WO2024022173A1 - Use of andrographolide and composition thereof in preparation of drug for treating hearing impairment - Google Patents

Abstract

Provided in the present application are the use of andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of a drug for treating hearing impairment, and the use of a pharmaceutical composition consisting of andrographolide or a pharmaceutically acceptable salt thereof or a solvate thereof, and other drugs for treating hearing impairment in the preparation of a drug for treating hearing impairment, wherein the hearing impairment comprises noise induced hearing loss, presbycusis, drug induced hearing loss, tinnitus, decreased hearing, etc.

Description

Application of Andrographolide and Its Composition in Preparing Medicinal Products for Treating Hearing Damage Technical field

The invention belongs to the field of pharmacy, and specifically relates to the application of andrographolide and its composition in the preparation of medicines for treating hearing loss.

Background technique

As my country's urbanization and aging trends gradually accelerate, the incidence of hearing loss is high and growing rapidly. It is estimated that the number of people suffering from hearing loss in my country is currently between 200 and 300 million, especially tinnitus, which is a disease characterized by conscious ringing in the ears or head without a corresponding sound source in the surrounding environment. , which can be caused by various factors such as emotional illness and environmental noise pollution. Many tinnitus patients are miserable and need medication.

Excessive exposure to noisy environments is one of the main causes of deafness and tinnitus. Noise-induced deafness refers to a slowly progressive sensorineural deafness that occurs due to long-term exposure of the auditory system to noise. Its early manifestation is auditory fatigue, which can be gradually recovered after getting out of the noisy environment as soon as possible, that is, the reversible hearing damage stage (with hidden hearing loss); if continued long-term exposure to the noisy environment will eventually lead to sensorineural deafness (permanent hearing loss) ). In addition to damaging the peripheral auditory nervous system, noise can also affect the central nervous system, inducing headaches, dizziness, insomnia, hypertension and other adverse reactions. The mechanisms related to damage to the auditory nervous system caused by noise mainly include: ① Noise induces oxidative stress damage and inflammatory response in the inner ear. Excessive noise exposure can lead to a significant increase in reactive oxygen species (ROS) in the inner ear and damage to hair cell mitochondria, accompanied by an increase in the expression of multiple inflammatory factors such as TNF-a. Subsequently, macrophages produce downstream inflammatory reactions in the sustained inflammatory response. Under the action of chemical factors and chemokines, it will continuously infiltrate into the cochlea, causing the hair cells in the cochlea to disappear; ② Noise-induced inner ear inflammatory reaction destroys the blood-lymph circulation barrier system (BLB). The capillary network of the stria vascularis on the lateral wall of the cochlea is parallel to the basal cells of the marginal cell nuclei and passes through the gaps between the intermediate cells. The capillaries are surrounded by the processes of the marginal cells and basal cells and are tightly connected to form between the blood circulation and the space within the stria vascularis. Blood-lymph circulation barrier system (BLB), BLB has the function of maintaining the potential in the cochlea, regulating ion transport in the inner ear, and regulating the balance of inner ear fluids. Destruction of BLB can lead to a variety of hearing disorders, such as autoimmune inner ear diseases, Meniere's disease, meningitis and other inner ear diseases; ③ Inner ear ribbon synaptic dysfunction. In the peripheral auditory system, cochlear ribbon synapses play a key role in the encoding and conduction of sound. Various hearing impairment factors (including noise, drugs, aging, etc.) can significantly damage the structure and function of cochlear ribbon synapses. Damage to this synaptic structure directly causes a decrease in the level of sound signal transmission to the brain, which in turn leads to abnormal hearing function.

Sudden deafness is a common ear disease, affecting 5 to 20 cases per 100,000 people every year. It is characterized by sudden, usually unilateral, sensorineural hearing loss, often accompanied by clinical symptoms such as tinnitus, ear blockage, dizziness, nausea, and vomiting.

Drug-induced hearing loss is a relatively common adverse reaction, which refers to hearing or vestibular system damage caused by therapeutic drugs. Hearing loss or disorder caused by system dysfunction. Clinical manifestations are often hearing loss, which may be accompanied by symptoms such as tinnitus, vertigo, and ataxia. In severe cases, it may lead to total deafness.

Presbycusis refers to the physiological phenomenon of hearing loss due to age.

Meniere's disease is an inner ear disease characterized by spontaneous intermittent vertigo that lasts for no less than 20 minutes, followed by tinnitus, fluctuating hearing loss, and a feeling of fullness in the ears.

