A targeted drug for treating glioma and a preparation method and application thereof

Abstract

The application discloses a targeted drug for treating glioma and a preparation method and application thereof. Alkaline copper carbonate, dopamine hydrochloride and ammonia water are mixed and dissolved, then defatted cotton is added, stirring is conducted until the defatted cotton is dissolved, and copper ammonia fiber solution is obtained by reaction at room temperature; a mixed solution of soluble copper ion salt, zinc ion salt and cobalt ion salt is prepared as a metal ion acid bath solution; the copper ammonia fiber solution is slowly injected into the metal ion acid bath solution, stirring reaction is completed, and then centrifugal separation is conducted; the obtained precipitate is added into a temozolomide solution, ultrasonic treatment is conducted under ice bath, and then centrifugal separation is conducted to obtain a precipitate; the precipitate is washed and freeze-dried to obtain polydopamine modified temozolomide adsorbed copper ammonia fiber; finally, the obtained polydopamine modified temozolomide adsorbed copper ammonia fiber is added into phosphate buffer solution containing Anti-ATRX antibody for incubation, and then washing, freeze-drying and grinding are conducted to obtain the product.

Description

Technical Field

[0001] This invention belongs to the field of tumor drugs and pharmaceuticals, specifically relating to a targeted drug for treating glioma, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the brain, exhibiting invasive growth. Current treatment primarily involves surgical resection combined with radiotherapy and chemotherapy. Malignant gliomas are characterized by a high recurrence rate and poor prognosis after surgery, and a truly effective treatment method remains lacking. Temozolomide is currently the first-line chemotherapy drug, and although it can improve the survival time and quality of life of glioma patients, its efficacy is still unsatisfactory. Insufficient drug uptake by tumor cells, low drug bioavailability, tumor chemotherapy resistance, and numerous side effects severely impact the effectiveness of chemotherapy. Therefore, the development of glioma-targeted drug delivery systems to increase local drug concentrations in gliomas and reduce systemic adverse reactions is of great significance. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a targeted drug for treating glioma and its preparation method, thereby achieving highly efficient targeted therapy for glioma.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for preparing a targeted drug for treating glioma includes the following steps:

[0006] (1) Mix and dissolve basic copper carbonate, dopamine hydrochloride and ammonia water, then add defatted cotton, stir until dissolved, and react at room temperature to obtain a copper ammonia fiber solution.

[0007] (2) Prepare a mixed solution of soluble copper ion salt, zinc ion salt and cobalt ion salt as a metal ion acid bath solution;

[0008] (3) Under stirring, the copper ammonia fiber solution obtained in step (1) is slowly injected into the metal ion acid bath solution in step (2). After the stirring reaction is completed, centrifuge to separate the precipitate and add it to the temozolomide solution. After ultrasonic treatment under ice bath, centrifuge to obtain the precipitate. After washing and freeze drying, polydopamine modified temozolomide adsorbed copper ammonia fiber is obtained.

[0009] (4) The polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step (3) was added to phosphate buffer containing Anti-ATRX antibody and incubated. Then it was washed, lyophilized and ground to obtain the final product.

[0010] Further, in step (1), the feeding ratio of basic copper carbonate, dopamine hydrochloride, ammonia and degreased cotton is (3.2-4.8)g:(0.038-0.19)g:40ml:(0.64-0.96)g, preferably 4.0g:0.085g:40ml:0.8g.

[0011] Furthermore, in step (1), air is introduced during the reaction at room temperature, with an air flow rate of 0.05-0.08 vvm, preferably 0.07 vvm; the air flow time is 2-8 h, preferably 6 h.

[0012] Further, in step (2), sulfuric acid, copper sulfate, zinc sulfate, and cobalt sulfate are added to water and stirred thoroughly until completely clear to obtain a metal ion acid bath solution; wherein, the feeding ratio of sulfuric acid, copper sulfate, zinc sulfate, cobalt sulfate, and water is (18-22) ml:(0.55-0.85) g:(0.08-0.29) g:(0.02-0.15) g:100 ml, preferably 20 ml:0.75 g:0.18 g:0.12 g:100 ml.

[0013] Further, in step (3), the feeding ratio of the cuprammonium fiber solution, the metal ion acid bath solution, and the temozolomide solution is 50ml:(200-550)ml:(10-20)ml, preferably 50ml:500ml:15ml; the concentration of the temozolomide solution is 1.5-2.5mg / ml, preferably 2.2mg / ml, and the solvent is water.

