Use of dihydroorotate dehydrogenase inhibitors in the preparation of megakaryocytes or platelets

Abstract

The present disclosure relates to the field of biological medicine, and provides a use of a dihydroorotate dehydrogenase inhibitor in the preparation of megakaryocytes, wherein the dihydroorotate dehydrogenase inhibitor is a compound represented by formula I, formula II or formula III, or a pharmaceutically acceptable salt of the compound. The dihydroorotate dehydrogenase inhibitor in the technical solution of the present disclosure can promote the differentiation of hematopoietic stem / progenitor cells into megakaryocytes and platelets, and increase the platelet production by about 2 times, thereby providing a promising strategy for the production of megakaryocytes and platelets in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedicine, in particular, the present disclosure relates to the use of dihydroorotate dehydrogenase inhibitors in the preparation of megakaryocytes or platelets. BACKGROUND

[0002] Megakaryocytes are rare large terminally differentiated blood cells in the hematopoietic system, mainly responsible for platelet production. The decrease in number or dysfunction of megakaryocytes can lead to low peripheral platelet count, causing or aggravating a series of clinical diseases including immune thrombocytopenia (ITP), chemotherapy-induced thrombocytopenia (CIT), aplastic anemia, congenital amegakaryocytic thrombocytopenia (CAMT), and bone marrow failure diseases. Patients face serious problems such as increased risk of bleeding, interruption of anti-tumor treatment, and decreased quality of life. The current research frontier is working to overcome the above-mentioned defects, among which megakaryocyte direct infusion as one of the most revolutionary strategies aims to return functional megakaryocytes obtained by in vitro differentiation. At present, the world's first megakaryocyte injection has been approved to enter clinical trials, marking a key breakthrough from concept to clinic in this field. However, this technology still faces the challenge of large-scale and high-quality in vitro production of megakaryocytes, such as how to efficiently expand megakaryocyte-biased hematopoietic stem cells (Mk-biased HSCs) and how to ensure the functional maturity and safety of the product cells.

[0003] Platelets are small pieces of cytoplasm with biological activity that are shed from the cytoplasm of megakaryocytes, and insufficient production or dysfunction of platelets can lead to bleeding disorders. Thrombocytopenia is a condition characterized by low platelet count and is a common complication of various clinical conditions such as hematological diseases, autoimmune diseases, infections, and chemotherapy. Impaired megakaryocyte differentiation and platelet production are key contributing factors to thrombocytopenia, as platelet production is closely related to the normal maturation and function of megakaryocytes. Current treatment options, including corticosteroids, thrombopoietin receptor agonists, and rituximab, have shown efficacy in treating thrombocytopenia. However, these treatments are not without limitations. For example, thrombopoietin receptor agonists can lose efficacy over time, and long-term use can increase the risk of thrombotic events, raising significant concerns. In addition, while glucocorticoids and rituximab are useful, they can have side effects, affecting their long-term use. Currently, the main means of treating severe thrombocytopenia (such as post-chemotherapy, aplastic anemia, immune thrombocytopenia, etc.) relies on platelet infusion donated by volunteers. However, this method faces significant challenges such as blood shortage, short shelf life, immune rejection, and potential infection risks.

[0004] In view of the problems in the prior art, it is important to find a new technology that can promote megakaryocyte and platelet production in vivo or in vitro. SUMMARY

[0005] Technical problems solved:

[0006] In view of the problems in the prior art, it is important to find a new technology that can promote megakaryocyte and platelet production in vivo or in vitro.

[0007] Specifically, the present inventors have creatively found in research that dihydroorotate dehydrogenase inhibitors can promote the differentiation of hematopoietic stem / progenitor cells into megakaryocytes or platelets in vivo or in vitro, providing a new idea and means to solve the above problems.

[0008] Technical solutions:

[0009] The application of dihydroorotate dehydrogenase (DHODH) inhibitors in the preparation of megakaryocytes, wherein the dihydroorotate dehydrogenase inhibitor is a compound represented by formula I, formula II or formula III, or a pharmaceutically acceptable salt of the compound,

[0010] ;

[0011] ;

[0012] .

