Small molecule compound 8-Me-PIQ and application thereof in in-vitro amplification of hematopoietic stem / progenitor cells

Abstract

The invention provides a small molecular compound 8-Me-PIQ and application of the small molecular compound 8-Me-PIQ in in-vitro amplification of hematopoietic stem / progenitor cells, and the compound is 8-methyl-N-(4-isopropyl phenyl)-[1, 2, 4] triazolo [4, 3-a] quinoxaline-4-amine. The compound is a safe and effective in-vitro amplification activator for human hematopoietic stem cells, can be applied to clinical HCT treatment in order to improve the transplantation efficiency of the hematopoietic stem cells, can promote the quantity amplification and colony formation of the hematopoietic stem cells cultured in vitro, can maintain the stemness and differentiation potential, and also can improve the transplantation efficiency of the hematopoietic stem cells. And the hematopoietic stem cells amplified by using the hematopoietic stem cells have long-term hematopoietic capability, can be suitable for various diseases needing hematopoietic stem cell transplantation treatment, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering and biomedicine technology, and in particular to a small molecule compound 8-Me-PIQ and an application thereof in in vitro amplification of hematopoietic stem cells and / or hematopoietic progenitor cells. Background Art

[0002] Hematopoietic stem cells (HSCs) are a type of multifunctional stem cells with the ability of self-renewal and multidirectional differentiation, which can form various mature blood cells. Hematopoietic stem cell transplantation (HCT) is an important method for treating more than 200 diseases, covering malignant and non-malignant blood system diseases, metabolic diseases and autoimmune diseases. Although it has been widely used in clinical practice, the treatment of hematopoietic stem cell transplantation is still limited by the number of donor HSCs. The number of HSCs after transplantation is closely related to the occurrence of complications such as infection and bleeding. Therefore, one of the urgent problems to be solved is to increase the number of hematopoietic stem cells and maintain their stemness.

[0003] At present, one of the important challenges in the research of the blood system is still the in vitro expansion of hematopoietic stem cells and hematopoietic progenitor cells, because as the in vitro culture time of hematopoietic stem cells increases, their transplantation ability or stemness gradually loses. Recently, Omisirge (omidubicel-onlv) was approved by the FDA for the treatment of blood cancer patients aged 12 years and above; Omisirge uses GamidaCell's proprietary nicotinamide (NAM) technology to expand the number of progenitor cells. On the one hand, the approval of this product demonstrates the feasibility of in vitro expansion of hematopoietic stem cells; on the other hand, the effects of this hematopoietic stem cell expansion product and other small molecule compounds for in vitro expansion of hematopoietic stem cells that are already under development, including SR1 and UM171, have not been widely recognized. Therefore, new small molecule compounds are urgently needed to be used to expand hematopoietic stem cells and hematopoietic progenitor cells to provide effective treatments for patients who need hematopoietic stem cell transplantation. Summary of the invention

[0004] The object of the present invention is to provide a small molecule compound or a salt thereof that can be used to effectively expand hematopoietic stem cells and hematopoietic progenitor cells, and the use of the small molecule compound or a salt thereof in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells. The compound can also be used for drug treatment of diseases related to HSC and / or HPC.

[0005] In order to achieve the above-mentioned purpose, the present invention provides an application of a small molecule compound or a salt thereof in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells, wherein the compound is 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine.

[0006] The present invention also provides the use of a small molecule compound or a salt thereof in in vitro expansion of hematopoietic stem cells and / or hematopoietic progenitor cells, wherein the compound is 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine.

[0007] The present invention also provides a pharmaceutical composition, which includes a hematopoietic stem cell and / or hematopoietic progenitor cell population expanded using the small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or its salt, and a pharmaceutically acceptable carrier.

[0008] The present invention also provides the use of a small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or a salt thereof in the preparation of a drug for hematopoietic dysfunction, malignant tumors, autoimmune diseases or hereditary immunodeficiency diseases.

[0009] As a preferred embodiment, the hematopoietic dysfunction, malignant tumor, autoimmune disease or inherited immunodeficiency disease includes bone marrow failure, lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

[0010] The present invention also provides a kit for amplifying hematopoietic stem cells and / or hematopoietic progenitor cells, the kit comprising a small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or a salt thereof.

