CN114569616A

CN114569616A - Small molecule composition and application thereof in preparation of medicine for treating neuroblastoma

Info

Publication number: CN114569616A
Application number: CN202210368116.5A
Authority: CN
Inventors: 江珏; 王颖; 岳明; 徐雪
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-06-03
Anticipated expiration: 2042-04-08
Also published as: CN114569616B

Abstract

The invention discloses a small molecule composition and application thereof in preparing a medicine for treating neuroblastoma, and Glycogen synthase kinase3 beta (Glycogen synthase kinase3 beta, GSK-3 beta) for promoting the degradation of MYC family member c-MYC has the function of promoting the progression of cancer in NB. The analysis of GSK-3 beta substrate protein shows that the levels of N-MYC and Cyclin E1 protein and mRNA are obviously reduced after GSK-3 beta is inhibited, while the level of anti-apoptosis protein MCL-1 is increased, and the cell proliferation is obviously slowed down on the cell function. However, it is possible that due to the enhanced MCL-1 protein level detail, cells do not undergo significant apoptosis under low concentration GSK-3 β inhibitor treatment. In subsequent experiments, the invention proves that the GSK-3 beta inhibitor and the MCL-1 inhibitor are synergistic to kill neuroblastoma for the first time.

Description

Small molecule composition and application thereof in preparation of medicine for treating neuroblastoma

Technical Field

The invention relates to the technical field of compositions for treating tumors clinically, in particular to application of a GSK-3 beta inhibitor and an MCL-1 inhibitor in high-efficiency killing of neuroblastoma.

Background

Neuroblastoma (NB) is a malignant embryonal tumor that originates in the neural crest and is the most common extracranial solid tumor in infants and young children. NB incidence is third place in the common tumors of children and is the major cause of death from cancer in children 1-5 years of age. According to the INRG (International neuroplastoma Risk Group Classification System), the five-year survival rate of low-Risk and medium-Risk patients is close to 80%, but high-Risk patients are still difficult to cure after multiple intensive treatments, more than 50% of patients relapse, and the five-year survival rate is only 40% -50%. CCLE database showed overexpression of MYCN (encoding N-MYC protein) in NB, with MYCN amplification in more than half of patients at high risk; whereas MYCN is a member of the MYC family of proto-oncogenes, the amplification of which is associated with a variety of tumors, especially neuroblastoma, MYCN amplification has been shown to be negatively correlated with patient survival (P <0.0001), positively correlated with late stage tumors and poor prognosis, and internationally used as a molecular biological marker for the diagnosis of NB. The TH-MYCN transgenic mouse model, MYCN and ALK zebra fish model demonstrate that MYCN can induce NB genesis. Given the key role of N-MYC in the development of NB, inhibition of its expression and function has been one of the research directions for targeted therapy of NB.

N-MYC acts as a transforming gene and oncogenic driver, which plays multiple roles in NB malignancies, and as a major transcriptional regulator, activating genes involved in self-renewal, proliferation, pluripotency, angiogenesis and metastasis, and suppressing those genes capable of promoting cell differentiation, cell cycle arrest and immune surveillance. Because the members of the entire family of MYCs are transcription factors, the protein structures are special, and direct targeting is difficult to form. Some of the small molecule compounds in previous studies, such as JQ1, 10058-F4, NY2267, and THZ1, were used to target members of the MYC family. However, due to the selectivity of such drugs for c-Myc or their functional efficiency in vivo is impaired, the use of these approaches has enormous challenges in treating MYCN-expanded NB. Thus, an increasing number of studies have considered N-MYC to be "non-druggable". Therefore, there is an urgent need to develop alternative treatment strategies for MYCN in NB, and the exploration of upstream regulators of MYCN expression is imperative.

Disclosure of Invention

In the research of targeting MYCN to treat NB, Glycogen synthase kinase3 beta (GSK-3 beta) which promotes the degradation of MYC family member c-MYC is found to have the function of promoting cancer progression in NB. The analysis of GSK-3 beta substrate protein shows that the levels of N-MYC and Cyclin E1 protein and mRNA are obviously reduced after GSK-3 beta is inhibited, the level of anti-apoptosis protein MCL-1 is increased, and the cell proliferation is obviously slowed down on the cell function. However, it is possible that the cells did not undergo significant apoptosis due to the enhanced MCL-1 protein level detail. In subsequent experiments, the invention proves that the GSK-3 beta inhibitor and the MCL-1 inhibitor are synergistic to kill neuroblastoma for the first time.

The protein level of Cyclin E1 is encoded by CCNE1 gene located at chromosome 19q12, is a main protein involved in regulating and controlling the G1/S phase restriction checkpoint of cells, normally Cyclin E1 is orderly expressed and degraded in the cell cycle, and mainly promotes the transformation of cells from G1 phase to S phase, and researches show that the continuous Cyclin E1 expression leads to the hyperphosphorylation of retinoblastoma protein (pRb), the abnormal phosphorylation of pRb leads to the uncontrolled proliferation of cells and even leads to the occurrence of tumors, and the abnormally expressed Cyclin E1 can be combined with cadherin 1, further, the mitotic anaphase promoting compound is inhibited, programmed degradation of various cell cycle regulating factors is disordered, DNA replication abnormality, centrosome aberration and chromosome instability are increased, the occurrence risk of chromosome breakage and translocation is increased, and malignant transformation of cells is promoted.

