A drug for preventing or treating glioma, its preparation method and application

Abstract

The present invention provides a drug for preventing or treating glioma, a preparation method and an application thereof. The drug comprises drug components: amitriptyline and doxorubicin. The present invention creatively uses the combined administration of doxorubicin and amitriptyline to synergistically resist glioma, achieving the effects of killing glioma and inhibiting the invasive growth of glioma. The present invention creatively discovers that the two drugs can synergistically inhibit the activity of glioma cells and can also significantly inhibit the secretion of exosomes by glioma cells. The combined administration combination has not been reported in the literature, and the synergistic effect of the two drugs exceeds expectations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a drug for preventing or treating glioma, a preparation method thereof, and an application thereof. Background Art

[0002] Glioma is one of the common malignant tumors. Most of them are primary intracranial malignant tumors, which are characterized by high incidence, high mortality, low cure rate, high recurrence rate, etc., and are one of the difficult problems in current tumor treatment. At present, the main treatment methods for glioma are surgery combined with radiotherapy and chemotherapy. However, due to the infiltrative and invasive growth of glioma, the boundary between the tumor and normal tissues is not clear. Therefore, the treatment effects of simple surgery and radiotherapy are not good, the prognosis is poor, and it is extremely easy to relapse. Moreover, due to the existence of the blood-brain barrier (BBB), most drugs are difficult to enter the brain to exert the best treatment effect. And the current drug delivery methods are mainly intravenous administration, arterial perfusion, opening the blood-brain barrier, and intracranial administration, etc. These drug delivery methods have problems such as non-aggregated drug distribution, great damage to blood vessels, non-selective opening increasing the probability of other substances entering the brain, and inconvenient drug administration.

[0003] Therefore, in recent years, the brain-targeted nano-drug delivery system has been one of the research hotspots for treating glioma. The brain-targeted nano-drug delivery system refers to the transport of drugs in the brain mainly through receptor-mediated and carrier-mediated means. However, there are still two aspects of technical difficulties to be overcome in the current brain-targeted nano-drug delivery system: First, the therapeutic effects of existing chemotherapy drugs themselves are not good; Second, the effects of existing nano-drug delivery systems in crossing the BBB are still not ideal, and their biocompatibility, targeted release property, and drug release rate also need to be improved. Therefore, the overall delivery effect is not ideal.

[0004] Therefore, how to overcome the above technical problems and achieve the improvement of the treatment effect for glioma from both the drug itself and the drug delivery system has become an urgent problem to be solved in this field. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a drug for preventing or treating glioma, a preparation method thereof, and an application thereof.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0007] In the first aspect, the present invention provides a drug for preventing or treating glioma, and the drug includes drug components: amitriptyline and doxorubicin.

[0008] Single chemotherapy drugs often have limitations in the treatment of gliomas. Co-delivery of two drugs can overcome some problems associated with single chemotherapy. With the appropriate combination of drugs, it is possible to improve the efficacy of chemotherapy, reduce the drug dosage, and reduce drug resistance through different modes of action. Doxorubicin (DOX) is an anthracycline broad-spectrum anticancer drug that acts on the cell nucleus. By acting on the DNA of tumor cells, it induces apoptosis and has a significant anticancer effect. Amitriptyline (AMI) is a tricyclic acid sphingomyelinase inhibitor, commonly used clinically as an antidepressant, and its mechanism of action is well-defined. Exosomes have been shown to play an important role in tumor development. Amitriptyline can disrupt the structure of acid sphingomyelinase and reduce the production of ceramide (ceramide plays an important role in the generation of exosomes), thereby reducing the secretion of exosomes. The combined administration of two drugs with different mechanisms of action can enhance the killing effect on gliomas and at the same time inhibit their recurrence.

[0009] The present invention creatively discovers that the two drugs, amitriptyline and doxorubicin, can synergistically inhibit the activity of glioma cells and can also significantly inhibit the secretion of exosomes by glioma cells. The above-mentioned combined administration combination has not been reported in the literature, and the synergistic effect of the two drugs exceeds expectations.

[0010] Preferably, the mass ratio of amitriptyline to doxorubicin is (0.01 - 0.02):(2 - 10).

[0011] At the above ratio, the combination index CDI of the two drugs is between 0.3 and 0.4, which is defined as having a significant synergistic effect in the art.

[0012] Specific values in the above (0.01 - 0.02) are, for example, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, etc.

[0013] Specific values in the above (2 - 10) are, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0014] Preferably, the drug also includes a nanocarrier, and the nanocarrier includes polyamidoamine (PAMAM-NH2) modified with poloxamer 188 (Pluronic F68).

