Intranasal administration of NGF for the treatment of sensorineural hearing loss

Abstract

The present invention relates to nerve growth factor (NGF) or a mutein thereof for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein said NGF is administered intranasally to said subject.

Description

[Technical Field]

[0001] The present invention relates to the prevention and treatment of sensorineural hearing loss. [Background technology]

[0002] Hearing loss is the fourth leading cause of disability worldwide, affecting 430 million people, or over 5% of the world's population. It is estimated that by 2050, over 700 million people will have disabling hearing loss (World Health Organization, 2021 https: / / www.who.int / news-room / fact-sheets / detail / deafness-and-hearing-loss).

[0003] The term hearing loss refers to a wide range of hearing impairments that can be divided into two major classes: (i) conductive hearing loss, which occurs when blockages or disease in the outer or middle ear prevent the transmission of sound energy to the inner ear, and (ii) sensorineural hearing loss (SNHL), which accounts for 90% of all cases of hearing loss and results from damage to the inner ear and auditory nerve. In particular, sensorineural hearing loss is primarily characterized by degeneration of two types of cells: cochlear hair cells, the primary mechanoreceptors that convert sound energy into neural signals, and / or spiral ganglion cells (SGCs), the auditory neurons that transmit signals from cochlear hair cells to the auditory nuclei in the brainstem via the auditory nerve.

[0004] Sensorineural hearing loss usually begins with degeneration of hair cells. These two types of cells are closely related, and degeneration of sensory hair cells leads to a reduction in synaptic signals that stimulate spiral ganglion neurons, leading to neurodegeneration (cochlear synaptopathy) (Cunningham et al., N. Engl. J. Med. 2017, 377(25): 2465-2473).

[0005] Hair cell degeneration in sensorineural hearing loss can have genetic or non-genetic etiologies. Non-genetic factors include noise exposure, viral or bacterial infections, ototoxic chemicals such as the chemotherapy drug cisplatin or aminoglycoside antibiotics, autoimmune diseases, and aging (Liu et al., Front. Neurosci. 2022, 16: art. 867453).

[0006] Despite the significant impact of hearing loss on all segments of the population, the only currently approved treatment option is the cochlear implant (CI), a hearing aid that works by directly stimulating spiral ganglion cell (SGC) cell bodies and possibly their central axons, partially restoring sensory function to patients. In contrast, there are no FDA-approved pharmacological treatments for the prevention or treatment of this disorder. In clinical practice, non-hereditary SNHL is typically treated with corticosteroids, particularly dexamethasone and prednisolone. However, this treatment has not proven to produce satisfactory results.

[0007] Therefore, there is a strong need for the development of new, more effective pharmacological treatments that target the inner ear and can prevent damage or replenish hair cell and cochlear neuronal populations, protecting and restoring hearing function.

[0008] Nerve growth factor (NGF) is a member of the neurotrophic factor family that is essential for the survival, differentiation, and maintenance of nerve cells. NGF has been identified as a promising therapeutic approach for the treatment of SNHL in several preclinical and clinical studies (Gao et al., Clinical and Experimental Otorhinolaryngology 2017, 10(4): 303-308).

[0009] As an alternative to NGF, muteins of NGF with one or more mutations in the amino acid sequence have also been developed, which retain the neurotrophic and neuroprotective properties of wild-type NGF while exhibiting reduced pain-inducing activity, thereby avoiding the hypernociceptive side effects of wild-type NGF.

[0010] However, a major challenge for the effective use of NGF or its mutant proteins in the prevention or treatment of hearing loss is the delivery of the protein to the inner ear compartment. The delivery of drugs, especially proteins such as NGF and its mutant proteins, to the inner ear compartment is hindered by the presence of several protective barriers.

[0011] Specifically, the blood-labyrinth barrier (BLB) separates the inner ear fluid from the blood circulation, limiting the possibility of systemic delivery. At the same time, topical delivery to the ear is hindered by barriers between different compartments within the ear: the tympanic membrane (TM) between the outer and middle ear, and the round window membrane (RWM) between the middle and inner ear.

[0012] To increase drug concentrations in the middle ear, intratympanic injection has been used to deliver active molecules directly into the middle ear, which then diffuses through the RWM to the inner ear. However, this delivery route requires invasive surgery, which can be distressing for patients and carries the risk of permanent perforation of the tympanic membrane. Summary of the Invention

[0013] The present inventors have surprisingly found that NGF or a biologically active mutein thereof can be delivered to the inner ear in effective concentrations by intranasal administration. Therefore, a first object of the present invention is NGF or a mutein thereof for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein the NGF or mutein is administered intranasally to said subject.

