CN115737819A

CN115737819A - Pharmaceutical composition, preparation method and application thereof

Info

Publication number: CN115737819A
Application number: CN202211190252.6A
Authority: CN
Inventors: 金必莲; 林珂锋; 陈宁; 靳迪; 杨舒尧; 谢芳; 郝宇超
Original assignee: Zhongai Digital Medical Technology Guangdong Co ltd
Current assignee: Guangdong Sainz Medical Laboratory Co.,Ltd.
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2023-03-07
Anticipated expiration: 2042-09-28
Also published as: CN115737819B

Abstract

The invention relates to a pharmaceutical composition, a preparation method and application thereof. The pharmaceutical composition comprises an NF-kB inhibitor and an AURKA inhibitor, wherein the molar ratio of the NF-kB inhibitor to the AURKA inhibitor is 1: (0.5-0.7). The synthetic lethal effect of GBM cells is induced by the combination of NF-kB inhibitor and AURKA inhibitor, more preferably the combination of CAPE and MLN8237, and the effective treatment strategy of GBM is revealed that when CAPE and MLN8237 are combined, the clone formation of GBM is reduced to 5%, the apoptosis is increased to about 65%, and the cells blocked to the G0/G1 stage are increased to 90%; CAPE (NF-kB inhibitor) and MLN8237 (AURKA inhibitor) mutually eliminate drug resistance, and remarkably increase the anti-tumor effect.

Description

Pharmaceutical composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition and a preparation method and application thereof.

Background

Gliomas are tumors that occur in neuroectoderm, and are also known as neuroectoderm tumors or neuroepithelial tumors. As a key therapeutic target for cancer, cell cycle disorders underlie uncontrolled cell proliferation, a characteristic of the malignant phenotype. Several key nuclear kinases responsible for cell cycle progression and cell division, including cyclin-dependent kinases (CDKs), aurora kinases (AURKs) and polo-like kinases (PLKs), have been investigated as drug targets. AURKA belongs to the family of serine/threonine kinases, the activation of which is essential for the regulation of the cell division process through mitosis. Activation of AURKA has been shown to play an important role in a variety of cancers, including glioblastoma. AURKA attracted the attention of researchers because nearly 50 AURKA specific inhibitors are undergoing clinical trials, which are almost underperforming in the clinic due to drug resistance. Therefore, the problem of drug resistance of AURKA inhibitors (AURKA) is urgently solved.

MLN8237 is a highly selective AURKA small molecule inhibitor and IC thereof ₅₀ Is 1nM. MLN8237 was developed as an enhancement to its precursor MLN8054, and the development of MLN8054 was terminated after the first phase of the study due to side effects of the central nervous system, including limited lethargy. MLN8237 has been shown to inhibit cell proliferation by impairing mitosis, inducing cell cycle arrest and autophagy, and accelerating apoptosis and senescence in cancer cells of various cancer types. Treatment of human epithelial ovarian and pancreatic cancer cells with MLN8237 also hampers the EMT process. Importantly, MLN8237 significantly increases the sensitivity of tumor cells to chemotherapeutic drugs or radiation. Also, MLN8237 down-regulates several oncogenic pathways in cancer cells, such as NF- κ B, AKT, and STAT3.

Caffeic acid phenethyl ester (CAPE for short) is one of the main components in propolis, and has wide pharmacological activity. CAPE has obvious anti-tumor effect, thus having wide application prospect in medicine. CAPE is reported to be involved in inducing death of natural cells and having cytotoxic effects on malignant lesion tissues, and the CAPE can improve inflammatory response caused by tumor factors (12-0-tetradecanoyl-fimbrol-13-acetate) at a trace concentration. CAPE has been shown to be effective in inhibiting adventitious carcinogens by studying the probability of development in animal experiments. In addition, research shows that CAPE has good inhibitory activity on HL-60, bel-7402 and He1a tumor cells, and the inhibitory rates respectively reach 56.05%, 48.78% and 56.05%. Moreover, CPAE can inhibit the synthesis of DNA, RNA and proteins by cancer cells. Domestic and foreign researches show that CAPE has extremely strong effect on melanoma, colon cancer and gastric cancer cell lines and shows the characteristic of inhibiting cancer cells.

In human glioma cell lines, NF- κ B can induce the expression of MGMT protein, resulting in cell lines resistant to alkylating agents such as MLN8237.CAPE can pass through a blood brain barrier, can be used as a specific inhibitor of NF kappa B, can generate a synergistic effect on an action mechanism when being combined with an AURKA inhibitor MLN8237, has the characteristics of low toxic and side effects and the like when being used as a natural product, and has the possibility of being combined with the MLN8237 to be used for treating glioma in clinic.

