A pharmaceutical composition for killing FLT3-ITD mutant AML cells and its application

Abstract

The present invention discloses a pharmaceutical composition for killing FLT3-ITD mutant AML cells and its application, and relates to the field of biomedicine technology. The present invention specifically discloses that a PARP inhibitor and a CREB inhibitor can be combined to synergistically and efficiently kill FLT3-ITD mutant AML cells and inhibit the proliferation of FLT3-ITD mutant AML cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a pharmaceutical composition for killing FLT3-ITD mutant AML cells and an application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, with extremely high relapse and mortality rates. FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that often mutates in AML. FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is the most common gene mutation in AML and is closely associated with malignant progression of the disease and poor prognosis. Although FLT3 inhibitors can effectively alleviate FLT3-ITD + Hematological indicators of AML patients, but the long-term treatment effect is still unsatisfactory. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a pharmaceutical composition for killing FLT3-ITD mutant AML cells, which can synergistically and efficiently kill FLT3-ITD mutant AML cells and inhibit the proliferation of FLT3-ITD mutant AML cells.

[0004] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition.

[0005] The present invention also provides application of the above-mentioned pharmaceutical composition.

[0006] The present invention also provides a medicine comprising the above-mentioned pharmaceutical composition.

[0007] The present invention also provides an in vitro non-therapeutic method for inhibiting the growth of leukemia cells.

[0008] According to the first aspect of the present invention, a pharmaceutical composition for killing FLT3-ITD mutant leukemia cells comprises: a poly ADP-ribose polymerase (PARP) inhibitor and a cAMP-response element binding protein 1 (CREB) inhibitor.

[0009] The pharmaceutical composition according to the embodiment of the present invention has at least the following beneficial effects: PARP is an enzyme involved in DNA damage repair. By blocking the activity of PARP, DNA damage cannot be repaired and can induce cancer cell death. CREB is an important transcription factor located in the downstream pathway of FLT3, mediating transcriptional responses to a variety of growth factors and stress signals. PARP inhibitors and CREB inhibitors can synergistically and efficiently kill FLT3-ITD mutant AML cells and inhibit the proliferation of FLT3-ITD mutant AML cells.

[0010] According to some embodiments of the present invention, the PARP inhibitor includes Olaparib, Niraparib, Fluzoparib, Pamiparib, Talazoparib, Rucaparib or at least one of the pharmaceutically acceptable salts, isomers, racemates, solvates or prodrugs of the above compounds.

[0011] According to some embodiments of the present invention, the PARP inhibitor is Olaparib or a pharmaceutically acceptable salt, isomer, racemate, solvate or prodrug thereof.

[0012] According to some embodiments of the present invention, the CREB inhibitor includes 666-15, KG-501, XX-650-23, niclosamide, CREB-IN-1 TFA or at least one of the pharmaceutically acceptable salts, isomers, racemates, solvates or prodrugs of the above compounds.

[0013] According to some embodiments of the present invention, the CREB inhibitor is 666-15 or a pharmaceutically acceptable salt, isomer, racemate, solvate or prodrug thereof.

[0014] According to some embodiments of the present invention, the molar ratio of the PARP inhibitor to the CREB inhibitor is (15-21): 1. For example, it can be 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, 20.5:1 or 21:1.

[0015] According to some embodiments of the present invention, based on the total weight of the active ingredients of the pharmaceutical composition, the mass percentage of the CREB inhibitor is 6% to 9%.

[0016] According to some embodiments of the present invention, based on the total weight of the active ingredients of the pharmaceutical composition, the mass percentage of the PARP inhibitor is 91% to 94%.

[0017] The method for preparing the above-mentioned pharmaceutical composition according to the second aspect of the present invention comprises the following steps: The PARP inhibitor and the CREB inhibitor are mixed to obtain the pharmaceutical composition.