[0014] A further object of the present invention is a pharmaceutical composition comprising NGF or a mutein thereof for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein the pharmaceutical composition is administered intranasally to said subject. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows NGF concentrations in the cochlear perilymph 2 hours after treatment in rhNGF-treated animals (NGF-treated animals) and PBS-treated animals (untreated animals), measured as described in Example 1. [Figure 2] 1 shows the NGF concentration in the cochlear perilymph at 12 hours in PBS-treated (PBS) animals, and at 12, 24, and 48 hours after treatment in rhNGF-treated (rhNGF) animals, measured as described in Example 1. [Figure 3] Baseline ABR threshold frequencies measured before any treatment are shown, as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). [Figure 4] Baseline DPOAE amplitudes measured before any treatment are shown at different frequencies as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). [Figure 5] 1 shows ABR thresholds at different frequencies measured after 2 months of treatment as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). [Figure 6] 1 shows DPOAE amplitudes at different frequencies measured after 2 months of treatment as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8+vehicle), or Group 3 (SAMP8+rhNGF). [Figure 7]1 shows plasma prestin concentrations measured after 2 months of treatment as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). [Figure 8] Shown are the total number of inner hair cells (IHCs) measured by immunohistological analysis in the middle turn of each cochlea, as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). [Figure 9] Shown are the total number of outer hair cells (OHCs) measured by immunohistological analysis in the middle turn of each cochlea, as described in Example 2, and data are shown for three experimental groups of mice: Group 1 (SAMR1), Group 2 (SAMP8 + vehicle), or Group 3 (SAMP8 + rhNGF). DETAILED DESCRIPTION OF THE INVENTION

[0016] A first object of the present invention is a nerve growth factor (NGF) or a mutein thereof for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein the NGF or mutein is administered intranasally to said subject.

[0017] As used herein, the terms "treatment" and "prevention" refer to elimination / amelioration of, or prevention / delay of onset of, a disorder or one or more symptoms associated therewith, respectively.

[0018] Preferably, the subject is a human subject. According to one embodiment, the subject has been diagnosed with sensorineural hearing loss, and the NGF or mutein thereof is used for treating the sensorineural hearing loss by intranasal administration to the subject.

[0019] According to an alternative embodiment, the subject has been identified as being at risk of developing sensorineural hearing loss, and the NGF or mutein thereof is used for the prevention of said sensorineural hearing loss by intranasal administration to the subject.

[0020] Preferably, the sensorineural hearing loss is a sensorineural hearing loss having a non-genetic etiology. Preferably, said sensorineural hearing loss having a non-genetic etiology is caused by noise exposure, bacterial or viral infection, treatment with ototoxic drugs, autoimmune disease, or aging.

[0021] Preferably, the ototoxic agent is selected from chemotherapeutic agents and aminoglycoside antibiotics, more preferably selected from cisplatin, carboplatin, and gentamicin.

[0022] Preferably, the NGF is human NGF. Preferably, the human NGF has the amino acid sequence of SEQ ID NO:1 below. SEQ ID NO:1: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRK AVR.

[0023] Alternatively, the human NGF has the amino acid sequence of SEQ ID NO:2 below. SEQ ID NO:2: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRK AVRRA.

[0024] Alternatively, the human NGF is a mixture of NGFs having the sequences of SEQ ID NO:1 and SEQ ID NO:2. Human NGF of SEQ ID NO: 2 has an amino acid sequence that differs only by the presence of two additional amino acids at the C-terminus from NGF of SEQ ID NO: 1. Because both forms of NGF are found in human cells, it is considered wild-type human NGF.

[0025] Therefore, when "human NGF" or "wild-type human NGF" is mentioned in this application, it means the human NGF of SEQ ID NO:1 or SEQ ID NO:2. Preferably, the NGF is produced by recombinant DNA technology, and is preferably human recombinant NGF (rhNGF). Methods for producing rhNGF are known to those skilled in the art, and are described, for example, in WO0022119A1 and WO2013092776A1. Preferably, the NGF has a purity of more than 70%, more preferably more than 80%, more than 90%, more than 95%, more than 98%, or more than 99%. The purity of NGF can be measured by conventional means known to those skilled in the art, for example, by HPLC analysis.

[0026] The term "NGF mutein" refers to a biologically active NGF mutein, meaning an NGF protein having an amino acid sequence with one or more amino acid mutations, preferably substitutions, such that the therapeutic activity of wild-type NGF is maintained.