MCL-1 is used as an anti-apoptosis protein to participate in the apoptosis, differentiation and cell cycle regulation of various cell lines, is vital to the survival and growth of cells, can cause neurodegenerative diseases due to insufficient expression of the protein, and can cause malignant tumors due to over-expression of the protein.

The technical scheme of the invention is as follows: use of a small molecule composition for the manufacture of a medicament for the treatment of neuroblastoma, said neuroblastoma being a neuroblastoma in a patient prior to chemo/radiotherapy treatment, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein said GSK-3 β inhibitor is LY2090314 which is: 6.4-102.5 parts by weight, the MCL-1 inhibitor is S63845 which is: 103.66-1658.52 parts by weight.

Further, the final concentration of LY2090314 in the small molecule composition in the solution state is: 12.5-200 nM, S63845 final concentration in solution: 125-2000 nM.

Use of a small molecule composition for the manufacture of a medicament for the treatment of neuroblastoma in a patient undergoing chemo/radiotherapy treatment, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein the GSK-3 β inhibitor is CT99021 which is: 1254.5-20072 parts by weight, wherein the MCL-1 inhibitor is S63845 which is: 5.39 to 82.92 parts by weight.

Further, the final concentration of CT99021 in the small molecule composition in a solution state is: 2.5-40 mu M, wherein the final concentration of S63845 in the solution state is as follows: 6.5-100 nM.

Further, the small molecule composition further comprises a pharmaceutically acceptable carrier or excipient: preservatives, antioxidants, flavouring agents, fragrances, cosolvents, emulsifiers, pH buffering substances, binders, fillers or lubricants.

Preferably, the cosolvent is a mixed solution containing PEG300 and Tween 80.

Preferably, the cosolvent is a mixed solution containing DMSO, PEG300 and Tween 80.

Preferably, the small molecule composition is prepared into a pharmaceutical dosage form comprising:

solid dosage forms, including powders, tablets, pills, capsules, sustained release formulations, controlled release formulations;

liquid dosage forms including injections, infusions, suspensions;

a gaseous formulation; or a semi-solid dosage form.

A pharmaceutical small molecule composition for use against neuroblastoma, said neuroblastoma being a neuroblastoma in a patient prior to treatment with chemo/radiotherapy, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein said GSK-3 β inhibitor is LY2090314 which is: 6.4-102.5 parts by weight, the MCL-1 inhibitor is S63845 which is: 103.66-1658.52 parts by weight.

A pharmaceutical small molecule composition for resisting neuroblastoma, the neuroblastoma being a neuroblastoma in a patient after undergoing chemo/radiotherapy treatment, the small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein the GSK-3 β inhibitor is CT99021 which is: 1254.5-20072 parts by weight, wherein the MCL-1 inhibitor is S63845 which is: 5.39 to 82.92 parts by weight.

The invention has the beneficial effects that: when the GSK-3 beta inhibitor LY2090314 and the MCL-1 inhibitor S63845 are used for treating neuroblastoma of a patient before radiotherapy treatment in a coordinated way, neuroblastoma apoptosis is obviously increased, the neuroblastoma has strong synergistic effect, the administration dosage is small, the target is specific, and the effect is better than that of the combined use of the GSK-3 beta inhibitor CT99021 and the MCL-1 inhibitor S63845;

when the GSK-3 beta inhibitor CT99021 and the MCL-1 inhibitor S63845 are used for synergistically treating the neuroblastoma of a patient treated by chemo-radiotherapy, the neuroblastoma apoptosis is obviously increased, and the neuroblastoma has strong synergistic effect and has better effect than that of the GSK-3 beta inhibitor LY2090314 and the MCL-1 inhibitor S63845 which are used in a combined way; after the Combination of the two medicines (Combination), the expression of the apoptosis protein clear caspase-3 is obviously activated, the expression of the anti-apoptosis protein MCL-1 is inhibited, the expression of the tumor cell proliferation protein PCNA is inhibited, and the expression of the oncoprotein N-MYC is inhibited.

In other malignancies, such as colorectal cancer or leukemia, no good combination of GSK-3 β inhibitor and MCL-1 inhibitor was found, suggesting selectivity specificity of the combination in neuroblastoma.

