Anti-leukemia pharmaceutical composition based on DNA damage activated NKT cells and application of anti-leukemia pharmaceutical composition

Abstract

The invention provides an anti-leukemia pharmaceutical composition based on DNA damage activated NKT cells and application of the anti-leukemia pharmaceutical composition. A mouse leukemia model is adopted as a research object, and experimental results show that in a C1498 homotransplantation tumor C57BL / 6 immune sound mouse leukemia model, the tumor inhibition rate of a daunorubicin and alpha-galactosylceramide combined treatment group reaches 67.4%, the survival time is remarkably prolonged, and obvious toxicity is not caused; in a C57 mouse leukemia model constructed by caudal vein injection of C1498, daunorubicin and alpha-galactosylceramide are combined to treat and reverse leukemia-related hepatosplenomegaly (the weight of liver and spleen is equivalent to that of a normal group), leukocyte proliferation is inhibited, and the survival state of mice is improved. The invention provides a new direction for treatment of leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-leukemia drug composition based on DNA damage to activate NKT cells and its application. In particular, the present invention discloses a combined application scheme of daunorubicin (DNR) and α-galactosylceramide (α-GalCer), which induces DNA damage in tumor cells and activates CD1d-dependent immunogenic signals through DNR, and synergizes α-GalCer to enhance the recognition and killing function of NKT cells on leukemia cells, forming a two-way action mechanism of "chemotherapy sensitization immune response-immunoenhanced chemotherapy effect". This scheme aims to solve clinical problems such as high chemotherapy resistance, significant immune escape and high toxicity of single therapy in the treatment of leukemia. It is suitable for the precision treatment of leukemia patients and can be extended to the combined immunotherapy intervention of other CD1d-positive blood system malignancies. Background Art

[0002] In recent years, CD1d-restricted natural killer T (NKT) cells have become a research hotspot for tumor immunotherapy due to their unique dual immunomodulatory functions. NKT cells directly kill tumor cells and secrete cytokines such as IFN-γ, activating dendritic cells, NK cells, and T cells to form a synergistic anti-tumor immune network. NKT cells have demonstrated potential in various mouse models and early clinical trials. However, the malignant tumor microenvironment often leads to NKT cell functional exhaustion, manifested by reduced secretion of effector molecules and loss of cytotoxicity, which greatly limits their clinical application.

[0003] α-Galactosylceramide (α-GalCer), a classic NKT cell agonist, activates NKT cells by binding to CD1d-antigen presentation complexes, significantly increasing their proliferation and promoting IFN-γ release. Phase I / II clinical trials have demonstrated a favorable safety profile for α-GalCer monotherapy in solid tumors, but its efficacy is limited by the tumor's immunosuppressive microenvironment and insufficient NKT cell persistence. Notably, recent studies have found that DNA-damaging agents can upregulate CD1d expression on the surface of tumor cells, suggesting that chemotherapy drugs may enhance the immunogenicity of tumor cells to NKT cells through epigenetic regulation. For example, daunorubicin (DNR), a first-line chemotherapy for acute myeloid leukemia (AML), directly induces tumor cell apoptosis, but it remains unclear whether the DNA damage response induced by DNR activates NKT cells through a CD1d-dependent pathway. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and innovatively propose a method for treating leukemia by combining the DNA damaging agent daunorubicin (DNR) with the NKT cell agonist (α-GalCer). Specifically, the present invention examines the effect of the combined use of the DNA damaging agent daunorubicin (DNR) and the NKT activator α-GalCer on the clearance of acute myeloid leukemia cells by NKT cells in vivo, to further explore whether DNA damage can activate NKT cells through CD1d, and whether α-GalCer can further amplify the activation effect and completely eliminate leukemia cells. This strategy is expected to break through the limitations of single therapy and provide a new target for overcoming leukemia immune escape.

[0005] The technical solution adopted in the present invention is as follows: Use of daunorubicin in combination with α-galactosylceramide in the preparation of an anti-leukemia pharmaceutical composition; Furthermore, the cells causing the leukemia are C1498, HL-60, THP-1, KG-1, Jurkat, REH, Nalm-6, K562, KBM-7, MEC-1, JVM-3, U937 or HEL; Furthermore, the anti-leukemia pharmaceutical composition comprises daunorubicin, α-galactosylceramide and pharmaceutically acceptable excipients; Furthermore, the dosage form of the anti-leukemia pharmaceutical composition is an injection, tablet, capsule, granule, powder, patch, suspension, syrup, oral solution, suppository or any combination thereof; Furthermore, the injection is administered via intraperitoneal injection.

