CN114848836B

CN114848836B - Conjugate and application thereof in treating inner ear diseases

Info

Publication number: CN114848836B
Application number: CN202110761572.1A
Authority: CN
Inventors: 庞嘉祺; 许小丁; 郑亿庆
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2023-09-15
Anticipated expiration: 2041-07-06
Also published as: CN114848836A

Abstract

The application discloses a conjugate and application thereof in inner ear diseases, wherein the conjugate is formed by combining inner ear targeting polypeptide and a main drug for diagnosing, preventing or treating the inner ear diseases, so that the main drug is assisted to target specific structures and cells in the inner ear or the inner ear by means of the inner ear targeting polypeptide, and enrichment of the main drug is realized to accurately act on the targeted inner ear structures or cells; the inner ear targeting polypeptide can improve the efficiency of drug delivery to the inner ear, thereby remarkably improving the content of the drug reaching the inner ear and improving the bioavailability of the drug in the inner ear. The application also provides application of the conjugate in treating inner ear diseases, and diagnosis, prevention or treatment of the inner ear diseases can be realized conveniently through the main drug in the conjugate. The present application more particularly provides for the delivery of conjugates targeted to the outer hair cells of the inner ear by locally delivering the conjugate a665-NAC. When the conjugate is applied to the inner ear with cisplatin effect, the hearing loss caused by cisplatin can be reduced, and the effect of preventing and treating cisplatin ototoxicity can be achieved.

Description

Conjugate and application thereof in treating inner ear diseases

Technical Field

The application relates to the field of pharmacy, in particular to a conjugate and application thereof in treating inner ear diseases.

Background

The inner ear diseases mainly comprise dizziness, deafness and the like, and are mainly caused by the damage of structures such as inner hair cells, auditory nerves and the like, for example, various sensorineural hearing losses are mainly caused by the damage of hair cells in cochlea, especially the damage of outer hair cells. The inner ear diseases not only can bring direct physical health problems to patients, but also can directly influence the normal life of the patients and bring inconvenience to the normal life of the patients, so that the treatment of the inner ear diseases is always an important research object in the field of the current otology diseases, and the patients with the inner ear diseases also need an effective inner ear disease treatment drug or scheme urgently.

At present, medicines are generally adopted for the treatment of inner ear diseases, but the distribution of the medicines to the inner ear is limited to a great extent due to the blood vessel barrier in the inner ear and the special anatomical physiological characteristics of deep embedded skull base bone, and the treatment effect is often limited. In particular the blood-labyrinth barrier in the inner ear, which limits the transport of various substances from the blood to the inner ear, when administered using a systemic system, only very small amounts of drug can reach the inner ear, far from the effective drug concentration required for the treatment of inner ear diseases. For example, yang et al injected dexamethasone 0.5mg/ml/kg intrapulmonary in guinea pigs, and the drug concentration in perilymph fluid was only 0.03mg/ml after 1 hour, whereas the drug concentration in plasma was 2.76mg/ml. When systemic system is adopted for administration, in order to achieve effective therapeutic concentration when the medicine reaches the inner ear, the systemic circulation medicine concentration is always required to be higher, and toxic and side effects on other organs and tissues of the body are easily caused. For this reason, in the prior art, local drug delivery treatment measures of the inner ear are proposed, the local drug delivery has the characteristics of "short distance", avoidance of "first pass" metabolism, avoidance of blood-labyrinth barrier, and significantly higher concentration of the inner ear drug than the whole body, and the current local administration mode, namely round window membrane administration route, namely tympanic injection, is more common. Because the round window membrane is a semi-permeable membrane separating the inner ear from the tympanic cavity of the middle ear, it is permeable to ions, molecules, and some drugs, and the use of the round window membrane route of administration aids in transporting sufficient drug to the inner ear and reduces the amount of drug exposed to other tissues. The current effective inner ear disease treatment measures mainly adopt a mode of local administration for treatment.

However, even though the problem of side effects of systemic administration and the problem of effective concentration of the drug can be solved by using a local administration mode, the problem of targeting of the drug still cannot be solved, and in the prior art, the drug can enter the inner ear through a round window membrane, but is diffused in the cochlea along with perilymph fluid, and cannot act on specific structures and cells of the inner ear in a targeting manner to protect or repair. Therefore, in order to improve the protective or therapeutic effect of the drug on the corresponding structure or cell, the concentration still needs to be increased to improve the opportunity of the drug on the corresponding structure or cell, and the drug action with high concentration still cannot be avoided; and the high-concentration medicine can stimulate the mucous membrane of the middle ear, so that the using effect is reduced, and the overall treatment effect is affected.

Therefore, there is a need for a targeting agent for treating inner ear diseases or a targeting drug delivery agent and system to solve the above problems.