[0018] Use of the above-mentioned pharmaceutical composition according to the third aspect of the present invention in any one of A1) to A7); A1) Preparation of products that inhibit the proliferation of leukemia cells; A2) Preparation of products for killing leukemia cells; A3) Preparation of products that inhibit leukemia cell colony formation; A4) Preparation of products that promote or induce apoptosis of leukemia cells; A5) Preparation of products that promote or induce cell cycle arrest in leukemia cells; A6) Preparation of products for the treatment or adjuvant treatment of leukemia; A7) Preparation of products that inhibit the occurrence and development of leukemia.

[0019] According to some embodiments of the present invention, the leukemia cells in any one of applications A1) to A5) are leukemia cells with FLT3-ITD mutations, and are acute myeloid leukemia cells.

[0020] According to some embodiments of the present invention, the leukemia cells in any one of applications A1) to A5) include at least one of MOLM-13 cells and MV-4-11 cells.

[0021] According to some embodiments of the present invention, the cell cycle arrest in A5) is G1 phase cell cycle arrest.

[0022] According to some embodiments of the present invention, the leukemia described in A6) or A7) is acute myeloid leukemia.

[0023] According to some embodiments of the present invention, the leukemia described in A6) or A7) is FLT3-ITD mutant leukemia.

[0024] According to some embodiments of the present invention, the product is a medicine or a kit.

[0025] A drug according to the fourth aspect of the present invention comprises the pharmaceutical composition described in the first aspect of the present invention, and an optional pharmaceutically acceptable carrier. Since the drug adopts all the technical solutions of the pharmaceutical composition of the above embodiment, it at least has all the beneficial effects brought by the technical solutions of the above embodiment.

[0026] According to some embodiments of the present invention, the pharmaceutically acceptable carrier includes at least one of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0027] According to some embodiments of the present invention, the dosage form of the drug is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, and the like.

[0028] According to some embodiments of the present invention, the drug may further include other anti-leukemia or leukemia cell proliferation-inhibiting substances, preferably so as not to affect the effect of the above-mentioned pharmaceutical composition.

[0029] According to the fifth aspect of the present invention, an in vitro non-therapeutic method for inhibiting the growth of leukemia cells comprises the following steps: contacting the leukemia cells with the pharmaceutical composition described in the first aspect or the drug described in the third aspect, thereby inhibiting the growth of the leukemia cells.

[0030] According to some embodiments of the present invention, the leukemia cells are leukemia cells with FLT3-ITD mutation.

[0031] According to some embodiments of the present invention, the leukemia cells are acute myeloid leukemia cells.

[0032] According to some embodiments of the present invention, in the method, the CREB inhibitor is administered at a concentration of 31.25 nM to 2000 nM; the PARP inhibitor is administered at a concentration of 0.5 μM to 40 μM.

[0033] According to some embodiments of the present invention, in the method, the CREB inhibitor is administered at a concentration of 60 nM to 280 nM; and the PARP inhibitor is administered at a concentration of 1 μM to 7 μM.

[0034] According to some embodiments of the present invention, the leukemia cells are MOLM-13 cells; the administration concentration of the CREB inhibitor is 31.25 nM~2000 nM; and the administration concentration of the PARP inhibitor is 0.625 μM~40 μM.

[0035] According to some embodiments of the present invention, the leukemia cells are MOLM-13 cells; the administration concentration of the CREB inhibitor is 60 nM~280 nM; and the administration concentration of the PARP inhibitor is 1 μM~7 μM.

[0036] According to some embodiments of the present invention, the leukemia cells are MOLM-13 cells; the administration concentration of the CREB inhibitor is 62.5 nM~250 nM; and the administration concentration of the PARP inhibitor is 1.25 μM~5 μM.

[0037] According to some embodiments of the present invention, the leukemia cells are MV-4-11 cells; the administration concentration of the CREB inhibitor is 25 nM~1300 nM; and the administration concentration of the PARP inhibitor is 0.4 μM~20 μM.

[0038] According to some embodiments of the present invention, the leukemia cells are MV-4-11 cells; the administration concentration of the CREB inhibitor is 31.25 nM~1000 nM; and the administration concentration of the PARP inhibitor is 0.5 μM~16 μM.

