A THERAPEUTIC COMPOSITION BASED ON THE CO-TRANSPORT OF ROYAL JELLY-DERIVED EXTRACELLULAR VESICS WITH MIR-1906 AND GALANGIN, SIMULTANEOUSLY SUPPRESSING CUPROPTOS, FERROPTOS, AND PYROPTOS IN ALZHEIMER'S DISEASE, AND THE METHOD OF ITS PREPARATION.

Abstract

The invention relates to a therapeutic compound obtained by co-loading hsa-miR-1906 mimic and galangin into extracellular vesicles (RJ-HDV) isolated from royal jelly, and to the method of preparing this compound. The median diameter of the vesicles in the compound is 89.1 nm, and the zeta potential is ≥32.1 mV; they contain CD63 and sintenin-1 but not calnexin. The compound reduces hsa_circ_0007556 (circAPP) levels by 75% and increases miR-1906 levels in the APPswe-SH-SY5Y Alzheimer's cell model; it eliminates 88% of the difference between diseased and healthy cells by simultaneously suppressing the coupeptosis, ferroptosis, and pyroptosis pathways. The effect obtained is 20 points above the expected value from the independent effects of the components. The compound maintains cell viability at 88% at the application dose.

Description

TARIFF ROYAL JELLY-DERIVED EXTRACELLULAR VESICLES WITH miR-1906 AND ALZHEIMER'S DISEASE BASED ON THE CO-TRANSPORT OF GALANGINE. IN HIS DISEASE, CUPROPTOS, FERROPTOS, AND PYROPTOS OCCUR SIMULTANEOUSLY. SUPPRESSIVE THERAPEUTIC COMPOSITION AND ITS PREPARATION METHOD TECHNICAL FIELD The invention involves a therapeutic compound for the treatment of the cellular pathology of Alzheimer's disease. This relates to a method for preparing this compound. More specifically, the invention involves extracellular vesicles isolated from royal jelly (thereof). (later to be referred to as RJ-HDV) contains hsa-miR-1906 mimic and a flavonol Obtained by co-loading galanji; hsa_circ_0007556 (circAPP) / miR-1906 Cuproptosis, ferroptosis, and NLRP3–gasdermin D-mediated pyroptosis via the regulatory axis. a therapeutic compound that simultaneously suppresses pathways and reduces amyloid-beta accumulation and relates to the production method of this compound. STATE OF THE ART Alzheimer's disease is associated with amyloid-beta (Aβ) deposition, tau pathology, and neuroinflammation. It is a characterized, progressive neurodegenerative disease. Current treatments are symptomatic. It does not stop the underlying cellular death mechanisms of the disease. In recent years, in addition to apoptosis, cuprotosis (copper-dependent, lipoylated neuronal loss) has been observed in neuronal loss. Cell death characterized by protein aggregation; FDX1, LIAS, and DLAT are key markers. Ferroptosis (iron-dependent lipid peroxidation; with loss of GPX4, increase in PTGS2 and 4-HNE) (observes) and pyroptosis (NLRP3 inflammasome–gasdermin D-mediated inflammatory cell death) It has been shown that these pathways play a role. These three pathways operate through different molecular mechanisms. And it cannot be suppressed adequately with a single active ingredient. In the current state of the technique, circular RNA derived from exons 4, 5, and 6 of the APP gene is used. circAPP (hsa_circ_0007556) suppresses miR-1906 by sponging, thereby inhibiting microglia. It has been reported to affect polarization and Alzheimer's pathology (Wu et al., 2025). However, this The study was conducted in mouse cell lines and the APP / PS1 mouse model, targeting the axis. No pharmaceutical formulation has been proposed. Galangin, a flavonol, has been shown to exhibit neuroprotective effects against Aβ-induced damage. It has been reported (Zhang et al., 2023). However, galangin has low solubility in water. It is a compound with limited bioavailability and restricted passage across the blood-brain barrier. Extracellular vesicles derived from royal jelly stimulate inflammatory cytokines in microglia. It has been shown to reduce (Zavala et al., 2025). However, RJ-HDVs act as miRNA transporters to neurons. 