A 10-hydroxy-2-decenoic acid nasal inclusion solution preparation composition and its application in the preparation of a drug for treating ischemic stroke

Abstract

The application discloses a 10-hydroxy-2-decenoic acid (10-HDA) nose solution agent for treating ischemic stroke or cerebral ischemia-reperfusion injury, and solves the problems of poor stability of 10-HDA ordinary aqueous solution and limited brain tissue delivery efficiency of oral suspension preparation. The application adopts hydroxypropyl-beta-cyclodextrin and other inclusion adjuvants to include 10-HDA, and cooperates with a dissolving agent, a surfactant and an isotonic solvent to prepare a nose inclusion solution agent, so as to improve the aqueous stability and nasal administration uniformity. Compared with the oral suspension preparation, the dosage form can improve the plasma and brain tissue exposure level under the same administration dose condition, can improve the neural function defect in the MCAO / R model earlier, can reduce the cerebral edema, blood-brain barrier injury and inflammatory reaction, and has the advantages of high brain tissue delivery efficiency, early pharmacodynamic effect and simple administration operation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation and biomedical technology, specifically relating to a nasal inclusion solution formulation composition with 10-hydroxy-2-decenoic acid as the active ingredient and hydroxypropyl-β-cyclodextrin as the inclusion excipient, and the application of the composition in the preparation of a drug for intranasal administration to treat ischemic stroke or cerebral ischemia-reperfusion injury. Background Technology

[0002] Stroke is a serious disease caused by the sudden rupture or blockage of cerebral blood vessels, resulting in acute brain damage. It mainly includes ischemic stroke and hemorrhagic stroke, with ischemic stroke accounting for approximately 70%–80% of all strokes. Stroke is characterized by rapid onset, high mortality, high disability rate, and high recurrence rate, and is one of the leading causes of death and long-term disability worldwide, imposing a heavy burden on society and families. Studies have shown that during cerebral ischemia and reperfusion, the structure and function of the blood-brain barrier are significantly disrupted, with a marked increase in permeability, leading to extravasation of plasma components, exacerbation of cerebral edema, and massive infiltration of inflammatory cells. This is an important pathological basis for the continuous deterioration of neurological function and hemorrhagic transformation. Therefore, stabilizing the integrity of the blood-brain barrier, inhibiting secondary inflammatory responses, and reducing cerebral edema as early as possible during the acute phase of stroke and the early stage of reperfusion are of significant clinical importance for improving the neurological prognosis of patients.

[0003] 10-Hydroxy-2-decenoic acid (10-HDA) is a characteristic unsaturated fatty acid component of royal jelly, possessing certain anti-inflammatory, antioxidant, and immunomodulatory activities. However, current technologies have not systematically elucidated the mechanism of action of 10-HDA in protecting the structure and function of the blood-brain barrier during the acute phase of stroke, especially under conditions of cerebral ischemia-reperfusion injury, and systematic studies on its application in acute stroke intervention are also lacking. Furthermore, for stroke emergency scenarios, there is a lack of suitable dosing strategies for 10-HDA that can produce earlier pharmacodynamic improvements, limiting its clinical application potential in stroke treatment.

[0004] Currently, oral administration is commonly used for drug intervention in stroke in clinical and experimental studies. However, oral administration can be affected by factors such as gastrointestinal absorption, metabolism, and the blood-brain barrier, limiting the systemic and brain tissue exposure of the active ingredient. Nasal administration, with its advantages of convenient administration and small dosage volume, is one of the research directions for drug delivery in central nervous system diseases. Therefore, it is necessary to develop a stable aqueous formulation suitable for 10-HDA nasal administration and evaluate its in vivo exposure and pharmacodynamic performance.

[0005] 10-HDA, a fatty acid compound, exhibits limited solubility and dispersion stability in aqueous systems. Direct preparation of ordinary aqueous solutions often results in turbidity, undissolved matter, crystallization, precipitation, or stratification, failing to meet the requirements for homogeneity, stability, and precise dosage volume in nasal drops. Current animal experiments frequently employ suspension media such as sodium carboxymethyl cellulose to prepare oral suspensions of 10-HDA. However, these formulations are heterogeneous systems, primarily suitable for gavage administration, and not for direct nasal administration. Furthermore, their central delivery efficiency in the acute phase of stroke remains insufficient. Therefore, developing a stable aqueous formulation of 10-HDA suitable for nasal administration, and improving its plasma exposure, brain tissue exposure, and pharmacodynamic improvement rate under the same dosage conditions, is a pressing technical challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to address the problems of limited water solubility of 10-HDA, poor stability of ordinary aqueous solutions, unsuitability of oral suspension formulations for nasal administration, and limited brain tissue delivery efficiency. This invention provides a 10-HDA nasal inclusion solution formulation composition to improve the solubility and dispersion of 10-HDA in aqueous systems, making it suitable for small-volume nasal administration, increasing plasma and brain tissue exposure levels after administration, thereby enhancing the therapeutic effect in the acute phase of stroke.

