CN117899071A - Small molecule medicine for treating malignant brain glioma and application thereof - Google Patents
Small molecule medicine for treating malignant brain glioma and application thereof Download PDFInfo
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Abstract
The invention discloses a small molecule medicine for treating malignant brain glioma, which comprises an active ingredient of phoxim. The small molecular medicine for treating malignant glioma can promote tumor blood vessel normalization, inhibit malignant glioma cell proliferation, and has small toxic and side effects and high targeting property.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a small-molecule medicine for treating malignant glioma and application of the small-molecule medicine for treating malignant glioma.
Background
Malignant glioma (Glioblastoma, GBM) is one of the most common invasive primary malignant tumors of the central nervous system, with high mortality. Patients generally have a median survival time of 12-18 months, a 2-year survival rate of 15% -20% and a 5-year survival rate of less than 5%. The high incidence rate and mortality rate of gliomas not only bring physiological and psychological harm to patients and family members, but also bring great economic pressure to families and society. There has been little improvement in the clinical therapeutic efficacy of malignant gliomas over the last 30 years, and new treatments and strategies are urgently needed for patients.
Current clinical standard treatment protocols for GBM patients involve maximal surgical excision followed by combined radiation and Temozolomide (TMZ) chemotherapy. TMZ is used as a first-line clinical medicine for treating malignant glioma, is a DNA alkylating agent for targeting proliferation cells, and has good curative effect in treating glioma. However, TMZ has strong toxic and side effects and is easy to resist, so that tumors recur, which greatly limits the clinical value of TMZ. Thus, there is an urgent need for new GBM chemotherapeutic agents.
Small molecule compounds are an important source of drugs for the treatment of various human diseases, including cancer. Sphingosine analog, siponimod (also known as BAF 312), is a second generation S1P receptor modulator approved by the united states food and drug administration for the treatment of relapsing multiple sclerosis. BAF312 is a second-generation S1P receptor agonist, selectively acts on S1P1 and S1P5 receptors, and has half maximal effector concentrations (concentration for 50%of maximal effect,EC50) of 0.39nM and 0.98nM, respectively, which are more than 1000-fold more selective than S1P2, S1P3, and S1P4 receptors. Previous studies have shown that BAF312, as an analog of S1P and a ligand of S1P1, competitively and selectively binds to S1P1, thereby promoting internalization and degradation of S1P 1. However, the action and the action mechanism of the traditional Chinese medicine composition in resisting malignant glioma are not reported at present. The invention provides application of sphingosine analogue BAF312 in anti-malignant glioma treatment and action mechanism thereof.
Disclosure of Invention
The invention provides a small molecular medicine for treating malignant glioma, which solves the defects of strong toxic and side effects and easy drug resistance of glioma medicines in the prior art.
In order to achieve the above purpose of the present invention, the following technical scheme is adopted:
A small molecule drug for treating malignant brain glioma, which comprises an active ingredient of simonamod.
A pharmaceutical composition comprising an effective dose of the small molecule drug for treating malignant glioma described above and a pharmaceutically acceptable carrier.
Further, the carrier includes one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a blending agent, a surfactant, a dispersing agent, or an antifoaming agent.
In the present invention, the medicament further comprises a pharmaceutically acceptable diluent.
Further, the diluent is one of distilled water, physiological sodium chloride or phosphate buffer saline and glucose solution.
Further, the pharmaceutical composition is administered in the gastrointestinal tract.
Further, the dosage form of the pharmaceutical composition is one of a tablet, a capsule, a granule, a pill and an oral liquid.
Further, the dosage of the small molecule drug for treating malignant brain glioma is 1mg/kg-5mg/kg.
In some embodiments of the invention, the pharmaceutical composition comprises a small molecule drug and temozolomide for treating malignant brain glioma.
An application of a small molecular medicine for treating malignant glioma in preparing malignant glioma medicine.
The invention has the following beneficial effects:
(1) The small molecular medicine for treating malignant glioma can promote tumor blood vessel normalization, inhibit malignant glioma cell proliferation, and has small toxic and side effects and high targeting property.
(2) The experimental result of the invention shows that BAF312 has no obvious systemic toxicity in mice. Our study shows that BAF312 small molecule compounds can be co-treated with TMZ for anti-malignant glioma treatment. May be a promising chemotherapeutic agent for the treatment of GBM.
Drawings
The technical scheme of the invention is further described below with reference to the specification, the drawings and the specific embodiments.
