A uf146 and abexinostat composition and its preparation for treating acute myeloid leukemia

Abstract

The present application relates to a kind of UF146 and abemaciclib composition and its preparation application for treating acute myeloid leukemia drug, belong to biological medicine technical field.To solve the problem of existing acute myeloid leukemia targeted drug clinical conversion is limited, the present application provides a kind of UF146 and abemaciclib composition, including independently packed UF146 injection preparation and abemaciclib oral preparation.The present application uses UF146 and abemaciclib in combination, so that abemaciclib originally in acute myeloid leukemia single drug activity is very limited to obtain significant anti-leukemia activity, while the effective concentration of UF146 is significantly reduced.The composition of the present application shows significant synergistic effect in a variety of acute myeloid leukemia cell lines and AML patient primary bone marrow cells, displays broad-spectrum anti-leukemia activity, and has excellent clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a composition of UF146 and abexicillin and its application in the preparation of drugs for the treatment of acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from undifferentiated myeloid hematopoietic stem / progenitor cells in the bone marrow, and is the most common type of acute leukemia in adults. Current clinical treatment primarily relies on chemotherapy, which suffers from insufficient specificity, significant side effects, and a high relapse rate, necessitating the development of new treatment strategies.

[0003] The CDK4 / 6 inhibitor abexib regulates cell cycle progression by specifically inhibiting CDK4 / 6 activity and has shown significant efficacy in HR+ / HER2- breast cancer. However, in AML, abexib's single-agent activity is extremely limited. Studies have shown that AML cells commonly exhibit multiple mechanisms, including RB pathway inactivation, CDK2 bypass activation, and natural drug resistance in leukemia stem cells. This makes simply blocking CDK4 / 6 insufficient to effectively curb the malignant proliferation of AML cells. Therefore, no CDK4 / 6 inhibitor monotherapy is currently approved for AML treatment.

[0004] Replacing abexib with other CDK4 / 6 inhibitors such as palbociclib or ribociclib is still limited by the core resistance mechanism of RB pathway inactivation, theoretically making it difficult to overcome the efficacy bottleneck of abexib in AML treatment. Combining abexib with traditional chemotherapy drugs, such as cytarabine or daunorubicin, may result in limited efficacy gains due to the cell cycle arrest induced by abexib, which could weaken the killing effect of chemotherapy drugs on proliferating cells. Furthermore, both drugs inhibit bone marrow hematopoiesis through different mechanisms of action, and their combined use can easily lead to additive toxicity, manifesting as severe bone marrow suppression and significantly increasing the patient's treatment risk. Therefore, such combination regimens are unlikely to achieve the clinical goal of "synergistic efficacy and reduced toxicity."

[0005] Ubiquitin-like protein containing PHD and ring finger domain 1 (UHRF1) is a key regulatory molecule for DNA methylation and histone modification, and has recently been identified as a potential therapeutic target for AML. Patent publication CN115245508A discloses the application of the small molecule inhibitor UF146, which specifically targets UHRF1, in AML. This patent reports that UF146 has an inhibitory effect on the proliferation of AML cells in vitro, and its IC50 in Kasumi-1 cells... 50At a concentration of approximately 3.58 μM, intraperitoneal injection at a dose of 2.5 mg / kg prolonged the survival of leukemia-infected mice. However, this effective concentration remains at the micromolar level, significantly higher than commonly used clinical anti-leukemia drugs. Furthermore, due to limitations in solubility, bioavailability, and pharmacokinetic characteristics, the in vivo exposure and tissue distribution of UF146 as a single drug are somewhat restricted. Further increasing in vivo exposure to enhance efficacy may pose safety risks. These factors collectively limit the clinical availability of UF146 as a single drug.

[0006] Studies suggest that PWWP2B can influence the expression of downstream cell cycle pathway-related genes by recruiting UHRF1, and that PWWP2B expression levels are positively correlated with the half-maximal inhibitory concentration (IC50) of CDK4 / 6 inhibitors. However, this research only focuses on correlation analysis at the basic scientific level and has not yet provided guidance for any specific combination therapy strategies for treating leukemia. Due to the structural and biological characteristics of the PWWP2B protein, no specific and highly effective small molecule drugs have been screened to date, making direct targeted intervention extremely difficult. Therefore, researchers cannot directly predict, based on the aforementioned correlation information, that targeting its downstream UHRF1 will enhance the anti-leukemia efficacy of CDK4 / 6 inhibitors.

[0007] In summary, theoretically effective drugs often cannot be effectively applied in clinical practice, and new strategies still need to be explored to overcome their clinical translation bottlenecks. Summary of the Invention

[0008] To address the limitations in the clinical translation of existing targeted drugs for acute myeloid leukemia, this invention provides a combination of UF146 and abexicillin, and its application in the preparation of drugs for treating acute myeloid leukemia.

[0009] The technical solution of the present invention:

[0010] A composition of UF146 and abexicillin, comprising separately packaged injectable formulations of UF146 and oral formulations of abexicillin, with the total two-day dose as the smallest unit of combination; the total molar ratio of UF146 to abexicillin based on the total two-day dose is 1:0.05~20, wherein the molecular formula of UF146 is C 12 H8ClNO3, structural formula is

[0011] The molecular formula of abexicillin is C1 27 H 32 F2N8, structural formula is .

[0012] Furthermore, the UF146 injectable formulation is a lyophilized powder injection or an injection solution, and the abecitabine oral formulation is a tablet, capsule, or oral solution.

[0013] Furthermore, the composition also contains pharmaceutically acceptable excipients.

[0014] Application of a combination of UF146 and abexilide in the preparation of a drug for treating acute myeloid leukemia.

[0015] Furthermore, the acute myeloid leukemia is either AML1-ETO fusion gene positive acute myeloid leukemia or MLL-AF9 fusion gene positive acute myeloid leukemia.

[0016] Furthermore, UF146 and abecitabine in the composition are administered simultaneously, separately, or sequentially.

