CN114246949A

CN114246949A - Application of ULK1 activator in preventing and/or treating hearing loss

Info

Publication number: CN114246949A
Application number: CN202111530607.7A
Authority: CN
Inventors: 吴皓; 董庭婷; 陶永; 冯宝怡; 郑晓飞; 金晨曦; 成祯哲; 刘祎晴
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-29
Anticipated expiration: 2041-12-14
Also published as: CN114246949B

Abstract

The invention belongs to the field of biomedicine, and discloses application of an ULK1 activator in preparation of a medicine for preventing and/or treating hearing loss, wherein the ULK1 activator comprises BL-918. According to the invention, through carrying out noise exposure experiment tests on a mouse model, the fact that the activity of ULK1 can be enhanced by administering ULK1 activator such as BL-918 before noise exposure, the autophagy level of spiral neurons in cochlea can be enhanced, hearing threshold shift caused by noise can be reduced, and the like, and the noise-induced hearing loss prevention effect is good for noise. As an ingredient of hearing protection, the ULK1 activator can also be administered as an effective treatment after hearing loss. The invention also discloses a pharmaceutical composition containing the ULK1 activator. The invention provides a new idea for preventing and treating hearing impairment, thereby having wide clinical application prospect.

Description

Application of ULK1 activator in preventing and/or treating hearing loss

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of an ULK1 activator in preventing and/or treating hearing loss, and more particularly to application of BL-918 in preventing and/or treating noise-induced hearing loss.

Background

Deafness is the most prevalent sensory organ disability worldwide, and as of 2020, 4.3 million people worldwide have moderate and above hearing impairment. Auditory abnormalities cause speech impairment, cognitive impairment, psychological occlusion and self-mutism in patients, and place a heavy burden on the family and society.

Of these, presbycusis (ARHL), an age-related hearing impairment, remains a major factor in causing hearing impairment. In the hearing impaired population, the proportion of the population above 60 years old is more than 42%. Presbycusis is a complex degeneration of the auditory system caused by multiple causes, with risk factors including cochlear aging, environmental factors, genetic susceptibility, family history and comorbidities, especially in noisy environments, noise exposure is the major cause of ARHL.

With the increasing unsafe use of audio equipment and the increasing daily and occupational exposure to noisy environments, the risk of noisy hearing impairment in young 12 to 35 years of age has increased. Not only can cognitive impairment, sleep impairment and cardiovascular health be caused by noise, but hearing impairment can be caused by prolonged exposure to noise at high and moderate sound intensities of 75-85 dB. Even studies have shown that after noise exposure, synapses in the auditory structures of the inner ear and spiral ganglia are lost, causing silent hearing impairment and speech recognition disorders.

Noisy hearing impairment (NIHL) refers to temporary or permanent hearing impairment caused by intense noise stimuli, which may be accompanied by damage to the inner and outer hair cells, the zonal synapses, or the Spiral Ganglia (SGN) of the cochlea. Mechanisms associated with noise-induced hearing impairment include abnormalities in ion channels, imbalances in calcium homeostasis, and impairment of oxidative stress, among others.

Autophagy is a major process of degradation of intracellular materials and organelles, and refers to a process in which various components in the cytoplasm are delivered to lysosomes and degraded in the lysosomes, ultimately maintaining the basic activity and function of cells. Autophagy plays an important role in the development of the cochlea, the maintenance of hair cell morphology and auditory function. Autophagy is also thought to be a protective mechanism that limits the triggering of pathological changes in tissues due to stress. Several studies have shown that autophagy plays a role in preventing ototoxic drug-induced hearing damage, such as that caused by neomycin, gentamicin, or cisplatin. One study has found that the autophagy signaling pathway is involved in the development and progression of NIHL by analyzing the plasma metabolomics of workers exposed to noise. In addition, studies in related rodents have shown that noise exposure promotes autophagy of cochlear hair cells. Some autophagy activators have been shown to reduce oxidative stress damage to the inner ear caused by noise. Rapamycin, a specific mTOR inhibitor, is involved in immunosuppression, affects transcription and protein synthesis, regulates cell growth, apoptosis, etc., and is also an autophagy activator, which has been shown to reduce the level of ROS in the inner ear, thus acting to alleviate oxidative stress injury in the cochlea caused by noise. In addition, peroxisomal phagocytosis, mediated by Pejvakin, can be considered a selective autophagy, and also plays a key role in controlling peroxisome levels and protecting auditory hair cells from noise (85). These studies suggest the importance of autophagy in protecting hearing from noise damage, and no drug is currently available that protects noise-induced hearing damage directly by enhancing autophagy.

