Supplementation of host response by targeting nitric oxide to the macrophage cytosol is efficacious in the hamster model of visceral leishmaniasis and adds to efficacy of amphotericin B

We investigated efficacy of nitric oxide (NO) against Leishmania donovani. NO is a mediator of host response to infection, with direct parasiticidal activity in addition to its role in signalling to evoke innate macrophage responses. However, it is short-lived and volatile, and is therefore difficult to introduce into infected cells and maintain inracellular concentrations for meaningful periods of time. We incorporated diethylenetriamine NO adduct (DETA/NO), a prodrug, into poly(lactide-co-glycolide) particles of similar to 200 nm, with or without amphotericin B (AMB). These particles sustained NO levels in mouse macrophage culture supernatants, generating an area under curve (AUC(0.08-24h)) of 591.2 +/- 95.1 mM x h. Free DETA/NO resulted in NO peaking at 3 h and declining rapidly to yield an AUC of 462.5 +/- 193.4. Particles containing AMB and DETA/NO were able to kill similar to 98% of promastigotes and similar to 76% of amastigotes in 12 h when tested in vitro. Promastigotes and amastigotes were killed less efficiently by particles containing a single druge- either DETA/NO (similar to 42%, 35%) or AMB (similar to 90%, 50%) alone, or by equivalent concentrations of drugs in solution. In a pre-clinical efficacy study of power >0.95 in the hamster model, DETA/NO particles were non-inferior to Fungizone (R) but not Ambisome (R), resulting in significant (similar to 73%) reduction in spleen parasites in 7 days. Particles containing both DETA/NO and AMB were superior (similar to 93% reduction) to Ambisome (R). We conclude that NO delivered to the cytosol of macrophages infected with Leishmania possesses intrinsic activity and adds significantly to the efficacy of AMB. (C) 2016 Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license.