WO2023051685A1 - Procédé d'amplification de progéniteurs érythroïdes ou d'érythroblastes, et application associée - Google Patents

Procédé d'amplification de progéniteurs érythroïdes ou d'érythroblastes, et application associée Download PDF

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WO2023051685A1
WO2023051685A1 PCT/CN2022/122562 CN2022122562W WO2023051685A1 WO 2023051685 A1 WO2023051685 A1 WO 2023051685A1 CN 2022122562 W CN2022122562 W CN 2022122562W WO 2023051685 A1 WO2023051685 A1 WO 2023051685A1
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alkyl
aryl
heterocyclyl
alkenyl
alkynyl
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李湘盈
吴顺康
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Peking University
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Peking University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the invention provides a method for expanding erythroid precursor cells or erythroblasts from various cell sources, promoting erythroid development, and finally producing mature denucleated erythrocytes and its application.
  • Blood transfusion is the area of greatest need.
  • autologous blood transfusion is a potential method to alleviate and restore blood loss during and after surgery. It's an important way to get enough red blood cells for those with rare blood types who don't have a readily available blood supply.
  • various diseases can be treated by the efficient expansion of red blood cells in vivo.
  • patients with bone marrow failure such as those with congenital pure red developmental aplastic anemia and Diamond-Blackfan anemia (DBA)
  • DBA Diamond-Blackfan anemia
  • the disease is currently Apart from high-dose hormones, blood transfusion, and bone marrow transplantation, there is no effective treatment.
  • EPO erythropoietin
  • CFU-E colony-forming unit erythroid progenitors
  • Some patients with chronic kidney disease will be unable to respond to conventional anemia treatments due to EPO resistance. So once a treatment that reduces EPO resistance is discovered, it could not only improve kidney inflammation in patients, but also alleviate symptoms of anemia and potentially improve patient outcomes and survival.
  • erythrocytes can serve as long-lived circulating delivery vehicles in vivo. Therefore, erythroid precursor cells can be genetically engineered so that they carry specific enzymes on their surface or inside them in genetically engineered red blood cell therapy. These engineered expanded erythrocytes can be or are designed as carriers of small molecule chemical drugs, or proteins (including various enzymes) to achieve various medical purposes of clinical treatment, for example, erythrocyte engineering can be used clinically to treat innate metabolic disorders Enzyme deficiency and other diseases that require long-term treatment in order to achieve long-term and sustained-release therapeutic effects.
  • BRAF kinase inhibitors are able to act through paradoxical activation of the MAPK pathway during erythroid development (i.e. BRAF inhibitors in cells with BRAF wild-type and upstream RAS protein activation, by binding to the wild-type BRAF ATP pocket, Keep BRAF in the activated conformation, greatly promote and stabilize the dimerization of BRAF and CRAF, so that CRAF can maintain the activated conformation, and then activate the downstream MAPK/ERK pathway, thereby promoting cell proliferation, differentiation and survival), and effectively expand the erythroid Precursor cells or erythroid cells (erythroid progenitors, EPs or erythroblasts, EBs), which can culture EPs in any erythroid medium, and the morphology, function and erythroid surface markers of EPs or EBs remain basically unchanged after treatment Change.
  • BRAF inhibitors in cells with BRAF wild-type and upstream RAS protein activation by binding to the wild-type BRAF ATP pocket, Keep BRAF in the activated conformation
  • BRAF inhibitors can greatly promote erythroid development and proliferation in conditions of cytokine deficiency such as pathological states. Moreover, when combined with existing erythropoietic agents such as TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor, BRAF Kinase inhibitors produce stronger amplification.
  • the present invention provides a medicament for the treatment of primary or secondary anemia, comprising a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
  • the medicament further comprises TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
  • the invention provides a kit comprising: a) a BRAF kinase inhibitor, and optionally b) erythropoietin.
  • the kit further includes: c) TGF- ⁇ inhibitor and/or SMAD2/3 inhibitor and/or glucocorticoid and/or erythropoietin and/or stem cell factor.
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts .
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia .
  • the invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2 /3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids in expanding erythroid precursor cells or erythroblasts.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or Or stem cell factor, or BRAF kinase inhibitor and TGF- ⁇ inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF- ⁇ inhibitor and SMAD2/3 inhibitor, and their combination with sugar A combination of corticosteroids.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin, or a BRAF kinase inhibitor agent and stem cell factor, or a combination of BRAF kinase inhibitor, erythropoietin, and stem cell factor.
  • the invention provides methods of expanding erythroid precursor cells or erythroblasts.
  • methods of expanding a population of erythroid precursor cells are also provided.
  • the EPs of the present invention have a small, round and non-adhesive uniform morphology.
  • the present invention also provides a method for preventing or treating related diseases, comprising importing a sufficient number of EPs amplified by the method, and carrying drugs to treat the diseases.
  • the present invention also provides a sufficient number of EPs or genetically modified EPs required for medicines for treating or preventing related diseases.
  • the present invention can be combined with the method WO2014183071 of the inventor's previous application for genetic modification of human EPs, which can be further differentiated into mature red blood cells with invertase or chemical drugs.
  • human EPs are expected to improve therapeutic efficacy and could be used clinically to treat genetic diseases such as phenylketonuria and even cancer.
  • Human EPs can also be engineered to produce the required enzymes on the surface of red blood cells for the development of new treatments for cancer and other diseases.
  • MEP megakaryocyte-erythroid progenitor
  • blood transfusions have become a very common therapy, and modified red blood cells have been used as natural drug delivery vehicles in the body, their clinical application is limited.
  • PBMC peripheral blood mononuclear cells
  • EPO resistance may be one of the causes of chronic inflammatory anemia. There are currently no existing treatments for EPO resistance.
  • the method provided by the present invention solves the above-mentioned problems.
  • the inventive method has at least the following advantages:
  • EPs in human PBMCs are amplified in large quantities, and the development and expression of red blood cells can be regulated by genetically modifying EPs;
  • Human EPs amplified in vitro can be used clinically, including but not limited to blood transfusion in various situations.
  • human EPs expanded in vitro can be used to treat anemia or excessive blood loss;
  • EPO concentration or EPO resistance
  • other cytokines necessary for erythroid growth it can promote the differentiation, maturation and denucleation of erythroid precursor cells or erythroblasts, and relieve EPO resistance or Symptoms of anemia caused by cytokine deficiency.
