CN114848836A - Conjugate and application thereof in treating inner ear diseases - Google Patents
Conjugate and application thereof in treating inner ear diseases Download PDFInfo
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- CN114848836A CN114848836A CN202110761572.1A CN202110761572A CN114848836A CN 114848836 A CN114848836 A CN 114848836A CN 202110761572 A CN202110761572 A CN 202110761572A CN 114848836 A CN114848836 A CN 114848836A
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- A61F11/00—Methods or devices for treatment of the ears or hearing sense; Non-electric hearing aids; Methods or devices for enabling ear patients to achieve auditory perception through physiological senses other than hearing sense; Protective devices for the ears, carried on the body or in the hand
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Abstract
The invention discloses a conjugate and application thereof to 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 inner ear by the inner ear targeting polypeptide, and the enrichment of the main drug is realized so as to accurately act on the targeted inner ear structures or cells; the efficiency of drug delivery to the inner ear can be improved by targeting the polypeptide to the inner ear, so that the content of the drug reaching the inner ear is remarkably improved, and the bioavailability of the drug in the inner ear is improved. The invention also provides the application of the conjugate in inner ear diseases, and the diagnosis, prevention or treatment of the inner ear diseases are conveniently realized through the main medicine in the conjugate. The invention more specifically contemplates a conjugate delivery, pharmaceutical process for targeting inner ear outer hair cells by local delivery of conjugate a 665-NAC. When the conjugate is applied to the inner ear with the effect of cisplatin, the conjugate can reduce the hearing loss caused by the cisplatin, and has the effects of preventing and treating the cisplatin ototoxicity.
Description
Technical Field
The invention 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 include vertigo, deafness and the like, which are mostly caused by the damage of structures such as inner ear hair cells, auditory nerves and the like, for example, the loss of various sensorineural hearing loss is mainly caused by the damage of hair cells in cochlea, especially the damage of outer hair cells. The inner ear diseases not only bring direct physical health problems to patients, but also directly affect the normal lives of the patients and bring inconvenience to the normal lives of the patients, so that the treatment of the inner ear diseases is always a key research object in the field of current otology diseases, and patients with the inner ear diseases also urgently need effective inner ear disease treatment medicines or schemes.
At present, conservative treatment is generally carried out on the diseases of the inner ear by adopting medicaments, but the distribution of the medicaments to the inner ear is limited to a great extent by the special anatomical physiological characteristics of the vascular barrier in the inner ear and the deep part of the bone deeply buried in the skull base, and the treatment effect is limited. In particular, the blood-labyrinthine barrier in the inner ear, which limits the transport of various substances from the blood to the inner ear, only very small amounts of drug reach the inner ear far short of the effective drug concentration required for the treatment of inner ear diseases when administered systemically. For example, Yang et al injected dexamethasone 0.5mg/ml/kg into guinea pigs at the heart, and after 1 hour the drug concentration in the perilymph fluid was only 0.03mg/ml, whereas the drug concentration in the plasma was 2.76 mg/ml. When systemic administration is adopted, in order to ensure that the medicine has effective treatment concentration when reaching the inner ear, the concentration of the systemic circulation medicine is always required to be higher, and toxic and side effects on other organs and tissues of the body are easily caused. Therefore, the prior art proposes a treatment measure for local drug delivery in the inner ear, the local drug delivery has the characteristics of short distance, avoidance of first-pass metabolism, avoidance of blood-labyrinth barrier and obviously higher inner ear drug concentration than the whole body, and the current common local administration mode is a round window membrane administration route, namely tympanogram injection. 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 can help deliver sufficient drug to the inner ear and reduce the amount of drug exposed to other tissues. That is, the currently effective treatment for inner ear diseases is mainly a treatment mode of local administration.
However, even though the method of local administration can solve the problem of side effect and effective concentration of the medicine of systemic administration, the problem of targeting of the medicine still cannot be solved, and in the prior art, the medicine can enter the inner ear through the round window membrane, but the medicine is diffused in the cochlea along with the perilymph fluid, and cannot be targeted to act on specific structures and cells of the inner ear for protection 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 chance of acting on the corresponding structure or cell, and the action of the drug at high concentration still cannot be avoided; and the high-concentration medicine can also stimulate the mucosa of the middle ear, the effect of the medicine is reduced, and the whole treatment effect is influenced.
Therefore, there is a need for a targeted drug or drug delivery system for treating inner ear diseases to solve the above problems.
