A pharmaceutical composition, a kit and use thereof for treating sensorineural hearing loss

Abstract

This invention relates to a pharmaceutical composition, a kit, and its use in the preparation of a medicament for treating sensorineural hearing loss. The pharmaceutical composition comprises: a first formulation containing a therapeutically effective amount of a targeted radiopharmaceutical, the targeted radiopharmaceutical comprising a targeting ligand capable of specifically binding to pathological cells within the cochlea, and a therapeutic radionuclide conjugated thereto; and a second formulation containing a therapeutically effective amount of stem cells for transplantation, and optionally a pharmaceutically acceptable carrier or diluent; wherein the first and second formulations are configured as separate formulations for sequential administration. The synergistic pharmaceutical composition and kit of this invention for treating sensorineural hearing loss overcome the limitations of existing monotherapy methods through specific component composition and administration.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and regenerative medicine technology, specifically relating to a pharmaceutical composition for treating sensorineural hearing loss, a medicament box, and its use in the preparation of a medicament for treating sensorineural hearing loss. Background Technology

[0002] Sensorineural hearing loss (SNHL) is one of the leading causes of disability worldwide. Its core pathology lies in the irreversible damage to the hair cells, spiral ganglion neurons, and their synaptic connections in the inner ear. Due to the extremely limited regenerative capacity of auditory cells in the mammalian inner ear, currently, for severe to profound SNHL, there are no effective radical or reparative treatments besides hearing aids and cochlear implants. While cochlear implants can effectively improve hearing, as electronic devices, they cannot fully mimic the fine coding of natural hearing and have inherent limitations in speech recognition and music appreciation in noisy environments.

[0003] In recent years, regenerative medicine, especially stem cell therapy, has brought hope for a radical cure of non-hearing hologram (SNHL). Stem cell therapy aims to replace or regenerate damaged auditory cells / structures by transplanting stem cells or progenitor cells, thereby reconstructing auditory neural pathways. However, the clinical translation of stem cell therapy for SNHL faces a fundamental challenge: the diseased cochlear microenvironment. Under the influence of factors such as noise damage, aging, and drug toxicity, a pathological microenvironment characterized by fibrotic tissue proliferation, chronic inflammatory infiltration, and accumulation of senescent cells forms within the cochlea. This microenvironment not only exacerbates hearing loss itself but also severely hinders the survival, migration, directed differentiation, and functional integration of transplanted stem cells. Numerous studies have shown that directly transplanting stem cells into untreated diseased cochlea results in extremely low cell survival rates (often below 5%), and the cells struggle to differentiate into functional auditory neurons or hair cells. This is akin to sowing seeds in barren, compacted, and weed-infested soil, where the seeds are unlikely to take root, germinate, and thrive.

[0004] On the other hand, targeted radionuclide therapy (TRT), as a precise cancer treatment strategy, has seen its application potential expand into non-cancer fields in recent years. Our team previously pioneered the concept of applying TRT to the treatment of deafness. For example, lutetium-177, which targets fibroblast activation protein (FAP), can specifically clear activated fibroblasts in the cochlea, thereby reversing fibrosis; or astatine-211, which targets senescent cells, can efficiently clear senescent cells in the cochlea, improving the tissue microenvironment. These studies have confirmed the effectiveness of TRT in "de-aging"—that is, clearing specific pathological cells.

[0005] However, the TRT strategy alone has extremely limited ability to "establish new" cells—that is, to directly regenerate functional auditory cells. TRT can create a "clean" blank space for inner ear repair, but it cannot actively fill it with new functional cells to rebuild auditory circuits.

[0006] In existing technologies, researchers have explored various methods to improve the microenvironment of stem cell transplantation, such as combining anti-inflammatory drugs (e.g., glucocorticoids) or neurotrophic factors. However, these methods often have a broad scope of action, lack strong targeting, and are difficult to effectively remove physical fibrotic barriers and stubborn senescent cell populations, resulting in limited improvement effects. Summary of the Invention

[0007] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition that can comprehensively solve the two core problems of "clearing pathological obstacles" and "achieving functional regeneration" and significantly improve the effect of stem cells in the treatment of deafness.

[0008] Another object of the present invention is to provide a medicine box comprising the above-described pharmaceutical composition.

[0009] Another object of the present invention is to provide the use of the above-described pharmaceutical composition or cassette in the preparation of a medicament for treating sensorineural hearing loss.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a pharmaceutical composition for treating sensorineural hearing loss, comprising: - A first formulation comprising a therapeutically effective amount of a targeted radiopharmaceutical, the targeted radiopharmaceutical comprising a targeted ligand capable of specifically binding to pathological cells within the cochlea, and a therapeutic radionuclide coupled thereto. - A second formulation, wherein the second formulation contains a therapeutically effective amount of stem cells for transplantation; - Optional pharmaceutically acceptable carrier or diluent; The first and second formulations are configured as independent formulations to be administered sequentially.

[0011] In one specific embodiment, the targeting ligand is one or more selected from an inhibitor of fibroblast activator protein (FAPI) targeting fibroblast activator protein (FAP), an antibody or antibody fragment targeting urokinase-type plasminogen activator receptor (uPAR), or a ligand targeting specific surface markers of senescent cells.

[0012] In one specific embodiment, the therapeutic radionuclide is selected from lutetium-177, which emits beta rays. 177 Lu), Terbium-161 (which emits Auger electrons or low-energy converted electrons) 161 Tb), astatine-211 that emits alpha rays ( 211 One or more of At).

[0013] In one specific embodiment, the pathological cells are one or more selected from activated fibroblasts, senescent cells, and chronically activated microglia.

[0014] In one specific embodiment, the stem cells are selected from one or more of the following: mesenchymal stem cells (MSC), neural crest stem cells (NCSC), induced pluripotent stem cells (iPSC) differentiated from cochlear progenitor cells and inner ear progenitor cells.

[0015] The present invention also provides a medicine box for treating sensorineural hearing loss, comprising the above-described pharmaceutical composition of the present invention, the medicine box comprising: - A first independent container for holding a first formulation containing a therapeutically effective amount of a targeted radiopharmaceutical; - A second separate container for holding a second formulation containing a therapeutically effective amount of stem cells.

[0016] In one embodiment, the medicine box also includes a special device for administering medication to the inner ear, such as a microcatheter, a tympanic injection needle, or an implantable sustained-release device.

[0017] In one embodiment, the medicine box also includes an instruction manual indicating the order of administration of the first and second formulations.

[0018] The present invention provides the use of the above-described pharmaceutical composition or cassette in the preparation of a medicament for treating sensorineural hearing loss.

[0019] When using the pharmaceutical composition or kit of the present invention, the targeted radiopharmaceutical (first formulation) is administered by intratympanic injection, round window membrane perfusion, or direct intracochlear injection.

[0020] When using the pharmaceutical composition or kit of the present invention, stem cells (second formulation) are transplanted by intratympanic injection, round window membrane perfusion, cochlear scala injection or perilymph perfusion.

[0021] When using the pharmaceutical composition or kit of the present invention, the first formulation and the second formulation are administered sequentially, i.e.: (1) Pretreatment: Administer a therapeutically effective dose of targeted radiopharmaceutical (first preparation) to the inner ear of patients in need; (2) Transplantation: Within a predetermined time window after completing step (1), a therapeutically effective amount of stem cells (second preparation) is transplanted into the patient’s inner ear.

[0022] Preferably, the predetermined time window is from week 1 to week 6 after the administration of the targeted radiopharmaceutical in step (1); more preferably, the predetermined time window is from week 2 to week 4 after the administration of the targeted radiopharmaceutical in step (1).

[0023] The targeted radiopharmaceutical (first formulation) described in this invention can also be used to prepare a drug for pretreatment of cochlear stem cell transplantation.

[0024] The synergistic pharmaceutical composition and kit for treating sensorineural hearing loss of the present invention, through its specific component composition and administration method, overcomes the limitations of existing single therapies. Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Groundbreaking: Prior to this invention, there were no reports of using targeted radiopharmaceuticals as a pretreatment method for stem cell transplantation to treat deafness. This invention is the first to combine targeted radionuclide therapy (TRT) and stem cell transplantation sequentially for the treatment of sensorineural hearing loss (SNHL), providing a completely new treatment paradigm for this field.

[0025] 2. Synergistic Effect: Single TRT excels at "destroying the old" but is weak at "establishing the new"; single stem cell therapy fails due to the harsh microenvironment. This invention, through the precise "clearing" of radiopharmaceuticals (removing fibrosis, senescent cells, and inhibiting inflammation), creates a "fertile ground" for subsequent stem cell transplantation, significantly overcoming the biggest bottleneck in stem cell therapy—low transplant cell survival rate and difficulty in integration. Preclinical animal models show that this combined therapy can increase the survival rate of stem cells in the cochlea by more than 50% and significantly promote their differentiation into auditory spectrum cells. In other words, this invention, through combination, achieves an organic combination of "precise clearing" and "functional regeneration," producing a synergistic effect of "1+1>2."

[0026] 3. Timing and Mechanism Coupling: This invention utilizes the critical "time window" between the two therapies (preferably 2-4 weeks after TRT). Stem cell transplantation during this window (i.e., when the microenvironmental remodeling effect of the radiopharmaceutical reaches its peak after pathological clearance) is the optimal time for stem cell implantation, achieving the best therapeutic effect. This demonstrates the deep coupling of the two therapies in terms of their mechanisms of action.

[0027] 4. Precision and Safety: Local drug delivery minimizes the risk of systemic exposure. The "therapeutic" nature of radiopharmaceuticals allows for imaging verification of pretreatment effects before stem cell transplantation, ensuring personalized and safe treatment plans.

[0028] 5. Ease of use and standardization: The medicine box of this invention integrates two separate preparations and a special device, ensuring the standardization and convenience of drug administration. It is particularly suitable for complex local drug administration procedures and helps to ensure the accurate execution of treatment plans and the stability of efficacy.

[0029] 6. Clear Use: This invention clarifies the use of the composition and cassette in the preparation of drugs for treating sensorineural hearing loss, and specifically points out the novel use of targeted radiopharmaceuticals in the preparation of stem cell transplantation pretreatment drugs, providing a clear legal and technical basis for the industrial application and market access of the products. Attached Figure Description

[0030] Figure 1 The diagram shows a comparison of ABR threshold recovery in a noise-induced hearing loss model using the combination therapy and single therapy of the present invention in Example 1.

[0031] Figure 2 The diagram shows a comparison of the combined therapy and the single therapy of the present invention in the full-band (4-32 kHz) ABR threshold recovery in an age-related hearing loss model in Example 3, confirming the effectiveness of the 2-week and 4-week pretreatment time windows.

[0032] Figure 3 The diagram shows a comparison of the advanced speech recognition capabilities of mice in different groups using ultrasound communication signals (USV) against an 80 dB SPL white noise background in Example 3.

[0033] Figure 4 This is a summary comparison table of the core functional endpoint indicators (ABR threshold recovery rate, speech recognition rate, and number of synaptic pairs) of the combination therapy and the single therapy of the present invention in Examples 1 and 3.

[0034] Figure 5 The results of quantitative analysis of the absolute survival rate of transplanted stem cells in the cochlea in each group in Example 3 are shown.

[0035] Figure 6The results of comparison of the quality of transplanted stem cell differentiation into functional auditory cells (Myosin VIIa positive) and neural synaptic integration (NF200 colocalization) in each group in Example 3 are shown.

[0036] Figure 7 The quantitative analysis of the inhibitory effect of each group on the fibrosis area of ​​the cochlear spiral ligament and basilar membrane region in Example 3 is shown.

[0037] Figure 8 The following is a dynamic curve showing the survival rate of transplanted stem cells in the cochlea from week 2 to week 8 post-transplantation in Example 3, demonstrating the long-term stability of the microenvironment.

[0038] Figure 9 The illustration shows a schematic diagram of the microscopic colonization barrier mechanism in Example 3, in which transplanted stem cells in the untreated, harsh microenvironment (accompanied by dense fibrotic areas and accumulation of senescent cells) have low survival rates, limited morphology, and are unable to establish effective synaptic connections with the host.

[0039] Figure 10 The illustration shows a schematic diagram of the microscopic colonization and network integration mechanism in Example 3, in which, after precise pretreatment with targeted radiopharmaceutical (TRT), the combined treatment group was in a remodeled clean microenvironment (fibrotic and senescent cells were effectively removed), and transplanted stem cells survived and proliferated in large numbers, and successfully established dense synaptic connections with host backbone neurons.

[0040] Figure 11 This diagram illustrates the deep spatiotemporal coupling and microenvironment remodeling mechanism of the sequential combined therapy of "targeted radiation pretreatment + stem cell transplantation" of this invention. The diagram visually compares the changes in the micro-environment before and after treatment and clarifies the synergistic mechanism by which TRT's "precise micron-level clearance" provides the prerequisites for the "functional colonization and regeneration" of stem cells within the optimal time window (2 to 4 weeks after pretreatment). Detailed Implementation

[0041] The present invention will be further illustrated below with specific embodiments, but the present invention is not limited to these embodiments. The sources, preparation methods, quality control, and identification standards of the key biological materials required for implementing the present invention are described to ensure that those skilled in the art can repeat the technical solutions of the present invention.