  • FIG. 1A Schematic representation of high-throughput screening (HTS) of small molecule expansion of erythroid precursor cells.
  • Figure 1B Results of high-throughput screening.
  • FIG. 1A Exploring the optimal concentration of GDC-0879 treatment.
  • Figure 2B Expansion effect of various BRAF inhibitors on erythroid precursor cells over a wide concentration range (one administration only).
  • Figure 2C Expansion effect of selected BRAF inhibitors on erythroid precursor cells over a broad concentration range (three doses).
  • Figure 3A Growth curves of three small molecules at optimal concentrations in erythroid differentiation system.
  • Figure 3B Growth curves of erythroblasts after small molecule treatment.
  • FIGS 4A-4E FACS results of GDC-0879 in differentiation medium.
  • Figures 5A-5D Colony formation results.
  • Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different Methocult H4435 administration groups and the control group
  • Figure 5B shows the results of erythrocyte-specific colony formation on the 14th day
  • Figure 5C shows The results of erythrocyte-specific colony formation on day 14 were detected under a bright-field microscope using a 5 ⁇ objective lens.
  • Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
  • Figures 6A-6E Effects of small molecules targeting BRAF on promoting proliferation of erythroid precursor cells.
  • Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 ⁇ M, and the treatment is 9 days), and
  • Figure 6B shows that other inhibitors of BRAF are effective in formazan containing only EPO.
  • Figure 6C shows the erythrocyte-specific colony formation on day 14 of other combinations containing BRAF inhibitors in methylcellulose medium containing EPO only
  • Fig. 6D shows the colony formation results under the light field microscope
  • Fig. 6E shows the colony forming unit counting results of each well of Fig. 6B and 6C.
  • Figures 7A-7E Effective dosing cycle assay.
  • Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell number statistics were performed on the 15th day
  • Figure 7B shows the 0-3 day treatment group and the control group Comparison of the number of cells on the 13th day
  • Figure 7C shows the FACS results of the experimental group and the control group on the 13th day in the differentiation medium
  • Figure 7D shows that the statistics of the enucleation rate of each group administered for 3 days on the 13th to 16th day ( Treatment group, 1 ⁇ M GDC-0879 treatment group)
  • Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to 16 (treatment group: 1 ⁇ M GDC-0879 treatment).
  • Figures 8A-8K In vitro expansion of erythroblasts in PBMCs.
  • Figure 8A shows the cells of the treatment group and the control group on the 9th day
  • Figure 8B shows the change in the number of erythroblasts in the PBMC of healthy donors (HD1-HD5) in vitro
  • Figure 8C shows the changes in the number of erythroblasts in the healthy donors (HD1-HD5) on the 9th day (HD1-HD5)
  • PBMCs were cultured in serum-free erythrocyte expansion medium on the 9th day of FACS results
  • Figure 8D shows the statistics of the FACS results of healthy donors (HD1-HD5) PBMCs on the 10th day cell group ratio
  • Figure 8E shows the statistics of the ratio of cell groups on the 10th day of FACS
  • Figure 8F shows the FACS results of PBMCs from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium
  • Figure 8G shows the results of healthy donors (Donor1
  • Figures 9A-9L Therapeutic effects of drugs under low EPO conditions.
  • Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium
  • Figure 9B shows the FACS results on day 14 after 5-10 days of culture without EPO
  • Figure 9C shows the 1/3 concentration FACS results on the 15th day after cultured under EPO conditions for 1-15 days
  • Figure 9D shows the erythrocyte denucleation rate under the conditions of various EPO concentrations from 13 to 19 days
  • Figure 9E shows the concentration of erythrocytes with 0% SCF or 5% EPO at low cytokine concentrations
  • Figure 9F shows 0% SCF (left) and 5% EPO ( Right)
  • Figure 9G and 9H
  • FIGS. 9I and 9J are the erythrocyte surface markers ( The expression of CD117, CD71 and CD235),
  • Figure 9K is a typical erythrocyte photo of samples cultured under different stress conditions,
  • Figure 9L representative Giemsa-benzidine stained erythrocyte images under different pressure culture conditions.
  • Figures 10A-10F Effects of in vitro treatment of anemia caused by defective pure red blood cell regeneration after hematopoietic stem cell transplantation.
  • Figure 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitor cells in erythrocyte development failure or pure erythrocyte aplasia after hematopoietic stem cell transplantation
  • Figure 10B shows the bone marrow single Growth curves of nucleated cells (BMMNCs)
  • Figure 10C shows the erythrocyte surface markers ( CD71, CD235 and Hoechst 33342)
  • Figure 10D is the representative Giemsa-benzidine stained erythrocytes obtained from EFAHSCT BMMNCs after erythroid culture
  • Figure 10E is 50000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium
  • Representative images in Figure 10F show the number of colonies of different lineages from 50,000 EFAHSCT-derived BMMNCs cultured
  • FIG. 11 SB-590885 potently expands erythroid precursors in peripheral mononuclear cells from DBA anemia patients.
  • Figure 12 Statistics of SB-590885 effectively expanding erythroid precursor cells in peripheral mononuclear cells of DBA anemia patients.
  • Figures 13A-13E BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation in MDS patient cells.
  • Figure 14 Expansion of hematopoietic stem progenitor cells (HSPC) by BRAF inhibitors.
  • FIG. 15 BRAF inhibitors promote hematopoiesis in mice (normal conditions).
  • FIGS 16A-16E BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
  • FIGS 17A-17B BRAF inhibitors promote hematopoiesis in mice (acute hemolysis model).
  • FIG. 18 BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation.
  • Figure 20 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is dependent on both upstream cytokine signaling and downstream signaling.
  • alkyl refers to a saturated linear or branched chain hydrocarbon group of 1 to 18 carbon atoms, wherein said alkyl group may be optionally substituted independently by one or more substituents described below.
  • the alkyl group may be monovalent, and examples thereof include C 1 -C 12 alkyl, C 1 -C 8 alkyl, C 1 -C 6 alkyl, such as methyl (Me, -CH 3 ), ethyl ( Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2-butyl (s
  • the alkyl group may have 2 monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Examples include, but are not limited to , methylene ( -CH2- ), 1,2-ethyl (-CH2CH2-) , 1,3-propyl ( -CH2CH2CH2- ) , 1, 4-Butyl (-CH 2 CH 2 CH 2 CH 2 -) etc.