[0013] Another aspect of the present disclosure provides the application of dihydroorotate dehydrogenase (DHODH) inhibitors in the preparation of platelets, wherein the dihydroorotate dehydrogenase inhibitor is a compound represented by formula I, formula II or formula III above, or a pharmaceutically acceptable salt of the compound.

[0014] Another aspect of the present disclosure provides the application of dihydroorotate dehydrogenase (DHODH) inhibitors in the preparation of a drug for treating diseases caused by megakaryocyte reduction, wherein the dihydroorotate dehydrogenase inhibitor is a compound represented by formula I, formula II or formula III above, or a pharmaceutically acceptable salt of the compound.

[0015] In some embodiments, the diseases caused by megakaryocyte reduction can include aplastic anemia, myelodysplastic syndrome, acquired pure megakaryocytic aplastic thrombocytopenia, leukemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, systemic sclerosis, or infectious diseases.

[0016] Another aspect of the present disclosure provides use of a dihydroorotate dehydrogenase (DHODH) inhibitor in the manufacture of a medicament for treating complications caused by thrombocytopenia, wherein the dihydroorotate dehydrogenase inhibitor is a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt of the compound.

[0017] In some embodiments, the complications caused by thrombocytopenia can include bleeding complications and secondary complications.

[0018] In some embodiments, the bleeding complications can include skin and mucosal bleeding, nosebleed, menorrhagia, gastrointestinal bleeding, intracranial hemorrhage, uncontrolled bleeding after surgery or trauma; and the secondary complications can include anemia, infection, and organ function impairment.

[0019] Another aspect of the present disclosure provides a cell culture medium, including a medium for hematopoietic stem / progenitor cell differentiation, wherein the cell culture medium further comprises a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt of the compound.

[0020] Another aspect of the present disclosure provides a method for producing megakaryocytes in vitro, comprising:

[0021] Step (1) obtaining hematopoietic stem / progenitor cells;

[0022] Step (2) subjecting the hematopoietic stem / progenitor cells to megakaryocyte lineage directed differentiation culture, wherein the cell culture medium is replaced at about 17-18 hours after the beginning of the culture, or a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt of the compound is added to the original culture medium, and the culture is continued to day 3;

[0023] Step (3) replacing the cell culture medium and continuing the culture;

[0024] Step (4) collecting the obtained megakaryocytes;

[0025] In some embodiments, the hematopoietic stem / progenitor cells carry surface markers of Lin - c-kit + and the final concentration of the compound or the pharmaceutically acceptable salt thereof is about 4 nM to about 2 μM.

[0026] Another aspect of the present disclosure provides a method for producing platelets in vitro, comprising:

[0027] Step (1) obtaining hematopoietic stem / progenitor cells;

[0028] Step (2) carries out megakaryocyte lineage-oriented differentiation culture on the hematopoietic stem / progenitor cells, and replaces the cell culture medium at about 17-18 hours after starting the culture, or adds the compound represented by the formula I, the formula II or the formula III, or the pharmaceutically acceptable salt of the compound, to the original culture medium, and continues the culture to the third day;

[0029] Step (3) replaces the cell culture medium and continues the culture;

[0030] Step (4) collects the obtained platelets;

[0031] The hematopoietic stem / progenitor cells carry Lin - c-kit + surface markers, and the final concentration of the compound or the pharmaceutically acceptable salt thereof is about 4 nM to about 2 μM.

[0032] In some embodiments, the final concentration of the compound or the pharmaceutically acceptable salt thereof can be about 0.5 μM to about 2 μM.

[0033] Beneficial effects:

[0034] The dihydroorotate dehydrogenase inhibitor in the technical solution of the present disclosure can promote the differentiation of hematopoietic stem / progenitor cells into megakaryocytes and platelets, and increase the platelet production by about 2 times, thereby providing a promising strategy for the production of megakaryocytes and platelets in vivo and in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure for the ratio of megakaryocytes on the third day after adding different concentrations of dihydroorotate dehydrogenase inhibitor PTC299 in the embodiment of the present disclosure;

[0036] Figure 2 Figure for the ratio and quantity of platelets on the fifth day after adding different concentrations of dihydroorotate dehydrogenase inhibitor PTC299 in the embodiment of the present disclosure;

[0037] Figure 3 Figure for the ratio of platelets on the fifth day after adding 0.5 μM dihydroorotate dehydrogenase inhibitor Teriflunomide in the embodiment of the present disclosure;

