Mesenchymal stem cell exosome loaded apt2 inhibitor of ml349 and preparation method and application thereof

Abstract

This invention relates to the field of pharmaceutical technology, disclosing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes, its preparation method, and its application. Inhibition of the APT2 target can treat Alzheimer's disease characterized by abnormal APT2 activation and decreased Tau protein palmitoylation levels. The nasal drop formulation comprises umbilical cord-derived mesenchymal stem cell exosomes, ML349, sterile PBS buffer, and a matching hyaluronic acid nasal pretreatment solution. The exosomes are purified using tangential flow filtration combined with size exclusion chromatography, and drug loading is achieved using pulsed ultrasound. This inhibitor, administered nasally, can bypass the blood-brain barrier and enter the brain. By inhibiting APT2 enzyme activity in the brain, it blocks the APT2-mediated Tau protein depalmitoylation process, upregulates Tau protein palmitoylation levels, inhibits neuroinflammation, and improves cognitive impairment in Alzheimer's disease model mice, exhibiting excellent biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to an APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a prevalent central nervous system degenerative disease in people over 65 years of age, characterized by cognitive impairment caused by progressive neuronal lesions. With the accelerating aging of the global population, elucidating the pathogenic molecular mechanisms of AD and developing novel intervention targets and efficient brain-targeted delivery strategies have become crucial issues urgently needing breakthroughs in neuroscience. Currently, the two recognized core pathological features of AD are senile plaques formed by abnormal deposition of β-amyloid protein (Aβ) and neurofibrillary tangles formed by excessive aggregation of Tau protein. The neurotoxicity mediated by these two factors is the main driver of neuronal loss. Under physiological conditions, Tau protein is highly expressed in neuronal axons, maintaining cytoskeletal stability and regulating axonal substance transport by binding to microtubules. An abnormal decline in the ability of Tau to bind to microtubules is considered a key precursor to intracellular Tau protein accumulation, driving the pathological progression of AD.

[0003] Mesenchymal stem cell exosomes are nanoscale extracellular vesicles secreted by cells. They have low immunogenicity and possess natural neuroprotective and immunomodulatory activities. When administered via the nasal cavity, they can bypass the blood-brain barrier and directly enter the brain through the olfactory nerve pathway and trigeminal nerve pathway. They can respond to inflammatory chemotactic signals in the brain and target and accumulate in central nervous system lesions, making them a highly promising drug delivery carrier for central nervous system diseases.

[0004] Currently, most existing research focuses on Tau protein hyperphosphorylation, which is the core reason for its dissociation from microtubules and the induction of tangles. However, whether other protein post-translational modifications besides phosphorylation participate in regulating this pathological process remains unclear and lacks systematic explanation and definitive verification. Among these, protein palmitoylation, as a reversible lipid post-translational modification, can participate in the progression of various neurological diseases by regulating protein subcellular localization, intermolecular interactions, and protein stability, representing a potential new direction for elucidating the pathological regulatory mechanism of Tau. Acylprotein thioesterase 2 (APT2 / LYPLA2) is one of the key enzymes regulating protein depalmitoylation. In the pathological state of Alzheimer's disease, APT2 is abnormally overactivated, catalyzing the depalmitoylation of Tau protein, resulting in a decrease in Tau protein palmitoylation modification levels, driving Tau protein aggregation and the outbreak of neuroinflammation in the brain, making it a highly valuable intervention target for Alzheimer's disease. Previous studies have only confirmed that APT2 is involved in physiological processes such as inflammation regulation and antiviral response. As a therapeutic target for AD, and the association between APT2 and its Tau protein modification and AD pathology, there have been no previous reports in the literature.

[0005] Existing patent CN119524136A discloses a novel use of a drug targeting APT2, achieving a systematic research breakthrough in the direction of APT2-targeted intervention for neurodegenerative diseases, filling a gap in the field, and clarifying the scientific basis for APT2 as a new target for AD intervention: This study confirmed in the brain tissue of AD model mice (5xFAD) and clinical AD patients that the palmitoylation level of Tau protein was significantly reduced compared with the normal control. Validation at both the cellular and animal levels showed that APT2 can directly mediate the depalmitoylation process of Tau protein. Overexpression of APT2 significantly downregulated the level of Tau palmitoylation, while knocking out the APT2 gene significantly increased the level of palmitoylation modification of Tau protein. At the same time, it significantly improved the spatial working memory and learning memory abilities of model mice, reduced the aggregation of Tau protein oligomers in the brain, upregulated the expression of synapse-related proteins to restore synaptic function, and reduced the activation levels of microglia and astrocytes and the expression of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, effectively alleviating neuroinflammation.

[0006] Therefore, developing APT2 inhibitors loaded with ML349 that can be efficiently delivered to brain lesions, have good biocompatibility, and can be administered non-invasively is of great practical significance. Summary of the Invention

[0007] In view of this, the present invention proposes an APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes, its preparation method and application, aiming to solve the problems of poor blood-brain barrier penetration, insufficient brain exposure when administered free of charge, and limited efficacy of cyclodextrin-loaded systems in the current technology of APT2 inhibitor ML349.

[0008] To achieve the above objectives, the present invention provides the following technical solution: An application of an APT2 target, wherein inhibiting the APT2 target can treat Alzheimer's disease characterized by abnormal APT2 activation and reduced Tau protein palmitoylation levels.

[0009] An APT2 inhibitor for inhibiting the APT2 target of mesenchymal stem cell exosomes loaded with ML349 as described in claim 1, said inhibitor comprising the following components in parts by weight: 0.1-2 parts of umbilical cord-derived mesenchymal stem cell exosomes, 0.01-0.2 parts of APT2 small molecule inhibitor ML349, and 97.8-99.89 parts of sterile PBS buffer.

[0010] Furthermore, the APT2 inhibitor is in the form of a nasal drop.

[0011] Furthermore, the APT2 inhibitor is also accompanied by a nasal pretreatment solution, which, by weight, contains 0.2 parts hyaluronic acid and the remainder is sterile PBS buffer.

[0012] A method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes includes the following steps: S1. Isolation and purification of exosomes from umbilical cord-derived mesenchymal stem cells to obtain purified exosomes from umbilical cord mesenchymal stem cells; S2. The purified umbilical cord mesenchymal stem cell exosomes were mixed with ML349 and placed in an ice bath; the mixture was treated with pulsed sonication; after each sonication cycle, the sample was cooled on ice. After S3 sonication, the mixture was incubated at room temperature for 4 hours. After incubation, it was centrifuged to precipitate the drug-loaded exosome complex. The precipitate was washed with pre-cooled PBS buffer and centrifuged again under the same conditions. The supernatant was discarded and the precipitate was resuspended in PBS. S4 The encapsulation efficiency of ML349 was determined, the pH and osmotic pressure of the system were adjusted, and the system was aseptically filtered at 0.22μm and aseptically dispensed to obtain the nasal drop formulation. At the same time, a matching 0.2% hyaluronic acid sterile aqueous solution for nasal pretreatment was prepared.