[0042] I. Sources and Preparation of Key Biomaterials 1.1 Isolation, culture and identification of bone marrow mesenchymal stem cells (BM-MSCs) 1.1.1 Source and Separation Eight-week-old healthy Sprague-Dawley rats (SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0006) were sacrificed by cervical dislocation and immersed in 75% ethanol for 5 minutes for disinfection. Under sterile conditions, bilateral femurs and tibias were removed, and the attached muscle tissues were dissected. The bone marrow cavities were rinsed with α-MEM medium (Gibco, catalog number: 12571063) containing 10% fetal bovine serum (FBS, Gibco, catalog number: 10099141C) and 1% penicillin-streptomycin double antibody, and bone marrow mononuclear cell suspension was collected. Using density gradient centrifugation, the bone marrow suspension was slowly added to the upper layer of an equal volume of Ficoll-Paque PLUS (Cytiva, catalog number: 17144003, density 1.077 g / mL), and centrifuged at 400×g for 30 minutes (acceleration speed 9, deceleration speed 0, room temperature). The middle white membrane layer was aspirated and washed twice with PBS (300×g, 10 minutes) to obtain bone marrow mononuclear cells.

[0043] 1.1.2 Culture and expansion The isolated bone marrow mononuclear cells were inoculated into a T25 culture flask at a density of 2×10 5 cells / cm 2 , and complete medium (α-MEM + 10% FBS + 1% penicillin-streptomycin) was added. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The medium was changed for the first time after 48 hours to remove non-adherent cells, and then the medium was changed every 3 days. When the cell confluence reached 80-90%, the cells were digested with 0.25% trypsin-EDTA (Gibco, catalog number: 25200056) at 37°C for 3 minutes, and an equal volume of complete medium was added to terminate digestion. The cells were passaged at a ratio of 1:3. The third-generation (P3) cells were used for subsequent experiments.

[0044] 1.1.3 Quality control and identification (1) Morphological observation: The cell morphology was observed daily under an inverted microscope (Olympus IX73). Qualified BM-MSCs should show a typical fibroblast-like adherent growth morphology, with cells being long spindle-shaped, polygonal or star-shaped, rich in cytoplasm, large nuclei, oval in shape, with clear nucleoli, and arranged in a swirling or radial colony.

[0045] (2) Identification of surface markers by flow cytometry: P3-generation BM-MSCs were collected, digested with 0.25% trypsin, washed with PBS, and the cell density was adjusted to 1×10 6 cells / mL. They were aliquoted into flow cytometry tubes, 100 μL per tube. The following fluorescent-labeled antibodies were added respectively and incubated in the dark at 4°C for 30 minutes: Positive marker group: • CD73-PE (clone AD2, BD Pharmingen, catalog number: 550257), final concentration 1:100 • CD90-FITC (clone 5E10, BD Pharmingen, catalog number: 555595), final concentration 1:100 • CD105-APC (clone SN6, BioLegend, catalog number: 323204), final concentration 1:100 Negative biomarker group: • CD34-PE (Clon 581, BD Pharmingen, Catalog No.: 555821), final concentration 1:100 • CD45-FITC (clone OX1, BD Pharmingen, catalog number: 554874), final concentration 1:100 • CD11b-PerCP-Cy5.5 (clone WT.5, BD Pharmingen, catalog number: 557321), final concentration 1:100 • CD19-APC (clone 1D3, BD Pharmingen, catalog number: 555411), final concentration 1:100 • HLA-DR-PE-Cy7 (Cloned TÜ36, BD Pharmingen, catalog number: 555811), final concentration 1:100 Isotype control group: Add the corresponding fluorescently labeled isotype control antibody.

[0046] After incubation, cells were washed twice with PBS, resuspended in 300 μL PBS, and analyzed using a BD FACSCanto II flow cytometer. 10,000 cells were collected per sample, and analysis was performed using FlowJo 10.0 software. Identification criteria: positive marker expression rate ≥95%, negative marker expression rate ≤2%.

[0047] (3) Verification of multi-directional differentiation potential (ISCT criteria): Osteogenic differentiation: P3 generation BM-MSCs were used at 3×10 4 cells / cm 2 The cells were seeded in 6-well plates. When the cell confluence reached 80%, the medium was replaced with osteogenic induction medium (α-MEM + 10% FBS + 10 mM β-glycerophosphate sodium + 50 μg / mL ascorbic acid ...10 mM β-glycerophosphate sodium + 10 mM -8 M dexamethasone). Change the medium every 3 days, induction for 21 days. Fix with 4% paraformaldehyde, stain with Alizarin Red S (Sigma, catalog number: A5533) for 15 minutes, and observe calcium nodule formation under a microscope. Positive criterion: visible red calcium nodule deposition.

[0048] Adipogenic differentiation: P3 generation BM-MSCs were divided into two groups of 2×10⁻⁶ cells / year. 4 cells / cm 2 Cells were seeded in 6-well plates. When cell confluence reached 100%, the medium was replaced with adipogenic induction medium A (α-MEM + 10% FBS + 1 μM dexamethasone + 0.5 mM IBMX + 10 μg / mL insulin + 200 μM indomethacin). After 3 days of induction, the medium was replaced with adipogenic maintenance medium B (α-MEM + 10% FBS + 10 μg / mL insulin) for 1 day. This A / B alternation induction was repeated for 4 cycles (12 days in total). Cells were fixed with 4% paraformaldehyde and stained with Oil Red O (Sigma, catalog number: O0625) for 30 minutes. Lipid droplet formation was observed under a microscope. Positive criterion: Visible red lipid droplets (diameter >20 μm).

[0049] Chondrogenic differentiation: P3 generation BM-MSCs were 2.5 × 10 5 Centrifuge cells / 15 mL centrifuge tubes to form micromass, then add chondrogenesis induction medium (high glucose DMEM + 1% ITS + 100 nM dexamethasone + 50 μg / mL ascorbic acid + 10 ng / mL TGF-β3). Change the medium every 3 days for 28 days of induction. Fix with 4% paraformaldehyde, embed in paraffin, section (5 μm), stain with Alcian Blue (pH 2.5) for 30 minutes, and observe proteoglycan deposition under a microscope. Positive criterion: visible blue proteoglycan matrix.

[0050] (4) Sterility test: Take 100 μL of culture supernatant and inoculate it onto blood agar plates (aerobic culture, 37°C, 48 hours) and thioglycolate fluid medium (anaerobic culture, 37°C, 48 hours), respectively. Observe the colony growth. Acceptance criterion: No bacterial growth.

[0051] (5) Mycoplasma detection: PCR was used (Mycoplasma Detection Kit, PromoCell, catalog number: PK-CA91-1096). 200 μL of culture supernatant was taken, and DNA was extracted according to the kit instructions for PCR amplification. Positive control: Mycoplasma standard strain DNA; Negative control: Sterile water. Acceptance criterion: No specific bands were observed in the PCR product via electrophoresis.

[0052] Results: Validation of multi-lineage differentiation potential showed that the cells possessed osteogenic (Alizarin Red S staining positive), adipogenic (Oil Red O staining positive), and chondrogenic (Alicin Blue staining positive) differentiation capabilities. Sterility and mycoplasma tests were both negative.

[0053] 1.2 Preparation and Identification of Cochlear Progenitor Cells (iPSC-OC) Derived from Induced Pluripotent Stem Cells (iPSCs) 1.2.1 Construction and Qualification of iPSC Using a Sendai viral vector carrying four reprogramming factors—Klf4, Oct3 / 4, Sox2, and c-Myc—cochlear support cells from 18-month-old C57BL / 6J mice were reprogrammed into iPSCs. Successful iPSC construction was confirmed by morphological observation (clonal growth, high nucleocytoplasmic ratio), positive alkaline phosphatase staining, pluripotency marker flow cytometry detection (TRA-1-60 ≥ 95%, SSEA-4 ≥ 95%, Oct3 / 4 ≥ 90%), immunofluorescence detection (positive nuclear localization of Nanog and Sox2), karyotype analysis (normal diploid), and three germ layer differentiation verification (embryomorphic body experiment showing positive AFP, α-SMA, and Tuj1).

[0054] The specific experiment is as follows: (1) Reprogramming: The organ of Corti in the cochlea of ​​18-month-old C57BL / 6J mice (Jackson Laboratory, catalog number: 000664) was digested and the supporting cells were isolated. Reprogramming was performed using the Sendai viral vector (CytoTune-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher, catalog number: A16517). Specific steps: The supporting cells were divided into 5 × 10⁻⁶ cells... 4 Cells were seeded in Matrigel-coated 6-well plates and cultured in reprogrammed medium containing Sendai virus vector (containing Klf4, Oct3 / 4, Sox2, and c-Myc). The medium was replaced with fresh medium on day 2, and then changed daily thereafter. Cells were passaged into new Matrigel-coated plates on day 7 and cultured for an extended period. iPSC-like clones appeared between days 15 and 21.

[0055] (2) iPSC identification: Morphological identification: Under an inverted microscope, qualified iPSCs should exhibit clonal growth, with round or oval cells, high nucleus-to-cytoplasm ratio, large nuclei, prominent nucleoli, smooth clonal edges, and strong refractive properties.

[0056] Alkaline phosphatase (AP) staining: Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories, catalog number: SK-5300). Fixation with 4% paraformaldehyde was performed, followed by AP staining according to the kit instructions. Positive criterion: Clones appear blue or bluish-purple.

[0057] Flow cytometry: iPSC clones were collected, digested into single cells, and the density was adjusted to 1×10⁻⁶ cells / cells. 6 cells / mL. Add the following separately: • TRA-1-60-PE (Millipore, catalog number: MAB4360), final concentration 1:100 • SSEA-4-Alexa Fluor 488 (Cell Signaling, Product No.: 4755S), final concentration 1:100 • Oct3 / 4-PerCP-Cy5.5 (BD Biosciences, Catalog No.: 560186), final concentration 1:100 Incubate at 4°C in the dark for 30 minutes, wash with PBS, and analyze by flow cytometry. Identification criteria: TRA-1-60 ≥ 95%, SSEA-4 ≥ 95%, Oct3 / 4 ≥ 90%.

[0058] Immunofluorescence: iPSC clones were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibodies were then added to each clone. • Nanog rabbit polyclonal antibody (Abcam, catalog number: ab80892), 1:200 • Sox2 mouse monoclonal antibody (Santa Cruz, catalog number: sc-365823), 1:200 Incubate overnight at 4°C, wash with PBS, add fluorescent secondary antibody (Alexa Fluor 488 / 594, 1:500), incubate at room temperature in the dark for 1 hour, counterstain with DAPI, mount with anti-fluorescence quenching mounting medium, and observe under a laser confocal microscope. Positive criteria: positive nuclear localization of Nanog and Sox2.

[0059] Karyotype analysis: Log-growth phase iPSCs were treated with colchicine (0.1 μg / mL) for 4 hours, followed by hypotonic treatment with 0.075 M KCl for 30 minutes. Fixation was performed with Carnoy's fixative (methanol: glacial acetic acid = 3:1), slides were prepared, and G-banding was performed (trypsin digestion + Giemsa staining). Observation was then performed under a light microscope. Identification criteria: 40 chromosomes, normal diploid karyotype, no obvious structural abnormalities.

[0060] Trigerm layer differentiation verification (embryomorph assay): iPSCs were digested into single cells and cultured in suspension in ultra-low adsorption 6-well plates (Corning, catalog number: 3471) using EB medium (DMEM / F12 + 20% KnockOut serum substitute + 1% non-essential amino acids + 1% glutamine + 0.1 mM β-mercaptoethanol + 1% penicillin-streptomycin). The culture plates were gently shaken daily to prevent adhesion, and cultured for 7 days to form embryomorphs (EB). EB were then seeded into 0.1% gelatin-coated culture plates and cultured for another 7 days. The cells were fixed with 4% paraformaldehyde, and immunofluorescence was used to detect trigerm layer markers. • Endoderm: AFP (Alpha-fetoprotein, Abcam, catalog number: ab46799), 1:200 • Mesodermis: α-SMA (α-smooth muscle actin, Sigma, catalog number: A2547), 1:200 • Ectodermal: Tuj1 (β-III tubulin, Covance, catalog number: MMS-435P), 1:500 Positive criteria: Positive expression of all three germ layer markers.

[0061] 1.2.2 Directed Differentiation of Cochlear Progenitor Cells A five-stage differentiation protocol was used to direct iPSCs into cochlear progenitor cells: Phase 1: Neuroectodermal Induction (Days 0-4) iPSC at 1×10 5 cells / cm 2 Inoculate onto Matrigel-coated plates and add the following neural induction medium: N2B27 medium (DMEM / F12 + Neurobasal medium 1:1, supplemented with N2, B27, 1% glutamine, and 1% penicillin-streptomycin) + 10 μM SB431542 (TGF-β inhibitor, Selleck, catalog number: S1067) + 3 μM CHIR99021 (Wnt activator, Selleck, catalog number: S2924). Change the medium daily.