[0038] Figure 4 Figure for the ratio of platelets on the fifth day after adding 10 nM dihydroorotate dehydrogenase inhibitor Brequinar in the embodiment of the present disclosure;

[0039] Figure 5 Dynamic figure for the platelet granule quantity after adding dihydroorotate dehydrogenase inhibitor in the mouse model of radiation-induced thrombocytopenia in the embodiment of the present disclosure;

[0040] Figure 6 Dihydroorotate dehydrogenase inhibitor dose response plot for megakaryocyte ratio in a radiation-induced thrombocytopenia mouse model. DETAILED DESCRIPTION

[0041] The present application discloses a kind of dihydroorotate dehydrogenase inhibitor in the application of megakaryocyte or platelet, those skilled in the art can learn from the content of this article, and appropriately improve process parameters to realize.For it needs to be pointed out specially, all similar substitutions and changes are obvious to those skilled in the art, they are all regarded as including in the present application, and relevant personnel obviously can change or appropriately change and combine described in this article based on not departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0042] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise specifically indicated, throughout the specification and claims, the terms "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements. The terms "a," "an" and "the" include plural references. The term "plurality" means two or more. The terms "such as," "for example," and the like are intended to mean exemplary implementations, and are not intended to limit the scope of the present disclosure.

[0043] In the present disclosure, when a range of values is provided, it is understood that, unless the context clearly indicates otherwise, the endpoints of the range are included and each intermediate value and any other specified or intervening value or sub-range of the specified range is encompassed.

[0044] In the present disclosure, the term "about" generally means a variation of 0.5-10% above or below the stated value, e.g., a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

[0045] In the present disclosure, “one implementation,” “one embodiment,” “some implementations,” “certain embodiments,” “related embodiments,” “certain embodiment,” “certain embodiments,” “additional embodiments,” or “further embodiments,” or “further implementations,” or “another embodiment,” “other embodiments,” means that at least one feature or characteristic described in relation to the embodiment is included in at least some implementations. Thus, the above phrases do not necessarily all refer to the same embodiment. In addition, particular features can be combined in any suitable manner in one or more implementations.

[0046] In the present disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Definitions of common terms in molecular biology can be found in Lewin’s GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. Definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: W. H. Freeman. Definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. Definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0047] Unless otherwise indicated, the experimental techniques utilized herein are according to conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, well known and within the skill of the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, John Wiley & Sons, Inc. (1999); Gait, ed., Oligonucleotide Synthesis: Methods, John Wiley & Sons, Inc. (1984); and the series Methods in Enzymology (Academic Press, Inc.).

[0048] Definitions:

[0049] The term "Dihydroorotate Dehydrogenase" (DHODH) is an enzyme that plays a key role in the biosynthetic pathway of pyrimidine nucleotides in living organisms. It catalyzes the oxidation of Dihydroorotate (DHO) to Orotate (OA) in mitochondria, which is a key step in pyrimidine synthesis. Inhibition of Dihydroorotate Dehydrogenase leads to pyrimidine depletion, blocking DNA / RNA synthesis, thus inhibiting rapidly proliferating cells, and is therefore considered a promising new anti-tumor target, and the development of its inhibitors has attracted increasing attention.

[0050] Dihydroorotate Dehydrogenase inhibitors are mainly divided into natural product inhibitors and small molecule inhibitors. Natural product inhibitors are less in number and complex in structure, for example, natural naphthoquinone and its derivatives shown in Formula IV.

[0051]

[0052] In some embodiments, the Dihydroorotate Dehydrogenase inhibitor is a small molecule inhibitor. For example, the compound PTC299 shown in Formula I, the compound Brequinar shown in Formula II, the compound Teriflunomide shown in Formula III, DSM502, AG-636, Lapachol, Izumerogant, Farudodstat, BAY-2402234, Leflunomide, L-Dihydroorotic acid, ML390, Vidofludimus, Orotic acid, Orotic acid zinc, Tenovin-6, Tenovin-1, etc.

