Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients.

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth.We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP).Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain.Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe.

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153 P. aeruginosa isolates from the United Kingdom (UK) (n = 58), Belgium (n = 44), and Germany (n = 51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum ß-lactamases (ESBL), metallo-ß-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-ß-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs.

A Randomised, Cross-Over Study to Estimate the Influence of Food on the 25-Hydroxyvitamin D3 Serum Level after Vitamin D3 Supplementation.

Vitamin D3 is known to be liposoluble and its release could be a factor limiting the rate of absorption. It was presumed that the presence of fat could favor absorption of vitamin D3. However, as bioavailability is related not only to the active molecules but also to the formulations and excipients used, the optimization of the pharmaceutical form of vitamin D3 is also important. The objective of this study was to evaluate if there is a food effect on absorption when a high dose of vitamin D3 is completely solubilized in an oily solution. In the present cross-over study, 88 subjects were randomized and received a single dose of 50,000 IU of vitamin D3 in fasting state or with a standardized high-fat breakfast. Assessment of serum concentrations of 25 hydroxyvitamin D3 (25(OH)D3) was performed three, five, seven, 14, 30 and 60 days after supplementation. In fed and fast conditions, the 25(OH)D3 serum concentrations were significantly higher than the baseline value three days after administration and remained significantly higher during the first month. No significant difference between fasting vs. fed conditions was observed. It is therefore concluded that the vitamin D3 absorption from an oily solution was not influenced by the presence or absence of a meal.

Budesonide/salmeterol in fixed-dose combination for the treatment of asthma.

Fixed dose combinations (FDC) of inhaled corticosteroid (ICS) and long-acting beta agonist (LABA) are well established in asthma treatment. The budesonide/salmeterol (B/S) FDC is now about to reach the market. It is provided as powder in hard capsules of two strengths: 120/20µg and 240/20µg when expressed as delivered doses, equivalent to 150/25µg and 300/25µg when expressed as nominal doses. Its development involved 9 pharmacokinetic (320 subjects), 3 phase II (123 subjects) and 4 phase III (1206 patients with different asthma severity) studies. Delivery is effectuated via low resistance inhaler device, Axahaler®, generating also fine particles targeting the small airways. B/S safety, assessed in 1401 subjects, did not outline novel concerns specific for this FDC. In conclusion, the B/S dry powder FDC can be used for asthma treatment in adults not adequately controlled on ICS alone, or to maintain control of ICS/LABA treated patients, in whom switching to alternative FDC is indicated.

A Randomized, Double-Blind, Parallel Study to Evaluate the Dose-Response of Three Different Vitamin D Treatment Schemes on the 25-Hydroxyvitamin D Serum Concentration in Patients with Vitamin D Deficiency.

Many people worldwide are vitamin D (VTD) deficient or insufficient, and there is still no consensus on the dose of VTD that should be administered to achieve a 25(OH)D concentration of 20 or 30 ng/mL. In this study, we aimed to determine an adapted supplementation of VTD able to quickly and safely increase the vitamin D status of healthy adults with low 25(OH)D. One hundred and fifty (150) subjects were randomized into three groups, each to receive, orally, a loading dose of 50,000, 100,000 or 200,000 IU of VTD3 at Week 0, followed by 25,000, 50,000 or 100,000 IU at Week 4 and Week 8. Whereas 25(OH)D baseline values were not different between groups (p = 0.42), a significant increase was observed at Week 12 (p < 0.0001) with a mean change from baseline of 7.72 ± 5.08, 13.3 ± 5.88 and 20.12 ± 7.79 ng/mL. A plateau was reached after eight weeks. No related adverse event was recorded. This study demonstrated a linear dose-response relationship with an increase in 25(OH)D levels proportional to the dose administered. In conclusion, a loading dose of 200,000 IU VTD3 followed by a monthly dose of 100,000 IU is the best dosing schedule to quickly and safely correct the VTD status.