[0007] The structural formula of the 10-hydroxy-2-decenoic acid (10-HDA) described in this invention is as follows:

[0008]

[0009] Formula I;

[0010] Molecular formula: C 10 H 18 O3; Molecular weight: 186.25.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] A nasal inclusion solution formulation composition of 10-hydroxy-2-decenoic acid, the formulation composition comprising 10-hydroxy-2-decenoic acid, hydroxypropyl-β-cyclodextrin, propylene glycol, Tween-80 and physiological saline solution, wherein the mass ratio of 10-hydroxy-2-decenoic acid to hydroxypropyl-β-cyclodextrin is 1:4 to 1:12.

[0013] Preferably, the mass ratio of the above-mentioned 10-hydroxy-2-decenoic acid to the hydroxypropyl-β-cyclodextrin is 1:6 to 1:10.

[0014] Preferably, the mass ratio of the above-mentioned 10-hydroxy-2-decenoic acid to hydroxypropyl-β-cyclodextrin is 1:8.

[0015] Preferably, the pH of the above-mentioned formulation composition is 4.5 to 6.5.

[0016] Preferably, the pH of the above-mentioned formulation composition is 5.0 to 6.0.

[0017] The above-mentioned formulation composition is a solution-type nasal drops.

[0018] Preferably, the above-mentioned formulation composition includes 0.5 g of 10-hydroxy-2-decenoic acid, 4.0 g of hydroxypropyl-β-cyclodextrin, 0.5 mL of propylene glycol and 0.05 mL of Tween-80, and is brought to a final volume of 10 mL with physiological saline solution, and the pH is adjusted to 5.0-6.0.

[0019] The above-mentioned 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition is used in the preparation of a medicament for intranasal administration to treat ischemic stroke or cerebral ischemia-reperfusion injury.

[0020] The above-mentioned ischemic stroke or cerebral ischemia-reperfusion injury is accompanied by neurological deficits, cerebral infarction, cerebral edema and / or blood-brain barrier damage.

[0021] The nasal delivery formulation composition comprises the following components:

[0022] The 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition comprises 10-hydroxy-2-decenoic acid, hydroxypropyl-β-cyclodextrin, propylene glycol, Tween-80, and physiological saline.

[0023] The 10-hydroxy-2-decenoic acid is the active ingredient in the formulation composition;

[0024] The hydroxypropyl-β-cyclodextrin is an inclusion excipient used to include 10-hydroxy-2-decenoic acid in an aqueous system to improve its solubility and dispersion.

[0025] The propylene glycol is used as a cosolvent and humectant.

[0026] Tween-80 is a surfactant used to improve the wettability and homogeneity of the formulation.

[0027] The physiological saline is used as the aqueous solvent and isotonic adjustment medium for the preparation;

[0028] In some embodiments of the present invention, the mass ratio of the 10-hydroxy-2-decenoic acid to the hydroxypropyl-β-cyclodextrin is 1:4 to 1:12.

[0029] In some preferred embodiments of the present invention, the mass ratio of the 10-hydroxy-2-decenoic acid to the hydroxypropyl-β-cyclodextrin is 1:6 to 1:10;

[0030] In a further preferred embodiment of the present invention, the mass ratio of the 10-hydroxy-2-decenoic acid to the hydroxypropyl-β-cyclodextrin is 1:8;

[0031] In some embodiments of the present invention, the formulation composition is a solution-type nasal drop, suitable for administration via nasal instillation;

[0032] In one specific embodiment of the present invention, the formulation composition comprises 0.5 g of 10-hydroxy-2-decenoic acid, 4.0 g of hydroxypropyl-β-cyclodextrin, 0.5 mL of propylene glycol and 0.05 mL of Tween-80, and is brought to a final volume of 10 mL with physiological saline.

[0033] In the above specific embodiments, the concentration of 10-hydroxy-2-decenoic acid in the formulation composition is 50 mg / mL, the concentration of hydroxypropyl-β-cyclodextrin is 400 mg / mL, and the mass ratio of 10-hydroxy-2-decenoic acid to hydroxypropyl-β-cyclodextrin is 1:8.

[0034] The above components, after mixing and inclusion treatment, form a homogeneous aqueous formulation. Under the observation conditions described in the examples, the resulting formulation is a clear, transparent or slightly opalescent homogeneous liquid, with no obvious precipitation, crystallization, or stratification observed.

[0035] The present invention also provides a method for preparing the above-mentioned nasal administration formulation composition, comprising the following steps:

[0036] Step (1) Dissolution and preparation of base solution:

[0037] Weigh 4.0 g of hydroxypropyl-β-cyclodextrin and place it in a clean beaker. Add 8 mL of physiological saline and stir under magnetic stirring (200-500 r / min, preferably 300 r / min) until completely dissolved to obtain a clear and transparent inclusion excipient solution.

[0038] Step (2) Inclusion reaction:

[0039] Accurately weigh 0.5 g of 10-hydroxy-2-decenoic acid and slowly add it to the above-mentioned inclusion excipient solution. Stir at 37°C for 2 h to include 10-HDA with hydroxypropyl-β-cyclodextrin, thereby improving the solubility and dispersion of 10-HDA in the aqueous system.

