CN108310386A - MTOR signal pathway inhibitors are preparing the purposes in preventing or treating nongenetic dysaudia drug - Google Patents

Abstract

The invention discloses the use of mTOR signaling pathway inhibitors in the preparation of drugs for preventing or treating non-hereditary hearing impairment. The mTOR signaling pathway inhibitors are selected from rapamycin, rapamycin analogs and second-generation mTOR signaling At least one of the pathway inhibitors; the rapamycin analog is selected from at least one of everolimus, temsirolimus and Deforolimus; the second generation mTOR signaling pathway inhibitor is selected from At least one of PI3K/mTOR dual inhibitors, selective mTORC1/2 inhibitors and ATP competitive mTOR kinase inhibitors. The inventors of the present application have discovered for the first time that mTOR signaling pathway inhibitors can effectively reduce the damage to inner ear hair cells and spiral ganglion cells caused by ototoxic drugs and other non-genetic factors, significantly improve the impaired hearing level, and effectively prevent and treat non-genetic factors. hearing impairment.

Description

Inhibitors of mTOR signaling pathway in the preparation of drugs for the prevention or treatment of non-hereditary hearing impairment use in things

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the use of mTOR signaling pathway inhibitors in the preparation of drugs for preventing or treating non-hereditary hearing impairment.

Background technique

According to the second national survey on the population of persons with disabilities, there are 27.8 million people with hearing and speech disabilities, surpassing physical disabilities and visual disabilities as the number one cause of disability. Sensorineural hearing impairment accounts for more than 70% of the hearing-impaired population, and conductive hearing impairment accounts for about 30%. Conductive hearing impairment can be improved by surgically repairing or reconstructing the sound transmission system or wearing hearing aids; while sensorineural hearing impairment is mainly due to hair cell lesions and/or spiral neuron damage, and mammalian cochlear hair cells and spiral nerves Damaged cells cannot regenerate. There are many factors that can cause damage to hair cells and spiral neurons, including genetic factors, aging, noise exposure, infection, and the use of ototoxic drugs.

However, the molecular mechanism of non-genetic hearing impairment is still unclear. There is evidence that it is related to the abnormal activity of calcium-dependent protease, inner ear microcirculation disturbance, inner ear cell free radical damage and cell apoptosis. Currently, there is no effective clinical treatment. The means to prevent and treat hearing impairment caused by non-genetic factors. Potential prevention and treatment methods include the use of glucocorticoids, antioxidants, neurotrophic factors combined with vasodilators, hyperbaric oxygen, acupuncture therapy, surgical treatment, and cochlear implantation, but there are large differences in the treatment effect and the effect is not obvious And other issues. It is an urgent need for clinical prevention and treatment to develop specific drugs or treatment methods for the pathogenesis of hearing impairment caused by non-genetic factors, especially drug-induced hearing impairment.

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase widely distributed in organisms. It plays a pivotal role in the process of apoptosis. mTOR regulates the translation of specific mRNAs by regulating the phosphorylation status of these major signaling proteins such as AKT, p70S6K and 4EBP1. Rapamycin is a macrolide antibiotic and a specific inhibitor of mTOR. It specifically binds to mTOR molecules by forming a complex with the cytoplasmic protein FKBP12, thereby regulating mTOR apoptosis and autophagy. Function, it can be used clinically for the treatment of tumor and fungal infection.

Contents of the invention

Based on this, the object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide the purposes of mTOR signaling pathway inhibitors in the preparation of drugs for the prevention or treatment of non-hereditary hearing impairment, said mTOR signaling pathway inhibitors are selected from Rapa At least one of mycin, rapamycin analogs and second-generation mTOR signaling pathway inhibitors.

More preferably, the mTOR signaling pathway inhibitor is rapamycin.

Preferably, the rapamycin analog is at least one selected from everolimus, temsirolimus and Deforolimus.