[0017] The beneficial effects of this invention are:

[0018] This invention, without altering the individual molecular structures of UF146 and abexicillin, enables the combined use of the two drugs to achieve significant anti-leukemic activity in abexicillin, which previously exhibited extremely limited single-drug activity in acute myeloid leukemia (AML), while simultaneously significantly reducing the effective concentration of UF146. Experimental data show that in Kasumi-1 cells, after fixing the abexicillin concentration, the required concentration of UF146 can be reduced by 5.6-fold, requiring only 17.9% of the single-drug dose to achieve a 30% tumor inhibition rate; after fixing the UF146 concentration, the required concentration of abexicillin can be reduced by 13.5-fold, requiring only 7.4% of the single-drug dose to achieve a 30% tumor inhibition rate. In various AML cell lines, including SKNO1, MOLM13, and THP-1, as well as primary bone marrow cells from AML patients, this combination exhibits a significant synergistic effect, with ZIP synergistic indices greater than 10 and combination index (CI) values ​​less than 1. In vivo experiments further confirmed that in the MLL-AF9 leukemia mouse model, the median survival of the combination therapy group was prolonged to 29.5 days, significantly better than the 24.5 days of the abexicillin monotherapy group and the 26 days of the UF146 monotherapy group. Furthermore, this composition was effective against both AML1-ETO fusion gene-positive and MLL-AF9 fusion gene-positive subtypes of acute myeloid leukemia, demonstrating broad-spectrum anti-leukemic activity. The pharmaceutical composition provided by this invention offers an effective solution to the technical problems of limited clinical translation of targeted drugs, insufficient efficacy of monotherapy, and difficulties in enhancing efficacy and reducing toxicity with combination chemotherapy, and has excellent clinical application prospects. Attached Figure Description

[0019] Figure 1The images show a comparison of in vitro colony formation in different AML cells from the control group, UHRF1 knockout group-1, and UHRF1 knockout group-2 in Example 1. A is a staining image of cell colony formation in each group, B is a comparison of the number of Kasumi-1 colonies, C is a comparison of the number of SKNO1 colonies, D is a comparison of the number of MOLM13 colonies, and E is a comparison of the number of THP-1 colonies.

[0020] Figure 2 The images show a comparison of AML progression in the control group and UHRF1 knockout group mouse models in Example 1. A is an in vivo fluorescence imaging image of leukemia cell proliferation and spread in mice, B is a comparison of fluorescence signal intensity in mice, and C is a comparison of survival rates of the model mice.

[0021] Figure 3 This is a heatmap showing the inhibition rate of cell activity of Kasumi-1 cells by the combined use of UF146 and abecitabine in Example 2.

[0022] Figure 4 The three-dimensional surface plot of the synergistic effect of UF146 and abecili in Kasumi-1 cells analyzed by ZIP model in Example 2;

[0023] Figure 5 This is a graph showing the relationship between the combined index of UF146 and abexicillin and the effect size, calculated using the Chou-Talalay method in Example 2.

[0024] Figure 6 The bar charts show the fold reduction in concentration of UF146 and abexicillin in Kasumi-1 cells during the combined administration of UF146 and abexicillin in Example 2. A represents the fold reduction in UF146 dose while maintaining the abexicillin concentration, and B represents the fold reduction in abexicillin dose while maintaining the UF146 concentration.

[0025] Figure 7 This is a heatmap showing the inhibition rate of cell viability of SKNO1 cells by the combined use of UF146 and abecitabine in Example 2.

[0026] Figure 8 The three-dimensional surface plot of the ZIP model analysis of the synergistic effect of UF146 and abecili in SKNO1 cells in Example 2;

[0027] Figure 9 This is a heatmap showing the inhibition rate of cell viability of MOLM13 cells by the combined use of UF146 and abecitabine in Example 3.

[0028] Figure 10 This is a three-dimensional surface plot of the synergistic effect of UF146 and abecili in MOLM13 cells analyzed by ZIP model in Example 3;

[0029] Figure 11 This is a heatmap showing the inhibition rate of cell viability of THP-1 cells by the combined use of UF146 and abecitabine in Example 3.

[0030] Figure 12 This is a three-dimensional surface plot of the ZIP model analysis of the synergistic effect of UF146 and abecicilline in THP-1 cells in Example 3;

[0031] Figure 13 This is a heatmap showing the inhibition rate of cell activity of primary bone marrow cells from AML patients by the combined use of UF146 and abecilibi in Example 3.

[0032] Figure 14 This is a three-dimensional surface plot of the synergistic effect of UF146 and abecitabine in primary bone marrow cells of AML patients analyzed using the ZIP model in Example 3.

[0033] Figure 15 Giemsa staining images of primary cells from AML patients in different treatment groups in Example 3;

[0034] Figure 16 Figure 4 shows the in vitro effects of UF146 and abecitabine in primary AE9a leukemia mouse cells in Example 4; A is a soft agar colony formation diagram of primary AE9a leukemia cells in different treatment groups; B is a comparison diagram of the number of colonies formed in primary AE9a leukemia cells in different treatment groups; C is a Giemsa staining diagram of primary AE9a leukemia cells in different treatment groups.

[0035] Figure 17 Figure 4 shows the in vitro effects of UF146 and abecitabine in primary MLL-AF9 leukemia mouse cells. A is a soft agar colony formation diagram of primary MLL-AF9 leukemia cells in different treatment groups; B is a comparison diagram of the number of colonies formed in primary MLL-AF9 leukemia cells in different treatment groups; C is a Giemsa staining diagram of primary MLL-AF9 leukemia cells in different treatment groups.

[0036] Figure 18 The following is a graph showing the results of the analysis of the antitumor effects of UF146 and abecitabine in MLL-AF9 leukemia mice in Example 4. A is a comparison of spleen morphology after treatment in each group of MLL-AF9 leukemia mice; B is a comparison of spleen weight after treatment in each group of MLL-AF9 leukemia mice.