[0027] Preferably, the mutein is a mutein of wild-type human NGF. Particularly preferred for use in accordance with the present invention are muteins of NGF characterized by greater than 70%, more preferably greater than 80%, even more preferably greater than 90%, and most preferably greater than 95% sequence identity with wild-type human NGF.

[0028] Preferably, the mutein is a mutein of wild-type human NGF and is characterized by at least one mutation, preferably an amino acid substitution in which proline at position 61 in the sequence of wild-type human NGF is replaced by another amino acid. In a particularly preferred embodiment, proline at position 61 is replaced by serine.

[0029] Preferably, the mutein is a mutein of human NGF and is characterized by at least one mutation in the amino acid sequence associated with reduced nociceptive activity. More preferably, the mutein is characterized by at least one mutation, preferably an amino acid substitution, at any of positions 95 to 101 of wild-type human NGF. Even more preferably, the mutein is characterized by a substitution of arginine at position 100 of wild-type human NGF. Most preferably, the substitution of arginine at position 100 of wild-type human NGF with glutamic acid.

[0030] Particularly preferred mutant proteins according to the present invention have the amino acid sequences of SEQ ID NOs: 3 to 6 below. SEQ ID NO:3: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKA VR. SEQ ID NO:4: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKA VRRA. SEQ ID NO:5: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDSNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKA VR SEQ ID NO:6: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKC RDSNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKA VRRA.

[0031] The above-mentioned muteins are particularly advantageous for use in the present invention, because they can reduce nociceptive sensitivity compared to the corresponding wild-type human NGF while maintaining the same biological activity as the wild-type human NGF. Preferably, the muteins of human NGF are produced by recombinant DNA technology. Methods for producing the muteins of rhNGF of the present invention by recombinant DNA technology are known to those skilled in the art, and are described, for example, in WO2019 / 207106.

[0032] Preferably, NGF or mutein for use according to the present invention is administered to a subject one to three times daily, with the treatment period being from 7 to 300 days, preferably from 60 to 240 days, more preferably from 100 to 200 days. Preferably, the amount of NGF or mutein thereof per administration is from 5 μg to 1 mg, more preferably from 10 μg to 400 μg, even more preferably from 15 μg to 200 μg.

[0033] When used for the treatment of SNHL, the effective amount of the NGF or mutant protein to be used in each administration, the duration of treatment, and the number of administrations per day will be selected by one skilled in the art based on the characteristics of the subject being treated, the severity of SNHL, and on the hearing tests performed during treatment.

[0034] When used for the prevention of SNHL, the effective amount of the NGF or mutant protein to be used in each administration, the duration of treatment, and the number of administrations per day will be selected by one skilled in the art based on the characteristics of the subject at risk of developing SNHL and a clinical assessment of the presence and duration of the risk of developing hearing loss as a result of one of the causes.

[0035] Evaluation of the above factors is within the knowledge and expertise of one of ordinary skill in the art. A further object of the present invention relates to a pharmaceutical composition for intranasal administration comprising the NGF or mutant protein as described above and at least one pharmaceutically acceptable excipient suitable for intranasal use.

[0036] Preferably, the pharmaceutical composition for intranasal administration of the present invention is a liquid intranasal composition. Preferably, the pharmaceutical composition of the present invention comprises an effective amount of the above-described NGF or mutant protein and at least one pharmaceutically acceptable excipient suitable for intranasal use, preferably selected from solvents, viscosity enhancing agents, mucoadhesive agents, buffers, antioxidants, preservatives, and penetration enhancers.

[0037] Preferably, the concentration of the NGF or variant protein in the liquid intranasal composition of the present invention is from 5 μg / ml to 1 mg / ml, more preferably from 10 μg / ml to 400 μg / ml, even more preferably from 15 μg / ml to 200 μg / ml.

[0038] Preferably, the solvent is water. Preferably, the mucoadhesive agent is glycerol, more preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v.

[0039] Preferably, the antioxidant is methionine, more preferably at a concentration of 0.005 mg / ml to 0.02 mg / ml, more preferably 0.01 mg / ml. Preferably, the surfactant is Kolliphor P188 (poloxamer 188), more preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v.

[0040] As used herein, the term "Kolliphor P188" refers to poloxamer 188, a block copolymer that is a synthetic copolymer of ethylene oxide and propylene oxide and is represented by the following chemical structure:

[0041] [ka]

[0042] where the a and b blocks have the following values:

[0043] [Table 1]

[0044] Preferably, the buffer is a phosphate buffer. Preferably, the penetration enhancer is n-dodecyl-β-D-maltoside, more preferably at a concentration of 0.1% w / v to 1% w / v, more preferably 0.5% w / v.