  • alkenyl refers to a straight or branched chain hydrocarbon radical of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp double bond, wherein said alkenyl radical may optionally be replaced by one or more
  • the substituents are independently substituted and include groups with cis and trans orientations, or "E" and "Z" orientations.
  • alkyl and alkenyl groups alone or as part of a larger group such as an alkoxy group (i.e. -OR group where R is the alkyl and alkenyl group) or as part of a larger group such as an alkylthio group moieties (ie -SR groups, where R is the alkyl and alkenyl groups described).
  • an alkoxy group i.e. -OR group where R is the alkyl and alkenyl group
  • alkylthio group moieties ie -SR groups, where R is the alkyl and alkenyl groups described.
  • alkynyl refers to a straight or branched chain hydrocarbon group of 2 to 18 carbon atoms, having at least one site of unsaturation, i.e. a carbon-carbon sp triple bond, wherein said alkynyl group may optionally be replaced by one or more The above substituents are independently substituted.
  • the alkynyl group may be monovalent, examples of which include C 2 -C 12 alkynyl, C 2 -C 8 alkynyl, C 2 -C 6 alkynyl, including but not limited to, ethynyl (-C ⁇ CH) and propynyl (propargyl, -CH 2 C ⁇ CH) and the like.
  • the alkynyl group may have two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Examples include, but are not limited to, ethynylene (-C ⁇ C-), propargylene ( -CH2C ⁇ C- ) , and 4-pentynylene ( -CH2CH2CH2C ⁇ C- ) wait.
  • aryl refers to a monovalent aromatic hydrocarbon radical having 6 to 20 carbon atoms, resulting from the removal of a hydrogen atom from a carbon atom of a parent aromatic ring system.
  • aryl groups include C 6-14 aryl, C 6-10 aryl, and some aryl groups in the exemplary structures are represented by "Ar".
  • Aryl includes bicyclic rings containing an aromatic ring fused to a non-aromatic or partially saturated ring.
  • Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3, 4-tetrahydronaphthyl, etc.
  • Aryl groups are optionally substituted independently with one or more substituents described herein.
  • heterocycle refers to ring atoms having 3 to 25 ring atoms (containing 2 to 20 carbon atoms and 1 to 5 ring atoms selected from N, O, P and heteroatoms of S), saturated, partially saturated (i.e., having one or more double and/or triple bonds within the ring) or aromatic carbocyclic groups in which at least one ring atom is independently selected from nitrogen, oxygen and sulfur and the remaining ring atoms are carbon, wherein one or more ring atoms are optionally independently substituted with one or more substituents described herein.
  • the heterocycle can be a monocyclic ring having 3 to 7 ring atoms (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a ring having 7 to 10 ring atoms (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclic [4,5], [5,5], [5,6] or [6,6 ]system.
  • Heterocycles are described in: Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A.
  • a heterocyclyl group can be a carbon-bonded group or a heteroatom-bonded group.
  • heterocycle includes heterocycloalkoxy.
  • Heterocyclyl also includes radicals in which heterocyclyl is fused to a carbocyclic, heterocyclic, aromatic or heteroaromatic ring.
  • heterocyclic groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidine Base, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrroliny
  • Spiro groups are also included within the scope of this invention.
  • the heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.
  • heteroaryl includes 1) monocyclic aromatic 5-, 6- and 7-membered rings containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur; and 2) 8 to 20 atom Fused ring systems wherein at least one aromatic ring contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • heteroaryl groups are pyridyl (including, for example, 2-hydroxypyridyl), imidazolyl, imidazopyridyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, thiazolyl, pyrazinyl, tetra Azolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolyl, indolyl, benzimidazolyl, benzofuryl , cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl , thiadiazoly
  • Heterocycles can be carbon-attached or nitrogen-attached (if applicable). For example, but not limited to: carbon-bonded heterocyclic ring binding position at 2, 3, 4, 5 or 6 positions of pyridine; 3, 4, 5 or 6 positions of pyridazine; 2, 4, 5 or 6 positions of pyrimidine ; 2, 3, 5 or 6 positions of pyrazine; 2, 3, 4 or 5 positions of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; 2, 4 or 5 of oxazole, imidazole or thiazole position; 3, 4 or 5 positions of isoxazole, pyrazole or isothiazole; 2 or 3 positions of aziridine; 2, 3 or 4 positions of azetidine; 2, 3, 4 positions of quinoline , 5, 6, 7 or 8 positions; or 1, 3, 4, 5, 6, 7 or 8 positions of isoquinoline.
  • nitrogen-bonded heterocyclic rings are bound at aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline , 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1-position of 1H-indazole; isoindole or 2-position for isoindoline; 4-position for morpholine; and 9-position for carbazole or ⁇ -carboline.
  • Carbocycle refers to a monocyclic non-aromatic, saturated or unsaturated ring having 3 to 12 carbon atoms, or a ring having 7 to A bicyclic non-aromatic, saturated or unsaturated ring of 12 carbon atoms.
  • Monocyclic carbocycles have 3 to 7, or 3 to 6, more usually 5 or 6 ring atoms.
  • Bicyclic carbocycles have 7 to 12 ring atoms, for example arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system; or have 9 or 10 ring atoms, arranged Form bicyclo[5,6] or [6,6] systems, or become bridged ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.
  • Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclo Hexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, Cyclodecyl, Cycloundecyl and Cyclododecyl.
  • protecting group refers to a substituent, typically used to block or protect a particular functional group while reacting other functional groups of a compound.
  • an "amino-protecting group” is a substituent attached to an amino group that blocks or protects the amino function in a compound.
  • Suitable amino protecting groups include acetyl, trifluoroacetyl, phthalimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc ).
  • hydroxyl protecting group refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality.
  • Suitable hydroxy protecting groups include acetyl, trialkylsilyl, dialkylphenylsilyl, benzoyl, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl group and tetrahydropyranyl group.
  • Carboxy protecting group refers to a carboxyl substituent that blocks or protects the carboxyl functionality.
  • Common carboxyl protecting groups include -CH2CH2SO2Ph , cyanoethyl , 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2 -(p-tosyl)ethyl, 2-(p-nitrophenylsulfinyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl and the like.
