A QL-H008 formulation for the treatment of glioma, its preparation method and application.

Abstract

This invention discloses a QL-H008 formulation for treating gliomas, its preparation method, and its application, and also discloses the application of compound QL-H008 in the preparation of drugs for treating gliomas. This invention utilizes an oral-nasal inhalation formulation, inhaling an aerosol containing QL-H008 and pharmaceutically acceptable salts through the mouth and nose to achieve the therapeutic effect on gliomas in situ, demonstrating the promising application of QL-H008 in the treatment of gliomas in situ.

Description

Technical Field

[0001] This invention relates to the application of a compound, specifically to a QL-H008 formulation for treating glioma, its preparation method, and its application. Background Technology

[0002] QL-H008 (generic name: V8, structure as shown in formula (1)) is a novel flavonoid compound obtained through group modification. QL-H008 has been shown to have good inhibitory effects on T-cell malignancies, liver cancer, multiple myeloma, and cervical cancer cells, and has not caused significant toxicity in in vivo experiments [Qing Y, Guo Y, Zhao Q, Hu P, Li H, Yu X, Zhu M, Wang H, Wang Z, Xu J, Guo Q, Hui H. Targeting lysosomal HSP70 induces acidsphingomyelinase-mediated disturbance of lipid metabolism and leads to cell death in T cell malignancies. Clin Transl Med. 2023 Mar; 13(3): e1229.]. However, there are currently no studies to prove whether QL-H008 has a therapeutic effect on glioma.

[0003] Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide the application of compound QL-H008 in the preparation of drugs for treating gliomas. Another objective of this invention is to provide a QL-H008 formulation, its preparation method, and its application in the preparation of drugs for treating gliomas.

[0005] Technical solution: Application of compound QL-H008 in the preparation of drugs for treating glioma.

[0006] The application described herein, wherein the compound QL-H008 has the structural formula shown in formula (1):

[0007]

[0008] The application described herein includes compound QL-H008 and its pharmaceutically acceptable salts.

[0009] The QL-H008 formulation is made by adding pharmaceutically acceptable excipients to the compound QL-H008.

[0010] The aforementioned QL-H008 formulation is an oral and nasal inhalation formulation with QL-H008 as the active ingredient.

[0011] The QL-H008 formulation is a liquid formulation, wherein the oral / nasal inhalation formulation is a liquid formulation.

[0012] The QL-H008 formulation includes QL-H008, pH adjuster acetic acid, sodium acetate, and water.

[0013] In the QL-H008 formulation, the water is purified water or water for injection.

[0014] The preparation method of the QL-H008 formulation includes the following steps:

[0015] (1) Dissolve the prescribed amount of sodium acetate and acetic acid in the prescribed amount of water; preferably, obtain a solution with a pH of 4.5;

[0016] (2) Add the prescribed amount of QL-H008 raw material and dissolve to obtain a yellow solution;

[0017] The content range of each component is as follows: QL-H008 content is 0.1-5.0% (w / v), sodium acetate content is 0.1%-0.5% (w / v), acetic acid content is 0.1%-0.5% (v / v), and pH value is 4.0-8.0.

[0018] The application of the QL-H008 formulation in the preparation of drugs for treating glioma.

[0019] The glioma mentioned is an orthotopic glioma model.

[0020] The glioma described is a mouse glioma model constructed by in situ inoculation with U87 MG-Luciferase.

[0021] QL-H008 or its formulations can significantly inhibit the growth of gliomas in situ in nude mice within the brain, significantly reduce the expression level of Ki67, a protein associated with glioma proliferation in nude mice, reduce the tumor area, and significantly prolong the survival of tumor-bearing mice.

[0022] This invention demonstrates for the first time, through a QL-H008 oral and nasal inhalation formulation, that QL-H008 and its inhalation formulation can be used to treat glioma.

[0023] Unlike hematologic malignancies and other solid tumors, the treatment of gliomas, which originate in the brain, requires drugs to overcome the blood-brain barrier. The blood-brain barrier's obstruction of drugs is one of the main reasons affecting the efficacy of glioma treatment. However, through improvements in dosage forms, especially the use of oral and nasal inhalation formulations, there are advantages in delivering drugs to the central nervous system for the treatment of brain tumors, including high bioavailability, fewer adverse reactions, and less invasiveness.Figure 1 ).

