CN117323361A

CN117323361A - Pharmaceutical composition for treating brain glioma and application thereof

Info

Publication number: CN117323361A
Application number: CN202311206069.5A
Authority: CN
Inventors: 吴蔚; 鲁萍; 王杰; 唐敬亮; 付四海; 魏康竞
Original assignee: Changsha City No1 Hospital
Current assignee: Changsha City No1 Hospital
Priority date: 2023-09-19
Filing date: 2023-09-19
Publication date: 2024-01-02

Abstract

The present disclosure provides a pharmaceutical composition for treating brain glioma and application thereof, and belongs to the field of medicine. The pharmaceutical composition comprises temozolomide and radix puerariae extract, wherein the mass ratio of temozolomide to radix puerariae extract is (4:1) - (1:1); the combination of temozolomide and radix puerariae extract has remarkable synergistic inhibition effect on brain glioma. The pharmaceutical composition can be applied to preparing medicines for treating brain glioma, and has wide application prospect.

Description

Pharmaceutical composition for treating brain glioma and application thereof

Technical Field

The present disclosure relates to the field of medicine, and in particular, to a pharmaceutical composition for treating glioma and application thereof.

Background

Gliomas refer to tumors that originate from brain glial cells, the most common primary intracranial tumor. The annual incidence rate of the glioma in China is 5-8/10 ten thousand, and the disease death rate of 5 years is inferior to that of pancreatic cancer and lung cancer in systemic tumor. Low-grade gliomas (LGG; WHO grade i, ii) progress relatively slowly, have a good prognosis, and have a median survival of 10-12.9 years; high-grade gliomas (HGG; WHO III, grade IV) tend to progress rapidly with a poor prognosis, with a median survival of only 18 months in glioblastomas (WHO grade IV).

The glioma treatment mainly comprises surgical excision and can relieve clinical symptoms and prolong the survival time by combining comprehensive treatment methods such as radiotherapy, chemotherapy and the like. Although radical surgery plus chemoradiotherapy is still a basic means for treating glioma, due to the invasive growth, the surgery is difficult to completely remove the tumor due to the radioactive damage to surrounding normal brain tissues, and has the advantages of easy recurrence, high death rate and no optimistic curative effect.

In 1976, the U.S. Food and Drug Administration (FDA) approved lomustine as a therapeutic drug for intracranial tumors. Only three drugs for treating brain cancer were formally approved 40 years later: in 1996, carmustine wafers were approved for recurrent glioma, and are currently approved as adjuvant therapy for newly diagnosed and operated glioma patients; temozolomide (TMZ) was approved in 1999 for 3-stage degenerative astrocytoma patients, after which the indication extended to newly diagnosed brain gliomas as maintenance therapy after radiotherapy; in 2009, bevacizumab was approved for accelerated use in glioma patients with worsening disease after treatment. At present, TMZ is a gold standard for clinically treating glioma, but TMZ is a cytotoxic drug, has a great side effect of killing normal growing mucous membrane cells and fast growing cells, and is easy to resist after a period of time.

The kudzu root is the dry root of the kudzu vine of the leguminous plant, is a common traditional Chinese medicine, has cool and sweet nature, and has the effects of relieving muscle and allaying fever, promoting the secretion of saliva or body fluid to quench thirst and raising yang to check diarrhea. Modern pharmacological research shows that the kudzuvine root has the functions of vasodilation, heart protection, neuroprotection, antioxidation, anti-inflammatory, anti-tumor, anti-alcohol, liver protection and insulin resistance alleviation. The kudzuvine root contains a plurality of compounds, wherein the content of puerarin is the largest. Recent researches show that puerarin has a certain therapeutic effect on colon cancer, and puerarin has a strong inhibition effect on the activity of glioma U251 cells under the induction of phorbol lipid, and can resist migration invasion of the U251 cells. The antagonism of puerarin on tumor cell migration invasion and the protection effect on normal cells can reduce the drug resistance of cytotoxic drugs and the killing of normal cells to a great extent, and puerarin can pass through the blood brain barrier, so that the puerarin has advantages in treating brain diseases, and the research of combining pueraria extract with temozolomide in treating brain glioma is not available at present.