[0014] Furthermore, in step (3), the stirring reaction time is at least 15 minutes, preferably 15-30 minutes, and most preferably 20 minutes; the ultrasonic treatment power is 1000-1200W, preferably 1120W.

[0015] Furthermore, in steps (3) and (4), phosphate buffer is used for washing, wherein the pH value of the phosphate buffer is 7.0-7.4, preferably 7.2, and the concentration is 0.10-0.12 mol / L, preferably 0.11 mol / L.

[0016] Further, in step (4), the ratio of polydopamine-modified temozolomide adsorbed cuprammonium fiber to phosphate buffer containing Anti-ATRX antibody is (280-450) mg:10 ml, preferably 340 mg:10 ml; the protein concentration in the phosphate buffer containing Anti-ATRX antibody is 5-15 μg / μl, preferably 12 μg / μl.

[0017] Furthermore, in step (4), the incubation conditions are incubation at 4-8℃ for 8-12 hours, preferably incubation at 5℃ for 10 hours.

[0018] Furthermore, the present invention also claims protection for a targeted drug for treating glioma prepared using the above-described preparation method.

[0019] Furthermore, the present invention also claims protection for the use of the above-mentioned targeted drug in the preparation of a drug for treating glioma.

[0020] Preferably, the glioma is a glioblastoma caused by a chemical carcinogen.

[0021] Beneficial effects:

[0022] (1) The cotton fiber dissolution process in the preparation of cuprammonium fiber of the present invention requires an alkaline environment, and the polydopamine modification also requires an alkaline environment. The present invention utilizes this characteristic to complete the cotton fiber dissolution and polydopamine modification in one step, which simplifies the preparation process.

[0023] (2) The cuprammonium fiber of the present invention forms a complex with copper ions, zinc ions and cobalt ions, adsorbs temozolomide with a urea-like structure, and can increase the drug concentration at the lesion site after tumor targeting, thereby obtaining better anti-tumor effect and reducing drug side effects.

[0024] (2) The present invention is based on the fact that the surface of the polydopamine-modified cuprammonium fiber has a variety of surface functional groups such as phenolic hydroxyl, carbonyl, and amino groups, which can non-specifically adsorb the antibody Anti-ATRX corresponding to the surface-specific antigen of glioma. ATRX is a classic glioma marker. By binding the Anti-ATRX antibody, the precise targeting of glioma can be achieved. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a schematic diagram of the urea-like structure of temozolomide.

[0027] Figure 2 It involves the construction of a mouse orthotopic glioma model.

[0028] Figure 3 These are the survival curves of tumor-bearing mice in each group.

[0029] Figure 4 These are photos of tumor-bearing mice with their tumors removed 10 days after treatment. Detailed Implementation

[0030] The present invention can be better understood from the following embodiments.

[0031] Example 1

[0032] The preparation method of the targeted drug for treating glioma in this embodiment includes the following steps:

[0033] 1. Pour 4.0g of basic copper carbonate, 0.085g of dopamine hydrochloride and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.8g of defatted cotton and stir until dissolved. Pass air through at 0.07vvm for 6 hours at room temperature to obtain a copper ammonia fiber solution.

[0034] 2. Add 20ml of sulfuric acid, 0.75g of copper sulfate, 0.18g of zinc sulfate, and 0.12g of cobalt sulfate to 100ml of water and stir thoroughly until completely clear to obtain a metal ion acid bath solution;

[0035] 3. Slowly inject 50 ml of the cuprammonium cellulose solution obtained in step 1 into 500 ml of the metal ion acid bath solution obtained in step 2, stirring continuously. After injection, stir for another 20 minutes, then centrifuge. Add the precipitate to 15 ml of 2.2 mg / ml temozolomide solution. Temozolomide has a urea-like structure, such as... Figure 1 As shown, after ultrasonic treatment at 1120W under ice bath conditions and centrifugation, a precipitate was obtained. After washing with 0.11mol / L phosphate buffer at pH 7.2, the precipitate was freeze-dried to obtain polydopamine-modified temozolomide-adsorbed cuprammonium fiber.