[0006] An anti-leukemia pharmaceutical composition based on DNA damage-activated NKT cells, comprising daunorubicin, α-galactosylceramide, and pharmaceutically acceptable excipients; Furthermore, the cells causing the leukemia are C1498, HL-60, THP-1, KG-1, Jurkat, REH, Nalm-6, K562, KBM-7, MEC-1, JVM-3, U937 or HEL; Furthermore, the dosage form of the anti-leukemia pharmaceutical composition is an injection, tablet, capsule, granule, powder, patch, suspension, syrup, oral solution, suppository or any combination thereof; Furthermore, the injection is administered via intraperitoneal injection.

[0007] The significant advantages of the present invention are: The present invention discloses a synergistic therapy for the treatment of acute myeloid leukemia (AML) based on the combination of daunorubicin (DNR) and α-galactosylceramide (α-GalCer). DNR induces DNA damage in tumor cells and upregulates CD1d expression, thereby enhancing tumor immunogenicity. At the same time, α-GalCer activates NKT cells to release IFN-γ and directly kill leukemia cells, forming a bidirectional mechanism of "chemotherapy-sensitized immunity-immune synergistic chemotherapy." In the C1498 homologous transplanted tumor C57BL / 6 immune-competent mouse leukemia model, the combined treatment group achieved a tumor inhibition rate of 67.4%, significantly prolonged survival time (*P<0.05), and did not cause obvious toxicity (body weight was stable); in the C57 mouse leukemia model constructed by tail vein injection of C1498, the combined treatment reversed leukemia-related hepatosplenomegaly (liver and spleen weight was equivalent to that of the normal group), inhibited leukocyte proliferation, and improved the survival status of the mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 :Progress in treatment of C1498 homograft tumor leukemia model in C57BL / 6 immune-competent mice.

[0009] Figure 2 : Tumor inhibition rates of four drug groups (Vehicle / Daunorubicin / α-GalCer / Daunorubicin+α-GalCer) in a C1498 homograft tumor leukemia model in immunocompetent C57BL / 6 mice.

[0010] Figure 3 : Tumor weights of four drug-treated groups (Vehicle / Daunorubicin / α-GalCer / Daunorubicin+α-GalCer) in a C1498 homograft tumor leukemia model in C57BL / 6 immunocompetent mice.

[0011] Figure 4 : Monitoring of mouse body weight during treatment in four drug-treated groups (Vehicle / Daunorubicin / α-GalCer / Daunorubicin+α-GalCer) in a C1498 homograft C57BL / 6 immunocompetent mouse leukemia model.

[0012] Figure 5 : The Kaplan-Meier method was used to analyze the survival time of mice in four drug-treated groups (Vehicle / Daunorubicin / α-GalCer / Daunorubicin+α-GalCer) in the C1498 homograft tumor C57BL / 6 immunocompetent mouse leukemia model (n=3).

[0013] Figure 6:2×10 6 The C1498 leukemia model was established by injecting murine acute myeloid leukemia cells (C1498) into C57BL / 6 mice via the tail vein. Seven days after injection, drug administration began: DNR at a dose of 2 mg / kg every two days, and α-GalCer at a dose of 10 μg / mouse every four days. Drug administration was stopped on day 21, and the mice were sacrificed for subsequent analysis.

[0014] Figure 7 Leukemia models in C57 mice were established by tail vein injection of C1498 and divided into the following groups: Normal (Control); Vehicle (AML model group); α-GalCer (AML model group treated with α-GalCer); DNR (AML model group treated with DNR); and DNR+α-GalCer (AML model group treated with both DNR and α-GalCer). White blood cell counts were compared between the groups.

[0015] Figure 8 : When the C57 mouse leukemia model was established by tail vein injection of C1498, the mice were killed on the 21st day and the changes in liver size in each group were measured.

[0016] Figure 9 : When the C57 mouse leukemia model was established by tail vein injection of C1498, the mice were killed on the 21st day and the changes in spleen size in each group were measured.

[0017] Figure 10 : In the experiment of establishing C57 mouse leukemia model by tail vein injection of C1498, the mice were killed on the 21st day and the changes of liver weight in each group were measured.

[0018] Figure 11 : When the C57 mouse leukemia model was established by tail vein injection of C1498, the mice were killed on the 21st day and the changes in spleen weight in each group were measured.

