Hepatocyte growth factor mimetic confers protection from aminoglycoside-induced hair cell death in vitro

Hearing loss and vestibular dysfunction are common debilitating disorders resulting from a wide variety of conditions including genetic factors, aging, and exposure to ototoxic drugs. Aminoglycoside antibiotics are one of the most prescribed classes of ototoxic drugs and are, in some cases, first-in-class treatment for severe mycobacterial infections like drug-resistant tuberculosis (Durante-Mangoni et al. 2009; Avent et al. 2011) and exacerbated respiratory infections in patients with cystic fibrosis (Garinis et al. 2017). Exposure to aminoglycosides results in incidences of hearing loss up to 33% and vestibular dysfunction up to 15% of treated patients (Chen et al. 2007). Despite the discovery of ototoxic side effects over 60 years ago, aminoglycosides are still prescribed for many indications and there are no approved therapeutic options to prevent ototoxicity (Hettig and Adcock 1946; Schatz et al. 2005; Mukherjea et al. 2015; Steyger 2021).

Strategies to prevent aminoglycoside-induced sensory deficits include the development of new, less ototoxic aminoglycosides and the identification of compounds that, when co-administered, ameliorate the ototoxic component of aminoglycosides while preserving anti-bacterial efficacy (e.g., Kruger et al. 2015; O'Sullivan et al. 2017; Chowdhury et al. 2018; Kitcher et al. 2019). Growth factors have been a source of many identified otoprotectants (Yagi et al. 1999; Shoji et al. 2000; Hayashi et al. 2013). For example, TrkB agonists, neurotrophin-3 (NT3), or brain-derived neurotrophic factor (BDNF) can stimulate recovery of synapse numbers in noise-damaged hair cells (Wan et al. 2014; Suzuki et al. 2016; Szobota et al. 2019; Fernandez et al. 2021; Mukherjee et al. 2022). Further research demonstrates that NT3 over-expression can attenuate age-related synaptopathy in mice and that either NT-3 or BDNF reduce spiral ganglion neuron loss following aminoglycoside exposure (Staecker et al. 1996; Agterberg et al. 2008; Cassinotti et al. 2022). Collectively, these studies suggest that trophic factors may be excellent therapeutic targets in the auditory periphery.

Hepatocyte growth factor (HGF) is a potent neurotrophic factor with known roles in cell metastasis and survival (Ebens et al. 1996; Miyazawa et al. 1998). The HGF receptor c-MET is expressed in the mammalian cochlea and homeostatic HGF signaling is critical for the development of non-sensory cochlear structures and for hair cell maintenance (Kikkawa et al. 2009; Shibata et al. 2016; Morell et al. 2020). Mutations in either the HGF ligand or its receptor c-MET have been linked to non-syndromic hearing loss in humans demonstrating the critical role of endogenous HGF signaling in the ear (Schultz et al. 2009; Mujtaba et al. 2015; Bousfiha et al. 2019). Given the known pro-survival capacity of HGF and its presence in the ear, prior studies have examined the otoprotective capacity of HGF signaling. Local application of HGF-containing hydrogels protected guinea pig hair cells from exposure to high-intensity noise and reduced threshold shifts (Inaoka et al 2009). Viral gene delivery of constitutively expressed HGF protected rat cochlear hair cells from kanamycin exposure in vivo (Oshima et al. 2004). HGF also protected neonatal mouse cochlear explants from neomycin exposure, further validating the protective role of HGF against aminoglycoside ototoxicity (Kikkawa et al. 2009). Here, we examine the otoprotective potential of a small molecule HGF mimetic.

MM-201 (previously Dihexa) is a synthetic HGF mimetic that is blood-brain barrier permeable, stable, and orally bioavailable (McCoy et al. 2013). MM-201 facilitates activation of the HGF ligand through dimerization at the hinge region (Benoist et al. 2014). Treatment with MM-201 reversed scopolamine-induced memory deficits in rats and increased dendritic spine density, demonstrating potential for neuroprotection or neural repair (McCoy et al. 2013). Previously, we reported that MM-201 robustly protected zebrafish lateral line hair cells from aminoglycoside exposure (Uribe et al. 2015). MM-201 exposure did not alter aminoglycoside uptake into hair cells and inhibition of the c-MET receptor or cell survival targets downstream of c-MET activation decreased MM-201 protection, strongly implicating protection via activation of intracellular substrates (Uribe et al. 2015). Here, we continue to examine the otoprotective effects of MM-201 using mouse ex vivo and in vivo models of ototoxicity. We also asked if MM-201 alters the bactericidal efficacy of aminoglycosides.

We demonstrate that MM-201 protects adult mouse utricular explant hair cells from neomycin and gentamicin toxicity. MM-201 protection is decreased following inhibition of c-MET's downstream target mTOR. MM-201 does not alter the bactericidal efficacy of aminoglycosides, suggesting promise as a co-treatment strategy. In vivo, MM-201 did not attenuate kanamycin-induced hearing loss. Further, systemic administration of MM-201 alone was ototoxic at the doses tested. These results suggest that dosing and delivery of MM-201 used here may not be optimal as an otoprotective therapy and that more studies are needed to identify efficacious in vivo administration strategies.

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