Drawings

FIG. 1A is a graph of the effect of different concentrations of the GSK-3 β inhibitor CT99021 on BE-2C cells;

FIG. 1B is a graph showing the effect of different concentrations of the GSK-3 β inhibitor LY2090314 on BE-2C cells;

FIG. 1C shows the effect of various concentrations of the GSK-3 β inhibitor CT99021 on Kelly cells;

FIG. 1D shows the effect of different concentrations of the GSK-3 β inhibitor LY2090314 on Kelly cells;

FIG. 1E is a graph of the effect of various concentrations of MCL-1 inhibitor S63845 on BE-2C cells;

FIG. 1F shows the effect of different concentrations of MCL-1 inhibitor S63845 on Kelly cells;

FIG. 2A is a synergistic killing of BE-2C cells by a GSK-3 β inhibitor and an MCL-1 inhibitor;

FIG. 2B is a synergistic killing of BE-2C cells by a GSK-3 β inhibitor and an MCL-1 inhibitor;

FIG. 2C shows synergistic killing of Kelly cells by GSK-3 β inhibitor and MCL-1 inhibitor;

FIG. 2D shows synergistic Kelly cell killing by a GSK-3 β inhibitor and an MCL-1 inhibitor;

FIG. 3A is a combination index of a GSK-3 β inhibitor and an MCL-1 inhibitor in conjunction in BE-2C cells;

FIG. 3B is a combination index of GSK-3 β inhibitor and MCL-1 inhibitor in Kelly cells;

FIG. 4A is a graph showing tumor volume changes after xenograft models demonstrate CT99021 and S63845 synergized against NB;

FIG. 4B is a graph showing the tumor appearance distribution of CT99021 and S63845 against NB in cooperation with a xenograft model;

FIG. 4C is a graph showing tumor weight distribution after xenograft models demonstrate CT99021 and S63845 synergize against NB;

FIG. 5 shows the expression levels of the relevant molecules in tumor tissues of immunohistochemical mice.

Detailed Description

The present invention is further illustrated by the following embodiments in combination with the drawings, and the embodiments are only used for further explaining the technical solutions of the present invention and should not be considered as limiting the scope of the present invention, and those skilled in the art should make insubstantial modifications or adjustments according to the above disclosure, which all belong to the scope of the present invention.

The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

Experimental materials and methods:

(1) MYCN expanded neuroblastoma cells Kelly, BE-2C (professor M. Celeste Simon, university of Pa., USA, present), in RPMI-1640+ 10% FBS +1 XP/S +2mM glutamine. The cells were cultured in a medium containing 5% CO₂At 37 ℃ in a sterile incubator, the medium was changed every 2 days and passaged in time. All cells were tested for mycoplasma regularly, and only mycoplasma negative cells were retained (following kit instructions, not described in detail). Media and PBS were pre-warmed to 37 ℃ prior to the experiment.

When the cells are frozen, the adherent cells are firstly washed 3 times by PBS, trypsinized until the cells are separated from a culture dish, a serum-containing culture medium is immediately added to stop digestion, the cells are centrifuged at 1000 Xg at room temperature for 5min, the supernatant is discarded, a cell freezing solution (90% FBS + 10% DMSO) is added, the cells are uniformly blown and transferred to a cell freezing tube, marked, slowly cooled and stored in a liquid nitrogen tank.

When the cells are recovered from the liquid nitrogen tank, the frozen tube is quickly placed in a water bath at 37 ℃, quickly thawed and transferred to a 15mL centrifuge tube, the culture medium is added, the mixture is gently mixed, the mixture is centrifuged at 1000 Xg at room temperature for 5min, the supernatant is discarded, and the mixture is placed in an incubator after the normal culture medium is added.

BE-2C is human neuroblastoma: BE-2C is a clone of SK-N-BE (2) neuroblastoma cell line, which was taken from bone marrow biopsies of children with diffuse neuroblastoma after repeated chemotherapy and radiotherapy in 1972, month 11. Various documents demonstrate MYCN amplification in BE-2C cells with high mRNA and protein expression.

Kelly is human neuroblastoma: kelly is a tumor cell of a pre-treatment neuron from a neuroblastoma patient. Several documents demonstrate MYCN amplification in Kelly cells with high mRNA and protein expression.

(2) Apoptosis detection

Apoptosis is a cell-mediated autonomous death in which Phosphatidylserine (PS) on the surface of a cell membrane is transferred from the inside of the cell membrane to the outside of the cell membrane, so that the PS is fully exposed on the outer surface of the cell membrane. Annexin V has high affinity to PS, and fluorescence-labeled Annexin V-FITC can detect apoptosis through the PS. PS is also exposed outside the cell membrane during cell necrosis, but the cell membrane integrity has been destroyed at this time, and the cell membrane structure is intact in the early stage of apoptosis. Propidium bromide (PI) binds nucleic acids but can only distinguish between apoptosis and necrosis by a cell membrane whose integrity is disrupted.

The method comprises the following steps:

(a) collecting cells: adherent cells: the medium supernatant was transferred to a centrifuge tube, the cells were washed 2 times with PBS (PBS and medium supernatant were combined), cells were trypsinized by adding pancreatin until the cells detached from the dish, and digestion was stopped by adding the PBS and medium supernatant mixture. Gently blow the cells to disperse the cells into single cells. Adherent cells: gently blow the cell culture solution to disperse the cells. Centrifuge at 1000 Xg for 5min at room temperature and discard the supernatant. For adherent cells, apoptotic cells will shed, so the culture supernatant is centrifuged together.

