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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals

Definitions

• the present invention relates to the prevention and treatment of sensorineural hearing loss.
• Hearing loss is the fourth leading cause of disability worldwide, affecting 430 million people, over 5% of the 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-hearinq-loss.).
• hearing loss refers to a vast array of hearing disorders that can be classified into two main classes: (i) conductive hearing loss, wherein the hearing loss results from obstruction or disease of the outer or middle ear that prevents transmission of sound energy to the inner ear, and (ii) sensorineural hearing loss (SNHL), which constitute 90% of all cases of hearing loss, wherein the hearing loss results from damage to the inner ear and the auditory nerve.
• conductive hearing loss wherein the hearing loss results from obstruction or disease of the outer or middle ear that prevents transmission of sound energy to the inner ear
• SNHL sensorineural hearing loss
• sensorineural hearing loss is mainly characterized by the degeneration of two types of cells: cochlear hair cells, which are the primary mechanoreceptors converting sound energy into neural signals, and/or auditory nerve neurons, the spiral ganglion cells (SGCs), that transmit signals from cochlear hair cells to the auditory nuclei of the brain stem through the auditory nerve.
• cochlear hair cells which are the primary mechanoreceptors converting sound energy into neural signals
• auditory nerve neurons the spiral ganglion cells (SGCs)
• SGCs spiral ganglion cells
• the degeneration of hair cells in sensorineural hearing loss may have genetic or non- genetic etiology.
• Non-genetic factors include noise exposure, viral or bacterial infections, ototoxic chemicals such as the chemotherapeutic drug cisplatin or aminoglycoside antibiotics, autoimmune diseases and aging (Liu et al, Front. Neurosci. 2022, 16: art. 867453).
• non-genetic SNHL is usually treated by administration of corticosteroids, in particular dexamethasone and prednisolone.
• corticosteroids in particular dexamethasone and prednisolone.
• this treatment has not proven to lead to satisfactory outcomes.
• Brain-derived neurotrophic factor is a member of the neurotrophin family. This signaling molecule has been well-documented for its ability to regulate neuronal plasticity, cell growth, proliferation, cell survival, and long-term memory.
• BDNF has been identified in preclinical and clinical studies as a promising therapeutic approach for the prevention and treatment of SNHL.
• the blood labyrinth barrier which separates the inner ear fluids from blood circulation, limits the possibility of systemic delivery.
• delivery by local administration into the ear is hindered by the 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.
• TM tympanic membrane
• RWM round window membrane
• the intratympanic injection of the protein has been used to deliver BDNF directly into the middle ear for subsequent diffusion into the inner ear across the RWM.
• this delivery route requires an invasive surgical procedure, which may distress patients and carries the risk of permanent tympanic membrane perforation.
• BDNF can be delivered at effective concentrations in the inner ear by intranasal administration.
• a first object of the invention is BDNF for use in the prevention or treatment of sensorineural hearing loss in a subject, wherein said BDNF is administered intranasally to said subject.
• a further object of the 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, wherein said composition is administered intranasally to said subject.
• Figure 1 shows the concentration of BDNF in the perilymph at different timepoints after administration of rhBDNF (BDNF) or of phosphate buffer (PBS), measured as described in Example 1 .
• BDNF rhBDNF
• PBS phosphate buffer
• Figure 2 shows the threshold shift of ABRs at low (6 kHz), mid (12, 16, and 20 kHz), and high (24 and 32 kHz) frequencies 7 days after the cisplatin treatment vs those measured at day 0, in mice treated with vehicle (grey line) or rhBDNF (black line), measured as described in Example 2.
• a first object of the invention is brain derived nerve 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.
• BDNF brain derived nerve factor
• treatment and prevention refer to the eradication/amelioration or prevention/delay in onset, respectively, of a disorder or of one or more of the symptoms associated thereof.
• said subject is a human subject.
• said subject has been diagnosed with a sensorineural hearing loss and said BDNF is for use in the treatment of said sensorineural hearing loss by intranasal administration to said subject.
• said subject has been identified as being at risk of developing a sensorineural hearing loss and said BDNF is for use in the prevention of said sensorineural hearing loss by intranasal administration to said subject.
• said sensorineural hearing loss is a sensorineural hearing loss of non-genetic etiology.
• said sensorineural hearing loss of non genetic etiology is caused by noise exposure, a bacterial or viral infection, the treatment with an ototoxic drug, an autoimmune disease or aging.
• said ototoxic drug is selected from chemotherapeutic drugs and aminoglycoside antibiotics, more preferably it selected from cisplatin, caroboplatin and gentamicin.
• BDNF is human BDNF, more preferably it is recombinant human BDNF (rhBDNF).
