Application of combined treatment of leukemia by using carotene and vinca

Abstract

The invention relates to a pharmaceutical composition which is used for treating leukemia and contains poison carotene and vinca, and also provides application of the pharmaceutical composition containing poison carotene and vinca to preparation of a medicine for treating leukemia, and the leukemia is preferably leukemia with resistance to vinca. 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] This invention belongs to the field of biomedical technology, specifically relating to the application of combined treatment of leukemia with beta-carotene and veneclade. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, characterized by rapid onset and poor prognosis. Patients with intermediate / high-risk cytogenetic or molecular abnormalities (such as FLT3-ITD, TP53 mutation, or complex karyotype) have a complete remission rate of only 50%–60%, a relapse rate exceeding 60% within 2 years, and a 5-year overall survival rate of 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 primary treatment for AML, but patients often relapse and die due to chemotherapy resistance. Patients with primary drug resistance cannot achieve remission through chemotherapy, thus losing 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, developing sensitization regimens for existing chemotherapy drugs to reverse chemotherapy resistance is urgently needed.

[0003] The standard induction therapy for AML uses a "7+3" regimen, which involves 7 consecutive days of intravenous infusion of cytarabine combined with 3 days of anthracycline drugs. Doxorubicin (DOX) is a representative core drug among anthracyclines, which kills leukemia cells by inducing double-strand breaks through DNA intercalation, topoisomerase II inhibition, and reactive oxygen species generation. However, this drug has high cardiotoxicity (Swain, SM et al., Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer, 97(11), 2869-2879), and drug resistance is a serious problem. Venetoclax (VEN) is a selective BCL2 inhibitor, and its combination with demethylating drugs such as azacitidine, decitabine, or low-dose cytarabine has become the standard regimen for patients over 65 years of age or who are not suitable for intensive chemotherapy. After veneclax was launched in 2020, the remission rate of patients increased from 28% to 66%, and the median survival increased from 9.6 months to 14.7 months (DiNardo, CD 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 is also struggling with drug resistance, and the severity of this problem is becoming increasingly apparent among patients. Currently, VEN has an efficacy rate of only 35.7% in treating refractory relapsed AML, with a median survival of only 4.7 months (Gaut, D. et al., Venetoclax combination therapy in relapsed / refractory acute myeloid leukemia: A singleinstitution experience. Leuk Res. 2020 Mar;90:106314.). The inability of patients to tolerate high-dose VEN is also becoming increasingly prominent in clinical practice. Therefore, developing new chemotherapy sensitizers or combination regimens and reducing the toxicity of existing regimens are crucial for improving the efficacy of AML treatment.

[0005] Thapsigargin (TG) is a highly specific endoplasmic reticulum calcium pump inhibitor. It induces apoptosis in tumor cells by disrupting cellular calcium homeostasis, demonstrating potential to overcome drug resistance in tumor treatment research. In a T-cell acute lymphoblastic leukemia model, TG monotherapy exhibits significant anti-T-ALL activity (Roti, G. et al., New Approaches to Target T-ALL. Front Oncol. 2014 Jul 8;4:170.). However, studies have found that TG can inhibit apoptosis induced by DOX in p53-deficient M1 cells, suggesting a cytoprotective effect under specific conditions (Lotem, J. et al., Different mechanisms for suppression of apoptosis by cytokines and calciummobilizing compounds. Proceedings of the National Academy of Sciences of the United States of America, 95(8), 4601-4606). These contradictory results indicate that the biological effects of TG are complex and may be jointly regulated by disease type, drug concentration, and the mechanisms of combination drugs.

[0006] Existing research has confirmed that TG has a clear potential to enhance the sensitization of AML under reasonable combination therapy: TG combined with cyclin-dependent kinase inhibitors can enhance the killing effect on myeloid leukemia U937 cells by arresting the cell cycle (Nguyen, TK et al., Dinaciclib (SCH727965) inhibits the unfolded protein response through a CDK1- and 5-dependent mechanism. Molecular cancertherapeutics, 13(3), 662-674.). The combination of TG and all-trans retinoic acid (ATA) can enhance the inhibitory effect on HL60 cells. ATA's core mechanism is the induction of cell differentiation (Schmidt-Mende, J. et al., Early mitochondrial alterations in ATRA-induced cell death. Cell death and differentiation, 13(1), 119-128.). The combination of TG and imatinib can improve the efficacy of chronic myeloid leukemia treatment. Imatinib is a tyrosine kinase inhibitor targeting the BCR::ABL fusion protein. The combination of TG and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can also sensitize apoptosis in Philadelphia chromosome-positive leukemia cells. TRAIL initiates extrinsic apoptosis by activating the death receptor pathway (Zhang, X. et al., Endoplasmic reticulum stress inducers, but not imatinib, sensitize Philadelphia chromosome-positive leukemia cells to TRAIL-mediated apoptosis. Leukemiaresearch, 35(7). 940-949.

