WO2020094789A1 - Methods and compositions for preventing and/or treating age-related hearing loss - Google Patents

Abstract

Inventors have shown that sub-lethal concentrations of H2O2-exposure initiated a DNA damage response illustrated by increased _ϒH2AX and 53BP1 expression and foci formation mainly in sensory hair cells, together with increased levels of p-Chk2 and p53. Interestingly, post mitotic cochlear cells exposed to H2O2 displayed key hallmarks of senescent cells, including dramatically increased levels of p21, p38 and p-p38 expression, concomitant with decreased p19 and BubR1 expression and positive senescence-associated β-galactosidase labelling. Surprisingly, inventors have observed that EUK-207 attenuated H2O2 –induced DNA damage and senescence phenotypes in cochlear cells in vitro. Furthermore, systemic administration of EUK-207 reduced age-related loss of hearing and hair cell degeneration in SAMP8 mice. Accordingly, the present invention relates to synthetic molecules having SOD-like and catalase-like activities such as EUK-207 and related compounds for use in the treatment of age-related hearing loss (ARHL) and/or progressive mitochondrial neurosensory hearing loss.

Description

METHODS AND COMPOSITIONS FOR PREVENTING AND/OR TREATING AGE-

RELATED HEARING LOSS

FIELD OF THE INVENTION:

The invention is in the field of hearing loss, in particular age-related hearing loss diseases such as presbycusis and progressive mitochondrial neurosensory hearing loss.

BACKGROUND OF THE INVENTION:

Age-related hearing loss (ARHL) or presbycusis is the third most prevalent chronic disease among older adults [1] It can vary in severity from mild to severe, the more severe forms affecting communication and contributing to social isolation, depression and possibly dementia [2] There is general agreement that cumulative effects of aging on hearing are exacerbated by genetic predisposition and environmental factors such as noise or drug exposure. With increasing noise pollution and life expectancy in our modem society, the prevalence of presbycusis is expected to grow dramatically. The precise mechanisms behind the age-related degeneration of the cochlear structures remain however unclear. This is in part due to the complexity of each causal factor and to the interaction of the different mechanistic pathways leading to ARHL.

Growing evidence links oxidative stress and mitochondrial dysfunction to ARHL [3,4] The presence of lipid peroxidation [4], oxidative mitochondrial DNA damage and glutathione- conjugated proteins [5,6] are each strongly associated with cochlear aging in mice. In addition, reduced expression of antioxidant enzymes such as catalase, mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and cytosolic SOD1 were observed in older mice [4- 6] Furthermore, mice lacking the antioxidant enzyme SOD1 displayed enhanced age-related cochlear hair cell loss, reduced thickness of the stria vascularis and severe degeneration of spiral ganglion neurons (SGNs) [7] On the contrary, an increase in SOD2 expression gradient in ganglion cells has been reported along the basal to apical axis of rodent and primate cochlea [8], consistent with the differential and decrease in vulnerability from high to low frequency regions in most ARHL. Finally, low serum levels of the ROS scavenger melatonin are significantly associated with the occurrence of high frequency hearing loss in the elderly [9]

It is generally accepted that oxidative stress may cause irreversible DNA damage [10]. Over 100 oxidative modifications to DNA have been identified, including adducts and both single-strand and double-strand breaks [11] In the cochlea, we have recently reported the activation of the DNA damage response in post-mitotic inner ear cells following treatment with the ototoxic anticancer drug cisplatin [12] However, the link between increased ROS, DNA damage and cochlear cell aging remains obscure. Understanding ROS-induced cochlear cell degeneration may lead to identifying key molecular targets to protect residual hearing in patients suffering ARHL.

SUMMARY OF THE INVENTION:

The invention relates to a compound having SOD-like and catalase-like activities and related compounds for use in the prevention and/or treatment of age-related hearing loss (ARHL) and progressive mitochondrial neurosensory hearing loss. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have shown that sub-lethal concentrations of H202-exposure initiated a DNA damage response illustrated by increased gH2AC and 53BP1 expression and foci formation mainly in sensory hair cells, together with increased levels of p-Chk2 and p53. Interestingly, post mitotic cochlear cells exposed to H202 displayed key hallmarks of senescent cells, including dramatically increased levels of p2l, p38 and p-p38 expression, concomitant with decreased pl9 and BubRl expression and positive senescence-associated b-galactosidase labelling. Surprisingly, inventors have observed that EUK-207 attenuated H202 -induced DNA damage and senescence phenotypes in cochlear cells in vitro. Furthermore, systemic administration of EUK-207 reduced age-related loss of hearing and hair cell degeneration in SAMP8 mice. Accordingly, the ROS-induced DNA damage responses drive cochlear cell senescence and contribute to accelerated age-related sensory hair cell degeneration and hearing loss. EUK-207 and likely other compounds having similar SOD-like and catalase-like activities could potentially postpone cochlear aging and prevent ARHL in humans.

In a first aspect, the invention relates to a compound having SOD-like and catalase-like activities for use in the prevention and/or treatment of age-related hearing loss (ARHL) and/or progressive mitochondrial neurosensory hearing loss.

In a particular embodiment, the invention relates to a method for preventing and/or treating age-related hearing loss (ARHL) and/or progressive mitochondrial neurosensory hearing loss in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a compound having SOD-like activity and catalase-like activities.

