A pharmaceutical composition for treating brain glioma and application thereof

Abstract

This disclosure provides a pharmaceutical composition for treating glioma and its application, belonging to the pharmaceutical field. The pharmaceutical composition comprises temozolomide and pueraria lobata extract, with a mass ratio of temozolomide to pueraria lobata extract of (4:1)–(1:1). The combined use of temozolomide and pueraria lobata extract exhibits a significant synergistic inhibitory effect on glioma. This pharmaceutical composition can be used in the preparation of drugs for treating glioma and has broad application prospects.

Description

Technical Field

[0001] This disclosure relates to the pharmaceutical field, specifically to a pharmaceutical composition for treating glioma and its application. Background Technology

[0002] Gliomas are tumors originating from glial cells in the brain and are the most common primary intracranial tumors. In my country, the annual incidence of gliomas is 5-8 per 100,000, and the 5-year mortality rate is second only to pancreatic and lung cancer among all cancers. Low-grade gliomas (LGG; WHO grades I and II) progress relatively slowly and have a good prognosis, with a median survival of 10–12.9 years. High-grade gliomas (HGG; WHO grades III and IV) often progress rapidly and have a poor prognosis; glioblastoma (WHO grade IV) has a median survival of only 18 months.

[0003] Treatment for gliomas primarily involves surgical resection, combined with radiotherapy, chemotherapy, and other comprehensive therapies to alleviate clinical symptoms and prolong survival. Although radical surgery combined with radiotherapy and chemotherapy remains the basic approach to treating gliomas, their invasive growth and radiation damage to surrounding normal brain tissue make complete surgical removal difficult, leading to a high recurrence rate, high mortality rate, and less than optimistic treatment outcomes.

[0004] In 1976, the U.S. Food and Drug Administration (FDA) approved lomustine as a treatment for intracranial tumors. In the 40 years since, only three drugs for treating brain cancer have been officially approved: in 1996, carmustine wafers were approved for recurrent gliomas and are now also approved as adjuvant therapy for newly diagnosed glioma patients who have undergone surgery; in 1999, temozolomide (TMZ) was approved for grade 3 anaplastic astrocytoma patients, and its indication was later expanded to newly diagnosed gliomas as maintenance therapy after radiotherapy; and in 2009, bevacizumab received accelerated approval for glioma patients whose condition has worsened after treatment. Currently, TMZ is the gold standard for glioma treatment in clinical practice, but TMZ is a cytotoxic drug that also kills normally growing mucosal cells and rapidly growing cells, resulting in significant side effects and a tendency to develop resistance over time.

[0005] Kudzu root, the dried root of the legume *Pueraria lobata*, is a commonly used traditional Chinese medicine. It is cool in nature and sweet in taste, and has the effects of relieving muscle tension and fever, promoting blood circulation, quenching thirst, and stopping diarrhea. Modern pharmacological studies have shown that kudzu root has vasodilatory, cardioprotective, neuroprotective, antioxidant, anti-inflammatory, anti-tumor, alcohol-relieving, liver-protective, and insulin-resistance-reducing effects. Kudzu root contains various compounds, with puerarin being the most abundant. Recent studies have found that puerarin has a certain therapeutic effect on colon cancer, and it also has a strong inhibitory effect on the activity of phorbol-induced glioma U251 cells, thus counteracting the migration and invasion of U251 cells. Puerarin's ability to counteract tumor cell migration and invasion, as well as its protective effect on normal cells, can significantly reduce drug resistance to cytotoxic drugs and the killing of normal cells. Furthermore, puerarin can cross the blood-brain barrier, offering advantages in the treatment of brain diseases. However, there are currently no studies on the combination of kudzu root extract and temozolomide for the treatment of glioma.

[0006] Previous research has shown that chronic inflammation is a major biological characteristic of glioblastoma. Glioblastoma disrupts the blood-brain barrier, leading to chronic neuroinflammatory conditions. Microglia are innate immune cells in the brain and can release various inflammatory factors and mediators, promoting inflammatory responses. On the one hand, studies have shown a positive correlation between the number of selectively activated M2 microglia and the grade of glioma, directly or indirectly contributing to glioma development, tumor proliferation, and tumor invasion. On the other hand, inhibiting microglia activation and reducing the release of inflammatory mediators can protect nerve cells. Summary of the Invention

[0007] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a pharmaceutical composition for treating glioma and its application. TMZ combined with kudzu root extract can inhibit the activation of microglia, thereby reducing neuroinflammation, enhancing the antitumor effect of TMZ while reducing toxicity to normal cells.