Disclosure of Invention
The invention combines the inner ear targeting polypeptide and the main drug for treating inner ear diseases to form a conjugate, thereby assisting the main drug to target specific structures and cells in the inner ear and the inner ear by means of the inner ear targeting polypeptide, realizing the enrichment of the main drug and accurately acting on the targeted inner ear structures or cells; the efficiency of drug delivery to the inner ear can be improved by targeting the polypeptide to the inner ear, so that the content of the drug reaching the inner ear is remarkably improved, and the bioavailability of the drug in the inner ear is improved. On the premise of improving the effectiveness of the drug in the inner ear, the drug delivery device is beneficial to reducing the total drug amount while achieving the same treatment effect as the prior art. Thereby avoiding the application of excessive amounts of the drug to achieve effective concentrations of the drug in the inner ear and avoiding possible side effects of the excessive amounts of the drug. The main drug which is part of the conjugate can be effectively transported to the inner ear, so that the total amount of the drug actually applied by using the conjugate method is less than that in the prior art on the premise of achieving the same drug amount or concentration of the inner ear, and the stimulation to structures such as middle ear mucosa can be further reduced, thereby improving the drug effect and improving the whole prevention or treatment effect. In addition, when the drug is a drug for diagnosing inner ear diseases, a conjugate formed by the drug and the inner ear targeting polypeptide can also be used for diagnosing the inner ear diseases. The invention more specifically relates to a conjugate A665-NAC formed by local delivery of polypeptide A665, NAC combination, to realize conjugate transportation and medicinal process of targeting inner ear and outer hair cell of inner ear. The targeting of the conjugate can be realized through the matching of the A665 and prestin protein expressed by outer hair cells, so that the efficiency of transporting the conjugate to regions of inner ears including Corti apparatus and the like is improved, the outer hair cells can be protected through NAC, when the conjugate is applied to the inner ears with the action of cisplatin, the effect of NAC antioxidant is effectively exerted, the cis-platinum ototoxicity caused by active oxygen increase is reduced, the apoptosis of the outer hair cells caused by cisplatin is reduced, the hearing loss caused by cisplatin is reduced, and the effects of preventing and treating the cis-platinum ototoxicity can be realized.
The invention provides a conjugate, which structurally comprises 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 targeted polypeptide is matched with protein expressed by cells in the inner ear to realize targeting, and the main drug acts on the inner ear structure or the inner ear cells to achieve the effect of preventing or treating corresponding inner ear diseases. Furthermore, when the main drug is a drug for transporting to the inner ear so as to obtain the diagnosis result of the inner ear disease, 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 can be efficiently transported to the inner ear and the target point, and the high-accuracy diagnosis result of the inner ear disease can be obtained. Further, the chemical bond includes a disulfide bond. The conjugate has targeting property, so that the required dosage is less, the targeting property is stronger, the conjugate can be directly enriched near a target site, and the conjugate is beneficial to 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 protein expressed by outer hair cells in the inner ear to realize targeting, is beneficial to carrying and transporting a main drug coupled with the inner ear targeting polypeptide A665, and acts on the inner ear, the outer hair cell concentration area or the outer hair cells in the inner ear to realize the corresponding main drug medicinal effect.
Further, the main drug is NAC, and the conjugate has a structure shown in the following formula:
NAC is a common antioxidant currently used clinically for expectorant treatment. NAC mitigates cisplatin-induced ototoxicity, and in one or more embodiments of the invention, protection of inner ear outer hair cells, and enhanced antioxidant capacity, can be achieved by the conjugate NAC component. Is helpful for preventing and treating inner ear diseases caused by damage and oxidation of outer hair cells. And the material for preparing the conjugate is relatively simple, the preparation cost of the polypeptide is low, and the conjugate is beneficial to batch production and clinical practical application.
The invention also provides the use of the above conjugate in the diagnosis, prevention or treatment of inner ear diseases. When the main drug component of the conjugate is a drug for diagnosing, preventing or treating inner ear diseases, the conjugate can realize effective delivery and drug action by combining the targeted inner ear polypeptide, thereby realizing the application of the conjugate in diagnosing, preventing or treating the inner ear diseases. Further, the inner ear disease is sensorineural hearing loss such as sensorineural deafness. When the inner ear targeting polypeptide is a polypeptide targeting inner ear outer hair cells, the coupled main drug can be transported to the area where the outer hair cells are located in a targeting manner, so that the main drug can more effectively act on the outer hair cells. In all sensorineural hearing loss diseases, a plurality of sensorineural hearing losses are caused by the damage of outer hair cells, and when the inner ear targeting polypeptide is the polypeptide targeting the outer hair cells and the main drug acts on the outer hair cells to protect or cure the outer hair cells, the application in preventing or treating the sensorineural hearing loss diseases can be realized. When the sensorineural hearing loss disease is not caused by outer hair cells, the effect of guiding the main drug and targeting can be achieved by setting the inner ear targeting polypeptide to target the corresponding pathological structure and the polypeptide of the cells. Therefore, the conjugate can be applied to the prevention or treatment of sensorineural hearing loss diseases. And when the main medicine is used 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 damage of the outer hair cells is a direct cause of various inner ear diseases, and the outer hair cells have a targeting protein such as prestin, which is more beneficial to the targeting and the action of the conjugate, so that the hearing loss diseases caused by the damage of the outer hair cells can be diagnosed, prevented or treated. Furthermore, the disease is sensorineural hearing loss caused by the damage of outer hair cells. Since the direct cause of various sensorineural hearing loss diseases is the damage of the outer hair cells, the outer hair cells are the earliest and the most serious damage region, and the outer hair cells cannot be regenerated, the targeted polypeptide of the inner ear in the conjugate is set as the polypeptide targeting the outer hair cells, so that the targeted transportation of the main drug is facilitated and the main drug acts on the outer hair cells, and the corresponding diagnosis, prevention or treatment effect of the main drug on the damage of the outer hair cells is realized.