[0037] Figure 19 This is a comparison of survival curves for each group of MLL-AF9 leukemia mice in Example 4. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0039] Statistical analysis

[0040] All embodiments of this invention used Graphpad Prism 8.0 to plot experimental data, and quantitative data were expressed as mean ± standard deviation. Unpaired t-tests were used between two groups, and one-way ANOVA was used between multiple groups. P < 0.05 indicated statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0041] Example 1

[0042] To clarify the biological function of the UHRF1 gene in acute myeloid leukemia (AML) and assess its potential as a therapeutic target, this embodiment systematically verified the regulatory effect of UHRF1 knockout on the malignant proliferation of AML cells and the progression of leukemia in vivo through in vitro cell experiments and in vivo animal models.

[0043] I. Soft agar colony formation experiment

[0044] (1) Experimental materials

[0045] Human AML cell lines Kasumi-1, SKNO1, MOLM13, THP-1, and lentiviral packaging cell line HEK293T were all purchased from ProCare Biotech.

[0046] The CRISPR / Cas9 lentiviral vector lentiCRISPR v2 was purchased from Addgene, and the sgRNA targeting UHRF1 was synthesized by Anhui General Biotechnology Co., Ltd., with the specific sequence as follows:

[0047] As shown in SEQ ID NO:1, sgUHRF1-1: 5'-AGGTTCGGACCATGGACGGG-3';

[0048] As shown in SEQ ID NO:2, sgUHRF1-2: 5'-CGCCGACACCATGTGGATCC-3'.

[0049] AML cells were cultured in RPMI 1640 cell culture medium containing 10% fetal bovine serum, and HEK293T cells were cultured in DMEM cell culture medium containing 10% fetal bovine serum. Both culture media were supplemented with 1% penicillin and streptomycin. After the cells and culture medium were thoroughly mixed, they were placed in cell culture dishes and finally placed in a cell culture incubator at a constant temperature of 37°C with 5% CO2.

[0050] The RPMI 1640 medium, DMEM medium, fetal bovine serum, penicillin-streptomycin antibiotics, agarose, puromycin, polybrene, and other reagents were all commercially available standard products.

[0051] (2) Methods for constructing stable cell lines

[0052] sgUHRF1-1 and sgUHRF1-2 were cloned into the lentiCRISPR v2 vector using BsmBI enzyme. Lentiviral expression vectors, packaging plasmids pMD2.G and psPAX2 were simultaneously transfected into HEK293T cells using PEI transfection reagent. Supernatant viral fluid was collected at 48 and 72 h post-transfection, filtered, and then infected with 8 μg / mL polybrene to infect human AML cell lines Kasumi-1, SKNO1, MOLM13, and THP-1, respectively. Positive cells were selected with the corresponding puromycin at 48 h post-infection to obtain stably knocked-out UHRF1 AML cell lines, which were then divided into a control group (transfected with empty vector), UHRF1 knockout group-1, and UHRF1 knockout group-2.

[0053] (3) Experimental methods and results of soft agar colony formation

[0054] Prepare a 3% agarose solution, autoclave it, and place it in a 42°C water bath. Preheat the complete culture medium in a 37°C water bath. Mix the 3% agarose solution and the preheated complete culture medium at a 1:3 ratio to achieve an agarose concentration of 0.75%. After thorough mixing, add 1.5 ml of this mixture to each well of a six-well plate to prepare the lower gel. Allow it to solidify at room temperature. Next, mix the 3% agarose solution and the preheated complete culture medium at a 1:7 ratio to achieve an agarose concentration of 0.36%. After thorough mixing, prepare the upper gel. Add 5000 cells per well according to the cell count concentration. Mix thoroughly and spread this mixture onto the lower agarose layer, adding 2 ml per well.

[0055] Gently place the six-well plate in the cell culture incubator. Add culture medium every 3 days, 200-300 μl each time. After 10-14 days, add dye, 300 μl per well, and incubate overnight at 37°C. Then, count the number of colonies formed and take pictures.

[0056] The results are as follows Figure 1As shown, after UHRF1 gene knockout, the number of colonies formed in AML1-ETO fusion AML cells Kasumi-1 and SKNO1, as well as MLL-AF9 fusion AML cells MOLM13 and THP-1, was reduced (all P<0.001 or P<0.0001), indicating that UHRF1 knockout can significantly inhibit the in vitro clonogenic ability of AML cells.

[0057] II. Construction and Processing of Human AML Cell Xenograft Mouse Model (CDX)

[0058] (1) Experimental materials

[0059] NCG immunodeficient mice, SPF grade, 5-6 weeks old, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0060] This experiment used Kasumi-1 cells to construct an AML model. The cells were divided into a control group and a UHRF1 (sgUHRF1-2) knockout group. All cells were transfected with a luciferase tag in advance to adapt for in vivo fluorescence imaging detection. The luciferase substrate, small animal imaging device, and other reagents and instruments were all commercially available standard products.

[0061] Animal experiments were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Harbin Medical University and strictly followed the ethical guidelines for laboratory animals.

[0062] (2) Methods for constructing CDX mouse models

[0063] At 5-6 weeks of age, NCG mice were injected with luciferase-tagged AML cells, including control and UHRF1 knockout groups, via the tail vein. These cells were then allowed to grow in the mice, and leukemia cells were observed to spread and proliferate within 1-3 weeks. The cells typically proliferated in the blood system or specific sites such as the spleen and bone marrow, thus mimicking human leukemia.

[0064] (3) In vivo fluorescence imaging detection method and results

[0065] On days 21, 28, 35, and 42 after modeling, mice were anesthetized and injected intraperitoneally with fluorescein substrate, 3 mg / mouse. The fluorescence signal intensity in vivo was detected using a small animal imaging system to assess the proliferation and spread of leukemia cells.

[0066] The results are as follows Figure 2 As shown in Figures A and B, the fluorescence signal of leukemia cells in the control group mice continued to increase over time, while the fluorescence signal intensity in the UHRF1 knockout group mice was significantly lower than that in the control group (P<0.05). This indicates that UHRF1 knockout can significantly inhibit the proliferation and spread of AML cells in vivo.

[0067] (4) Survival statistics

[0068] The survival time of mice in each group was recorded, survival curves were plotted, and the effect of UHRF1 knockout on the survival of AML mice was analyzed.