[0008] To achieve the above objectives, the technical solution adopted in this disclosure is as follows: a pharmaceutical composition for treating glioma is provided, comprising temozolomide and pueraria lobata extract, wherein the mass ratio of temozolomide to pueraria lobata extract is (4:1)-(1:1).

[0009] In one embodiment, the mass ratio of temozolomide to kudzu root extract is 2:1.

[0010] This disclosure describes the combined use of temozolomide and pueraria lobata extract as active ingredients for the treatment of glioma. The combination of the two has a synergistic effect, and the combined use of temozolomide and pueraria lobata extract has a significant inhibitory effect on cultured glioma cells in vitro, which is stronger than when either is used alone.

[0011] The active ingredients of kudzu root extract are puerarin, daidzin and daidzein, genistein, genistein, 3'-hydroxypuerarin, and 3'-methoxypuerarin. The inventors have found that when the kudzu root extract is puerarin, the drug composition has a better therapeutic effect on glioma.

[0012] Furthermore, the mass ratio of temozolomide to kudzu root extract is a key factor affecting the efficacy of the pharmaceutical composition. In this disclosure, the mass ratio of temozolomide to kudzu root extract can be 4:1, 3.8:1, 3.6:1, 3.4:1, 3.2:1, 3:1, 2.8:1, 2.6:1, 2.4:1, 2.2:1, 2:1, 1.8:1, 1.6:1, 1.4:1, 1.2:1, or 1:1, and this disclosure is not limited to these. To further improve the efficacy of the pharmaceutical composition, a mass ratio of temozolomide to kudzu root extract of 2:1 is preferred.

[0013] In one embodiment, the pharmaceutical composition further includes an aqueous solution of hydroxypropyl methylcellulose.

[0014] In one embodiment, the mass percentage concentration of hydroxypropyl methylcellulose (HPMC) in the aqueous hydroxypropyl methylcellulose solution is 0.2%.

[0015] Hydroxypropyl methylcellulose aqueous solution can completely dissolve temozolomide and kudzu root extract to improve the dispersion uniformity of the pharmaceutical composition.

[0016] The mass percentage concentration of hydroxypropyl methylcellulose in aqueous solution is a factor affecting the solubility of temozolomide and kudzu root extract. The solubility of temozolomide and kudzu root extract is optimal when the mass percentage concentration of hydroxypropyl methylcellulose in aqueous solution is 0.2%.

[0017] On the other hand, a method for preparing the pharmaceutical composition is provided, comprising the following steps: adding temozolomide and kudzu root extract to a sterile centrifuge tube, then adding an aqueous solution of hydroxypropyl methylcellulose with a mass percentage concentration of 0.2%, and vortexing and sonicating until the solution is clear to obtain the pharmaceutical composition.

[0018] In one embodiment, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0019] In one embodiment, the dosage form of the pharmaceutical composition is an injection, tablet, capsule, granule, suspension, emulsion, solution, sol, lyophilized powder for injection, gel, aerosol, microcapsule, microsphere, liposome, micelle, sustained-release formulation, or controlled-release formulation.

[0020] On the other hand, the use of the pharmaceutical composition in the preparation of a drug for treating glioma is provided.

[0021] In one embodiment, the glioma includes astrocytoma, oligodendroglioma, and ependymoma.

[0022] Compared with the prior art, the beneficial effects of this disclosure are as follows: a pharmaceutical composition for treating glioma, comprising temozolomide and pueraria lobata extract, wherein the mass ratio of temozolomide to pueraria lobata extract is (4:1)-(1:1); the combined use of temozolomide and pueraria lobata extract has a significant synergistic inhibitory effect on glioma. The pharmaceutical composition of this invention can be used in the preparation of drugs for treating glioma and has broad application prospects. Attached Figure Description

[0023] Figure 1 The effect of different concentrations of TMZ on LPS-induced NO release from microglia;

[0024] Figure 2 The effect of different concentrations of kudzu root extract on LPS-induced NO release from microglia;

[0025] Figure 3 The effect of different concentrations of puerarin on LPS-induced NO release from microglia;

[0026] Figure 4 The effect of different concentrations of the drug composition from Example 2 on LPS-induced NO release from microglia;

[0027] Figure 5 The effect of different concentrations of the drug composition in Example 4 on LPS-induced NO release from microglia;

[0028] Figure 1-5 In the diagram, # indicates a significant difference compared to the blank control group; * indicates a significant difference compared to the model group. Detailed Implementation

[0029] To better illustrate the purpose, technical solutions, and advantages of this disclosure, the following description, in conjunction with specific embodiments and comparative examples, aims to provide a detailed understanding of the content of this disclosure, rather than limiting it. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this disclosure. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this disclosure are all commonly used reagents and instruments.