In summary, due to the extremely complex anatomical structure of the ear and the existence of an independent blood-lymph circulation barrier system, it has become an important obstacle that restricts drug molecules from entering the inner ear to treat related diseases, including deafness, tinnitus, vertigo, and a series of hearing and balance diseases. Medically related inner ear diseases are difficult and difficult diseases worldwide. At present, there are no effective measures for drug delivery in the inner ear except surgery, which has many inconveniences and high risks in terms of preventive drug administration and multiple drug administration. At present, there is no effective treatment for deafness and tinnitus. Except for the administration of hormones, microcirculation-improving drugs, or thrombolytic drugs in the acute phase (within 1-2 weeks of onset), the therapeutic effect is limited, especially for the treatment of noise-induced and presbycusic deafness and tinnitus.

Andrographolide, the structure is shown in the figure below.

Andrographolide molecular formula C20H30O5, also known as Andrographis B, is the main active ingredient of the natural plant Andrographis paniculata. It has the effects of heat-removing, detoxifying, anti-inflammatory and analgesic. It has special effects on bacterial and viral upper respiratory tract infections and dysentery. It is known as a natural Antibiotic drugs. Currently, pharmaceutical preparations containing andrographolide include: andrographolide tablets, capsules, dropping pills, etc.

At present, there are no reports on the use of andrographolide and its composition for treating hearing loss or hearing impairment.

Contents of the invention

The present invention is aimed at the current situation that there is no effective drug for treating hearing loss in the prior art, and develops a drug for treating hearing loss and a composition thereof.

In a first aspect, the present invention provides the use of andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of a medicine for treating hearing loss.

In a second aspect, the present invention also provides an andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof, combined with another drug, or a composition prepared with another drug, in the preparation of a treatment Application of medicines in hearing impairment.

Among them, the other medicine includes compound salvia miltiorrhiza, ginkgo leaf extract or preparations thereof, puerarin, ferulic acid, anisodamine, α-lipoic acid, glutathione, N-acylcysteine, Acetyl levocarnitine, water-soluble coenzyme Q10, erythropoietin, adenosine triphosphate, buflodil, dizocycline, nimodipine, carbamazepine, lidocaine, Prozac, Paxil Roxetine, sertraline, citalopram, doxepin.

Preferably, the other drug is compound salvia miltiorrhiza.

Among them, compound salvia miltiorrhiza is compound salvia miltiorrhiza extract or its preparation.

Compound Salvia miltiorrhiza preparations are Chinese patent medicines that are already on the market. They mainly include: Compound Salvia miltiorrhiza dropping pills, Compound Salvia miltiorrhiza tablets, Compound Salvia miltiorrhiza capsules and other dosage forms. They are composed of Salvia miltiorrhiza, Panax notoginseng and Borneol. They are mainly used for qi stagnation and blood stasis caused by Chest obstruction, symptoms include chest tightness and precordial tingling; treatment of angina pectoris in coronary heart disease.

The preparation method of the compound salvia miltiorrhiza extract can be obtained with reference to the existing technology.

The weight ratio of andrographolide of the present invention, or its pharmaceutically acceptable salt, or its solvate to compound salvia miltiorrhiza is 1: (0.2-10.8).

Preferably, the weight ratio of andrographolide of the present invention, or its pharmaceutically acceptable salt, or its solvate to compound salvia miltiorrhiza is 1: (0.4-2.5).

On the one hand, the andrographolide of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof, is composed of another drug The composition may also include a pharmaceutically acceptable carrier and be prepared into a compound pharmaceutical preparation.

Wherein, the preparation form may be tablets, capsules, pills, oral liquids, buccal preparations, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, Ointments, plasters, creams, sprays, drops and patches.

On the other hand, in the composition of the andrographolide of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, and other drugs for treating hearing loss, the andrographolide, or a pharmaceutically acceptable salt thereof The salt, or its solvate, and another drug can also be independently prepared into appropriate preparation forms, and then combined and packaged together.

For example, andrographolide dropping pills, andrographolide tablets, andrographolide capsules, andrographolide dispersible tablets, andrographolide soft capsules, etc. Compound Danshen Dropping Pills, Compound Danshen Tablets, Compound Danshen Capsules, Compound Danshen Injection, etc. The two are packaged together. It can be taken at the same time or at different times. It can be taken 1-5 times a day, and the dose taken each time is the effective dose of the drug. The most preferred method of the present invention is to take andrographolide preparation, or andrographolide preparation + compound salvia miltiorrhiza preparation. . If you take andrographolide dropping pills, you can take them orally, 1 bag at a time, 3 times a day, 0.6g per bag, containing 0.15g of andrographolide. If you take Compound Danshen Dropping Pills, you can take them orally, 10 pills at a time, 3 times a day. The specification is 27 mg × 180 pills.

The medicines described in the present invention are all finished products on the market and can be purchased from the market. If taken according to the instructions of the present invention, the therapeutic effect can be achieved.

In a third aspect, the present invention provides the use of a composition of andrographolide and compound salvia miltiorrhiza in preparing medicine for treating hearing loss.

