Use of Lycium barbarum glycopeptide in preparing medicine for preventing or treating drug-induced deafness

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of Lycium barbarum glycopeptide in the preparation of a drug for preventing or treating drug-induced deafness. Preferably, the drug is an aminoglycoside antibiotic. The experimental results show that Lycium barbarum glycopeptide has a protective effect on neomycin-induced HEI-OC1 cell damage and mouse hearing loss. It can be seen that Lycium barbarum glycopeptide, as a product extracted from traditional Chinese medicinal materials, can be effectively used for the prevention and treatment of drug-induced deafness, and has the prospect of being developed into a drug for the prevention and treatment of drug-induced deafness, providing more treatment options for the clinical prevention or treatment of drug-induced deafness, and has important social benefits and economic value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically relates to the use of Lycium barbarum glycopeptide in preparing a medicine for preventing or treating drug-induced deafness. Background Art

[0002] Hearing impairment is the most prevalent ear disease in the world. According to statistics from the World Health Organization (WHO), there are currently about 466 million people suffering from hearing impairment in the world, and this number will rise to more than 900 million by 2050. With the aging of the population, abuse of ototoxic drugs, noise and environmental pollution, the number of people with deafness and hearing loss has been increasing year by year. Deafness has become a global health issue that affects social politics and the economy. Among them, sensorineural hearing loss accounts for about 63% of deaf patients, and damage to cochlear hair cells is one of the main causes of sensorineural hearing loss. At present, although hearing aids and cochlear implants commonly used in clinical practice have improved patients' hearing to a certain extent, they have certain limitations and cannot fundamentally solve the problem. Moreover, their effectiveness depends entirely on the number and quality of hair cells. Therefore, finding the cause and mechanism of hair cell damage is crucial to the prevention and treatment of sensorineural hearing loss.

[0003] Hair cell damage caused by aminoglycosides is the main factor of drug-induced deafness. Aminoglycoside antibiotics include streptomycin, neomycin, gentamicin, kanamycin, etc. They are a class of glycoside antibiotics composed of aminosugars and aminocyclyls connected by oxygen bridges. They are mainly used clinically to prevent and treat various systemic infectious diseases. Aminoglycoside antibiotics all have varying degrees of ototoxic effects. The deafness caused by them accounts for the vast majority of drug-induced deafness and is the leading cause of drug-induced deafness. Studies have shown that the mechanism of ototoxicity caused by aminoglycoside antibiotics is mainly that aminoglycoside antibiotics enter the mitochondria of vestibular and cochlear hair cells through ion channels or transporters and accumulate, inhibiting the synthesis of mitochondrial proteins and oxidative phosphorylation of vestibular and cochlear hair cells, causing mitochondrial dysfunction, further leading to cell apoptosis and hearing loss.

[0004] Although the ototoxic effects of aminoglycoside antibiotics have been widely studied, there are no effective treatments or drugs in clinical practice. Therefore, it is of great scientific significance to find effective protective drugs for the clinical prevention and treatment of aminoglycoside antibiotic ototoxicity.

[0005] Lycium barbarum L. is a dried mature fruit of Lycium barbarum L., a plant of the Solanaceae family. It has the effects of nourishing the liver and kidneys, improving the eyesight, and is also used for deficiency of essence, back pain, vertigo and tinnitus. Lycium barbarum polysaccharide is a natural macromolecular water-soluble polysaccharide extracted from Lycium barbarum. Studies have found that Lycium barbarum polysaccharide has pharmacological effects such as immunomodulation, hypoglycemic and lipid-lowering, anti-aging, anti-oxidation, and anti-tumor. Other studies have shown that Lycium barbarum polysaccharide can protect neuronal cell damage and play a neuroprotective role such as anti-depression. Lycium barbarum glycopeptide (LBGP) extracted from Lycium barbarum polysaccharide has attracted people's attention in recent years. Compared with Lycium barbarum polysaccharide, Lycium barbarum glycopeptide removes inorganic salts and various monosaccharides. Studies have shown that Lycium barbarum glycopeptide has strong immune activity and anti-aging effects.

[0006] At present, there are no reports on the use of Lycium barbarum glycopeptide to prevent or treat drug-induced deafness. Summary of the invention

[0007] In order to solve the problem of insufficient clinical drugs that can effectively prevent or treat drug-induced deafness, the inventors adopted modern drug research methods to conduct in-depth development and utilization of natural products, combined with a large number of pharmacodynamic experimental studies, and provided the use of Lycium barbarum glycopeptide in the preparation of drugs for preventing or treating drug-induced deafness.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] The present invention provides use of Lycium barbarum glycopeptide in preparing a medicine for preventing or treating drug-induced deafness.