[0019] Note: Figures 2 to 5 In the figure, the black line represents the saline group, the red line represents the solvent control group, the green line represents the α-GalCer group, the yellow line represents the DNR group, and the blue line represents the DNR+α-GalCer group. DETAILED DESCRIPTION

[0020] Experimental materials involved in this invention: 1. Cell line: Murine acute myeloid leukemia cell line C1498 was purchased from ATCC cell bank.

[0021] 2. Experimental animals: SPF (special pathogen free) - level female C57BL / 6 mice, 6 - 8 weeks old, weighing about 18 - 20 g, were purchased from Shanghai Slack Experimental Animal Co., Ltd., with the license number: SCXK(Shanghai)2017 - 0005. They were housed in the SPF barrier of the Experimental Animal Center of Fujian Medical University, and the license number of the animal experiment facility is SYXK(Fujian)2016 - 0007. All animals were adaptively housed for one week before the experiment. The light - dark cycle of the housing environment was 12 / 12 h (light time: 08:00 - 20:00), with a constant temperature of (23 ± 2)°C and a constant humidity of (50 ± 5)%. Six mice were housed in each cage, with free access to water and food. The animal experiments of this project were approved by the Experimental Animal Ethics Committee of Fujian Medical University (No.: IACUC FJMU 2023 - 0111) and were conducted in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments guidelines) operating guidelines.

[0022] 3. Experimental reagents: In this invention, i.P. refers to intraperitoneal injection.

[0023] Examples: 1. Cultivation of cell lines: The murine acute myeloid leukemia cell line C1498 was routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin, and was placed in an incubator at 37°C and 5% CO2 with saturated humidity for cultivation.

[0024] 2. Experiment on the C57BL / 6 immunocompetent mouse leukemia model with C1498 allograft tumors: A cell suspension of 2 million C1498 cells was inoculated subcutaneously into the right upper limb of 6 C57BL / 6 mice (referred to as the first generation). When the tumor mass grew to 1000 mm 3 at that time, the first passage was carried out: 2 - 3 mm 3 tumor masses from the first - generation tumor - bearing mice were respectively inoculated subcutaneously into 50 C57BL / 6 mice (referred to as the second - generation mice). When the tumor masses of the mice grew to 100 mm 3Tumor-bearing mice were randomly divided into five groups, each with 10 mice: a saline group (0.9% sodium chloride injection, iP), a solvent control group (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, iP), an α-GalCer group (injection concentration of 0.1 mg / mL, iP), a DNR group (injection concentration of 0.5 mg / mL, 2 mg / kg, iP), and a DNR (injection concentration of 0.5 mg / mL, iP) + α-GalCer group (injection concentration of 0.1 mg / mL, iP). Each group received different drug treatments every two days (DNR at a dose of 2 mg / kg every two days, and α-GalCer at a dose of 10 μg / mouse every four days). Tumor length and width were measured every two days, and tumor volume was calculated using the formula. The survival time was analyzed using the Kaplan-Meier method and the tumor growth curve and mouse survival curve were plotted using GraphPad Prism software. 2 × (length)] / 2. Changes in mouse body weight were assessed to determine whether the drug caused systemic toxicity.

[0025] 3. Experiment on establishing C57 mouse leukemia model by tail vein injection of C1498: (1) Take out the cultured C1498 mouse leukemia cells from the incubator and count them using a cell counting instrument. The number of cells must reach 2×10 6 Transfer the cells to a 50 mL tube and centrifuge at 300 g for 10 minutes.

[0026] (2) Wash twice with PBS, resuspend and place on ice.

[0027] (3) C57BL / 6 mice were placed in a restrainer and injected into the tail vein under sterile conditions in a laminar flow operating table.

[0028] (4) Grasp the distal end of the mouse tail and disinfect it with 70% ethanol gauze. Check for bubbles in the syringe and then slowly inject 100 μL of C1498 cell suspension (approximately 2×10 6 cells) were injected into the tail vein.

[0029] (5) After injection, disinfect the injection site with a cotton ball soaked in 70% ethanol and apply pressure with a sterile gauze sponge to control bleeding. Return the mice to their cages and carefully monitor their diet and health status over the next few days.

[0030] (6) One week after inoculation of C1498 cells into the tail vein, the AML model was randomly divided into 4 groups, with 6 mice in each group, and treated with the following drugs: Vehicle (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, iP); α-GalCer (injection concentration 0.1 mg / mL, iP); DNR (injection concentration 0.5 mg / mL, iP); DNR (injection concentration 0.5 mg / mL, iP) + α-GalCer (injection concentration 0.1 mg / mL, iP). DNR was administered at a dose of 2 mg / kg every 2 days, and α-GalCer was administered at a dose of 10 μg / mouse every 4 days. The drug administration was stopped on day 21, and the mice were sacrificed for subsequent analysis.