Preferably, the second-generation mTOR signaling pathway inhibitor is selected from at least one of PI3K/mTOR dual inhibitors, selective mTORC1/2 inhibitors and ATP-competitive mTOR kinase inhibitors;

The PI3K/mTOR dual inhibitor is selected from at least one of PI-103, GNE-477, NVP-BEZ235, BGT226, XL765, SF-1126 and WJD008;

The selective mTORC1/2 inhibitor is selected from at least one of pp242, Torin1, way-600, wye-687, wye-354, ink128 and AZD8055;

The ATP competitive mTOR kinase inhibitor is selected from at least one of NVPBBD130, Ku0063794, WJD008 and PKI402.

Preferably, the drug is in oral dosage form or injection dosage form.

Preferably, the oral dosage form is tablet, granule, capsule, powder, solution, emulsion or suspension.

Another object of the present invention is to provide a drug for preventing or treating non-hereditary hearing impairment, which can effectively reduce the damage of non-genetic factors such as ototoxic drugs to inner ear hair cells and spiral ganglion cells, and significantly improve the damaged hearing loss. listening level.

In order to achieve the above purpose, the technical solution adopted by the present invention is: a drug for preventing or treating non-hereditary hearing impairment, the drug comprising an mTOR signaling pathway inhibitor and a pharmaceutically acceptable carrier.

Preferably, the mTOR signaling pathway inhibitor is selected from at least one of rapamycin, rapamycin analogs and second-generation mTOR signaling pathway inhibitors.

Preferably, the rapamycin analog is at least one selected from everolimus, temsirolimus and Deforolimus.

Preferably, the second-generation mTOR signaling pathway inhibitor is selected from at least one of PI3K/mTOR dual inhibitors, selective mTORC1/2 inhibitors and ATP-competitive mTOR kinase inhibitors;

The PI3K/mTOR dual inhibitor is selected from at least one of PI-103, GNE-477, NVP-BEZ235, BGT226, XL765, SF-1126 and WJD008;

The selective mTORC1/2 inhibitor is selected from at least one of pp242, Torin1, way-600, wye-687, wye-354, ink128 and AZD8055;

The ATP competitive mTOR kinase inhibitor is selected from at least one of NVPBBD130, Ku0063794, WJD008 and PKI402.

The pharmaceutically acceptable carrier is a conventional adjuvant for preparing medicines in the field, which can be selected according to needs, and the medicines can be prepared into various required dosage forms, and the preparation method is a conventional technology in the field.

Preferably, the drug is in oral dosage form or injection dosage form.

Preferably, the oral dosage form is tablet, granule, capsule, powder, solution, emulsion or suspension.

The non-hereditary hearing impairment in the present invention includes the hearing impairment caused by non-genetic factors such as the use of ototoxic drugs, noise exposure, infection and aging.

The rapamycin described in the present invention (English name is Rapamycin, Sirolimus, RAPA; also known as Sirolimus in Chinese) is a macrolide antibiotic with a molecular formula of C ₅₁ H ₇₉ NO ₁₃ and a molecular weight of 914.17. It is a white solid crystal with a melting point of 183-185°C, lipophilic, soluble in organic solvents such as methanol, ethanol, acetone, and chloroform, slightly soluble in water, and almost insoluble in ether. Its molecular structure formula is:

The rapamycin, rapamycin analogs and second-generation mTOR signaling pathway inhibitors described in the present invention can all be purchased from the market.

Compared with the prior art, the beneficial effects of the present invention are as follows: the inventors of the present application have found through a large amount of research and analysis that the mTOR signaling pathways such as rapamycin, rapamycin analogs and second-generation mTOR signaling pathway inhibitors Specific inhibitors can effectively reduce the damage of non-genetic factors such as ototoxic drugs, noise exposure, infection and aging to inner ear hair cells and spiral ganglion cells, significantly improve the impaired hearing level, and effectively prevent and treat non-genetic factors. Genetic hearing impairment provides a new option for the clinical prevention and treatment of non-hereditary hearing impairment.

Description of drawings

Figure 1 is a graph showing the loss of cochlear spiral ganglion cells caused by gentamicin in vivo.

Figure 2 is a graph showing the degeneration of cochlear spiral ganglion cells cultured in vitro caused by gentamicin.

Figure 3 is a graph showing the results of acute in vivo injection of gentamicin that can activate the mTOR signaling pathway.

Fig. 4 is a diagram showing the effect of different concentrations of rapamycin on the spiral ganglion cells of various parts of the cochlea cultured in vitro.