Disclosure of Invention

The conjugate is formed by combining the inner ear targeting polypeptide and the main drug for treating the inner ear diseases, so that the inner ear targeting polypeptide is used for assisting the main drug to target the inner ear and specific structures and cells in the inner ear, enrichment of the main drug is realized, and the targeted inner ear structure or cells are precisely acted; the inner ear targeting polypeptide can improve the efficiency of drug delivery to the inner ear, thereby remarkably improving the content of the drug reaching the inner ear and improving the bioavailability of the drug in the inner ear. On the premise of improving the effectiveness of drug delivery to the inner ear, the method is beneficial to reducing the total dosage while achieving the same therapeutic effect as the prior art. Thereby avoiding the administration of an excess of the drug in order to achieve an effective concentration in the inner ear and avoiding the possible side effects of an excess of the drug. The main medicine serving as a part of the conjugate can be effectively transported to the inner ear, so that the total amount of the medicine actually applied by the conjugate can be smaller than that in the prior art on the premise of reaching the same dosage or concentration of the inner ear, the irritation to structures such as mucous membranes of the inner ear can be further reduced, the medicine effect is improved, and the overall prevention or treatment effect is improved. In addition, when the drug is a drug for diagnosing an inner ear disease, the conjugate formed by the drug and the inner ear targeting polypeptide can also be used for diagnosing the inner ear disease. The present application more specifically contemplates the delivery of conjugates a665-NAC that are formed by local delivery of polypeptides a665, NAC to achieve conjugate transport, pharmaceutical procedures that target inner ear, outer hair cells of the inner ear. Targeting of the conjugate can be achieved through coordination of A665 and prestin proteins expressed by outer hair cells, so that the efficiency of delivering the conjugate to the inner ear including the Corti device and other areas can be improved, and protection of outer hair cells can be achieved through NAC.

The application provides a conjugate, which structurally comprises two components of an inner ear targeting polypeptide and a main drug, wherein the inner ear targeting polypeptide is connected with the main drug through a chemical bond, the inner ear targeting polypeptide is suitable for targeting the main drug to the inner ear, and the main drug is a drug for diagnosing, preventing or treating inner ear diseases. Further, the conjugate is administered topically. The inner ear targeting polypeptide is matched with proteins expressed by cells in inner ears to realize targeting, and the main medicine acts on inner ear structures or inner ear cells to achieve the effect of preventing or treating corresponding inner ear diseases. Furthermore, when the main drug is a drug for being transported to the inner ear so as to obtain the inner ear disease diagnosis result, the corresponding conjugate can also utilize the inner ear targeting polypeptide component in the structure to realize the targeting guiding process, so that the coupled main drug is efficiently transported to the inner ear and the target point, and the inner ear disease diagnosis result with high accuracy can be obtained. Further, the chemical bond includes a disulfide bond. The conjugate has targeting property, so that the dosage required by the conjugate is less, the targeting property is stronger, the conjugate can be directly enriched near a target site, and the conjugate is favorable for accurately acting on the target site.

Further, the inner ear targeting polypeptide is a665, and the amino acid sequence of a665 is: leu-Ser-Thr-His-Thr-Thr-Glu-Ser-Arg-Ser-Met-Val. The inner ear targeting polypeptide A665 can be matched with prestin proteins expressed by outer hair cells in the inner ear to realize targeting, is favorable for carrying a main drug coupled with the prestin proteins to transport and act on the inner ear, the outer hair cell concentration area or the outer hair cells in the inner ear, and realizes corresponding main drug medicinal effects.

Further, the main drug is NAC, and the conjugate has a structure shown in the following formula:

NAC is a common antioxidant and is currently used clinically for phlegm reduction therapy. NAC can reduce cisplatin-induced ototoxicity, and in one or more embodiments of the present application, protection of the outer hair cells of the inner ear and enhanced antioxidant capacity can be achieved by a conjugate NAC moiety. Is helpful for preventing and treating inner ear diseases caused by injury and oxidation of outer hair cells. The material for preparing the conjugate is relatively simple, the preparation cost of the polypeptide is low, and the mass production and the clinical practical application are realized.

The application also provides the application of the conjugate in diagnosis, prevention or treatment of inner ear diseases. When the main medicine component of the conjugate is a medicine for diagnosing, preventing or treating inner ear diseases, the conjugate can realize effective delivery and medicine effect by combining the targeted inner ear polypeptide, thereby realizing the purpose of the conjugate in diagnosing, preventing or treating inner ear diseases. Further, the inner ear disease is sensorineural hearing loss disease such as sensorineural deafness. When the inner ear targeting polypeptide is a polypeptide targeting the outer hair cells of the inner ear, the coupled main drug can be targeted and transported to the region where the outer hair cells are located, so that the main drug can more effectively act on the outer hair cells. In all sensorineural hearing loss diseases, various sensorineural hearing losses are caused by outer hair cell injury, and when the inner ear targeting polypeptide is a polypeptide targeting outer hair cells and the main drug acts on the outer hair cells to protect or cure the outer hair cells, the sensorineural hearing loss diseases can be prevented or treated. When the sensory acoustic nerve hearing loss disease is not caused by outer hair cells, the effect of guiding the main medicine and targeting can be achieved by setting the inner ear targeting polypeptide as the polypeptide targeting the corresponding pathological structure and cells. Therefore, the conjugate can be applied to preventing or treating sensorineural hearing loss diseases. And when the main medicine is a medicine for obtaining the diagnosis result of the sensorineural hearing loss disease, the application in diagnosis can be realized.

