Over the last 20 years there has been increasing evidence to indicate an antiinflammatory action of macrolide antibiotics in addition to their well-documented antibiotic effect. Evidence from both diffuse panbronchiolitis and chronic rhinosinusitis has illustrated an overall decrease in the production of inflammatory cytokines. In addition in-vitro experiments have also shown a change in biofilm production, microbiological virulence factor production, oxidative burst function and mucociliary clearance. Chronic rhinosinusitis is believed to be mediated by numerous factors including, bacterial load, mucociliary dysfunction, and mucosal inflammation. Macrolides, particularly nasally administered therefore have the potential to alter the disease contribution of each of these factors and bypass side effects from oral administration.
Nasal formulations have demonstrated considerable promise in several previous experiments and provide a potential mechanism to target drug delivery and site of action while minimising unwanted side effects. At present there are several limitations in producing a topical solution of clarithromycin. These include a pH dependent solubility curve and the fact that it is insoluble in water.
Therefore the aim of this study was to firstly confirm and demonstrate an immunomodulating ability of the macrolide clarithromycin in an ‘infected’ single cell based in vitro study. An immortalized respiratory type epithelium, A549 was used to simulate nasal mucosa given the reproducibility and previously documented similarities with harvested nasal mucosa. From this experiment the cytokine responses of respiratory type epithelial cells were observed after exposure to multiple common virulence factors and treatment with clarithromycin. Immuno-modulation of Leukotriene B4, Nitrate, IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were all examined following treatment with clarithromycin at several time points. As previous studies have shown, a biphasic response in IL-8 and IL-6 was observed after exposure to the majority of virulence factors tested following clarithromycin treatment. Treatment of a549 cells to a variety of virulence factors tended to inhibit the production of TNF-α, IL-10, IL- 12p70, IL-1β, Nitrate and Leukotriene B4 at nearly all times points studied. Individual 3 responses were variable and appeared to be dependent on the type of virulence factor used to expose the cells to.
The second aim of this research project was to identify a mechanism that would enable clarithromycin to be solubilised in solution. From previous experimental research cyclodextrins were selected as the complexation molecule to enable solubilisation. Three naturally occurring cyclodextrins α, β, γ- cyclodextrins were selected and subsequent toxicity studies were performed on a549 cells prior to complexation with clarithromycin. Based on these toxicity studies two different concentration of α and β- cyclodextrins were selected to form complexes with clarithromycin for further studies.
Complexation of clarithromycin and α and β- cyclodextrins was achieved through previously validated methods. Complexation of each cyclodextrin and clarithromycin formulation was confirmed the different thermal properties of the complexes when compared to the physical mixtures of components when studied with differential scanning calorimetry.
Finally the third aim of this project was to examine the immuno-modulating ability of the produced cyclodextrin-clarithromycin complexes. From the previous experiments the Staphylococcus aureus virulence factor, Protein A was seen to give the most reproducible inflammatory response that could be modulated by clarithromycin. This was then used to stimulate a549 cells to initiate an inflammatory response and evaluate the effect of clarithromycin-cyclodextrin complexes on the production of several cytokines. In addition complexes containing 250μg/ml, 100μg/ml and 10μg/ml of clarithromycin were evaluated with each cyclodextrin concentration to examine if a dose dependent response was observed.
Results indicated that treatment with β-cyclodextrin without clarithromycin complexation at 0.5mM resulted in a significant reduction in IL-6, IL-8, Nitrate and Leukotriene B4. In relation to clarithromycin-cyclodextrin complexes, there appeared no dose dependent response with the 250μg/ml complexes often exacerbating the cytokine production. Significant immuno-modulation of the cytokine response was 4 observed with the 0.5mM β-cyclodextrin complexes containing both 100μg/ml and 10μg/ml clarithromycin after 4 hours of treatment.
In conclusion this study confirms the immuno-modulating ability of macrolides in a single cell based model. In addition there appears to be a biphasic response of cytokine production following clarithromycin that is unique to each individual virulence factor. Finally multiple formulations of cyclodextrin and clarithromycin were developed that appeared to significantly alter cytokine response after exposure to the virulence factor Protein A.