The Endothelin-A Receptor Antagonist Zibotentan Induces Damage to the Nasal Olfactory Epithelium Possibly Mediated in Part through Type 2 Innate Lymphoid Cells

Abstract

Zibotentan, an endothelin-A receptor antagonist, has been used in the treatment of various cardiovascular disorders and neoplasia. Castrated athymic nude mice receiving zibotentan for a preclinical xenograft efficacy study experienced weight loss, gastrointestinal bloat, and the presence of an audible respiratory click. Human side effects have been reported in the nasal cavity, so we hypothesized that the nasal cavity is a target for toxicity in mice receiving zibotentan. Lesions in the nasal cavity predominantly targeted olfactory epithelium in treated mice and were more pronounced in castrated animals. Minimal lesions were present in vehicle control animals, which suggested possible gavage-related reflux injury. The incidence, distribution, and morphology of lesions suggested direct exposure to the nasal mucosa and a possible systemic effect targeting the olfactory epithelium, driven by a type 2 immune response, with group 2 innate lymphoid cell involvement. Severe nasal lesions may have resulted in recurrent upper airway obstruction, leading to aerophagia and associated clinical morbidity. These data show the nasal cavity is a target of zibotentan when given by gavage in athymic nude mice, and such unanticipated and off-target effects could impact interpretation of research results and animal health in preclinical studies.

Keywords: ILC2; TH2-mediated; endothelin-A receptor antagonist; gavage injury; nasal; olfactory epithelium; zibotentan.

Figure 1:

Body weight measurements of zibotentan-treated and vehicle control male castrated and intact male athymic nude mice. A linear mixed effect model regression was used to determine the effect of time, castration controlled for time, and drug treatment controlled for time on weight gain in mice across the various treatments. Castrated animals showed reduced weight gain compared to intact animals over time (p = 1.39e⁻⁰⁷). Zibotentan-treated animals showed reduced weight gain compared to vehicle treated animals over time (p = 9.68e⁻⁰⁹). Castration and zibotentan combination treatment had also had a significant effect leading to weight decline compared to either treatment alone (p = 2.03e⁻⁰⁹). Regression model output can be found in Table 2.

Figure 2:

Radiograph of a castrated zibotentan-treated athymic nude male mouse. There is diffuse gas distention within the stomach, small and large intestines.

Figure 3.

Characteristic histopathologic features of olfactory epithelial lesions initiated by administration of zibotentan by gavage in male castrated athymic nude mice. Olfactory epithelial hyaline droplet accumulation (arrowheads) within the dorsal meatus at level III (A) and ethmoid turbinates at level IV (B), with diffuse loss of olfactory epithelial layers. (C) Degeneration and necrosis of olfactory epithelium with accompanying cellular debris and apoptotic bodies (arrowheads). (D) Atrophy of olfactory nerves (arrowheads) accompanying severe olfactory epithelial atrophy and necrosis. (E) Accumulation of luminal protein adjacent to areas of severe hyaline droplet accumulation. (F) Severe mixed inflammatory cell infiltrates expanding the submucosa of the ethmoid turbinates, underlying zones of olfactory epithelial hyalinosis.

Figure 4.

Characteristic histopathologic features of respiratory epithelial lesions initiated by administration of zibotentan by gavage in male castrated athymic nude mice. (A) Zones of respiratory epithelial hyaline droplet accumulation along the nasal septum (arrowheads), accompanied by mixed inflammatory infiltrates (B) expanding the submucosa (asterisk). Respiratory epithelial hyperplasia was evidenced by multifocal folding and undulation, or zones of mucosal expansion by numerous proliferative tubular profiles admixed with hyalinosis and inflammatory cell infiltrates (C). Squamous metaplasia (D) was observed as conversion of respiratory epithelium to squamous morphology (arrowheads), often associated with erosion and variable inflammatory infiltrates. Hyperplasia of nasal associated lymphoid tissue (NALT) was characterized by expansion of the submucosa at multiple levels of the nose with multifocal large sheets of well differentiated lymphoid cells (E), compared to normal NALT in control animals typically observed as small foci of lymphocytes within the submucosa (F) of the ventral meatus in level IV.

Figure 5.

Scatter plots of severity scores for olfactory epithelial lesions based on statistically significant lesion incidences. Severity scores are elevated in both intact and castrated animals for (A) hyaline droplet accumulation, (B) atrophy, (C) respiratory metaplasia, (D) necrosis, and (E) nerve atrophy in the olfactory epithelium of animals treated with zibotentan compared to vehicle controls.

Figure 6.

Immunohistochemical expression of YM1/CHI3L3, MBP, and OMP in zibotentan-treated and vehicle control male athymic nude mice. In olfactory epithelium of zibotentan-treated mice (A,C,E), there was diffuse positive cytoplasmic immunoreactivity to anti-YM1/CHI3L3 antibody in areas of olfactory epithelial hyalinosis (A), positive immunoreactivity of eosinophils to anti-MBP antibody (C), and diffuse loss of immunoreactivity to anti-OMP antibodies corresponding with loss of olfactory epithelium and nerves (E) compared to vehicle control animals (B, D, F).

Figure 7.

Immunohistochemical expression of Caspase-3, IL33, and GATA3 in zibotentan-treated and vehicle control male athymic nude mice. In olfactory epithelium of zibotentan-treated mice (A,C,E), there was punctate immunoreactivity of scattered apoptotic bodies to anti-Caspase-3 (CSP3) antibodies (A) and diffuse loss of nuclear immunoreactivity to ant-IL33 antibodies within degenerate olfactory epithelium compared to vehicle control animals (B,D). In areas of inflammation, there was multifocal immunoreactivity to anti-GATA3 antibodies (E) compared to vehicle control animals (F).