Further, the inner ear disease is a hearing loss disease caused by damage of outer hair cells. The outer hair cell injury is a direct cause of various inner ear diseases, and the outer hair cell has a prestin targetable protein, which is more beneficial to the targeting and the action of the conjugate, so as to realize the diagnosis, prevention or treatment of hearing loss diseases caused by the outer hair cell injury. Further, it is a sensorineural hearing loss disease caused by outer hair cell damage. The direct etiology of various sensorineural hearing loss diseases is the damage of outer hair cells, and the outer hair cells are the earliest and most seriously damaged areas, and the outer hair cells cannot be regenerated, so that the main medicine is enabled to be transported in a targeting way and act on the outer hair cells in a targeting way by setting the inner ear targeting polypeptide in the conjugate as the polypeptide of the targeting outer hair cells, and the effect of diagnosing, preventing or treating the damage of the outer hair cells corresponding to the main medicine is realized.

Further, the sensorineural hearing loss disease is a hearing loss disease caused by cisplatin ototoxicity. Cisplatin is the most common clinical chemotherapeutic drug, and because cisplatin can enter the inner ear to generate cytotoxicity, clinical multi-pass chemotherapy can lead to the occurrence of deafness. There are many mechanisms of cytotoxicity caused by cisplatin, and it is thought that there is an increase in active oxygen by DNA hybridization, DNA damage, and cisplatin. In one or more embodiments of the application, NAC is selected as the primary drug and the inner ear targeting polypeptide is set as the target outer hair cell A665, so that the application of the conjugate A665-NAC to auditory cells can reduce the ototoxicity caused by cisplatin, reduce the active oxygen increased by cisplatin, provide antioxidant capacity, resist cisplatin-induced apoptosis and enhance the resistance to cisplatin cytotoxicity. Therefore, the conjugate can be applied to hearing loss diseases caused by cisplatin ototoxicity, so that diagnosis, prevention or treatment can be realized by combining with a main drug. Further, there is provided the use of a conjugate for protecting an outer hair cell; further, the outer hair cells under the action of cisplatin are protected.

The application also provides a composition comprising a conjugate as described above. The composition can be applied at least to the treatment of inner ear diseases.

Further, the kit also comprises other medicines compatible with the conjugate and pharmaceutically acceptable carriers and/or auxiliary materials. The synergistic therapeutic effect is realized by matching other medicines with the conjugate. And the transportation of the conjugate is realized through the carrier, which is favorable for improving the transportation efficiency and the action effect of the conjugate, such as the carrier or auxiliary materials which can prolong the residence time of the conjugate at the administration position in the prior art.

Further, the pharmaceutically acceptable auxiliary material is matrigel. Matrigel is an injectable gel, which is injected into tympanic cavity of middle ear by tympanostomy after carrying medicine, stays in middle ear and forms storage effect, and can effectively control sustained release of medicine into inner ear. And the matrigel has no extra covalent cross-linking agent and toxic substances, and has good biocompatibility and high safety. The matrigel is used as auxiliary materials to assist the delivery of the conjugate, which is beneficial to prolonging the stay time of the conjugate at the administration position, such as the stay in the middle ear, so as to realize continuous conjugate release, facilitate the conjugate to enter the inner ear, increase the drug concentration in the inner ear and improve the bioavailability of the conjugate. In one embodiment of the application, the conjugate is carried by a matrigel such that the conjugate is retained in the middle ear and enters the inner ear, and further in one embodiment of the application, the conjugate A665-NAC is carried by a matrigel such that the A665-NAC is targeted to the inner ear Corti device region concentrated in the outer hair cells for controlled release and targeted transport of the conjugate.

The present application also provides a topical drug delivery system comprising a delivery device configured to release a composition to treat sensorineural hearing loss disease when the delivery device is inserted into the middle and/or inner ear, and a composition as described above. Still further, insertion of the delivery device into the middle and/or inner ear retains the composition for a sufficient period of time to achieve therapeutic concentrations of the inner ear drug. Namely, the local drug delivery is realized through the delivery device, so that the drug delivery effect is further improved on the basis of targeting of the composition, the conjugate is concentrated in the local area to avoid the influence of the conjugate on other organs and tissues as much as possible, and the local drug delivery is beneficial to reducing the total drug quantity, so that the toxic and side effects possibly caused by excessive drug quantity are avoided, and the action effect of the conjugate is improved. Still further, insertion of the delivery device into the middle ear administers the drug via the round window membrane, and the composition passes through the round window membrane into the inner ear upon release of the composition.

