Abstracts: International Society for Aerosols in Medicine 19th International Congress Chapel Hill,North Carolina,USA April 6–10,2013

1 Infectious Disease Aerobiology, Microbiology Division, Tulane National Primate Research Center, Tulane University School of Medicine, NEW ORLEANS, LA, United States of America 2 Department of Microbiology and Immunology, Tulane University School of Medicine, NEW ORLEANS, LA, United States of America O-04 ANIMAL MODELS FOR ASSESSING AEROSOLIZED VACCINES

Mucosal vaccination has been a goal for many dedicated to eliciting dual immunity in target populations. The prospect of achieving a serological response while concomitantly inducing an antibody response within the mucosal tissues takes on special importance when vaccinating against respiratory pathogens. Few vaccine products contain the particular componentry to stimulate both serological and mucosal immune response; past studies have shown that vaccines usually will bias the response based either upon the vaccine platform and/or the selected adjuvant. Demonstration of dual immunity - or eliciting an appropriate mucosal response in an animal model that is consistent with the predictive clinical response is a technical challenge that few vaccines candidates have successfully achieved. One such example is the ongoing development of a vaccine for protection against aerosolized ricin toxin, an extremely toxic plant-derived lectin that continues to be a concern as biological threat (bioterrorism) agent. Aerosol exposure to ricin causes an overwhelmingly local mucosal response in the lower respiratory system, involving indiscriminate apoptosis, loss of vascular integrity, and eventual alveolar flooding from an exceedingly small inhaled dose (>5 μg/kg). Experimental vaccine(s) and therapeutics under development have all targeted a common localized response in lung mucosa mediated through antibody-antigen interaction in order to achieve protection. The leading candidates under development for protection against ricin have been evaluated in the nonhuman primate (rhesus) disease model and have shown mixed utility to prevent intoxication. Study results suggest that optimization of delivery and formulation of vaccines and antibody-based therapies that are specifically formulated to elicit a mucosal response may be required to maximize effectiveness and achieve better protection in the respiratory system against an agent such as ricin toxin.

1 Department of Pharmaceutics, Virginia Commonwealth University, RICHMOND, VA, United States of America 2 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, RICHMOND, VA, United States of America O-07 BYPASSING UPPER AIRWAY AEROSOL DEPOSITION WITH ENHANCED CONDENSATIONAL GROWTH

Aerosol delivery to the lungs during non-invasive ventilation (NIV) is characterized by poor delivery efficiency (<1–10%) due to high depositional losses in the delivery tubing, cannula and nasal airways. In part, this is due to the particle size of the aerosols generated by commercial nebulizers. Enhanced condensation growth (ECG) is a technique that employs submicrometer drug aerosols to minimize depositional losses within the delivery setup and nasal airways. Condensational growth of these aerosols in the presence of co-administered heated and humidified air produces micrometer-sized droplets in the lungs allowing airway deposition to occur. In this study, in vitro experiments and computational fluid dynamic (CFD) simulations were used to compare the delivery of conventional nebulized aerosols with ECG aerosols generated from the Aeroneb Lab nebulizer and dried in a prototype dryer system to produce submicrometer particles. In vitro lung delivery efficiency of these aerosols was assessed and deposition in the delivery setup and nasal model determined with steady state and transient inhalation profiles. For both steady state and transient inhalation, deposition in the delivery tubing and cannula was low (∼10%) for the submicrometer ECG aerosols. Delivery through the nose to the airways during steady state flow using ECG was about 90% of the recovered dose. A series of strategies including streamlining the cannula geometry and inhalation synchronized aerosol administration were combined with the ECG approach to improve lung delivery of the drug during transient inhalation. The use of the ECG approach may allow efficient pharmaceutical aerosol delivery to patients receiving NIV.

Acknowledgements: NIH

1 Parion Sciences, Inc., DURHAM, NC, United States of America 2 University of North Carolina, CHAPEL HILL, NC, United States of America 3 Cambridge Consultants, CAMBRIDGE, United Kingdom 4 Georgia State University, ATLANTA, GA, United States of America 5 RTI International, RESEARCH TRIANGLE PARK, NC, United States of America O-08 AEROSOL DELIVERY THROUGH HIGH FLOW NASAL CANNULAE

Trans-nasal pulmonary aerosol delivery via nasal cannula offers benefits over the oral administration for a range of patients and healthcare care settings. Trans-nasal delivery originated during non-invasive ventilation with high flow therapy, utilizing high flow nasal cannulae. However, the clinical utility of trans-nasal aerosol delivery has been historically limited by low pulmonary deposition (1–4% in Chua et al. 1994), significant rainout in the nasal cannula, and the absence of available delivery systems capable of efficiently delivering aerosols though ergonomic, comfortable cannulae. Over the last decade, novel approaches and evaluation of novel delivery systems identified critical factors for improving trans-nasal delivery. First, the potential for efficient aerosol delivery via nasal cannula and loss of efficiency due in part to large particles was described by Corcoran et al. Second, Ari and Fink demonstrated the dilutive effects of increasing gas flow and the benefits of improved laminar flow with heliox at higher flow. Third, Bennett and Zeman, while exploring the nasal deposition of fine particles, described the potential for decreased nasal deposition and improved overall pulmonary deposition with 1μm and 2μm monodisperse carnauba wax particles. Lastly, the in-silico and in-vitro work of Longest and Hindle demonstrated the potential of enhanced condensational growth and excipient enhanced hygroscopic growth to improve trans-nasal pulmonary deposition efficiency. Building on these concepts and integrating them with additional innovative technologies, a novel trans-nasal pulmonary aerosol delivery (tPAD) platform with nasal cannulae optimized for aerosol conductance was developed by Parion Sciences. The tPAD platform achieved ∼ten-fold higher pulmonary deposition efficiency (∼38% of the emitted dose) compared to the established trans-nasal pulmonary deposition efficiency values, with minimal deposition in the nose in healthy adult subjects. These advances support the future clinical use of the trans-nasal aerosol delivery to the lung with a variety of therapeutic agents to treat a broad range of pulmonary disorders.

1 US Environmental Protection Agency, CHAPEL HILL, NC, United States of America 2 US Environmental Protection Agency, RESEARCH TRIANGLE PARK, NC, United States of America 3 University of North Carolina, CHAPEL HILL, NC, United States of America O-17 CONTROLLED EXPOSURES OF HUMAN VOLUNTEERS TO DIESEL ENGINE EXHAUST: BIOMARKERS OF EXPOSURE AND HEALTH OUTCOMES

Combustion of diesel fuel contributes to ambient air pollutant fine particulate matter (PM) and gases. Fine PM exposure has been associated with increased mortality due to adverse cardiac events, and morbidity, such as increased hospitalization for asthma symptoms and lung infections. Controlled exposure studies with diesel exhaust (DE) have been used, in part, to examine if epidemiological observations between PM exposure and health effects had biological plausibility. In our facility, exposures of healthy, young adult human volunteers were exposed to either 100 μg/m3 DE to identify markers of exposure and effect. At 100 μg/m3 exposure, small changes in plasma cytokine and clotting factors were observed without lung function changes. Urinary naphthalene and phenanthrene appear to surrogates of the exposure. For 300 μg/m3 DE exposures, we examined lung function effects with both concurrent and sequential 300 ppb ozone (O3) exposure. Subjects received either air FA, O3, DE, or DE+O3 on Day 1, followed by only O3 exposures on Day 2. Exposures were for 2 hr with intermittent, moderate exercise. For lung function on Day 1, FA and DE did not induce FEV1 changes, O3 decreased FEV1 16±3%, while DE+O3 decreased FEV1 22±4%. On Day 2 after the O3 challenge, subjects who were exposed to DE on Day 1 had a greater FEV1 decrement (18±4%) compared to the FEV1 decrements of subjects (11±3%) receiving air exposure on Day 1. Lung function recovered to approximately pre-exposure values within 4 hr at similar rates in all groups on both Days 1 and 2. These data suggest that simultaneous DE+O3 exposure can synergistically decrease FEV1, and sequential DE followed by O3 exposures can induce a greater FEV1 decrement than FA/O3 exposures.

Disclaimer: This is an abstract of a proposed presentation and may not represent official US EPA policy.

Funding by US EPA; Protocols approved by the UNC Biomedical IRB.

1 Boehringer Ingelheim Pharma GmbH & Co. KG, INGELHEIM, Germany 2 Johannes Gutenberg-University Mainz, MAINZ, Germany O-20 DEPOSITION STUDIES IN INFANTS AND YOUNG CHILDREN FOR DEVICE AND FORMULATION DEVELOPMENT

The therapeutic success of inhaled medications relies on three major columns: the inhalation device, the pharmaceutical formulation and the patient. While the first two parameters are extensively investigated from the very beginning of the drug product development process, the specific human anatomy and breathing behavior is often not taken into account. Currently used cascade impaction methods of the USP/Ph.Eur. for determination of the particle size distribution of pharmaceutical aerosols are highly effective quality control tools but not intended to resemble the human in vivo conditions. Achieving good correlations between in vitro methods and in vivo performance is a major challenge but worthwhile to design safe and efficient inhaled medications for pediatric application. Therefore idealized/realistic replicas of the infant mouth, nose and throat region in combination with an electronic lung to mimic inhalation profiles were used to assess performance differences of several inhalation devices (MDIs, SMI, DPIs, VHCs) and powder formulations in children aged 1–5 years. In addition, studies with monodisperse aerosol particles (3, 5 and 7μm) at different constant air flow rates were conducted to determine the optimal particle size to enter the pediatric lung. The whole data set was compared to the results using an adult model. The pediatric models showed a higher sensitivity to the different devices and formulations, especially the ones <3 years of age. Aerosol particles ≤3 μm had the optimal size to deposit in the lung of very young children.

1 Respiratory Medicine, Bern University Hospital, BERN, Switzerland 2 Telethon institute for Child Health Research, PERTH, Australia 3 School of Veterinary and Biomedical Sciences, Faculty of Health Sciences, Murdoch University, PERTH, Australia 4 Centre for Child Health Research University of Western Australia, PERTH, Australia 5 Adolphe Merkle Institute, University of Fribourg, FRIBOURG, Switzerland O-30 DELIVERY OF ANTIGENS FOR VACCINATION: RECENT ADVANCES AND CHALLENGES

Nanocarriers have been proposed for promising novel diagnostic, therapeutic approaches, as well as for vaccination strategies in a variety of human diseases. Specifically, the delivery of nano-sized carriers to the lung has been receiving increasing interest due to the large surface area provided by the gas exchange region, limited local proteolytic activity, non-invasive administration, and thin anatomical barriers for systemic access. Pulmonary antigen presenting cells (APC) such as macrophages and dendritic cells are considered as sentinels of the immune system due to their strategic localization, their phagocytic activity and ability to present antigen. In particular, respiratory tract dendritic cells, as key APC in the lung, constitute an ideal target for vaccine delivery. To improve efficiency of vaccination and develop new strategies, an in-depth characterization of APC populations throughout different respiratory tract compartments is essential. Furthermore, nanocarrier size, material and surface properties are key factors when designing new carriers, as these parameters not only determine deposition within different respiratory tract compartments, but will also influence interaction with lung tissue components and immune cells. Clarifying which APC/dendritic cell populations primarily interact with nanocarriers and traffic these from different respiratory tract compartments to lung draining lymph nodes is paramount to understanding related downstream inflammatory and immune responses. Such data will be fundamental to rationally design future novel particulate systems in the nano-size range for novel applications in the respiratory tract, such as pulmonary vaccination.

Acknowledgements: Grant funding by the Swiss National Science Foundation and the Swiss Respiratory Society

1 University of North Carolina, CHAPEL HILL, NC, United States of America 2 North Carolina State University, RALEIGH, NC, United States of America O-31 TAILORING MACROPHAGE UPTAKE OF INHALED PARTICLES FOR PULMONARY DELIVERY APPLICATIONS

There is a significant unmet need in the delivery of respiratory drugs that target or de-target macrophages depending on the desired site of action. Alveolar macrophages present a significant barrier to efficient pulmonary drug delivery applications because of i) their rapid and undesirable clearance of certain deposited drugs in the lung whose site of action involves non-macrophage targets, like aerosolized therapeutics targeted to the alveoli and epithelial cells; or ii) by their protection and harboring of disease targets, such as infectious bacteria like tuberculosis, which makes it difficult to treat effectively. We have been interested in designing particles for use in dry powder inhalers that have the ability to target or de-target alveolar macrophages in order to increase their therapeutic index. Using PRINT^®, a top-down nano- and micro-molding particle fabrication technique based on soft lithography, we were able to generate unique micron-sized particle geometries with aerodynamic diameters between 1–5 μm but with volumes that ranged between 5 – 45 μm³ which was achieved by controlling particle shapes. Over two-fold reduction in particle uptake by alveolar macrophages with select shapes was observed indicating the potential for tailored avoidance of macrophage clearance in the lung. These shaped particles were also shown to remain in the lung for an extended period of time without inducing inflammatory cytokine release, which holds promise for localized and sustained pulmonary therapeutics. Integrating drug delivery design with particle geometry engineering to modulate phagocytic tendencies may lead to novel therapeutic and diagnostic paradigms for a wide range of diseases.

Acknowledgements: Funding by the NIH Pioneer Award and NSF Graduate Research Fellowship

1 Comprehensive Pneumology Center, Institute for Lung Biology and Disease, Helmholtz Zentrum München, NEUHERBERG, Germany 2 PARI Pharma GmbH, MUNCHEN, Germany 3 Department of Nuclear Medicine, LMU Medical Center Grosshadern, MUNCHEN, Germany 4 Department of Pulmonary Medicine, Asklepios Hospital München-Gauting, GAUTING, Germany 5 Department of Otolaryngology, Head and Neck Surgery, LMU Medical Center Grosshadern, MUNCHEN, Germany O-32 DRUG DELIVERY TO PARANASAL SINUSES USING PULSATING AEROSOLS

Chronic rhinosinusitis (CRS) is a common chronic disease of the upper airways with considerable impact on quality of life. Since current approaches to treat CRS using topical and systemic steroids and antibiotics often fail, functional endonasal sinus surgery (FESS) has been the primary approach for treating CRS. The paranasal sinuses are involved in CRS, but they cannot be reached by conventional nebulizers or nasal pump sprays. Pulsating aerosols are a new drug delivery technology reaching the paranasal sinuses of CRS patients enabling new topical therapy options for CRS.

^99mTc-DTPA vibrating aerosols were applied in CRS patients before and 2–3 months after FESS. The deposition distribution in the upper airways was measured by gamma camera imaging. The mass median diameter (MMD) was 3.0μm with a geometric standard deviation of 1.6. Before FESS, 56.7+/−13.3% of the nebulizer output deposited in the nasal cavity. 4.0+/−1.7% of the deposited fraction was found in the paranasal sinuses. No aerosol deposition was detected in the lung. At least two months after FESS 46.7+/−12.7% of the nebulizer output deposited in the nasal cavity and 6.1+/−2.2% of the deposited fraction was found in the paranasal sinuses. Nasal deposition decreased after FESS due to increased nasal calibres after surgery. Moreover, sinus deposition significantly increased after FESS, which is also an indication for resolved nasal obstructions.

Vibrating aerosols can deliver significant doses into posterior nasal spaces and paranasal sinuses, providing alternative conservative therapy options before and after sinus surgery. Patients with chronic lung diseases may also benefit from vibrating aerosols, since these diseases partly manifest in the upper airways.

Acknowledgement: The study was supported by Pari GmbH, Starnberg, Germany and by the Bayerische Forschungsstiftung (AZ 914-10).

1 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, RICHMOND, VA 2 Department of Pharmaceutics, Virginia Commonwealth University, RICHMOND, VA O-33 AEROSOL TRANSPORT THROUGHOUT THE CONDUCTING AIRWAYS: ASSESSING CONVENTIONAL AND NEW DELIVERY STRATEGIES WITH COMPUTATIONAL MODELS

Predicting the performance of respiratory drug delivery strategies is difficult due to challenges associated with simulating pharmaceutical aerosols and the complexities of the respiratory tract. Computational fluid dynamics (CFD) provides an effective method to address factors associated with the physics of pharmaceutical aerosols. A new stochastic individual path (SIP) modeling approach offers an effective method to capture delivery throughout the lung in three-dimensions. In this study, a CFD and SIP modeling approach is combined with in vitro experiments in order to predict the performance of both conventional and new pharmaceutical aerosol delivery systems. The in vitro experiments are used to both validate the aerosol deposition predictions and to provide necessary initial information for the computational model. For all delivery strategies considered, model predictions of deposited drug mass match in vitro experimental results very closely. Considering conventional inhalers, the model results provide new insights into where drugs are delivered in the conducting airways. For example, a softmist inhaler platform is shown to improve delivery efficiency of drug by over an order of magnitude in the lower conducting airways compared with a high quality DPI. Considering new delivery strategies, condensational growth is shown to be an effective method for eliminating extrathoracic deposition and potentially targeting the site of deposition. In summary, a combination of CFD and SIP modeling together with in vitro experiments provides new insights into delivery with conventional inhalers and forms the basis for developing novel highly effective strategies capable of targeting deposition to and within the lungs.

Acknowledgements: NIH and US FDA

1 Medical Gases Group, Air Liquide Santé International, LES-LOGES-EN-JOSAS, France 2 Department of Mechanical Engineering, Lafayette College, EASTON, PA, United States of America 3 Delaware Research and Technology Center, American Air Liquide, NEWARK, DE, United States of America 4 School of Health Sciences, University of Southampton, SOUTHAMPTON, United Kingdom 5 Department of Medical Physics and Bioengineering, Southampton University Hospitals, NHS Trust, SOUTHAMPTON, United Kingdom 6 Department of Anesthesia (Bioengineering), MGH/Harvard, BOSTON, MA, United States of America O-34 USING HELIUM-OXYGEN TO IMPROVE REGIONAL DEPOSITION OF INHALED PARTICLES

Helium-oxygen has been used for decades as a respiratory therapy conjointly with aerosols. It has been shown, under some conditions, as a means to provide more peripheral particle deposition. If this greater outer deposition is thought to be along a serial path, then in contrast we can also consider deposition along parallel paths that are quite different, especially in a heterogeneous, pathological lung. It is in this context that it is hypothesised that helium-oxygen can improve regional deposition, leading to more homogenous deposition, by increasing ventilation to obstructed lung regions. This hypothesis is based on mathematical and in vitro modelling of the fundamental mechanisms of fluid flow and aerosol deposition in the respiratory tract, specifically related to inertial flow resistance caused by turbulence and changes in airway direction. Ongoing studies using 3D imaging techniques provide visual examples of this improvement in asthmatics. Further analysis is also underway to assess these effects quantitatively. Thus helium-oxygen might be able to improve the efficacy of those inhaled drugs that would benefit from a more peripheral and homogenous deposition distribution to ventilation deficient lung regions.

1 University Hospital Southampton, SOUTHAMPTON, United Kingdom 2 Johns Hopkins Medical Institutions, BALTIMORE, MD, United States of America, ³University of Pittsburgh, PITTSBURGH, PA, United States of America 4 University of Western Australia, PERTH, Western Australia 5 McMaster University, HAMILTON, Ontario, Canada 6 Scientific Consultant, HUNSTANTON, Norfolk, United Kingdom O-37 CURRENT METHODS FOR LUNG IMAGING OF AEROSOL DEPOSITION FROM MEDICAL INHALERS: GUIDELINES FROM THE ISAM TASK FORCE REPORT

Assessing deposition of inhaled drug products using radionuclide imaging is useful in estimating the amount of medication inhaled and its topographical distribution. In addition, because imaging enables comparison of two aerosolized products in terms of both whole lung dose and deposition pattern, imaging has potential application in assessing bioequivalence. However, to date, imaging studies have been conducted without common standards and this has led regulatory agencies to question the value of imaging data. To address this issue, the ISAM Regulatory Affairs Networking Group convened the Sub-committee for the Standardization of Lung Imaging Techniques in early 2010. The aims were (i) To identify key areas in radionuclide imaging that could be standardized, (ii) To determine the essential information to allow standardization and (iii) To publish a consensus statement that incorporated practical recommendations. This paper describes the content of this consensus statement. It provides practical guidance on standardizing radiolabel validation and image acquisition and analysis, using planar (2D), SPECT and PET imaging modalities. Studies with standardized imaging techniques could make government regulators more confident in using them to support claims of bioequivalence and therapeutic equivalence, and would allow more direct comparisons of data from different laboratories. Combining data from studies conducted by different groups could lead to multi-center studies and more rapid development of devices for inhaled therapies. The consensus statement has been published as a supplement in the Journal of Aerosol Medicine and Pulmonary Drug Delivery entitled “Standardization of Lung Imaging Techniques for Aerosol Deposition Assessment of Orally Inhaled Products”.