[0013] Furthermore, the specific steps for isolating and purifying umbilical cord-derived mesenchymal stem cell exosomes in step S1 are as follows: S1-1 Sample pretreatment: Collect the supernatant of umbilical cord mesenchymal stem cells after serum-free culture for 24 to 48 hours, centrifuge the supernatant at 300×g for 10 minutes at 4℃, then take the supernatant and centrifuge it at 2000×g for 20 minutes at 4℃. Filter the supernatant through a 0.22μm filter to complete the pretreatment. S1-2, TFF tangential flow filtration concentration: Place the inlet and reflux ends of the hollow fiber column into the pretreated feed container, and the permeate end into the waste liquid tank. Then start the peristaltic pump to start concentration. Set the inlet pressure to 30 psi, the outlet pressure to 10 psi, and the transmembrane pressure to about 20 psi. Continue concentration until the volume is reduced to one-twentieth of the original volume. S1-3, TFF washing and filtration: Immediately after concentration, start continuous washing and filtration. Adjust the inflow rate of sterile PBS buffer to match the outflow rate at the permeate end to achieve equal volume replacement. Continuously wash and filter 6 times the dialysis volume. After washing and filtration, collect the concentrate at the choked end for SEC loading. S1-4, SEC size exclusion chromatography purification: Select an appropriate qEV column according to the sample volume, equilibrate the SEC column to room temperature, remove the sliding cap at the bottom of the column, rinse the column with at least 10 mL of PBS elution buffer and observe whether the buffer flow rate is stable, then slowly add TFF concentrate to the top of the SEC column to start elution. The first 3 mL of elution is the void volume. Continue to add PBS for elution, collect the liquid after the void volume, and subsequently determine the exosome-enriched fractions by nanoparticle tracking analysis or 280 nm absorbance detection and combine them. S1-5. Final processing and storage: Combine the exosome enrichment fractions eluted from SEC, sterilize them using a 0.22μm filter, aliquot them into sterile cryovials according to experimental requirements, and store them at -80℃.

[0014] Furthermore, the ultrasonic power in step S2 is 130W; the pulse mode is 10s of ultrasound followed by 10s of pause, repeated 3 times, with a total pulse processing time of 2min; after each ultrasonic cycle, the sample is placed on ice to cool for 2min.

[0015] Furthermore, the centrifugation parameters in step S3 are: rotation speed 14000g, temperature 4℃, and time 30min.

[0016] Furthermore, the amount of pre-cooled PBS buffer added in step S3 is 250 μL.

[0017] Furthermore, in step S4, the pH of the system is adjusted to 5.5-6.5; the osmotic pressure is 250-350 mOsm / kg.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, umbilical cord mesenchymal stem cell exosomes are administered via nasal drops. Only two drops are needed to detect significant fluorescent signals of the drug carrier in the hippocampus and cerebral cortex. The drug enters the brain directly via the naso-brain pathway, bypassing the blood-brain barrier and the first-pass effect of the liver. The exosomes actively target AD lesions in response to intracranial inflammatory signals, achieving effective inhibition of the APT2 target in the brain.

[0019] The umbilical cord mesenchymal stem cell exosomes used in this invention have immunomodulatory and neuroprotective activities, which synergize with the pharmacological effect of ML349 targeting APT2. Compared with ML349 encapsulated with hydroxypropyl-β-cyclodextrin (core indicators improved by about 30%), the palmitoylation level of Tau protein in the formulation of this invention was increased by 45%-50%; the levels of IL-1β and TNF-α mRNA were downregulated by 50-60%, and the therapeutic effect was significantly improved.

[0020] The nasal drops in this invention are a non-invasive drug delivery method, which is easy to operate; the hyaluronic acid pretreatment further enhances mucosal penetration; the exosomes have low immunogenicity, high biocompatibility, and good in vivo safety.

[0021] This invention employs tangential flow filtration combined with size exclusion chromatography to purify exosomes, balancing yield and purity; the ultrasonic pulsed drug delivery process is stable and controllable, suitable for research and pilot-scale production; and the nasal drop formulation is physicochemically adapted to the physiological conditions of the nasal cavity.

[0022] Animal experiments in this invention have confirmed that the inhibitor simultaneously improves molecular pathology, cellular inflammation, and behavioral function: it reduces Tau oligomerization, inhibits microglia and astrocyte activation, and upregulates synaptic proteins NeuN and Syn-1; Y maze and Morris water maze tests have confirmed that it can significantly reverse short-term working memory and spatial learning memory deficits in 5xFAD model mice. Attached Figure Description

[0023] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 A schematic diagram of the multidimensional detection results after oral administration of free ML349 to 5xFAD Alzheimer's disease model mice.

[0024] Figure 2 LC-MS / MS quantitative standard curve for tail vein injection of free ML349.

[0025] Figure 3 This is a schematic diagram illustrating the central nervous system intervention effect of hydroxypropyl-β-cyclodextrin encapsulated with ML349 and its nasal delivery on Alzheimer's disease.

[0026] Figure 4 A schematic diagram of the structure of ML349 loaded on exosomes of umbilical cord mesenchymal stem cells and a visualization of the results of WB and qPCR detection in brain tissue.

[0027] Figure 5 This is a visualization of the Y-maze experiment using umbilical cord mesenchymal stem cell exosomes loaded with ML349.

[0028] Figure 6 This is a visualization of the results of a water maze experiment using umbilical cord mesenchymal stem cell exosomes loaded with ML349.

[0029] Figure 7 A schematic diagram illustrating the intrabrain delivery of ML349 via exosomes of umbilical cord mesenchymal stem cells. Detailed Implementation

[0030] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0031] An application of an APT2 target, wherein inhibiting the APT2 target can treat Alzheimer's disease characterized by abnormal APT2 activation and reduced Tau protein palmitoylation levels.

[0032] An APT2 inhibitor for inhibiting the APT2 target of mesenchymal stem cell exosomes loaded with ML349 as described in claim 1, said inhibitor comprising the following components in parts by weight: 0.1-2 parts of umbilical cord-derived mesenchymal stem cell exosomes, 0.01-0.2 parts of APT2 small molecule inhibitor ML349, and 97.8-99.89 parts of sterile PBS buffer.

[0033] In this invention, the APT2 inhibitor is a nasal drop formulation.

[0034] In this invention, the APT2 inhibitor is also accompanied by a nasal pretreatment solution, which, by mass fraction, consists of 0.2 parts hyaluronic acid and the remainder is sterile PBS buffer.

[0035] A method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes includes the following steps: S1. Isolation and purification of exosomes from umbilical cord-derived mesenchymal stem cells to obtain purified exosomes from umbilical cord mesenchymal stem cells; S2. The purified umbilical cord mesenchymal stem cell exosomes were mixed with ML349 and placed in an ice bath; the mixture was treated with pulsed sonication; after each sonication cycle, the sample was cooled on ice. After S3 sonication, the mixture was incubated at room temperature for 4 hours. After incubation, it was centrifuged to precipitate the drug-loaded exosome complex. The precipitate was washed with pre-cooled PBS buffer and centrifuged again under the same conditions. The supernatant was discarded and the precipitate was resuspended in PBS. S4 The encapsulation efficiency of ML349 was determined, the pH and osmotic pressure of the system were adjusted, and the system was aseptically filtered at 0.22μm and aseptically dispensed to obtain the nasal drop formulation. At the same time, a matching 0.2% hyaluronic acid sterile aqueous solution for nasal pretreatment was prepared.

