Application of PGG in the preparation of drugs for treating acute leukemia

Abstract

This invention relates to the application of PGG in the preparation of drugs for treating acute leukemia, belonging to the field of biomedical technology. To address the problems of narrow application range and easy development of drug resistance in existing acute leukemia treatments, this invention provides the application of PGG in the preparation of drugs for treating acute leukemia. Experiments have shown that PGG can inhibit the viability, proliferation, and colony formation of human acute myeloid leukemia cells (MOLM-13) and human acute lymphoblastic leukemia cells (Jurkat), and induce their apoptosis. In animal models, intraperitoneal injection of PGG can prolong the survival days of mice with acute leukemia and reduce the infiltration of leukemia cells in the liver, spleen, and bone marrow tissues. In this invention, PGG directly inhibits the viability of acute leukemia cells and can simultaneously inhibit AML and ALL, without relying on specific gene mutations, resulting in a lower risk of drug resistance, more flexible clinical use, and broader patient coverage, showing promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of PGG in the preparation of drugs for treating acute leukemia. Background Technology

[0002] 1,2,3,4,6-Penta-O-galloyl-beta-D-glucopyranose (PGG) is a typical hydrolyzed gallic tannin, widely found in various plants such as pomegranate, mango, peony, and lacquer tree, and possesses a variety of biological activities including antioxidant, anti-inflammatory, and antitumor effects. In the field of tumor research, PGG has been reported to have antitumor activity against various solid tumors, such as colorectal cancer, renal cell carcinoma, and prostate cancer. In the field of hematologic malignancies, the application of PGG in the drug-resistant chronic myeloid leukemia (CML) cell line K562 / ADR has been reported, showing that PGG can inhibit P-glycoprotein (P-gp)-mediated drug efflux, thereby reversing multidrug resistance.

[0003] Chronic myeloid leukemia (CML) is characterized by an abnormal increase in relatively mature cells, with a slow disease progression. The characteristic BCR-ABL fusion gene is the primary driving event. Its pathogenesis and drug targets are relatively singular, and its cellular sensitivity to apoptosis signals, metabolic characteristics, and drug responsiveness differ significantly from those of acute leukemia (ALL). Acute leukemia (ALL) is a group of malignant clonal hematopoietic tumors originating from hematopoietic stem cells / progenitor cells. It includes acute myeloid leukemia (AML) originating from myeloid hematopoietic stem cells / progenitor cells and acute lymphoblastic leukemia (ALL) originating from lymphoid progenitor cells. Its core characteristics are the massive proliferation of abnormal primitive and immature cells, impaired differentiation, and abnormal apoptosis, which inhibits normal hematopoietic function. The disease progresses rapidly, and without timely intervention, the short-term mortality rate is high. Therefore, the treatment of ALL aims to rapidly kill the rapidly proliferating malignant clones and restore normal hematopoiesis.

[0004] Because research on PGG has been limited to its application as a drug resistance reversal agent in drug-resistant cells of chronic myeloid leukemia, and because there are fundamental differences between acute leukemia and chronic leukemia in terms of cell differentiation, proliferation kinetics, driver genes and treatment strategies, there have never been reports of PGG being used to treat acute leukemia. Summary of the Invention

[0005] To address the issues of limited application and drug resistance in existing treatments for acute leukemia, this invention provides the application of PGG in the preparation of drugs for treating acute leukemia.

[0006] The technical solution of the present invention: The application of PGG in the preparation of drugs for treating acute leukemia, wherein the acute leukemia is acute myeloid leukemia and / or acute lymphoblastic leukemia, and the molecular formula of PGG is C1. 41 H 32 O 26 CAS No. 14937-32-7, structural formula is .

[0007] Furthermore, the acute myeloid leukemia mentioned above refers to adult acute myeloid leukemia, namely, non-acute promyelocytic leukemia.

[0008] Furthermore, the adult acute myeloid leukemia is the human acute myeloid leukemia cell line MOLM-13.