[0011] 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxalin-4-amine, its English name: 8-methyl-N-(4-propan-2-ylphenyl)-[1,2,4]triazolo[4,3-a]quinoxalin-4-amine (abbreviated as 8-Me-PIQ), chemical formula:

[0012] C(N(C(C1=CC2)=CC=2C)C2)(C(=N1)NC(C=C1)=CC=C1C(C)C)=NN=2

[0013] Molecular formula: C 19 H 19 N 5 ; Molecular weight: 317.4. The structural formula is as follows:

[0014]

[0015] The "salt" of the compound in the present invention is preferably a pharmaceutically acceptable salt, including acid addition salts and base addition salts.

[0016] The "hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC)" in the present invention refer to human hematopoietic stem cells and / or progenitor cells, including hematopoietic stem cells and / or progenitor cells derived from bone marrow, hematopoietic stem cells and / or progenitor cells derived from mobilized peripheral blood, hematopoietic stem cells and / or progenitor cells derived from umbilical cord blood, and hematopoietic stem cells and / or progenitor cells derived from embryonic stem cells or induced pluripotent stem cells.

[0017] The present invention provides a method for increasing HSC and / or HPC, the method comprising contacting a starting cell population in the presence of a compound provided by the present invention. In an embodiment of the present invention, the starting cell population is in vivo, in vitro or ex vivo. In addition, in an embodiment of the present invention, the starting cell population comprises CD34+ cells collected from mobilized peripheral blood (mPB), bone marrow (BM) or umbilical cord blood (UCB). + cell.

[0018] According to one aspect of the present invention, the present invention provides a method for treating hematopoietic dysfunction, malignant tumors, autoimmune diseases or inherited immunodeficiency diseases in a subject, the method comprising administering HSCs expanded using the compounds disclosed herein to a subject in need of such treatment.

[0019] In an embodiment of the present invention, the hematopoietic dysfunction, malignancy, autoimmune disease or inherited immunodeficiency disease includes bone marrow failure, various congenital diseases of global concern (such as sickle cell anemia and thalassemia), lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

[0020] The present invention includes administering HSC and / or HPC expanded using the compounds of the present invention to patients suffering from any of the above diseases / malignancies, and also includes cell populations obtained after expansion using the methods according to the present invention. Hematopoietic stem cells and hematopoietic progenitor cells can be harvested from adult, cord blood, fetal or embryonic sources, and the method of cell expansion using the present invention can lead to an increase in the number of progenitor cells, which can be used to accelerate the time of implantation of neutrophils or platelets, for example. Such methods include: contacting a starting population containing HSC and / or HPC with an agent capable of increasing the number of HSC and / or HPC.

[0021] The present invention relates to a method for expanding HSCs and / or HPCs, the method comprising: (a) providing a starting cell population comprising HSCs and / or HPCs, and (b) contacting the starting cell population ex vivo under conditions suitable for expanding HSCs and / or HPCs, such as contacting the starting cell population ex vivo in the presence of a compound or composition of the present invention.

[0022] The amount of umbilical cord blood from a newborn is often insufficient to treat an adult or older child. An advantage of the expansion method using the compounds or compositions of the present invention is that it is possible to generate sufficient amounts of HSCs and / or HPCs from one umbilical cord blood unit. Thus, in one embodiment, the starting cell population is derived from a CD34-enriched + In a related embodiment, the starting cell population is derived from one or two umbilical cord blood units. In another embodiment, the starting cell population is derived from a CD34-enriched neonatal umbilical cord blood cell population. + In a related embodiment, the starting cell population is derived from human mobilized peripheral blood cells isolated from a patient.

[0023] The starting cell population may preferably contain at least 50% CD34 + cells, in some embodiments, greater than 90% CD34 + Cells. The culture conditions for expansion of the starting cell population will vary depending on the starting cell population, the desired final number of cells, and the desired final ratio of HSCs and / or HPCs.

[0024] In one embodiment, a sample enriched for CD34 + The culture conditions include the use of other cell expansion factors generally known in the art for HSC / HPC expansion, such as cytokines and growth factors.

[0025] The above-mentioned chemical components and biological components can be used not only by adding them to a culture medium, but also by fixing them on the surface of a substrate or support for culture, more specifically, by dissolving the components to be used in a suitable solvent, coating the substrate or support with the resulting solution, and then washing away the excess components. Such components to be used can be added to a substrate or support previously coated with a substance to which the components are bound.