Disclosure of Invention

Based on the above, the invention aims to provide a pharmaceutical composition, which can remarkably enhance the anti-tumor effect by jointly inhibiting GBM synthesis death caused by NF-kB and AURKA through the combination of NF-kB inhibitor and AURKA inhibitor.

The first object of the present invention is to provide a pharmaceutical composition comprising an NF- κ B inhibitor and an AURKA inhibitor, wherein the molar ratio of the NF- κ B inhibitor to the AURKA inhibitor is (0.5-0.7): 1.

further, the NF- κ B inhibitor is CAPE (caffeic acid phenethyl ester) and the AURKA inhibitor is MLN8237.CAPE (caffeic acid phenethyl ester) is one of the main components of propolis, and has pharmacological effects of resisting oxidation, resisting inflammation, resisting tumor, etc. The inventors investigated that CAPE binding to MLN8237 can significantly increase the antitumor effect of MLN8237.

Further, the molar ratio of the NF- κ B inhibitor to the AURKA inhibitor is 0.625:1.

it is another object of the present invention to provide a method for preparing the pharmaceutical composition of any one of the above, which is obtained by mixing the NF- κ B inhibitor and the AURKA inhibitor in a molar ratio.

Another object of the present invention is to provide a use of any one of the above pharmaceutical compositions in the preparation of a medicament for treating glioblastoma.

Further, the glioblastoma comprises LN229 and/or U251.

Further, the NF- κ B inhibitor and the AURKA inhibitor in the pharmaceutical composition induce glioblastoma lethality through combined inhibition of NF- κ B and AURKA.

Further, the NF- κ B inhibitor and the AURKA inhibitor of the pharmaceutical composition trigger glioblastoma lethality through combined inhibition of p65 and AURKA co-expression.

It is specifically noted that the focus of the present invention is to find the antitumor effect of caffeic acid phenethyl ester in combination with MLN8237, not the molar ratio between the above components, since both are known compounds, and the dosage in mammals has specific effective numerical range, therefore, the present invention is not limited to the above mentioned molar ratio range values, including all dosage ranges that can be safely used in mammals.

Compared with the prior art, the invention has the following beneficial effects:

GBM synthetic lethality was triggered by combined inhibition of NF-. Kappa.B and AURKA. The anti-tumor effect can be significantly enhanced by the combination of the NF- κ B inhibitor and the AURKA inhibitor, more preferably the combination of CAPE and MLN8237. The calculation of the Combined Index (CI) can result in the remarkable synergy (CI > 1) when CAPE is combined with MLN8237, and not only the superposition of the respective results. Applicants have found that the combination of an NF-. Kappa.B inhibitor and an AURKA inhibitor induces synthetic lethality in GBM cells, revealing an effective therapeutic strategy for GBM. The inventor researches the antitumor effect of CAPE combined with MLN8237, and finds that when CAPE or MLN8237 is used alone, the clone formation of GBM is reduced to about 40%, the apoptosis is increased to about 30%, and the number of cells blocked to the G0/G1 stage is increased to about 40%; when CAPE and MLN8237 are used together, the colony formation of GBM is reduced to 5%, the apoptosis is increased to about 65%, and the number of cells blocked to the G0/G1 stage is increased to 90%; CAPE (NF-kB inhibitor) and MLN8237 (AURKA inhibitor) mutually eliminate drug resistance, and remarkably increase the anti-tumor effect.

Drawings

FIG. 1 is the expression levels of AURKA or P65 in normal tissues and GBM in the GEPIA database;

FIG. 2 is a Kaplan-Meier survival curve analysis of AURKA or p65 patients with different levels of gene expression in the CGGA database;

FIG. 3 is a correlation between AURKA or p65 expression and WHO ranking in CGGA databases;

FIG. 4 is a Kaplan-Meier survival curve for p65 and AURKA in three expression sets;

FIG. 5 is a graph of the relationship and clinical case characteristics between three expression sets in the CGGA database;

FIG. 6 is a Western blot showing protein expression of p65 and p-p65 in five conventional GBM cell lines;

FIG. 7 is a volcano plot of RNA-seq analyzed differential regulatory gene expression between WT and p65 KO LN229 cells;

FIG. 8 is a bubble diagram of GO pathway analysis showing differential genes by RNA-seq analysis between WT and p65 KO LN229 cells;

FIG. 9 is a heat map of a G2/M-associated differential gene analysis to identify top10 between WT and p65 KO in LN229 cells by RNA-seq;