[0039] According to some embodiments of the present invention, the leukemia cells are MV-4-11 cells; the administration concentration of the CREB inhibitor is 50 nM to 650 nM; and the administration concentration of the PARP inhibitor is 0.8 μM to 10 μM.

[0040] According to some embodiments of the present invention, the leukemia cells are MV-4-11 cells; the administration concentration of the CREB inhibitor is 62.5 nM~500 nM; and the administration concentration of the PARP inhibitor is 1 μM~8 μM.

[0041] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The effect of 666-15 on the clone-forming ability of MOLM-13 cells; Figures A and B are photos of cell clone-forming unit (CFU) detection (scale bar: 500 μm) and their quantitative and statistical analysis results, respectively; Figure 2 The effect of 666-15 on the cell cycle and apoptosis level of MOLM-13 cells; Figures A and B are the results of apoptosis detection and their statistical results, and Figures C and D are the results of cell cycle detection and their statistical results; Figure 3 The results of pathway and process enrichment analysis and protein interaction network analysis in Example 3; wherein, Figure A is the results of pathway and process enrichment analysis of intersection genes using the Metascape program, and Figure B is the results of protein interaction network analysis of genes related to "DNA damage response" and "cell cycle" in Figure A using the STRING program; Figure 4Figure 6 shows the killing effect of 666-15 and Olaparib on different leukemia cells; Figure A and Figure B show the killing effect of different concentrations of 666-15 and Olaparib on MOLM-13 cells and the analysis results of Combination Index (CI); Figure C and Figure D show the killing effect of different concentrations of 666-15 and Olaparib on MV-4-11 cells and the analysis results of Combination Index. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0044] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0045] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0046] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0047] 666-15 was purchased from Shanghai Taoshu Biotechnology Co., Ltd. with a purity of 99.77%; Olaparib was purchased from Shanghai Taoshu Biotechnology Co., Ltd. with a purity of 100%.

[0048] Cell lines MOLM-13 and MV-4-11 were cultured and subcultured according to the protocols of the suppliers (ATCC or DSMZ). MOLM-13 was cultured in RPMI 1640 medium (Gibco, ThermoFisher, USA) containing 10% FBS and penicillin and streptomycin antibiotics. MV-4-11 cells were cultured in IMDM medium (Gibco, ThermoFisher, USA) containing 10% FBS and penicillin and streptomycin antibiotics. MOLM-13 cells and MV-4-11 cells were cultured in an incubator at 37°C, 5% CO2 and 95% air (20% O2). Genetically authenticated, microbial-free cells in the exponential growth phase were used for all experiments.

[0049] Example 1 MOLM-13 cells were seeded at 50 cells / well in a methylcellulose-based semisolid medium (MethoCult, Stem Cell Technologies), and treated with 666-15 at concentrations of 0.5 μM and 1 μM in a 24-well plate as the experimental group, and treated with an equal amount of DMSO as the control group (CTL). After 9 days of culture, the cells were stained (crystal violet staining solution, Beyotime) and recorded with a LEICA DMi1 microscope to detect the cell clone formation ability.

[0050] The results are as follows Figure 1 shown.

[0051] 666-15 can significantly inhibit the growth of MOLM-13 cells.

[0052] Example 2 (1) Cell apoptosis experiment MOLM-13 cells were treated with DMSO or 1 μM 666-15 in vitro for 48 h. MOLM-13 cells were collected and washed with PBS buffer. After resuspending in Annexin V binding buffer, MOLM-13 cells were stained with Annexin V-fitc and propyl iodide staining solution (Beyotime, China) at room temperature for 15 min. MOLM-13 cells were then washed with PBS buffer and analyzed by flow cytometry (Fortessa Cell Analyzer, BD Biosciences). Flow cytometry data were analyzed by FlowJo (Version X.0.7, Tree Star, Inc.).