[0026] When culturing hematopoietic stem cells and / or hematopoietic progenitor cells, different types of culture media can be used for expansion, including natural, semi-synthetic or synthetic culture media; liquid culture media are currently commonly used to expand and culture hematopoietic stem and progenitor cells. These culture media are used to provide a mixture of various nutrients and cytokines required for cell expansion, usually including sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, cytokines, hormones, antibiotics, serum, fatty acids or sugars, etc. During the culture process, other chemical or biological components can be added alone or in combination as needed. These additional components may include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, fetal bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines or growth factors, etc. The addition of these components helps to provide cells with a suitable growth environment and the required nutrients, promoting the growth and expansion of stem cells.

[0027] In which the starting cell population consists essentially of CD34-rich cells from one or two cord blood units or from mobilized PB cells or from harvested bone marrow + In a specific embodiment of the cell composition, the cells are grown under conditions for HSC and / or HPC expansion, for example, 2 to 21 days and / or until a specified multiple expansion, and a characteristic cell population is obtained. In a specific embodiment, the cells are grown in vitro under conditions for HSC and / or HPC expansion for no more than 21 days, 14 days, 10 days or 7 days. The cell population is then washed to remove the compound or composition of the present invention, and / or other components in the cell culture, and resuspended in a suitable cell suspension medium for short-term use or resuspended in a long-term storage medium, such as a medium suitable for cryopreservation.

[0028] The present invention also provides a cell population with expanded HSC and / or HPC, which can be obtained by the above-mentioned expansion method. In a specific embodiment, such a cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition. The present invention also provides a cell population or a composition thereof with expanded HSC and / or HPC for autologous or allogeneic stem cell transplantation in a mammalian subject.

[0029] The composition is formulated in any conventional manner for use in the methods described herein. In an embodiment, the composition of the present invention comprises HSC and / or HPC groups amplified using the compounds described herein. The composition of the present invention may also comprise a pharmaceutically acceptable carrier. The administration of the composition is by any known effective route by those skilled in the art. For example, the composition is administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, percutaneously, in vitro, intranasally or topically.

[0030] The preferred method of administration is intravenous infusion. The number of cells infused will take into account various factors, such as sex, age, weight, type of disease or condition, stage of condition, percentage of desired cells in the cell population, and number of cells required to produce therapeutic benefit. In one embodiment, the infused cells are all derived from expanded umbilical cord blood cells from a single newborn.

[0031] The expanded HCS and / or HPC can be infused by drip (e.g., in the case of treating leukemia) into a patient who has been pretreated with anticancer drugs, whole body irradiation or immunosuppressive drugs to eradicate cancer cells or promote the implantation of donor cells. As described above, the present invention makes it possible to expand HCS and / or HPC, and to perform transplantation therapy safely and easily in a short time by using the expanded HCS and / or HPC.

[0032] The present invention also provides a kit comprising one or more containers filled with one or more compounds of the present invention, and optionally comprising a solution and a buffer. The kit optionally includes an amplified population of stem cells manufactured by the above method, and may include a container or composition for manufacturing an amplified population of HSC and / or HPC. In particular, the present invention provides a kit for ex vivo expansion of hematopoietic stem cells, comprising a compound of the present invention and instructions for using such a compound in a HSC and / or HPC expansion method, and optional one or more cell expansion factors, or a culture medium for cell growth, particularly a culture medium for HSC and / or HPC growth as described above.

[0033] The advantage of the present invention is that the present invention provides the use of a novel small molecule compound 8-Me-PIQ in the in vitro expansion culture of hematopoietic stem cells. The compound is a safe and effective in vitro expansion activator of human hematopoietic stem cells, which can be used in clinical HCT treatment to improve the transplantation efficiency of hematopoietic stem cells. It can not only promote the expansion of the number of in vitro cultured hematopoietic stem cells, colony formation, maintenance of stemness and differentiation potential, and long-term hematopoietic ability, but also can be applied to a variety of diseases that require hematopoietic stem cell transplantation treatment, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1.8-Me-PIQ can promote the in vitro expansion of functional hematopoietic stem cells from human umbilical cord blood. (A) The expansion effect of some small molecule compound activators on functional hematopoietic stem cells; (B) The expansion fold of total cells cultured in vitro for 7 days under different concentrations of 8-Me-PIQ (n=3); (C) The expansion fold of CD34 cells cultured in vitro for 7 days under different concentrations of 8-Me-PIQ + Cell expansion times, (n=3); (D) CD34 + Flow cytometry display of cell ratio (n=3); (E) CDD34 after 7 days of in vitro expansion + Statistical graph of the number of HSC cells (n=3); n represents biological replicates, One-way ANOVA with Tukey's multiple comparison test was used to compare statistical significance, Error bars represent Mean±SEM, *p<0.05, ***p<0.001.