  • protecting groups and their use see TW Greene and P. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons, New York, 1999; and P. Kocienski, Protecting Groups, 3rd ed., Verlag, 2003.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being activated enzymatically or hydrolytically or by Conversion to the more active parent form. See, eg, Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery” Directed Drug Delivery , Borchardt et al., (ed.), pp. 247-267, Humana Press (1985).
  • Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs and others that can be Conversion of 5-fluorouridine prodrugs to more active non-cytotoxic drugs.
  • Examples of cytotoxic drugs that can be derivatized as prodrugs for use in the present invention include, but are not limited to, those chemotherapeutic drugs described above.
  • alkyl, alkenyl, cycloalkyl, and cycloalkenyl groups described herein can be optionally substituted with the following substituents: aryl, heteroaryl, heterocyclyl, C 1-6 alkoxy, C 1-6 alkylthio, aryl C 1-6 alkoxy, aryl C 1-6 alkylthio, amino, mono- or di-C 1-6 alkylamino, aminosulfonyl, cycloalkyl, Cycloalkenyl, carboxyl and its ester, amide, ureido, guanidino, C 1-6 alkyl guanidino, amidino, C 1-6 alkyl amidino, C 1-6 acyloxy, hydroxyl and halogen or Any of their combined substituents.
  • Another substituent may also be a cyano group.
  • Preferred optional substituents include water-soluble groups; those of ordinary skill in the art are familiar with suitable water-soluble moieties, including hydroxyl, amino groups. More preferred optional substituents include amino, mono- or di-C 1-6 alkyl, amino, amino-containing heterocyclyl or hydroxy or any combination thereof.
  • the aryl, heterocyclyl, and heteroaryl groups described herein can be optionally substituted with the following substituents: halogen, hydroxy, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1-6 alkyl , halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, nitro, cyano, azido , amino, mono- and di-NC 1-6 alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, carbamoyl, mono- and di-NC 1-6 alkyl Carbamoyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, urea, carbamic acid Est
  • heteroC 1-6 alkyl- refers to a C 1-6 carbon chain in which the terminal carbon atoms of the chain are replaced by heteroatoms selected from N, O or S, for example, C 1-6 alkylamino, C 1 -6 alkoxy, C 1-6 alkylthio.
  • C 1-6 alkylheteroC 1-6 alkyl refers to a C 3-13 alkyl chain in which one carbon atom is replaced by a heteroatom selected from N, O or S, for example, C 1-6 Alkylamino C 1-6 alkyl or C 1-6 alkylaminodiC 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl-, C 1-6 alkylthio C 1- 6 alkyl-, or C 1-6 alkylthio diC 1-6 alkyl.
  • stereoisomer refers to compounds that have identical chemical constitution but differ in the arrangement of the atoms or groups in space.
  • tautomer or “tautomeric form” refers to structural isomers of different energies, which are interconvertible via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers involve interconversion by recombination of some of the valence electrons.
  • the term "pharmaceutically acceptable” means that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients making up the formulation and/or the mammal receiving its treatment.
  • the term "pharmaceutically acceptable salt” refers to those carboxylate, amino acid addition salts of the compounds of the present invention, which are suitable within the scope of sound medical judgment for use in contact with patient tissues without undue toxicity, irritation Effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are valid for their intended application, including, where possible, zwitterionic forms of the compounds of the invention.
  • isotopic derivative refers to a compound in which one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature. All isotopes of any particular atom or element specified and their use are intended to be within the scope of the compounds of the present invention. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I and 125 I.
  • solvate refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
  • hydrate refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined.
  • a hydrate of a compound can be represented, for example, by the general formula RxxH2O , where R is the compound, and x is a number greater than zero.
  • a given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R ⁇ 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)).
  • the compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
  • polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • More expansion of erythroid progenitors and erythroblasts in CD34+ derived cells obtained from bone marrow, umbilical cord blood or iPSC/ESC is also of scientific interest, especially the expansion of erythroid precursors allows us to obtain more erythroid lineage stem/progenitor cells, thereby enriching our understanding of the mechanisms underlying erythroid development.
  • EPs or EBs are derived from stem cells in donor PBMCs.
  • the stem cells are genetically modified prior to or during culture.
  • the stem cells are hematopoietic stem cells.
  • the hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
  • the subject of the experiment is a mammal. In another example, the subject is a human, mouse or rat.
  • erythroid precursor cells or erythroblasts can be cultured in vitro in culture medium.
  • Media include erythroid differentiation medium or serum-free erythroid differentiation medium and expansion medium.
  • the erythrocyte differentiation medium comprises IMDM, 10% FBS, 5% human serum, 300 ⁇ g/ml holo-transferrin, 2 IU/ml heparin, 10 ⁇ g/ml insulin, 2 mM L-glutamine; then supplemented with 3 IU /ml erythropoietin, 50ng/ml human stem cell factor and 10ng/ml interleukin (IL)-3.
  • erythrocyte differentiation and expansion in serum-free medium includes SFEM II supplemented with 3 IU/mL erythropoietin, 50 ng/ml human stem cell factor, 10 ng/ml interleukin (IL)-3 and Insulin-like growth factor (IGF)-1.
  • SFEM II supplemented with 3 IU/mL erythropoietin, 50 ng/ml human stem cell factor, 10 ng/ml interleukin (IL)-3 and Insulin-like growth factor (IGF)-1.
  • preferred BRAF kinase inhibitors are compounds of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • the BRAF kinase inhibitor induces paradoxical activation of MAPK;
  • the BRAF kinase inhibitor promotes RAF protein dimerization;
  • the BRAF kinase inhibitor induces ⁇ C-helix and DFG domain in BRAF kinase IN conformation of at least one of them, and the IN conformation of R506;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • TGF- ⁇ inhibitors currently on the market and in clinical practice are well known to those skilled in the art, and all of them can be used for the purpose of the present invention.
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • the SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
  • Glucocorticoid is a kind of adrenocortical hormone, which is a steroid hormone secreted by the zona fascicularis in the middle layer of the adrenal cortex, and can also be synthesized artificially in vitro by chemical methods. It plays an important role in regulating the body's development, growth, metabolism, and immune function. It is the most important regulatory hormone for the body's stress response, and it is also the most widely used and effective anti-inflammatory and immunosuppressant clinically. In urgent or critical situations, glucocorticoids are often the drug of choice.