Fig. 5 is a graph showing the protective effect of rapamycin on degeneration of cochlear spiral ganglion cells cultured in vitro caused by gentamicin.

Fig. 6 is a graph showing that 50 μM gentamicin can cause significant loss of hair cells.

Fig. 7 is a diagram showing the effect of different concentrations of rapamycin on hair cells in various parts.

Fig. 8 is a graph showing the results of rapamycin alleviating the loss of cochlear hair cells caused by gentamicin.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples.

Example 1

This example takes rapamycin as an example to study the protective effect of mTOR signaling pathway inhibitors on cochlear spiral ganglion cells.

1. Gentamicin causes loss of cochlear spiral ganglion cells in vivo

Twenty-eight healthy C57B6/J mice were purchased from the Experimental Animal Center of Southern Medical University, 7-9 weeks old, regardless of sex, and randomly divided into two groups, the control group and the model group. Mice were kept in a quiet, warm animal breeding room, given sufficient food and water. Mice were cared for according to Animal Care and Use Committee guidelines. The mice in the control group were intraperitoneally injected with 5ml/kg normal saline for 15 consecutive days. The mice in the model group were injected with 40 mg/kg furosemide intraperitoneally for 15 consecutive days, and then 200 mg/kg gentamicin sulfate was injected intraperitoneally 20 minutes later. The auditory brainstem response test (ABR) test was performed before administration and after 15 days of continuous administration (Fig. 1A). The results showed that compared with the control group, the ABR threshold of the gentamicin-administered group (post-Gent.) mice was significantly increased (Fig. 1B), indicating that gentamicin can induce ototoxicity. Based on the successful establishment of the ototoxicity-induced ototoxicity model by gentamicin, the injury of the two main cells involved in the ototoxicity induced by gentamicin, namely hair cells and spiral neurons, was observed. First, after the cochleas of mice in the control group and the model group were separated, 4% paraformaldehyde and 20mM EDTA were added for fixation and decalcification, and then the cochlear basement membrane was separated for rhodamine staining to observe the loss of hair cells. The results are shown in the figure, compared with the control group, the hair cells on the basement membrane of the cochlear apex, middle and floor of the cochlea in the gentamicin (Gent.) administration group did not lose (Fig. 1C and D). Secondly, after the cochleas of the mice in the control group and the model group were separated, they were fixed and decalcified by adding 4% paraformaldehyde and 4% EDTA, and treated with 20% and 30% hypertonic sucrose solutions for 24 hours respectively. After the specimens were frozen and sectioned, they were fixed with 4% formaldehyde for 10 min, stained with Nissl, and imaged with a pathological analysis microscope. It was found that administration of gentamicin caused the loss of cochlear spiral ganglion cells compared with the control group (Fig. 1E and F).

2. Gentamicin can cause degeneration of cochlear spiral ganglion cells cultured in vitro

Six C57B6/J suckling mice from healthy C57B6/J pregnant mice on day 0-1 were purchased from the Experimental Animal Center of Southern Medical University. Mid-bottom sites were cultured with neuronal medium (neurobasal+2% B27). The cultured tissue blocks were randomly divided into the control group (control) and the gentamicin group (Gent.). into neuronal culture medium containing 50 μM gentamicin. After 72 hours, the culture was terminated, fixed with 4% paraformaldehyde, stained with TUJ-1 antibody (1:1000), observed and photographed with a laser confocal microscope. The results are shown in Figure 2, 50 μM gentamicin can significantly reduce the density and length of spiral ganglion cell axons, especially in the middle and bottom.