[0030] The raw materials used in the embodiments and comparative examples are described below, but are not limited to these materials:

[0031] Temozolomide: Purchased from SIGMA-ALDRICH;

[0032] Kudzu root extract: purchased from Wuhan Baolai Biotechnology Co., Ltd.;

[0033] Puerarin: Purchased from SIGMA-ALDRICH.

[0034] Examples 1-5 and Comparative Examples 1-4

[0035] The mass ratios of TMZ and kudzu root extract in the pharmaceutical compositions of Examples 1-5 and Comparative Examples 1-4 are shown in Table 1. The preparation methods of the pharmaceutical compositions of Examples 1-5 and Comparative Examples 1-4 are as follows:

[0036] Weigh 1.0g of HPMC and dissolve it in 400mL of sterile water at about 70℃. Stir overnight, and the next day, bring the volume up to 500mL and store at 4℃.

[0037] Weigh TMZ and puerarin into a sterile centrifuge tube, slowly add 75 mL of 0.2% HPMC along the tube wall, vortex and sonicate until the solution is clear, and store at 4°C in the dark. The total mass of TMZ and puerarin is 15 mg.

[0038] Table 1

[0039]

[0040]

[0041] Example 6

[0042] This embodiment tests the inhibitory effect of the pharmaceutical compositions of Examples 1-5 and Comparative Examples 1-4 on the in vitro proliferation of human glioma U-87 cells.

[0043] The testing method is as follows:

[0044] Materials: Human U-87 cell line was purchased from PerkinElmer.

[0045] Test reagents: EMEM culture medium, fetal bovine serum, and penicillin antibodies were purchased from GIBCO.

[0046] Cell line and cell culture: Human glioma U-87 cells were cultured in EMEM containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator using standard methods.

[0047] Complete culture medium: EMEM medium + 10% fetal bovine serum + 1% antibiotics.

[0048] Experimental procedure:

[0049] (1) Collect human glioma U-87 cells in the logarithmic growth phase and adjust the cell suspension to 1×10⁻⁶ with complete culture medium. 5 Cells / mL. Take three 96-well plates, add 100 μL of physiological saline to each well on the periphery of the plate, and add 1×10⁶ cells to each of the other wells using a pipette. 5 100 μL of cell suspension per cell / mL was prepared to achieve a cell count of 2.0 × 10⁶ cells per well. 4 Then, incubate the samples in a 37°C, 100% relative humidity, 5% CO2 incubator for 24 hours.

[0050] (2) The experimental group used complete culture medium to dilute the drug compositions of Examples 1-5 and Comparative Examples 1-4 50 times and added them to cells at 25 μL / well. The control group was added with an equal volume of complete culture medium. Each drug composition was in 6 replicates. The cells were incubated at 37°C, 100% relative humidity and 5% CO2 for 72 hours.

[0051] (3) Add 10 μL of CCK-8 detection reagent to each well and incubate in a 37℃ incubator for 3 hours;

[0052] (4) After shaking, the absorbance at 450 nm wavelength was measured on SpectraMax M5 MicroplateReader. The absorbance at 650 nm was used as a reference to calculate the cell inhibition rate. The calculation formula is: cell inhibition rate = (1 - average OD value of experimental group / average OD value of control group) × 100%.

[0053] The experimental results are shown in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] As can be seen from the experimental data in Table 2, the pharmaceutical composition obtained in the embodiments of this disclosure has a significant inhibitory effect on the in vitro proliferation of human glioma U-87 cells.

[0058] Example 7

[0059] This embodiment tests the growth-inhibiting effect of the pharmaceutical compositions of Examples 1-5 and Comparative Examples 1-4 on the human glioma U-87MG-Luc nude mouse xenograft tumor in situ model.

[0060] The testing method is as follows:

[0061] Materials: Human glioma U-87MG-Luc cell line was purchased from PerkinElmer.

[0062] Test reagents: EMEM culture medium, fetal bovine serum, and Matrigel were all purchased from GIBCO.