• the BDNF for use according to the invention is administered from one to three times a day for a period of treatment of between 7 and 300 days, preferably between 60 and 240 days, more preferably between 100 and 200 days.
• the amount of BDNF per each administration is between 5 pg and 1 mg, more preferably between 10 pg and 400 pg, even more preferably between 15 pg and 200 pg.
• the effective amount of BDNF used in each administration, the duration of the treatment and the number of administrations for day are selected by the skilled person based on the characteristics of the subject to be treated, the severity of the SNHL and on the basis of the auditory tests carried out during the treatment.
• the effective amount of BDNF used in each administration, the duration of the treatment and the number of administrations per day are selected by the skilled man based on the characteristics of the subject at risk of developing SNHL and evaluation of the entity and duration of the risk of developing SNHL as a result of one of said causes.
• a further object of the present invention relates to a pharmaceutical composition for intranasal administration comprising BDNF, as defined above.
• the pharmaceutical composition for intranasal administration of the invention is a liquid intranasal composition.
• the pharmaceutical composition according to the present invention comprises an effective amount of BDNF and at least one pharmaceutically acceptable excipient, preferably selected from solvents, thickening agents, mucoadhesive agents, buffers, antioxidants, preservatives, and penetration enhancers.
• BDNF BDNF
• at least one pharmaceutically acceptable excipient preferably selected from solvents, thickening agents, mucoadhesive agents, buffers, antioxidants, preservatives, and penetration enhancers.
• the concentration of BDNF in the liquid intranasal composition according to the invention is between 5 pg/ml and 1 mg/ml, more preferably between 10 pg/ml and 400 pg/ml, even more preferably between 15 pg/ml and 200 pg/ml.
• said solvent is water.
• said antioxidant is methionine, more preferably at a concentration between 0.005 mg/ml and 0.02 mg/ml, more preferably of 0.01 mg/ml.
• said surfactant is Kolliphor P188 (Poloxamer 188), more preferably at a concentration between 0.05 % w/v and 0.2% w/v, more preferably of 0.1% w/v.
• Kerphor 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: where in a and b blocks have the following values:
• said buffer is phosphate buffer.
• said penetration enhancer is n-Dodecyl-p-D-maltoside, more preferably at a concentration between 0.1 % w/v and 1% w/v, more preferably of 0.5% w/v.
• a particularly preferred liquid intranasal composition according to the invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer and water.
• Another particularly preferred liquid intranasal composition according to the invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer, Kolliphor P188 (Poloxamer 188), L-Methionine and water.
• Another particularly preferred liquid intranasal composition according to the invention comprises, preferably consists of, BDNF, sodium chloride, phosphate buffer, Kolliphor P188 (Poloxamer 188), L-Methionine, n-Dodecyl-p-D-maltoside and water.
• liquid intranasal composition comprises, preferably consists of, the following components:
• -BDNF preferably at a concentration between 5 pg/ml and 1 mg/ml, more preferably between 10 pg/ml and 400 pg/ml, even more preferably between 15 pg/ml and 200 pg/ml
• -NaH2PO4 * H2O preferably at a concentration between 5 and 8 mg/ml, more preferably of 6.9 mg/mL
• -NaCI preferably at a concentration between 5 and 6.5 mg/ml, more preferably of 5.84 mg/mL,
• -Poloxamer 188 preferably at a concentration between 0.05 % w/v and 0.2% w/v, more preferably of 0.1 % w/v,
• -L-Methionine preferably at a concentration 0.005 mg/ml and 0.02 mg/ml, more preferably of 0.01 mg/ml,
• n-Dodecyl-p-D-maltoside preferably at a concentration between 0.1 % w/v and 1 % w/v, more preferably of 0.5% w/v, -Water.
• the pharmaceutical composition according to the invention may be suitably formulated using appropriate methods known in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton Pa.
• the pharmaceutical composition of the invention is for use in the prevention or treatment of sensorineural hearing loss in a subject, as above described, wherein said composition is administered intranasally to the subject.
• 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 BDNF, as described above, in a therapeutically effective amount.
• said BDNF is administered as described above.
• said BDNF used in the method of the invention is in form of a pharmaceutical composition, as above defined.
• rhBDNF recombinant human BDNF
• mice were divided in six groups:
• Group 1 control group, treated with PBS and sampling carried out at 2 hours after treatment.
• Groups 2-6 rhBDNF-treated groups with sampling of the periplymph at different timepoints:
• the animals were treated as follows.
• a micropipette was filled with 10 pl of a solution of rhBDNF (expressed in E.coli at Dompe farmaceutici SpA) at 2mg/ml in sodium phosphate buffer 50mM, 100mM NaCI, pH 7.2, (groups 2-6) or with PBS (group 1 ) and the tip of the filled pipette was placed near the mouse's left nostril, which was kept at a 45 degree angle, and a droplet of the content released which was immediately inhaled by the mouse. A second droplet was released for the mouse to inhale it through the same nostril about 2-3 sec later. After administration, the mice were held in this position for 15 sec.