In the fourth aspect, the hearing impairment according to the present invention includes noise-induced hearing impairment, drug-induced hearing impairment, and sudden hearing loss. Deafness, sensorineural deafness, presbycusis, Meniere's disease, symptoms include tinnitus, hearing loss, etc.

Description of drawings

Figure 1 ABR thresholds of mice treated with drugs in each group to evaluate the degree of hearing damage

Figure 2 Immunofluorescence synaptic staining analysis

Figure 3 Synapse count analysis

Figure 4 Liquid chip technology detects different cytokine levels in the cochlear endolymph of each drug treatment group

Figure 5 Evaluation of the protective effects of andrographolide, compound salvia miltiorrhiza alone and their combination on STZ-induced HEI-OC1 cell death

Detailed ways

Experimental example 1:

1. Experimental purpose

To explore the evaluation of the effect of andrographolide, andrographolide + compound salvia miltiorrhiza combination in the treatment of permanent hearing loss in mice after noise.

2. Experimental materials

2.1 Test product

Test substances: Compound Danshen Dropping Pills (Batch No.: 190117), Andrographolide Dropping Pills (Batch No.: 190909)

Storage conditions: room temperature, store at 4°C after dissolving the drug

Provided by: Tasly

2.2 Reagents

Normal saline (batch number: E1903021, manufacturer: Huaren Pharmaceutical Co., Ltd.)

Dexamethasone (batch number: 1912252211, manufacturer: Chenxin Pharmaceutical Co., Ltd.)

2.3 Experimental animals

Strain and grade: C57 Bl/6 J mice

Level: SPF level

Weight: 4-6 weeks old, 15±2g

Quantity: 10 pieces/group

Source: Beijing Speifu Laboratory Animal Technology Co., Ltd., production unit license number: SCXK (Beijing) 2016-0006. Experimental animal quality certificate number: 110011211101474482.

2.4 Main instruments

Table 1 Main instruments

3. How to administer the test substance.

3.1 Ways

Intragastric or intraperitoneal injection

3.2 Method

4. Experimental methods

4.1 Model preparation

C57 Bl/6 J mice were raised in the animal room for 2-5 days. Starting from about 5 weeks old, 10 mice in each group (initial screening click hearing range <30dB SPL) were exposed to 120dB white noise for 2 hours, and therapeutic administration was given on the same day after the noise treatment. (administered by gavage), the same intensity of noise exposure was performed again the next day, and the drug was administered after the second exposure. The administration was then continued for 13 days (a total of 15 days of administration), and the day of secondary noise treatment was designated as NE0d. Then brainstem evoked potential tests (ABR) were conducted on NE4d, NE7d, and NE14d respectively, and the mice were recorded at 4K, 8K, and 16K respectively. , auditory threshold under stimulation of 24K short pure tone or click compound sound. (10 mice in each group, 20 hearing threshold data, calculate the average hearing threshold, and use T test statistics to calculate the significance of differences between groups)

4.2 Experimental grouping and administration

The experiment was divided into 5 groups, namely model group, Compound Danshen Dropping Pills group, andrographolide Dropping Pills group, Compound Danshen Dropping Pills + Andrographolide Dropping Pills group, and positive drug (dexamethasone acetate) group, with 10 animals in each group. See Table 2 for details.

The daily dosage of Andrographolide Dropping Pills for humans is 1 bag (0.6g/bag) once, 3 times a day. The equivalent dosage for mice is (1×3×600mg)/60kg×12.3=369mg /kg, then the dose of the andrographolide dropping pill group is set to the clinical equivalent dose of 369mg/kg.

According to the proportion screening experiment, the Compound Danshen Dropping Pills were used in this experiment to be 3 times the clinical equivalent dose. The equivalent dose in mice is (3×3×27×10)/60kg×12.3=498mg/kg, then the compound The administration of Danshen Dropping Pills + Andrographolide Dropping Pills group The dosage is: Compound Danshen Dropping Pills 498mg/kg + Andrographolide Dropping Pills 369mg/kg.

The Compound Danshen Dropping Pills group also used the corresponding clinical equivalent dose 3 times, that is, 498mg/kg.

Dexamethasone acetate (DEX), a commonly used clinical therapeutic drug, was selected as a positive control. The daily dosage for humans is 0.75 to 3.00 mg (1 to 4 tablets) once, 2 to 4 times a day. The maintenance dose is approximately 0.75 mg (1 tablet) per day, which converted into the equivalent dose for mice is (1 × 2 × 750 mg)/60 kg × 12.3 = 308 mg/kg.

Table 2. Animal experiment grouping methods and administration methods

4.3 Auditory brainstem responses (ABRs)

(1) The time point is the day of secondary noise treatment as NE0d, and then detection is performed at NE4d, NE7d, and NE14d respectively.

(2) Anesthetize the mice and weigh them before testing. Prepare 1% sodium pentobarbital in physiological saline as an anesthetic, and administer 0.1ml/10g intraperitoneally for anesthesia.