Compared with the prior art, the application has the beneficial effects that: the inner ear targeting polypeptide of the conjugate and the medicine for diagnosing, preventing or treating inner ear diseases can realize the effects of targeting delivery and targeting effect, improve the delivery efficiency of the main medicine part in the conjugate and improve the main medicine content delivered to the inner ear, thereby reducing the total medicine amount required to be applied for achieving effective medicine amount and avoiding toxic and side effects caused by excessive medicine. At the same time, the reduction of the applied dose also helps to reduce the irritation to other parts, such as middle ear mucosa, thereby improving the drug effect. Because of the targeting, the effectiveness of drug delivery to the inner ear can be improved, and the main drug can act on target sites and target cells, so that the targeting treatment is realized, the target sites are precisely acted, and the drug effect of the main drug is improved. And when the conjugate is applied locally, the toxic and side effects on other organs and tissues, which are possibly caused by systemic administration, can be avoided, and the possible adverse effects caused by the drug are further reduced. The conjugate can stay for a sufficient time at the administration position through the matrigel administration, so that the sustained release and the action of the conjugate are facilitated, and the overall drug action effect is further improved. The application also provides an A665-NAC conjugate, which can improve the efficiency of delivery to the inner ear, and enriches the conjugate in the outer hair cell concentration area by utilizing the protein expressed by the A665 and the targeted outer hair cells; so that the carrying primary NAC acts on the outer hair cells to protect the outer hair cells, such as: under the environment of cisplatin action, the antioxidation capability of inner ear hair cells is enhanced, and the cytotoxic side effect of cisplatin is resisted; reduce cell injury and apoptosis caused by cisplatin, and relieve hearing loss caused by cisplatin ototoxicity.

Drawings

FIG. 1 shows the structural formula of conjugate A665-NAC and RP-HPLC chromatography, mass spectrometry results;

FIG. 2 is a graph showing the ability of A665-NAC to enter auditory cell HEI-OC 1;

FIG. 3 is a graph showing that A665-NAC enhances the ability of HEI-OC1 cells to resist cisplatin cytotoxicity;

FIG. 4 is a graph of A665-NAC enhancing HEI-OC1 cell antioxidant capacity;

FIG. 5 is a graph showing that A665-NAC enhances viability of cells in primary tissue under the action of cisplatin;

FIG. 6 is a graph showing that A665-NAC reduces apoptosis of cells in primary tissue under the action of cisplatin;

FIG. 7 is a graph showing that A665-NAC reduces the amount of active oxygen generated by cells in primary tissue under the action of cisplatin;

figure 8 is a schematic representation of matrigel placement in the round window niche of the middle ear cavity and a665-NAC penetration schematic representation;

FIG. 9 shows matrigel controlled release A665-NAC assay results;

FIG. 10 is a graph showing the result of detecting the safety of use of a matrigel carrier;

FIG. 11 is a graph showing the results of a delivery test of matrigel bearing A665-NAC into the inner ear;

FIG. 12 is a graph showing the results of reducing cisplatin-induced hearing loss in an animal model of A665-NAC application;

FIG. 13 is a graph showing the results of reducing cisplatin-induced hair cell damage in an animal model of A665-NAC application.

Detailed Description

The drawings are for illustrative purposes only and are not to be construed as limiting the application. For better illustration of the following embodiments, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the actual product dimensions; it will be appreciated by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.

Example 1

Synthesis preparation of A665-NAC

In this example, A665-NAC is prepared by conventional polypeptide solid phase synthesis techniques, and the targeting polypeptide A665 is coupled to NAC by covalent bonding, and the structural formula of the prepared A665-NAC conjugate is shown in FIG. 1A.

Specifically, the preparation process comprises the following steps:

1. peptide sequence: leu-Ser-Thr-His-Thr-Thr-Glu-Ser-Arg-Ser-Met-Val-Cys (Ac-Cys); 2. description of preparation process: adopting a solid-phase organic synthesis method, adopting Fmoc-protected amino acid strategy and SPPS solid-phase synthesis technology to complete main chain assembly, cracking, and purifying after oxidation reaction to obtain the target product.

The preparation process specifically comprises the following steps:

(1) Solid phase synthesis: and (3) sequentially condensing amino acid links from the C end to the N end (from right to left) by using Fmoc-Cys (Trt) -CTC resin according to the peptide sequence and using an Fmoc-protected amino acid strategy, and forming the linear peptide resin after the linear peptide A2-A13 is condensed.