[0009] Furthermore, the acute lymphoblastic leukemia is the human acute lymphoblastic leukemia cell line Jurkat.

[0010] Furthermore, the drug uses PGG as the sole active ingredient or one of its active ingredients.

[0011] Furthermore, the dosage form of the drug is an injectable formulation or an oral formulation.

[0012] Furthermore, the drug is an inhibitor of acute leukemia cell proliferation.

[0013] Furthermore, the drug is an apoptosis inducer for acute leukemia cells.

[0014] The beneficial effects of this invention are: This invention is the first to apply PGG to the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Experiments have demonstrated that PGG can inhibit the viability, proliferation, and colony formation of human AML-13 and human ALL-Jurkat cells, and induce apoptosis. In animal models, intraperitoneal injection of PGG can prolong the survival time of mice with AML and reduce the infiltration of leukemia cells in the liver, spleen, and bone marrow. PGG, as a natural plant component, is widely available and easily accessible. Unlike existing technologies where PGG acts as a drug resistance reversal agent, this invention directly inhibits the viability of acute leukemia cells and can simultaneously inhibit AML and ALL, without relying on specific gene mutations, resulting in a lower risk of drug resistance, more flexible clinical use, and broader patient coverage, demonstrating promising clinical application prospects. Attached Figure Description

[0015] Figure 1 This is a comparison of the changes in cell viability of human acute leukemia cells MOLM-13 and Jurkat under different concentrations of PGG treatment in Example 1 over 72 hours. A represents MOLM-13 cells, and B represents Jurkat cells. Figure 2 This is a comparison of the proliferation counts of human acute leukemia cells MOLM-13 and Jurkat under different concentrations of PGG treatment in Example 2. A represents MOLM-13 cells, and B represents Jurkat cells. Figure 3 The images show a comparison of clone formation of human acute leukemia cells MOLM-13 and Jurkat under different concentrations of PGG treatment in Example 3. A is a comparison of the clone morphology of MOLM-13 cells, B is a comparison of the clone number of MOLM-13 cells, C is a comparison of the clone morphology of Jurkat cells, and D is a comparison of the clone number of Jurkat cells. Figure 4 The graph shows the apoptosis ratio of human acute leukemia cells MOLM-13 and Jurkat under different concentrations of PGG treatment in Example 4. A is a flow cytometry scatter plot of MOLM-13 cells, B is a comparison of the ratio of early apoptosis to late apoptosis of MOLM-13 cells, C is a flow cytometry scatter plot of Jurkat cells, and D is a comparison of the ratio of early apoptosis to late apoptosis of Jurkat cells. Figure 5 The image shows a comparison of the survival time of mice with acute leukemia after intraperitoneal injection of PGG in Example 5. A is the survival curve of mice with acute myeloid leukemia and B is the survival curve of mice with acute lymphoblastic leukemia. Figure 6 The images show pathological HE staining of the liver, spleen, and femoral bone marrow of the acute myeloid leukemia model mice after intraperitoneal injection of PGG in Example 6. A shows HE staining images and magnified views of the liver in the control and experimental groups; B shows the spleen staining images in the control and experimental groups; and C shows HE staining images and magnified views of the femoral bone marrow in the control and experimental groups. Detailed Implementation

[0016] 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.

[0017] All experimental data from this invention embodiment were statistically analyzed and plotted using GraphPad Prism 9.5 software. The independent samples t-test was used to compare the means between two groups. Survival analysis and plotting were performed using GraphPad Prism 9.5 software. The survival rate and median survival time of each group were calculated using the Kaplan-Meier method, and survival curves were plotted. A p-value < 0.05 was considered statistically significant. .

[0018] Example 1 To verify the inhibitory effect of 1,2,3,4,6-pentagalloglucopyranoside (PGG) on the viability of human acute myeloid leukemia cells MOLM-13 and human acute lymphoblastic leukemia cells Jurkat, and to determine the half-maximal inhibitory concentration (IC50) of PGG on the two cell types. 50 The cell viability detection experiment of this embodiment was carried out.