  • Glucocorticoid drugs mainly act on the downstream pathway of intracellular glucocorticoid receptors, increase the production of red blood cells and hemoglobin, increase platelets and fibrinogen, increase the amount of monocytes and neutrophils entering the blood circulation, and lymphocytes and decreased eosinophils and basophils.
  • Glucocorticoids are clinical drugs currently used to treat anemia, especially refractory anemia, bone marrow failure anemia (such as DBA anemia), which can expand erythroid precursor cells (BFU-E and CFU-E) in vitro , can also be used in vivo to treat anemia and promote the production of red blood cells in the body.
  • Anemia diseases are mainly classified into anemia caused by decreased or defective red blood cell production, anemia caused by destruction of red blood cells, and anemia caused by excessive blood loss.
  • the anemia caused by reduced or defective erythropoiesis may be primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome, dyskeratosis congenita, Fanconi anemia, Anemia due to congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia myelodysplastic syndrome.
  • the anemia caused by the reduction or defect of red blood cell production can also be secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency anemia, iron deficiency anemia, chronic Inflammatory anemia, anemia of endocrine disease, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary aplastic anemia is caused by: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO incompatibility, stem cell transplantation, pyoderma gangrenosum), lymphoproliferative diseases (such as Chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin lymphoma, etc.), other hematological malignancies (such as
  • the anemia caused by the destruction of red blood cells may be primary, including hereditary spherocytosis, hereditary ellipsocytosis, abeta lipoproteinemia, anemia caused by enzyme deficiency (such as pyruvate kinase and hexose Kinase deficiency leads to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress).
  • enzyme deficiency such as pyruvate kinase and hexose Kinase deficiency leads to glycolysis deficient anemia, glucose 6-phosphate dehydrogenase deficiency and glutathione synthetase deficiency, anemia due to increased oxidative stress.
  • the anemia caused by the destruction of red blood cells can also be secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic diseases (such as hemolytic disease of newborns), red blood cell Mechanical injury (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), infection-induced hemolytic anemia (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia e.g, cold agglutinin hemolytic anemia, hemolytic diseases (such as hemolytic disease of newborns), red blood cell Mechanical injury (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), infection-induced hemolytic anemia (eg, malaria infection).
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood sampling in laboratory tests, and optionally combined with insufficient erythropoiesis), external/internal injuries (acute blood loss caused by various traumas or operations), gastrointestinal lesions Acute bleeding (eg, varicose lesions, peptic ulcers) or chronic bleeding (eg, angiodysplasia), chronic bleeding due to gynecological disorders, acute or chronic bleeding due to cancer (including colorectal and bladder cancers, especially in late stage), blood-feeding intestinal nematode infections (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws and medical procedures.
  • intestinal nematode infections such as hookworm and whipworm causing hemorrhagic anemia
  • iatrogenic anemia blood loss from repeated blood draws and medical procedures.
  • anemic disease may be resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • the present invention provides a medicament for treating primary or secondary anemia, which comprises a BRAF kinase inhibitor and one or more pharmaceutically acceptable excipients.
  • the drug further comprises a TGF- ⁇ inhibitor and/or a SMAD2/3 inhibitor and/or a glucocorticoid.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase
  • the IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives:
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof:
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof:
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other currently available drugs for the treatment of anemia.
  • the invention provides a kit comprising:
  • TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor are examples of TGF- ⁇ inhibitors and/or SMAD2/3 inhibitors and/or glucocorticoids and/or erythropoietin and/or stem cell factor.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and Use of SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF- ⁇ inhibitors and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of a medicament for expanding erythroid precursor cells or erythroblasts use.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives: Luspatercept, Sotatercept, SIS3 HCl , Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and a TGF-beta inhibitor, or a BRAF kinase inhibitor and a SMAD2/3 inhibitor, or a BRAF kinase inhibitor, a TGF-beta inhibitor Agents and SMAD2/3 inhibitors, and their combination with glucocorticoids in the preparation of medicines for the treatment of primary or secondary anemia.
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • the present invention provides a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and/or stem cell factor, or a BRAF kinase inhibitor and a TGF- ⁇ inhibitor, or a BRAF kinase inhibitor and SMAD2/3 inhibitors, or BRAF kinase inhibitors, TGF-beta inhibitors and SMAD2/3 inhibitors, and their use in combination with glucocorticoids for expanding erythroid precursor cells or erythroblasts.
  • said BRAF kinase inhibitor delays the progression of terminal phase differentiation of erythrocytes.
  • said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
  • the erythroid precursor cells are genetically modified erythroid precursor cells or drug-loadable erythroid precursor cells.
  • said genetically modified erythroid precursor cells are capable of treating genetic diseases, such as phenylketonuria and cancer.
  • said erythroid precursor cells or erythroblasts are derived from blood sources, including blood and blood products of mammals such as mice, rats, humans, and the like.
  • said erythroid precursor cells or erythroblasts are CD34+ derived cells obtained from bone marrow, umbilical cord blood or embryonic/induced pluripotent stem cells (iPSC/ESC).
  • erythroid precursor cells or erythroblasts are derived from in vitro differentiated and expanded human erythroid precursor cells and stem cells from peripheral blood mononuclear cells (PBMC), or from PBMC of stem cells.
  • PBMC peripheral blood mononuclear cells
  • stem cells are hematopoietic stem cells.
  • hematopoietic stem cells are derived from bone marrow or umbilical cord blood.
  • stem cells are genetically modified before or during culturing.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, Regorafenib, or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M.
  • erythropoietin is also used concomitantly.
  • the invention provides a method of expanding erythroid precursor cells or erythroblasts comprising administering to a subject a BRAF kinase inhibitor, or a BRAF kinase inhibitor and erythropoietin and /or stem cell factor, or BRAF kinase inhibitor and TGF- ⁇ inhibitor, or BRAF kinase inhibitor and SMAD2/3 inhibitor, or BRAF kinase inhibitor, TGF- ⁇ inhibitor and SMAD2/3 inhibitor, and their combination Combinations of glucocorticoids.