(3) Audiometry environment The audiometry was conducted in a shielded room that was isolated from electronic and sound interference. In order to prevent experimental animals from hypothermia under anesthesia, the mice were placed on a 37°C constant temperature heating plate, and the indoor temperature was maintained at around 23°C. to avoid electromyographic interference caused by muscle tremors. During the listening test, the listening room was kept dark. In order to reduce external acoustic and electrical interference, other electrical equipment in the room was turned off, and the internal and external soundproof doors were closed at the same time.

(4) Connect the electrodes After the mouse is anesthetized, use alcohol cotton balls to disinfect the needle electrodes. Place the recording electrode under the skin at the intersection between the front edge of the auricles on both sides of the mouse and the middle of the skull. Place the reference electrode under the skin behind the test ear. Place the ground electrode under the skin behind the contralateral ear. Detect the resistance of each connected electrode and test the resistance. It should be less than 3kΩ. If the resistance is too high, readjust the electrode insertion depth to reduce the resistance. The speaker is about 0.5cm away from the external auditory canal of the test ear. The speaker needs to be calibrated regularly with a sound level meter.

(5) The audiometric system uses TDT audiometric equipment. After the system is stable, open the SigGen/Biosig (Tucker-Davis Technologies, USA) audiometric software. The stimulus sounds are short sounds (Click) and short pure tones (Tone burst). For short pure tones, 4kHz, 8kHz, 16kHz, and 24kHz are selected as the listening frequency points. The rise/fall time (Rise/Fall time): 0.5ms, duration Duration: 5 ms, the total number of superpositions is 1024, the band-pass filtering is 300~3000 Hz, and the scanning duration is 10 ms. The stimulus intensity starts from 90dB, gradually decreases by 10dB until no repeated ABR waveform is detected, and then increases upward by 5dB until repeated ABR waveforms can be detected. This stimulus intensity is the hearing threshold of the mouse.

(6) Record experimental data and record the hearing threshold of mice at each frequency.

4.4 Cochlear basilar membrane tissue spreading and immunofluorescence staining

(1) Temporal bone anatomy After the NE14d hearing function test is completed, the mice are killed and quickly decapitated. The skull is cut open along the midline of the skull, the brain tissue is removed, the auditory vesicle is separated and the cochlea is removed.

(2) Cochlear fixation: Place the cochlea in a petri dish containing 0.01 mmol/L phosphate buffer solution (PBS) pre-cooled at 4°C, wash away the blood, and remove muscle tissue and blood vessels. Under a stereoanatomy microscope, use a fine needle to drill holes in the cochlear roof, remove the malleus, incus and stapes, and puncture the round window and oval window membranes. Use 4% paraformaldehyde solution (PFA) pre-cooled at 4°C to lavage from the top of the cochlea. Blood or flocculent lymph can be seen from the round window and oval window. Place the perfused cochlear specimen in 4% PFA solution to continue fixation and keep it in the refrigerator at 4°C overnight.

(3) The day after cochlear decalcification, take out the specimen and put it into a petri dish containing 0.01mmol/L PBS. Wash it three times on a shaker for 5 minutes each time, and then place it in 10% ethylenediaminetetraacetic acid (EDTA). Decalcify in the solution for 2 hours at room temperature. The decalcification process can be carried out on a shaker. The decalcified cochlear specimens were then washed three times with PBS for 5 min each time.

(4) Basement membrane separation Under a stereodissecting microscope, use microscopic fine forceps to separate the decalcified cochlear bony labyrinth from the membranous labyrinth from the cochlear top to the cochlear bottom in a petri dish containing 0.01 mmol/L PBS solution, and remove Vestibular membrane and tegmental membrane, and carefully peel off the cochlear basilar membrane. Use a razor blade to cut the basilar membrane and divide it into three sections: top, middle and bottom. The dissected cochlear basilar membrane specimen can be placed in a centrifuge tube with 0.01mmol/L PBS solution and stored in a 4°C refrigerator for later use.

(5) Membrane rupture and punching and sheep serum sealing. Soak the separated basement membrane in PBS containing 0.3% Triton X-100, and rupture and punch the membrane at room temperature for 30 minutes. Finally, the 0.3% Triton X-100 working solution was aspirated, replaced with PBS containing 5% sheep serum, and blocked at room temperature for 1 hour.

(6) Immunofluorescence staining: Place the specimen in the prepared primary antibody CtBP2 (BD Biosciences, 612044, 1:500 dilution) solution, place it in a 4°C refrigerator for overnight incubation, take out the specimen the next day, and rinse three times with PBS. Each session lasts 5 minutes. Add the immunofluorescence secondary antibody corresponding to the primary antibody and phalloidin (1:500), and incubate for 2 hours on a shaker at room temperature in the dark.