1 35 an application regarding its use and loading together with a flavonol It is unknown. Free miRNA mimics are rapidly degraded by nucleases and efficiently cross the cell membrane. It cannot pass through and can generate immunogenicity in systemic administration. Synthetic liposomes and Lipid nanoparticle carriers, however, have disadvantages such as cytotoxicity and liver accumulation. It carries. THE TECHNICAL PROBLEM THAT THE INVENTION INTENDED TO SOLVE In the current state of the technique, coupeptosis occurs concurrently with Alzheimer's disease. By initiating all ferroptosis and pyroptosis pathways in a single application, while preserving cell viability. There is no suppressive compound. Furthermore, the circAPP / miR-1906 axis is pharmacologically active in human neuronal cells. No biocompatible and naturally derived carrier system has been identified that enables this targeting. The invention aims to solve these problems by (i) using a naturally derived, biocompatible carrier. (ii) RJ-HDV, which is sponged by circAPP, miR-1906 and (iii) a multi-target By combining galangin, a flavonol, into a single compound, the individual effects of the components can be combined. It provides a significantly greater (synergistic) therapeutic effect than the sum of its components. BRIEF DESCRIPTION AND PURPOSE OF THE INVENTION The composition subject to the invention was obtained from royal jelly by sequential centrifugation and size exclusion chromatography (SEC). isolated cells with a median diameter of 89.1 nm, a modal diameter of 89.5 nm, and a zeta potential of −32.1 mV. external vesicles; into these vesicles, the hsa-miR-1906 mimic is loaded using an ultrasonic method. and contains galangin. The compound was used in the APPswe-SH-SY5Y Alzheimer's cell model, which is derived from human neuroblastoma. When applied, it reduces the difference between diseased and healthy cells by 88% (13 phenotypic) The average of the parameters (95% confidence interval 87–89%) eliminates the possibility of a pathway-based solution. The recovery rates were 90% in cuproptosis, 89% in pyroptosis, 87% in amyloid pathology, and in ferroptosis. It is 86%. The effect of the composition is the value expected from the independent effects of its components (Bliss independence). (model) is above 20 points (observed 88%; expected 68%; 95% confidence interval 18–22 points). This indicates that the combination exhibits a synergistic effect. The compound maintains cell viability at a level of 87.6% at a dose of 5 µg / mL (IC₅₀ = (27.4 µg / mL) and shows lower cytotoxicity than its components. Another technical effect of the invention is that no single component alone can increase the circAPP level of the composition. It suppresses to the extent that it cannot provide: circAPP, while 0.88 is in carrier control, triple composition. 2 In the application, it decreased to a level of 0.22 (75% reduction; P = 1.1 × 10⁸), whereas single agents⁻ And binary combinations do not produce this effect. BRIEF DESCRIPTION OF THE FIGURES Figure 1 — Physicochemical characterization of extracellular vesicles (1) derived from royal jelly. Figure In 1a, the horizontal axis represents the particle diameter (nm), and the vertical axis represents the particle concentration per 1 nm interval. (×10⁹ particles / mL) shows; (16) particle size distribution curve, (17) modal diameter. Figure In 1b, the horizontal axis represents the zeta potential (mV), and the vertical axis represents the particle number weighted by... It shows the percentage frequency; (18) zeta potential distribution, (19) weighted average zeta potential Figure 2 — Cytotoxicity of components and combinations in healthy neuronal cells. Horizontal axis concentration (logarithmic; µg / mL, nM for free mimicry), vertical axis no application Shows cell viability (%) compared to control. (20) dose-response curve, (21) 50% viability level, (22) study dose used in subsequent experiments. The curves are (1), (2), (3), (4), (5) and respectively. (6) is shown. Figure 3 — Response of the regulatory axis to treatment. The numbers on the horizontal axis in both figures. The application groups are shown on the vertical axis, indicating the relative expression level compared to the healthy control. The level of hsa_circ_0007556 is given in Figure 3a (10), and