[0045] A particularly preferred liquid intranasal composition according to the present invention comprises, and preferably consists of, the NGF or mutein thereof, sodium chloride, phosphate buffer, and water. Another particularly preferred liquid intranasal composition of the present invention comprises, and preferably consists of, the NGF or mutein thereof as described above, sodium chloride, phosphate buffer, Kolliphor P188 (poloxamer 188), L-methionine, and water.

[0046] Another particularly preferred liquid intranasal composition according to the present invention comprises, and preferably consists of, the NGF or mutant protein, sodium chloride, phosphate buffer, Kolliphor P188 (poloxamer 188), L-methionine, glycerol, n-dodecyl-β-D-maltoside, and water.

[0047] Preferably, the liquid intranasal composition of the present invention comprises the following components: - NGF or a mutant protein thereof as defined above, preferably at a concentration of 5 μg / ml to 1 mg / ml, more preferably 10 μg / ml to 400 μg / ml, even more preferably 15 μg / ml to 200 μg / ml, NaH2PO4·H2O, preferably at a concentration of 5 to 8 mg / ml, more preferably 6.9 mg / ml; NaCl, preferably at a concentration of 5 to 6.5 mg / ml, more preferably 5.84 mg / ml; - poloxamer 188, preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v; L-methionine, preferably at a concentration of 0.005 mg / ml to 0.02 mg / ml, more preferably 0.01 mg / ml; - optionally n-dodecyl-β-D-maltoside, preferably at a concentration of 0.1% to 1% w / v, more preferably 0.5% w / v, and / or glycerol, preferably at a concentration of 0.05% to 0.2% w / v, more preferably 0.1% w / v, - water It comprises, and preferably consists of,

[0048] Pharmaceutical compositions of the present invention may be suitably formulated using any suitable method known in the art or by the methods disclosed in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton Pa.

[0049] Preferably, the pharmaceutical composition of the present invention is used for the prevention or treatment of sensorineural hearing loss in a subject, as described above, and said composition is administered intranasally to the subject. In a further aspect, the present invention relates to a method for the prevention or treatment of sensorineural hearing loss in a subject, comprising intranasally administering to the subject a therapeutically effective amount of NGF or a mutein thereof as described above.

[0050] Preferably, in the methods of the present invention, the NGF or mutant protein is administered as described above. Preferably, the NGF or mutant protein used in the methods of the present invention is in the form of a pharmaceutical composition as described above.

[0051] The present invention is further illustrated by the following examples, which do not limit the scope of the invention as claimed. [Example]

[0052] The bioavailability of rhNGF (having the sequence of SEQ ID NO: 1) in the inner ear (perilymph) after a single intranasal administration was evaluated in 10-week-old C57BL6 male mice.

[0053] Mice were randomly divided into six groups. Two control groups (n = 3) received vehicle, and cochlear perilymph was collected 2 and 12 hours after vehicle administration. Four treatment groups received a single 10 μL dose of rhNGF (SEQ ID NO: 1) solution at a concentration of 2 mg / mL in phosphate buffer, and samples were collected at 2 hours (n = 4), 12 hours (n = 4), 24 hours (n = 4), and 48 hours (n = 4). For intranasal treatment, the tip of a filled pipette was placed near the left nostril of the mouse, held at a 45-degree angle, and a droplet of the contents was released, allowing the mouse to immediately inhale. A second droplet was released into the same nostril approximately 2–3 seconds later, allowing the mouse to inhale. After administration, the mouse was held in this position for 15 seconds.

[0054] All mice were euthanized by cervical dislocation, and the tympanic bullae were removed. Each bulla was opened, the cochlea was washed with PBS, and 1 μl of cochlear perilymph was collected from each bulla using a capillary micropipette. The perilymph was immediately frozen in a low-binding Eppendorf tube and stored at −20°C until ELISA analysis.

[0055] Cochlear perilymph samples were diluted 1 / 10 and analyzed by ELISA (Novus Biological, Ref. NBP2-62776). Duplicate measurements were performed on the right and left cochlea. The manufacturer's protocol was optimized. Standard references were diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg / mL to generate a standard curve.

[0056] Different concentrations of standard working solution were added to the first two rows. Each concentration was added to two adjacent wells, and samples were added to the remaining wells (10 μl / well). The plate was covered with the sealer provided with the kit and incubated at 37°C for 90 minutes. The liquid in each well was then removed, and 100 μL of biotinylated detection antibody working solution was immediately added to each well.