Past studies have shown that chronic inflammation is a major feature of glioblastoma biology, with the blood brain barrier being destroyed by glioblastoma, resulting in chronic neuroinflammation. Microglia are innate immune cells in the brain that can release a variety of inflammatory factors and mediators of inflammation to promote inflammatory responses. In one aspect, studies have shown that the number of selectively activated M2 microglia is positively correlated with glioma grade, directly or indirectly leading to glioma, tumor proliferation and tumor invasion. On the other hand, inhibiting activation of microglia, reducing release of inflammatory mediators can act to protect nerve cells.

Disclosure of Invention

The purpose of the present disclosure is to overcome the deficiencies of the prior art and provide a pharmaceutical composition for treating glioma and applications thereof. TMZ and radix Puerariae extract can inhibit activation of microglial cells, thereby reducing neuroinflammation, improving anti-tumor effect of TMZ, and reducing toxicity to normal cells.

In order to achieve the above purpose, the technical scheme adopted by the present disclosure is as follows: provides a pharmaceutical composition for treating brain glioma, which comprises temozolomide and radix puerariae extract, wherein the mass ratio of temozolomide to radix puerariae extract is (4:1) - (1:1).

In one embodiment, the mass ratio of temozolomide to kudzuvine root extract is 2:1.

The temozolomide and the radix puerariae extract are combined to be used as active ingredients for treating the glioma, the combination of the temozolomide and the radix puerariae extract is in a synergistic relationship, and the combination of the temozolomide and the radix puerariae extract has obvious inhibition effect on glioma cells cultured in vitro, and the effect is stronger than that of the temozolomide and the radix puerariae extract when the temozolomide and the radix puerariae extract are used singly.

The active ingredients of the pueraria extract are puerarin (puearin), daidzein and daidzein, genistein, genistin, 3'-hydroxy puerarin (3' -hydroxy puearin), 3'-methoxy puerarin (3' -methoxypuearin), and the inventor finds that the pharmaceutical composition has better therapeutic effect on brain glioma when the pueraria extract is puerarin.

In addition, the mass ratio of temozolomide to kudzuvine root extract is a key factor affecting the efficacy of the pharmaceutical composition, in the present disclosure, the mass ratio of temozolomide to kudzuvine root extract may 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, 1:1, and the present disclosure is not limited thereto; in order to further improve the efficacy of the pharmaceutical composition, it is preferable that the mass ratio of temozolomide to pueraria extract is 2:1.

In one embodiment, the pharmaceutical composition further comprises an aqueous hydroxypropyl methylcellulose solution.

In one embodiment, the hydroxypropyl methylcellulose (HPMC) in the aqueous hydroxypropyl methylcellulose solution has a mass percent concentration of 0.2%.

The hydroxypropyl methylcellulose aqueous solution can completely dissolve temozolomide and radix Puerariae extract to improve the dispersion uniformity of the pharmaceutical composition.

The mass percentage concentration of the hydroxypropyl methylcellulose in the hydroxypropyl methylcellulose aqueous solution is a factor influencing the solubility of the temozolomide and the radix puerariae extract, and when the mass percentage concentration of the hydroxypropyl methylcellulose in the hydroxypropyl methylcellulose aqueous solution is 0.2%, the solubility of the temozolomide and the radix puerariae extract is optimal.

In another aspect, a method of preparing the pharmaceutical composition is provided, comprising the steps of: adding temozolomide and radix Puerariae extract into a sterile centrifuge tube, adding hydroxypropyl methylcellulose water solution with the mass percentage concentration of 0.2% of hydroxypropyl methylcellulose, and performing vortex ultrasonic treatment until the solution is clarified to obtain the pharmaceutical composition.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

In one embodiment, the pharmaceutical composition is in the form of injection, tablet, capsule, granule, suspension, emulsion, solution, sol, lyophilized powder for injection, mucilage, aerosol, microcapsule, microsphere, liposome, micelle, sustained release preparation or controlled release preparation.

In another aspect, the application of the pharmaceutical composition in preparing a medicament for treating glioma is provided.

In one embodiment, the brain glioma comprises an astrocytoma, an oligodendroglioma, a ependymoma.