3. Acute in vivo injection of gentamicin can activate mTOR signaling pathway

Thirty-six healthy C57B6/J mice were purchased from the Experimental Animal Center of Southern Medical University, 7-9 weeks old, regardless of sex, and randomly divided into two groups, the control group and the model group. After the mice in the model group were anesthetized, the auditory bulb of one side of the ear was exposed from the neck, the auditory bulb was opened, and 20 μl of gentamicin sulfate (500 μM) was injected into it, while the mice in the vehicle control group were injected with 20 μl of 0.9% NaCl solution. The contralateral untreated cochlea served as a control. After 1 hour, the mice were sacrificed acutely, and the cochlea was removed for subsequent experiments. In order to observe the expression of important factors related to the mTOR signaling pathway (AKT, 4EBP1 and p70S6K) in tissue cells, the cochlea of mice in the control group and the model group were fixed and removed by adding 4% paraformaldehyde and 4% EDTA. Calcium, and then treated with 20% and 30% hypertonic sucrose solution for 24h respectively. After the specimens were frozen and sectioned, they were fixed with 4% formaldehyde for 10 min, stained with p-AKT, p-4EBP1 and p-p70S6K antibodies, and imaged with a pathological analysis microscope. It was found that, compared with the control group, administration of gentamicin could significantly increase the expression of p-AKT, p-4EBP1 and p-p70S6K proteins (Fig. 3A). In order to further observe the effects of acute injection of gentamicin on the expression of AKT, 4EBP1 and p70S6K proteins in the cochlea, the cochlear axis of the mice in the control group and the model group were separated and added to the protein lysate for lysing, and the total protein extracted was analyzed. Western blot analysis. The results are shown in Figure 3B and F, compared with the contralateral non-administered or 0.9% saline group, 500 μM gentamicin for 1 h can increase the expression of p-AKT, p-4EBP1 and p-p70S6K in spiral ganglion cells. The protein was expressed and was statistically significant (p<0.01, Figure 3C and G). At the same time, 500 μM gentamicin could increase the protein expression of p-AKT and p-4EBP1 in a time-dependent manner and had statistical significance (p<0.01, Figure 3D and E).

4. Effects of different concentrations of rapamycin on various parts of cochlear spiral ganglion cells cultured in vitro

The cochlear spiral ganglion tissue blocks were cultured according to the method of Experiment 2, and the available tissues were randomly divided into a control group (control) and a rapamycin group (rapa.) according to the location. Starting from taking out the tissue culture, the control group was the same amount of neuron culture medium as the experimental group, and the different concentrations of rapamycin groups were added with neuron culture medium containing rapamycin at concentrations of 5 μM and 15 μM respectively. After 72 hours, the culture was terminated, fixed with 4% paraformaldehyde, stained with TUJ-1 antibody (1:1000), observed and photographed with a laser confocal microscope. The results are shown in FIG. 4A , from left to right are the top, middle, and bottom, and the concentration of rapamycin from top to bottom is 5 μM and 15 μM (n=6). It can be seen from the figure that in the control group, the cilia in various parts of the 5μM group were basically not missing, and the structure was basically complete; while 15μM rapamycin could cause a significant decrease in the density and length of the axons of the spiral ganglion cells, especially in the middle and Bottom, the statistical results shown in Figure 4B and C are consistent with the results in Figure 4A and have statistical significance (p<0.01). These results indicated that high concentrations of rapamycin had toxic effects on helical nerve cells, but no toxic effects were observed below 5 μM. Therefore, our subsequent experiments used 5 μM rapamycin to observe its protective effect on gentamicin-induced degeneration of cochlear spiral ganglion cells. Apical, Middle, and Basal represent the top, middle, and bottom of the cochlea, respectively.

5. Rapamycin has a protective effect on the degeneration of cochlear spiral ganglion cells cultured in vitro induced by gentamicin

According to the experimental method of experiment 2, the cochlear spiral ganglion tissue blocks were cultured, and the available tissues were randomly divided into control group (control), gentamicin group (Gent.) and rapamycin-genta Mycin combination group (Rapa.+Gent.). From taking out the tissue culture, the control group is the same amount of neuron culture medium as the experimental group, and the rapamycin-gentamicin combined action group (Rapa.+Gent.) is added with 5μM rapamycin neuron Culture medium, after 10h, the gentamicin group (Gent.) replaced the culture medium with neuron culture medium containing 50 μM gentamicin, and the rapamycin-gentamicin combined action group (Rapa.+Gent.) Change to neuron culture medium containing 5 μM rapamycin and 50 μM gentamycin and continue to culture for 62 h. After 72 hours, the culture was terminated, fixed with 4% paraformaldehyde, stained with TUJ-1 antibody (1:1000), observed and photographed with a laser confocal microscope. The results are shown in Figure 5A, from left to right are top, middle, bottom, and from top to bottom are control group (control), gentamicin group (Gent.) and rapamycin-gentamicin Combined effect group (Rapa.+Gent.) (n=6). It can be seen from the figure that there is basically no loss of cilia in various parts of the control group, and the structure is basically complete, while 50 μM gentamicin can cause a significant decrease in the density and length of the axons of the spiral ganglion cells, especially in the middle and bottom, ahead of time. Adding rapamycin can significantly alleviate the damage effect of gentamicin on the density and length of spiral ganglion cell axons. The statistical results are shown in Figure 5B and C, and it was found that the trend was consistent with the result in Figure 5A, and it was statistically significant (p<0.001). Apical, Middle, and Basal represent the top, middle, and bottom of the cochlea, respectively.