1 Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, HANNOVER, Germany 2 MTF MediTech Franken GmbH, ECKENTAL, Germany 3 Takeda Pharmaceuticals Int'l, ZURICH, Switzerland 4 Nycomed: a Takeda Company, KONSTANZ, Germany O-39 PERFORMANCE TESTING OF A NOVEL CONTINUOUS POWDER AEROSOLIZER (CPA) FOR INHALATIVE ADMINISTRATION OF SURFACTANT TO PRETERM NEONATES

The surfactant Continuous Powder Aerosolization (CPA) technology is intended for continuous administration of an aerosol for inhalation by preterm neonates on non-invasive ventilatory support. The CPA allows efficient aerosolization of dry surfactant powder, generating an aerosol of high concentration. In a subsequent humidification step, aerosol particles are heated and covered with a surface layer of water. The wet surfactant aerosol is then conducted via a tube to the patient interface (e.g., nasal prongs).

Performance testing of nebulizers currently follows DIN EN 13544-1. Since this norm applies to nebulizers designed for adults, a new test strategy for the special requirements of a device for neonates had to be implemented. In particular, these requirements had to take into consideration neonatal respiratory physiology and operation of the CPA system in a ventilatory circuit under elevated temperatures.

With the newly developed test rig, performance of the CPA can be characterized by measuring the aerosol output and output rate at the exit of the aerosol tube as well as at the outlet of the nasal prongs. Performance at the outlet of the nasal prongs is measured making use of an artificial lung with a hard-coded breathing profile of a preterm neonate. Results obtained with the test rig show that 50 to 60% of the surfactant leaving the aerosolizer as dry powder reach the exit of the aerosol tube as humidified with a temperature of 37 °C. Total delivery of surfactant at the prong exit is about 20% when applying the hard-coded breathing profile.

Acknowledgements: This work was funded by Nycomed GmbH; Nycomed: a Takeda Company, Konstanz, Germany.

1 School of Pharmacy, Royal College of Surgeons in Ireland, DUBLIN 2, Ireland 2 School of Biology and Environment Science, University College Dublin, DUBLIN 4, Ireland. 3 Centre for Infection and Immunity, Queen's University Belfast, BELFAST, United Kingdom 4 Respiratory Research Division, RCSI, Education and Research Centre, Beaumont Hospital, DUBLIN 9, Ireland O-41 BIOENGINEERING OF siRNA FOR TREATMENT OF INFLAMMATORY LUNG DISEASE

Inflammatory lung disease is associated with the activation of inflammation pathways inciting secretion of cytokine and chemokine proteins, and progressive recruitment of macrophages, mast cells, and neutrophils to the lungs. siRNA offers a highly specific means of impeding this pathway and combined with inhalation represents a unique means of targeting inflammation. Alveolar macrophages (AMs) are a key cellular component of inflammatory lung disease and therefore an attractive target for anti-inflammatory siRNA. We bioengineered siRNA-loaded poly(lactic-co-glycolic acid) PLGA microparticles (MPs) for optimal inhalation and transfection of AMs. The process of siRNA encapsulation in MPs was optimised using a double emulsion technique and the resulting particles characterised for size, shape, aerosol characteristics, encapsulation efficiency and siRNA integrity. The cell uptake of siRNA-loaded MPs was determined by flow cytometry, confocal laser scanning microscopy and high content analysis (HCA) with MPs capable of transfecting up to 55% of cells. Anti-TNFα siRNA-MPs were prepared to study the functionality of encapsulated siRNA in Lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation. The MPs were able to decrease TNFα expression by 45% over 48 hrs in differentiated THP-1 compared to negligible knockdown using commercial transfection reagents and offered significant, controlled release siRNA knockdown of TNFα in primary monocytes for up to 72 hours. Cytotoxicity and immunogenicity screens showed cells remained viable and MPs did not cause significant immunogenicity compared to commercial controls which showed 4.7 fold increase in TNFα expression. Furthermore, intratracheal dosing of siRNA-MPs into LPS-inflammation mouse models showed the formulation to be well tolerated.

Acknowledgments: Grant funding by Science Foundation Ireland SFI 07/SRC/B1154 and Health Research Board (HRB) of Ireland PHD/2007/11

1 Defence Science and Technology Laboratory, PORTON DOWN, SALISBURY, WILTSHIRE, United Kingdom 2 Aradigm Corporation, HAYWARD, CA, United States of America O-42 EFFICACY OF INHALED LIPOSOME-ENCAPSULATED CIPROFLOXACIN AGAINST YERSINIA PESTIS

Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative post-exposure therapeutic agent against Yersinia pestis, the causative agent of plague. The high mortality rate of pneumonic plague and the potential for person to person transmission led to the classification of Y. pestis, by the US Centers for Disease Control, as a Category A agent of concern.

The efficacy of CFI against Y. pestis strain CO92 was determined using a mouse model. Mice were challenged by the inhalational route and treated 24 or 48 hours post challenge with 50 mg/kg orally delivered ciprofloxacin, 50 mg/kg CFI delivered via intranasal instillation to the lung or CFI delivered in an aerosol. A single dose of oral ciprofloxacin administered at 24 hours post-challenge did not prevent mortality. However, it did increase the mean time to death to 5 days compared to 3 days for untreated mice. Conversely, a single dose of CFI administered as an aerosol at 24 hours post-challenge provided approximately 70% protection and significantly improved survival when compared to a single dose of ciprofloxacin (P<0.001). A single dose of CFI delivered by the intranasal route provided 100% protection and significantly improved survival compared to a single dose of ciprofloxacin (P<0.0001).

Consequently, this preliminary study suggests that CFI is a promising prophylaxis for use in the event of a deliberate release of Y. pestis.

© Crown copyright 2012.

1 Creare Inc., HANOVER, NH, United States of America 2 Centers for Disease Control and Prevention, ATLANTA, GA, United States of America O-43 THREE-DIMENSIONAL DEPOSITION IMAGING USING DUAL ENERGY COMPUTED TOMOGRAPHY

Aerosol delivery effectiveness depends on efficient deposition of the agent on the targeted airway tissues and the spatial distribution of the delivered material. Influenza vaccine delivery targets the mucosal tissue surfaces beyond the nasal valve and above the vocal cords. Delivery efficiency can be assessed as the fraction of the dose deposited in the target area and the fraction of the target area reached by the dose.

Current quantitative techniques for characterizing nasal airway deposition involve radiolabelled tracer techniques—such as gamma scintigraphy—performed in humans, animals, or airway phantoms. Gamma scans provide planar images from which the signal intensity can be measured. By overlaying the images of anatomic structures, which are not visible in the scan, a proportional deposition by section of the nasal airway can be determined. The spatial deposition information from gamma scintigraphy is limited to reasonable two-dimensional quantitative analysis. We present a preliminary demonstration of a three-dimensional technique for imaging deposition for aerosol delivery systems. The technique is based on Dual Energy Computed Tomography (CT). Dual energy CT is similar to conventional CT imaging, except that two measurements are made simultaneously at different energies. The two measurements allow for significantly greater discrimination between different materials, allowing CT contrast agents to be quantitatively imaged. We demonstrated the feasibility of the technique in a nasal airway phantom using both a dry powder inhaler and a spray device. The technique provided quantitative, 3-D deposition patterns with high-spatial resolution. This technique also has potential for use with animals or humans.

OPEN IN VIEWER

Dual Energy CT Scan of Dry Powder Inhaler (DPI) Deposition in a Child Nasal Airway Phantom

Acknowledgements: SBIR funding provided by US Centers of Disease Control and Prevention

1 Massachusetts General Hospital and Harvard Medical School, BOSTON, MA, United States of America 2 Massachusetts Institute of Technology and Massachusetts General Hospital, BOSTON, MA, United States of America O-44 AEROSOL IMAGING WITH PET-CT: FROM DEPOSITION TO TISSUE DOSING

Whether developing new aerosol compounds or validating drug delivery systems it can be important to evaluate the delivered drug in pharmacological terms. Two such parameters are the concentration on the inner surface of the airways (CIS) and the dose per milligram of lung parechema (LPD). While two-dimensional imaging provides indices of central to peripheral deposition, limited spatial resolution and lack of anatomical information prevent assessment of CIS or LPD. Here we present a method to estimate global and lobar CIS and LPD using the fusion of PET deposition and CT anatomical imaging. We evaluated these parameters in the PET-CT deposition images of 11 bronchoconstricted asthmatics in whom we expected an amplified regional contrast of CIS and LPD. Estimates of aerosol deposition in central airways per unit airway volume were on average 22 times greater than estimations from raw PET data. This deposition distributed onto the airways' surface with an average lobar CIS of 47E-6 of the total lung deposition (TLD) per square mm. CIS was highly variable among subjects (COV=0.42) and among lobes (COV=0.43). Average lobar LPD was 0.9E-12 TLD per mg. Although less variable than CIS amongst subjects (COV=0.15), it was similarly variable amongst lobes (COV=0.43). We conclude that the analysis of PET-CT aerosol deposition images has the ability to yield regional pharmacological parameters that may be important in drug translational development and in the evaluation of aerosol delivery systems.

Acknowledgments: Sponsored by NIH grants HL86717 and HL68011, and by support from Air Liquide

1 Battelle Memorial Institute, COLUMBUS, OH, United States of America 2 Oak Ridge National Laboratory, OAK RIDGE, TN, United States of America 3 Pacific Northwest National Laboratory, RICHLAND, WA, United States of America 4 Lawrence Livermore National Laboratory, LIVERMORE, CA, United States of America O-46 IN VIVO INHALATION EXPOSURES TO SUPER-PARAMAGNETIC IRON-OXIDE NANO-PARTICLES (SPIONP) FOLLOWED BY MAGNETIC PARTICLE DETECTION (MPD) AND ACCELERATOR MASS SPECTROMETRY (AMS) ANALYSIS

SPIONP were synthesized by chemical co-precipitation and surface-modified with −COOH groups through covalent bonding (¹⁴C labeling was also developed). Nano-aerosol was generated out of water suspension SPIONP (15 nm core) using Collison nebulizer under high pressure (∼80 psi) helium, mixed with oxygen, dried with HEPA filtered air, and delivered to the multi-tier nose-port exposure carousel. Aerosol concentration (∼10⁷ particles/cm³) and particle size (∼70 nm CMAD) were monitored using Scanning Mobility Particle Sizer (SMPS). Micro-Orifice Uniform-Deposition Impactor and Scanning Electron Microscopy were provided to characterize nano-particles sampled from the exposure atmosphere. Spatial uniformity (3–6% RSD) of the nano-aerosol distribution across the nose-only exposure unit was confirmed using SMPS. Temporal stability (11–20% RSD) gravimetric filter analysis was conducted each hour of exposure.

Two 4-hours inhalation exposures of male BALB/C mice (30 mice/study) were conducted. One exposure was followed by MPD and transcriptomics analysis. The second exposure was followed by pharmacokinetic and biodistribution analysis using ultrasensitive AMS. Respiratory physiology measurements were conducted and total inhaled mass was estimated. Blood, lung, heart, liver, brain, head, and other tissues were analyzed. Both studies demonstrated similar lung clearance (∼55–70% after 2 days post-exposure and ∼36% after 7 days post-exposure). Small amounts of SPIONP were detected in the liver, spleen, upper GI and olfactory bulb over 7 days post-exposure using AMS. 815 genes were significantly changed in SPIONP exposed mice at the end of the 4-hour exposure period. Data demonstrated strong transcriptional response that persisted through 6 hour post-exposure and gradually declined over the next 7 days.

Acknowledgements: This work was supported by the Battelle Fundamental and Applied Systems Toxicology (B-FAST) Team.

1 Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, D-66123 Saarbrücken, Germany 2 Department of Biochemistry and Molecular Biology I, Universidad Complutense, E-28040 Madrid, Spain 3 Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz-Center for Infectious Diseases (HZI), Saarland University, D-66123 Saarbrücken, Germany O-48 ADSORPTION OF PULMONARY SURFACTANT PROTEINS TO NANOSCALE DRUG CARRIERS: IMPLICATIONS FOR TARGETING ALVEOLAR MACROPHAGES

With respect to deposition of nanoparticulate drug carriers in the peripheral lungs, the primarily encountered biological matter is the lung surfactant layer, which besides phospholipids contains also proteins. In particular the pulmonary collectins surfactant protein A (SP-A) and D (SP-D) can bind a variety of structural patterns and favor the alveolar macrophage (AM) clearance of foreign material. In first studies with lung surfactant proteins and other proteins we have addressed the interactions with metal oxide nanoparticles (NPs).^1–3 In a more recent study, we investigated the involvement of SP-A and SP- D in triggering the uptake of differently modified NPs (starch, ST; and phosphatidylcholine, PL) by macrophages in an immortalized murine AM cell model (MH-S cells). Using flow cytometry and confocal microscopy, we could demonstrate that SP-A or SP-D both can exert strongly stimulating effects on the NP uptake by AM. By means of gel electrophoresis, these effects were shown to be due to adsorption of SP-A or SP-D. Moreover, the agglomeration of NPs was influenced upon protein adsorption, which also contributed to the extent of NP uptake by AM. In addition, we could show that the presence of artificial surfactant lipids or isolated native surfactant lipids essentially modulates surfactant protein-mediated effects regarding macrophage uptake of NPs.⁴

Our ongoing work underscores the need to thoroughly investigate such bio-nano interactions in the lungs, as they might profoundly determine the fate of NPs deposited in the lung. Current work focuses on controlling interactions between nanoparticulate drug carriers and endogenous compounds of the lung lining fluid that govern macrophage internalization. This approach holds promise for more selective and efficient treatment of macrophage-related infections, as occurring e.g. in early tuberculosis.

References:

1. Ruge CA, Kirch J, Cañadas O, Schneider M, Pérez-Gil J, Schaefer UF et al. Uptake of Nanoparticles by Alveolar Macrophages Is Triggered by Surfactant Protein A. Nanomedicine : Nanotechnology, Biology, and Medicine 2011; 7 (6): 690–693.

2. Schulze C, Schaefer UF, Ruge CA, Wohlleben W, Lehr C-M. Interaction of metal oxide nanoparticles with lung surfactant protein A. European Journal of Pharmaceutics and Biopharmaceutics 2011; 77 (3): 376–383.

3. Schaefer J, Schulze C, Marxer EEJ, Schaefer UF, Wohlleben W, Bakowsky U et al. Atomic Force Microscopy and Analytical Ultracentrifugation for Probing Nanomaterial Protein Interactions. ACS Nano 2012; 6 (6): 4603–4614.

4. Ruge CA, Schaefer UF, Herrmann J, Kirch J, Cañadas O, Echaide M et al. The Interplay of Lung Surfactant Proteins and Lipids Assimilates the Macrophage Clearance of Nanoparticles. PLoS ONE 2012; 7 (7): e40775.

1 Drug Delivery, Helmholtz-Institute for Pharmaceutical Research Saarland, Saarland University, SAARBRUECKEN, Germany 2 Gene Regulation and Differentiation, Helmholtz-Centre for Infection Research, BRAUNSCHWEIG, Germany 3 SHG Clinics, Department of Cardiothoracic Surgery, Voelklingen Heart Center, VOELKLINGEN, Germany O-49 IMMORTALIZATION OF PRIMARY HUMAN ALVEOLAR EPITHELIAL CELLS: DEVELOPMENT OF A NEW IN VITRO MODEL OF THE AIR-BLOOD BARRIER

Barrier properties like functional tight junctions and hence high transepithelial electrical resistance (TEER) values are of utmost importance in drug delivery research. Till date there is no cell line available that reflects the crucial barrier properties of human primary alveolar type I (AT I) cells. By lentiviral transfection we immortalized the primary AT I cells, which were developed as a model for the human air-blood barrier (ABB) in our lab years ago. It has been reported, that the loss of cellular identity and the correlated cellular functions is a big disadvantage of transformation and particularly the use of classical transformation genes like hTert or SV40LTAg often causes cellular de-differentiation. However, to overcome this obstacle a set of 33 so called “mild proliferators” was chosen in our study, which can under certain circumstances also enable cell proliferation. The cell lines underwent characterization regarding their epithelial origin, the expression of lung-cell-specific markers and their barrier properties by morphological studies, immunofluorescence staining techniques, real-time PCR and TEER measurement. The new alveolar cell lines conserved epithelial characteristics, have an immortal life-span (sigmoid growth curve, currently passage 30, freezable/thawable), show tight junction expression (TEER>1000 Ω·cm²) and exhibit AT I-like marker expression. We assume that these new alveolar cell lines could provide considerable advantages in terms of reflecting the in vivo situation of the air-blood barrier and could serve as an in vitro model for various applications. This system could allow standardized toxicity and transport studies for newly developed compounds and delivery systems and could be helpful in elucidating infection pathways across the respiratory tract in the context of aerosol transmitted infectious diseases (e.g. swine flu, tuberculosis, etc.) and their treatments.

1 Department of Pharmaceutics, Virginia Commonwealth University, RICHMOND, VA, United States of America 2 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, RICHMOND, VA, United States of America O-50 STREAMLINED DESIGNS TO REDUCE AEROSOL DEPOSITION LOSSES IN CIRCUITS USED FOR INVASIVE AND NON-INVASIVE VENTILATION

Pharmaceutical aerosol delivery to the lungs during invasive and non-invasive ventilation is associated with dose variability and drug depositional losses. In part, these losses are due to regions of sudden flow contraction and expansion, and angle changes within the circuit. To address this issue, key components of two delivery circuits were produced using streamlined designs that originated from computational fluid dynamics simulations. Aerosol drug deposition was determined in a model invasive delivery circuit consisting of an Aeroneb adult T-adapter connected to a wye connector and then an endotracheal tube (7–9 mm diameter). Similarly, deposition was determined in a model non-invasive delivery circuit consisting of an Aeroneb neonate T-adapter and an Optiflow nasal cannula connected using 20 cm of tubing (10 mm diameter). Albuterol sulfate aerosols were generated using the Aeroneb Lab Nebulizer and delivered to the circuits with humidified air (>90%RH, 25°C) at 30LPM. Commercial and custom-made streamlined T-adapters, wye connectors and cannula designs were tested in each system. For the invasive ventilation circuit, deposition in the commercial wye connector was high (33.8±1.2%). The streamlined wye connector reduced deposition to 5.1±0.8%. As the endotracheal tube size was increased, this 6 fold reduction in depositional losses resulted in higher delivery fractions of the drug to the patient. For the non-invasive ventilation circuit, there was significant deposition in the neonate T-adapter (30.6±1.9%). A streamlined design T-adapter resulted in deposition losses of 5.7±0.4%. The combination of streamlined T-adapter and cannula improved total delivery fraction through the non-invasive circuit by a factor of approximately 2x.

Acknowledgements: NIH

1 The University of Oklahoma Health Sciences Center, OKLAHOMA CITY, OK, United States of America 2 RTI International, RESEARCH TRIANGLE PARK, NC, United States of America O-51 PHARMACOKINETICS OF CPZEN-45, A NOVEL ANTI-TUBERCULOSIS DRUG IN MALE GUINEA PIGS

CPZEN-45 is a derivative of caprazamyin which has been shown, in vitro, to kill mycobacteria. The purpose of the present study was to determine the disposition of CPZEN-45 after administration of solutions by the IV and SC routes and aerosol powder by the pulmonary route. The day before the study, animals underwent cannulation of the jugular vein for blood sampling. Blood samples were collected at predetermined time points. Bronchoalveolar lavage (BAL) was performed after the last blood sample. Lung and spleen tissues were resected and frozen until analysis. CPZEN-45 concentrations in blood, BAL and tissues were determined by HPLC. Plasma concentration versus time curves after IV and pulmonary administration were statistically similar, suggesting that the drug is rapidly and efficiently absorbed after pulmonary administration, which reflected in the time after maximum plasma concentration (Tmax). The route of administration also influenced favorably the CPZEN concentrations in the airways (BAL concentrations) and in the lung tissue at the end of the blood sampling period. Non-compartmental analysis of plasma concentration versus time also revealed a favorable influence of the route of administration on key pharmacokinetic parameters for CPZEN-45. The potential of the pulmonary route of administration to administer CPZEN-45 is supported by drug concentrations in the lungs, the primary site of tuberculosis infection, and by the pharmacokinetic parameters.