As used herein, the terms “treating” or“treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term“age-related hearing loss” also called as presbycusis refers to an age-related decline of auditory function, such as increased hearing thresholds and poor frequency resolution. The primary pathology of presbycusis includes the hair cells, stria vascularis, and afferent spiral ganglion neurons as well as the central auditory pathways. Pesbycusis may take different forms, such as a low frequency hearing loss or a flat hearing loss affecting all frequencies of hearing. Each of these hearing losses have been identified with different pathological changes occurring over time in different structures of the inner ear. ARHL affects one-third of the population after 65 years of age and 60% of the population between 70 and 80 years. ARHL typically occurs slowly over time, and can be identified by default without a known cause. However, some risk factors have been identified as contributing to ARHL including, but not limited to, malnutrition, cardiovascular disease, diabetes, smoking, exposure to heavy metals, and exposure to noise or ototoxic drugs. As used herein, the term“progressive mitochondrial neurosensory hearing loss” refers to a mitochondrial dysfunction which causes hearing loss both in isolation (non- syndromic) and as a feature of systemic mitochondrial disease (syndromic). The cells of the auditory sensory axis, including the cochlea hair cells, stria vascularis (that maintain the endocochlear potential) and auditory neurones are metabolically active and therefore are enriched in mitochondria.

As used herein, the term“subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with age-related hearing loss (ARHL) and/or progressive mitochondrial neurosensory hearing loss.

As used herein, the term“compound having SOD-like and catalase-like activities” refers to compounds capable of transforming oxygen free radicals into oxygen, and hydrogen peroxide into oxygen and water, as described in Doctrow et al., 2002. In a particular embodiment, said compound is EUK-207 and its related compounds. More particularly, EUK-207 related compounds have the general formula I as described in US Patent 6,589,948:

Formula I

wherein:

M is a metal;

A is an anion;

XI and X2 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; Yl, Y2, Y3, Y4, Y5, and Y6 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; Rl, R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryl, substituted aryl, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; with the proviso that one of Rl or R2 may be covalently linked to one of R3 or R4 forming a five- or six-membered ring selected from the group consisting of cycloalkyls, heterocycles, aryls and heteroaryls;

Z is a bridging group selected from the group consisting of

— (CH2)m— , wherein m is equal to or greater than 1 ;

— (CR5R6)m— , wherein each R5 and R6 is independently selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, acyl and amino; and m is equal to or greater than 1; and — (CR7R8)m— R9— (CR10R1 l)p—

wherein:

R7, R8, R10 and Rl l are independently selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, acyl and amino;

R9 is a member selected from the group consisting of alkyls, substituted alkyls, cycloalkyls, substituted cycloalkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls and heteroatoms; and

m and p are independently selected and are equal to 1, 2, 3 or 4; and

Ql and Q2 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; and

n is 0, 1, or 2.

In a particular embodiment, in formula I, M is a metal, preferably a transition metal, and A is an anion, preferably a halogen or an organic anion (e.g., acetate). Examples of suitable transition metals include, but are not limited to, Mn, Cr, Fe, Zn, Cu, Ni, Co, Ti, V, Ru and Os. Examples of suitable anions include, but are not limited to, PF6, (Aryl)4, BF4, B(Aryl )4, halogen, acetate, acetyl, formyl, formate, triflate, tosylate or, alternatively, the anion can be an oxygen atom typically bound via a double bond to the metal, i.e., M. XI and X2 are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys.“Yl, Y2, Y3, Y4, Y5 and Y6, in Formula I, are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys. Rl, R2, R3 and R4 are independently selected and are functional groups including, but not limited to, hydrogen, halogens, alkyls, substituted alkyls, aryl, substituted aryl, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys; with the proviso that one of Rl or R2 may be covalently linked to one of R3 or R4 forming a cyclic structure. Z, in Formula I, is a bridging group. Ql and Q2, in Formulae I and II, are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys. The index“n” is 0, 1 or 2.

As used herein, the term“EUK-207” refers to a synthetic superoxide dismutase/catalase mimetic (40). EUK-207 is well known in the art, has the molecular formula: C24H27MnN208; and CAS No.478020-50-7. EUK-207 has the following structure in the art:

Formula II

As used herein, the term "effective amount" refers to a quantity sufficient of the EUK- 207 to achieve treatment of ARHL. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, an effective amount of EUK-207 for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In some embodiments, a single dosage of peptide ranges from 0.1- 10,000 micrograms per kg body weight. In some embodiments, aromatic- cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., EUK-207) into the subject, such as by, intravenous, intramuscular, enteral, subcutaneous, parenteral, systemic, local, spinal, nasal, topical or epidermal administration (e.g., by injection or infusion). In a particular embodiment, the administration of EUK-207 compound is systemic. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

In a second aspect, the invention relates to a pharmaceutical composition comprising a compound having SOD-like activity and catalase-like activities for use in the prevention and/or treatment of ARHL and/or progressive mitochondrial neurosensory hearing loss.

The pharmaceutical composition according to the invention wherein said compound is EUK-207.

