Application of indolepropionic acid in preparation of medicine for preventing and / or treating hearing loss

Abstract

The invention discloses application of indolepropionic acid in preparation of a medicine for preventing and / or treating hearing loss. The invention discloses application of indolepropionic acid in the field of hearing loss for the first time, and the application comprises the steps of reducing an ABR threshold value of a mouse with hearing loss and relieving cochlea morphological injury of the mouse with hearing loss; experimental research proves that indolepropionic acid can significantly improve the symptom of hearing impairment of mice, and indolepropionic acid can be used for research and development of novel drugs for treating or protecting hearing impairment, and has wide medical prospects and economic values.

Description

Technical Field The present invention belongs to the technical field of new drug uses, and relates to the use of indolepropionic acid in preparing a drug for preventing and / or treating hearing loss, and specifically relates to the use of indolepropionic acid or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof in preparing a drug for preventing and / or treating hearing loss. Background Art

[0001] Hearing loss is the fourth leading cause of disability in the world. Currently, more than 1.5 billion people are affected worldwide, accounting for 20% of the world's population. Among them, about 1.16 billion people suffer from hearing loss, and 466 million people suffer from moderate or severe hearing loss (including 432 million adults and 34 million children). The World Health Organization (WHO) predicts that by 2050, more than 900 million people in the world will suffer from disabling hearing loss. Among all types of hearing loss, sensorineural hearing loss accounts for about 90%. The main causes of sensorineural hearing loss include genetic factors, noise exposure, aging, environmental risk factors, and the use of ototoxic drugs. These factors usually have an additive effect, which increases the incidence of hearing loss to a certain extent. Its pathophysiological mechanism usually involves a decrease in the number of cochlear hair cells, a thinning of the thickness of the vascular stria, and damage and loss of the spiral ganglion of the inner ear, which ultimately causes hearing loss characterized by an increased hearing threshold. For the treatment of sensorineural hearing loss, alternative treatment devices such as hearing aids and cochlear implants are currently used in clinical practice. Although these alternative therapies can improve patients' hearing to a certain extent, their effectiveness depends on the patient's residual functional hair cells and spiral neurons, so the hearing aid effect varies greatly among different patients. Some studies use gene therapy and stem cell therapy to restore the function of hair cells, but their safety and ethical feasibility remain to be studied. Since mammalian cochlear hair cells are terminal cells and do not have the ability to divide, it is difficult to regenerate after loss, and there is still no targeted effective drug. Therefore, it is particularly important to study safe and effective drugs for the protection or treatment of hearing loss.

[0002] Indolepropionic acid is a tryptophan metabolite of the intestinal symbiont Clostridium sporogenes and one of the most abundant tryptophan catabolites in the human intestine. Studies have shown that indolepropionic acid improves intestinal blood barrier function and plays a key role in maintaining intestinal mucosal balance; in addition, indolepropionic acid can also regulate vasodilation response and effectively reduce the risk of cardiovascular disease. There are patents related to the use of indolepropionic acid in the prevention and treatment of rheumatoid arthritis, prevention and treatment of ischemic heart disease, treatment of nephritis, and treatment of autism. However, other applications of indolepropionic acid still need to be further expanded. Summary of the invention

[0003] The first object of the present invention is to overcome the shortcomings of current treatment methods and provide the use of indolepropionic acid in the preparation of a drug for preventing and / or treating hearing loss.

[0004] Furthermore, the indolepropionic acid is the original indole-3-propionic acid drug, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof.

[0005] Furthermore, the hearing loss is sensorineural hearing loss.

[0006] Furthermore, the hearing loss is caused by damage to hair cells, supporting cells and nerve fibers.

[0007] Furthermore, indolepropionic acid can significantly reduce the ABR (Auditory brainstem response) threshold of hearing-impaired mice.

[0008] Furthermore, IPA was able to reduce damage to hair cells, supporting cells, spiral ganglion cells, stria vascularis, spiral ligament, and nerve fibers.