[0069] The results are as follows Figure 2 As shown in Figure C, compared with the control group, the survival time of AML cell recipient mice in the UHRF1 gene knockout group was significantly prolonged (P=0.0091), indicating that UHRF1 gene knockout has a significant inhibitory effect on leukemia cells in mice.

[0070] The experimental results of this embodiment demonstrate that UHRF1 plays a key pro-cancer role in AML, and its knockout can significantly inhibit the malignant proliferation and disease progression of AML cells in vitro and in vivo, confirming that UHRF1 is a potential key target for AML treatment.

[0071] Example 2

[0072] To verify the combined antitumor effect of UF146 and abecitabine in AML1-ETO fusion-type acute myeloid leukemia, this example uses Kasumi-1 and SKNO1 AML1-ETO fusion cells as research subjects to conduct a synergistic effect analysis of the combined drug use.

[0073] I. Kasumi-1 cell combination drug experiment

[0074] (1) Experimental materials

[0075] Kasumi-1 cells were selected, and the source was the same as in Example 1.

[0076] The experimental reagents include UF146, abecitabine, and CellTiter-Glo assay reagent.

[0077] UF146 was purchased from ProbeChem (Shanghai, China), catalog number PC-49681, with a purity >98%. It was dissolved in DMSO before use.

[0078] Abecili was purchased from TargetMol (Shanghai, China), catalog number T2381, with a purity of 99.87%. It was dissolved in sterile PBS before use.

[0079] The CellTiter-Glo test kit is supplied by Promega, product code G7570.

[0080] The experimental apparatus included a GloMax Navigator chemiluminescence analyzer, which was used with SoftMax Pro software. Data analysis software employed included SynergyFinder and CalcuSyn.

[0081] The culture medium used for cell culture is a conventional complete culture medium.

[0082] (2) Drug synergistic experiment of Kasumi-1 cells

[0083] Cells were seeded in 96-well plates at a seeding density of 3,000 to 10,000 cells per well, with 100 μL of culture medium added to each well. UF146 concentration gradients of 0, 312.5, 625, 1250, 2500, and 5000 nM and abexicillin concentration gradients of 15.625, 31.25, 62.5, 125, and 250 nM were established, and cells were treated with different concentration combinations for 72 h.

[0084] ① After treatment with different concentrations of drugs, add an equal volume of CellTiter-Glo reagent to each well, place the culture plate on a shaker and incubate with horizontal shaking at 100 rpm for 10 min. Quantitatively detect the luminescence signal using a GloMax Navigator chemiluminescence analyzer (with SoftMaxPro software). Cell viability was normalized to 100% using the untreated control group as a baseline, and the cell viability inhibition rate was calculated.

[0085] The results are as follows Figure 3 As shown, the cell activity inhibition rate increased with increasing concentrations of both UF146 and abexicillin. At abexicillin concentration of 125 nM, the inhibition rate reached 66.13% when combined with UF146 at 2500 nM, significantly higher than the combined effect of either drug alone. At abexicillin concentration of 62.5 nM, the inhibition rate also reached 61.81% when combined with UF146 at 2500 nM, indicating that high-efficiency inhibition can be achieved with low-dose combination therapy, providing a basis for reducing toxicity. This suggests that the combination of UF146 and abexicillin significantly enhances the inhibitory effect on Kasumi-1 cells compared to either drug alone, exhibiting a clear synergistic effect, and achieving potent inhibition without requiring extremely high doses.

[0086] ②The SynergyFinder software was used to evaluate the synergistic effect of the two drugs based on the Zero Interaction Potential (ZIP) model. A ZIP synergy index greater than 10 was considered synergistic, an index between -10 and 10 was considered additive, and an index less than -10 was considered antagonistic.

[0087] The results are as follows Figure 4 As shown, the ZIP synergy index of the two drugs was 13.82, with a P-value of 3.66e-12, indicating that the two drugs have a significant synergistic effect.

[0088] ③ The Chou-Talalay synergy index method was used, and the synergy index was calculated and the Fa-CI effect curve was plotted using CalcuSyn software. Fa represents the effect value (cell growth inhibition rate) under drug action, and CI is a quantitative indicator of the synergistic effect of combined drug use. CI less than 1 is considered a synergistic effect, CI equal to 1 is an additive effect, CI greater than 1 is an antagonistic effect, and the smaller the CI value, the stronger the synergistic effect.

[0089] The results are as follows Figure 5 As shown in Table 1, the CI values ​​for the combination of UF146 and abexicillin at all concentration combinations were significantly less than 1. Within the core therapeutic range of Fa 0.2–0.8, the CI values ​​remained stably within the strong synergistic range of 0.3–0.7, with only a slight increase in CI values ​​during periods of high inhibitory effect, but still well below 1. These results fully demonstrate that UF146 and abexicillin possess broad-spectrum, stable, and potent synergistic antitumor activity in RB-deficient AML cells, significantly reducing the effective dose of single drugs and expanding the therapeutic window, providing solid experimental evidence for the clinical application of this combination regimen.

[0090] Table 1

[0091]

[0092] ④ Conduct analysis on the fold reduction in concentration of combined drugs.

[0093] The effective concentration EC30 value corresponding to a 30% tumor inhibition rate under single-drug action was obtained through dose-response curve fitting analysis. Specifically, the EC30 value of Kasumi-1 cells treated with UF146 alone was 1745.2 nM, and the EC30 value of abexicillin alone was 211.3 nM. This was used as a control baseline with a fold reduction of 1. By comparing the effective concentrations of the combined drug and the single drug under the same tumor inhibition efficiency, the drug dose reduction factor was calculated. A bar graph was plotted with the drug dose reduction factor as the ordinate and different concentration gradients of the combined drug as the abscissa. A bar value greater than 1 indicates that the combination of the two drugs can significantly reduce the dosage under the premise of equivalent antitumor effect.