The EUK-207 compound as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Inventors have shown that compounds having SOD-like and catalase-like activities such as EUK-207 are able to postpone cochlear aging and prevent ARHL in humans.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: EUK-207 upregulates antioxidant response elements and suppresses DNA damage and senescence phenotype. A. Representative western blot analysis using antibodies against FOX03a, Nrf2, gH2AC, pl9, p2l and b-actin in whole cochlear extracts. B- F. Histograms representing the levels of FOX03a, Nrf2, gH2AC, pl9 and p2l in control (Ctrl), 10 mM EUK-207 alone, and 0.5 mM H202 alone or in combination with 10 mM EUK-207 exposed groups (n=6 cochleae per condition) b-actin served as a loading control. Data are expressed as mean ± SEM. Kruskal- Wallis test was followed by post hoc Dunn's test (*P < 0.04, **P<0.0l, *** P < 0.001 vs. Ctrl; ## P < 0.01, vs. H202 0.5 mM). Data information: all experiments were performed in triplicate. Figure 2: Pharmacological mitigation of ROS prevents loss of hearing and hair cells in adult mice. A. ABR thresholds recorded before (plot (3)) and after 2 months (plot (2)) and 3 months (plot (1)) of mannitol treatment (left panel), or before (plot (3)) and after 2 months (plot (2)) and 3 months (plot (1)) of EUK-207 treatment (right panel). B. Mean ABR threshold from 2 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (h=10 per group). One-way ANOVA test was followed by post hoc Tukey’s test (**P < 0.008,

< 0.001, vs. mannitol). C. Cytocoehleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1 , 2.6, 3.5 or 4.1 mm from the cochlear apex from mannitol-treated (white bars) and EUK-207-treated (black bars) SAMP8 mice (n=5 per group). Data are expressed as mean ± SEM. Kruskal- Wallis test was followed by post hoc Dunn's test (*P < 0.04, ***P < 0.001 vs. mannitol treated group).

EXAMPLE:

Material & Methods

Animals

Neonate Swiss mice were purchased from Janvier Laboratories. The senescence accelerated prone 8 (SAMP8) and senescence-accelerated resistant 1 mice (SAMR1 , serves as a control) of both sexes were purchased from Harlan SARL Laboratory. Mice were housed in pathogen-free animal care facilities accredited by the French Ministry of Agriculture and Forestry (C-34- 172-36; December 19, 2014).

Drug preparation

H202 was purchased from Acros Organics (#202460250). The salen-manganese (Mn) SOD-catalase mimetic, EUK-207 was a gift from Dr Bernard Malfroy-Camine, of MindSet Rx, Arlington, MA, USA.

For in vitro experiments, H202 was freshly prepared in culture medium to final concentrations ranging from 0-1.25 mM. The EUK-207 was prepared at 10 mM in pure water and freshly diluted in culture medium to a final concentration of 10 mM. This final concentration was chosen based on our preliminary evaluations of the dose-response effects of EUK-207, at concentrations ranging from 0-30 mM, on H202-induced gH2AC expression on day 3 in H202- exposed cochlear explants. For in vivo experiments, EUK-207 was freshly prepared at 1.5 mM in 5% mannitol and was administered through a subcutaneously implanted Alzet micro-osmotic pump (DURECT Corporation, 0.1 Imΐ per hour, 28 days #1004) at a dose of 0.2 mg/kg/day.

In vitro assessments

Organ of Corti, and whole cochlea cultures Mouse whole cochleae and organ of Corti explants were collected from postnatal day 3 mice and prepared according to the procedures described previously [12,13] The whole cochleae were kept in suspension, and the organ of Corti explants in adherent conditions in a 6-well culture plate containing 2ml/well of culture medium. Culture medium consisted of Dulbecco's modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12, #21331020) containing 2 mM L-glutamine (#25030024), N-2 complement (#17502048) at IX and insulin transferrin delenium (#41400045) at IX purchased from Gibco Life technologies, and 8.25 mM D-glucose (#G6l52) and 30 U/ml penicillin G (#P3032) from Sigma- Aldrich.

H202-induced oxidative stress

The oxidizing agent used to induce oxidative stress in vitro H202 was chosen based on its wide use in a variety of cell types [14,15] Here, cochlear samples were firstly exposed to culture medium alone for 24 hours in a humidified incubator (37°C, 5% C02). To probe the dose-effect of H202, the culture medium was then replaced with fresh medium containing H202 at various concentrations (0, 0.25, 0.4, 0.5, 0.75, 1 and 1.25 mM) for 5 hours before being replaced again with the culture medium alone and maintained for 5 days. The culture medium was renewed once on day 2 or 3 following its replacement. To determine time-dependent cochlear cell degeneration, organ of Corti explants were exposed to medium containing H202 at its half maximal effective concentration (EC50) and also at the concentration just below the EC50 (0.5 mM) for 5 hours and then maintained in the culture medium alone for 0, 1, 3 and 5 days. All control samples were maintained in culture medium alone and were run concurrently alongside the experimental cultures.

Dose-dependent cytotoxicity and apoptosis

To evaluate the toxicity of H202, cultured organ of Corti explants were immuno labelled with a rabbit polyclonal antibody against myosin 7A (1/300, Proteus Biosciences Inc #25-6790) and a mouse monoclonal antibody against neurofilament (NF 200, 1/600, Sigma-Aldrich #N0l42) to label hair cells and spiral ganglion neurons, respectively. Hair cell counting (6 cochleae per condition and per time point) was performed using standard techniques [13]. Considering the resistance of apical turn hair cells to H202 cytotoxicity, counting concerned only a 1.5 mm length of the cochlear duct at the basal turn (4 to 5.5 mm from the apex, see [12])·

The TUNEL kit (DeadEnd™ fluorometric TUNEL System, Promega #G3250,) was used to identify apoptotic DNA fragmentation. The cochlear samples were counterstained with a rabbit polyclonal antibody against myosin 7A and a mouse monoclonal antibody against neurofilament. All secondary antibodies were used at a dilution of 1/1000. This included donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (Molecular Probes #A-21202, #A-21206, #A-10037, #A-l0042,). Fluorescent tags were visualized using a confocal microscope (LSM 5 Live Duo, Zeiss). In control specimens without primary antibodies, neither Alexa 488 nor 568 fluorescent tags were observed. All experiments were performed in triplicate.