The following amino acids were coupled in sequence: a-02Fmoc-Val-OH, A-03Fmoc-Met-OH, A-04Fmoc-Ser (tbu) -OH, A-05Fmoc-Arg (Pbf) -OH, A-06Fmoc-Ser (tbu) -OH, A-07Fmoc-Glu (Otbu) -OH, A-08Fmoc-Thr (tbu) -OH, A-09Fmoc-Thr (tbu) -OH, A-10 Fmoc-His (Trt) -OH, A-11Fmoc-Thr (tbu) -OH, A-12Fmoc-Ser (tbu) -OH, A-13Fmoc-Leu-OH. The peptide resin was washed, transferred out and dried to constant weight, and cleaved.

(2) Peptide resin cleavage:

preparation of a cracking reagent: the amount of cleavage reagent used was calculated as 1g peptide resin to 10 ml.+ -. 2 ml: TFA, H2O and EDT, tis=95:1:2:2, and the required cracking reagent H2O, TFA, EDT and TIS are sequentially arranged in a cracking reaction bottle, and the temperature of the cracking reagent is controlled between 0 and 10 ℃; adding a cracking reagent into the peptide resin under stirring, and after the system temperature is stable; then the temperature is controlled between 25 ℃ and 30 ℃ and stirring is carried out for 2.5 hours.

The lysate is filtered off, precipitated with 5 times the volume of the concentrate of glacial ethyl ether, the precipitate is filtered off and dried under reduced pressure at room temperature, the crude product is obtained.

(3) Oxidation reaction:

grinding the crude product, preparing purified water, slowly adding the ground crude product under stirring, simultaneously dropwise adding an acetonitrile water solution, adding 1 time of Ac-Cys after the crude product is completely dissolved, dropwise adding 10 times of iodomethanol solution after the crude product is completely dissolved, and stirring for reacting for 2 hours.

(4) And (3) purifying and freeze-drying:

the purification adopts Shimadzu semi-preparation, uses 5cm,10um and C-18 column packing, uses proper gradient to separate and purify at normal temperature, collects target products, analyzes and detects, classifies, and finally obtains A665-NAC. And (3) separating and purifying the target substance with unqualified purity again by using a 2cm,5um and C-18 column with a proper gradient, and performing reduced pressure freeze drying on the qualified main peak to obtain the target substance.

For further verification, the detection of the present example was also performed by reverse phase high performance liquid chromatography/mass spectrometry, and the results are shown in fig. 1B and 1C, and a single peak chemical substance is obtained, which indicates that the conjugate has high purity and monodispersity, i.e., is synthesized as a single substance. Wherein 1B and 1C are RP-HPLC chromatograms and mass chromatograms respectively.

Example 2

Detection of the ability of A665-NAC to enter auditory cell HEI-OC1

To examine the ability of A665-NAC to enter auditory cells, HEI-OC1 cells expressing the A665 target transmembrane protein prestin were selected as subjects in this example to study the ability of A665-NAC to enter auditory cells.

In this example, the inventors added fluorescein Cy5.5 to A665-NAC, random peptide-NAC (designated RP-NAC), and noted that fluorescein-bearing A665-NAC was Cy5.5-A665-NAC, and fluorescein-bearing RP-NAC was Cy5.5-RP-NAC. After attaching HEI-OC1 cell plates overnight, 5mM of Cy5.5-A665-NAC/Cy5.5-RP-NAC was co-cultured on ice for 5 minutes, then the plates were changed, washed 3 times with PBS, and examined by flow cytometry after digestion with pancreatin.

The results are shown in FIG. 2, where Ctrl is the control group, RP-NAC is the random peptide-NAC treated group, and A665-NAC is the corresponding A665-NAC treated group. In this example, a group of experiments was also provided, in which 5mM A665 was used to treat auditory cells for 5 minutes, and then A665-NAC was added to the cells and incubated on ice for 5 minutes, the group being the group A665 Block A665-NAC in FIG. 2; the group is intended to achieve a blocking effect by reducing subsequent A665-NAC binding targets by first using A665 targeted binding targets to verify the targeting of A665-NAC.

In FIG. 2, (A) shows the results of using flow cytometry to detect control RP-NAC, A665 Block A665-NAC experimental groups. (B) fluorescence intensity analysis showing flow results. (C) Shown is the observed in confocal fluorescence microscopy of HEI-OC1 cells, cy5.5 orange, green cytoskeleton, blue nucleus, scale 5 μm, N=3, p <0.001.

As can be seen from fig. 2A, 2B, 2C, a665-NAC is able to enter auditory cell HEI-OC1 more rapidly than RP-NAC, a665 assists NAC in entering auditory cell HEI-OC1 rapidly and in large amounts, exhibiting targeting. And the A665 Block A665-NAC group had a reduced ability to enter cells compared to A665-NAC, indicating that if pre-treated with A665 5mM for 5 minutes and co-incubated on A665-NAC ice for 5 minutes, the ability of A665-NAC to enter cells was reduced.