[0040] Step (3) Solubilization and Volume Adjustment:

[0041] After the reaction was complete, the system was cooled to room temperature, 0.5 mL of propylene glycol and 0.05 mL of Tween-80 were added, and the mixture was stirred for 10 min. Then, physiological saline was added to bring the total volume to 10 mL to ensure the system was well mixed.

[0042] Step (4) pH measurement and adjustment:

[0043] The pH value of the above solution is determined using a calibrated pH meter. If the pH value is not in the range of 4.5 to 6.5, an appropriate amount of dilute hydrochloric acid solution or sodium hydroxide solution (such as 0.1 mol / L or 0.01 mol / L) is added for fine adjustment, and the solution is stirred until it is fully mixed until the pH reaches the target range; preferably, the pH is adjusted to 5.0 to 6.0.

[0044] Step (5) Microfiltration:

[0045] The obtained formulation was filtered through a 0.22 μm microporous membrane to remove visible or trace amounts of undissolved matter, resulting in a clear and transparent 10-HDA nasal solution.

[0046] Step (6) Dispensing and Storage:

[0047] Under aseptic conditions, the filtrate was dispensed into brown, light-protected spray bottles or microsyringe-compatible storage tubes. The resulting formulation was stored at 4°C in the dark for subsequent animal experiments.

[0048] Under the observation conditions described in this embodiment, the formulation can maintain good appearance uniformity, with no obvious precipitation, crystallization or stratification observed.

[0049] In animal experiments, the oral and nasal formulations are administered at equal doses based on 10-HDA, with a preferred dose of 150 mg / kg. The oral formulation is administered by gavage, while the nasal formulation is administered by intranasal drops in multiple doses into both nostrils. The volume of nasal administration is controlled according to the nasal cavity tolerance of mice, with 10 μL per side per dose to avoid aspiration and choking.

[0050] The 10-HDA nasal encapsulation solution prepared using the above formulation and method can improve the plasma and brain tissue exposure levels of 10-HDA. In the MCAO / R ischemic stroke model, the nasal formulation showed earlier improvement in neurological function compared to the oral formulation, and could reduce blood-brain barrier permeability, alleviate cerebral edema, and inhibit inflammatory responses, demonstrating advantages such as high brain tissue delivery efficiency and earlier pharmacodynamic onset.

[0051] Beneficial effects:

[0052] This invention improves the solubility and appearance stability of 10-HDA in aqueous systems, making it suitable for nasal drops or sprays; compared with oral suspensions of the same dose of 10-HDA, the nasal formulation increases plasma and brain tissue exposure levels and shows neurological function improvement at an earlier observation time point.

[0053] The 10-HDA nasal inclusion solution formulation of this invention better improves neurological deficits, reduces cerebral infarction and cerebral edema, and improves blood-brain barrier and brain tissue inflammation-related indicators in the MCAO / R model. These effects are accompanied by increased expression of ZO-1, Occludin, and Claudin-5, and decreased expression of MMP-9. Attached Figure Description

[0054] Figure 1 This is a comparative graph of plasma and brain tissue drug exposure levels under different formulations administered according to application 10-HDA. (A) Plasma drug concentration-time curve; (B) Plasma AUC statistical results; (C) Brain tissue drug concentration-time curve; (D) Brain tissue AUC statistical results. ig indicates gavage administration, and in indicates nasal administration. One-way ANOVA analysis was used. P < 0.05 P<0.01, P<0.001, P<0.0001;

[0055] Figure 2 This figure shows the results of the nasal formulation of 10-HDA in this application improving neurological deficits and motor coordination in MCAO / R mice earlier than the oral formulation. (A) Dynamic changes in neurological deficit scores at different administration time points; on day 3 of administration, the difference between the nasal formulation group and the model group was statistically significant, while the difference between the oral formulation group and the model group was not statistically significant; (B) Neurological deficit scores on day 5 of administration; (C) Latency of the rotarod test. One-way ANOVA analysis was used. P<0.05, P<0.01, P<0.001, P<0.0001;

[0056] Figure 3 This application describes how 10-HDA reduced cerebral infarction area and neuronal apoptosis in mice with MCAO / R modeling of ischemic stroke. Images show (A, B, C) TTC staining and percentages of cerebral infarction and cerebral edema; and (D, E) images and percentages of neuronal apoptosis. One-way ANOVA analysis was used. P<0.05, P<0.01, P<0.001, P<0.0001;

[0057] Figure 4This application describes how 10-HDA reduces blood-brain barrier permeability in mice with ischemic stroke induced by MCAO / R. The results include: (A) brain tissue water content; (B) Western blot images of ZO-1, Occludin, and Claudin-5; (C) relative quantitative analysis of ZO-1 expression; (D) relative quantitative analysis of Occludin expression; (E) relative quantitative analysis of Claudin-5 expression; (F) Western blot images of MMP-9; and (G) relative quantitative analysis of MMP-9 expression. One-way ANOVA analysis was used. P<0.05, P<0.01, P<0.001, P<0.0001;

[0058] Figure 5 This application describes how 10-HDA improves neuroinflammation in mice with ischemic stroke induced by MCAO / R, specifically by reducing the levels of IL-6, IL-1β, and TNF-α in brain tissues of (A, B, and C) mice. One-way ANOVA analysis was used. P<0.05, P<0.01, P<0.001, P<0.0001. Specific Implementation

[0059] The present invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the following embodiments are only for illustrating the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents, consumables, and instruments used in the implementation process can be conventional commercial products in the field or their equivalent substitutes.