[0007] The combination of TG and VEN has been preliminarily explored in multiple myeloma (MM) (Gomez-Bougie, P. et al., Repression of Mcl-1 and disruption of the Mcl-1 / Bak interaction in myeloma cells couple ER stress to mitochondrial apoptosis. Cancer Letters, 383(2), 204-211.), but the core therapeutic targets and pathological features of the two diseases are fundamentally different. MM treatment focuses on proteasome inhibitors, immunomodulators, and anti-CD38 monoclonal antibodies, targeting plasma cell protein metabolism and the immune microenvironment; AML focuses on myeloid progenitor cell proliferation, differentiation, and apoptosis pathways. Therefore, the synergistic effect observed in MM cannot be directly used as evidence for the efficacy of TG in AML.

[0008] Therefore, using TG to sensitize AML core drugs VEN and DOX, especially to overcome VEN resistance, is a critical clinical issue that urgently needs to be addressed. Re-validating the sensitizing effect of TG on DOX can resolve contradictions in previous studies; however, research on its sensitizing effect on VEN and resistant strains remains lacking. This study aims to explore combination regimens to enhance efficacy and reduce toxicity, providing a new treatment strategy for AML drug-resistant patients. Summary of the Invention

[0009] The purpose of this invention is to overcome the above-mentioned defects of the prior art and to provide the following technical solution.

[0010] According to a first aspect of the present invention, a pharmaceutical composition for treating leukemia is provided, the pharmaceutical composition comprising carotenoid and veneclade, wherein the molar concentration ratio of carotenoid to veneclade is 1:0.25-1600; preferably, the molar concentration ratio of carotenoid to veneclade is 1:20-1000; more preferably, the molar concentration ratio of carotenoid to veneclade is 1:100.

[0011] Furthermore, the content of carotene in the pharmaceutical composition is 0.025-0.1 μM; preferably, it is 0.025-0.04 μM, 0.04-0.05 μM or 0.05-0.1 μM; more preferably, it is 0.025 μM, 0.040 μM, 0.050 μM or 0.100 μM; even more preferably, it is 0.05 μM.

[0012] Furthermore, the content of veneclade in the pharmaceutical composition is 0.01-80 μM; preferably, it is 0.01-40 μM, 0.01-5 μM, 2-5 μM, 0.1-40 μM, 5-40 μM, 10-40 μM, 5-80 μM, or 50-80 μM; more preferably, it is 0.01 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 25 μM, 40 μM, 50 μM, or 80 μM; even more preferably, it is 5 μM.

[0013] Furthermore, the content of carotenoids in the pharmaceutical composition is 0.025-0.1 μM; and / or the content of veneclade in the pharmaceutical composition is 0.01-80 μM.

[0014] Furthermore, the content of carotene in the pharmaceutical composition is 0.04-0.05 μM; and / or the content of veneclade in the pharmaceutical composition is 0.1-40 μM, preferably, the content of veneclade in the pharmaceutical composition is 10-40 μM.

[0015] Furthermore, the content of carotene in the pharmaceutical composition is 0.025-0.04 μM; and / or the content of veneclade in the pharmaceutical composition is 0.01-5 μM, preferably, the content of veneclade in the pharmaceutical composition is 2-5 μM.

[0016] Furthermore, the content of carotene in the pharmaceutical composition is 0.05-0.1 μM; and / or the content of veneclade in the pharmaceutical composition is 5-80 μM, preferably, the content of veneclade in the pharmaceutical composition is 50-80 μM.

[0017] Furthermore, the content of carotenoids in the pharmaceutical composition is 0.05 μM; and / or the content of veneclade in the pharmaceutical composition is 5 μM.

[0018] Furthermore, the carotenoid and veneclade in the pharmaceutical composition are administered sequentially or simultaneously.

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

[0020] Furthermore, the leukemia includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia; preferably, the leukemia is an acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia resistant to Venecella; more preferably, the leukemia is acute myeloid leukemia; even more preferably, the leukemia is an acute myeloid leukemia resistant to Venecella.