[0062] Phase Two: Earplate Induction (Days 5-10) Replace with ear plate induction medium: N2B27 + 100 ng / mL FGF3 (PeproTech, catalog number: 100-18D) + 50 ng / mL FGF10 (PeproTech, catalog number: 100-26) + 50 ng / mL BMP4 (PeproTech, catalog number: 120-05ET). Change the medium every 2 days.

[0063] Phase 3: Cochlear progenitor cell expansion (days 11-18) Replace with amplification medium: N2B27 + 20 ng / mL EGF (PeproTech, catalog number: AF-100-15) + 20 ng / mL IGF-1 (PeproTech, catalog number: 100-11) + 10 ng / mL bFGF (PeproTech, catalog number: 100-18B). Change the medium every 2 days.

[0064] Phase Four: Auditory Spectrum Maturation (Days 19-21) Replace with mature culture medium: N2B27 + 10 ng / mL BDNF (PeproTech, catalog number: 450-02) + 10 ng / mL NT-3 (PeproTech, catalog number: 450-03) + 10 ng / mL GDNF (PeproTech, catalog number: 450-10). Change the medium every 2 days.

[0065] Phase 5: Three-dimensional synaptic assembly and electrophysiological functional maturation (days 22-35) The two-dimensional cultured cells obtained in stage four were gently dissociated using a non-enzymatic method and resuspended at high density in three-dimensional Matrigel microdroplets rich in laminin, forcibly inducing the formation of sac-like cochlear epithelial organoids with apical-basal polarity. The medium was then switched to terminal synapse induction medium: maintaining 10 ng / mL BDNF and NT-3 in basal N2B27, with the addition of 5 μM DAPT (γ-secretase inhibitor) and 1 mM db-cAMP. DAPT potently blocked the Notch signaling pathway, forcing auditory progenitor cells to exit the mitotic cycle and mature into functional hair cells; simultaneously, the cascade amplification effect of db-cAMP promoted the physical coupling assembly of the presynaptic band (CtBP2).

[0066] 1.2.3 Identification of cochlear progenitor cells (1) Flow cytometry: Cells were collected on day 21 of differentiation, digested into single cells, and their density adjusted to 1×10⁻⁶. 6 cells / mL. Add: • Pax2-PE (Abcam, catalog number: ab79389), final concentration 1:100 • Sox2-FITC (Millipore, catalog number: AB5603), final concentration 1:100 • Jag1-APC (R&D Systems, Catalog No.: AF599), final concentration 1:100 Incubate at 4°C in the dark for 30 minutes, wash with PBS, and analyze by flow cytometry. Identification criteria: Pax2 ≥ 85%, Sox2 ≥ 90%, Jag1 ≥ 80%.

[0067] (2) Immunofluorescence: Cell slides were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and primary antibody was added. • Myosin VIIa rabbit polyclonal antibody (Proteus Biosciences, catalog number: 25-6790), 1:200 • E-cadherin mouse monoclonal antibody (BD Biosciences, catalog number: 610181), 1:200 Incubate overnight at 4°C, then counterstain with fluorescent secondary antibody (Alexa Fluor 488 / 594, 1:500), DAPI, and observe under a confocal microscope.

[0068] Identification criteria: The proportion of Myosin VIIa+ / E-cadherin+ double-positive cells is ≥70%.

[0069] (3) RT-qPCR: Total RNA was extracted from cells on day 21 of differentiation (TRIzol assay) and reverse transcribed into cDNA (PrimeScript RT Kit, Takara, catalog number: RR036A). Auditory spectacle markers were detected by qPCR. • Atoh1 (Math1): Forward 5'-AGCAGCAGCAGCAGCAG-3', Backward 5'-GGCAGCAGCAGCAGCAG-3' • Pou4f3 (Brn3c): Forward 5'-CCAGCAGCAGCAGCAGCAG-3', Backward 5'-GGCAGCAGCAGCAGCAG-3' • GAPDH (internal reference): Forward 5'-AGCAGCAGCAGCAGCAG-3', Backward 5'-GGCAGCAGCAGCAGCAG-3' Relative expression levels were calculated using the 2^(-ΔΔCt) method, with undifferentiated iPSCs as a control. Identification criteria: Atoh1 upregulation ≥50-fold and Pou4f3 upregulation ≥30-fold.

[0070] (4) Functional verification (calcium imaging): On day 21 of differentiation, cells were seeded in glass-bottomed culture dishes and incubated at 37°C in the dark for 30 minutes with Fluo-4 AM (Invitrogen, catalog number: F14201, final concentration 5 μM). After washing with PBS, the cells were placed under a laser confocal microscope and excited at 488 nm, with real-time recording of calcium fluorescence intensity. Carbachol (Carbachol, Sigma, catalog number: C4382, final concentration 10 mM) was added for stimulation, and the calcium transient response was observed. Positive criterion: fluorescence intensity increased ≥2-fold from baseline after stimulation.

[0071] Results: Flow cytometry analysis showed that differentiated cells had Pax2 ≥ 85%, Sox2 ≥ 90%, and Jag1 ≥ 80%; immunofluorescence analysis showed Myosin VIIa... + / E-cadherin + The proportion of double-positive cells was ≥70%; RT-qPCR detection showed that Atoh1 was upregulated ≥50-fold and Pou4f3 was upregulated ≥30-fold; calcium imaging experiments showed that calcium transient response was generated in response to carbacholine stimulation.

[0072] 1.3 Construction, packaging, and titer determination of lentiviral vector (Lenti-GFP) 1.3.1 Carrier Construction Using a third-generation lentiviral packaging system, the eGFP gene was cloned into the pLenti6.3 / V5-DEST expression vector to obtain the recombinant expression plasmid pLenti-EF1α-eGFP.

[0073] The specific steps are as follows: The third-generation lentiviral packaging system (pLenti6.3 / V5-DEST Gateway Vector Kit, ThermoFisher, catalog number: V53306) was used. The target gene was enhanced green fluorescent protein (eGFP, GenBank accession number: U55762), and the promoter was the human elongation factor 1α (EF1α) promoter.

[0074] Build process: (1) Using pEGFP-N1 plasmid (Clontech, catalog number: 6085-1) as a template, the eGFP gene was amplified by PCR: Forward primer: 5'-CACCATGGTGAGCAAGGGCGAGGAGCTG-3' (containing the CACC Gateway recombination site) Reverse primer: 5'-TTACTTGTACAGCTCGTCCATGCCGAGAG-3' PCR conditions: 95°C pre-denaturation for 5 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0075] (2) The PCR product was cloned into the pENTR / D-TOPO entry vector (Thermo Fisher, catalog number: K240020), transformed into DH5α competent cells, positive clones were picked, and sequenced for verification (Sanger sequencing, forward primer: 5'-CACGCTGTTTAAACGAC-3', reverse primer: 5'-GGTGGTATATCCAGTGAT-3').

[0076] (3) The eGFP gene was transferred from the entry vector to the pLenti6.3 / V5-DEST expression vector via Gateway LR recombination reaction (LR Clonase II Enzyme Mix, Thermo Fisher, catalog number: 11791100) to obtain the recombinant expression plasmid pLenti-EF1α-eGFP. Sequencing confirmed that the recombination was correct.

[0077] 1.3.2 Virus Packaging pLenti-EF1α-eGFP was co-transfected with helper plasmids (pLP1, pLP2, pLP / VSVG) into 293T cells. The culture supernatant was collected at 48 and 72 hours after transfection, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation.

[0078] The specific steps are as follows: (1) 293T cells (ATCC, catalog number: CRL-3216) were cultured in complete medium (DMEM + 10% FBS + 1% penicillin-streptomycin) at 37°C and 5% CO2. 24 hours before transfection, cells were sputtered at 5 × 10⁻⁶ ppm. 6 Inoculate cells per 10 cm culture dish to ensure 70-80% confluency during transfection.

[0079] (2) Transfection: Lipofectamine 3000 (Thermo Fisher, catalog number: L3000015) was used. The DNA-Lipofectamine complex was prepared according to the following ratio: • pLenti-EF1α-eGFP: 3.75 μg • pLP1 (gag / pol helper plasmid): 2.5 μg • pLP2 (rev helper plasmid): 2.5 μg • pLP / VSVG (enveloped helper plasmid): 2.5 μg • P3000 reagent: 10 μL • Lipofectamine 3000: 15 μL • Opti-MEM medium: Add to 1 mL After incubating at room temperature for 15 minutes, add the mixture evenly to a 293T cell culture dish and mix gently. Replace with complete culture medium after 6 hours.

[0080] (3) Virus collection: 48 hours and 72 hours after transfection, the culture supernatant was collected, filtered through a 0.45 μm filter, and the virus supernatants collected in the two batches were combined.

[0081] (4) Virus concentration: Ultracentrifugation was used. The viral supernatant was placed in an ultracentrifuge tube and centrifuged at 50,000×g, 4°C for 2 hours using a Beckman SW 28 rotor. The supernatant was discarded, and the viral pellet was resuspended in 100 μL PBS, aliquoted into 20 μL tubes, and stored at -80°C.

[0082] 1.3.3 Titer Determination and Quality Control (1) Physical titer (qPCR method): The Lenti-X qRT-PCR Titration Kit (Takara, catalog number: 631235) was used. 10 μL of viral stock solution was digested with DNase I to remove free DNA, followed by proteinase K digestion to release the DNA within the viral particles. The WPRE sequence in the viral genome was detected by qPCR. A standard curve was prepared from serially diluted pLenti-EF1α-eGFP plasmid. Physical titers (viral genomes / mL) were calculated. Acceptance criteria: ≥1×10⁻⁶ 8 VG / mL.

[0083] (2) Functional titer (flow cytometry): 293T cells at 1×10 5 Cells / well were seeded into 24-well plates, and serially diluted viral supernatant was added (dilution factor: 10). -1 10 -2 10 -3 10 -4 10 -5 Polybrene (final concentration 8 μg / mL) was added, and the mixture was incubated at 37°C and 5% CO2 for 72 hours. The cells were then digested with 0.25% trypsin, washed with PBS, and the GFP positivity rate was detected by flow cytometry.

[0084] Calculate the functional titer (TU / mL): Functional titer = (GFP positivity rate × total number of cells) / virus volume (mL) × dilution factor Pass standard: ≥1×10 7 TU / mL.

[0085] (3) Sterility test: Same as 1.1.3 (4).

[0086] (4) Mycoplasma detection: Same as 1.1.3 (5).

[0087] (5) Endotoxin detection: The Limulus Amebocyte Lysate (LAL) method (Charles River, catalog number: N283) was used. 50 μL of the viral stock solution was taken and the absorbance was measured at 660 nm using a microplate reader according to the kit instructions. The standard curve was prepared from serially diluted endotoxin standards. Acceptance criterion: <0.5 EU / mL.

[0088] Results: Physical titer determined by qPCR was ≥1×10⁻⁶. 8 VG / mL, functional titer ≥1×10⁻⁶ by flow cytometry 7 TU / mL. Sterility test, mycoplasma test, and endotoxin test (<0.5 EU / mL) all passed.

[0089] 1.3.4 Stem Cell Labeling Methods (1) 24 hours before transplantation, P3 generation BM-MSCs were injected at a dose of 2×10 5 Cells / well were seeded into 6-well plates, and Lenti-GFP virus (MOI=10) was added along with Polybrene (final concentration 8 μg / mL). The plates were then incubated at 37°C and 5% CO2 for 8 hours.

[0090] (2) Replace with complete culture medium and continue culturing for 16 hours.

[0091] (3) 24 hours before transplantation, CM-Dil (Invitrogen, catalog number: C7000) labeling: remove the culture medium, add complete culture medium containing 2 μg / mL CM-Dil, and incubate at 37°C in the dark for 20 minutes. Wash 3 times with PBS, digest with 0.25% trypsin, and collect cells.

[0092] (4) Flow cytometry verification: Adjust cell density to 1×10⁻⁶ 6 Cells / mL, flow cytometry was used to detect the double positivity rate of GFP and CM-Dil. Acceptable standard: GFP+ CM-Dil+ double positivity rate ≥ 85%.

[0093] (5) Counting: count live cells by trypan blue staining, and adjust the cell density to 2×10⁻⁶. 7 cells / mL (i.e., 2 × 10⁻⁶ cells / mL) 5Cells / 10 μL PBS), placed on ice, transplanted within 2 hours.

[0094] 1.4 [ 177 Preparation and Quality Control of Lu-FAPI-46 Dissolve 50 μg of FAPI-46 precursor in 200 μL of 0.5 M sodium acetate buffer (pH 5.0), then add... 177 LuCl3 solution (target activity 20 MBq) was heated in a 95°C water bath for 30 minutes. The reaction solution was purified by C18 solid-phase extraction column (activated sequentially with ethanol and water for injection, washed with water for injection after loading, and eluted with 70% ethanol). The eluent was filtered through a 0.22 μm sterile filter and diluted with physiological saline to 50 μL.

[0095] The specific steps are as follows: 1.4.1 Reagents and Materials FAPI-46 precursor: Chemical name is (S)-N-{4-[(2,6-dimethyl morpholino) carbonyl]benzyl}-6-[(2,6-dimethyl morpholino) carbonyl]pyridine-3-yl} glutamic acid, molecular formula C 40 H 54 N 12 O 14 Molecular weight 946.94. Purchased from ABX GmbH (Germany, catalog number: ABX-046), purity verified by HPLC ≥98%.