Pharmacokinetic evaluation in mice of amorphous itraconazole-based dry powder formulations for inhalation with high bioavailability and extended lung retention.

Three Itraconazole (ITZ) dry powders for inhalation (DPI) were prepared by spray-drying a mannitol solution in which the ITZ was in suspension (F1) or was in solution without (F2) or with phospholipid (PL) (F3). These powders were endotracheally insufflated in vivo at a single dose of 0.5mg/kg for pharmacokinetic profile (lung and plasma concentration) determination in ICR CD-1 mice. ITZ was crystalline in F1 and assumed to be amorphous in the F2 and F3 formulations. F2 and F3 formulations allowed the in vitro formation of an ITZ supersaturated solution with a maximum solubility of 450±124ng/ml (F2) and 498±44ng/ml (F3), in contrast to formulation F1 (<10ng/ml). As a result of these higher solubilities, absorption into the systemic compartment after endotracheal administration was faster for formulations F2 and F3 (shorter tmax) and in larger quantities compared to the F1 formulation (plasmatic AUC0-24h of 182ngh/ml, 491.5ngh/ml and 376.8ngh/ml, and tmax of 60min, 30min and 5min for F1, F2 and F3, respectively). PL increased the systemic bioavailability of ITZ (determined by the AUCplasma to AUClung ratio) as a consequence of their wetting and absorption enhancement effect. ITZ lung concentrations after pulmonary administration remained higher than the targeted dose, based on the minimal inhibitory concentrations for Aspergillus fumigatus (2µg/glung), 24h post-administration for both F1 and F2 formulations. However, this was not the case for formulation F3, which exhibited a faster elimination rate from the lung, with an elimination half-life of 4.1h vs. 6.5h and 14.7h for F1 and F2, respectively.

Efficacy and safety of a 12-week therapy with a new formulation of fluticasone propionate at doses of 125 and 250 µg administered through a new generation cyclohaler twice daily, in comparison to fluticasone propionate 500 µg dry powder inhaler twice daily in patients with moderate asthma.

INTRODUCTION: Inhaled fluticasone is used in the treatment of chronic bronchial asthma. Its high efficacy and good safety profile have been proven by clinical trials and observations. Its unique pharmacokinetic properties make it distinguishable from other drugs from this group. In vitro tests run on an artificial model of the airways and pharmacokinetic studies conducted on healthy volunteers have shown that the new formulation of this drug is outstanding due a twofold better lung deposition, compared to the reference medicine. The aim of this study was to evaluate the efficacy and safety of the new formulation of fluticasone propionate administered through new generation cyclohaler (CNG), compared to original fluticasone administered through dry powder inhaler (DPI) in patients with chronic moderate asthma. MATERIAL END METHODS: The study included 457 patients. 376 subjects were randomized to one out of the three groups: 127 subjects--to the group treated with the new formulation of fluticasone at a dose of 125 µg BID, 125 subjects--to the group treated with new formulation of fluticasone at a dose of 250 µg BID, and 124 subjects--to the group treated with the reference drug--fluticasone DPI 500 µg BID. At the beginning of the study, the groups did not differ in demographical or clinical aspects. Active therapy lasted 12 weeks. The primary endpoint was a mean change in morning PEF during a 12-week course of therapy (ΔmPEF of 15 L/min was considered as statistically significant). Additionally, other functional parameters of the respiratory system--clinical symptoms and the use of rescue drugs were studied. During the whole study the safety of patients was monitored by recording adverse events; in addition, a systemic exposure to fluticasone was evaluated by testing the changes of cortisol in serum and in a 24-hour collection of urine in a subgroup consisting of 45 patients. Statistical analysis was conducted on both groups: intention-to-treat (ITT) and per protocol (PP). RESULTS: In PP as well as in ITT analysis, a mean change in morning PEF at the end of the therapy in comparison with the initial period was statistically significant in all therapeutic groups. The efficacy of the treatment with fluticasone at doses of 125 µg BID and 250 µg and the reference medicines did not differ statistically significantly after a 12-week course of therapy or during the whole period of treatment. During the study, significant improvement in the range of other functional parameters such as evening PEF, FEV1, clinical symptoms and the use of rescue drugs was observed in all therapeutic groups, without significant differences in efficacy between the study groups. The comparison of efficacy of fluticasone at a dose of 125 µg BID with the generic product at a dose of 250 µg BID showed a weak dose-response relationship concerning the change in morning PEF, which arises from the almost flat dose-response curve in the range of medium and high doses for this drug. No significant quantitative or qualitative differences were shown between the groups in the recorded adverse events, qualified as related to treatment with fluticasone. There were no significant changes revealed in cortisol concentration in serum or in a 24-hour collection of urine between the initial level and the final visit in any of the groups. CONCLUSIONS: Fluticasone administered through the new generation cyclohaler, compared to original fluticasone DPI, allows a twofold reduction in drug dose, retaining in new formulation clinical efficacy that corresponds to the reference drug at twice the dose. New formulation of fluticasone administered through the new generation cyclohaler has a safety profile clinically comparable to the reference drug.

