Application of poison carotene and doxorubicin in combined treatment of leukemia

Abstract

The invention relates to a pharmaceutical composition containing poison carotene and doxorubicin for treating leukemia, and also provides application of the pharmaceutical composition containing poison carotene and doxorubicin to preparation of a medicine for treating leukemia, and the leukemia is preferably leukemia with resistance to Vickers. The pharmaceutical composition can synergistically kill leukemia cells and promote apoptosis, has low cytotoxicity, and can significantly reduce expression of Venokra drug resistance related proteins.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to the application of a combination of toxic carotene and doxorubicin in the treatment of leukemia. BACKGROUND

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, with acute onset and poor prognosis. The complete remission rate of patients with medium / high-risk cytogenetic or molecular abnormalities (such as FLT3-ITD, TP53 mutation, complex karyotype) is only 50%-60%, the relapse rate within 2 years is more than 60%, and the 5-year overall survival rate is less than 30% (Shimony, S. et al. Acute Myeloid Leukemia: 2025 Update on Diagnosis, Risk-Stratification, and Management. American journal of hematology, 100(5), p860-891; National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Acute Myeloid Leukemia. Version 1.2025. Plymouth Meeting (PA): National Comprehensive Cancer Network; 2025 [cited 2026 Feb 3]. Available from: https: / / www.nccn.org). Chemotherapy is the main treatment for AML, but patients often relapse and die due to chemotherapy resistance. Patients with primary drug resistance cannot achieve remission through chemotherapy and lose the opportunity for bone marrow transplantation, with a four-year survival rate of only 23% (Othus M et al. Fate of patients with newly diagnosed acute myeloid leukemia who fail primary induction therapy. Biol Blood Marrow Transplant. 2015 Mar; 21(3):559-64.). Therefore, it is urgent to develop a sensitization regimen of chemotherapeutic drugs to reverse chemotherapy resistance.

[0003] The standard induction therapy for AML adopts the "7+3" regimen, that is, intravenous infusion of cytarabine for 7 consecutive days combined with 3 days of anthracycline drugs. Doxorubicin (DOX) is a representative core drug of anthracycline drugs, which exerts its effect on killing leukemia cells by DNA intercalation, topoisomerase II inhibition and active oxygen generation induced double-strand break. However, the drug has high cardiotoxicity (Swain, S. M. et al. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer, 97(11), 2869-2879) and serious drug resistance problem. Venetoclax (VEN) is a selective BCL2 inhibitor, which has become a standard regimen for patients over 65 years old or not suitable for intensive chemotherapy when combined with demethylation drugs such as azacitidine, decitabine or low-dose cytarabine. Since the launch of Venetoclax in 2020, the remission rate of patients has increased from 28% to 66%, and the median survival time has increased from 9.6 months to 14.7 months (DiNardo, C. D. et al. Azacitidine and Venetoclax in Previously Untreated Acute Myeloid Leukemia. N Engl J Med. 2020 Aug 13;383(7):617-629.).

[0004] However, VEN also cannot escape the fate of drug resistance, and the serious drug resistance problem of patients is increasingly prominent. At present, the effective rate of VEN for refractory and relapsed AML is only 35.7%, and the median survival is shortened to 4.7 months (Gaut, D. et al. Venetoclax combination therapy in relapsed / refractory acute myeloid leukemia: A single institution experience. Leuk Res. 2020 Mar;90:106314.). The situation that patients cannot tolerate high-dose VEN is also highlighted in clinical practice, therefore, it is crucial to develop new chemotherapy sensitizers or combination regimens, and reduce the toxicity of existing regimens to improve the efficacy of AML treatment.

[0005] Thapsigargin (TG) is a highly specific endoplasmic reticulum calcium pump inhibitor. It induces tumor cell apoptosis by disrupting cellular calcium homeostasis, and shows potential to overcome drug resistance in tumor treatment research.

[0006] In T-cell acute lymphoblastic leukemia model, TG monotherapy has significant anti-T-ALL activity (Roti, G. et al. New Approaches to Target T-ALL. Front Oncol. 2014 Jul 8;4: 170.). But some studies found that TG can inhibit DOX-induced apoptosis in P53-deficient M1 cells, suggesting that it has a cytoprotective effect under certain conditions (Lotem, J. et al. Different mechanisms for suppression of apoptosis by cytokines and calcium mobilizing compounds. Proceedings of the National Academy of Sciences of the United States of America, 95(8), 4601-4606). The above contradictory results show that the biological effects of TG are complex, and may be regulated by disease type, drug concentration and combined drug mechanism.