[0053] In some embodiments, the small molecule inhibitors described above can be used individually or in combination in appropriate ratios. In some embodiments, the small molecule inhibitors described above can be used at a concentration of about 4 nM to about 2 μΜ, for example, about 4 nM, about 8 nM, about 20 nM, about 40 nM, about 80 nM, about 100 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1000 nM, about 1200 nM, about 1400 nM, about 1600 nM, about 1800 nM, about 2 μΜ. In other embodiments, the small molecule inhibitors described above can be used at a concentration of about 500 nM to about 2 μΜ.

[0054] The term "pharmaceutically acceptable salt" refers to a salt form of an active pharmaceutical ingredient (API, typically an organic molecule with pharmacological activity) with a suitable acid or base that is suitable for use in pharmaceutical formulations and administration to a living organism. Pharmaceutically acceptable salts of a drug can optimize solubility (affecting absorption), stability (extending shelf life), hygroscopicity (facilitating production), crystallinity (facilitating purification), etc. In some embodiments, the pharmaceutically acceptable salt is an acid or base salt of the compound. For example, the acid salt includes but is not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, besylate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate or nitrate; and the base salt includes but is not limited to, sodium salt, calcium salt, potassium salt, zinc salt or meglumine salt.

[0055] The term "complication" refers to a secondary, independent, usually adverse disease or pathological state that is induced in the course of development or treatment of a primary disease or medical treatment. It is not a primary part of the disease, although it can be caused by the disease or an independent cause.

[0056] The term "cell culture medium" refers to any nutritive solution used to grow cells. Cell culture media typically provide one or more of the following components: an energy source (e.g., in the form of a carbohydrate, such as glucose); one or more essential amino acids (e.g., all essential amino acids, the twenty basic amino acids plus cysteine); vitamins and / or other organic compounds that are required in low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that are required in very low concentrations. In some embodiments, the cell culture medium is further supplemented with necessary substances for differentiation of hematopoietic stem / progenitor cells into megakaryocytic lineage cells, such as, but not limited to, TPO (thrombopoietin), SCF, IL-3, IL-6, etc.

[0057] The term "Hematopoietic Stem and Progenitor Cells (HSPCs)", i.e. hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), is a type of multipotent stem cells that exist in bone marrow, peripheral blood and umbilical cord blood, have self-renewal ability and can differentiate into all types of mature blood cells (such as red blood cells, white blood cells, platelets) and immune cells, and is a core cell population for maintaining lifelong hematopoietic function of the body. They form progenitor cells of various lineages (such as myeloid progenitor cells, lymphoid progenitor cells) through stage-by-stage and multi-level proliferation and differentiation, and ultimately generate functionally specific blood cells. Among them, hematopoietic progenitor cells are progenitor cells of various blood cells that hematopoietic stem cells proliferate and differentiate into under the regulation of certain microenvironments and factors, and they are also quite primitive cells with proliferation ability, but have lost the ability of multi-directional differentiation, and can only proliferate and differentiate into one or several blood cell lines, so they are also called committed stem cells (committed stem cell). Human hematopoietic stem cells and hematopoietic stem and progenitor cells usually express CD34 surface markers.

[0058] The present disclosure provides a method for producing megakaryocytes or platelets in vitro:

[0059] In some embodiments, a method for producing megakaryocytes in vitro is provided, the method comprising:

[0060] Step (1) obtaining hematopoietic stem / progenitor cells;

[0061] Step (2) subjecting the hematopoietic stem / progenitor cells to megakaryocyte lineage directional differentiation culture, replacing the above-mentioned cell culture medium at about 17-18 hours after starting the culture, or adding the compound represented by Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt of the compound, to the original culture medium, and continuing the culture to the third day;

[0062] Step (3) replacing the above-mentioned cell culture medium and continuing the culture;

[0063] Step (4) collecting the obtained megakaryocytes;

[0064] wherein the hematopoietic stem / progenitor cells carry Lin - c-kit + surface markers, and the final concentration of the compound or the pharmaceutically acceptable salt thereof is about 4 nM to about 2 μM.

[0065] In some embodiments, the hematopoietic stem / progenitor cells can be derived from fetal liver, spleen (mouse), placenta and umbilical cord, liver or bone marrow, or can be derived from in vitro artificial / engineered cells, such as differentiated from pluripotent stem cells (examples of pluripotent stem cells, such as human embryonic stem cells isolated or obtained from fertilized human embryos less than 14 days without in vivo development; induced pluripotent stem cells), or directly reprogrammed from somatic cells.

