Intranasal administration of BDNF for the treatment of sensorineural hearing loss

Abstract

The present invention relates to brain-derived neurotrophic factor (BDNF) for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein said BDNF 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 in the world, affecting 430 million people, more than 5% of the world's population. It is estimated that by 2050, more than 700 million people will have 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 variety of hearing impairments that can be divided into two main classes: (i) conductive hearing loss, which results from blockages or disease in the outer or middle ear that prevent the transmission of sound energy to the inner ear, and (ii) sensorineural hearing loss (SNHL), which accounts for 90% of all hearing loss cases and results from damage to the inner ear and auditory nerve. Specifically, sensorineural hearing loss is characterized by the degeneration of two main 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 through the auditory nerve to the auditory nuclei in the brainstem.

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

[0005] Hair cell degeneration in sensorineural hearing loss can be of genetic or non-genetic etiology, including 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 that hearing loss has on the general population, the only currently approved treatment option is the cochlear implant (CI) hearing aid, which works by directly stimulating the neuronal cell bodies (soma) of spiral ganglion cells (SGCs) and possibly their central axons, providing patients with partial restoration of sensory function. Currently, there are no FDA-approved drug therapies for the prevention or treatment of this group of disorders.

[0007] In clinical practice, non-hereditary SNHL is usually treated with the administration of corticosteroids, particularly dexamethasone and prednisolone, however, this treatment has not proven to produce satisfactory results.

[0008] Therefore, there is a strong need to develop new and more effective drug therapies that can target the inner ear and restore hearing function by preventing damage to or replenishing hair cell and neuronal populations within the cochlea.

[0009] Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, a signaling molecule well documented for its ability to regulate neuroplasticity, cell growth, proliferation, cell survival, and long-term memory.

[0010] BDNF has been identified in preclinical and clinical studies as a promising therapeutic approach for the prevention and treatment of SNHL. Preclinical studies have demonstrated that BDNF is effective in rescuing spiral ganglion neurons after hair cell injury, and in animal models of cochlear synaptopathy, local administration of BDNF to the cochlea has been shown to restore ribbon synapses and their function (Liu et al., Front. Neurosci. 2022, 16:art.867453; Foster at al., J. Acoust. Soc. Am. 2022, 151(6):3937-3946; Foster at al., Pharmacol Res Perspect. 2022, 10(3):e00970).

[0011] Additionally, a clinical trial is underway to evaluate the safety, tolerability, and efficacy of OTO-413 administered as an intratympanic injection for the treatment of speech hearing impairment in noise (https: / / clinicaltrials.gov / ct2 / show / NCT04129775?term=otonomy&draw=3).

[0012] However, a major challenge for the effective use of BDFN in the treatment of hearing impairment is the delivery of the protein to the inner ear compartment. The delivery of drugs, especially large molecules such as proteins, to the inner ear compartment is hindered by the presence of several protective barriers.

[0013] The blood-labyrinth barrier (BLB), which separates the inner ear lymphatic fluid from the blood circulation, limits the possibility of systemic delivery. At the same time, topical delivery into the ear is hindered by barriers between different compartments of the ear: the tympanic membrane (TM) between the outer and middle ear, and the round window membrane (RWM) between the middle and inner ear.

[0014] To achieve higher drug concentrations in the middle ear, ongoing clinical trials using BDNF have used intratympanic injection of the protein to deliver BDNF directly into the middle ear, where it then diffuses across the RWM into the inner ear. However, this delivery route requires an invasive surgical procedure that can be painful for patients and carries the risk of permanent tympanic membrane perforation.

[0015] Another method used in preclinical studies to deliver BDNF to the inner ear has been the implantation of osmotic minipumps into the scala tympani, however, again, the clinical application of this method is limited by the fact that it requires patients to undergo an invasive surgical procedure. [Prior art documents] [Non-patent literature]

[0016] [Non-Patent Document 1] Cunningham al.,N.Engl.J.Med.2017,377(25):2465-2473 [Non-patent document 2] Liu et al,Front.Neurosci.2022,16:art.867453 [Non-patent document 3] Foster at al,J.Acoust.Soc.Am.2022,151(6):3937-3946 [Non-patent document 4] Foster at al,Pharmacol Res Perspect.2022,10(3):e00970 Summary of the Invention

[0017] The present inventors have now surprisingly found that BDNF can be delivered to the inner ear in effective concentrations by intranasal administration. Accordingly, a first object of the present invention is BDNF for use in the prevention or treatment of sensorineural hearing loss in a subject, said BDNF being administered intranasally to said subject.