[0007] The existing researches have confirmed that TG has clear sensitization potential for AML under reasonable combined drug regimen: TG combined with cyclin-dependent kinase inhibitor can block cell cycle and enhance the killing effect on myeloid leukemia U937 cells (Nguyen, T. K. et al., Dinaciclib (SCH727965) inhibits the unfolded protein response through a CDK1- and 5-dependent mechanism. Molecular cancer therapeutics, 13(3), 662-674.). TG combined with all-trans retinoic acid can enhance the inhibitory effect on HL60 cells, and all-trans retinoic acid induces cell differentiation as the core mechanism (Schmidt-Mende, J. et al., Early mitochondrial alterations in ATRA-induced cell death. Cell death and differentiation, 13(1), 119-128.); TG combined with imatinib can improve the efficacy of chronic myeloid leukemia, and imatinib is a tyrosine kinase inhibitor targeting BCR::ABL fusion protein; TG combined with tumor necrosis factor-related apoptosis-inducing ligand (TNF-Related Apoptosis-Inducing Ligand, TRAIL) can also sensitize Philadelphia chromosome-positive leukemia cells to apoptosis, and TRAIL activates the death receptor pathway to initiate exogenous apoptosis (Zhang, X. et al., Endoplasmic reticulum stress inducers, but not imatinib, sensitize Philadelphia chromosome-positive leukemia cells to TRAIL-mediated apoptosis. Leukemia research, 35(7), 940-949.).

[0008] Therefore, it is an urgent clinical key problem to use TG to sensitize the core drugs VEN and DOX for AML, especially to overcome VEN resistance. Reverification of the sensitization effect of TG on DOX can respond to the contradictions in previous studies; and the sensitization research of TG on VEN and drug-resistant strains is still blank. The present study intends to explore the combined regimen in order to enhance the effect and reduce the toxicity, and to provide a new treatment strategy for AML drug-resistant patients. SUMMARY

[0009] The purpose of the present application is to overcome the above-mentioned defects of the prior art, and the following technical solutions are provided.

[0010] According to a first aspect of the present application, there is provided a pharmaceutical composition for treating leukemia, the pharmaceutical composition comprising a toxic carotene and doxorubicin. Preferably, the molar concentration ratio of the toxic carotene to doxorubicin is 1:0.5-20; more preferably, the molar concentration ratio of the toxic carotene to doxorubicin is 1:1-10; more preferably, the molar concentration ratio of the toxic carotene to doxorubicin is 1:5.

[0011] Further, the content of the toxic carotene in the pharmaceutical composition is 0.025-0.1 μM; preferably, 0.025-0.04 μM, 0.04-0.05 μM or 0.05-0.1 μM; more preferably, 0.025 μM, 0.040 μM, 0.050 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.10 μM; more preferably, 0.04 μM.

[0012] Further, the content of the doxorubicin in the pharmaceutical composition is 0.025-1.0 μM; preferably, 0.05-0.4 μM, 0.025-0.50 μM, 0.025-0.1 μM or 0.05-1.0 μM, more preferably, 0.2-0.4 μM; more preferably, 0.025 μM, 0.05 μM, 0.075 μM, 0.10 μM, 0.20 μM, 0.25 μM, 0.30 μM, 0.40 μM, 0.50 μM, 0.60 μM, 0.70 μM, 0.75 μM, 0.80 μM, 0.90 μM, 1.0 μM; more preferably, 0.20 μM.

[0013] Further, the content of the toxic carotene in the pharmaceutical composition is 0.025 μM-0.1 μM; and / or the content of the doxorubicin in the pharmaceutical composition is 0.025 μM-1 μM.

[0014] Further, the content of the toxic carotene in the pharmaceutical composition is 0.04 μM-0.05 μM; and / or the content of the doxorubicin in the pharmaceutical composition is 0.05 μM-0.4 μM, preferably, the content of the doxorubicin in the pharmaceutical composition is 0.2-0.4 μM.