[0063] Cell line and cell culture: Human glioma U-87MG-Luc cells were cultured in EMEM containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator using standard methods.

[0064] Model establishment: Male BALB / c nude mice (approximately 15-18g in weight) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a specific pathogen-free (SPF) environment. Glioma cells were routinely passaged and cultured. U-87MG-Luc cells in the logarithmic growth phase were resuspended in EMEM medium, and Matrigel gel was added at a 1:1 mass ratio to prepare a glioma cell suspension with a cell concentration of 3 × 10⁻⁶ cells / mL. 7 Cells / mL; suspend the cells (1.5 × 10⁻⁶) 5 (1 cell) was stereotactically inoculated into the cranium of each mouse.

[0065] Drug intervention:

[0066] Three days after inoculation, the bioluminescence value of the model mice was measured. The model mice were randomly divided into 10 groups of 6 mice each based on their bioluminescence value. The grouping date was recorded as Day 0. Drug administration began according to the weight of each animal. The dosage, drug concentration, administration volume, route of administration, and frequency are shown in Table 3. The blank control group received a 0.2% (w / w) aqueous solution of hydroxypropyl methylcellulose. The experimental period was 40 days. Mouse weight was measured twice weekly. Mice were observed at the cage edge daily. If any mice showed signs of near-death or death, the frequency of clinical observation was increased, and the number of deaths was recorded. Bioluminescence intensity was measured on Day 0, Day 7, Day 14, Day 21, and Day 35, for a total of 5 times. During the drug administration period, if the weight of any mouse decreased by more than 15% compared to Day 0 (BWL ≥ 15%), drug administration was discontinued until the mouse's weight recovered (BWL < 15%), at which point administration was resumed.

[0067] Evaluation indicators:

[0068] The tumor growth inhibition rate (TGI%) is calculated as follows: (TFt-TFc) / TFc×100%; where TFc is the bioluminescence value obtained in each measurement of the negative control group, and TFt is the bioluminescence value obtained in each measurement of the drug-treated group.

[0069] During the experiment, the mortality of mice in each group was recorded daily. If any mice died, the survival time of the mice in each group was compared. The median survival time (MST) was used to evaluate the survival time of each group.

[0070] The formula for calculating the change in mouse body weight (%) is as follows: (BWt-BW0) / BW0×100%; where BW0 is the mouse body weight measured when the mice are divided into cages for administration (i.e., Day 0), and BWt is the mouse body weight at each measurement.

[0071] The test results are shown in Table 4.

[0072] Table 3

[0073]

[0074] Note: po means oral administration, qd*5days means once a day for five consecutive days.

[0075] Table 4

[0076] Group MST / day TGI% Day 21 Weight Change Rate Blank control 25 / -12.74％ Example 1 35 88.52％ -5.49％ Example 2 / 86.41％ 3.22％ Example 3 32 77.95％ 3.96％ Example 4 35 70.83％ -1.05％ Example 5 32 69.58％ 2.79％ Comparative Example 1 28 70.20％ -8.36％ Comparative Example 2 26 56.74％ -6.95％ Comparative Example 3 29 69.24％ -11.83％ Comparative Example 4 26 35.17％ -9.28％

[0077] Note: At the end of the experiment in Example 2, most mice were alive and less than half died, so the median survival time (MST) could not be calculated.

[0078] The experimental data in Table 4 demonstrate that the combined use of temozolomide and puerarin has a significant inhibitory effect on human glioma U-87MG-Luc cells and prolongs the survival of mice with orthotopic tumor implantation.

[0079] Example 8

[0080] Effects of Pueraria lobata extract and puerarin on LPS-induced release of inflammatory mediators in microglia

[0081] The testing method is as follows:

[0082] Materials: Mouse microglia N9 cell line, purchased from Shanghai Baili Biotechnology Co., Ltd.

[0083] Test reagents:

[0084] Lipopolysaccharide (LPS): Purchased from SIGMA (USA);

[0085] DMEM medium: purchased from GIBCO (USA);

[0086] Fetal bovine serum: purchased from GIBCO (USA);

[0087] Sodium penicillin for injection: purchased from Harbin Pharmaceutical Group General Factory, A110405414;

[0088] Streptomycin sulfate for injection: purchased from Shandong Lukang Pharmaceutical Co., Ltd., 091201;

[0089] Nitric oxide assay kit: purchased from Beyotime Biotechnology Research Institute, product number S0021;

[0090] Dimethyl sulfoxide (DMSO): purchased from Amresco.