(b) Cell fixation: cell fixative (10mM HEPES (pH7.4), 150mM NaCl, 2.5mM CaCl) was added₂) Adjusting the cell density to about 1X 10⁶/mL。

(c) Adding 5 μ L Annexin V-FITC reagent, and incubating for 10min at room temperature in dark place; then 5. mu.L of PI reagent was added and incubated at room temperature for 5min in the dark.

(d) Flow cytometry detection: annexin V-FITC is FL1 channel, PI is FL2 channel, and cells without apoptosis induction are used as a control to adjust fluorescence compensation.

Statistical analysis: all data are shown as Mean ± standard error (Mean ± SEM), t-test and two-way ANOVA analysis were performed using IBM SPSS Statistics 19 software. P <0.05, p <0.01, p < 0.001.

(3) CT99021 is an effective GSK-3 alpha and GSK-3 beta inhibitor, and can also target CDKs and the like; CT99021 acts on GSK-3 with 500 times higher selectivity than that of the closest homologues CDC2, ERK2 and other protein kinases, and no cross-effect is generated. CT99021 also acts as an activator of Wnt/beta-catenin and induces autophagy.

(4) LY2090314 is a potent GSK-3 inhibitor, acting on GSK-3 α/β. LY2090314 has high selectivity to GSK3 and low inhibitory activity against most other kinases. In vitro studies, LY2090314 selectively inhibits the activity of GSK-3 by blocking ATP binding.

(5) S63845 is a highly selective and potent MCL-1 inhibitor. There was no significant binding ability to other Bcl-2 members, such as Bcl-2 or Bcl-xL. S63845 is a small molecule with high affinity specific binding to BH3 of MCL 1. It has been demonstrated that S63845 can potently kill MCL-1 dependent cancer cells, and multiple myeloma, leukemia and lymphoma cells are studied and used more frequently.

Example 1

The preparation of the small molecule composition for inducing the apoptosis of the neuroblastoma and the culture medium and the reagent thereof comprises the following steps:

1. small molecule composition for neuroblastoma apoptosis:

(1) small molecule composition 1

GSK-3 β inhibitors: CT99021, final concentration 1.25. mu.M. The relative molecular weight of 501.8 corresponds to 0.62725. mu.g/mL.

(2) Small molecule composition 2

GSK-3 β inhibitors: CT99021, final concentration 2.5. mu.M. The relative molecular weight of 501.8 corresponds to 1.2545. mu.g/mL.

(3) Small molecule composition 3

GSK-3 β inhibitors: CT99021, final concentration 5. mu.M. The relative molecular weight of 501.8 corresponds to 2.509. mu.g/mL.

(4) Small molecule composition 4

GSK-3 beta inhibitors: CT99021, final concentration 10. mu.M. The relative molecular weight of 501.8 corresponds to 5.018. mu.g/mL.

(5) Small molecule composition 5

GSK-3 β inhibitors: CT99021, final concentration 20. mu.M. The relative molecular weight of 501.8 corresponds to 10.036. mu.g/mL.

(6) Small molecule composition 6

GSK-3 β inhibitors: CT99021, final concentration 40. mu.M. The relative molecular weight of 501.8 corresponds to 20.072. mu.g/mL.

(7) Small molecule composition 7

GSK-3 β inhibitors: LY2090314, final concentration 3.125 nM. The relative molecular weight of 512.53 corresponds to 1.60165 ng/mL.

(8) Small molecule composition 8

GSK-3 β inhibitors: LY2090314, final concentration 6.25 nM. The relative molecular weight of 512.53 corresponds to 3.2033 ng/mL.

(9) Small molecule composition 9

GSK-3 β inhibitors: LY2090314, final concentration 12.5 nM. The relative molecular weight of 512.53 corresponds to 6.4066 ng/mL.

(10) Small molecule composition 10

GSK-3 β inhibitors: LY2090314, final concentration 25 nM. The relative molecular weight of 512.53 corresponds to 12.8133 ng/mL.

(11) Small molecule composition 11

GSK-3 β inhibitors: LY2090314, final concentration 50 nM. The relative molecular weight of 512.53 corresponds to 25.6265 ng/mL.

(12) Small molecule composition 12

GSK-3 β inhibitors: LY2090314, final concentration 100 nM. The relative molecular weight of 512.53 corresponds to 51.253 ng/mL.

(13) Small molecule composition 13

GSK-3 β inhibitors: LY2090314, final concentration 200 nM. The relative molecular weight of 512.53 corresponds to 102.506 ng/mL.

(14) Small molecule composition 14

MCL-1 inhibitors: s63845, final concentration 6.25 nM. The relative molecular weight of 829.26 corresponds to 5.1829 ng/mL.

(15) Small molecule composition 15

MCL-1 inhibitors: s63845, final concentration 12.5 nM. The relative molecular weight of 829.26 corresponds to 10.3658 ng/mL.

(16) Small molecule compositions 16

MCL-1 inhibitors: s63845 at final concentration of 25 nM. The relative molecular weight of 829.26 corresponds to 20.7315 ng/mL.