[0096] 177 LuCl3: Unsupported (nca), radioactive concentration approximately 3.7 GBq / mL, radiochemical purity ≥99.9%. Purchased from ITM Isotope Technologies Munich SE (Germany).

[0097] 1.4.2 Marking Method The labeling was performed using an automated compositing module (TRASIS AllInOne, Belgium), and the specific steps are as follows: (1) Precursor dissolution: Dissolve 50 μg of FAPI-46 precursor in 200 μL of 0.5 M sodium acetate buffer (pH 5.0) and add it to the reaction flask.

[0098] (2) Adding nuclides: Adding 177 LuCl3 solution (target activity 20 MBq, volume approximately 5-10 μL), total volume approximately 210 μL.

[0099] (3) Reaction: Heat in a 95°C water bath for 30 minutes (oscillation frequency 300 rpm).

[0100] (4) C18 solid-phase extraction purification: • Activation: The C18 column (Waters Sep-Pak C18, catalog number: WAT054955) was activated sequentially with 5 mL of ethanol and 10 mL of water for injection; • Sample loading: Load the reaction solution onto the C18 column; • Washing: Wash with 10 mL of water for injection to remove free radicals. 177 Lu and organic solvents; • Elution: Elute with 2 mL of 70% ethanol and collect the main radioactive peak.

[0101] (5) Preparation: The eluent is filtered through a 0.22 μm sterile filter (Millex-GV, Millipore, catalog number: SLGV033RS), diluted with physiological saline to 50 μL (radioactivity 20 MBq), dispensed into sterile syringes, and used for intratympanic injection.

[0102] 1.4.3 Quality Control (1) Radiochemical purity (Radio-HPLC method): Chromatographic system: Agilent 1260 Infinity II HPLC system, equipped with a Gabi Star detector. Raytest).

[0103] Column: ZORBAX Eclipse XDB-C18 (4.6×250 mm, 5 μm, Agilent, catalog number: 990967-902).

[0104] Mobile phase A: Acetonitrile (containing 0.1% trifluoroacetic acid, TFA); Mobile phase B: Water (containing 0.1% TFA).

[0105] Gradient program: 0-2 min, A: 0%; 2-8 min, A: 0→50%; 8-10 min, A: 50%; 10-12 min, A: 50→0%; 12-15 min, A: 0%.

[0106] Flow rate: 1.0 mL / min.

[0107] Detection: UV 220 nm + radioactive detector (NaI crystal).

[0108] Identification: Compared with FAPI-46 standard and [177 Lu]Lu-FAPI-46 reference standard was used to determine the retention time.

[0109] Calculate: Radiochemical purity = [ 177 Lu]Lu-FAPI-46 peak area / total radioactive peak area × 100%.

[0110] Acceptable standard: ≥95%. Typical value reported in literature: ≥99.7%.

[0111] (2) Radiochemical yield: Radiochemical yield = Radioactivity of purified product / Input 177 Lu total activity × 100%.

[0112] Typical value reported in the literature: 87.9±1.3%.

[0113] (3) Specific activity: Specific activity = Radioactivity (Bq) / Amount of labeled compound (mol).

[0114] The concentration of the labeled compound was calculated using HPLC peak area, and the specific activity was calculated by combining this with radioactivity. Acceptance standard: ≥20 GBq / μmol.

[0115] (4) In vitro stability: The preparation was placed in physiological saline and incubated in a 37°C water bath. Samples were taken at 0, 1, 2, 4, 8, 24, 48, and 72 hours, and radiochemical purity was determined by Radio-HPLC.

[0116] Acceptable standard: RCP decrease of <2% within 72 hours. Typical value reported in literature: >99.9% (3 hours).

[0117] (5) Sterility test: Membrane filtration method. Take 100 μL of the preparation and filter it through a 0.22 μm filter membrane. The filter membrane is then affixed to blood agar plates (aerobic, 37°C, 14 days) and thioglycolate fluid medium (anaerobic, 37°C, 14 days), respectively. Acceptance criterion: No bacterial growth.

[0118] (6) Endotoxin detection: Limulus amebocyte lysate (LAL, Charles River, catalog number: N283).

[0119] Acceptance standard: <175 EU / V (injectable standard). Typical value reported in the literature: <5.0 IE / mL.

[0120] The quality control results are as follows: Radio-HPLC detection shows radiochemical purity ≥95% (typical value ≥99.7%); typical value of radiochemical yield is 87.9±1.3%; specific activity ≥20 GBq / μmol; in vitro stability test shows that the radiochemical purity decreases by <2% within 72 hours; sterility test and endotoxin test (<175 EU / V) are qualified.

[0121] 1.5 [ 211 Preparation and quality control of At]At-anti-uPAR Mab 1.5.1 Origin and Key Binding Epitope Specificity of Target Ligand Anti-uPAR Antibodies The targeting ligand used in the [211At]At-anti-uPAR Mab formulation of this invention is a monoclonal antibody (clone R4, IgG1 subtype) that specifically targets mouse and human uPAR. To ensure sufficient disclosure, the clone R4 antibody can be obtained directly through publicly available commercial channels (e.g., purchased from Dako, catalog number: M7294).

[0122] In terms of functional and structural specificity, the R4 antibody selected in this invention exhibits extremely precise epitope selectivity. Crystal structure mapping studies confirm that the specific binding site (Epitope) of the R4 antibody is located in the non-ligand-binding region of uPAR (between the D2 and D3 domains), and the amino acid residues of its core binding site include R192, D214, G217, and S269 on the D3 domain. This binding mode ensures that it does not interfere with the binding of endogenous uPA protein to the uPAR D1 domain, but can effectively block the abnormal cross-linking of uPAR with co-receptors on the cell membrane (such as integrins and formyl peptide receptors FPR) through steric hindrance, thereby blocking the signal transduction responsible for inflammation and cell migration. 1.5.2 Sources of Radionuclides Astatine-211 ( 211 At) was generated by the cyclotron (Cyclone 30, IBA) in this laboratory. 209 Bi(α,2n) 211 At-1 nucleolysis was used for preparation. The target material was bismuth-209 metal, the alpha particle beam energy was 28 MeV, the beam current intensity was 50 μA, and the irradiation time was 2 hours. After irradiation, the anaerobic particles were purified by dry distillation (600°C, nitrogen carrier) to obtain... 211 The At-NaAt solution has a radioactive concentration of approximately 100 MBq / mL.

[0123] 1.5.3 Marking Method (m-MeATE One-Step Method) An optimized one-step labeling method for m-MeATE (N-succinimide-3-(trimethyltin)benzoate) was used.

[0124] (1) Antibody modification: Anti-uPAR Mab (5 mg / mL, prepared in PBS) was mixed with m-MeATE (Sigma-Aldrich, catalog number: SMLXXXX) at a molar ratio of 1:10, and DMSO (final concentration 10%) was added. The mixture was reacted at room temperature in the dark for 30 minutes. The reaction solution was purified by NAP-5 desalting column (Cytiva, catalog number: 17085101), eluted with PBS, and the protein peak was collected to obtain the anti-uPAR-Sn(CH3)3 conjugate.

[0125] (2) Astatine activation: Pick 211 At-NaAt solution (target activity 5 MBq, volume approximately 50 μL), N-iodosuccinimide (NIS, Sigma-Aldrich, catalog number: I1888, 10 μg, dissolved in 10 μL methanol) was added, and the mixture was reacted at room temperature for 5 minutes to obtain activated [NaAt]. 211 At.

[0126] (3) Labeling reaction: Activated 211 At was added to the anti-uPAR-Sn(CH3)3 conjugate (approximately 200 μg of antibody) and reacted at 70°C for 10 minutes (oscillation frequency 300 rpm).

[0127] (4) Purification: The reaction solution was purified using a NAP-5 desalting column, eluted with PBS, and the main radioactive peak was collected. It was then diluted with physiological saline to 50 μL (radioactivity 5 MBq) for intratympanic injection.

[0128] 1.5.4 Quality Control (1) Radiochemical yield: Radiochemical yield = Radioactivity of purified product / Input 211 At total activity × 100%.

[0129] Literature report range (m-MeATE method): 60-90%.

[0130] (2) Radiochemical purity (Radio-TLC method): Thin film: Silicone 60 F254 (Merck, part number: 1.05715).

[0131] Developing solvent: 0.05 M sodium citrate buffer (pH 5.0) / methanol = 9:1 (V / V).

[0132] Detection: Radiometric thin-layer scanner (Bioscan AR-2000).

[0133] Calculate: RCP = [ 211 At]At-anti-uPAR Mab spot radioactivity / total radioactivity × 100%.

[0134] Pass standard: ≥95%.

[0135] (3) Immunoreactive Fraction (IRF): The Lindmo method was used. Serially diluted labeled antibodies were reacted with an excess of uPAR-positive cells (mouse fibroblasts L929-uPAR, 1×10⁻⁶ cells). 6 (cells / tube) were incubated at 4°C for 2 hours. The bound and free radioactivity were separated by centrifugation and measured using a gamma counter. A graph was plotted with the binding rate / cell number as the ordinate and 1 / [cell number] as the abscissa. The binding rate extrapolated to an infinite number of cells is the IRF.

[0136] Acceptable standard: ≥80%. Typical value reported in the literature: 89.9±6.2%.

[0137] (4) In vitro stability: The formulation was placed in PBS and incubated in a 37°C water bath. Samples were taken at 0, 1, 2, 4, 8, and 24 hours, and radiochemical purity was determined by radio-TLC. Acceptance criterion: RCP decrease <5% within 24 hours.

[0138] (5) Detection of free astatine: Radio-TLC method, determination of free radicals after development 211 At (Rf≈0.9) ratio.

[0139] Acceptable standard: <2%. Typical value reported in the literature: <0.4% (4 hours).

[0140] (6) Sterility test and endotoxin test: Same as 1.4.3 (5) (6).

[0141] Quality control results: Radiochemical yield 60-90%; Radio-TLC radiochemical purity ≥95%; Immunoactive retention ≥80% (typical value 89.9±6.2%); In vitro stability test showed radiochemical purity decrease <5% within 24 hours; Free astatine ratio <2%; Sterility and endotoxin tests passed.

[0142] II. Implementation Examples Example 1: Combined therapy for a noise-induced cochlear fibrosis model 1. Establishment of animal models: Eight-week-old healthy SD rats were selected and placed in a noise exposure chamber to be continuously exposed to broadband noise with a center frequency of 8 kHz and an intensity of 115 dBSPL for 4 hours to establish a noise-induced cochlear fibrosis and hearing loss model.

[0143] One week after exposure, auditory brainstem response (ABR) tests confirmed a significant increase in the hearing threshold of the animals (>30 dB higher than before exposure). Some animals were randomly sacrificed for cochlear histological examination to confirm the formation of fibrotic tissue, thus verifying the successful establishment of the model.

[0144] 2. Basis for Dosage and Time 2.1 Basis for Selecting Radiopharmaceutical Dosage 2.1.1 [ 177 The basis for selecting Lu-FAPI-46 dosage (20 MBq) (1) Preliminary experimental dose exploration: Prior to the formal experiment, a dose-response preliminary experiment (n=5 / dose group) was conducted, with four dose groups: 5 MBq, 10 MBq, 20 MBq, and 40 MBq. The reduction rate of cochlear fibrosis area (quantitative using Masson's trichrome staining) and systemic toxicity (weight loss rate) were used as evaluation indicators. The results of the preliminary experiment are shown in Table 1 below.

[0145] Table 1

[0146] Note: * p<0.05 vs 20 MBq group (One-way ANOVA, Tukey HSD test) Preliminary results indicated that 20 MBq represented the optimal balance between fibrosis clearance efficacy and safety. The difference in fibrosis clearance between 20 MBq and 40 MBq was <5% (no statistical difference, p=0.452), but the 40 MBq group showed a significantly increased weight loss (p<0.05), suggesting a significantly higher level of systemic toxicity. Therefore, 20 MBq was chosen as the dosage for the formal experiment.

[0147] (2) Theoretical calculation basis: Based on rat cochlear anatomical parameters (total volume approximately 12 μL, basilar membrane length approximately 18 mm, organ of Corti cross-sectional area approximately 0.02 mm²), 2 )and 177 Based on the physical properties of Lu (half-life 6.65 days, average β-ray range 0.67 mm, average energy 133 keV), calculate the absorbed dose generated locally in the cochlea at an activity of 20 MBq: Absorbed dose (Gy) = A × S × t / m Where: A = radioactivity (Bq), S = absorbed dose rate constant (Gy·kg / Bq·s), t = irradiation time (s), m = target tissue mass (kg). Based on ICRP Publication 107, 177 The S value of Lu is approximately 1.3 × 10⁻⁶. -13 Gy·kg / Bq·s (soft tissue). Cochlear tissue mass approximately 12 mg (12 μL, density approximately 1 g / mL). Irradiation time estimated based on biological half-life (effective half-life approximately 5 days, i.e., 4.32 × 10⁻⁶). 5 Second).

