A pharmaceutical composition containing 8-chloroadenosine and homoharringtonine for the co-treatment of AML

Abstract

The present invention relates to a pharmaceutical composition for treating acute myeloid leukemia. The molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is (30-100):1. The 8-chloroadenosine and homoharringtonine in the pharmaceutical composition have a synergistic killing effect on AML cell lines and primary cells of patients, and can synergistically inhibit cell proliferation and promote cell apoptosis. Therefore, the pharmaceutical composition of the present invention has good application prospects in treating patients with acute myeloid leukemia.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a pharmaceutical composition for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is the most common hematologic malignancy in adults. It is a malignant disease characterized by impaired differentiation and maturation of hematopoietic stem / progenitor cells, which inhibits normal bone marrow hematopoiesis. It is characterized by high heterogeneity, rapid progression, and a poor prognosis. AML primarily occurs in middle-aged and elderly individuals, with a median age of onset exceeding 60 years. The global burden of AML is increasing due to factors such as an aging population and environmental pollution. Despite continuous improvements in precise diagnosis and prognostic stratification systems for AML and the introduction of new targeted drugs, the early remission rate for AML has not significantly improved, and the overall prognosis remains poor, with a 5-year overall survival rate of less than 30% and a median survival of only 4 months in elderly patients. Therefore, the exploration of effective therapeutic agents and novel regimens with minimal toxicity and side effects is currently a major research focus in the treatment of AML.

[0003] Homoharringtonine (HHT), a uniquely Chinese alkaloid extracted from Cephalotaxus chinensis or its relatives in the Cephalotaxaceae family, is currently used to treat diseases such as AML and chronic myeloid leukemia. It boasts multiple targets, low toxicity, and cost-effectiveness. The HHT-based "HAA" regimen (HHT + cytarabine + aclarubicin) and "HAD" regimen (HHT + cytarabine + daunorubicin) proposed by Chinese researchers for the treatment of AML have demonstrated superior efficacy compared to the internationally standard DA or IA regimens (anthracycline combined with cytarabine) and have become first-line treatment options for adult AML in my country. The anti-leukemia mechanisms of HHT are not fully understood. Currently, the main known mechanisms include: inhibition of the phosphatidylinositol-3-kinase / protein kinase B (PI3K / AKT) pathway, inducing apoptosis in AML cells; inhibition of myeloid cell leukemia sequence 1 (MCL-1) and C-MYC protein synthesis, thereby inhibiting leukemia cell proliferation and inducing apoptosis; and upregulation of the pro-apoptotic protein BAX, thereby inducing apoptosis. However, the clinical efficacy of HHT alone is limited, and its toxicity is increased when combined with chemotherapy drugs, making it difficult for elderly patients to tolerate treatment. Therefore, exploring new HHT-based regimens, such as combining HHT with targeted drugs, traditional Chinese medicine monomers, and small molecule compounds, is a new direction for optimizing AML treatment options.

[0004] 8-Chloroadenosine (8-Cl-Ado) is a nucleoside analog synthesized by Chinese researchers in the 1990s. Early studies demonstrated that 8-Cl-Ado's antitumor mechanism involves its conversion to energy donors such as AMP, ADP, and ATP after intracellular phosphorylation, dynamically interfering with intracellular ATP formation and thereby inducing apoptosis in tumor cells. Since its introduction, 8-Cl-Ado has demonstrated excellent antitumor activity in a variety of tumors, including AML, multiple myeloma, mantle cell lymphoma, breast cancer, and non-small cell lung cancer. Toxicological studies have demonstrated that the acute toxicity of 8-Cl-Ado is significantly lower than that of known effective anticancer drugs, suggesting that 8-Cl-Ado is a promising therapeutic for AML with minimal side effects. One of the antitumor mechanisms of 8-Cl-Ado is its inhibition of mTOR (Mechanistic target of rapamycin) expression in tumor cells, which, however, leads to compensatory activation of the PI3K / AKT signaling pathway, resulting in resistance to 8-Cl-Ado.