After continuing to observe the adherent cells for 30 minutes using a confocal microscope, a665-NAC was found to enter more cells than RP-NAC; whereas the A665 Block A665-NAC group had a reduced ability of A665-NAC to enter HEI-OC1 after 5 minutes of blocking the targeting point with A665. The above results indicate that A665 enhances the ability of NAC to enter HEI-OC1 auditory cells and is targeted.

Example 3

A665-NAC enhances the antioxidant capacity of HEI-OC1 against cisplatin cytotoxicity.

1. Cisplatin-induced cell damage detection

In this example, cisplatin (CDDP) was used to treat HEI-OC1 cells for 24 hours with 30. Mu.M Cisplatin to achieve 50% cell damage, as shown in FIG. 3A. Whereas with either RP-NAC or A665-NAC 2.5mM with CDDP, there was less cellular damage than CDDP treatment alone, and A665-NAC was less damaging than the RP-NAC group, indicating that A665-NAC was able to resist the cytotoxic effects of CDDP and was stronger than the RP-NAC.

And the inventors compared the cell survival at various concentrations and found that A665-NAC was more protective than RP-NAC at 10mM at 2.5mM, as shown in FIG. 3B.

The inventor also obtains HEI-OC1 cell early apoptosis and apoptosis conditions caused by cisplatin of each experimental group through flow detection, as shown in figures 3C and 3D, CDDP can induce apoptosis to die, and RP-NAC and A665-NAC carrying NAC can both relieve apoptosis conditions caused by cisplatin, and because A665 has targeting and can be better combined, A665-NAC group can obviously relieve apoptosis conditions caused by cisplatin, and early apoptosis cell proportion is obviously reduced compared with other groups. That is, the results above demonstrate that A665-NAC is able to rapidly enter HEI-OC1 cells and enhance its ability to resist CDDP cytotoxicity.

Fig. 3: (A) results of cell activity experiments; (B) treatment results of different concentrations of A665-NAC, RP-NAC; (C) flow-testing early apoptosis results caused by cisplatin; (D) flow-through detection of apoptosis. N=3, p <0.05, p <0.001.

2. Reactive oxygen species detection

In this example, the DCFDA probe was also used to detect reactive oxygen species (Reactive Oxygen Species, ROS), and it was found that the CDDP treated cells had significantly higher ROS content, while the group treated with A665-NAC+CDDP had significantly lower levels than the group treated with RP-NAC, as shown in the first line of FIG. 4. Mitochondria are organelles of ROS production, and this example also uses mitoSOX to detect the level of ROS produced by mitochondria, and a665-NAC was found to reduce elevated levels of ROS in CDDP, as shown in the second line of fig. 4. The results indicate that A665-NAC can enhance the antioxidant capacity of cells and resist the effects of cisplatin on the active oxygen layer.

Fig. 4 top row: the DCFDA reactive oxygen species probe appears green in the presence of ROS, scale 10 μm. The second row of fig. 4: the mitoSOX probe appears orange in the presence of ROS, scale 20 μm.

Example 4

The A665-NAC enhances the antioxidant capacity of primary tissue of cochlear hair cells against cisplatin cytotoxicity.

To further verify on a cellular basis that a665-NAC also works to enhance antioxidant and resistance to cisplatin cytotoxicity in complex cochlear tissues, this example is based on detection of primary cochlear hair cell tissue. The inventor extracts cochlear basement membrane tissue of a milk mouse born for 3 days for primary culture, treats the primary basement membrane tissue by CDDP after overnight adherence, and discovers that obvious damage appears in the areas of the top, middle and bottom gyres. In the groups A665-NAC+CDDP and RP-NAC+CDDP, the surviving cells are obviously more than that of the CDDP group alone, and the cell survival condition based on the control group can be seen that cisplatin treatment can cause the loss or apoptosis of cells in primary tissues, and when A665-NAC can obviously reduce the loss and death of hair cells caused by cisplatin, the effect is obvious compared with that of the RP-NAC group. A665-NAC has a more pronounced hair cell protective effect than RP-NAC, and differences are statistically significant (FIG. 5); hair cells were labeled with the hair cell marker myosin VIIa (green fluorescence) and cells to enter apoptosis were represented by clear-caspase 3 (red fluorescence). The results suggest that A665-NAC reduced CDDP-induced apoptotic cells (FIG. 6).

When the mitochondrial production of ROS was assayed by mitoSOX, A665-NAC was found to reduce elevated levels of ROS in CDDP (FIG. 7). The results show that A665-NAC is equally effective in primary tissue, enhancing the antioxidant capacity of cochlear hair cell primary tissue against cisplatin cytotoxicity.