[0060] The "effective dose" described in this specification refers to the dosage that enables 10-HDA to produce a detectable pharmacodynamic effect in animal models of ischemic stroke or cerebral ischemia-reperfusion injury. The effective dose is not fixed and its specific value can be adjusted based on factors such as the species, body weight, disease state, dosing time window, and dosing regimen of the experimental subjects. Those skilled in the art can reasonably determine this dose without departing from the spirit of this invention.

[0061] The phrase "earlier onset of action" or "earlier pharmacodynamic onset of action" in this specification refers to the fact that, under the same dosage conditions, compared with the oral formulation of 10-HDA, the nasal formulation of 10-HDA in this application shows improvements in neurological deficit scores, motor coordination, or other pharmacodynamic indicators at an earlier observation time point in the MCAO / R ischemic stroke animal model. "Earlier onset of action" does not refer to an earlier time to peak plasma or brain tissue drug concentration (Tmax), but rather to an earlier time to improvement of the pharmacodynamic endpoint.

[0062] Example 1: Preparation of oral suspension and nasal solution of 10-HDA

[0063] a) Preparation process of 10-HDA oral suspension:

[0064] 10-Hydroxy-2-decenoic acid 0.5 g (Active drug)

[0065] 10 mL sodium carboxymethyl cellulose suspension medium

[0066] Preparation process: Weigh 0.5 g of 10-HDA, add an appropriate amount of 0.5% sodium carboxymethyl cellulose solution and grind to disperse. Transfer to a volumetric container and make up to 10 mL with 0.5% sodium carboxymethyl cellulose solution. Vortex or sonicate to form a uniform suspension. Shake well before use.

[0067] b) Preparation process of 10-HDA nasal solution:

[0068] 10-Hydroxy-2-decenoic acid 0.5 g (Active drug)

[0069] Hydroxypropyl-β-cyclodextrin 4.0 g, including excipients and solubilizers

[0070] Propylene glycol 0.5 mL, as a solubilizer and humectant;

[0071] Tween-80 0.05 mL surfactant

[0072] Adjust the volume of physiological saline to 10 mL; solvent and isotonic adjuster;

[0073] Preparation process: Weigh 4.0 g of hydroxypropyl-β-cyclodextrin into a clean container, add 8 mL of physiological saline, and stir until completely dissolved under magnetic stirring. Weigh 0.5 g of 10-hydroxy-2-decenoic acid and slowly add it to the above hydroxypropyl-β-cyclodextrin solution. Stir continuously at 37℃ for 2 h to encapsulate 10-hydroxy-2-decenoic acid with hydroxypropyl-β-cyclodextrin, thereby improving the solubility and dispersion of 10-hydroxy-2-decenoic acid in the aqueous system. After the system cools to room temperature, add 0.5 mL of propylene glycol and 0.05 mL of Tween-80, continue stirring for 10 min, and then bring the volume to 10 mL with physiological saline. Measure the pH value of the above solution using a calibrated pH meter, and finely adjust by adding an appropriate amount of dilute hydrochloric acid solution or sodium hydroxide solution (e.g., 0.1 mol / L or 0.01 mol / L), stirring until fully mixed, until the pH is adjusted to 5.0–6.0. A homogeneous 10-hydroxy-2-decenoic acid nasal inclusion solution was obtained by filtration through a 0.22 μm microporous membrane to remove visible or trace amounts of undissolved matter. The resulting formulation was stored at 4°C in the dark for use in animal experiments.

[0074] Example 2: Comparison of in vivo exposure and brain tissue delivery of oral and nasal formulations of 10-HDA

[0075] Male C57BL / 6 mice aged 6–8 weeks were randomly divided into an oral 10-HDA formulation group and a nasal 10-HDA formulation group, with n=6 in each group. Both groups were administered 150 mg / kg of the active ingredient 10-HDA. The final concentration of 10-HDA in both the oral suspension and nasal inclusion solution formulations was 50 mg / mL, corresponding to a dosing volume of 3 mL / kg. The oral formulation group was administered via gavage; the nasal formulation group was administered via bilateral nasal drops, 10 μL per nostril per administration, alternating between nostrils, until the calculated total dosing volume was reached. The time taken to complete all nasal drops was used as the starting point for post-administration timing. Plasma and brain tissue samples were collected at 0, 2, 5, 10, 20, 40, 60, and 120 min after drug administration. The concentration of 10-HDA was determined by LC-MS / MS, and the AUC of plasma and brain tissue was calculated using a non-compartmental model.