[0036] 4. Add 340 mg of polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step 3 to 10 ml of Anti-ATRX antibody at pH 7.2 and 0.11 mol / L phosphate buffer containing 12 μg / μl protein. After incubation at 5°C for 10 h, wash with pH 7.2 and 0.11 mol / L phosphate buffer, freeze-dry and grind to obtain the targeted drug for treating glioma.

[0037] Example 2

[0038] The preparation method of the targeted drug for treating glioma in this embodiment includes the following steps:

[0039] 1. Pour 3.2g of basic copper carbonate, 0.038g of dopamine hydrochloride and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.64g of defatted cotton and stir until dissolved. Pass air through at 0.05vvm for 8 hours and react at room temperature to obtain a copper ammonia fiber solution.

[0040] 2. Add 18ml of sulfuric acid, 0.55g of copper sulfate, 0.08g of zinc sulfate, and 0.02g of cobalt sulfate to 100ml of water and stir thoroughly until completely clear to obtain a metal ion acid bath solution;

[0041] 3. Slowly inject 50 ml of the cuprammonium fiber solution obtained in step 1 into 200 ml of the metal ion acid bath solution obtained in step 2, stirring while injecting. After the injection is completed, stir for another 15 min, then centrifuge. Add the precipitate to 10 ml of 1.5 mg / ml temozolomide solution, sonicate at 1000 W under ice bath conditions, then centrifuge again to obtain the precipitate. Wash with pH 7.0 0.1 mol / L phosphate buffer, and freeze dry to obtain polydopamine modified temozolomide adsorbed cuprammonium fiber.

[0042] 4. Add 280 mg of polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step 3 to 10 ml of pH 7.0 0.1 mol / L phosphate buffer containing 5 μg / μl protein of Anti-ATRX antibody. After incubation at 8°C for 8 h, wash with pH 7.0 0.1 mol / L phosphate buffer, freeze-dry and grind to obtain the targeted drug for treating glioma.

[0043] Example 3

[0044] The preparation method of the targeted drug for treating glioma in this embodiment includes the following steps:

[0045] 1. Pour 4.8g of basic copper carbonate, 0.19g of dopamine hydrochloride and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.96g of defatted cotton and stir until dissolved. Pass air through at 0.08vvm for 2 hours and react at room temperature to obtain a copper ammonia fiber solution.

[0046] 2. Add 22ml of sulfuric acid, 0.85g of copper sulfate, 0.29g of zinc sulfate, and 0.15g of cobalt sulfate to 100ml of water and stir thoroughly until completely clear to obtain a metal ion acid bath solution;

[0047] 3. Slowly inject 50 ml of the cuprammonium fiber solution obtained in step 1 into 550 ml of the metal ion acid bath solution obtained in step 2 while stirring. After the injection is completed, stir for another 30 min, then centrifuge. Add the precipitate to 20 ml of 2.5 mg / ml temozolomide solution, sonicate at 1200 W under ice bath conditions, and then centrifuge to obtain the precipitate. Wash with pH 7.4 0.12 mol / L phosphate buffer, and freeze dry to obtain polydopamine modified temozolomide adsorbed cuprammonium fiber.

[0048] 4. Add 450 mg of polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step 3 to 10 ml of Anti-ATRX antibody at pH 7.4 and 0.12 mol / L phosphate buffer containing 15 μg / μl protein. After incubation at 4°C for 12 h, wash with pH 7.4 and 0.12 mol / L phosphate buffer, freeze-dry and grind to obtain the targeted drug for treating glioma.

[0049] Comparative Example 1 (without metal ions)

[0050] The preparation method of this comparative pharmaceutical formulation includes the following steps:

[0051] 1. Pour 4.0g of basic copper carbonate, 0.085g of dopamine hydrochloride and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.8g of defatted cotton and stir until dissolved. Pass air through at 0.07vvm for 6 hours at room temperature to obtain a copper ammonia fiber solution.

[0052] 2. Add 20ml of sulfuric acid to 100ml of water and stir thoroughly until completely clear to obtain an acid bath solution;

[0053] 3. Slowly inject 50 ml of the cuprammonium fiber solution obtained in step 1 into 500 ml of the acid bath solution obtained in step 2 while stirring. After the injection is completed, stir for another 20 min, then centrifuge. Add the precipitate to 15 ml of 2.2 mg / ml temozolomide solution, sonicate at 1120 W under ice bath conditions, and then centrifuge to obtain the precipitate. Wash with pH 7.2 0.11 mol / L phosphate buffer, and freeze dry to obtain polydopamine modified temozolomide adsorbed cuprammonium fiber.