FIG. 10 shows RT-qPCR detection of mRNA levels of candidate genes in WT and p65 KO LN229 cells;

FIG. 11 shows the Western blot results showing AURKA expression after 24h incubation of LN229 and U251 cells with 5. Mu.M BAY11 or 3. Mu.M CAPE;

FIG. 12 is the score of the synergistic effect of increasing doses of MLN8237 in combination with increasing doses of CAPE on LN229 or U251 cell lines for 24 hours;

FIG. 13 is a clonogenic assay for LN229 or U251 cells with CAPE, MLN8237 alone and CAPE + MLN8237 in combination;

FIG. 14 is an apoptosis assay of LN229 or U251 cells with CAPE, MLN8237 alone and CAPE + MLN8237 in combination;

FIG. 15 is a cell cycle analysis of LN229 or U251 cells with CAPE, MLN8237 alone and CAPE + MLN8237 in combination;

FIG. 16 shows CCK8 measurements in LN229 cell line after combination of MLN8237 and CAPE;

FIG. 17 shows the CCK8 assay after combined use of MLN8237 and CAPE in the U251 cell line;

FIG. 18 is a semilog dose response curve and calculated EC for MLN8237 and CAPE in LN229 cell line ₅₀ A value;

FIG. 19 is a semi-logarithmic dose response curve and calculated EC for MLN8237 and CAPE in the U251 cell line ₅₀ The value is obtained.

Detailed Description

Example 1

In this example, bioinformatics analysis was performed using the data of glioblastoma in the GEPIA and CGGA databases, and the analysis software used was the R language to verify the performance of the evaluation criteria.

Analysis was performed using a glioblastoma database, in which:

referring to FIG. 1, by analyzing the data in the GEPIA database, it was found that the expression level of AURKA or P65 (NF-. Kappa.B P65) in GBM was significantly up-regulated compared to normal tissues.

Referring to FIG. 2, the genes of AURKA or p65 patients were divided into high expression and low expression groups according to their gene expression level by analyzing the data in the CGGA database. The prognosis of the AURKA high expression group is worse than that of the AURKA low expression group, and the prognosis of the p65 high expression group is worse than that of the p65 low expression group. The immediate survival analysis curve shows that the AURKA high expression and the P65 high expression indicate poor prognosis (P < 0.05).

Referring to fig. 3, by analyzing the data in the CGGA database, the expression of AURKA or p65 was significantly high in GBM in all three WHO grades (WHO ii, WHO iii, WHO iv), and the expression level of AURKA or p65 was higher as the WHO grade was higher. I.e. there is a correlation between AURKA or p65 expression levels and WHO grading, and AURKA or p65 expression is significantly correlated with poor prognosis of GBM.

Referring to FIG. 4, patients were divided into three groups based on the co-expression of p65 and AURKA: high expression group (both p65 and AURKA are high expression, i.e. p65+/AURKA +); a middle expression group (p 65 or AURKA is high expression, namely p65-/AURKA + or p65+/AURKA +); low expression group (p 65 and AURKA are low expression, p 65-/AURKA-); referring to FIG. 4, the analysis shows that survival (OS) is associated with co-expression of p65 and AURKA, where the Overall Survival (OS) for the high expression group (p 65+/AURKA +) is worse than for the medium expression group (p 65-/AURKA + or p65+/AURKA +), and the Overall Survival (OS) for the medium expression group (p 65-/AURKA + or p65+/AURKA +) is worse than for the low expression group (p 65-/AURKA-).

Analysis of fig. 3 and 4 reveals that AURKA or p65 expression is significantly associated with poor prognosis of GBM, including Overall Survival (OS) and WHO grading.

Referring to fig. 5, analysis of the three co-expressed groups of p65 and AURKA in the CGGA database with respect to clinical pathology revealed that co-expression of p65 and AURKA was associated with pathological tissues, and WHO stratification; among them, patients with glioblastoma and WHO iv had higher expression levels of p65 and AURKA.

From the above analysis, co-expression of NF-. Kappa.B and AURKA was associated with the prognosis of GBM. Our analysis shows that OS and other clinicopathologic features are associated with the co-expression of AURKA and p 65.

Example 2

This example demonstrates that AURKA is regulated by NF-. Kappa.B pathway in GBM by GO analysis of altered genes.

In primary GBM tissues, recent studies have shown that both phosphorylated p65 and total p65 expression levels are enhanced compared to control tissues.

In order to confirm the activation state of NF-. Kappa.B in GBM, see FIG. 6, protein expression of p65 and p-p65 was detected in 5 GBM conventional cell lines (U251, LN229, U118, T98G, U87), indicating that NF-. Kappa.B is active in GBM.