[0018] A further object of the present invention is a pharmaceutical composition comprising BDNF and at least one pharmaceutically acceptable excipient for use in the prevention or treatment of sensorineural hearing loss in a subject, said composition being administered intranasally to said subject. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows the concentration of BDNF in the perilymph at various times after administration of rhBDNF (BDNF) or phosphate buffered saline (PBS), measured as described in Example 1. ***: p<0.0002 vs. PBS; ****: p<0.0001 vs. PBS. [Figure 2]Figure 2 shows ABR threshold shifts at low (6 kHz), mid (12, 16, and 20 kHz), and high (24 and 32 kHz) frequencies 7 days after cisplatin treatment in vehicle-treated (gray line) or rhBDNF-treated (black line) mice, measured as described in Example 2, compared to those measured on day 0. DETAILED DESCRIPTION OF THE INVENTION

[0020] A first object of the present invention is a brain-derived neurotrophic factor (BDNF) for use in the prevention or treatment of sensorineural hearing loss in a subject, said BDNF being administered intranasally to said subject. As used herein, the terms "treatment" and "prevention" refer respectively to eradicating / ameliorating or preventing / delaying the onset of a disorder or one or more symptoms associated therewith.

[0021] Preferably, the subject is a human subject. According to one embodiment, the subject has been diagnosed with sensorineural hearing loss, and the BDNF is used to treat the sensorineural hearing loss by intranasal administration to the subject.

[0022] In an alternative embodiment, the subject has been identified as being at risk for developing sensorineural hearing loss, and the BDNF is used to prevent said sensorineural hearing loss by intranasal administration to the subject. Preferably, the sensorineural hearing loss is sensorineural hearing loss of non-genetic etiology.

[0023] Preferably, said sensorineural hearing loss of non-genetic etiology is caused by noise exposure, bacterial or viral infection, treatment with ototoxic drugs, autoimmune disease or aging. Preferably, the ototoxic drug is selected from chemotherapeutic drugs and aminoglycoside antibiotics, more preferably selected from cisplatin, carboplatin and gentamicin.

[0024] Preferably, the BDNF is human BDNF, more preferably recombinant human BDNF (rhBDNF). Preferably, BDNF for use according to the present invention is administered 1 to 3 times daily for a treatment period of 7 to 300 days, preferably 60 to 240 days, more preferably 100 to 200 days.

[0025] Preferably, the amount of BDNF per administration is 5 μg to 1 mg, more preferably 10 μg to 400 μg, and even more preferably 15 μg to 200 μg. For the treatment of SNHL, the effective amount of BDNF to be used in each administration, the duration of treatment, and the number of daily administrations will be selected by one skilled in the art based on the characteristics of the subject being treated, the severity of the SNHL, and based on the hearing tests performed during treatment.

[0026] In the case of prevention of SNHL, the effective amount of BDNF 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 an assessment of the characteristics of the subject at risk of developing SNHL as a result of one of the above causes and the nature and duration of the risk of developing SNHL.

[0027] 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 BDNF as defined above.

[0028] Preferably, the pharmaceutical composition for intranasal administration of the present invention is a liquid intranasal composition. Preferably, the pharmaceutical composition according to the present invention comprises an effective amount of BDNF and at least one pharmaceutically acceptable excipient, preferably selected from a solvent, a viscosity increasing agent, a mucoadhesive agent, a buffering agent, an antioxidant, a preservative, and a penetration enhancer.

[0029] Preferably, the concentration of BDNF in the liquid intranasal composition according to the present invention is from 5 μg / ml to 1 mg / ml, more preferably from 10 μg / ml to 400 μg / ml, and even more preferably from 15 μg / ml to 200 μg / ml.

[0030] Preferably, the solvent is water. Preferably, the antioxidant is methionine, more preferably methionine at a concentration of 0.005 mg / ml to 0.02 mg / ml, more preferably 0.01 mg / ml.