Key Words: Tuberculosis, Pharmacokinetics, CPZEN-45, pulmonary route, guinea pig model

1 Institute of Anatomy, University of Bern, BERN, Switzerland 2 Institute of Aerosol and Sensor Technology, University of Applied Sciences Northwestern Switzerland, WINDISCH, Switzerland 3 Department of Chemistry, University of Cambridge, CAMBRIDGE, United Kingdom O-52 EFFECTS OF INHALED NANOPARTICLES ON THE SUSCEPTIBLE POPULATION – IN VITRO EVOLUTION

Inhalation of engineered nanoparticles (ENPs) in industrial processes, in consumer products, but also in medicine poses a largely unknown risk. Thereby, persons with pre-existing lung disease are more vulnerable.

Based on previous work a chamber for efficient deposition of (nano) particles out of a continuous air-stream on cell cultures has been developed. It allows simultaneous particle deposition on 24 individual cell cultures by electrostatic deposition under controlled conditions (85–95% RH and 37°C). The new chamber has been characterized in terms of deposition efficiency, spatial distribution of particles and compatibility for cell exposures. In first experiments, re-differentiated human bronchial epithelia (HBE) with established air-liquid interface and the bronchial cell line BEAS-2B were exposed to Ag- and C-nanoparticles.

Particles were evenly distributed on inserts except for the edge, where fewer particles were deposited. Particle deposition efficiency was in the range of 15%. The exposure treatment itself was not cytotoxic, as lactate dehydrogenase release in BEAS-2B cells exposed to inert particles was not different from unexposed controls. Preliminary results from Ag- and C-nanoparticles exposures indicate a greater sensitivity of HBE from a donor with cystic fibrosis compared to healthy HBE and BEAS-2B cells.

Thus, the Nano Aerosol Chamber for In-Vitro Toxicology (NACIVT, www.nacivt.ch) combined with advanced cell models, i.e. HBE from healthy and diseased donors provides a highly realistic in vitro system for safety testing of ENPs. Moreover, the combined system allows studies with novel therapeutic aerosols with particle diameters up to micrometer size.

Acknowledgements: Funding by the Swiss National Science Foundation

1 Medical Gases Group, Air Liquide Santé International, LES LOGES-EN-JOSAS, France 2 Delaware Research and Technology Center, American Air Liquide, NEWARK, DE, United States of America 3 Lafayette College, EASTON, PA, United States of America 4 EA6305, CEPR, INSERM 1100, Faculty of Medicine, University of Tours, TOURS, France 5 Department of Anesthesia (Bioengineering), MGH/Harvard, BOSTON, MA, United States of America P-002 AEROSOL DELIVERY FROM A VIBRATING MESH NEBULIZER WITH HOLDING CHAMBER IN HELIUM/OXYGEN VERSUS AIR

Helium/oxygen mixtures have been used to improve aerosol delivery by increasing deposition in peripheral airways. In addition, the influence of the aerosol size distribution on deposition pattern is well-known. Therefore, in assessing aerosol delivery systems with helium-oxygen, knowledge of gas property effects on aerosol generation is important. These effects on aerosol generation must be accounted for when gauging changes in deposition pattern arising from altered fluid and particle transport. In the present study, an aerosol delivery system consisting of a vibrating mesh nebulizer and holding chamber was assessed prior to use in an aerosol deposition study with helium/oxygen. Laser diffraction experiments were conducted to investigate the influence of breathing pattern and carrier gas (air versus helium/oxygen 78/22%) on size distributions of saline droplets delivered from Aeroneb Solo nebulizers used with an Idehaler holding chamber. Differences in the volume median diameter (VMD) and geometric standard deviation (GSD) between various breathing patterns studied were small compared to differences between individual nebulizers. Further, average VMDs and GSDs were not significantly different between helium/oxygen and medical air. Accordingly, the aerosol delivery system can be used to directly study the effects of helium/oxygen on regional deposition patterns. In particular, the potential for helium/oxygen to increase delivery of therapeutic aerosols to obstructed airways through increased ventilation of obstructed lung regions holds promise for the treatment of severe respiratory disease.

Acknowledgements: Funded by Air Liquide

1 The University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States of America 2 RTI International, Research Triangle Park, NC, United States of America P-003 DEVELOPMENT OF AN HPLC METHOD TO DETERMINE CPZEN-45 IN BIOLOGICAL MATRICES

CPZEN-45 is a new drug entity considered for the treatment of multi-drug resistant (MDR) tuberculosis. A reverse-phase high-performance liquid chromatographic (HPLC) method was developed and validated to determine CPZEN-45 concentrations in biological matrices. CPZEN-45 was extracted from guinea pig's plasma, bronchoalveolar lavage (BAL), lung and spleen tissues by sequential extraction with acetonitrile and quantified by a Waters HPLC coupled with a ZORBAX Bonus-RP (4.6×75 mm), guard column and UV detection at 263 nm. The mobile phase was 20:80 acetonitrile: water with 0.05% TFA. The CPZEN-45 peak was eluted at 5.1 min with no interference with the peaks of impurities from plasma, BAL, lung or spleen tissues. Recovery of CPZEN-45 from guinea pig plasma, BAL, lung and spleen was >95%. The limit of quantification was 0.29 μg/ml which was more than 5 and 21 times lower than the reported minimal inhibitory concentration (MIC), 1.56 μg/ml for Mycobacterium tuberculosis H37Rv and 6.25 μg/ml for MDR-TB, respectively. Limit of detection was 0.05 μg/ml. The HPLC method appeared to be sensitive, reproducible and accurate for quantification of CPZEN-45 in guinea pig's plasma, BAL, lung and spleen tissues to be used in subsequent drug disposition studies in the animal model.

Acknowledgement: Grant funding by the NIH (1R01AI091882-01)

1 National Taiwan University, TAIPEI, Taiwan 2 National Taiwan University Hospital, TAIPEI, Taiwan P-004 CHARACTERIZATION OF VIBRATING MESH AEROSOL GENERATORS

Vibrating mesh nebulizers have been reported to have increased output efficiency, minimal residual volume, and high percentage of particles in the emitted respirable and fine particle fraction. This work aimed to investigate and identify the major operating parameters of vibrating mesh nebulizers and their effects on the characteristics of aerosol output. The vibrating mesh plates were customarily made to contain 279∼4606 tapered holes. The aperture size was uniform on each plate and varied from 3 to 12 μm. The aperture distance also varied from 75 to 450 μm, to examine the potential of droplet coagulation. The aperture plates vibrated at a fixed frequency (100∼300 kHz), which caused the ejection of liquid droplets. These nebulizers were mainly evaluated with 0.9% sodium chloride solution. A syringe pump was employed to carry the solution to the vibrating mesh plate. An aerosol size spectrometer (Welas 3000) was employed to measure the aerosol number concentration and size distribution. The droplet size was found to increase with increasing aperture diameter. The distance between apertures and the frequency applied to the mesh plate did not significantly affect the aerosol concentration and size distribution. For each vibrating mesh of different aperture size and aperture number, there is an optimal vibrating frequency to stably deliver maximum amount of aerosol output. This maximum feeding rate increased with increasing aperture number and applied electric current, but the aerosol size distribution remained the same. Vibrating mesh aerosol generators can be orientation independent, if equipped with capillary transport device, such as fibrous absorbent materials.

Acknowledgements: Financial support by National Science Council, Taiwan.

1 Department of Medicine, University of California, San Diego, La Jolla, CA 2 University of North Carolina at Chapel Hill, Chapel Hill, NC 3 Department of Radiology, University of California, San Diego, La Jolla, CA P-007 EFFECT OF GRAVITY ON THE REGIONAL DISTRIBUTION OF PARTICLES DEPOSITED IN THE HUMAN LUNG

The deposition of >0.5 μm particles in the lung is strongly influenced by gravitational sedimentation, with deposition being reduced in low gravity compared to normal gravity (1G). Reduced sedimentation also affects the distribution of inhaled aerosol and as a consequence deposition patterns. Using gamma scintigraphy, we measured regional deposition and retention of radiolabeled particles (^99mTc-labeled sulfur colloid, MMAD=5.1μm, GSD=1.6) in five healthy volunteers. Particles were inhaled in a controlled fashion (0.5 L/s, 15 breaths/min) during multiple periods of microgravity (μG) aboard the NASA Microgravity Research Aircraft and in 1G on a separate occasion. In both cases, deposition scans were obtained immediately post-inhalation and at 1h30, 4h and 22h post-inhalation. The distribution of deposited particles in the large (DE_aw,large), intermediate (DE_aw,interm), and small airways (DE_aw,small) as well as in the alveolar region (DE_alv) was derived from these data and is listed in the table as mean±SD (*=p<0.001 when compared to 1G data). The data show a profound shift in the distribution of deposited particles away from the lung periphery towards the large airways when particles are inhaled in μG. This is the direct result of the absence of gravitational sedimentation that has the greatest effect in the distal lung where airway size is small and residence time high, with a consequent increase in the deposition fraction in the central airways where particles deposited mainly by inertial impaction.

Acknowledgements: This study was funded by the National Space Biomedical Research Institute through NASA NCC 9–58.

1 National Taiwan University, TAIPEI, Taiwan 2 National Taiwan University Hospital, TAIPEI, Taiwan 3 Institute of Occupational Safety and Health, NEW TAIPEI CITY, Taiwan 4 Chung Hwa University of Medical Technology, TAINAN, Taiwan P-008 AEROSOL GENERATION AND DEPOSITION IN THE HUMAN LUNG

Human lung is not only an aerosol collector but also an aerosol generator. Previous lung deposition studies have shown that aerosol deposition efficiency were strongly dependent on particle size, breathing pattern, aerosol charge distribution and lung morphometric parameters. On the other hand, the generation rate of exhaled breath aerosols (EBA) was mainly controlled by the tidal volume. The main objective of this work was to study the relationship between the exhaled breath aerosols and the lung deposition efficiency. The experimental system consisted of a test chamber, a mouthpiece, a pneumotachograph flow meter, and a particle counter. Both tests shared the same experimental apparatus except the aerosol generating system. For regional lung deposition measurement, a stable aerosol output was essential, while aerosol-free air was supplied to the chamber when conducting the EBA measurements. The volunteers were asked to follow sinusoidal breathing patterns which were generated by using a piston-cylinder breathing simulator. The subjects were instructed to perform percentage of forced vital capacity (20, 40, 60%FVC) and fixed tidal volume (500, 750 and 100 mL) with different breathing frequency (10, 12, 14, 15 breath/min). The results showed that the EBA counts increased with increasing tidal volume, but nearly not affected by the breathing frequency. The regional deposition data showed that local deposition efficiency increased with penetration volume. The correlation analysis showed that total lung deposition efficiency increased with increasing EBA counts, a somewhat confusing phenomenon, indicating that a subject who collects aerosols more efficiently, generates more aerosol particles through tidal breathing.

Acknowledgements: Financial support by the Institute of Occupational Safety and Health, Taiwan.

1 Université Catholique de Louvain, Louvain Drug Research Institute, BRUSSELS, Belgium 2 Ludwig Institute for Cancer Research, BRUSSELS, Belgium 3 UCB Pharma, SLOUGH, United Kingdom P-010 PEGYLATION OF ANTI IL-17A ANTIBODY CONSTRUCTS FOR THE TREATMENT OF BRONCHIAL HYPERREACTIVITY

The PEGylation of the F(ab’)₂ anti IL-17A antibody fragment and the biological characterization of the generated conjugate are presented. This modification aims to generate an optimized candidate therapeutic for the treatment of bronchial hyperreactivity (BHR). The murine anti IL-17A antibody was initially digested by pepsin to produce the F(ab’)₂ fragment. Then, N-hydroxysuccinimide (NHS) activated ester-PEG of 40 kDa was added to react with the fragment available primary amines. Factors affecting the degree and the yield of PEGylation, including pH, incubation time, PEG:antibody molar ratio and protein concentration were tested. The extent of modification was evaluated by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Cation Exchange Chromatography (CEC) was used for removal of PEG excess and purification of PEGylated species. The efficacy of the generated anti IL-17A constructs in binding to and inhibiting the biological activity of IL-17A was assessed and compared to the one of the full-length antibody. Optimal conditions were observed when anti IL-17A F(ab')₂ (4mg/ml) at pH 8.2 was allowed to react with branched PEG40-NHS activated ester at a PEG:antibody molar ratio of 6:1 at room temperature, under agitation for 30 min. Upon purification, 34% unreacted, 42% mono-PEGylated and 24% multi-PEGylated F(ab’)₂ fragment were obtained. The mono-PEGylated fragment appeared to have a slight decrease in its binding activity (4-fold) and its in vitro inhibitory potency (2-fold) rendering it a good candidate for in vivo studies. Further investigation is needed for the determination of the pharmacokinetic profile and in vivo therapeutic efficacy in BHR models.

Acknowledgements: Grant funding by the Walloon Region, Belgium

1 Applied Research Associates, Inc., Raleigh, NC, United States of America 2 British American Tobacco, Southampton, United Kingdom P-011 COMPONENT-SPECIFIC MODELING OF CIGARETTE SMOKE PARTICLE DEPOSITION IN THE RESPIRATORY TRACT

Assessment of the dose and site of deposition of inhaled cigarette particles and associated carcinogenic components in the lung aids in the interpretation of biological response and adverse effects. Available deposition models developed for environmental aerosols are not capable of reliable predictions of smoke particle deposition in the lung mainly to due to the differences in the respiratory maneuver and their physico-chemical and aerodynamic properties. A mechanistic model for cigarette smoke particle deposition was developed that included cigarette-specific colligative and non-colligative effects on deposition. Using Maxwell's equation for diffusion, particle size change by hygroscopicity and phase change of semi-volatile components were calculated. In addition, the particle size change model included particle coagulation which depended on particle concentration and airway deposition. The cigarette smoke particles were found to grow rapidly by absorbing water vapor and the diameter increased by 50–100%. The non-protonated component of nicotine in the cigarette smoke was found to rapidly undergo phase change from the particle phase to the gas phase. A multi-component, mechanistic model of inhaled cigarette smoke particle transport was developed which accounted for particle size change and hydrodynamic interactions of the particles. Particle deposition was higher in the tracheobronchial region than in the alveolar region. Model predictions agreed with available measurements in the literature for both the oral cavity and lung. The proposed model is a powerful tool for studying the fate of individual smoke constituents.

Acknowledgement: This study was funded by British American Tobacco

1 Novartis Pharmaceutical Corp., San Carlos, CA, US 2 Department of Pharmacy, the University of Sydney, Sydney, Australia P-012 IN-VITRO ASSESSMENT OF DOSE DELIVERY PERFORMANCE OF ENGINEERED DRY POWDERS FOR INHALATION

The dose delivery performance of inhalable porous particles as a function of powder properties was assessed using the “Alberta” throat (DeHaan and Finlay et al., 2001). Engineered placebo powders were prepared using the PulmoSphere™ technology, which is based on spray-drying an emulsion feedstock to produce porous particles with well-controlled size and density. These placebo powders are useful surrogates for a class of potent active formulations. As part of study design, particles with range of particle size and density were manufactured, representing a particle design space relevant to dry powder inhalers. The Alberta throat provides an in-vitro estimate of ”lung dose”, and was used to study the effect of flow rate, particle size and density on dose delivery performance using the Simoon blister-based inhaler. Commercially available inhalers with lactose-blend formulations were also evaluated to provide a comparative benchmark. The in-vitro lung doses measured with the Alberta throat were compared to predictions from semi-empirical numerical models. Data from the Alberta throat and numerical models were used to rank order dose delivery performance over a pressure drop range of 1–6 kPa. Powder fluidization and aerosol emission for the PulmoSphere powders was favored by low particle density and large geometric diameter, while improved lung dose and flow rate dependence were favored by powders with median particle diameter Ⅵ2.5 microns. In comparison, traditional lactose blend formulations showed significantly lower lung dose and increased airflow dependence. The rank-ordered in-vitro results for the different formulation types are consistent with published in-vivo results from clinical trials with similar formulations (Duddu et al., 2002).

References

1. DeHaan, W. H., Finlay, W. H. In Vitro Monodisperse Aerosol Deposition in a Mouth and Throat with Six Different Inhalation Devices. Journal of Aerosol Medicine. 14: 361–367 (2001).

2. Duddu, S. P., Sisk, S. A., Walter, Y. H., Tarara, T. E., Trimble, K. R., Clark, A. R., Eldon, M. A., Elton, R. C., Pickford, M., Hirst, P. H., Newman, S. P., Weers, J. G. Improved Lung Delivery from a Passive Dry Powder Inhaler Using an Engineered PulmoSphere^® Powder. Pharmaceutical Research. 19: 689–695 (2002).

1 Dept of Physics, Faculty of Science, UAE University, Al-Ain, P.O. Box: 17551, United Arab Emirates 2 Department of Electrical Engineering, University of Southampton, Southampton SO17 1BJ, United Kingdom P-014 ELECTROSTATIC FORCES AND DEPOSITION MECHANISMS OF CHARGED AEROSOL PARTICLES IN LUNG AIRWAYS: “IMAGE” AND SPACE-CHARGE EFFECTS

Electrical charge as inhaled particles may increase particle retention in the lung. All therapeutic aerosols produced by devices such as nebulizers, metered-dose inhalers and dry powder dispersers consist of charged particles. Charged particle deposition in the lungs, during the administration of therapeutic aerosols, can be controlled by adopting various strategies. These include prescribed breathing patterns, pulsing of aerosol during inhalation and by control of particle size and charge. For inhalation of charged aerosols of low particle number concentration deposition force, for particles close to the airway wall, is dominated by the electrostatic “image” force. If particle number concentration is sufficiently high, space-charge depositional forces become dominant. Space charge is often the important deposition mechanism in the early airway generations whereas the “image” mechanism tends to become more important in the smaller airways. In this work we look critically at the space-charge and image charge expressions that have been used to predict charged particle deposition in the lungs and we derive new expressions accounting for airflow.

1 DTF-Aerodrug, Faculté de Médecine, F-37032 TOURS, France 2 CEA Le Ripault, F-37260 MONTS, France 3 ANSYS France, F-78180 MONTIGNY LE BRETONNEUX, France 4 Dantec Dynamics, F-91620 NOZAY, France 5 Centre d'Etude des Pathologies Respiratoires INSERM U1100/EA6305, Université François Rabelais de Tours, Faculté de Médecine, F-37032 TOURS, France P-015 COMPARISON OF NUMERICAL SIMULATION TO EXPERIMENTS FOR A JET NEBULIZER

The purpose of this study was to design a numerical model simulating the nebulization process inside a jet nebulizer and to compare it with experimental data obtained from various measurement methods. Such a model allows a better understanding of the atomization process and a determination of the relevant physical parameters influencing the nebulizer output. A model based on the Updraft nebulizer (Hudson) was designed with ANSYS Workbench and meshed in 4 μm cells. Geometry and boundary conditions were set in ANSYS Fluent with experimental data and the relevant model parameters were determined through an axisymmetric 2D simulation. 3D calculations were made at two air flow rates (2L/min and 8L/min) to account for different turbulence regimes. Experimental methods such as laser diffractometry, phase Doppler anemometry and CCD camera were used to characterize the spray output. Image acquisitions made with CCD camera showed similar liquid patterns as numerical results, such as film formations or droplet spreading. VMD predicted by numerical results are close to distributions obtained from image processing (26 μm vs. 21 μm at 2L/min; 17 μm vs. 14 μm at 8L/min). However, size is overestimated in relation to PDA and laser diffractometry, due to different sizing method and size range. Numerical simulation then provides a good modeling of the phenomena causing droplet atomization and could help understanding nebulizer output with defined parameters.

1 Inamed GmbH, Gauting, Germany 2 Novartis Pharma AG, Basel, Switzerland P-016 IN VITRO DOSE DELIVERY PERFORMANCE OF GLYCOPYRRONIUM USING REPRESENTATIVE INSPIRATORY FLOW PROFILES DERIVED FROM COPD PATIENTS

The aim of this study was to evaluate the dose delivery characteristics from the low resistance Breezhaler^® dry powder inhaler used with the once-daily LAMA glycopyrronium (Seebri^®) in an in-vitro study. The dose delivery characteristics of 50 μg drug in inhalation capsules were measured on different simulated COPD patient breathing patterns.

Seven representative patient inhalation flow profiles were selected from 26 COPD patients to cover the range of patients' parameters and inspiratory flow profile variables caused by disease severities, from moderate to severe. Based on the results of the aerodynamic particle size distribution analysis, the theoretical respiratory tract deposition for each of the simulated patient breathing patterns was estimated with the ICRP 66 deposition model.