[0015] Polyamide-amine (PAMAM-NH2) dendrimer, a type of cationic polymer material, is commonly used in drug delivery systems. Due to the presence of surface amino groups, polyamide-amine has many unique advantages: the amino groups are relatively reactive, prone to chemical reactions, and easy to connect ligands; it has a proton sponge effect and can escape the endosome-lysosome system. PAMAM dendrimers have hydrophobic cavities and surface functional groups. Since the amino groups on the outer surface of the whole generation of polyamide-amine are positively charged, it shows certain cytotoxicity and hemolytic toxicity, and its surface functional groups can be modified to load other therapeutic substances such as drugs. Poloxamer 188 (Pluronic F68) has strong hydrophilicity and a long-circulation effect similar to polyethylene glycol, which can increase the circulation time of the drug delivery system in vivo. Poloxamer 188 is used to modify polyamide-amine to improve its biocompatibility and enhance the drug delivery effect.

[0016] In the present invention, poloxamer 188 (Pluronic F68) which itself has the property of reducing drug resistance is used to modify PAMAM to neutralize the positive charge on its surface, increase the biocompatibility of the carrier, and reduce its toxicity to the human body. In particular, Pluronic F68 can adsorb apolipoprotein A-I (Apo A-I) in plasma, and Apo A-I can specifically bind to the scavenger receptor class B type I (SR-BI) which is abundantly present on the brain capillary endothelial cells of the blood-brain barrier (BBB). Therefore, this design of the present invention achieves the purpose of crossing the BBB through receptor-mediated means.

[0017] Preferably, the poloxamer 188-modified polyamide-amine is prepared by a preparation method including the following steps:

[0018] (1) Succinic anhydride (SA), poloxamer 188, a solvent and the catalyst triethylamine are mixed and reacted to obtain F68-SA;

[0019] (2) F68-SA, NHS (N-hydroxysuccinimide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), triethylamine and a solvent are mixed and reacted to activate the carboxyl group to obtain an activated product;

[0020] (3) The polyamide-amine is mixed with the activated product and reacted to obtain the product.

[0021] Theoretically, the poloxamer 188-modified polyamide-amine prepared by any method can be used as a nanocarrier. However, when the nanocarriers prepared by different methods are loaded with drugs, the encapsulation efficiency and drug loading amount are different. By using the above preparation method of the present invention, each parameter cooperates with each other, and the drug loading effect of the obtained nanocarrier is better than that of other preparation methods.

[0022] Preferably, the temperature of the reaction in step (1) is 60 - 80 °C, such as 60 °C, 62 °C, 64 °C, 66 °C, 68 °C, 70 °C, 72 °C, 74 °C, 76 °C, 78 °C, 80 °C, etc., and the reaction time is 20 - 30 h, such as 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, etc.

[0023] Preferably, the molar ratio of succinic anhydride to poloxamer 188 in step (1) is (1 - 3):(1 - 3).

[0024] Specific values in the above (1 - 3) are, for example, 1, 1.2, 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3, etc.

[0025] Preferably, the solvent in step (1) includes tetrahydrofuran.

[0026] Preferably, after the reaction in step (1), it further includes removing the solvent and drying.

[0027] Preferably, the temperature of the reaction in step (2) is 15 - 30 °C, such as 15 °C, 17 °C, 20 °C, 22 °C, 25 °C, 27 °C, 30 °C, etc., and the reaction time is 3 - 6 h, such as 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, etc.

[0028] Preferably, the molar ratio of F68 - SA, NHS, EDC to triethylamine in step (2) is (60 - 70):(2 - 6):(2 - 6):(2 - 4).

[0029] Specific values in the above (60 - 70) are, for example, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, etc.

[0030] Specific values in the above (2 - 6) are, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0031] Specific values in the above (2 - 4) are, for example, 2, 2.2, 2.5, 2.7, 3, 3.2, 3.5, 3.7, 4, etc.

[0032] Preferably, the solvent in step (2) includes dimethyl sulfoxide.

[0033] Preferably, the temperature of the reaction in step (3) is 15-40°C, such as 15°C, 17°C, 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, 40°C, etc., and the reaction time is 60-90 h, such as 60 h, 62 h, 65 h, 67 h, 70 h, 72 h, 75 h, 77 h, 80 h, 82 h, 85 h, 87 h, 90 h, etc.

[0034] Preferably, the molar ratio of the polyamide-amine to poloxamer 188 is (60-70):(0.5-1.5).

[0035] The specific values in the above (60-70) are, for example, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, etc.

[0036] The specific values in the above (0.5-1.5) are, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0037] Preferably, drying is further included after the reaction in step (3).

[0038] Preferably, the drying method includes freeze-drying.

[0039] Preferably, purification is further included before drying.

[0040] Preferably, the purification method includes dialysis.