(17) Small molecule composition 17

MCL-1 inhibitors: s63845 at final concentration of 50 nM. The relative molecular weight of 829.26 corresponds to 41.463 ng/mL.

(18) Small molecule composition 18

MCL-1 inhibitors: s63845 at final concentration of 100 nM. The relative molecular weight of 829.26 corresponds to 82.926 ng/mL.

(19) Small molecule composition 19

MCL-1 inhibitors: s63845, final concentration 125 nM. The relative molecular weight of 829.26 corresponds to 103.6575 ng/mL.

(20) Small molecule composition 20

MCL-1 inhibitors: s63845 at final concentration of 250 nM. Relative molecular weight 829.26, corresponding to 207.315 ng/mL.

(21) Small molecule composition 21

MCL-1 inhibitors: s63845, final concentration 500 nM. Relative molecular weight 829.26, corresponding to 414.63 ng/mL.

(22) Small molecule composition 22

MCL-1 inhibitors: s63845 at final concentration 1000 nM. The relative molecular weight of 829.26 corresponds to 829.26 ng/mL.

(23) Small molecule composition 23

MCL-1 inhibitors: s63845, final concentration 2000 nM. The relative molecular weight of 829.26 corresponds to 1.6585. mu.g/mL.

(24) Small molecule compositions 24

GSK-3 β inhibitors: CT99021, final concentration 5. mu.M; this corresponds to 2.509. mu.g/mL.

MCL-1 inhibitors: s63845 at final concentration of 25 nM. Corresponding to 20.7315 ng/ml.

(25) Small molecule composition 25

GSK-3 β inhibitors: CT99021, final concentration 5. mu.M; this corresponds to 5.018. mu.g/mL.

MCL-1 inhibitors: s63845 at final concentration of 250 nM. Equivalent to 207.315ng/ml

(26) Small molecule composition 26

GSK-3 β inhibitors: LY2090314, final concentration 25 nM; equivalent to 12.8133ng/ml

MCL-1 inhibitors: s63845 at final concentration of 25 nM. Equivalent to 20.7315ng/ml

(27) Small molecule composition 27

MCL-1 inhibitors: s63845, final concentration 500 nM. Equivalent to 207.315ng/ml

Each specific small molecule composition was dissolved in cell-grade dimethyl sulfoxide (DMSO) to make a concentrated sample.

2. Tumor cell apoptosis culture medium configuration

The samples prepared in the above experimental step 1 were added to a medium containing RPMI-1640+ 10% FBS +1 XP/S +2mM glutamine to obtain a tumor cell apoptosis medium (i.e., medium 1 was at the same final concentration as the compound of composition 1, medium 2 was at the same final concentration as the compound of composition 2, …, and medium 27 was at the same final concentration as the compound of composition 27). An equivalent amount of DMSO lacking only the small molecule composition was also added to the medium as a control. The sample volume is no more than 0.1% of the culture medium.

3. Injection reagent configuration for tumor cell apoptosis

Adding the sample prepared in the experimental step 1 into a cosolvent containing PEG300 or PEG300 and Tween 80 which have no toxic or side effect on organisms for dissolving to prepare an injection reagent for the experimental animal for tumor cell apoptosis; meanwhile, compared with the reagent for injection of experimental animals, the cosolvent which only lacks the small molecular composition is prepared as a control. The dose of CT99021 in mice in the single administration group and the combination group is 15mg/kg, the dose of S63845 is 10mg/kg, the average body weight of the mice is 20g, and the administration volume of each animal is 200 mu L. The dissolution method of CT99021 in vivo experiment of mouse is to use 4% DMSO + 30% PEG300+ 66% ddH₂O is taken as a cosolvent, and the dissolution method of LY2090314 is to utilize 5% DMSO + 45% PEG300+ 50% ddH₂O isCosolvent, S63845 was dissolved by using 5% DMSO + 30% PEG300+ 5% Tween 80+ 60% ddH₂O is a cosolvent. The specific operation method comprises the following steps: the mice in the single administration group were treated in a total of 5 mice, and the number of mice was 6. Dissolving 1.8mg of CT99021 in 48 mu L of DMSO solution, adding 360 mu L of PEG300, mixing uniformly, clarifying, adding 792 mu L of sterile water, mixing uniformly, clarifying, wherein the concentration of a working solution is 1.5 mg/mL; dissolving 1.2mg of S63845 drug in 60 mu of LDMSO solution, adding 360 mu of PEG300, uniformly mixing and clarifying, adding 60 mu of Tween 80, uniformly mixing and clarifying, adding 720 mu of sterilized water, uniformly mixing and clarifying, wherein the concentration of the working solution is 1 mg/mL. The quality of CT99021 and S63845 in the combined administration group are respectively the same as those of the single administration group, wherein the dosage modes of the CT99021 and the S63845 in the single administration group are the same, and the concentration of the working solution is respectively 1.5mg/mL and 1 mg/mL. The combined group is administered with CT99021 first and S63845 after the mice are stabilized for half an hour, and the administration volume of the combined group is 400 mu L.

