Pharmaceutical composition for treating drug-resistant brain glioma

Abstract

The invention discloses a pharmaceutical composition for treating drug-resistant brain glioma, and belongs to the technical field of biological medicines.The pharmaceutical composition comprises at least one blood-brain barrier opener and at least one drug-resistant brain glioma chemotherapy drug, the blood-brain barrier opener is arginine or hydroxyurea, and the drug-resistant brain glioma chemotherapy drug is a drug-resistant brain glioma chemotherapy drug. The drug-resistant brain glioma chemotherapeutic drug comprises doxorubicin hydrochloride, mitoxantrone or methotrexate. The blood-brain barrier opener and the drug-resistant brain glioma chemotherapeutic drug are combined for treatment of drug-resistant brain glioma, the blood-brain barrier opener arginine or hydroxyurea can obviously increase the accumulation amount of the chemotherapeutic drug in the brain, and the pharmaceutical composition can effectively inhibit the growth of the drug-resistant brain glioma and prolong the lifetime of a model animal. According to the technical scheme, a new solution is provided for overcoming the blood-brain barrier and treating the drug-resistant brain glioma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a drug composition for treating drug-resistant brain glioma. BACKGROUND

[0002] Brain glioma is the most common primary malignant tumor of the central nervous system, among which glioblastoma is highly invasive and has a poor prognosis. The standard treatment regimen includes surgical resection, radiotherapy and temozolomide chemotherapy. However, the high heterogeneity and infiltrative growth of tumors make it difficult to completely resect them by surgery; radiotherapy can damage adjacent normal brain tissue; more importantly, the problem of temozolomide resistance is prominent - more than 50% of patients are initially insensitive to temozolomide, and long-term use of temozolomide can easily induce acquired drug resistance. Patients with temozolomide-resistant brain glioma lack effective treatment methods and have a significantly shortened survival period. Therefore, developing effective therapies for drug-resistant brain glioma is an urgent clinical problem.

[0003] Researchers have conducted a large number of studies on the treatment of drug-resistant brain glioma, and the current strategy has been expanded from traditional chemotherapy drugs to multi-modal combined intervention: Chinese patent document with publication number CN117843635A discloses a HOTAIR inhibitor and its medical use. The HOTAIR inhibitor inhibits the proliferation, migration and invasion of malignant tumor cells by targeting HOTAIR, which means interfering with the binding of HOTAIR to EZH2 and / or interfering with the recruitment of PRC2 by HOTAIR. The HOTAIR inhibitor combined with temozolomide can be used to treat temozolomide-resistant brain glioma. Chinese patent document with publication number CN110354096A discloses a brain-targeting drug delivery system that can improve the microenvironment of drug-resistant brain glioma and reverse drug resistance. The invention synthesizes a new cationic polymer PASP-g-PEI by grafting small molecular weight polyethyleneimine PEI through a disulfide bond on the side chain of polyaspartic acid PASP. The siPD-L1 is compressed with siPD-L1, and the complex formed by siPD-L1 and PASP-g-PEI is used as the core. The outer shell is composed of a lipid film of 2-deoxyglucose Glu modified distearoyl phosphatidyl ethanolamine-polyethylene glycol DSPE-PEG, cholesterol and dioleoyl phosphatidyl ethanolamine DOPE, etc. The core-shell lipid polymer nanoparticle TMZ / siPD-L1@GLPN(PASP-g-PEI) co-loaded with siPD-L1 and TMZ is prepared by the film dispersion method. It is transported through the blood-brain barrier and enters the drug-resistant brain glioma cells mediated by glucose transporters, and inhibits the growth of drug-resistant brain glioma based on double pathways. However, the above-mentioned methods may have problems such as difficulty in obtaining raw materials or complex preparation methods.