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages: QL-H008 has the application of treating orthotopic gliomas. In a mouse glioma model constructed by orthotopic inoculation of U87 MG-Luciferase, oral and nasal inhalation of QL-H008 can significantly inhibit the growth of orthotopic gliomas in nude mice, reduce the tumor area, and significantly prolong the survival of tumor-bearing mice. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrating the use of QL-H008 aerosol for oral and nasal inhalation therapy for glioma.

[0026] Figure 2 The aerodynamic particle size distribution of solution 1 inhaled through the mouth and nose is shown in the diagram.

[0027] Figure 3 A schematic diagram of a new generation pharmaceutical disc impactor (NGI) assembly;

[0028] Figure 4 Schematic diagram of a small animal's oral and nasal inhalation exposure system;

[0029] Figure 5 This is the drug delivery dose monitoring data for QL-H008 nasal and oral inhalation solution;

[0030] Figure 6 A graph showing the aerosol stability monitoring during administration of QL-H008 via oral and nasal inhalation for the treatment of glioma.

[0031] Figure 7 The effect of oral and nasal inhalation of QL-H008 on the bioluminescence intensity of gliomas;

[0032] Figure 8 The graph shows the inhibitory effect of inhaled QL-H008 on the bioluminescence intensity of gliomas after 14 days of treatment, compared to the negative control group.

[0033] Figure 9 The pathological effects of inhaled QL-H008 via the mouth and nose on glioma tissue and normal brain tissue compared to the negative control group (T represents the tumor area and N represents the normal tissue area);

[0034] Figure 10 The effect of oral and nasal inhalation of QL-H008 on the survival of mice with orthotopic glioma. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] The reagents and materials described in the examples are all commercially available. QL-H008 was provided by China Pharmaceutical University.

[0037] Example 1

[0038] A method for preparing QL-H008 oral and nasal inhalation solution

[0039] To prepare a sustainable and stable oral and nasal inhalation solution, the solubility of QL-H008 in different media was systematically studied.

[0040] Test method: Take an appropriate amount of the active pharmaceutical ingredient (batch number: 20230403) and place it in 10-20 ml of medium. Observe the dissolution phenomenon by sonication. After centrifugation at 13000 rpm for 10 min, take the supernatant and filter it through a 0.45 μm PES filter membrane. Take the filtrate and perform content determination by HPLC.

[0041] Experimental results:

[0042] medium Saturated solubility (mg / ml) 0.1M hydrochloric acid 0.08 0.01M hydrochloric acid 6.73 pH 4.5 acetate buffer 11.95 pH 6.8 phosphate buffer 0.02 water 0.01

[0043] Studies have shown that QL-H008 has high solubility in pH 4.5 acetate buffer. Therefore, this invention uses pH 4.5 acetate buffer to prepare a QL-H008 solution for inhalation, wherein the content of QL-H008 is 0.1-5.0% (w / v), the content of sodium acetate is 0.1%-0.5% (w / v), and the content of acetic acid is 0.1%-0.5% (v / v). To ensure solution stability and inhalation safety, the pH range of this solution is 4.0-8.0.

[0044] The prescription in this example consists of the following:

[0045] Composition Inhalation solution 1 (23051101) via mouth and nose QL-H008 2mg Sodium acetate 3mg acetic acid 0.002m1 Water for Injection 1ml

[0046] Preparation method: Dissolve sodium acetate and acetic acid in water, add the prescribed amount of QL-H008, and sonicate to prepare an oral and nasal inhalation solution.

[0047] According to the requirements of General Chapter 0951 (Determination of Aerodynamic Properties of Fine Particles in Inhalation Preparations) of the 2020 edition of the Chinese Pharmacopoeia, a new generation pharmaceutical disc impactor (NGI) was used to detect and analyze the aerodynamic particle size of oral and nasal inhalation solution 1 (see...). Figure 2 ).

[0048] Experimental methods (see) Figure 3 Assemble the new generation pharmaceutical disc impactor with the artificial larynx and pre-cool it in a 5°C cooling device for 90 minutes. Place a filter membrane in the micro-orifice collector (MOC). Connect the new generation pharmaceutical disc impactor to the vacuum pump. Connect the artificial larynx to the flow meter, turn on the vacuum pump, and adjust the flow rate to 15 L·min⁻¹ (±5%). Remove the flow meter. 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 QL-H008 and place it in the nebulizer. Turn on the compressor and start timing. After 600 seconds, turn off the compressor, remove the nebulizer, and turn off the vacuum pump. Accurately add 10 mL of collection liquid to the collection trays of stages 1-7 and MOC, respectively, cover, and shake for 10 minutes. These are stages 1-8. Use the collection liquid to clean the adapter and L-shaped connecting tube, cleaning to 100 ml. This is stage 9. The atomizing cup was cleaned with the collected solution, and then rinsed with solvent to a final volume of 100 ml, which was designated as stage 10. The dosage of each stage was determined by HPLC.