This example selects LN229 with high expression of p65 and U251 with low expression of p65 as cell lines for subsequent studies. This example uses RNA-seq to identify differentially expressed genes in LN229 cells after p65 knock-out (p 65 KO) and GO analysis, see fig. 7 and 8, where highly expressed genes were found to be most significantly concentrated in the cell cycle pathway (-greater log10p values are more significant), especially in the transformation of the G2/M phase of the cell cycle.

In the cell cycle, the regulation of kinases plays a crucial role. The mRNA levels of three kinases in the top ten genes were also examined for foldchange values, as shown in FIG. 9, and the mRNA levels of candidate genes in WT and p65 KO LN229 cells were also examined by RT-qPCR, as shown in FIG. 10, and analysis of FIGS. 9 and 10 shows that only AURKA is significantly affected by NF-. Kappa.B.

This example shows that the levels of AURKA are reduced in both cell lines following inhibition of NF-. Kappa.B, as shown in FIG. 11, by treating GBM cells with the NF-. Kappa.B inhibitors BAY11 and CAPE.

The results of the above studies indicate that AURKA is regulated by NF-kB pathway in GBM.

Example 3

CAPE, MLN8237 used alone, and CAPE + MLN8237 in combination were used in viability assays for cells observed on LN229 or U251 cell lines.

This example used increasing doses of MLN8237 in combination with increasing doses of CAPE to treat LN229 or U251 cell lines for 24 hours and proliferation was detected using the CCK8 kit, and a synergistic effect score was calculated by Combenefit. The results are shown in FIG. 12, where MLN8237 (AURKA inhibitor) and CAPE (NF-. Kappa.B inhibitor) were found to be able to eliminate drug resistance from each other. In this example, the molar ratio between CAPE and MLN8237 was (0.5-0.7): 1, more preferably the molar ratio of CAPE to MLN8237 is 0.625:1.

this example also allows observation of cell viability by CAPE, MLN8237 alone, and CAPE + MLN8237 in combination on LN229 cell line. The survival rates of the cells were observed on LN229 and U251 cell lines using CAPE (concentration: 3. Mu.M), MLN8237 (concentration: 5. Mu.M) alone and CAPE (concentration: 3. Mu.M) + MLN8237 (concentration: 5. Mu.M) in combination, and as a result, referring to FIGS. 13-15, it was found that CAPE and MLN8237 had substantially no effect on LN229 cell lines when used alone, but CAPE and MLN8237 had significant growth inhibitory (decrease) and survival rate-reducing effects on LN229 cell lines when used in combination, and the combination of CAPE and MLN8237 at different concentrations had significant growth inhibitory (decrease) and survival rate-reducing effects.

Wherein:

referring to fig. 13, when the cell lines LN299 and U251 were treated with the combination of CAPE and MLN8237, the ability to clonally form the cell lines LN299 and U251 was significantly inhibited, compared to the treatment of LN299 and U251 with CAPE or MLN8237 alone.

Referring to FIG. 14, the combined use of CAPE and MLN8237 to treat LN299 and U251 cell lines significantly increased apoptosis in LN299 and U251 cell lines as compared to CAPE or MLN8237 alone to treat LN299 and U251 cell lines.

Referring to FIG. 15, when CAPE and MLN8237 were used in combination to treat LN299 and U251 cell lines, the percentage of G0/G1 phase in the cell cycle of LN299 and U251 cell lines was significantly increased, the cell cycle was retarded, and LN229 and U251 cell proliferation was significantly inhibited, as compared to the treatment of LN299 and U251 cells with CAPE or MLN8237 alone.

Compared with the control group, when CAPE or MLN8237 is used alone, the clone formation is reduced to about 40%, the apoptosis is increased to about 30%, and the cells blocked to the G0/G1 phase are increased to 40%. When CAPE and MLN8237 were used together, colony formation decreased to 5%, apoptosis increased to about 65%, and cells arrested in the G0/G1 phase increased to 90%, and MLN8237 (AURKA inhibitor) and CAPE (NF-. Kappa.B inhibitor) mutually abolished resistance. The results of the studies in FIGS. 13-15 show that the combined use of CAPE and MLN8237 can produce a synthetic lethal effect on GBM compared to CAPE or MLN8237 alone.

The experimental methods and procedures involved in this example:

1. clone formation experiments

1) When the cell density meets the requirement of passage, the passage treatment is carried out while the cell counting is carried out, 500-100 cells are selected per hole according to the cell characteristics such as the cell growth speed and the like, then the cells are evenly paved in a 6-hole plate, and then the cells are continuously put back to a cell culture box for culture.