[0031] Preferably, the surfactant is Kolliphor P188 (Poloxamer 188), more preferably Kolliphor P188 at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v.

[0032] 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 represented by the following chemical structure:

[0033] [ka]

[0034] In the formula, the a and b blocks have the following values:

[0035] [Table 1]

[0036] Preferably, the buffer is a phosphate buffer. Preferably, the penetration enhancer is n-dodecyl-β-D-maltoside, more preferably n-dodecyl-β-D-maltoside at a concentration of 0.1% w / v to 1% w / v, more preferably 0.5% w / v.

[0037] A particularly suitable liquid intranasal composition according to the present invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer and water. Another particularly suitable liquid intranasal composition according to the present invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer, Kolliphor P188 (Poloxamer 188), L-methionine and water.

[0038] Another particularly suitable liquid intranasal composition according to the present invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer, Kolliphor P188 (Poloxamer 188), L-methionine, n-dodecyl-β-D-maltoside and water.

[0039] Preferably, the liquid intranasal composition according to the present invention comprises, preferably consists of, the following ingredients: BDNF, 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 * HO, preferably NaHPO4 at a concentration of 5-8 mg / mL, more preferably 6.9 mg / mL * H2O, 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% w / v to 1% w / v, more preferably 0.5% w / v ·water.

[0040] Pharmaceutical compositions according to 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.

[0041] Preferably, the pharmaceutical composition of the present invention is for use in 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 also relates to a method for preventing or treating sensorineural hearing loss in a subject, the method comprising intranasally administering to the subject a therapeutically effective amount of BDNF as described above.

[0042] Preferably, in the method according to the invention, said BDNF is administered as described above. Preferably, the BDNF used in the method of the present invention is in the form of a pharmaceutical composition as defined above.

[0043] The present invention is further described in the following examples, which do not limit the scope of the invention as defined in the claims. [Example]

[0044] Example 1 The bioavailability of recombinant human BDNF (rhBDNF) in the inner ear (perilymph) after a single intranasal administration was evaluated in 10-week-old C57BL6 mice (Envigo Labs, France).

[0045] The mice were divided into six groups. Group 1 (n=4): control group, treated with PBS, sample collection was performed 2 hours after treatment. Groups 2–6: rhBDNF-treated groups, perilymph samples collected at different time points.

[0046] Group 2 (n=4): treated with rhBDNF, sample collection was performed at 2 hours. Group 3 (n=4): treated with rhBDNF, sample collection was performed at 6 hours. Group 4 (n=4): treated with rhBDNF, sample collection was performed at 12 hours.

[0047] Group 5 (n=4): treated with rhBDNF, sample collection was performed at 24 hours. Group 6 (n=4): treated with rhBDNF, sample collection was performed at 48 hours. The animals were treated as follows:

[0048] A micropipette was filled with 10 μl of 2 mg / ml rhBDNF (expressed in E. coli by Dompe farmaceutici SpA) solution in 50 mM sodium phosphate buffer and 100 mM NaCl (pH 7.2) (Groups 2–6) or PBS (Group 1), and the tip of the filled pipette was placed near the left nostril of the mouse at a 45-degree angle. One drop of the contents was immediately inhaled by the mouse. Approximately 2–3 seconds later, a second drop was released through the same nostril for the mouse to inhale. After administration, the mouse remained in this position for 15 seconds.

[0049] All mice were sacrificed by cervical dislocation at the appropriate time points, and the tympanic bulla was removed. Each tympanic bulla was opened, the cochlea was washed with PBS, and 1 μl of cochlear perilymph was collected from each cochlea using a capillary micropipette. The perilymph was immediately frozen in a low-binding Eppendorf tube and stored at −20°C until ELISA analysis. The contralateral cochlea was used as a duplicate.

[0050] Cochlear perilymph samples were diluted 1 / 10 and 10 μl per well was analyzed by ELISA (Thermo Fisher, Ref. EH42RB). To generate a standard curve, dilutions of the rhBDNF standard reference solution were performed at 0, 0.066 ng / mL, 0.160 ng / mL, 0.410 ng / mL, 1.020 ng / mL, 2.560 ng / mL, 6.40 ng / mL, and 16 ng / mL. The resulting standard curve for rhBDNF was R 2 A linear regression with a slope of 0.1209±0.0005206 and a mean of 0.9997 allows interpolation of the curve to determine the concentration of rhBDNF in each mouse sample.