[0049] Experimental results: See [link to results] Figure 2 .

[0050] Figure 2 This indicates that the median particle size of the oral and nasal inhalation solution in aerosol form is 4.18 μm, and the proportion of inhalable fine particles (FPF%) is 58%, which is conducive to QL-H008 fully exerting its efficacy.

[0051] Example 2

[0052] Stability Study of a QL-H008 Nasal Inhalation Solution

[0053] Prepare QL-H008 nasal and oral inhalation solution according to the table below:

[0054] Composition Nebulized inhalation solution 2 (20230417) QL-H008 6mg Sodium acetate 3mg acetic acid 0.002ml Water for Injection 1ml

[0055] Preparation process: Dissolve sodium acetate and acetic acid in water, add the prescribed amount of QL-H008, and sonicate to prepare an oral and nasal inhalation solution.

[0056] The QL-H008 nasal and oral inhalation solution 2 was stored at room temperature and refrigerated, respectively, and its content was determined by HPLC at 0, 1, 4, and 9 days. The results of its content stability are shown in the table below, indicating that the QL-H008 nasal and oral inhalation solution was stable at room temperature for 9 days.

[0057]

[0058]

[0059] Example 3

[0060] An experiment on a small animal oral-nasal inhalation exposure system for QL-H008 oral-nasal inhalation solution.

[0061] Samples: Nasal and oral inhalation solution 3 (23041902, 2 mg / ml) and nasal and oral inhalation solution 4 (23041903, 6 mg / ml) prepared according to the formulation process of Examples 1 and 2.

[0062] Small animal oral-nasal inhalation exposure experiments were conducted using a small animal oral-nasal inhalation exposure system to inhale solutions 3 and 4 (see schematic diagram). Figure 4 The small animal oral-nasal inhalation exposure system is a universal method for oral-nasal inhalation experiments. The drug is passed through an aerosol generator to produce a uniform aerosol, which then reaches an exposure tower, allowing different animals to inhale the drug through their mouth and nose. This process avoids exposing other parts of the animal's body to the aerosol. It can be used for pharmacodynamic studies and preliminary toxicological studies of liquid drugs administered through oral-nasal inhalation.

[0063] To investigate whether stable aerosol concentrations can be obtained for inhalation solutions of different concentrations, this embodiment collected drug concentrations in the aerosol at different time periods. The aerosol concentrations at different concentrations and time periods are shown in the table below. Figure 5 The results showed that stable aerosols could be obtained from drug solutions of different concentrations within 60 minutes.

[0064]

[0065] Example 4

[0066] Main pharmacodynamic effects of QL-H008 (inhaled via oral and nasal route) on a nude mouse glioma model induced by in situ inoculation with U87 MG-luciferase.

[0067] 1. Experimental Materials

[0068] (1) Drugs

[0069] The QL-H008 solution for oral and nasal inhalation prepared in Example 3.

[0070] (2) Laboratory animals

[0071] BABL / c nude mice, 5 weeks old, male, housed in an SPF environment at a temperature of 20-25℃ and a relative humidity of 30-70%, with a 12:12h light / dark lighting condition; free access to water and food.

[0072] (3) Reagents

[0073] 1) D-fluorescein potassium salt: purchased from Shanghai Yisheng Biotechnology Co., Ltd., stored at -20℃.

[0074] 2) DMEM / F12 medium: purchased from Gibco, USA. Before use, weigh 12g of DMEM / F12 medium and 2.438g of NaHCO3, dissolve in 1L of ultrapure water, stir well, filter through a 0.22μm filter, and store at 4℃.

[0075] 3) Isoflurane: Purchased from Shenzhen Ruiwode Life Technology Co., Ltd. Store at room temperature away from light.