[0036] In this invention, the specific steps for isolating and purifying the exosomes of umbilical cord-derived mesenchymal stem cells in step S1 are as follows: S1-1 Sample pretreatment: Collect the supernatant of umbilical cord mesenchymal stem cells after serum-free culture for 24 to 48 hours, centrifuge the supernatant at 300×g for 10 minutes at 4℃, then take the supernatant and centrifuge it at 2000×g for 20 minutes at 4℃. Filter the supernatant through a 0.22μm filter to complete the pretreatment. S1-2, TFF tangential flow filtration concentration: Place the inlet and reflux ends of the hollow fiber column into the pretreated feed container, and the permeate end into the waste liquid tank. Then start the peristaltic pump to start concentration. Set the inlet pressure to 30 psi, the outlet pressure to 10 psi, and the transmembrane pressure to about 20 psi. Continue concentration until the volume is reduced to one-twentieth of the original volume. S1-3, TFF washing and filtration: Immediately after concentration, start continuous washing and filtration. Adjust the inflow rate of sterile PBS buffer to match the outflow rate at the permeate end to achieve equal volume replacement. Continuously wash and filter 6 times the dialysis volume. After washing and filtration, collect the concentrate at the choked end for SEC loading. S1-4, SEC size exclusion chromatography purification: Select an appropriate qEV column according to the sample volume, equilibrate the SEC column to room temperature, remove the sliding cap at the bottom of the column, rinse the column with at least 10 mL of PBS elution buffer and observe whether the buffer flow rate is stable, then slowly add TFF concentrate to the top of the SEC column to start elution. The first 3 mL of elution is the void volume. Continue to add PBS for elution, collect the liquid after the void volume, and subsequently determine the exosome-enriched fractions by nanoparticle tracking analysis or 280 nm absorbance detection and combine them. S1-5. Final processing and storage: Combine the exosome enrichment fractions eluted from SEC, sterilize them using a 0.22μm filter, aliquot them into sterile cryovials according to experimental requirements, and store them at -80℃.

[0037] In this invention, the ultrasonic power in step S2 is 130W; the pulse mode is 10s of ultrasound, 10s of pause, repeated 3 times, with a total pulse processing time of 2min; after each ultrasound cycle, the sample is placed on ice to cool for 2min.

[0038] In this invention, the centrifugation parameters in step S3 are: rotation speed 14000g, temperature 4℃, and time 30min.

[0039] In this invention, the amount of pre-cooled PBS buffer added in step S3 is 250 μL.

[0040] In this invention, the pH of the system in step S4 is adjusted to 5.5-6.5; the osmotic pressure is 250~350 mOsm / kg.

[0041] The present invention also provides the application of the APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes as described in the above technical solution, specifically the application of the APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes in the prevention and / or treatment of Alzheimer's disease.

[0042] In this invention, the application specifically refers to the use of the inhibitor, with APT2 as the drug intervention target, in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease related to abnormal APT2 activation and reduced Tau protein palmitoylation levels.

[0043] In this invention, the nasal drop formulation of the umbilical cord-derived mesenchymal stem cell exosomes loaded with the APT2 small molecule inhibitor ML349 is pretreated with hyaluronic acid in the nasal cavity before administration to improve the nasal mucosa penetration efficiency. After administration via the nasal cavity, the formulation bypasses the blood-brain barrier and enters the brain directly by relying on the olfactory nerve pathway and the trigeminal nerve pathway. The mesenchymal stem cell exosomes can respond to inflammatory chemotactic signals in the brain and actively target and accumulate in the lesion area of ​​Alzheimer's disease.

[0044] In this invention, the drug-loaded exosomes release the loaded small molecule ML349 at the central lesion site. ML349 acts as an inhibitor of APT2 (acylprotein thioesterase 2 / LYPLA2) enzyme activity, binding to the catalytic domain of APT2 protein. Without changing the total APT2 protein expression level, it directly inhibits the catalytic activity of APT2 thioesterase, blocks the APT2-mediated depalmitoylation process of Tau protein, and increases the palmitoylation modification level of Tau protein in the brain. Palmitoylated Tau protein can stably bind to microtubules, reduce the aggregation of Tau protein oligomers, and alleviate Tau-related pathological damage.

[0045] At the cellular level, inhibition of APT2 activity can suppress excessive activation of microglia and astrocytes, significantly downregulate the transcriptional expression of pro-inflammatory factors in the brain such as IL-1β, IL-6, and TNF-α, and alleviate excessive neuronal inflammation in the central nervous system; at the same time, it upregulates the expression of neuronal and synapse-related proteins such as NeuN and Syn-1, protects neuronal integrity, and maintains synaptic structure and function.

[0046] Furthermore, the exosomes of umbilical cord-derived mesenchymal stem cells possess natural neuroprotective and immunomodulatory activities, and produce a synergistic pharmacological effect with ML349's targeted inhibition of APT2. Compared with free small molecules and cyclodextrin-encapsulated systems, the therapeutic effect is significantly improved. The above-mentioned multiple pathological improvements at the molecular and cellular levels jointly reverse the impairment of short-term working memory and spatial learning memory in Alzheimer's disease model mice, and achieve intervention and treatment of Alzheimer's disease related to abnormal APT2 activation and reduced Tau protein palmitoylation.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1: Isolation and culture of exosomes from umbilical cord-derived mesenchymal stem cells (1) Sample pretreatment: Collect the supernatant of umbilical cord mesenchymal stem cells after serum-free culture for 24 to 48 hours; then centrifuge the supernatant at 300×g for 10 minutes at 4°C; then take the supernatant and centrifuge it at 2000×g for 20 minutes at 4°C; finally, filter the supernatant through a 0.22μm filter to complete the pretreatment. (2) TFF tangential flow filtration concentration: Place the inlet and reflux ends of the hollow fiber column into the pretreated feed container, and the permeate end into the waste container. Then start the peristaltic pump to begin concentration, setting the inlet pressure to 30 psi, the outlet pressure to 10 psi, and the transmembrane pressure (TMP) to around 20 psi (which can be fine-tuned according to the equipment). Continue concentration until the volume is reduced to one-twentieth of the original volume (e.g., concentrating 2L to 100mL). During the concentration process, a stable flow rate must be maintained to avoid membrane blockage due to concentration polarization. (3) TFF washing (buffer replacement) Immediately after concentration, begin continuous filtration; then adjust the inflow rate of sterile PBS buffer to match the outflow rate of the permeate end (waste liquid) to achieve equal volume replacement; after continuous filtration of 6 times the dialysis volume (DV), collect the concentrate from the choked end for SEC loading. (4) SEC size exclusion chromatography purification Select the appropriate qEV column based on the sample volume. For example, qEVOriginal or qEV10 can be used for small-scale research samples; qEV10 or qEV100 can be used for pilot-scale amplification; and qEV100 or the Zenco automated system can be used for large-scale production. Afterward, equilibrate the SEC column to room temperature, remove the sliding cap at the bottom of the column, rinse the column with at least 10 mL of PBS elution buffer, and observe whether the buffer flow rate is stable. Then, slowly add TFF concentrate to the top of the SEC column. The recommended loading volume for qEV columns is 500 μL. If using other SEC columns, the loading volume should not exceed 10% to 20% of the total column bed volume. Start elution. The first 3 mL of eluent is the void volume, which does not contain exosomes and should be discarded. Continue elution with PBS and collect the remaining 1.5 mL of liquid after the void volume, which is the high-purity exosomes. Alternatively, you can collect one fraction every 500 μL and then determine and combine the exosome-enriched fractions by nanoparticle tracking analysis (NTA) or 280 nm absorbance detection. (5) Final processing and storage: The exosome enrichment fractions eluted from SEC were combined and then sterilized by filtration through a 0.22 μm filter. The fractions were then aliquoted into sterile cryovials according to experimental requirements and finally stored at -80°C to avoid repeated freeze-thaw cycles.