Further, the sensorineural hearing loss disease is a hearing loss disease caused by cisplatin ototoxicity. Cisplatin is the most common chemotherapy drug clinically, and because cisplatin can enter the inner ear to generate cytotoxicity, clinical multi-course chemotherapy can cause deafness. Cisplatin causes cytotoxicity through many mechanisms, and it is considered that cisplatin causes active oxygen increase due to binding to DNA, DNA destruction, and cisplatin. In more than one embodiment of the invention, the main drug is selected to be NAC, the inner ear targeting polypeptide is set to be A665 targeting outer hair cells, and the formed conjugate A665-NAC can be applied to auditory cells to relieve the ototoxicity caused by cisplatin, reduce active oxygen increased by the cisplatin, provide antioxidant capacity, resist apoptosis induced by the cisplatin and enhance the capacity of resisting the cytotoxicity of the cisplatin. Therefore, the conjugate can be applied to the hearing loss diseases caused by cisplatin ototoxicity, so as to realize diagnosis, prevention or treatment by combining with the main drug. Further, there is provided the use of a conjugate for protecting outer hair cells; further, the outer hair cells under the action of cisplatin were protected.
The invention also provides a composition comprising a conjugate as described above. The composition can be applied at least for the treatment of inner ear diseases.
Further, other medicines compatible with the conjugate and pharmaceutically acceptable carriers and/or auxiliary materials are also included. The conjugate is matched with other medicines, so that the synergistic treatment effect is realized. And the delivery of the conjugate is realized through the carrier, which is beneficial to improving the delivery efficiency and the action effect of the conjugate, for example, the carrier or the auxiliary material which can increase the residence time of the conjugate at the administration position in the prior art is adopted.
Further, the pharmaceutically acceptable auxiliary material is matrigel. The matrigel is injectable gel, is injected into the tympanic cavity of the middle ear through tympanocentesis after carrying the medicine, stays in the middle ear and forms a storage effect, and can effectively control the medicine to be continuously released into the inner ear. And the matrigel has no additional covalent cross-linking agent and toxic substances, has good biocompatibility and high safety. The matrigel is taken as an auxiliary material to assist the delivery of the conjugate, which is beneficial to prolonging the retention time of the conjugate at the administration part, such as retention in the middle ear, so as to realize the sustained release of the conjugate, facilitate the entry of the conjugate into the inner ear, increase the drug concentration in the inner ear and improve the bioavailability of the conjugate. In one embodiment of the invention, the conjugate is carried by matrigel, allowing the conjugate to be retained in the middle ear and enter the inner ear, and further, in one embodiment of the invention, the conjugate A665-NAC is carried by matrigel, allowing the A665-NAC to be targeted to enrich the region of the inner ear Corti organ where the outer hair cells are concentrated, so as to achieve controlled release and targeted transport of the conjugate.
The present invention also provides a topical drug delivery system comprising a delivery device and a composition as described above, the delivery device being configured to release the composition upon insertion of the delivery device into the middle and/or inner ear to treat sensorineural hearing loss disorders. Still further, insertion of the delivery device into the middle and/or inner ear allows the composition to be retained for a sufficient period of time to allow the inner ear drug concentration to reach a therapeutic concentration. The local drug delivery is realized through the delivery device, so that the drug delivery effect is further improved on the basis that the composition has targeting property, the conjugate is concentrated on the local part 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 amount, thereby avoiding the toxic and side effects possibly caused by excessive drug amount and improving the action effect of the conjugate. Further, the delivery device is inserted into the middle ear and administered by the round window membrane route, and the composition is delivered through the round window membrane into the inner ear.