[0005] Currently, there are no reports on the combination of 8-chloroadenosine and homoharringtonine for the treatment of AML. Summary of the Invention

[0006] To solve the above technical problems, the present invention includes the following aspects:

[0007] A first aspect of the present invention provides a pharmaceutical composition for treating acute myeloid leukemia, comprising 8-chloroadenosine and homoharringtonine.

[0008] Preferably, the molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is (30-100):1.

[0009] More preferably, the molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is (50-80):1.

[0010] More preferably, the molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is (60-70):1.

[0011] Most preferably, the molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is 62.5:1 or 70:1.

[0012] Preferably, the concentration of 8-chloroadenosine in the pharmaceutical composition is 10-1000 nM, and the concentration of homoharringtonine in the pharmaceutical composition is 1-20 nM.

[0013] More preferably, the concentration of 8-chloroadenosine in the pharmaceutical composition is 62.5 nM, 125 nM, 150 nM, 210 nM, 250 nM, 280 nM, 350 nM, 375 nM, 420 nM, 490 nM, 500 nM, 625 nM, 750 nM, 875 nM or 1000 nM.

[0014] More preferably, the concentration of homoharringtonine in the pharmaceutical composition is 1 nM, 2 nM, 2.5 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 10 nM, 12 nM, 14 nM or 16 nM.

[0015] Further preferably, the concentration of 8-chloroadenosine in the pharmaceutical composition is 62.5 nM, and the concentration of homoharringtonine is 1 nM; or, the concentration of 8-chloroadenosine is 125 nM, and the concentration of homoharringtonine is 2 nM; or, the concentration of 8-chloroadenosine is 150 nM, and the concentration of homoharringtonine is 2.5 nM; or, the concentration of 8-chloroadenosine is 210 nM, and the concentration of homoharringtonine is 3 nM; or, the concentration of 8-chloroadenosine is 250 nM, and the concentration of homoharringtonine is 4 nM; or, the concentration of 8-chloroadenosine is 280 nM, and the concentration of homoharringtonine is 4 nM; or, the concentration of 8-chloroadenosine is 350 nM, and the concentration of homoharringtonine is 5 nM; or, the concentration of 8-chloroadenosine is or, the concentration of 8-chloroadenosine is 375 nM and the concentration of homoharringtonine is 6 nM; or, the concentration of 8-chloroadenosine is 420 nM and the concentration of homoharringtonine is 6 nM; or, the concentration of 8-chloroadenosine is 490 nM and the concentration of homoharringtonine is 7 nM; or, the concentration of 8-chloroadenosine is 500 nM and the concentration of homoharringtonine is 8 nM; or, the concentration of 8-chloroadenosine is 625 nM and the concentration of homoharringtonine is 10 nM; or, the concentration of 8-chloroadenosine is 750 nM and the concentration of homoharringtonine is 12 nM; or, the concentration of 8-chloroadenosine is 875 nM and the concentration of homoharringtonine is 14 nM; or, the concentration of 8-chloroadenosine is 1000 nM and the concentration of homoharringtonine is 16 nM.

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0017] Preferably, the pharmaceutical composition is an oral preparation or a parenteral preparation.

[0018] Preferably, the pharmaceutical composition is in the form of tablets, capsules, solutions, injections or intravenous infusions.

[0019] The second aspect of the present invention provides use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating acute myeloid leukemia.

[0020] The third aspect of the present invention provides use of the above-mentioned pharmaceutical composition in the preparation of a drug for inhibiting the proliferation of acute myeloid leukemia cells.

[0021] Preferably, the acute myeloid leukemia cells are MV4-11 or MOLM-13 cell lines.

