Pharmacokinetics of Colistin Methansulphonate (CMS) and Colistin after CMS Nebulisation in Baboon Monkeys.

PURPOSE: The objective of this study was to compare two different nebulizers: Eflow rapid® and Pari LC star® by scintigraphy and PK modeling to simulate epithelial lining fluid concentrations from measured plasma concentrations, after nebulization of CMS in baboons. METHODS: Three baboons received CMS by IV infusion and by 2 types of aerosols generators and colistin by subcutaneous infusion. Gamma imaging was performed after nebulisation to determine colistin distribution in lungs. Blood samples were collected during 9 h and colistin and CMS plasma concentrations were measured by LC-MS/MS. A population pharmacokinetic analysis was conducted and simulations were performed to predict lung concentrations after nebulization. RESULTS: Higher aerosol distribution into lungs was observed by scintigraphy, when CMS was nebulized with Pari LC® star than with Eflow Rapid® nebulizer. This observation was confirmed by the fraction of CMS deposited into the lung (respectively 3.5% versus 1.3%).CMS and colistin simulated concentrations in epithelial lining fluid were higher after using the Pari LC star® than the Eflow rapid® system. CONCLUSIONS: A limited fraction of CMS reaches lungs after nebulization, but higher colistin plasma concentrations were measured and higher intrapulmonary colistin concentrations were simulated with the Pari LC Star® than with the Eflow Rapid® system.

The airways, a novel route for delivering monoclonal antibodies to treat lung tumors.

PURPOSE: Lung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited, with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer. We have shown that cetuximab, a chimeric anticancer anti-EGFR mAb, is suitable for airway delivery as it resists the physical constraints of aerosolization, and have evaluated the aerosol delivery of cetuximab in vivo. METHODS: We developed an animal model of lung tumor sensitive to cetuximab by injecting Balb/c Nude mice intratracheally with A431 cells plus 10 mM EDTA and analyzed the distribution, pharmacokinetics and antitumor efficacy of cetuximab aerosolized into the respiratory tract. RESULTS: Aerosolized IgG accumulated durably in the lungs and the tumor, but passed poorly and slowly into the systemic circulation. Aerosolized cetuximab also limited the growth of the mouse tumor. Thus, administering anticancer mAbs via the airways is effective and may limit systemic side effects. CONCLUSION: Delivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.

New ipratropium formulation to decrease nebulization time.

A new anticholinergic aerosol containing 0.5mg ipratropium bromide dissolved in 1mL of solution has been produced with the purpose of decreasing nebulization time for patients compared to the traditional formulation which is twice as voluminal (0.5mg/2mL, Boehringer-Ingelheim, France). The aim of this study was to compare aerosol characteristics (inhaled mass, particle size distribution and nebulization time) of these two formulations of ipratropium bromide, nebulized alone and with terbutaline (5mg/2mL, Astra Zeneca, Sweden), to determine whether the new formulation was equivalent to the old one. Four different jet nebulizers were used: PariLC+, Atomisor NL9M, Sidestream and Mistyneb. Statistical analysis of the results showed that for all types of nebulizer, the inhaled mass of ipratropium bromide 0.5mg/1mL was significantly lower than the inhaled mass of ipratropium bromide 0.5mg/2mL, and that there was no statistical difference between the inhaled mass of ipratropium bromide 0.5mg/1mL+terbutaline 5mg/2mL and the inhaled mass of ipratropium bromide 0.5mg/2mL+terbutaline 5mg/2mL. The study also showed that the new formulation of ipratropium bromide (0.5mg/1mL) mixed with terbutaline allowed a 26% decrease in nebulization time compared to the old formulation (0.5mg/2mL) mixed with terbutaline without changing aerosol characteristics (inhaled mass and particle size distribution). This leads to the conclusion that a 2mL minimum volume is required for nebulization, and that nebulization of ipratropium bromide 0.5mg/1mL alone must be avoided.

Relationship between ozone and temperature during the 2003 heat wave in France: consequences for health data analysis.

BACKGROUND: PAPRICA is a research program designed to estimate the impact on the health of patients with chronic respiratory insufficiency of a prevention strategy based on notification of ozone pollution. The first year of this study was conducted during the 2003 heat wave, and high temperatures were therefore considered as a confounding factor in the data analysis. The aim of the present study was to assess the relationship between ozone and temperature in order to propose a methodology to distinguish between the effects of ozone and temperature on the impact of a prevention strategy with regard to ozone pollution. METHODS: Multivariate analyses were used to identify associated climate and ozone pollution profiles. This descriptive method is of great value to highlight the complexity of interactions between these parameters. RESULTS: Ozone concentration and temperature were strongly correlated, but the health impact of ozone pollution alone will be evaluated by focusing on situations characterized by ozone concentrations above 110 mug/m3/8h (air quality guidelines to protect human health defined by the French legislation) and temperatures lower than 26 degrees C, below the discomfort threshold. CONCLUSION: The precise relationship between ambient ozone concentration and temperature identified during the PAPRICA 2003 study period will be used in analysing the PAPRICA health data.