During autophagy, UNC-51-like kinase 1(ULK1) is an important kinase for the initiation of autophagy and plays a key role in the initiation of autophagy. Complexes of ULK1 with FIP200, ATG13, and ATG101 can initiate autophagosome formation. Activation of ULK1 is regulated by mTORC1 and AMPK. It has been reported that impairment of the AMPK-mTORC1-ULK1 signaling pathway may play a key role in the degeneration of the auditory cortex. BL-918 is a potent activator of ULK1, and BL-918 has been shown to be effective in preventing the phenotype of mouse parkinsonian model induced by 1-methyl-4-phenylpyridine ions by activating ULK1 to enhance autophagy in SH-SY5Y cells.

Disclosure of Invention

To solve the problems of the prior art, the present invention aims to provide the application of BL-918 in preventing and/or treating hearing loss, and discloses a novel drug for effectively preventing and/or treating noise-induced hearing loss by enhancing autophagy.

The technical scheme of the invention is as follows:

the invention provides application of an ULK1 activator in preventing and/or treating noise-induced hearing loss, protecting hearing and/or treating hearing loss.

Preferably, the ULK1 activator is BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof.

Preferably, the ULK1 activator is used to prevent hearing impairment.

Further, the ULK1 activator was administered 2 days prior to noise exposure.

Further, the administration mode of the ULK1 activator is round window membrane penetration administration.

Further, the ULK1 activator is administered at a dose of 2-200uM in a volume of 2 ul. Preferably, the ULK1 activator is administered at a dose of 200uM in a volume of 2 ul.

Further, the ULK1 activators are useful for enhancing ULK1 activity in the cochlea, for enhancing autophagy levels in spiral neurons in the cochlea, for reducing noise-induced upward hearing threshold shifts, for increasing cochlear hair cell survival rates after noise exposure, or for protecting noise-induced reduction of neurosynaptic, among others.

The invention also provides a pharmaceutical composition, which comprises BL-918 or its pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate as described above, and a pharmaceutically acceptable carrier and a medium.

By the scheme, the invention at least has the following advantages:

1. the invention provides a novel small molecule compound which has a prevention and protection effect on hearing impairment, particularly noise-induced hearing impairment, and provides a novel treatment means and approach for the noise-induced hearing impairment. A single administration of BL-918 prior to noise exposure may reduce the threshold shift in mouse hearing after noise.

2. BL-918 is a novel ULK1 activator, and no relevant research report exists at present.

3. BL-918 has good effect of preventing noise hearing loss on noise of a plurality of frequencies, especially has good prevention effect on diseases such as noise deafness and cannon-shocking deafness, and can be used as a medicine and/or health care product for preventing noise hearing loss.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to be implemented according to the content of the description, the following description is given with reference to the detailed drawings and the preferred embodiments of the present invention.

Drawings

FIG. 1 shows cochlear tissue pULK1 of mice in the noise-free control group and noise-free drug-spraying group of example 1^S757Relative levels of protein.

Fig. 2 shows the spiral neuron autophagosome and autophagososome in cochlea of mice in the noise control group and the noise reduction group in example 2, and statistics.

FIG. 3 shows basement membrane staining and statistics of mice in the control group, BL-918 treatment group, NK treatment group and NK + BL-918 treatment group of example 3.

Fig. 4 shows ABR thresholds 3, 7, and 14 days after noise exposure for the noise-free group and the noise control group and the noise PBS group in example 4.

Fig. 5 shows ABR thresholds 3, 7 and 14 days after noise exposure for the noise-free group and the noise control group and the noise reduction group in example 5.

Fig. 6 shows ABR thresholds 3, 7 and 14 days after noise exposure for the noise-free group and the noise control group and the noise reduction group in example 6.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following specific examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Through years of research on the effect of ULK1, the inventor discovers the relationship between the expression of ULK1 and hearing loss, surprisingly discovers that BL-918 can be used as an activator of ULK1 by activating ULK1, and further proves that BL-918 can be used as an activator of ULK1 to play roles in preventing hearing loss and protecting hearing and repair the damaged hearing. The invention particularly provides application of BL-918 in preventing hearing loss, and provides a useful reference for developing a medicine for treating sensorineural hearing loss.