[0066] In some embodiments, the hematopoietic stem / progenitor cells carry at least surface markers of Lin - c-kit + . Wherein, Lin - is "lineage negative". Lin - means that the cells do not express markers of mature cells (e.g., T cells, B cells, granulocytes, macrophages, red blood cells, etc.), so as to remove mature cells and most progenitor cells that have been committed to differentiation, thereby enriching more primitive cells. c-kit (also known as CD117) is an important cell surface receptor and is the receptor of stem cell factor (SCF). In the hematopoietic system, c-kit is highly expressed on very primitive hematopoietic stem cells and multipotent progenitor cells, and its expression gradually decreases as the cells mature. c-kit + is used to screen cells with stem cell characteristics.

[0067] In some embodiments, the megakaryocytic lineage directed differentiation culture of the hematopoietic stem / progenitor cells can be generally performed by using conventional procedures in the prior art. For example, (1) resuspend the obtained hematopoietic stem / progenitor cells in serum-free medium (SFM); (2) add an appropriate dose (e.g., 50-100 ng / mL) of TPO (thrombopoietin) to the above-mentioned medium for directed culture; (3) SCF, IL-3, IL-6, etc. can also be used in the above-mentioned medium; (4) detect and sort CD41 + CD42d + megakaryocytes or CD41 + CD42d + , CD42d + CD61 + platelets by flow cytometry.

[0068] In some embodiments, the cell culture medium containing the dihydrofolate dehydrogenase inhibitor in the present disclosure can be replaced for continued differentiation culture at about 17-18 hours after the start of culture. The above-mentioned about 17-18 hours can be about 17 hours, about 17.1 hours, about 17.2 hours, about 17.3 hours, about 17.4 hours, about 17.5 hours, about 17.6 hours, about 17.7 hours, about 17.8 hours, about 17.9 hours, about 18 hours.

[0069] Embodiments:

[0070] In order for those skilled in the art to better understand the technical solutions of the present disclosure, the present disclosure will be further described in detail below in combination with specific embodiments.

[0071] Example 1: Obtaining megakaryocytes and platelets in vitro

[0072] 1) Preparation of mouse embryonic E13.5-E15.5 fetal liver hematopoietic stem progenitor cells.

[0073] a. Obtain mouse embryonic fetal liver using a body microscope, and obtain total fetal liver cell suspension by filter screen grinding;

[0074] b. Add 2 ml of red blood cell lysis solution to the sample, mix thoroughly, stand for 4 min, then add 2 ml of PBE buffer (PBS+2% fetal bovine serum+0.04% 0.5 mol EDTA) for resuspension;

[0075] c. Wash the sample at 400 g for 5 min; completely remove the supernatant, try to collect all the cells, and add 1 ml of PBE for resuspension;

[0076] d. Incubate the cells at 4°C with a lineage series antibody mixture (including CD3, CD11b, CD45R / B220, Gr-1, Ter119, CD117 (c-kit)) for 30 min;

[0077] e. Obtain Lin - c-kit + HSPCs by sorting with a BD FACS Aria Fusion.

[0078] 2) Cell culture

[0079] a. Resuspend the Lin - c-kit + cells in serum culture medium Glibco DMEM added with mouse thrombopoietin (mTPO) at a starting density of 1×10 5 / mL, and inoculate in a 24-well plate, and incubate at 37°C in a 5% CO2 incubator;

[0080] b. 17 hours Add 0.5 μM, 1 μM, 2 μM final concentration of dihydrofolate dehydrogenase inhibitor PTC299 in different experimental groups respectively.

[0081] c. On the third day, replace the above culture solution with the full amount, and add dihydrofolate dehydrogenase inhibitor PTC299 to the original culture medium, and continue to incubate until the fifth day; detect CD41 + CD42d + Megakaryocytes, detect CD41 + CD42d + , CD42d + CD61 + Platelets.

[0082] 3) Detect the obtained megakaryocytes.

[0083] a. On the third day of culture, collect the cell suspension, add 1 μl anti-c-Kit-APC, 1 μl anti-CD41-PE, 1 μl anti-CD42d-Percp-cy5.5, 1 μl anti-CD61-Pe-cy7 to each group, and incubate in the dark for 30 min.