Compared with the prior art, the beneficial effects of the present disclosure are: a pharmaceutical composition for treating brain glioma comprises temozolomide and radix Puerariae extract, wherein the mass ratio of temozolomide to radix Puerariae extract is (4:1) - (1:1); the combination of temozolomide and radix puerariae extract has remarkable synergistic inhibition effect on brain glioma. The pharmaceutical composition can be applied to preparing medicines for treating brain glioma, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the effect of varying concentrations of TMZ on LPS-induced microglial NO release;

FIG. 2 shows the effect of varying concentrations of Pueraria lobata extract on LPS-induced microglial NO release;

FIG. 3 shows the effect of varying concentrations of puerarin on LPS-induced microglial NO release;

FIG. 4 is a graph showing the effect of various concentrations of the pharmaceutical composition of example 2 on LPS-induced microglial NO release;

FIG. 5 is a graph showing the effect of various concentrations of the pharmaceutical composition of example 4 on LPS-induced microglial NO release;

in fig. 1-5, # indicates that there was a significant difference compared to the placebo group; * Indicating that there was a significant difference compared to the model group.

Detailed Description

For a better understanding of the objects, technical solutions and advantages of the present disclosure, the present disclosure will be further described with reference to specific examples and comparative examples, which are intended to be in detail, not to be limiting of the present disclosure. All other embodiments, which can be made by one of ordinary skill in the art without making any inventive effort, are intended to be within the scope of the present disclosure. The experimental reagents and apparatus to which the present disclosure is directed are common reagents and apparatus unless otherwise indicated.

The following description of the raw materials used in the examples and comparative examples is provided, but is not limited to these materials:

temozolomide: purchased from SIGMA-ALDRICH;

radix Puerariae extract: purchased from wuhanbao leys, biotechnology limited;

puerarin: purchased from SIGMA-ALDRICH.

Examples 1 to 5 and comparative examples 1 to 4

The mass ratios of TMZ and Pueraria lobata extracts in the pharmaceutical compositions of examples 1-5 and comparative examples 1-4 are shown in Table 1, and the pharmaceutical compositions of examples 1-5 and comparative examples 1-4 were prepared as follows:

1.0g of HPMC is weighed and dissolved in 400mL of sterilized water with the temperature of about 70 ℃, stirred overnight, and stored at 4 ℃ after the next day and constant volume to 500 mL;

weighing TMZ and puerarin, placing into a sterile centrifuge tube, slowly adding 75mL of 0.2% HPMC along the tube wall, performing vortex ultrasound until the solution is clear, and preserving at 4deg.C in dark, wherein the total mass of TMZ and puerarin is 15mg.

TABLE 1

Example 6

This example tests the pharmaceutical compositions of examples 1-5 and comparative examples 1-4 for inhibition of proliferation of human glioma U-87 cells in vitro.

The test method is as follows:

materials: human U-87 cell lines were purchased from Perkinelmer.

Test reagent: EMEM medium was purchased from GIBCO company, fetal bovine serum was purchased from GIBCO company, and double antibody was purchased from GIBCO company.

Cell strain and cell culture: human glioma U-87 cells were cultured with EMEM containing 10% fetal bovine serum at 37℃on 5% CO ₂ Culturing in a constant temperature incubator.

Complete medium: EMEM medium +10% foetal calf serum +1% diabody.

The experimental process comprises the following steps:

(1) Collecting human glioma U-87 cells in logarithmic growth phase, and regulating cell suspension to 1×10 with complete culture medium ⁵ And each mL. Three 96-well plates were used, 100. Mu.L of physiological saline was added to each well on the periphery of the cell plate, and the other wells were added to each well with a pipette at a cell concentration of 1X 10 ⁵ mu.L of each cell suspension per mL was adjusted to 2.0X10 cells per well ⁴ And then at 37 ℃,100% relative humidity, 5% CO ₂ Incubating for 24 hours in an incubator;

(2) The experimental groups diluted the pharmaceutical compositions of examples 1-5 and comparative examples 1-4 50-fold with complete medium, cells were added at 25 μl/well, the control group was added with an equal volume of complete medium, and a total of 6 duplicate wells per group of pharmaceutical composition; incubation in a 5% CO2 incubator at 37 ℃,100% relative humidity for 72 hours;

(3) Adding 10 mu L of CCK-8 detection reagent into each hole, and placing the mixture in a 37 ℃ incubator for incubation for 3 hours;

(4) After shaking, absorbance at a wavelength of 450nm is measured on a SpectraMax M5 MicroplateReader, and the cell inhibition rate is calculated by taking absorbance at 650nm as a reference, wherein the calculation formula is as follows: cell inhibition = (1-average OD value of experimental group/average OD value of control group) X100%.

The experimental results are shown in table 2.

TABLE 2

From the experimental data in Table 2, the pharmaceutical composition obtained in the embodiment of the disclosure has obvious inhibition effect on the proliferation of human glioma U-87 cells in vitro.