[0010] Preferably, the present invention provides the use of Lycium barbarum glycopeptide as the sole active ingredient in the preparation of a drug for preventing or treating drug-induced deafness.

[0011] As an optional mode, in the above use, the drug causing the drug-induced deafness is an aminoglycoside antibiotic.

[0012] As an optional mode, in the above use, the aminoglycoside antibiotic is selected from one or more of the following: streptomycin, neomycin, gentamicin or kanamycin.

[0013] As an optional mode, in the above use, the drug-induced deafness is caused by hair cell damage.

[0014] As an optional mode, in the above use, the symptoms of drug-induced deafness include one or more of the following: tinnitus, hearing loss or hearing loss.

[0015] As an optional mode, in the above use, the wolfberry glycopeptide has a sugar content of ≥60%, a uronic acid content of ≥5%, and a protein content of 20-33% by weight, and the wolfberry glycopeptide is in the form of a powder with a purity of ≥90%.

[0016] As an optional mode, in the above use, the molecular weight of the Lycium barbarum glycopeptide is 88 kD as determined by SDS-PAGE, the sugar content is 70% by weight, and the sugar composition is ArA:GA1:GlC=2.5:1.0:1.0 by molar ratio.

[0017] As an optional mode, in the above-mentioned use, the medicine comprises a preventive or therapeutically effective amount of Lycium barbarum glycopeptide and a pharmaceutically acceptable carrier.

[0018] As an optional mode, in the above-mentioned use, the drug is an oral preparation, an injection or an external preparation.

[0019] Preferably, the oral preparation is selected from oral liquid, tablet, powder, capsule or granule.

[0020] Also preferably, the injection is selected from water injection or sterile powder for injection.

[0021] Also preferably, the external preparation is selected from drops, sprays or gels.

[0022] As an optional mode, in the above-mentioned use, the drug is suitable for the following various administration modes, which may be oral administration, parenteral administration or intraaural administration.

[0023] Preferably, the parenteral administration includes intravenous injection, intramuscular injection, subcutaneous injection and the like.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention combines my country's advantages in natural product research and discovers for the first time a new use of the natural product Lycium barbarum glycopeptide in preventing or treating drug-induced deafness. The experimental results show that Lycium barbarum glycopeptide has a protective effect on neomycin-induced HEI-OC1 cell damage and mouse hearing loss. It can be seen that Lycium barbarum glycopeptide, as a product extracted from traditional Chinese medicinal materials, can be effectively used in the prevention and treatment of drug-induced deafness, and has the prospect of being developed into a drug for the prevention and treatment of drug-induced deafness, providing more treatment options for the clinical prevention or treatment of drug-induced deafness, and has important social benefits and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 : Lycium barbarum glycopeptide (LBGP) protects HEI-OC1 cells from neomycin-induced damage ( ). *P<0.05, ****P<0.0001.

[0028] Figure 2 Lycium barbarum glycopeptide protects against neomycin-induced hearing loss in mice ). *P<0.05, **P<0.01, ****P<0.0001. Figure 2 A represents the in vivo animal experiment flow chart, Figure 2 B represents ABR detection of hearing in different groups of mice.

[0029] Figure 3 :Lycium barbarum glycopeptide protects against neomycin-induced cochlear hair cell loss ). *P<0.05, ***P<0 .001, ****P<0 .0001. Figure 3 A represents the immunofluorescence image of mouse cochlear hair cells, magnification 100x, Myosin 7a marks hair cells, Phalloidin marks cilia, and DAPI marks cell nuclei. Figure 3 B is a statistical graph showing the cochlear hair cell counts. DETAILED DESCRIPTION

[0030] The inventors used modern drug research methods to deeply develop and utilize natural products. Through a large number of screenings, they first discovered that Lycium barbarum glycopeptide can prevent or treat drug-induced deafness, especially drug-induced deafness caused by aminoglycoside antibiotics. On this basis, the present invention was completed.

[0031] The dosage form of the drug of the present invention is preferably an oral preparation, an injection or an external preparation.

[0032] Preferably, the oral preparation is selected from oral liquid, tablet, powder, capsule or granule. Also preferably, the injection is selected from water injection or sterile powder for injection. Also preferably, the external preparation is selected from drops, spray or gel.

