CN114469950A - Application of chelidonine in preparation of FLT3-ITD mutant acute myelogenous leukemia treatment drug - Google Patents

Abstract

The invention relates to the field of new application of medicines, in particular to application of a small molecular compound Chelidonine (Chelidonine) in preparation of FLT3-ITD mutant Acute Myeloid Leukemia (AML) treatment medicines. In the invention, chelidonine inhibits FLT3-ITD mutant MV4 in vitro; 11 cells proliferate, induce apoptosis, and target AML mutated at FLT 3-ITD. In vivo experiments, the composition can reduce the proportion of bone marrow hCD45+ cells, reduce the infiltration of bone marrow and spleen, increase the expression of liver and spleen apoptosis protein Caspase3, prolong the survival time of NSG mice, has an in vivo anti-tumor effect, can be used as a new means for AML drug intervention of FLT3-ITD mutation, and brings a new hope for AML treatment of FLT3-ITD mutation.

Description

Application of chelidonine in preparation of FLT3-ITD mutant acute myelogenous leukemia treatment drug

Technical Field

The invention relates to the technical field of new application of medicaments, in particular to application of a small molecular compound Chelidonine (Chelidonine) in preparation of FLT3-ITD mutant acute myelogenous leukemia treatment medicaments.

Background

Acute leukemia is one of the hematological malignancies that seriously threaten human health, of which Acute Myelogenous Leukemia (AML) is the most common type of adult leukemia, accounting for about 70%. However, the long-term survival rate of AML patients under the age of 60 is only 35-40%, and that of AML patients over the age of 60 is reduced to 5-15%. For elderly AML patients who are unable to tolerate intensive chemotherapy, the median survival time is only 5-10 months.

The FLT3 mutation in AML patients mainly includes internal tandem repeat (ITD) mutation and Tyrosine Kinase Domain (TKD) point mutation. The FLT3-ITD mutation is the most common tumor-driving mutation in AML patients, with an incidence of about 25%, which predicts a shorter survival time, a higher risk of relapse, and an unfavorable prognosis that cannot be overcome by hematopoietic stem cell transplantation. Thus, NCCN and ELN guidelines classify AML patients positive for the FLT3-ITD mutation, especially high FLT3-ITD mutation load, as high risk AML. The high incidence and poor prognosis of this mutation suggest that it is expected to be an important potential target for AML treatment. The first generation tyrosine kinase inhibitors aiming at FLT3 mutation comprise Sorafenib and Midostaurin, and the clinical curative effect is unsatisfactory due to the problems of low specificity, limited pharmacokinetics, obvious adverse reaction and the like. The second generation FLT3 inhibitors Quizartinib and Gilteritinib with stronger specificity and better tolerance show good response in the early treatment stage of AML patients with relapsing refractory FLT3-ITD mutation, but the drug action lasts for a short time, and many patients have drug resistance after being treated for weeks or months and the disease relapses again. Therefore, actively exploring more efficient targeted drugs and overcoming drug resistance is a practical problem in clinical.

Chelidonine (Chelidonine) is an isoquinoline alkaloid extracted from the plant Chelidonium majus, and is widely distributed in Asia and Europe. Chelidonine, of formula: c₂₀H₁₉NO₅Molecular weight is 353.37, and structural formula is shown in formula I:

in solid tumors such as melanoma, head and neck tumors, digestive tract tumors, breast cancer and the like, Chelidonine has the effects of inhibiting cell proliferation, promoting apoptosis and inhibiting cell migration and invasion. It has been found that Chelidonine induces apoptosis by competitively inhibiting STK19, resulting in the inhibition of RAF-MEK-ERK and PI3K-AKT pathways downstream of the RAS, thereby inhibiting proliferation of NARS-mutated liver and lung Cancer cells (Qian L, Chen K, Wang C, Chen Z, Meng Z, Wang P.Targeting NRAS-Mutant Cancer with the Selective STK19 Kinase Inhibitor or Chelidonine.Clin Cancer Res.2020Jul 1; 26(13):3408 3419.). In vivo experiments also confirmed that Chelidonine can inhibit tumor growth with little toxicity. However, the role of Chelidonine in FLT3-ITD mutated AML has not been studied.

