Pharmacological inhibition of NAD-dependent enzymes in lung inflammation

(eng) Several enzymes are dependent of nicotinamide adenine dinucleotide (NAD) including mono-ADP-ribosyltransferases, poly-ADP-ribose polymerases, ADP-ribose cyclases and sirtuins. These enzymes have several substrates and regulate a multitude of cellular processes including apoptosis and gene transcription. NAD-dependent enzymes have been implicated in the modulation of immune responses. The pulmonary route has been used for local administration of drugs for many years in order to treat lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pulmonary drug delivery allows local drug targeting and reduced systemic side effects. The objective of this thesis was to evaluate whether pharmacologic inhibitors of NAD-dependent enzymes could attenuate LPS-induced lung inflammation following pulmonary delivery. We aimed to evaluate their efficacy and associated mechanism of action as well as to develop a formulation that would efficiently and safely deliver them into the lung in a mouse model of LPS-induced lung inflammation. The pulmonary administration of nicotinamide, an end product inhibitor of NAD-dependent enzymes, accelerated the resolution of LPS-induced lung inflammation by decreasing airway neutrophilia and lung injury but without affecting the production of chemokines implicated in neutrophil recruitment. Therefore, we evaluated the impact of nicotinamide on both neutrophil chemotaxis and apoptosis. Nicotinamide decreased neutrophil chemotaxis in vitro. In addition, nicotinamide completely inhibited the pro-survival effect of G(M)-CSF-treated neutrophils in vitro and in vivo in a mouse model of LPS-induced lung inflammation. Another study showed that two sirtuin inhibitors, cambinol and sirtinol, decreased LPS-induced production of cytokines by macrophages in vitro but stronger effects were observed with cambinol. These results were in agreement with in vivo studies where sirtinol did not attenuate LPS-induced lung inflammation, whereas cambinol significantly decreased lung inflammation. Cambinol anti-inflammatory activity was associated with decreased NF-κB activity but was independent of the MAPK pathway. Finally, in order to study the effect of pulmonary cambinol delivery in a mouse model of LPS-induced lung inflammation, cambinol was encapsulated in polymeric micelles of PEG-p(CL-co-TMC). These polymeric micelles successfully increased cambinol solubility in aqueous medium by four orders of magnitude. Moreover, cambinol polymeric micelles were well tolerated in the lung. Yet, contrary to the intraperitoneal injection of cambinol in vehicle (cremophor EL and ethanol), cambinol-loaded polymeric micelles failed to decrease LPS-induced lung inflammation by both intraperitoneal or intratracheal injection. In conclusion, this thesis shows that the pharmacological inhibition of NAD-dependent enzymes is a promising tool for the treatment of lung inflammatory conditions. Although further research is needed in order to clarify nicotinamide mechanism of action, in this work we show that nicotinamide accelerates the resolution of lung inflammation in part due to the induction of neutrophil apoptosis. These data suggest that nicotinamide could be an interesting pharmacological candidate for the treatment of several neutrophilic lung diseases (e.g. acute lung injury and chronic obstructive pulmonary disease). Our results point out cambinol, a pharmacological inhibitor of sirtuins, as an anti-inflammatory molecule both in vitro and in vivo. Although, cambinol is a promising tool for the treatment of pulmonary inflammatory conditions, new strategies should be exploited in order to efficiently deliver cambinol into the lungs. Altogether, this thesis opens promising perspectives for the pulmonary delivery of NAD-dependent enzymes inhibitors in the treatment of pulmonary inflammatory conditions.