the level of hsa-miR-1906 is given in Figure 3b (11). Figure 4 — Markers of the coupeptose pathway (12). Figure 4a FDX1 mRNA, Figure 4b LIAS mRNA (relative expression), Figure 4c FDX1 protein and Figure 4d DLAT protein (ng / mL) levels. It shows. Figure 5 — Markers of the ferroptosis pathway (13). Figure 5a PTGS2 mRNA, Figure 5b SLC7A11 mRNA (relative expression), Figure 5c GPX4 protein and Figure 5d 4-hydroxynonenal adducts (ng / mL) It shows their levels. Figure 6 — Markers of the pyroptosis pathway (14). Figure 6a NLRP3 mRNA, Figure 6b GSDMD mRNA (relative expression), Figure 6c gasdermin D protein (ng / mL) and Figure 6d interleukin-1 beta (pg / mL) It shows their levels. Figure 7 — Amyloid-beta (15) release (pg / mL). Numbers on the horizontal axis represent the application groups. It shows; (24) diseased level, (25) healthy control level. Figure 8 — Pathway-based recovery rate (23). Numbers on the horizontal axis are respectively cuprotosis (12), ferroptosis (13), pyroptosis (14), amyloid-beta (15) and their average (23) It shows; the six columns above each number are respectively (1), (2), (3), (5), (6) and (4) It belongs to the applications. The vertical axis shows the correction rate (%); (25) healthy control level. 3 Figure 9 — Schematic diagram of the mechanism of action of the invention (figure to be published with the abstract). (26) within the cell, triple composition (4) prints hsa_circ_0007556 (10) via the regulatory axis and hsa-miR- It releases 1906 (11); in this way, cuproptosis (12), ferroptosis (13), pyroptosis (14) and amyloid- beta (15) pathways are suppressed, from diseased level (24) to healthy control level (25) improvement (23) is achieved. REFERENCE MARKS IN THE FIGURES 1. Royal jelly-derived extracellular vesicles (RJ-HDV) 2. hsa-miR-1906 mimicry 3. Galangin 4. Triple composition (RJ-HDV + hsa-miR-1906 mimic + galanjin) 5. RJ-HDV + hsa-miR-1906 mimic binary combination 6. RJ-HDV + galangin binary combination 7. Healthy control cells (SH-SY5Y) 8. Disease model cells (APPswe-SH-SY5Y) 9. Carrier control (dimethyl sulfoxide) 10. hsa_circ_0007556 (circAPP) level Level 11 hsa-miR-1906 12. Cuprotose pathway 13. Ferroptosis pathway 14. Pyroptosis pathway 15. Amyloid-beta 16. Particle size distribution curve 17. Modal particle diameter 18. Zeta potential distribution 19. Weighted average zeta potential 20. Dose-response curve 21. 50% cell viability level 22. Study dose 23. Correction rate 24. Diseased level 25. Healthy control level 26. Cell DETAILED DESCRIPTION OF THE INVENTION Definitions In this specification, the term 'RJ-HDV' refers to a lipid bilayer-enclosed, 2-diameter, isolated from royal jelly. It is in the 50–200 nm range and carries extracellular vesicle markers such as CD63 and sintenin-1. It is used for nanovesicles. The term 'triple compound' refers to RJ-HDV, hsa-miR-1906 mimicry, and It refers to the formulation containing galangin. 'Recovery rate', carrier (DMSO). It indicates the percentage of the difference between controlled and healthy SH-SY5Y control that is closed with treatment. 4 35 Isolation of extracellular vesicles Royal jelly samples are stored at +4 °C. Sequential centrifugation procedures are applied: 10 at 1,000 × g. 10 minutes (removal of large particles), 10 minutes at 2,000 × g (removal of smaller debris) (removal of large vesicles and protein clumps) and at 10,000 × g for 30 minutes. (removal). The resulting supernatant is passed through a 0.22 µm membrane filter. Purification is performed by size exclusion chromatography. Cold phosphate buffered salt solution (PBS) 10 mL of filtered sample is loaded into a qEV10 column balanced with PBS and eluted. This is carried out. The collected fractions are centrifuged at 4,000 × g at 20 °C for approximately 40 minutes. It is concentrated to 2 mL. The eluate collected after vesicle fractionation is separated from the vesicle. The purified supernatant is stored as a control. Characterization of vesicles Nanoparticle tracking analysis (NTA) revealed a single, narrow particle population: modal. Diameter 89.5 nm, numerical weighted average 