[0049] Example 2: Preparation of ML349-loaded nasal drops containing mesenchymal stem cell exosomes (1) Take the umbilical cord mesenchymal stem cell exosomes obtained in Example 1, mix them with ML349, and place them in an ice bath; (2) The mixture containing exosomes was placed in an ice bath and subjected to ultrasonic treatment using an ultrasonic cell disruptor to promote the rupture of the exosome membrane structure or the release of its contents. The ultrasonic parameters were set as follows: ultrasonic power 130W, pulse mode (ultrasound for 10 seconds, stop for 10 seconds, cycle 3 times), and the total treatment time was 2 minutes. After each treatment cycle, the sample was placed on ice to cool for 2 minutes. (3) After ultrasonic treatment, the mixture is transferred to room temperature and incubated for 4 hours; (4) After incubation, place the sample in a pre-cooled centrifuge at 4°C and centrifuge at 14000×g for 30 minutes to precipitate exosomes and their bound complexes; (5) Add 250 μL of pre-cooled PBS buffer to the precipitate, gently blow or vortex to wash the precipitate, and then centrifuge again at 14000×g for 30 minutes at 4°C. (6) Discard the supernatant, resuspend the washed precipitate in 250 μL PBS buffer, and gently pipette until evenly dispersed; (7) Detect the encapsulation rate; adjust the pH to 6.0 and the osmotic pressure to 300 mOsm / kg; filter with 0.22 μm sterile, and dispense sterilely to obtain the ML349 nasal drop formulation loaded with mesenchymal stem cell exosomes; the matching pretreatment solution: 0.2% sterile aqueous solution of hyaluronic acid, used for nasal pretreatment before administration.

[0050] Comparative Example 1: Hydroxypropyl-β-cyclodextrin-encapsulated ML349 nasal drops (1) Dissolve HP-β-CD to prepare blank inclusion vector. Accurately weigh 1.0 g of HP-β-CD, pour it into a dry beaker, and add about 8 mL of physiological saline.

[0051] Stir the solution at low speed (300~500 rpm) with a magnetic stirrer. Place the solution in a water bath at 30~35℃ and stir for 15~20 minutes until HP-β-CD is completely dissolved. (2) Add ML349 to carry out inclusion reaction. If ML349 is poorly soluble in physiological saline: Take a calculated amount of ML349 (about 0.72 g), add 0.5~1 mL of anhydrous ethanol (volume ≤ 1% of the total solution, to avoid irritating the nasal mucosa), vortex to dissolve, and then slowly drip it into the HP-β-CD solution; If ML349 can be directly dispersed: add ML349 powder directly to HP-β-CD solution and continue stirring for 30-60 minutes (inclusion equilibrium time). (3) Volume control and system conditioning Transfer the inclusion solution along a glass rod to a 100mL volumetric flask, rinse the beaker 2-3 times with a small amount of physiological saline, and pour all the rinsing solution into the volumetric flask. Add physiological saline until the concave meniscus is level with the 10mL mark, then invert and shake well 3-5 times. pH adjustment: Adjust the pH of the solution to 5.5~6.5 using hydrochloric acid or sodium hydroxide solution; Osmotic pressure adjustment: Measure with an osmometer. If it deviates from 250~350 mOsm / kg, add a small amount of sodium chloride (about 0.5~0.9g) to adjust it to the standard. (4) Removal and aseptic treatment of free drugs To separate free ML349: Pour the solution into a dialysis bag (pre-soaked and activated with physiological saline), place it in a large amount of physiological saline for dialysis for 2-4 hours (room temperature or 4°C), and change the dialysis solution 1-2 times; Aseptic treatment: The dialysis solution was sterilized by filtration through a 0.22 μm filter membrane, and the filtrate was collected as the final inclusion complex solution; hydroxypropyl-β-cyclodextrin-encapsulated ML349 nasal drops were obtained.

[0052] Comparative Example 2 ML349 alone.

[0053] Performance testing: The performance of the ML349-loaded nasal drops formulation from intermediate mesenchymal stem cell exosomes in Example 2, the ML349-loaded nasal drops formulation from Comparative Example 1 (hydroxypropyl-β-cyclodextrin), and ML349 alone in Comparative Example 2 were tested: (1) Effects of APT2 gene knockout (APT2-KO) on AD animal models Construction of APT2 knockout mice (APT2-KO): APT2-KO mice were constructed using CRISPR / Cas9 technology. Cas9 mRNA and gRNA were prepared via in vitro transcription, thoroughly mixed, and microinjected into the cytoplasm of mouse zygotes. After injection, the zygotes were cultured in vitro to the two-cell stage and then transplanted into ICR surrogate mothers. Genotyping of offspring: Genomic DNA was extracted from F0 mice after birth, and genotyping was performed using PCR. The results confirmed the existence of APT2-KO positive gene knockout mice. APT2-KO mice were crossed with 5xFAD Alzheimer's disease model mice to obtain 5XFAD-APT2-KO double-gene modified mice, which were used for subsequent drug efficacy evaluation.

[0054] Construction of cells overexpressing APT2 (APT2-HA): HEK-293T cells were co-transfected with the APT2-HA overexpression plasmid and the Tau-Flag plasmid using Lip3000 transfection reagent; the control group was transfected with only the Tau-Flag plasmid.

[0055] Molecular-level experiments showed that knocking out the APT2 gene significantly increased the palmitoylation level of Tau protein in the mouse brain; while overexpression of the APT2 gene in cells significantly decreased the palmitoylation level of Tau protein, confirming that APT2 is a key enzyme mediating the depalmitoylation of Tau protein.

[0056] Y-maze results showed that, compared with wild-type mice, the number of arm insertions and the spontaneous alternation rate were significantly reduced in 5xFAD model mice; the above indicators were significantly restored in 5XFAD+APT2-KO mice, suggesting that knocking out APT2 can improve short-term working memory impairment in Alzheimer's disease model mice.

[0057] Morris water maze test results showed that 5xFAD model mice had significant spatial learning and memory impairment, with decreased average swimming speed, fewer platform crossings, and prolonged escape latency. After APT2 knockout, the behavioral indicators of 5XFAD+APT2-KO mice were significantly restored, and their spatial learning and memory abilities were improved.