[0084] b. After centrifuging the sample at 400g for 5 min, discard the supernatant, and resuspend in 200 μl PBE.

[0085] c. Detect CD41 + CD42d + Megakaryocyte ratio by flow cytometry (BD LSR Fortessa X-20).

[0086] d. The results are shown in Figure 1 , dihydrofolate dehydrogenase inhibitor PTC299 significantly increases the ratio of CD41 + CD42d + Megakaryocytes, with an increase of about 1.5 times.

[0087] 4) Detect the obtained platelet particles.

[0088] a. On the fifth day of culture, collect the cell suspension, add 1 μl anti-c-Kit-APC, 1 μl anti-CD41-PE, 1 μl anti-CD42d-Percp-cy5.5, 1 μl anti-CD61-Pe-cy7 to each group, and incubate in the dark for 30 min.

[0089] b. Centrifuge the sample at 800 rpm for 5 min, and collect the supernatant.

[0090] c. Centrifuge the supernatant at 3000-3500 rpm for 4 min, discard the supernatant, and add 100 μl PBE and 100 μl beads to resuspend.

[0091] d. Flow cytometry (BD LSR Fortessa X-20) detection of CD41 + CD61 + , CD42d + CD61 + , and the ratio and number of platelet granules.

[0092] e. The results are shown in Figure 2 Compared with the control group, the dihydroorotate dehydrogenase inhibitor PTC299 significantly increased the ratio of CD41 + CD61 + , CD42d + CD61 + platelets, and doubled the number of CD41 + CD61 + , CD42d + CD61 + platelets.

[0093] Example 2: Obtaining megakaryocytes and platelets in vitro

[0094] 1) Preparation of mouse embryonic E13.5-E15.5 fetal liver hematopoietic stem progenitor cells.

[0095] a. Obtain the total cell suspension of the mouse embryonic fetal liver by using a body microscope and a filter screen to grind the fetal liver;

[0096] b. Add 2 ml of red blood cell lysis solution to the sample, mix thoroughly, and stand for 4 min, then add 2 ml of PBE buffer (PBS + 2% fetal bovine serum + 0.04% 0.5 mol EDTA) to resuspend;

[0097] c. Wash the sample at 400 g for 5 min; completely remove the supernatant and collect all the cells, then add 1 ml of PBE to resuspend;

[0098] d. Incubate the cells with a lineage series antibody mixture (including CD3, CD11b, CD45R / B220, Gr-1, Ter119, CD117 (c-kit)) at 4°C for 30 min;

[0099] e. Obtain Lin - c-kit + HSPCs by sorting with a BD FACS Aria Fusion.

[0100] 2) Cell culture

[0101] a. Resuspend Lin - c-kit + cells in serum medium Glibco DMEM with added mouse thrombopoietin (mTPO) at a starting density of 1x10 5 / mL, and seed in 24-well plates, and incubate at 37℃, 5% CO2 incubator.

[0102] b. Add 0.5 μM dihydroorotate dehydrogenase inhibitor Teriflunomide or 10 nM dihydroorotate dehydrogenase inhibitor Brequinar after 18 hours.

[0103] c. Replace the medium on the third day, and add dihydroorotate dehydrogenase inhibitor Teriflunomide or Brequinar to the original medium, and continue incubation until the fifth day; detect CD41 + CD42d + , CD42d + CD61 + platelets on the fifth day.

[0104] 3) Flow cytometry detection of platelet particles obtained

[0105] a. Collect cell suspension on the fifth day of culture, and add 1 μl anti-c-Kit-APC, 1 μl anti-CD41-PE, 1 μl anti-CD42d-Percp-cy5.5, and 1 μl anti-CD61-Pe-cy7 to each group, and incubate in the dark for 30 min.

[0106] b. Centrifuge the sample at 800 rpm for 5 min, and collect the supernatant.

[0107] c. After centrifuging the supernatant at 3000-3500 rpm for 4 min, discard the supernatant, and add 100 μl PBE and 100 μl beads to resuspend.

[0108] d. Detect the proportion of CD41 + CD61 + , CD42d + CD61 + platelet particles by flow cytometry (BD LSR Fortessa X-20).