89.3 ± 6.2 nm, D10 / D50 / D90 values ​​respectively. The particle sizes are 80.6 / 89.1 / 95.5 nm and the spacing is 0.17. All detected particles are between 50–132 nm. It is within the range and the particle concentration is 4.92 × 10¹⁰ after a 1:500 dilution correction. particles / mL (Figure 1a). The particle-weighted zeta potential, measured by electrophoretic light scattering, is −32.1 ± 6.0 mV. It is compatible with a colloidally stable, negatively charged vesicle suspension (Figure 1b, 1c). In Western blot analysis, extracellular vesicle markers CD63 and sintenin-1 (32–33 kDa) RJ- Detected in the HDV fraction, but not in the vesicle-free supernatant. The endoplasmic reticulum marker calnexin, however, showed the opposite distribution; RJ-HDV It was not detected in the fraction but was detected in the supernatant (Figure 1e–1g). These findings indicate that the preparation It shows that it does not contain endoplasmic reticulum contamination. Loading of active ingredients into vesicles Galangin (CAS 548-83-4; molecular weight 270.24 g / mol) 10 mM in dimethyl sulfoxide It is prepared as a stock solution at a certain concentration. hsa-miR-1906 mimics 5′- It has the sequence UGAGGAGAUCGUCGAGGUUGG-3′ (SEQ ID NO: 1). RJ-HDV, galangin, and miR-1906 are mixed in ratios of 1:1, 1:2, 1:4, 1:8, and 1:10, and YH- 250 ultrasonic cell disruptors are subjected to sonication at a power of 75 W. The sonication... The suspensions are then immediately cooled on ice and left for 30 minutes; after that The vesicle is incubated at 37°C to restore membrane integrity. Unloaded Free galangin and mimic are removed by centrifugation at 12,000 × g for 10 minutes. Loading efficiency. (YV) is calculated using the following formula: YV (%) = (loaded substance − unloaded substance) / loaded item × 100. The mimic is encapsulated within a vesicle, with RNase administration versus RNase + membrane disruptor administration. This is confirmed by comparison: RNase application alone has little effect on the signal, while membrane signaling with RNase is significantly affected. The combined application of the disruptor significantly reduces the signal. Dose determination and biocompatibility. Cytotoxicity was measured as 0–100 in SH-SY5Y cells after 24 hours of application using the MTT method. Evaluated in the range of µg / mL (0–100 nM for free mimicry). Four-parameter logistic analysis. IC₅₀ values ​​obtained with the model were 27.4 µg / mL for the ternary compound and 21.0 µg / mL for RJ-HDV. galangin 20.6 µg / mL, RJ-HDV+galangin 13.9 µg / mL, RJ-HDV+miR-1906 7.5 The concentration is 6.2 nM for µg / mL and for the free miR-1906 mimic (Figure 2c). The triple compound is the lowest. It is a preparation that exhibits cytotoxicity. Based on these data, the study dose is 5 µg / mL for all vesicle-based preparations and galangin. For free mimicry, the concentration was determined to be 2 nM. At these doses, cell viability ranged from 82.7% to 88.5%. It is among them and is above the 80% threshold accepted for biocompatibility (Figure 2d). Thus The pathway changes reported below have been shown not to be secondary to cell death. Cell model and application groups As a healthy control, the human neuroblastoma cell line SH-SY5Y was used for amyloid-based Alzheimer's disease. SH-SY5Y-APPswe cells, which stably express the APP Sweden mutation, are used as a model. The line was used. Nine groups, with n = 7 independent biological replicates in each group. The following were evaluated: SH-SY5Y, APPswe, carrier (DMSO), RJ-HDV, miR-1906, galanjin, RJ- HDV+miR-1906, RJ-HDV+galangine and triple combination. Example 1 — Effect on the regulatory axis The triple combination treatment reduced the circAPP level from 0.88 in carrier control to 0.22 ± 0.07. This effect was reduced to the level of RJ-HDV+miR-1906 (0.73; P = 1.5 × 10⁸). RJ-HDV+galangine (0.81; P = 5.1 × 10⁷) was significantly higher than all other groups⁻ It is large. Galangin alone did not alter the circAPP level (0.90). In contrast, miR- The 1906 level is 2.75 times higher when mimicry is applied alone, and