[0035] Figure 2 .8-Me-PIQ-cultured hematopoietic stem cells have stronger transplantation ability. (A) CD34 of freshly isolated, control group and 8-Me-PIQ-cultured hematopoietic stem cells after 7 days in vitro + The number of colony-forming units formed by cells (n=3); (BC) Human CD45 in the bone marrow of the first transplant recipient mice + The proportion of cells, representative flow cytometry display (C), and corresponding statistical results (B), (n = 10 NSG mice); n represents biological replicates, One-way ANOVA with Tukey's multiple comparison test was used to compare statistical significance, Error bars represent Mean ± SEM, *p < 0.05, ***p < 0.001.

[0036] Figure 3 The freshly isolated group and the in vitro cultured group showed the frequency of functional hematopoietic stem cells SRC. DETAILED DESCRIPTION

[0037] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0038] Example 1. In vitro expansion and functional evaluation of hematopoietic stem cells based on 8-Me-PIQ compounds

[0039] Human umbilical cord blood derived CD34 + Isolation of cells

[0040] Collect fresh human umbilical cord blood and process within 24 hours: pour 30 mL into each 50 mL centrifuge tube, add 20 mL PBS to wash, centrifuge at 500g for 10 min, discard the supernatant and mix thoroughly.

[0041] Add 15 mL of Ficoll solution (GE Healthcare, Piscataway, NJ, USA) to a new 50 mL centrifuge tube, mix the washed umbilical cord blood thoroughly along the tube wall, and perform density gradient centrifugation at 500 g for 30 min. After centrifugation, the liquid is divided into three layers. The upper supernatant is plasma and PBS, the middle layer has a white flocculent mixture, which is mononuclear cells (MNCs), and the lower layer is red blood cells and granulocytes. Use a pipette to aspirate the liquid in the middle white film layer, try to aspirate as much as possible, be careful not to aspirate the cells in the lower layer, put it into a new centrifuge tube, centrifuge at 500 g for 10 min, and discard the supernatant.

[0042] Sorting and collecting CD34 + cell:

[0043] Resuspend the cells in 1 mL of MACS buffer, take 10 μL of the cell suspension and dilute it, then count it with a cell counting plate, and use an immunomagnetic bead sorting kit (Miltenyi 130-046-703, CD34 MicroBead Kit, human) for 1^10 8 Add 100 μL FCR blocking reagent and CD34 microbeads to the cells, mix thoroughly, and incubate in a 4°C refrigerator for 30 min.

[0044] After incubation, add 10 mL of MACS buffer, centrifuge at 500 g for 10 minutes at room temperature, discard the supernatant, and resuspend the cells with 1 mL of MACS buffer.

[0045] Place the LS column (Miltenyi 130-042-401, LS Columns) on the magnetic bead sorting rack, first wet and equilibrate the LS column with 3 mL of MACS buffer, then add the cell suspension and wait for it to drip naturally. When the cell suspension is completely drained, add 3 mL of MACS buffer to wash the column, and repeat three times.

[0046] Remove the LS column from the magnetic field and place it on top of a sterile 15 mL centrifuge tube. Add 5 mL of MACS buffer and insert the corresponding piston. Push the cell suspension in the LS column quickly and forcefully to collect the dripping CD34 +Cells. Centrifuge at room temperature at 1500 rpm for 10 min, discard the supernatant, resuspend the cells in 1 mL of culture medium, and take 10 μL of the cell suspension to count on a counting plate. + DMSO or corresponding concentrations of 8-Me-PIQ were added to the cells for in vitro culture and expansion.

