Use of oral-nasal inhalation type wogonin formulation in the preparation of drugs treating brain glioma

Abstract

The use of an oral-nasal inhalation type wogonin formulation in the preparation of drugs treating brain glioma. The effective component of the formulation is wogonin and pharmaceutically acceptable salts thereof, and the formulation further comprises an osmotic adjusting agent sodium chloride, an amino acid substance capable of achieving relatively high solubility of wogonin and water. The formulation is administrated in a nasal delivery mode using nebulization inhalation.

Description

Application of a wogonin preparation for oral and nasal inhalation in the preparation of a drug for treating brain glioma Technical Field

[0001] The present invention relates to a use of a pharmaceutical preparation, and in particular to the use of a wogonin preparation for oral and nasal inhalation in the preparation of a medicine for treating brain glioma. Background Art

[0002] The annual incidence of glioma in my country is 5 to 8 per 100,000, and the 5-year mortality rate is second only to pancreatic cancer and lung cancer among systemic tumors. Glioma originates from tumors of brain glial cells and is the most common primary intracranial tumor, accounting for approximately 40%-50% of central nervous system tumors. The 2021 edition of the WHO Central Nervous System Tumor Classification divides gliomas into grades 1 to 4, with grades 1 and 2 being low-grade gliomas and grades 3 and 4 being high-grade gliomas. The treatment of gliomas is mainly surgical resection, combined with comprehensive treatment methods such as radiotherapy and chemotherapy. Postoperative radiotherapy for glioblastoma (GBM, grade 4) combined with synchronous temozolomide and adjuvant temozolomide chemotherapy is the standard treatment for newly diagnosed GBM in adults. High-grade gliomas have a poor prognosis and a low survival rate. The 5-year survival rate of low-grade gliomas is 80% overall.

[0003] Wogonin is an active monomer in the traditional Chinese medicine Scutellaria baicalensis, and studies have found that it has pharmacological activity in treating brain gliomas [Tsai CF, Yeh WL, Huang SM, Tan TW, Lu DY. Wogonin induces reactive oxygen species production and cell apoptosis in human glioma cancer cells. Int J Mol Sci. 2012; 13(8): 9877-9892.]. However, for the treatment of brain diseases, drugs must be able to overcome the barrier of the blood-brain barrier.

[0004] In the prior art, there is no research on treating brain glioma by inhaling wogonin through the mouth and nose.

[0005] Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a wogonin preparation for oral and nasal inhalation for use in the preparation of drugs for treating brain gliomas.

[0007] Technical solution: Use of the wogonin preparation for oral and nasal inhalation of the present invention in the preparation of drugs for treating brain gliomas.

[0008] In the application, wogonin includes wogonin as shown in formula (1) and pharmaceutically acceptable salts thereof.

[0009] The application is that the preparation is a preparation containing wogonin prepared by adding pharmaceutically acceptable excipients to wogonin and its pharmaceutically acceptable salts.

[0010] In the application, the wogonin preparation for oral and nasal inhalation is an oral and nasal inhalation preparation that can be inhaled after being atomized.

[0011] In the application, the oral and nasal inhalation preparation is a liquid preparation.

[0012] The application described herein comprises ingredients of wogonin, an isotonicity regulator, sodium chloride, an amino acid substance capable of achieving a higher solubility of wogonin, and water.

[0013] In the application, the amino acid substances that can achieve higher solubility of wogonin include arginine and lysine.

[0014] In the application, the water is purified water or water for injection.

[0015] The application, the preparation method of the wogonin preparation for oral and nasal inhalation comprises the following steps:

[0016] Dissolve arginine in water until clear; add the prescribed amount of the raw material wogonin, heat, and dissolve until clear; or use citric acid to adjust the pH value;

[0017] The content range of each component is as follows: wogonin content is 0.1-5.0% (w / v), arginine content is 2%-10% (w / v), citric acid content is 0.01%-0.05% (w / v), and pH value is 7.0-11.0.

[0018] In the application, the pH value is 9-10.

[0019] The brain glioma is an in situ brain glioma.

[0020] The brain glioma is a mouse brain glioma model constructed by in situ inoculation of U87 cells.