The delivered dose ranged from 38 μg to 43 μg per capsule as collected by the Next Generation Impactor. Fine particle mass (<5μm) ranged between 19 μg to 25 μg. This is equivalent to 38% to 51% of the nominal dose of 50 μg. The MMAD ranged from 2.7 μm to 2.9 μm and the GSD from 1.9 to 2.0.

In summary, the Breezhaler^® inhalation device delivers a consistent dose and high fine particle fraction of glycopyrronium across a broad range of simulated inspiratory flow profiles representative of COPD patients of different disease severities. The results of the theoretical respiratory tract deposition calculation provided a good indication that a pretty consistent glycopyrronium dose at the intrathoracic target site is achieved following simulated COPD patient inhalation profiles including the lower measured flow rates and inspiratory volumes.

1 Center for Environmental Medicine, Asthma and Lung Biology 2 Curriculum in Toxicology 3 Department of Pediatrics, University of North Carolina at Chapel Hill P-018 LOWER NATURAL KILLER CELL CYTOTOXICITY IN SMOKERS MAY BE MEDIATED BY EPITHELIAL CELLS

Cigarette smoking is a risk factor for viral infections, yet the mechanisms underlying this susceptibility remain unknown. Natural killer (NK) cells play an important role in fighting influenza and signals from epithelial cells (EC) likely regulate NK cell activity. We have previously shown that markers of cytotoxicity are reduced in nasal NK cells from smokers after influenza infection. This study investigated if human EC from smokers and nonsmokers differentially affect NK cell activity in the context of influenza infection.

EC were infected with influenza and subsequently co-cultured with NK cells. NK cells were also incubated with apical washes and basolateral supernatants of EC to identify the role of soluble mediators.

At baseline, co-culturing NK cells either directly with smoker EC or stimulating with basolateral supernatants from these cells reduced markers of cytotoxicity. Similarly, after influenza infection markers of cytotoxicity as well as levels of IFN-γ, IL-4 and granzyme B were decreased in NK cells incubated with basolateral supernatants of smoker EC.

Co-culturing NK cells with EC from smokers reduced markers of cytotoxicity in NK cells through a potential mechanism involving EC-derived soluble mediators. Thus, EC-mediated modification of NK cell activity may contribute to the higher susceptibility to influenza infections seen in smokers.

Acknowledgments: The work was supported by Swiss National Science Foundation, NIH, EPA and FAMRI.

1 University catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery Group, BRUSSELS, Belgium 2 WIV-ISP-Site Ukkel, O.D. Communicable and Infectious diseases, Immunology, BRUSSELS, Belgium P-020 IMMUNIZATION AND CHALLENGE STUDIES OF A BCG BOOST COMPOSED OF AG85A AND CpG AND DELIVERED TO THE LUNGS

The current Bacille Calmette and Guérin (BCG) vaccine fails to protect adults against pulmonary tuberculosis. Boosting BCG with subunit vaccines could increase protection against tuberculosis. Pulmonary vaccination is a promising route to deliver vaccines as it acts at the site of infection. In the present study, BALB/c mice were vaccinated with BCG by the subcutaneous route. Three months later, mice were boosted by intratracheal instillation in the deep lungs of the subunit vaccine composed of recombinant antigen 85A (Ag85A) of Mycobacterium tuberculosis (M.tb) alone or combined with the mucosal adjuvant CpG. Mice were then divided in two groups, one for the immunization study and the other one for the challenge study. In the immunization study, sera, spleens and broncho-alveolar lavages were taken three weeks after boost. In the challenge study, mice were challenged intratracheally with a virulent strain of M. tb six weeks after boost. Mycobacterial load was quantified four and twelve weeks after challenge, both in lungs and spleens. BCG boosted with Ag85A with or without CpG stimulated specific humoral as well as cellular immune responses by increasing IgG titres in sera and splenocyte proliferation in vitro, respectively. Moreover, the boost of Ag85A and CpG showed a Th-1 phenotype by inducing high in vitro production of IFN-γ and IL-2 by splenocytes. However, mycobacterial load did not decrease due to the boost, either in spleens or in lungs. The pulmonary delivery of Ag85A with CpG provides good immune responses but needs further optimization to achieve good protection.

Acknowledgements: Grant funding by the Fonds de la Recherche Scientifique Médicale

1 CNRS, IRPHE UMR7342, 13384, Marseille, France 2 Aix-Marseille Univ, IRPHE UMR7342, 13384, Marseille, France 3 Centrale Marseille, IRPHE UMR7342, 13451, Marseille, France P-021 LOCAL AEROSOL DEPOSITION AND GAS-FLOW IN VITRO MEASUREMENTS USING PIV-LIF TECHNIQUES

During the last decades, numerous in vitro experiments have been proposed to mimic airflow dynamics and deposition patterns of inhaled aerosols in models of lower airways. These experimental developments are needed to assess and validate the computational works employed to quantify two-phase flows features in complex realistic geometries with high spatio-temporal resolutions. So far, most in vitro studies have focused on airflow dynamics characterization. Previous aerosol deposition experiments have mainly provided total or regional deposition efficiency in human or animal models. Yet, the individual particle trajectories and local deposition patterns have been barely investigated, and tracking particle dynamics together with local flow structures still represents a challenging task.

The objective of the present work is to characterize unsteady local deposition patterns of aerosols within in vitro simple geometries, as a function of airflow properties. The instantaneous flow velocity fields are obtained using 2D Digital Particle Image Velocimetry (DPIV). The measurement of aerosol concentration fields on the wall is achieved using Rhodamine water solution to provide tracer particles and the Planar Laser Induced Fluorescence (PLIF) technique. Two experimental phases will be presented. The first phase will be dedicated to the case of a round turbulent two-phase jet impinging on a plate under steady flow conditions. This standard case will allow the setting and optimization of the experimental set-up. The second phase will study the case of a two-phase flow generated in a bend geometry using a controlled mechanical ventilation. The deposition patterns obtained for different breathing conditions will be discussed.

1 DTF-Aerodrug, Faculté de médecine, F-37032 Tours, France. 2 EA6305, CEPR, Faculté de médecine, Université François Rabelais F-37032 Tours, France. 3 DTF-medical, F-42003 Saint Etienne, France. 4 Ecole Nationale Supérieure des Mines, Centre ingénierie et santé, F-42003 Saint Etienne, France. 5 Université Jean Monnet, LINA EA4624, IFR143, F-42003 Saint Etienne, France. 6 Service de Pneumologie, CHRU de Tours, F-37044 Tours, France. 7 Service de pneumologie, Cliniques Universitaires Saint-Luc, UCL, 1200 Brussels, Belgium. 8 Service d'ORL et de chirurgie cervico-faciale, Centre hospitalier Emile-Roux, F-43012 Le Puy-en-Velay, France. 9 Service de Médecine Nucléaire, Cliniques Universitaires Saint-Luc, UCL, 1200 Brussels, Belgium. P-022 IN VITRO COMPARISON OF AEROSOL DEPOSITION IN NASAL CAVITIES

Intranasal aerosols deposition inhaled through the nose is often studied using in vitro nasal cavities models. But these models have not been validated as relevant models to predict in vivo deposition. This study compares in vivo nasal aerosol deposition to in vitro nasal aerosol deposition with two nasal cast models: A plastinated head model (NC1) and its replica in plastic (NC2). Two types of nebulizers were used with ^99mTc-DTPA in 7 healthy volunteers, NC1 and NC2. An in vitro experimental setup was used to reproduce in vivo aerosol inhalation with NC1 and NC2. Aerosol deposition was quantified by gamma camera in nasal, ethmoid and maxillary sinuses (MS) regions and was expressed in term of nebulizer charge. The distribution of aerosol deposited was determined along the 3 axes of nasal cavities (x, y and z).

There was no statistical difference in terms of nasal, ethmoid and maxillary sinuses (MS) aerosol deposition between in vivo and NC1 (p<0.05) but there is a difference between in vivo and NC2 (p>0.05). The mean of all NC1 aerosol distributions was included in the standard deviation (SD) of in vivo aerosols distributions while the mean of NC2 aerosol distributions was excluded from the in vivo SD, exclusively for x-axis.

Depending on its quality, a nasal cast model can be a relevant model to predict aerosol produced by nebulizers.

1 Imperial College London and Royal Brompton Hospital, LONDON, UK 2 University Hospital UZ Brussel, BRUSSELS, Belgium P-024 THE MULTIPLE BREATH WASHOUT TEST AS A TOOL FOR ASSESSING PERIPHERALLY TARGETED THERAPEUTIC DRUGS: ESTABLISHING A NORMAL RANGE IN HEALTHY VOLUNTEERS

Background: Considerable effort has gone into developing new aerosol formulations and delivery devices to target drugs to the lung periphery. It is essential that this region be adequately assessed in order to determine the efficacy of these treatments but conventional spirometry tests are unable to assess changes in response to treatment targeted to this region. The multiple breath washout (MBW) test has been advocated for small airways detection in obstructive lung disease and uses the principle of washing resident N₂ from the lungs by consecutive breaths of oxygen to derive S_cond and S_acin. S_cond and S_acin are markers of ventilation heterogeneity in the conducting and acinar lung regions respectively and have previously been shown to be increased in diseased lungs. To establish the method more widely, values in healthy subjects are required for comparison.

Methods: MBW tests were performed on 60 never-smoker healthy subjects (age range 25 to 65 years). S_cond and S_acin and lung clearance index (LCI) were measured.

Conclusion: MBW parameters were measured in healthy subjects over a wide age range. A small age related increase in parameters was noted. This is an important consideration in the light of an aging population. This data will be pooled with data from other centres worldwide to produce a database of normal values.

1 Cardiothoracic Anesthesia and Intensive Care, Lund University Hospital, LUND, Sweden 2 Pediatric Anesthesia and Intensive Care, Lund University Hospital, LUND, Sweden 3 PARI Pharma GmbH, Moosstrasse 3, STARNBERG, Germany P-025 COMPARISON OF IN-VIVO AND IN-VITRO LUNG DEPOSITION OF AEROSOLIZED SURFACTANT USING A NEONATAL NEBULIZER BASED ON EFLOW TECHNOLOGY

Surfactant has a proven effect against RDS when instilled endotracheally. It would be advantageous if it could instead be inhaled as an aerosol, avoiding the need for tracheal intubation. PARI Pharma has developed a neonatal nebuliser system incorporating eFlow^® technology for this specific use and it has been tested with the pulmonary surfactant marketed as Curosurf® (Chiesi Parma, Italy). We assessed the lung deposition of surfactant in-vivo and in-vitro.

For the in-vivo assessment, one-day-old piglets inhaled nebulised surfactant mixed with technetium-99-labelled nanocolloid. A gamma camera measured the lung deposition. For the in-vitro assessment, we employed the PrINT cast model of a preterm baby at conditions close to reality, using an incubator, humidified air, a bubble CPAP system and a breathing pattern, typical of a preterm baby.

Both in-vivo and in-vitro, nasal prongs, a mask or a tracheal tube served as nebuliser-patient interface.

Lung deposition of nebulised Curosurf® is shown in Table 1.

Lung deposition was comparable in-vivo and in-vitro and the in-vitro set-up should therefore be useful in experiments aiming at optimization of the lung deposition. The deposition that we found might be sufficient for treatment of RDS. Efficacy studies in preterm babies are needed to prove this.

1 Medical Gases Group, Air Liquide Santé International, LES LOGES-EN-JOSAS, France 2 Lafayette College, EASTON, PA, United States of America 3 National Institute of Health Research Biomedical Research Unit in Respiratory Disease, SOUTHAMPTON, UK 4 Department of Medical Physics and Bioengineering, University Hospital Southampton NHS Foundation Trust, SOUTHAMPTON, UK 5 Faculty of Health Sciences, University of Southampton, SOUTHAMPTON, UK P-027 COMPARISON OF 2D PLANAR GAMMA CAMERA AND 3D SPECT MEASUREMENTS WITH 1D SIMULATION RESULTS OF AEROSOL DEPOSITION IN HEALTHY HUMANS

An analytical, mechanistic model was used to mimic aerosol inhalation experiments on healthy adult male subjects, simulating particle deposition within the lungs for two aerosols (MMAD=5.3μm and 3.1μm) and two carrier gases (air and a 78/22% helium-oxygen mixture). Simulations were performed using asymmetric lung morphologies based on subject's morphometric data extracted from High Resolution Computed Tomography images (e.g., length and diameter of the first airway generations) and lobar volumes, completed for deeper generations by symmetric subtrees. The gas ventilation distribution is calculated using a model that includes resistance, inertance, and compliance of the airways. Comparisons with experimental measurements have been done for tracheo-bronchial and pulmonary deposition, based on 2D gamma camera acquisitions just after inhalation and after 24h. 3D SPECT data have also been analysed to estimate the fraction of aerosol deposition in each of the five lobes. The results show a good correlation between experiments and simulations for tracheo-bronchial deposition, e.g., for the large aerosol the mean fraction as percentage of inhaled is 16.8±2.1 (experiment) vs. 15.4±1.6 (simulation) with air (n=7) and 15.4±1.6 vs. 12.4±3.7 with helium-oxygen (n=2) as the carrier gas. However the pulmonary deposition is systematically underestimated by our simulations suggesting that the ventilation distribution model needs improvement. Nevertheless, the high complexity of the human lung morphology is well described in terms of both inter-subject and intra-subject variability (e.g., distinction between the right and left lungs), as shown in the figure. This preliminary work suggests modelling deposition in heterogeneous lungs is possible.

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1 University of Iowa, College of Pharmacy, IOWA CITY, IA, United States of America 2 University of Iowa, College of Engineering, IOWA CITY, IA, United States of America P-028 DISPERSION COMPOUNDS ENHANCE ANTIBIOTIC EFFECTIVENESS AGAINST PSEUDOMONAS AERUGINOSA BIOFILMS

Pseudomonas aeruginosa infections are the leading cause of mortality among people with cystic fibrosis. The formation of bacterial microenvironments called biofilms provides protection, enabling the bacteria to resist the host's immune system and treatment by antibiotics, and leads to chronic infection. Thus, current treatments are ineffective in completely eradicating the infection. The goal of this study was to assess the effectiveness of a combination therapy, containing a nutrient dispersion compound and an antibiotic, on the eradication of P. aeruginosa biofilms in vitro. We hypothesized that nutrient dispersion compounds would entice bacteria to exit the biofilm as planktonic-like bacteria and enhance the eradication of the bacteria with traditional antibiotics. Young mucoid P. aeruginosa biofilms were grown in the MBEC peg assay (Innovotech) for 24 hours. Mature biofilms were grown in Lab-Tek chambered coverglass for 4 days. Then bacterial biofilms were treated for 24 hours with combinations of antibiotic (ciprofloxacin, amikacin, tobramycin, polymyxin B, colistin, colistin methanesulfonate and erythromycin) and dispersion compound (sodium citrate, succinic acid, xylitol, and glutamic acid). Young biofilms were quantified for incidence of biofilm growth by optical density and mature biofilms were quantified for the percent live biofilm bacteria in confocal microscopy images. Young biofilm growth was reduced with 15 out of 24 combinations, while the mature biofilms were susceptible to 4 out of 26 combinations. Our studies indicated that the biofilms comprised mainly bacteria with low metabolic activity and that access to these bacteria was enhanced by the disruption of the biofilm matrix upon bacterial dispersion.

Acknowledgements: Grant funding by the PhRMA Foundation.

1 Aerosol Research Laboratory, University of Alberta, EDMONTON, Canada 2 Medical Gases Group, Air Liquide Santé International, LES LOGES-EN-JOSAS, France 3 Lafayette College, EASTON, PA, United States of America P-029 SIMULATION OF HYGROSCOPIC AEROSOL DEPOSITION IN THE RESPIRATORY TRACT: HELIUM-OXYGEN VS. AIR AS THE CARRIER GAS

The size and temperature of the droplets of hygroscopic aerosols, as well as the thermodynamic state of the carrier gas, change due to transport of water vapor and heat between dispersed droplets, carrier gas, and airway walls. A coupled numerical model is developed to examine the effect of using helium-oxygen instead of air on hygroscopic size changes and deposition of inhaled aqueous solution aerosols. Transport properties of helium-oxygen are remarkably higher, e.g., coefficient of water vapor diffusion in helium-oxygen is 2.3 times more than air. This gives rise to a noteworthy increase in hygroscopic size changes. Polydisperse aerosols with initially isotonic droplets and lognormal size distributions are considered. In each lung generation, coefficients of heat and mass transfer are estimated based on realistic assumptions. Evaporation and condensation are simulated by solving the two-way coupled heat and mass transfer differential equations for both the continuous and dispersed phases. Once the droplet sizes are known, deposition is calculated based on previously published equations. The results include regional deposition data for a variety of size distributions for both helium-oxygen and air, as well as size and temperature variations in the respiratory tract of the dispersed droplets and carrier gas. The results reveal that helium-oxygen tends toward less deposition in the extrathoracic airways and more drug delivery to the lungs, which could have strong implications in aerosol inhalation therapies. Furthermore, from a hygroscopic point of view, these differences in deposition distribution are greater as the number concentration of droplets decreases.

Acknowledgements: Funded by Air Liquide

1 School of Paediatrics and Child Health, University of Western Australia, PERTH, W.A., Australia. 2 Philips Respironics, Respironics New Jersey, Inc., PARSIPPANY, NJ, USA. P-032 LUNG DEPOSITION OF ^99MTC-SALBUTAMOL FROM A PRESSURISED METERED DOSE INHALER AND A VALVED HOLDING CHAMBER, USED WITH FACEMASK OR MOUTHPIECE IN CHILDREN WITH STABLE ASTHMA-A PILOT STUDY

Early research in children has indicated that oral inhalation via mouthpiece was more efficient than the combination of oral and nasal inhalation which occurs when using a facemask. Changes in inhaler formulations as well as spacer and facemask design have highlighted the need for new comparative studies of VHC use, particularly focusing on the age at which children can be taught to transition from facemask to mouthpiece. Nine children aged 4–5 years (5 male) with stable asthma were recruited. A transmission scan of each patient was taken, using a 37MBq ^99mTc flood source. 2–3 actuations of a ^99mTc-labelled albuterol pMDI (ProAir, TEVA) were administered through an antistatic VHC (OptiChamber Diamond, Philips Respironics) either with a facemask (LiteTouch, Philips Respironics) or mouthpiece, while the patient's inhalation pattern was simultaneously recorded (Fig. 1). Anterior and posterior planar scintigraphic scans were taken immediately after administration. Mean (SD) lung deposition (% label dose) was 17.4 (9.4)% with the facemask and 21.3 (8.4)% with the mouthpiece (p>0.05). Peripheral lung deposition was consistently higher with the facemask than the mouthpiece (0.9 (0.7) vs 0.6 (0.2)), at levels approaching significance (p=0.07). Oropharyngeal/gastrointestinal (OG) deposition was significantly higher with the facemask; 24.3 (8.9)% vs 19.7 (3)% (p=0.05); this difference was more marked in children aged 4.5 years and above (27.2 (7.5)% vs 19.8 (3.4)% (p<0.02)). Lung deposition with the facemask appears higher than previously reported, due to a combination of factors including the inhaler formulation, and use of an antistatic spacer with a flexible, well-fitted facemask.

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Figure 1:

Breathing pattern recorded using a datalogger, during administration of radiolabelled albuterol, also showing shaking of pMDI and synchronisation of firing with inhalation

Acknowledgements: IRB approval for this study was obtained from the Princess Margaret Hospital Ethics Committee (1868/EP). This work was supported by a grant from the PMH Foundation (Perth, Western Australia). Partial sponsorship was provided by Philips Respironics (USA).

1 School of Pharmacy, Royal College of Surgeons in Ireland, Dublin, Ireland 2 Aerogen Ltd. Galway Business Park, Galway, Ireland 3 School of Pharmacy, Trinity College, Dublin, Ireland P-034 INVESTIGATION OF EXCIPIENT PROPERTIES ON OUTPUT RATES IN A VIBRATING MESH NEBULIZER

To date, there have been a number of studies investigating the effectiveness of vibrating mesh nebulizers in delivering test formulations, however there has not been any comprehensive analysis of vibrating mesh nebulizers' ability to successfully nebulize solutions across a wide range of different physicochemical properties. To address this, a range of approved pharmaceutical excipients, including preservatives, surfactants and viscosity enhancing agents, were tested for their effects on fluid physicochemical properties including surface tension and viscosity. Viscosity was measured using a Brookfield Viscometer with an ultra low adapter attached. Surface tension was examined using a LAUDA TD1C ring/plate tensiometer (Lauda GmbH, Germany). The output rate for each fluid was then determined in mg/min by nebulizing 1g of each sample using the Aeroneb Pro nebulizer (Aerogen Limited, Galway, Ireland).