[0076] This embodiment establishes a quantitative detection method for 10-HDA based on LC-MS / MS. Plasma and brain tissue samples were collected at different time points after drug administration. Protein precipitation was performed using a methanol solution containing an internal standard, and the supernatant was collected after centrifugation for analysis.

[0077] Specifically, 50 μL of plasma sample was added to 250 μL of methanol protein precipitant containing the internal standard chlorzoxazone. After vortexing for 5 min, the sample was centrifuged at 18,000 rpm for 5 min at 4℃. The supernatant was collected and centrifuged again. Finally, the supernatant was transferred to a vial with an injection volume of 5 μL. Brain tissue sample was homogenized with the addition of the internal standard protein precipitant by weight, and then processed according to the above centrifugation and sampling steps. The supernatant was used for LC-MS / MS analysis. The LC conditions were as follows: an Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm) was used; the column temperature was 40℃; the injection plate temperature was 4℃; mobile phase A was 0.1% formic acid-0.02% ammonium formate aqueous solution, and mobile phase B was methanol; gradient elution was used at a flow rate of 0.3 mL / min, and the injection volume was 5 μL. Mass spectrometry detection conditions were as follows: detection was performed using an electrospray ionization source in negative ion mode (ESI-) and multiple reaction monitoring mode (MRM). The monitoring ion pair for 10-HDA was m / z 185.00→139.00, the declustering voltage DP was -70 V, and the collision energy CE was -22 V; the monitoring ion pair for the internal standard chlorzoxazone was m / z 168.00→132.00, the DP was -80 V, and the CE was -25 V.

[0078] Experimental results showed that there was no significant difference in the time to peak concentration (Tmax) between the nasal and oral formulations of 10-HDA; compared with the oral formulation, the nasal formulation of 10-HDA significantly increased the concentration of 10-HDA in mouse plasma and brain tissue, and significantly increased the AUC levels in plasma and brain tissue. Figure 1 (AD), indicating that the nasal formulation of 10-HDA can effectively increase the systemic and intracranial exposure levels of the drug. This demonstrates that the nasal formulation of 10-HDA significantly improves the overall drug exposure level.

[0079] Example 3 Comparison of appearance and dispersion state of different formulations of 10-HDA

[0080] To compare the stability of oral and nasal formulations of 10-HDA, oral suspensions of 10-HDA, ordinary aqueous solutions of 10-HDA, and nasal inclusion solutions of 10-HDA prepared in the examples of this application were prepared. Each formulation was placed in a clean, transparent container and stored at room temperature in the dark for one week. The initial appearance, appearance after storage, precipitation, crystallization, layering, and redispersion were observed. The results are shown in Table 1.

[0081] Table 1. Appearance and dispersion of the three formulations before and after one week of storage at room temperature in the dark:

[0082]

[0083] The above results indicate that the 10-HDA nasal inclusion solution formulation described in this application maintained good appearance uniformity during the observation period, with no obvious precipitation, crystallization, or stratification observed.

[0084] Example 4: 10-HDA nasal formulation improves neurological deficits in MCAO / R mice

[0085] It should be noted that this embodiment compares a sham surgery group, an MCAO / R model group, an oral 10-HDA formulation group, and a nasal 10-HDA formulation group, primarily to evaluate the overall efficacy differences of different 10-HDA-containing formulations in an ischemic stroke model. This embodiment does not evaluate the pharmacological effects of the nasal excipients themselves.

[0086] Step 1: Experimental grouping and drug administration regimen:

[0087] Male C57BL / 6 mice aged 6–8 weeks were randomly divided into a sham-operated group, an MCAO / R model group, a 10-HDA oral formulation group, and a 10-HDA nasal formulation group, with n=6 in each group.

[0088] a) Sham surgery group: An equal volume of blank solvent was administered after the sham surgery;

[0089] b) MCAO / R model group: After MCAO / R modeling, an equal volume of blank solvent is provided;

[0090] c) 10-HDA oral formulation group: After MCAO / R modeling, 10-HDA oral suspension was administered at a dose of 150 mg / kg based on 10-HDA, via gavage.

[0091] d) 10-HDA nasal formulation group: After MCAO / R modeling, the 10-HDA nasal inclusion solution formulation of this application was administered at a dose of 150 mg / kg based on 10-HDA, via bilateral nasal drops.

[0092] In this embodiment, the final concentration of 10-HDA in both the oral suspension and the nasal inclusion solution is 50 mg / mL. Based on a dosage of 150 mg / kg, the administration volume is 3 mL / kg, or 0.03 mL / 10 g. For a mouse weighing 20 g, the required amount of 10-HDA for a single administration is 3 mg, corresponding to a total formulation volume of 60 μL.

[0093] For the oral formulation of 10-HDA, the calculated volume was administered via a single gavage. For the nasal formulation of 10-HDA, the calculated total volume was instilled into both nostrils in multiple doses, 10 μL per nostril each time, alternating between nostrils. If necessary, the entire dose was administered in multiple doses to avoid aspiration and choking. The time taken to complete the entire nasal administration was used as the starting point for post-administration timing for the nasal formulation.