The invention provides application of an ULK1 activator in preventing and/or treating noise-induced hearing loss, protecting hearing and/or treating hearing loss. The ULK1 activator can specifically promote the transcription or translation of the ULK1 gene, or can specifically promote the expression or activity of the ULK1 protein, thereby enhancing the autophagy level in spiral neurons in cochlea and achieving the aim of protecting hearing.

Preferably, the ULK1 activator is BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof. BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof can play a role in protecting hearing by taking ULK1 as a drug action target.

The chemical formula of the BL-918 is C₂₃H₁₅F₈N₃OS, the structure is shown in formula I below:

the invention also provides application of the pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate of BL-918 in preparation of a medicine or a kit for preventing and/or treating hearing loss.

In the application of the present invention, the hearing impairment is sensorineural hearing impairment, and is selected from at least one of the following: ototoxic drugs, noise, hereditary, senile, metabolic, autoimmune diseases and tumor-induced hearing impairment. Wherein the hearing impairment caused by the noise is noise hearing impairment, including noise deafness and cannon-shot deafness; the BL-918 can reduce threshold movement of hearing after noise exposure, improve activity of ULK1, improve autophagy level in spiral neurons in cochlea, improve survival rate of cochlear hair cells after noise exposure, protect noise-induced reduction of nerve synapses, and the like.

The noise is broad-spectrum noise with the frequency of 4-60 kHz and/or the intensity of 90-150 dB.

The administration mode of the BL-918 is round window membrane permeation administration; and/or, the BL-918 is administrated in a dose of 2-200uM and a volume of 2 ul.

In the application of the invention, the BL-918 is a single effective component of a pharmaceutical preparation. In some embodiments, the pharmaceutical preparation comprises other pharmaceutical ingredients related to hearing protection besides the BL-918 ingredient, and is prepared from BL-918 and pharmaceutical excipients. In a specific embodiment, the application of BL-918 in preparing a medicament for preventing and/or treating hearing loss specifically refers to: BL-918 is used as a main effective component of a medicament for preparing a medicament for preventing and/or treating hearing impairment.

In the application of the present invention, the activation or promotion effect of BL-918 on ULK1 includes but is not limited to: promote the gene transcription or expression of ULK1, activate the activity of ULK1, activate or promote the function of ULK 1. For example, BL-918 can increase the level of ULK1 in a cell by 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% without affecting other functions of the cell, as compared to a control.

The invention also provides a pharmaceutical composition for preventing and/or treating hearing impairment, which comprises ULK1 activator BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof as described above, and a pharmaceutically acceptable carrier or vehicle.

Further, the acceptable carrier, medium such as sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), preservative, surfactant (PEG, Tween, etc.), chelating agent (EDTA, etc.), binder, etc. Moreover, other low molecular weight polypeptides may also be present; proteins such as serum albumin, gelatin, and immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; saccharides or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol and sorbitol. When an aqueous solution for injection is prepared, for example, physiological saline, an isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, may be used in combination with an appropriate solubilizing agent such as alcohol (ethanol, etc.), polyhydric alcohol (propylene glycol, PEG, etc.), nonionic surfactant (Tween 80, HCO-50), etc.

In the pharmaceutical composition of the present invention, the BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof may be a single active ingredient, or may be combined with other active ingredients to form a combined preparation.

The amount of active ingredient (ULK1 activator BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof) in the pharmaceutical composition of the present invention is generally a safe and effective amount which should be adjusted by those skilled in the art, for example, the amount of active ingredient administered will generally depend on the body weight of the patient, the type of application, the condition and severity of the disease, for example, the amount of active ingredient administered will generally be 1-1000 mg/kg/day, 20-200 mg/kg/day, 1-3 mg/kg/day, 3-5 mg/kg/day, 5-10 mg/kg/day, 10-20 mg/kg/day, 20-30 mg/kg/day, 30-40 mg/kg/day, 40-60 mg/kg/day, 60-80 mg/kg/day, 80-100 mg/kg/day, 100-150 mg/kg/day, 150-200 mg/kg/day, 200-300 mg/kg/day, 300-500 mg/kg/day, or 500-1000 mg/kg/day.