[0004] The blood-brain barrier is a physiological barrier formed by tight junction proteins, which strictly limits more than 98% of chemotherapeutic drugs into the brain parenchyma. Existing strategies to open the blood-brain barrier, such as using drug hyperosmolar mannitol and vasoactive peptides or focused ultrasound, have obvious limitations: (1) safety risks, non-selective opening may cause brain edema or neurotoxicity; (2) poor timeliness, the opening time window is difficult to match the drug metabolism kinetics; (3) complex operation, often requiring invasive equipment or special drug delivery devices, such as intracranial injection. Therefore, developing a safe, reversible and time-controllable blood-brain barrier opening method is crucial for improving the efficiency of drug delivery to the brain to treat drug-resistant glioma. SUMMARY

[0005] In view of the treatment problem of temozolomide-resistant glioma, the blood-brain barrier opening agent (arginine or hydroxyurea) and the drug-resistant glioma chemotherapy drug are used in combination to treat drug-resistant glioma, and the arginine or hydroxyurea significantly improves the accumulation of the chemotherapy drug in the brain. The drug composition can effectively inhibit the growth of drug-resistant glioma and prolong the survival period of the model animal.

[0006] The specific technical solutions adopted are as follows: The application provides application of arginine in preparation of a preparation for promoting a glioma chemotherapy drug to pass through a blood-brain barrier. The arginine, as a blood-brain barrier opening agent, can improve the accumulation of the glioma chemotherapy drug in the brain and improve the treatment effect.

[0007] The application provides application of hydroxyurea in preparation of a preparation for promoting a glioma chemotherapy drug to pass through a blood-brain barrier. The hydroxyurea, as a blood-brain barrier opening agent, can also improve the accumulation of the glioma chemotherapy drug in the brain and improve the treatment effect.

[0008] Further, the glioma chemotherapy drug is a chemotherapy drug for sensitive glioma or drug-resistant glioma (specifically temozolomide-resistant glioma).

[0009] The application further provides a drug composition for treating drug-resistant glioma, which comprises at least one blood-brain barrier opening agent and at least one drug-resistant glioma chemotherapy drug. The blood-brain barrier opening agent is arginine or hydroxyurea, and the drug-resistant glioma chemotherapy drug comprises doxorubicin hydrochloride, mitoxantrone or methotrexate.

[0010] Further, the drug-resistant glioma is temozolomide-resistant glioma, the blood-brain barrier opening agent is arginine, and the drug-resistant glioma chemotherapy drug is doxorubicin hydrochloride. In a temozolomide-resistant glioma model, arginine combined with doxorubicin hydrochloride can effectively inhibit tumor growth, with an inhibition rate of greater than 89%, prolong the median survival period of the animal, and reduce systemic toxicity.

[0011] Preferably, the mass ratio of the blood-brain barrier opening agent and the drug-resistant brain glioma chemotherapy drug is 5-1000:1.

[0012] Further preferably, when the blood-brain barrier opening agent is arginine, the mass ratio of arginine and the drug-resistant brain glioma chemotherapy drug is 5-1000:1, most preferably 100-500:1; when the blood-brain barrier opening agent is hydroxyurea, the mass ratio of hydroxyurea and the drug-resistant brain glioma chemotherapy drug is 5-500:1, most preferably 50-100:1.

[0013] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent, and is prepared into a pharmaceutically acceptable dosage form.

[0014] The blood-brain barrier opening agent and the drug-resistant brain glioma chemotherapy drug are administered simultaneously or separately.

[0015] Further, the dosage of the pharmaceutical composition for mice is: the dosage of arginine is 1 mg-20 g / time / day, the dosage of doxorubicin hydrochloride is 0.06 mg-20 mg / time / day, and the method of use is to orally or intravenously inject arginine first, and then intravenously inject doxorubicin hydrochloride; the dosage of the pharmaceutical composition for mice is: the dosage of hydroxyurea is 1 mg-2.4 g / time / day, the dosage of doxorubicin hydrochloride is 0.06 mg-20 mg / time / day, and the method of use is to orally or intravenously inject hydroxyurea first, and then intravenously inject doxorubicin hydrochloride. The dosage for mice can be converted to the dosage for humans, and the dosage for humans can be adjusted according to the human condition and the application dosage requirements of arginine and hydroxyurea.

[0016] The application further provides the use of the pharmaceutical composition in the preparation of a drug-resistant brain glioma treatment product.

[0017] Compared with the prior art, the application has the following beneficial effects: (1) The experimental results show that arginine and hydroxyurea have excellent blood-brain barrier opening efficiency, and arginine is particularly excellent. The intravenous injection of 200 mg / kg arginine can increase the accumulation of brain chemotherapy drugs by 0.07 times, which is better than the traditional opening agent such as mannitol, which can increase the accumulation of brain drugs by about 1.07 times when 500 g / kg is intravenously administered.