Carrier-free combination for dry powder inhalation of antibiotics in the treatment of lung infections in cystic fibrosis.

The aim of the study was to develop an efficient combination antibiotic formulation containing tobramycin and clarithromycin as a dry powder for inhalation. A carrier-free formulation of the two drugs was produced by spray-drying and characterised for its aerodynamic behaviour by impaction tests with an NGI and release profiles. The particle size distribution, morphological evaluation and crystallinity state were determined by laser diffraction, scanning electron microscopy and powder X-ray diffraction, respectively. Drug deposition profiles were similar for the two antibiotics, which has a synergistic effect, allowing them to reach the target simultaneously at the expected dose. The release profiles show that tobramycin and clarithromycin should probably dissolve without any difficulties in vivo in the lung as 95% of tobramycin and 57% of clarithromycin mass dissolved in 10min for the spray-dried formulation. The FPF increased from 35% and 31% for the physical blend for tobramycin and clarithromycin, respectively, to 65% and 63% for the spray-dried formulation. The spray-dried formulation shows particularly high deposition results, even at sub-optimal inspiratory flow rates, and therefore, represents an attractive alternative in the local treatment of lung infection such as in cystic fibrosis.

New co-spray-dried tobramycin nanoparticles-clarithromycin inhaled powder systems for lung infection therapy in cystic fibrosis patients.

The aim of the study was to produce easily dispersible and porous agglomerates of tobramycin nanoparticles surrounded by a matrix composed of amorphous clarithromycin. Nanoparticles of tobramycin with a median particle size of about 400 nm were produced by high-pressure homogenisation. The results from the spray-dried powders showed that the presence of these nanoparticles enhanced powder dispersion during inhalation. Moreover, local drug deposition profiles were similar for the two antibiotics, allowing them to reach the target simultaneously. The dissolution-release profiles showed that tobramycin and clarithromycin might dissolve without any difficulties in the lung. The fine particle fraction increased from 35% and 31% for the physical blend for tobramycin and clarithromycin, respectively, to 63% and 62% for the spray-dried formulation containing nanoparticles. These new formulations, showing high lung deposition properties, even at sub-optimal inspiratory flow rates, represent a great possibility for advancing pulmonary drug administration and local therapy of lung infections.

New inhalation-optimized itraconazole nanoparticle-based dry powders for the treatment of invasive pulmonary aspergillosis.