[0021] The present invention has the following beneficial effects:

[0022] 1. The pharmaceutical composition of the present invention can synergistically kill leukemia cells and promote cell apoptosis, wherein carotenoid significantly increases the killing ability of Venecra in leukemia cells, and the two have a significant synergistic effect.

[0023] 2. The pharmaceutical composition of the present invention can kill leukemia cells at a low cytotoxic dose, achieving up to 90% reduction in the dosage of Venecella, especially in leukemia cells resistant to Venecella, and can significantly reduce the expression of Venecella resistance-related proteins. Attached Figure Description

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

[0025] Figure 2 Sensitizing effect of a single dose of TG and VEN combination therapy in the MOLM13-R2 cell line. Note: Significance symbols in the figure correspond to the comparison between the single-drug group and the combination group.

[0026] Figure 3 Sensitizing effect of a single dose of TG and VEN combination therapy in the MOLM13-WT cell line. Note: Significance symbols in the figure correspond to comparisons between the single-drug group and the combination group.

[0027] Figure 4 The sensitizing effect of a single dose of TG and VEN in the U937 cell line. Note: Significance symbols in the figure correspond to the comparison between the single-drug group and the combination group.

[0028] Figure 5 The sensitizing effect of multiple doses of TG combined with VEN in the MOLM13-R2 cell line.

[0029] Figure 6 The sensitizing effect of multiple doses of TG combined with VEN in the MOLM13-WT cell line.

[0030] Figure 7The sensitizing effect of multiple doses of TG and VEN combined in the U937 cell line.

[0031] Figure 8 The VEN reduction required to achieve 80% inhibition rate in MOLM13-R2 cells under different TG concentrations.

[0032] Figure 9 The VEN reduction required to achieve 90% inhibition rate in MOLM13-WT cells at different TG concentrations.

[0033] Figure 10 The VEN reduction required to achieve a 70% inhibition rate in U937 cells at different TG concentrations.

[0034] Figure 11 Synergistic index of HSA model in MOLM13-R2 cell line when TG and VEN are used in combination.

[0035] Figure 12 Synergistic index of TG and VEN combined therapy in HSA model of MOLM13-WT cell line

[0036] Figure 13 Synergistic index of HSA model in U937 cell line when TG and VEN are used in combination.

[0037] Figure 14 Apoptosis flow cytometry of TG and VEN alone and in combination in MOLM13-R2 cell line.

[0038] Figure 15 Statistical analysis of early and late apoptosis in MOLM13-R2 cell lines in each treatment group. Note: Drug concentrations in each group are consistent with Table 2 and... Figure 14 correspond.

[0039] Figure 16 Apoptosis flow cytometry of TG and VEN alone and in combination in MOLM13-WT cell line.

[0040] Figure 17 Statistical analysis of early and late apoptosis in MOLM13-WT cell lines in each treatment group. Note: Drug concentrations in each group are consistent with Table 2 and... Figure 16 correspond.

[0041] Figure 18 LDH release from MOLM13-R2 after treatment with different concentrations of TG for 72 h. Note: Significance symbols in the figure correspond to comparisons between experimental groups and control groups.

[0042] Figure 19LDH release from MOLM13-WT after treatment with different concentrations of TG for 72 h. Note: Significance symbols in the figure correspond to comparisons between experimental groups and the control group.

[0043] Figure 20 LDH release from U937 after treatment with different concentrations of TG for 72 h. Note: Significance symbols in the figure correspond to comparisons between experimental groups and control groups.

[0044] Figure 21 Effects of different doses of TG on the viability of HS-5 cell lines. Note: Significance symbols in the figure correspond to comparisons between experimental groups and control groups.

[0045] Figure 22 Expression of VEN-related resistance proteins in MOLM13-WT and MOLM13-R2 cell lines in each treatment group. Detailed Implementation

[0046] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0047] Example 1: Cell culture and induction and maintenance of VEN-resistant strains

[0048] Drug-resistant cell lines were constructed using a concentration gradient induction method: VEN was used to induce the IC50 expression of the parental cell line MOLM13-WT. 50 At the initial induction concentration, after 24 hours of continuous treatment, the drug-containing medium was discarded by centrifugation, and the cells were cultured in standard complete medium until stable proliferation was achieved. Subsequently, the drug concentration was gradually increased to obtain the stable drug-resistant strain MOLM13-R2. The drug resistance index (RI = IC50 of drug-resistant strain) was used as the criterion. 50 / parental strain IC 50 Quantitatively assess drug resistance levels.