[0057] The plate was re-covered with the sealer provided with the kit and incubated at 37°C for 1 hour. After incubation, the solution in each well was aspirated, and 350 μL of wash buffer was added and aspirated. This wash step was repeated three times. 100 μL of HRP conjugate working solution was added to each well. The plate was re-covered with the sealer provided with the kit and incubated at 37°C for 30 minutes. After washing, 90 μL of substrate reagent was added to each well and incubated at 37°C for 15 minutes. Finally, 50 μL of stop solution was added to each well, and the optical density of each well was immediately measured using a microplate reader set at 450 nm.

[0058] The results of the analysis are reported in Figures 1 and 2. Descriptive statistics by group were expressed as mean ± SEM for continuous variables. Statistical significance was determined using t-tests to compare treated and untreated control animals. Statistical analysis was performed using GraphPad Prism version 5.02 for Windows (GraphPad Software, La Jolla, California, USA). A P value of less than 0.05 was considered significant.

[0059] As can be seen in Figures 1 and 2, human NGF concentrations in the perilymph of the compound-treated group were significantly increased compared to the untreated control group. Two hours after a single intranasal NGF administration, the compound was detected in the perilymph at an average of 162.7 pg / mL, compared with an average of 27.3 pg / mL in the untreated control group (Figure 1). Peak concentrations were reached 12 hours after treatment, with an average of 832.201 pg / mL in the treated group and 7.331 pg / mL in the untreated control group (Figure 2).

[0060] The data obtained indicate that intranasal administration of NGF is an effective method for protein delivery to the inner ear. [Example]

[0061] The efficacy of intranasally administered rhNGF (sequence of SEQ ID NO: 1) was tested using the Senescence-Accelerated Mouse Prone (SAMP8) strain, a preclinical mouse model of age-related hearing loss (ARHL). This strain is widely used in aging research to study phenotypes such as peripheral neuropathy, hearing loss, immune dysfunction, osteoporosis, blindness, and brain atrophy. Premature aging in SAMP8 mice is accompanied by oxidative stress, altered levels of antioxidant enzymes, and an increase in senescent cells, leading to chronic inflammation, faithfully mimicking human aging. Senescence-Accelerated Resistant (SAMR1) mice were used as normal aging controls.

[0062] One-month-old SAMR1 and SAMP8 male mice (n=24) were purchased from Envigo, France. Mice were housed in Makrolon cages with filter hoods in rooms where the air was constantly filtered to prevent contamination. They were housed two per cage during the experiment, kept at a constant temperature, and maintained on a 12-hour day / 12-hour night cycle.

[0063] The mice were then divided into three groups and treated as follows: Group 1: SAMR1 mice without treatment (SAMR1) (negative control, n=8) Group 2: SAMP8 mice treated with vehicle (SAMP8 + vehicle) (n = 8) Group 3: SAMP8 mice treated with 10 μl of a 2 mg / ml rhNGF solution in saline buffer (carrier) administered intranasally (SAMP8+rhNGF) (n=8).

[0064] All animals underwent assessment of auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOE) on day 1 prior to treatment according to the procedures described below. As shown in Figures 3 and 4, similar ABR thresholds and DPOAE amplitudes were observed at baseline among the three groups at the analyzed frequencies, indicating no hearing impairment at this time point.

[0065] Intranasal administration was performed once daily for 60 days starting from the animals' age of 1 month, following the same procedure as described in Example 1. No deaths or clinical pathological signs were observed during this study.

[0066] On day 60, which corresponds to the animals being 3 months old, several tests described below were performed to determine the effect of treatment on hearing loss. Statistical significance of the obtained data was determined by two-way or one-way ANOVA, followed by Bonferroni or Tukey's multiple comparison post-hoc test, to compare between groups assuming normal distribution of variables and homogeneity of variance. Statistical analysis was performed using GraphPad Prism version 5.02 for Windows (GraphPad Software, La Jolla, California, USA). A P value of less than 0.05 was considered significant. Descriptive statistics by group were expressed as mean ± SEM for continuous variables.

[0067] Auditory brainstem response (ABR) ABR is an electrical potential recorded from scalp electrodes, with the first ABR wave representing the total activity of auditory nerve fibers in contact with the inner hair cells. For ABR studies, mice were anesthetized with a ketamine / xylazine mixture and their body temperature was regulated using a heating pad set at 37°C. Each mouse was then fitted with an earphone in its left ear, with the active electrode at the vertex of the skull, the reference electrode subcutaneously over the mastoid bone, and the ground electrode on the skin of the neck. Stimuli consisted of tone pips at five frequencies (2 kHz, 4 kHz, 6 kHz, 12 kHz, 16 kHz, and 24 kHz) varying in sound pressure level from 0 to 90 dB to cover the mouse's hearing frequency range. ABR measurements were performed individually for each animal using the OtoPhyLab system. Evoked potentials were extracted using a signal averaging technique for each noise level, and ABR thresholds for each frequency were determined using OtoPhyLab software.