(7) After incubation with secondary antibody, slides were washed three times with PBS (5 min/time), sealed with fluorescent mounting agent DAPI (ZSGB-BIO, ZLI-9557), and stored in a 4°C refrigerator.

4.5 Laser confocal microscopy detection

(1) A 63x oil immersion high-resolution confocal microscope (Zeiss LSM710, Germany) was used to observe the cochlear tissue after immunofluorescence staining.

(2) Select the excitation light of the corresponding wavelength according to the selected fluorescent secondary antibody for incubation, find the appropriate specimen position, and After all the hair cells are placed in the center of the field of view, the appropriate laser intensity, layer and other indicators are selected.

(3) Scan the sample from top to bottom in layers with an interval of 0.35 μm/layer. Finally, superimpose the layer scan images in the z-axis direction to generate the final final image.

4.6 Luminex liquid phase suspension chip

(1) Sample collection and preparation After the NE14d hearing function test is completed, the mice are killed and quickly decapitated. After taking out the two cochleae, use sharp tweezers to break the cochlear tip under a stereoscopic dissecting microscope, and use a 20 μl micropipette to aspirate the cochlear endolymph liquid. The lymph fluid was collected into EP tubes and centrifuged. Centrifuge at 10,000 rpm for 10 minutes, take the supernatant, and detect the mass according to Null μg. Use Sample Diluent: RIPA (medium) = 24:1, and add 0.5% BSA to make up the volume to 50 μL before testing.

(2) Sample incubation: Take the microbeads and shake them on the oscillator at 1,400rpm for 30s and then dilute them (dilute with Assay Buffer). The diluted microbeads are shaken again under the same conditions for 30s. Add 50 μL of sample to each well of a 96-well plate, and at the same time add 50 μL of standard and Blank prepared in advance, affix a sealing film, shake on a plate shaker at 850 rpm, and incubate for half an hour at room temperature in the dark.

(3) Incubate the detection antibody. After incubation, remove the sample and wash it 3 times with a plate washer; use Antibody Diluent to dilute the Detection Antibody according to the instructions; add 25 μL of the diluted Detection Antibody to each well, attach a sealing film and shake on a 850rpm plate shaker. , incubate for half an hour at room temperature and away from light.

(4) After color development and incubation, remove the sample and wash it 3 times with a plate washer; use Assay Buffer to dilute Streptavidin-PE according to the instructions; add 50 μL of diluted Streptavidin-PE to each well, affix a sealing film and place on an 850rpm plate shaker Shake and incubate for 10 minutes at room temperature in the dark; wash 3 times with a plate washer; add 125 μL AssayBuffer to each well and resuspend, shake under the same conditions for 30 seconds; open the Bio-Plex machine in advance for calibration, and then place the 96-well plate for reading. .

(5) Concentration calculation Calculate the standard curve formula based on the concentration and color values of the standard product and blank product. According to the standard curve formula and the reading value detected after the sample is colored, the concentration of the sample can be calculated by putting the obtained results into the formula, which can be used for comparison between samples.

5. Detection indicators

ABR threshold, synaptic staining and counting, inflammation-related indicators

6. Statistics

All data are used Indicates that the significance of differences between groups was compared using t test or one-way analysis of variance.

7.Experimental results

As shown in Table 3 and Figure 1: Among the tested drug groups, andrographolide dripping pills, compound salvia miltiorrhiza dripping pills, andrographolide dripping pills and compound salvia miltiorrhiza dripping pills combined group showed a more obvious hearing protection effect. The effect is even better at 4k and 8k frequencies In the DEX positive drug control group.

Table 3 ABR thresholds of mice treated with drugs in each group to evaluate the degree of hearing damage

Note: The value in the table represents the decibel value at which animals can hear the sound at this frequency. The normal range for humans is 25-30. The higher the value, the greater the decibel the sound needs to be heard. That is, the louder the sound, the greater the sound, indicating hearing impairment. Click refers to a short sound, a mixed sound, with a very wide spectrum, ranging from 125-8kHz. Compared with the model group, * indicates P<0.05, which is a significant difference; ** indicates P<0.01, which is very significant.

As shown in Figure 2: the andrographolide dropping pill group, combination group and DEX group showed better synaptic protection effect, and the number of remaining synapses after noise (Ctbp2 red fluorescence signal) was much higher than the model group, and the synapse count The levels showed that the andrographolide dropping pill group and the combination group were basically equivalent to the positive drug DEX.