[0018] (2) The combination of the blood-brain barrier opening agent and the drug-resistant brain glioma chemotherapy drug can significantly improve the delivery of drugs in the brain. In the glioma model, the combination of arginine and doxorubicin hydrochloride can increase the distribution of drugs in the brain by 0.87 times, effectively improving the delivery efficiency of chemotherapy drugs that are usually difficult to penetrate the blood-brain barrier.

[0019] (3) The drug composition of the present application, the blood brain barrier opening agent and the drug-resistant brain glioma chemotherapy drug play an anti-tumor synergistic effect, and in the glioma model, the combination of arginine and doxorubicin significantly inhibits the growth of drug-resistant glioma and prolongs the survival period of animals.

[0020] (4) Arginine and hydroxyurea, as a blood brain barrier opening agent based on physiological mechanism, have low risk of damage to normal brain tissue, can avoid brain edema caused by high-osmotic mannitol and other strategies, and have good biological safety.

[0021] (5) The technical scheme of the present application is simple to administer, and can be intravenously injected or orally administered with a blood brain barrier opening agent combined with intravenous injection of a chemotherapy drug, without the need for complex equipment, and has high clinical transformation potential, providing an effective strategy for overcoming the blood brain barrier of drug-resistant brain glioma and drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 In order to observe the fluorescence intensity related results of Claudin-5 under the inverted laser confocal microscope, wherein A is a schematic diagram of a blood brain barrier cell model and a tumor-blood brain barrier cell model, B is a laser confocal microscope observation of the expression amount of Claudin-5 protein in the tight junction protein of the blood brain barrier before and after administration of arginine 0h, 0.5h, 1h, 2h, 4h, and C is the fluorescence semi-quantitative analysis result (n = 3) of Image J.

[0023] Figure 2 In order to observe the fluorescence intensity related results of Claudin-5 under the inverted laser confocal microscope, wherein A is a schematic diagram of a blood brain barrier cell model and a tumor-blood brain barrier cell model, B is a laser confocal microscope observation of the expression amount of Claudin-5 protein in the tight junction protein of the blood brain barrier before and after administration of arginine 0h, 0.5h, 1h, 2h, 4h, and C is the fluorescence semi-quantitative analysis result (n = 3) of Image J.

[0024] Figure 3 In order to observe the fluorescence intensity related results of Claudin-5 under the inverted laser confocal microscope, wherein A is a schematic diagram of a blood brain barrier cell model and a tumor-blood brain barrier cell model, B is a laser confocal microscope observation of the expression amount of Claudin-5 protein in the tight junction protein of the blood brain barrier before and after administration of arginine 0h, 0.5h, 1h, 2h, 4h, and C is the fluorescence semi-quantitative analysis result (n = 3) of Image J.

[0025] Figure 4Fig. 2 shows the results of in vivo fluorescence imaging of the distribution of doxorubicin hydrochloride in the brain of normal mice and tumor-bearing mice after administration of the optimal dose of arginine and hydroxyurea, wherein A is a schematic diagram of fluorescence imaging of doxorubicin hydrochloride in the brain of mice after administration of normal saline, 2 mg / kg doxorubicin hydrochloride, 2 mg / kg doxorubicin hydrochloride + 200 mg / kg arginine, and 2 mg / kg doxorubicin hydrochloride + 1 g / kg arginine for 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h, and B is the results of semi-quantitative analysis of fluorescence imaging.

[0026] Figure 5 Fig. 3 shows the pharmacodynamic evaluation of the combination of blood-brain barrier opening agents and doxorubicin hydrochloride for the treatment of drug-resistant glioma, wherein A is a small animal live imaging diagram of tumor-bearing mice on the 4th day, 7th day, 9th day, 11th day, and 13th day after tumor implantation, B is the results of semi-quantitative analysis of small animal live imaging of tumor-bearing mice on the 4th day, 7th day, 9th day, 11th day, and 13th day after tumor implantation, and C is the body weight change of tumor-bearing mice on the 4th day, 6th day, 8th day, 10th day, and 12th day after tumor implantation.