  • said erythroid precursor cells are selected from the group consisting of erythroid colony forming units (CFU-E), burst forming unit erythroid progenitors (BFU-E) and proerythroblasts.
  • the subject is a mammal, such as a mouse, a rat, a human, or the like.
  • anemia is anemia caused by decreased or defective erythropoiesis.
  • anemia caused by reduced or defective erythropoiesis is primary, including primary aplastic anemia, Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome , dyskeratosis congenita, Fanconi anemia, congenital dyserythropoietic anemia (CDA), thalassemia, sickle cell anemia, anemia caused by myelodysplastic syndrome.
  • DBA Diamond-Blackfan anemia
  • CDA congenital dyserythropoietic anemia
  • thalassemia thalassemia
  • sickle cell anemia anemia caused by myelodysplastic syndrome.
  • anemia caused by reduced or defective erythropoiesis is secondary, including anemia caused by secondary myelodysplastic syndrome, secondary aplastic anemia, vitamin deficiency type anemia, iron deficiency anemia, chronic inflammatory anemia, endocrine disease anemia, secondary anemia with insufficient EPO secretion due to renal failure, poor blood lineage reconstitution after hematopoietic stem cell transplantation; preferably, wherein said secondary regeneration Obstacle anemia is caused by the following reasons: autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, etc.), anemia caused by other immune mechanisms (such as ABO blood group incompatibility, stem cell transplantation, gangrenous pyoderma, etc.) disease), lymphoproliferative diseases (such as chronic lymphocytic leukemia, LGL leukemia, Hodgkin's disease, non-Hodgkin
  • anemia is anemia due to destruction of red blood cells.
  • anemia due to destruction of red blood cells is primary, including hereditary spherocytosis, hereditary elliptocytosis, abeta lipoproteinemia, enzyme deficiency Anemias resulting from (eg, glycolysis deficient anemia due to pyruvate kinase and hexokinase deficiencies, glucose 6-phosphate dehydrogenase and glutathione synthase deficiencies, anemia due to increased oxidative stress).
  • anemia caused by red blood cell destruction is secondary, including antibody-mediated mild autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • antibody-mediated mild autoimmune hemolytic anemia eg, cold agglutinin hemolytic anemia, hemolytic disease (eg, hemolytic disease of the newborn), mechanical damage to red blood cells (eg, microangiopathic hemolytic anemia, including thrombotic thrombocytopenic purpura and disseminated intravascular coagulation), and hemolytic anemia due to infection (eg, malaria infection).
  • anemia is anemia caused by excessive blood loss.
  • the anemia caused by excessive blood loss includes anemia in premature infants (such as frequent blood collection as detected by the laboratory, and optionally combined with insufficient erythropoiesis), external/internal injuries (various trauma or Acute blood loss due to surgery), acute bleeding due to gastrointestinal lesions (eg, variceal lesions, peptic ulcer) or chronic blood loss (eg, angiodysplasia), chronic blood loss due to gynecological diseases, cancer (including colorectal cancer and bladder cancer) Acute or chronic blood loss from cancer, especially in advanced stages), infection with blood-feeding intestinal nematodes (such as hookworm and whipworm causing hemorrhagic anemia), iatrogenic anemia, blood loss from repeated blood draws, and medical procedures.
  • the disease is resistant to erythropoietin or glucocorticoids or other drugs for the treatment of anemia.
  • said BRAF kinase inhibitor induces paradoxical activation of MAPK; preferably, said BRAF kinase inhibitor promotes RAF protein dimerization; preferably, said BRAF kinase inhibitor induces BRAF kinase The IN conformation of at least one of the ⁇ C-helix and DFG domain, and the IN conformation of R506; preferably, the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs thereof and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, BRAF inhibitor 1 (compound 13), RAF709, L-779450, LY3009120, Belvarafenib (HM95573), RO5126766 (CH5126766), TAK-632, PLX-4720, Agerafenib(RXDX-105), Regorafenib(BA
  • the BRAF kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives: GDC-0879, SB-590885, SB-682330, Dabrafenib, PLX -4720, Sorafenib (BAY 43-9006), BRAF Inhibitor 1 (Compound 13), Vemurafenib (PLX4032), Doramapimod (BIRB 796), Encorafenib (LGX818), BGB659, TAK-632, LY3009120, GW5074, L-779450, Regorafenib or ZM336372;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: GDC-0879, SB-590885, SB-682330 or BMS- 908662;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879, SB-590885 or SB-682330;
  • the BRAF kinase inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: GDC-0879 or SB-590885.
  • the BRAF kinase inhibitor is a compound of the formula:
  • R 20 and R 21 and the atoms to which they are attached form a heterocyclic ring, wherein the heterocyclic ring is optionally substituted by one or more groups independently selected from: F, Cl, Br, I, C 1 -C 8 Alkyl, C 2 -C 8 alkenyl and C 2 -C 8 alkynyl;
  • R 23 is H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl, C 2 -C 20 heterocyclyl or protecting group;
  • R a and R b are independently H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 20 aryl or C 2 -C 20 heterocyclyl ;
  • Y is independently O, S, NR 20 , + N(O)R 20 , N(OR 20 ), + N(O)(OR 20 ), or N-NR 20 R 21 ;
  • the protecting group is selected from trialkylsilyl, dialkylphenylsilyl, benzoyloxy, benzyl, benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, benzene Diformimido, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyloxycarbonyl (Fmoc) and tetrahydropyranyl, or their stereoisomers, mutual Isomers, pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives.