The cochlear ribbon synapse is the first synaptic structure in the auditory conduction system that transmits sound to the central nervous system. It is called a ribbon synapse because of its ribbon-like spatial distribution. The function of the cochlear ribbon synapse determines Determine the quality and quantity of sound transmitted to the center. The maintenance of cochlear ribbon synaptic function relies on the transport of vesicles, which is a highly energy-consuming quantum release process that requires continuous energy supply from the mitochondria of inner hair cells. When the auditory system is subjected to overloaded noise stimulation, mitochondrial oxidative damage increases, affecting the production of adenosine triphosphate (ATP), which in turn induces an excessive immune response in the inner ear, thus affecting the function of the cochlear ribbon synapse. This may be noise-induced Pathophysiological mechanisms related to cochlear ribbon synapses in sensorineural hearing loss (NIHL). However, the specific mechanism of ribbon synapse damage in cochlear hair cells during strong noise stimulation requires further study. This study refers to the fact that dexamethasone (DEX) is a commonly used drug in the clinical treatment guidelines for sudden deafness, and the main effect of this drug is anti-inflammatory. Therefore, in terms of synaptic function testing, it was also found that DEX has better synaptic protection. At the same time, the results show that Andrographolide Dropping Pills and Compound Danshen Dropping Pills also have a certain degree of synaptic protection. In terms of synaptic protection, Andrographolide Dropping Pills are better than Compound Danshen Dropping Pills. And it is equivalent to the positive drug DEX. Considering that systemic administration of DEX has a great risk of side effects and affects a variety of normal human metabolic physiological functions, the research results have laid the necessary theoretical foundation for the further development of safer clinical medication regimens (traditional Chinese medicine). Figure 3 shows the statistical results of synapse counting in Figure 2.

In view of the above speculation, this application adopts the latest liquid chip technology (this technology has been widely used in the detection of biological body fluid samples such as tumors, endocrinology, autoimmunity, etc., and some people also use it for cytokine profile judgment) for high-throughput exploration. Quantitative analysis of different inflammation and chemokines in the cochlea of mice in each experimental group.

This application uses liquid chip technology to monitor 17 different types of inflammatory factors. It is found that the indicators of various inflammatory factors in the cochlear lymph fluid of mice in the model group (Nacl) after noise increased significantly, while Compound Danshen Dropping Pills and Andrographolide Drops Both pills have a certain degree of anti-inflammatory effect, andrographolide dripping pills are more obvious, and the combination of the two shows a better synergistic effect on some inflammatory indicators. See Table 4 and Figure 4.

Table 4 Different cytokine levels in cochlear endolymph in each drug treatment group (unit pg/ml)

Note: The control group is normal mice.

8.Conclusion

The present invention evaluates the effectiveness of the combination of andrographolide and andrographolide + compound salvia miltiorrhiza in protecting the hearing function of mice after noise exposure.

Overall, each drug test group showed a certain degree of hearing protection effect, and both groups of drugs had a certain inhibitory effect on noise-mediated inner ear inflammation, thereby achieving hearing protection function.

However, it is preliminarily speculated that the treatment mechanisms of the two are different, each has its own focus, and is highly complementary to a certain extent. Therefore, their combined use or separate use is expected to become a standardized medication guide for the clinical treatment of sudden deafness.

Andrographolide has shown good therapeutic effects on noise-induced sensorineural hearing loss, whether used alone or in combination with compound salvia miltiorrhiza dropping pills. It is expected that the existing clinical guidelines for sudden deafness will be supplemented in the future.

Experimental example 2

In order to further study the effect of combined use of andrographolide and compound salvia miltiorrhiza in the treatment of hearing loss, the dosage ratio of the two was further screened.

Cell test: 10uM STZ streptozotocin (Streptozotocin)-induced HEI-OC1 cell death model (Raza H, John A. Streptozotocin-induced cytotoxicity, oxidative stress and mitochondrial dysfunction in human hepatoma HepG2 cells. Int J Mol Sci. 2012; 13(5):5751-5767.doi:10.3390/ijms13055751.Epub 2012 May 11.PMID:22754329; PMCID:PMC3382802.)

The HEI-OC1 mouse cochlear hair cell line is one of the few mouse auditory cell lines used for research purposes. The cells are an in vitro system for screening ototoxic drugs and have been used to study drug-activated apoptotic pathways, autophagy, senescence, cell protection mechanisms, inflammatory responses, cell differentiation, and genetic and epigenetic effects of drugs. , role of hypoxia, oxidation and endothelial cells, expression of molecular channels and receptors in the cochlea. (Kalinec G, Thein P, Park C, Kalinec F. HEI-OC1 cells as a model for investigating drug cytotoxicity. Hear Res. 2016 May; 335:105-117.doi:10.1016/j.heares.2016.02.019.Epub 2016 Feb 27.PMID:26930622.)

