Preparation method of medicine for overcoming non-BCR-ABL1 dependent drug resistance of chronic granulocytic leukemia

Abstract

The invention provides the cabazitaxel-loaded bone marrow mesenchymal stem cell exosome pharmaceutical composition, drug loading is realized through ultrasonic treatment, and the cabazitaxel-loaded bone marrow mesenchymal stem cell exosome pharmaceutical composition can target CML-LSCs and inhibit the self-renewal ability of the CML-LSCs, so that non-BCR-ABL1 dependent drug resistance is overcome. In-vivo and in-vitro experiments prove that the pharmaceutical composition has a remarkable inhibition effect on drug-resistant CML cells and CML-LSCs, and a new strategy is provided for treatment of CML drug-resistant patients.

Description

Technical Field

[0001] This invention belongs to the field of drug development, specifically relating to a method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia. Background Technology

[0002] Chronic myeloid leukemia (CML) is a myeloproliferative disorder of leukemia, accounting for 15%-20% of adult leukemia cases. It is primarily caused by chromosomal translocations and is characterized by a significant increase in granulocytes (including mature and immature granulocytes), marked splenomegaly, and the vast majority of cells possessing relatively specific pH-marked chromosomes. The disease progresses slowly. Its characteristic oncoprotein, BCR-ABL1, is a hallmark of CML, and the discovery of tyrosine kinase inhibitors has revolutionized the treatment of this disease.

[0003] The use of tyrosine kinase inhibitors allows most patients to achieve survival rates similar to the general population after treatment. However, in clinical practice, 20%-30% of patients still develop resistance to tyrosine kinase inhibitors. Among these resistant cases, 70% do not show BCR-ABL1 gene mutations, creating a clinical challenge of non-BCR-ABL1-dependent resistance. Effective Western medicine treatments are still lacking for these patients.

[0004] BCR-ABL1 resistance mechanisms can be broadly classified into two categories: BCR-ABL1-dependent and BCR-ABL1-independent. The former primarily involves mutations in the tyrosine kinase domain (such as the T315I mutation) and overexpression of the BCR-ABL1 protein. Notably, 70% of resistant patients do not exhibit BCR-ABL1 point mutations, highlighting the clinical importance of BCR-ABL1-independent resistance mechanisms. This type of resistance manifests as the continued proliferation of leukemia cells despite effective inhibition of BCR-ABL1 kinase activity by tyrosine kinase inhibitors. The core mechanisms include leukemia stem cell self-renewal, activation of alternative proliferative pathways, and abnormal epigenetic modifications, with leukemia stem cells being widely recognized as the source of resistance.

[0005] The reasons for leukemia stem cells' resistance to tyrosine kinase inhibitors are as follows: ① Under tyrosine kinase inhibitor treatment, BCR-ABL1 can maintain its survival through bypass signals; ② Its genetic instability allows it to survive under continuous drug stress and undergo clonal evolution, ultimately leading to uncontrolled disease.

[0006] Although the academic community has reached a consensus on the central role of leukemia stem cells in non-BCR-ABL1-dependent drug resistance, the clinical safety and efficacy of the findings mentioned above still require large-scale validation, and there are currently no approved targeted drugs for leukemia stem cells. Therefore, a new technology is needed to improve this situation. Summary of the Invention

[0007] To address the problems in the prior art, this invention provides a method for preparing a drug that overcomes non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia. In vitro and in vivo experiments have confirmed that the drug overcomes non-BCR-ABL1-dependent drug resistance and has a significant inhibitory effect on drug-resistant CML cells and CML-LSCs, providing a new strategy for the treatment of drug-resistant CML patients.

[0008] This invention is implemented through the following technical solution: a drug preparation method for overcoming non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia, including the construction of a drug targeting leukemia stem cells, the specific steps of which are as follows: S1 sample pretreatment: using bone marrow mesenchymal stem cell culture supernatant as raw material, exosomes are extracted by differential centrifugation and ultracentrifugation;

[0009] Preparation of S2 exosome drug formulation: Cabazitaxel was used as the active ingredient and loaded into the exosomes obtained in step S1 by sonication. Free drug was removed by low-speed centrifugation and washing with PBS to obtain the exosome drug formulation.

[0010] Furthermore, the exosome drug formulation is specifically a cabazitaxel formulation encapsulated in exosomes, the particle size and potential of which are confirmed by electron microscopy and zeta potential detection.

[0011] Furthermore, the half-maximal inhibitory concentration (IC50) of cabazitaxel in the drug-resistant cell lines K562-IMR and KCL22-IMR were 11.49 nM and 7.179 nM, respectively.

[0012] Furthermore, the ultrasonic processing parameters can be adjusted as follows: frequency 40kHz, pulse mode (2 seconds on / 1 second off), intensity 20mW / cm². 2 Time: 10-15 minutes.

[0013] Furthermore, the exosome drug formulation can be used in combination with other resistance reversal agents (such as the JAK inhibitor ruxolitinib) to enhance efficacy.