[0109] e. The results are as follows: Figure 3As shown, compared with the control group, the dihydroorotate dehydrogenase inhibitor Teriflunomide significantly increased the proportion of CD41 + CD61 + , CD42d + CD61 + platelets.

[0110] f. The results are shown in Figure 4 As shown, compared with the control group, the dihydroorotate dehydrogenase inhibitor Brequinar increased the proportion of CD41 + CD61 + , CD42d + CD61 + platelets.

[0111] Example 3: Construction of a mouse model of radiation-induced thrombocytopenia

[0112] After a week of adaptation, C57BL / 6 mice were irradiated with 4.5 Gy of X-rays to establish a mouse model of thrombocytopenia.

[0113] Example 4: Platelet recovery experiment

[0114] 1) The mice in Example 3 were randomly divided into 3 groups, IR group (X-ray + PBS), dihydroorotate dehydrogenase inhibitor low-dose group (X-ray + 3 mg / kg), and dihydroorotate dehydrogenase inhibitor high-dose group (X-ray + 4.5 mg / kg).

[0115] The dihydroorotate dehydrogenase inhibitor selected in this example is PTC299.

[0116] 2) The peripheral blood platelets of the mouse model in Example 3 were counted.

[0117] a. Blood samples were collected from the tip of the mouse tail into 1.5 ml anticoagulant tubes.

[0118] b. After mixing, 10 μl of whole blood was added to 240 μl of PBS to dilute the sample, and the number of platelets was measured using a Myriad automatic blood cell counter.

[0119] c. The results are shown in Figure 5 As shown, compared with the PBS group, the addition of dihydroorotate dehydrogenase inhibitor (PTC299) significantly promoted the increase in the number of peripheral blood platelets in mice with radiation-induced thrombocytopenia.

[0120] Dynamic monitoring showed that the platelet count of mice receiving high-dose dihydroorotate dehydrogenase inhibitor (PTC299) treatment was significantly higher than that of the control group after 4 days of treatment (day 13). This increase persisted for up to 8 days (day 17) after treatment, indicating that dihydroorotate dehydrogenase inhibitors (PTC299) can effectively accelerate platelet recovery in radiation-induced thrombocytopenia.

[0121] Example 5: Bone marrow megakaryocyte number recovery experiment

[0122] 1) The mice in Example 3 were randomly divided into 3 groups, IR group (X-ray + PBS), dihydroorotate dehydrogenase inhibitor low-dose group (X-ray + 3 mg / kg), and dihydroorotate dehydrogenase inhibitor high-dose group (X-ray + 4.5 mg / kg).

[0123] The dihydroorotate dehydrogenase inhibitor selected in this example is PTC299.

[0124] 2) Flow cytometry was performed on the bone marrow megakaryocytes of the mice in Example 3.

[0125] a. On day 13, the mice were sacrificed, and the femur and tibia of the mice were stripped.

[0126] b. PBE buffer was drawn into a 1 ml syringe to flush the bone marrow.

[0127] c. The sample was centrifuged at 350g for 5 min, the supernatant was discarded, 2 ml of red blood cell lysis solution was added, and it was left to stand for 4 min, 2 ml of PBE buffer was added to stop the red blood cell lysis, and it was centrifuged at 350g for 5 min.

[0128] e. The supernatant was discarded, and the cells were resuspended in PBE buffer.

[0129] f. Anti-CD41-FITC and anti-CD42d-APC were added to the bone marrow cells at a volume ratio of 1:500, and incubated in the dark at 4°C for 30 minutes.

[0130] g. The proportion of bone marrow megakaryocytes was detected by BD LSRFortessa X-20 analytical flow cytometer.

[0131] h. The results are shown in Figure 6 As shown, compared with the DMSO group, the addition of dihydroorotate dehydrogenase inhibitor (PTC299) can significantly promote the generation of CD41 + CD42d + megakaryocytes in mice with radiation-induced thrombocytopenia.