1Test products and configurations

Test substances: Compound Danshen Dropping Pills (Batch No.: 190117), Andrographolide Dropping Pills (Batch No.: 190909)

(1) Prepare compound salvia miltiorrhiza dropping pill solution (CDDP) of different concentrations

Method: Dissolve 9 compound Salvia miltiorrhiza dropping pills (27 mg each) in 10 ml ddH ₂ O and filter with a 0.22 μm microporous filter membrane to obtain mother liquor C0 with a concentration of 24.3 mg/ml. Take the mother solution and dilute it 5 times in sequence to obtain C1, C2, C3, C4 and C5 series working solutions. The concentrations are shown in the table below:

(2) Prepare andrographolide solutions of different concentrations ((Andro)

Method: Dissolve 1 bag of andrographolide dropping pills (600 mg per bag) in 32 ml ddH ₂ O, and filter with a 0.22 μm microporous filter membrane to obtain the mother liquor A0, with a concentration of 18.75 mg/ml. Take the mother solution and dilute it 5 times in sequence to obtain A1, A2, A3, A4, and A5 series working solutions. The concentrations are shown in the table below:

2Experimental purpose

The protective effects of different concentrations of CDDP and Andro on STZ (10 μM)-induced HEI-OC1 cell death were examined.

3 Experimental methods

1) Prepare HEI-OC1 cell suspension and count.

2) Inoculate cell suspension in a 96-well plate, approximately 100 μl per well, approximately 5,000 cells, and 5 replicates per group.

3) Place the culture plate into an incubator and pre-culture for 24 hours (33°C, 10% CO ₂ ).

4) Add different concentrations of toxic substances (STZ) and protective drugs (CDDP, Andro) to each well of the culture plate.

5) Place the culture plate in the incubator for 24 hours and replace with fresh culture medium.

(If the substance to be tested has oxidizing or reducing properties, the fresh medium can be replaced before adding CCK-8 to remove the influence of the substance to be tested. If the effect is relatively small or has no effect, the medium can be deducted directly without changing the medium. The blank after adding the drug can be absorbed).

6) Add 10μl CCK-8 solution to each well.

7) Place the culture plate into the incubator and incubate for 4 hours.

8) Use a microplate reader to measure the absorbance (OD) at 450nm.

(If you do not want to measure the OD value temporarily, you can add 10μl 0.1M HCL solution or 1% w/v SDS solution to each well and store it at room temperature in the dark. This will ensure that the OD value does not change within 24 hours.)

Vitality calculation:

Cell viability (%)=[A(drug added)-A(blank)]/[A(Control)-A(blank)]×100%

A (drug addition): OD value of the well with cells, CCK-8 solution and drug solution

A (Control): OD value of wells with cells, CCK-8 solution and no drug solution

A (blank): OD value of wells without cells

Cell viability: cell proliferation activity

Cell Counting Kit-8 (CCK-8 for short) is a detection reagent based on WST-8 that is widely used in cell proliferation and cytotoxicity. WST-8 [Chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzene disulfonate)-2H- Tetrazolium monosodium salt] is a compound similar to MTT. It is used by mitochondria in the presence of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The dehydrogenase in it reduces it to the highly water-soluble orange-yellow formazan product. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of living cells, so this feature can be used to directly analyze cell proliferation and toxicity. .

4Experimental results

After preliminary screening of the cell viability of each single drug group, the C1 group, C2 group, A3 group and A4 group with better effects were screened out, and the two were combined in pairs. The experimental results of each group are shown in Table 5 and Figure 5.

Table 5 Cell viability of each single drug group and combination group

The STZ-induced HEI-OC1 cell death test data shows that, as shown in Table 5 and Figure 5: at the half-lethal induction concentration, taking the CCK8 absorbance value of normal cultured OC1 cells (control) as 100% as a reference, 10uM STZ induces killer cells for 24 hours. Afterwards, the cell viability was about 65%. Compared with C1, C2, A3, A4 and other different concentrations used alone, as well as the combination of the two, each group showed different degrees of antioxidant protective effects, especially A3+C1 and A4 The +C1 drug combination showed a significant protective effect. Compared with the STZ group, the A3+C1 group, P<0.0001, showed an almost complete reversal of the STZ-induced cell oxidative stress death effect; the A4+C1 group, compared with the STZ group, P =0.0338, which has a significant improvement effect. The results of this experiment indicate to a certain extent that the combination of the two drugs has a better anti-oxidative stress effect than the drug alone.

It can be seen that when the ratio of andrographolide to compound salvia miltiorrhiza ranges from 1:32.4 to 162 (converted into a clinical human dose of 1:1 to 1:5, that is, the weight ratio is 1:0.4 to 1:2.5), The combination of the two has a significant improvement effect on the oxidative stress-induced cell death effect of STZ.

Example 1

Preparation of andrographolide preparations

Andrographis paniculata leaves are soaked in 95% ethanol, and the resulting ethanol-soaked liquid is decolorized with activated carbon. The concentrated liquid after the decolorizing liquid is distilled to recover the ethanol is left to obtain coarse crystals. Add 15 times the amount of 95% ethanol to the coarse crystals and heat to dissolve them. The activated carbon is decolorized and served while hot. Filter and leave to recrystallize to obtain a light yellow recrystallized product, which is then washed and refined with distilled water, chloroform, and methanol to obtain the finished product of andrographolide.