The active ingredient or pharmaceutical composition containing the active ingredient provided by the present invention may be adapted to any form of administration, and may be administered orally or parenterally, for example, by pulmonary, nasal, rectal and/or intravenous injection, more specifically intradermal, subcutaneous, intramuscular, intraarticular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or parenteral administration; the injection administration includes intravenous injection, intramuscular injection, subcutaneous injection and the like, transdermal administration and the like.

As used herein, the dosage form of the pharmaceutical composition is selected from: injection, injectable sterile powder, tablet, pill, capsule, lozenge, spirit, powder, granule, syrup, solution, tincture, aerosol, powder spray, or suppository. One skilled in the art can select a suitable formulation according to the administration mode, for example, a formulation suitable for oral administration may be a formulation including, but not limited to, pills, tablets, chewables, capsules, granules, solutions, drops, syrups, aerosols or dusts, etc., a formulation suitable for parenteral administration may be a formulation including, but not limited to, solutions, suspensions, reconstitutable dry preparations or sprays, etc., a formulation suitable for rectal administration may be usually a suppository, and a formulation suitable for injection may be an injection, a sterile powder for injection, etc.

The present invention further provides a method for the therapeutic or prophylactic treatment of hearing impairment, which comprises administering to a subject (e.g., a mammal) in need thereof an effective amount of BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph, solvate or pharmaceutical composition. The method may also be in vitro or non-therapeutic.

The subject or individual to be treated therapeutically or prophylactically is preferably a mammal, such as, but not limited to, a human, a primate, a livestock animal (e.g., sheep, cattle, horse, donkey, pig), a pet animal (e.g., dog, cat), a laboratory test animal (e.g., mouse, rabbit, rat, guinea pig, hamster) or a captured wild animal (e.g., fox, deer). The subject is preferably a primate. The subject is most preferably a human. The subject may be a hearing impaired patient or an individual in whom prevention of hearing impairment is desired.

The BL-918 or the pharmaceutical composition can be administered to a subject before, during, or after receiving a hearing impairment treatment.

In the present invention, the salt or ester of ULK1 activator BL-918 may be used in the form of a pharmaceutically or physiologically acceptable salt or ester. By "pharmaceutically acceptable salt" is meant, in general, any salt which is physiologically tolerable (in general, it is meant that it is non-toxic, in particular as a result of counterions) when used in a suitable manner in therapy, in particular when applied or used in humans and/or mammals. These physiologically acceptable salts may be formed with cations or bases and in the context of the present invention, especially when administered in humans and/or mammals, they are to be understood as being salts formed by at least one compound provided according to the invention, usually an acid (deprotonated), such as an anion, and at least one physiologically tolerated cation, preferably an inorganic cation. In some embodiments, the salt, ester of BL-918 includes, but is not limited to, salts or esters with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Salts of halides are also suitable. Other salts include: salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium.

In the present invention, the term "prodrug" refers to a compound that, when administered by an appropriate method, undergoes a metabolic or chemical reaction in the human body to convert the active ULK1 activator or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof.

In the present invention, the active compound BL-918 may also be used in combination with other therapeutic agents. For example in combination with one or more ingredients useful for the protection of hearing selected from the group consisting of: zuoci pill, liuwei dihuang pill, nicergoline tablet, oryzanol, vincamine sustained release capsule, mecobalamin dispersible tablet, vinpocetine injection, cerebroside carnosine injection, troxerutin injection, deproteinized calf blood extract injection, folium Ginkgo extract tablet, citicoline sodium capsule, etc.

In the present invention, when the active compound is used in combination with other therapeutic agents, the active compound is co-administered with the other therapeutic agents. "coadministration" means simultaneous administration via the same or different routes, or sequential administration via the same or different routes, in the same formulation or in two different formulations. By "sequential" administration is meant having a time difference in seconds, minutes, hours, or days between administration of two or more different compounds.

In the present invention, the ULK1 activator, the medicament or the pharmaceutical composition comprising the same plays a role in protecting hearing by at least one of:

enhancing the activity of ULK 1;

enhancing the level of autophagy in spiral neurons in the cochlea;

reducing noise-induced upward hearing threshold shifts;

improving the survival rate of cochlear hair cells after noise exposure;

protection against noise-induced reduction of the synapses, etc.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

References herein to "comprising," "including," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; i.e., meaning "including but not limited to".

