantly reduced lung tumour burden and induced global demethylation of the epigenome at one-third of the comparable effective systemic dose. High commonality for demethylation of genes was seen in tumours sampled throughout lung lobes and across treated animals receiving the aerosolised drug.Collectively, these findings show that aerosolised 5-azacytidine targets the lung, effectively reprogrammes the epigenome of tumours, and is a promising approach to combine with other drugs for treating lung cancer.