Christian Beaulac

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.

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.

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.

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 Bactericidal Evaluation of a Low Phase Transition Temperature Liposomal Tobramycin Formulation as a Dry Powder Preparation Against Gram Negative and Gram Positive Bacteria

AbstractIn previous studies, delivery of a liquid preparation of encapsulated tobramycin in fluid liposomes, called Fluidosomes, has showed a marked improvement in the bactericidal activity against in-vitro and in-vivo extracellular infections. To examine the possibility of developing aerosol treatment using dehydrated Fluidosomes for the treatment of chronic pulmonary infections, freeze-dried preparations of tobramycin and Fluidosomes were tested against cultures of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burk-holderia cepacia, Escherichia coli and Staphylococcus aureus. Bacterial colonies were enumerated 0, 1, 3, 6 and 16 h after the addition of the antibiotic. Sixteen hours post-treatment, the growth of P. aeruginosa, S. maltophilia, B. cepacia and E. coli in the presence of sub-minimal inhibitory concentrations of tobramycin was significantly lowered respectively by 17 (P < 0.01), 40 (P < 0.001), 47 (P < 0.001), and 50 (P < 0.001) times in comparison with growth in the presence of free a...