[0015] Further, the content of the toxic carotene in the pharmaceutical composition is 0.025-0.04 μM; and / or the content of the doxorubicin in the pharmaceutical composition is 0.025-0.1 μM, preferably, the content of the doxorubicin in the pharmaceutical composition is 0.1 μM.

[0016] Further, the content of the toxicophore in the pharmaceutical composition is 0.05 μM-0.1 μM; and / or the content of the doxorubicin in the pharmaceutical composition is 0.05 μM-1.0 μM, preferably, the content of the doxorubicin in the pharmaceutical composition is 1.0 μM.

[0017] Further, the content of the toxicophore in the pharmaceutical composition is 0.04 μM, and the content of the doxorubicin in the pharmaceutical composition is 0.20 μM.

[0018] Further, the toxicophore and the doxorubicin in the pharmaceutical composition are sequentially administered or simultaneously administered.

[0019] According to another aspect of the present application, there is provided a use of any of the aforementioned pharmaceutical compositions in the preparation of a medicament for treating leukemia. Further, the toxicophore and the doxorubicin in the pharmaceutical composition are sequentially administered or simultaneously administered.

[0020] Further, the leukemia includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia and hairy cell leukemia; preferably, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia and hairy cell leukemia resistant to venetoclax; more preferably, the leukemia is acute myeloid leukemia; more preferably, the leukemia is acute myeloid leukemia resistant to venetoclax.

[0021] The present application has the following advantages:

[0022] 1. The pharmaceutical composition of the present application can synergistically kill leukemia cells and promote apoptosis, wherein the toxicophore significantly increases the killing ability of doxorubicin on leukemia cells, and the two have a significant synergistic effect.

[0023] 2. The pharmaceutical composition of the present application can kill leukemia cells, especially leukemia cells resistant to venetoclax, at a low cytotoxicity dose, which can significantly reduce the expression of venetoclax resistance-related proteins. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : Expression of anti-apoptotic protein MCL-1 in MOLM13-WT and MOLM13-R2.

[0025] Figure 2 : Synergistic effect of multiple doses of TG combined with DOX on MOLM13-R2 cell line.

[0026] Figure 3Sensitization effect of TG combined with DOX multi-dose in MOLM13-WT cell line.

[0027] Figure 4 Sensitization effect of TG combined with DOX multi-dose in U937 cell line.

[0028] Figure 5 DOX reduction required for 80% inhibition rate of MOLM13-R2 cells under different TG concentrations.

[0029] Figure 6 DOX reduction required for 90% inhibition rate of MOLM13-WT cells under different TG concentrations.

[0030] Figure 7 DOX reduction required for 80% inhibition rate of U937 cells under different TG concentrations.

[0031] Figure 8 HSA model synergy index of TG combined with DOX in MOLM13-R2 cell line.

[0032] Figure 9 HSA model synergy index of TG combined with DOX in MOLM13-WT cell line.

[0033] Figure 10 HSA model synergy index of TG combined with DOX in U937 cell line.

[0034] Figure 11 LDH release of MOLM13-R2 treated with different concentrations of TG for 72 h. Note: The significance symbols in the figure correspond to the comparison between each experimental group and the control group.

[0035] Figure 12 LDH release of MOLM13-WT treated with different concentrations of TG for 72 h. Note: The significance symbols in the figure correspond to the comparison between each experimental group and the control group.

[0036] Figure 13 LDH release of U937 treated with different concentrations of TG for 72 h. Note: The significance symbols in the figure correspond to the comparison between each experimental group and the control group.

[0037] Figure 14 Effect of each dose of TG on the cell viability of HS-5 cell line. Note: The significance symbols in the figure correspond to the comparison between each experimental group and the control group. DETAILED DESCRIPTION

[0038] The application will be further described in details by specific examples below, but the scope of protection of the application is not limited thereto. Unless otherwise specified, the reagents, methods and devices used in the application are the conventional reagents, methods and devices in the technical field. Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial channels.

[0039] Example 1 Cell culture and VEN-resistant strain induction and maintenance

[0040] The resistant cell strain was constructed by concentration gradient induction method: the IC 50 of VEN to the parent strain MOLM13-WT was used as the starting induction concentration, and after 24 hours of continuous treatment, the drug-containing medium was centrifuged and discarded, and the cells were cultured in regular complete medium until stable proliferation, and then the drug concentration was gradually increased to obtain the stable resistant strain MOLM13-R2. The resistance level was quantitatively evaluated by the resistance index (RI = resistant strain IC 50 / parent strain IC 50 ).