[0047] In vitro experiments:

[0048] Cell culture: Stem Cell Expansion Medium (Sigma, S0912) + 100 ng / mL stem cell growth factor (SCF) (R&D Systems, #7466-SC-010 / CF) + 100 ng / mL thrombopoietin (TPO) (R&D Systems, #288-TP-200 / CF) + 50 ng / mL Fms-like tyrosine kinase 3 ligand (Flt3L) (BioLegend, #710802) + 50 IU / mL penicillin + 50 μg / mL streptomycin. Cell culture conditions were 5% O 2 , 5% CO 2 Freshly isolated CD34 + The cell suspension was evenly spread in a 24-well plate, and 500 μL of cell suspension was added to each well (the cell concentration was adjusted so that the total number of cells in each well was 1*10^ 5 ), 8-Me-PIQ small molecule drug concentration was added to each well at 1 μM. The culture plate was placed at 37°C and 5% CO 2 Culture in an incubator for 7 days.

[0049] To investigate the amplification effect of different drug concentrations: 8-Me-PIQ backbone compound was purchased from TargetMol Chemicals Inc., with a stock concentration of 10 mM. It was diluted with DMSO to concentrations of 10 mM, 5 mM, 1 mM, 750 μM, 500 μM, and 200 μM. + The cells were resuspended in the prepared culture medium and plated in a 24-well plate. 500 μL of cell suspension was added to each well (the cell concentration was adjusted so that the total number of cells in each well was 10*10^ 4 0.5 μL of drug mixture was added to each well, so that the final drug concentration gradient in the culture medium was 10 μM, 5 μM, 1 μM, 750 nM, 500 nM, and 200 nM.

[0050] Colony formation assay: Collect freshly isolated CD34 +The cells were centrifuged at 500g for 5min, the supernatant was removed, and the cells were resuspended in an appropriate amount of PBS buffer. The residual culture medium and compounds (DMSO for control and 1μM for 8-Me-PIQ) in the cell suspension were washed off, and the cells were centrifuged at 500g for 5min. The supernatant was discarded, and 150μL of expansion medium was taken to resuspend the cells. The cell suspension and the semi-solid culture medium H4434 were fully vortexed and mixed, and the mixture was allowed to stand at room temperature for 20 minutes until the bubbles in the semi-solid culture medium and cell mixture disappeared. Planting cells: After replacing the needle of the 18G syringe with a 2mL syringe, 1mL of CFU culture medium after mixing cells was slowly added to a 35mm cell culture dish. Three samples were repeated for each group, and then sterile water after high pressure was added to the wells around the 24-well plate to prevent excessive evaporation of the culture medium from affecting the cell state. Statistics: Place in a 37℃ constant temperature incubator and wait for 14 days. After 14 days, the clone formation was observed under a microscope and clone images were taken under 4x, 10x, and 20x microscopes (need to reflect the clone density and size). After the shooting is completed, draw grids evenly on the bottom of the dish and count the number of clones under a microscope.

[0051] In vivo experiments: hematopoietic stem cell transplantation in NSG immunodeficient mice

[0052] Drinking water treatment: The recipient mice were 6-8 weeks old NSG mice. One week before the transplantation experiment, 0.01% antibiotics were added to the drinking water of the mice to prevent gastrointestinal infection.

[0053] Mouse irradiation: NSG mice were prepared and irradiated with a sublethal dose of 1.3 Gy using a RadSource RS2000 X-ray irradiator.

[0054] Cell collection: 8-12 hours after mouse irradiation, fresh CD34 + Cells or cells cultured with DMSO and 1 μM 8-Me-PIQ were collected into centrifuge tubes, centrifuged at 1500 rpm for 8 min, and the supernatant was discarded. The cell pellet was resuspended in PBS buffer and counted according to 5×10^ 5 Add PBS buffer to 300 μL of cells / mL and place the cell suspension into a sterile flow cytometry tube in preparation for transplantation.

[0055] Tail vein injection: Wipe the mouse tail with alcohol, disinfect and fully expose the capillaries, and inject the cell suspension through the tail vein of the mouse, 300 μL per mouse and punch holes in the mouse ears to mark the number. After the transplantation, the NSG mice were raised in an SPF-grade mouse room.

[0056] Cell reconstruction ratio detection: After 16 weeks, the reconstruction ratio of donor cells in mice was detected by flow cytometry.

[0057] Immunofluorescence staining and flow cytometry analysis of cells

[0058] After the cells were collected, they were centrifuged at 300 g for 10 minutes, washed twice with pre-cooled PBS, resuspended in 500 μL PBS, added with fluorescent antibodies and stained at 4°C for 30 minutes, washed twice with pre-cooled PBS, fixed with 1% formaldehyde, and analyzed by flow cytometry.

