Pharmaceutical composition for treating acute myelogenous leukemia and pharmaceutical application thereof

Abstract

The invention discloses a pharmaceutical composition for treating acute myelogenous leukemia and pharmaceutical application thereof, and relates to the technical field of biological medicine. The pharmaceutical composition comprises eltrombopag and an MEK inhibitor. The research finds that eltrombopag can inhibit AML cell proliferation and induce AML cell apoptosis by regulating the steady state of copper ions in cells and down-regulating the expression of key molecules of an MAPK signal channel on the premise of not depending on the hematopoietic promotion function of eltrombopag. A further research shows that after the eltrombopag and the MEK inhibitor are combined for use, the significant synergistic effect of inhibiting cell viability and inducing apoptosis is shown in various AML cell lines. The pharmaceutical composition can improve the treatment effect on acute myelogenous leukemia, provides a new targeted treatment strategy for patients with acute myelogenous leukemia, and is especially suitable for patients with insufficient response to a single drug MEK inhibitor or drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a pharmaceutical composition for treating acute myeloid leukemia and its pharmaceutical use. Background Art

[0002] Acute myeloid leukemia (AML) is a type of malignant clonal disease derived from hematopoietic stem cells. Its main characteristics are abnormal bone marrow proliferation and abnormal proliferation of leukemia cells in the bone marrow and peripheral blood, which leads to the suppression of normal hematopoietic function. The current first-line treatments for AML mainly include induction chemotherapy (such as the "3+7" regimen), targeted drug therapy (such as FLT3, IDH inhibitors) and allogeneic hematopoietic stem cell transplantation. However, in the face of relapsed or refractory AML, these regimens have limited efficacy and are often accompanied by high toxicity and drug resistance. Clinical treatment still faces huge challenges.

[0003] MEK (mitogen-activated protein kinase) is a key kinase in the MAPK signaling pathway and a crucial signaling node in regulating tumor cell proliferation, differentiation, and apoptosis. MEK inhibitors have shown potential therapeutic value in various malignancies, but single-agent MEK inhibitors are still limited by poor efficacy and strong drug resistance. Summary of the Invention

[0004] The present invention aims to provide a pharmaceutical composition for treating acute myeloid leukemia and its pharmaceutical use, thereby overcoming the aforementioned problems of the prior art. This pharmaceutical composition can improve the therapeutic efficacy of acute myeloid leukemia and provide a new targeted therapy strategy for patients with acute myeloid leukemia. It is particularly suitable for patients who have an inadequate response to or develop resistance to single-agent MEK inhibitors.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a pharmaceutical composition for treating acute myeloid leukemia, comprising eltrombopag and a MEK inhibitor.

[0007] Furthermore, the MEK inhibitor is U0126.

[0008] The present invention also provides use of the above-mentioned pharmaceutical composition in preparing medicine for treating acute myeloid leukemia.

[0009] The present invention also provides a medicine for treating acute myeloid leukemia, the active ingredient of which includes the above-mentioned pharmaceutical composition.

[0010] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0011] The present invention also provides the use of eltrombopag in preparing an auxiliary drug for enhancing the inhibitory effect of MEK inhibitors on acute myeloid leukemia tumor cells.

[0012] Furthermore, the MEK inhibitor is U0126.

[0013] Furthermore, the auxiliary drug enhances the inhibitory effect of the MEK inhibitor on acute myeloid leukemia tumor cells by regulating the copper ion homeostasis in acute myeloid leukemia tumor cells and downregulating the expression of key proteins in the MAPK pathway.

[0014] The present invention also provides an auxiliary drug for enhancing the inhibitory effect of MEK inhibitors on acute myeloid leukemia tumor cells, wherein the active ingredient includes eltrombopag.

[0015] Furthermore, the auxiliary medicine also includes pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects:

[0017] The present study found that eltrombopag can induce apoptosis and inhibit proliferation in AML cells by regulating copper homeostasis and the MAPK signaling pathway, independent of its TPO-R agonist mechanism. In Kasumi-1 and KG-1 cell models, eltrombopag demonstrated significant anti-tumor effects. Furthermore, in THP-1 cells, it was confirmed that eltrombopag can downregulate the expression of key MAPK pathway proteins (including KRas, Raf, MEK1 / 2, and ERK1 / 2), induce increased expression of apoptotic factors associated with the mitochondrial pathway (such as Bax and Caspase-3), and downregulate Bcl-2.