[0058] Brain tissue protein detection: Compared with 5xFAD model mice, 5XFAD+APT2-KO mice showed reduced Tau oligomer expression and upregulated synaptic protein Synapsin expression in the brain; microglia marker Iba1 and astrocyte marker GFAP expression were downregulated, indicating that knocking out APT2 can alleviate abnormal Tau protein aggregation, repair synaptic function, and inhibit excessive glial cell activation.

[0059] The pro-inflammatory factor assay used ZDHHC23-KO as a control: IL-1β, IL-6, and TNF-α were not significantly downregulated in the brains of 5XFAD+ZDHHC23-KO mice; however, the expression of these pro-inflammatory factors was significantly reduced in 5XFAD+APT2-KO mice. These results indicate that simply regulating palmitoyltransferase ZDHHC23 cannot alleviate neuroinflammation, while knocking out APT2 can effectively inhibit excessive neuroinflammation in the brains of model mice.

[0060] In summary, knocking out APT2 can improve learning and memory deficits in Alzheimer's disease model mice, reduce Tau pathological accumulation, repair synaptic function, and alleviate central nervous system inflammation, demonstrating that APT2 can serve as an effective intervention target for Alzheimer's disease.

[0061] (2) 5xFAD mice were orally administered ML349: The pathological amelioration effect of ML349 in a 5xFAD Alzheimer's disease model mouse was verified by oral administration of ML349 (Comparative Example 2). Two dosing cycles (1 month, 1M and 2 months, 2M) were performed, and the results were analyzed from four dimensions: Tau protein palmitoylation modification, glial cell activation, neuronal / synaptic integrity, and inflammatory factor transcription levels. Brain tissue was treated with RIPA buffer (P0013B, Beyotime) supplemented with a protease inhibitor (P1006, Beyotime) to obtain protein lysates. The protein lysates were then alkylated with 50 mM n-ethylmaleimide (NEM) for 90 min at room temperature to block free thiols. Excess NEM was removed from the samples by chloroform-methanol extraction, and the samples were washed twice with methanol. Next, the samples were incubated with 2 M hydroxylamine (NH2OH) at room temperature for 120 min to cleave the cys-palmitoyl thioester bond, and then incubated with 4 mM biotin-hpdp to biotinylate the protein. After chloroform-methanol precipitation and two methanol washes, biotinylated proteins were purified using streptavidin agarose affinity chromatography and incubated overnight at 4°C. The bound proteins were eluted with wash buffer and precipitated with methanol / chloroform. The precipitated proteins were dissolved in 50 μL of Laemmle loading buffer (2.1% SDS, 66 mM Tris, 26% glycerol w / v, 50 mM DTT, pH 7.5) for SDS-PAGE analysis, or dissolved in 50 µL of 8 μM urea for subsequent mass spectrometry analysis.

[0062] The test results are as follows: Figure 1As shown, (A) is: using the ABE experiment to verify that oral administration of ML349 (concentration after dissolution: 20 mg / kg, administered for 1 month and 2 months respectively) did not significantly improve the palmitoylation modification of Tau in 5xFAD mice; (B) is: using the WB experiment to verify that after oral administration of ML349 to 5xFAD mice (1 month and 2 months), the IBA1 (microglia) level in brain tissue did not change much, while the GFAP (glial cells) level increased, and the neuronal proteins such as NeuN did not change; using the RT-PCR experiment to verify that after oral administration of ML349 to 5xFAD mice (1 month and 2 months), the transcriptional levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α in brain tissue were not uniformly affected (IL-6 increased after 2 months of oral administration); the transcriptional level of the anti-inflammatory factor IL10 did not show a uniform trend with the increase of administration time.

[0063] Depend on Figure 1 The results showed that oral administration of free ML349 did not significantly increase the palmitoylation level of Tau protein in the brain tissue of 5xFAD model mice, nor did it significantly downregulate the protein expression of microglia and astrocyte activation markers. Furthermore, the transcriptional levels of various pro-inflammatory factors did not show a consistent improvement trend with increasing administration time. These results suggest that oral administration of free ML349 alone is not an ideal intervention for AD pathological phenotypes. Considering the delivery characteristics of drugs in the central nervous system, the underlying reason is speculated to be the low blood-brain barrier penetration efficiency of free ML349, resulting in insufficient effective drug exposure in the brain after oral administration, making it difficult to fully exert the biological regulatory effect of targeting APT2.

[0064] (3) ML349 was injected into the tail vein of 5xFAD mice: In Comparative Example 2, ML349 was administered via tail vein injection, and the exposure level of the drug to brain tissue after systemic circulation was dynamically monitored. Plasma and brain tissue drug concentrations at multiple time points after administration were detected by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the brain-plasma drug concentration ratio was calculated to quantitatively reflect the blood-brain barrier permeability of ML349. The results are as follows Figure 2 As shown, the pharmacokinetic quantitative analysis results indicate that ML349 has extremely low blood-brain barrier penetration efficiency after entering the systemic circulation via tail vein injection. Its brain-plasma drug concentration ratio is only 0.74% (14.6 / 1965=0.74%), which is far below the brain exposure threshold required for the central nervous system drug to exert its therapeutic effect. It is insufficient to reach an effective concentration in the brain to inhibit APT2 enzyme activity.

[0065] Depend on Figure 2It can be seen that the experimental method of high performance liquid chromatography-tandem mass spectrometry was used to verify the amount of ML349 in plasma (p) and cerebrospinal fluid (csf) after tail vein injection. The results can not only clarify the brain entry efficiency of free ML349, but also provide a pharmacokinetic reference for the subsequent optimization of different delivery systems. Moreover, the results directly elucidate the core mechanism that the pathological phenotype in the brain was not significantly improved after oral administration of free ML349, and clarify that insufficient blood-brain barrier permeability is the key bottleneck restricting its central therapeutic effect.

[0066] (4) Inflammatory response in the brain of 5xFAD mice after administration of hydroxypropyl-β-cyclodextrin-encapsulated ML349 nasal drops. Using hydroxypropyl-β-cyclodextrin (HP-β-CD) to encapsulate ML349 nasal drops (Comparative Example 1), 5xFAD heterozygous mice and wild-type (WT) mice were used as research subjects. Hyaluronic acid nasal pretreatment was combined throughout the process to improve mucosal penetration efficiency. First, ML349 encapsulated in 10% HP-β-CD was prepared, and its encapsulation rate and solubility were characterized. Simultaneously, HP-β-CD blank solvent and hyaluronic acid pretreatment solutions of corresponding concentrations were prepared. WT mice and 5xFAD mice were randomly divided into two subgroups: the HP-β-CD solvent control group and the HP-β-CD-encapsulated ML349 administration group. All animals underwent nasal pretreatment with hyaluronic acid before nasal administration for one month. After the intervention, the central biological effects of ML349 under this delivery strategy were systematically verified by detecting three dimensions: Tau protein palmitoylation modification level, glial activation and neuronal synaptic damage, and inflammatory factor transcriptional expression. Specific experimental results are as follows: Figure 3 As shown.