[0054] 4. Add 340 mg of polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step 3 to 10 ml of Anti-ATRX antibody at pH 7.2 and 0.11 mol / L phosphate buffer containing 12 μg / μl protein. After incubation at 5°C for 10 h, wash with pH 7.2 and 0.11 mol / L phosphate buffer, freeze-dry and grind to obtain the drug formulation.

[0055] Comparative Example 2 (without polydopamine modification)

[0056] The preparation method of this comparative pharmaceutical formulation includes the following steps:

[0057] 1. Pour 4.0g of basic copper carbonate and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.8g of defatted cotton and stir until dissolved. Pass air through at 0.07vvm for 6 hours at room temperature to obtain a copper ammonia fiber solution.

[0058] 2. Add 20ml of sulfuric acid, 0.75g of copper sulfate, 0.18g of zinc sulfate, and 0.12g of cobalt sulfate to 100ml of water and stir thoroughly until completely clear to obtain a metal ion acid bath solution;

[0059] 3. Slowly inject 50 ml of the cuprammonium fiber solution prepared in step 1 into 500 ml of the metal ion acid bath solution prepared in step 2 while stirring. After the injection is completed, stir for another 20 min, then centrifuge. Add the precipitate to 15 ml of 2.2 mg / ml temozolomide solution, sonicate at 1120 W under ice bath conditions, and then centrifuge to obtain the precipitate. Wash with pH 7.2 0.11 mol / L phosphate buffer, and freeze dry to obtain temozolomide adsorbed cuprammonium fiber.

[0060] 4. Add 340 mg of temozolomide-adsorbed cuprammonium cellulose obtained in step 3 to 10 ml of Anti-ATRX antibody at pH 7.2 0.11 mol / L. After incubation at 5°C for 10 h, wash with pH 7.2 0.11 mol / L phosphate buffer, freeze-dry and grind to obtain the drug formulation.

[0061] Comparative Example 3 (without targeted antibody)

[0062] The preparation method of this comparative pharmaceutical formulation includes the following steps:

[0063] 1. Pour 4.0g of basic copper carbonate, 0.085g of dopamine hydrochloride and 40ml of ammonia into a beaker and stir thoroughly until dissolved. Add 0.8g of defatted cotton and stir until dissolved. Pass air through at 0.07vvm for 6 hours at room temperature to obtain a copper ammonia fiber solution.

[0064] 2. Add 20ml of sulfuric acid, 0.75g of copper sulfate, 0.18g of zinc sulfate, and 0.12g of cobalt sulfate to 100ml of water and stir thoroughly until completely clear to obtain a metal ion acid bath solution;

[0065] 3. Slowly inject 50 ml of the cuprammonium fiber solution obtained in step 1 into 500 ml of the metal ion acid bath solution obtained in step 2 while stirring. After the injection is completed, stir for another 20 min, then centrifuge. Add the precipitate to 15 ml of 2.2 mg / ml temozolomide solution, sonicate at 1120 W under ice bath conditions, and then centrifuge to obtain the precipitate. Wash with pH 7.2 0.11 mol / L phosphate buffer, and freeze dry to obtain polydopamine modified temozolomide adsorbed cuprammonium fiber.

[0066] 4. Add 340 mg of polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step 3 to 10 ml of pH 7.2 0.11 mol / L phosphate buffer, incubate at 5°C for 10 h, wash with pH 7.2 0.11 mol / L phosphate buffer, freeze dry and grind to obtain the drug formulation.

[0067] Mouse glioma model construction

[0068] 5 μl of GL261 glioma cell suspension (containing 5 × 10⁻⁶ cells) 4 (cells) were injected into the cerebral cortex of healthy male C57BL / 6 mice (6-8 weeks old, weighing 20-22g) using a mouse stereotaxic instrument to construct a mouse orthotopic glioma model. Figure 2 ).

