A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations.

Absorption studies with aerosol formulation delivered by metered dose inhalers across cell- and tissue-based in vitro models of the pulmonary epithelia are not trivial due to the complexity of the processes involved: (i) aerosol generation and deposition, (ii) drug release from the carrier, and (iii) absorption across the epithelial air-blood barrier. In contrast to the intestinal mucosa, pulmonary epithelia are only covered by a thin film of lining fluid. Submersed cell culture systems would not allow to studying the deposition of aerosol particles and their effects on this delicate epithelial tissue. We developed a new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to mimic the inhalation of a single metered aerosol dose and its subsequent deposition on filter-grown pulmonary epithelial cell monolayers exposed to an air-liquid interface. The reproducibility of deposition of these dry powder aerosols and subsequent drug transport across Calu-3 monolayers with commercially available dry powder inhalers containing salbutamol sulphate or budesonide could be demonstrated. In the context of developing new dry powder aerosol formulations, PADDOCC appears as a useful tool, allowing reducing animal testing and faster translation into clinical trials.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier.

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

The Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) in vitro system: design and experimental protocol.

The development of aerosol medicines typically involves numerous tests on animals, due to the lack of adequate in vitro models. A new in vitro method for testing pharmaceutical aerosol formulations on cell cultures was developed, consisting of an aerosolisation unit fitting a commercial dry powder inhaler (HandiHaler(c), Boehringer Ingelheim, Germany), an air-flow control unit (Akita(c), Activaero, Germany) and a custom-made sedimentation chamber. This chamber holds three Snapwell(c) inserts with monolayers of pulmonary epithelial cells. The whole set-up, referred to as the Pharmaceutical Aerosol Deposition Device On Cell Cultures (PADDOCC) system, aims to mimic the complete process of aerosol drug delivery, encompassing aerosol generation, aerosol deposition onto pulmonary epithelial cells and subsequent drug transport across this biological barrier, to facilitate the investigation of new aerosol formulations in the early stages of development. We describe here, the development of the design and the protocol for this device. By testing aerosol formulations of budesonide and salbutamol sulphate, respectively, reproducible deposition of aerosol particles on, and the integrity of, the pulmonary cell monolayer could be demonstrated.

Pulmonary drug delivery: medicines for inhalation.

Mankind has inhaled substances for medical and other reasons for thousands of years, notably resulting in the cultural manifestations of tobacco and opium smoking. Over the course of time concepts of pulmonary application, including inhalation devices and drug formulations, have been and still are being continuously developed. State of the art instruments even allow for individualized drug application by adaptation of the inhalation procedure to the breathing pattern of the patient. Pulmonary drug delivery offers promising advantages in comparison to "classical" drug administration via the oral or transcutaneous routes, which is also reflected by an increasing interest and number of marketed products for inhalation therapy. However, the lungs' efficient clearance mechanisms still limit the benefit of many therapeutic concepts. In consequence the objective of current research and development in pulmonary drug delivery is to overcome and to control drug clearance from the intended target site. Here, several of the most auspicious future drug delivery concepts are presented and discussed in order to give the reader an insight into this emerging field of medicine.

Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles.

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

Drug transport across pulmonary epithelial cell monolayers: effects of particle size, apical liquid volume, and deposition technique.

BACKGROUND: Pulmonary cell cultures are increasingly used to predict in vivo drug absorption after inhalation, similar to intestinal cell culture models that have already been well established to predict oral drug absorption. In contrast to the intestinal barrier, however, the so-called air-blood barrier of the lung is covered only with a thin film of liquid, on which the aerosol particles are deposited. The aim of this study was to investigate the relevance of this apical liquid film on the drug absorption rate when deposited as a dry powder formulation on pulmonary epithelial cells in vitro. METHODS: Budesonide and salbutamol sulfate were chosen as model drugs, and for each drug three generic aerosol powder formulations were used. Filter-grown monolayers of the human bronchial epithelial cell line Calu-3 were used as a model, using various volumes of apical liquid. RESULTS AND CONCLUSIONS: Although proven to be bioequivalent in vivo for each of the two drugs, the generic dry powder fomulations showed strikingly different epithelial transport rates in vitro, depending on the amount of apical liquid and the deposition technique, and suggesting that the dissolution of the aerosol particles in the apical liquid volume was rate limiting for the overall absorption rate. However, we found that the absorption rates of the formulations were similar after aerosolization and deposition in a multistage liquid impinger, which simulates more realistically the detachment of the drug crystals from the carrier lactose and their aerodynamic particle size-dependent deposition in the respiratory tract following inhalation from a dry powder inhaler. These data demonstrate the need for improved in vitro test systems to allow deposition of aerosol particles on the air-liquid interface cultivated cell monolayers by simultaneously taking into account aerodynamic properties.

