Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model

Abstract

Objective: More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin-mediated laryngeal damage in vivo.

Methods: Drug and pepsin binding and inhibition were screened by high-throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis.

Results: HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC₅₀ in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin-mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis.

Conclusions: Fosamprenavir and darunavir, FDA-approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects.

Level of evidence: NA. Laryngoscope, 133:S1-S11, 2023.

Keywords: LPR; Laryngopharyngeal reflux; darunavir; fosamprenavir; pepsin.

Figure 1.

Treatment schema of in vivo mouse study.

Figure 2.

Binding (a) and activity (b) curves of pepsin with HIV protease inhibitors.

Figure 3.. Pepsin and HIV protease inhibitor structural data.

Left panels: The active site of porcine pepsin with HIV protease inhibitor bound. The 2Fo-Fc electron density map contoured at 1.0σ is shown as magenta mesh and the 2Fo-Fc simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. Right panels: Schematic view of the active site with ritonavir bound showing potential hydrogen bonding interactions as green, dashed lines. Electron density maps were generated via POVSCRIPT and POV-Ray and schematic representation by MarvinSketch, and Adobe Illustrator.

Figure 4.. Laryngeal epithelial damage by pepsin and acid in vivo.

Representative specimens from treatment groups. Paired images at 40x (a-d) and 200x (e-h) magnification collected rostral to vocal folds, representing larynx: pH7 (a,e), pH4 (b,f), 0.3 mg/ml pepsin at pH7 (c,g), and 0.3 mg/ml pepsin at pH4 (d,h). (a,e) Normal respiratory columnar epithelium (arrow) about one cell layer thick with basal polarization of the nuclei and ciliated apical surfaces. (b,f) Reactive epithelium characterized by thickening (fat arrow) and focal squamous epithelia (long arrow) with loss of cilia. In other areas, relative thickening of the mucosa with moderately increased nuclear to cytoplasmic (N:C) ratio and irregular, condensed chromatin is seen. (c,g) Thickened respiratory epithelium with pseudostratification of the epithelial cells. Keratinization (arrow) is present in multiple foci. Significant increase in the N:C ratio with loss of nuclear polarization and reduction in the apical cilia is evident in several regions of this treatment group. (d,h) Respiratory epithelium is necrotic (arrow) and replaced by an inflammatory exudate. A brisk, acute inflammatory infiltrate infiltrates the submucosal area. Scale bars a-d = 100um; e-h = 50um.

Figure 5.. Fosamprenavir gavage and aerosol and darunavir aerosol prevent pepsin-mediated laryngeal damage in vivo.

Representative specimens at 400x. Solvent control group laryngeal epithelium was characterized by a single layer of respiratory epithelium with no reactive changes. In mice treated with pepsin-pH7, the laryngeal epithelium exhibited reactive epithelial changes and apoptotic debris. Fosamprenavir gavage and aerosol protected against pepsin-mediated laryngeal damage as indicated by normal histology in mice receiving fosamprenavir gavage or aerosol with saline (solvent), or fosamprenavir gavage or aerosol with pepsin-pH7. Darunavir gavage elicited mild reactivity (rare intraepithelial lymphocytes) in the saline treatment group; the darunavir gavage group with pepsin-pH7 appeared similar. Darunavir aerosol provided mild protection against pepsin-mediated damage. Epithelial injury was still present (mildly increased intraepithelial inflammatory cells and reactive epithelial cells), however no apoptosis was observed. Scale bar=200um.