[0009] The second object of the present invention is to provide a pharmaceutical composition comprising an active ingredient, indolepropionic acid, and a pharmaceutically acceptable carrier.

[0010] Furthermore, the indolepropionic acid is the original indole-3-propionic acid drug, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof.

[0011] Furthermore, in the pharmaceutical composition, the effective concentration of indolepropionic acid is 40 mg / kg.

[0012] Furthermore, the pharmaceutical composition adopts any pharmaceutically acceptable dosage form.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time that indolepropionic acid can significantly reduce the ABR threshold of hearing-impaired mice, maintain the integrity of cochlear tissue morphology, and exert a significant therapeutic effect. It has broad application prospects in hearing loss and broadens the application scenarios of indolepropionic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The auditory brainstem response (ABR) after drug administration was measured by short stimulus (Click) and 8kHz, 16 kHz, 32kHz stimulus (Tone) for the blank group, TCP group, and indolepropionic acid treatment group. Compared with the blank group: ** P<0.01, *** P<0.001; compared with the TCP group: # P<0.05, ## P<0.01, ### P<0.001.

[0015] Figure 2 The results of hematoxylin and eosin staining of cochlear tissues in the blank group, TCP group and indolepropionic acid treatment group after drug administration under different treatment conditions are shown. A is the hematoxylin and eosin staining results of auditory hair cells (HCs), spiral neurons (SGNs), vascular striae (SV) and spiral ligament (SL) of cochlear tissues under different treatment conditions, B is a schematic diagram of the cross-sectional structure of the cochlea, C is the change in the number of SGNs in cochlear tissues under different treatment conditions, and D is the change in the thickness of SV in cochlear tissues under different treatment conditions.

[0016] Figure 3 The immunofluorescence staining results of the cochlear basilar membrane hair cells in the blank group, TCP group and indolepropionic acid treatment group after drug administration are shown. A is the morphological result of the cochlear basilar membrane HCs in the basal gyrus, and B is the quantitative result of the cochlear basilar membrane HCs in the basal gyrus.

[0017] Figure 4 The immunofluorescence staining results of spiral neurons (SGNs) and nerve fibers (NF) in the basilar membrane of the cochlea in the blank group, TCP group and indolepropionic acid treatment group after drug administration are shown. A is the morphological result of SGNs in the basal gyrus, and B is the morphological result of NF in the basal gyrus. DETAILED DESCRIPTION

[0018] The above contents of the present invention are further described below through specific embodiments. The following examples are used to illustrate the technical contents of the present invention, but are not intended to limit the contents of the present invention. All changes or equivalent substitutions made based on the present invention should fall within the protection scope of the present invention.

[0019] Example 1. Animal grouping, construction of hearing loss model and drug administration C57BL / 6 mice, 6-8 weeks old, male, after 1 week of adaptive culture, ABR analysis was performed to screen experimental mice with normal hearing.

[0020] The mice were randomly divided into three groups: blank group, TCP group and indolepropionic acid (IPA) treatment group, with 8 mice in each group.

[0021] 3,5,6-Trichloro-2-pyridinol (TCP) has been shown to be neurotoxic and auditory cytotoxic, which can cause hearing loss in mice. Therefore, TCP was used to construct a hearing loss model in this example.

[0022] 50mg / kg TCP; 50mg / kg TCP + 40mg / kg indolepropionic acid were dissolved in corn oil respectively. Mice in the indolepropionic acid treatment group were given 50mg / kg / day TCP and 40mg / kg / day indolepropionic acid, and mice in the TCP group were given 50mg / kg / day TCP. The blank group was given the same volume of corn oil for continuous gavage for 21 days.