[0021] The nasal cavity has its own unique anatomical advantages: the trigeminal nerve in the respiratory area is connected to the central nervous system in the brain, and there are porous capillaries in the olfactory area that connect the brain tissue to the external environment, which is the weakest part of the blood-brain barrier. At the same time, the nasal cavity is connected to the oral cavity through the pharyngeal cavity. When aerosols are inhaled through the mouth, part of the aerosols can reach the nasal cavity through the pharyngeal cavity and enter the brain. Therefore, compared with simple nasal administration, the oral and nasal inhalation method can further increase the amount of drug delivered. Therefore, the oral and nasal inhalation method (Figure 1) has many advantages of delivering drugs to the central nervous system to treat brain tumors, crossing the blood-brain barrier, improving bioavailability, reducing adverse reactions, and reducing trauma.

[0022] Beneficial Effects: Compared with existing technologies, the present invention has the following advantages: wogonin is applicable to the treatment of orthotopic brain gliomas. In a mouse glioma model established by orthotopic inoculation with U87MG-Luciferase, oral and nasal inhalation of wogonin significantly inhibited the growth of orthotopic gliomas in the brain of nude mice, reduced the tumor area, and significantly prolonged the survival of tumor-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the treatment process from inhalation of the preparation to reaching the ward;

[0024] FIG2 is a graph showing the aerodynamic particle size distribution of an inhalation preparation;

[0025] Figure 3 is a schematic diagram of the next generation pharmaceutical disc impactor (NGI) assembly;

[0026] Figure 4 is a schematic diagram of the oral and nasal inhalation exposure system for small animals;

[0027] FIG5 is a graph showing the dosage per unit time of wogonin solution for oral and nasal inhalation in an exposure tower;

[0028] FIG6 is a graph showing changes in aerosol concentration of wogonin inhaled through the mouth and nose to treat brain glioma;

[0029] FIG7 shows the effect of oral and nasal inhalation of wogonin on the growth level of glioma transfected with fluorescein in mice;

[0030] FIG8 is a statistical diagram and significant difference analysis of FIG7;

[0031] FIG9 shows the pathological effects of oral and nasal inhalation of wogonin on brain glioma tissue and normal brain tissue compared with the negative control group (T represents the tumor area, and N represents the normal tissue area);

[0032] FIG10 shows the effect of oral and nasal inhalation of wogonin on the survival of mice with orthotopic glioma model. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Example 1

[0035] Aerodynamic particle size distribution characteristics of a wogonin solution for oral and nasal inhalation

[0036] Wogonin is a flavonoid compound with the common characteristics of flavonoids: poor water solubility.

[0037] In this study, we attempted to increase the water solubility of wogonin by adding arginine, thereby preparing a solution suitable for oral and nasal inhalation. Studies have shown that a uniform solution can be obtained when the wogonin content in the formulation is 0.1-5.0% (w / v) and the arginine content is 2% to 10% (w / v). Furthermore, to ensure that the pH value of the solution remains stable within the range of 7.0 to 11.0, a certain amount of citric acid (0.01% to 0.05% (w / v)) may be added to the formulation.

[0038] Prescription in the present embodiment is as follows:

[0039] Preparation method: Dissolve arginine in water, stir until dissolved, then add the prescribed amount of raw materials to obtain a bright yellow solution.

[0040] According to the requirements of General Chapter 0951 (Determination of Aerodynamic Characteristics of Fine Particles of Inhalation Preparations) of the 2020 edition of the Chinese Pharmacopoeia, a new generation of pharmaceutical disc impactor (NGI) was used to detect and analyze the aerodynamic particle size of oral and nasal inhalation solution 1 (see Figure 2).

[0041] Experimental Method (see Figure 3): Assemble the next-generation pharmaceutical disc impactor with the artificial larynx and precool it in a 5°C cooling unit for 90 minutes. Place a filter membrane in the micro-orifice collector (MOC) and connect the next-generation pharmaceutical disc impactor to a vacuum pump. Connect the artificial larynx to a flowmeter, start the vacuum pump, adjust the flow rate to 15 L / min⁻¹ (±5%), and remove the flowmeter. Connect the compressor, nebulizer, mouthpiece, and adapter in sequence, and then connect the adapter to the artificial larynx. Accurately measure 2 mL of the drug solution (wogonin) and place it in the nebulizer. Start the compressor and time the time. After 600 seconds, turn off the compressor, remove the nebulizer, and turn off the vacuum pump. Accurately add 10 mL of the collection solution to each of the 1st to 7th and MOC collection plates, cover, and shake for 10 minutes. These serve as stages 1 to 8. Use the collection solution to clean the adapter and L-shaped connecting tube until the volume is reduced to 100 mL. This serves as stage 9. The nebulizer cup was cleaned with the collected solution and then washed with solvent to 100 ml, which was used as the 10th level. The drug content of each level was detected by HPLC.

