The Role of Pepsin in Gastroesophageal Reflux Disease and its Extraesophageal Manifestations

: Towards a Novel Therapeutic Approach

Abstract

Introduction: Gastroesophageal reflux disease is a common disorder that reduces quality of life and productivity. The gastric protease, pepsin (optimal pH ~2), elicits significant injury in the context of acid reflux. Yet, given the ease of monitoring and modifying refluxate acidity and the acid-dependent activity of pepsin, acid has become the primary diagnostic and therapeutic target for gastroesophageal reflux disease. Growing awareness of extraesophageal syndromes linked to high pH (≥4) refluxate, persistent symptoms in medically acid-suppressed patients, and the rising incidence of reflux- attributed cancer despite widespread use of acid-suppressing medications, has called to question the primacy of acid in reflux disease. Above pH 6.5, pepsin is enzymatically inactive but endocytosed by aerodigestive tract epithelia and retained in acidic intracellular vesicles (pH 4-5) permitting its reactivation. The work herein examines the repercussions of pepsin pH 4-7 on aerodigestive tract epithelia and the preclinical efficacy of a pepsin-inhibiting therapeutic for reflux disease.
Methods: Pepsin protein and mRNA were assessed in specimens from patients with suspected reflux-attributed disease and healthy counterparts via Western blot and reverse transcriptase polymerase chain reaction. Effects of pepsin pH 4-7 were evaluated via real-time polymerase chain reaction, colony formation, flow cytometry, wound-healing, and RNA sequencing in vitro. Existing protease inhibitors were screened for pepsin inhibition and protection against peptic injury in a mouse and cell culture model.
Results: Pepsin was common in disease specimens, absent in controls. Pepsin pH 7 elicited proinflammatory and cancer-associated gene expression profiles and increased cell proliferation, migration, and colony formation in vitro. Fosamprenavir prevented laryngeal inflammation by pepsin pH 7 in vivo and epithelial barrier disruption by pepsin pH 4 in vitro.
Conclusions: This work supports the contribution of pepsin pH 4-7 to aerodigestive tract inflammatory injury and neoplasia and investigation of fosamprenavir as a novel pepsin- inhibiting therapeutic for reflux disease.

Date of Award	8 Oct 2025
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Murphy Wan (Supervisor) & James Brown (Supervisor)