[0041] In a second aspect, the present invention provides a preparation method of a drug for preventing or treating glioma as described in the first aspect, and the preparation method includes: mixing a drug component, a nanocarrier and a solvent, and reacting to obtain the drug.

[0042] Preferably, the reaction is carried out in the dark, the reaction temperature is 33-40°C, such as 33°C, 34°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc., and the time is 18-30 h, such as 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, etc.

[0043] Preferably, the molar amount ratio of the drug component to the nanocarrier is (5-20):1, preferably (8-12):1.

[0044] The specific values in the above (5-20) are, for example, 5, 6, 7, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0045] When the molar ratio of the drug component to the nanocarrier is (8 - 12):1, the comprehensive results of the drug encapsulation efficiency and drug loading are better.

[0046] Preferably, the solvent includes methanol.

[0047] Preferably, after the reaction, it further includes removing the solvent and purification.

[0048] Preferably, the purification method includes dialysis.

[0049] In a third aspect, the present invention provides the use of the drug as described in the first aspect in the preparation of a drug for preventing or treating glioblastoma.

[0050] In a fourth aspect, the present invention provides the use of the drug as described in the first aspect in the preparation of a drug for inhibiting the activity of glioblastoma cells.

[0051] In a fifth aspect, the present invention provides a method for inhibiting the activity of glioblastoma cells, the method comprising: administering amitriptyline and doxorubicin to glioblastoma cells.

[0052] Preferably, the mass ratio of amitriptyline to doxorubicin is (0.01 - 0.02):(2 - 10).

[0053] In a sixth aspect, the present invention provides the use of the drug as described in the first aspect in the preparation of a drug for targeting the blood - brain barrier.

[0054] In a seventh aspect, the present invention provides a method for targeting the blood - brain barrier, the method comprising administering a drug encapsulated by a nanocarrier to a target animal model, and the nanocarrier includes polyamidoamine modified with poloxamer 188.

[0055] Preferably, the preparation method of the polyamidoamine modified with poloxamer 188 is the same as that described in the first aspect.

[0056] The numerical ranges described in the present invention not only include the above - listed point values, but also include any point values between the above - mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the ranges.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) The present invention creatively uses the combination of doxorubicin and amitriptyline for co - administration to synergistically treat glioblastoma, achieving the effects of killing glioblastoma and inhibiting the invasive growth of glioblastoma. The present invention creatively discovers that the two drugs can synergistically inhibit the activity of glioma cells and can also significantly inhibit the secretion of exosomes by glioma cells. The co - administration combination has not been reported in the literature, and the synergistic effect of the two drugs exceeds expectations.

[0059] (2) By simply modifying the surface of PAMAM-NH2 with Pluronic F68, a nano-drug carrier can be obtained, which can endow the carrier with multiple functions simultaneously: ① long circulation; ② high safety and biocompatibility; ③ inhibiting the passive physical barrier of the BBB and weakening the active barrier of the BBB; ④ significantly increasing the drug uptake by cells and having a good drug sustained-release effect. The design of the nano-drug carrier is simple and low-cost, making it more suitable for clinical applications and providing new ideas for the design of an intelligent nano-drug delivery system for anti-glioblastoma.

[0060] The present invention provides a Pluronic F68-modified PAMAM dendrimer loaded with doxorubicin and amitriptyline (the two drugs synergistically inhibit the activity of glioma cells and synergistically inhibit the secretion of exosomes by glioma cells), and constructs a new nano co-delivery system PAMAM-NH2-Pluronic F68 / DOX / AMI, achieving multiple goals of crossing the passive physical barrier and active barrier of the BBB, efficiently killing glioblastoma, and inhibiting the invasive growth of glioblastoma. Brief Description of the Drawings

[0061] Figure 1 It is a result diagram of the synergistic inhibition of the activity of glioblastoma cells by amitriptyline and doxorubicin.

[0062] Figure 2 It is a drug sustained-release effect diagram of the drug system PAMAM-F68-AMI+DOX.

[0063] Figure 3 It is the drug uptake result of glioblastoma cells. Detailed Embodiments

[0064] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0065] In the following embodiments, unless otherwise specified, all reagents and consumables are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0066] Example 1

[0067] This example examines the synergistic effect of AMI-DOX on glioma cells

[0068] (1) AMI-DOX inhibits the secretion of exosomes by C6 cells

[0069] Take 15 mL of C6 cells (rat glioma cells) in the logarithmic growth phase, at 2×10 6Inoculate at a density of [number] cells per dish in a culture dish and culture in a carbon dioxide incubator at 5% CO2 and 37°C for 24 hours. A total of three groups are set up (a control group without drugs, 10 μg / mL AMI, 10 μg / mL AMI + 0.06 μg / mL DOX), with 6 dishes in each group. After overnight incubation for the cells to adhere, add 15 mL of the prepared culture medium containing different drugs, collect the cell supernatant after culturing for 48 hours, extract exosomes, and measure the concentration using a nanodrop.