[0059] Antibodies to the following surface markers were used: Hematopoietic stem cells: CD34 - APC (581, BD Bioscience), CD133-BV421 (293C3, BD), ADGRG1-PE (4C3, BioLegend), CD19-PE (HIB19, BD), CD33-PEcy7 (WM53, BD) and CD45-APC (HI30, BD).

[0060] NSG mouse bone marrow and peripheral blood cells: anti-CD45-APC (HI30, BD), anti-CD19-PE (HIB19, BD), anti-CD33-PEcy7 (WM53, BD), anti-CD3-FITC (UCHT1, Biolegend).

[0061] Counting bone marrow reconstitution cells by limiting dilution

[0062] The number of transplanted bone marrow repopulating cells (SCID) (Dou Latov et al., 2012; Guo et al., 2018) was calculated using the limiting dilution method reported in 2012 and 2018. CD34 + The cells were injected into the tail vein of NSG donor mice, which had been previously irradiated with a sublethal dose. Sixteen weeks after transplantation, the mice were sacrificed and samples were collected for staining and flow cytometry to determine the expression of human CD45 + The SRC frequency of functional hematopoietic stem cells was calculated using L-Calc software (Stem Cell Technologies Inc, Vancouver, BC, Canada), and the graph was drawn using ELDA software (bioinf.wehi.edu.au / software / elda / ).

[0063] Statistical analysis

[0064] The data in this article were analyzed using statistical software GraphPad Prism 5.0., and expressed as mean ± standard deviation (SD) or standard error (SEM). The statistical analysis of the low-level and high-level MitoROS groups was performed using a two-sided T test, and P < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001).

[0065] Example 2. Screening of small molecule compounds as functional hematopoietic stem cell expansion activators

[0066] The small molecule compound skeleton comes from the Mini Scaffold (L5600, TopScience) Mini skeleton library. A total of more than 5,000 compounds were screened. After premixing them in groups of 10, they were added to hematopoietic stem cells from human umbilical cord blood at a concentration of 1 μM. 1 μM DMSO was added to the control group. After 7 days of in vitro culture, the cells in the wells were phenotyped by flow cytometry to detect the effect of small molecule compounds on the expansion of CD34+ cell populations. Through screening experiments, we found that the small molecule compound 8-Me-PIQ can expand functional HSCs under in vitro stress culture conditions ( Figure 1 A).

[0067] Example 3.8-Me-PIQ can promote the in vitro expansion of functional hematopoietic stem cells from human umbilical cord blood

[0068] In order to achieve the best effect, the amplification effect of 8-Me-PIQ at different concentration gradients was compared. Five concentration gradients of 0, 0.2, 0.5, 1, 5, and 10 μM were selected in the concentration range of 0.1 μM-10 μM. CD34 + Hematopoietic stem cells were cultured in vitro for 7 days and the effects of 8-Me-PIQ on CD34 + Effect of cell proliferation in vitro. It was found that compared with the control group, when the 8-Me-PIQ concentration was greater than 1.5 μM, the total number of cells began to decrease, but there was no statistical significance ( Figure 1 B) Flow cytometry was used to detect CD34 + The proportion of cells was found to be increased with in vitro culture. + The cell ratios were significantly increased, based on the total cell number and CD34 + The proportion of cells was further calculated as CD34 + Cell proliferation ( Figure 1 C), 8-Me-PIQ achieved the best amplification effect at 1 μM.

[0069] Freshly isolated CD34 + Compared with the cells cultured with 8-Me-PIQ, the CD34 + The number of cells increased by 22.59 times. The number of functional hematopoietic stem cells increased by 2.34 times ( Figure 1 D, E).

[0070] In addition, in order to further explore the effect of 8-Me-PIQ on the amplified CD34 + We analyzed the short-term proliferation and differentiation capacity of CD34 cells after fresh isolation, control group, and 8-Me-PIQ treatment for 7 days in vitro culture. + The cells were subjected to colony formation assay. The results showed that the number of CFU-GM in the 8-Me-PIQ group was significantly higher than that in the Vehicle group ( Figure 2 A) These results indicate that 8-Me-PIQ can significantly promote CD34 + The in vitro expansion of HSC cells can also significantly promote the in vitro expansion of CFU-GM clones to generate HPCs.