[0018] Further investigation of the synergistic effect of eltrombopag and the MEK inhibitor U0126 in THP-1 cells revealed that the combination treatment group exhibited significantly enhanced effects in both cell viability inhibition and apoptosis induction compared to either monotherapy group. qRT-PCR and Western blot experiments demonstrated that the combination group further reduced the expression levels of key proteins in the MAPK pathway, demonstrating a synergistic signaling blockade mechanism. These findings reveal for the first time that eltrombopag can enhance the anti-AML cell activity of MEK inhibitors, such as U0126, and provide a novel strategy for combined targeted therapy in acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Results of CCK-8 assay for THP-1 (A), Kasumi-1 (B), and KG-1 (C) cell viability after treatment with different concentrations of eltrombopag for 24, 48, and 72 hours;

[0021] Figure 2 Figures 2 and 3 are EdU immunofluorescence staining images and histograms of the proportion of positive cells in THP-1, Kasumi-1, and KG-1 cells after eltrombopag treatment; A and B are EdU immunofluorescence staining images and histograms of the proportion of positive cells in THP-1 cells; C and D are EdU immunofluorescence staining images and histograms of the proportion of positive cells in Kasumi-1 cells; E and F are EdU immunofluorescence staining images and histograms of the proportion of positive cells in KG-1 cells;

[0022] Figure 3 The original flow cytometric graphs show that different doses of eltrombopag induced apoptosis in THP-1, Kasumi-1, and KG-1 cells using Annexin V-FITC / PI flow cytometry.

[0023] Figure 4 Statistical bar graphs of apoptosis in THP-1 (A), Kasumi-1 (B) and KG-1 (C) cells after induction by different doses of Eltrombopag;

[0024] Figure 5 Figure 2 shows the cell cycle detection results of THP-1, Kasumi-1 and KG-1 cells after Eltrombopag treatment;

[0025] Figure 6 Cell cycle statistics of THP-1 (A), Kasumi-1 (B) and KG-1 (C) cells after Eltrombopag treatment;

[0026] Figure 7Figure 2 shows the effects of eltrombopag, U0126, and their combined treatment on the cell viability of three cell types at 48 h and 72 h. A and B are statistical graphs of THP-1 cell viability at 48 h and 72 h, respectively; C and D are statistical graphs of KG-1 cell viability at 48 h and 72 h, respectively; E and F are statistical graphs of Kasumi-1 cell viability at 48 h and 72 h, respectively. ELT stands for eltrombopag.

[0027] Figure 8 The results of qRT-PCR detection of the mRNA expression levels of KRas (A), Raf (B), MEK1 / 2 (C), and ERK1 / 2 (D), key factors of the MAPK signaling pathway, in THP-1 cells after treatment with eltrombopag, U0126, and their combination; ELT stands for eltrombopag;

[0028] Figure 9 Figure 1 shows the results of qRT-PCR detection of the mRNA expression levels of apoptosis-related genes Bcl-2 (A), Bax (B), and Caspase-3 (C) in THP-1 cells after treatment with eltrombopag, U0126, and their combination. ELT stands for eltrombopag.

[0029] Figure 10 The results of Western blot analysis of key factors of the MAPK signaling pathway in THP-1 cells after eltrombopag, U0126, and their combined treatment; A is a Western blot analysis; B and E are statistical graphs of the expression levels of KRas, Raf, MEK1 / 2, and ERK1 / 2, respectively; ELT represents eltrombopag;

[0030] Figure 11 Figure 3 Western blot analysis of apoptosis-related genes in THP-1 cells after treatment with eltrombopag, U0126, and their combination. A is a Western blot analysis graph; B and D are statistical graphs of the expression levels of Bcl-2, Bax, and Caspase-3, respectively. ELT represents eltrombopag. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Terminology Notes:

[0037] Eltrombopag, English name Eltrombopag, CAS number 496775-61-2, structural formula is as follows:

[0038]

[0039] U0126 is a MEK inhibitor with CAS number 109511-58-2 and the structural formula is as follows:

[0040]

[0041] Example 1

[0042] 1. Materials and Methods

[0043] Three human acute myeloid leukemia cell lines, THP-1, Kasumi-1 and KG-1, were selected and treated with different concentrations of Eltrombopag (1, 5, 10, 25, 50, 100 μg / mL) for 24 h, 48 h and 72 h, after which cell viability, cell proliferation, apoptosis and cell cycle were detected.