[0027] Figure 6 Fig. 4 shows the survival cycle and median survival period of tumor-bearing mice in the control group and the experimental group. DETAILED DESCRIPTION

[0028] In order to make the objectives, characteristics and advantages of the present application more obvious and easy to understand, the following will be described in detail through specific embodiments. In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the concept of the present application, so the present application is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present application can be combined accordingly without mutual conflict.

[0029] The operation methods not specified in the following examples are generally performed according to conventional conditions or according to the conditions recommended by the manufacturers. The contents not described in detail in the specification belong to the prior art known to those skilled in the art. The experimental materials used in the following examples can be purchased from conventional biochemical reagent companies, unless otherwise specified.

[0030] In the following examples, the application is first screened for drug sensitivity of chemotherapeutic drugs: through in vitro pharmacodynamic evaluation, the chemotherapeutic drugs with significant inhibitory activity on temozolomide-resistant glioma cells are screened; secondly, the research on blood-brain barrier opening agent based on physiological mechanism is carried out: based on the scientific mechanism that arginine can down-regulate the expression of tight junction proteins, the time-effect and dose-effect relationship of opening blood-brain barrier is systematically studied, and the efficiency is compared and optimized with other opening agents such as hydroxyurea; finally, the synergistic antitumor drug composition is constructed: the optimized blood-brain barrier opening agent is combined with the screened effective chemotherapeutic drugs to improve the target delivery efficiency of drugs in tumor sites.

[0031] Example 1 Screening of sensitive chemotherapeutic drugs for drug-resistant glioma In this study, the thiazolyl blue colorimetric method was used to evaluate the cytotoxicity of drugs. Exponentially growing GL261 cells and GL261 / TR cells were inoculated in 96-well plates at a density of 5 10 3 cells / well and cultured at 37℃ and 5% CO2. When the cell adhesion density reached 70-80%, a series of drug concentration gradients were added, and blank cells without drug were set as controls. The same method was used for parallel 3 groups. 37℃, 5% CO2 continued to incubate for 48 h, 20 μL of 5 mg / ml thiazolyl blue solution was added to each well. 37℃, 5% CO2 continued to incubate for 4 h, the culture solution was discarded, 200 μL of dimethyl sulfoxide was added to each well, and 37℃ constant temperature shaking table was shaken for 15-30 min until the crystal violet was completely dissolved. The absorbance value was measured at 570 nm by ultraviolet-visible spectrophotometer. The cell survival rate was calculated according to the following formula: cell survival rate (%) = A 给药 / A 空白 100%, wherein A 给药 is the absorbance value of the drug group, and A 空白 is the absorbance value of the cells without drug.

[0032] IC50 value greater than 500 μM is defined as no significant toxicity. As shown in Table 1, the chemotherapeutic drugs have obvious inhibitory effect on sensitive and temozolomide-resistant glioma, and hydroxyurea, methotrexate and doxorubicin hydrochloride are preferred.

[0033] Table 1 Effect of chemotherapeutic drugs on sensitive glioma cells GL261 strain and temozolomide-resistant glioma cells GL261 / TR strain respectively determined by thiazolyl blue colorimetric method

[0034] Example 2 Arginine regulates tight junction protein expression to open blood-brain barrier The blood-brain barrier cell model was constructed: exponentially growing bEnd.3 cells were inoculated in 96-well plates at a density of 8 10 4 The cells were seeded at a density of 1 cell per well in 24-well plates covered with small discs and cultured at 37 °C with 5% CO2. Subsequent experiments could be performed once the bEnd.3 cell adhesion density reached 70-80%.

[0035] Construction of a tumor-blood-brain barrier cell model: GL261 / TR cells with exponential growth were used to construct a tumor-blood-brain barrier cell model. 10 4 The cells were seeded at a density of cells / well in 24-well Transwell chambers of a plate, with 1 mL of serum-containing culture medium added to the outer chamber. The plates were then incubated at 37 °C and 5% CO2 for 24 hours. The Transwell chamber containing GL261 / TR cells was then transferred to the lower chamber of a 24-well plate containing bEnd.3 cells, and co-cultured at 37 °C and 5% CO2 for another 12 hours before proceeding with subsequent experiments.

