Demonstration of a fusion mechanism between a fluid bactericidal liposomal formulation and bacterial cells.

It was previously demonstrated that fluid liposomal-encapsulated tobramycin, named Fluidosomes, was successful in eradicating mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection, whereas free antibiotic did not reduce colony-forming unit (CFU) counts (C. Beaulac et al., Antimicrob. Agents Chemother. 40 (1996) 665-669; C. Beaulac et al., J. Antimicrob. Chemother. 41 (1998) 35-41). These liposomes were also shown to be bactericidal in in vitro tests against strong resistant P. aeruginosa 64 microg/ml). The time needed to reach the maximal fusion rate was about 5 h for the resistant strain comparatively to much shorter time for the sensitive strain. The specific characteristics of Fluidosomes could help overcome bacterial resistance related to permeability barrier and even enzymatic hydrolysis considering the importance of synergy in the whole process of antibiotic resistance.

Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by Pseudomonas aeruginosa.

Eradication of mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection has been previously demonstrated following the intratracheal administration of Fluidosomes, a low phase transition temperature (low T(C)) liposomal tobramycin preparation administered in liquid form (Beaulac et al., Antimicrob. Agents Chemother., 40, 665-669, 1996). In the present work, the same liposomal formulation was administered as a dry powder aerosol to an animal model of chronic pulmonary infection in view of a possible clinical development in cystic fibrosis patients. Chronic infection was established by intratracheal administration of 10(5) cfu of a mucoid variant of P. aeruginosa, PA 508, prepared in agar beads. Sixteen hours after one aerosol treatment, the cfu counts performed on lungs (pair) treated with liposomal tobramycin were of 4.31x10(5) cfu/lungs comparatively to 1.32x10(8) and 3.02x10(8) cfu/lungs respectively in untreated and in lungs treated with free antibiotic. Considering the quantity of liposome-tobramycin that has reached the lungs, the results suggest that aerosolization of low T(C) liposomal tobramycin used as a dry powder preparation could be an effective way of treating chronic pulmonary infection caused by Pseudomonas.

Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal-tobramycin formulation for the treatment of chronic infections in lungs.

In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria. One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P. aeruginosa and other related bacteria. Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated. The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t. ) immunizations in BALB/c mouse. No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera. These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.

In-vitro bactericidal efficacy of sub-MIC concentrations of liposome-encapsulated antibiotic against gram-negative and gram-positive bacteria.

It has been shown previously that tobramycin encapsulated in fluid liposomes (composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG)) eradicated mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection. Exponential cultures of P. aeruginosa, Stenotrophomonas maltophila, Burkholderia cepacia, Escherichia coli and Staphylococcus aureus were treated with (i) free tobramycin, (ii) sub-MIC tobramycin encapsulated in DPPC/DMPG liposomes, (iii) control liposomes without antibiotic or (iv) control liposomes combined with free tobramycin. Bacterial colonies were counted 0, 1, 3, 6 and 16 h after addition of antibiotic. After 3 h, the growth of B. cepacia, E. coli and S. aureus was reduced 129, 84 and 566 times respectively in cultures treated with encapsulated antibiotic compared with those treated with free antibiotic. Six hours and 16 h after treatment, the maximal reduction of growth between strains treated with liposome-encapsulated tobramycin and free tobramycin was 84, 129, 166, 10(5) and 10(4) times respectively for P. aeruginosa, B. cepacia, E. coli, S. maltophilia and S. aureus. The liposomes were stable at 4 degrees C and at room temperature for the whole period studied. At 37 degrees C, equivalent stability was observed for the first 16 h of the study. Administration of antibiotic encapsulated in DPPC/DMPG liposomes may thus greatly improve the management of resistant infections caused by a large range of microorganisms. The strong bactericidal activity of the encapsulated antibiotic at sub-MIC doses of the strains tested cannot be explained only as a result of prolonged residence time of liposome-encapsulated tobramycin and the resulting release of entrapped antibiotic at the bacterial site; rather, direct interaction of chemoliposomes and bacteria, probably by a fusion process, may explain the bactericidal effect of the sub-MIC antibiotic doses used.

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay.