[0044] Cell viability assay: CCK-8 method, 96-well plate, 1×10 cells per well 4 .

[0045] Cell proliferation detection: EdU staining (Apollo567 labeling) and observation under a fluorescence microscope.

[0046] Cell apoptosis detection: Annexin V-FITC / PI flow cytometry analysis.

[0047] Cell cycle detection: PI single staining flow cytometry was used to detect the distribution of G0 / G1, S, and G2 / M phases.

[0048] 2. Experimental Results

[0049] Figure 1 Eltrombopag showed that it inhibited cell viability in three AML cell lines in a dose- and time-dependent manner. Figure 2 EdU fluorescence imaging and histograms showed that eltrombopag significantly inhibited DNA synthesis. Figure 3-Figure 4 It showed that Eltrombopag significantly induced early and late apoptosis, and the apoptosis rates of the three cell lines were significantly increased. Figure 5-Figure 6 Eltrombopag induces G0 / G1 arrest and reduces the proportion of cells in the S phase, suggesting that it acts through cell cycle blockade. The test results of the effects of eltrombopag, U0126 and their combination on the cell viability of the three cell lines at 48h and 72h are shown in Figure 7 .

[0050] Example 2 Study on the Mechanism of Eltrombopag in Regulating MAPK Pathway and Apoptosis Factor Expression and the Synergistic Inhibitory Effect of Eltrombopag Combined with MEK Inhibitor U0126 in THP-1 Cells

[0051] 1. Materials and Methods

[0052] THP-1 cells were divided into four groups: blank control, eltrombopag (15 μg / mL), U0126 (20 μM), and combination group (7.5 μg / mL + 10 μM). After treatment with each reagent for 48 hours, total RNA and total protein were extracted. qRT-PCR was used to measure the mRNA levels of KRas, Raf, MEK1 / 2, ERK1 / 2, Bcl-2, Bax, and Caspase-3; Western blot was used to measure the expression of these proteins, with β-actin used as an internal control.

[0053] 2. Experimental Results

[0054] Figure 8 and Figure 9 Results showed that eltrombopag significantly downregulated the mRNA levels of MAPK pathway genes and regulated the expression of apoptosis-related genes (upregulating Bax and Caspase-3 and inhibiting Bcl-2). Figure 10 and Figure 11 The WB results of (ELT group vs control group) showed that Eltrombopag significantly inhibited the expression of KRas, Raf, MEK1 / 2, and ERK1 / 2, and promoted the upregulation of Bax and Caspase-3, suggesting that it may induce apoptosis through the dual mechanism of "copper regulation + MAPK inhibition".

[0055] Figure 8 and Figure 9 It showed that the expression of KRas, Raf, MEK and ERK genes was downregulated more significantly in the combined group. Figure 10 and Figure 11 Western blot results showed that the combination of eltrombopag and U0126 can synergistically reduce the expression of key proteins in the MAPK pathway and the anti-apoptotic factor Bcl-2, while further enhancing the expression of pro-apoptotic factors (Bax, Caspase-3).

[0056] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: These include eltrombopag and MEK inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that The MEK inhibitor is U0126.

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a medicament for treating acute myeloid leukemia.

4. A drug for treating acute myeloid leukemia, characterized in that: The active ingredient comprises the pharmaceutical composition according to claim 1 or 2.

5. The drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The use of eltrombopag in the preparation of auxiliary drugs to enhance the inhibitory effect of MEK inhibitors on acute myeloid leukemia tumor cells.

7. The use according to claim 6, characterized in that The MEK inhibitor is U0126.

8. The use according to claim 7, characterized in that The auxiliary drug enhances the inhibitory effect of the MEK inhibitor on acute myeloid leukemia tumor cells by regulating the copper ion homeostasis in acute myeloid leukemia tumor cells and downregulating the expression of key proteins in the MAPK pathway.

9. An auxiliary drug for enhancing the inhibitory effect of MEK inhibitors on acute myeloid leukemia tumor cells, characterized in that: The active ingredient includes eltrombopag.

10. The auxiliary drug according to claim 9, characterized in that The auxiliary drug also includes pharmaceutically acceptable excipients.

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