Preparation method of andrographolide dropping pills: Add andrographolide into the molten matrix, stir well, drop the pills into pills, cool them in a coolant and then dry them.

Example 2

Preparation of compound salvia miltiorrhiza preparation

440-460 parts of Salvia miltiorrhiza, 135-146 parts of Panax notoginseng and 6-10 parts of Borneol. Add appropriate amount of sodium bicarbonate to Salvia miltiorrhiza and Panax notoginseng, heat and extract twice, 1 to 2 hours each time, boil and filter the extracts, and combine The filtrate is concentrated; add 95% ethanol to the concentrated solution to make The alcohol concentration is 68%, let it stand for 10 hours, and filter the supernatant. Ethanol is recovered from the filtrate and concentrated. After the extract is mixed with borneol and auxiliary materials, polyethylene glycol-6000 is added, mixed, heated, and the materials are dissolved to form dropping pills.

Example 3

A compound composition of andrographolide and compound salvia miltiorrhiza.

Put 1 part by weight of the Andrographolide Dropping Pills of Example 1 and 2.5 parts by weight of the Compound Danshen Dropping Pills of Example 2 into a capsule to obtain a compound capsule.

Example 4

A compound composition of andrographolide and compound salvia miltiorrhiza.

Add 2.5 parts by weight of the finished andrographolide product of Example 1 and 1 part by weight of the Salvia miltiorrhiza notoginseng extract and borneol fine powder of Example 2 into the molten matrix, stir evenly, drop into pills, and cool in a coolant After drying, it is made into pills.

Example 5

Combination packaging of andrographolide preparation and compound salvia miltiorrhiza preparation.

The andrographolide dripping pills of Example 1 and the compound Salvia miltiorrhiza dripping pills of Example 2 are packed in bottles or bags respectively. The number of dripping pills in each bottle or bag is 10-100. In the bottles or bags of the two drugs, The bags are packaged together. When using, take out the two medicines separately and take them at the same time.

Example 6

A combination of andrographolide preparation and Ginkgo biloba preparation.

Example 7

A combination of andrographolide and puerarin preparations.

Example 8

A combination of andrographolide preparations and ferulic acid preparations.

Example 9

A combination of andrographolide and anisodamine preparations.

Example 10

A combination of andrographolide preparations and chemical drug preparations, the chemical drugs being selected from the group consisting of: α-lipoic acid, glutathione, N-acylcysteine, acetyl L-carnitine, water-soluble coenzyme Q10, and erythropoietin , adenosine triphosphate, buflodil, dizocycline, nimodipine, carbamazepine, lidocaine, Prozac, paroxetine, sertraline, citalopram, doxepin.

Claims (10)

Use of andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof, in the preparation of a medicament for treating hearing loss. The use of andrographolide, or a pharmaceutically acceptable salt thereof, or a solvate thereof, in combination with another drug, or a composition prepared with another drug, in the preparation of a drug for treating hearing loss. The application according to claim 2, wherein the other drug is selected from: compound salvia miltiorrhiza, ginkgo leaf extract or preparations thereof, puerarin, ferulic acid, anisodamine, α-lipoic acid, glutathione Peptides, N-acylcysteine, acetyl levocarnitine, water-soluble coenzyme Q10, erythropoietin, adenosine triphosphate, buflodil, dizocycline, nimodipine, carbamazepine, lidocaine, Youjie, paroxetine, sertraline, citalopram, doxepin. The application according to claim 3, wherein the other drug is Compound Salvia Miltiorrhizae. The application according to claim 4, wherein the ratio of the andrographolide, or its pharmaceutically acceptable salt, or its solvate, to the compound salvia miltiorrhiza is 1:0.2-10.8. The application according to claim 5, wherein the ratio of the andrographolide, or its pharmaceutically acceptable salt, or its solvate, to the compound salvia miltiorrhiza is 1:0.4-2.5. The application according to any one of claims 2, wherein the composition prepared from the andrographolide, or its pharmaceutically acceptable salt, or its solvate and another drug, also includes a pharmaceutically acceptable A carrier exists in the form of a preparation. The application according to claim 2, wherein the andrographolide, or its pharmaceutically acceptable salt, or its solvate, is combined with another drug, including independently preparing the two drugs into appropriate preparations. form, and then combined and packaged together. The application according to claim 7 or 8, wherein the preparations include tablets, capsules, pills, oral liquids, buccal agents, granules, pills, powders, ointments, elixirs, suspensions, and powders , solutions, injections, suppositories, ointments, plasters, creams, sprays, drops and patches. The application according to claim 1 or 2, wherein the hearing impairment includes noise-induced hearing impairment, drug-induced hearing impairment, sudden deafness, sensorineural deafness, presbycusis, and Meniere's disease, Symptoms include tinnitus and hearing loss.