[0094] Abecitabine concentrations were fixed at 15.625, 31.25, 62.5, 125, and 250 nm. Then, the concentration multiples of UF146 corresponding to the achievement of a 30% tumor inhibition rate with combined drug therapy were analyzed and a bar chart was plotted. The results are as follows: Figure 6 As shown in Figure A and Table 2, when the concentration of abexicillin is ≥125 nM, the concentration of UF146 can be reduced by 5.6 times, and only 17.9% of the dose is needed to achieve a tumor inhibition rate of 30%.

[0095] The concentrations of UF146 were fixed at 312.5, 625, 1250, 2500, and 5000 nm. Then, the concentration multiples of abexicillin corresponding to the achievement of a 30% tumor inhibition rate with combined drug therapy were analyzed and a bar chart was plotted. The results are as follows: Figure 6 As shown in Figure B and Table 3, when the UF146 concentration is ≥1250 nM, the required concentration of abexicillin can be reduced by 13.5 times, requiring only 7.4% of the dose of the single drug to achieve a 30% tumor inhibition rate. These results indicate that combination therapy can maintain good antitumor activity while reducing the dosage of the single drug, helping to reduce drug toxicity and side effects, and improving clinical safety and patient tolerability.

[0096] Table 2

[0097]

[0098] Table 3

[0099]

[0100] II. SKNO1 Cell Combined Drug Experiment

[0101] (1) Experimental materials

[0102] SKNO1 cells were used, from the same source as in Example 1. The reagents, instruments, and analytical software used were consistent with those used in the Kasumi-1 cell experiment.

[0103] (2) Drug synergistic experiment of SKNO1 cells

[0104] Cells were seeded into 96-well plates at a seeding density of 3,000 to 10,000 cells per well, with 100 μL of culture medium added to each well. UF146 concentration gradients of 0, 78.125, 156.25, 312.5, 625, and 1250 nM and abexicillin concentration gradients of 0, 7.8125, 15.625, 31.25, 62.5, and 125 nM were established, and cells were treated with different concentration combinations for 72 h.

[0105] ① After treatment with different concentrations of drugs, add an equal volume of CellTiter-Glo reagent to each well, place the culture plate on a shaker and incubate with horizontal shaking at 100 rpm for 10 min. Quantitatively detect the luminescence signal using a GloMax Navigator chemiluminescence analyzer (with SoftMaxPro software). Cell viability was normalized to 100% using the untreated control group as a baseline, and the cell viability inhibition rate was calculated.

[0106] The results are as follows Figure 7As shown, the cell activity inhibition rate increases with the simultaneous increase of the concentrations of UF146 and abexicillin. At a concentration of 62.5 nM abexicillin combined with 625 nM UF146 achieves an inhibition rate of 84.38%, higher than the combined effect of the two drugs used alone. At a concentration of 31.25 nM abexicillin combined with 625 nM UF146 also achieves an inhibition rate of 75.99%. This indicates that the combined use of UF146 and abexicillin significantly enhances the inhibitory effect on SKNO1 cells compared to either drug alone, exhibiting a clear synergistic effect, and achieving potent inhibition without requiring extremely high doses.

[0107] ② The synergistic effect of the two drugs was evaluated using SynergyFinder software based on the Zero Interaction Performance (ZIP) model. The results are as follows: Figure 8 As shown, the ZIP synergy index of the two drugs was 14.64, with a P-value of 2.89e-08, indicating that the two drugs have a significant synergistic effect.

[0108] ③ Conduct analysis on the fold reduction in concentration of combined drugs.

[0109] The effective concentration EC30 value corresponding to a 30% tumor inhibition rate under single-drug action was obtained by dose-response curve fitting analysis. Specifically, the EC30 value of SKNO1 cells treated with UF146 alone was 482.36 nM, and the EC30 value of abexicillin alone was 68.52 nM. This was used as a control baseline with a fold reduction of 1. By comparing the effective concentrations of combined and single-drug administration under the same tumor inhibition efficiency, the drug dose reduction fold was calculated.

[0110] The concentrations of abexicillin were fixed at 7.8125, 15.625, 31.25, 62.5, and 125 nm. Then, the concentration multiples of UF146 corresponding to the combination therapy achieving a 30% tumor inhibition rate were analyzed. The results are shown in Table 4. When the concentration of abexicillin was 31.25 nM, the concentration of UF146 used could be reduced by 3.05 times, and only 32.8% of the dose of the drug alone was needed to achieve a 30% tumor inhibition rate.

[0111] The concentrations of UF146 were fixed at 78.125, 156.25, 312.5, 625, and 1250 nm. Then, the concentration multiples of abexicillin corresponding to the combined use of drugs to achieve a 30% tumor inhibition rate were analyzed. The results are shown in Table 5. When the concentration of UF146 is 312.5 nM, the concentration of abexicillin can be reduced by 3.76 times, and only 26.5% of the dose of abexicillin alone is needed to achieve a 30% tumor inhibition rate.

[0112] Table 4

[0113]

[0114] Table 5

[0115]

[0116] The results of this embodiment demonstrate that UF146 combined with abexicillin exhibits a significant synergistic effect in AML1-ETO fusion-type acute myeloid leukemia cells. This combination regimen significantly reduces the single-drug dose by 3 to 13 times while maintaining good antitumor activity, greatly reducing the toxicity risk associated with high-dose single-drug therapy and significantly extending the treatment safety window. Compared with single-drug therapy, the combined application of UF146 and abexicillin achieves an antitumor effect greater than the sum of its parts (1+1>2), providing a novel, low-toxicity, and highly effective treatment strategy for patients with AML1-ETO fusion-type acute myeloid leukemia, and possessing extremely high clinical translational value.

[0117] Example 3

[0118] To verify the combined antitumor effect of UF146 and abecilibus in MLL-AF9 fusion acute myeloid leukemia and to further evaluate the applicability of this combination regimen to leukemia cells derived from clinical patients, this embodiment uses two MLL-AF9 fusion cell lines, MOLM13 and THP-1, as well as primary bone marrow cells from AML patients as research subjects to systematically conduct a synergistic effect analysis of the combined drug use.