Fig. 5: (A) Hair cell loss and death due to cisplatin in the different groups. The green is a hair cell marker Myosin VIIa, hair cells can be specifically marked, and the scale is 20 mu m. (B) Top, middle, bottom, statistical analysis of panel a, n=3. P <0.001.

Fig. 6: the expression of caspase3, which is activated by cisplatin in different groups, is green as a hair cell marker Myosin VIIa, which can specifically label hair cells, and the scale is 20 μm.

Fig. 7: the amount of active oxygen released by mitochondria in different groups is green, which is a hair cell marker Myosin VIIa, hair cells can be specifically marked, and the scale is 20 mu m.

Example 5

A665-NCA delivery and efficacy detection in an in vivo animal environment

Tympanic administration is a mode of administration by middle ear, allowing the drug to pass through the round window membrane into the inner ear, reach the inner ear lymph fluid, and contact the target cells. In clinical treatment, in order to increase the content of the drug reaching the inner ear, administration is often performed by means of tympanic injection, which is a topical administration; in this example, administration was performed in a manner simulating clinical drum injection, i.e., by bolus administration, in mice, as shown in fig. 8. And based on the mode of local administration, the delivery and effect of A665-NCA in the animal's internal environment was tested.

Meanwhile, the embodiment also adopts a matrigel bearing mode for administration, and matrigel release conjugate detection and use safety detection are also carried out before matrigel bearing is adopted. Specifically, the matrigel used was corning matrigel (no phenol red, 356237) without explanation in the above and below examples.

And (3) release detection: dissolving A665-NAC in saline, mixing with matrigel to give final concentration of A665-NAC of 5mM, and applying when matrigel forms gel; HEI-OC1 cells were placed in a transwell lower chamber, after overnight adherence, matrigel mixed with Cy5.5-A665-NAC was placed in an upper chamber, after mixing for 1h, 2h and 6h, the upper chamber was removed, washed with PBS, cells were digested with pancreatin, and flow cytometry was performed. As a result, as shown in FIG. 9, a large amount of Cy5.5-A665-NAC in matrigel was taken up by HEI-OC1 cells at 1 h.

And (3) safety detection:

in mice, there was no significant difference in the pre-operative detection of binaural ABR threshold following the follicular drug administration procedure. The left ear is used for promoting the auditory bulb to be administrated, matrix glue is placed, the right ear is used as a control group, and no operation is performed. ABR results were retested 1 day and 7 days after surgery, respectively, and it was found that the post-surgery 1 day binaural ABR thresholds were significantly different at each frequency, considering that matrigel blocked the middle ear and ossicular chain activity was blocked. After 7 days of matrigel application, retest ABR found no significant difference in ABR threshold for both ears, and the results are shown in fig. 10. The middle ear placement matrigel is proved to have long-term hearing loss of animals, and the matrigel is proved to have use safety.

Delivery and efficacy detection of a665-NCA in an in vivo animal environment: cy5.5-A665-NAC or Cy5.5-RP-NAC was administered to the left ear auditory bulb using matrigel, right ear prosthetic surgery. As a result, as shown in fig. 11, since cy5.5 appears blue-green under white light, it is seen that the blue-green color appears near the portion of the inner ear's fundus back near the round window niche, as shown in fig. 11A, suggesting that cy5.5-a665-NAC, cy5.5-RP-NAC enter the inner ear; fixing cochlea, decalcification, slicing, and finding that Cy5.5 is most concentrated near Corti's device in the area where hair cells of cochlea are located under confocal microscope, and less concentrated in spiral ganglion area. In the Corti organ region, cy5.5-a665-NAC aggregates more than cy5.5-RP-NAC, whereas in the spiral ganglion region cy5.5-RP-NAC aggregates more than cy5.5-a665-NAC, suggesting that a665 can target peripheral hair cells, aggregating in the Corti organ region where CDDP lesions were earliest, as shown in fig. 11B.

Fig. 8: the A665-NAC is carried by matrigel and placed in a round window niche of the middle ear cavity; the a665-NAC permeates through the round window membrane into the periauricular lymph fluid to reach the target auditory cells, hair cells.

Fig. 9: the matrigel can be slowly released in vitro; (A) Flow cytometry suggests that matrigel may release a665-NAC at 1 h; and (B) carrying out statistical analysis on the streaming result. N=3.

Fig. 10: matrix glue is placed in the middle ear to prevent the hearing of animals from being damaged for a long time. Matrigel placement in the middle ear retest hearing cues no damage to ABR thresholds of mice 4k,8k,16k, and 32k after 7 days, n=3.