[0076] 2. Experimental Methods:

[0077] Take healthy Luciferase-labeled U87 MG cells, resuspend them in serum-free DMEM / F12 medium, count them, and then incubate them at 2×10⁻⁶ cells / day. 5 A mouse orthotopic glioma model was established by inoculating the right caudate nucleus of nude mice with a specific number of luciferase substrates. Seven days after inoculation, the nude mice were anesthetized with isoflurane and injected intraperitoneally with 0.2 mL of luciferase substrate D-luciferin potassium at a concentration of 30 mg / mL. Ten minutes later, bioluminescence in vivo imaging was used to detect tumor formation in the nude mice's brains, and the bioluminescence intensity in the brains of the nude mice was quantitatively calculated using Living Image software. Twelve mice with bioluminescence intensities greater than 1 × 10⁻⁶ were selected. 8 Nude mice were randomly divided into two groups: a model control group and a test sample group, with six mice in each group. The grouping is shown in the table below:

[0078] Table 1 Grouping of experimental animals

[0079]

[0080] After randomization, the mice were given their first dose the following day. The test sample group was administered the drug via a small animal oral-nasal inhalation exposure system, once daily for 14 consecutive days. In vivo imaging of nude mice was performed on day 0 and day 14 of drug administration.

[0081] During the experiment, to monitor drug stability during administration, the concentration of aerosol drugs was continuously monitored.

[0082] During the experiment, the mental state, feeding, and activity levels of the nude mice were observed daily, and their survival status, time of death, and number were recorded. Kaplan-Meier survival curves for the nude mice were plotted using the ggsurvplot function of the survival package in R software (version 4.2.2), and the survival differences between the two groups were analyzed using the Log-rank test.

[0083] After the mice died, their brains were harvested. The brain tissue was fixed with 4% paraformaldehyde, then paraffin sections were prepared and subjected to Ki67 immunohistochemical staining.

[0084] After the mice died, their brains were harvested. The brain tissue was fixed with 4% paraformaldehyde and then sectioned in paraffin. After dewaxing and hydration, the sample sections were stained with hematoxylin and eosin.

[0085] 3. Experimental Results:

[0086] During administration, the concentration of aerosols in the small animal's oral and nasal inhalation exposure system was continuously monitored (see [link to relevant documentation]). Figure 6 The results showed that the aerosol concentration remained stable during the 14-day administration period.

[0087] Figures 7-8 The results showed that on day 0 of drug administration, there was no significant difference in bioluminescence intensity in the heads of nude mice between the model control group and the QL-H008 group. On day 14 of drug administration, the bioluminescence intensity in the heads of nude mice in the model control group significantly increased, and there was a significant difference between the model control group and the QL-H008 group. These results indicate that oral and nasal inhalation of QL-H008 can significantly inhibit the growth of gliomas in situ within the skull of nude mice.

[0088] Figure 9 The results showed that the area of ​​glioma tissue in the QL-H008 group was significantly reduced compared with that in the model control group, indicating that oral and nasal inhalation of QL-H008 can reduce the volume of orthotopic gliomas in nude mice.

[0089] Figure 10 The results showed that the median survival time of nude mice in the model control group was 33 days, while that in the QL-H008 group was 43.5 days. Inhalation of QL-H008 via the mouth and nose significantly prolonged the survival of nude mice with orthotopic glioma.

[0090] In conclusion, oral and nasal inhalation of QL-H008 has shown good efficacy in the treatment of glioma in situ.

Claims

1. Application of compound QL-H008 in the preparation of drugs for treating glioma; the structural formula of compound QL-H008 is shown in formula (1): 。 2. The application according to claim 1, characterized in that, The compound QL-H008 includes compound QL-H008 and its pharmaceutically acceptable salt.

3. Application of QL-H008 formulation in the preparation of drugs for treating glioma; the QL-H008 formulation is made by adding pharmaceutically acceptable excipients to compound QL-H008. The formulation is a liquid formulation for oral and nasal inhalation with QL-H008 as the active ingredient, including QL-H008, pH adjuster acetic acid, sodium acetate, and water. The content range of each component is as follows: QL-H008 content is 0.1-5.0% (w / v), sodium acetate content is 0.1%-0.5% (w / v), acetic acid content is 0.1%~0.5% (v / v), and pH value is 4.0~8.

0.

4. The application according to claim 3, characterized in that, The water in the QL-H008 formulation is purified water or water for injection.

5. The application according to any one of claims 3 to 4, characterized in that, The preparation method of QL-H008 formulation includes the following steps: (1) Dissolve the prescribed amount of sodium acetate and acetic acid in the prescribed amount of water; (2) Add the prescribed amount of QL-H008 raw material and dissolve it to obtain a yellow solution.

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