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH 2 , CO, S or NH, or the group XR 1 is hydrogen;
  • Y 1 and Y 2 are independently N or CH;
  • R 1 is hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or Heteroaryl C 1-6 alkyl, any of which can be optionally substituted; in addition, when X is CH , then R 1 can be hydroxyl or C 1-6 alkoxy, which can be optionally substituted replace;
  • R 2 is H, C 1-6 alkyl, C 2-6 alkenyl, C 3-7 cycloalkyl, C 5-7 cycloalkenyl, heterocyclyl, aryl or heteroaryl, any of which can be optionally replaced;
  • Ar is a group of formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • R 15 is O or N-OH
  • One of X 1 and X 2 is N, and the other is NR 6 , wherein R 6 is hydrogen or C 1-6 alkyl;
  • the BRAF kinase inhibitor is a compound of the formula:
  • X is O, CH2 , CO, S or NH, or XR1 is H;
  • Y1 and Y2 are independently selected from CH or N;
  • R 1 is H, C 1-6 alkyl, C 3-7 cycloalkyl, aryl, aryl C 1-6 alkyl, heterocyclyl, heterocyclyl C 1-6 alkyl, heteroaryl or HeteroarylC 1-6 alkyl, except H, which may be optionally substituted;
  • R is H, or optionally substituted aryl or heteroaryl
  • Ar is the following formula a) or b):
  • A represents a fused 5- to 7-membered ring, optionally containing up to two heteroatoms selected from O, S and NR 5 , wherein R 5 is hydrogen or C 1-6 alkyl, said ring is either Optionally substituted by up to two substituents selected from the group consisting of halogen, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy or keto;
  • R 3 and R 4 are independently selected from: hydrogen, halogen, C 1-6 alkyl, aryl, aryl C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkoxy C 1 -6 alkyl, halogenated C 1-6 alkyl, aryl C 1-6 alkoxy, hydroxyl, nitro, cyano, azido, amino, monosubstituted or disubstituted -NC 1-6 alkyl Amino, acylamino, arylcarbonylamino, acyloxy, carboxyl, carboxyl salt, carboxyl ester, aminosulfonyl, monosubstituted or disubstituted -NC 1-6 alkylaminosulfonyl, C 1-6 alkoxycarbonyl , aryloxycarbonyl, ureido, guanidino, C 1-6 alkylguanidino, amidino, C 1-6 alkylamidino, sulfon
  • One of X 1 and X 2 is selected from O, S or NR 11 , the other is CH, wherein R 11 is hydrogen, C 1-6 alkyl, aryl or aryl C 1-6 alkyl;
  • TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotopic derivatives thereof: LY2109761, Galunisertib (LY2157299) , LY364947, SB431542, LDN-193189, SB525334, SB505124, GW788388, RepSox(E-616452), K02288, BIBF-0775, TP0427736, A-83-01, LDN-214117, SD-208, Vactosertib( , LDN-212854, Dorsomorphin (Compound C) or LY3200882;
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: LY2109761 or Galunisertib (LY2157299);
  • the TGF- ⁇ inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Galunisertib (LY2157299).
  • said SMAD2/3 inhibitor is selected from the following compounds or pharmaceutically acceptable salts, solvates, hydrates, polymorphs and isotope derivatives thereof: Luspatercept, Sotatercept, SIS3HCl, Alantolactone, Halofuginone, and AUDA.
  • said BRAF kinase inhibitor is used at a concentration of 1 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M.
  • erythropoietin and/or stem cell factor are also used concomitantly.
  • the differentiation medium includes: IMDM, 10% FBS, 5% human serum, 300 ⁇ g/ml holo-transferrin (Sigma-Aldrich, St.Louis, MO, USA, Cat T0665), 2IU/ml heparin (Sigma-Aldrich, St.
  • Serum-free differentiation medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 3IU/mL erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml Human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)-3 (StemCell, Vancouver, BC, CA, Cat 78042) and insulin-like growth factor (IGF)-1 ( 40ng/ml, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11).
  • SFEM II SteCell Technologies, Vancouver, BC, Canada, Cat 09655
  • 3IU/mL erythropoietin Amgen, Thousand Oaks, CA, Cat 55513-144-10
  • 50ng/ml Human stem cell factor StemCell, Vancouver, BC, CA, Cat 78062
  • 10ng/ml interleukin (IL)-3 Stem
  • Hematopoietic stem and progenitor cell serum-free medium includes: SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655), supplemented with 100 ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 100 ng/ml FLT3-L (StemCell, Vancouver, BC, CA, Cat 78137), 50ng/ml thrombopoietin (TPO) (StemCell, Vancouver, BC, CA, Cat 78210), 10 ⁇ g/ml human low density lipoprotein (Low density lipoprotein) ) (StemCell, Vancouver, BC, CA, Cat 02698).
  • SFEM II SteCell Technologies, Vancouver, BC, Canada, Cat 09655
  • TPO thrombopoietin
  • Fresh umbilical cord blood was mixed with phosphate buffered solution (DPBS) at a ratio of 1:1, and then carefully poured into a 50mL SepMate separation tube filled with lymphocyte separation agent (Lymphoprep) in the lower layer with a pipette, and centrifuged in a precooled to 4°C Centrifuge at 1200g for 10min. Then the supernatant serum and peripheral mononuclear cells were transferred to a new 50mL tube and centrifuged at 2000rpm for 10min.
  • DPBS phosphate buffered solution
  • BRAF inhibitor solution Dissolve SB-590885 in 2% Cremophor EL, 2% N,N-dimethylacetamide, pH 5.0, to 30mg/ml.
  • GDC-0879 was dissolved in 50% PGE 300 and 50% PBS to 50mg/ml.
  • Encorafenib was dissolved in 50% PGE 300 and 50% PBS to 30mg/ml.
  • SB-590885 is administered by intraperitoneal injection, once a day unless otherwise mentioned.
  • GDC-0879 and Encorafenib were administered by gavage.
  • mice To induce acute hemolytic anemia: Dissolve phenylhydrazine in PBS and inject 60 mg/kg intraperitoneally into mice.
  • Example 1 High-throughput screening of small molecule expansion of erythroid precursor cells
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 02690) for 3 days, and then screened the compound library.
  • SFEM II StemCell Technologies, Vancouver, BC, CA, Cat 09655
  • CC100 StemCell Technologies, Vancouver, BC, CA, Cat 02690
  • GDC-0879 is a potent BRAF inhibitor with IC 50 of 0.13nM.
  • FIG. 1A it is a schematic diagram of high-throughput screening (HTS).
  • HTS high-throughput screening
  • Example 2-1 Explore the optimal concentration of GDC-0879 treatment
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cell density was kept below 100,000/ml every time the medium was changed.
  • Add 20% volume of Cell to each well on the ninth day Cell Viability reagent after incubating at 37°C in the dark for 2 hours, detect the absorbance at 560nM and 590nM on the BioTek multi-functional microplate detector, calculate the ratio of 560/590 in each well, subtract the background ratio and calculate for each experimental group Ratio relative to the mean of the control group.