Reference herein to a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is effective to treat the disease condition as set forth above.

References herein to "therapeutic" and "prophylactic" are to be understood in their broadest sense. The term "therapeutic" does not necessarily imply that the mammal is receiving treatment until complete recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not ultimately be infected with the disease condition. Thus, treatment and prevention includes alleviation of the symptoms of, or prevention or reduction of the risk of, a particular disorder. The term "prevention" is to be understood as reducing the severity of the onset of a particular condition. Treatment may also reduce the severity or frequency of acute episodes of an existing condition.

Reference herein to an "cell" that is impaired by hearing should be understood to be any cell that has been impaired by hearing, or any cell that may suffer from hearing loss.

The compounds of the invention and methods thereof are useful for preventing and/or treating hearing impairment, not only in the early stages of hearing impairment to prevent expansion of the impairment, but also for repair after hearing impairment, and as a prophylactic treatment prior to or after exposure to noise or drugs for a period of time; preferably, the BL-918 of the present invention is used for preventing hearing impairment.

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1 evaluation of the Activity of BL-918 on the cochlear ULK1 in mice

When the Ser757 site of ULK1 protein is in a phosphorylated state, the interaction between ULK1 and AMPK is inhibited, thereby inhibiting the activity of ULK 1. Thus, pULK1 was tested^S757The protein level may reflect ULK1 activity, pULK1^S757Reduction in protein levels can reflect the degree of activation of ULK 1.

Preparing 10mM BL-918 storage liquid: 1mg of BL-918 powder was dissolved in 0.1875ml of DMSO liquid.

Preparing 200uM BL-918 solution: 2uL of 10mM BL-918 stock solution was diluted to 100uL with PBS to obtain 200uM BL-918 solution.

Experimental grouping and experimental procedure:

selecting 3 healthy and normal-hearing male wild type 4-week-old C57BL6/J inbred line mice with the weight of 16-18 g. The left and right ears of the mouse are respectively used as a noise-free control group and a noise-free drug-mixing group. The method specifically comprises the following steps:

noise-free control group: the left ear of each mouse was left without any treatment.

The noiseless medicine-beating group: the drug is administered through the round window membrane of the cochlea on the right side of the mouse, namely the round window membrane is exposed through the entrance way behind the ear after the mouse is anesthetized, a proper amount of gelatin sponge (the drug administration concentration is 200uM, the volume is 2ul) of BL-918 solution is sucked and placed in the round window niche, and the auditory bulb is sealed and sewed. And (5) when the mice revive, putting the mice back into the cage for feeding after the mice are ensured to be in a good state. 2 days after administration, mouse cochlear tissue was sampled, ground, lysed using RIPA lysate (Thermo Fisher Scientific,89900) to extract total protein, and subjected to immunoblotting after protein quantification by BCA method (picyunnan, P0012). Using housekeeping gene protein beta-actin as internal reference, pULK1 was observed^S757Protein and beta-actin protein expression level, calculating pULK1^S757The ratio of the beta-actin to the drug is calculated by taking the noiseless control group as 1, and pULK1 of the noiseless drug group is calculated^S757Relative quantification of (c).

As shown in fig. 1, pULK1 in the cochlea of noiseless laxative group^S757The relative protein level is obviously reduced compared with that of a noise-free control group, which indicates that the activity of ULK1 in the cochlea of the noise-free drug combination is higher, and BL-918 can enhance the activity of ULK1 in the cochlea.

Example 2 assessment of the ability of BL-918 to activate the level of autophagy of the cochlea in mice

CAG-RFP-EGFP-LC3 transgenic mice have CAG promoter/enhancer sequences that drive the Red Fluorescent Protein (RFP), Enhanced Green Fluorescent Protein (EGFP), and microtubule-associated protein 1 light chain 3 α (Map1LC3a or LC3) genes. LC3 can be used as a marker of a common autophagosome, and the expression level of the LC3 directly reflects the autophagy level of a target region. The two fluorophores RFP and EGFP are not stable to acidic environments, RFP is stable at acidic pH, and EGFP is easily quenched in acidic lysosomal environments. After the autophagosome is combined with a lysosome, the autophagosome forms the autophagososome, and the EGFP fluorescence is quenched due to the reduction of the pH value, so that the autophagosome can be distinguished from the autophagososome. That is, yellow fluorescence (RFP and EGFP colocalization) shows autophagosomes, and only red fluorescence, but no green fluorescence, shows autophagosomes. The CAG-RFP-EGFP-LC3 mouse is commonly used for researching autophagy conditions in mouse cells, and can visually detect autophagy levels in different cells in the mouse.