Compared with the prior art, the invention 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 action, improve the delivery efficiency of the main medicine part in the conjugate and improve the content of the main medicine delivered to the inner ear, thereby reducing the total medicine amount required for reaching the effective medicine amount and avoiding the toxic and side effects caused by excessive medicine. Meanwhile, the reduction of the applied medicine amount is also beneficial to reducing the stimulation to other parts, such as middle ear mucosa, thereby improving the medicine effect. Due to the targeting property, the drug delivery efficiency to the inner ear can be improved, and the main drug can act on the target site and the target cell, so that the targeted therapy is realized, the drug can accurately act on the target site, and the drug effect of the main drug is improved. And when the conjugate is locally applied, the toxic and side effects on other organs and tissues possibly caused by systemic administration can be avoided, and the adverse effects possibly caused by the drug can be further reduced. The conjugate can be retained at the administration position for a sufficient time by matrix gel administration, so that the sustained release and the action of the conjugate are facilitated, and the overall drug action effect is further improved. The invention also provides an A665-NAC conjugate, which can improve the delivery efficiency to the inner ear, and the A665-NAC conjugate is enriched in the concentrated region of the outer hair cells by utilizing the protein expressed by the A665 and the targeted outer hair cells; thus allowing the primary NAC carried to act on the outer hair cells to protect them, such as: under the environment of cisplatin action, the antioxidant capacity of inner ear hair cells is enhanced, and the toxic and side effects of cisplatin cells are resisted; reducing cell damage and apoptosis caused by cisplatin, and relieving hearing loss caused by cisplatin ototoxicity.
Drawings
FIG. 1 shows the structural formula of the conjugate A665-NAC and RP-HPLC chromatography and mass spectrum results;
FIG. 2 shows the results of the measurement of the ability of A665-NAC to enter the auditory cell HEI-OC 1;
FIG. 3 is a graph of A665-NAC enhancing the cytotoxic potency of HEI-OC1 cells against cisplatin;
FIG. 4 is a665-NAC enhancing antioxidant capacity of HEI-OC1 cells;
FIG. 5 is a665-NAC enhancing cell viability in primary tissues under cisplatin action;
FIG. 6 is a665-NAC reduces apoptosis of cells in primary tissues under the action of cisplatin;
FIG. 7 is a665-NAC reducing reactive oxygen species generated by cells in primary tissues under the action of cisplatin;
FIG. 8 is a schematic illustration of matrigel administration and A665-NAC penetration in the middle ear cavity round window niche;
FIG. 9 shows the results of a matrigel controlled release A665-NAC assay;
FIG. 10 shows the result of safety test of matrigel-loaded application;
FIG. 11 is a graph showing the results of a matrigel-borne A665-NAC delivery test into the inner ear;
FIG. 12 is a graph of the results of reducing cisplatin-induced hearing loss in an A665-NAC application animal model;
FIG. 13 is a graph of the reduction of cisplatin-induced hair cell damage in an animal model of A665-NAC application.
Detailed Description
The drawings are only for purposes of illustration and are not to be construed as limiting the invention. For the purpose of better illustrating the following embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
Example 1
Synthetic preparation of A665-NAC
In this example, A665-NAC was prepared by conventional solid phase polypeptide synthesis techniques, targeting polypeptide A665 was conjugated to NAC by covalent binding, and the A665-NAC conjugate was prepared as 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 the preparation process: the target product is obtained by adopting a solid-phase organic synthesis method, utilizing Fmoc-protected amino acid strategy and SPPS solid-phase synthesis technology to complete main chain assembly, cracking, and purifying after oxidation reaction.
The preparation process specifically comprises the following steps:
(1) solid-phase synthesis: the linear peptide resin is formed by sequentially condensing amino acid chains from the C end to the N end (from right to left) by Fmoc-protected amino acid strategy by using Fmoc-Cys (Trt) -CTC resin according to the peptide sequence and 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-06 Fmoc-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-13 Fmoc-Leu-OH. The peptide resin is washed, transferred out, dried to constant weight and ready for cleavage.
(2) Cleavage of peptide resin:
preparing a cracking reagent: the amount of lysis reagent was calculated as 1g peptide resin to 10 ml. + -.2 ml: putting a required cracking reagent H2O, TFA, EDT and TIS into a cracking reaction bottle in sequence, wherein TFA is H2O, EDT is Tis is 95:1:2:2, and the temperature of the cracking reagent is controlled to be 0-10 ℃; adding a cracking reagent into the peptide resin under stirring until the temperature of the system is stable; then the temperature is controlled at 25-30 ℃ and the stirring reaction is carried out for 2.5 hours.
Filtering out the lysate, precipitating with 5 times of the amount of the concentrated solution of ethyl acetate, filtering out the precipitate, and drying at room temperature under reduced pressure to obtain a crude product.
(3) And (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, after the crude product is completely added and dissolved, adding 1 time of Ac-Cys, after the crude product is completely dissolved, dropwise adding 10 times of iodine methanol solution, and stirring for reacting for 2 hours.
(4) And (3) purification and freeze-drying:
the purification is carried out by adopting Shimadzu semi-preparation and using a 5cm, 10um and C-18 column packing, carrying out separation and purification by using a proper gradient at normal temperature, collecting a target product, analyzing, detecting and classifying to obtain the A665-NAC. And 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 present example also performs detection 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 is high in purity and monodispersity, i.e., is synthesized as a single substance. Wherein 1B and 1C are respectively an RP-HPLC chromatogram and a mass spectrogram.