The protective effect of other mTOR signaling pathway inhibitors (including rapamycin analogues and second-generation mTOR signaling pathway inhibitors) of the present invention on cochlear spiral ganglion cells is similar to that of rapamycin, and the specific data are omitted.

Example 2

This example takes rapamycin as an example to study the protective effect of mTOR signaling pathway inhibitors on cochlear hair cells.

1. Gentamicin can induce the loss of cochlear hair cells cultured in vitro

Twenty C57B6/J suckling mice born 2-3 days old from healthy C57B6/J pregnant mice were purchased from the Experimental Animal Center of Southern Medical University. . After 12 hours, the tissues with good adherent growth and clearly visible cell outlines were selected for further experiments. The available tissues were randomly divided into control group and gentamicin group according to the site. The control group was the same amount of complete culture solution as the experimental group, and the gentamicin group was the complete culture solution containing 50 μM gentamicin. The culture was terminated after 48 hours, fixed with 4% paraformaldehyde, stained with Phalloidin (1:200) in the dark for 30 minutes, rinsed with PBS, mounted, observed and photographed with a fluorescent microscope. The results are shown in Fig. 6, 50 μM gentamicin can cause significant loss of hair cells, especially in the middle and bottom. A, B, and C are respectively the top, middle, and bottom of the control group (n=6); D, E, and F are respectively the top, middle, and bottom (n=8) of the experimental group (50 μM gentamicin). 50μM gentamicin for 48 hours can cause the obvious loss of outer hair cells in the middle and bottom.

2. Effects of different concentrations of rapamycin on cochlear hair cells in various parts cultured in vitro

The cochlear basement membrane was cultured according to the experimental method in the previous step, and after 12 hours, the adherent tissues were randomly divided into a control group and a rapamycin group according to their locations. The control group was the same amount of complete culture solution as the experimental group, and the different concentrations of rapamycin groups were respectively added with complete culture solutions containing rapamycin concentrations of 1 μM, 10 μM, and 50 μM. The culture was terminated after 48 hours, fixed with 4% paraformaldehyde, stained with Phalloidin (1:200) in the dark for 30 minutes, rinsed with PBS, mounted, observed and photographed with a fluorescent microscope. The results are shown in Figure 7, from left to right are top, middle, bottom, and the concentration of rapamycin from top to bottom is 1 μM, 10 μM, 50 μM (n≥4). It can be seen from the figure that in the control group, 1 μM group, and 10 μM group, there was basically no loss of cilia in various parts, and the structure was basically complete; while 50 μM rapamycin could kill almost all hair cells. These results indicated that high concentrations of rapamycin had toxic effects on hair cells, but lower than 10 μM had no toxic effects. Our subsequent experiments used 1 μM rapamycin to observe its protective effect on gentamicin-induced loss of cochlear hair cells. A, M, and B represent the top, middle, and bottom of the cochlea, respectively.