[0069] Glioma treatment trial

[0070] Mice were randomly divided into a model group and six treatment groups, with 40 mice in each group. The model group received a fasting injection of saline as a control, while the six treatment groups received an intraperitoneal injection of 10 mg / kg of the drug formulations from Examples 1-3 and Comparative Examples 1-3, respectively, once every one day. Survival curves were plotted. Figure 3 As shown. On day 10, six mice from each of the model group and treatment group were sacrificed, their brains were perfused, and the tumors were removed and photographed. The results are shown in [the table below]. Figure 4 .

[0071] Depend on Figure 3 and Figure 4 It can be seen that the targeted drug prepared in the embodiments of the present invention can effectively reduce tumor volume and inhibit tumor growth, indicating that temozolomide is targeted to the lesion site, and the survival time of tumor-bearing mice is also greatly extended. This shows that the targeted drug prepared in the embodiments of the present invention can achieve targeted treatment of glioma. In the preparation process of Comparative Example 1, due to the lack of metal ions, a large amount of temozolomide could not be loaded, and the tumor growth could not be effectively inhibited. Therefore, the tumor continued to grow, and the survival time of mice was also shortened compared with the Example group. In the preparation processes of Comparative Examples 2 and 3, polydopamine modification and targeting antibody were lacking, respectively, which led to the inability of temozolomide to be released on the target, resulting in enhanced side effects of temozolomide and weakened tumor inhibition effect. The results also showed that the tumor volume inhibition was not significant. Although the survival time of mice was slightly longer than that of the model group, it was greatly shortened compared with the Example group.

[0072] This invention provides a targeted drug for treating gliomas, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a targeted drug for treating glioma, characterized in that, Includes the following steps: (1) Mix and dissolve basic copper carbonate, dopamine hydrochloride and ammonia water, then add defatted cotton, stir until dissolved, and react at room temperature to obtain a copper ammonia fiber solution; (2) Prepare a mixed solution of soluble copper ion salt, zinc ion salt and cobalt ion salt as a metal ion acid bath solution; (3) Under stirring, the copper ammonia fiber solution obtained in step (1) is slowly injected into the metal ion acid bath solution in step (2). After the stirring reaction is completed, centrifuge to separate the precipitate and add it to the temozolomide solution. After ultrasonic treatment under ice bath, centrifuge to obtain the precipitate. After washing and freeze drying, polydopamine modified temozolomide adsorbed copper ammonia fiber is obtained. (4) The polydopamine-modified temozolomide-adsorbed cuprammonium fiber obtained in step (3) was added to phosphate buffer containing Anti-ATRX antibody and incubated. Then it was washed, lyophilized and ground to obtain the final product.

2. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (1), the feeding ratio of basic copper carbonate, dopamine hydrochloride, ammonia and degreased cotton is (3.2-4.8)g:(0.038-0.19)g:40ml:(0.64-0.96)g.

3. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (1), air is introduced during the reaction at room temperature, with an air flow rate of 0.05-0.08 vvm and an air flow time of 2-8 h.

4. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (2), sulfuric acid, copper sulfate, zinc sulfate, and cobalt sulfate are added to water and stirred thoroughly until completely clear to obtain a metal ion acid bath solution; wherein, the feeding ratio of sulfuric acid, copper sulfate, zinc sulfate, cobalt sulfate, and water is (18-22) ml: (0.55-0.85) g: (0.08-0.29) g: (0.02-0.15) g: 100 ml.

5. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (3), the feeding ratio of the cuprammonium fiber solution, the metal ion acid bath solution, and the temozolomide solution is 50ml:(200-550)ml:(10-20)ml; the concentration of the temozolomide solution is 1.5-2.5mg / ml.

6. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (3), the stirring reaction time is at least 15 minutes, and the ultrasonic treatment power is 1000-1200W.

7. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In both steps (3) and (4), phosphate buffer solution is used for washing. The pH value of the phosphate buffer solution is 7.0-7.4 and the concentration is 0.10-0.12 mol / L.

8. The method for preparing the targeted drug for treating glioma according to claim 1, characterized in that, In step (4), the ratio of polydopamine-modified temozolomide-adsorbed cuprammonium fiber to phosphate buffer containing Anti-ATRX antibody is (280-450) mg: 10 ml; the protein concentration in the phosphate buffer containing Anti-ATRX antibody is 5-15 μg / μl; and the incubation conditions are incubation at 4-8℃ for 8-12 h.

9. A targeted drug for treating glioma prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the targeted drug of claim 9 in the preparation of a drug for treating glioma.

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