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

In vitro kinetics of drug release and pulmonary retention of microencapsulated antibiotic in liposomal formulations in relation to the lipid composition.

In previous in-vivo studies, we demonstrated that liposomal entrapment of tobramycin resulted in an increased availability of the antibiotic in the lungs without increasing bactericidal efficacy (Omri et al. 1994). With the aim of developing liposomal formulations allowing more efficient liposome-bacteria interactions, we studied the influence of lipid composition on both drug release and pulmonary retention of encapsulated tobramycin. The phase transition temperatures of nine liposome-tobramycin formulations consisting of two synthetic phospholipids (distearoyl phosphatidylcholine (DPSC) or dipalmitoyl phosphatidylcholine (DPPC) with dimyristoyl phosphatidyl-glycerol (DPMG) or dimyristoyl phosphatidylcholine (DMPC) were determined by differential scanning calorimetry. Liposomes, varying in terms of membrane fluidity and charge were submitted to in-vitro and in-vivo kinetic studies while retention and release of tobramycin were measured by high-performance liquid chromatography (HPLC). Five less fluid liposome formulations showed absence or very low tobramycin release in in-vitro tests and long term pulmonary retention of tobramycin. Four fluid liposome formulations showed in vitro tests modulated tobramycin release while pulmonary retention of tobramycin was dependent of the presence of charged phospholipids. Administration of charged fluid liposomes in mice showed a low level of tobramycin in the kidneys; non-charged fluid liposomes exhibited a relatively high level of tobramycin retention in the kidneys.

Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection.

Despite controversies associated with forms and value of antibiotic therapy for cystic fibrosis patients, antibiotherapy remains a cornerstone in the management of those patients. Locally administered liposome-encapsulated antibiotics may offer advantages over free antibiotics, including sustained concentration of the antibiotic, minimal systemic absorption, reduced toxicity, and increased efficacy. We evaluated the efficacy of free and encapsulated tobramycin in fluid and rigid liposomal formulations administered to rats chronically infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(5) CFU of a mucoid variant of P. aeruginosa PA 508 prepared in agar beads. Antibiotic treatments were given intratracheally at time intervals of 16 h. After the last treatment, lung bacterial counts were determined and tobramycin levels in the lungs and kidneys were evaluated by high-performance liquid chromatographic analysis and microbiological assay. Two independent experiments showed that animals treated with encapsulated tobramycin in fluid liposomes had a number of CFU less than the minimal CFU number required to be statistically acceptable compared with > or = 10(6) CFU per pair of lungs for animals treated with encapsulated tobramycin in rigid liposomes, free antibiotic, or liposomes without tobramycin. Tobramycin measured in the lungs at 16 h after the last treatment following the administration of encapsulated antibiotic was still active, and its concentration was > or = 27 micrograms/mg of tissue. Low levels of tobramycin were detected in the kidneys (0.59 to 0.87 micrograms/mg of tissue) after the administration of encapsulated antibiotic, while 5.31 micrograms/mg of tissue was detected in the kidneys following the administration of free antibiotic. These results suggest that the local administration of fluid liposomes with encapsulated tobramycin could greatly improve the management of chronic pulmonary infection in cystic fibrosis patients.

Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa.

The pulmonary residence time of free and liposome-encapsulated tobramycin was studied with uninfected rats and rats infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(8) CFU of P. aeruginosa PA 508 prepared in agar beads. After 3 days, a single dose (300 micrograms) of free or liposome-encapsulated tobramycin was given intratracheally to both infected and uninfected rats. At various time intervals (0.25 to 16 h) after drug instillations, the remaining tobramycin was evaluated in blood, lungs, and kidneys by a microbiological assay. Intratracheal instillation of liposome-encapsulated tobramycin resulted in high and sustained levels of tobramycin in lungs of uninfected and infected rats over the 16-h period studied; however, the tobramycin levels were two times higher in uninfected rats. There was no tobramycin detected in the blood or kidneys from these animals. In contrast, the intratracheally instilled free tobramycin was cleared within 3 and 1 h from the lungs of uninfected and infected animals, respectively. These data suggest that the encapsulation of tobramycin in liposomes can result in a significant increase of its residence time within lungs. This study also shows that pulmonary infection was associated with a lowering of tobramycin levels in lungs.