[0119] I. Experiment on combined drug use in MOLM13 cells

[0120] (1) MOLM13 cells were selected, and the source was the same as in Example 1. The reagents, instruments and analysis software used were consistent with those used in the Kasumi-1 cell experiment.

[0121] (2) Drug synergistic experiment of MOLM13 cells

[0122] Cells were seeded in 96-well plates at a seeding density of 3,000 to 10,000 cells per well, with 100 μL of culture medium added to each well. UF146 concentration gradients of 0, 312.5, 625, 1250, 2500, and 5000 nM and abexicillin concentration gradients of 0, 15.625, 31.25, 62.5, 125, and 250 nM were established, and MOLM13 cells were treated with different concentration combinations for 72 h.

[0123] ① After treatment with different concentrations of drugs, add an equal volume of CellTiter-Glo reagent to each well, place the culture plate on a shaker and incubate with horizontal shaking at 100 rpm for 10 min. Quantitatively detect the luminescence signal using a GloMax Navigator chemiluminescence analyzer (with SoftMaxPro software). Cell viability was normalized to 100% using the untreated control group as a baseline, and the cell viability inhibition rate was calculated.

[0124] The results are as follows Figure 9 As shown, the cell activity inhibition rate increased with increasing concentrations of both UF146 and abexicillin. At 250 nM abexicillin combined with 2500 nM UF146, the inhibition rate reached 74.87%, higher than the combined effect of the two drugs alone. At 125 nM abexicillin combined with 2500 nM UF146, the inhibition rate also reached 62.05%. This indicates that the combined use of UF146 and abexicillin significantly enhances the inhibitory effect on MOLM13 cells compared to either drug alone, exhibiting a clear synergistic effect, and achieving potent inhibition without requiring extremely high doses.

[0125] ② The synergistic effect of the two drugs was evaluated using SynergyFinder software based on the Zero Interaction Performance (ZIP) model. The results are as follows: Figure 10 As shown, the ZIP synergy index of the two drugs was 12.12, with a P-value of 4.78e-07, indicating that the two drugs have a significant synergistic effect.

[0126] ③ Conduct analysis on the fold reduction in concentration of combined drugs.

[0127] The effective concentration EC30 value corresponding to a 30% tumor inhibition rate under single-drug action was obtained by dose-response curve fitting analysis. Specifically, the EC30 value of MOLM13 cells treated with UF146 alone was 421.78 nM, and the EC30 value of abexicillin alone was 62.35 nM. This was used as a control baseline with a fold reduction of 1. By comparing the effective concentrations of combined and single-drug administration under the same tumor inhibition efficiency, the drug dose reduction fold was calculated.

[0128] The concentrations of abexicillin were fixed at 7.81, 15.63, 31.25, 62.5, and 125 nm. Then, the concentration multiples of UF146 corresponding to the combined use of the drugs to achieve a 30% tumor inhibition rate were analyzed. The results are shown in Table 6. When the concentration of abexicillin is ≥31.25 nM, the concentration of UF146 can be reduced by 2.5 to 3 times, and only 34 to 38% of the dose of the drug alone is needed to achieve a 30% tumor inhibition rate.

[0129] The concentrations of UF146 were fixed at 78.13, 156.25, 312.5, 625, and 1250 nm. Then, the concentration multiples of abexicillin corresponding to the combined use of the drugs to achieve a 30% tumor inhibition rate were analyzed. The results are shown in Table 7. When the concentration of UF146 is ≥312.5 nM, the concentration of abexicillin can be reduced by 3 to 4 times, and only 25 to 30% of the dose of the drug alone is needed to achieve a 30% tumor inhibition rate.

[0130] Table 6

[0131]

[0132] Table 7

[0133]

[0134] II. Experiment with combined drug therapy using THP-1 cells

[0135] (1) THP-1 cells were selected, and the source was the same as in Example 1. The reagents, instruments and analysis software used were consistent with those used in the Kasumi-1 cell experiment.

[0136] (2) Drug synergistic experiment of THP-1 cells

[0137] Cells were seeded in 96-well plates at a seeding density of 3,000 to 10,000 cells per well, with 100 μL of culture medium added to each well. UF146 concentration gradients of 0, 312.5, 625, 1250, 2500, and 5000 nM and abexicillin concentration gradients of 0, 7.8125, 15.625, 31.25, 62.5, and 125 nM were established, and THP-1 cells were treated with different concentration combinations for 72 h.

[0138] ① After treatment with different concentrations of drugs, add an equal volume of CellTiter-Glo reagent to each well, place the culture plate on a shaker and incubate with horizontal shaking at 100 rpm for 10 min. Quantitatively detect the luminescence signal using a GloMax Navigator chemiluminescence analyzer (with SoftMaxPro software). Cell viability was normalized to 100% using the untreated control group as a baseline, and the cell viability inhibition rate was calculated.

[0139] The results are as follows Figure 11 As shown, the cell activity inhibition rate increased with increasing concentrations of both UF146 and abexicillin. At abexicillin concentration of 125 nM, combined with UF146 at 5000 nM, the inhibition rate reached 50.8%, higher than the combined effect of the two drugs alone. At abexicillin concentration of 62.5 nM, combined with UF146 at 5000 nM, the inhibition rate also reached 45.07%. This indicates that the combined use of UF146 and abexicillin significantly enhances the inhibitory effect on THP-1 cells compared to either drug alone, exhibiting a significant synergistic effect, and achieving potent inhibition without requiring extremely high doses.

[0140] ② The synergistic effect of the two drugs was evaluated using SynergyFinder software based on the Zero Interaction Performance (ZIP) model. The results are as follows: Figure 12 As shown, the ZIP synergy index of the two drugs was 12.05, with a P value of 1.11e-05, indicating that UF146 combined with abexicillin has a significant synergistic effect on THP-1 cells.