Disclosure of Invention

The invention aims to provide a new application of a small molecular compound Chelidonine (Chelidonine), in particular to an application of treating FLT3-ITD mutant Acute Myelogenous Leukemia (AML), and provides a new way for improving the prognosis of an AML patient with FLT3-ITD mutation.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention acts on FLT3-ITD mutant AML cell line MV4 by in vitro application of Chelidonine; 11, while Kasumi-1 and K562 cell lines without the FLT3 mutation were used as negative controls. The effect of Chelidonine treatment on FLT3-ITD mutant cell proliferation and apoptosis was observed. The invention detects the cell proliferation condition after the action of Chelidonine with different concentrations by a CCK-8 method. After the treatment for 24h and 48h by using Chelidonine with different concentrations, the apoptosis ratio is determined by analyzing AnnexinV/7-AAD through flow cytometry. Furthermore, we treated MV4 with FLT3-ITD mutation with Chelidonine 2.5. mu.M and 10. mu.M, respectively; 11 cell line 24h, using Western blot to detect the expression of apoptosis proteins Caspase3, Caspase 7 and PARP in the medicine group and the control group. In order to provide more theoretical support for discussing the treatment of FLT3-ITD mutated AML by Chelidonine.

To investigate the effect of Chelidonine on FLT3-ITD mutant cells in vivo, we used FLT3-ITD mutant cell line MV 4; 11 into immunodeficient NSG mice to construct MV 4; 11CDX leukemia model. The tail vein injection cell line is d0, and Chelidonine is injected into abdominal cavity every day after d4 days, the dosage is 20mg/kg, and the continuous use is carried out for 14 days. Mice body weight, hair, acroparalysis and changes in activity were observed and recorded daily. MV 4; the proportion of the cells which are positive to human CD45 (hCD45+) in the flow detection in 11 cells is more than 99 percent. Therefore, in NSG mice, the ratio of the tissue hCD45+ detected by flow is used as an index for judging the leukemia, and the pathogenesis is comprehensively judged by combining the results of hCD45 immunohistochemistry and HE staining.

MV4 for tail vein injection in this experiment; 11 cell count 1X 10⁶A/only. MV 4; the CDX model of 11 cells was divided into 4 groups. The experimental group 1 is a Chelidonine medication group, 7 Chelidonine medication groups are observed and survival time is recorded; experiment group 2 is a non-drug group, each group contains 7 patients, and observation records are recordedSurvival time; experimental group 3 was a treatment group with Chelidonine, experimental group 4 was a non-treatment group, and these two groups were periodically sacrificed by 6 mice each, and when mice in experimental group 3 or group 4 first developed symptoms of disease, all mice in both groups were sacrificed. Improving living cells of peripheral blood, bone marrow, liver and spleen to complete hCD45+ flow detection, taking partial tissues of liver, spleen and bone marrow to perform pathological section, completing HE staining, hCD45 immunohistochemistry and immunofluorescence detection of apoptosis protein Caspase3, and freezing and storing residual cell sediment of liver, spleen and bone marrow. After the mice of the experimental group 1 and the experimental group 2 died, pathological sections of the liver, the spleen and the bone marrow are reserved, and HE staining and hCD45+ immunohistochemistry are completed to verify that the death of the mice is caused by the morbidity. Statistical analysis is carried out on each group of data by adopting one-way variance analysis, and NSG mouse survival curves of a Chelidonine treatment group and a control group are drawn by adopting a Kaplan-Meier method.

The above experimental results demonstrate that Chelidonine can inhibit FLT3-ITD mutated MV4 in vitro; 11 cells proliferate, inducing apoptosis. In vivo experiments prove that the Chelidonine can reduce the proportion of bone marrow hCD45+ cells, reduce the infiltration of bone marrow and spleen, increase the expression of liver and spleen apoptosis protein Caspase3, prolong the survival time of NSG mice, and prompt that the Chelidonine has in vivo anti-tumor effect and can be used as a new means for AML drug intervention of FLT3-ITD mutation.

In a first aspect of the invention, the application of Chelidonine (Chelidonine) in preparing a medicine for treating FLT3-ITD mutant Acute Myeloid Leukemia (AML) is provided.