[0014] Furthermore, the exosome drug formulation is used in the preparation of a drug for treating chronic myeloid leukemia that is not BCR-ABL1 dependent.

[0015] Furthermore, the exosome drug formulation exerts its anti-drug resistance effect by inhibiting leukemia stem cell self-renewal-related proteins (NANOG, β-catenin, OCT-4) and drug resistance proteins (MRP1, P-gp).

[0016] Furthermore, the exosome drug formulation was verified by HE staining to significantly reduce liver and spleen indices and inhibit leukemia cell infiltration in a leukocyte bone marrow transplantation model.

[0017] Furthermore, the half-maximal inhibitory concentration (IC50) of the drug on drug-resistant cell lines was determined experimentally, and the inhibitory effect of the exosomal drug formulation on CML-LSCs was evaluated by colony formation assay, apoptosis detection, and mitochondrial membrane potential analysis.

[0018] Beneficial effects:

[0019] This invention provides a drug composition for bone marrow mesenchymal stem cell exosomes loaded with cabazitaxel. Drug loading is achieved through ultrasound treatment, enabling the composition to target CML-LSCs and inhibit their self-renewal capacity, thereby overcoming non-BCR-ABL1-dependent drug resistance. In vitro and in vivo experiments have demonstrated that this drug composition has a significant inhibitory effect on drug-resistant CML cells and CML-LSCs, providing a new strategy for the treatment of drug-resistant CML patients. Attached Figure Description

[0020] Figure 1 This is a comparative analysis chart showing that drug screening has revealed Cabazitaxel as an effective drug for CML after resistance to imatinib.

[0021] Figure 2 This is a diagram illustrating the construction of exosome-encapsulated Cabazitaxel targeting leukemia stem cells and the verification analysis of its anti-drug resistance effect.

[0022] Figure 3 This is an analysis of the effect of exosome-encapsulated Cabazitaxel targeting leukemia stem cells on promoting apoptosis and decreasing mitochondrial membrane potential in K562-IMR and KCL22-IMR cells.

[0023] Figure 4 This is an analysis diagram showing how exosomes encapsulating Cabazitaxel, a drug targeting leukemia stem cells, inhibit the self-renewal of leukemia stem cells. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the embodiments described in the specification.

[0025] Example 1: Preparation and characterization of exosomes loaded with cabazitaxel

[0026] Exosome extraction:

[0027] Collect the culture supernatant of bone marrow mesenchymal stem cells and remove impurities by centrifuging at 4℃, 300×g for 10 min and at 4℃, 2000×g for 10 min respectively.

[0028] The supernatant was centrifuged at 100,000×g for 120 min at 4 °C to obtain exosome precipitate. After washing with 200 μl PBS, the precipitate was centrifuged again and finally resuspended in 100 μl PBS.

[0029] Drug loading:

[0030] Exosomes were mixed with 1 μM cabazitaxel solution and subjected to sonication in an ice bath (frequency 40 kHz, pulse wave mode, pulse burst width 200 μs, repetition frequency 1 kHz, intensity 20 mW / cm²). 2 (Exposure time 15 minutes).

[0031] After sonication, the mixture was incubated at 4°C for 1 hour to restore the exosome membrane structure. Then, unloaded exosomes were removed by centrifugation at 10,000×g for 10 min. After washing with PBS, drug-loaded exosomes were obtained.

[0032] Example 2: In vitro efficacy evaluation of the drug composition: CCK8 assay for IC50:

[0033] K562-IMR and KCL22-IMR cells (10,000 cells / well) were seeded in 96-well plates and treated with exosomes loaded with cabazitaxel at different concentrations (0-100 nM) for 48 hours.

[0034] Add 10 μl of CCK8 reagent to each well and continue culturing for 1-4 hours. Then, measure the OD value at 450 nm using a microplate reader and calculate the cell viability.

[0035] Results: The IC50 of cabazitaxel-loaded exosomes against K562-IMR cells was 11.49 nM, and against KCL22-IMR cells it was 7.179 nM, which were significantly lower than those against the free drug group. Western blot analysis confirmed that the expression of drug resistance proteins (MRP1, P-gp) was downregulated.

[0036] Cloning experiments:

[0037] 25,000 drug-resistant cells were resuspended in soft agar medium, seeded in 96-well plates, and cultured at 37°C for 14 days.

[0038] Microscopic counting of colonies (cell count > 50) showed that exosomes loaded with cabazitaxel significantly inhibited the clonogenic ability of drug-resistant cells.

[0039] Apoptosis detection:

[0040] Drug-resistant cells (200,000 cells / well) were seeded in 6-well plates and treated with exosomes loaded with different concentrations of drugs for 48 hours.

[0041] Resuspend cells in PBS, add Annexin V-FITC and PI, incubate in the dark for 10-15 minutes, and then detect cell apoptosis rate by flow cytometry.