[0141] III. Experimental Study on Combined Drug Use with Bone Marrow Cells in AML Patients

[0142] (1) Bone marrow cell samples from AML patients

[0143] Bone marrow cell samples from AML patients were collected by the Department of Hematology, First Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all participants, and the research protocol was approved by the Scientific and Clinical Research Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

[0144] Patient-derived bone marrow cells were cultured in Opti-MEM medium containing 20% ​​FBS, 100 ng / mL hSCF, 100 ng / mL hFLT3, 20 ng / mL hTPO, 20 ng / mL hIL6, and 10 ng / mL hIL3. All media were supplemented with 1% penicillin and streptomycin. After thorough mixing with the medium, the cells were placed in cell culture dishes and then incubated at a constant temperature of 37°C in a cell culture incubator containing 5% CO2.

[0145] (2) Drug synergistic experiment on bone marrow cells of AML patients

[0146] Cells were seeded in 96-well plates at a seeding density of 3,000 to 10,000 cells per well, with 100 μL of culture medium added to each well. UF146 concentration gradients of 0, 312.5, 625, 1250, 2500, and 5000 nM and abexicillin concentration gradients of 0, 62.5, 125, 250, 500, and 1000 nM were used to treat AML patient bone marrow cells for 72 hours.

[0147] ① After treatment with different concentrations of drugs, add an equal volume of CellTiter-Glo reagent to each well, place the culture plate on a shaker and incubate with horizontal shaking at 100 rpm for 10 min. Quantitatively detect the luminescence signal using a GloMax Navigator chemiluminescence analyzer (with SoftMaxPro software). Cell viability was normalized to 100% using the untreated control group as a baseline, and the cell viability inhibition rate was calculated.

[0148] The results are as follows Figure 13 As shown, the cell activity inhibition rate increases with the simultaneous increase of UF146 and abexicillin concentrations. At abexicillin concentration of 500 nM, combined with UF146 at 2500 nM, the inhibition rate reaches 78.72%, higher than the combined effect of the two drugs used alone. At abexicillin concentration of 250 nM, combined with UF146 at 2500 nM, the inhibition rate also reaches 72.78%. This indicates that the combined use of UF146 and abexicillin significantly enhances the inhibitory effect on AML patient cells compared to either drug alone, demonstrating a clear synergistic effect, and achieving potent inhibition without requiring extremely high doses.

[0149] ② The synergistic effect of the two drugs was evaluated using SynergyFinder software based on the Zero Interaction Performance (ZIP) model. The results are as follows: Figure 14 As shown, the ZIP synergy index of the two drugs was 10.66, with a P value of 8.74e-07, indicating that UF146 combined with abexicillin has a significant synergistic effect on AML patient cells.

[0150] (3) Giemsa staining

[0151] Cells from AML patients treated with the drug were collected to prepare smears, including a control group (abecicalb=0 nM, UF146=0 nM), an abecicalb group (abecicalb=250 nM), a UF146 group (UF146=5000 nM), and an abecicalb + UF146 group (abecicalb=250 nM, UF146=5000 nM). 5-10 μl of fresh specimen was placed on one end of a glass slide, and the slide was gently pushed at a 30-45° angle to form a thin blood film, free of air bubbles and cell overlap. The slide was then allowed to air dry at room temperature. The completely dried smear was placed on a staining rack, and 3-5 drops of Giemsa-mixed staining solution were added to cover the blood film. The slide was allowed to stand for a few seconds to allow initial dye penetration. Then, twice the volume of phosphate buffer was added, and the mixture was gently blown to mix. The slide was allowed to stand for 5 minutes. Rinse the back of the slide slowly with running water, air dry at room temperature, and then photograph it under a microscope. Observe the cell morphology characteristics under an optical microscope (×1000 oil immersion) to evaluate the effect of the drug on primary leukemia cells.

[0152] The results are as follows Figure 15 As shown, compared with the control group and the two single-drug groups, the combination drug group showed more obvious cell differentiation characteristics, such as smaller nuclear area, increased cytoplasm, significantly reduced nucleus-to-cytoplasm ratio, and gradually irregular nuclear shape.

[0153] In summary, UF146 combined with abexicillin also has a significant synergistic effect in MLL-AF9 fusion acute myeloid leukemia cells, and also has a synergistic effect of 1+1>2 in bone marrow cells of acute myeloid leukemia patients, further verifying the applicability of UF146 combined with abexicillin.

[0154] Example 4

[0155] This embodiment aims to verify the anti-tumor effect of UF146 combined with abexicillin at both in vitro and in vivo levels using AE9a and MLL-AF9 leukemia mouse models, and to evaluate the intervention effect of the two drugs in combination on leukemia cell proliferation, differentiation and disease progression.

[0156] I. Constructing a mouse model of leukemia (ex vivo):

[0157] Bone marrow cells were collected from wild-type (WT) C57 mice (purchased from Suzhou Cyagen Biosciences Co., Ltd.) after they reached adulthood. Lentiviral cells were obtained by co-transfecting HEK293T cells with the AE9a or MLL-AF9 target plasmid (purchased from Addgene) and the helper packaging plasmids psPAX2 and pMD2.G. The obtained lentiviruses were then used to infect bone marrow cells. The AE9a and MLL-AF9 genes are typical oncogenic fusion genes in acute myeloid leukemia (AML) and were used to construct a mouse model of leukemia, simulating two clinically prevalent and refractory AML subtypes.

[0158] Mouse-derived bone marrow cells were cultured in Opti-MEM medium containing 10% FBS, 10 ng / mL SCF, 10 ng / mL IL-3, 10 ng / mL IL-6, and FLT3. All media were supplemented with 1% penicillin and streptomycin. After thorough mixing with the medium, the cells were placed in cell culture dishes and then incubated at a constant temperature of 37°C in a cell culture incubator containing 5% CO2.

[0159] Successfully infected mouse bone marrow cells were screened and then divided into groups of 2 × 10⁻⁶ cells. 5 / dose was injected into wild-type (WT) C57 mice via tail vein to construct AE9a and MLL-AF9 models, respectively. The AE9a model simulates t(8;21) AML, which is clinically common and prone to relapse; the MLL-AF9 model simulates MLL rearranged AML, which is highly malignant and difficult to treat.