Measurement of oxidative stress

Oxidative stress was studied in the whole cochlea homogenates as previously described [4] Catalase and superoxide dismutase (SOD) activities were measured as previously described by Marklund [16]. Lipid peroxidation was assessed using the thiobarbituric acid-reactive substances method, and was expressed in nmol/mg malondialdehyde (MDA) [17]. Protein concentrations were measured using the BCA protein assay kit (Pierce #23250,). All experiments were performed in triplicate.

Cellular localization of DNA damage foci and foci counting

Immunocytochemistry was employed to localize DNA damage foci in cultured organ of Corti using mouse monoclonal antibody against phospho-H2AX (1/500, Serl39, Merck Millipore #05-636) or rabbit polyclonal antibody against 53BP1 (1/100, Novus Biologicals #NB 100-305). The cochlear samples were counterstained with a rabbit polyclonal antibody against myosin 7A or mouse monoclonal antibody against parvalbumin (1/500, SWANT #PV235) to label hair cells. All secondary antibodies were used at a dilution of 1/1000. The Hoechst 33342 dye (0.002% wtvol in PBS IX, Thermo Fisher Scientific #62249) was used to stain DNA. Fluorescent tags were visualized using a confocal microscope (LSM 5 Live Duo, Zeiss). No fluorescent signal was detected in control specimens without primary antibodies.

The number of foci (i.e. gH2AC or 53BP1) per nucleus, was computed using algorithms with Matlab custom-made software (MathWorks Company) that allow 3D rendering and visualization of“isosurfaces” enveloping all pixel clusters with intensities greater than a user- defined criterion value in each corresponding image channel (for more details see [12]). All experiments were performed in triplicate.

Expression of apoptosis, oxidative stress and autophagy-related proteins, DNA damage response and senescence markers

Evaluation of apoptosis, oxidative stress, autophagy, DNA damage responses and cellular senescence-like state in the cochlear protein extracts from H202 exposed and cultured whole cochleae, were performed using western blotting technique as described previously [4] Antibodies used included those recognizing Bax (1/1000, Abeam #7977), BC12 (1/1000, Santa Cruz #sc-492) and cleaved caspase 3 (1/1000, Aspl75 Cell-signaling #9661), SOD2 (1/1000, Abeam #abl3533), catalase (1/1000, Abeam #16731), p66Shc (1/1000, Abeam #ab33770), phospho-p66Shc (S36, 1/1000, Abeam #54518), p-Beclinl Ser93 (1/1000, Cell Signaling #14717), LC3-II (1/800, Cell Signaling #2775), Rab7 (1/800, Santa Cruz Biotechnology #sc- 376362), p62 (1/1000, MBL International #PM045), phospho-H2AX (1/1500, Serl39, Cell Signaling #2577), 53BP1 (1/1000, Novus Biologicals #100-305), DDB2 (1/800, Invitrogen #PA5-3736l), phospho-Chkl (1/1000, Ser345, Cell Signaling #2348), phospho-Chk2 (1/1000, Thr68, Cell Signaling #2661), p53 (1/1500, Cell Signaling #2524), p2l (1/2000, Cell Signaling #2946), p38 (1/500, Elabscience #E-AB-32459), p-p38 (Thrl80/Tyrl82, 1/1000, Cell Signaling #9211), pl6 (1/1000, BD Pharmingen #551154), pl9 (1/1000, Abeam #ab80), BubRl (1/1000, Abeam #abl83496), FOX03a (1/1000, Cell Signaling #2497) and Nrf2 (1/1000, Santa Cruz # sc-365949). b-actin (1/10000, Sigma-Aldrich #Al978) served as a loading control. Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1/3000, Jackson ImmunoResearch #115-001-003) or goat anti-rabbit IgG antibodies (1/3000, Jackson ImmunoResearch #111-001-003). All experiments were performed in triplicate. Image scans of western blots were used for semi-quantitative analysis.

Senescence-associated beta-galactosidase activity assay

Senescence-associated beta-galactosidase (SA-P-gal) activity was measured according to the manufacturer’s protocol (Cell Signaling #9860). Briefly, cochlear explants were fixed in 4% paraformaldehyde in phosphate-buffered saline IX (PBS) for 15 minutes at room temperature and washed 2 times in PBS, before being incubated overnight at 37°C in a dry incubator with fresh b-galactosidase staining solution at pH 6.0.

Pharmacological mitigation of ROS

To probe the impact of ROS mitigation on H202-induced DNA damage and senescence-like phenotype, whole cochleae were exposed to either culture medium alone or medium containing 10 mM EUK-207 for 24 hours in a humidified incubator (37°C, 5% C02). The culture medium was then replaced with fresh medium containing H202 at a concentration of 0.5 mM for 5 hours. The cochlear samples were further grown for 3 additional days in culture medium either alone or with EUK-207. All control samples were maintained in culture medium and were run concurrently with experimental cultures. The samples were then collected for the evaluation of oxidative stress, DNA damage and cellular senescence-like state using western blotting and immuno labelling techniques.