[0068] The results obtained for each group of animals are shown in FIG. As expected, at 3 months of age, the SAMP8 + vehicle group exhibited significantly higher ABR thresholds at 6, 12, 16, and 24 kHz compared with the SAMR1 control group (Figure 4, SAMP8 + vehicle vs. SAMR1). SAMP8 animals treated with rhNGF exhibited significantly lower ABR thresholds at 12, 16, and 24 kHz compared with the SAMP8 + vehicle group, demonstrating that intranasally administered NGF has a substantial protective effect against age-induced hearing impairment (Figure 3, SAMP8 + NGF vs. SAMP8 + vehicle).

[0069] Distortion Product Otoacoustic Emissions (DPOAEs) DPOAEs are acoustic signals generated and amplified by the cochlear epithelium and provide an index of cochlear function. They are related to the health of outer hair cells (OHCs), which amplify cochlear vibrations induced by sound stimulation. They are independent of IHCs or auditory nerve fibers. For DPOAE measurements, mice were anesthetized with a ketamine / xylazine mixture, and a probe (OtoPhyLab) was inserted into the left ear canal. The primary tone F2 was set at five frequencies (4 kHz, 6 kHz, 12 kHz, 16 kHz, and 24 kHz) at a sound pressure level of 58 dB. The frequency ratio F2 / F1 was set to 1.2. At all frequencies (F2), the DPOAE system input was received, digitized, and evaluated using the microphone output. The amplitude of the frequency components at the distortion component frequencies was measured and expressed.

[0070] The results obtained are shown in FIG. As expected, at 3 months of age, the SAMP8 + vehicle group exhibited significantly lower DPOAE amplitudes at 6 kHz, 12 kHz, and 16 kHz compared with the SAMR1 control group (Figure 6, SAMP8 + vehicle vs. SAMR1). SAMP8 animals treated with rhNGF exhibited significantly higher DPOAE amplitudes at 6 kHz, 12 kHz, and 16 kHz compared with the SAMP8 + vehicle group (Figure 6, SAMP8 + rhNGF vs. SAMP8 + vehicle), confirming that intranasally administered nerve growth factor plays a direct or indirect protective role on outer hair cell function, leading to improved hearing in aged animals.

[0071] Prestin quantification Prestin is a protein recognized as a biomarker of cochlear damage. Therefore, the levels of this protein were assessed in animals at the end of treatment. Two milliliters of blood was collected from each animal by cardiac puncture and collected in tubes containing EDTA as an anticoagulant. Within 30 minutes of collection, the samples were centrifuged at 1,000 × g (or 3,000 rpm) for 15 minutes at 2–8°C. The supernatant (plasma) was stored at -80°C until analysis. Prestin quantification for each animal was performed in duplicate using an ELISA method (Mybiosource. Ref. SLC26A5. Cat Number MBS286559).

[0072] The results obtained are shown in FIG. As expected, at 3 months of age, the SAMP8 + vehicle group exhibited significantly higher plasma prestin concentrations than the SAMR1 control group (Figure 7, SAMP8 + vehicle vs. SAMR1), indicating cochlear damage and supporting the electrophysiological and otoacoustic results. SAMP8 animals treated with rhNGF exhibited significantly lower plasma prestin concentrations than the SAMP8 + vehicle group (Figure 7, SAMP8 + rhNGF vs. SAMP8 + vehicle). Taken together, these data confirm that intranasally administered nerve growth factor plays a protective role against age-induced hearing loss.

[0073] Cochlear cytocochleogram and ribbon synapses The total number of inner hair cells (IHCs) and outer hair cells (OHCs) in the middle turn of each cochlea was quantified by immunohistological analysis. Specifically, the left cochlea of all animals was removed, fixed overnight in paraformaldehyde solution, and then decalcified in EDTA for 7 days. The membranous and sensory spirals, including the organ of Corti, were removed, and hair cells were immunolabeled with anti-myosin VIIa antibody. The middle region of Corti was mounted on glass slides, images were acquired using a confocal microscope, and the images were saved in TIFF format. The total number of inner and outer hair cells was counted in the middle region. For ribbon synapse immunostaining, one right cochlea from each group was removed, fixed, and decalcified as described above. Ribbon synapses in the middle region were immunolabeled with anti-GluR2 antibody, and cochlear neurons were immunolabeled with anti-Tuj1 antibody. The samples were mounted on glass slides, images were acquired using a confocal microscope, and the images were saved in TIFF format.