[0022] The technical effects produced by the present invention are:

[0023] The present invention unexpectedly discovered that when 8-chloroadenosine and homoharringtonine are combined in a certain molar ratio (for example, 30:1-100:1), they have a synergistic killing effect on AML cell lines and primary cells of patients, and can synergistically inhibit cell proliferation and promote cell apoptosis. Therefore, the pharmaceutical composition of the present invention has good application prospects in the treatment of patients with acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the cell survival rate of MV4-11 cell line treated with different concentrations of 8-Cl-Ado or HHT alone;

[0025] Figure 2 is the cell survival rate of MOLM-13 cell line treated with different concentrations of 8-Cl-Ado or HHT alone;

[0026] Figure 3 The IC values of MV4-11 or MOLM-13 cell lines treated with 8-Cl-Ado or HHT monotherapy for different time periods are shown in Table 1. 50 value;

[0027] Figure 4 is the cell survival rate of MV4-11 or MOLM-13 cell lines treated with different concentrations of 8-Cl-Ado, HHT alone or in combination;

[0028] Figure 5 is the cell survival rate of primary AML cell lines treated with different concentrations of 8-Cl-Ado, HHT alone or in combination;

[0029] Figure 6 The cell proliferation of MV4-11 or MOLM-13 cell lines after treatment with 8-Cl-Ado, HHT alone or in combination;

[0030] Figure 7 Flow cytometry was used to detect cell apoptosis in MV4-11 cell lines after treatment with 8-Cl-Ado, HHT alone or in combination;

[0031] Figure 8 Flow cytometry was used to detect cell apoptosis in MOLM-13 cell lines after treatment with 8-Cl-Ado, HHT alone or in combination.

[0032] Unless otherwise specified, in the figures of the present invention, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates not significant. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0034] Experimental Example 1: 8-Cl-Ado and HHT single-agent inhibition of MV4-11 and MOLM-13 cell activity test

[0035] 1. Test method

[0036] According to 2×10 5 MV4-11 and MOLM-13 cells were seeded in 24-well plates at a density of 1 ml per well. 8-Cl-Ado and HHT were treated with a concentration gradient of 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, 1600 nM, and 3200 nM, respectively. A control group containing only 1 ml of cells was set up and mixed thoroughly. After 24, 48, and 72 hours, the plates were transferred to 96-well plates, with 100 μl per well and three replicates for each concentration. 20 μl of MTS working solution was added to each well, and the cells were incubated in an incubator for 4 hours. The OD value at a wavelength of 490 nm was then measured using a microplate reader. The cell inhibition rate was calculated as follows: 1-(OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group). The half-maximal inhibitory concentration (IC) of the drug was calculated using GraphPad Prism 8.0.2. 50 Each group of experiments was repeated 3 times independently.

[0037] 2. Test results

[0038] like Figure 1 As shown, the IC of 8-Cl-Ado for MV4-11 50 The IC values of HHT against MV4-11 cell line were 1070 nM (24 h), 317.5 nM (48 h) and 146 nM (72 h), respectively. 50The values were 11.37nM (24h), 5.733nM (48h) and 4.962nM (72h) respectively. Within the same time period, as the concentration of 8-Cl-Ado and HHT increased, cell viability decreased, indicating that 8-Cl-Ado and HHT alone had a killing effect on MV4-11 cells in a concentration-dependent manner.

[0039] like Figure 2 As shown, the IC of 8-Cl-Ado on MOLM-13 cell line 50 The IC values of HHT for MOLM-13 were 1548 nM (24 h), 847.1 nM (48 h) and 127 nM (72 h), respectively. 50 The concentrations of 8-Cl-Ado and HHT were 4.730nM (24h), 2.167nM (48h) and 2.138nM (72h) respectively. 8-Cl-Ado and HHT alone also had a killing effect on MOLM-13 cells in a concentration-dependent manner.

[0040] like Figure 3 As shown in Figure 2, the IC values of 8-Cl-Ado and HHT on MV4-11 and MOLM-13 cells increased with time. 50 The values gradually decreased, indicating that the killing effects of 8-Cl-Ado and HHT alone on MV4-11 and MOLM-13 cell lines were time-dependent.