[0036] To construct blood-brain barrier and tumor-blood-brain barrier cell models: 1 ml of 1 mM arginine solution was added to each well, and the cells were incubated at 37 ℃ and 5% CO2 for 0.5 h, 1 h, 2 h, and 4 h, respectively. Blank blood-brain barrier and tumor-blood-brain barrier cell models were set up as control groups. Two parallel groups were constructed using the same method.

[0037] Immunofluorescence staining: After drug administration, discard the culture medium and wash three times with PBS. Add 200 μL of 4% paraformaldehyde to each well for fixation for 30 min, and wash three times with PBS. Add 20 μL of 3% BSA solution to each well for blocking for 1 h, add proportionally diluted Claudin-5 primary antibody, incubate overnight at 4°C, and wash three times with PBS. Add the corresponding fluorescent secondary antibody, incubate at room temperature for 3-4 h, and wash three times with PBS. Add 2 μL of 1 mg / ml Hochest to each well, counterstain cell nuclei with DAPI for 5 min, and wash three times with PBS. Remove the small discs from the 24-well plate, place them on a glass slide with glycerol, and mount. All procedures must be performed in the dark. Observe the Claudin-5 fluorescence intensity under an inverted laser confocal microscope, acquire images, and take photographs. Calculate the fluorescence intensity per unit area using the following formula: Fluorescence intensity per unit area (times) = Luminescent signal intensity of the treatment group / Luminescent signal intensity of the blood-brain barrier group.

[0038] Depend on Figure 1 According to the AC analysis, arginine intervention significantly downregulated Claudin-5 protein expression in bEnd.3 cells within the tumor-blood-brain barrier, with the expression level decreasing in a time-dependent manner 0.5 h after administration. These results indicate that arginine can downregulate the expression of the tight junction protein Claudin-5, leading to pathological "disruption" of the blood-brain barrier function.

[0039] Example 3 Dose-effect relationship of hydroxyurea in regulating opening of blood-brain barrier Exponentially growing Gl261 / TR-luci cells were trypsinized, and the cells were collected by centrifugation at 1000 rpm for 3 min, and the supernatant was discarded. The cell pellet was washed with PBS pre-cooled at 4°C, and centrifuged, and the supernatant was discarded. The cell pellet was resuspended by adding serum-free medium pre-cooled at 4°C, and placed on ice for standby. Male C57BL / 6 mice weighing 20-22 g were used as model animals, and the brain of each mouse was shaved. With the aid of a brain stereotaxic apparatus, 1×10 5 Gl261 / TR-luci cells were inoculated into the striatum of each mouse (0.8 mm anterior to the bregma, 1.0 mm right, and 2.5 mm deep), to construct a mouse glioma model expressing luciferase.

[0040] On the fourth day after modeling, the mice were imaged, and were randomly divided into four groups according to the tumor size. Another group of normal mice without modeling was set up. Each group of mice was orally administered with 200 μL of PBS, 60 mg / kg hydroxyurea, 120 mg / kg hydroxyurea, and 480 mg / kg hydroxyurea, respectively. Three parallel administration groups were set up.

[0041] Two hours after administration, 2 mg / kg doxorubicin hydrochloride was injected into the tail vein of each mouse. One hour later, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate, the chest cavity was opened, the heart was exposed, the right auricle was cut, and 50 mL of normal saline and 50 mL of 4% paraformaldehyde were sequentially perfused through the left ventricle. The brain tissue was immersed in 10 mL of 4% paraformaldehyde for fixation, and fluorescence imaging was performed using a live fluorescence imager. The images were collected and photographed. Under dark conditions, slice 1-slice 4 of the brain tissue was sectioned, and slice 2 of the brain tissue was subjected to fluorescence imaging, and the images were collected and photographed.

[0042] Bioluminescence refers to a luminescence phenomenon generated by bioluminescence, and the excitation energy required comes from enzymatic reactions in the organism, which is autofluorescence in the animal body, and does not require an excitation light source. Generally, mammalian bioluminescence is generally to integrate the Firefly Luciferase gene, i.e., the luciferase gene, into the chromosomal DNA of the cells to be observed, so as to express luciferase, and to culture a cell strain capable of stably expressing luciferase. When the cells divide, transfer, and differentiate, luciferase is also continuously and stably expressed. The GL261-Luciferase cells containing fluorescent markers are inoculated in situ into the brain of nude mice, and the substrate luciferin is injected intraperitoneally, so that stable luminescence can be generated within 5-15 min, and the luminescence intensity is linearly related to the number of labeled cells. Therefore, the tumor size can be evaluated based thereon, and the mice can be uniformly grouped. The relative fluorescence intensity is calculated according to the following formula: relative fluorescence intensity (times) = luminescence signal intensity of the administration group / luminescence signal intensity of the control group.