[0094] The dosing regimen is as follows: the first dose is administered within 0–30 min after the start of MCAO / R reperfusion; thereafter, the dose is administered once daily for 7 consecutive days.

[0095] Step 2: Establishing a C57BL / 6 mouse model of ischemic stroke induced by middle cerebral artery occlusion / reperfusion (MCAO / R):

[0096] a) Anesthetize the mice by inhalation of isoflurane (induction concentration 4%, maintenance concentration 1%-1.5%), and fix them in a supine position on the operating table. Keep the mice warm during the modeling process.

[0097] b) Prepare and disinfect the skin above the sternum and neck. Make a longitudinal incision of about 1 cm along the midline of the neck. Under the observation of a stereomicroscope, bluntly dissect the muscles, fascia and nerves with ophthalmic forceps, and free the left common carotid artery (CCA). Tie a slipknot in the silk thread.

[0098] c) Dissect upwards along the CCA to free the internal carotid artery (ICA) and external carotid artery (ECA). Ligate the distal end of the ECA together with the thyroid artery using a suture, and insert a thread between the ligation point and the bifurcation of the CCA. Sew the thread and tie a loose knot.

[0099] d) Between the loose knot and the ligation site, make a small oblique cut with micro-scissors, insert the suture plug, and then tighten the loose knot with such force that the suture plug just slides inside the blood vessel and the opening breaks without bleeding.

[0100] e) Cut the ECA close to the distal end of the ligation point, adjust the angle to insert the suture into the ICA, and continue to push it forward along the ICA until you feel slight resistance. Stop pushing the suture at this point, and its front end will completely block the origin of the middle cerebral artery. Tie a slipknot at the cut end of the ECA to fix the suture.

[0101] f) Suture the subcutaneous tissue and skin layer by layer, and disinfect. After 1 hour of ischemia, pull the suture plug outward, allowing its tip to retract into the ECA to ensure reperfusion of blood flow from the CCA to the middle cerebral artery. Re-suture and disinfect, and administer 1 ml of normal saline intraperitoneally for fluid replacement.

[0102] g) Except for not blocking blood flow to the middle cerebral artery, the sham-operated mice underwent the same anesthesia, incision, vascular separation, and suturing procedures as the MCAO / R group. In the MCAO / R group, the suture embolus was removed and reperfusion was performed 1 hour after ischemia; the sham-operated group did not undergo effective embolization.

[0103] Step 3: Improving the Behavioral Characteristics of Mice with Ischemic Stroke Using 10-HDA Nasal Formulation

[0104] a) Mouse neurological function score:

[0105] Neurological deficits in mice were assessed on days 1, 2, 3, 4, and 5 after MCAO / R reperfusion, and changes in scores at different time points were recorded. The Bederson modified 9-point scale was used for scoring, with higher scores indicating more severe neurological deficits. The scoring criteria were as follows: 0 points: No symptoms of neurological damage; 1 point: Contralateral forelimb curled up when tail is lifted, or unable to fully reach the affected forelimb; 2 points: Contralateral shoulder adducted when tail is lifted; 3 points: Resistance decreases when pushing towards the opposite side; 4 points: Can move spontaneously in all directions, but only turns towards the opposite side when tail is detached; 5 points: Spontaneous movement in circles or only towards the opposite side; 6 points: No voluntary movement, only movement upon stimulation; 7 points: No voluntary movement, no movement even upon stimulation; 8 points: Death related to cerebral ischemia.

[0106] The experimental results showed that on the 3rd day after administration, compared with the MCAO / R model group, the neurological deficit score of the 10-HDA nasal formulation group was significantly reduced, and the difference was statistically significant. P<0.05; however, there was no statistically significant difference between the oral 10-HDA group and the MCAO / R model group (P>0.05). On day 5 of administration, the neurological deficit scores of both the oral 10-HDA group and the nasal 10-HDA group were lower than those of the model group, with the improvement being more significant in the nasal 10-HDA group. Figure 2 AB).

[0107] b) Rotating bar experiment:

[0108] The rotarod test is designed to assess limb motor abilities in mice. Mice are placed on a rotating rod, with the rotation speed increasing from 4 rpm to 40 rpm over 300 seconds. The time it takes for the mouse to fall off the rod is recorded as the fall latency. Mice in each group were trained three times daily for three days prior to modeling, with 15-minute intervals between each test. The average of the three results from the day before modeling was taken as the baseline (Pre). The rotarod test was performed on day 5 after modeling, with each mouse tested three times. The average value was taken as the rotarod latency for that animal.

[0109] The experimental results showed that on the fifth day of administration, the latency time of the rotarod test in the nasal formulation of 10-HDA was significantly increased, which was superior to that in the oral formulation of 10-HDA. Figure 2 (C) The above results indicate that, under the same 10-HDA dosage, the nasal formulation produces detectable neurological function improvement at an earlier observation time point than the oral formulation.