Among them, FLT3-ITD mutated AML was diagnosed based on bone marrow morphological, cytogenetic, immunological and molecular biological criteria. Chelidonine (Chelidonine), of the formula: c₂₀H₁₉NO₅The molecular weight is 353.37, the structural formula is shown in formula I, and the molecular weight is from ChemFaces company.

Furthermore, the medicine is a medicine for targeted inhibition of proliferation and apoptosis induction of the FLT3-ITD mutant acute myeloid leukemia cells.

Furthermore, when the Chelidonine (Chelidonine) concentration is more than or equal to 2.5 mu M, the proliferation of FLT3-ITD mutant cells is obviously inhibited, the expression of apoptosis proteins Caspase3, Caspase 7 and PARP is induced, and the apoptosis of the cells is induced.

Furthermore, the Chelidonine (Chelidonine) can reduce the proportion of myeloid leukemia cells in vivo, reduce the leukemia cell infiltration of bone marrow and spleen tissues, reduce the expression of bone marrow hCD45+, increase the expression of liver and spleen apoptosis protein Caspase3 and prolong the survival time.

In a second aspect of the invention, the invention provides a FLT3-ITD mutated acute myeloid leukemia targeted therapy drug, and the active ingredient of the drug is Chelidonine (Chelidonine).

Furthermore, the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material.

Further, the drug administration mode can be injection or oral administration.

In a third aspect of the invention, there is provided the use of Chelidonine (Chelidonine) in the preparation of an FLT3-ITD mutation inhibitor.

In a fourth aspect of the invention, the use of Chelidonine (Chelidonine) as an FLT3-ITD mutation inhibitor for the preparation of a medicament for the treatment of Acute Myeloid Leukemia (AML) and other related diseases is provided.

The invention has the advantages that:

in the invention, Chelidonine can inhibit FLT3-ITD mutant MV4 in vitro; 11 cells proliferate, inducing apoptosis. While the effect on FLT3 wild-type cells was not evident, suggesting that it could target AML mutated for FLT 3-ITD. In vivo experiments prove that the Chelidonine can reduce the proportion of bone marrow hCD45+ cells, reduce the infiltration of bone marrow and spleen, increase the expression of liver and spleen apoptosis protein Caspase3, prolong the survival time of NSG mice, and prompt that the Chelidonine has in vivo anti-tumor effect and can be used as a new means for AML drug intervention of FLT3-ITD mutation. As a very poor prognostic class of AML, Chelidonine is expected to bring new hopes for the treatment of FLT3-ITD mutated AML.

Drawings

FIG. 1 shows Chelidonine on FLT3-ITD mutant cell line MV 4; 11 and FLT3 wild type cell lines Kasumi-1 and K562 cell proliferation. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 2. varying concentrations of Chelidonine versus FLT3-ITD mutant cell line MV 4; 11 and FLT3 wild type cell lines Kasumi-1 and K562 apoptosis. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 3, varying concentrations of Chelidonine treated FLT3-ITD mutant cell line MV 4; cell apoptosis after 1124 h. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 4. treatment of FLT3-ITD mutant cell line MV4 with varying concentrations of Chelidonine; apoptosis after 1148 h. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 5 apoptosis following treatment of FLT3 wild-type cell line K56224 h with varying concentrations of Chelidonine. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 6 apoptosis following treatment of FLT3 wild-type cell line K56248 h with varying concentrations of Chelidonine. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 7 apoptosis of Chelidonine treated FLT3 wild-type cell line Kasumi-124 h at various concentrations. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 8 apoptosis following treatment of FLT3 wild type cell line Kasumi-148 h with varying concentrations of Chelidonine. The study has a total of 8 groups of chelidones with different concentrations, which are respectively as follows: 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M.

FIG. 9 shows that Chelidonine can induce FLT3-ITD mutant cell line MV 4; 11 expression of apoptosis-related proteins. Chelidonine concentrations were 2.5. mu.M and 10. mu.M, respectively, and MV4 was treated; 11 cells for 24 h.

FIG. 10.MV 4; 11CDX model, the ratio of the cells in bone marrow hCD45+ of the Chelidonine treated and control NSG mice, suggesting that Chelidonine can reduce the ratio of myeloid leukemia cells.