From these results an insight into the impact of fluid properties on device performance could be derived and enable the considered selection of formulation parameters for future applications of the Aeroneb Pro. Output rates for each sample were plotted as a function of (density x surface tension)/viscosity versus output to normalize the data. Fluids with viscosities between 0.9667 – 2.73cP and surface tensions between 31.933 – 70.7mN/m were successfully nebulized. With the information gathered it is possible to more accurately predict how a formulation will perform with the Aeroneb Pro nebulizer and to tailor formulations to ensure optimal device performance.

Acknowledgement: This work was supported by Aerogen Ltd., Galway, Ireland

1 Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland 2 Centre for Research on Adaptive Nanostructures and Nanodevices, Trinity College Dublin, Dublin, Ireland 3 Department of Chemistry, Trinity College Dublin, Dublin, Ireland 4 School of Pharmacy, University College Cork, Cork, Ireland 5 Dublin City University, Dublin, Ireland 6 Tyndall National Institute, University College Cork, Cork, Ireland 7 Aerogen, IDA Business Park, Dangan, Galway, Ireland P-036 MAGNETIC CORE-SHELL NANOPARTICLES FOR DRUG DELIVERY BY NEBULIZATION

Aerosolized therapeutics has emerged as a promising alternative to systemic drug delivery for the treatment of various diseases including lung cancer. The main aim of our work is the development of a biocompatible nanocarrier system suitable for drug delivery by nebulization. Magnetite Fe₃O₄ nanoparticles (MNPs) were selected because of their excellent physicochemical properties and their successful use as a clinical reagent for diagnostics and drug delivery. To improve the dispersibility and biocompatibility, MNP core particles were modified by surface coating with biocompatible polymers poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol or dextran, and their physicochemical properties were characterized. Utilizing a series of standardized biological tests we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed in vitro when applied to the cultured human lung epithelial cells. Moreover, these MNPs were well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To examine the therapeutic efficacy of the developed MNP preparations, a poorly soluble flavonoid quercetin with reported anti-cancer and anti-inflammatory potential was loaded in the polymeric shell of PLGA-coated MNPs. These drug-loaded MNPs were applied to the human lung carcinoma cell line A549 following a single round of nebulization, imitating aerosol delivery to the lungs in the clinical setting. The quercetin-loaded MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting. The present study has implications for targeted delivery of nanocarrier-based drugs and poorly soluble medicinal compounds via the inhalation route.

1 Huntingdon Life Sciences Ltd., 100 Mettlers Road, East Millstone, New Jersey, US. 2 Janssen Research and Development, L.L.C., Raritan, New Jersey, US. 3 Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Huntingdon, Cambs UK. P-038 RESPIRATORY INDUCTANCE PLETHYSMOGRAPHY WITH CONCURRENT CARDIOVASCUALAR ASSESSMENT AS A COMPREHENSIVE APPROACH TO SAFETY ASSESSMENT IN INHALATION STUDIES

These data represent a series of studies designed to provide a comprehensive assessment of the utility of Respiratory Inductance Plethysmography (RIP) for the generation of nonclinical cardiorespiratory safety pharmacology data in dogs and non-human primates (NHP). RIP calibration methods, including the use of facemask in conscious and sedated animals as well as the use of intubated anesthetized animals for comparison are described. Accuracy of the calibrations was assessed by CO₂ rebreathe maneuvers. In addition, the ability of the RIP system to accurately assess subtle changes in breathing parameters was assessed by measurement of the effects of both positive (respiratory excitatory) and negative (respiratory inhibitory) challenges. The utility of the phase angle parameter for use as an index of airway resistance changes was also assessed via the response to a methacholine challenge. Finally, telemetry-derived cardiovascular parameters (ECG, blood pressure, and heart rate) and RIP-derived respiratory parameters (respiration rate, tidal volume, and minute volume) were determined simultaneously in a group of NHPs for 24 hr pretest, 2 hr predose and 24 hr postdose for a test article dose. Analysis of data following a single dose of the test article demonstrated test article-related changes in cardiovascular parameters for up to 23 hours after dose. These data show that the integrated assessment of cardiovascular and respiratory parameters is achievable continuously for at least 24 hr postdose. The use of RIP to assess the effects of a novel compound on the respiratory system complements, but does not interfere with, the cardiovascular assessment of new drugs.

1 Massachusetts Institute of Technology and Massachusetts General Hospital, BOSTON, MA, USA 2 Massachusetts General Hospital and Harvard Medical School, BOSTON, MA, USA 3 Air Liquide R&D, JOY EN JOSAS, FRANCE P-040 RELATIONSHIP BETWEEN REGIONAL VENTILATION AND AEROSOL DEPOSITION IN BRONCHOCONSTRICTED SUBJECTS WITH ASTHMA

This work evaluated the extent that the deposition of aerosols is related to the heterogeneous ventilation of bronchoconstricted asthmatics. 11 mild-to-moderate asthmatic subjects were imaged with PET-CT (Biograph 64; Siemens AG) in the supine position. HRCT images of the chest were obtained after methacholine challenge and at the end of the study. The specific deposition of aerosol in the periphery (sD_P) was imaged with dynamic PET during and after the inhalation of ¹³N-NH3 labeled aerosol delivered via mouthpiece from a vibrating mesh nebulizer (Aeroneb Solo, 4.5 μm VMD) used with an Idehaler holding chamber. Regional ventilation per unit of gas volume in (sV) was assessed from the washout rates of ¹³N-NN gas following a bolus injection of the tracer in saline solution. Peripheral regions were identified as those PET voxels within a lobe that would not reflect deposition within the central airways. Heterogeneity in both sV (COV=0.25±0.09) and sD_P (COV=.38±0.16) were observed between the subjects. Considering all lobes, the distribution of normalized peripheral sD_P correlated with the normalized sV (R=0.57). Those subjects breathing with faster breathing frequencies during inhalation tended to have poorer relationships between sV and sD_P (R=−0.89). Understanding the ventilation deposition relationship at a regional level may help to develop strategies for inhaled therapies and provide validation for CFD models of aerosol deposition.

Acknowledgments: Sponsored by NIH grants HL86717 and HL68011, and by support from Air Liquide and Airogen

1 Massachusetts General Hospital and Harvard Medical School, BOSTON, MA, USA 2 Massachusetts Institute of Technology and Massachusetts General Hospital, BOSTON, MA, USA 3 Air Liquide R&D, JOY EN JOSAS, FRANCE P-041 EFFECT OF CARRIER GAS ON THE RELATIONSHIP BETWEEN REGIONAL VENTILATION AND AEROSOL DEPOSITION IN BRONCHOCONSTRICTED SUBJECTS WITH ASTHMA

In severe asthma, bronchodilators are sometimes administered with a mixture of helium and oxygen (HeO₂) to lower central airways resistance and improve peripheral aerosol deposition. CFD models, however, suggest that reduction of turbulent mixing by HeO₂ could increase heterogeneous deposition of aerosol among lung regions. Here we evaluated the heterogeneity of specific ventilation (sV) and peripheral aerosol deposition (sD_P) with the two carrier gases in bronchoconstricted asthmatics, and looked at how these measures were co-varied. 21 mild-to-moderate asthmatics subjects were studied following a methacholine challenge (10 of them while breathing HeO₂). The lobar distributions of sD_P of ¹³N-NH3-labeled inhaled aerosol (Aeroneb SoloTM, 4.5μm VMD), and specific ventilation (sV), assessed from the washout rate of ¹³N-N gas, were imaged with PET-CT. sD_P, sV and central-to-peripheral ratio of deposition (C/P) were evaluated for each lobe. Both carrier gassed had similarly heterogeneous sV and sD_P. Although both carrier gasses had similar average C/P between lobes, the C/P stratification was more uniform with HeO₂ than air (P<0.0001). HeO₂ deposition also tended to correlate better with ventilation. With air, but not with HeO2, breathing at faster frequencies during nebulization led to a poor sDp-sV relationship. Although limited power may have obscured potential differences of HeO₂ on sDp and sV heterogeneity and C/P, we found no evidence of a more peripheral or heterogeneous deposition with HeO₂.

Acknowledgments: Sponsored by NIH grants HL86717 and HL68011, and by support from Air Liquide and Aerogen

1 Massachusetts Institute of Technology and Massachusetts General Hospital, BOSTON, MA, USA 2 Massachusetts General Hospital and Harvard Medical School, BOSTON, MA, USA P-042 ANALYSIS OF AEROSOL DEPOSITION USING ANATOMICALLY DERIVED REGIONS OF INTEREST (ROIs): BEYOND BLACK AND WHITE

Defining the anatomical location of central airways is needed for analysis of aerosol deposition images. When structural information is not available empirical ROIs are used that may not accurately sample the central airways anatomy. We present here a method to define anatomically based ROIs derived from CT scans of 24 individuals. For each individual, ROIs sampling lung periphery (P), intrapulmonary central airways (C) and the extrapulmonary airways (E) were defined (indROIs). Central regions were those likely to sample deposition within a fixed portion of the central airways. indROIs were scaled to a standard lung size and shape and averaged across subjects to create aveROIs. In contrast to conventional black-and-white ROIs, aveROIs are grayscale images where each voxel expresses its likelihood of being an indROI voxel. The aveROIs were rescaled to the individuals' lung outline, and the depositions sampled by the aveROIs were compared against those sampled by the indROIs. Total deposition per subject were similar between indROIs and aveROIs (within 16%) but the deposition assigned to the P, C and E were not; aveROIs sampled 26% less P deposition, and 79% and 47% more for C and E, respectively. 2D projections of indROIs and PET deposition (simulating scintigraphy) yielded similar differences. However, visual inspection suggests that central aveROIs included deposition immediately adjacent to the central indROIs and thus the aveROIs may overcome limitations of black-and-white ROI definition. More importantly, anatomically based aveROIs may be useful to analyze and compare deposition images lacking detailed structural information.

Acknowledgments: Sponsored by NIH grants HL86717 and HL68011, and by support from Air Liquide.

1 University of Alberta, EDMONTON, AB, Canada 2 Chiesi Ltd., CHIPPENHAM, WILTS, United Kingdom P-043 EFFECT OF AMBIENT TEMPERATURE AND HUMIDITY ON THE IN VITRO REGIONAL LUNG DEPOSITION OF SOLUTION AND SUSPENSION MDIS

The effect of ambient conditions on in vitro mouth-throat and lung deposition was investigated for the following pressurized metered dose inhaler (pMDI) formulations: 1) beclomethasone dipropionate (BDP) solution in 13% w/w ethanol, 1.3% w/w glycerol and HFA 134a propellant solution (“BDP HFA 134a”); 2) BDP solution in 13% w/w ethanol and HFA 227 propellant solution ("BDP HFA 227"); 3) Flixotide Evohaler™ (fluticasone propionate 250μg/dose suspension in HFA 134a). The testing apparatus was an Alberta Idealised Throat connected to an inline filter and a vacuum pump. The apparatus, and the pMDIs prior to discharge, were kept within an environmental chamber. Single doses (3 replicates) from each pMDI formulation were collected in the apparatus according to an experimental matrix, containing the following variables: relative humidity (RH) (0%, 35%, 80%); temperature (20°C, 40°C); air flow rate (28.3, 60, 90 L/min). The results suggest that delivered lung dose fractions from the formulations are affected by relative humidity, while air flow rate and temperature have a limited and inconsistent effect. For the solution formulations, an increase in RH from 30 to 80% had a general decreasing trend in fractional delivered lung dose (35% decrease the worst case). Flixotide™ suspension exhibits different behavior, with lung dose fraction falling sequentially (50% decrease the overall worst case), as RH is increased from 0% to 30% to 80%. The in vitro lung delivery of pMDI formulations is affected by ambient conditions, implicating variability in inhaler performance when used in different climates, or when used outside of air conditioned rooms.

1 Dept. of Mechanical and Aerospace Engineering, University of California at San Diego, La Jolla, CA, USA 2 Medicine, University of California at San Diego, La Jolla, CA, USA 3 Radiology, University of California at San Diego, La Jolla, CA, USA 4 INRIA, Paris-Roquencourt, Le Chesnay CEDEX, FRANCE P-044 SPATIAL DISTRIBUTION OF AEROSOLS IN HEALTHY RAT LUNGS: FINDINGS FROM NUMERICAL AND EXPERIMENTAL MODELS

Understanding the fate of inhaled aerosols in the lung may help in assessing their toxic or therapeutic effect. We used a combination of experimental studies and computational simulations to assess the distribution of inhaled particles in healthy rat lungs. Airflow simulations were performed (using an in-house finite element solver (simtk.org)), by coupling realistic 3D airways to 0D models representing the downstream lung. The resistance and capacitance parameters were estimated from the time varying tracheal pressure measured during the aerosol exposure and from the breathing pattern used in the experiments (Oakes et al. 2012 Summer Bioengineering Conference). Using Lagrangian particle tracking, trajectories of 0.95 μm-diameter particles were calculated in the 3D model during inspiration. The distribution of inhaled particles in each lobe of the lungs was derived from these predictions and compared to experimental data of aerosol deposition obtained from magnetic resonance (MR) images of rat lungs ventilated in a controlled fashion (TV=2ml, 80 breath/min) with 0.95 μm particles (Oakes et al. 2011 Am. J. Respir and Crit Care Med., 183:A2463). Good agreement was found between the percent of particles delivered to each lobe from the numerical simulations and the percent of particles deposited in each lobe calculated from the MR images (see Figure 1). Assuming that deposition at the lobar level is proportional to the number of particles delivered to each lobe, our data suggest that the numerical model accurately predicts particle distribution. This type of approach can be extended to disease models and their effect on aerosol deposition.

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Figure 1:

Deposition fraction in the 5 lobes of a healthy rat lung. Error bars represent the standard deviation between rats.

Acknowledgements: This work was supported by grant 1R21HL087805-02 from NHLBI (NIH), National Science Foundation Graduate Fellowship (J. Oakes), Burroughs Wellcome Fund Travel Grant (J. Oakes), team INRIA grant, and the Burroughs Wellcome Fund (A. Marsden).

1 Institute of Pharmacy and Biochemistry, J Gutenberg University, MAINZ, Germany 2 Boehringer Ingelheim, INGELHEIM, Germany 3 Dept of Pneumology and Allergy, Medical Clinic III, Bethanien Hospital, MOERS, Germany P-049 IN VITRO DOSE COMPARISON OF RESPIMAT^® SOFT MIST INHALER™ WITH DRY POWDER INHALERS FOR COPD MAINTENANCE THERAPY

A head-to-head comparison of contemporary inhalers in real patients is difficult because of the variability inevitably introduced by the patients. Therefore in-vitro data on the dose passing the mouth-throat region was acquired using the realistic Alberta throat model. Three inhalers (Respimat^®, Breezhaler^®, and Genuair^®) were compared. From the investigation, important input is obtained for consideration in the patient leaflet or other recommendations for use. The inhaler outlets were centered with the mouth cavity and aligned with their axis parallel to the “tongue”. Passing the air from the throat through a mixing inlet, the size classification was performed in a Next Generation Impactor at constant flow (100 L/min) while the flow through the inhaler was variable. Drug quantitation was performed using HPLC. The inhalers have different air flow resistances (Respimat: 22300 Sqrt(Pa)*s*m⁻³, Breezhaler: 36200 Sqrt(Pa)*s*m⁻³, Genuair: 58400 Sqrt(Pa)*s*m⁻³. As the generation of aerosol is independent of air flow in the Respimat inhaler, only the simulation of very fast inhaling patients creates drug impact in the throat, while in most dry powder inhalers, a relatively high air flow is required for de-agglomeration of the drug particles. This in turn entails higher deposition of all constituents of the powder formulations in the mouth-throat region. The data suggest that the Respimat Soft Mist Inhaler is an excellent inhaler which by its unique spray technology makes use of a large amount (approx. 50–70% in vitro) of the metered drug which compares well to 44–63% in vivo (P Brand, 2007, Pneumologie, Vol. 61).

Acknowledgements: Warren Finlay provided 3d-Data of his idealized mouth-throat model.

1 Pharmacy, University of Huddersfield, HUDDERSFIELD, United Kingdom 2 Respiratory, Tripoli Medical Centre, TRIPOLI, Libyan Arab Jamahiriya P-051 IMPROVEMENT OF DRY POWDER INHALER (DPI) USE BY ASTHMATICS RECOVERING FROM AN ACUTE EXACERBATION

During acute exacerbations patient inspiratory effort will be reduced and thus their ability to create sufficient turbulent energy inside a DPI to de-aggregate the metered dose may be reduced. We have measured the inhalation pressure profiles of 18 asthmatics, mean (SD) age 42.0(11.8) years, on days 1–4 following their admission with an acute exacerbation. These measurements have been made using placebo versions of a Diskus (DSK), Easyhaler (EASY) and Turbuhaler (TBH); inhalers with medium, medium/high and high resistance.

All parameters improved during the recovery phase after their acute exacerbation. Although PIF was lower for the high resistance DPI the pressure changes and acceleration were greater suggesting better de-aggregation. During patient use these parameters should be the focus and not flow. PIF should not be used to compare patient use with each device.

1 Pharmacy, University of Huddersfield, HUDDERSFIELD, United Kingdom 2 Paediatrics, Leeds General Infirmary, LEEDS, United Kingdom 3 Respiratory, Airedale General Hospital, STEETON, United Kingdom 4 Respiratory, Bradford Royal Infirmary, BRADFORD, United Kingdom P-052 INHALATION CHARACTERISTICS OF PATIENTS WHEN THEY USE DIFFERENT DRY POWDER INHALERS (DPIs)

During routine practice emphasis is placed on the peak inhalation flow (PIF) when patients use a DPI. However other aspects of the patient's inhalation manoeuvre are important. We have measured the inhalation pressure profiles of asthmatic children (CHILD; n=16, FEV₁ 79% predicted), asthmatic adults (ADULT; n=53, FEV₁ 72%) and COPD (n=29, FEV₁ 42%) patients when they inhaled through placebo versions of an Aerolizer (AERO), Diskus (DSK), Turbuhaler (TBH) and Easyhaler (EASY) using their 'real life' DPI inhalation technique. These are low, medium, medium/high and high resistance DPIs. A summary of the inhalation characteristics is presented below.

Although peak inhalation flows were faster for the DPI with lowest resistance the pressure change corresponding to the peak turbulent energy and the acceleration of the pressure change at the start of the inhalation was greatest for the DPI with the highest resistance suggesting better de-aggregation.

1 Pharmacy, University of Huddersfield, HUDDERSFIELD, United Kingdom P-053 FINE PARTICLE DOSE EMISSION OF A COMBINATION OF SALMETEROL (SX) AND FLUTICASONE PROPIONATE (FP) IN A DRY POWDER INHALER IS DEPENDENT OF THE ACCELERATION OF FLOW AND NOT INHALED VOLUME

Flow dependent dose emission is widely reported but the effect of the inhaled volume (IV) and the acceleration of the inhalation flow (ACC) has not been rigorously examined. We have adapted our mixing inlet Andersen Cascade Impactor method (Nadarassan et al, Eur J Pharm Sci. 2010;39(5):348–54) to study the effect of IV and ACC on the dose emission characteristics of SX and FP from an Advair® 500/50 Diskus (also known as Seretide® Accuhaler, GlaxoSmithKline). The method uses inhalation flow at 60L/min with the input of supplementary flow at the same rate. An inhalation profile is drawn from the supplementary arm which is replayed at the induction port with the inhaler in situ. Inhalation profiles ranging from 240–1000 ml with peak inhalation flows ranging from 20–60L/min have been used. 10 doses were used for each determination. A summary of the data (n=3 determinations) is shown in the table above.

Total dose emission for the lowest to highest ACC ranged from 38.9–41.0μg for SX and 413.3–460.8μg for FP

The results show the influence of ACC rather than IV with this product.