[0041] Experimental Example 2: Synergistic Inhibitory Effect of 8-Cl-Ado Combined with HHT on MV4-11 and MOLM-13 Cell Lines

[0042] 1. Test method

[0043] According to the IC values of 8-Cl-Ado and HHT alone in MV4-11 and MOLM-13 cell lines 50 For MV4-11 cells, 8-Cl-Ado concentrations of 375nM, 500nM, 625nM, 750nM, and 875nM were used, respectively, in combination with 6nM, 8nM, 10nM, 12nM, and 14nM HHT; for MOLM-13 cells, 8-Cl-Ado concentrations of 350nM, 420nM, and 490nM were used, respectively, in combination with 5nM, 6nM, and 7nM HHT. A control group containing only 1ml of cells was set up and mixed thoroughly. The cell plating density was 2×10 5cells / ml, and transferred to a 96-well plate 24 and 48 hours after plating, with 3 replicates for each concentration. Add 20 μl of MTS working solution to each well, place it in an incubator and culture for 4 hours, and then use a microplate reader to detect its OD value at a wavelength of 490 nm. According to the above cell survival rate calculation formula, the cell survival rate of each group was calculated. CalcuSyn software was used to calculate the combination index (CI value, CI>1 indicates antagonism, CI=1 indicates addition, and CI<1 indicates synergy). Each group of experiments was repeated 3 times independently.

[0044] 2. Test results

[0045] Table 18-Cl-Ado combined with HHT to kill MV4-11 and MOLM-13 cell lines combined index

[0046]

[0047] like Figure 4 As shown, 8-Cl-Ado and HHT, when used alone or in combination, inhibited cell growth in MV4-11 and MOLM-13 cell lines to varying degrees, and the combined treatments were significantly more potent in killing cells than either single-drug group. The CI values for the combined treatments of 8-Cl-Ado and HHT were all less than 1, indicating that the combination had a synergistic killing effect on MV4-11 and MOLM-13 cell lines (Table 1).

[0048] Experimental Example 3: Synergistic Inhibitory Effect of 8-Cl-Ado Combined with HHT on Primary AML Cell Lines

[0049] 1. Test method

[0050] For primary AML cells (AML#1), 8-Cl-Ado concentrations of 62.5nM, 250nM, 500nM, and 1000nM were used, respectively, in combination with HHT concentrations of 1nM, 4nM, 8nM, and 16nM. For AML#2 and AML#3, 8-Cl-Ado concentrations of 62.5nM, 125nM, 250nM, and 500nM were used, respectively, in combination with HHT concentrations of 1nM, 2nM, 4nM, and 8nM. A control group containing only 1ml of cells was set up and mixed thoroughly. The cell plating density was 1×10 6 Cells were plated at 400 cells / ml and transferred to 96-well plates 24 and 48 hours after plating, with triplicate wells set up for each concentration. 20 μl of MTS working solution was added to each well. After incubation for 4 hours, the cells were assayed using a microplate reader at 490 nm. Cell viability was calculated for each group using the aforementioned cell viability formula, and the CI value was calculated using CalcuSyn software. Each experiment was repeated three times.

[0051] 2. Test results

[0052] Table 28-Combination index of killing primary AML cells by Cl-Ado combined with HHT

[0053] cell lines <![CDATA[ED 50 ]]> <![CDATA[ED 75 ]]> <![CDATA[ED 90 ]]> AML#1 0.76439 0.56332 0.45358 AML#2 0.96876 0.76331 0.60650 AML#3 0.53835 0.39443 0.31474

[0054] like Figure 5 As shown, 8-Cl-Ado and HHT, alone or in combination, inhibited the growth of newly diagnosed primary AML cells to varying degrees, and the combined group had a stronger cell-killing effect than either single-agent group. The CI values for the combined 8-Cl-Ado and HHT treatment were all less than 1, indicating that the combination had a synergistic killing effect on primary AML cells (see Table 2).