In vitro and in vivo performance of biocompatible negatively-charged salbutamol-loaded nanoparticles.

The development and performance of a novel nanoparticle-based formulation for pulmonary delivery has been characterized chronologically through the particle preparation process, in vitro testing of drug release, biocompatibility, degradation, drug transport in cell culture, and in vivo bronchoprotection studies in anaesthetised guinea pigs. This study demonstrates excellent agreement of the in vitro and in vivo experiments undertaken to prove the feasibility of the design, thereby serving as an example highlighting the importance of in vitro test methods that predict in vivo performance. Nanoparticles were prepared from the newly designed negatively-charged polymer poly(vinyl sulfonate-co-vinyl alcohol)-g-poly(d,l-lactic-co-glycolic acid) loaded with salbutamol free base. Average particle sizes of blank and drug-loaded nanoparticles prepared at the various stages of the investigations were between 91 and 204nm; average zeta potential values were between -50.1 and -25.6mV. Blank nanoparticles showed no significant toxicity, and no inflammatory activity was detected in Calu-3 cells. Sustained release of salbutamol from the nanoparticles was observed for 2.5h in vitro, and a prolonged effect was observed for 120min in vivo. These results demonstrate good agreement between in vitro and in vivo tests and also present a promising foundation for future advancement in nanomedicine strategies for pulmonary drug delivery.

Inhalative nanomedicine--opportunities and challenges.

Inhalation therapy is still limited by the low bioavailability of the administered drugs. Advantages of the pulmonary administration site like large resorption area, low enzymatic equipment, and circumvention of the first pass effect are set into perspective by the rigid barrier properties of the alveolar region. As a consequence, the systemic bioavailability of peptides and proteins is still relatively limited, even when administered by modern pharmaceutical aerosol technologies. In the context of advanced pulmonary drug therapy the use of nanoparticles as alternative to micronsized drug formulation could be of special interest, because nanoparticles seem to overcome some cellular barriers quite efficiently. Besides such outstanding permeation properties, nanoparticles may also hold promises to escape from pulmonary clearance mechanisms and to allow for cell-specific targeting within the lung. Such opportunities and challenges of inhalative nanomedicine are reviewed in this short review.

A novel cell compatible impingement system to study in vitro drug absorption from dry powder aerosol formulations.

A modified Astra type multistage liquid impinger (MSLI) with integrated bronchial cell monolayers was used to study deposition and subsequent drug absorption on in vitro models of the human airway epithelial barrier. Inverted cell culture of Calu-3 cells on the bottom side of cell culture filter inserts was integrated into a compendial MSLI. Upside down cultivation did not impair the barrier function, morphology and viability of Calu-3 cells. Size selective deposition with subsequent absorption was studied for three different commercially available dry powder formulations of salbutamol sulphate and budesonide. After deposition without size separation the absorption rates from the aerosol formulations differed but correlated with the size of the carrier lactose particles. However, after deposition in the MSLI, simulating relevant impaction and causing the separation of small drug crystals from the carrier lactose, the absorption rates of the three formulations were identical, confirming the bioequivalence of the three formulations.

In vitro systems for studying epithelial transport of macromolecules.

Biological barriers, typically, represented by epithelial tissues are the main hindrance against uncontrolled uptake of a variety of substances. However, the delivery across a biological barrier is a crucial factor in the development of drugs. As the permeability of macromolecular drugs is very limited, new delivery strategies have to be developed and further improved. Thereby, nanoparticle carriers offer an enormous potential for the controlled delivery of active substances into the organism. Besides an intensive study for the reason of risk assessment and toxicology, the possible transport enhancement caused by nanoparticles must be quantified. A powerful tool for these studies is in vitro cell culture models imitating the more complex in vivo situation under controlled conditions. We use polyethylenimine as model enhancer mimicking toxicological effects and altered barrier function in the epithelial in vitro model, Calu-3. Cytotoxicity assays based on different mechanisms and transport properties of a low-permeability marker with and without delivery enhancer are described.

Pulmonary cell culture models to study the safety and efficacy of innovative aerosol medicines.