Validation in adult SAMP8 in vivo

Functional assessment The auditory function was assessed by recording auditory brainstem responses (ABRs) in SAMP8 and SAMR1 mice at the age of 1, 3, 6 and 12 months (n=l4 animals per age and per strain). ABR reflect the synchronous activation of auditory neurons from the cochleae up to the colliculi in response to incoming sound. The recordings were carried out under anaesthesia with Rompun 2% (3 mg/kg) and Zoletil 50 (40 mg/kg) in a Faraday shielded anechoic sound proof cage. Rectal temperature was measured with a thermistor probe, and maintained at 38.5° C ± 1 using a heated under blanket. ABRs were recorded from three subcutaneous needle electrodes placed at the vertex (active) and on the pinna of the tested ear, as well as in the neck muscles (ground) of the mice. The acoustic stimuli generated by a NI PXI-4461 signal generator (National Instruments) consisted of 10 ms tone-bursts with a 1 ms rise and fall time delivered at a rate of lO/s. Sound was delivered by a JBL 075 loudspeaker (James B. Lansing Sound) positioned at 10 cm from the tested ear, under calibrated free-field conditions. Cochlear amplification (20,000) was achieved via a Grass P511 differential amplifier, averaged 1000 times (Dell Dimensions). Amplitude-intensity functions of the ABRs were obtained at each frequency tested (2, 4, 6.3, 8, 10, 12.5, 16, 20, 25, and 32 kHz) by varying the intensity of the tone bursts from 0 to 100 dB SPL, in 5dB incremental steps. The ABR thresholds were defined as the minimum sound intensity necessary to elicit a well-defined and reproducible wave II. Recordings and analysis were performed blindly.

Molecular assessment

Evaluation of oxidative stress, DNA damage responses and cellular senescence-like state in the cochlear protein extracts from cochleae of SAMP8 and SAMR1 mice aged 1, 6 and 12 months (n=l6 cochleae per age and per strain), were performed using western blotting technique as described previously [4] Antibodies used included those recognizing Nrf2 (1/1000, Santa Cruz # sc-365949), SOD2 (1/1000, Abeam #abl3533), p66Shc (1/1000, Abeam #ab33770), phospho-p66Shc (S36, 1/1000, Abeam #54518), phospho-Chk2 (1/1000, Thr68, Cell Signaling #2661), p53 (1/1500, Cell Signaling #2524), phospho-p53 (1/1500, Serl5, Cell Signaling #9289), p2l (1/2000, Cell Signaling #2946), pl6 (1/1000, BD Pharmingen #551154), BubRl (1/1000, Abeam #abl83496) and pl9 (1/1000, Abeam #ab80). b-actin (1/10000, Sigma- Aldrich #Al978) served as a loading control. Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1/3000, Jackson ImmunoResearch #115-001-003) or goat anti-rabbit IgG antibodies (1/3000, Jackson ImmunoResearch #111- 001-003). All experiments were performed in triplicate. Image scans of western blots were used for semi-quantitative analysis.

Age-related hearing impairment and molecular correlation To compare the time course of the hearing impairments found in SAMP8 and SAMR1 mice, the mean ABR threshold evoked by the range of tested frequencies from 2 kHz to 32 kHz for each strain and at each time point was calculated. A linear regression was then performed to determine the threshold elevation per month. To decipher the molecular determinants of the accelerated age-related hearing impairment in SAMP8 mice, we used the mean values of ABR thresholds and protein levels in SAMR1 as a reference and subtracted these from the values for SAMP8 mice. The differences in threshold and protein level (meanSAMP8 - meanSAMRl) where then averaged and used for statistical analysis and to calculate SEM.

Pharmacological counteraction of ROS

Finally, the impact of ROS mitigation on premature ARHL was also investigated in SAMP8 mice. The 6 month-old male and female SAMP8 mice were randomly divided into 2 groups: i) control 5% mannitol (vehicule, h=10) and ii) EUK-207 (h=10). The EUK-207 was dissolved in 5% mannitol to a final concentration of 1.5 mM, filter-sterilized and administered at a dose of 0.2 mg/kg/day with use of a subcutaneous Alzet micro-osmotic pump, continuing for up to 84 days. This dose of EUK-207 was selected according to the effective EUK-207 dose range reported previously [18]. Control mice received pumps filled with 5% mannitol. Mice received new pumps every month.

The auditory brainstem responses (ABRs) were recorded prior to pump implantation, and 2 and 3 months afterwards. Sensory hair cell loss was evaluated using scanning electron microscopy (Hitachi S4000). The cochleae from the different groups (n=5 per group) were processed and evaluated using previously reported standard techniques [19,20] Hair cell counting was performed in four different 300 pm long segments of the organ of Corti, centred at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end and corresponding to the frequencies of 8, 16, 25 and 32 kHz, respectively [21]

Statistics

Data are expressed as mean ± SEM. Normality of the variables was tested by the Shapiro-Wilks test. If conditions for a parametric test were met, the significance of the group differences was assessed with a one-way ANOVA; once the significance of the group differences (P < 0.05) was established, Tukey's post hoc tests were subsequently used for pairwise comparisons. If not, Kruskal- Wallis tests were used to assess the significance of differences among several groups; if the group differences were significant (P < 0.05), Dunn's tests were then used for post hoc comparisons between pairs of groups. For in vivo mouse studies, based on data from our previous reports [22,23] or from preliminary experiments, we calculated the sample size using G* Power 3.1.9.2 to ensure adequate power of key experiments for detecting pre-specified effect sizes. The statistical test used and associated P values are indicated in the legends for each figure.

Results

In vitro assessments

To probe a direct causal relationship between increased ROS and premature occurrence of cochlear cell senescence- like phenotype, we adopted an available in vitro model of oxidative stress [24,25], which consists of exposing cochlear explants or whole cochleae to a series of H20 concentrations in culture.

