Efficacy and safety of liposomal clarithromycin and its effect on Pseudomonas aeruginosa virulence factors

Abstract

We investigated the efficacy and safety of liposomal clarithromycin formulations with different surface charges against clinical isolates of Pseudomonas aeruginosa from the lungs of cystic fibrosis (CF) patients. The liposomal clarithromycin formulations were prepared by the dehydration-rehydration method, and their sizes were measured using the dynamic-light-scattering technique. Encapsulation efficiency was determined by microbiological assay, and the stabilities of the formulations in biological fluid were evaluated for a period of 48 h. The MICs and minimum bactericidal concentrations (MBCs) of free and liposomal formulations were determined with P. aeruginosa strains isolated from CF patients. Liposomal clarithromycin activity against biofilm-forming P. aeruginosa was compared to that of free antibiotic using the Calgary Biofilm Device (CBD). The effects of subinhibitory concentrations of free and liposomal clarithromycin on bacterial virulence factors and motility on agar were investigated on clinical isolates of P. aeruginosa. The cytotoxicities of the liposome preparations and free drug were evaluated on a pulmonary epithelial cell line (A549). The average diameter of the formulations was >222 nm, with encapsulation efficiencies ranging from 5.7% to 30.4%. The liposomes retained more than 70% of their drug content during the 48-h time period. The highly resistant strains of P. aeruginosa became susceptible to liposome-encapsulated clarithromycin (MIC, 256 mg/liter versus 8 mg/liter; P < 0.001). Liposomal clarithromycin reduced the bacterial growth within the biofilm by 3 to 4 log units (P < 0.001), significantly attenuated virulence factor production, and reduced bacterial twitching, swarming, and swimming motilities. The clarithromycin-entrapped liposomes were less cytotoxic than the free drug (P < 0.001). These data indicate that our novel formulations could be a useful strategy to enhance the efficacy of clarithromycin against resistant P. aeruginosa strains that commonly affect individuals with cystic fibrosis.

Fig 1

Liposomal clarithromycin activity on P. aureginosa PA-13572 biofilm (MBEC assay). Free (F-CAM) or liposomal formulations were introduced to mature biofilm at concentrations of 32 mg/liter (a), 64 mg/liter (b), and 128 mg/liter (c). Untreated biofilm acted as a control. The data represent three independent experiments in triplicate and are shown as means ± SEM. P values were considered significant compared with the control: ***, P < 0.001.

Fig 2

Effects of subinhibitory concentrations of liposomal clarithromycin formulations on growth of PA-13572 at 1/2 the MIC (a), 1/4 the MIC (b), and 1/8 the MIC (c). Experiments were tested three times in triplicate, with means shown (the error bars were deleted for clarity of the graphs). Shown are control (filled circles), negatively charged liposomal clarithromycin (filled squares), positively charged liposomal clarithromycin (filled triangle), uncharged liposomal clarithromycin (open circle), and free clarithromycin (open squares).

Fig 3

Effects of subinhibitory concentrations (1/8 the MIC) of free and liposomal clarithromycin on PA-13572 virulence factor production. (a) Lipase. (b) Chitinase. (c) Elastase. (d) Protease. The results represent the means and SEM of three independent experiments in triplicate. For lipase, chitinase, and elastase experiments, the results were normalized by dividing the OD by the OD₆₀₀ (cell density). P values were considered significant compared with the control: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig 4

Effect of a subinhibitory concentration of liposomal clarithromycin on P. aeruginosa motility. Free or liposomal clarithromycin at 1/8 the MIC was added to agarose plates, and motility was examined. Twitching (1% agarose [wt/vol]) (a), swarming (0.5% agarose [wt/vol]) (b), and swimming (0.3% agarose [wt/vol]) (c) were measured with digital calipers. P values were considered significant compared with the control and between groups: ***, P < 0.001.