[0041] The IC 50 of MOLM13-R2 to VEN was detected before the experiment to verify the stability of the resistance. The cells were treated in two groups, one group was maintained in a low concentration of VEN to maintain the resistance phenotype, and the other group was drug-free for 3 days to eliminate the short-term effect of the drug.

[0042] The U937 cell line, MOLM13-WT cell line, VEN-resistant strain MOLM13-R2 induced from MOLM13-WT, and HS-5 cell line (human bone marrow stromal cell line) were all placed in RPMI-1640 medium containing 10% fetal bovine serum and cultured in a cell incubator at 37°C and 5% CO2, and the cells in the logarithmic growth phase were used for subsequent experiments.

[0043] The results showed that the IC 50 value of MOLM13-R2 was 12.92 μM, which was significantly higher than that of MOLM13-WT, 22.87 times that of MOLM13-WT (Table 1). The Western Blot results showed that the expression of VEN resistance marker anti-apoptotic protein MCL-1 in MOLM13-R2 was significantly higher than that in MOLM13-WT, indicating that MOLM13-R2 had strong resistance to VEN ( Figure 1 ).

[0044] Table 1 Analysis of VEN resistance of cell lines

[0045]

[0046] Note: IC 50 Data are expressed as mean ± SD (n = 3). The resistance multiple was calculated from the average IC 50 .

[0047] Example 2 Cell viability assay

[0048] MOLM13-WT, MOLM13-R2, U937 cells were seeded at 8000 cells per well in 96-well plates, with 3 replicates per group. TG, DOX single agent or combination were added, with final drug concentrations as shown in Table 2. Negative control group was added with PBS equal to the volume of drug, and blank control group was added with medium equal to the volume of cell suspension. Cells were incubated in 37°C incubator for 72 hours. After incubation, MTS reagent (Promega, G3581) was used to detect cell relative viability. After 2.5 hours incubation at 37°C in dark, absorbance at 490 nm was measured using a microplate reader, and inhibition rate was calculated.

[0049]

[0050] Example 3 Cell apoptosis assay

[0051] Annexin V-FITC / PI double staining method (Link Biological, AP101) was used to detect cell apoptosis according to the instructions. First, cells were collected and resuspended in 400 μL of 1x binding buffer. 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube, and incubated at room temperature for 5 minutes in the dark. Finally, the proportion of apoptosis was detected by flow cytometry. FlowJo was used to analyze the flow cytometry results.

[0052] Example 4 Cell cytotoxicity assay

[0053] MOLM13-WT cells and its drug-resistant strain MOLM13-R2 in logarithmic growth phase were seeded at 8000 cells per well in 96-well plates, with 3 replicates per group. TG was added in complete medium gradient dilution, and incubated for 72 hours. After incubation, lactate dehydrogenase (LDH) detection kit (Tongren Chemical, CK12) was used to detect cell cytotoxicity. 1x LDH detection working solution was added to each well according to the instructions, and incubated at room temperature for 30 minutes in the dark. Stop solution was added immediately, and the absorbance value was measured at 490 nm using a microplate reader to calculate the cytotoxicity.

[0054] Example 5 Western blot

[0055] The protein sample was the supernatant obtained by centrifugation. The protein concentration of each group was determined by BCA protein detection kit. After denaturation, SDS-PAGE was used to separate the protein. After electrophoresis, the PVDF membrane was transferred. 5% skim milk TBST solution was used for room temperature blocking for 1 hour. According to the antibody instructions, the primary antibody was diluted to the appropriate concentration, and incubated at 4°C overnight, and washed with TBST for three times. According to the species of the primary antibody, the corresponding secondary antibody was added after dilution, and incubated at room temperature for 1 hour, and washed with TBST for three times. Subsequently, the membrane was treated with ECL luminescent liquid, and the chemiluminescence imaging instrument was used for exposure and photography to record the results. Image Lab was used to analyze the protein immunoblotting image.

[0056] Example 6 Statistical analysis

[0057] The data is expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 9.5, including one-way ANOVA and two-way ANOVA. A p-value less than 0.05 was considered statistically significant. Asterisks (*), (**), (***), and (****) represent p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. All results are from three independent repeats.