[0053] (2) Cell cycle experiment MOLM-13 cells were treated with DMSO or 1 μM 666-15 in vitro for 24 h. MOLM-13 cells were collected, washed with PBS buffer, fixed with 70% ethanol at 4°C overnight, and stained with PI (Beyotime, China). After washing with PBS buffer, MOLM-13 cells were analyzed by flow cytometry (Beckman Coulter, USA). Cell cycle data were analyzed and compared using FlowJo (Version X.0.7, Tree Star, Inc.).

[0054] The results are as follows Figure 2 shown.

[0055] Treatment with 666-15 resulted in apoptosis and G1 cell cycle arrest in MOLM-13 cells.

[0056] Example 3 The hTFtarget portal and TF-Target Finder were used to identify putative CREB target genes (n=6511) in blood and bone marrow samples (non-redundant ChIP-seq datasets of human transcription factors were screened using public databases such as NCBI Gene Expression Omnibus (GEO), NCBI Sequence Read Archive (SRA), and ENCODE); overlapping CREB-related genes (from the LinkedOmics database, genes that were positively correlated with CREB expression in blood samples of AML patients (correlation coefficient>0.3)) and CREB target genes (n=956), and pathway and process enrichment analysis was performed using the Metascape program. Protein interaction network analysis of candidate genes involved in "DNA damage response" and "cell cycle" was performed using the STRING program. Systematic signaling pathway clustering analysis was performed on CREB target genes (n=6511, hTFtarget portal and TF-Target Finder databases) and genes that were positively correlated with CREB expression (n=3342, LinkedOmics database).

[0057] The datasets for the analyses involved are publicly available in the SangerBox program (http: / / vip.sangerbox.com / home.html), Kaplan-Meier Plotter (https: / / kmplot.com / ), LinkedOmics (http: / / www.linkedomics.org), hTFtarget (http: / / bioinfo.life.hust.edu.cn / hTFtarget), TF-Target Finder (https: / / jingle.shinyapps.io / TF_Target_Finder / ), Metascape (https: / / metascape.org / gp / index.html), and STRING (https: / / cn.string-db.org / ).

[0058] The results are as follows Figure 3 shown.

[0059] The results of Metascape online clustering analysis showed that genes regulated downstream of CREB were enriched in important signaling pathways such as "DNA damage response".

[0060] Example 4 MOLM-13 cells (0.15×10 6 / mL, 80 μL) was inoculated into a 96-well plate. In the experimental groups, the cells in 666-15 group were treated with the specified concentrations of 666-15, and the final concentrations of 666-15 were 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, and 2000 nM, respectively; the cells in Olaparib group were treated with the specified concentrations of Olaparib, and the final concentrations of Olaparib were 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; the cells in 666-15+Olaparib group were treated with the specified concentrations of 666-15 and Olaparib, and the final concentrations of 666-15 and Olaparib were 31.25 nM and 0.625 μM, 62.5 nM and 1.25 μM, 125 nM and 2.5 μM, 250 nM and 5 μM, 500 nM and 10 μM, 1000 nM and 20 μM, 2000 nM and 40 μM. The control group was treated with an equal amount of DMSO; the blank group did not contain MOLM-13 cells. After 72 h of treatment, cell viability was detected by MTT assay (Beyotime, China). The absorbance OD value was detected at 570 nm, and three biological replicates were performed to calculate the cell survival rate.

[0061] MV-4-11 cells (0.15 × 10 6 / mL, 80 μL) was inoculated into a 96-well plate. In the experimental groups, cells in 666-15 group were treated with specified concentrations of 666-15, and the final concentrations of 666-15 were 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, and 2000 nM, respectively; cells in Olaparib group were treated with specified concentrations of Olaparib, and the final concentrations of Olaparib were 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM, respectively; cells in 666-15+Olaparib group were treated with specified concentrations of 666-15 and Olaparib, and the final concentrations of 666-15 and Olaparib were 31.25 nM and 0.5 μM, 62.5 nM and 1 μM, 125 nM and 2 μM, 250 nM and 4 μM, 500 nM and 8 μM, 1000 nM and 2000 nM, respectively. nM and 16 μM, 2000 nM and 32 μM. The control group was treated with an equal amount of DMSO; the blank group did not contain MV-4-11 cells. After 72 h of treatment, cell viability was detected by MTT assay (Beyotime, China). The absorbance OD value was detected at 570 nm, and three biological replicates were performed.