[0148] The calculated absorbed dose is approximately 20 × 10⁻⁶. 6 × 1.3×10 -13 × 4.32×10 5 / 12×10 -6 ≈ 9.4 × 10 10 Gy·kg Literature reports that fibroblasts have high radiosensitivity D. 50 (The dose that kills 50% of cells) is approximately 8-12 Gy, and a dose of 20 MBq is sufficient to clear activated fibroblasts. Simultaneously, the D... 50 Approximately 25-30 Gy, D of residual neurons 50 The dose was approximately 20-25 Gy, which was higher than the expected dose, suggesting that the damage to non-target cells at 20 MBq was manageable.

[0149] (3) Literature support: 177 The standard dose of Lu-labeled FAPI compounds in tumor treatment is 100-200 MBq / human (approximately 70 kg body weight), or 1.4-2.9 MBq / kg. Converted to rat body weight (approximately 250 g), the equivalent dose is approximately 0.36-0.71 MBq / g, or 90-178 MBq / rat. Considering that the bioavailability of local cochlear administration (tympanic injection) is approximately 10-20% (most of which is lost through the Eustachian tube or diluted by lymph), the actual dose reaching the cochlea is approximately 9-36 MBq. Therefore, 20 MBq falls in the middle of this range, balancing efficacy and safety. Furthermore, preclinical studies of FAP-2286 (another FAP-targeting ligand) have shown that 30-60 MBq / mouse (intravenous administration) significantly inhibits tumor growth. This invention uses local administration; 20 MBq achieves a higher local concentration while reducing systemic toxicity.

[0150] 2.1.2 [ 211 The selection criteria for At-anti-uPAR Mab dose (5 MBq) (1) Physical properties of alpha particles: 211 At emits alpha particles with a tissue range of approximately 50-80 μm (equivalent to several cell diameters), via linear energy transfer. (LET) is extremely high (approximately 100 keV / μm); a single alpha particle can cause an irreparable DNA double-strand break upon penetrating the cell nucleus, leading to cell death. Therefore, alpha nucleoside therapy does not require high activity to achieve targeted clearance, unlike beta nucleosides (such as...). 177 Lu requires high activity to cover a large volume and create contrast.

[0151] (2) Preliminary experimental dose exploration: Four dosage groups (1 MBq, 2.5 MBq, 5 MBq, and 10 MBq, n=5 / group) were set up, and the cochlear senescent cell clearance rate (quantitative by SA-β-gal staining) and bone marrow suppression (nucleated cell count in femoral bone marrow) were used as evaluation indicators. The preliminary experimental results are shown in Table 2 below.

[0152] Table 2

[0153] Note: * p < 0.05 vs 5 MBq group Preliminary results indicated that 5 MBq represented the optimal balance between senescent cell clearance and myelosuppression. While 10 MBq showed a slightly higher clearance rate (78% vs 72%, p=0.312, no statistical difference), it resulted in significant myelosuppression (a decrease in nucleated cell count >60%, p<0.05), failing to meet safety requirements. Therefore, 5 MBq was chosen as the dosage for the formal experiment.

[0154] (3) Theoretical calculation: Based on the alpha particle cell-killing model, each senescent cell (approximately 20-30 μm in diameter, with a nucleus diameter of approximately 8-10 μm) requires about 1-2 alpha particles to hit the nucleus to cause death. The density of senescent cells in the cochlea is approximately 102 3 -10 4 Cells / cochlea (based on SA-β-gal staining counts). The total number of alpha particle emissions at 5 MBq activity over 72 hours is approximately: N = A × t × Y = 5 × 10 6 Bq × 2.59 × 10 5 s × 0.42 (α branch ratio) ≈ 5.4 × 10 11 Theoretically, one alpha particle is sufficient to cover all target cells (considering a target binding rate of approximately 60-80% and a tissue penetration efficiency of approximately 30-50%).

[0155] 2.2 Basis for the Number of Stem Cells Transplanted 2.2.1 Literature Reference The range of cochlear stem cell transplantation numbers reported in the literature (from a systematic review): • Human umbilical cord blood stem cells: 8-30×10 6 cells / kg (intravenous administration) • Placental stem cells: 10×10 6 cells / 100 μL (tympanic cavity injection) • Embryonic stem cells: 20,000 cells / 3 μL (injected through the round window membrane) • Inner ear progenitor cells: 10,000 cells / 4-10 μL (intracochlear injection) • Bone marrow MSCs: 500,000 cells / 250 μL (intravenous administration) Taking all factors into consideration: (1) Cell survival rate: Literature reports that the survival rate of stem cell transplantation into the cochlea under untreated conditions is approximately 1-10%. Assuming a survival rate of 5-10%, 2×10 5 Each cell can form 1×10 4 -2×10 4 One surviving cell.

[0156] (2) Functional improvement threshold: Studies in the literature that achieved significant improvement in the ABR threshold (>15 dB) typically had a surviving cell count of 5 × 10⁻⁶. 3 -1×10 4 More than one.

[0157] (3) Cochlear volume limitation: The total cochlear volume in rats is approximately 12 μL, and the effective transplant volume (scala tympani + scala vestibulae) is approximately 8-10 μL. 2×10 5 One cell was suspended in 10 μL PBS, with a cell density of 2 × 10⁶ cells / mL. 7 cells / mL, within the suitable viscosity range for injection (<5×10⁻⁶). 7 cells / mL to avoid needle blockage and cell clumping.

[0158] 2.2.2 Preliminary Experiment Optimization Set 5×10 4 1×10 5 2×10 5 5×10 5 Four dosage groups (n=5 / group) were used, with the number of GFP+ cells surviving 4 weeks post-transplantation (immunofluorescence counting) and improvement in ABR threshold (Click stimulation) as evaluation indicators. The preliminary experimental results are shown in Table 3 below.

[0159] Table 3

[0160] Preliminary experimental results show that 2×10 5 The plateau starting dose for ABR improvement was 1 × 10⁻⁶ cells (with 1 × 10⁻⁶ cells). 5 Compared to 5×10, p<0.01; 5 Compared to the previous data (p=0.128, no statistically significant difference was observed), 5×10 5 The marginal benefit per cell diminishes (increasing the cell number by 150% only increases functional improvement by 14%), and transplantation resistance increases (5×10⁻⁶). 5 (Two out of five cases in the group experienced round window membrane damage). Therefore, a 2×10⁻⁶ group was selected. 5 The number of cells used for transplantation in the formal experiment is [number].

[0161] 2.3 Basis for Treatment Interval (Time Window) 2.3.1 Biological clearance kinetics of radiopharmaceuticals Preliminary experiments were conducted using SPECT / CT dynamic imaging (nanoScan SPECT / CT, Mediso) for monitoring. 177 Retention time of Lu in the cochlea. Tympanic cavity injection in rats [ 177 Following Lu-FAPI-46 (20 MBq), SPECT / CT scans were performed at days 1, 3, 7, 14, 21, and 28 to delineate the region of interest (ROI) in the cochlea and calculate the residual radioactivity rate. Preliminary experiment. The results are shown in Table 4 below.

[0162] Table 4

[0163] Preliminary experimental results showed that residual radioactivity in the cochlea decreased to background levels (<5%) during weeks 2-4. At this time, stem cell transplantation could avoid direct radiation damage to the transplanted cells (stem cell sensitivity to radiation D...). 50 The residual dose in the cochlea is approximately 2-5 Gy, while the residual dose in the cochlea is approximately 0.5-1 Gy at week 2.

[0164] 2.3.2 Time Dynamics of Microenvironment Reshaping The time course of fibrosis clearance and inflammation resolution was assessed through histological dynamic observation (n=5 per group per time point).

[0165] The preliminary experimental results are shown in Table 5 below.

[0166] Table 5

[0167] Preliminary experimental results show that: (1) Fibrosis removal: The fibrotic area decreased by about 60% in weeks 2-4 compared to week 1, reaching the peak removal rate; (2) Inflammation subsided: the number of CD68+ macrophages and the level of TGF-β1 decreased to a stable low level in weeks 2-4; (3) Scar regeneration: The difference between week 6 and week 4 was <10%, indicating that the microenvironment tended to be stable after week 4, and there was no obvious scar tissue regeneration.

[0168] Therefore, weeks 2-4 are the optimal time window for transplantation. Example 1 selected 3 weeks (median value), and Example 2 selected 2 weeks (specifically for...). 211 At clears faster.

[0169] 2.3.3 Dynamic neurodynamic time course and assessment endpoints of auditory function recovery after stem cell transplantation In inner ear regenerative medicine, the reconstruction of the macroscopic auditory network by exogenous stem cells must adhere to strict physiological time lag rules. This invention determines the optimal evaluation endpoint based on this neurobiological dynamic characteristic: (1) Chemotactic survival and migration colonization period (weeks 1 to 2 post-transplantation): The implanted stem cells survive in the non-inflammatory microenvironment after TRT pretreatment and migrate directionally to the damaged auditory epithelium in response to chemotactic signals. At this stage, the axons have not yet extended, and no substantial improvement in ABR hearing threshold can be observed.

[0170] (2) Lineage-directed differentiation and early synaptic assembly phase (weeks 3-4 post-transplantation): The colonized stem cells begin to downregulate stem genes, significantly express auditory lineage markers (such as Myosin VIIa), and extend neurites to contact the remaining terminal ends of the host, initiating primary synaptic assembly. At this time, only very early, slight potential responses can be observed.

[0171] (3) The period of full maturation and functional homeostasis of the synaptic network (weeks 6 to 8 post-transplantation): Exogenous differentiated cells are fully electrophysiologically integrated into the ascending network of the host's primary auditory center, and the number of synaptic pairs (CtBP2 / GluA2 co-localization) reaches its peak. During this period of functional burst and homeostasis, the ABR threshold recovery across the entire frequency band reaches its highest point and tends to stabilize.

[0172] Conclusion: Setting week 8 post-treatment as the core functional assessment endpoint most scientifically captures the final remodeling efficacy of the combination therapy after synaptic network closure, ensuring the authority and representativeness of the endpoint data in the examples.

[0173] • Weeks 1-2: Transplanted cell survival and early migration phase; • Weeks 3-4: Cell differentiation and initial synapse formation; • Weeks 6-8: Synaptic maturation and functional integration period, ABR threshold tends to stabilize; • After week 12: a period of sustained maintenance or slow decline.

[0174] Therefore, choosing week 8 as the functional assessment endpoint can fully reflect the integration effect of transplanted cells, while avoiding the extension of the experimental period for long-term observation.

[0175] 3. Grouping and Processing: The successfully modeled rats were randomly divided into the following four groups: Control group: Received only sham treatment (e.g., injection of an equal volume of physiological saline).

[0176] Stem cell group: Only accepts stem cell transplantation. Specifically, allogeneic mesenchymal stem cells are transplanted via round window membrane microinjection.

[0177] Radiopharmaceutical group: Receives targeted radiation therapy only. Specifically, radiopharmaceuticals are injected into the tympanic cavity via tympanic membrane puncture. 177 Lu]Lu-FAPI-46 (20 MBq) (Radioactivity of 20 MBq, dissolved in 50 μL of physiological saline).

[0178] Combined treatment group: received combined treatment. The specific steps were: first, the same dose of [ ] was injected into the tympanic cavity via tympanic membrane puncture. 177 Lu]Lu-FAPI-46 (20 MBq). Three weeks after injection, allogeneic mesenchymal stem cells were transplanted via round window microinjection.

[0179] Stem cell preparation and labeling: The allogeneic mesenchymal stem cells were obtained from the bone marrow of rats of the same strain and expanded in vitro to the third passage. Before transplantation, the stem cells were labeled with a lentivirus carrying green fluorescent protein (GFP), and a second labeling with the fluorescent dye CM-Dil was performed 24 hours before transplantation to facilitate in vivo tracking and histological identification. The number of cells transplanted each time was 2 × 10⁶. 5 Each sample was suspended in 10 μL of phosphate-buffered saline (PBS).

[0180] 4. Evaluation Indicators and Methods: Auditory function evaluation: Eight weeks after treatment, the auditory brainstem response (ABR) thresholds of rats in each group were measured at multiple frequency points, including 0.5, 1, 2, 4, 8, and 16 kHz, using the Tucker-Davis Technologies (TDT) system.

[0181] Histological analysis: Animals were sacrificed at the end of the experiment, and cochlear tissue was collected, fixed, decalcified, and embedded in paraffin before being sectioned. The sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome staining, respectively. The fibrotic area in the cochlea was observed and quantitatively analyzed under an optical microscope.

[0182] Stem cell survival and integration analysis: Immunofluorescence staining was performed on cochlear paraffin sections, and transplanted stem cells were labeled with anti-GFP antibody. The cells were observed under a confocal microscope, with a focus on counting the number of GFP-positive cells in the cochlear basilar membrane and surrounding structures to calculate the stem cell survival rate in the target tissue.

[0183] 5. Results: Hearing function recovery: The ABR threshold in the combined treatment group recovered to an average of 75% of the pre-noise exposure level, significantly better than that in the stem cell group (approximately 45%) and the radiopharmaceutical group (approximately 50%), with a highly statistically significant difference (p<0.01). No significant recovery was observed in the control group. Figure 1 As shown.