The preparation method of 200uM BL-918 solution is the same as that of example 1.

Experimental grouping and experimental procedure:

3 healthy and normal-hearing male 4-week-old CAG-RFP-EGFP-LC3 transgenic mice with the weight of 16-18 g are selected. The left ear and the right ear of the mouse are respectively used as a noise control group and a noise medicine-beating group. The method specifically comprises the following steps:

noise control group: the left ear of each mouse was left without any treatment.

A noise medicine-making set: the drug is administered through the round window membrane of the cochlea on the right side of the mouse, namely the round window membrane is exposed through the entrance of the ear after the mouse is anesthetized, gelatin sponge (with the administration concentration of 200uM and the volume of 2ul) which absorbs a proper amount of BL-918 solution is placed in the round window niche, the auditory bulb is sealed, and the suture is carried out. And (5) when the mice revive, putting the mice back into the cage for feeding after the mice are ensured to be in a good state.

After 2 days of dosing, all mice were exposed to a narrow frequency noise environment with a frequency of 8-16kHz, 104dB SPL for 2 hours, during which the mice were free to move in the noise cage compartment. The material was harvested from 2 groups of mouse cochlea 1 hour after the noise exposure of the mice, the tissue was fixed in 4% paraformaldehyde in the dark for 16 hours at 4 ℃ and then decalcified with 0.5M ethylenediaminetetraacetic acid (EDTA). Then placing the cochlear tissue in sucrose with the mass percent of 15% and 30% in sequence for tissue gradient dehydration for 1 day respectively, and then placing the cochlear tissue into an OCT embedding medium for freezing for 12 hours at the temperature of minus 20 ℃. The mouse cochlea was then subjected to cochlear freezing sectioning with a section thickness of 10 um. Then, immunofluorescent staining is carried out on the cochlear frozen section, RFP and EGFP signals are autofluorescence, staining is not needed, and cochlear spiral neuron cells are displayed by using TUJ1(801202, Biolegend) antibody. Changes in the number of autophagosomes (yellow signal) and autophagosomes (RFP signal only) in helical neurons in the mouse cochlea were observed under confocal microscopy.

As shown in fig. 2, the numbers of autophagosomes and autophagosomes in spiral neurons in cochlea of the noise reduction drug group are both significantly increased compared with those of the control side, suggesting that BL-918 can enhance the autophagy level in spiral neurons of the cochlea of a mouse.

Example 3 BL-918 evaluation of protective action against nerve injury of mouse culture basement membrane

The noise impairment mechanism is thought to be a kind of neuronal excitatory impairment. N-methyl-D-aspartate (NMDA) and Kainic Acid (KA) are potent agonists of excitatory amino acid receptors in nerve cells. Nmda (tocris) in combination with ka (sigma) (NK) can cause excitotoxicity of nerve cells, thereby causing nerve damage to the cultured cochlear basement membrane. This example uses NK to simulate the damage of noise to spiral neurons in vitro.

General medium: DMEM/F12 medium (Gibco,11320033) was added with 1x B27(Gibco,17504044) and 1x N2(Gibco, A1370701).

The medium containing 5uM BL-918 was obtained by adding 5uM BL-918 to a normal medium to a final concentration.

The medium containing NK (containing 0.5mM NMDA and 0.5mM KA) was a medium obtained by additionally adding NMDA and 0.5mM KA to a normal medium to a final concentration of 0.5 mM.

The medium containing NK (containing 0.5mM NMDA and 0.5mM KA) and 5uM BL-918 was obtained by additionally adding NMDA, 0.5mM KA and 5uM BL-918 to a common medium to a final concentration of 0.5 mM.

6 wild-type post-natal day 2 (P2) C57BL6/J inbred mice were selected, the cochlear basilar membranes of the left and right ears were dissected and cultured at 37 ℃ under 5% CO2 for 24 hours, and 12 cochlear basilar membranes were randomly divided into four groups: control group, BL-918 treatment group, NK + BL-918 treatment group.