[0019] I. Experimental Materials Experimental drug: PGG, purchased from TargetMol Biotechnology, catalog number T3794; Dissolving reagents: dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS); Cell lines: Human acute myeloid leukemia cells MOLM-13 and human acute lymphoblastic leukemia cells Jurkat. MOLM-13 cells were purchased from Pronosun Biotechnology Co., Ltd. (catalog number CL-0681), and Jurkat cells were purchased from Pronosun Biotechnology Co., Ltd. (catalog number CL-0315). Both were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and incubated at 37°C in a humidified incubator with 5% CO2. The medium was changed / passaged every 2–3 days at a passage ratio of 1:2–1:6 to maintain the cells in the logarithmic growth phase for future use.

[0020] II. Experimental Methods (1) Preparation and storage of PGG solution: PGG powder was dissolved in organic solvent DMSO to prepare a 100mM PGG storage solution; the storage solution was diluted to the required target concentration with phosphate buffer before use. Both PGG powder and the prepared storage solution were stored in a clean environment at -80℃, and the diluted working solution was prepared fresh each time.

[0021] (2) Gradient concentration settings: Set the PGG drug concentration gradient to 0 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM.

[0022] (3) Cell culture for drug administration: MOLM-13 human acute myeloid leukemia cells and Jurkat human acute lymphoblastic leukemia cells in the logarithmic growth phase were selected and cell culture medium containing different gradient concentrations of PGG were added for intervention treatment. During the culture process, the cell culture medium containing the corresponding concentration of PGG was changed daily for 72 hours.

[0023] (4) Cell viability assay: After culture, the cell viability level of each group was detected using the CellTiter-Glo® cell viability assay kit, and the PGG half-maximal inhibitory concentration (IC50) for the two cell types was calculated. 50 .

[0024] III. Experimental Results like Figure 1 As shown, with the gradual increase of PGG concentration, the cell viability of both MOLM-13 and Jurkat human acute leukemia cell lines showed a significant decreasing trend, exhibiting a clear dose-dependent inhibitory effect; the IC50 of PGG on MOLM-13 cells was calculated. 50 The IC50 concentration of the active ingredient in Jurkat cells is approximately 9.38 μM. 50 It is approximately 0.98 μM.

[0025] The results of this embodiment demonstrate that PGG can significantly inhibit the cell viability of human acute myeloid leukemia MOLM-13 cells and human acute lymphoblastic leukemia Jurkat cells, and has a strong inhibitory effect on both types of acute leukemia cells.

[0026] Example 2 To verify the inhibitory effect of PGG on the in vitro proliferation and growth of human acute myeloid leukemia cells MOLM-13 and human acute lymphoblastic leukemia cells Jurkat, the cell proliferation experiment of this embodiment was carried out.

[0027] The source, preparation, and culture methods of PGG and human acute leukemia cells in this embodiment are the same as those in Example 1.

[0028] I. Experimental Methods MOLM-13 and Jurkat cells in the logarithmic growth phase were collected, and the cell density was adjusted to 5 × 10⁶ cells per well. 4 Cells were seeded; complete cell culture media containing different concentrations of PGG were prepared for drug treatment.

[0029] MOLM-13 cells were cultured at concentrations of 0 μM, 5 μM, and 10 μM; Jurkat cells were cultured at concentrations of 0 μM, 1 μM, and 2 μM. Fresh cell culture medium containing the corresponding concentration of PGG was added daily during the culture process. Cell counts were performed on days 2, 4, 6, and 8 of each group to record changes in cell number.

[0030] II. Experimental Results like Figure 2 As shown, compared with the blank control, all PGG treatment concentrations significantly slowed down the proliferation rate of MOLM-13 and Jurkat acute leukemia cells. At each detection time point on days 2, 4, 6, and 8, the number of cells in the PGG treatment group was significantly lower than that in the blank control group at the same time point, and showed a certain concentration-dependent inhibitory trend.