PURPOSE: Itraconazole (ITZ) dry powders for inhalation (DPI) composed of nanoparticles (NP) embedded in carrier microparticles were prepared and characterized. METHODS: DPIs were initially produced by reducing the ITZ particle size to the nanometer range using high-pressure homogenization with tocopherol polyethylene 1000 succinate (TPGS, 10% w/w ITZ) as a stabilizer. The optimized nanosuspension and the initial microsuspension were then spray-dried with different proportions of or in the absence of mannitol and/or sodium taurocholate. DPI characterization was performed using scanning electron microscopy for morphology, laser diffraction to evaluate the size-reduction process, and the size of the dried NP when reconstituted in aqueous media, impaction studies using a multistage liquid impactor to determine the aerodynamic performance and fine-particle fraction that is theoretically able to reach the lung, and dissolution studies to determine the solubility of ITZ. RESULTS: Scanning electron microscopy micrographs showed that the DPI particles were composed of mannitol microparticles with embedded nano- or micro-ITZ crystals. The formulations prepared from the nanosuspension exhibited good flow properties and better fine-particle fractions, ranging from 46.2% ± 0.5% to 63.2% ± 1.7% compared to the 23.1% ± 0.3% that was observed with the formulation produced from the initial microsuspension. Spray-drying affected the NP size by inducing irreversible aggregation, which was able to be minimized by the addition of mannitol and sodium taurocholate before the drying procedure. The ITZ NP-based DPI considerably increased the ITZ solubility (58 ± 2 increased to 96 ± 1 ng/mL) compared with that of raw ITZ or an ITZ microparticle-based DPI (<10 ng/mL). CONCLUSION: Embedding ITZ NP in inhalable microparticles is a very effective method to produce DPI formulations with optimal aerodynamic properties and enhanced ITZ solubility. These formulations could be applied to other poorly water-soluble drugs and could be a very effective alternative for treating invasive pulmonary aspergillosis.

New respirable and fast dissolving itraconazole dry powder composition for the treatment of invasive pulmonary aspergillosis.

PURPOSE: Novel itraconazole (ITZ)-based dry powders for inhalation (DPI) were optimized for aerodynamic and dissolution properties and contained excipients that are acceptable for inhalation. METHODS: The DPI were produced by spray drying solutions. The drug content, crystallinity state, and morphological evaluation of the dry powders were determined by high performance liquid chromatography, powder X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy, respectively. A particle size analysis was conducted using laser light scattering. The aerodynamic behaviors of the powders were characterized by impaction tests. ITZ dissolution rates were evaluated using a dissolution method adapted to inhaled products. RESULTS: The DPI presented very high fine particle fractions that ranged from 46.9% to 67.0% of the nominal dose. The formulations showed very fast dissolution rates compared to unformulated crystalline ITZ with the possibility of modulating the dissolution rate by varying the quantity of phospholipids (PL) incorporated. ITZ remained amorphous while the mannitol was crystalline. The α, ß and δ-mannitol polymorph ratios varied depending on the formulation compositions. CONCLUSION: This formulation strategy could be an attractive alternative for treating invasive pulmonary aspergillosis. The ITZ and PL content are key characteristics because of their influence on the dissolution rate and aerosol performance.

In vitro and in vivo evaluation of a dry powder endotracheal insufflator device for use in dose-dependent preclinical studies in mice.

The aim of this study was to evaluate the ability of the Penn-Century Dry Powder Insufflator for mice (DP-4M) to reproducibly, uniformly, and deeply deliver dry powders for inhalation in the mouse lung. Itraconazole-based dry powder formulations produced by spray-drying were different in terms of composition (different ratios of drug and mannitol, with or without phospholipids), but relatively similar in terms of particle size and mass median aerodynamic diameter. The ability of the dry powder insufflator to disaggregate each formulation was the same, indicated by the absence of a statistically significant difference between the particle size distribution parameters, as measured by laser scattering. The emitted fraction varied in vivo compared to the in vitro condition. Fluorescent particle distribution in the lungs was uniform and reached the alveolar spaces, as visualized by fluorescent microscopy. In terms of drug recovery in lung tissue, a minimum administered powder mass (in this case ∼1 mg) was necessary to recover at least 30% of the emitted dose in the lung and to obtain reproducible pulmonary concentrations. To reduce the dose administered in the lung, it was preferable to dilute the active ingredient within the carrier instead of reducing the dry powder mass inserted in the sampling chamber. Dry powder insufflators are devices usable in dose-dependent preclinical trials but have critical parameters to efficiently deliver reproducible doses depending on the type of formulation.