FIG. 11.MV 4; results of bone marrow/spleen/liver HE staining in the 11 cell line mouse CDX model, the Chelidonine treated group and the control group suggest that Chelidonine can reduce leukemic cell infiltration of tissues.

FIG. 12.MV 4; results of immunohistochemistry on bone marrow/spleen hCD45 in the 11 cell line mouse CDX model, the Chelidonine treated group and the control group suggest that Chelidonine can reduce bone marrow and spleen hCD45 expression.

FIG. 13.MV 4; results of immunofluorescence of spleen Caspase3 in a cell line mouse CDX model, a Chelidonine treatment group and a control group indicate that Chelidonine can induce the expression of spleen apoptosis protein Caspase 3.

FIG. 14.MV 4; results of immunofluorescence of liver Caspase3 in a cell line mouse CDX model, a Chelidonine treatment group and a control group suggest that Chelidonine can induce the expression of liver apoptosis protein Caspase 3.

FIG. 15.MV 4; 11 cell line mouse CDX model, Chelidonine treatment group and control group NSG mouse survival curves.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1: in vitro experiments

Chelidonine inhibition of FLT3-ITD mutant cell line proliferation

To determine the effect of Chelidonine on cell proliferation in the presence of FLT3-ITD mutation, we selected MV4 in the presence of FLT3-ITD mutation; 11 cell lines were used as subjects, while 2 cell lines K562 and Kasumi-1 without FLT3-ITD were used as controls. The above 3 cell lines were treated with 8 groups of Chelidonine at different concentrations and cell proliferation was measured by CCK-8, the results are shown in FIG. 1.

According to the detection result of CCK-8, the Chelidonine concentration is less than or equal to 1 mu M, and the proliferation of cells is not influenced. But the Chelidonine concentration is more than or equal to 2.5 mu M, and the FLT3-ITD mutant MV4 can be obviously inhibited; proliferation of 11 cell lines with a 24h IC50 of 2.8. mu.M. And 2 cell lines without FLT 3-related mutations with 24h IC 50: k5624.9. mu.M, Kasumi-15.8. mu.M. Indicating that Chelidonine has targeted inhibition effect on FLT3-ITD mutant cell proliferation.

Chelidonine Induction of FLT3-ITD mutant apoptosis

We performed AnnexinV/7-AAD flow cytometry analysis after Chelidonine treatment for 24h and 48h, respectively, and the results are shown in FIGS. 2-8. According to the detection result, the Chelidonine concentration is less than or equal to 1 mu M, and the apoptosis of the cells is not influenced. When the Chelidonine concentration is more than or equal to 2.5 mu M, the MV4 of the cells can be obviously increased; apoptosis of 11 cells (FIGS. 2-4). After 24h, the MV4 was treated with Chelidonine 2.5. mu.M; after 11 cells, apoptosis increased 28.3% over the control group (FIGS. 2-3). Whereas in cell lines K562 and Kas μmi-1 lacking the FLT3 mutation, apoptosis increased by 4.4% and 3.4% respectively upon 2.5 μ M treatment with Chelidonine for 24h, and there was no significant increase in apoptosis even at a concentration of 10 μ M (FIG. 2, FIG. 5, FIG. 7). While Chelidonine treated for 48h, whether FLT3-ITD mutant MV 4; there was a variable increase in apoptosis in 11 cells, also K562 or Kasumi-1 wild-type to FLT3, which was thought to be associated with the cytotoxic effects of Chelidonine (FIG. 2, FIG. 4, FIG. 6, FIG. 8).

According to the flow detection result, when the Chelidonine concentration is more than or equal to 2.5 mu M, the apoptosis of the cells can be obviously increased. We applied Chelidonine 2.5. mu.M and 10. mu.M, respectively, to cell line MV4 which harbored FLT 3-related mutations; 1124 h, Western blot is adopted to detect the expression of apoptosis proteins Caspase3, Caspase 7 and PARP of the medicine group and the control group. As a result, it was found that Chelidonine induced the expression of apoptotic proteins, and the results were consistent with those of flow-type assay (FIG. 9).