Example 2
Detection of the ability of A665-NAC to enter auditory cells 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 investigate 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 the HEI-OC1 cell plates were attached overnight, 5mM Cy5.5-A665-NAC/Cy5.5-RP-NAC were co-cultured on ice for 5 minutes, then subjected to a change of medium and washed 3 times with PBS, and subjected to flow cytometry after trypsinization.
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 addition, a group of experimental groups is also provided in the embodiment, after auditory cells are treated by 5mM A665 for 5 minutes in advance, A665-NAC and the auditory cells are added for co-culture for 5 minutes on ice, and the group is the group A665 Block A665-NAC in figure 2; this group is intended to verify the targeting of a665-NAC by first using a665 targeted binding target, reducing subsequent a665-NAC binding targets, and achieving a blocking effect.
In FIG. 2, (A) shows the results of the experimental group of comparison RP-NAC, A665 Block A665-NAC using flow cytometry. (B) Fluorescence intensity analysis showing flow results. (C) Showing the entry into the HEI-OC1 cells as observed by confocal fluorescence microscopy, Cy5.5 was orange, the cytoskeleton was green, the nucleus was blue, and the scale was 5 μm, N-3, p < 0.001.
As can be seen from fig. 2A, 2B, 2C, a665-NAC was able to enter auditory cells HEI-OC1 more rapidly than RP-NAC, a665 helped NAC enter auditory cells HEI-OC1 rapidly and in large quantities, showing targeting. And the A665 Block A665-NAC group had a reduced cell entry capacity compared to A665-NAC, indicating that the A665-NAC entry capacity was reduced if pre-treatment with A6655 mM for 5 minutes, followed by co-incubation with A665-NAC on ice for 5 minutes.
After the adherent cells are continuously observed by using a confocal microscope for 30 minutes of co-culture, the A665-NAC enters the cells more than the RP-NAC; whereas the A665 Block A665-NAC group, when first blocked the target site with A665 for 5 minutes, the A665-NAC's ability to enter HEI-OC1 decreased. The above results indicate that a665 can enhance NAC entry into HEI-OC1 auditory cells, and is targeted.
Example 3
A665-NAC enhances HEI-OC1 antioxidant capacity and is resistant to cisplatin cytotoxicity.
First, cisplatin-induced cell damage detection
Cytotoxicity experiments were performed with Cisplatin in this example, and HEI-OC1 cells were treated with Cisplatin (Cisplatin, CDDP) at 30 μ M for 24 hours with 50% cell damage, as shown in FIG. 3A. Whereas treatment with RP-NAC or A665-NAC at 2.5mM in combination with CDDP resulted in less cell damage compared to treatment with CDDP alone, and A665-NAC was less damaging than the group of RP-NAC, indicating that A665-NAC is capable of resisting the cytotoxic effects of CDDP and is more potent than RP-NAC.
And the inventors compared the cell survival at different concentrations and found that A665-NAC was more protective at 2.5mM than RP-NAC at 10mM, as shown in FIG. 3B.
The inventor also obtains the early apoptosis and apoptosis of HEI-OC1 cells caused by cisplatin from each experimental group through flow detection, as shown in figures 3C and 3D, CDDP can induce apoptosis and die, RP-NAC and A665-NAC carrying NAC can reduce the apoptosis caused by cisplatin, and because A665 has targeting property and can be combined better, the A665-NAC group can obviously reduce the apoptosis caused by cisplatin, and the proportion of early apoptotic cells is obviously reduced compared with other groups. That is, the above results indicate that A665-NAC is able to rapidly enter HEI-OC1 cells and enhance their ability to defend against CDDP cytotoxicity.
FIG. 3: (A) cell activity assay results; (B) processing results of A665-NAC and RP-NAC with different concentrations; (C) detecting early apoptosis results caused by cisplatin in a flow mode; (D) and (3) flow-detecting the apoptosis condition. N-3, p <0.05, p < 0.001.
Second, active oxygen detection
In this example, DCFDA probe was also used to detect Reactive Oxygen Species (ROS), and it was found that the ROS content in the cells was significantly increased during CDDP treatment, whereas the A665-NAC + CDDP treatment group was significantly decreased and was more greatly decreased than the RP-NAC group, as shown in the first row of FIG. 4. Mitochondria are ROS-producing organelles, and this example also measures mitochondria-produced ROS content using mitoSOX, and a665-NAC was found to be able to reduce CDDP-elevated ROS levels, as shown in the second row of fig. 4. The results show that the A665-NAC can enhance the antioxidant capacity of cells and resist the influence of cisplatin on the level of active oxygen.