[0071] Example 4.8-Me-PIQ in vitro cultured HSCs have hematopoietic activity and self-renewal ability

[0072] To investigate whether the HSC population expanded in vitro by 8-Me-PIQ treatment is still active and functional in vivo, we transplanted freshly isolated CD3 cells from the control group (DMSO) and 8-Me-PIQ group cultured in vitro for 7 days into sublethally irradiated immunodeficient NSG mice. + The results showed that compared with freshly isolated CD34 + Cell groups and DMSO-treated CD34 + Compared with the cell group, 8-Me-PIQ expanded umbilical cord blood CD34 + CD45 + The cell chimeras were significantly higher than those in the control group and the freshly isolated group ( Figure 2 BC). This result indicates that 8-Me-PIQ-expanded cells can promote the expansion and implantation of HSCs in vivo.

[0073] We also performed bone marrow transplantation experiments in NSG immunodeficient mice to compare the freshly isolated and in vitro expanded control and 8-Me-PIQ treated human umbilical cord blood CD34 + The results of limiting dilution analysis showed that the bone marrow transplantation ability of umbilical cord blood CD34+ cells was significantly reduced after in vitro culture ( Figure 3). The analysis results showed that freshly isolated human umbilical cord blood CD34 + The frequency of SRC in the functional hematopoietic stem cells was 1 / 92241, and the frequency of SRC in the control group decreased to 1:823991. However, the frequency of SRC in the CD34 cells cultured in vitro treated with 8-Me-PIQ was significantly decreased. + The frequency of functional hematopoietic stem cells SRC in cells was 1:163691 (Table 1, Table 2). These results fully indicate that human umbilical cord blood CD34 + After the stem cells were expanded and cultured in vitro, the number of functional hematopoietic stem cells with blood reconstruction ability increased significantly compared with the control group. 8-Me-PIQ can be used as an activator drug for the in vitro expansion of functional hematopoietic stem cells.

[0074] Table 1. Statistics of transplanted mice in limiting dilution transplantation experiment

[0075]

[0076] Table 2. Frequency of functional hematopoietic stem cells SRC in fresh human umbilical cord blood CD34+ cells, control group, and SUT group

[0077]

[0078] The present invention provides a mini-framework compound 8-Me-PIQ, which can significantly promote the in vitro expansion of CD34+ hematopoietic stem cells and hematopoietic progenitor cells in a serum-free hematopoietic stem cell culture system containing 100 ng / mL of three cytokines, SCF, TPO, and Flt3L, within a concentration range of 100 nanomolar to 10 micromolar. The compound is a safe and effective in vitro expansion activator of human hematopoietic stem cells, which can be used in clinical HCT treatment to improve the transplantation efficiency of hematopoietic stem cells.

[0079] Establish an in vitro expansion system of hematopoietic stem cells with the small molecule compound or its skeleton derivative compound and its targeted gene or protein for application in hematopoietic stem cell transplantation therapy. Use the small molecule compound or its skeleton derivative compound and its targeted gene or protein-related signal pathway to achieve the maintenance of long-term hematopoietic function of hematopoietic stem cells. Use the small molecule compound to expand hematopoietic stem cells to facilitate differentiation and produce downstream mature blood cells including red blood cells, platelets, T cells, B cells, NK cells and other cell populations, which can be further applied to congenital diseases (thalassemia, etc.), autoimmune diseases, or other malignant tumors.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. For example, any modifications, equivalent substitutions, improvements, etc. made within the design concept of compound derivatives should be included in the scope of protection of the present invention.

Claims

1. Use of a small molecule compound or its salt in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells, It is characterized in that The compound is 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine.

2. Use of small molecule compounds or their salts in in vitro expansion of hematopoietic stem cells and / or hematopoietic progenitor cells, It is characterized in that The compound is 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine.

3. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises a hematopoietic stem cell and / or hematopoietic progenitor cell population expanded by using the small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or its salt, and a pharmaceutically acceptable carrier.

4. Use of the small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or its salt in the preparation of drugs for hematopoietic dysfunction, malignant tumors, autoimmune diseases or hereditary immunodeficiency diseases.

5. The use according to claim 4, It is characterized in that The hematopoietic dysfunction, malignancy, autoimmune disease or inherited immunodeficiency disease includes bone marrow failure, lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

6. A kit for expanding hematopoietic stem cells and / or hematopoietic progenitor cells, It is characterized in that The kit comprises a small molecule compound 8-methyl-N-(4-isopropylphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline-4-amine or a salt thereof.

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