In vitro study and semiempirical model for aerosol delivery control during mechanical ventilation.

The object of this study was to evaluate in vitro the influence of various ventilatory parameters on the delivery of synchronized nebulization of terbutaline during mechanical ventilation and to determine a semiempirical model to control the quantity of aerosol delivered into the patient's lung. An ATOMISOR NL9 M jet nebulizer (La Diffusion Technique Francaise, France) was filled with terbutaline (Bricanyl, Astra-Zeneca, Sweden) and connected to the inspiratory line of a Horus ventilator (Taema, France). Nebulization was synchronized with the inspiratory phase. We assessed at the end of the endotracheal tube the quantity of terbutaline (terbutaline mass output) and the volume median diameter (VMD) by diffraction-laser method. There was a negative correlation between terbutaline mass output and inspiratory air flow ( r =-0.95, p <0.0001) and between VMD and inspiratory air flow ( r =-0.96, p <0.0001). Moreover, positive end-expiratory pressure levels between 0 cm and 8 cm of water did not significantly change the terbutaline output mass ( p =0.22). Total nebulization time and terbutaline mass output calculated by the mathematical model showed good agreement with experimental data. In conclusion, our semiempirical model allows calculation of the duration of the nebulization required to deliver a given mass of terbutaline into patient lungs.

Residual gravimetric method to measure nebulizer output.

The aim of this study was to assess a residual gravimetric method based on weighing dry filters to measure the aerosol output of nebulizers. This residual gravimetric method was compared to assay methods based on spectrophotometric measurement of terbutaline (Bricanyl, Astra Zeneca, France), high-performance liquid chromatography (HPLC) measurement of tobramycin (Tobi, Chiron, U.S.A.), and electrochemical measurements of NaF (as defined by the European standard). Two breath-enhanced jet nebulizers, one standard jet nebulizer, and one ultrasonic nebulizer were tested. Output produced by the residual gravimetric method was calculated by weighing the filters both before and after aerosol collection and by filter drying corrected by the proportion of drug contained in total solute mass. Output produced by the electrochemical, spectrophotometric, and HPLC methods was determined after assaying the drug extraction filter. The results demonstrated a strong correlation between the residual gravimetric method (x axis) and assay methods (y axis) in terms of drug mass output (y = 1.00 x -0.02, r(2) = 0.99, n = 27). We conclude that a residual gravimetric method based on dry filters, when validated for a particular agent, is an accurate way of measuring aerosol output.

Particle deposition in a child respiratory tract model: in vivo regional deposition of fine and ultrafine aerosols in baboons.

To relate exposure to adverse health effects, it is necessary to know where particles in the submicron range deposit in the respiratory tract. The possibly higher vulnerability of children requires specific inhalation studies. However, radio-aerosol deposition experiments involving children are rare because of ethical restrictions related to radiation exposure. Thus, an in vivo study was conducted using three baboons as a child respiratory tract model to assess regional deposition patterns (thoracic region vs. extrathoracic region) of radioactive polydisperse aerosols ([d16-d84], equal to [0.15 µm-0.5 µm], [0.25 µm-1 µm], or [1 µm-9 µm]). Results clearly demonstrated that aerosol deposition within the thoracic region and the extrathoraic region varied substantially according to particle size. High deposition in the extrathoracic region was observed for the [1 µm-9 µm] aerosol (72% ± 17%). The [0.15 µm-0.5 µm] aerosol was associated almost exclusively with thoracic region deposition (84% ± 4%). Airborne particles in the range of [0.25 µm-1 µm] showed an intermediate deposition pattern, with 49% ± 8% in the extrathoracic region and 51% ± 8% in the thoracic region. Finally, comparison of baboon and human inhalation experiments for the [1 µm-9 µm] aerosol showed similar regional deposition, leading to the conclusion that regional deposition is species-independent for this airborne particle sizes.

Evaluation of lung tolerance of ethanol, propylene glycol, and sorbitan monooleate as solvents in medical aerosols.