Solid dispersions of itraconazole for inhalation with enhanced dissolution, solubility and dispersion properties.

The purpose of this study was to produce a dry powder for inhalation (DPI) of a poorly soluble active ingredient (itraconazole: ITZ) that would present an improved dissolution rate and enhanced solubility with good aerosolization properties. Solid dispersions of amorphous ITZ, mannitol and, when applicable, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) were produced by spray-drying hydro-alcoholic solutions in which all agents were dissolved. These dry formulations were characterized in terms of their aerosol performances and their dissolution, solubility and physical properties. Modulate differential scanning calorimetry and X-ray powder diffraction analyses showed that ITZ recovered from the different spray-dried solutions was in an amorphous state and that mannitol was crystalline. The inlet drying temperature and, indirectly, the outlet temperature selected during the spray-drying were critical parameters. The outlet temperature should be below the ITZ glass transition temperature to avoid severe particle agglomeration. The formation of a solid dispersion between amorphous ITZ and mannitol allowed the dry powder to be produced with an improved dissolution rate, greater saturation solubility than bulk ITZ and good aerosol properties. The use of a polymeric surfactant (such as TPGS) was beneficial in terms of dissolution rate acceleration and solubility enhancement, but it also reduced aerosol performance. For example, significant dissolution rate acceleration (f(2)<50) and greater saturation solubility were obtained when introducing 1% (w/w) TPGS (mean dissolution time dropped from 50.4 min to 36.9 min and saturation solubility increased from 20 ± 3 ng/ml to 46 ± 2 ng/ml). However, the fine particle fraction dropped from 47 ± 2% to 37.2 ± 0.4%. This study showed that mannitol solid dispersions may provide an effective formulation type for producing DPIs of poorly soluble active ingredients, as exemplified by ITZ.

Safety of a fixed-dose combination of fenofibrate/pravastatin 160 mg/40 mg in patients with mixed hyperlipidaemia: a pooled analysis from a database of clinical trials.