1 Novartis Pharmaceuticals, SAN CARLOS, CA, United States of America 2 Georgia State University, ATLANTA, GA, United States of America P-054 IMPROVING IN VITRO MODELS FOR AEROSOL DELIVERY OF ANTIBIOTICS DURING MECHANICAL VENTILATION

In vitro estimates of aerosol drug dose delivered to the lungs during mechanical ventilation are influenced by the bench model used. For example, in vitro models of mechanical ventilation have typically demonstrated a 40% to 50% reduction in delivered dose for heated and humidified (wet) versus non-humidified (dry) inspiratory gas. Placement of an aerosol generator between the ventilation circuit and an endotracheal tube (ETT; 8.0 mm ID) resulted in delivery of more vancomycin under wet (68% at 80 lpm inspiratory flow) than dry conditions, but this was likely due to drug raining out in the airway and dripping into the inspiratory filter.

Since efficacy of aerosol vs. liquid drug may differ in vivo, we modified the model by a) placing an active humidifier between the test lung and the inspiratory filter (simulating exhaled heat and humidity), b) elevating the inspiratory filter, and c) adding traps to collect non-aerosol drug. Vancomycin was aerosolized with a vibrating mesh nebulizer placed distal to the ETT during simulated adult mechanical ventilation using a PB 7200AE ventilator. Mass balance was determined by HPLC.

Using the modified model the differences in mean inhaled dose (mean±SD) between wet and dry conditions at 80 lpm (20±2% wet and 23±1% dry) and at 40 lpm (46±1% wet and 47±4% dry) are eliminated. In summary, using active humidification during exhalation, elevating the distal airway tip, and using traps to collect rainout improved both sensitivity and specificity of in vitro aerosol delivery measurements during mechanical ventilation.

Study supported by Nektar Therapeutics

1 Novartis Pharmaceuticals, SAN CARLOS, CA, United States of America 2 Nektar Therapeutics, South San Francisco, CA, United States of America P-055 IN VITRO ASSESSMENT OF AEROSOLIZED AMIKACIN LUNG DOSE DELIVERED BY NKTR-061 PDDS CLINICAL DURING ON-VENTILATOR AND OFF-VENTILATOR USE

NKTR-061 (BAY41-6551, Amikacin Inhale) is a drug-device combination product being co-developed by Bayer HealthCare and Nektar to treat intubated and mechanically ventilated patients with Gram-negative pneumonia. The product uses a proprietary vibrating mesh nebulizer system (PDDS Clinical) with amikacin sulfate formulated for inhalation (3.2mL of 125mg/mL) for a 10-day bid course of therapy. It is designed for use with mechanical ventilators for intubated patients. For patients who are extubated before completing the course of therapy, a Handheld (Off-ventilator) device has also been configured. This study was intended to determine the in vitro lung doses of the two delivery systems.

An in vitro on-ventilator model was used to estimate the lung dose (ELD) of aerosolized amikacin post-endotracheal tube during mechanical ventilation. In comparison, the ELD for the Off-ventilator device was calculated from the fine particle fraction (FPF_<5μm) post-mouthpiece, multiplied by the in vitro delivered dose post-mouthpiece. FPF_<5μm reflects lung deposition observed during Phase 2 clinical trials. Eighty one nebulizers (VMD: 4.4±0.5μm; output rates: 0.23±0.10mL/min) were tested for each system. Amikacin sulfate content of each dose was determined by HPLC. Results were analyzed using a least squares fit with 95% confidence limits. The average ELDs were 50±9% (On-ventilator) and 49±11% (Off-ventilator) of the nominal dose.

These results support using the PDDS Clinical with two delivery systems to administer aerosolized amikacin to treat intubated and mechanically ventilated patients with Gram-negative pneumonia. The PDDS Clinical allows patients who no longer require intubation to achieve a comparable pulmonary without dose adjustment when extubated.

1 Comprehensive Pneumology Center, Institute for Lung Biology and Disease, Helmholtz Zentrum München, Neuherberg, Germany 2 Fisher&Paykel Healthcare, Auckland, New Zealand 3 Department of Nuclear Medicine, LMU Medical Center Grosshadern, München, Germany 4 Department of Pulmonary Medicine, Asklepios Hospital München-Gauting, Gauting, Germany P-056 UPPER AIRWAY DEAD SPACE CLEARANCE KINETICS AND EFFICIENCY DURING NASAL HIGH FLOW

It has been suggested that Nasal High Flow (NHF) might clear anatomical dead space in upper airways. Individuals with high dead space to tidal volume ratio ventilation (i.e. patients with chronic obstructive pulmonary disease, COPD) may benefit from dead space reduction by decreasing carbon dioxide re-breathing and increasing oxygen delivery to the lung.

Airways were filled with radioactive 81mKr-gas through the nose and a gamma camera was used to study the upper airway clearance in 10 healthy volunteers during breath holding. Following inhalation of 81mKr-gas bolus a nasal cannula with NHF at 15, 30 and 45 L/min was inserted in the nostrils and the kinetics and compartmental efficiency of Kr-gas clearance from upper airways was investigated. Respiration and breath holding was monitored with a pressure belt. Clearance was also studied in an upper airway model with infrared-CO₂ absorption imaging using a high speed camera. After filling the model with CO₂ clearance was studied by flushing air into the model through a cannula at different flow rates (15–45 L/min).

Kr-gas clearance half time in the anterior and posterior nasal cavity was 0.95±0.40s and 1.50±0.49s, respectively at 15 L/min NHF and decreased with an increase of NHF. NHF 45 L/min caused clearance of Kr-gas down to the larynx. In the upper airway model clearance half time was 0.55±0.1s at 15 L/min NHF and similarly decreased with increasing NHF.

The study confirms upper airway clearance during NHF and it is flow dependant. The effect of breathing pattern on the efficiency of dead space clearance needs to be investigated.

1 Trinity College Dublin, DUBLIN, Ireland 2 Georg-August-Universität, GÖTTINGEN, Germany P-058 BETA-2-RECEPTOR AGONISTS INHIBIT BUT ARE NOT TRANSPORTED BY ORGANIC CATION TRANSPORTERS

Beta 2-agonists are cations at physiological pH. Here, we studied if β₂-agonists are substrates and/or inhibitors of putative organic cation transporters (OCTs). Uptake of [³H]-salbutamol into alveolar (A549) and bronchial (Calu-3) epithelial cells was assessed in the presence of OCT modulators. The inhibitory potential of β₂-agonists (i.e., salbutamol and formoterol) on [¹⁴C]-tetraethylammonium (TEA) uptake was also studied. In HEK-293 transfectants [³H]-1-methyl-4-phenylpyridinium (MPP⁺) was used as substrate. [³H]-salbutamol transmonolayer transport studies were carried out in A549 and Calu-3 cells, respectively. TEA uptake into A549 cells was reduced to 15.4% (formoterol) and 54.5% (salbutamol). TEA uptake into Calu-3 cells was 72.6% (formoterol) and 83.6% (salbutamol) of control, respectively. [³H]-MPP⁺ uptake into HEK-293 cells was inhibited by both β₂-receptor agonists, however, formoterol showed a much stronger inhibition than salbutamol. In OCT-transfected HEK-293 expression systems, β₂-agonists exerted the strongest inhibition on OCT1 followed by OCT3. OCT2 were only marginally affected and only at non-physiological concentrations. [³H]-salbutamol uptake was not inhibited by typical OCT substrates in A549 and Calu-3 cell lines. [³H]-salbutamol transport across Calu-3 cell monolayers revealed P_app values of 6.8±0.9·10⁻⁷ cm/s (A-to-B) and 6.2±1.4·10⁻⁶ cm/s (B-to-A). OCT mediated uptake was attenuated by β₂-agonists in respiratory epithelial cells. HEK-293 data suggested OCT1 to be mainly involved. However, no OCT mediated uptake was observed. Transport of salbutamol was net-secretive across Calu-3 cells, due to a yet to be identified efflux transporter.

Acknowledgements: Grant funding by Science Foundation Ireland

1 InspiRx Inc., NEW BRUNSWICK, NJ, United States of America 2 McMaster Faculty of Health Sciences, HAMILTON, ONTARIO, Canada 3 Ziv Medical Center, SAFED, Israel 4 Technosaf, PARDES HANNA-KARKUR, Israel P-061 INSPIRALERT™ AND SEALVISION™: SIMPLE AND UNIQUE OPTIMIZED BIOFEEDBACK IN AN IMPROVED MDI AEROSOL DELIVERY SYSTEM

Rationale: More efficacious aerosol delivery is achieved when patients understand how to best use their delivery devices. Current valved holding chamber (VHC) audible reeds sound at flow velocities much higher than appropriate for optimal aerosol delivery. Most current facemasks do not provide a visual indicator of critical face to mask seal. The InspiraChamber^® VHC's InspirAlert™ audible signal and the SealVision™ visual seal indicator on SootherMask™ and InspiraMask™ are designed to provide optimized biofeedback.

Methods and Results: InspiraChamber^® was compared to AeroChamber Plus Flo-Vu^® (AC) and OptiChamber Advantage^® (OC) with 5 pMDIs to evaluate varying air entrainment. A vacuum was applied to each VHC mouthpiece and flows at which their audible signal sounded were recorded. Each chamber+pMDI combination was tested 3 times by 3 individuals (n=9). Masks were applied to a surface providing a seal representative of that anticipated when applied to a child's face and the flow required to readily visualize (=/> 2mm excursion) the SealVision™ recorded. The InspirAlert™ audible signal sounded at flows significantly more clinically appropriate (∼30L/min) than AC and OC (both reed devices), averaging 34% lower (p<0.001). The SealVision™ visual indicator was readily visible with minimal inspiratory effort at flow velocity as low as 0.05 L/sec.

Conclusions: The InspiraChamber^® VHC's InspirAlert™ is a unique cylindrical solid-state whistle that indicates too-rapid inhalation at inspiratory flows, significantly more clinically appropriate than both AC and OC. SootherMask™ and InspiraMask™ are first to provide SealVision™, a low-resistance visual indicator confirming adequate face to mask seal at low (pediatric) flow velocities.

1 Department of Pharmaceutical Sciences, College of Pharmacy, The University of New Mexico, Health Sciences Center, Albuquerque, NM 87131. 3 Biomedical Sciences Graduate Program, The University of New Mexico, Health Sciences Center, Albuquerque, NM 87131. 3 Nanoscience and Microsystems Graduate Program, The University of New Mexico, Health Sciences Center, Albuquerque, NM 87131. P-062 INHALED DRY POWDER BCG FOR UNIFORM PROTECTION AGAINST TUBERCULOSIS IN ENVIRONMENTAL MYCOBACTERIA ENDEMIC REGIONS

The current vaccine against Mycobacterium tuberculosis, the Bacille Calmette Guerin (BCG), has a highly variable efficacy rate of 0–80%. This variability has been shown to be the result of, at least in part, human ingestion of environmental mycobacteria (EM) that are found in soil and water. Recent literature suggests that the lung airway acts as an isolated immune environment in the body, which may makes it the ideal target for vaccination without EM interference. In addition, while intradermal vaccination generates immune cells systemically, there is little to no protection generated in the lung. It is only with inhaled vaccines that a robust pulmonary immune response is generated in the airways. We exploit this phenomenon using an inhalable dry powder BCG to vaccinate mice which have been chronically exposed to EM prior to pulmonary vaccination. Live BCG was spray dried using a lung compatible excipient to yield dry powders with a mass median aerodynamic diameter (MMAD) of 3–6 μm. The MMAD of the BCG dry powder was confirmed using a Next Generation Impactor. The viability and immunogenicity of the BCG dry powder were analyzed using a plating method and an in vitro macrophage infection assay, respectively. Mice were chronically exposed to EM before being vaccinated with our BCG dry powder. Murine airways were assayed for BCG specific immune cells using flow cytometry and ELISAs. In conclusion, this protocol provides an optimized method for preparing a live BCG vaccine as an inhalable immunization delivery platform for EM exposed mice.

Acknowledgments: Grant funding by the Gates Foundation Grand Challenges Exploration

1 DTF-Aerodrug, Faculté de Médecine, F-37032 TOURS, France 2 Université François Rabelais de Tours, Faculté de Pharmacie, EA 6306 IMT, Fédération GICC UMR 7292, F-37000 TOURS, France 3 Centre d'Etude des Pathologies Respiratoires INSERM U1100/EA6305, Université François Rabelais de Tours, Faculté de Médecine, F-37032 TOURS, France P-063 USE OF SURFACTANTS TO AVOID AGGREGATION OF MONOCLONAL ANTIBODIES DURING THE AEROSOLIZATION PROCESS

The airways are a promising alternative to the systemic route for the delivery of local-acting monoclonal antibodies (Mabs). Inhalation allows the non-invasive delivery of drugs into the lungs, but implies drug dispersion through the airways as an aerosol. It is well known that Mabs subjected to stresses, such as aerosolization, can aggregate, thereby potentially affecting their efficacy and safety. We previously showed that mesh nebulizers are the most reliable devices to efficiently administer liquid formulations of Mabs into the lungs and limit formation of massive insoluble subvisible aggregates. In this study, we evaluated the ability of surfactants, which are usually added to stabilize injectable Mab products, to avoid generation of small size and subvisible aggregates during nebulization. We compared aggregation of a human polyclonal IgG upon mesh-nebulization in the presence of various concentrations of surfactants using Diffusion Light Scattering (DLS) and microscopy. Viscosity of formulations and aerosol aerodynamical characteristics in terms of distribution and diameter were also evaluated. Although surfactants significantly reduced formation of antibody particles from a range of 10-4 to 10-1 % (v:v), high concentrations critically altered flow rate of mesh nebulizers. Moreover, levels of IgG in the formulation also impacted on aggregation. Altogether our results show that addition of surfactants is valuable to stabilize IgG during aerosolization process. However, considering the lack of consistent data on the safety of surfactants delivered through the pulmonary route and concentration impact on aerosol flow rate, levels of surfactants would rather be maintained as low as possible.

Acknowledgements: Grant funding by the French army.

1 Centre d'Etude des Pathologies Respiratoires INSERM U1100/EA6305, Université François Rabelais de Tours, Faculté de Médecine, F-37032 TOURS, France. 2 laboratoire Protec'Som, F-50700 VALOGNES, France. 3 DTF-Aerodrug, Faculté de Médecine, F-37032 TOURS, France. P-064 STUDY OF THE PERFORMANCES OF A NEW SPACER IN MECHANICAL VENTILATION

The objective of this study was to evaluate the performances of a new spacer called Combihaler (Protec'som, France) to improve drug delivery either from nebulizer or pMDI.

To assess the Combihaler chamber in clinical conditions, assembly includes a respirator (Volume controlled, Vc=450mL, f=15/min, PEEP=6, P max=19, Ti / Ttot=40/60) and a model of adult lung Dual TTL 5600i (Michigan Instruments). Ventilation parameters were measured with and without the new spacer. A filter was placed after the endotracheal tube to measure aerosol delivery. Amikacin (1g/8ml) was nebulized with an Aeroneb (Aerogen, Ireland) and a T piece or a Combihaler. Salbutamol was delivered with a pMDI (Salbutamol) and a T-piece (Allegiance Healthcare Corporation) or a Combihaler. Drug deposited on filter was assayed. Amikacin was measured with an electrochemical tracer and salbutamol was measured by spectrophotometry.

The use of the Combihaler didn't change the ventilation parameters (p=0.82). The mass of amikacin deposited on the filter was twice higher with the Combihaler chamber compared with the Aerogen T-adapter (394.4±8.9 mg vs 142.4±4.9 mg). The mass of salbutamol deposited on the filter was increased with Combihaler chamber in comparison with T-piece (62.7±0.7 μg vs 18.8±1.9 μg).

In conclusion, the Combihaler chamber didn't modify ventilator parameters and increased drug delivery by mesh nebulizer and pMDI.

1 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, Richmond, VA 2 Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA P-065 GENERATION AND EFFECTIVE DELIVERY OF SUBMICROMETER AEROSOLS THROUGH HIGH FLOW NASAL CANNULA DURING NONINVASIVE VENTILATION

Enhanced condensational growth (ECG) has been shown to improve pulmonary aerosol delivery through a nasal cannula interface by introducing submicrometer particles that combine with heated and humidified air in the nasopharynx to cause aerosol size increase. An aerosol delivery system capable of producing submicrometer aerosol particles with minimal system losses is required for the successful implementation of ECG. Computational fluid dynamics (CFD) was used with in vitro experimental results to evaluate aerosols generated using an existing radial aerosol mixer design and an improved aerosol mixer with a compact heat-transfer region. Aerosol delivery through the commercially available Optiflow nasal cannula, a divided (D) nasal cannula, and a divided and streamlined (DS) nasal cannula was also considered. The improved mixer was shown to reliably produce submicrometer aerosols (900 nm) with device depositional losses that were 3 times lower than the radial design at flow rates of 10 and 15 LPM. The DS cannula was demonstrated to reduce depositional losses by a factor of 2–3 in comparison to the Optiflow and D cannulas at flow rates of 10 and 15 LPM. The DS cannula was also shown to be capable of delivering 80% or more of conventional size particles to the nose at flow rates up to 15 LPM. The optimized system has an overall high delivery efficiency of about 90% and it effectively produced submicrometer particles. The system may be combined with the ECG concept to improve drug delivery when used with non-invasive ventilation and high flow therapy.

Acknowledgements: NIH

1 University of Iowa, IOWA CITY, IA, United States of America P-066 DEVELOPMENT OF ERYTHROMYCIN-LOADED PLGA NANOPARTICLES FOR TARGETED DELIVERY TO THE LUNG EPITHELIUM

The goal of this study was to develop erythromycin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with optimized particle size, maximum drug loading, and surface properties tailored to target the lung epithelium. PLGA nanoparticles were fabricated using the solvent diffusion method. Formulation and process parameters (injection needle diameter and concentrations of PLGA, erythromycin and the surfactant poly(ethylene maleic anhydrite)) were varied in a factorial design to control particle size and % drug encapsulation. Particles were characterized to determine their geometric size by dynamic light scattering, zeta potential by electrophoretic light scattering, and % drug encapsulated and release rate by UV spectroscopy. PLGA concentration had the largest effect on particle size, increasing the size from 70 to 3000 nm with an increase in PLGA concentration from 3.3 – 33.3 mg/ml. The nanoparticle fabrication process was optimized to fabricate particles with narrow unimodal particle size distribution at low PLGA concentration and heated aqueous phase. Drug loading up to 62% was achieved. To enhance specific uptake of the particles into lung cells, particles were coated with a bacterial ligand, lipooligosaccharide from non-typeable Haemophilus influenzae (NTHi LOS), using the carbodiimide coupling reaction. The coating was detected by IR and NMR spectroscopy and quantified via ELISA. Chemical conjugation of NHTi LOS was detected via amide bonds using IR spectroscopy and signature 1H NMR signals of oligosaccharides in LOS. Successful formulation of these nanoparticles will provide a sustained-release formulation for targeted delivery of antimicrobials to treat epithelial penetrating infections.

Acknowledgements: Grant funding by the National Institutes of Health (1R2HL11387601).

1 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, RICHMOND, VA, United States of America 2 Department of Pharmaceutics, Virginia Commonwealth University, RICHMOND, VA, United States of America P-067 DEVELOPMENT OF A NEW HIGH PERFORMANCE DPI

The purpose of this study was to develop a capsule-based dry powder inhaler (DPI) that could effectively deaggregate a submicrometer dry powder formulation for the excipient enhanced growth delivery approach. A submicrometer combination particle formulation was produced using a spray dryer and its aerosol performance in prototype DPIs was characterized using the Next Generation Impactor (NGI). Capsule operating chamber (CoC) with various sized and aligned air-inlets were studied in combination with an optimized mouthpiece design. The air-inlets investigated were a single inlet (CoC-S), double inline inlet (CoC-DIL), and double staggered inlet with different levels of offset. Air-inlet diameters were selected corresponding to a 4 kPa pressure drop at a flow rate of approximately 45 Lmin⁻¹. Capsule wall impactions with each CoC were analyzed using a high speed camera. Powder was aerosolized through the DPIs at 4 kPa using the NGI and a square inhalation waveform. Decrease in capsule aperture increased mean percent fine particle fraction less than 1 μm (expressed as % of emitted dose (FPF_<1μm/ED). Capsule-to-wall impactions were not observed with CoC-S and as the degree of air-inlet staggering increased, impactions decreased. Maximum capsule-wall impactions were observed with CoC-DIL which was the best performing device. The resulting ED, FPF_<1μm/ED and FPF_<5μm/ED with CoC-DIL from three replicates (mean±SD) were 63.9±2.9%, 30.9±0.6% and 93.8±0.7%, respectively. Future studies will seek to improve the ED by reducing retention in the capsule and flow passage. This study showed that the CoC-DIL was capable of effective deaggregation of the submicrometer powder formulation.