[0051] Various concentrations of standard working solutions were added to the first two columns. Each concentration was added to two adjacent wells, and the samples to be analyzed were added to the other wells (10 μl / well). The plate was covered with the seal provided in the kit and incubated overnight at 4°C. Washing and incubation procedures were performed according to the manufacturer's instructions. At the end of the procedure, 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.

[0052] The results of the analysis are reported in Figure 1. As can be seen in Figure 1, a gradual and significant increase in rhBDNF concentrations was observed in the perilymph of the rhBDNF-treated group compared with the PBS-treated control group from 2 to 48 hours after treatment. The rhBDNF concentration peaked at 12 hours after administration, reaching 9.91 ng / mL ± 1.02. A subsequent decrease in rhBDNF concentration was observed at 24 and 48 hours after treatment, reaching concentrations (mean ± SEM) of 9.33 ng / mL ± 0.59 and 8.20 ng / mL ± 0.47, respectively. The data obtained demonstrate that intranasal administration of rhBDNF results in delivery of the protein to the cochlear perilymph, with peak concentrations at 12 hours and accumulating for up to 48 hours after treatment, suggesting that this administration route is suitable for targeting hearing impairment.

[0053] Example 2 The efficacy of recombinant human BDNF (rhBDNF) administered via the intranasal route was tested in a cisplatin-induced hearing loss mouse model.

[0054] Specifically, adult male Wistar rats (Catholic University Laboratories, 200-250 g), 2 months old, with normal Preyer's reflexes were used in this study. Experiments were conducted on a total of 13 animals randomly assigned to two experimental groups. Group 1: Animals treated with cisplatin and phosphate buffer pH 7.2 (vehicle); ("Cis+vehicle" group; n=6), Group 2: animals treated with cisplatin and recombinant hBDNF (“Cis+rhBDNF” group; n=7).

[0055] All animals were treated with cisplatin (catalog no. P4394, Sigma-Aldrich, St. Louis, MO, USA) at time 0. Specifically, cisplatin was diluted (1 mg / ml) in sterile saline (freshly prepared, protected from light). To facilitate drug dissolution, the solution was heated and stirred for 20 minutes. Under deep anesthesia, a single dose of 12 mg / kg cisplatin was delivered intraperitoneally (ip) using an infusion pump (Axon Instruments, Foster City, CA, USA) at a rate of 8 ml / h over approximately 30 minutes. To limit the side effects of cisplatin, animals were overdosed with saline (subcutaneous injection, 15 ml daily).

[0056] Next, rhBDNF (expressed in Escherichia coli by Dompe farmaceutici SpA) (2.4 mg / mL) (Group 2) or vehicle (Group 1) was administered intranasally daily for 7 days. Administration began 1 hour after cisplatin treatment (Day 0). Using the dominant (non-paralyzed) hand, a micropipette (Gilson) was filled with 40 μl of rhBDNF or vehicle. The tip of the filled pipette was placed at a 45-degree angle into the rat's nostril. Each administration consisted of the slow release of one drop of drug or vehicle into each nostril twice (40 μl per nostril, 80 μl per animal). The animal was allowed to suck up the drops, taking care not to let them enter the cleft. The immobilized animals were placed in a supine-like position to facilitate drug entry and absorption into the nostrils. At the end of each administration, the rats were returned to their cages and their nostrils were allowed to dry completely.

[0057] To assess the hearing loss induced by cisplatin treatment and the effects of rhBDNF administration, auditory function was estimated by ABR recording. In all animals, ABRs were measured at low (6 kHz), mid (12, 16, and 20 kHz), and high (24 and 32 kHz) frequencies on days 0 and 7 of treatment. In all animals, ABRs were assessed bilaterally before treatment to confirm normal hearing and reassessed at all time points to assess the effects of treatment on hearing.