[0067] in, Figure 3 (B) Represents the effect of intranasal administration on the palmitoylation level of Tau protein in the brain. Palmitoylated Tau protein in the brain was enriched using the acyl-biotin exchange method (ABE method), and the level of palmitoylated Tau protein was detected by Western blotting with Tau1 antibody. The experiment included a hydroxylamine-treated group (+HA, specifically cleaving palmitoylation sites to achieve biotinylated enrichment) and a hydroxylamine-free negative control group (-HA) to verify the enrichment specificity. Total protein and GAPDH were used as the total Tau loading control and internal control, respectively. The results showed that compared with the WT group, the palmitoylated Tau level in the solvent control group of the 5xFAD model was significantly reduced, while the palmitoylated Tau level in the brain of 5xFAD mice was increased, indicating that the drug can effectively cross the blood-brain barrier to exert its target regulatory effect and reverse the abnormal palmitoylation modification of Tau protein.

[0068] Figure 3(C) Represents the effect of intranasal administration on glial activation, neuronal and synapse-related proteins. Western blotting was used to detect markers of neuroinflammation, Tau pathology, and neuronal synaptic integrity in the mouse brain, with GAPDH as the internal control. The detected markers included microglia activation marker IBA1, astrocyte activation marker GFAP, neuronal nuclear antigen NeuN, normal conformation Tau protein (Tau1), pathological Tau oligomer marker T22, and synaptic protein Syn-1. Results showed that compared with the WT group, the levels of IBA1 and GFAP proteins were significantly upregulated in the 5xFAD solvent control group, while the expression of NeuN, Tau1, and Syn-1 proteins was significantly downregulated, reflecting significant glial cell overactivation, Tau pathological progression, and neuronal synaptic damage in the model. After intranasal administration of the ML349 formulation, the levels of glial activation markers decreased, and the expression of neuronal and synapse-related proteins increased, confirming that this administration regimen can effectively improve central nervous system pathological damage in the AD model and exert a neuroprotective effect. However, the effect was not obvious.

[0069] Figure 3 (D) Represents the effect of intranasal administration on the transcriptional levels of inflammatory factors in brain tissue. Real-time quantitative PCR (qPCR) was used to detect the relative mRNA expression levels of inflammation-related factors in mouse brain tissue, quantifying the anti-inflammatory effect of intranasal administration. The detection indicators included pro-inflammatory factors IL-1β, IL-6, TNF-α, and the anti-inflammatory factor IL-10. Results showed that compared with the WT group, the mRNA levels of all pro-inflammatory factors were significantly increased in the 5xFAD solvent control group, with compensatory upregulation of the anti-inflammatory factor IL-10. After intervention with HP-β-CD-loaded ML349, the transcriptional levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α were downregulated to varying degrees, with the downregulation effect of TNF-α being the most significant, but the decrease was relatively small, approximately 30%.

[0070] Depend on Figure 3 It is known that although hydroxypropyl-β-cyclodextrin-encapsulated ML349 administered intranasally can have a certain intervention effect on pathological phenotypes such as abnormal palmitoylation of Tau and neuroinflammation in the brain of AD model, the improvement of core indicators is only about 30%, which is difficult to achieve a full reversal of central pathology.

[0071] (5) Fluorescent tracer for intrabrain delivery of ML349 carried by umbilical cord mesenchymal stem cell exosomes Atto655, a fluorescent dye, was loaded onto mesenchymal stem cell exosomes and administered to 5xFAD mice via intranasal instillation only twice. Brain tissue was harvested after administration and frozen sections were prepared. The distribution of fluorescence signals within the brain tissue was observed using a fluorescence microscope. The results showed that significant Atto655 fluorescence signals were detected in brain regions such as the olfactory bulb, hippocampus, and cortex, confirming that exosomes can efficiently bypass the blood-brain barrier and enter the brain via the intranasal route. Effective enrichment of brain tissue was achieved with only two intranasal instillations, further demonstrating the central delivery advantages of EVs as a carrier of ML349. Figure 7 As shown, Fluorescence imaging of frozen sections of brain tissue in the cerebral cortex and hippocampus (DAPI-labeled cell nuclei appear blue, and Atto655-traced exosomes appear red): There was almost no red fluorescence signal in the cortex and hippocampus of wild-type WT mice, while obvious Atto655 red fluorescence distribution could be observed in the cerebral cortex and hippocampus of 5xFAD model mice.

[0072] Depend on Figure 7 It can be seen that after only two nasal infusions, exosomes can be efficiently transported to key brain regions such as the cerebral cortex and hippocampus through the nasal cavity-brain pathway, which directly verifies that mesenchymal stem cell exosomes have excellent nasal delivery capabilities to the brain, and provides direct evidence for their use in loading ML349 to achieve central targeted therapy.

[0073] (6) Direct detection of APT2 target in brain tissue In this invention, ML349 was effectively delivered to brain lesions via nasal instillation, inhibiting the activity of the APT2 target enzyme in the brain. Specifically, the experimental animal groups were consistent with the previous pharmacodynamic studies: wild-type WT control group, 5xFAD model group, 5xFAD + empty EVs group, and 5xFAD + EVs + ML349 administration group. The inhibitor administration intervention was performed using the method described in Example 2 for one month. After animal sacrifice, target brain tissue from the hippocampus and cerebral cortex of mice was rapidly isolated, flash-frozen in liquid nitrogen, and stored at -80°C. Each brain tissue sample was divided into two parts: one part was used for Western blotting to detect the total protein expression of APT2 (LYPLA2); the other part was used to prepare a brain tissue protein lysate, and the APT2 enzyme activity assay kit was used to determine the catalytic activity of the brain tissue APT2 enzyme. Repeated freeze-thaw cycles were prohibited for the enzyme activity assay samples to prevent enzyme inactivation.

[0074] Western blot (WB) detection procedure: Total protein was extracted from brain tissue, subjected to SDS-PAGE electrophoresis and membrane transfer, and APT2 total protein expression was detected using APT2 / LYPLA2 specific antibody. GAPDH was used as an internal control.

[0075] APT2 enzyme activity assay: The APT2 hydrolase activity of brain tissue samples in each group was quantitatively detected strictly in accordance with the kit instructions.

[0076] The results showed that the APT2 enzyme activity in the brain tissue of 5xFAD model mice was significantly higher than that in the wild-type WT group, and the total APT2 protein expression level showed an upward trend. The empty exosome EVs intervention group had no significant inhibitory effect on APT2 protein expression and APT2 enzyme activity. After intervention with EVs-ML349 nasal drops, the APT2 enzyme activity in the brain tissue decreased significantly, while the total APT2 protein expression level did not change significantly.

[0077] Furthermore, these results are consistent with the mechanism of action of ML349: ML349 is an APT2 enzyme activity inhibitor that acts on the catalytic site of the APT2 protein, inhibiting the enzyme's catalytic function without degrading the APT2 protein. This confirms that after the drug-loaded exosomes are delivered into the brain via the nose, ML349 can directly inhibit the activity of the APT2 target enzyme in the brain, blocking APT2-mediated Tau protein depalmitoylation, thereby achieving a series of pharmacological effects including upregulating Tau palmitoylation, inhibiting neuroinflammation, protecting neuronal synapses, and improving cognitive function.