[0031] (7) On the 21st day, the number of white blood cells in the mice was detected by white blood cell counting; the behavior, weight, and fur of the mice were observed to see if there were any leukemia-like signs; after the mice were killed, the size and weight of the liver and spleen of the mice were dissected and compared.

[0032] Experimental test results characterization: 1. C1498 homograft tumor leukemia model experiment in C57BL / 6 immune-competent mice: The present invention constructs a C1498 homologous transplant tumor C57BL / 6 immune-competent mouse leukemia model to verify whether DNA damage combined with α-GalCer has a synergistic anti-tumor effect in vivo. The tumors in the control group of tumor-bearing mice grew rapidly. DNR or α-GalCer monotherapy also had a certain inhibitory effect on tumor growth, while the combined treatment of DNR and α-GalCer significantly inhibited tumor growth and reduced tumor weight, with a tumor inhibition rate of 67.4%, and a significant tumor inhibition effect. The weight of the mice was monitored during the treatment process. By evaluating the changes in the weight of the mice, it was found that the weight of the mice was relatively stable during the treatment process, indicating that the DNR and α-GalCer combination group did not cause obvious systemic toxicity. The Kaplan-Meier method was used to analyze the survival time of mice in each treatment group (n=3). The results represent the mean ± SD of three independent experiments (*P<0.05, **P<0.01, ***P<0.001). The results showed that the survival time of the DNR+α-GalCer group was prolonged compared with the mice in other groups.

[0033] 2. Tail vein injection of C57 mouse leukemia model: To further evaluate the efficacy of the combined treatment with DNR and α-GalCer, we established a C1498 leukemia model by injecting C1498 leukemia cells into the tail vein of C57BL / 6 mice to mimic the clinical pathogenesis of AML. One week after inoculation of C1498 cells, the AML model group was randomly divided into four groups of six mice each and treated with the corresponding drug concentrations. White blood cell counts on day 21 revealed that combined treatment with DNR and α-GalCer significantly inhibited leukocyte proliferation in AML mice. Compared with the AML model group receiving saline alone, the mice in the combined treatment group were more active, had stable weights, smooth fur, and showed no obvious leukemia-like signs. Further analysis of the mice after treatment revealed that the liver and spleen of the AML model group receiving saline alone were significantly enlarged compared to the normal group. However, hepatosplenomegaly was significantly reduced in all treatment groups, particularly in the DNR + α-GalCer group, and was comparable to that of the normal group. In addition, the liver and spleen weights of the DNR and α-GalCer combination group were significantly lower than those in the AML model group.

[0034] The above data indicate that DNA damage-activated NKT cell activity has a good anti-tumor effect in vivo, and combined treatment with DNR and α-GalCer is beneficial for alleviating the progression of AML.

Claims

1. Use of daunorubicin in combination with α-galactosylceramide in the preparation of an anti-leukemia pharmaceutical composition.

2. The use according to claim 1, characterized in that: The cells causing the leukemia are C1498, HL-60, THP-1, KG-1, Jurkat, REH, Nalm-6, K562, KBM-7, MEC-1, JVM-3, U937 or HEL.

3. The use according to claim 1, characterized in that: The anti-leukemia pharmaceutical composition comprises daunorubicin, alpha-galactosylceramide and pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The dosage form of the anti-leukemia pharmaceutical composition is injection, tablet, capsule, granule, powder, patch, suspension, syrup, oral solution, suppository or any combination thereof.

5. The use according to claim 4, characterized in that: The administration route of the injection is intraperitoneal injection.

6. An anti-leukemia pharmaceutical composition based on DNA damage-activated NKT cells, characterized by: The anti-leukemia pharmaceutical composition comprises daunorubicin, alpha-galactosylceramide and pharmaceutically acceptable excipients.

7. The anti-leukemia pharmaceutical composition according to claim 6, characterized in that: The cells causing the leukemia are C1498, HL-60, THP-1, KG-1, Jurkat, REH, Nalm-6, K562, KBM-7, MEC-1, JVM-3, U937 or HEL.

8. The anti-leukemia pharmaceutical composition according to claim 6, characterized in that: The dosage form of the anti-leukemia pharmaceutical composition is injection, tablet, capsule, granule, powder, patch, suspension, syrup, oral solution, suppository or any combination thereof.

9. The anti-leukemia pharmaceutical composition according to claim 8, characterized in that: The administration route of the injection is intraperitoneal injection.

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