BACKGROUND: Aerosol therapy is an expanding technique allowing administration of drugs acting locally in the bronchial tree and lungs or acting systemically after absorption through the respiratory tract. However, the choice of solvents and adjuvants is a critical step in the formulation process of new drugs. Pulmonary tolerance of ethanol, propylene glycol and sorbitan ester was evaluated in a rat model of intratracheal administration using a Microsprayer in a 4-day toxicity study. METHODS: Four groups of Sprague-Dawley rats (11 rats per group, n = 44) have received, on 4 consecutive days 150 microL of solutions containing the solvents, by intratracheal route using a IA-1B-2 inches-Microsprayer (PennCentury, Philadelphia, PA). Once a day, the rats received deionized water (control) or ethanol 10% or propylene glycol 30% or sorbitan monooleate 10%. All rats were sacrificed 24 h after the fourth administration. Biochemical analysis on bronchoalveolar lavage (BAL) fluid was performed on seven rats per group. The respiratory tract of the remaining four rats/group was examined histologically. RESULTS: Biochemistry and histopathology findings demonstrated that under the conditions tested, deionized water, 10% ethanol, and 30% propylene glycol were tolerated in a qualitatively similar way presenting limited cellular reaction. In contrast, 10% sorbitan monooleate produced an accumulation of foamy macrophages in the lungs and a higher degree of inflammation. In addition, animals in this group showed higher polymorphonuclear neutrophil recruitment and total proteins levels in BAL fluid. CONCLUSION: The overall results recommended ranking the vehicles according to the degree of inflammation which was induced: deionized water <10% ethanol < or =30% propylene glycol <10% Tween 80.

Differences in transcriptomic profile and IgA repertoire between jejunal and ileal Peyer's patches.

In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

Ultra-early weaning in piglets results in low serum IgA concentration and IL17 mRNA expression.

In pigs raised for meat production, weaning is a critical period because of related physiological perturbations and negative consequences on performance. Previous studies have shown that early weaning could either impair development of mucosal barrier function or boost intestinal immunologic parameters. In order to obtain further knowledge about the impact of ultra-early weaning on the porcine immune system development, three groups of piglets were weaned at different ages and compared to the unweaned control group. Lower IgA concentrations in ultra-early and early weaned piglets than in other piglets were identified in serum. In the mesenteric lymph node (MLN), significant differences in the mRNA expression of IL17a, TGF beta and FOXP3 were found between specific groups. Indeed, IL17a mRNA was mainly detected in ultra-early weaned piglets while FOXP3 and TGF beta mRNA were associated to both ultra-early weaned and suckling piglets. Reduced serum IgA concentration and MLN induction of a Th17 cytokine in ultra-early weaned piglets could be related to alterations of the mucosal barrier functions consecutive to the milk deprivation. All together, our findings suggest a crucial role for endogenous milk factors onto the onset of IgA synthesis.

Lung deposition of HFA beclomethasone dipropionate in an animal model of bronchopulmonary dysplasia.

The best delivery of a drug in ventilated neonates is obtained when using a small particle diameter solution administered via a spacer. Lung deposition of hydrofluoroalkane beclomethasone dipropionate (QVAR, 1.3 microm particles), delivered via an Aerochamber-MV15, was measured in piglets under conditions mimicking ventilated severely ill neonates (uncuffed 2.5 mm endotracheal tube; peak pressure 16 cm H2O; respiratory rate 40/min). After determining the mass and particle size distribution of the 99mTc-labeled and unlabeled drug, three lung deposition studies were performed: after 1 h of ventilation (controls, n = 18), after 48 h aggressive ventilation inducing an acute lung injury (nine piglets out of the controls), and after increasing the pressure to 24 cm H2O during drug delivery (five piglets out of the nine with acute lung injury). All piglets were then killed for lung histology. Results (median, range), expressed as a percentage of the delivered dose, were compared using an inferential or the Friedman test. While lung deposition was low, it was greater (p = 0.003) in controls (2.66%, 0.50-7.70) than in piglets with histologically confirmed acute lung injury (0.26%, 0.06-1.28) or under a high-pressure ventilation (1.01%, 0.30-2.15). Lung deposition of QVAR in an animal model of ventilated neonates is low, variable, and dramatically affected by lung injury.

Expression of the human kallikrein genes 10 (KLK10) and 11 (KLK11) in cancerous and non-cancerous lung tissues.

Only one transcript for KLK10 was identified by RT-PCR in lung tissue, whereas KLK11 expressed at least four alternative transcripts. Quantitative analysis of KLK10 and KLK11 expression levels was assessed by real-time PCR, in a cohort of 47 patients with non-small-cell lung cancer (NSCLC). Expression levels of these genes were widely distributed in the population studied. Multivariate analysis revealed a correlation between KLK10 over-expression and the squamous cell carcinoma histotype (p=0.034). There was no correlation between gene expression and patient survival. Overall, both genes behaved similarly (p<0.001). These results suggest a co-regulation of KLK10 and KLK11 expression in lung and a lack of KLK10 suppressor role in NSCLC. Finally, our findings indicate that these genes are likely involved in normal physiology processes in bronchus.