[0033] The drug of the present invention is suitable for the following various administration modes, which can be oral administration, parenteral administration or intraaural administration. The parenteral administration includes intravenous injection, intramuscular injection and subcutaneous injection.

[0034] The pharmaceutically acceptable carrier refers to a conventional drug carrier in the field of pharmaceutical preparations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0035] The filler of the present invention includes but is not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricant includes but is not limited to magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binder includes but is not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the disintegrant includes but is not limited to starch effervescent mixture, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agent includes but is not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan, etc.; the solvent includes but is not limited to water, balanced salt solution, etc. The above-mentioned various dosage forms can be prepared according to conventional processes in the field of pharmaceutical preparations.

[0036] "Lycium barbarum glycopeptide" can be extracted and separated from plants such as wolfberry containing the active ingredient by conventional natural product extraction methods in the art, or it can be purchased from commercially available products. In particular, Lycium barbarum glycopeptide can be prepared by the preparation method described in CN107021995B. Lycium barbarum glycopeptide, as the most effective active ingredient in wolfberry pulp, is the essence of wolfberry. Modern pharmacological experiments have fully confirmed that Lycium barbarum glycopeptide can significantly improve the body's immune function, participate in the immune regulation of organisms, participate in various activities of life cells, and has certain effects of lowering blood pressure, lowering blood lipids, lowering blood sugar, anti-inflammatory, anti-oxidation, anti-aging, and anti-tumor. The high biological activity of Lycium barbarum glycopeptide makes it hundreds of times more immune active than the oral polysaccharides commonly used in clinical practice. There are also experiments that further prove that Lycium barbarum glycopeptide has multiple pharmacological activities such as anti-oxidation, anti-aging immunoregulation, and inhibition of tumor cell replication.

[0037] In the medical use described in this article, the administration time, number of administrations and frequency of administration of Lycium barbarum glycopeptide, etc., need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0038] The treatment plan for animals can be applied to humans, and the effective doses of all drugs for humans can be converted by the effective doses of the drugs for animals. This is also easy to achieve for ordinary technicians in this field.

[0039] In order to better understand the essence of the present invention, the pharmacodynamic experiments and their results are used in the following specific implementation section to further illustrate the new use of Lycium barbarum glycopeptide in the pharmaceutical field for preventing or treating drug-induced deafness.

[0040] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0043] Example 1: Protective effect of Lycium barbarum glycopeptide on neomycin-induced HEI-OC1 cell damage

[0044] 1.1 Experimental methods

[0045] HEI-OC1 mouse cochlear hair cell line (isolated and cultured in the laboratory) was grown to a confluent state in a culture dish, digested with 0.25% EDTA-containing trypsin solution, repeatedly pipetted to a cell suspension, diluted to 1×105 cells / mL with DMEM high-glucose medium containing 10% FBS, and 100 μL per well was inoculated in a 96-well culture plate, cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and replaced with serum-free medium for assimilation culture for 1 hour before the experiment.

[0046] Different concentrations of Lycium barbarum glycopeptide (1, 5, 10, 50, 100, 200 μg / mL) were added and incubated for 24 hours, and then neomycin (2 mM) was added and incubated with Lycium barbarum glycopeptide for 24 hours. CCK8 solution (1:10 dilution) was added and cultured in a cell culture incubator at 37°C and 5% CO2 for 30 minutes, and the absorbance value was read at 450 nm.

[0047] Statistical analysis methods: Graphpad prism 9.0 software was used to calculate statistically significant differences by one-way analysis of variance (ANOVA) and Dunnett's test, and P values ​​< 0.05 were considered significant.

[0048] 1.2 Experimental Results

[0049] like Figure 1As shown in the figure, 24 hours after neomycin modeling, the survival rate of HEI-OC1 cells was significantly reduced, while Lycium barbarum glycopeptide (LBGP) at concentrations of 50μg / mL, 100μg / mL, and 200μg / mL could significantly improve neomycin-induced cell damage (*P<0.05 or ****P<0.0001) and promote the survival of HEI-OC1 cells, among which LBGP 100μg / mL had the best protective effect (****P<0.0001).