[0042] Results: Exosomes loaded with cabazitaxel significantly induced apoptosis in drug-resistant cells in a dose-dependent manner, and mitochondrial membrane potential assays showed an increased proportion of JC-1 monomers.

[0043] Example 3: In vivo efficacy evaluation of drug composition: Construction of CML mouse model:

[0044] The retrovirus (MSCV-BCR-ABL1-IRES-EGFP plasmid) was packaged in Plat-E cells, and after infecting bone marrow cells, 1×10⁻⁶ cells were used. 6 A CML model was established by injecting cells into recipient mice that had been irradiated with radiation (500 cGy, twice).

[0045] Drug intervention:

[0046] Mice were divided into a control group, a free cabazitaxel group, and a cabazitaxel-loaded exosome group, and were administered the drugs by gavage for 14 consecutive days (high dose 6.3 g / kg, low dose 3.15 g / kg).

[0047] The survival rate of mice was observed, the peripheral blood leukocyte (WBC) index and liver and spleen index were detected, and the infiltration of leukemia cells in the liver and spleen was observed by HE staining.

[0048] Results: The survival rate of mice loaded with cabazitaxel exosomes was significantly improved, the WBC index and liver and spleen index were reduced, and the infiltration of leukemia cells in the liver and spleen was reduced.

[0049] CML-LSCs detection:

[0050] Mouse bone marrow cells were collected, cleaved, and stained with flow cytometry antibody solution (LSK:GFP). + Lin - The proportion of CML-LSCs was detected using APC, Sca-1-PE-CF594, and c-Kit-PE.

[0051] Results: Exosomes loaded with cabazitaxel significantly reduced the proportion of CML-LSCs, and Western blot analysis confirmed the downregulation of self-renewal-related proteins (NANOG, β-catenin, OCT-4).

[0052] Example 4: Detection of drug resistance-related proteins using Western blot analysis:

[0053] Proteins from drug-resistant cells treated with different concentrations and for different times were extracted, and the expression of drug resistance-related proteins (MRP1, P-gp) and self-renewal-related proteins (NANOG, β-catenin, OCT-4) was detected.

[0054] Results: Exosomes loaded with cabazitaxel significantly downregulated the expression of drug resistance-related proteins and self-renewal-related proteins, further validating their inhibitory effect on drug resistance and CML-LSCs.

[0055] As described in Examples 1-4, this invention provides a bone marrow mesenchymal stem cell exosome drug composition loaded with cabazitaxel. Drug loading is achieved through ultrasound treatment, enabling the composition to target CML-LSCs and inhibit their self-renewal capacity, thereby overcoming non-BCR-ABL1-dependent drug resistance. In vitro and in vivo experiments have confirmed that this drug composition has a significant inhibitory effect on drug-resistant CML cells and CML-LSCs, providing a new strategy for the treatment of drug-resistant CML patients.

Claims

1. A method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia, characterized in that: The specific steps for constructing drugs targeting leukemia stem cells are as follows: S1 sample pretreatment: Exosomes were extracted from the culture supernatant of bone marrow mesenchymal stem cells by differential centrifugation and ultracentrifugation. Preparation of S2 exosome drug formulation: Cabazitaxel was used as the active ingredient and loaded into the exosomes obtained in step S1 by sonication. Free drug was removed by low-speed centrifugation and washing with PBS to obtain the exosome drug formulation.

2. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The exosome drug formulation is specifically a cabazitaxel formulation encapsulated in exosomes, and its particle size and potential are confirmed by electron microscopy and zeta potential detection.

3. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The half-maximal inhibitory concentrations (IC50) of cabazitaxel in the drug-resistant cell lines K562-IMR and KCL22-IMR were 11.49 nM and 7.179 nM, respectively.

4. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The ultrasonic processing parameters can be adjusted as follows: frequency 40kHz, pulse mode (2 seconds on / 1 second off), intensity 20mW / cm². 2 Time: 10-15 minutes.

5. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The exosome drug formulation can be used in combination with other resistance reversal agents (such as the JAK inhibitor ruxolitinib).

6. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The application of the exosome drug formulation in the preparation of a drug for treating chronic myeloid leukemia that is not BCR-ABL1 dependent.

7. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The exosome drug formulation exerts its anti-drug resistance effect by inhibiting leukemia stem cell self-renewal-related proteins (NANOG, β-catenin, OCT-4) and drug resistance proteins (MRP1, P-gp).

8. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The exosome drug formulation was verified by HE staining to significantly reduce liver and spleen indices and inhibit leukemia cell infiltration in a leukemia bone marrow transplantation model.

9. The method for preparing a drug to overcome non-BCR-ABL1-dependent drug resistance in chronic myeloid leukemia according to claim 1, characterized in that: The half-maximal inhibitory concentration (IC50) of the exosomal drug formulation against drug-resistant cell lines was determined experimentally, and the inhibitory effect of the exosomal drug formulation on CML-LSCs was evaluated by colony formation assay, apoptosis detection, and mitochondrial membrane potential analysis.

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