[0132] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Use of a dihydroorotate dehydrogenase (DHODH) inhibitor for the preparation of megakaryocytes, characterized in that, The dihydrodihydrofolate dehydrogenase inhibitor is a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound, ； ； The megakaryocytes are prepared in vitro. The method for preparing comprises: Step (1) obtaining hematopoietic stem / progenitor cells; Step (2) culturing the hematopoietic stem / progenitor cells in a serum medium added with mouse thrombopoietin for megakaryocyte lineage directional differentiation, replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin at 17-18 hours after starting the culture, and continuing the culture to the third day; Step (3) replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin, and continuing the culture; Step (4) collecting the obtained megakaryocytes; wherein said hematopoietic stem / progenitor cells carry the surface markers Lin - c-kit + at a final concentration of 4 nM to 2 μM of said compound or a pharmaceutically acceptable salt thereof.

2. Use of a dihydroorotate dehydrogenase (DHODH) inhibitor for the preparation of platelets, characterized in that, The dihydrodihydrofolate dehydrogenase inhibitor is a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound; and the platelets are prepared in vitro. The method for preparing comprises: Step (1) obtaining hematopoietic stem / progenitor cells; Step (2) culturing the hematopoietic stem / progenitor cells in a serum medium added with mouse thrombopoietin for megakaryocyte lineage directional differentiation, replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin at 17-18 hours after starting the culture, and continuing the culture to the third day; Step (3) replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin, and continuing the culture; Step (4) collecting the obtained platelets; wherein said hematopoietic stem / progenitor cells carry the surface markers Lin - c-kit + at a final concentration of 4 nM to 2 μM of said compound or a pharmaceutically acceptable salt thereof.

3. Use of a dihydroorotate dehydrogenase (DHODH) inhibitor for the preparation of a medicament for the treatment of a disease associated with a reduction in megakaryocytes, characterized in that, The dihydrodihydrofolate dehydrogenase inhibitor is a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound; and the platelets are prepared in vitro. The disease associated with megakaryocyte reduction comprises aplastic anemia or acquired pure megakaryocyte reduction thrombocytopenia.

4. Use of a dihydroorotate dehydrogenase (DHODH) inhibitor for the preparation of a medicament for the treatment of complications resulting from thrombocytopenia, characterized in that, The dihydrodihydrofolate dehydrogenase inhibitor is a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound; and the platelets are prepared in vitro. The complications caused by platelet reduction comprise hemorrhagic complications and secondary complications. The hemorrhagic complications comprise skin and mucous membrane hemorrhage, nosebleed, menorrhagia, gastrointestinal hemorrhage, intracranial hemorrhage, and uncontrolled bleeding after surgery or trauma; and the secondary complications comprise anemia, infection, and organ function impairment.

5. A method for producing megakaryocytes in vitro, characterized in that, The method comprises: Step (1) obtaining hematopoietic stem / progenitor cells; Step (2) culturing the hematopoietic stem / progenitor cells in a serum medium added with mouse thrombopoietin for megakaryocyte lineage directional differentiation, replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin at 17-18 hours after starting the culture, and continuing the culture to the third day; Step (3) replacing the cell culture medium containing a compound as shown in Formula I or Formula III, or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin, and continuing the culture; Step (4) collecting the obtained megakaryocytes; wherein said hematopoietic stem / progenitor cells carry the surface markers Lin - c-kit + at a final concentration of 4 nM to 2 μM of said compound or a pharmaceutically acceptable salt thereof.

6. A method for producing platelets in vitro, characterized in that, The method comprises: Step (1) obtaining hematopoietic stem / progenitor cells; Step (2) culturing the hematopoietic stem / progenitor cells in a serum medium supplemented with mouse thrombopoietin for megakaryocyte lineage-oriented differentiation, replacing the cell culture medium containing the compound of Formula I or III as defined in claim 1 or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin at 17-18 hours after the beginning of the culture, and continuing the culture to day 3; Step (3) replacing the cell culture medium containing the compound of Formula I or III as defined in claim 1 or a pharmaceutically acceptable salt of the compound and mouse thrombopoietin, and continuing the culture; Step (4) collecting the obtained platelets; wherein said hematopoietic stem / progenitor cells carry the surface markers Lin - c-kit + at a final concentration of 4 nM to 2 μM of the compound or a pharmaceutically acceptable salt thereof.

7. The method of claim 6, wherein, The final concentration of the compound or a pharmaceutically acceptable salt thereof is 0.5-2 μM.

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