1 Department of Mechanical and Nuclear Engineering, Virginia Commonwealth, University, Richmond, Virginia, USA 2 Department of Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia, USA P-068 DELIVERY OF AEROSOLS FROM NASAL HIGH FLOW CANNULA TO THE RESPIRATORY TRACT USING CONDENSATIONAL GROWTH TECHNIQUES

Aerosol drug delivery during non-invasive ventilation (NIV) including high flow therapy (HFT) through a nasal cannula interface is known to be inefficient due to high depositional losses. To improve respiratory drug delivery during NIV, the concept of enhanced condensational growth (ECG) was recently proposed in which submicrometer aerosols are delivered to one nostril and warm air saturated with water vapor is delivered to the other nostril. Using a similar excipient enhanced growth (EEG) approach, submicrometer particles composed of a drug and hygroscopic excipient are delivered to both nostrils through a nasal cannula interface. The submicrometer particles are inhaled through the cannula interface and nasal passages resulting in low depositional losses. Subsequent aerosol growth in the tracheobronchial (TB) airways occurs for ECG and EEG, due to mixing with the inhaled saturated air or the natural relative humidity of the airways, respectively. The objective of this study is to evaluate the growth and deposition characteristics of nasally administered submicrometer aerosols throughout the conducting airways for ECG and EEG delivery methods. Computational fluid dynamics simulations were validated based on in vitro experiments in a characteristic nasal airway model extending through the trachea. Results indicate that the ECG and EEG approaches both produce very low nasal depositional losses and increased the aerosol size to 2 μm and above within the conducting airways. Ultimately, the proposed technology will provide a highly effective method for delivering pharmaceutical aerosols to patients for local or systemic therapy during NIV.

Acknowledgments: NIH

1 School of Pharmacy, Royal College of Surgeons in Ireland, Dublin 2 2 Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, Dublin 2 3 Aerogen Ltd., Galway Business Park, Galway, Ireland P-082 CONVERGENT PEGYLATED NANOPARTICLE-NEBULIZER PLATFORM FOR RESPIRATORY DELIVERY OF siRNA

For efficient intracellular delivery of siRNA to airway cells, it must be encapsulated in nanoparticles. To this end, we have developed “muco-inert” particles using highly PEGylated (80%) poly(ethyleneimine)-poly(ethyleneglycol) (PEI-PEG(10kDa)) to encapsulate siRNA that effectively transfect airway cells[1]. To harness these siRNA-nanoparticles for treatment of respiratory disease an effective means of delivering them via inhalation is required. Herein, Aerogen^®'s vibrating mesh nebulizer technology was harnessed to aerosolize PEGylated siRNA-nanoparticles and determine their bioactivity post-nebulization.

Nebulized siRNA-nanoparticle droplets were analyzed for volumetric median diameter (VMD) and fine particle fraction (FPF). Reporter gene knockdown (luciferase and GAPDH) knockdown was assessed pre- and post-nebulization in an undifferentiated and fully differentiated, mucous covered Calu-3 monolayers. Finally, siRNA-nanoparticles were nebulized through a twin-stage impinger (TSI) onto polarized Calu-3 monolayers as an in vitro model of lung deposition.

Nebulization of nanoparticles resulted in 57% FPF and VMD of ∼5μm. PEGylated siRNA nanoparticles (PEI-PEG siRNA) applied directly to polarized Calu-3 monolayers resulted in superior GAPDH knockdown compared to PEI (47.2%±4.02 vs. 24.2%±11.24). Furthermore, gene knockdown was better retained post-nebulization by PEI-PEG-siRNA nanoparticles than PEI-siRNA (29.9±8% vs. 2.44±16.5%). Crucially, in the TSI model, knockdown up to 57±9.3% in differentiated monolayers was observed using PEI-PEG siRNA-nanoparticles which was significantly higher than PEI, which failed to elicit any knockdown at this dose. Using the TSI model, only negligible knockdown was ever observed in PEI-siRNA nanoparticle treated cultures (1.62±6.49%).

PEGylated nanoparticles can be effectively nebulized for pulmonary delivery of siRNA using the Aeroneb technology. This work provides an integrated nanomedicine-device combination for future in vivo pre-clinical and clinical studies of inhaled RNA therapeutics.

Acknowledgements: This work was supported by grant SFI 07/SRC/B1154.

1. Hibbitts, A., et al., Screening of siRNA nanoparticles for delivery to airway epithelial cells using high-content analysis. Therapeutic Delivery, 2011. 2(8): p. 987–999.

1 INSERMU110/EA6305, Faculté de médecine de Tours, Tours, guillel@free.fr 2 Service de pneumologie et d'explorations fonctionnelles, CHRU Tours, Tours 3 GICC, UMR CNRS 6239, Service de pharmacologie, CHRU Tours, Tours 4 Centre de Recherches Biologiques, Baugy 5 Faculté de médecine de Tours, PPF animalerie, Tours 6 Laboratoire Novaxia, Saint-Laurent-Nouan 7 Service de biochimie, Service d'hématologie, CHRU Tours, Tours P-084 DELIVERY OF MONOCLONAL ANTIBODY THROUGH THE PULMONARY ROUTE IN NON-HUMAN PRIMATES

Introduction: Monoclonal antibodies (mAbs) have revolutionized the treatment of various affections and they have a tremendous opportunity to deliver in patients with lung diseases. Pulmonary route is a promising alternative to systemic injection for local-acting agents. Despite the potential opportunity of the pulmonary route for mAb delivery to the lungs, it remains essentially unexplored. In this study, we evaluated cetuximab, an anti-EGFR antibody, administration through pulmonary route in cynomolgus macaques.

Methods: Two control macaques received intravenous cetuximab. Six macaques received 6-fold concentrated cetuximab through the airways using a Microsprayer®. Blood samples were collected several times over a 45-days period. Tolerance was evaluated on clinical and biological parameters. Plasmatic cetuximab and anti-cetuximab antibodies were measured by ELISA.

Results: No clinical or biological toxicity was observed. Mean systemic absorption of aerosolized cetuximab was estimated at 0.35% of the delivery dose. Anti-cetuximab antibodies were observed 15 days after aerosol administration for all the monkeys. No immunogenicity was observed in the monkeys that received cetuximab through the intravenous route. Two monkeys received cetuximab through pulmonary route once a week for 4 weeks and plasmatic cetuximab level was undetectable after the first two administrations. Microscopic and DLS analysis revealed high amount of aggregates in the preparation of cetuximab after concentration. We hypothesized that aggregates may be involved in immunogenicity observed after airways delivery. To confirm this hypothesis, eight mice received unconcentrated cetuximab via Microsprayer® and no immunogenicity was detected.

Conclusion: mAb systemic absorption is weak after administration through pulmonary route. Immunogenicity seems to be related to aggregates.

1 Stony Brook University Medical Center, NY 2 New York University Medical Center, NY P-085 CLINICAL ENDPOINTS FOLLOWING INHALED INTERFERON IN IDIOPATHIC PULMONARY FIBROSIS (IPF)

Background: Systemic antifibrotic agents to treat IPF are being tested in phase 3 trials. Endpoints have included 6 min walk and FVC, reporting small changes over time compared to placebo. In a phase 2 trial, testing therapy with inhaled interferon gamma (IFN-γ), we analyzed full pulmonary function tests (PFTs) before and after therapy.

Methods: Ten patients with IPF received inhaled IFN-γ, (InterMune) via I-neb (Philips Respironics) 100 μg 3x/week for 80 weeks. Full PFTs were available for approximately 20–50 weeks before Rx and monthly during Rx. Linear mixed models for modeling longitudinal data were used to test if the PFT change over time was significantly different before and after inhaled interferon. Autoregressive dependence structure with order one was consistently selected as the best one to model the intra-patient correlation over time. Normality assumption was confirmed. Significance level was set at 0.05 and all analysis was done in SAS 9.3. Eighty-nine observations from 10 patients were used to build models.

Results: The change over time in DLCO was significantly different before and after interferon treatment. More specifically, DLCO decreased over time before treatment but increased after treatment (p-value=0.03). Changes in TLC, FRC, RV and FVC were not statistically significant in this small pilot study.

Conclusions: Future randomized, controlled, phase 3 trials, comparing the difference in PFT behavior longitudinally may be more sensitive to drug effect and serve as a valuable clinical endpoint.

Acknowledgements: supported in part by Philips Respironics and InspiRx, Nostrum Pharmaceuticals, LLC.

1 Aerogen Limited, IDA Business Park, Dangan, Galway, Ireland 2 Department Viroscience, Erasmus MC, Rotterdam, The Netherlands P-086 DOSE NORMALIZATION OF AEROSOL DELIVERY TO THE RESPIRATORY TRACT OF NON-HUMAN PRIMATES

Uniformity of dosing across subjects and administration routes is a crucial requirement in pre-clinical and clinical studies. With this in mind, we have developed a protocol for dose normalization of an aerosolized measles virus vaccine administered to cynomolgus monkeys (Macaca fascicularis) by inhalation. Initially, macaque breathing regimen (tidal volume, breathing frequency and I:E ratio) were measured using plethysmography in 9 monkeys across a range of 3.3–6.5 kg body weight, using a Paediatric pneumotachometer. Subsequently, these regimen were reproduced on a breathing simulator attached to an absolute filter. The effect of the breathing regimen on the percentage inhaled dose delivered was determined using albuterol. Albuterol was nebulized using a vibrating mesh nebulizer (Aeroneb Pro, Aerogen, VMD=3.5μm) and the percentage inhaled dose was determined by extraction of drug from the filter, using spectrophotometric analysis at 276nm. Tidal volumes ranged from 24 to 46 ml (average 29.5±7.1). Breathing frequencies ranged from 19 to 31 breaths per minute (average 25.7±4.5). I:E ratios ranged from 0.7 to 1.6 (average 1.1±0.3). As expected, increased body weight was associated with larger tidal volumes, increased breathing frequency and larger I:E ratios. High tidal volumes were associated with increased percentage inhaled dose. Varying breath rate and I:E ratio did not significantly affect dosing efficiency. Animals in a weight range of 3.5–4.5 kg showed little variation in breathing regimen, resulting in a percentage inhaled dose of 24.8±2.3. This would suggest that within this range the need for titration of doses is negated and this will be determined in future in vivo studies.

1 Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Huntingdon, Cambs, PE28 4HS, UK. 2 Huntingdon Life Sciences Ltd., 100 Mettlers Road, East Millstone, New Jersey 08875, USA. 3 GlaxoSmithKline (GSK Inhaled Sciences), Park Road, Ware, Herts, SG12 0DP, UK. P-087 CAPSULE BASED AEROSOL GENERATION - TEST ARTICLE CONSERVATION DURING THE CONDUCT OF IN-VIVO LEAD OPTIMIZATION RESPIRATORY SAFETY ASSESSMENT STUDIES

In both clinical and non-clinical environments the inhaled delivery of drug substances to conscious subjects will typically require larger quantities of the test article than any other route of administration. This difference is greatest for dry powder formulations in which charge and proximity effects are most pronounced, but it is also due to the losses and inefficiencies in the delivery device and aerosol delivery system. Minimizing such losses, particularly when conducting in-vivo lead optimization studies in rodents, provides a means of reducing the cost of drug development. Decreasing the amount of test article required enables the conduct of investigations utilizing inhaled route earlier in a development program, when the test article is most likely to have limited availability and greatest cost.

Over a period of three years GSK Inhaled Sciences has collaborated with HLS to identify aerosol generation methods to minimize powder usage for inhaled delivery to conscious non-clinical species. The principle alternative delivery methodology that has been commonly employed, intra-tracheal insufflation, achieves particulate deposition dissimilar to conscious inhaled delivery and can produce artifactual toxicological and pharmacological outcomes.

The results of work sponsored by GSK Inhaled Sciences to characterize an existing capsule-based aerosol generator and design and manufacture an alternative instrument are presented. If further work is successfully concluded, the instrument developed will facilitate the use of the inhaled route earlier in in-vivo lead optimization studies, so increasing the quality of candidate drugs and reducing compound attrition precipitated by findings in later more resource intensive in-vivo studies.

1 Aerogen Limited, IDA Business Park, Dangan, Galway, Ireland. P-088 EVALUATION OF VIBRATING MESH NEBULIZER, JET NEBULIZER AND pMDI PERFORMANCE DURING SIMULATED ADULT AND PAEDIATRIC CPAP AND NIPPV

The opportunity for concurrent aerosol delivery during CPAP and NIPPV is limited as a result of inappropriate device design and the importance of how and where the aerosol is introduced into the system. Here we report the performance of a vibrating mesh nebulizer (Nivo nebulizer, Philips), jet nebulizer (Ventstream, Respironics) and pMDI (100 mcg Ventolin Evohaler, GlaxoSmithKline) during simulated Adult and Paediatric CPAP and NIPPV. CPAP was simulated for both adult (BPM 15, Tv 500mL, I:E 1:1, CPAP 4 cmH₂O) and paediatric (BPM 25, Tv 155mL, I:E 1:2, CPAP 4 cmH₂O) patients using a BiPAP/CPAP ventilator (V60 BiPAP/CPAP, Philips). NIPPV also was simulated for adults (BPM 15, IPAP 20 cmH2O, EPAP 5 cmH2O, Max Vol. 500mL) and paediatrics (BPM 25, IPAP 15 cmH2O, EPAP 5 cmH2O, Max Vol. 155mL). Salbutamol (2.5ml of a 1mg/ml conc.) was nebulized and the percentage inhaled dose characterized following elution from a distal filter and spectrophotometric analysis (276nm).

The Nivo achieved the highest respirable dose of the three devices tested in both adult and child CPAP and NIPPV settings. It should also be noted that although the Nivo and pMDI achieved comparable delivery efficiencies in NIPPV the pMDI delivers a much lower dose as each activation only delivers 100 mcg.

1 Respiratory department, Tripoli Medical Centre, Tripoli, Libya 2 School of Pharmacy, Tripoli University, Tripoli, Libya P-093 KNOWLEDGE OF HEALTH CARE PROFESSIONALS ON THE USE OF DIFFERENT INHALER DEVICES

Aerosol inhalation as a method of drug delivery to the respiratory tract has become well established in the treatment of asthma and COPD. There are many types of inhaler devices (IDs) now available for delivering treatment. Inefficient inhaler technique is common patient problem resulting in poor drug delivery, decreases disease control and increases inhaler use. The aim of the study was to evaluate practical knowledge and understanding of the use of IDs among the health care professionals (HCPs). 170 HCPs from different hospitals, and private pharmacies were included in the study. Fig 1 shows the knowledge of HCPs on the important steps of using different IDs. It shows that the physicians, mainly consultants, have more knowledge on the use of IDs than the other HCPs and this may be due to the recurrent visits of medical representatives of the pharmaceutical company to the physicians only. It also shows that the pharmacists have poor knowledge, which may contribute to that only 59% of the people working in the private pharmacy are pharmacists and the others are from other different specialties. From the results, we conclude that most of the HCPs have poor knowledge on the use of the different ID, which may affect the efficacy of treatment of patients with asthma and COPD. The deficiencies noted in the present study highlight the need for a system to provide information to the HCP. One possibility is mandatory continuing medical education programs to maintain proficiency.

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1 Gilead Sciences Inc., SEATTLE, WA, United States of America 2 Parion Sciences Inc., DURHAM, NC, United States of America 3 Nucleus Network, Ltd., MELBOURNE, Australia 4 Cardeas Pharma, SEATTLE, WA, United States of America 5 Division of Nephrology, University of Alabama at Birmingham, BIRMINGHAM, AL, United States of America 6 Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, CHAPEL HILL, NC, United States of America P-095 ACUTE HYPERKALEMIA ASSOCIATED WITH INHALATION OF A POTENT ENaC INHIBITOR

Inhaled epithelial sodium channel (ENaC) blockers are designed to increase airway surface liquid volume, thereby benefiting cystic fibrosis patients. This randomized, double-blind, placebo-controlled, parallel-group, residential, Phase 1 study evaluated the safety, tolerability, and pharmacokinetics of multiple doses of ENaC blocker (GS-9411) in healthy adult volunteers. Inhaled GS-9411 (2.4, 4.8, 9.6 mg) or placebo was dosed twice daily (14 days). 86.1% of treated participants completed dosing (n=31/36). Cough and dizziness (27.8% participants each; most of mild severity) were the most commonly reported adverse events, and occurred both in placebo and GS-9411 treatment groups.

Serum potassium levels exceeded the upper limit of normal (>5 mmol/L), 4 hours after the morning dose in GS-9411 (n=16/24) and placebo (n=4/12) treatment groups (38 incidences total). Retesting revealed potassium levels had returned to normal within 2–3 hours. Arrhythmias were not observed for GS-9411-treated participants and electrocardiographic changes were not considered clinically significant. In urine electrolyte analyses, obtained 0–6 hours after the Day 1 morning dose, mean sodium/potassium ratios significantly increased from values 0–6 hours before dosing. Increased urine sodium/potassium ratios corresponded with high urine concentrations of active GS-9411 metabolites, which presumably inhibited sodium reabsorption in the kidney, leading to the observed transient hyperkalemia in these participants. Inhaled GS-9411 was well tolerated except for the emergence of transient clinically significant hyperkalemia. Design of new ENaC blockers should seek to provide a sustained improvement in mucociliary clearance, while reducing renal exposure to ENaC blockade.

Acknowledgements: This study was sponsored by Gilead Sciences, Inc.

1 MicroDose Therapeutx, MONMOUTH JUNCTION, NJ, United States of America 2 Moerae Matrix, MORRISTOWN, NJ, United States of America P-097 AEROSOL PERFORMANCE OF SPRAY DRIED MMI-0100 FROM THE MICRODOSE INHALER FOR TREATMENT OF PULMONARY FIBROSIS

MMI-0100, a cell-permeant peptide inhibitor of MAPKAP kinase 2 (MK2), is in preclinical development for treatment of idiopathic pulmonary fibrosis (IPF) and other, acute fibrotic indications. In the industry standard bleomycin murine model of pulmonary fibrosis, MMI-0100 has been demonstrated to inhibit the development of fibrosis (prevention) as well as to arrest the progression of established fibrosis (treatment). It was of interest to determine whether dry powder inhalation utilizing MicroDose Therapeutx (MDTx) delivery technology would also be feasible as a commercial MMI-0100 product formulation. MMI-0100 was prepared by aqueous spray drying as neat peptide or co-formulated with trehalose. Range-finding spray drying experiments were focused towards the generation of particles having a D₅₀ of 1.1 microns (laser diffraction), consistent with peripheral airways deposition. Spray dried MMI-0100 was characterized via scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffraction. Pulmonary delivery was optimized with the MDTx Dry Powder Inhaler (DPI) over a loaded dose range of 5 mg to 10 mg of the neat peptide, and generated consistent aerosol performance with small particle size (MMAD=∼2.1 microns, GSD=1.5), high efficiency (Fine Particle FractionⅥ70%), that is deemed appropriate for the IPF patient population. Finally, stability studies at accelerated conditions (40 °C/75% RH) for 4 weeks confirmed the absence of physical and chemical changes for spray dried MMI-0100.

Overall, the results demonstrate that MMI-0100 can be spray dried to a sufficiently small particle size that is stable and can be efficiently delivered using the MDTx DPI.

1 Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland 2 PARI Pharma GmbH, Starnberg, Germany P-099 BIOKINETIC STUDIES OF LIPOSOMAL CICLOSPORIN A IN HUMAN LUNG CELLS IN VITRO USING AN AIR-LIQUID CELL EXPOSURE SYSTEM

Ciclosporin A (CsA) is an immunosuppressive drug widely used to reduce rejection of transplanted organs such as the lungs. To reduce severe side effects targeted delivery of CsA to the site where the drug is needed would be most desirable. However, interaction of CsA with lung cells and its molecular mechanisms are poorly understood.

The behaviour of liposomal CsA (L-CsA) at the epithelial barrier was studied in vitro by exposing human lung epithelial cells (A549 and 16HBE140 cell lines) to L-CsA at the air-liquid interface by using a sophisticated exposure system. Uptake into cells and translocation of L-CsA was determined by HPLC-MS/MS. Cell viability, epithelial tightness and cell morphology were assessed as well as the release of the (pro)-inflammatory chemokine interleukin-8 (IL-8).