Example 7

This example tests the growth inhibitory effect of the pharmaceutical compositions of examples 1-5 and comparative examples 1-4 on in situ model of human glioma U-87MG-Luc nude mice xenograft tumors.

The test method is as follows:

materials: human glioma U-87MG-Luc cell lines were purchased from Perkinelmer.

Test reagent: EMEM medium was purchased from GIBCO, fetal bovine serum was purchased from GIBCO, matrigel gum was purchased from GIBCO.

Cell strain and cell culture: human glioma U-87MG-Luc cells were cultured with EMEM containing 10% fetal bovine serum at 37℃on 5% CO ₂ Culturing in a constant temperature incubator.

And (3) establishing a model: BALB/c male nude mice (weighing about 15-18 g) were supplied by Peking Vitrendylar laboratory animal technologies Inc., and kept in a pathogen free specific grade (specefic pathogenfree, SPF) environment. Taking U-87MG-Luc cells in logarithmic growth phase, re-suspending in EMEM culture medium, adding Matrigel, and preparing glioma cell suspension with mass ratio of Matrigel to EMEM culture medium of 1:1 and cell concentration of 3×10 ⁷ cells/mL; cell suspension (1.5X10) ⁵ Individual cells) were stereotactically inoculated into the cranium of each mouse.

Drug administration intervention:

after 3 days of inoculation, bioluminescence values of model-producing mice were measured, the model-producing mice were randomly divided into 10 groups of 6 animals each according to the bioluminescence values, the group diary was Day 0, and administration was started according to the weight of each animal, and the administration dose, the drug concentration, the administration volume, the administration route and the administration frequency were as shown in table 3, wherein the drug of the blank control group was an aqueous hydroxypropyl methylcellulose solution having a mass percentage concentration of 0.2%. The experimental period was 40 days, and the body weight of the mice was measured 2 times a week during the experimental period. Cage side observations, 1 time per day, if mice were dying or dead, the frequency of clinical observations could be appropriately increased and scored as the number of mice dying. Bioluminescence intensities were measured 5 times in Day 0, day7, day14, day21, and Day 35. During the administration period, the individual mice body weight was reduced by more than 15% compared with Day 0 (BWL. Gtoreq.15%), and the treatment was stopped until the mice body weight was recovered (BWL < 15%), and administration was resumed.

Evaluation index:

the tumor growth inhibition (TGI%) was calculated as follows: (TFt-TFc)/TFc×100%; wherein TFc is the bioluminescence value obtained at each measurement of the negative control group, and TFt is the bioluminescence value at each measurement of the administration group.

The death of each group of mice was recorded daily during the test, and if mice were to die, the difference in survival time of each group of mice was compared. Each group of time to live was evaluated as median time to live (median survival time, MST).

The weight change (%) of the mice was calculated as follows: (BWt-BW 0)/BW 0X 100%; where BW0 is the weight of the mice measured at the time of divided cage administration (i.e., day 0), and BWt is the weight of the mice at each measurement.

The test results are shown in Table 4.

TABLE 3 Table 3

Note that: p.o. is oral, qd 5days once daily for five consecutive days.

TABLE 4 Table 4

Group of	MST/day	TGI％	Day21 rate of body weight change
				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％

Note that: example 2 most mice survived at the end of the experiment, and half the mice died, median Survival Time (MST) could not be calculated.

The test data in Table 4 show that the combination of temozolomide and puerarin has obvious inhibition effect on human glioma U-87MG-Luc cells, and prolongs the survival time of in-situ-implanted tumor mice.

Example 8

Influence of Pueraria lobata extract and puerarin on LPS-induced microglial inflammatory mediator release

The test method is as follows:

materials: the mouse microglial cell line N9 was purchased from Shanghai Bayer Biotechnology Co.

Test reagent:

lipopolysaccharide (LPS): purchased from SIGMA corporation (united states);

DMEM medium: purchased from GIBCO corporation (united states);

fetal bovine serum: purchased from GIBCO corporation (united states);

penicillin sodium for injection: purchased from the pharmaceutical general factory of the Harrow pharmaceutical, A110405414;

streptomycin sulfate for injection: purchased from Shandong Lukang medicine Co., ltd., 091201;

nitric oxide detection kit: purchased from the Biyun institute of biotechnology, product number S0021;

dimethyl sulfoxide (DMSO): purchased from amerco corporation;

tetramethylazo salt (MTT): purchased from amerco corporation.