[0110] Example 5: 10-HDA nasal formulation reduces cerebral infarction area, cerebral edema, and neuronal apoptosis in MCAO / R mice.

[0111] a) TTC staining and statistical analysis of infarct volume percentage and edema volume percentage:

[0112] After neurological function assessment, mice were euthanized under anesthesia with 3% sodium pentobarbital. The heart was ruptured to release blood, and the skin at the back of the neck was cut to expose the head and neck. The cervical spinal cord was severed at the cervical vertebrae. Ophthalmic scissors were used to longitudinally cut the skull bones outside the brainstem and cerebellum. The skull bones were then dissected with dental forceps to separate the dura mater from the surface of the brain, taking care not to scratch the brain tissue. The removed brain tissue was frozen at -80°C for 10 minutes, then cut into 1mm thick serial sections (generally 7 sections, 4 anterior to the anterior fontanelle and 3 posterior). The sections were immediately placed in 10ml of 2,3,5-triphenyltetrazolium (TTC) staining solution (prepared with PBS) and incubated at 37°C in the dark for 20 minutes, with gentle shaking every 5 minutes to ensure thorough staining. Normal brain tissue stained bright red, while infarcted areas appeared pale white. After staining, the brain tissue was fixed with 4% neutral paraformaldehyde and then photographed grossly. Image-ProPlus (version 6.0) software was used to calculate infarct volume and edema volume. The specific calculation formulas are: Infarct volume percentage = 100 * (volume of the brain on the side of injury - volume of the brain on the side of injury without infarction) / (volume of the brain on the side of injury * 2), and edema volume percentage = 100 * (volume of the brain on the side of injury - volume of the brain on the side of injury) / volume of the brain on the side of injury.

[0113] Experimental results showed that under MCAO / R conditions, mouse brain tissue exhibited obvious pale infarct areas, and the infarct area and cerebral edema area were significantly increased. After intervention with 10-HDA preparation, the infarct and edema areas in mice were significantly reduced, and the nasal 10-HDA preparation showed a more significant improvement effect than the oral preparation. Figure 3 (AC) indicates that 10-HDA preparations can effectively improve MCAO / R-induced cerebral infarction and edema, and reduce the area of ​​brain injury, with nasal preparations showing better intervention effects.

[0114] b) Level of neuronal apoptosis in the brain:

[0115] Mice were euthanized by anesthesia with 3% sodium pentobarbital and perfused with PBS buffer at 4°C to drain blood. They were then perfused with 4% paraformaldehyde solution for 8 minutes to fix the vascular intima. Brain tissue was harvested and fixed with 4% paraformaldehyde solution at room temperature for 24 hours. The tissue was then dehydrated with 15% and 30% sucrose solutions. The dehydrated tissue was embedded in OCT gel and cut into 50 μm slices using a cryostat. The slices were washed three times with PBS for 5 minutes each time. The brain slices were blocked at room temperature for 2 hours with a blocking solution containing 0.3% Triton and 5% BSA. NeuN antibody (…) Incubate overnight at 4°C. After washing with PBS, sections are inoculated with secondary antibody (…). Incubate at room temperature for 1 hour. Then, stain using the TUNEL staining kit (Roche, 11684817910) according to the manufacturer's instructions. Cell nuclei are stained with DAPI, and images are observed and captured under a fluorescence microscope (OLYMPUS, BX51). Positive cell counts are analyzed using Image Pro Plus software.

[0116] Experimental results showed that under MCAO / R conditions, the number of TUNEL-positive apoptotic cells in mouse brain tissue increased significantly. After intervention with 10-HDA, the proportion of positive apoptotic cells in brain tissue decreased significantly, and the nasal 10-HDA formulation showed a more significant improvement than the oral formulation. Figure 3 DE indicates that 10-HDA formulations can effectively inhibit MCAO / R-induced neuronal apoptosis and reduce brain cell apoptosis damage, with the nasal formulation showing better anti-apoptotic effects.

[0117] Example 6: 10-HDA nasal formulation improves blood-brain barrier damage in MCAO / R mice

[0118] a) Measurement of brain tissue water content:

[0119] Seventy-two hours after MCAO / R modeling, mice were deeply anesthetized and euthanized, and their heads were quickly severed to obtain intact brain tissue. The brain was divided into left and right hemispheres along the sagittal line, with the left hemisphere being the ischemic side. The left hemisphere was immediately weighed using an electronic balance and recorded as wet weight. It was then quickly placed in a 100°C oven and weighed again after 24 hours, recorded as dry weight. Brain water content was calculated using the following formula: Brain water content = (Wet weight - Dry weight) / Wet weight × 100%.

[0120] Experimental results showed that under MCAO / R conditions, the water content of mouse brain tissue was significantly increased. After intervention with 10-HDA, the water content of brain tissue was significantly reduced, and the nasal 10-HDA formulation showed a more significant improvement effect, suggesting that 10-HDA can effectively alleviate MCAO / R-induced cerebral edema in mice, and the nasal formulation is more effective than the oral formulation. Figure 4 A).