[0042] Experimental results: See Figure 2.

[0043] Figure 2 shows that the median particle size of the oral and nasal inhalation solution in the aerosol state is 4.3 μm, and the proportion of inhalable fine particles (FPF%) is 57%, which is conducive to the full exertion of the efficacy of wogonin.

[0044] Example 2

[0045] Stability study of a wogonin solution for oral and nasal inhalation containing an isotonicity regulator

[0046] This example provides a stability study of a wogonin oro-nasal inhalation solution containing an isotonicity regulator.

[0047] The prescription and preparation method are as follows:

[0048] Preparation: Dissolve arginine and sodium chloride (if specified in the recipe) in the prescribed amount of water and stir to obtain a bright yellow solution.

[0049] In order to prevent the addition of isotonic regulator sodium chloride from affecting the oral and nasal inhalation solution of wogonin, the present invention compared the changes in wogonin content with and without the addition of sodium chloride. The content results on day 0 and day 10 are shown in the following table.

[0050] The results showed that the addition of sodium chloride did not affect the content of wogonin in oral and nasal inhalation solution.

[0051] Example 3

[0052] Small animal oronasal inhalation exposure experiment of a wogonin oronasal inhalation solution

[0053] In order to investigate whether a wogonin oronasal inhalation solution can produce a stable and sustained aerosol, this example provides a small animal oronasal inhalation exposure experiment of a wogonin oronasal inhalation solution.

[0054] The prescription and preparation method are as follows:

[0055] Preparation: Dissolve arginine in water, stir until dissolved, then add the prescribed amount of API and dissolve until dissolved. If necessary, adjust the pH to 9-10 with citric acid to obtain a bright yellow solution.

[0056] In this example, a small animal oro-nasal inhalation exposure system was used for oro-nasal inhalation studies. The small animal oro-nasal inhalation exposure system (see Figure 4) is a universal oro-nasal inhalation experimental method. The drug is passed through an aerosol generator to produce a uniform aerosol. Once in the exposure tower, the aerosol is inhaled orally by different animals while minimizing exposure to any part of the animal's body. This system can be used for pharmacodynamic studies of liquid drugs and preliminary toxicology studies.

[0057] The above-mentioned oral and nasal inhalation solutions 4 and 5 were used in a small animal oral and nasal inhalation exposure system to generate aerosols. Filter paper was used to collect the aerosols at different time periods, and the concentrations were determined by HPLC. Aerosol concentrations at different concentrations and time periods are shown in the table below and Figure 5. The results showed that stable aerosols were generated with the wogonin inhalation solutions at various concentrations.

[0058] Example 4

[0059] Study on the main pharmacodynamic effects of wogonin by oral and nasal inhalation on the nude mouse glioma model inoculated with U87MG-luciferase

[0060] 1. Experimental Materials

[0061] (1) Drugs

[0062] The wogonin solution for oral and nasal inhalation prepared in Example 3.

[0063] (2) Experimental animals

[0064] BABL / c nude mice, 5 weeks old, male, were housed in an SPF environment at a temperature of 20-25°C, a relative humidity of 30-70%, and a 12:12 h light-dark cycle; they had free access to water and food.

[0065] (3) Reagents

[0066] 1) D-luciferin potassium salt: purchased from Shanghai Yisheng Biotechnology Co., Ltd. and stored at -20°C.

[0067] 2) DMEM / F12 medium: purchased from Gibco, USA. Before use, weigh 12 g of DMEM / F12 medium and 2.438 g of NaHCO3, dissolve in 1 L of ultrapure water, stir well, filter through a 0.22 μm filter, and store at 4°C.

[0068] 3) Isoflurane: purchased from Shenzhen Ruiwode Life Science Technology Co., Ltd. and stored at room temperature in the dark.