[0070] The exosome extraction method is as follows: (1) Centrifuge 90 mL of the cell supernatant at 4000 g for 30 minutes at 4°C to remove large cell debris and other impurities; (2) Pass the centrifuged supernatant through a sterile filter membrane with a pore size of 0.22 μm to remove large-sized extracellular vesicles and cell debris and other impurities; (3) Transfer the filtered supernatant to a 100KDa ultrafiltration tube, ultrafilter at 4°C and 4000 g for 30 minutes to concentrate the sample, then add PBS buffer for ultrafiltration to clarify the concentrate, and transfer it to a centrifuge tube; (4) Add an exosome extraction reagent to the concentrate at a ratio of 5:1 (operate according to the EXO-Quick kit instructions), mix well, and let it stand overnight at 4°C; (5) After overnight incubation, centrifuge the mixture at 4°C and 1500 g for 30 minutes, remove the supernatant, add sterile injection water and pipette evenly, centrifuge at 4°C and 1500 g for 30 minutes, and the lower white precipitate is the exosome.

[0071] The experimental results are shown in Table 1:

[0072] Table 1

[0073]

[0074]

[0075] This result shows that AMI can inhibit the secretion of exosomes, and AMI + DOX has a better effect on inhibiting exosomes in C6.

[0076] (2) AMI-DOX synergistically inhibits the activity of C6 cells

[0077] Digest and resuspend C6 cells (rat glioma cells) in the logarithmic growth phase and inoculate them in a 96-well plate at a density of 8×10 3 cells per well, and culture in a carbon dioxide incubator at 5% CO2 and 37°C for 24 hours. Add different concentrations of doxorubicin and amitriptyline respectively for a cytotoxicity test. The results show that the half-maximal inhibitory concentration IC50 of AMI and DOX against glioma cells are 0.06 μg / mL and 12 μg / mL respectively.

[0078] Based on the above IC50 values (0.06 mg / mL AMI: 12 mg / mL DOX is 100% AMI: 100% DOX), a series of ratios were set to make AMI:DOX be: 80%:20%, 60%:40%, 40%:60%, and 20%:80%, respectively. After inoculating C6 cells into a 96-well cell culture plate, the above different ratios of doxorubicin and amitriptyline were added respectively. After co-incubating for 48 h, the OD values of the drugs used alone and in combination were measured respectively, the combination index CDI was calculated, and the results were processed with CalcuSyn 2.0 software to optimize the concentration and ratio for the two drugs to exert the best synergistic effect. The obtained results will be applied to the investigation of the synergistic mechanism. The CDI calculation formula is: CDI = OD(A + B) / [OD(A)×OD(B)];

[0079] Synergy evaluation index: CDI < 0.7 indicates that drugs A and B have a significant synergistic effect; CDI < 1 indicates that the two drugs have a synergistic effect; CDI = 1 indicates that the two drugs have an additive effect; CDI > 1 indicates that the two drugs have an antagonistic effect.

[0080] The inhibition rate results of each drug ratio for C6 cells are as Figure 1 shown, and the combination index CDI results are shown in Table 2;

[0081] Table 2

[0082]

[0083]

[0084] The results showed that the CDI values corresponding to the four ratios were between 0.3 and 0.4, indicating that AMI and DOX had a significant synergistic effect on inhibiting the activity of C6 cells. When the concentration ratio was 40% AMI + 60% DOX, the synergistic effect was the best.

[0085] Example 2

[0086] In this example, the nano-carrier PAMAM-F68 (PAMAM-NH2-Pluronic F68), that is, PAMAM modified with Pluronic F68, was synthesized.

[0087] (1) Synthesis method:

[0088] 1. Weigh 0.05 g of succinic anhydride (SA, 0.5 mmol) and 4.15 g of Pluronic F68 (0.5 mmol, purchased from Aladdin), dissolve them in a 100 mL round-bottom flask containing 20 mL of anhydrous tetrahydrofuran (molar ratio SA:F68 = 1:1), add 3 drops of triethylamine as a catalyst, and reflux and stir at 70 °C for 24 h.

[0089] 2. After the reaction is completed and cooled, the solvent is removed by rotary evaporation under reduced pressure, and 20 mL of distilled water is added to fully dissolve the product.

[0090] 3. Divide the above solution into two portions and transfer them to two dialysis bags (MWCO = 1000) with a disposable dropper, and purify and dialyze in distilled water in a 1000 mL beaker for 48 h.