Competition between elastase and related proteases from human neutrophil for binding to alpha1-protease inhibitor.

The protease-antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to alpha1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of alpha1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.

KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer.

Emerging data indicate that serine proteases of the kallikrein family (KLK) are implicated in various human diseases, including carcinoma; however, kallikrein gene expression has never been investigated in lung cancer. Using RT-PCR and Western blotting, we demonstrated the expression of both KLK5 and KLK7, and their respective proteins (hK5 and hK7) in tumoral and nontumoral lung tissues. Quantitative gene expression was then analyzed in a cohort of 56 patients with non-small cell lung cancer by real-time RT-PCR. KLK5 expression is significantly more expressed in squamous cell carcinoma than in matched nonmalignant lung tissue (P=0.02), whereas expression of KLK7 was decreased in adenocarcinoma (P=0.003). Multivariate analysis revealed diverse correlations between the KLK5 and KLK7 expression levels in nonmalignant and malignant tissues, and clinical parameters, including histotype, metastatic status, and grade. Our findings provide new insight into kallikrein gene expression in hormone-independent carcinoma. Altogether, our results suggest that variability in KLK5 and KLK7 gene expression might be involved in lung tumorigenesis and useful for clinical purposes.

Aerosol deposition in neonatal ventilation.

Lung deposition of inhaled drugs in ventilated neonates has been studied in models of questionable relevance. With conventional nebulizers, pulmonary deposition has been limited to 1% of the total dose. The objective of this study was to assess lung delivery of aerosols in a model of neonatal ventilation using a conventional and novel electronic micropump nebulizer. Aerosol deposition studies with 99mTc diethylenetriamine pentaacetate (99mTc-DTPA) were performed in four macaques (2.6 kg) that were ventilated through a 3.0-mm endotracheal tube (with neonatal settings (peak inspiratory pressure 12-14 mbar, positive end-expiratory pressure 2 mbar, I/E ratio 1/2, respiratory rate 40/min), comparing a jet-nebulizer MistyNeb (3-mL charge, 4.8 microm), an electronic micropump nebulizer operating continuously [Aeroneb Professional Nebulizer (APN-C); 0.5-mL charge, 4.6 microm], and another synchronized with inspiration [Aeroneb Professional Nebulizer Synchronized (APN-S); 0.5-mL charge, 2.8 microm]. The amount of radioactivity deposited into lungs and connections and remaining in the nebulizer was measured by a gamma counter. Despite similar amounts of 99mTc-DTPA in the respiratory circuit with all nebulizers, both APN-S and APN-C delivered more drug to the lungs than MistyNeb (14.0, 12.6, and 0.5% in terms of percentage of nebulizer charge, respectively; p = 0.006). Duration of delivery was shorter with APN-C than with the two other nebulizers (2 versus 6 and 10 min for the APN-S and the MistyNeb, respectively; p < 0.001). Electronic micropump nebulizers are more efficient to administer aerosols in an animal model of ventilated neonates. Availability of Aerogen's electronic micropump nebulizers offers new opportunities to study clinical efficacy and risks of aerosol therapy in ventilated neonates.

Relationship between occupational risk factors and severity markers of systemic sclerosis.

OBJECTIVE: To investigate a potential association between occupational risk factors and severity markers of systemic sclerosis (SSc) defined by diffuse cutaneous extent, pulmonary involvement, and immunologic profile, i.e., presence of antitopoisomerase I antibody (anti-topo I). METHODS: Occupational exposures were assessed in 105 patients with SSc from 1998 to 2002. Exposures to silica dust, welding fumes, solvents, and epoxy resins were investigated. A group of 39 exposed SSc patients and a group of 66 unexposed ones were identified and compared according to severity markers of SSc. The stage of cutaneous extent was defined according to the classification of Leroy, as limited scleroderma (lSSc) or diffuse scleroderma (dSSc). Respiratory status was defined by pulmonary function tests and high resolution computed tomography. Immunological profile was determined by the presence of anti-topo I or anticentromere antibodies (ACA). Statistical relationships between occupational exposures and severity markers of SSc were evaluated using a multiple correspondence analysis and Fisher's exact test. RESULTS: Diffuse scleroderma affected mainly patients exposed during their occupational life to toxic agents. There were significant or close to significant associations between toxic exposure and dSSc (p = 0.06), pulmonary involvement (p = 0.10), and negative ACA (p = 0.03). The most incriminated products seemed to be epoxy resins (p = 0.06), white spirit (p = 0.07), aromatic solvents (p = 0.07), and silica coupled to welding fumes (p = 0.10). CONCLUSION: Our results indicate that occupational toxic factors have an influence on the severity of SSc.