FIG. 4, first line: DCFDA reactive oxygen species probe appeared green in the presence of ROS, with a scale of 10 μm. Second row of fig. 4: the mitobox probe appeared orange in the presence of ROS, with a scale of 20 μm.
Example 4
A665-NAC enhances the antioxidant capacity of primary tissues of cochlear hair cells and resists cisplatin cytotoxicity.
In order to further verify that A665-NAC can also play a role in enhancing antioxidation and resisting cisplatin cytotoxicity in complex cochlear tissues on a cellular basis, the detection is carried out on the basis of cochlear hair cell primary tissues in the embodiment. The inventors extracted cochlear basement membrane tissue from 3-day-old newborn mice for primary culture and treated the primary basement membrane tissue with CDDP after overnight adherence, and found that significant lesions were present in the apical, middle and basal gyrus regions. In the A665-NAC + CDDP and RP-NAC + CDDP groups, the number of the surviving cells is remarkably larger than that of the CDDP group alone, and based on the cell survival condition of the control group, the cisplatin treatment can cause the cell loss or apoptosis of primary tissues, and when the A665-NAC can remarkably reduce the hair cell loss and death caused by the cisplatin, the effect is more remarkable than that of the RP-NAC group. A665-NAC has a more pronounced hair cell protection effect than RP-NAC, with the differences being statistically significant (FIG. 5); hair cells were labeled with hair cell marker myostatin VIIa (green fluorescence) and clear-caspase 3 (red fluorescence) was used to represent cells that were about to enter apoptosis. The results suggest that A665-NAC reduced CDDP-induced apoptotic cells (FIG. 6).
When mitobox was used to measure mitochondrial produced ROS levels, a665-NAC was found to be able to reduce CDDP elevated ROS levels (fig. 7). The above results indicate that a665-NAC is equally effective in primary tissues, enhances the antioxidant capacity of cochlear hair cell primary tissues, and is resistant to cisplatin cytotoxicity.
FIG. 5: (A) loss of hair cells and death from cisplatin in different groups. Green is a hair cell marker, Myosin VIIa, which can specifically mark hair cells, and the scale is 20 μm. (B) Statistical analysis of top, middle and bottom in graph a, n is 3. P < 0.001.
FIG. 6: caspase3 expression in different groups of cisplatin-induced hair cell activation, green as hair cell marker Myosin VIIa, specifically labeled hair cells, with 20 μm scale.
FIG. 7: the number of active oxygen released by mitochondria in different groups is green, which is a hair cell marker Myosin VIIa, and the hair cells can be specifically marked with a scale of 20 mu m.
Example 5
A665-NCA delivery and effect detection in animal body environment
Tympanogram is a mode of administration in the middle ear, in which a drug is allowed to pass through the round window membrane into the inner ear, and reach the lymph fluid of the inner ear, thereby contacting target cells. In clinical treatment, in order to increase the amount of drug reaching the inner ear, the drug is often administered by tympanogram, which is a local administration; in this example, administration was performed in a manner simulating clinical drum injection in mice, i.e., in an acoustic bleb, as shown in fig. 8. And the delivery and the effect test of the A665-NCA in the environment of the animal body are carried out based on the mode of local administration.
Meanwhile, the embodiment also adopts a matrix glue bearing mode for drug administration, and matrix glue release conjugate detection and use safety detection are also carried out before matrix glue bearing is adopted. Specifically, the matrigel used is corning (corning) matrigel (no phenol red, 356237), on the premise that the above and following examples are not described.
And (3) release detection: dissolving A665-NAC in saline, mixing with matrigel to make A665-NAC concentration 5mM, and using when matrigel is formed; HEI-OC1 cells were placed in the lower transwell chamber and adhered overnight, matrigel mixed with Cy5.5-A665-NAC was placed in the upper chamber and after mixing for 1h, 2h and 6h, the upper chamber was removed, washed with PBS, trypsinized, and tested by flow cytometry. As a result, as shown in FIG. 9, it was found that a large amount of Cy5.5-A665-NAC was taken up by HEI-OC1 cells in the matrigel at 1 hour.
Safety detection:
the auditory blister drug administration operation is performed in the mouse, and the binaural ABR threshold is detected before the operation without obvious difference. The left ear was administered by tympanocystia, matrigel was placed in the left ear, and the right ear was used as a control group without surgery. The ABR results are respectively retested in 1 day and 7 days after operation, and the obvious difference of the ABR threshold values of ears in 1 day after operation in each frequency is found, and the ABR results are considered to be caused by the blockage of the middle ear by matrix glue and the obstruction of the activity of the ossicular chain. After 7 days of matrigel application, repeated measurements of ABR revealed no significant difference in ABR thresholds in both ears, with results as shown in figure 10. The matrigel placed in the middle ear can not damage the hearing of animals for a long time, and the matrigel has use safety.