[0091] 4. After dialysis, sprinkle PEG10000 (polyethylene glycol, purchased from Mackin) on the dialysis bag to concentrate the liquid in the bag to about 10 mL, and then dispense the concentrated liquid into vials (about 2 mL per vial). First, pre-freeze at -20 °C for about 2 h, then overnight in an -80 °C refrigerator, and then lyophilize for 48 h to obtain a white powder, which is Pluronic F68-SA.

[0092] 5. Weigh 1.8685 g of Pluronic F68-SA, 0.0012 g of NHS (N-hydroxysuccinimide), and 0.0015 mL of triethylamine into a round-bottom flask, add 20 mL of anhydrous dimethyl sulfoxide (DMSO), and dissolve 0.002 g of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) in 2 mL of anhydrous DMSO. Then add the dissolved EDC solution to the mixed solution containing Pluronic F68-SA, NHS, and triethylamine (molar ratio of Pluronic F68:NHS:EDC:triethylamine = 64:3:3:3, and the ratio of NHS to EDC can be appropriately increased), and stir and react in a water bath at about 22 °C for 4 h to activate the carboxyl group.

[0093] 6. Take 307.5 μL of PAMAM-NH2 solution (using methanol as the solvent, containing 50 mg of PAMAM-NH2, PAMAM-NH2-G4.0 is purchased from Weihai Chenyuan New Materials Co., Ltd.) into a vial, evaporate the methanol to dryness, then add 3 mL of anhydrous DMSO to dissolve it, and then suck it out with a syringe and slowly add it drop by drop to the activated product obtained in step 5 (molar ratio of Pluronic F68:PAMAM-NH2 = 64:1), and stir at room temperature (25 °C) for 3 d.

[0094] 7. Divide the solution obtained in step 6 above into two portions and transfer them to two dialysis bags (MWCO = 10000, the dialysis bags need to be cut and activated in advance: boil in boiling water for 15 min and use after cooling), and dialyze in distilled water for 3 d.

[0095] 8. Then, sprinkle PEG10000 on the dialysis bag to concentrate the liquid inside the bag to approximately 10 mL. Then, aliquot the concentrated liquid into vials and pre-freeze it at -20 °C for about 2 h, followed by overnight storage in an -80 °C refrigerator, and lyophilize for 48 h to obtain a white solid, which is PAMAM-NH2-Pluronic F68.

[0096] (2) Characterization results:

[0097] Characterize the nano-carrier PAMAM-NH2-Pluronic F68, as well as the raw materials PAMAM-NH2 and the intermediate product Pluronic F68-SA, to prove the successful synthesis of the nano-carrier.

[0098] 1. Particle size and zeta potential investigation

[0099] Take an ultrapure aqueous solution containing an appropriate amount of the sample to be measured, dilute it appropriately, filter it through a 0.22 μM filter membrane, and use a particle size analyzer to measure the particle size distribution and zeta potential at a measurement temperature of 25 °C.

[0100] The results show that the particle size of PAMAM-NH2-Pluronic F68 is about 104.54 nm, and the zeta potential is about -0.6 mV. Among them, compared with unmodified PAMAM-NH2, the particle size of PAMAM-NH2-Pluronic F68 has increased. The possible reason is that the Pluronic molecules can form a hydrophilic layer on the surface of PAMAM-NH2, causing an increase in particle size. In addition, the zeta potential of the modified PAMAM-NH2 has decreased significantly. The reason is that the Pluronic molecules covalently bound to the surface of PAMAM-NH2 reduce the density of the surface amino groups, and the outer hydrophilic layer of PAMAM-NH2 also masks a part of the positive charges on the surface of PAMAM-NH2. This result confirms the successful synthesis of the nano-carrier.

[0101] 2. Infrared characterization

[0102] Mix the powder of the sample to be measured with dried potassium bromide powder at a ratio of 1:100, grind it, and press it into a transparent thin film with a thickness of about 1 mm for testing with an infrared spectrum.

[0103] The results show that in the infrared spectrum of PAMAM-NH2-Pluronic F68, characteristic peaks of carbonyl groups appear at 1651.45 cm -1 and 1557.36 cm -1 , and a stretching vibration peak of ether -C-O- appears at 1107.7 cm -1 , indicating the successful synthesis of the target product.

[0104] 3. NMR characterization

[0105] Dissolve an appropriate amount of the sample to be tested with heavy water (D2O) or DMSO, and then analyze it using a nuclear magnetic resonance spectrometer. Calculate the substitution degree of the amino group of PAMAM-NH2 based on the characteristic proton peaks of Pluronic and PAMAM-NH2.