[0069] 2. Experimental methods:

[0070] Take the Luciferase-labeled U87MG cells in good condition, resuspend them in serum-free DMEM / F12 medium, count them, and use 2×10 5 The number of cells was inoculated into the right caudate nucleus of the brain of nude mice to establish an orthotopic glioma model in mice. Seven days after inoculation, the nude mice were anesthetized with isoflurane and intraperitoneally injected with 0.2 mL of luciferase substrate D-luciferin potassium salt at a concentration of 30 mg / mL. After 10 minutes, the tumor formation in the brain of nude mice was detected by bioluminescence imaging, and the bioluminescence intensity in the brain of nude mice was quantitatively calculated using Living Image software. Twelve mice with bioluminescence intensity greater than 1×10 8 The nude mice were randomly divided into two groups: model control group and test sample group, with 6 mice in each group. The grouping is shown in the table below:

[0071] Table 1 Experimental animal groups

[0072] After randomization, the first dose was administered the next day. The test sample group was administered via a small animal oral and nasal inhalation exposure system once daily for 14 consecutive days. In vivo imaging of nude mice was performed on days 0 and 14 of administration.

[0073] During the experiment, to monitor the stability of the drug during administration, the aerosol drug concentration was continuously monitored during the administration process.

[0074] During the experiment, the mice's mental state, food intake, and exercise were observed daily. Survival status, death time, and number of mice were recorded. Kaplan-Meier survival curves were plotted using the ggsurvplot function in the survival package in R software (version 4.2.2). Survival differences between the two groups were analyzed using the log-rank test.

[0075] After the mice died, the brains were removed. The brain tissue was fixed with 4% paraformaldehyde and then paraffin-embedded. The sections were dewaxed, hydrated, and then stained with hematoxylin-eosin.

[0076] 3. Experimental results:

[0077] During the administration period, the aerosol concentration of the small animal oral and nasal inhalation exposure system was continuously monitored as shown in Figure 6. The results showed that the aerosol concentration was stable during the administration period.

[0078] The results in Figures 7 and 8 show that on day 0 of administration, there was no significant difference in head bioluminescence intensity between the model control group and the wogonin group. On day 14 of administration, head bioluminescence intensity in the model control group increased significantly and was significantly different from that in the wogonin group. These results demonstrate that oronasal inhalation of wogonin significantly inhibits the growth of orthotopic gliomas in the brains of nude mice.

[0079] The results in Figure 9 show that the area of ​​brain glioma tissue in the wogonin group was significantly reduced compared with the model control group, indicating that oral and nasal inhalation of wogonin can reduce the volume of in situ glioma in nude mice.

[0080] The results in Figure 10 show that the median survival time of nude mice in the model control group was 37.5 days, and the median survival time of nude mice in the wogonin group was 51 days. Oral and nasal inhalation of wogonin can significantly prolong the survival of nude mice with in situ glioma models.

[0081] In conclusion, oral and nasal inhalation of wogonin has a good therapeutic effect on in situ brain glioma.

Claims

1. Use of a baicalin preparation for oral and nasal inhalation in the preparation of a drug for treating brain glioma.

2. The use according to claim 1, characterized in that: Wogonin includes wogonin represented by formula (1) and pharmaceutically acceptable salts thereof.

3. The use according to claim 1, characterized in that: The preparation is a preparation containing wogonin, which is prepared by adding pharmaceutically acceptable excipients to wogonin and pharmaceutically acceptable salts thereof.

4. The use according to claim 1, characterized in that: The wogonin preparation for oral and nasal inhalation is an oral and nasal inhalation preparation that can be inhaled after being atomized.

5. The use according to claim 4, characterized in that: The oral and nasal inhalation preparation is a liquid preparation.

6. The use according to claim 3, characterized in that: The ingredients of the preparation include wogonin, isotonicity regulator sodium chloride, amino acid substances that can achieve higher solubility of wogonin, and water.

7. The use according to claim 6, characterized in that: The amino acid substances that can achieve higher solubility of wogonin include arginine and lysine.

8. The use according to claim 6, characterized in that: The water is purified water or water for injection.

9. The use according to claim 1, characterized in that: The preparation method of the wogonin preparation for oral and nasal inhalation comprises the following steps: Dissolve arginine in water until it becomes clear; add the prescribed amount of raw material wogonin, heat it, and dissolve it; or adjust the pH value with citric acid; The content range of each component is as follows: wogonin content is 0.1-5.0% (w / v), arginine content is 2%-10% (w / v), citric acid content is 0.01%-0.05% (w / v), and pH value is 7.0-11.

0.

10. The application according to claim 9, characterized in that: The pH value is 9-10.

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