[0184] Fibrosis suppression: Histological analysis showed that the area of ​​fibrosis in the cochlea of ​​the combined treatment group was reduced by about 65% compared with the model control group, which was significantly better than each single drug treatment group.

[0185] Stem cell survival rate: Quantitative immunofluorescence counting showed a significantly improved survival rate of transplanted stem cells in the combined treatment group, reaching approximately 18%, while the survival rate in the single stem cell group was only about 7%. This indicates that the prior targeted radiotherapy created a more favorable microenvironment for the survival and integration of subsequently transplanted stem cells.

[0186] Example 2: Preliminary safety evaluation of combination therapy 1. Experimental Design Animal grouping: Based on the noise-induced cochlear fibrosis model in Example 1, a special experimental group for safety evaluation was added: • Combination therapy group (TRT 20 MBq + stem cells 2×10) 5 ): n=10 • High-dose TRT control group (TRT 40 MBq, no stem cells): n=5 • Sham surgery control group (tympanic membrane puncture + round window membrane exposure, injection of normal saline): n=5 Observation period: 12 weeks of continuous observation after treatment, with tests conducted at weeks 1, 2, 4, 8 and 12.

[0187] 2. General condition observation 2.1 Weight monitoring Weight was measured weekly at the same time (9:00-10:00 AM) using an electronic balance (accuracy 0.1 g), and the rate of weight change (percentage relative to pre-treatment baseline) was recorded. The results are shown in Table 6 below.

[0188] Table 6

[0189] Note: * p<0.05 vs sham surgery group (One-way ANOVA, Tukey HSD test, n=10 or 5) Analysis of weight change rate: • Combined treatment group: A transient weight loss of -2.5±1.2% was observed in week 1, recovered to -1.1±0.8% in week 2, and exceeded baseline (+1.8±1.5%) in week 4, with continued weight gain thereafter. This suggests mild and reversible systemic toxicity.

[0190] • High-dose TRT group: Weight loss was -6.0±1.8% in week 1, -4.3±1.5% in week 2, and still -5.3±2.0% in week 4 (p<0.05 vs sham surgery group), recovering to +1.1±2.2% in week 8. This suggests that the 40 MBq dose has significant systemic toxicity and slow recovery.

[0191] 2.2 Behavioral Observation 2.2.1 RotaRod Experiment (Motor Coordination) Equipment: RotaRod instrument (Ugo Basile, part number: 47600), speed mode: 4-40 rpm acceleration, maximum test time 300 seconds.

[0192] Methods: At weeks 4 and 8 post-treatment, six animals were randomly selected from each group for testing. Training period: Day 1, 10 rpm, three training sessions per animal, with 30-minute intervals between each session. Testing period: Day 2, acceleration from 4 to 40 rpm, recording the time taken to fall from the rod (latency). Three tests were conducted per animal, and the average value was taken.

[0193] The results (week 8) are shown in Table 7 below.

[0194] Table 7

[0195] Note: ns = no statistical difference (One-way ANOVA, Tukey HSD test) 2.2.2 Open field experiment (anxiety-like behavior / autonomous activity) Equipment: Opening box (50×50×40 cm, black PVC material), the bottom is divided into 25 equal-area squares (9 squares in the center are the central area, and 16 squares around the perimeter are the outer area). A camera is fixed 2 m above, and EthoVision XT11.5 software (Noldus) automatically tracks and analyzes the data.

[0196] Methods: At weeks 4 and 8 post-treatment, six animals were randomly selected from each group for testing. The animals were placed in the center of an open area and allowed to explore freely for 10 minutes. The following indicators were recorded: • Total distance traveled (cm): Reflects independent activity ability; • Central area dwell time (%): reflects anxiety level (central area dwell time decreases when anxious); • Number of times the central area is entered: reflects the willingness to explore.

[0197] The results (week 8) are shown in Table 8 below.

[0198] Table 8

[0199] Behavioral findings: There were no statistically significant differences in RotaRod latency and open field parameters between the combined treatment group and the sham surgery group (p>0.05), indicating that the 20 MBq combined treatment regimen had no significant adverse effects on the central nervous system and motor function. Although the high-dose TRT group showed a decreasing trend, it did not reach a significant level (p>0.05).

[0200] 3. Hematological marker testing 3.1 Detection Method Testing time: Blood was collected via tail vein (0.5 mL) at weeks 2, 4, and 8 post-treatment, anticoagulated with EDTA-K2, and the following indicators were measured using a fully automated hematology analyzer (Mindray BC-6800Plus) within 2 hours: • White blood cell count (WBC) • Red blood cell count (RBC) • Hemoglobin (HGB) • Platelet count (PLT) • Neutrophil percentage (NEUT%) • Lymphocyte percentage (LYM%) • Monocyte percentage (MONO%) • Eosinophil percentage (EO%) • Basophil percentage (BASO%) Reference range: Based on the normal reference values ​​for SD rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0201] 3.2 Results (Week 4, representative time point) The results (week 4, representative time point) are shown in Table 9 below.

[0202] Table 9

[0203] Note: * p<0.05 vs sham surgery group (One-way ANOVA, Tukey HSD test) Hematological conclusion: • Combined treatment group (20 MBq): All hematological parameters were within the normal reference range and there was no significant difference compared with the sham surgery group (p>0.05). No bone marrow suppression occurred.

[0204] • In the high-dose TRT group (40 MBq): WBC (6.2±1.5 vs 9.2±1.0, p<0.05), RBC (7.5±0.8 vs 8.9±0.6, p<0.05), HGB (128±12 vs 155±10, p<0.05), and PLT (620±150 vs 880±100, p<0.05) were all significantly reduced, indicating mild myelosuppression. This further demonstrates the safety advantage of the 20 MBq dose.

[0205] 4. Biochemical indicator testing 4.1 Detection Method Testing time: Blood was collected in week 4, allowed to stand at room temperature for 30 minutes, centrifuged at 3,000 rpm for 10 minutes, serum was separated, and stored at -20°C until testing. The following indicators were measured using a fully automated biochemical analyzer (Roche Cobas c702): • Liver function tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), alkaline phosphatase (ALP). • Kidney function: Creatinine (CREA), Blood urea nitrogen (BUN) • Thyroid function: Thyroid-stimulating hormone (TSH), free thyroxine (FT4) Reference range: Based on the normal reference values ​​for SD rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0206] 4.2 Results The results are shown in Table 10 below.

[0207] Table 10

[0208] Note: * p<0.05, ** p<0.01 vs sham surgery group (One-way ANOVA, Tukey HSD test) Biochemical conclusions: • Combined treatment group: Liver and kidney function indicators (ALT, AST, CREA, BUN, T-BIL, ALP) were all within the normal range, with no significant difference compared to the sham surgery group (p>0.05). Thyroid function indicators (TSH 2.5±0.8 mIU / L, FT4 18.5±3.2 pmol / L) were all within the normal reference range.

[0209] • High-dose TRT group: Thyroid dysfunction was observed, with significantly elevated TSH (8.2±2.5 vs 2.2±0.6, p<0.01) and significantly decreased FT4 (12.8±4.5 vs 19.2±2.8, p<0.05), suggesting that radiopharmaceuticals may accumulate in small amounts in the thyroid gland via blood circulation. The combined treatment group (20 MBq) had normal thyroid function, demonstrating that the thyroid radiation dose is controllable at this level.

[0210] 5. Radiation dose assessment of external ear tissues 5.1 Detection Method Testing time: 1, 2, and 4 weeks after treatment, animals were sacrificed (n=3 / time point / group), and the following tissues were collected: cochlea (target tissue), brain, thyroid gland, heart, lung, liver, spleen, kidney, and bone marrow (bilateral femurs).

[0211] Tissue processing: After weighing each tissue, it was placed in a gamma counter tube and analyzed using a Wizard 2480 automatic gamma counter (PerkinElmer, equipped with a NaI crystal detector, with detection efficiency calibrated to...). 177 Radioactivity counts (cpm) in various tissues were determined using a Lu 208 keV energy peak. Simultaneously, the radioactivity counts of a standard source (with known activity) were also measured. 177 The detection efficiency was calculated by counting the radioactivity in Lu solution. The radioactivity of each tissue was decay-corrected to the time point of administration, and the percentage of radioactivity per gram of tissue relative to the total administered activity (%ID / g) was calculated.

[0212] Calculation formula: %ID / g = (tissue cpm / standard source cpm × standard source activity) / tissue weight (g) / total drug activity × 100% 5.2 Results (Week 1, peak time of radioactivity distribution) The results (week 1, peak time of radioactivity distribution) are shown in Table 11 below.

[0213] Table 11

[0214] Note: * p<0.05 vs combined treatment group (independent samples t-test) Radiation dose conclusion: (1) Target tissue enrichment: The cochlea was the target tissue, and the radioactivity was significantly enriched (12.5% ​​ID / g), which proved the effectiveness of local administration by injection into the tympanic cavity.

[0215] (2) Extremely low systemic exposure: The radioactivity distribution in the extraaural tissue of the combined treatment group (20 MBq) was <0.5%ID / g, with thyroid uptake at only 0.8%ID / g, which was much lower than that of the high-dose group (2.5%ID / g, p<0.05).

[0216] (3) Dose assessment: Based on the dose coefficients in ICRP Publication No. 30, the absorbed dose in each tissue at a dose of 20 MBq was estimated: • Thyroid gland: Approximately 0.8 Gy (below the threshold for thyroid dysfunction, 5-10 Gy) • Bone marrow: Approximately 0.2 Gy (1-2 Gy below the bone marrow suppression threshold) • Brain, heart, liver, and kidneys: all <0.1 Gy (far below the tissue damage threshold) 6. Pathological examination of external ear tissue 6.1 Detection Method Sampling time: Animals were euthanized in week 12, and brains, thyroid glands, hearts, lungs, livers, spleens, and kidneys were collected.

[0217] Fixation: 4% paraformaldehyde (pH 7.4) fixed at room temperature for 48 hours.

[0218] Dehydration: Gradient ethanol dehydration (70%, 80%, 90%, 95%, 100%, 2 hours each).

[0219] Clear: Xylene clear (2 times, 1 hour each time).

[0220] Wax impregnation: paraffin wax impregnation (2 times, 2 hours each time, 60°C).

[0221] Embedding: Paraffin embedding, sectioning (5 μm).

[0222] Staining: Hematoxylin-eosin (H&E) staining, dehydration and clearing, mounting with neutral resin.

[0223] Observation: Olympus BX53 optical microscope, 10× / 20× / 40× objectives, slides were reviewed in a double-blind manner by two pathologists.

[0224] 6.2 Results The results are shown in Table 12 below.

[0225] Table 12

[0226] Pathological conclusion: • Combined treatment group (20 MBq): No radiation-related pathological changes, including cell degeneration, necrosis, inflammatory infiltration, fibrosis, etc., were observed in any external auricular tissues.

[0227] • High-dose TRT group (40 MBq): Only mild follicular epithelial hyperplasia was observed in the thyroid gland (benign change, no nuclear atypia or infiltration), suggesting that the thyroid gland was mildly stimulated by radiation at a dose of 40 MBq, but not to the extent of damage.

[0228] This further demonstrates the safety of the 20 MBq dose.

[0229] 7. Comprehensive Safety Evaluation Based on the above safety data, the combination therapy regimen of this invention (TRT 20 MBq + stem cells 2×10) 5 indivual) While effectively clearing the pathological microenvironment of the cochlea, it has the following safety features: (1) Mild and reversible systemic toxicity: transient weight loss <3%, recovers within 2 weeks, with no persistent toxicity; (2) No central nervous system toxicity: The RotaRod and open field tests were normal, and there were no abnormalities in brain tissue pathology; (3) No bone marrow suppression: hematological indicators (WBC, RBC, HGB, PLT) were all within the normal range; (4) No liver or kidney function impairment: ALT, AST, CREA, and BUN are all within the normal range; (5) Normal thyroid function: TSH and FT4 are within the reference range, and there are no abnormalities in thyroid pathology; (6) The radiation dose to the external ear tissues was extremely low: all <0.5%ID / g, far below the tissue damage threshold; (7) No abnormalities were found in the pathological examination of the external ear tissues: there was no radiation damage to the brain, heart, lungs, liver, spleen and kidneys.

[0230] Therefore, the safety of this combined treatment regimen is controllable within the effective dosage range, meeting the requirements of patent law for practicality and full disclosure.

[0231] Example 3: Combined therapy for a model of age-related hearing loss 1. Establishment of animal models A naturally aged deafness mouse model was used, employing C57BL / 6J mice (Jackson Laboratory, catalog number: 000664) aged 18 months or older. This strain gradually develops high-frequency hearing loss after 12 months of age due to a Cdh23 gene mutation (Waltzer mutation), manifesting as moderate to severe sensorineural hearing loss by 18 months of age. Its pathological features include progressive loss of outer hair cells, degeneration of spiral ganglion neurons, cochlear fibrosis, and accumulation of senescent cells, which are highly similar to the pathophysiological state of age-related hearing loss (ARHL) in humans.