[0106] The results showed that the characteristic peaks in the 1H-NMR spectrum of PAMAM-NH2-Pluronic F68 were attributed as follows: 1.06 ppm (-CH3), 2.36 ppm (-NCH2CH2CO-), 2.51 ppm (-CONHCH2CH2N), 2.73 ppm (-CONHCH2CH2N-), 2.95 ppm, (-NCH2CH2CO-), 3.20 ppm (-CONHCH2CH2NH2, -NCH2CH2NHCO-), 3.43 ppm (-CH2CH(CH3)-O-), 3.60 ppm (-OCH2CH2OH).

[0107] Calculate the substitution degree of the surface amino group of PAMAM from the peak integral area ratio of the methylene (-CH2-) characteristic peak adjacent to the carbonyl group in the PAMAM molecule and the methyl characteristic peak in the Pluronic molecule. The substitution degree of PAMAM-F68 was obtained as 23.15%, and the theoretical molecular weight was 138669.4.

[0108] Based on the above results, it can be seen that the nano-carrier PAMAM-F68 was successfully synthesized in this example.

[0109] Example 3

[0110] In this example, the drug system PAMAM-F68-AMI+DOX was synthesized.

[0111] (1) Synthesis method

[0112] DOX free stock solution: Weigh 5 mg of doxorubicin hydrochloride, dissolve it in an appropriate amount of 10 mL of a methanol-acetone (1:1, v / v) mixed solvent, add 25 mL of triethylamine, stir and react at room temperature for 12 h to remove the hydrochloride salt. Rotate and evaporate the solvent and excess triethylamine at 37 °C, and redissolve it with an appropriate amount of organic solvent (methanol) to prepare a DOX free stock solution with a concentration of 1 mg / mL.

[0113] AMI free stock solution: Weigh 5 mg of amitriptyline hydrochloride, dissolve it in an appropriate amount of 10 mL of a methanol-acetone (1:1, v / v) mixed solvent, add 25 mL of triethylamine, stir and react at room temperature for 12 h to remove the hydrochloride salt. Rotate and evaporate the solvent and excess triethylamine at 37 °C, and redissolve it with an appropriate amount of organic solvent (methanol) to prepare an AMI free stock solution with a concentration of 1 mg / mL.

[0114] AMI + DOX stock solution: Take a certain amount of free AMI stock solution and free DOX stock solution respectively. The ratio of AMI to DOX is 40% AMI + 60% DOX.

[0115] Precisely measure an appropriate amount of dehydrochlorinated DOX free stock solution / AMI free stock solution / AMI + DOX stock solution into a 50 mL round-bottom flask. Weigh the corresponding amount of PAMAM-F68 polymer according to the molar ratios of the nanocarrier PAMAM-F68 to the drugs AMI / DOX / AMI + DOX being 1:5, 1:10, and 1:20 respectively (each drug has 3 ratios), and place it in an appropriate amount of methanol solution. Add it to the round-bottom flask and stir in the dark at 37 °C for 24 h. Remove the organic solvents triethylamine and methanol under reduced pressure (rotary evaporation), add a small amount (5 mL) of distilled water, and dissolve it by sufficient hydration. Remove the unencapsulated free drugs by dialysis (NWCO = 1000 D), and dialyze with distilled water for 12 h to obtain the drug systems PAMAM-F68-AMI, PAMAM-F68-DOX, and PAMAM-F68-AMI + DOX.

[0116] (2) Drug encapsulation efficiency and drug loading

[0117] Take out the solution in the dialysis bag, dilute it five times with methanol, and determine the drug content by HPLC (high-performance liquid chromatography) method. Calculate the drug encapsulation efficiency and drug loading.

[0118] Encapsulation efficiency EE% = encapsulated drug concentration / drug added concentration × 100%;

[0119] Drug loading DL% = encapsulated drug mass / total mass of drug system × 100%;

[0120] The results are shown in Table 3.

[0121] Table 3

[0122]

[0123] Generally speaking, when the molar ratio of the carrier to the drug is 1:10, the results of drug encapsulation efficiency and drug loading are better.

[0124] (2) Investigation of physicochemical properties

[0125] The physicochemical properties of the drug systems PAMAM-F68-AMI, PAMAM-F68-DOX, and PAMAM-F68-AMI+DOX with a carrier-to-drug molar ratio of 1:10 were investigated respectively. The results showed that the particle size of PAMAM-F68-AMI was 169.66±8.82 and the zeta potential was 5.4±1.56; the particle size of PAMAM-F68-DOX was 146.47±12.12 and the zeta potential was 1.87±0.47; the particle size of PAMAM-F68-AMI+DOX was 178.58±7.52 and the zeta potential was 2±0.85. The particle sizes of all the preparations were less than 200 nm, meeting the particle size requirements for nanodrugs.