[0023] Example 2: ABR analysis of changes in hearing threshold of mice under different drug administration conditions 24 hours after the end of drug administration, the mice in each exposure group were anesthetized and ABR tests were performed using short sounds (Click) and pure tones of 8, 16 and 32 kHz (Tone), respectively. The stimulus intensity was from 90 dB SPL to 10 dB SPL, starting from 90 dB, and decreased by 10 dB every time until the characteristic wave II disappeared. The sound intensity when the wave was no longer detected was recorded as the ABR threshold.

[0024] Example 3: Histopathological examination of mouse cochlea After ABR detection, mice were anesthetized and decapitated. Then the left and right temporal bones of mice were dissected as a whole, the bilateral auditory bullae were quickly removed, and the cochlear tissues were collected and separated; fixation: fixed with 4% paraformaldehyde for 24 hours; decalcification: decalcified with 0.5M EDTA for 3-5 days; sectioning: the direction was parallel to the direction of the cochlear modiolus, paraffin embedded, and sectioned; staining: conventional hematoxylin and eosin staining, sealed with coverslips for preservation.

[0025] Example 4: Immunofluorescence staining of mouse cochlear basilar membrane Fixation: Place the segmented cochlear basilar membrane in a 96-well plate and fix it with 4% paraformaldehyde for 30 minutes; Washing: Wash with PBS solution three times, 15 minutes each time; Blocking: Add 100µl of blocking solution containing 0.03% saponin and 10% horse serum in 0.1% PBST to each well of the 96-well plate; Incubate with primary antibody: Prepare antibody diluent with 2mg / mL BSA and 3% horse serum in 0.1% PBST, add hair cell specific marker antibody Myosin7a (rabbit source) (1:1000) and spiral ganglion cell specific marker antibody Tuj1 (mouse source) (1:500), and incubate at 4°C for 24 hours; Incubate with secondary antibody: After washing, add goat anti-rabbit Alexa Fluor 488 (1:100) and goat anti-mouse Alexa Fluor 488 (1:100) to the antibody diluent. 594 (1:100), incubate at room temperature for 2h; sealing: after washing, add appropriate amount of sealing medium containing DAPI and cover with coverslip.

[0026] Statistical analysis and graphics were performed with the aid of SPSS 24.0 and GraphPad Prism 7, and all data are presented as mean ± standard deviation. The Shapiro-Wilk test was used to assess data distribution, and the Levene test was used to assess variance homogeneity. For normally distributed data with equal variances, we used an unpaired two-tailed t-test; for data with unequal variances, we used the Welch t-test; for data with non-normal distribution, we used the Wilcoxon rank sum test. A P value of less than 0.05 was considered statistically significant.

[0027] result like Figure 1 As shown in A in the figure, the ABR threshold of the blank group under short sound (Click) was 22.50±4.08dB. In contrast, the TCP model group can raise the threshold under Click to 40.00 ± 5.78dB. This shows that the hearing loss model was successfully constructed and the clinical manifestations were typical. However, the ABR threshold of the indolepropionic acid treatment group was 27.38 ± 6.65dB. This shows that compared with the TCP group, the TCP-exposed mice treated with 40mg / kg indolepropionic acid had significantly improved hearing function. As shown in Figure 1 As shown in Figure B, the center of the noise band was further adjusted from 32 kHz to 16 kHz, and the frequency range of the test was finally expanded to 8 kHz. The results also showed that the ABR threshold of the indolepropionic acid treatment group was significantly lower than that of the TCP group. That is, 40 mg / kg indolepropionic acid can alleviate the hearing loss caused by TCP exposure.