BACKGROUND: Pulmonary cell culture models for the development of new aerosol medicines are attracting increasing interest. Ease of handling, ethical acceptance and high explanatory power are the main advantages of cell culture systems in pulmonary drug research. OBJECTIVE: Pulmonary cell culture models are described and evaluated regarding their suitability for the biopharmaceutical characterisation of innovative aerosol medicines. METHODS: The review focuses on the peculiarities of the pulmonary cell culture models arising from the specialised pulmonary epithelia, the clearance systems in the lung and the limited but functionalised lung fluid layers. Additional aerosol deposition systems suitable for the close to in vivo simulation of aerosol delivery on pulmonary cell cultures are described. RESULTS: Suitable cell culture models of the cellular part of the human air-blood barrier are established and well characterised. However, the physical barriers on top of the cellular barriers - surfactant, mucus and so on - and their influence on the safety and efficacy of aerosol medicines, are still underestimated.

Sulfur isotopes link overwinter habitat use and breeding condition in Double-crested Cormorants.

North American Double-crested Cormorant (Phalacrocorax auritus) populations have increased greatly. Both breeding and overwintering ground factors have likely contributed to these increases. However, demonstrating how overwintering conditions may affect breeding birds has not been possible because of the difficulty in linking breeding birds to their wintering grounds. Here, we demonstrate the utility of stable sulfur isotopes to elucidate overwintering habitat use by cormorants breeding on Lake Erie. Sulfur isotopes in feathers grown on overwintering grounds provided insights into the degree to which birds used freshwater vs. marine environments. The proportion of birds utilizing freshwater habitats increased through time. This change may have reflected increases in freshwater aquaculture (i.e., catfish) in the U.S. south. Examination of body condition in birds returning to breed on Lake Erie indicated that those individuals that solely used marine habitats for at least a portion of the overwintering period were in poorer condition than birds using freshwater. Enhanced foraging opportunities at aquaculture facilities may improve the fitness of individuals that have returned to breed after overwintering at such locations. This study is the first to demonstrate a linkage between overwinter habitat use and breeding ground parameters in Double-crested Cormorants. These results underscore that factors throughout the Mississippi flyway are likely acting together to regulate cormorant populations.

Temporal trends of young-of-year fishes in Lake Erie and comparison of diel sampling periods.

We explored temporal trends of young-of-year (YOY) fishes caught in bottom trawl hauls at an established offshore monitoring site in Lake Erie in fall during 1961-2001. Sampling was conducted during morning, afternoon, and night in each year. Catches per hour (CPH) of alewife (Alosa pseudoharengus) YOY were relatively low and exhibited no temporal trend. This result was consistent with the species' intolerance to Lake Erie's adverse winter water temperatures. Gizzard shad (Dorosoma cepedianum) YOY decreased sharply after 1991, which was consistent with recent oligotrophication of the lake. Following the establishment in 1979 and rapid increase of white perch (Morone americana) YOY, white bass (Morone chrysops) and freshwater drum (Aplodinotus grunniens) YOY decreased. Trout-perch (Percopsis omiscomaycus) YOY decreased during 1986-1991, but recovered to previous levels during 1991-2001. The recovery coincided with the resurgence of mayflies (Ephemoptera) in the lake. CPH of spottail shiner (Notropis hudsonius) and emerald shiner (N. atherinoides) YOY exhibited no temporal trend between 1961 and the late 1970s to early 1980s. CPH of yellow perch (Perca flavescens) YOY decreased during 1961-1988, and walleye (Sander vitreum) YOY increased overall during the time series. These observations were consistent with published studies of adults in the region. CPH of 4 of the 10 species of YOY considered were greatest during night. CPH for walleye YOY was higher in the morning than in the afternoon, but there was no significant difference between night and morning abundances. The results suggest that (1) CPH of YOY fishes may be a useful monitoring tool for Lake Erie, and (2) offshore monitoring programs that do not include night sampling periods may underestimate recruitment for several common species.

Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers.

In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive directions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent permeability coefficients (P(app); x10(-7)cm/s, mean+/-S.D.) for GLP-1 (6.13+/-0.87 (absorptive) versus 1.91+/-0.51 (secretive)), HSA (2.45+/-1.02 versus 0.21+/-0.31), TF (0.88+/-0.15 versus 0.30+/-0.03), and IgG (0.36+/-0.22 versus 0.15+/-0.16) were all strongly direction-dependent, i.e., net absorptive, while PTH (2.20+/-0.30 versus 1.80+/-0.77), GH (8.33+/-1.24 versus 9.02+/-3.43), and INS (0.77+/-0.15 versus 0.72+/-0.36) showed no directionality. Trichloroacetic acid precipitation analysis of tested molecules collected from donor and receiver fluids exhibited very little degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.