Apoptosis of NB cells by tumor apoptosis medium

4.1 apoptotic culture of NB cells

MYCN-expanded NB (BE-2C, Kelly) cells in log phase of growth were suspended in the tumor apoptosis medium described above and plated as treatment group.

Tumor apoptosis medium was obtained by adding cell-grade dimethyl sulfoxide (DMSO) to the medium to make DMSO concentration 0.1% by volume, and MYCN-expanded NB (BE-2C, Kelly) cells in log phase of growth were suspended in the above tumor apoptosis medium and plated as a control group.

After culturing at 37 ℃ for 48 hours in the treatment group and the control group, all cells (including supernatant) in each well were collected according to step (a) of the materials and methods, washed twice with PBS, and cell-fixing solution (10mM HEPES (pH7.4), 150mM NaCl, 2.5mM CaCl) was added₂) Adjusting the cell density to about 1X 10⁶Adding 5 mu L Annexin V-FITC reagent into the mixture, and incubating the mixture for 10min at room temperature in a dark place; then 5. mu.L of PI reagent was added and incubated at room temperature for 5min in the dark. And (4) operating a flow cytometer, analyzing by combining Flowjo software according to experimental data to obtain the percentage of the apoptotic cells, repeating the experiment, and calculating the statistical difference.

The experimental results are as follows:

as shown in fig. 1A-1B, according to the results of the cell experiment: when NB cells are treated by different concentrations of GSK-3 beta inhibitor CT99021 (the concentrations are 2.5 mu M, 5 mu M, 10 mu M, 20 mu M and 40 mu M in BE-2C cell culture medium and 2.5 mu M, 5 mu M, 10 mu M, 20 mu M and 40 mu M in Kelly cell culture medium), the apoptosis gradually increases along with the increase of the drug concentration, and obvious inflection points are appeared, the dosage is more, and the action targets are possibly not specific enough. The addition of a trace amount (2.5 mu M or 5 mu M) of CT99021 can cause the apoptosis of neuroma cells, and the addition of a certain amount (20 mu M or 40 mu M) of CT99021 can induce the obvious apoptosis of BE-2C cells and Kelly cells.

As shown in fig. 1C to fig. 1D, according to the results of the cell experiment: when NB cells were treated with different concentrations of the GSK-3 inhibitor LY2090314 (3.125nM, 6.25nM, 12.5nM, 25nM, 50nM in BE-2C cell culture medium and 12.5nM, 25nM, 50nM, 100nM, 200nM in Kelly cell culture medium), it was found that apoptosis increased gradually with increasing drug concentration, and the amount administered was very low, and the target was probably specific. The addition of a small amount (3.125nM or 6.5nM) of LY2090314 can significantly apoptosis BE-2C cells, and the addition of a certain amount (25nM or 50nM) of LY2090314 to BE-2C cells already causes apoptosis in many neuroma cells. Addition of a small amount (12.5nM) of LY2090314 did not significantly induce apoptosis in Kelly cells, and treatment of Kelly cells with addition of a certain amount (200nM) of LY2090314 induced significant apoptosis.

As shown in fig. 1E-1F, according to the results of the cell experiment: when NB cells were treated with different concentrations of MCL-1 inhibitor S63845 (6.25 nM, 12.5nM, 25nM, 50nM, 100nM in BE-2C cell culture medium, 125nM, 250nM, 500nM, 1000nM, 2000nM in Kelly cell culture medium), it was found that BE-2C was more sensitive to S63845, and that the apoptosis rate was concentration-dependent, while Kelly exhibited significant resistance to S63845. The addition of a trace amount (6.5nM and 12.5nM) of S63845 did not significantly induce apoptosis in BE-2C cells, and the addition of a certain amount (25nM, 50nM and 100nM) of S63845 to BE-2C cells induced significant apoptosis.

As shown in FIGS. 1A to 1B, and as shown in FIGS. 1C to 1D, according to the results of the cell experiments, when NB cells were treated with a GSK-3. beta. inhibitor (CT99021 at a concentration of 5. mu. M, LY2090314 at 25nM), as shown in FIGS. 1E to 1F, apoptosis was significantly increased when NB cells were treated with an MCL-1 inhibitor (25nM in BE-2C cell culture medium and 500nM in Kelly cell culture medium). As shown in FIGS. 2A-2D, apoptosis increased significantly when NB cells were treated with a GSK-3 β inhibitor in combination with an MCL-1 inhibitor.

Joint indices were analyzed using the Calcusyn software: BE-2C and Kelly cells are treated by combining CT99021, LY2090314 and S63845 at different concentrations, the apoptosis rate of each group is detected in a flow mode, and then the drug combination index CI <1 is found to show a synergistic effect by using Calcusyn software analysis. When the combination index CI <0.3, the two drugs interact strongly synergistically (fig. 3A-3B).

In vitro results show that in BE-2C, the joint index CI value of CT99021/S63845 is smaller, and the synergistic efficiency is better; in Kelly, the combination index CI of LY2090314/S63845 is extremely small, which proves that the effect of different drug combination combinations in different tumor cells is different, and provides a theoretical basis for different clinical treatment schemes.