H202 induces dose-dependent outer hair cell apoptosis

We firstly determined the survival of cochlear cells under increasing concentrations of H202. Our results show that a 5 hour exposure to H202 in the millimolar range led to a concentration-dependent loss of outer hair cells (OHCs) in the basal turn of the organ of Corti 5 days later, in contrast to an only slight loss of inner hair cells (IHCs) (data not shown). A concentration of 1.25 mM provoked 77% of OHC and 20% of IHC loss (data not shown). The counting of hair cells remaining after exposure to 0.75 mM H202 (EC50) and 0.5 mM (the concentration just below EC50) revealed a time-dependent OHC loss. Indeed, a concentration of 0.75 mM H202 led to a loss of 22.8% ± 1.4% and 50.2 % ± 1.52 % OHCs at 3 and 5 days after exposure, respectively (data not shown), while 0.5 mM provoked a respective loss of only 11.6% ± 2% and 28.4 % ± 4.7 %. By contrast, IHCs showed only slight alterations up to 5 days after exposure at all concentrations. We therefore performed all molecular assessments within 3 days after H202 exposure.

To determine the nature of the H202-induced hair cell loss, we probed for apoptotic markers at 3 days after H202 exposure. Large amounts of OHCs and some IHCs displayed TUNEL-positive nuclei (data not shown) after incubation with the higher H202 concentration (> 0.75 mM), when compared with controls or the sub-lethal concentration (0.5 mM, data not shown). Concomitantly, western blots from protein extracts of H202-exposed cochleae showed significant caspase 3 activation in the cochleae exposed to >0.5 mM of H202 and higher levels of Bax protein in those exposed to >0.75 mM of H202 (data not shown). By contrast, Bcl-2 protein levels remained unaltered (data not shown). Taken together, these data demonstrate that H202 induces a concentration-dependent hair cell loss with massive OHC apoptosis after exposure to high concentrations (>0.75 mM). In an in vivo situation, ROS levels are presumed very low due to their short half-life and the existence of various antioxidant enzymes [26] On this basis, we chose H202 concentrations < 0.5 mM for subsequent experiments.

Increased oxidative stress and upregulation of autophagy To confirm the occurrence of oxidative stress in cochlear tissues after exposure to H202 at concentrations < 0.5 mM, we assessed the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (Cat), lipid peroxidation (data not shown), and the expression levels of SOD2, Cat, p66Shc and serine 36-phosphorylated p66Shc. P66Shc is a redox protein playing an important role in the regulation of the cellular response to oxidative stress [27] Three days after H202 exposure, cochleae treated with 0.5 mM H202 showed significantly increased SOD activity (data not shown) and lipid peroxidation (MDA, data not shown) but significantly decreased catalase activity data not shown). Western blot analysis revealed significantly higher levels of SOD2, Cat, p66Shc and p-p66Shc in the cochleae exposed to 0.4- 0.5 mM H202 when compared with control-cultured cochleae (data not shown). Collectively, these results show that events related to oxidative stress occur after exposure to H202 at low concentrations.

Considering the link between oxidative stress and autophagy [28], we examined autophagy induction by monitoring the levels of proteins involved in autophagosome formation and autophagic flux, the dynamic process of autophagosome synthesis and degradation. H202- exposed cochleae displayed significantly increased levels of phosphorylated Beclin 1, a major player in the autophagic initiation process [29] (data not shown); LC3-II (data not shown), a hallmark of autophagosome formation; Rab7, a small GTP-binding protein that plays a role in maturation of late autophagic vacuoles [30]; and finally p62, an ubiquitin-binding scaffold protein and a subtract of autophagy (data not shown). Taken together, these results indicate that H202 induced cochlear cell oxidative stress and triggered an autophagic response.

DNA damage responses

To determine the nature of DNA damage induced by H202 in hair cell nuclei, we used immuno labelling to detect and localize H2AX phosphorylation (gH2AC) and 53BP1, two hallmarks of DNA damage. H202-intoxicated organ of Corti (data not shown) exhibited significantly higher levels (p<0.00l) of gH2AC foci formation in both outer hair cells (4.6l±0.55 foci per nucleus) and inner hair cells (4.2l±0.22 per nucleus) when compared to control organ of Corti cultures (data not shown, 3.22±0.l8 versus 2.88±0.27 foci per nucleus for OHC and IHC, respectively). While control organ of Corti cultures displayed almost no 53BP1 foci indicative of DNA double-strand breaks [31,32] (data not shown), the 0.5 mM H202 intoxicated cochleae showed a large number of 53BP1 foci (4.27±l .27 and 4.25±0.56 foci per nucleus for IHC and OHC, respectively) (data not shown). Concomitantly, western blots from protein extracts of H202-exposed cochleae revealed strong expression of both gH2AC and 53BP1 when compared with control-cultured cochleae (data not shown). Altogether, these results indicate the occurrence of DNA damage, including DNA double- strand breaks in H202 intoxicated cochleae.

DNA double-strand breaks are potent inducers of DNA damage response (DDR), characterized by activation of DDR proteins, including the cell cycle checkpoint proteins like checkpoint kinase 1 (p-Chkl), checkpoint kinase 2 (p-Chkl) and p53 [33] Once activated, p53 plays an important role in modulating distinct cell fate decisions [34-36] It enhances DNA repair pathways through the upregulation of several repair proteins like the damage-specific DNA binding protein 2 (DDB2), which is required for nucleotide excision repair [37] We found significant increases in the levels of DDB2, p-Chk2 and p53 in H202-exposed cochleae when compared with the control condition (data not shown). By contrast, the increase in the level of p-Chkl after H202 challenge did not reach significance (data not shown). These results indicate that H202 exposure induces DNA damage and the activation of the Chk2-p53 pathway in post mitotic cochlear cells.