FIG. 1 is a schematic diagram of constructing an in situ model and administration of a malignant brain glioma mouse using GL261 cells of the mouse glioma cell line;
FIG. 2 shows the effect of BAF312 on glioma cell proliferation;
FIG. 3 shows the in situ tumor Collagen IV/CD31/DAPI co-IF staining;
FIG. 4 is a Claudin-5/CD31/DAPI co-IF staining of in situ tumors;
FIG. 5 shows in situ tumor Occludin/CD31/DAPI co-IF staining;
FIG. 6 shows ZO-1/CD31/DAPI co-IF staining of in situ tumors;
FIG. 7 shows in situ tumor PDGFR beta/CD 31/DAPI co-IF staining;
FIG. 8 shows the effect of BAF312 on the number of immune cells in a tumor;
FIG. 9 shows the inhibition of glioblastoma cell growth by BAF312 in combination with TMZ.
Detailed Description
According to the invention, a glioma model is constructed, and in-vivo experiments of mice prove that sphingosine analog, octabolmod (BAF 312), has the effect of inhibiting malignant glioma cells from proliferation, is mainly realized by inducing cell cycle arrest to promote apoptosis, and verifies the effect of combining Temozolomide (TMZ) with the effect of treating glioma.
1. Effect of small molecule medicine on malignant brain glioma
(1) Construction of glioma model
The glioma model was constructed as shown in fig. 1, C57BL/6J mice were selected as in vivo experimental study subjects, the glioma cell line GL261 cells (1×10 4/μl,3×10 4/unit) were implanted into the right striatum (right side of bregma, 1.8 mm, depth about 2.8 mm) of the mice by in situ injection, needles were withdrawn at a rate of 0.5 mm/30 s, then sutured with 4-0 sutures, and after one week the mice were randomly divided into control and experimental groups (n=5-6), and the same manner was used for the intragastric administration: BAF312 mg/kg (labeled BAF-1mg in the figure) and BAF312 5mg/kg (labeled BAF-5mg in the figure); control group: 0mg/kg, i.e. only solvent PBS was taken. The administration was started 1 time per day for 7 days after 15 days of molding, and the mice were anesthetized and drawn for immunofluorescence analysis after 7 days of administration to find the most effective drug concentration.
(2) Immunofluorescent staining
Deeply anesthetized C57BL/6J mice require heart perfusion with approximately 20 ml pre-chilled PBS followed by brain seed tumor tissue. Tissues were washed once with PBS, fixed with 4% pfa at room temperature for 1 hour, then dehydrated with 15% sucrose at room temperature for 1-2 hours, and then transferred to 30% sucrose for dehydration overnight at 4 ℃. After dehydration, OCT is used for embedding, and the frozen is used at the temperature of minus 80 ℃. OCT embedded tissue was cut into 10 μm sections using a cryostat and placed at-80℃for use. Sections were removed, dried at room temperature for about 1 hour, washed 3 times with PBS for 10 minutes/time, incubated with blocking solution (PBS containing 5% goat serum+1% BSA+0.3% Triton+0.1% NaN 3) for 1 hour at room temperature, followed by overnight incubation of primary antibodies (PBS containing 1% BSA+0.3% Triton+0.1% NaN 3, first group: hamster CD31:1/500, rat TER119:1/200, rabbit Collagen IV:1/200; the second group hamster CD31 1/500, rat PDGFRbeta 1/100, rabbit Ki67 1/50, the third group hamster CD31 1/500, rat CD144 1/100, rabbit Claudin-5:1/300, the fourth group hamster CD31 1/500, rat CD3 1/100, rabbit Caveolin-1:1/1000, the fifth group hamster CD31 1/500, rat CD8 1/100, rabbit Occludin:1/100, the sixth group hamster CD31:1/500, rat CD4:1/100, rabbit ZO-1:1/100), followed by shaking 3 times with PBS, 10 minutes/times, room temperature secondary antibodies (PBS containing 1% BSA+0.3% Triton+0.1% NaN 3, anti-488:1/500, anti-594:1/500, anti-hamster 647:1/500), 3 times with DAP, and finally sealing with a microscope for 4 minutes after quenching, sealing with a fluorescent tablet at 4℃after 3 times. The fluorescence results are shown in FIGS. 2-8.
Experimental results:
FIG. 2A is an in situ tumor CD31/DAPI co-IF staining. Scale bar: 80 μm. The tumor vessel density was quantified by calculating the ratio of CD31 staining positive area to DAPI area, see fig. 2B. Data are presented as mean ± SEM, * P <0.05 compared to control. When the BAF312 concentration reaches 5mg/kg, the tumor vascular density is obviously increased compared with the control group. FIG. 2C is an in situ tumor Ki-67/CD31/DAPI co-IF staining, scale bar: 80 μm. Ki-67 positive cells were quantified by calculating the ratio of the Ki-67 IF staining positive area to the DAPI area, see FIG. 2D. For each group (n=4-6), data are expressed as mean ± SEM, P <0.01 compared to control group. When the BAF312 concentration reaches 5mg/kg, the proliferation of tumor cells is obviously reduced compared with the control group. FIG. 2E is an orthotopic tumor TER119/CD31/DAPI co-IF staining, scale bar: 80 μm. The extent of red blood cell leakage within the tumor was determined by calculating the intensity of TER119 IF staining and the area of the CD31 region, see FIG. 2F. For each group (n=4-6), data are expressed as mean±sem, * P <0.05 compared to the control group. When the BAF312 concentration reached 5mg/kg, the red blood cell leakage was significantly reduced compared to the control group. The results of fig. 2 demonstrate that BAF312 can inhibit glioblastoma cell proliferation, promote tumor vascular density increase, and reduce red blood cell leakage.