[0028] Depend on Figure 2 As can be seen in A, the hair cells (HCs, indicated by red arrows) and the corresponding supporting cells (SCs, indicated by yellow arrows) in the blank group are intact, the Deiter SCs in the 50mg / kg TCP group are lost (indicated by white asterisks), and the 50mg / kg TCP + 40mg / kg IPA group is consistent with the blank group, and neither HCs nor Deiter SCs are lost. Figure 2 A and Figure 2 As shown in Figure C, the 50mg / kg TCP group caused shrinkage, deformation and loss of the nuclei of spiral neurons (SGNs) in the cochlea of ​​mice, but the nucleus morphology and number of SGNs in the 50mg / kg TCP+40mg / kg IPA group did not change significantly compared with the blank group. Comparison of the thickness of the vascular striae (SV) in different groups, Figure 2 A and Figure 2D in the figure shows that the thickness of the SV of the cochlear basement membrane of mice in the 50mg / kg TCP group was significantly thinner than that in the blank group, while the thickness of the SV of the cochlear basement membrane of mice in the 50mg / kg TCP+40mg / kg IPA group was no different from that in the blank group. In addition, the type I to V fiber cells in the lateral spiral ligament (SL) of the blank group were intact, while the type I and type V fiber cells in the basal gyrus SL of the 50mg / kg TCP group were lost, but the type I to V fiber cells in the basal gyrus SL of the 50mg / kg TCP+40mg / kg IPA group were not significantly lost. In general, indolepropionic acid pretreatment can effectively counteract the loss of supporting cells (SCs), deformation and loss of cochlear spiral neurons (SGNs), thinning of vascular stria (SV), and loss of type I and type V fiber cells in the spiral ligament (SL) caused by TCP exposure, protecting the integrity of cochlear tissue morphology.

[0029] Depend on Figure 3 It can be seen that compared with the blank group, the loss of outer hair cells (OHCs) in the 50mg / kg TCP group was obvious and their arrangement was disordered; in contrast, the loss of OHCs in the 50mg / kg TCP+40mg / kg IPA group was significantly reduced, but the degree of disorder was reduced. In other words, IPA has a protective effect on the damage of the hair cells (HCs) of the mouse cochlear basilar membrane caused by TCP.

[0030] Depend on Figure 4 As can be seen in A, compared with the blank group, the spiral neurons (SGNs) in the 50mg / kg TCP group were lost in multiple places (white asterisks), while the loss of SGNs in the 50mg / kg TCP+40mg / kg IPA group was significantly reduced; Figure 4 As shown in B, there was loss and breakage of nerve fibers (NF) in the 50mg / kg TCP group, and the NF arrangement density decreased, while the NF loss in the 50mg / kg TCP + 40mg / kg IPA group was significantly reduced. The above results indicate that IPA can protect the morphological changes of spiral neurons (SGNs) and nerve fibers (NF) induced by TCP.

[0031] The results showed that indolepropionic acid can significantly improve the clinical symptoms of hearing loss model animals and has a significant therapeutic effect on hearing loss. Therefore, indolepropionic acid has a good research prospect in the preparation of drugs for the treatment of hearing loss and is expected to become a new type of drug for the treatment of hearing loss.

Claims

1. Use of indolepropionic acid in the preparation of a drug for preventing and / or treating hearing loss.

2. The use according to claim 1, characterized in that: The hearing loss is sensorineural hearing loss.

3. The use according to claim 1, characterized in that: The hearing loss is caused by damage to hair cells, supporting cells and nerve fibers.

4. The use according to claim 1, characterized in that: Indolepropionic acid can significantly reduce the ABR threshold of hearing loss.

5. The use according to claim 1, characterized in that: Indolepropionic acid can relieve damage to cochlear hair cells, supporting cells, spiral neurons, vascular striae, spiral ligaments and nerve fibers.

6. The use according to claim 4 or 5, characterized in that: The indolepropionic acid is the original indolepropionic acid drug, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof.

7. A pharmaceutical composition, characterized in that The invention comprises an active ingredient, indolepropionic acid, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The indolepropionic acid is the original indolepropionic acid drug, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof.

9. The pharmaceutical composition according to claim 7, characterized in that In the pharmaceutical composition, the effective concentration of indolepropionic acid is 40 mg / kg.

10. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition adopts any pharmaceutically acceptable dosage form.

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