Subcutaneous tumor-bearing experiment of NB cells by tumor cells

And (3) selecting the BE-2C cells with higher malignancy degree to construct a xenograft tumor model.

20 thymus Nude mice (BALB/c Nude, abbreviated as Nude mice) with the age of 5-6 weeks are selected and adapted to one week in SPF level environment. BE-2C cells grown in log phase were collected by trypsinization, resuspended in PBS, counted and cell density adjusted. 2X 10 subcutaneous injections were administered to each nude mouse in the axilla on one side⁶The body weight and state of the animals are closely observed and recorded, the tumor formation time is about 5-7 days, the long diameter (d) and the short diameter (r) of the tumor body are measured and recorded by using a vernier caliper, and the tumor volume V is calculated to be 1/2 Xd Xr²The tumor diameter of the tumor body reaches about 5mm or the tumor volume reaches about 50mm³Monitoring and treatment can be started.

6. Evaluation of the combined effects of CT99021 and S63845 against neuroblastoma in mice

The nude mice treated in the experimental step 5 are divided into four groups randomly, and the co-solvent which is only lack of the small molecular composition and is prepared simultaneously is used as a control (Ctrl), a CT99021 injection reagent, a S63845 injection reagent, a CT99021 injection reagent and a S63845 injection reagent, the nude mice are treated by injecting the medicine into the abdominal cavity for 1 to 2 weeks, and the injection frequency is twice a week.

The state and the growth condition of the tumor of the nude mice are observed every 2-3 days during the monitoring period, the weight of the nude mice is weighed and recorded by an electronic balance, and the tumor diameter length (the long diameter d and the short diameter r) of the nude mice is measured and recorded in vitro by a vernier caliper.

Maximum tumor volume (V: 1/2 × d × r)²) Approximately 1000mm³In the process, orbital blood is taken to detect blood routine, all nude mice are killed by dislocation of cervical vertebrae, tumors are taken out, the tumors are photographed, data are recorded, and then the nude mice are fixed in paraformaldehyde with the volume concentration of 4% for immunohistochemical analysis.

Immunohistochemical experimental procedure and analytical method:

the basic principle is as follows: the principle of specific combination of antigen and antibody is applied, and the reaction part of antigen and antibody is displayed by means of histochemical method, so that the distribution and content of some chemical components can be determined in situ in cell or tissue. The specific experimental steps are as follows:

(1) tissue fixation: fixing the tissue sample with 4% paraformaldehyde for no more than 48 hr.

(2) Dehydration, paraffin embedding and sectioning: is delivered to Wuhan hundred kilometric biotechnology limited company.

(3) Dewaxing and hydrating: the tissue slices were dewaxed in an oven at 60 ℃ for 1 hour, followed by hydration with xylene (10min) -absolute ethanol (5min) -90% ethanol (5min) -80% ethanol (5min) -70% ethanol (5 min).

(4) Antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH 6.0) in a microwave oven for antigen repairing, stopping heating for 8min until the tissue slices are boiled, maintaining the temperature for 8min, and turning to the low-medium heat for 7min to prevent the buffer solution from excessively evaporating during the process, so as to prevent dry slices. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

(5) Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

(6) And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the tissues, and blocking for 30min at room temperature. (primary antibodies were goat-derived blocked with rabbit serum and other sources blocked with BSA).

(7) Adding a primary antibody: gently throwing off the confining liquid, dropwise adding primary antibody prepared by PBS according to a certain proportion on the slice, wherein the dilution ratio of the primary antibody is MCL-11: 100, N-MYC 1:200, PCNA 1:1000 and cleaned Caspase-31: 50, incubating for 16h at 4 ℃, and flatly placing the slice in a wet box for incubating overnight at 4 ℃ (adding a small amount of water in the wet box to prevent the antibody from evaporating).

(8) Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section was slightly spun off, a secondary antibody (HRP-labeled) against the corresponding species was added dropwise to the ring to cover the tissue, and the mixture was incubated at room temperature for 1 hr.

(9) DAB color development: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.

(10) Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.

(11) Dewatering and sealing: putting the slices into 70% ethanol-80% ethanol-90% ethanol-anhydrous ethanol for 5min respectively; xylene (2), each for 10 min; and (5) dehydrating and transparent, taking out the slices from the dimethylbenzene, slightly drying the slices, and sealing the slices by using sealing glue.

(12) Microscopic examination and image acquisition and analysis.

Analyzing the immunohistochemical result of the paraffin section:

hematoxylin staining cell nucleus is blue, and DAB shows that positive expression is brown yellow. The stronger the expression, the more pronounced the area and intensity of the brown-yellow color.

The statistical analysis mouse weight and tumor diameter, the experimental results show: compared with the nude mice in the single-drug administration group, the nude mice treated by the combination of CT99021 and S63845 have the advantages of obviously reduced growth rate of unilateral transplanted tumor, reduced volume, obviously reduced tumor weight and statistical difference (fig. 4A-4C).