Medicament:

minocycline (MINO), available from SIGMA Inc. (USA).

Preparation of drugs and reagents:

the test substance is completely dissolved in DMSO solution to prepare mother solution with a certain concentration, and the final concentration of DMSO is controlled to be less than or equal to 0.1% when the test substance and cells are co-cultured in an experiment;

MINO and LPS are respectively dissolved in PBS buffer solution to prepare mother solution with certain concentration, and the mother solution is diluted in use;

PBS：NaCl 8.0g，KCl 2.0g，Na ₂ HPO ₄ ·12H ₂ O 3.492g，KH ₂ PO ₄ 0.2 g is dissolved in 1L double distilled water;

MTT:250mg MTT was dissolved in 50ml PBS buffer, stirred on a magnetic stirrer for 30min, sterilized by filtration through a 0.22 μm filter, and sub-packaged.

Cell culture:

all glassware and metal instruments (flasks, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 119 ℃ for 30min to thoroughly remove the contamination. A cell culture solution containing 10% fetal bovine serum, 100U/mL penicillin/streptomycin and 1mM sodium pyruvate was prepared based on DMEM medium. N9 microglial cells at about 5X 10 ⁵ The concentration of cells/mL was 37℃and 5% CO ₂ Subculturing in culture flask until the adherent cells occupy about bottom surface of culture flaskThe product is 70-80%, the adherent cells are digested by pancreatin, and the adherent cells are passaged to another culture flask. Experiments were performed with N9 microglia following passage 3 after thawing by liquid nitrogen cryopreservation.

Effect on LPS-induced N9 microglial NO release:

taking N9 microglial cells in logarithmic growth phase at 5×10 ⁵ cells/mL were inoculated in 96-well plates (100. Mu.L/well), incubated for 24 hours, then different concentrations of the test substance or minocycline positive drug (MINO) were added, pre-protected for 18 hours, LPS (final concentration 1. Mu.g/mL) was added, and incubated for 48 hours with a blank. Three parallel wells were provided for each concentration. After the incubation, 50. Mu.L of the supernatant culture solution is taken, 50. Mu.L of Griess reagent I is added, 50. Mu.L of Griess reagent II is added, the absorbance value is measured at 540nm of an enzyme-labeled instrument, and NO in the supernatant is calculated ^2- And calculates an IC50 value.

Data statistics:

experimental data are all expressed as mean ± standard error, and data statistics are performed using SPSS17.0 statistical software. The differences between groups were analyzed by one-way variance analysis, with P <0.05 being significant differences. The test results are shown in FIGS. 1-5.

As can be seen from FIGS. 1 and 5, TMZ has NO inhibitory effect on the release of N9 microglial NO induced by LPS, and the release of microglial NO can be inhibited only when the concentration of radix Puerariae extract is not less than 25 mu M; puerarin can inhibit release of microglial NO at certain concentration, and TMZ and Puerarin extract or Puerarin composition can significantly inhibit release of microglial NO at various concentrations, thereby reducing neuroinflammation; and under the same concentration, the inhibition effect of the composition is better than that of puerarin alone. TMZ and radix Puerariae extract or puerarin can be combined to improve anti-tumor effect of TMZ and reduce toxicity to normal cells.

Finally, it should be noted that the above embodiments illustrate the technical solution of the present disclosure and not limit the scope of the present disclosure, and although the present disclosure has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present disclosure may be modified or equally substituted without departing from the spirit and scope of the technical solution of the present disclosure.

Claims

1. The pharmaceutical composition for treating the glioma is characterized by comprising temozolomide and radix puerariae extract, wherein the mass ratio of temozolomide to radix puerariae extract is (4:1) - (1:1).

2. The pharmaceutical composition according to claim 1, wherein the mass ratio of temozolomide to kudzuvine root extract is 2:1.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises hydroxypropyl methylcellulose.

4. A pharmaceutical composition according to claim 3, wherein the hydroxypropyl methylcellulose is present in a concentration of 0.2% by mass.

5. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable adjuvant.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is in the form of an injection, a tablet, a capsule, a granule, a suspension, an emulsion, a solution, a sol, a lyophilized powder for injection, a cement, an aerosol, a microcapsule, a microsphere, a liposome, a micelle, a sustained release formulation or a controlled release formulation.

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

8. The use according to claim 7, wherein the brain glioma comprises astrocytoma, oligodendroglioma, ependymoma.