Medicine for treating Philadelphia chromosome positive acute lymphocytic leukemia and application thereof

Abstract

The invention provides an application of a novel HDAC1 inhibitor, namely low-concentration epirubicin, in treatment of Philadelphia chromosome positive acute lymphocytic leukemia (Ph + ALL). Through anti-tumor activity screening and bioinformatics analysis, it is found that low-concentration epirubicin has a remarkable inhibiting effect on Ph + ALL cells, and dose and time dependence is achieved. Experimental results show that the low-concentration epirubicin can effectively inhibit proliferation, DNA synthesis and clone formation ability of leukemia cells and induce ferroptosis of the cells, and the specific mechanism comprises induction of cell lipid peroxidation and intracellular Fe < 2 + > overload so as to cause Fenton reaction. In addition, in-vivo experiments further prove the anti-tumor effect of low-concentration epirubicin in an NOG mouse model. The research not only reveals the potential of low-concentration epirubicin as a novel drug for treating Ph + ALL, but also provides a theoretical basis and experimental support for developing a novel anti-tumor drug based on a ferroptosis mechanism.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a medicine for treating Philadelphia chromosome-positive acute lymphoblastic leukemia and application thereof. Background Art

[0002] Philadelphia chromosome-positive acute lymphoblastic leukemia is a high-risk subtype of ALL, with an incidence of 20% to 25% in adult ALL and up to 50% in patients over 50 years of age. + ALL has a poor prognosis, with a 5-year survival rate of less than 20% with chemotherapy alone. Although the emergence of tyrosine kinase inhibitors (TKIs) has improved the + Although the efficacy of treatment for ALL patients has been improved, frequent relapses remain a major problem in clinical treatment. Therefore, it is necessary to find new drugs, new targets and new mechanisms for the treatment of Ph. + The treatment of leukemia is particularly important.

[0003] In order to solve the above problems, the applicant proposes a targeted drug for treating Philadelphia chromosome-positive acute lymphoblastic leukemia and its application. Summary of the invention

[0004] The present invention aims to provide a method for treating Ph + Targeted drugs for ALL and their applications to solve the problems in the existing technologies.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for treating Ph + Epirubicin is a targeted drug for ALL, with an effective concentration of 0.01-30 μM.

[0006] Optionally, the epirubicin is dissolved in dimethyl sulfoxide and diluted with physiological saline to a desired concentration.

[0007] A kind of epirubicin in the preparation of + The invention relates to a method for treating ALL cell proliferation in a drug, wherein the epirubicin is used in a dosage effective for inhibiting the proliferation of leukemia cells.

[0008] Optionally, the epirubicin inhibits the proliferation of leukemia cells by inducing ferroptosis, specifically by inducing cellular lipid peroxidation and Fenton reaction caused by overload of intracellular free Fe2+.

[0009] Optionally, it is characterized in that after administration, epirubicin can cause morphological changes in the mitochondria of leukemia cells, which is specifically manifested as a reduction in mitochondrial volume and a reduction in mitochondrial ridges.

[0010] Optionally, the method further includes detecting the levels of reactive oxygen species and lipid peroxides in leukemia cells to evaluate the therapeutic effect of the drug.

[0011] Optionally, the method further includes detecting changes in free Fe2+ levels and unstable iron pools in leukemia cells to confirm the effect of the drug in inhibiting the proliferation of leukemia cells by inducing the ferroptosis pathway.

[0012] Beneficial effects: Improve therapeutic effect:

[0013] The present invention has confirmed through in vitro cell experiments and in vivo animal model experiments that low concentrations of epirubicin can significantly inhibit Ph + Proliferation of ALL cells. Compared with traditional treatments, low-concentration epirubicin achieves better therapeutic effects while reducing the amount of drug used, showing superior anti-tumor activity.

[0014] Enhanced drug safety:

[0015] Using low-concentration epirubicin as a therapeutic drug can significantly reduce the toxic side effects that may be caused by excessively high drug concentrations, such as cardiotoxicity, bone marrow suppression, etc., thereby improving the safety of treatment and patient tolerance.