[0049] Pre-experiment testing of MOLM13-R2's IC for VEN 50 To verify the stability of drug resistance, cells were divided into two groups: one group was treated with a low concentration of VEN to maintain the drug resistance phenotype, and the other group was cultured for 3 days after drug withdrawal to eliminate the short-term effects of the drug.

[0050] U937 cell line, MOLM13-WT cell line, MOLM13-R2 (a VEN-resistant strain induced by 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 passaged in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for subsequent experiments.

[0051] The results show that the MOLM13-R2 IC 50 The value was 12.92 μM, significantly higher than MOLM13-WT, and 22.87 times higher than MOLM13-WT (Table 1). Western blot results showed that the expression of the anti-apoptotic protein MCL-1, a marker of VEN resistance, was significantly higher in MOLM13-R2 than in MOLM13-WT, indicating that MOLM13-R2 has strong resistance to VEN. Figure 1 ).

[0052]

[0053] Note: IC 50 Data are represented as mean ± SD (n=3). Resistance folds are derived from the mean IC50. 50 The calculation yielded the result.

[0054] Example 2 Cell viability detection

[0055] MOLM13-WT, MOLM13-R2, and U937 cells were seeded at 8000 cells / well in 96-well plates, with 3 replicates per group. TG and VEN, either alone or in combination, were added; the final drug concentrations are shown in Table 2. The negative control group received an equal volume of PBS as the drug, while the blank control group received an equal volume of culture medium as the cell suspension. Cells were cultured at 37°C for 72 hours. After culture, relative cell viability was assessed using MTS reagent (Promega, G3581). Cells were then incubated at 37°C in the dark for 2.5 hours, and the absorbance at 490 nm was measured using a microplate reader to calculate the inhibition rate.

[0056]

[0057] Example 3 Apoptosis Detection

[0058] Apoptosis was detected using the Annexin V-FITC / PI double staining method (Linken Biotech, AP101) according to the manufacturer's instructions. First, cells were collected and resuspended in 400 μL of 1× binding buffer. 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube, and the cells were incubated at room temperature in the dark for 5 minutes. Finally, the apoptosis rate was detected by flow cytometry. FlowJo was used for flow cytometry analysis.

[0059] Example 4 Cytotoxicity Detection

[0060] Logarithmically growing MOLM13-WT cells and its drug-resistant strain MOLM13-R2 were seeded at 8000 cells / well in 96-well plates, with three replicates per group. The test drug TG was added in serially diluted complete culture medium, and the cells were incubated for 72 hours. After incubation, cytotoxicity was assessed using a lactate dehydrogenase (LDH) assay kit (Tongren Chemical, CK12). 1×LDH working solution prepared according to the manufacturer's instructions was added to each well, and the cells were incubated at room temperature in the dark for 30 minutes. Stop solution was then added, and the absorbance was immediately measured at 490 nm using a microplate reader to calculate cytotoxicity.

[0061] Example 5: Western blotting of proteins

[0062] Cell lysis buffer was added to each group of cells, and the protein sample was obtained as the supernatant after centrifugation. Protein concentrations in each group were determined using a BCA protein assay kit. After quantitative denaturation, proteins were separated by SDS-PAGE, transferred to PVDF membranes after electrophoresis, and blocked at room temperature for 1 hour in 5% skim milk TBST solution. Primary antibodies were diluted to appropriate concentrations according to the antibody instructions, incubated overnight at 4°C, and washed three times with TBST. Secondary antibodies were selected based on the species of the primary antibody, diluted, added, and incubated at room temperature for 1 hour, followed by three washes with TBST. Subsequently, the membranes were treated with ECL chemiluminescence buffer, exposed using a chemiluminescence imager, and the results were recorded. Image Lab was used to analyze the protein immunoblotting images.

[0063] Example 6 Statistical Analysis

[0064] Data are 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 (****) indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. All results were obtained from three independent replicates.

[0065] Example 7: TG significantly increased the killing ability of VEN against leukemia cells.