First results showed that aerosolization of different amounts of L-CsA were homogenous and reproducible. Cell viability after 24h post-exposure was not impaired and immune fluorescence stainings revealed the typical epithelial cell morphology in control as well as in L-CsA exposed cells. IL-8 levels were not elevated under any conditions. After 24h only low L-CsA concentrations were found inside the cells and most of the L-CsA was translocated across the epithelial barrier. The applied exposure system combined with lung epithelial cells cultured at the air-liquid interface offers an excellent tool to study the effects of inhalable substances such as CsA under realistic conditions. Further studies are ongoing by comparing different lung cell models as well as different CsA formulations.

Acknowledgements: This study was financed by PARI Pharma GmbH and the Adolphe Merkle Foundation.

1 Vall d'Hebron University Hospital, Barcelona, Spain 2 La Fe University Hospital, Valencia, Spain 3 Virgen de la Arrixaca University Hospital, Murcia, Spain 4 Cruces University Hospital, Baracaldo, Spain 5 La Princesa University Hospital, Madrid, Spain 6 12 de Octubre University Hospital, Madrid, Spain 7 La Paz University Hospital, Madrid, Spain 8 Ramón y Cajal University Hospital, Madrid, Spain 9 Virgen de Candelaria University Hospital, Sta. Cruz de Tenerife, Spain 10 Niño Jesús Hospital, Madrid, Spain P-100 MONITORING ADHERENCE TO THE INHALED ANTIBIOTIC TREATMENT AND LUNG FUNCTION IN PATIENTS WITH CYSTIC FIBROSIS (CF)

RATIONALE: Ineb AAD records information on how the device is used. It's authorized for inhaled administration of colistimethate sodium (CMS) in CF patients in Spain (Promixin®, Profile, UK). The main aim of the study was to describe the adherence to the inhaled antibiotic treatment and lung function in CF patients.

METHODS: Retrospective study carried out at 10 CF Spanish centers. Patients with CF colonized with Pseudomonas aeruginosa (PA), in treatment with CMS administered by Ineb for at least 12 months, were included. At baseline, lung function, anthropometric data and long-term treatments were recorded. During 12 months, lung function, antibiotic courses and microbiology in respiratory samples were obtained from medical records. Information about Ineb use was downloaded to calculate adherence to the treatment.

RESULTS: Data from 108 patients with CF were analyzed (mean age: 24.5±10.2 years; range: 4–57; 38.9% female). Mean inhaled antibiotic adherence for this sample of CF patients was 65.9±34.7%. The evolution of % predicted FEV1 in the total population and in each level of adherence to the treatment over 12 months is described in next table.

CONCLUSIONS: Mean annual decline of lung function in this population of patients receiving continuous inhaled CMS was 1.6% and seems to be influence by of the level of adherence to the inhaled antibiotic treatment in the lung function of the patients.

1 Research Unit of Experimental Physiology, Cruces University Hospital, Bilbao, Spain 2 Neonatal Unit, Cruces University Hospital, Bilbao, Spain P-101 SURFACTANT AEROSOL TREATMENT OF PRETERM LAMBS WITH SEVERE RESPIRATORY DISTRESS SYNDROME

Background: Tracheal bolus Surfactant (SF) instillation is the standard method used to treat RDS. However, it needs tracheal intubation and is linked to “peridosing adverse events”. In order to avoid side effects, SF aerosolization has been proposed as an alternative to intratracheal instillation.

Aim: To compare the acute and sustained effects of aerosolized vs. bolus SF dosing on gas exchange, lung mechanics and hemodynamics in premature lambs with severe RDS.

Methods: 21 preterm lambs (85%gestation) were randomly assigned to receive aerosolized-SF (Curosurf®, 200 mg/kg during 20 min) via an intratracheal aerosol generating catheter coupled to a jet nebulizer (SF-Aero), an intratracheal bolus of SF (SF-Bolus) or just intermittent mechanical ventilation (control). Gas exchange, lung mechanics and cerebral hemodynamic were measured for 6 hrs. Mean±SD, ANOVA, p<0.05.

Results: After 60 min, both SF-treated lamb groups showed a significant improvement over controls in terms of gas exchange and lung mechanics (p<0.05). In the SF-bolus group PaCO2 and CBF increased (p<0.05) immediately after the SF bolus (at 15 min: PaCO2 101±21 mmHg CBF 231±52 ml/min) whereas in the SF-Aero group there was a gradual decrease of PaCO2 and CBF (at 15 min: PaCO2 76±24 mmHg CBF 178±76 ml/min).

Conclusion: In preterm lambs with RDS, SF aerosolization improves pulmonary mechanics and gas exchange without the evidenced acute haemodynamic changes observed in the SF-Bolus group.

1 Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, GRONINGEN, The Netherlands 2 Beatrix Children's Hospital, University Medical Center Groningen, GRONINGEN, The Netherlands P-104 INVESTIGATION OF THE APPLICABILITY OF DRY POWDER INHALATION IN SCHOOL CHILDREN

Children are an important target group for inhalation therapy, but little is known about their intellectual and inspiratory capacities to operate dry powder inhalers (DPIs). Most studies so far have focused either on a specific DPI, or on (single) inhalation parameters and how these are affected by airflow resistance, which leaves many questions for the development of new devices for the pediatric population unanswered. In this study, we investigated the applicability of DPIs in children by a more systematic approach by using a test inhaler with variable airflow resistance and exchangeable mouthpiece design. The test inhaler was instrumented to record inhalation curves and equipped with fiberglass optics (sinuscope) to study the passageway for the aerosol through the mouth cavity. We examined for 103 school children (aged 4–12 years) whether they were able to use the test inhaler according to the instructions given. Nearly all (91) were able to perform one or more correct inhalations. We computed PIFs, inhaled volumes, and inhalation times from in total 256 recordings, and tested relationships with height and airflow resistance upon significance. We also noted preferences for airflow resistance and mouthpiece design, and investigated how these variables affect the inhalation maneuver. Ultimately, we aim to use the data from this study not only for prescribing the most appropriate type of currently marketed DPI to children, but particularly to design a new DPI that performs well within the range of inspiratory capacities (PIF and inhaled volume) to be expected for children.

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Figure 1.

Colistimethate sodium activity (bioassay; dashed lines) or equivalent (HPLC; solid lines) for each breathing pattern, plotted by nebulizer.

1 MAP Pharmaceuticals, Inc; Mountain View, CA, USA 2 University of Western Australia; Perth, WA, AUS P-108 EVALUATION OF A BREATH SYNCHRONIZED pMDI IN CHILDREN WITH ASTHMA AGED 3–11 YEARS

Introduction: Many children use traditional press-and-breath pressurized metered dose inhalers (pMDIs) that require bulky spacer devices to reduce oropharyngeal deposition. A breath synchronized pMDI has been developed that removes the need for a spacer while simultaneously coordinating drug delivery while the child inhales.

Methods: 70 children with asthma aged 3–11 were provided with simple instructions to perform single inhalations maneuvers through two configurations of a breath synchronized pMDI, the TEMPO^® inhaler. One configuration had a circular mouthpiece evaluated in several clinical trials with adults (TEMPO-A); the second configuration had a proprietary pediatric oval mouthpiece (TEMPO-P). Inhalers were attached to a pneumotach (Hans Rudolph, USA) connected to a spirometer (Korr Medical, USA) at the mouthpiece of the device. Inhalation profiles were recorded and the ability to trigger the TEMPO's self-actuating mechanism was quantified.

Results: Subjects aged 3–5 years had low to moderate success triggering both TEMPO-A (16–48%) and TEMPO-P (20–47%). Subjects aged 6–8 years had a good to high degree of success at triggering both TEMPO-A (68–86%) and TEMPO-P (68–77%). Subjects aged 9–11 years had a high degree of trigger success with both TEMPO-A (80%) and TEMPO-P (92%).

Conclusion: With minimal training and a set of basic instructions, children with asthma age 6–11 have demonstrated the ability to successfully use a breath synchronized pMDI with two different mouthpiece configurations. Children aged 5 and under demonstrated moderate success and may require more training as they transition off nebulizers.

1 Trudell Medical International (TMI), LONDON, ON, Canada 2 Western University, LONDON, ON, Canada 3 Robarts Research Institute, LONDON, ON, Canada P-109 DEVELOPMENT AND INITIAL VALIDATION OF THE CHILD AEROSOL DELIVERY TO ANATOMICAL MODEL (ADAM-III)

The ADAM series of face models was developed to provide clinically pertinent apparatuses for the evaluation of valved holding chambers, and nebulizers equipped with a facemask as patient interface. The child model had open oral and nasopharyngeal airways, obtained from a 4-year old by magnetic resonance imaging. This approach permitted the realization of the facial profile together with the underlying bone structure. The model was constructed at TMI from the 3-dimensional imaging data and the same processes for the infant model were used to create the overlying soft facial tissues. Previous studies had confirmed the mechanical responsiveness to the application of a facemask with a known applied force was within clinical expectations. The model (with surfactant-coated airways simulating the mucosa) was validated by delivering a known mass of salbutamol (1000 μg) via pMDI with antistatic AeroChamber Plus^® VHC/inspiratory flow indicator with child facemask applied to the face with a force of 1.6 kg, with a filter located at the model exit (equivalent to just above the carina) to collect delivered aerosol. The model was coupled to a breathing simulator (ASL 5000, IngMar Medical, Pittsburgh, PA) mimicking small child tidal breathing (tidal volume=155 ml, duty cycle=33 %, rate/min=25). Salbutamol (μg) was recovered from the pMDI (209.8±16.3) within the VHC and facemask (347.6±2.6), on the face (19.1±5.8), from the model airways (39.8±8.7), at the model exit (247.9±49.8), and assayed by a validated HPLC-UV spectrophotometric method. Total mass recovery was 86.4±4.0%.

1 University of Florida, Gainesville, FL, United States of America. 2 Boehringer Ingelheim Pharma GmbH & Co. KG, Germany. P-110 DEVELOPMENT OF AN IN VITRO TEST METHOD FOR DISSOLUTION OF INHALED CORTICOSTEROIDS

In vitro dissolution testing on an inhaled drug can provide some predictive estimates of its behavior in vivo and thus we aim to develop a robust in vitro test that could be used for IVIVC as well as quality testing. In order to simulate the fluid-capacity-limited dissolution of inhaled drug products, a suitably modified Transwell® system was developed and used to perform in vitro dissolution experiments. In order to mimic the in vivo conditions, the polycarbonate membrane in the Transwell® was replaced with a filter paper, which eliminated the role of diffusion as the rate limiting factor. Size differentiated fractions of drugs, obtained using a cascade impactor, were used for experiments. The rates with which a corticosteroid solution and the corresponding dry powder particles entered the receptor compartment differed significantly (f2=29.7) only when filter paper was used instead of the polycarbonate membrane. Thus we confirmed that the rate-limiting step in this system was dissolution as opposed to diffusion. Experiments were also done to study the sensitivity of this in vitro method to differences in drug loading and particle size and to explore the effect of stirring on the dissolution profiles. The dissolution data for both, hydrophilic and hydrophobic, drugs, in 0.5% SDS solution in PBS as the medium, were in agreement with their respective physicochemical properties and were strongly related to their in vivo absorption rates measured in pharmacokinetic studies.

1 Department of Pharmaceutics, University of Florida, Gainesville, FL, USA 2 Department of Statistics, University of Florida, Gainesville, FL, USA P-111 EVALUATION OF THE FEASIBILITY OF PHARMACODYNAMIC BIOEQUIVALENCE STUDIES OF INHALED CORTICOSTEROIDS THROUGH MONTE CARLO SIMULATIONS

Background: To establish bioequivalence (BE) of generic inhaled corticosteroids (ICS) at the site of action, the FDA recommends pharmacodynamic/clinical studies (e.g. monitoring effects on exhaled nitric oxide (eNO)).

Aim: To evaluate the feasibility of such studies, we developed a clinical trial simulation model that was able to predict the effects of ICS on eNO incorporating its variability, and the subsequent outcome of BE studies.

Methods: Crossover studies were simulated for sample sizes ranging from 16–64 patients and a wide range of doses of identical test (T) and reference (R) products. Within- and between-subject variability estimates were obtained from literature. The mean responses for R and T doses were obtained from an Emax model with relevant E_max and ED₅₀ values and data were analyzed on the dose scale as recommended by the FDA. The study power, defined as the % of the simulated datasets showing bioequivalence, was then calculated via bootstrap procedure (R 2.14.1).

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Results: Higher power was obtained when the test dose was close to the ED₅₀. When the BE criteria was 0.8–1.2, the highest power was 45% using a sample size of 64 patients (a large sample size considering the strict exclusion criteria for such studies). For relaxed BE criteria of 0.7–1.3, it was 80%.

Significance: Our findings suggest that a large number of subjects are required to show BE using traditional criteria of 0.8–1.2, warranting a revision of the BE criteria. This simulation approach can be applied to optimize BE studies design.

1 Adolphe Merkle Institute, University of Fribourg, MARLY, FR, Switzerland 2 Bern University for Applied Sciences, NIDAU, BE, Switzerland 3 Technik Thermische Maschinen, NIEDERROHRDORF, AG, Switzerland 4 Swiss Federal Laboratories for Materials Science and Technology (EMPA), DÜBENDORF, ZH, Switzerland P-117 A NEW STANDARD APPROACH FOR RAPID AND RELIABLE SCREENING OF EXHAUST EMISSION TOXICITY IN 3D LUNG MODELS IN VITRO

The finding that air pollution profoundly affects human respiratory health has led to increasingly stringent exhaust emission legislations for internal combustion engines. As a consequence, new strategies for improving engine combustion and for exhaust after-treatment have been developed

Estimating their actual effects on emission toxicity however, is very difficult. Since standardized protocols for in vitro testing are lacking, it relies in many aspects on epidemiological studies, which suffer from the inability to differentiate effects of different emission sources, are very time-consuming and cost-intensive.

We have established and optimized an exposure system for exhaust toxicity assessment in human lung cells in vitro which allows taking complete exhaust samples at the tailpipe of an engine of choice and their immediate use for exposure experiments under any desired condition. Using a 3D model of the human airway epithelium as a biological system (consisting of epithelial cells (cell line 16HBE14o^-) and human blood derived macrophages and dendritic cells) and a valuable battery of tests to determine different endpoints such as cytotoxicity, oxidative stress and (pro)inflammation, we have performed exposure experiments using a diesel passenger car operated under various settings (e.g. diesel particle filter, biodiesel, fuel additives, different lubrication oils) as a test vehicle.

We will present data that show that because of its versatility, its high robustness and its sensitivity, the combination of our air-liquid exposure system and 3D lung cell culture model provides an adequate tool for fast and reliable investigation of exhaust toxicity as an alternative standard protocol for animal testing.

Acknowledgments: The project is funded by the Swiss Federal Office for the Environment, VSS lubes and Swissoil.

1 Adolphe Merkle Institute, Université de Fribourg, MARLY, Switzerland 2 Walter Brendel Centre, Ludwig-Maximilians-Universität München, MUNICH, Germany 3 Inorganic Chemistry and Center for Nanointegration, Universität Duisburg-Essen, ESSEN, Germany 4 Institute of Lung Biology & Disease, Helmholtz Zentrum München, MUNICH, Germany P-118 EFFECTS OF SILVER NANOPARTICLES ON PRECISION-CUT SLICES FROM RAT LUNGS AND AN IN VITRO MODEL OF THE HUMAN EPITHELIAL AIRWAY BARRIER

Silver nanoparticles (Ag-NPs) are widely known for their antimicrobial properties. However, the interaction of Ag-NPs with mammalian systems is currently not fully understood. Exposure via inhalation is of primary concern for humans in occupational settings. Here, potential cytotoxic and (pro)-inflammatory effects of 70nm Ag-NPs were investigated by aerosolization onto the surface of a human epithelial airway barrier in vitro composed of epithelial as well as two types of immune cells, i.e. monocyte-derived macrophages and dendritic cells. Different Ag-NP concentrations (0.22–22 mM) and post-exposure times (4 and 24 hours) were applied. Cell viability and (pro)-inflammatory responses were investigated using LDH- and ELISA-assays. To compare these in vitro findings with an ex vivo approach, 250μm precision-cut slices from rat lungs were exposed to 5–30 μg/ml 70nm Ag-NPs under submerged culture conditions in vitro. For both approaches no cytotoxicity was observed. Furthermore, there was no elevated release of the (pro)-inflammatory markers TNF-α and IL-8. Taken together, Ag-NPs do not exert cytotoxic or (pro)-inflammatory effects in both ex vivo cultured precision-cut slices from rat lungs and an in vitro model of the human epithelial airway barrier. In conclusion, these findings demonstrate that both complex cell culture models represent validated techniques to significantly reduce the number of animal experiments.

Acknowledgements: Grant funding by the Federal Office of Public Health Switzerland, the Swiss National Science Foundation, the Deutsche Forschungsgemeinschaft (DFG SPP 1313) and the Adolphe Merkle Foundation.

1 Applied Research Associates, RALEIGH, NC, United States of America 2 British American Tobacco, Group R&D, Regents Park Road, Southampton, SO, 15 8TL, UK P-122 CFD SIMULATIONS OF MAINSTREAM CIGARETTE SMOKE PARTICLE GROWTH AND DEPOSITION IN THE ORAL AIRWAYS

An accurate quantification of cigarette smoke deposition in lung airways depends on the amount depositing in the mouth. Significant deposition (>20%) of mainstream cigarette smoke was measured in the upper airways, yet the reasons for this high deposition are not fully understood. A 3D computational fluid dynamics (CFD) model of the mouth and throat region was developed from CT scans. Steady-state airflow rates of 1.05 and 2.5 L/min were used to simulate low-flow puffing scenarios (ANSYS Fluent, v14.0). Transport and deposition of sub-micrometer particles released from a cigarette-sized inlet at the mouth opening was simulated. Predicted deposition of 200–500 nm particles in the oral cavity due to inertial impaction, sedimentation, and diffusion was <6%. The CFD model was used to simulate the size change of particles due to coagulation and condensation of water vapor, and also to study deposition by thermophoresis. At concentrations of 10^9 particles/ml, particles were predicted to increase in size by approximately 50% due to coagulation. With relative humidity in the mouth cavity>90%, particles were predicted to increase in size by ∼1.5-fold due to hygroscopic growth. At a smoke temperature of 40C, thermophoretic effects on particle deposition were negligible. Deposition of 200–500 nm particles with these additional deposition mechanisms implemented still resulted in oral deposition<10%. These simulation results suggest that high deposition in the upper airways could be due to other factors such as cloud effect or enhanced coagulation during mouth-hold and needs further investigation.

This study was funded by British American Tobacco.

1 Institute of Anatomy, University of Bern, BERN, Switzerland 2 Laboratory of Atmospheric Chemistry, Paul Scherrer Institute, VILLIGEN, Switzerland 3 Department of Civil and Environmental Engineering, University of Southern California, LOS ANGELES, CA, United States of America 4 Department of Chemistry, University of Cambridge, CAMBRIDGE, United Kingdom P-125 EFFECTS OF SECONDARY ORGANIC AEROSOLS FROM GASOLINE EXHAUST ON HEALTHY AND DISEASED RESPIRATORY EPITHELIA

Gasoline exhaust is an important source of atmospheric particles and supposedly contributes to respiratory diseases from inhalation exposure. Persons with pre-existing lung disease are more vulnerable. This study aimed at investigating acute responses of the respiratory epithelium after exposure to photochemically aged gasoline aerosols using advanced technologies from aerosol science and in-vitro toxicology. Exhaust of a gasoline car (EURO 5 standard) was introduced into a smog chamber and secondary organic aerosol (SOA) was produced upon irradiation with artificial sunlight by photochemical processes. Air from the smog chamber was then transferred via a versatile aerosol concentration enrichment system (VACES) into the aerosol deposition chamber. Adjusting the enrichment of particles by up to a factor of 50, the dose of deposited particles on cell cultures could be varied over a broad range. SOA was deposited out of a continuous air-stream simultaneously on 12 individual cell cultures by electrostatic deposition under controlled conditions (85–95% RH and 37°C). Re-differentiated human bronchial epithelia (HBE) with established air-liquid interface and the bronchial cell line BEAS-2B were exposed to the aerosols for 2 hours. First results give evidence for cytotoxic effects of gasoline SOA. In addition, different responses of cell cultures from a donor with lung disease were observed compared to healthy HBE and BEAS-2B cells. The combined technologies of aerosol science and in-vitro toxicology provide a highly realistic system for toxicity testing of environmental aerosols.

Acknowledgements: Grant funding by the Swiss National Science Foundation