[0091] Tetramethylazoazole salt (MTT): purchased from Amresco.

[0092] drug:

[0093] Minocycline (MINO), purchased from SIGMA (USA).

[0094] Preparation of drugs and reagents:

[0095] The test substance was completely dissolved in DMSO solution to prepare a stock solution of a certain concentration. During the experiment, the final concentration of DMSO was controlled to be ≤0.1% when the test substance was co-cultured with cells.

[0096] MINO and LPS were dissolved in PBS buffer to prepare stock solutions of a certain concentration, which were then diluted before use.

[0097] PBS: Dissolve 8.0g NaCl, 2.0g KCl, 3.492g Na2HPO4·12H2O, and 0.2g KH2PO4 in 1L of double-distilled water;

[0098] MTT: Dissolve 250 mg MTT in 50 ml PBS buffer, stir on a magnetic stirrer for 30 min, filter through a 0.22 μm filter membrane for sterilization, and dispense.

[0099] Cell culture:

[0100] All glassware and metal instruments (culture flasks, pipettes, solution bottles, etc.) used in cell culture and model establishment were autoclaved at 119°C for 30 minutes to thoroughly remove contaminants. A cell culture medium containing 10% fetal bovine serum, 100 U / mL penicillin / streptomycin, and 1 mM sodium pyruvate was prepared using DMEM medium as a base. N9 microglia were cultured at approximately 5 × 10⁶ cells / mL. 5 Cells / mL were cultured at 37°C in 5% CO2 flasks. By day 3, adherent cells covered approximately 70-80% of the bottom area of ​​the flask. The adherent cells were then digested with trypsin and passaged to another flask. Experiments were performed using N9 microglia from passage 3 onwards after cryopreservation and thawing in liquid nitrogen.

[0101] Effects of LPS on NO release induced by N9 microglia:

[0102] N9 microglia in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells. 5 Cells / mL were seeded in 96-well plates (100 μL / well). After 24 hours of incubation, different concentrations of the test substance or positive control drug minocycline (MINO) were added. After 18 hours of pre-protection, LPS (final concentration 1 μg / mL) was added, and the plates were incubated for 48 hours. A blank control was also included. Each concentration was tested in triplicate. After incubation, 50 μL of the supernatant was collected, and 50 μL of Griess reagent I and then 50 μL of Griess reagent II were added. The absorbance was measured at 540 nm using a microplate reader, and the NO concentration in the supernatant was calculated. 2- And calculate the IC50 value.

[0103] Data statistics:

[0104] Experimental data are expressed as mean ± standard error and statistically analyzed using SPSS 17.0 software. One-way ANOVA was used to assess differences between groups, with P < 0.05 considered statistically significant. Test results are as follows: Figure 1-5 As shown.

[0105] from Figure 1 and Figure 5 The study showed that TMZ had no inhibitory effect on LPS-induced NO release from N9 microglia; only when the concentration of kudzu root extract was ≥25 μM could it inhibit NO release from microglia. Puerarin could inhibit NO release from microglia at certain concentrations, while the combination of TMZ with kudzu root extract or kudzu root extract significantly inhibited NO release from microglia at all concentrations, thereby reducing neuroinflammation; and at the same concentration, the inhibitory effect of the combination was better than that of kudzu root extract alone. The combination of TMZ with kudzu root extract or kudzu root extract can enhance the antitumor effect of TMZ while reducing its toxicity to normal cells.

[0106] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of this disclosure and not to limit the scope of protection of this disclosure. Although this disclosure has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this disclosure without departing from the substance and scope of the technical solutions of this disclosure.

Claims

1. A pharmaceutical composition for treating glioma, characterized in that, It includes an active ingredient, which is composed of temozolomide and kudzu root extract in a mass ratio of (4:1) to (1:1).

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of temozolomide to kudzu root extract is 2:

1.

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes hydroxypropyl methylcellulose.

4. The pharmaceutical composition according to claim 3, characterized in that, The hydroxypropyl methylcellulose has a mass percentage concentration of 0.2%.

5. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The dosage form of the pharmaceutical composition is injection, tablet, capsule, granule, suspension, emulsion, solution, sol, or aerosol.

7. Use of the pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament for treating glioma.

8. The application according to claim 7, characterized in that, The gliomas mentioned include human gliomas.

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