[0074] As expected, at 3 months of age, the SAMP8 + vehicle group showed significantly fewer IHCs and OHCs than the SAMR1 control group, confirming cochlear damage due to aging (Figures 8 and 9, SAMP8 + vehicle vs. SAMR1, respectively). SAMP8 animals treated with rhNGF showed significantly more IHCs and OHCs than the SAMP8 + vehicle group (Figures 7 and 8, SAMP8 + rhNGF vs. SAMP8 + vehicle, respectively). Taken together, these results confirm that intranasal administration of nerve growth factor is effective against age-related hearing loss.

[0075] Scanning electron microscope image of the cochlea Three right cochleae from each group were removed from the temporal bone and fixed overnight at room temperature in 2.5% glutaraldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES free acid, 20 mM EGTA, 2 mM MgCl2). The stria vascularis, tectorial membrane, and Reissner's membrane were then removed by microdissection. After rinsing with PHEM buffer, the specimens were dehydrated in graded ethanol concentrations (30-100%), subjected to CO2 critical point drying, coated with gold-palladium, and examined using a Hitachi S4000 scanning electron microscope. Images were saved in TIFF format.

[0076] In the middle turn of the cochlea, scanning electron microscopy was used to determine the morphology of the stenocilia of outer and inner hair cells. Loss of outer hair cells was confirmed in vehicle-treated SAMP8 animals, but not in SAMR1 control or rhNGF-treated SAMP8 animals. Fusion of stenocilia of inner hair cells was observed in vehicle-treated SAMP8 animals, whereas normal stenocilia morphology was observed in rhNGF-treated SAMP8 animals. The morphology of stenocilia in the SAMP8 + rhNGF group was similar to that of SAMR1 control animals, confirming the histological protective effect of intranasal administration of nerve growth factor.

[0077] Image analysis of ribbon synapses Imaging analysis of ribbon synapses and cochlear neurons was performed using immunohistochemical labeling of the middle turn of the cochlea.

[0078] As expected, at 3 months, the cochleae of vehicle-treated SAMP8 mice had fewer ribbon synapses and neurons than SAMR1 mice.In rhNGF-treated SAMP8 mice, the number of ribbon synapses and efferent and afferent cochlear nerves was observed to be similar to that of SAMR1 control animals, confirming the histological protective effect of intranasal administration of nerve growth factor.

[0079] conclusion As expected, at 3 months, vehicle-treated SAMP8 mice exhibited elevated ABR thresholds, decreased DPOAE amplitudes, elevated plasma prestin concentrations, and significant cochlear hair cell loss, confirming the presence of hearing impairment due to accelerated aging in this mouse model. No differences were observed in SAMR1 control animals. SAMP8 mice treated once daily with rhNGF for 2 months exhibited significantly lower ABR thresholds, increased DPOAE amplitudes, decreased plasma prestin concentrations, and cochlear hair cell loss at 3 months compared with the vehicle group. Furthermore, histological characterization of the cochlea by SEM and immunohistochemistry demonstrated a lack of fusion of inner hair cell stenocilia and an increase in the number of ribbon synapses and cochlear neurons after compound treatment, supporting the electrophysiological, otoacoustic, and functional data. Taken together, these results confirm that nerve growth factor administered via the intranasal route exerts a significant protective effect against age-related hearing impairment in the preclinical accelerated aging model SAMP8. [Example]

[0080] The inventors have prepared the pharmaceutical compositions of the following examples, which are obtained according to the preparation methods disclosed below. Composition 1 rhNGF having the sequence of SEQ ID NO: 1 (0.6 mg / mL) 6.9 mg / mL NaH2PO4 H2O 5.84mg / mL NaCl 1mg / mL Kolliphor P188 5mg / mL n-dodecyl-β-D-maltoside 0.01mg / mL L-methionine ·Water for injection Dissolve 0.69 g of NaH2PO4·H2O, 0.58 g of NaCl, and 0.1 g of Kolliphor P188 in 100 mL of water. Wait for complete dissolution, then add 0.5 g of n-dodecyl-β-D-maltoside under magnetic stirring. After complete dissolution, add 0.001 g of L-methionine. Adjust the pH to 7 with NaOH.

[0081] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophin was added to the composition to a final concentration of 0.6 mg / mL. Composition 2 rhNGF having the sequence of SEQ ID NO: 1 (0.6 mg / mL) 6.9 mg / mL NaH2PO4 H2O 5.84mg / mL NaCl 5mg / mL n-dodecyl-β-D-maltoside 0.01mg / mL L-methionine ·Water for injection Dissolve 0.69 g of NaH2PO4·H2O and 0.58 g of NaCl in 100 mL of water. Wait for complete dissolution, then add 0.5 g of n-dodecyl-β-D-maltoside under magnetic stirring. After complete dissolution, add 0.001 g of L-methionine. Adjust the pH to 7 with NaOH.

