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.

Multisite Analysis of Lesions in the Respiratory Tract of the Rat and Nonhuman Primate (Cynomolgus Monkey) Exposed to Air, Vehicle, and Inhaled Small Molecule Compounds.

This paper presents a review of the nature, range, and incidences of background pathology findings in the respiratory tract of cynomolgus monkeys and rats. Data were collected from 81 inhalation studies and 133 non-inhalation studies evaluated at 3 geographically distinct contract research organization facilities. The inhalation studies were comprised of 44 different small molecule pharmaceuticals or chemicals which were also analyzed in order to understand the patterns of induced changes within the respiratory tract. The lung was the most frequently affected organ in both species, with increased alveolar macrophages being the most common background and test article-related finding. In the upper respiratory tract (URT), inflammatory cell infiltrates were the most common background findings in the nasal cavity in monkeys. Induced URT findings were more frequent in rats than monkeys, with squamous metaplasia in the larynx, and goblet cell hyperplasia in the nasal cavity being the most common. Overall, the data revealed a limited pattern of response to inhaled molecules in the respiratory tract, with background and test article-related findings often occurring in the same regions. It is hoped that these data will assist in the interpretation of findings in the respiratory tract induced by novel inhaled small molecule entities.

Femoral Head Growth Plate Dysplasia and Fracture in Juvenile Rabbits Induced by Off-target Antiangiogenic Treatment.

Epiphyseal growth plate dysplasia (chondrodysplasia) might be considered as the pathognomonic feature of antiangiogenic treatment in preclinical species as it is reliably and dose-responsively induced in rodents and monkeys with vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor (FGF) receptor inhibitors, matrix metalloproteinase inhibitors, and vascular targeting agents. Here we report epiphyseal growth plate dysplasia in juvenile rabbits treated with an oral spleen tyrosine kinase inhibitor induced by off-target antiangiogenic inhibition of VEGF and FGF family kinase receptors. Epiphyseal growth plate dysplasia resulted in weakening and fracturing of the femoral head physis in 6 of 10 male and 1 of 10 female animals as well as microfracturing and dysplasia of the distal femoral articular cartilage in 1 male animal. Fracture lines ran through the zone of hypertrophic cartilage (as well as adjacent zones), were orientated parallel to the physeal plane, and often involved displacement of the femoral head. We would suggest that the high prevalence of growth plate fracture in the rabbit may represent a potential additional adverse risk to those already established for children treated with antiangiogenic therapy.

Comparison of computerized image analysis with traditional semiquantitative scoring of Perls' Prussian Blue stained hepatic iron deposition.

Image analysis is now routinely employed as a tool in toxicologic pathology to help quantitate end points of efficacy and safety. It is regarded as a proficient and a sensitive technique to generate numerical data that can be easily interrogated for statistical evaluation. Traditional semiquantitative pathology scoring on the other hand is sometimes regarded as less accurate due to the limitations of the scoring systems employed and the day-to-day variations often noted between pathologists. We therefore decided to generate an optimized histochemical staining and image analysis protocol to compare the accuracy of semiquantitative scoring with computerized image analysis. In order to achieve this, we describe a standardized protocol for staining and image analysis that eliminates or minimizes as many sources of error as possible. The results of this experiment demonstrate that despite consistent variations in scoring between two independent pathologists, correlation with image analysis data of 0.91 to 0.95 (Spearman's Rho test) was achieved. These data indicate that either image analysis or traditional semiquantitative scoring can generate accurate data. As a result of this, it appears that it is equally safe to employ either method dependent upon the complexity and the practicality of the task at hand provided that the experimental conditions are rigorously optimized and rigidly adhered to.