[0050] Example 2: Protective effect of Lycium barbarum glycopeptide on neomycin-induced hearing loss in mice

[0051] 2.1 Animal grouping and experimental methods

[0052] C57BL / 6J mice (purchased from Nanjing Qinglongshan Animal Breeding Farm) were randomly divided into 5 groups: blank control group, model group (neomycin group), low-dose Lycium barbarum glycopeptide group (5 mg / kg), medium-dose Lycium barbarum glycopeptide group (10 mg / kg), and high-dose Lycium barbarum glycopeptide group (20 mg / kg). Mice in the Lycium barbarum glycopeptide administration group were gavaged for 14 days, and then neomycin (100 mg / kg) was intraperitoneally injected, and furosemide (200 mg / kg) was intraperitoneally injected 2 hours later. Two days later, the mice were subjected to auditory brainstem response (ABR) audiometry and cochlear hair cell immunofluorescence counting analysis.

[0053] 2.2 Auditory Brainstem Response (ABR)

[0054] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital according to their body weight. The anesthetized mice were placed in a shielded room with a 37°C constant temperature pad, and the detection electrode was inserted into the subcutaneous position of the center of the mouse's head, the reference electrode was inserted into the subcutaneous position below the mouse's left auricle, and the ground electrode was inserted into the skin of the mouse's outer thigh. The sound wave device was placed in the mouse's left ear, and then 4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz pure tones were used for stimulation, and the sound wave intensity was gradually reduced from 90dB to 10dB until no repeatable waveform was recorded. The stimulation intensity at which a repeatable waveform was finally recorded was the mouse's hearing threshold.

[0055] 2.3 Immunofluorescence counting of mouse cochlear hair cells

[0056] After the mice were killed, they were quickly decapitated, the auditory bulla was opened, and the cochlear tissue obtained was fixed with 4% paraformaldehyde (PFA) for 24 hours and then decalcified with EDTA. After complete decalcification, the cochlea was placed in PBS and the basement membrane was peeled off under a dissecting microscope. Immunofluorescence staining was performed with hair cell markers (Myosin 7a) and cilia markers (Phalloidine). The hair cells in the mouse cochlea were counted and statistically analyzed under a ZEISS900 laser confocal microscope.

[0057] Statistical analysis methods: Graphpad prism 9.0 software was used to calculate statistically significant differences by one-way analysis of variance (ANOVA) and Dunnett's test, and P values ​​< 0.05 were considered significant.

[0058] 2.4 Experimental Results

[0059] like Figure 2 As shown in A, P28 mice were given Lycium barbarum glycopeptide by oral gavage for 2 weeks and then intraperitoneally injected with neomycin + furosemide to establish an acute hearing loss model. Two days later, the hearing level of the mice was tested by ABR and the hair cell count was performed by immunofluorescence.

[0060] like Figure 2 As shown in B, the ABR thresholds of mice at different frequencies were significantly increased after neomycin modeling, and the oral gavage group of Lycium barbarum glycopeptide had a significant improvement effect, among which the high-dose group (20 mg / kg) had the best effect.

[0061] like Figure 3 As shown in the results, by extracting mouse cochlear tissue and performing basement membrane spreading, immunofluorescence results showed that the cochlear outer hair cells (OHC) in the neomycin (Neo) injury group were almost completely missing, while oral gavage of different doses of Lycium barbarum glycopeptide groups all had a certain improvement effect, among which the high-dose Lycium barbarum glycopeptide group (20 mg / kg) had the most obvious improvement effect.

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of Lycium barbarum glycopeptide in the preparation of a drug for preventing or treating drug-induced deafness, characterized in that: The drug causing the drug-induced deafness is an aminoglycoside antibiotic; The aminoglycoside antibiotic is neomycin; The drug-induced deafness is caused by hair cell damage.

2. The use according to claim 1, characterized in that: The symptoms of drug-induced deafness include one or more of the following: tinnitus, hearing loss or hearing loss.

3. The use according to any one of claims 1 to 2, characterized in that: In terms of weight percentage, the wolfberry glycopeptide has a sugar content of ≥60%, a uronic acid content of ≥5%, and a protein content of 20-33%. The wolfberry glycopeptide is in the form of a powder with a purity of ≥90%.

4. The use according to any one of claims 1 to 2, characterized in that: The medicine comprises a preventive or therapeutic effective amount of Lycium barbarum glycopeptide and a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The medicine is an oral preparation, an injection or an external preparation.

6. The use according to claim 5, characterized in that: The oral preparation is selected from oral liquid, tablet, powder, capsule or granule; the injection is selected from water injection or sterile powder for injection; the external preparation is selected from drops, spray or gel.

7. The use according to any one of claims 5 to 6, characterized in that: The medicament is for oral administration or parenteral administration or intraaural administration.

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