[0031] The results of this embodiment demonstrate that PGG can effectively inhibit the in vitro proliferation of human acute myeloid leukemia MOLM-13 cells and human acute lymphoblastic leukemia Jurkat cells, and significantly inhibit the growth of acute leukemia cells.

[0032] Example 3 To verify the inhibitory effect of PGG on the clonogenic ability of human acute myeloid leukemia cells MOLM-13 and human acute lymphoblastic leukemia cells Jurkat, the soft agar clonogenic assay of this embodiment was carried out.

[0033] The source, preparation, and culture methods of PGG and human acute leukemia cells in this embodiment are the same as those in Example 1.

[0034] I. Experimental Methods MOLM-13 and Jurkat cells in the logarithmic growth phase were used for soft agar colony formation experiments; a supergel system containing 0.38% low-melting-point agarose gel and the corresponding concentration of PGG was prepared.

[0035] Drug concentration settings: MOLM-13 cells were set to 0 μM and 5 μM; Jurkat cells were set to 0 μM and 1 μM. 3000 cells were seeded per well, and the culture plates were incubated continuously at 37°C in a 5% CO2 incubator for 14 days. During the culture period, 200 μL of cell culture medium containing the corresponding concentration of PGG was added to each well every 2 days to prevent the culture system from drying out. After the culture cycle, 300 μL of iodine chloride nitroxytetrazolium solution was added to each well for staining, and the cell clone counts were recorded for each group.

[0036] II. Experimental Results The results are as follows Figure 3 As shown, compared with the blank control, PGG treatment significantly reduced the clonogenic ability of MOLM-13 and Jurkat acute leukemia cells. The number of cell clones in the PGG treatment group was significantly less than that in the control group, which was statistically significant.

[0037] The results of this embodiment demonstrate that PGG can significantly inhibit the clonogenic ability of human acute myeloid leukemia MOLM-13 cells and human acute lymphoblastic leukemia Jurkat cells.

[0038] Example 4 To verify the effect of different concentrations of PGG on inducing apoptosis in human acute myeloid leukemia cells MOLM-13 and human acute lymphoblastic leukemia cells Jurkat, flow cytometry apoptosis detection experiments were conducted in this embodiment.

[0039] The source, preparation, and culture methods of PGG and human acute leukemia cells in this embodiment are the same as those in Example 1.

[0040] I. Experimental Methods MOLM-13 and Jurkat cells in the logarithmic growth phase were seeded into complete culture medium containing different concentrations of PGG for drug treatment: MOLM-13 cells were set at concentrations of 0 μM and 5 μM; Jurkat cells were set at concentrations of 0 μM and 2 μM. Cells were cultured continuously in an incubator for 48 h. After culture, cells from each group were collected, and the apoptosis rate of each group was detected by flow cytometry according to the Annexin V-APC / PI apoptosis detection kit instructions. The proportions of cells undergoing early and late apoptosis were statistically analyzed.

[0041] II. Experimental Results The results are as follows Figure 4 As shown, compared with the blank control, PGG treatment can significantly upregulate the proportion of early and late apoptosis in MOLM-13 and Jurkat cells, suggesting that PGG can effectively induce programmed apoptosis in acute leukemia cells.

[0042] The results of this embodiment demonstrate that PGG can significantly induce apoptosis in human acute myeloid leukemia MOLM-13 cells and human acute lymphoblastic leukemia Jurkat cells, providing direct molecular mechanism evidence for its anti-acute leukemia activity.

[0043] Example 5 To verify the effect of PGG administration in vivo on improving the survival of mice with acute leukemia, this in vivo pharmacodynamic study was conducted.