Control group: no treatment is carried out;

BL-918 processing group: incubate with 5uM BL-918 in medium for 20 hours;

NK treatment group: incubating with medium containing NK (containing 0.5mM NMDA and 0.5mM KA) for 2 hours, and then replacing with common medium for 18 hours;

NK + BL-918 processing group: first, incubation was performed for 2 hours with medium containing both NK (0.5mM NMDA and 0.5mM KA) and 5uM BL-918, followed by a change to medium containing 5uM BL-918 and incubation continued for 18 hours.

The cultured basement membrane was then fixed and immunostained to observe the attachment of inner hair cells, nerve fibers and inner hair cells, and inner hair cells were visualized using Myosin VIIa (#25-6790, Proteus BioSciences Inc) and NF-H (# AB5539, Millipore) to show nerve fibers attached to inner hair. And calculating the number of nerve fibers linked by each inner hair cell so as to evaluate the damage condition of the basal lamina nerve.

As shown in fig. 3, the ordinate means the number of nerve fibers connected per inner hair cell. As a result, the numbers of nerve fibers connected to inner hair cells in the BL-918-treated group and the control group were similar, indicating that BL-918 is not toxic in vivo. Compared with the control group, the number of nerve fibers connected with each inner hair cell of the basement membrane of the mice in the NK treatment group is obviously reduced, which indicates that the NK treatment damages the nerve fiber connection of the inner hair cells of the basement membrane of the mice, and is similar to the previous report. The number of nerve fibers connected with each inner hair cell in the NK + BL-918 treatment group is obviously increased compared with that in the NK treatment group, and the fact that BL-918 can protect nerve fiber damage caused by NK is proved.

Example 4 evaluation of the protective Effect of PBS buffer on noise-induced hearing impairment in mice

Selecting 8 healthy and normal-hearing male wild type 4-week-old C57BL6/J inbred line mice with the weight of 16-18 g.

The method specifically comprises the following steps:

noise-free group: the left ear hearing was tested in 4 mice without any treatment.

The other 4 mice were noise group, and the left and right ears were noise control group and noise administration group, respectively. Noise control group: the left ear of another 4 mice was not treated with drug and noise exposure was performed 2 days later. The 4 mice were right-ear as noise PBS group: the drug is administered through a round window membrane of a cochlea on the right side of a mouse, namely the round window membrane is exposed through the retroauricular approach after the mouse is anesthetized, gelatin sponge (the administration volume is 2ul) which absorbs a proper amount of 1xPBS buffer solution is placed in a round window niche, the auditory vesicle is sealed, and the suture is carried out. And (3) waiting for the mice to wake up, returning the mice to a cage for feeding after the mice are ensured to be in good state, and exposing noise after 2 days.

Noise group mice noise exposure method: mice were exposed to a narrow frequency noise environment with a frequency of 8-16kHz at an intensity of 104dB SPL for 2 hours 2 days after dosing, during which the mice were free to move in the noise cage compartment.

Cochlear hearing thresholds were measured in mice by performing an auditory brainstem response test. And then measuring the hearing threshold values of the left cochlea and the right cochlea of the mice in the noise group on 3 rd, 7 th and 14 th days after the noise exposure of the mice respectively, namely detecting the hearing threshold value change of the noise control group and the noise reduction group to measure the noise damage degree and the medicine protection effect.

As shown in fig. 4, at 3, 7, and 14 days after noise exposure, no noise protection was observed for the noisy PBS dosing group.

Example 5 evaluation of protective Effect of high concentration of BL-918 on noise-induced hearing impairment of mice

The method specifically comprises the following steps:

The other 4 mice were noise group, and the left and right ears were noise control group and noise administration group, respectively. Noise control group: the left ear of another 4 mice was not treated with drug and noise exposure was performed 2 days later. The right ear of the 4 mice served as the noise-fighting group: the administration is carried out through the round window membrane of the cochlea on the right side of the mouse, namely the round window membrane is exposed through the entrance of the ear after the mouse is anesthetized, a proper amount of gelatin sponge (the administration concentration is 200uM, the volume is 2uL, the preparation method is the same as that of the example 1) of BL-918 solution is absorbed and placed in the round window niche, the auditory vesicle is sealed, and the suture is carried out. And (3) waiting for the mice to wake up, putting the mice back into a cage to be raised after the mice are ensured to be in good state, and exposing noise after 2 days.