The weight of the nude mice of the control group and the treatment group is not obviously changed during the intraperitoneal injection of the medicament, the blood routine detection result is not abnormal, and no medicament toxic or side effect is generated. The nude mice have normal diet, sensitivity to stimulation and normal excretion during the culture period, and have no abnormal death condition. The immunohistochemical results shown in fig. 5 show that: the control group does not activate the expression of apoptosis protein clear caspase-3 (blue), does not express anti-apoptosis protein MCL-1 (blue), expresses tumor cell proliferation protein PCNA (brown), expresses oncoprotein N-MYC (brown);

the expression of the inactivated apoptosis protein clear caspase-3 (blue) in the CT99021 group is singly used, the expression of the anti-apoptosis protein MCL-1 (anaglyph) is not inhibited, the expression of the tumor cell proliferation protein PCNA (anaglyph and a small amount of blue are alternated), and the expression of the oncoprotein N-MYC (light anaglyph) is weakly inhibited;

the expression of the S63845 group weakly activated apoptosis protein clear caspase-3 (alternate colors of full-color and blue) is singly used, the expression of the anti-apoptosis protein MCL-1 (brown) is not inhibited, the expression of the tumor cell proliferation protein PCNA (blue) is inhibited, and the expression of the oncoprotein N-MYC (brown) is not inhibited;

after the Combination of the two medicines (Combination), the expression (brown) of the apoptosis protein clear caspase-3 is obviously activated, the expression (blue) of the anti-apoptosis protein MCL-1 is inhibited, the expression (blue) of the tumor cell proliferation protein PCNA is inhibited, and the expression (dark blue and brown alternate) of the oncoprotein N-MYC is inhibited.

In vivo experiments show that when two groups of drugs (GSK-3 beta inhibitors CT99021 and MCL-1 inhibitor S63845) with higher synergistic index are selected to treat neuroblastoma BE-2C nude mouse models, compared with a control group and a single drug group, the combined inhibitor has obvious synergistic killing effect on neuroblastoma cells, and the drugs are low in toxicity and have no obvious damage to mice.

In vitro experiments show that CT99021, LY2090314 and S63845 have synergistic effect of killing neuroblastoma cells. There are several GSK-3 inhibitors on the market, most of them have not been tested clinically, and most of them have other multiple targets besides targeting GSK-3 β. The LY2090314 target used in this example is relatively specific and has been in phase I clinical trials at present.

Claims

1. Use of a small molecule composition for the manufacture of a medicament for the treatment of neuroblastoma, said neuroblastoma being a neuroblastoma in a patient prior to chemo/radiotherapy treatment, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein said GSK-3 β inhibitor is LY2090314 that is: 6.4-102.5 parts by weight, the MCL-1 inhibitor is S63845 which is: 103.66-1658.52 parts by weight.

2. The use of claim 1, wherein the neuroblastoma is a neuroblastoma in a patient after chemo/radiotherapy treatment, and the small molecule composition comprises a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein the GSK-3 β inhibitor is CHIR99021 which is: 1254.5-20072 parts by weight, the MCL-1 inhibitor is S63845 which is: 5.39 to 82.92 parts by weight.

3. The use of claim 1, wherein the final concentration of LY2090314 in the small molecule composition in solution is: 12.5-200 nM, S63845 final concentration in solution: 125-2000 nM.

4. The use of claim 2, wherein the final concentration of CT99021 in the small molecule composition in solution is: 2.5-40 mu M, wherein the final concentration of S63845 in the solution state is as follows: 6.5-100 nM.

5. The use of any one of claims 1 to 4, wherein the small molecule composition further comprises a pharmaceutically acceptable carrier or excipient: preservatives, antioxidants, flavouring agents, fragrances, cosolvents, emulsifiers, pH buffering substances, binders, fillers or lubricants.

6. The use of claim 5, wherein the cosolvent is a mixed solution containing PEG300 and Tween 80.

7. A pharmaceutical small molecule composition for use against neuroblastoma, wherein said neuroblastoma is a neuroblastoma in a patient prior to chemo/radiotherapy treatment, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein said GSK-3 β inhibitor is LY2090314 which is: 6.4-102.5 parts by weight, the MCL-1 inhibitor is S63845 which is: 103.66-1658.52 parts by weight.

8. The small molecule composition according to claim 7, wherein the neuroblastoma is a neuroblastoma in a patient after treatment with chemo/radiotherapy, said small molecule composition comprising a GSK-3 β inhibitor and a MCL-1 inhibitor, wherein the GSK-3 β inhibitor is CT99021 which is: 1254.5-20072 parts by weight, the MCL-1 inhibitor is S63845 which is: 5.39 to 82.92 parts by weight.

9. The small molecule composition according to claim 7, wherein the final concentration of LY2090314 in the small molecule composition in solution is: 12.5-200 nM, S63845 final concentration in solution: 125-2000 nM.

10. The small molecule composition according to claim 8, wherein the final concentration of CT99021 in the small molecule composition in the solution state is: 2.5-40 mu M, wherein the final concentration of S63845 in the solution state is as follows: 6.5-100 nM.