[0044] I. Experimental Materials and Model Construction Experimental drug: PGG, purchased from TargetMol Biotechnology, catalog number T3794; Experimental animals: C57BL / 6 wild-type mice were used to construct an acute myeloid leukemia (AML) model, and cNKG immunodeficient mice were used to construct an acute lymphoblastic leukemia (ALL) model, with 5 mice in each group; The method for constructing the AML model mouse is as follows: Bone marrow cells were collected from wild-type C57BL / 6J mice (Cyagen Biosciences, catalog number C001089); then, Lin cells rich in hematopoietic stem cells / progenitor cells were isolated using the Mouse Lineage Cell Removal Kit (Medrin, catalog numbers 130-090-858 and 130-042-401). - Cell population; subsequently, retroviruses containing the MLL-AF9 fusion gene plasmid (Addgene, catalog number #71443) were used to target Lin. - Cells were infected; after infection, GFP-positive successfully infected cells were sorted by flow cytometry to obtain mouse hematopoietic stem cells expressing the MLL-AF9 fusion gene; finally, the cells were injected into recipient mice via tail vein to establish an AML leukemia model.

[0045] The method for constructing the ALL model mouse is as follows: Bone marrow or peripheral blood samples were collected from ALL patients, and mononuclear cells were isolated using density gradient centrifugation. The isolated mononuclear cells were then injected via tail vein into cNKG immunodeficient mice that had been pretreated with 2.5 Gy irradiation.

[0046] During the growth period after inoculation, peripheral blood was collected from mice periodically, and the expression ratio of the human-specific surface marker hCD45 was dynamically detected by flow cytometry to assess the engraftment and expansion of leukemia cells in mice. When the proportion of human cells in the peripheral blood of mice reached a high level, or when mice showed obvious symptoms of leukemia such as weight loss and decreased activity, they were euthanized, and their bone marrow and spleen were collected to obtain human ALL cells.

[0047] The harvested human ALL cells were then inoculated again via the tail vein into a new batch of cNKG immunodeficient mice irradiated with a dose of 2.5 Gy for in vivo passage and expansion. After three or more stable passages, a stable ALL PDX model was obtained that could be used for subsequent experiments.

[0048] II. Dosing Regimen Administration method: Intraperitoneal injection; Dosage: The PGG treatment group received 10 mg / kg body weight per dose; the control group received an equal volume of drug-free solvent. Dosing cycle: Starting from the 3rd day of modeling, the drug was administered every other day for a total of 6 times; the volume and frequency of administration were exactly the same in both groups.

[0049] III. Experimental Methods During and after drug administration, the general condition of the mice was observed daily, and the survival status of the mice was continuously recorded until all mice died. The Kaplan-Meier method was used to plot survival curves, and the Log-rank method was used for statistical testing.

[0050] IV. Experimental Results The results are as follows Figure 5 As shown, compared with the control group, intraperitoneal injection of PGG significantly prolonged the survival time of AML and ALL model mice, and the difference was statistically significant. This suggests that PGG has a clear anti-acute leukemia activity in vivo, and the therapeutic effect can last until after drug withdrawal.

[0051] The results of this embodiment demonstrate that in vivo administration of PGG can effectively prolong the survival of mice with acute leukemia, providing crucial in vivo pharmacodynamic evidence for its clinical application.

[0052] Example 6 To verify the inhibitory effect of PGG administration in vivo on intramedullary and extramedullary infiltration of leukemia cells in a mouse model of acute myeloid leukemia, this embodiment of the pathological and histological study was conducted.

[0053] I. Experimental Materials and Model Construction Experimental drug: PGG, purchased from TargetMol Biotechnology, catalog number T3794; Experimental animals: C57BL / 6 wild-type mice, 3 mice per group; Model construction: Mouse hematopoietic stem cells (mouseHSCs MLL-AF9) expressing the MLL1-AF9 fusion gene were injected into the tail vein of mice to construct an acute myeloid leukemia model.

[0054] II. Dosing Regimen Administration method: Intraperitoneal injection; Dosage: The PGG treatment group received 10 mg / kg body weight per dose; the control group received an equal volume of drug-free solvent. Dosing cycle: Starting from the 3rd day of modeling, the drug was administered every other day for a total of 6 times. The volume and frequency of administration were exactly the same in both groups.