The delivery and effect test of A665-NCA in the environment of animals are as follows: Cy5.5-A665-NAC or Cy5.5-RP-NAC was administered to the left ear in the form of an auricular bullae using matrigel, and sham treatment of the right ear. As a result, as shown in FIG. 11, since Cy5.5 exhibits blue-green color under white light, it can be seen that blue-green color appears near the bottom of the inner ear back to the portion near the round window niche, as shown in FIG. 11A, indicating that Cy5.5-A665-NAC, Cy5.5-RP-NAC entered the inner ear; fixing the cochlea row, decalcifying, slicing, and finding that Cy5.5 is most gathered near the organ of Corti in the area where the hair cells of the cochlea are located and is less gathered in the spiral ganglion area under a confocal microscope. In the region of the organ of Corti, Cy5.5-A665-NAC aggregated more than Cy5.5-RP-NAC, while in the region of the spiral ganglion Cy5.5-RP-NAC aggregated more than Cy5.5-A665-NAC, suggesting that A665 could target the outer hair cells, aggregating in the region of the organ of Corti where CDDP injury first occurred, as shown in FIG. 11B.
FIG. 8: A665-NAC is carried by matrigel and is arranged in the round window niche of the middle ear cavity; A665-NAC permeates through the round window membrane into perilymph fluid to the target auditory cell, hair cell.
FIG. 9: the matrigel can be slowly released in vitro; (A) flow cytometry suggested that matrigel released a665-NAC at 1 h; (B) and carrying out statistical analysis on the flow type result. N is 3.
FIG. 10: placing matrigel in the middle ear for a long period of time does not impair animal hearing. Matrigel placement in middle ear hearing was repeated 7 days later suggesting no damage to ABR thresholds of mice 4k, 8k, 16k and 32k, N3.
FIG. 11: matrigel-bearing a665-NAC can enter the inner ear through a round window membrane; (A) the picture of the cochlea after the operation is the left ear and the right ear of the same mouse, and the circle is the position of the round window niche; in contrast to the sham group, blue-green shadows (indicated by arrows) were visible in the inner ear in Cy5.5-A665-NAC group; (B) cochlear section map, OHC three rows of outer hair cells and IHC one row of inner hair cells, this is the region of the organ of Corti. SG is the spiral ganglion region and N is the spiral ganglion cell. Cy5.5 is red fluorescence; the scale is 10 μm; n is 3.
Example 6
A665-NAC effect detection in cisplatin hearing impairment animal model
Testing ABR threshold value before cisplatin modeling, rejecting ABR abnormal mice, and randomly dividing into blank group, cis-platinum group, RP-NAC group and A665-NAC group. Both RP-NAC and A665-NAC groups were administered by left-handed audiovesicular administration. All mice were subjected to the sham operation group on the right ear without any matrigel.
Cisplatin modeling is 45 days, cisplatin is injected into the abdominal cavity, the cisplatin is administrated in 3 cycles, the ABR threshold values of the cisplatin group are obviously higher than those of the blank group at 4k, 8k, 16k and 32k, the result is shown in figure 12B, the difference has statistical significance, and the success of cisplatin modeling is prompted. Specifically, cisplatin used in this example is cisplatin (seleck, S1166).
The ABR thresholds for the right ear of mice in the RP-NAC group and A665-NAC group were not significantly different from those of mice in the cisplatin group, as shown in FIG. 12C; comparing the ABR threshold differences between the left and right ears of mice in the RP-NAC and A665-NAC groups, and making a statistical comparison, it was found that in mice modeled after systemic cisplatin administration, the administration of A665-NAC to the left ear gave better protection than RP-NAC to the left ear, with significant statistical differences at 4k, 8k, 16k, and 32k, as shown in FIG. 12C.
Further, cochlear basement membrane sections were also taken and stained with the hair cell marker Myosin VIIa. The cisplatin group was found to have significant outer hair cell loss in the apical, middle, and basal gyrus, with loss of basal gyrus being most significant. The A665-NAC group protected hair cell survival better than the RP-NAC group with statistical differences, and the results are shown in FIG. 13.
FIG. 12: A665-NAC reduced the cisplatin-induced hearing impairment better. (A) Schematic administration diagram of left and right ear operation; (B) ABR threshold difference before and after cisplatin molding of each frequency between blank group and cis-platinum group (left ear); n is 6; (C) the cisplatin, RP-NAC and A665-NAC groups (right ear) had differences in ABR thresholds before and after cisplatin modelling at each frequency. N is 6; (D) right ear ABR threshold-left ear ABR threshold is the difference in hearing threshold, and the RP-NAC group and the a665-NAC group compare the threshold differences, i.e., the lower the left ear ABR threshold, the greater the difference. N6, p <0.05, p < 0.01.