when applied together with RJ-HDV. It increased 7.07 times, which shows that RJ-HDV enhances mimicry transmission. Example 2 — Cuprotose pathway The triple combination reduced FDX1 mRNA levels from 7.07 to 1.79 (P = 4.4 × 10⁵), and LIAS mRNA levels⁻ from 8.20 to 1.69 (P = 1.6 × 10⁴), increasing FDX1 protein from 90.7 ng / mL to 34.1 ng / mL (P = 9.8 ×⁻ ⁹) and reduced DLAT protein from 23.5 ng / mL to 4.1 ng / mL (P = 5.7 × 10 ¹²). Pathway⁻ ⁻ The recovery rate is 90%, significantly exceeding the 60% value in the best pair combination. It is located on (Figure 4). Example 3 — Ferroptosis pathway 6 The triple combination increased GPX4 protein from 25.1 ng / mL to 83.5 ng / mL (P = 6.5 × 10⁻¹) and⁻ This value has become indistinguishable from the healthy SH-SY5Y level (84.6 ng / mL). PTGS2 mRNA, SLC7A11 mRNA and 4-HNE levels were decreased by 80%, 77%, and 65%, respectively. Pathway The recovery rate is 86% (Figure 5). Example 4 — Pyroptosis pathway The triple compound contains 80% NLRP3 mRNA, 81% GSDMD mRNA, and 80% gasdermin D protein. and reduced IL-1β release by 87% (P = 1.6 × 10⁹ or less for all). Pathway⁻ The recovery rate is 89% (Figure 6). Example 5 — Amyloid-beta and integrated phenotype Amyloid-beta release decreased from 535 pg / mL to 89 pg / mL under carrier control. decreased (83% decrease; P = 5.4 × 10⁹). Based on z-scores of thirteen phenotypic parameters⁻ The calculated multivariate disease distance was reduced by 86% compared to carrier control. (Figure 7). Example 6 — Synergy analysis Expected rate of recovery when components act independently (BLISS independence) The model score was 68%, while the observed value was 88%. The 20-point difference is statistically significant. It is significant (95% confidence interval 18–22 points). The triple combination also shows the best pairwise combination of 26. The score exceeds 27 points. Synergy was most clearly observed in the cuprotosis pathway (+27 points; Figure 8). Technical effects provided by the invention (i) Simultaneous suppression of three separately regulated cell death pathways with a single application; (ii) a synergistic improvement significantly greater than that expected from the independent effects of its components; (iii) suppression of circAPP to an extent not achievable by single components; (iv) synthetic carriers (v) use of a naturally derived and biocompatible carrier system compared to the study dose Maintaining over 80% cell viability; (vi) galangin, which has low water solubility. Transported within vesicles and delivered to the cell. INDUSTRIAL APPLICABILITY The invention relates to a therapeutic compound aimed at the cellular pathology of Alzheimer's disease. It can be produced and used in the pharmaceutical industry. The compound also includes cuprotose, ferroptose, and For research purposes in other neurodegenerative conditions in which pyroptosis pathways play a co-role. It can be used as a reference composition. 7 Production method, standard laboratory and production equipment (centrifuge, size exclusion It can be implemented with a chromatography column (ultrasonic cell scavenger) and is scalable. It is of a certain quality. REFERENCES Wu, D.-P., Wei, Y.-S., Hou, L.-X. et al. Circular RNA APP contributes to Alzheimer's disease pathogenesis by modulating microglial polarization via miR-1906 / CLIC1 axis. Alzheimer's Research & Therapy, 17, 44 (2025). Urdánoz-Casado, A., Sánchez-Ruiz de Gordoa, J., Robles, M. et al. circRNA from APP Gene Changes in Alzheimer's Disease Human Brain. International Journal of Molecular Sciences, 24(5), 4308 (2023). Zhang, R., Lu, J., Pei, G., Huang, S. Galangin Rescues Alzheimer's Amyloid-β Induced Mitophagy and Brain Organoid Growth Impairment. International Journal of Molecular Sciences, 24(4), 3398 (2023). Zavala, G., Berríos, P., Sandoval, F. ve ark. Royal jelly derived extracellular vesicles modulate microglial nanomechanics and inflammatory responses. Nanoscale (2025). Théry, C. ve ark. Minimal information for studies of extracellular vesicles 2018 (MISEV2018). Journal of Extracellular Vesicles, 7 (2018). 8