2) The cells were subjected to fluid exchange treatment every 3-4 days, and when the culture reached about 14 days, or when the clones in the well plate reached an appropriate size, the well plate was taken out of the incubator.

3) Old medium was aspirated off with a suction pump, washed 2-3 times with PBS, and then cells were fixed by adding 500. Mu.l of 4% paraformaldehyde under the conditions: standing for 30 minutes at room temperature, and discarding paraformaldehyde after standing.

4) After methanol is removed, 0.1 percent of crystal violet dye solution is added for dyeing, and the conditions are as follows: standing at room temperature for 15min, recovering crystal violet dye solution, washing with double distilled water, and air drying the pore plate.

5) And (4) photographing the dried pore plate, and storing an image for subsequent analysis.

2. Apoptosis assay

1) GBM cells in logarithmic growth phase were treated by changing the medium while 10. Mu.M etoposide was added to treat the cells for 24 hours.

2) Cells were digested with the appropriate pancreatin, then centrifuged at 300g for 5 minutes, the supernatant was discarded, the cells were washed once with PBS, and then the cells were resuspended and counted.

3) Take 5X 10 ⁵ The resuspended cells were centrifuged at 500g for 5 minutes, the supernatant was discarded and washed once with PBS, and then 500. Mu.L of diluted 1 × Annexin V Binding Buffer was added to resuspend the cells.

4) Add 5. Mu.L of AV staining solution and 5. Mu.L of PI staining solution.

5) After gently vortexing and mixing by a vortexer, incubation was performed for 30 minutes at room temperature in the dark.

6) After the reaction is finished, the detection is carried out by a flow analyzer immediately, and a filter membrane with 300 meshes is used for filtration treatment before the detection on a machine.

3. Cell cycle experiments

1) Establishing a detection model by using a proper method, establishing a negative control group at the same time, and collecting cells; calculating the volume of the required staining working solution according to the number of samples, and incubating 500 mu L of the working solution in each sample; rnase a: PI working solution is prepared according to the following steps of 1:9 volumes of the solution are prepared into dyeing working solution.

2) After washing the cells with PBS, 1 × 106 cells were fixed with 1mL 70% glacial ethanol at 4 ℃ overnight.

3) 1500rpm,5min, discard the supernatant (ethanol), resuspend in 1.5mL tubes with 1mL PBS (working solution and standard tubes were prepared).

4) 1500rpm,3min, speed centrifuge 30s,1500rpm,3min, and when all the precipitates were stably attached to the bottom side of the tube, the supernatant (PBS) was discarded.

5) Add 500. Mu.L of PI/RNase A staining solution prepared in advance, and keep out of the sun for 30min at room temperature (at this time, open the flow machine downstairs).

6) And (5) detecting by using a computer, and recording red fluorescence at the 488nm excitation wavelength.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, to those skilled in the art, changes and modifications may be made without departing from the spirit of the present invention, and it is intended that the present invention encompass such changes and modifications.

Claims

1. A pharmaceutical composition characterized by: comprises an NF-kB inhibitor and an AURKA inhibitor, wherein the molar ratio of the NF-kB inhibitor to the AURKA inhibitor is (0.5-0.7): 1.

2. the pharmaceutical composition of claim 1, wherein: the NF- κ B inhibitor is CAPE, and the AURKA inhibitor is MLN8237.

3. The pharmaceutical composition of claim 1, wherein: the molar ratio of the NF- κ B inhibitor to the AURKA inhibitor is 0.625:1.

4. a process for preparing a pharmaceutical composition according to claims 1-3, characterized in that: is obtained by mixing the NF-kB inhibitor and the AURKA inhibitor according to a molar ratio.

5. Use of a pharmaceutical composition according to claims 1-3 for the manufacture of a medicament for the treatment of glioblastoma.

6. The use of the pharmaceutical composition of claim 5 for the treatment of glioblastoma, wherein: the glioblastoma comprises LN229 and/or U251.

7. The use of the pharmaceutical composition of claim 5 for the treatment of glioblastoma, wherein: the NF-kB inhibitor and the AURKA inhibitor in the pharmaceutical composition induce glioblastoma lethality by combined inhibition of NF-kB and AURKA.

8. The use of the pharmaceutical composition of claim 7 for the treatment of glioblastoma, wherein: the NF-kappa B inhibitor and the AURKA inhibitor in the pharmaceutical composition induce glioblastoma lethality by jointly inhibiting p65 and BRCA1 co-expression.