• mice All mice were sacrificed by cervical dislocation at the relevant timepoints and the tympanic bullas were isolated. Each tympanic bulla was opened, the cochlea was cleaned with PBS and 1 pl of cochlear perilymph was sampled from each cochlea using a capillary micropipette and immediately frozen in a low-binding Eppendorf tube and stored at -20 °C before ELISA analysis. The contralateral cochlea was used as duplicated.
• the cochlear perilymph samples were diluted at 1/10 and 10 pl per well analyzed by ELISA (Thermo Fisher, Ref. EH42RB).
• Dilutions of the rhBDNF standard reference 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 to perform the standard curve.
• the standard curve of rhBDNF obtained presented a linear regression of R square 0.9997 and slope of 0.1209 ⁇ 0.0005206 allowing to perform the interpolation of the curve to determine the rhBDNF concentration in each mouse sample.
• each concentration of the solution was added into two wells side by side and the samples to be analyzed to the other wells (10 pl/well).
• the plate was covered with sealer provided in the kit and incubated overnight at 4 °C. The washing and incubation procedure was performed following the manufaturer’s instructions.
• 50 pl of Stop Solution was added to each well and the optical density of each well was immediately determined using a micro-plate reader set to 450 nm.
• rhBDNF recombinant human BDNF administered via the intranasal route
• rhBDNF recombinant human BDNF
• mice Male adult Wistar rats (Catholic University Laboratories, 200-250 g), 2 months of age, with normal Preyer's reflex, were used in this study.
• cisplatin Cat. No. P4394, Sigma-Aldrich, St. Louis, MO, USA
• cisplatin was diluted in sterile saline (1 mg/ml), and prepared freshly, protected by light. To facilitate drug dissolution, the solution was heated and stirred for a period of 20 minutes. Under deep anesthesia, a single cisplatin dose of 12 mg/kg was delivered intraperitoneally (i.p.) at a rate of 8 ml/h with an infusion pump (Axon Instruments, Foster City, CA, USA) over about 30 minutes. The animals were hyper-hydrated with saline solution (subcutaneous injection, 15 ml daily) to limit cisplatin side effects.
• rhBDNF expressed in E.coli at Dompe farmaceutici SpA (2.4mg/mL) (Group 2) or vehicle (Group 1 ) was then administered by intra-nasal route daily for 7 days, starting 1 hour after the cisplatin treatment (day 0).
• the micropipette (Gilson) was filled with 40pl of rhBDNF or vehicle.
• the tip of the filled pipette was placed in the rat's nostril, kept at a 45-degree angle.
• Each administration involved the gradual release of two single drops of the drug or vehicle in the nostrils (40pl per nostril, 80p I animal). The drops were sucked up by the animal taking care to prevent its transfer into the oral opening.
• the immobilized animal was positioned in a supine-like position to facilitate the entry and absorption of the drug in the nostril.
• the rat was placed in its own cage to allow the complete drying of the nostril.
• hearing function was estimated by ABR recordings.
• ABRs were measured at low (6 kHz), mid (12, 16, and 20 kHz), and high (24 and 32 kHz) frequencies at day 0 and 7 days from treatment onset.
• ABRs were assessed bilaterally before treatment to assure normal hearing and reassessed at all- time points to evaluate the effect of treatments on hearing.
• the ABRs recordings were performed as follows. All animals were mildly anesthetized (ketamine, 35 mg/kg and medetomidine-domitor, 0.25 mg/kg) and placed in the anechoic room. Three stainless steel recording electrodes were subcutaneously inserted posterior to the tested pinna (active), vertex (reference), and contralateral pinna (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. Tone bursts of pure tones from 6 to 32 kHz (1 ms rise/fall time, 10 ms total duration, 20/s repetition rate) were presented monaurally. Responses were filtered (0.3-3 kHz), digitized, and averaged (across 500 discrete samples at each frequency-level combination). Threshold value was defined as the lowest stimulus level that yielded a repeatable waveform-based onset.
• the inventors have prepared the following example pharmaceutical compositions, obtained according to the below-disclosed preparation methods.
• composition was filtered with 0.22pm filter.
• composition 2 The neurotrophin was added to the composition at final concentration 0.6 mg/mL.
• composition was filtered with 0.22pm filter.
• the neurotrophin was added to the composition at final concentration 0.6 mg/mL.

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• 2022
  • 2022-08-05 EP EP22189067.6A patent/EP4316462A1/en not_active Withdrawn
• 2023
  • 2023-08-04 JP JP2025507001A patent/JP2025525990A/ja active Pending
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