FIG. 13: in cisplatin ototoxic hearing impaired mice, A665-NAC protected cochlear hair cells more effectively, reducing cisplatin-induced death. The scale bar is 20 μm. N is 3. And the two groups are CDDP + RP-NAC and CDDP + A665-NAC, p is less than 0.05, and p is less than 0.01. # is two comparison sets of CDDP and CDDP + A665-NAC, # p < 0.05. & is a comparison of CDDP and CDDP + RP-NAC, & p < 0.05.
The above experimental data show that a665 can assist NAC in entering hair cells, can target outer hair cells more effectively in vitro and in vivo, and reduce cisplatin-induced cell death, thereby reducing hearing loss.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
1. The conjugate is characterized by 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, and the main drug is a drug for diagnosing, preventing or treating inner ear diseases.
2. A conjugate according to claim 1, wherein 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.
3. A conjugate according to claim 2, wherein the primary agent is NAC and the conjugate has the formula:
4. use of a conjugate according to any one of claims 1 to 3 for the diagnosis, prevention or treatment of inner ear diseases.
5. The use according to claim 4, wherein the inner ear disease is a hearing loss disease caused by damage to outer hair cells.
6. The use according to claim 4, wherein the inner ear disease is a hearing loss disease caused by cisplatin ototoxicity.
7. A composition comprising a conjugate according to any one of claims 1 to 3.
8. The composition of claim 7, further comprising other drugs compatible with the conjugate and pharmaceutically acceptable carriers and/or excipients.
9. The composition of claim 7, wherein the pharmaceutically acceptable excipient is matrigel.
10. A topical drug delivery system comprising a delivery device and a composition according to any one of claims 7 to 9, the delivery device being configured to release the composition to treat an inner ear disease when the delivery device is inserted into the middle and/or inner ear.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001080832A2 (en) * | 2000-04-26 | 2001-11-01 | Oregon Health Sciences University | Administration of a thiol-based chemoprotectant compound |
| CA2825792A1 (en) * | 2002-01-04 | 2003-07-17 | Sound Pharmaceuticals Incorporated | Otoprotectant compositions of glutathione peroxidase mimics for ameliorating hearing loss |
| US20030191064A1 (en) * | 2001-01-23 | 2003-10-09 | Kopke Richard D. | Methods for preventing and treating loss of balance function due to oxidative stress |
| WO2010117997A2 (en) * | 2009-04-07 | 2010-10-14 | University Of Kansas | Peptide targeting of inner ear cells |
| WO2014197909A1 (en) * | 2013-06-07 | 2014-12-11 | The Administrators Of The Tulane Educational Fund | Analogs of pituitary adenylate cyclase-activating polypeptide (pacap) and methods for their use |
| WO2016137279A1 (en) * | 2015-02-27 | 2016-09-01 | 경북대학교 산학협력단 | Peptide for targeting autophagic cells and use thereof |
-
2021
- 2021-07-06 CN CN202110761572.1A patent/CN114848836B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001080832A2 (en) * | 2000-04-26 | 2001-11-01 | Oregon Health Sciences University | Administration of a thiol-based chemoprotectant compound |
| US20030191064A1 (en) * | 2001-01-23 | 2003-10-09 | Kopke Richard D. | Methods for preventing and treating loss of balance function due to oxidative stress |
| CA2825792A1 (en) * | 2002-01-04 | 2003-07-17 | Sound Pharmaceuticals Incorporated | Otoprotectant compositions of glutathione peroxidase mimics for ameliorating hearing loss |
| WO2010117997A2 (en) * | 2009-04-07 | 2010-10-14 | University Of Kansas | Peptide targeting of inner ear cells |
| WO2014197909A1 (en) * | 2013-06-07 | 2014-12-11 | The Administrators Of The Tulane Educational Fund | Analogs of pituitary adenylate cyclase-activating polypeptide (pacap) and methods for their use |
| WO2016137279A1 (en) * | 2015-02-27 | 2016-09-01 | 경북대학교 산학협력단 | Peptide for targeting autophagic cells and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| KAYYALI M N, BRAKE L, RAMSEY A J, ET AL.: "A novel nano-approach for targeted inner ear imaging", JOURNAL OF NANOMEDICINE & NANOTECHNOLOGY, vol. 8, pages 1 - 7 * |
| MEHMET AKIF SOMDA 等: "Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy", BRAZILIAN JOURNAL OF OTORHINOLARYNGOLOGY, vol. 86, pages 1 - 4 * |
| SUROVTSEVA E V, JOHNSTON A H, ET AL.: "Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 424, pages 3 * |
| 杨卫平;胡博华;陈光地;ERIC C BIELEFELD;DONALD HENDERSON;: "N乙酰基-L-半胱氨酸对苯乙烯致耳蜗毛细胞损伤的防护作用", 临床耳鼻咽喉头颈外科杂志, no. 04 * |
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