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We tested whether GV1001 has any ototoxic side effects at different doses and whether it protects hearing in an aminoglycoside-induced ototoxicity mouse model.GV1001, a novel peptide vaccine currently being examined in a Phase 3 clinical trial to treat pancreatic cancer, also has anti-inflammatory and antioxidant effects.In the first experiment, C57/BL6 mice were injected with GV1001 preparations at concentrations of 0.1 to 100 mg/kg for 7 days to evaluate the toxicity of GV1001 on the inner ear and kidneys. In the second experiment, the protective effect of GV1001 was tested in an ototoxicity mouse model that was generated by injecting 800 mg/kg kanamycin (KM) for 2 weeks. The hearing threshold and hair cell loss were compared between the KM + GV1001 group (treated with 10 mg/kg GV1001 for 2 wk) and the KM + saline group. The hearing threshold was measured before, and 7, 14, and 21 days after the initial treatment. The blood urea nitrogen level was measured.No ototoxicity or renal toxicity was found following treatment with different doses of GV1001 (0.1–100 mg/kg). The KM + saline group showed impaired auditory function and markedly disoriented and missing cochlear hair cells, while the KM + GV1001 group showed significant hearing and hair cell preservation in comparison (p < 0.05).GV1001 itself did not have any detrimental effects on the inner ear or kidney. In the KM induced ototoxicity model, concomitant administration of GV1001 protected against cochlear hair cell damage and preserve hearing.