[0043] Mouse brain slice area as Figure 2 As shown in A, the result is as follows Figure 2 As shown in B, both medium and high doses of hydroxyurea increased the fluorescence signal value of doxorubicin in the mouse brain. Specifically, compared with the saline group, the fluorescence signal value increased by 0.85 times after intervention with 120 mg / kg hydroxyurea. Figure 2 (C in the middle).

[0044] Example 4: Dose-response relationship of arginine in regulating blood-brain barrier opening A mouse glioma model expressing parasitic luciferase was constructed according to the method in Example 3. On the 4th day after modeling, the mouse tumors were imaged, and the mice were randomly divided into 4 groups according to tumor size. The mice had their brains shaved. Each group of mice was injected intravenously with 200 μL of PBS, 2 mg / kg doxorubicin hydrochloride, 2 mg / kg doxorubicin hydrochloride + 200 mg / kg arginine, and 2 mg / kg doxorubicin hydrochloride + 1 g / kg arginine, respectively. Three parallel administration groups were set up. The drugs were administered every other day for three consecutive times. Two hours after the third administration, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. The thoracic cavity of the mice was opened, the heart was exposed, the right atrial appendage was cut open, and 50 mL of physiological saline and 50 mL of 4% paraformaldehyde were perfused sequentially through the left ventricle. The brain tissue was taken and fixed by immersing it in 10 mL of 4% paraformaldehyde. Fluorescence imaging was performed using a live fluorescence imaging system, and images were acquired and photographed. Under light-protected conditions, corresponding portions of brain tissue slices 1-4 were sectioned, and brain tissue from slice 2 was used for fluorescence imaging with doxorubicin hydrochloride. Images were acquired and photographed. The relative fluorescence intensity was calculated using the corresponding formula.

[0045] The results are as follows Figure 3 As shown in Figure A, both low-dose and high-dose arginine intervention increased the fluorescence signal value of doxorubicin in the mouse brain. Specifically, compared with the group without the open-label agent, the fluorescence signal value increased 2.07 times after 200 mg / kg arginine intervention. Figure 3 (B in the middle).

[0046] Example 5: Blood-brain barrier opener regulates the intracerebral distribution of doxorubicin hydrochloride. A mouse glioma model expressing parasitic luciferase was constructed according to the method described in Example 3. On the 4th day after modeling, the mouse tumors were imaged, and the mice were randomly divided into 4 groups according to tumor size. Each group of mice was injected with an equal dose of physiological saline via tail vein and orally with physiological saline, injected with 2 mg / kg doxorubicin hydrochloride via tail vein, injected with 2 mg / kg doxorubicin hydrochloride via tail vein and orally with 200 mg / kg arginine, or injected with 2 mg / kg doxorubicin hydrochloride via tail vein and orally with 120 mg / kg hydroxyurea. Three parallel administration groups were set up. Administration was performed every other day. After the third administration, at 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h, fluorescence imaging of the mouse brain was performed to detect the content of doxorubicin hydrochloride in the brain, i.e., the total fluorescence efficiency and distribution.

[0047] Fluorescence imaging results as follows Figure 4 As shown in A, the semi-quantitative results are as follows: Figure 4 As shown in B, compared with the mice in the group without the opening agent, both the hydroxyurea and arginine intervention groups increased the fluorescence signal value of doxorubicin hydrochloride in the brain by 0.40 and 0.87 times, respectively, indicating that opening the blood-brain barrier helps to increase the distribution of doxorubicin hydrochloride in the brain.