[0016] Mechanisms of induced ferroptosis:

[0017] This invention first revealed that low concentration of epirubicin can induce Ph + ALL cells undergo ferroptosis. This discovery is the first major finding in the field of ferroptosis in ALL cells. + It provides new ideas and targets for the treatment of ALL, and also lays the foundation for the future development of new anti-tumor drugs based on the ferroptosis mechanism.

[0018] Optimizing treatment strategies:

[0019] Combined with the experimental results, the present invention proposes the potential of low-concentration epirubicin as a new treatment strategy. This strategy can be used not only as a single therapy, but also in combination with other chemotherapeutic drugs or targeted drugs to improve the overall treatment effect and possibly reduce the occurrence of drug resistance.

[0020] Broaden the prospects of clinical application:

[0021] The result of the present invention is Ph + The clinical treatment of ALL provides a new option, which is expected to improve the prognosis and quality of life of patients. At the same time, since epirubicin is a drug that has been marketed, its safety and effectiveness have been widely recognized, which will accelerate the clinical application process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It shows the changes in cell survival rate of K562 and SUP-B15 cells after different concentrations of epirubicin acted on them for different periods of time;

[0023] Figure 2 It shows the changes in the cell DNA synthesis capacity of K562 and SUP-B15 cells after 24 hours of exposure to different concentrations of epirubicin;

[0024] Figure 3 It shows the changes in the clone formation ability of K562 and SUP-B15 cells after being treated with different concentrations of epirubicin;

[0025] Figure 4 It shows the changes in mitochondrial morphology of K562 and SUP-B15 cells after 24 hours of exposure to different concentrations of epirubicin;

[0026] Figure 5 It shows the changes of intracellular reactive oxygen species in K562 and SUP-B15 cells after 24 hours of exposure to different concentrations of epirubicin;

[0027] Figure 6 It shows the changes of lipid peroxidation in K562 and SUP-B15 cells after 24 hours of exposure to different concentrations of epirubicin;

[0028] Figure 7 The results show that the free Fe in K562 and SUP-B15 cells was activated by different concentrations of epirubicin for 24 hours. 2+ Level changes;

[0029] Figure 8 It shows the changes in the level of labile iron pool in K562 and SUP-B15 cells after 24 hours of exposure to different concentrations of epirubicin;

[0030] Fig. 9 To demonstrate the in vivo anti-leukemia pharmacodynamics evaluation of different concentrations of epirubicin. DETAILED DESCRIPTION

[0031] The following describes the preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0032] Example 1

[0033] In the process of implementing the present invention, we designed a series of detailed experiments to verify the efficacy of low-concentration epirubicin in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + The following is a detailed description of the experimental steps and results.

[0034] Experimental Materials and Methods

[0035] Cell lines and reagents

[0036] Cell lines: Two cell lines were used in the experiment, K562 (human chronic myeloid leukemia cell line) and SUP-B15 (human acute lymphoblastic leukemia cell line), both purchased from ATCC, USA.

[0037] Drugs and reagents:

[0038] Epirubicin (Epi): purchased from Selleck, USA, dissolved in dimethyl sulfoxide (DMSO), prepared into a 10 mmol / L stock solution, and stored at -20°C. During the experiment, diluted with physiological saline to the required concentration.

[0039] Culture medium: IMDM and 1640 complete medium, which is prepared by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody to IMDM or 1640 basal medium.

[0040] Experimental kits: including CCK-8 kit, EdU kit, flow cytometry kit (DCFH-DA, BODIPY C11, CA-AM, etc.), FerroOrange stain, etc.

[0041] Cell recovery, passaging and cryopreservation

[0042] Cell Recovery:

[0043] Prepare complete medium and preheat in a 37°C water bath.

[0044] Take the cells out of the liquid nitrogen tank and quickly thaw them in a water bath.

[0045] Transfer the cell suspension to a centrifuge tube in a clean bench, discard the supernatant after centrifugation, resuspend the cells in complete culture medium, and transfer to a culture flask.

[0046] The cells were cultured in an incubator at 37°C and 5% CO2.

[0047] Cell passaging:

[0048] When the cell density reached 80-90%, subculture was performed.