FIG. 3A is an in situ tumor Collagen IV/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in Collagen IV was judged by calculating the ratio of the Collagen IV staining positive area to the CD31 staining positive area, see FIG. 3B. Data are presented as mean ± SEM, P >0.05 compared to control. Collagen IV showed no significant change at BAF312 concentration of 1mg/kg and 5 mg/kg. FIG. 4A is an in situ tumor Claudin-5/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in Claudin-5 was judged by calculating the ratio of Claudin-5 staining positive area to CD31 staining positive area, see FIG. 4B. Data are presented as mean ± SEM, P >0.05 compared to control. Claudin-5 showed no significant change in BAF312 concentration of 1mg/kg and 5 mg/kg. FIG. 5A is an in situ tumor Occludin/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in Occludin was judged by calculating Occludin the ratio of the staining positive area to the CD31 staining positive area, see fig. 5B. Data are presented as mean ± SEM, ** P <0.01 compared to control. Occludin at BAF312 concentrations of 1mg/kg and 5mg/kg were significantly reduced. FIG. 6A is an in situ tumor ZO-1/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in ZO-1 was judged by calculating the ratio of ZO-1 staining positive area to CD31 staining positive area, see FIG. 6B. Data are presented as mean ± SEM, * P >0.05 compared to control. ZO-1 had no significant change at BAF312 concentrations of 1mg/kg and 5 mg/kg. FIG. 7A is an in situ tumor PDGFRbeta/CD 31/DAPI co-IF staining (FIG. I), scale bar: 80 μm. The change in pdgfrβ was judged by calculating the ratio of ZO-1 staining positive area to CD31 staining positive area, fig. 7B. Data are presented as mean ± SEM, ** P <0.01 compared to control. PDGFRbeta coverage increases significantly at BAF312 concentration of 5 mg/kg. The results in FIGS. 3-7 demonstrate that BAF312 treatment can reduce Occludin expression levels and increase pericyte coverage.
FIG. 8A is an in situ tumor CD3/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in CD3 number was judged by calculating the ratio of CD3 number to DAPI staining positive area, fig. 8B. Data are presented as mean ± SEM, * P <0.05 compared to control. The amount of CD3 was significantly reduced at a BAF312 concentration of 1 mg/kg. FIG. 8C is an in situ tumor CD4/CD31/DAPI co-IF staining, scale bar: 80 μm. The change in CD4 number was judged by calculating the ratio of CD4 number to DAPI staining positive area, fig. 8D. Data are presented as mean ± SEM, * P >0.05 compared to control. CD4 did not significantly change at either BAF312 concentrations of 1mg/kg or 5 mg/kg. FIG. 8E is an orthotopic tumor CD8/CD31/DAPI co-IF staining (panel E), scale bar: 80 μm. The change in CD4 number was judged by calculating the ratio of CD8 number to DAPI staining positive area, see fig. 8F. Data are presented as mean ± SEM, * P <0.05 compared to control. The amount of CD8 was significantly reduced at a BAF312 concentration of 5 mg/kg. The results in fig. 8 demonstrate that BAF312 can reduce the number of immune cells in tumors.
2. Effect of small molecule medicine combined with TMZ administration on malignant brain glioma
After the most effective drug concentration is determined. Glioma models were constructed using a BAF312 and TMZ single/combined dosing regimen, as shown in fig. 4A. Mouse glioma (GL 261) cells (PBS resuspended, 1×10 4/μl,3×10 4/mouse) were pushed into the right striatum (right 1.8 mm of bregma, depth about 2.8 mm) of the mice by in situ injection, needles were withdrawn at 0.5 mm/30 s, then sutured with 4-0 sutures, and after two weeks, the mice were randomly divided into 4 groups (n=4-6), and the same manner of intragastric administration was used. V-V group: the solvent PBS alone was taken, and the molding was started 1 time per day for 7 days, and 1 time every 2 days for 7 days for 5 days. V-TMZ: the solvent PBS was administered first, and the molding was started 1 time per day for 7 days, and the administration of TMZ 100mg/kg was started 1 time every 2 days for 7 days for 5 days. BAF312-V group (labeled BAF-V in the figure): BAF312 5mg/kg was administered 1 time per day for 7 days, and 1 time every two days for 5 days for 7 days after 15 days of molding. BAF312-TMZ group (labeled BAF-TMZ in the figure): BAF312 5mg/kg was administered 1 time per day for 7 days, and BAF312 5mg/kg+TMZ 100mg/kg was administered 1 time per 2 days for 5 days from 15 days for molding. After the end of the post-administration for 1 day, the mice were anesthetized and the materials were taken for tumor volume statistics and immunofluorescence analysis, and immunofluorescence analysis was performed as described above.