[0082] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophin was added to the composition to a final concentration of 0.6 mg / mL. Composition 3 rhNGF having the sequence of SEQ ID NO: 1 (0.6 mg / mL) 6.9 mg / mL NaH2PO4 H2O 5.84mg / mL NaCl 1mg / mL Kolliphor P188 5mg / mL n-dodecyl-β-D-maltoside 2mg / mL glycerol 0.01mg / mL L-methionine ·Water for injection Dissolve 0.69 g of NaH2PO4·H2O, 0.58 g of NaCl, and 0.1 g of Kolliphor P188 in 100 mL of water. Wait for complete dissolution, then add 0.5 g of n-dodecyl-β-D-maltoside under magnetic stirring. Next, add 0.2 g of glycerol. After all excipients are completely dissolved, add 0.001 g of L-methionine. Adjust the pH to 7 with NaOH.

[0083] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophin was added to the composition to a final concentration of 0.6 mg / mL. Composition 4 rhNGF having the sequence of SEQ ID NO: 1 (0.6 mg / mL) 6.9 mg / mL NaH2PO4 H2O 5.84mg / mL NaCl 1mg / mL Kolliphor P188 2mg / mL glycerol 0.01mg / mL L-methionine ·Water for injection Dissolve 0.69 g of NaH2PO4·H2O and 0.58 g of NaCl in 100 mL of water. Wait for complete dissolution, then add 0.5 g of n-dodecyl-β-D-maltoside under magnetic stirring. Then add 0.2 g of glycerol. After all excipients are completely dissolved, add 0.001 g of L-methionine. Adjust the pH to 7 with NaOH.

[0084] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophin was added to the composition to a final concentration of 0.6 mg / mL.

Claims

1. A pharmaceutical composition comprising nerve growth factor (NGF) or a mutant protein thereof having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and at least one pharmaceutically acceptable excipient, for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein the pharmaceutical composition is administered intranasally to the subject.

2. The pharmaceutical composition for use according to claim 1, wherein the sensorineural hearing loss has a non-hereditary etiology.

3. The pharmaceutical composition for use according to claim 2, wherein the sensorineural hearing loss having a non-hereditary etiology is caused by noise exposure, bacterial or viral infection, treatment with ototoxic drugs, autoimmune disease, or aging.

4. The pharmaceutical composition for use according to claim 1, wherein the NGF is human NGF.

5. The pharmaceutical composition for use according to claim 4, wherein the human NGF has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

6. The pharmaceutical composition for use according to claims 1 to 5, wherein the NGF or mutant protein is administered once to three times a day over a treatment period of 7 to 300 days, preferably 60 to 240 days, more preferably 100 to 200 days.

7. The pharmaceutical composition for use according to claims 1 to 5, wherein the amount of NGF per administration is 5 μg to 1 mg, more preferably 10 μg to 400 μg, and even more preferably 15 μg to 200 μg.

8. The pharmaceutical composition for use according to claim 1, wherein the NGF or its mutant protein is present in the composition at a concentration of 5 μg / ml to 1 mg / ml, more preferably 10 μg / ml to 400 μg / ml, and even more preferably 15 μg / ml to 200 μg / ml.

9. A pharmaceutical composition for use according to claim 1, comprising, preferably comprising, NGF or its mutant protein, sodium chloride, phosphate buffer, and water.

10. Preferably, a concentration of 5 μg / ml to 1 mg / ml, more preferably 10 μg / ml to 400 μg / ml, and even more preferably 15 μg / ml to 200 μg / ml of NGF or a mutant protein having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, Preferably, a concentration of NaH of 5 to 8 mg / ml, more preferably 6.9 mg / mL. ２ PO ４ ・H ２ O and, NaCl at a concentration of 5 to 6.5 mg / mL, more preferably 5.84 mg / mL, Preferably, poloxamer 188 at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v, Preferably, L-methionine at a concentration of 0.005 mg / ml to 0.02 mg / ml, more preferably 0.01 mg / ml, Optionally, n-dodecyl-β-D-maltoside in a concentration preferably 0.1% w / v to 1% w / v, more preferably 0.5% w / v, and / or glycerol in a concentration preferably 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v, Water and A pharmaceutical composition containing, preferably comprising, these.

11. The pharmaceutical composition for use according to claim 4, wherein the NGF is recombinant human NGF.

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