3. Rapamycin has a protective effect on the loss of cochlear hair cells in vitro cultured by gentamicin

According to the experimental method in the previous step, the cochlear basement membrane was cultured. After 12 hours, the available tissues were randomly divided into control group (control), gentamicin group (Gen) and rapamycin-gentamycin combined action group (1 μM Rapa + 50 μM Gen). The combined administration group was pretreated with the culture solution containing 1 μM rapamycin for 24 hours, and the gentamicin group was treated with the culture solution without any drug. The culture solution of damycin was continued for 48 hours, and the gentamicin group was directly replaced with the culture solution containing 50 μM gentamycin for 48 hours, and the control group was the same amount of complete culture solution as the experimental group. The culture was terminated after 48 hours, fixed with 4% paraformaldehyde, stained with Phalloidin (1:200) in the dark for 30 minutes, rinsed with PBS, mounted, observed and photographed with a fluorescent microscope. The results are shown in Figure 8. Compared with the control group (control), the administration of 50 μM gentamicin (50 μM Gen) can lead to the loss of cochlear hair cells, especially in the bottom of the cochlea, while adding 1 μM rapamycin for pretreatment Hair cells in the middle and bottom were significantly increased after treatment (1 μM Rapa+50 μM Gen, n=6). It shows that rapamycin can alleviate the loss of cochlear hair cells caused by gentamicin. A, M, and B represent the top, middle, and bottom of the cochlea, respectively.

The protective effect of other mTOR signaling pathway inhibitors (including rapamycin analogs and second-generation mTOR signaling pathway inhibitors) described in the present invention on cochlear hair cells is similar to that of rapamycin, and the specific data are omitted.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The purposes of mTOR signaling pathway inhibitor in the preparation prevention or treatment non-hereditary hearing impairment medicine, it is characterized in that, described mTOR signaling pathway inhibitor is selected from rapamycin, rapamycin analogue and second generation At least one of mTOR signaling pathway inhibitors. 2. The use according to claim 1, characterized in that the rapamycin analog is at least one selected from everolimus, temsirolimus and Deforolimus. 3. The use according to claim 1, wherein the second-generation mTOR signaling pathway inhibitor is selected from PI3K/mTOR dual inhibitors, selective mTORC1/2 inhibitors and ATP-competitive mTOR kinase inhibitors at least one of The PI3K/mTOR dual inhibitor is selected from at least one of PI-103, GNE-477, NVP-BEZ235, BGT226, XL765, SF-1126 and WJD008; The selective mTORC1/2 inhibitor is selected from at least one of pp242, Torin1, way-600, wye-687, wye-354, ink128 and AZD8055; The ATP competitive mTOR kinase inhibitor is selected from at least one of NVPBBD130, Ku0063794, WJD008 and PKI402. 4. The use according to any one of claims 1-3, characterized in that, the drug is in an oral dosage form or an injection dosage form. 5. The use according to claim 4, wherein the oral dosage form is tablet, granule, capsule, powder, solution, emulsion or suspension. 6. A medicament for preventing or treating non-hereditary hearing impairment, characterized in that the medicament comprises an mTOR signaling pathway inhibitor and a pharmaceutically acceptable carrier. 7. The medicine for preventing or treating non-hereditary hearing impairment according to claim 6, wherein the mTOR signaling pathway inhibitor is selected from rapamycin, rapamycin analogues and second-generation mTOR signaling at least one of pathway inhibitors. 8. The medicine for preventing or treating non-hereditary hearing impairment according to claim 7, wherein the rapamycin analog is selected from at least one of everolimus, temsirolimus and Deforolimus A sort of. 9. The medicament for preventing or treating non-hereditary hearing impairment according to claim 7, wherein said second-generation mTOR signaling pathway inhibitor is selected from PI3K/mTOR dual inhibitors, selective mTORC1/2 inhibitors and at least one of ATP-competitive mTOR kinase inhibitors; The PI3K/mTOR dual inhibitor is selected from at least one of PI-103, GNE-477, NVP-BEZ235, BGT226, XL765, SF-1126 and WJD008; The selective mTORC1/2 inhibitor is selected from at least one of pp242, Torin1, way-600, wye-687, wye-354, ink128 and AZD8055; The ATP competitive mTOR kinase inhibitor is selected from at least one of NVPBBD130, Ku0063794, WJD008 and PKI402. 10. The medicament for preventing or treating non-hereditary hearing impairment according to any one of claims 6 to 9, characterized in that, the medicament is an oral dosage form or an injection dosage form; preferably, the oral dosage form is a tablet, Granules, capsules, powders, solutions, emulsions or suspensions.