[0062] Cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0063] The cell viability obtained at different concentrations was input into Graphpad Prism 9.5.1 to generate dose-response curves (GraphPad Prism 9.5.1) and predict the drug half-maximal inhibitory concentration (IC 50 666-15 and Olaparib were used alone or in combination at a certain concentration, and the cell survival rate at the corresponding drug concentration was input into CompuSyn (Cambridge, UK) software to calculate the potential synergistic or additive effects, and the combination index was input to represent the effect of the double drug combination. CI values ​​<1, =1, and >1 were defined as drug synergy, additive, and antagonism, respectively.

[0064] The results are as follows Figure 4 shown.

[0065] 666-15 alone can inhibit MOLM-13 cells (IC 50 =0.14 μM) and MV-4-11 cells (IC 50 =0.79 μM). Olaparib alone can inhibit the growth of MOLM-13 cells (IC 50 =10 μM) and MV-4-11 cells (IC 50 =2.8 μM). The combination of 666-15 and Olaparib can act synergistically (Combination Index<1) and kill MOLM-13 cells and MV-4-11 cells more efficiently. Among them, the synergistic effect of the drug combination (666-15 concentration 125 nM combined with Olaparib concentration 2.5 μM) on killing FLT3-ITD mutant MOLM-13 cell lines was the most significant (CI=0.56); the synergistic effect of the drug combination (666-15 concentration 125nM combined with Olaparib concentration 2 μM) on killing FLT3-ITD mutant MV-4-11 cell lines was the most significant (CI=0.29).

[0066] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A pharmaceutical composition, characterized in that include: PARP inhibitors and CREB inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that The PARP inhibitor includes at least one of olaparib, niraparib, fluzoparib, pamiparib, talazoparib, rucaparib or a pharmaceutically acceptable salt, isomer, racemate, solvate or prodrug of the above compounds; And / or, the CREB inhibitor includes 666-15, KG-501, XX-650-23, niclosamide, CREB-IN-1 TFA or at least one of the pharmaceutically acceptable salts, isomers, racemates, solvates or prodrugs of the above compounds.

3. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the PARP inhibitor to the CREB inhibitor is (15-21):

1.

4. The method for preparing the pharmaceutical composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: The PARP inhibitor and the CREB inhibitor are mixed to obtain the pharmaceutical composition.

5. Use of the pharmaceutical composition according to any one of claims 1 to 3 in any one of A1) to A7); A1) Preparation of products that inhibit the proliferation of leukemia cells; A2) Preparation of products for killing leukemia cells; A3) Preparation of products that inhibit leukemia cell colony formation; A4) Preparation of products that promote or induce apoptosis of leukemia cells; A5) Preparation of products that promote or induce cell cycle arrest in leukemia cells; A6) Preparation of products for the treatment or adjuvant treatment of leukemia; A7) Preparation of products that inhibit the occurrence and development of leukemia.

6. The use according to claim 5, characterized in that: The leukemia cell described in any one of A1) to A5) is a leukemia cell with FLT3-ITD mutation; And / or, the leukemia cells in any one of applications A1) to A5) include at least one of MOLM-13 cells and MV-4-11 cells.

7. The use according to claim 5, characterized in that: The cell cycle arrest described in A5) is a G1 phase cell cycle arrest.

8. The use according to claim 5, characterized in that: The leukemia described in A6) or A7) is FLT3-ITD mutant leukemia.

9. A drug, characterized in that The pharmaceutical composition comprises the pharmaceutical composition according to any one of claims 1 to 3, and an optional pharmaceutically acceptable carrier.

10. An in vitro non-therapeutic method for inhibiting the growth of leukemia cells, characterized in that: The method comprises the following steps: contacting the leukemia cells with the pharmaceutical composition according to any one of claims 1 to 3 or the drug according to claim 9, thereby inhibiting the growth of the leukemia cells.

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