[0160] All animal studies were conducted in accordance with guidelines approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

[0161] II. In vitro pharmacodynamic evaluation

[0162] The experimental cells were obtained from primary myeloid leukemia cells from AE9a and MLL-AF9 model mice.

[0163] Primary bone marrow cells from two leukemia model mice were isolated, seeded in complete culture medium and cultured routinely, and randomly divided into a blank solvent control group, an abecicilline monotherapy group, a UF146 monotherapy group, and a combination of UF146 and abecicilline group.

[0164] In vitro cell drug administration concentrations were uniformly set: the final concentration of abexicillin monotherapy was 250 nM, and the final concentration of UF146 monotherapy was 5000 nM; the combination therapy groups used the same concentration ratio, i.e., UF146 5000 nM combined with abexicillin 250 nM. Each group was added to the corresponding formulation, and the control group received the same amount of DMSO solvent as the drug-treated groups, ensuring that the solvent concentration and culture conditions were completely consistent across groups to eliminate interference from experimental variables.

[0165] Cells from each group were continuously cultured in a 37℃, 5% CO2 incubator for 72 h. After treatment, a soft agar colony formation assay was performed to detect the colony-forming ability of each group of cells. Simultaneously, cell smears were collected from each group, stained with Giemsa stain according to standard procedures, and cell morphological changes were observed under a light microscope to analyze cell differentiation.

[0166] The results of the clonogenic assay of primary myeloid leukemia cells in AE9a and MLL-AF9 model mice are as follows: Figure 16 Figures A and B Figure 17 As shown in Figures A and B, compared with the control group and the two single-drug treatment groups, the UF146 combined with abexicillin group can significantly inhibit the clonogenic ability of AE9a and MLL-AF9 mouse primary leukemia cells.

[0167] The Giemsa staining morphological observation results of primary myeloid leukemia cells from AE9a and MLL-AF9 model mice are as follows: Figure 16 Figure C Figure 17 As shown in Figure C, the cells in the control group and the single-drug group showed obvious malignant characteristics, while the cells in the combined-drug group showed typical differentiation phenotypes such as cell nucleus shrinkage, cell body cytoplasm increase, nuclear-cytoplasm ratio decrease, and irregular nuclear morphology. The degree of cell differentiation was significantly better than that of the other groups.

[0168] III. In vivo animal efficacy evaluation

[0169] (1) Experimental methods

[0170] Three days after successfully establishing the MLL-AF9 leukemia mouse model, the model mice were randomly divided into a blank solvent control group, an abecicilline monotherapy group, a UF146 monotherapy group, and a combination of UF146 and abecicilline.

[0171] Administration methods for each group:

[0172] Control group: Administered the same amount of 50% PEG400 + 49% saline + 1% DMSO as the treatment group via intraperitoneal injection.

[0173] Abexicillin monotherapy group: Abexicillin, 50 mg / kg, was administered by gavage once daily.

[0174] UF146 monotherapy group: UF146 was administered intraperitoneally at a dose of 2.5 mg / kg every other day.

[0175] The UF146 combined with abexicillin group: Abexicillin was administered by gavage combined with UF146 by intraperitoneal injection. The specific dosage of abexicillin was 50 mg / kg once a day; the specific dosage of UF146 was 2.5 mg / kg once every other day. Based on the total dosage over two days, the total molar ratio of UF146 to abexicillin was 1:20.

[0176] Five mice were sacrificed after 15 days of continuous drug administration. Their spleens were dissected, separated, and weighed to evaluate the degree of organ infiltration by leukemia cells. The remaining mice continued to be administered the drug, and the survival time of each group was recorded throughout the process. The median survival time was calculated, and survival curves were plotted to analyze the effect of the drug on the survival time of the model mice.

[0177] Spleen weighing and morphological analysis results are as follows: Figure 18 As shown, compared with the single-drug treatment group, the spleen weight of mice in the combination drug group was significantly reduced, suggesting that the infiltration of leukemia cells into the spleen was significantly inhibited.

[0178] Survival statistics as follows Figure 19 As shown, the median survival time of mice in the control group was 21.5 days, the median survival time of mice in the abecitabine monotherapy group was 24.5 days, the median survival time of mice in the UF146 monotherapy group was 26 days, and the median survival time of mice in the combination therapy group reached 29.5 days. The survival time of mice in the combination therapy group was significantly prolonged compared with the monotherapy groups. This indicates that the combination of UF146 and abecitabine has excellent anti-tumor effects in leukemia mice.

[0179] This embodiment, through in vitro cell experiments and in vivo animal model experiments, validated that, compared with monotherapy, UF146 combined with abexicillin effectively inhibited the proliferation of AE9a and MLL-AF9 leukemia cells, blocked tumor organ infiltration, and significantly prolonged the survival of diseased mice, demonstrating outstanding synergistic therapeutic effects in animals. This combination regimen provides a new reference direction for the clinical treatment of acute myeloid leukemia.

Claims

1. A composition of UF146 and abexicillin, characterized in that, The product comprises separately packaged UF146 injectable formulation and abexicillin oral formulation, with the total two-day dosage as the smallest unit of combination; based on the total two-day dosage, the total molar ratio of UF146 to abexicillin is 1:0.05~0.4, and the molecular formula of UF146 is C 12 H8ClNO3, structural formula is The molecular formula of abexicillin is C1 27 H 32 F2N8, structural formula is .

2. The UF146 and abexicillin composition according to claim 1, characterized in that, The UF146 injectable formulation is a lyophilized powder injection or an injection solution, and the abecicilline oral formulation is a tablet, capsule, or oral solution.

3. The UF146 and abexicillin composition according to claim 1 or 2, characterized in that, The composition also contains pharmaceutically acceptable excipients.

4. The use of the composition of UF146 and abexicillin as described in any one of claims 1-3 in the preparation of a drug for treating acute myeloid leukemia, characterized in that, The acute myeloid leukemia mentioned is either AML1-ETO fusion gene positive acute myeloid leukemia or MLL-AF9 fusion gene positive acute myeloid leukemia.

5. The application according to claim 4, characterized in that, UF146 and abecitabine in the composition are administered simultaneously or sequentially.

Images