[0126] (3) Drug release

[0127] Appropriately take 1 mL each of PAMAM-F68-AMI+DOX, AMI, and DOX and place them into dialysis bags (MWCO = 3500 D). The drug concentration in PAMAM-F68-AMI+DOX was obtained based on the peak area of the free drug measured. The concentrations of both AMI and DOX were 1 mg / mL. Place the three samples into dialysis bags respectively and put them into conical flasks containing 20 mL of phosphate buffer solution with pH 5.0 and pH 7.4. At a constant temperature of 37 °C and 120 r / min, 2 mL was aspirated at certain intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96 h), and then 2 mL of PBS was added. The aspirated liquid medicine was detected by high performance liquid chromatography, substituted into the standard curve equation, and the drug release amount at different times was calculated. The results are shown in Figure 2 (P-F-A+D in the figure represents PAMAM-F68-AMI+DOX), indicating that the sustained release effect of the drug system is excellent.

[0128] Example 4

[0129] This example investigated the therapeutic effect of the PAMAM-F68-AMI+DOX drug system on glioblastoma

[0130] (1) Inhibiting the activity of C6 cells

[0131] After digesting and resuspending C6 cells (in the logarithmic growth phase), inoculate them at 8×10 3Inoculate at a density of cells / holes in a 96-well plate and culture in a carbon dioxide incubator at 5% CO₂ and 37 °C for 24 h. Dilute the drug systems PAMAM-F68-AMI, PAMAM-F68-DOX, and PAMAM-F68-AMI+DOX (the molar ratio of the carrier to the drug is 1:10 during preparation) to different concentrations in the culture medium, filter and sterilize with a 0.22 μm microporous filter membrane, aspirate 100 μL of the solution at each concentration and add it to the small holes, make six parallel replicates, add 100 μL of DMEM culture medium to the control group, and continue to culture in a 5% CO₂ incubator at 37 °C for 48 h. After the time is up, add CCK8 reagent to each well, continue to place it in the incubator for 4 h, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the inhibition rate of the drug system on the activity of C6 cells.

[0132] The results show that the drug carrier PAMAM-F68-AMI+DOX has a stronger killing effect on C6 cells than PAMAM-F68-AMI or PAMAM-F68-DOX, and this result is consistent with the conclusion of the synergistic effect of AMI+DOX obtained in Example 1.

[0133] (2) Uptake of drugs by C6 cells

[0134] Inoculate C6 cells in a 6-well plate covered with a coverslip (2×10 5 cells per well), place it in a CO₂ incubator and culture for 24 h, remove the old culture medium, add DOX, PAMAM-DOX (PAMAM loaded with DOX, the difference in the preparation method compared with PAMAM-F68-DOX is only that PAMAM-F68 is replaced with PAMAM at an equimolar concentration, and the others remain unchanged), and PAMAM-F68-DOX (concentration 20 μg / mL) to each well respectively. After incubating for 48 h, take out the 6-well plate, aspirate the liquid, rinse it 3 times with PBS, add 0.5 mL of trypsin to each well for digestion, terminate the digestion when the cytoplasm begins to retract and become round, and gently pipette until the cells fall off, transfer them to a centrifuge tube, centrifuge at 1000 r / min for 5 min, remove the upper layer of the culture medium, add 1 mL of PBS buffer solution to resuspend the cells, then continue to centrifuge at 1000 r / min for 5 min, add 1 mL of PBS buffer solution to resuspend the cells and detect them with a flow cytometer. Compare the uptake amount of drugs by the cells. The results are shown in Figure 3 .

[0135] The results show that the uptake rate of C6 for DOX and PAMAM-F68-DOX is the highest, and almost complete uptake occurs at about 8 h, indicating that the drug is not affected by C6 cell uptake after being encapsulated by PAMAM-F68. The uptake rate of C6 cells for PAMAM-F68-DOX is significantly higher than that of PAMAM-DOX, indicating that PAMAM can significantly improve the uptake of the preparation by C6 cells after being modified with Pluronic F68.

[0136] (3) Therapeutic effect of PAMAM-F68-AMI+DOX drug system on glioma (animal experiment)

[0137] A C6-bearing mouse model was constructed and randomly divided into 2 groups to conduct experiments A and B respectively:

[0138] Experiment A: The mice were randomly divided into 3 groups and equal amounts of DiR-labeled PAMAM-F68-AMI+DOX, PAMAM-AMI+DOX and free AMI+DOX were injected into the tail vein. After 2, 4, 12, 24 and 48 h of injection, an in vivo imaging system was used to analyze the distribution of PAMAM-F68 and PAMAM in the mice.

[0139] The experimental results were as follows: After 2 h, the intensity of the fluorescence signal in the brain of the PAMAM-F68 group was stronger than that of the PAMAM group. As time extended, the fluorescence signal in the glioma region of the PAMAM-F68 group gradually increased, and its intensity was still higher than that of the PAMAM group. This result confirmed that PAMAM-F68 could adsorb apolipoprotein in plasma and target the scavenger receptor on the BBB, thereby achieving targeting of the blood-brain barrier.