Senescence-like phenotype in post mitotic cochlear cells

To assess whether DDR induces a senescence-like phenotype in post mitotic cochlear cells in vitro, we examined the accumulation of p2l , one of the downstream effectors of p53. We showed that 0.5 mM H202 resulted significant accumulation of p2l (data not shown), suggesting the activation of p53-p2l pathway. Considering both p2l -dependent and - independent p38MAPK activation may occur in senescence [38], we probed the expression level of p38 and its phosphorylation on threonine-l80 and tyrosine-l82. Consistent with the activation of r53-r21, H202 exposed cochleae showed increased levels of p38 and phospho- p38 (data not shown). H202 exposure did not significantly affect the expression level of pl6, a cell cycle inhibitor. However, the cell cycle regulator pl9 and the mitotic checkpoint protein BubRl were significantly repressed (data not shown). Concomitantly, H202 exposed cochleae showed increased activity of SA-P-gal, a widely accepted general marker of the senescent phenotype [39] (data not shown). Altogether, these data firmly demonstrate the occurrence of a senescence-like phenotype in post mitotic cochlear cells after H202 challenge.

Pharmacological mitigation of ROS with EUK-207

To probe the impact of ROS mitigation on H202-induced DNA damage and senescence-like phenotype, we aimed to counteract excessive ROS by pre- and post-treating cochleae with 10 mM EUK-207, a potent synthetic superoxide dismutase/catalase mimetic that scavenges superoxide and hydrogen peroxide [40] As a readout, we first analysed the expression of Foxo3a, a forkhead transcription factor of the Foxo class, which is known to reduce the level of cellular oxidative stress by directly increasing mRNA and protein levels of MnSOD and catalase [41] We found that treatment with EUK-207 alone significantly increased the levels of Foxo3a (Figures 1A-B). We also established that EUK-207 alone increased Nrf2 levels (Figures 1A, 1C), but significantly reduced gH2AC levels (Figures 1A, 1D). Interestingly, a combined treatment with 10 mM EUK-207 and 0.5 mM H202 significantly increased the expression levels of Foxo3a and Nrf2 (Figures 1A-C) when compared with control or H202 alone. By contrast, this combination efficiently attenuated the induction of gH2AC by H202 (Figures 1A, 1D). Finally, we also report that this combined treatment significantly increased pl9 expression (Figures 1A, 1E), but reduced p2l level (Figures 1A, 1F). Taken together, these results demonstrate that by counteracting excessive ROS, EUK-207 may attenuate oxidative stress-induced DNA damage and senescence phenotype.

In vitro validation in adult SAMP8

Molecular pathway validation

The senescence accelerated mouse prone 8 (SAMP8) mice has been shown to display premature hearing loss and cochlear degeneration associated with oxidative stress and altered levels of antioxidant enzymes [4] Here, we probe whether cochlear cells engender similar DNA damage responses and senescence signatures during premature aging in vivo. We first analysed the nuclear factor erythroid 2-related factor 2 (Nrf2), emerging as a regulator of cellular resistance to oxidants [42] Western blot analyses revealed a decrease in Nrf2 with age in both strains. However, SAMP8 mice systematically showed significantly lower expression levels compared to the control senescence accelerated mouse resistant 1 (SAMR1) mouse strains at all ages analysed. Interestingly, this much decreased Nff2 expression was detectable from 1 month of age in SAMP8 mice (data not shown). Similarly, while SAMP8 mice displayed a significantly lower level of MnSOD (SOD2) expression early from 1 month of age and that was maintained to 12 months, SAMR1 mice only showed such decrease in SOD2 at 12 months of age (data not shown). SAMP8 mice showed significantly higher levels of p66Shc and phorphorylated p66Shc (p-p66Shc) when compared with SAMR1 mice of the same age (data not shown). They also showed dramatic increases in the amounts of p66Shc and p-p66Shc during the aging process. In contrast, SAMR1 mice showed reduced levels of p-p66Shc at 12 months compared to 1 month of age (data not shown). Taken together, these results indicate the early occurrence (1 month-old) and a higher level of oxidative stress in the cochleae of SAMP8 mice.

To further explore the potential appearance of DDR in SAMP8 mice, we assessed DDR proteins. Western blot analysis demonstrated dramatic increases in p-Chk2 levels from as early as 6 months in SAMP8 mouse cochleae compared to a much lower yet significant increase only from 12 months of age in SAMR1 mice (data not shown). Similarly, while SAMP8 mice showed significantly increased p53 and p-p53 levels from 6 months, SAMR1 mice displayed a significant increase of p-p53 from 12 months only (data not shown). Altogether, these results point towards the activation of a DNA damage response during cochlear aging, with more severe effects in SAMP8 compared to SAMR mice.

In addition, while both strains displayed an increase in senescence-like features, characterized by an increase in the levels of p2l and pl6 during aging. Here again, changes were more pronounced in SAMP8 than in SAMR1 mice (data not shown). SAMP8 mice also showed slightly decreased levels of BubRl at 12 months, and a significantly increased level of pl9 was only observed in SAMP8 mice at 6 months (data not shown).