[0058] ABR recordings were performed as follows. All animals were lightly anesthetized (ketamine 35 mg / kg and medetomidine-domitol 0.25 mg / kg) and placed in an anechoic chamber. Three stainless steel recording electrodes were inserted subcutaneously behind the test auricle (active), the parietal region (reference), and the contralateral auricle (ground). A PC-controlled TDT System 3 (Tucker Davis Technologies, Alachua, FL, USA) data acquisition system with real-time digital signal processing was used for ABR recording and auditory stimulus generation. Pure tone bursts (1 ms rise / fall time, 10 ms total duration, 20 / s repetition rate) ranging from 6 to 32 kHz were presented monaurally. Responses were filtered (0.3–3 kHz), digitized, and averaged (across 500 separate samples at each frequency level combination). Threshold was defined as the lowest stimulus level at which a repeatable waveform-based onset was obtained.

[0059] The results obtained are shown in FIG. As can be seen, in the cisplatin and vehicle treated groups, an increase in threshold shift of approximately 25 dB was observed at all frequencies analyzed, whereas treatment with rhBDNF reduced hearing loss by approximately 15 dB at low and mid frequencies and 10 dB at high frequencies.

[0060] These data demonstrate that noninvasive intranasal administration of rhBDNF induced recovery of auditory function in an animal model of cisplatin ototoxicity. Example 3 The inventors have prepared the following example pharmaceutical compositions, which were obtained according to the manufacturing methods disclosed below.

[0061] Composition 1 rhBDNF (0.6 mg / mL) 6.9mg / mL NaH2PO4 * H2O 5.84mg / mL NaCl 1mg / mL Kolliphor P188 5mg / mL n-dodecyl-β-D-maltoside 0.01mg / mL L-methionine WFI 0.69g of NaH2PO4 in 100mL of water * In HO, 0.58 g of NaCl and 0.1 g of Kolliphor P188 were dissolved. After complete solubilization, 0.5 g of n-dodecyl-β-D-maltoside was added under magnetic stirring. After complete solubilization, 0.001 g of L-methionine was added. The pH was adjusted to 7 with NaOH.

[0062] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophic factors were added to the composition at a final concentration of 0.6 mg / mL. Composition 2 rhBDNF (0.6 mg / mL) 6.9mg / mL NaH2PO4 * H2O 5.84mg / mL NaCl 5mg / mL n-dodecyl-β-D-maltoside 0.01mg / mL L-methionine WFI 0.69g of NaH2PO4 in 100mL of water * 0.58 g of NaCl was dissolved in H2O. After complete solubilization, 0.5 g of n-dodecyl-β-D-maltoside was added under magnetic stirring. After complete solubilization, 0.001 g of L-methionine was added. The pH was adjusted to 7 with NaOH.

[0063] After complete dissolution, the composition was filtered through a 0.22 μm filter. Neurotrophic factors were added to the composition at a final concentration of 0.6 mg / mL.

Claims

1. A pharmaceutical composition comprising brain-derived neurotrophic factor (BDNF) 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 according to claim 1, wherein the sensorineural hearing loss is due to a non-genetic etiology.

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

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the BDNF is human BDNF, more preferably recombinant human BDNF.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered once to three times a day for a treatment period of 7 to 300 days, preferably 60 to 240 days, and more preferably 100 to 200 days.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the amount of BDNF per dose is 5 μg to 1 mg, more preferably 10 μg to 400 μg, and even more preferably 15 μg to 200 μg.

7. the below described: BDNF, preferably 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. NaH2PO4 ＊ H2O, preferably at a concentration of 5-8 mg / ml, more preferably 6.9 mg / mL of NaH2PO4 ＊ H2O, 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 at 0.01 mg / ml. Optionally, n-dodecyl-β-D-maltoside, preferably at a concentration of 0.1% w / v to 1% w / v, more preferably 0.5% w / v. ·water A pharmaceutical composition containing, preferably comprising, these.

8. The pharmaceutical composition according to claim 7, wherein the BDNF is human BDNF, more preferably recombinant human BDNF.

9. The pharmaceutical composition according to any one of claims 1 to 3, further comprising at least one pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, wherein the BDNF is contained 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.

11. The pharmaceutical composition according to claim 9, comprising, preferably comprising, BDNF, sodium chloride, phosphate buffer and water.

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