Fig. 11: matrigel bearing a665-NAC can pass through the round window membrane into the inner ear; (A) A post-operation cochlea picture, which is the left ear and the right ear of the same mouse, and the circle is the position of a round window niche; in contrast to the sham-operated group, the Cy5.5-A665-NAC group was seen with a blue-green shade (indicated by the arrow) in the inner ear; (B) Cochlea slice map, OHC is three rows of outer hair cells, IHC is one row of inner hair cells, this is the organ region of Corti. SG is the spiral ganglion region and N is the spiral ganglion cell. Cy5.5 is reddish fluorescence; the scale is 10 μm; n=3.

Example 6

Detection of A665-NAC Effect in cisplatin Hearing impairment animal models

Pre-molding ABR thresholds were tested prior to cisplatin molding, and ABR abnormal mice were knocked out, randomly divided into a blank group, cisplatin group, RP-NAC group, a665-NAC group. Both RP-NAC and A665-NAC groups were administered left-hand on auditory vesicles. All mice were treated with the sham procedure group, with the procedure without matrigel.

Cisplatin molding was performed for 45 days, and cisplatin was injected intraperitoneally, and administered in 3 cycles, with cisplatin groups having significantly higher ABR thresholds than the blank groups of 4k,8k,16k, and 32k, and the difference was statistically significant, as shown in fig. 12B, suggesting that cisplatin molding performed. Specifically, cisplatin used in this example is cisplatin (seleck, S1166).

The mouse right ear ABR thresholds for RP-NAC group and a665-NAC group were not significantly different from the cisplatin group mouse right ear ABR thresholds, as shown in fig. 12C; comparing the difference in ABR threshold values between the left and right ears of mice in the RP-NAC group and the A665-NAC group, and performing a statistical comparison, it was found that in mice in which the systemic cisplatin administration was modelled, the left ear had better protection against A665-NAC than the left ear against RP-NAC, and the differences were statistically significant at 4k,8k,16k and 32k, as shown in FIG. 12C.

Further, this example also takes a cochlear basement membrane patch and stains with the hair cell marker Myosin VIIa. Cisplatin groups were found to have significant outer hair cell loss in the top, middle and bottom loops, with bottom loop loss being most pronounced. The A665-NAC group was better able to protect hair cell survival than the RP-NAC group, with statistical differences, and the results are shown in FIG. 13.

Fig. 12: the A665-NAC better reduced cisplatin-induced hearing impairment. (a) schematic administration of left and right ear surgery; (B) Blank and cisplatin groups (left ear) have different ABR thresholds before and after cisplatin molding at each frequency; n=6; (C) Cisplatin group, RP-NAC group, and A665-NAC group (right ear) were inferior in ABR threshold before and after cisplatin molding at each frequency. N=6; (D) The right ear ABR threshold-left ear ABR threshold is the hearing threshold difference, and the difference between the thresholds of the RP-NAC group and the a665-NAC group is compared, i.e. the lower the left ear ABR threshold, the greater the difference. N=6, p <0.05, p <0.01.

Fig. 13: in cisplatin ototoxic hearing impaired mice, A665-NAC more effectively protected cochlear hair cells, reducing cisplatin-induced death. The scale is 20. Mu.m. N=3. * For the comparison of CDDP+RP-NAC and CDDP+A665-NAC, p <0.05, p <0.01.# is a comparison of two groups CDDP and CDDP+A665-NAC, with #p <0.05. Comparison of CDDP and CDDP+RP-NAC, & p <0.05.

The above experimental data indicate that a665 can assist NAC in entering hair cells, can target outer hair cells more effectively, both in vitro and in vivo, and can reduce cell death caused by cisplatin, thereby reducing hearing loss.

It should be understood that the foregoing examples of the present application are merely illustrative of the present application and are not intended to limit the present application to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present application should be included in the protection scope of the claims of the present application.

Claims

1. The conjugate is characterized by structurally comprising an inner ear targeting polypeptide and a main drug, wherein the inner ear targeting polypeptide is connected with the main drug through a chemical bond, the inner ear targeting polypeptide is suitable for targeting the main drug to the inner ear, the main drug is a drug for treating inner ear diseases caused by cisplatin ototoxicity, the inner ear targeting polypeptide is A665, and the amino acid sequence of A665 is: leu-Ser-Thr-His-Thr-Thr-Glu-Ser-Arg-Ser-Met-Val, wherein the main drug is NAC, and the conjugate has a structure shown in the following formula:

2. use of a conjugate according to claim 1 for the preparation of a medicament for the treatment of inner ear diseases caused by cisplatin ototoxicity.

3. The use according to claim 2, wherein the inner ear disease is a hearing loss disease caused by damage to outer hair cells.

4. A composition comprising a conjugate of claim 1.

5. A composition according to claim 4, further comprising additional agents compatible with the conjugate and pharmaceutically acceptable carriers and/or excipients.

6. The composition of claim 5, wherein the pharmaceutically acceptable adjuvant is matrigel.