[0078] (7) Effects of ML349 nasal drops containing mesenchymal stem cell exosomes on cognitive impairment and brain inflammation in 5xFAD mice Using the APT2 inhibitor containing ML349 mesenchymal stem cell exosomes from Example 2, the aforementioned dosage form, a nasal drop, was used in 5xFAD model mice for in vivo efficacy evaluation, and its efficacy was compared with that of the HP-β-CD loading system. The experiment first isolated and cultured umbilical cord-derived mesenchymal stem cell exosomes, and completed intracellular loading of ML349 using ultrasonic co-incubation to characterize drug loading, encapsulation efficiency, cell viability, and in vitro drug release characteristics. Subsequently, the animals were divided into a wild-type control group, a 5xFAD group, a 5xFAD empty EVs group, and a 5xFAD+EVs+ML349 administration group, with an intervention period of one month. After the intervention, the central therapeutic effect of this cell-mediated drug delivery system was systematically verified by detecting three core dimensions: Tau protein palmitoylation level, glial activation and neuronal synaptic damage, and inflammatory factor transcription. The results are as follows: Figure 4 As shown, Figure 4 (B) represents the effect of intranasal administration on the palmitoylation level of Tau protein in the brain. The palmitoylated protein in the brain was enriched using the acyl-biotin exchange method (ABE). Compared with wild-type mice, the palmitoylation level of Tau protein in the brain of 5xFAD model mice was abnormally downregulated. Empty exosome intervention had no significant reversal effect on this abnormal modification, while exosomes loaded with ML349 significantly increased the palmitoylation level of Tau protein. This verifies the specific regulatory activity of ML349 on the APT2 target and also confirms that drug-loaded exosomes can effectively penetrate the blood-brain barrier and exert pharmacological effects in the brain.

[0079] Figure 4(C) Western blot results showed that, compared with the WT group, the 5xFAD model group exhibited downregulated expression of microglial activation markers IBA1 and Tau protein monomers, significantly downregulated expression of neuronal nuclear antigen NeuN, and an increasing trend in astrocyte activation marker GFAP, indicating clear neuroinflammation, Tau pathological progression, and neuronal synaptic damage. Empty exosome intervention could mildly alleviate the above pathological phenotypes; after exosome loading with ML349, microglial activation levels and pathological Tau content were further reduced, and the expression of neuronal and synapse-related proteins further increased, demonstrating a superior neuroprotective effect compared to exosomes alone.

[0080] Figure 4 (D) qPCR results showed that, compared with the WT group, the mRNA levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, as well as the anti-inflammatory factor IL-10, were significantly increased in the 5xFAD empty exosome group, reflecting an excessive inflammatory activation state in the brain. After intervention with exosomes loaded with ML349, the transcriptional levels of each pro-inflammatory factor were significantly downregulated, and the anti-inflammatory factor IL-10 also decreased synchronously, confirming that this drug delivery system can effectively inhibit excessive inflammatory responses in the brain and exert a clear anti-inflammatory biological activity.

[0081] Depend on Figure 4 In 5xFAD Alzheimer's disease model mice, compared with intervention using empty mesenchymal stem cell exosomes, exosome-loaded ML349 significantly improved multidimensional central nervous system pathological phenotypes: the mRNA levels of core pro-inflammatory factors IL-1β and TNF-α in the brain decreased by 50%–60%, and IL-6 decreased by 30%–35%; the protein levels of microglial activation markers IBA1 and pathological Tau oligomer T22 decreased by about 30%, and the astrocyte activation marker GFAP decreased by 20%–25%; at the same time, the palmitoylation level of Tau protein increased by 45%–50%, and the expression of neuronal and synapse-related proteins increased synchronously. The overall improvement was significantly better than the previous intervention effect of about 30% achieved by HP-β-CD-loaded intranasal administration, confirming that exosome carriers can effectively improve the central delivery efficiency and therapeutic benefit of ML349.

[0082] The inhibitor prepared in Example 2 of this invention is administered via nasal instillation. The drug carrier can be directly transported into the brain through the nasal cavity via the olfactory nerve pathway and the trigeminal nerve pathway. This method avoids the blood-brain barrier, gastrointestinal degradation, and the first-pass effect of the liver, making it a non-invasive drug delivery method that is easy to operate. The umbilical cord mesenchymal stem cell exosomes themselves have immunomodulatory and neuroprotective activities, which can synergistically enhance the pharmacological effects of ML349 in inhibiting APT2, further amplifying the therapeutic effect.

[0083] (8) Overall therapeutic value of ML349 nasal drops encapsulated with mesenchymal stem cell exosomes Using two classic behavioral experiments, the Y-maze and the Morris water maze, functional verification was conducted from the dimensions of short-term working memory and spatial learning memory, as detailed below: The experiment included a wild-type control group (WT), a 5xFAD model group, an empty exosome intervention group (5xFAD+EVs), and an exosome-loaded ML349 intervention group (5xFAD+EVs+ML349). The Y-maze experiment utilized the spontaneous exploration behavior of rodents in new environments, recording the mice's trajectories in the three arms of the maze. The spontaneous alternation rate reflected short-term working memory levels, and the total number of arm entries reflected the mice's autonomous activity ability. The Morris water maze experiment established spatial reference memory through continuous hidden platform training. In the spatial exploration test, the number of times mice crossed the original platform area, the escape latency upon reaching the original platform position, and the average swimming speed were recorded to evaluate spatial memory retention ability and spatial learning efficiency, respectively, and to exclude the interference of differences in motor function on the experimental results. Results are as follows: Figure 5-6 As shown, Depend on Figure 5 The Y-maze test results showed that, compared with wild-type mice, the total number of arm entries and the spontaneous alternation rate of 5xFAD model mice were significantly reduced, indicating that the model had a clear decline in the willingness to engage in spontaneous activities and short-term working memory impairment. The intervention with empty exosomes had no significant effect on improving either indicator. After intervention with exosomes loaded with ML349, the total number of arm entries of mice significantly increased, the spontaneous alternation rate was also significantly restored, and the short-term working memory deficit was effectively reversed.

[0084] Depend on Figure 6 The Morris water maze experiment showed that 5xFAD mice exhibited significant spatial learning and memory cognitive impairments. During the orientation navigation phase, the escape latency decreased very slowly with training. During the spatial exploration phase, the number of times mice crossed the original platform decreased significantly, while the latency to reach the original platform increased. Their swimming trajectory exhibited aimless, edge-walking characteristics, accompanied by a decrease in spontaneous swimming speed. The intervention with empty exosomes had limited effect on improving these cognitive deficits, with no statistically significant differences in any of the indicators compared to the model group. However, after ML349-loaded exosome intervention, the escape latency during orientation navigation significantly shortened with training, spatial learning efficiency significantly improved, the number of times mice crossed the original platform increased, the latency to reach the original platform shortened during the spatial exploration phase, and the proportion of exploration within the target quadrant significantly increased. Furthermore, the improvement in cognitive indicators far exceeded the recovery in swimming speed, confirming that this intervention specifically improves the spatial learning acquisition and memory retention abilities of the AD model, rather than relying on improvements in motor skills.

