Aspiration and Inspiration: Using Bronchoalveolar Lavage for Toxicity Assessment.

Bronchoalveolar lavage (BAL) is a simple procedure that is used to investigate drug efficacy or lung toxicity. It is sensitive to lung changes and less invasive than histological evaluation. It can be performed repeatedly at interim time points or as a terminal procedure. Airborne contaminants and purposely inhaled compounds, resident and inflammatory cells, as well as different cellular soluble products can be harvested in bronchoalveolar fluid (BALF) and measured. Bronchoalveolar lavage can also be an important tool to understand drug exposure and its metabolism in the lung, although it should be rigorously performed and interpreted with caution, especially in the context of regulated toxicology studies. This review focuses on the methods and uses of BAL in animal research, primarily in the pharmaceutical industry, as well as for the assessment of drugs, pollutants, and chemical lung toxicity. Methods of collecting and analyzing BALF and parameters affecting variability are discussed in detail. Improved automated methods for cell counting and analysis of the inflammatory cellular differential using hematology analyzers, common markers of lung injury, and new methodologies are described. Correlation between BALF and histological evaluation should not be considered as repetitive but as complementary assessments in the context of efficacy and toxicity studies.

BSTP Review of 12 Case Studies Discussing the Challenges, Pathology, Immunogenicity, and Mechanisms of Inhaled Biologics.

The inhalation route is a relatively novel drug delivery route for biotherapeutics and, as a result, there is a paucity of published data and experience within the toxicology/pathology community. In recent years, findings arising in toxicology studies with inhaled biologics have provoked concern and regulatory challenges due, in part, to the lack of understanding of the expected pathology, mechanisms, and adversity induced by this mode of delivery. In this manuscript, the authors describe 12 case studies, comprising 18 toxicology studies, using a range of inhaled biotherapeutics (monoclonal antibodies, fragment antigen-binding antibodies, domain antibodies, therapeutic proteins/peptides, and an oligonucleotide) in rodents, nonhuman primates (NHPs), and the rabbit in subacute (1 week) to chronic (26 weeks) toxicology studies. Analysis of the data revealed that many of these molecules were associated with a characteristic pattern of toxicity with high levels of immunogenicity. Microscopic changes in the airways consisted of a predominantly lymphoid perivascular/peribronchiolar (PV/PB) mononuclear inflammatory cell (MIC) infiltrate, whereas changes in the terminal airways/alveoli were characterized by simple ("uncomplicated") increases in macrophages or inflammatory cell infiltrates ranging from mixed inflammatory cell infiltration to inflammation. The PV/PB MIC changes were considered most likely secondary to immunogenicity, whereas simple increases in alveolar macrophages were most likely secondary to clearance mechanisms. Alveolar inflammatory cell infiltrates and inflammation were likely induced by immune modulation or stimulation through pharmacologic effects on target biology or type III hypersensitivity (immune complex disease). Finally, a group of experts provide introductory thoughts regarding the adversity of inhaled biotherapeutics and the basis for reasonable differences of opinion that might arise between toxicologists, pathologists, and regulators.

Symposium Summary: "Breathe In, Breathe Out, Its Easy: What You Need to Know About Developing Inhaled Drugs".

Developing inhaled drugs requires knowledge of lung anatomy, cell biology, respiratory physiology, particle physics, and some plumbing. Although dose makes the poison, in the context of an inhaled drug, the "dose" is not easily defined. This lack of clarity around dose poses issues and challenges in the design of inhalation toxicology programs. To better understand dose, the influence of ventilation is discussed as are the perturbations in pulmonary function observed with inhalation exposure that can affect dose. Methods for determining inhaled drug deposition to arrive at an estimate of lung dose are examined. Equally important to understanding dose are the techniques used to deliver aerosols to animals. With a better understanding of dose and inhalation exposure, species-specific histopathologic lesions, both common background and toxicologically significant lesions, are reviewed. Finally, insight into how regulators synthesize and evaluate these complex findings to assess clinical safety risks is presented.

Pathogenesis of mucous cell metaplasia in a murine asthma model.

Increased mucus production in asthma is an important cause of airflow obstruction during severe exacerbations. To better understand the changes in airway epithelium that lead to increased mucus production, ovalbumin-sensitized and -challenged mice were used. The phenotype of the epithelium was dramatically altered, resulting in increased numbers of mucous cells, predominantly in the proximal airways. However, the total numbers of epithelial cells per unit area of basement membrane did not change. A 75% decrease in Clara cells and a 25% decrease in ciliated cells were completely compensated for by an increase in mucous cells. Consequently, by day 22, 70% of the total epithelial cell population in the proximal airways was mucous cells. Electron microscopy illustrated that Clara cells were undergoing metaplasia to mucous cells. Conversely, epithelial proliferation, detected with 5-chloro-2-deoxyuridine immunohistochemistry, was most marked in the distal airways. Using ethidium homodimer cell labeling to evaluate necrosis and terminal dUTP nick-end labeling immunohistochemistry to evaluate apoptosis, this proliferation was accompanied by negligible cell death. In conclusion, epithelial cell death did not appear to be the stimulus driving epithelial proliferation and the increase in mucous cell numbers was primarily a result of Clara cell metaplasia.

Interleukin-9 induces mucous cell metaplasia independent of inflammation.

Interleukin-9 (IL-9) has been strongly implicated in the pathogenesis of asthma, including the overproduction of mucus, in humans and in animal models. We evaluated the inflammatory changes associated with the upregulation of mucus production by examining the time course of inflammation after daily intratracheal IL-9 administration to naive C57Bl6 mice for 9 d. IL-9 induced an asthmatic phenotype, which in general took several days to develop, as assessed by the measurement of airway hyperresponsiveness, pulmonary inflammation, and serum immunoglobulin E. However, within 24 h of a single dose of IL-9, muc5ac mRNA upregulation occurred, and increased numbers of periodic acid Schiff/Alcian blue-positive mucous cells appeared. This response occurred before the development of an inflammatory cell influx and was the result of epithelial metaplasia. It seemed that IL-9 evoked mucous cell metaplasia independent of IL-13 because mRNA tissue evaluation indicated that muc5ac upregulation preceded any increase in IL-13 mRNA expression or detectable levels of IL-13 in the brochoalveolar lavage fluid. Therefore, the upregulation of IL-13 by IL-9 may be responsible for the amplification of mucus production but is not required for its initiation. IL-9 seems to directly stimulate mucous cell metaplasia without the requirement of inflammatory cell influx.