[0140] Experiment B: The mice were randomly divided into 3 groups and treated with PAMAM-F68-AMI+DOX, PAMAM-AMI+DOX and free AMI+DOX nanoparticles respectively. Administration was via tail vein injection, once every other day, for a total of 6 times. The dosage was uniformly (5 mg AMI + 0.6 mg DOX) / kg. The treated C6-bearing mice were kept under constant temperature conditions, and the bedding of the mice was changed in time, and drinking water and mouse food were supplemented. The body weight of the mice was weighed every other day and the change in body weight was observed, and at the same time, the survival status of the mice was observed and recorded every day.

[0141] The experimental results were as follows: The median survival time of the PAMAM-F68-AMI+DOX group was significantly longer than that of the PAMAM-AMI+DOX and AMI+DOX groups. This result further confirmed the synergistic effect of AMI and DOX in the treatment of glioma.

[0142] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a drug for preventing or treating glioma, its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0143] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0144] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A drug for preventing or treating glioma, characterized in that, The drug comprises drug components: amitriptyline and doxorubicin; the mass ratio of amitriptyline to doxorubicin is (0.01 - 0.02):(2 - 10); The drug further comprises a nanocarrier, and the nanocarrier comprises polyamidoamine modified with poloxamer 188.

2. The drug for preventing or treating glioma according to claim 1, wherein, The polyamidoamine modified with poloxamer 188 is prepared by a preparation method comprising the following steps: (1) Mix succinic anhydride, poloxamer 188, a solvent and the catalyst triethylamine, and react to obtain F68-SA; (2) Mix F68-SA, NHS, EDC, triethylamine and a solvent, and react to activate the carboxyl group to obtain an activated product; (3) Mix polyamidoamine and the activated product, and react to obtain the product.

3. The drug for preventing or treating glioma according to claim 2, wherein The temperature of the reaction in step (1) is 60 - 80 °C, and the reaction time is 20 - 30 h.

4. The drug for preventing or treating glioma according to claim 2, characterized in that, In step (1), the molar ratio of succinic anhydride to poloxamer 188 is (1 - 3):(1 - 3).

5. The drug for preventing or treating glioma according to claim 2, wherein The solvent in step (1) is tetrahydrofuran.

6. The drug for preventing or treating glioma according to claim 2, wherein After the reaction in step (1), it further comprises removing the solvent and drying.

7. The drug for preventing or treating glioma according to claim 2, characterized in that, The temperature of the reaction in step (2) is 15 - 30 °C, and the reaction time is 3 - 6 h.

8. The drug for preventing or treating glioma according to claim 2, characterized in that, In step (2), the molar ratio of F68-SA, NHS, EDC to triethylamine is (60 - 70):(2 - 6):(2 - 6):(2 - 4).

9. The drug for preventing or treating glioma according to claim 2, wherein The solvent in step (2) is dimethyl sulfoxide.

10. The drug for preventing or treating glioma according to claim 2, wherein, The temperature of the reaction in step (3) is 15 - 40 °C, and the reaction time is 60 - 90 h.

11. The drug for preventing or treating glioma according to claim 2, wherein The molar ratio of polyamidoamine to poloxamer 188 is (60 - 70):(0.5 - 1.5).

12. The drug for preventing or treating glioma according to claim 2, wherein After the reaction in step (3), it further comprises drying.

13. The drug for preventing or treating glioma according to claim 12, wherein, The drying method is freeze-drying.

14. The drug for preventing or treating glioma according to claim 12, wherein Before drying, it further comprises purification.

15. The drug for preventing or treating glioma according to claim 14, wherein, The purification method is dialysis.

16. A method for preparing a drug for preventing or treating glioma as described in any one of claims 1-15, characterized in that, The preparation method comprises: mixing the drug components, the nanocarrier and a solvent, and reacting to obtain the product.

17. The preparation method according to claim 16, characterized in that, The reaction is carried out in the dark, the temperature of the reaction is 33 - 40 °C, and the time is 18 - 30 h.

18. The preparation method according to claim 16, characterized in that, The molar amount ratio of the drug components to the nanocarrier is (5 - 20):

1.

19. The preparation method according to claim 18, characterized in that, The molar amount ratio of the drug components to the nanocarrier is (8 - 12):

1.

20. The preparation method according to claim 16, characterized in that, The solvent is methanol.

21. The preparation method according to claim 16, characterized in that, After the reaction, it further comprises removing the solvent and purification.

22. The preparation method according to claim 21, characterized in that, The purification method is dialysis.

23. Use of the drug according to any one of claims 1 - 15 in the preparation of a drug for preventing or treating glioblastoma.

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