[0085] Based on the above, it can be seen that oral administration of free ML349: In 5xFAD mice, oral administration of ML349 for 1 month and 2 months did not significantly increase the level of palmitoylation of Tau protein in the brain; IBA1 and GFAP glial markers were not significantly downregulated, and the transcription of pro-inflammatory factors did not show consistent improvement; oral administration resulted in insufficient brain exposure and weak efficacy. 2) Tail vein injection of free ML349: LC-MS / MS pharmacokinetic analysis showed that the brain-plasma drug concentration ratio was only 0.74%; the cerebrospinal fluid drug concentration was far below the effective concentration required to inhibit APT2, making it difficult to exert a central therapeutic effect. 3) Hydroxypropyl-β-cyclodextrin-encapsulated ML349 intranasal administration: Although it can achieve partial drug delivery to the brain and has a slight improvement in AD pathology, the improvement in core pathological indicators is only about 30%; it has the defects of weak lesion targeting, short brain retention time, and no therapeutic gain of the carrier, resulting in limited therapeutic effect.

[0086] In summary, addressing the bottlenecks of low blood-brain barrier penetration efficiency of ML349 and limited central nervous system improvement after intranasal administration of hydroxypropyl-β-cyclodextrin, this invention constructs an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes. In 5xFAD Alzheimer's disease model mice, this inhibitor was demonstrated to be highly efficient in brain penetration via intranasal administration, effectively inhibiting APT2 enzyme activity in brain tissue, blocking APT2-mediated depalmitoylation of Tau protein, reversing abnormal Tau protein palmitoylation at the molecular pathological level, inhibiting excessive activation of microglia and astrocytes, downregulating the expression of core pro-inflammatory factors such as IL-1β and TNF-α (with a reduction of 50%–60%), and alleviating neuronal and synaptic damage. Functionally, it significantly improved short-term working memory and spatial learning memory in mice. The overall intervention effect was significantly superior to that of empty exosomes and previous HP-β-CD intranasal administration regimens, achieving multi-dimensional therapeutic benefits from APT2 target inhibition, molecular pathology, and cognitive function, providing a new and feasible strategy for central nervous system targeted therapy of Alzheimer's disease.

[0087] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. An application of the APT2 target, characterized in that, Inhibiting the APT2 target can treat Alzheimer's disease characterized by abnormal APT2 activation and reduced Tau protein palmitoylation levels.

2. An APT2 inhibitor for inhibiting the APT2 target in mesenchymal stem cell exosomes loaded with ML349 as described in claim 1, characterized in that, The inhibitor comprises the following components in parts by weight: 0.1-2 parts of umbilical cord-derived mesenchymal stem cell exosomes, 0.01-0.2 parts of APT2 small molecule inhibitor ML349, and 97.8-99.89 parts of sterile PBS buffer.

3. The APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes according to claim 2, characterized in that, The APT2 inhibitor is a nasal drop formulation.

4. The APT2 inhibitor loaded with ML349 on mesenchymal stem cell exosomes according to claim 2, characterized in that, The APT2 inhibitor is also accompanied by a nasal pretreatment solution, which, by weight, consists of 0.2 parts hyaluronic acid and the remainder being sterile PBS buffer.

5. A method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to any one of claims 2-4, characterized in that, Includes the following steps: S1. Isolation and purification of exosomes from umbilical cord-derived mesenchymal stem cells to obtain purified exosomes from umbilical cord mesenchymal stem cells; S2. The purified umbilical cord mesenchymal stem cell exosomes were mixed with ML349 and placed in an ice bath; the mixture was treated with pulsed sonication; after each sonication cycle, the sample was cooled on ice. After S3 sonication, the mixture was incubated at room temperature for 4 hours. After incubation, it was centrifuged to precipitate the drug-loaded exosome complex. The precipitate was washed with pre-cooled PBS buffer and centrifuged again under the same conditions. The supernatant was discarded and the precipitate was resuspended in PBS. S4 The encapsulation efficiency of ML349 was determined, the pH and osmotic pressure of the system were adjusted, and the system was aseptically filtered at 0.22μm and aseptically dispensed to obtain the nasal drop formulation. At the same time, a matching 0.2% hyaluronic acid sterile aqueous solution for nasal pretreatment was prepared.

6. The method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to claim 5, characterized in that, The specific steps for isolating and purifying umbilical cord-derived mesenchymal stem cell exosomes in step S1 are as follows: S1-1 Sample pretreatment: Collect the supernatant of umbilical cord mesenchymal stem cells after serum-free culture for 24 to 48 hours, centrifuge the supernatant at 300×g for 10 minutes at 4℃, then take the supernatant and centrifuge it at 2000×g for 20 minutes at 4℃. Filter the supernatant through a 0.22μm filter to complete the pretreatment. S1-2, TFF tangential flow filtration concentration: Place the inlet and reflux ends of the hollow fiber column into the pretreated feed container, and the permeate end into the waste liquid tank. Then start the peristaltic pump to start concentration. Set the inlet pressure to 30 psi, the outlet pressure to 10 psi, and the transmembrane pressure to about 20 psi. Continue concentration until the volume is reduced to one-twentieth of the original volume. S1-3, TFF washing and filtration: Immediately after concentration, start continuous washing and filtration. Adjust the inflow rate of sterile PBS buffer to match the outflow rate at the permeate end to achieve equal volume replacement. Continuously wash and filter 6 times the dialysis volume. After washing and filtration, collect the concentrate at the choked end for SEC loading. S1-4, SEC size exclusion chromatography purification: Select an appropriate qEV column according to the sample volume, equilibrate the SEC column to room temperature, remove the sliding cap at the bottom of the column, rinse the column with at least 10 mL of PBS elution buffer and observe whether the buffer flow rate is stable, then slowly add TFF concentrate to the top of the SEC column to start elution. The first 3 mL of elution is the void volume. Continue to add PBS for elution, collect the liquid after the void volume, and subsequently determine the exosome-enriched fractions by nanoparticle tracking analysis or 280 nm absorbance detection and combine them. S1-5. Final processing and storage: Combine the exosome enrichment fractions eluted from SEC, sterilize them using a 0.22μm filter, aliquot them into sterile cryovials according to experimental requirements, and store them at -80℃.

7. The method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to claim 5, characterized in that, The ultrasonic power in step S2 is 130W; the pulse mode is 10s of ultrasound, 10s of pause, repeated 3 times, with a total pulse processing time of 2min; after each ultrasonic cycle, the sample is placed on ice to cool for 2min.

8. The method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to claim 5, characterized in that, The centrifugation parameters in step S3 are: rotation speed 14000g, temperature 4℃, and time 30min.

9. The method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to claim 5, characterized in that, The amount of pre-cooled PBS buffer added in step S3 is 250 μL.

10. The method for preparing an APT2 inhibitor loaded with ML349 from mesenchymal stem cell exosomes according to claim 5, characterized in that, In step S4, the pH of the system is adjusted to 5.5-6.5; the osmotic pressure is 250-350 mOsm / kg.

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