[0049] Collect the cell suspension into a centrifuge tube, discard the supernatant after centrifugation, and wash once with PBS.

[0050] Add culture medium to resuspend the cells and subculture into new culture flasks at appropriate ratios.

[0051] Cryopreservation of cells:

[0052] Prepare cell freezing solution (FBS:DMSO=9:1).

[0053] Collect the cell suspension into a centrifuge tube, discard the supernatant after centrifugation, resuspend the cells with freezing solution, and transfer to a freezing tube.

[0054] After marking the information on the cryotube, place it in a cryobox, store it at -80℃ overnight, and then transfer it to a liquid nitrogen tank for long-term storage.

[0055] Cell viability assay (CCK-8 method)

[0056] The cells were seeded in 96-well plates at a rate of 1×10^4 cells / well, and different concentrations of epirubicin were added for 24, 48, and 72 hours.

[0057] Three replicate wells were set up for each group, 10 μL of CCK-8 reagent was added to each well, and after further culturing for 2 hours, the absorbance value at 450 nm was detected using an enzyme-labeled instrument.

[0058] The cell survival rate was calculated as [(OD(450) of the experimental group wells - OD(450) of the blank wells) / (OD(450) of the control group - OD(450) of the blank wells)] × 100%.

[0059] EdU assay to detect cell DNA synthesis ability

[0060] The cells were seeded in 6-well plates at 1×10^6 cells / well, and different concentrations of epirubicin were added for 24 hours.

[0061] After incubation with EdU working solution, the cells were fixed and permeabilized, and then Click reaction solution and Hoechst 33342 were added for staining.

[0062] The cells were observed under an upright fluorescence microscope, and 5 fields of view were randomly selected to take pictures and calculate the positive cell rate.

[0063] Soft Agar cell soft colony formation experiment

[0064] Prepare the lower layer gel and the upper layer gel, mix the cell suspension with the upper layer gel and pour into the culture plate.

[0065] After solidification, the cells were placed in a 37°C incubator for 2-3 weeks. After cell clones were formed, MTT solution was added for incubation and the cells were photographed and counted.

[0066] Transmission electron microscopy to detect mitochondrial morphology

[0067] The cells were seeded in 6-well plates and collected after 24 hours of exposure to epirubicin.

[0068] The cells were fixed with electron microscopy fixative and the cell morphology was detected by transmission electron microscopy.

[0069] Flow cytometry was used to detect intracellular ROS and L-ROS levels

[0070] The cells were seeded in 6-well plates, and different concentrations of epirubicin were added and the cells were collected after 24 hours.

[0071] After staining with DCFH-DA and BODIPY C11, the levels of intracellular ROS and L-ROS were detected by flow cytometry.

[0072] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0073] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A drug epirubicin for treating Philadelphia chromosome-positive acute lymphoblastic leukemia, with an effective concentration of 0.01-30 μM.

2. The drug according to claim 1, wherein the epirubicin is dissolved in dimethyl sulfoxide and diluted with physiological saline to a desired concentration.

3. Use of epirubicin in the preparation of a drug for inhibiting the proliferation of Philadelphia chromosome-positive acute lymphoblastic leukemia cells, Features The epirubicin is used at a dosage effective to inhibit the proliferation of leukemia cells.

4. The use according to claim 3, Features The epirubicin inhibits the proliferation of leukemia cells by inducing ferroptosis, specifically by inducing cellular lipid peroxidation and the Fenton reaction caused by overload of free Fe2+ in the cells.

5. The use according to claim 3 or 4, Features After administration, epirubicin can cause morphological changes in the mitochondria of leukemia cells, which are specifically manifested in the reduction of mitochondrial volume and mitochondrial ridges.

6. Use according to any one of claims 3 to 5, Features The method also includes detecting the levels of reactive oxygen species and lipid peroxides in leukemia cells to evaluate the therapeutic effect of the drug.

7. The use according to any one of claims 3 to 6, further comprising detecting changes in the free Fe2+ level and the unstable iron pool in leukemia cells to confirm the effect of the drug in inhibiting the proliferation of leukemia cells by inducing the ferroptosis pathway.

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