BACKGROUND: Fenofibrate can be prescribed concomitantly with an HMG-CoA reductase inhibitor (statin) to improve achievement of lipid goals in patients with atherogenic mixed hyperlipidaemia. However, some safety concerns, particularly an increased risk of myopathy, have been reported when these drugs are taken together. OBJECTIVE: The aim of this analysis was to assess the general safety and tolerability of a fenofibrate/pravastatin (FF/PRA) 160 mg/40 mg fixed-dose combination (FDC) capsule based on a pooled database of phase III clinical trials in patients with mixed hyperlipidaemia. METHODS: Safety data were pooled from five phase III studies (four double-blind with an uncontrolled extension and one open) of ≥12 to 64 weeks' duration. Adverse event (AE) profiles of FF/PRA 160 mg/40 mg (n=645 in the double-blind cohort) were evaluated relative to comparators (statins, n=519 or fenofibrate, n=122). Absolute incidence rates were calculated in both the double-blind cohort and the all-studies cohort (FF/PRA 160 mg/40 mg, n=1566) for all AEs, drug-related AEs, serious AEs, discontinuations due to AEs, AEs of specific interest including abnormal laboratory data, and deaths. RESULTS: The frequency and/or intensity of overall AEs, drug-related AEs, serious AEs and discontinuations due to AEs were not significantly increased for the FDC (36.0%, 12.3%, 1.7% and 5.1%, respectively) versus the statin (28.7%, 8.9%, 0.8% and 2.7%, respectively) and fenofibrate (59.0%, 21.3%, 0% and 4.9%, respectively) monotherapies. No deaths were reported during the course of treatment in clinical trials. Nevertheless, three deaths were reported more than 30 days after the patients completed the study; none of these deaths were assessed as being related to FF/PRA 160 mg/40 mg treatment. Among the AEs of special interest, no myopathy or rhabdomyolysis were reported; no patients were considered to have experienced a drug-induced liver injury; no case of pancreatitis occurred in the double-blind cohort and four patients reported pancreatitis in the all-studies cohort, two of them being study-treatment related; no case of pulmonary embolism was reported in the double-blind cohort and two patients presented with pulmonary embolism, unrelated to the study drug, in the all-studies cohort; there were more cases of decreased creatinine clearance in the FF/PRA 160 mg/40 mg group (1.7%) than in the statin group (0.6%). CONCLUSION: Within the limitations of this database (notably low percentage of very elderly patients, limited sample size of patients with mild renal insufficiency, and mode of selection in the clinical trials), no particular safety concern was raised with FF/PRA 160 mg/40 mg in the double-blind cohort as compared with statin and fenofibrate monotherapies. The acceptable long-term safety profile of FF/PRA 160 mg/40 mg was confirmed with a low frequency of AEs of interest, comparable to that observed in the 12-week double-blind cohort. Emergent effects possibly related to FF/PRA 160 mg/40 mg were mainly those attributable to fenofibrate (decrease in creatinine clearance and pancreatitis).

Efficacy of the combination of tobramycin and a macrolide in an in vitro Pseudomonas aeruginosa mature biofilm model.

Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 µg/ml tobramycin was administered with azithromycin at 2 µg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 µg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 µg/ml combined with clarithromycin at 200 µg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections.

Evaluation of long-term co-administration of tobramycin and clarithromycin in a mature biofilm model of cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonisation and chronic lung infection associated with biofilm formation is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. There is thus an urgent need to develop alternative ways to treat biofilm-associated clinical infections. A kinetic study of twice-daily co-administration of the antibiotics tobramycin and clarithromycin was performed over 28 days on 12-day-old mature P. aeruginosa biofilms formed on microplate pegs for 23 clinical isolates of various phenotypes and genotypes to simulate the treatment of CF patients with inhaled tobramycin through aerosolisation (TOBI). Drug activity was assessed by enumeration of persistent bacteria before, during and after treatment. A mature (12-day-old) biofilm model was chosen because a previous study suggested that such a biofilm was a more realistic in vitro model than a 24-h-old biofilm. Synergistic activity of the drug combination was confirmed on biofilms of 9/23 P. aeruginosa isolates. Of these nine isolates, total destruction of the biofilm was observed for five of them. Combination treatment was superior or equivalent to treatment with tobramycin alone, as activity was observed on 47.8% of the isolates with the combination versus 26.1% with tobramycin alone. No correlation was observed between drug susceptibility profiles and the phenotype or genotype of the isolates.

Preparation and characterization of spray-dried tobramycin powders containing nanoparticles for pulmonary delivery.

Using high-pressure homogenization and spray-drying techniques, novel formulations were developed for manufacturing dry powder for inhalation, composed of a mixture of micro- and nanoparticles in order to enhance lung deposition. Particle size analysis was performed by laser diffraction. Spray-drying was applied in order to retrieve nanoparticles in dried-powder state from tobramycin nanosuspensions. The aerolization properties of the different formulations were evaluated by a multi-stage liquid impinger. Suspensions of nanoparticles of tobramycin containing Na glycocholate at 2% (w/w) relative to tobramycin content and presenting a mean particle size about 200 nm were produced. The results from the spray-dried powders showed that the presence of nanoparticles in the formulations improved particle dispersion properties during inhalation. The fine particle fraction (percentage of particles below 5 microm) increased from 36% for the raw micronized tobramycin material to about 61% for the most effective formulation. These new nanoparticle-containing tobramycin DPI formulations, based on the use of very low level of excipient and presenting high lung deposition properties, offer very important perspectives for improving the delivery of drugs to the pulmonary tract.