Correlation between molecular events and hearing loss

The ABR threshold showed no significant difference between SAMP8 and SAMR1 mice at 1 month (data not shown). By contrast, mice of both strains after 1 month of age showed an age-related hearing loss that was twice as severe in SAMP8 (5 dB/month) than in SAMR1 mice (2.8 dB/month, data not shown). To facilitate the comparison between the two strains of mice, we expressed all the functional data obtained in SAMP8 with respect to those in SAMR1. We then expressed the molecular events as a function of hearing threshold shift with respect to the control SAMR1. When the molecular event was close to zero, hearing loss was considered mainly due to age and independent of the strain of mouse. In contrast, a positive or negative shift indicated a correlation between molecular event and the phenotype of SAMP8 (i.e. accelerated hearing loss).

The SAMP8 strain displayed a deficit in antioxidant proteins Nfr2 and SOD2 and an excess of the pro-oxidant molecule p66shc at all ages. The levels of pro-oxidant molecules p66shc and p-p66shc increased concomitantly with the degradation of hearing in S AMP8, while the deficit in antioxidants remained relatively stable until 6 months of age (data not shown). The differences between the strains in terms of levels of the antioxidant molecules Nrf2 and SOD2 decreased at 12 months of age (data not shown), probably due to the age-related repression of these molecules in both strains. While both strains displayed comparable levels of DDR proteins (p-Ch2, p53, p-p53) at one month, only SAMP8 mice thereafter showed a dramatic increase in levels to reach a plateau at 6 months (data not shown). Finally, pro- senescence proteins (p2l, pl6) increased continually with age in SAMP8 mice (data not shown). By contrast, the mitotic checkpoint protein BubRl and the cell cycle regulator pl9 remained close to zero, except at 6 months where pl9 had increased before falling back to zero at 12 months with a net result of no significant difference between strains at 12 months (data not shown).

Collectively, these in vivo data support our in vitro results indicating that an early increase in the level of ROS triggers the activation of DNA damage responses and premature occurrence of senescence-like state in post-mitotic cochlear cells and an accelerated hearing loss in SAMP 8 mice.

EUK-207 slows down age-related hearing loss in SAMP8 mice

With a view to developing pharmacological therapies in vivo, we began treatment of groups of SAMP8 mice from 6 months of age with either EUK-207 dissolved in mannitol or with mannitol alone as a control, delivered by subcutaneously implanted osmotic pumps (see Mat &Methods). In contrast with mannitol-treated control SAMP8 mice, two and three month treatments with EUK-207 significantly prevented the age-related ABR threshold increase. The mean thresholds from EUK-207-treated and mannitol-treated SAMP8 mice were 55.7 dB ± 3.5 and 75.1 dB ± 4.7, respectively (p<0.00l), at the end of 3 months treatment (Figures 2A-B). Consistent with this finding, hair cell counting using scanning electron microscopy at the end of 3 months of treatment showed greater OHC survival in the basal region of EUK-207-treated mice. OHC survival rates from EUK-207 treated mice and mannitol treated mice were 92.2% ± 1.3 and 57.12% ± 3.44, respectively, at a frequency of 32 kHz (Figure 2C). Altogether, these results indicate that the mitigation of excessive ROS with EUK-207 prevented accelerated age- related hearing loss and sensory hair cell loss in SAMP8 mice.

Conclusion

The in vitro and in vivo data presented here provide evidence that: i) ROS is one of the main culprits behind age-related sensory hair cell degeneration; ii) ROS-induced DDR driving senescence-like features may account for premature cochlea aging; and iii) pharmacological scavenging of superoxide and hydrogen peroxide can mitigate ARHL in SAMP8 mice.

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A compound having SOD-like and catalase-like activities and related compounds for use in the prevention and/or treatment of age-related hearing loss (ARHL) and/or progressive mitochondrial neurosensory hearing loss.

2. The compound for use according to claim 1 wherein, said compound has the general formula I:

wherein:

M is a metal;

A is an anion;

XI and X2 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys;

Yl, Y2, Y3, Y4, Y5, and Y6 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys;

Rl, R2, R3 and R4 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryl, substituted aryl, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; with the proviso that one of Rl or R2 may be covalently linked to one of R3 or R4 forming a five- or six-membered ring selected from the group consisting of cycloalkyls, heterocycles, aryls and heteroaryls;

Z is a bridging group selected from the group consisting of — (CH2)m— , wherein m is equal to or greater than 1 ;

— (CR5R6)m— , wherein each R5 and R6 is independently selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, acyl and amino; and m is equal to or greater than 1; and

— (CR7R8)m— R9— (CR10R1 l)p—

wherein:

R7, R8, R10 and Rl l are independently selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, acyl and amino;

R9 is a member selected from the group consisting of alkyls, substituted alkyls, cycloalkyls, substituted cycloalkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls and heteroatoms; and

m and p are independently selected and are equal to 1, 2, 3 or 4; and

Ql and Q2 are independently selected from the group consisting of hydrogen, halogens, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid ester radicals, alkoxys, aryloxys and acyloxys; and

n is 0, 1, or 2.

3. The compound for use according to claim 1 wherein said compound is EUK-207.

4. The compound for use according to claim 1, wherein, the EUK-207 is administered by systemic administration.

5. A pharmaceutical composition comprising a compound having SOD-like activity and catalase-like activities for use in the prevention and/or treatment of ARHL and/or progressive mitochondrial neurosensory hearing loss.

6. The pharmaceutical composition according to claim 1 wherein said compound is EUK- 207.

7. A method for preventing and/or treating ARHL and/or progressive mitochondrial neurosensory hearing loss in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of a compound having SOD-like activity and catalase-like activities.

8. The method according to claim 7, wherein said compound is EUK-207.