[0066] In all cell lines, the combination of TG and VEN was more effective than either TG or VEN alone. Figures 2-4In the MOLM13-R2 cell line, the combined use of 0.04-0.05 μM TG and 0.1-40.0 μM VEN achieved synergistic effects, with cell inhibition rates reaching 80% or higher when 0.04-0.05 μM TG was combined with 10.0-40.0 μM VEN. In the MOLM13-WT cell line, the combined use of 0.025-0.04 μM TG and 0.01-5.0 μM VEN achieved synergistic effects, with cell inhibition rates reaching 80% or higher when 0.025-0.04 μM TG was combined with 2.0-5.0 μM VEN. In the U937 cell line, the combined use of 0.05-0.1 μM TG and 5.0-80.0 μM VEN achieved synergistic effects, with cell inhibition rates reaching 80% or higher when 0.05-0.1 μM TG was combined with 10.0-40.0 μM VEN. When TG is used in combination with 50.0-80.0 μM VEN, the cell inhibition rate can reach 70% or more. Figures 5-7 When an 80% inhibition rate was achieved, in the VEN-resistant strain MOLM13-R2, 5 μM VEN combined with 0.05 μM TG resulted in an 87.5% reduction in VEN dosage; when a 90% inhibition rate was achieved, in MOLM13-WT, 0.5 μM VEN combined with 0.04 μM TG resulted in a 90% reduction in VEN dosage. Figures 8-9 When an inhibition rate of 70% was achieved, the combination of 0.1 μM TG and 0.05 μM TG was significantly more effective in U937, and the combination of 25 μM VEN and 0.1 μM TG resulted in a 68.75% reduction in VEN dosage. Figure 10 In three cell lines, HSA model analysis revealed that TG, at both IC10 and IC30 levels, produced a synergistic effect when combined with different concentrations of VEN. Figures 11-13 ).

[0067] Example 8: TG combined with VEN promotes cell apoptosis

[0068] The proportion of apoptotic cells was analyzed by flow cytometry. The results showed that, compared with monotherapy, the combined application of TG and VEN significantly increased the apoptotic proportion in MOLM13-R2 and MOLM13-WT. In the VEN-resistant strain MOLM13-R2, the proportion of apoptotic cells increased after the combination of VEN and TG, increasing by 55.92% and 56.42% respectively compared with VEN monotherapy. Figures 14-15 Among the studies, the apoptosis level reached its highest when the concentrations of VEN and TG were 10 μM and 0.05 μM, respectively. In MOLM13-WT, the proportion of apoptotic cells increased after the combined use of VEN and TG. When the concentrations of VEN and TG were 0.4 μM and 0.04 μM, respectively, the proportion of apoptotic cells increased by 71.64% compared to VEN alone, reaching the highest apoptosis level. Figures 16-17 ).

[0069] Example 9: Low cytotoxicity of TG single drug

[0070] Based on the aforementioned determined concentrations of TG used on the three groups of tumor cells, the following results indicate that the single-drug concentration of TG did not reach the tumor cell killing concentration and had no destructive effect on normal stromal cells. LDH assay results showed that in MOLM13-R2, treatment with TG at concentrations of 0.04 μM and 0.05 μM for 72 hours did not result in a statistically significant difference in LDH release compared to the untreated group. Figure 18 In MOLM13-WT and U937 cells, the corresponding IC10 concentrations of TG (Table 2) did not cause significant changes in LDH release; however, at their corresponding IC30 concentrations (Table 2), the LDH release values ​​of both cell lines were higher than those in the untreated group. Figures 19-20 MTS experiments showed that TG did not significantly inhibit cell viability after 72 hours of treatment at these sensitizing concentrations. Figure 21 ).

[0071] Example 10: TG downregulates VEN resistance protein expression

[0072] The expression changes of the VEN resistance-associated protein MCL-1 were detected by Western blotting. The results showed that in both MOLM13-WT and MOLM13-R2 cell lines, the expression level of the anti-apoptotic protein MCL-1 was downregulated in the combination therapy group. Figure 22 ).

[0073] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating leukemia, characterized in that: The pharmaceutical composition comprises carotenoid and veneclade, wherein the molar ratio of carotenoid to veneclade is 1:0.25-1600.

2. The pharmaceutical composition according to claim 1, characterized in that: The molar ratio of the toxic carotenoid to the venerealis is 1:20-1000.

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

100.

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 veneclade is 0.01-80 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 veneclade is 0.1-40 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 veneclade is 5-80 μM.

7. The pharmaceutical composition according to claim 5, characterized in that: The content of the carotenoid is 0.05 μM; and / or the content of the veneclade is 5 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, or 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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