[0058] Example 7 TG significantly increases the killing ability of DOX on leukemia cells

[0059] TG combined with DOX showed stronger cell inhibition than single drug: in MOLM13-R2 cell line, 0.04-0.05 μM TG combined with 0.05-0.4 μM DOX can achieve synergistic effect, and when 0.04-0.05 μM TG combined with 0.25-0.4 μM DOX, the cell inhibition rate can reach 80% or more; in MOLM13-WT cell line, 0.025-0.04 μM TG combined with 0.025-0.1 μM DOX can achieve synergistic effect, and when 0.025-0.04 μM TG combined with 0.1 μM DOX, the cell inhibition rate can reach more than 60%; in U937 cell line, 0.05-0.1 μM TG combined with 0.05-1.0 μM DOX can achieve synergistic effect, and when 0.05-0.1 μM TG combined with 1.0 μM DOX, the cell inhibition rate can reach more than 80% ( Figures 2-4 ). When the cell inhibition rate reaches 80%, in MOLM13-R2, 0.2 μM DOX combined with 0.04 μM TG can achieve 50% drug reduction of DOX ( Figure 5); 0.2 μM DOX and 0.025 μM TG combination can reach 20% of DOX dose reduction in MOLM13-WT when the cell inhibition rate reaches 90% ( Figure 6 ); 1 μM DOX and 0.05 μM TG combination can reach 50% of DOX dose reduction in U937 when the cell inhibition rate reaches 80% ( Figure 7 ). In the three cell lines, HSA model analysis, TG combined with DOX treatment showed positive synergistic effect ( Figures 8-10 ).

[0060] Example 8 TG single drug cytotoxicity is low

[0061] Based on the foregoing determined TG concentration for use in three groups of tumor cells, the following results show that the TG single drug concentration does not reach the tumor cell killing concentration, and also has no destructive effect on normal stromal cells. The LDH experiment results show that in MOLM13-R2, the LDH release amount is not significantly statistically different compared with the non-drug group when treated with TG at 0.04 μM and 0.05 μM concentrations for 72 hours ( Figure 11 ). In MOLM13-WT and U937 cells, the respective IC10concentration of TG does not cause significant changes in LDH release; at the respective IC30concentration, the LDH release values of the two cells are higher than the non-drug group ( Figures 12-13 ). The MTS experiment results show that TG does not show significant cell viability inhibition at these sensitizing concentrations for 72 hours of treatment ( Figure 14 ).

[0062] The foregoing is a further detailed description of the present application in combination with specific preferred embodiments, which cannot be construed as limiting the specific embodiments of the present application to the foregoing. For those of ordinary skill in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can also be made, which should be considered to fall within the scope of protection determined by the claims submitted.

Claims

1. A pharmaceutical composition for treating leukemia, characterized in that: The pharmaceutical composition comprises carotenoids and doxorubicin, wherein the molar ratio of carotenoids to doxorubicin is 1:0.5-20.

2. The pharmaceutical composition according to claim 1, characterized in that: The molar ratio of the toxic carotenoid to doxorubicin is 1:1-10.

3. The pharmaceutical composition according to claim 2, characterized in that: The molar ratio of the toxic carotenoid to doxorubicin is 1:

5.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that: The content of the carotenoid is 0.025-0.1 μM; and / or the content of the doxorubicin is 0.025-1 μM.

5. The pharmaceutical composition according to claim 4, characterized in that: The content of the carotenoid is 0.04-0.05 μM; and / or the content of the doxorubicin is 0.05-0.4 μM.

6. The pharmaceutical composition according to claim 4, characterized in that: The content of the carotenoid is 0.05-0.1 μM; and / or the content of the doxorubicin is 0.05-1.0 μM.

7. The pharmaceutical composition according to claim 4, characterized in that: The content of the carotenoid is 0.04 μM; and / or the content of the doxorubicin is 0.2 μM.

8. Use of the pharmaceutical composition according to any one of claims 1-7 in the preparation of a medicament for treating leukemia.

9. The use according to claim 8, characterized in that: The leukemias mentioned include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.

10. The use according to claim 9, characterized in that: The leukemia mentioned refers to acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia that are resistant to Venecra.

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