Experimental results: FIG. 9B AF312 inhibits glioblastoma cell growth, achieving the same effect as TMZ. Fig. 9A is a model of in situ construction of a malignant glioma mouse combination using mouse GL261 cells. Fig. 9B is a graph showing changes in body weight of mice during BAF312 and TMZ dosing. Fig. 9C is an in situ tumor image of 4 groups of different dosing regimens mouse glioblastoma. Fig. 9D shows that both the in situ tumor volume of the group treated with TMZ or BAF312 alone (V-TMZ and BAF-V) and the group treated with TMZ in combination with BAF312 (BAF-TMZ) were reduced compared to the control group, the tumor volume mean of control group V-V was taken as reference ratio 1, the volume ratio was obtained by the volume mean of the remaining 3 groups/the tumor volume mean of V-V, and independent sample analysis was performed, the differences were statistically significant (P < 0.05), and the tumor volume differences between the 3 experimental groups were statistically non-significant (P > 0.05). It was demonstrated that BAF312 inhibited glioblastoma cell growth, achieving the same effect as TMZ.
Example 1
A small molecule drug for treating malignant brain glioma, comprising the active ingredient sphingosine analogue, simonamod. In this example, sphingosine analogs can normalize tumor vessels and inhibit proliferation of malignant glioma cells.
Example 2
A pharmaceutical composition comprising an effective dose of the above-described small molecule drug for the treatment of malignant glioma. Depending on the desired formulation, pharmaceutically acceptable, non-toxic carriers or diluents are also included. Such diluents are distilled water, physiological sodium chloride or phosphate buffered saline, dextrose solution, or the like. Such pharmaceutically acceptable carriers may be buffers, emulsifiers, suspending agents, stabilizers, preservatives, excipients, fillers, coagulants and harmonizing agents, surfactants, dispersing agents or antifoaming agents. In addition, the pharmaceutical compositions or formulations may also include other carriers, adjuvants or nontoxic, non-therapeutic stabilizers and the like.
Example 3
An application of a small molecular medicine for treating malignant glioma in preparing malignant glioma medicine. Specifically, the pharmaceutical composition comprises a small molecule drug for treating malignant glioma and temozolomide. The small molecular medicine for treating malignant glioma and temozolomide can be combined for treating malignant glioma.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A small molecule drug for treating malignant brain glioma, which is characterized by comprising an active ingredient of phoxim.
2. A pharmaceutical composition comprising an effective dose of the small molecule drug for treating malignant glioma described above and a pharmaceutically acceptable carrier.
3. The pharmaceutical composition of claim 2, wherein the carrier comprises one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a reconciling agent, a surfactant, a dispersing agent, or an antifoaming agent.
4. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable diluent.
5. The pharmaceutical composition of claim 5, wherein the diluent is one of distilled water, physiological sodium chloride or phosphate buffered saline, dextrose solution.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is administered parenterally.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is in the form of one of a tablet, a capsule, a granule, a pill, and an oral liquid.
8. The pharmaceutical composition according to claim 2, wherein the small molecule drug dose for treating malignant glioma is 1mg/kg-5mg/kg.
9. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition comprises a small molecule drug and temozolomide for treating malignant glioma.
10. Use of the small molecule drug for treating malignant glioma according to claim 1 in the preparation of a malignant glioma drug.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311722287.4A CN117899071A (en) | 2023-12-14 | 2023-12-14 | Small molecule medicine for treating malignant brain glioma and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311722287.4A CN117899071A (en) | 2023-12-14 | 2023-12-14 | Small molecule medicine for treating malignant brain glioma and application thereof |
Publications (1)
| Publication Number | Publication Date |
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| CN117899071A true CN117899071A (en) | 2024-04-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311722287.4A Pending CN117899071A (en) | 2023-12-14 | 2023-12-14 | Small molecule medicine for treating malignant brain glioma and application thereof |
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| Country | Link |
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| CN (1) | CN117899071A (en) |
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- 2023-12-14 CN CN202311722287.4A patent/CN117899071A/en active Pending
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