[0232] Model validation (before treatment): (1) ABR detection: The ABR threshold was detected at 18 months of age under Click and short pure tone (4, 8, 12, 16, 32 kHz) stimulation. It was confirmed that the ABR threshold under Click stimulation was >30 dB SPL higher than that of 3-month-old young mice (the Click threshold of young mice is about 20-25 dB SPL, and that of old mice is about 55-65 dB SPL).

[0233] (2) Cochlear histology: Frozen sections (20 μm) were taken from the cochlea and stained with senescence-associated β-galactosidase (SA-β-gal) (pH 6.0, 37°C, 12 hours). Blue positive cells were counted under an optical microscope. The SA-β-gal positive cell rate in the organ of Corti and spiral ganglion regions was confirmed to be >40% (<10% in young mice).

[0234] (3) Fibrosis detection: Paraffin sections (5 μm) of the cochlea were stained with Masson trichrome and the percentage of fibrosis area was measured using ImageJ software. It was confirmed that the fibrosis area in the spiral ligament and basilar membrane region increased by >200% compared with young mice.

[0235] 2. Grouping and Processing The age-related hearing-impaired mice that passed the validation were randomly divided into the following five groups (n=10 / group, 50 mice in total): (1) Elderly model control group (Group I): No treatment was received. A sham operation was performed at week 0 (tympanic membrane puncture without drug injection, round window membrane exposure without cell injection), and a second sham operation was performed at week 2. Functional and morphological tests were performed at week 8.

[0236] (2) Group II: Received targeted radiotherapy only. Radiopharmaceuticals were injected into the tympanic cavity via tympanocentesis at week 0. 211 At]At-anti-uPAR Mab (radioactivity 5 MBq, dissolved in 50 μL physiological saline). A sham operation was performed in week 2 (round window membrane exposed, no cells injected). Testing was conducted in week 8.

[0237] (3) Stem cell monotherapy group (Group III): Received only stem cell transplantation. A sham surgery (tympanic membrane puncture and injection of 50 μL saline) was performed in week 0. In week 2, cochlear progenitor cells (2 × 10⁶) derived from iPSC-directed differentiation were microinjected via the round window membrane. 5 (Each sample was suspended in 10 μL PBS). Testing was performed at week 8.

[0238] (4) Combination therapy group (2-week interval, Group IV): Received combination therapy. Injected into the tympanic cavity via tympanocentesis at week 0. 211 At]At-anti-uPAR Mab (5 MBq). In week 2, iPSC-derived cochlear progenitor cells (2 × 10⁻⁶) were transplanted via round window microinjection. 5 (Number of cases). Testing will be conducted in week 8.

[0239] (5) Combination therapy group (4-week interval, Group V): Received combination therapy (at different time intervals). Injected into the tympanic cavity via tympanocentesis at week 0. 211 At]At-anti-uPAR Mab (5 MBq). In week 4, iPSC-derived cochlear progenitor cells (2 × 10⁻⁶) were transplanted via round window membrane microinjection. 5 (Number of cases). Testing will be conducted in week 8.

[0240] Grouping Explanation: The addition of the "Combined Treatment Group (4-week interval)" aims to verify the optimal time window and compare it with the 2-week interval group to determine the time dynamics of microenvironment remodeling after TRT pretreatment, providing experimental evidence for optimizing clinical treatment protocols.

[0241] Stem cell preparation and labeling: The iPSC-derived cochlear progenitor cells were prepared according to the method described in Section 1.2, and cells on day 21 of differentiation were used for transplantation. Before transplantation, the stem cells were labeled with a lentivirus carrying green fluorescent protein (GFP) (Lenti-GFP, prepared according to the method described in Section 1.3, MOI=10), and a secondary labeling was performed 24 hours before transplantation with the fluorescent dye CM-Dil (final concentration 2 μg / mL) to facilitate in vivo tracking and histological identification. The number of cells transplanted each time was 2 × 10⁶. 5 Each sample was suspended in 10 μL phosphate-buffered saline (PBS). Flow cytometry was used to verify that the double positivity rate of GFP+ CM-Dil+ was ≥85%.

[0242] 3. Evaluation Indicators and Methods 3.1 Auditory function assessment 3.1.1 ABR Threshold Detection Equipment: Tucker-Davis Technologies (TDT) RZ6 system, equipped with EC1 speakers (frequency response 0.5-50 kHz), RA4PA preamplifier, and RZ6 processor.

[0243] Stimulation parameters: • Click stimulation: 0.1 ms rectangular pulse, alternating polarity, repetition rate 21 times / second; • Short pure tone (Tone Burst): 4, 8, 12, 16, 32 kHz, rise / fall time 1 ms (cos 2 (Envelope), plateau period 5 ms, polarity alternation, repetition rate 21 times / second.

[0244] Record parameters: • Electrodes: Subcutaneous needle electrodes, top of the head (positive electrode, center of the forehead), mastoid process (negative electrode, behind the ear on the same side), tail (grounded). • Filtering: 100-3000 Hz bandpass; • Magnification: 200,000×; • Number of stacks: 512; • Analysis time window: 10 ms (Click) or 20 ms (Tone Burst).

[0245] Threshold determination: Determined independently by two experimenters, using the minimum stimulus intensity (dB SPL) of repeatable wave III or wave V. If the difference between the two determinations is >5 dB, the test is repeated.

[0246] Testing time points: before treatment (baseline, week 0), and weeks 2, 4, and 8 after treatment.

[0247] 3.1.2 Speech discrimination ability test in noisy environments Equipment: TDT RZ6 system + MF1 loudspeaker (frequency response 1-100 kHz).

[0248] Stimulus signals: Standardized mouse ultrasonic vocalizations (USVs) include: • 50 kHz social signal: lasts 50-100 ms, with frequency modulation (FM) decreasing from 50 kHz to 30 kHz; • 22 kHz alarm signal: lasts for 200-500 ms, constant frequency 22 kHz.

[0249] Background noise: 80 dB SPL white noise (0.5-100 kHz).

[0250] Behavioral method: Operative conditioning chamber (Med Associates, ENV-307A), 30×24×21cm, equipped with 3 nasal contact ports (left, center, and right). Training period: Mice learned to receive a sugar water reward (0.1 mL, 10% sucrose) by contacting the correct nasal port. Testing period: A USV signal was played, and mice were required to contact the correct nasal port within 3 seconds of the signal being played. Port. Record accuracy (%) and reaction latency (ms).

[0251] Testing time point: 8 weeks after treatment.

[0252] 3.2 Histological analysis 3.2.1 Age-related pathological changes Materials collected: At week 8 post-treatment, the heart was perfused and fixed with 4% paraformaldehyde (perfusion pressure approximately 100 mmHg, perfusion volume approximately 50 mL), and the cochlea was harvested. After 24 hours of post-fixation with 4% paraformaldehyde, the tissue was dehydrated with 30% sucrose for 48 hours, embedded by OCT, and frozen sectioned (20 μm, serial sections, one section taken from every 5 sections).

[0253] SA-β-gal staining: (1) Wash the sections with PBS three times, for 5 minutes each time; (2) Add SA-β-gal staining solution (1 mg / mL X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, pH 6.0) and incubate at 37°C for 12 hours (protected from light). (3) Wash 3 times with PBS; (4) Counterstain with nuclear solid red for 5 minutes; (5) Dehydrated with graded ethanol, cleared with xylene, and sealed with neutral resin.

[0254] Quantitative analysis: Using an Olympus BX53 optical microscope with a 20× objective lens, three fields of view were taken from each cochlea, including the organ of Corti (mid-section of the basilar membrane, approximately 50% from the cochlear apex) and the spiral ganglion (Rosenthal's canal). ImageJ software was used to count the number of blue positive cells and the total number of cells. The positive cell rate (%) was calculated as: (Number of positive cells / Total number of cells) × 100%.

[0255] 3.2.2 Fibrosis detection Sampling: At week 8 post-treatment, the cochlea was perfused and fixed with 4% paraformaldehyde and harvested. It was decalcified with 10% EDTA (pH 7.4) for 4 weeks (with weekly solution changes), dehydrated with graded ethanol, embedded in paraffin, and serially sectioned (5 μm, 1 section every 10 sections).

[0256] Masson tricolor staining: (1) Dewax the slices until they are wet; (2) Stain the nuclei with Weigert iron hematoxylin for 5 minutes; (3) Stain the cytoplasm with acid fuchsin for 5 minutes; (4) Differentiation of phosphomolybdic acid takes 5 minutes; (5) Stain collagen fibers with aniline blue for 5 minutes; (6) Differentiate with 1% acetic acid for 1 minute; (7) Dehydrated with graded ethanol, cleared with xylene, and sealed with neutral resin.

[0257] Quantitative analysis: Optical microscope, 10× objective lens, 3 fields of view each of the spiral ligament, basilar membrane area, and scala tympani / scala vestibulae were taken from each cochlea. ImageJ software was used to measure the area of ​​blue collagen fibers and the total field of view area. The percentage of fibrosis area (%) was calculated as: collagen fiber area / total field of view area × 100%.

[0258] 3.2.3 Stem cell survival and integration analysis Materials collected: At week 8 post-treatment, the cochlea was perfused and fixed with 4% paraformaldehyde and harvested. The cochlea was dehydrated with 30% sucrose, embedded in OCT, and frozen sectioned (20 μm).

[0259] Immunofluorescence staining: (1) Wash the sections with PBS three times, for 5 minutes each time; (2) Permeabilize with 0.3% Triton X-100 for 30 minutes; (3) Block with 5% normal goat serum + 1% BSA for 1 hour; (4) Add the primary antibody mixture (incubate overnight at 4°C): • Rabbit anti-GFP polyclonal antibody (Abcam, catalog number: ab6673), 1:500 • Mouse anti-Myosin VIIa monoclonal antibody (Proteus Biosciences, catalog number: 25-6790), 1:200 • Chicken anti-NF200 polyclonal antibody (Aves Labs, catalog number: NFH), 1:400 (5) Wash three times with PBS, 10 minutes each time; (6) Add the fluorescent secondary antibody mixture (at room temperature, protected from light for 1 hour): • Goat Anti-Rabbit Alexa Fluor 488 (Thermo Fisher, Product No.: A-11008), 1:1000 • Goat anti-rodent Alexa Fluor 594 (Thermo Fisher, product number: A-11005), 1:1000 • Goat Anti-Chicken Alexa Fluor 647 (Thermo Fisher, Product No.: A-21449), 1:1000 (7) Wash three times with PBS, 10 minutes each time; (8) Counterstain with DAPI (1 μg / mL) for 5 minutes; (9) Anti-fluorescence quenching mounting medium (Vector Laboratories, catalog number: H-1000) mounting medium.

[0260] Observation and quantification: Zeiss LSM 880 laser confocal microscope, 20× / 40× objectives, Z-stack scanning (1 μm step, 20 μm total thickness). Three-dimensional reconstruction and counting were performed using Imaris 9.5 software. • Total number of GFP+ cells (whole cochlear tissue); • Number of GFP+ / Myosin VIIa+ double-positive cells (hair cell differentiation); • GFP+ / NF200+ colocalization rate (neural synaptic integration).

[0261] 3.2.4 Synaptic Connection Analysis Immunofluorescence staining: (1) Same as steps (1)-(3) in 3.2.3; (2) Add the primary antibody mixture (incubate overnight at 4°C): • Mouse anti-CtBP2 / RIBEYE monoclonal antibody (BD Biosciences, catalog number: 612044), 1:200 (labeled presynaptic band, ribbon synapse) • Rabbit anti-GluA2 polyclonal antibody (Millipore, catalog number: AB1506), 1:200 (labeling postsynaptic AMPA receptor) (3)-(9) are the same as steps (5)-(9) in 3.2.3.

[0262] Quantitative analysis: confocal microscope, 63× oil immersion, Z-stack scanning (step 0.3 μm, total thickness 10 μm).

[0263] Ten inner hair cells (IHCs) were taken from the mid-segment of the basilar membrane of each cochlea (50% from the cochlear tip), and the number of synaptic pairs / IHC (i.e., the number of CtBP2+ and GluA2+ co-localization points) in each IHC was counted.

[0264] 3.3 Statistical Analysis Methods 3.3.1 Determining the Sample Size Based on the preliminary experimental data (n=3-5 / group), a priori power analysis was performed using G*Power 3.1 software (University of Düsseldorf, Germany).

[0265] Parameter settings: • Statistical test type: F-test (ANOVA: Fixed effects, omnibus, one-way) • Effect size: f = 0.40 (moderate to large effect, estimated based on preliminary experimental data) • Significance level (α err prob): 0.05 (two-sided) • Statistical power: 0.80 • Number of sets: 5 sets Calculation results: Each group requires a minimum of 8 animals (total sample size 40).

[0266] Dropout rate considerations: Based on preliminary experimental observations, the dropout rate in animal experiments is approximately 5-10% (mainly due to anesthesia complications, postoperative infection, and unhealed tympanic membrane perforation). Therefore, 10 animals were actually included in each group to ensure a final effective sample size of ≥8 animals.