[0055] III. Experimental Methods Animal handling: The mice were observed daily. When the model mice became weak and near death, all experimental animals were euthanized and their livers, spleens, and femurs were removed. Tissue fixation and processing: The liver, spleen, and femur were fixed overnight in 4% paraformaldehyde solution; the liver and spleen tissues were directly subjected to routine paraffin embedding and sectioning; the femur tissue was decalcified before being paraffin embedded and sectioned. Staining and analysis: Paraffin sections from each group were stained with H&E, dehydrated and cleared, and mounted. Digital images of the pathological sections were acquired using a whole-slide scanning system to analyze the degree of leukemia cell infiltration and histopathological changes.

[0056] IV. Experimental Results like Figure 6 As shown, compared with the control group, intraperitoneal injection of PGG significantly reduced intramedullary and extramedullary infiltration of leukemia cells: Liver tissue: In the control group, a large number of densely packed, deeply stained leukemia cells were observed infiltrating the central vein and hepatic sinusoids. The lobular structure was disrupted, normal hepatocytes were compressed and destroyed, and large areas of abnormal cell aggregation appeared locally. In the experimental group, the number of leukemia cells in the hepatic sinusoids was significantly reduced, the lobular structure was basically intact, the infiltrative foci around the central vein were significantly reduced, and the morphology and distribution of normal hepatocytes were clearer. This indicates that the degree of leukemia cell infiltration in the central vein and between the hepatic lobules was significantly reduced in the PGG-treated group.

[0057] Spleen tissue: In the control group, the white pulp of the spleen exhibited disordered structure and blurred boundaries, with extensive infiltration and destruction of normal splenic corpora by leukemia cells. The red pulp region was occupied by abnormal cells, resulting in a loose and severely damaged overall structure. In the experimental group, the white pulp (splenic corpora) of the spleen had clear boundaries and intact morphology. Leukemia cell infiltration within the red pulp was reduced, the boundary between the red and white pulp structures was clearer, and the normal spleen tissue structure was protected. This indicates that the degree of damage to the normal spleen structure caused by leukemia cells was significantly alleviated in the PGG treatment group.

[0058] Femoral bone marrow tissue: In the control group, the bone marrow cavity was almost entirely filled with a large number of deeply stained leukemia cells, with an extremely low proportion of hematopoietic cells, and the normal hematopoietic structure around the bone trabeculae was completely destroyed. In the experimental group, the density of leukemia cells in the bone marrow cavity was significantly reduced, and residual normal hematopoietic cells were visible around the bone trabeculae, with a looser cell arrangement, and the normal structure of the bone marrow was partially restored. This indicates that the number of leukemia cells infiltrating the bone marrow cavity was significantly reduced in the PGG treatment group.

[0059] The results of this embodiment demonstrate that in vivo administration of PGG can effectively inhibit the spread and infiltration of acute leukemia cells in vivo, significantly reduce the degree of infiltration of leukemia cells in the liver, spleen and bone marrow tissues, reduce the damage to the normal structure of the spleen, protect the target organ tissue structure, and thus reduce the pathological damage of leukemia to important organs.

Claims

1. The application of PGG in the preparation of drugs for treating acute leukemia, characterized in that, The acute leukemia is acute myeloid leukemia and / or acute lymphoblastic leukemia, and the molecular formula of PGG is C 41 H 32 O 26 The structural formula is 。 2. The application according to claim 1, characterized in that, The acute myeloid leukemia mentioned refers to adult acute myeloid leukemia.

3. The application according to claim 2, characterized in that, The adult acute myeloid leukemia mentioned is the human acute myeloid leukemia cell line MOLM-13.

4. The application according to claim 1, characterized in that, The acute lymphoblastic leukemia mentioned is the human acute lymphoblastic leukemia cell line Jurkat.

5. The application according to any one of claims 1-4, characterized in that, The drug has PGG as its sole active ingredient or as one of its active ingredients.

6. The application according to claim 5, characterized in that, The drug is available in either injectable or oral formulation.

7. The application according to claim 6, characterized in that, The drug is an inhibitor of acute leukemia cell proliferation.

8. The application according to claim 6, characterized in that, The drug is an apoptosis inducer for acute leukemia cells.

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