Claims

REQUESTS 1. It is a therapeutic compound whose characteristic is that it consists of extracellular vesicles (RJ-) isolated from royal jelly. HDV), the hsa-miR-1906 mimic loaded into these vesicles, and also these vesicles It contains galangin along with the loaded galangin.

2. The composition is based on claim 1 and its characteristic is; nanoparticle tracking of the said extracellular vesicles. The median diameter (D50) measured by analysis was 89 ± 10 nm, the modal diameter was 90 ± 10 nm, and the distribution was... its width (span) must be less than 0.

30.

3. The composition is according to claim 1 or 2, and its characteristic is that the extracellular vesicles in question are zeta- Its potential is between -25 mV and -40 mV.

4. It is a composition according to any of the previous requirements, and its characteristic is that it is an extracellular component. The vesicles contain CD63 and sintenin-1 markers but no calnexin.

5. It is a combination according to any of the previous requests, and its characteristic is; the aforementioned hsa-miR-1906 The sequence of mimicry is given by SEQ ID NO: 1, 5′-UGAGGAGAUCGUCGAGGUUGG-3′ It is having.

6. It is a compound according to any of the previous requirements, and its characteristic is; extracellular vesicles, galangin. and the hsa-miR-1906 mimic is found in a mass ratio between 1:1 and 1:

10.

7. It is a composition according to any of the previous requirements, and its characteristic is; to the cell culture medium. When applied, the extracellular vesicle concentration ranged from 1 µg / mL to 10 µg / mL, hsa-miR- The concentration of the mimicry spectrum in 1906 is between 0.5 nM and 5 nM.

8. It is a combination according to any of the previous requirements, and its characteristic is; Alzheimer's disease. In cellular pathology, the coupptosis, ferroptosis, and pyroptosis pathways occur simultaneously. It is used for suppression.

9. Composition according to any of the previous requirements, its characteristic is; amyloid precursor protein Sweden to reduce hsa_circ_0007556 (circAPP) levels in neuronal cells carrying the mutation and is used to increase the level of hsa-miR-1906.

10. A method for preparing a composition according to any of claims 1 to 9, and its characteristic is... (a) the royal jelly sample is heated at 1,000 × g for 10 minutes, then 2,000 (b) centrifugation at 10,000 × g for 10 minutes and at 10,000 × g for 30 minutes; (b) the resulting supernatant (c) size exclusion of the filtered sample after passing through a 0.22 µm membrane filter. (d) purification by chromatography and collection of vesicle-containing fractions; (e) concentration of the fractions by centrifugation; extracellular vesicles obtained (f) mixing with galangin and hsa-miR-1906 mimics; sonication of the mixture by ultrasonic treatment. (g) cooling the suspension on ice after sonication and then 9 Incubate at 37 °C to restore vesicle membrane integrity; (h) Removal of unloaded free galangin and mimic by centrifugation.

11. Method according to claim 10, its characteristic is that the sonication process in step (f) has a power of 75 W. The procedure is to perform it and the waiting time on ice in step (g) is 30 minutes.

12. Method according to claim 10 or 11, its characteristic is; loading efficiency, YV (%) = (loaded The amount of material loaded is determined using the formula: (matter - unloaded material) / loaded material × 100.

13. The method is according to any of claims 10 to 12, and its characteristic is; hsa-miR-1906 mimicry. It was found to be encapsulated within a vesicle, and only with the sample treated with RNase, and with RNase and membrane disruptor. This is verified by comparing the examples applied together.

14. According to any of claims 1 to 9, the composition is associated with cellular Alzheimer's disease. Its use in the preparation of a drug for the treatment of its pathology.

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