[0121] b) Western Blot detection Occludin, Claudin-5, and MMP-9 protein levels:

[0122] Brain tissue from the infarcted area was added to an appropriate amount of RIPA lysis buffer containing protease inhibitors (Roche, 04693132001) and phosphatase inhibitors (Roche, 4906837001), sonicated, centrifuged, and the supernatant was collected to obtain total protein. Protein concentration was determined using a BCA protein assay kit (Thermo, 23225). Equal amounts of protein samples were added to loading buffer and separated by 10% SDSPAGE electrophoresis. After electrophoresis, the protein was transferred to a PVDF (Millipore, IPVH00010) membrane. After transfer, the PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 hour. The PVDF membrane was washed three times with TBST for 5 minutes each time, and incubated overnight at 4°C with primary antibody. After TBST washing, the corresponding species-specific secondary antibody (Jackson ImmunoResearch) was added and incubated at room temperature for 1 hour. The light was developed using an ECL luminescent substrate (BioRad, 1705062), and the signal was collected using a BioRad gel imaging system (ChemiDoc XRS+).

[0123] Experimental results showed that MCAO / R modeling significantly downregulated the expression levels of blood-brain barrier tight junction proteins ZO-1, Occludin, and Claudin-5 in mouse brain tissue. After treatment with 10-HDA, the expression levels of these tight junction proteins significantly rebounded, with the upregulation effect of the nasal 10-HDA formulation being more pronounced. Figure 4 (BE) indicates that the nasal formulation of 10-HDA better repairs blood-brain barrier structural damage caused by MCAO / R. Simultaneously, the expression of the damage-related protein MMP-9 in mouse brain tissue was significantly upregulated under MCAO / R conditions. After intervention with the 10-HDA formulation, the abnormally high expression of MMP-9 was significantly inhibited, and the inhibitory effect of the nasal formulation of 10-HDA was superior to that of the oral formulation. Figure 4 FG), Ming 10-HDA nasal formulation better reduces the degree of blood-brain barrier damage.

[0124] Example 7: 10-HDA nasal formulation inhibits inflammatory response in MCAO / R mouse brain tissue.

[0125] RT-qPCR detection of mRNA levels of inflammatory cytokines IL-6, IL-1β, and TNF-α:

[0126] Total RNA was extracted from tissues and cells using Trizol (Sigma, T9424). The obtained RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme, R323-01). RT-qPCR was performed using the cDNA as a template with ChamQ SYBR Master Mix (Vazyme, Q311-03) and a LightCycler 480 qPCR system (Roche Holding AG). The relative expression levels of each gene mRNA were analyzed using GAPDH as an internal control. Primer sequences for the target genes are shown in Table 2 below.

[0127] Table 2: Primer sequences of related genes

[0128]

[0129] Experimental results showed that under MCAO / R conditions, the mRNA expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse brain tissue were significantly increased. After intervention with 10-HDA, the expression levels of these inflammatory factors were significantly reduced, and the nasal 10-HDA formulation showed a more significant improvement. Figure 5 (AC) indicates that 10-HDA formulations can effectively inhibit MCAO / R-mediated intracranial inflammatory responses and reduce inflammatory damage to brain tissue, with nasal formulations showing better anti-inflammatory effects than oral formulations.

[0130] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A nasal inclusion solution formulation composition of 10-hydroxy-2-decenoic acid, characterized in that, The formulation composition is for intranasal administration and consists of 10-hydroxy-2-decenoic acid, hydroxypropyl-β-cyclodextrin, propylene glycol, Tween-80, and physiological saline, wherein the mass ratio of 10-hydroxy-2-decenoic acid to hydroxypropyl-β-cyclodextrin is 1:6 to 1:

10.

2. The 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition according to claim 1, characterized in that, The mass ratio of the 10-hydroxy-2-decenoic acid to the hydroxypropyl-β-cyclodextrin is 1:

8.

3. The 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition according to claim 1, characterized in that, The pH of the formulation composition is 4.5 to 6.

5.

4. The composition according to claim 3, characterized in that, The pH of the formulation composition is 5.0 to 6.

0.

5. The 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition according to claim 1, characterized in that, The formulation composition comprises 0.5 g of 10-hydroxy-2-decenoic acid, 4.0 g of hydroxypropyl-β-cyclodextrin, 0.5 mL of propylene glycol, and 0.05 mL of Tween-80, and is diluted to 10 mL with physiological saline. The pH is 5.0–6.0, and the formulation composition is a solution-type nasal drop.

6. The use of the 10-hydroxy-2-decenoic acid nasal inclusion solution formulation composition of claim 1 in the preparation of a medicament for intranasal administration to treat ischemic stroke or cerebral ischemia-reperfusion injury.

7. The application according to claim 6, characterized in that... The drug, at the same dosage, has a better effect on reducing cerebral infarction volume, improving neurological deficits, and alleviating cerebral edema compared with oral administration.

8. The application according to claim 7, characterized in that, The drug, at the same dosage, increases plasma and brain tissue exposure levels of 10-hydroxy-2-decenoic acid compared to oral administration.

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