[0055] Experimental Example 4: 8-Cl-Ado combined with HHT synergistically inhibits the proliferation of MV4-11 and MOLM-13 cell lines

[0056] 1. Test method

[0057] 1×10 5 MV4-11 and MOLM-13 cells were seeded in 96-well plates at a density of 150 cells / ml. 8-Cl-Ado was used at a concentration of 150 nM in combination with 2.5 nM HHT. A blank control group, 8-Cl-Ado alone group, HHT alone group, and a combination of the two drugs were set up. Three replicate wells were set up in each group, with 100 μl of cell solution per well. 20 μl of MTS was added to each well of each group at 0, 24, 48, 72, and 96 hours, respectively. Four hours later, the OD value at a wavelength of 490 nm was measured using a microplate reader. The cell proliferation fold of each group at each time point was calculated using the formula (OD value at each time point / OD value at 0 hour). Cell growth curves were plotted using Graphpad Prism 8.0.2 software. Each experiment was repeated three times independently.

[0058] 2. Test results

[0059] like Figure 6 As shown in the figure, compared with the control group, each single drug group and the combination drug group had different degrees of inhibitory effect on the proliferation of MV4-11 and MOLM-13 cell lines. The inhibitory effect of the combination drug group was significantly stronger than that of each single drug group and was time-dependent.

[0060] Experimental Example 5: 8-Cl-Ado combined with HHT synergistically induces apoptosis in MV4-11 and MOLM-13 cell lines

[0061] 1. Test method

[0062] According to 2×10 5MV4-11 and MOLM-13 cells were seeded in 24-well plates at a density of 1 ml per well. For MV4-11 cells, 8-Cl-Ado concentrations of 125 nM, 250 nM, 500 nM, and 1000 nM were used in combination with HHT at concentrations of 2 nM, 4 nM, 8 nM, and 16 nM, respectively. For MOLM-13 cells, 8-Cl-Ado concentrations of 210 nM, 280 nM, 350 nM, and 420 nM were used in combination with HHT at concentrations of 3 nM, 4 nM, 5 nM, and 6 nM, respectively. A blank control group, an 8-Cl-Ado monotherapy group, an HHT monotherapy group, and a combination of the two drugs were set up. After incubating the 24-well plates in an incubator for 48 hours, cells from each group were collected into centrifuge tubes and centrifuged at 1500 rpm for 5 minutes at room temperature. The culture medium was discarded and the cells were washed once with PBS, and any remaining PBS was aspirated as much as possible. To each tube, add 250 μl of 1× Binding Buffer, 12 μl of PI, and 6 μl of Annexin V FITC. Incubate at room temperature in the dark for 15 minutes before analysis by flow cytometry. Repeat each experiment three times.

[0063] 2. Test results

[0064] Table 3 Combination index of 8-Cl-Ado combined with HHT to induce apoptosis in MV4-11 and MOLM-13 cell lines

[0065] cell lines <![CDATA[ED 50 ]]> <![CDATA[ED 75 ]]> <![CDATA[ED 90 ]]> MV4-11 0.79725 0.51596 0.33402 MOLM-13 0.87099 0.71718 0.60446

[0066] like Figure 7-8 As shown, with increasing drug concentrations in each group, MV4-11 and MOLM-13 cells showed increasingly pronounced apoptosis, and the apoptotic effect of the two-drug combination group was significantly greater than that of each single-drug group. In both MV4-11 and MOLM-13 cell lines, the CI values were less than 1, indicating that 8-Cl-Ado combined with HHT synergistically induces apoptosis in AML cell lines (see Table 3).

[0067] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The active ingredients in the pharmaceutical composition consist of 8-chloroadenosine and homoharringtonine, wherein the molar ratio of 8-chloroadenosine to homoharringtonine is (60-70):

1.

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of 8-chloroadenosine to homoharringtonine in the pharmaceutical composition is 62.5:1 or 70:

1.

3. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The pharmaceutical composition is an oral preparation or a parenteral preparation.

5. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The dosage form of the pharmaceutical composition is tablet, capsule or solution.

6. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The dosage form of the pharmaceutical composition is injection.

7. The pharmaceutical composition according to any one of claims 1 to 2, characterized in that The dosage form of the pharmaceutical composition is intravenous infusion.

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

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