Example 2: in vivo experiments

In an in vivo experiment, MV4 is constructed; 11CDX mouse model, NSG mouse d0 tail vein injection cell number is 1X 10⁶The experiment is divided into 4 groups, the experiment group 1 is a Chelidonine medication group, 7 Chelidonine medication groups are arranged in each group, and the survival time is observed and recorded; the experimental group 2 is a non-drug group, 7 drugs in each group are observed and the survival time is recorded; experimental group 3 is a Chelidonine medication group, and experimental group 4 is a non-medication group, and the two groups are regular medication groupsThe group was sacrificed. After sacrifice, disease status and tumor burden were examined to determine the in vivo therapeutic effect of Chelidonine. The initial onset time of the CDX model in the NSG mouse is d16 after transplantation, and the weight is reduced by 9% at 24 h. After periodic sacrifice, 2 mice from the 6 NSG mice in the Chelidonine treatment group demonstrated morbidity with a bone marrow hCD45+ ratio of 3.48% and 8.59%, respectively. Whereas 6 mice not treated with Chelidonine developed disease with a 10% -31% ratio of bone marrow hCD45+ (figure 10). The bone marrow hCD45+ cell proportion of two groups of mice killed regularly and the flow detection result show that Chelidonine can reduce the bone marrow leukemia cell proportion. HE staining of bone marrow, spleen, and liver indicated that Chelidonine could reduce leukemic cell infiltration of tissues (fig. 11). Immunohistochemistry results for hCD45 in bone marrow and spleen tissues showed that Chelidonine could reduce hCD45+ expression (FIG. 12). Meanwhile, the tissue immunofluorescence results show that the expression of Caspase3 of the Chelidone medicine group is increased in liver and spleen tissues, and the Chelidone is suggested to be capable of inducing apoptosis in vivo (FIGS. 13-14). In addition to reducing tumor burden, Chelidonine can prolong survival in the CDX model of NSG mice. At MV 4; in the 11-cell CDX mouse model, the survival time of the CDX mice was prolonged and statistically different in the Chelidonine-treated group compared to the non-drug-administered intervention group (FIG. 15).

Taken together, the results indicate that Chelidonine can inhibit FLT3-ITD mutant MV4 in vitro; 11 cells proliferate and induce apoptosis. In vivo experiments prove that the Chelidonine can reduce the proportion of bone marrow hCD45+ cells, reduce the infiltration of bone marrow and spleen, increase the expression of liver and spleen apoptosis protein Caspase3, prolong the survival time of NSG mice, and prompt that the Chelidonine has in vivo anti-tumor effect and can be used as a new means for AML drug intervention of FLT3-ITD mutation.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims (9)

1. Application of chelidonine in preparation of FLT3-ITD mutant acute myelogenous leukemia treatment drugs.

2. The use of chelidonine according to claim 1 for the manufacture of a medicament for the treatment of acute myeloid leukemia with FLT3-ITD mutation, wherein said medicament is a medicament for the targeted inhibition of proliferation and apoptosis in acute myeloid leukemia with FLT3-ITD mutation.

3. Use of chelidonine according to claim 2 for the manufacture of a medicament for the treatment of FLT 3-ITD-mutated acute myeloid leukemia, wherein the concentration of chelidonine is greater than or equal to 2.5 μ M.

4. The use of chelidonine according to claim 1 for the manufacture of a medicament for the treatment of FLT 3-ITD-mutated acute myeloid leukemia, wherein said chelidonine reduces the proportion of myeloid leukemia cells, reduces leukemia cell infiltration in bone marrow and spleen tissues, reduces expression of hCD45+, increases expression of Caspase3, and prolongs survival time of NSG mice in vivo.

5. The FLT3-ITD mutant drug for targeted therapy of acute myeloid leukemia is characterized in that the active ingredient of the drug is chelidonine.

6. The medicament of claim 5, further comprising a pharmaceutically acceptable carrier or excipient.

7. The medicament of claim 5, wherein the administration is by injection or orally.

8. Application of chelidonine in preparation of FLT3-ITD mutation inhibitor.

9. Application of chelidonine as FLT3-ITD mutation inhibitor in preparing acute myelogenous leukemia therapeutic drugs and other related diseases.

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