As shown in fig. 5, 3, 7, and 14 days after the noise exposure, the hearing threshold shift of the inner ear of the mice in the noise reduction group is observed to be reduced, and compared with the hearing threshold shift of the noise control group, the hearing threshold shift of about 20dB SPL is reduced, which proves that 200uM BL918 has a protective effect on the noise hearing damage of the inner ear of the mice.

Example 6 evaluation of protective Effect of Low concentration BL-918 on noise-induced hearing impairment in mice

Preparing a 2uM BL-918 solution: 2ul of 10mM BL-918 stock solution was diluted to 10000ul with PBS to obtain 200uM BL-918 solution.

The method specifically comprises the following steps:

The other 4 mice were noise group, and the left and right ears were noise control group and noise administration group, respectively. Noise control group: the left ear of another 4 mice was not treated with drug and noise exposure was performed 2 days later. The right ear of the 4 mice served as the noise-fighting group: the administration is carried out through the round window membrane of the cochlea on the right side of the mouse, namely the round window membrane is exposed through the entrance of the ear after the mouse is anesthetized, a proper amount of gelatin sponge (the administration concentration is 2uM, the volume is 2ul, the preparation method is the same as that of the example 1) of BL-918 solution is absorbed and placed in the round window niche, the auditory vesicle is sealed, and the suture is carried out. And (3) when the mice revive, putting the mice back into the cage to be raised after the mice are ensured to be in good state, and exposing noise after 2 days.

As shown in fig. 6, 3, 7, and 14 days after the noise exposure, the hearing threshold shift of the inner ear of the mice in the noise reduction group is observed to be reduced, and compared with the noise control group, the hearing threshold shift of about 20dB SPL is reduced, which proves that 2uM BL918 has a protective effect on the noise hearing damage of the inner ear of the mice.

The above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; variations and advantages that may occur to those skilled in the art may be made without departing from the spirit and scope of the inventive concept, which is intended to be covered by the claims.

Claims

Use of an ULK1 activator for the preparation of a medicament for the prevention and/or treatment of hearing impairment.
2. The use of claim 1, wherein said ULK1 activator is BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof, wherein said BL-918 has a chemical formula C₂₃H₁₅F₈N₃OS, the structure is shown in formula I below:
3. the use of claim 1, wherein the hearing impairment is sensorineural hearing impairment.
4. The use of claim 3, wherein the sensorineural hearing impairment is selected from at least one of the following: hearing impairment due to noise, ototoxic drugs, hereditary, senile, metabolic, autoimmune diseases and tumors; preferably, the sensorineural hearing impairment is noise-induced hearing impairment and/or NK-induced hearing impairment.
5. The use of claim 4, wherein the noise-induced hearing impairment is noise-induced hearing impairment, including noise-induced deafness and cannon-induced deafness; and/or the noise refers to the noise with the frequency of 4-60 kHz and the intensity of 90-150 dB.
6. The use according to any one of claims 1 to 5, wherein the medicament acts to protect hearing by at least one of:

enhancing the activity of ULK 1;

enhancing the level of autophagy in spiral neurons in the cochlea;

reducing noise-induced upward hearing threshold shifts;

improving the survival rate of cochlear hair cells after noise exposure;

the noise-induced reduction of the synapses is preserved.
7. The use according to any one of claims 1 to 6, wherein the ULK1 activator is administered by round window membrane permeation; and/or, the BL-918 is administrated in a dose of 2-200uM and a volume of 2 ul.
8. A pharmaceutical composition for preventing and/or treating hearing impairment, comprising an ULK1 activator as described in claim 1 or 2, and a pharmaceutically acceptable carrier, vehicle.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is administered in combination with at least one of a hearing protection related drug.
10. The pharmaceutical composition of claim 8 or 9, wherein the drug acts to protect hearing by at least one of:

enhancing the activity of ULK 1;

enhancing the level of autophagy in spiral neurons in the cochlea;

reducing noise-induced upward hearing threshold shifts;

improving the survival rate of cochlear hair cells after noise exposure;

the noise-induced reduction of the synapses is preserved.