Effect of antibiotic co-administration on young and mature biofilms of cystic fibrosis clinical isolates: the importance of the biofilm model.

The prognosis of patients with cystic fibrosis (CF) has improved dramatically over the last three decades although the majority of patients still die in early adulthood. Infection with Pseudomonas aeruginosa has generally been associated with declining lung function and increased mortality in patients. This study aimed to investigate the in vitro activity of tobramycin/clarithromycin combination on biofilms of clinical isolates of P. aeruginosa, meticillin-susceptible and -resistant Staphylococcus aureus, and Burkholderia cepacia. First, the impact of antibiotic co-administration on biofilms at different stages of maturation, i.e. during early formation and on 24-h-old and 12-day-old biofilms, was compared. The 24-h-old biofilms were found to behave differently compared with those aged 12 days, which were more resistant to antibiotics. A kinetic study of antibiotic co-administration twice a day for 9 days on 12-day-old P. aeruginosa biofilms was then performed to simulate the effect of treatment of CF patients by inhaled tobramycin through aerosolisation (TOBI). The results obtained support a synergistic activity of tobramycin/clarithromycin combination on biofilms of P. aeruginosa PY02 and PA01, with a logarithmic bacterial decrease of 3.37 and 3.96, respectively. On the other hand, increased resistance to each of the antibacterial agents used alone was observed. This study highlights the importance of the biofilm stage for in vitro investigations and enabled the development of an in vitro model of mature biofilm that is more appropriate to mimic in vivo conditions in CF patients.

Spray-dried carrier-free dry powder tobramycin formulations with improved dispersion properties.

Tobramycin was spray dried at different temperatures from different water to isopropanol feed ratios (0:100-20:80) in order to obtain dry powder formulations for inhalation. The spray-dried powders were characterized for their physicochemical properties including crystallinity, morphology, density, water content, and particle size distribution using X-ray powder diffraction, scanning electron microscopy, tapped density measurements and laser diffraction. Aerosol performance was studied by dispersing the powders into a Multi-Stage Liquid Impinger with an Aerolizer device. The results indicate that formulations spray dried at temperatures below 200 degrees C exhibited poor powder flow properties and were therefore unlikely to display optimal aerosolization characteristics. Nevertheless, it is interesting to note that the presence of water in the suspensions used for spray-drying markedly enhanced the fine particle fraction, which was about 37% for the raw tobramycin and about 57% for a powder obtained from a suspension containing 2% (v/v) water. Overall, this latter formulation was shown to keep its initial particle size distribution and aerodynamic behaviour for 12 months of storage at 40 degrees C and 75% RH. These new carrier-free formulations provide an attractive alternative for delivering high doses of antibiotics directly to the site of infection while minimising systemic distribution.

Correlations between cascade impactor analysis and laser diffraction techniques for the determination of the particle size of aerosolised powder formulations.

The purpose of the study was to examine the suitability of the Spraytec laser diffraction technique for measuring the size distribution of aerosol particles generated from dry powder inhalators. A range of formulations with different dispersion properties were produced by spray-drying. The percentage of particles below 5.0 microm of these formulations was measured by laser diffraction (Mastersizer 2000 and Spraytec and inertial impaction (MsLI and NGI) using various inhaler devices and at different flow rates between 30 and 100 l/min. Linear relationships and correlations (R(2)>0.9) existed between the results obtained from, on one hand, the Mastersizer 2000 and the Spraytec, and, on the other hand, the MsLI and the Spraytec regardless of flow rates and inhaler devices. The Spraytec could be a reliable technique for the development, evaluation and quality control of dry powder aerosol formulations.