[0048] Example 6: Pharmacodynamics of a blood-brain barrier opener combined with doxorubicin hydrochloride for the treatment of drug-resistant gliomas A mouse glioma model expressing parasitic luciferase was constructed according to the method described in Example 3. On day 4 after modeling, the mouse tumors were imaged and randomly divided into 4 groups according to tumor size. Each group of mice was injected with an equal dose of PBS via tail vein and then administered physiological saline by gavage. Other groups received injections of 2 mg / kg doxorubicin hydrochloride via tail vein, 2 mg / kg doxorubicin hydrochloride via tail vein and 200 mg / kg arginine via gavage, or 2 mg / kg doxorubicin hydrochloride via tail vein and 120 mg / kg hydroxyurea via gavage. Five parallel administration groups were set up. Mouse body weight was continuously monitored, and the bioluminescent signal intensity of the orthotopic glioma was recorded. The median survival of the model animals was calculated according to the Kaplan-Meier method. The relative tumor size was calculated using the following formula: Relative tumor size (times) = Bioluminescent signal intensity on day n / Bioluminescent signal intensity on day 4.

[0049] like Figure 5 The results of the AB study showed that, compared with the doxorubicin hydrochloride treatment group, the treatment group based on hydroxyurea and arginine as blood-brain barrier openers exhibited superior inhibitory effects on glioma growth. Compared with the saline control group, the relative fold increase in mean bioluminescence intensity of tumors in the model animals treated with hydroxyurea combined with doxorubicin hydrochloride decreased from 2321 to 872, while the relative fold increase in mean bioluminescence intensity of tumors in the model animals treated with arginine combined with doxorubicin hydrochloride decreased from 2321 to 253. Therefore, arginine intervention had the most significant enhancing effect on glioma growth inhibition in the doxorubicin hydrochloride treatment group.

[0050] During treatment, the mice's body weight changed as follows Figure 5 As shown in C, the body weight of the model animals in the saline, doxorubicin hydrochloride, hydroxyurea combined with doxorubicin hydrochloride, and arginine combined with doxorubicin hydrochloride treatment groups decreased by 30.5%, 28.7%, 24.1%, and 18.5%, respectively. Animal lifespan results are as follows... Figure 6 As shown, both hydroxyurea and arginine combined with doxorubicin hydrochloride treatments prolonged the survival time of the animals. Compared with the saline group, the survival curves of the model animals in the hydroxyurea combined with doxorubicin hydrochloride treatment group and the arginine combined with doxorubicin hydrochloride treatment group were extended from 16.0 days to 18.0 days and 19.0 days, respectively.

[0051] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of arginine in the preparation of formulations that promote the crossing of chemotherapeutic drugs for gliomas across the blood-brain barrier.

2. Application of hydroxyurea in the preparation of formulations that promote the crossing of chemotherapeutic drugs for gliomas across the blood-brain barrier.

3. The application according to claim 1 or 2, characterized in that, Chemotherapy drugs for gliomas are chemotherapy drugs that target sensitive or drug-resistant gliomas.

4. A pharmaceutical composition for treating drug-resistant glioma, characterized in that, The components include at least one blood-brain barrier opener and at least one drug-resistant glioma chemotherapy drug. The blood-brain barrier opener is arginine or hydroxyurea, and the drug-resistant glioma chemotherapy drug includes doxorubicin hydrochloride, mitoxantrone, or methotrexate.

5. The pharmaceutical composition according to claim 4, characterized in that, Drug-resistant gliomas are gliomas resistant to temozolomide. The blood-brain barrier opener is arginine, and the chemotherapy drug for drug-resistant gliomas is doxorubicin hydrochloride.

6. The pharmaceutical composition according to claim 4, characterized in that, The mass ratio of blood-brain barrier openers to chemotherapy drugs for drug-resistant gliomas is 5-1000:

1.

7. The pharmaceutical composition according to claim 4, characterized in that, When the blood-brain barrier opener is arginine, the mass ratio of arginine to drug-resistant glioma chemotherapy drugs is 5-1000:

1. When the blood-brain barrier opener is hydroxyurea, the mass ratio of hydroxyurea to drug-resistant glioma chemotherapy drugs is 5-500:

1.

8. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier or diluent, and is formulated into a pharmaceutically acceptable dosage form.

9. The pharmaceutical composition according to claim 4, characterized in that, Blood-brain barrier openers and chemotherapy drugs for drug-resistant gliomas are administered simultaneously or alone.

10. The use of the pharmaceutical composition according to any one of claims 4-9 in the preparation of a treatment product for drug-resistant glioma.

Images