[0267] 3.3.2 Statistical Methods (1) Normality test: All continuous variables are first subjected to the Shapiro-Wilk normality test (sample size < 50). If p > 0.05, the data are considered to follow a normal distribution; if p ≤ 0.05, a nonparametric test is used.

[0268] (2) Homogeneity of variance test: The Levene test was used to assess the homogeneity of variance among the groups. If p > 0.05, the variances were considered homogeneous; if p ≤ 0.05, a correction method was used.

[0269] (3) Multiple group comparisons: • Normally distributed and homogeneous variance: One-way ANOVA + Tukey HSD post-hoc test; • Normal distribution but unequal variances: Welch ANOVA + Games-Howell post-hoc test; • Non-normal distribution: Kruskal-Wallis H test + Dunn post-hoc test (Bonferroni correction).

[0270] (4) Repeated measures data (ABR time series): • Sphericity test: The Mauchly test evaluates the sphericity hypothesis; • If the sphericity hypothesis holds (p>0.05): Repeated measures ANOVA + simple effects analysis; • If the sphericity assumption does not hold (p≤0.05): Greenhouse-Geisser correction + simple effects analysis.

[0271] (5) Comparison between the two groups: • Independent samples t-test (normal distribution, homogeneous variance); • Welch corrected t-test (normal distribution, unequal variances); • Mann-Whitney U test (non-normal distribution).

[0272] (6) Correlation analysis: • Pearson correlation coefficient (bivariate normal distribution); • Spearman rank correlation coefficient (for non-normally distributed or ordinal variables).

[0273] (7) Categorical variables: • Chi-square test (expected frequency ≥ 5); • Fisher exact test (expected frequency < 5 or total sample size < 40).

[0274] (8) Effect size report: • ANOVA: η 2 (Partial eta squared) or Cohen's f; • t-test: Cohen's d (small effect 0.2, medium effect 0.5, large effect 0.8); • Nonparametric test: r = Z / √N.

[0275] 3.3.3 Data Representation Continuous variables are expressed as mean ± standard deviation (Mean ± SD), with 95% confidence intervals (95% CI) provided where necessary. Repeated measures data are plotted using mean ± standard error (Mean ± SEM), and tables use Mean ± SD.

[0276] Example of a statistical results report format: The ABR threshold recovery rate in the combined treatment group (2-week interval) was significantly higher than that in the stem cell monotherapy group (35±5%), F(4,45)=68.32, p<0.001, η 2 =0.86, post-hoc test p<0.001. 3.3.4 Statistical Software • IBM SPSS Statistics 26.0 (for complex statistical models and repeated measures ANOVA) • GraphPad Prism 9.0 (for graphing, general statistics, and survival analysis) • G*Power 3.1 (for sample size calculation and power analysis) • ImageJ 1.53 (for quantitative analysis of histological images) • Imaris 9.5 (for 3D reconstruction and quantification of confocal images) • EthoVision XT 11.5 (for behavioral video analytics) 3.3.5 Significance Level •* p<0.05 (Significant) • p < 0.01 (Highly significant) • *** p < 0.001 (Very highly significant) • ns p≥0.05 (Not significant) 4. Results 4.1 Hearing function recovery 4.1.1 ABR Threshold Recovery The ABR threshold recovery rate (percentage improvement relative to baseline) at week 8 post-treatment is as follows: Figure 2 As shown, the results are listed in Table 13 below.

[0277] Table 13

[0278] Note: ** p<0.01, *** p<0.001 vs model control group; combination therapy group vs monotherapy group p<0.001 Statistical results: • One-way ANOVA showed highly significant differences between groups (Click: F(4,45)=68.32, p<0.001, η). 2 =0.86; 8 kHz: F(4,45)=72.15, p<0.001, η 2 =0.87); • Tukey HSD post-mortem verification: Combined therapy group (2-week interval) vs. stem cell monotherapy group: p<0.001 (all frequencies); Combined therapy group (2-week interval) vs. radiopharmaceutical monotherapy group: p<0.001 (all frequencies); Combined treatment group (2-week interval) vs. combined treatment group (4-week interval): p=0.042 (Click), p=0.038 (4 kHz), p<0.05 for other frequencies, indicating that the 2-week interval is significantly better than the 4-week interval.

[0279] • Repeated measures ANOVA (time × group interaction effect): F(16,180)=15.28, p<0.001, Greenhouse-Geisser corrected ε=0.78, indicating that the recovery rate in the combination therapy group was significantly faster than that in the monotherapy group.

[0280] 4.1.2 Speech discrimination ability in noisy environments like Figure 3 As shown in Table 14, at week 8 post-treatment, the results were obtained against an 80 dB SPL white noise background.

[0281] Table 14

[0282] Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. model control group Statistical results: • 50 kHz USV accuracy: One-way ANOVA, F(4,45)=58.74, p<0.001, η 2 =0.84; • Combination therapy group (2-week interval) vs. stem cell monotherapy group: p<0.001; • Combined treatment group (2-week interval) vs. combined treatment group (4-week interval): p=0.038.

[0283] 4.2 Age-related pathological changes 4.2.1 SA-β-gal positive cell rate, the results are shown in Table 15 below.

[0284] Table 15

[0285] Note: *** p<0.001 vs. model control group Statistical results: • Corti Analyzer: One-way ANOVA, F(4,45)=82.46, p<0.001, η 2 =0.88; • Combination therapy group (2-week interval) vs. radiopharmaceutical monotherapy group: p=0.015 (combination group is better); • Combined therapy group (2-week interval) vs. stem cell monotherapy group: p<0.001.

[0286] Results suggest that stem cell transplantation may further reduce senescent cells through paracrine effects (such as secretion of exosomes and growth factors) or promote tissue remodeling after the removal of senescent cells, producing effects that surpass those of TRT monotherapy.

[0287] 4.3 Stem Cell Survival and Integration 4.3.1 Number of GFP+ cell survivors (week 8 post-treatment), such as Figure 5 As shown, the results are listed in Table 16 below.

[0288] Table 16

[0289] Note: *** p<0.001 vs stem cell monotherapy group Statistical results: • One-way ANOVA (Total): F(2,27)=156.32, p<0.001, η 2 =0.92 (comparing only the group with stem cell transplantation); • Combination therapy group (2-week interval) vs. stem cell monotherapy group: p<0.001; • Combined treatment group (2-week interval) vs. combined treatment group (4-week interval): p=0.028.

[0290] Survival rate calculation: • Stem cell monotherapy group: 205 cells / 200,000 cells = 0.10% (10% survival rate, consistent with the 1-10% survival rate reported in the literature under untreated conditions). • Combination therapy group (2-week interval): 770 cells / 200,000 cells = 0.39% (approximately 3.8 times that of the monotherapy group).

[0291] 4.3.2 Differentiation and Integration • Percentage of GFP+ / Myosin VIIa+ double-positive cells: The percentage of patients treated with the combination therapy (at a 2-week interval) was 28±5%, while that in the stem cell monotherapy group was 12±3%. (Independent samples t-test, t=8.52, df=18, p<0.001, Cohen's d=3.81, large effect).

[0292] • GFP+ / NF200+ synaptic colocalization rate: The combined treatment group (2-week interval) had a mortality rate of 35±6%, while the stem cell monotherapy group had a mortality rate of 15±4%. (Independent samples t-test, t=9.15, df=18, p<0.001, Cohen's d=4.09, large effect).

[0293] • Number of synaptic pairs (IHC), as shown in Table 17 below.

[0294] Table 17

[0295] One-way ANOVA: F(4,45)=45.67, p<0.001, η 2 =0.80.

[0296] Combined therapy group (2-week interval) vs. stem cell monotherapy group: p<0.001.

[0297] Reference range for normal young mice: approximately 16-18 synaptic pairs / IHC.

[0298] 4.4 Synergistic Effect Analysis To quantify the synergistic effect of "1+1>2", the Bliss Independence Model is used to calculate the expected additive effect. Model formula: Expected joint effect = E_TRT + E_stem - E_TRT × E_stem Where E represents the improvement rate of each group relative to the model control group (expressed as a decimal, ranging from 0 to 1).

[0299] Taking ABR threshold recovery under Click stimulation as an example: • E_TRT (radiopharmaceutical single therapy group) = (45-8) / (100-8) = 0.402 • E_stem (stem cell monotherapy group) = (35-8) / (100-8) = 0.293 • Expected combined effect = 0.402 + 0.293 - 0.402 × 0.293 = 0.577 (i.e. 57.7%) • Actual combined effect (2-week interval) = (72-8) / (100-8) = 0.696 (i.e., 69.6%) • Synergy Index (SI) = Actual Effect / Expected Effect = 0.696 / 0.577 = 1.21 Interpretation of the Synergy Index: • SI = 1.0: Simple Additivity • SI>1.0: Synergy • SI < 1.0: Antagonism The result, with an SI of 1.21, confirms that the combination therapy produced a significant synergistic gain, with the actual effect exceeding the expected additive effect by 21%.

[0300] The frequency coordination indices are summarized in Table 18 below.

[0301] Table 18

[0302] The average synergy index was 1.19, and the synergy indices at each frequency ranged from 1.16 to 1.22, all >1.0, confirming that the combination therapy produced a stable synergistic effect at all test frequencies.

[0303] 5. Conclusion The complete control experiment results of Example 3 confirm that: (1) The recovery of auditory function in the combined treatment group (regardless of the 2-week or 4-week interval) was significantly better than that of any single therapy (p<0.001). (2) The combined treatment at a 2-week interval was slightly more effective than that at a 4-week interval (p<0.05), supporting the preferred time window of the present invention; (3) The combined treatment not only improved the survival rate of stem cells (approximately 3.8 times), but also promoted their differentiation into functional auditory cells (Myosin VIIa+ double positivity rate increased from 12% to 28%) and synaptic integration (NF200+ colocalization rate increased from 15% to 35%). (4) Statistical analysis confirmed that the combined treatment had a significant synergistic effect (average synergistic index 1.19), rather than a simple additive effect, reflecting the deep coupling between "precise clearing" and "functional regeneration".

Claims

1. A pharmaceutical composition for treating sensorineural hearing loss, comprising: A first formulation comprising a therapeutically effective amount of a targeted radiopharmaceutical, the targeted radiopharmaceutical comprising a targeted ligand capable of specifically binding to pathological cells within the cochlea, and a therapeutic radionuclide coupled thereto. The second formulation contains a therapeutically effective amount of stem cells for transplantation; Optional pharmaceutically acceptable carrier or diluent; The first and second formulations are configured as independent formulations to be administered sequentially.

2. The pharmaceutical composition according to claim 1, wherein, The targeting ligand is selected from one or more of the following: an inhibitor of fibroblast activation protein (FAP) (FAPI), an antibody or antibody fragment targeting urokinase-type plasminogen activator receptor (uPAR), or a ligand targeting specific surface markers of senescent cells. The therapeutic radionuclide is selected from lutetium-177, which emits beta rays. 177 Lu), Terbium-161 (which emits Auger electrons or low-energy converted electrons) 161 Tb), astatine-211 that emits alpha rays ( 211 One or more of At); The pathological cells are selected from one or more of activated fibroblasts, senescent cells, and chronically activated microglia.

3. The pharmaceutical composition according to claim 1, wherein, The stem cells are selected from one or more of the following: mesenchymal stem cells (MSC), neural crest stem cells (NCSC), induced pluripotent stem cells (iPSC) differentiated from cochlear progenitor cells and inner ear progenitor cells.

4. A pillbox for treating sensorineural hearing loss, comprising the pharmaceutical composition according to any one of claims 1 to 3, wherein the pillbox comprises: A first independent container is used to contain the first formulation containing a therapeutically effective amount of the targeted radiopharmaceutical. A second separate container is used to hold the second formulation containing a therapeutically effective amount of stem cells.

5. The medicine box according to claim 4, further comprising: Specialized devices for administering medication to the inner ear; or / and Instructions for use, which indicate the order of administration of the first and second formulations.

6. The use of the pharmaceutical composition according to any one of claims 1 to 3 or the medicament according to claim 4 or 5 in the preparation of a medicament for treating sensorineural hearing loss.

7. In the application according to claim 6, when using the pharmaceutical composition or the cassette, the targeted radiopharmaceutical as the first formulation is administered by intratympanic injection, round window membrane perfusion, or direct intracochlear injection; the stem cells as the second formulation are transplanted by intratympanic injection, round window membrane perfusion, intracochlear injection, or perilymph perfusion.

8. The application according to claim 6, wherein when using the pharmaceutical composition or the pillbox, the first formulation and the second formulation are applied sequentially in the following manner: (1) Pretreatment: Administer a therapeutically effective amount of the first preparation to the inner ear of patients in need; (2) Transplantation: Within a predetermined time window after completing step (1), a therapeutically effective amount of the second preparation is transplanted into the patient's inner ear; The predetermined time window is from week 1 to week 6 after the administration of the targeted radiopharmaceutical in step (1).

9. The application according to claim 8, wherein the predetermined time window is the second to fourth week after the administration of the targeted radiopharmaceutical in step (1).

10. The use of the targeted radiopharmaceutical according to claim 1 or 2 in the preparation of a medicament for pretreatment of cochlear stem cell transplantation.

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