  • the experiments used concentrations of 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M and 20 ⁇ M GDC-0879 and concentrations of 0.1 ⁇ M, 1 ⁇ M Galunisertib (LY2157299) and combinations of 0.1 ⁇ M GDC-0879 and 1 ⁇ M Galunisertib.
  • GDC-0879 has a wide range of effective concentrations. In differentiation medium, GDC-0879 at 100nM to 10 ⁇ M is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 1-10 ⁇ M, and the erythroid expansion of cord blood-derived CD34+ cells can reach more than 5 times.
  • TGF- ⁇ inhibitors were previously reported as potential erythroblast stimulators.
  • Galunisertib (LY2157299) is an oral, selective TGF- ⁇ receptor type I (TGF- ⁇ RI) kinase inhibitor with IC50 of 56nM.
  • GDC-0879 combined with Galunisertib showed about 6-fold amplification on the 9th day. Medium and small molecules were changed every 3 days.
  • GDC GDC-0879
  • Gal Galunisertib
  • Dex dexamethasone.
  • Example 2-2 Explore the optimal concentration of BRAF inhibitor treatment
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • Figure 2B shows the effect of a single initial dose of BRAF inhibitor on the expansion of erythroid precursors only.
  • Figure 2B shows that after 3 days of expansion culture, 200 ⁇ L of differentiation medium containing 300 CD34+ cells was added to a 96-well plate, and then small molecule compounds were added to each well. Three biological replicates were prepared for each small molecule in this screening, and culture was continued for 7 days without changing the medium thereafter.
  • Figure 2B shows the expansion effect of various BRAF inhibitors on erythroid precursor cells in a wide range of concentrations (administered only once).
  • Fig. 2B shows the results obtained by the 96-well plate assay that different concentrations of BRAF inhibitors stimulate the proliferation of erythroid progenitor cells on Day 7.
  • Vemu Vemurafenib; Enco: Encorafenib; Dabra: Dabrafenib; SB: SB-590885; PLX: PLX-8394; GDC: GDC-0879.
  • Statistical data histograms are mean ⁇ SD, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • SB and GDC are BRAF inhibitors that can better promote the proliferation of early erythroid stem progenitor cells.
  • Figure 2C shows the expansion effect of selected BRAF inhibitors on erythroid precursor cells over a wide range of concentrations (three doses).
  • GDC-0879 has a wide effective concentration range. GDC-0879 at 100 nM to 10 ⁇ M in differentiation medium is most effective for the expansion of erythroid precursor cells. The optimal effective concentration of GDC-0879 is in the range of 300nM-10 ⁇ M, and the erythroid expansion of cord blood-derived CD34+ cells can reach 5-10 times.
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 200,000/ml when changing the medium.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Figure 3A shows the effect of BRAF inhibitors on the expansion of erythroblasts.
  • Figure 3A is the growth curves of three small molecules in the erythroid differentiation system at the optimal concentration. Three biological replicates were set for each condition, and statistical data are mean ⁇ SD, ***: P ⁇ 0.001, ****: P ⁇ 0.0001.
  • Fig. 3B shows the synergistic effect of BRAF inhibitors and TGF- ⁇ inhibitors on the expansion of erythroblasts.
  • Fig. 3B The growth curve of erythroblasts after small molecule treatment in differentiation medium is shown in Fig. 3B.
  • Fig. 3B The growth curve of erythroblasts after small molecule treatment in differentiation medium is shown in Fig. 3B.
  • red blood cells expanded about 10 times.
  • Administration of GDC-0879 in combination with Galunisertib resulted in approximately 12-fold expansion of red blood cells at day 12.
  • GDC GDC-0879
  • Gal Galunisertib
  • LY LY2109761.
  • Data are presented as mean ⁇ SD.
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
  • the FACS results in Figure 4A show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days).
  • the FACS results in Figure 4B show the effects of different concentrations of GDC-0879 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) in the differentiation medium on day 6 (small molecule treatment for 6 days).
  • CD235a erythroid surface marker CD235a
  • the main cell group in the 1 ⁇ M GDC-0879 treatment group was still CD235a-negative, while the main cell group in the control group was CD235a-positive, that is, GDC-0879 delayed the terminal differentiation process of erythrocytes, allowing more self-renewal of erythroblasts.
  • Figure 4C shows the effects of different concentrations of GDC-0879 in the differentiation medium on day 12 on the development of erythrocytes and the expression of the erythroid surface marker CD235a (Glycophorin A) (small molecule treatment for 6 days).
  • CD71 expression decreased on day 12 until erythrocytes matured.
  • the treatment group still had high CD71 expression cell population, indicating that the red blood cell differentiation in the GDC-0879 treatment group was delayed.
  • Figure 5A shows that BRAF inhibitors promote PBMC-derived colony formation.
  • PBMC Resuscitate PBMC from a healthy donor one day before the experiment, and overnight in the differentiation medium.
  • 100,000 PBMC cells per well (35 mm) are seeded in methylcellulose containing cytokines necessary for the development of myeloid cells
  • Methocult H4435 StemCell, Vancouver, BC, CA, Cat H4435
  • the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed, counted and area counted using a Leica inverted phase-contrast microscope at 14 days.
  • Figure 5A is the statistics of the number of erythrocyte colonies (middle) and the area of a single erythrocyte colony (right) in different administration groups of Methocult H4435 and the control group.
  • GDC GDC-0879;
  • Dex Dexamethasone. Three biological repetitions were set for each condition, and statistical data are mean ⁇ SD; ns: no significant difference, P>0.05; ****: P ⁇ 0.0001.
  • Figure 5B shows that BRAF inhibitors promote erythropoiesis of CD34+ cells derived from umbilical cord blood (EPO only medium).
  • the CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium. On the first day, 600 CD34+ cells per well (35 mm) were inoculated in the methylcellulose medium Methocult H4330 containing only EPO (StemCell, Vancouver, BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope.
  • Figure 5B shows the results of erythrocyte-specific colony formation at day 14.
  • 600 CD34+ cells were pre-cultured overnight in erythroid differentiation medium containing only EPO, and cultured in 35 mm wells in methylcellulose medium containing only EPO the next day, and on the 14th day of culture Take photos of the medium.
  • no large BFU-E colonies could be seen with naked eyes and pictures, but large BFU-E colonies could be seen with the naked eye in the GDC-0879 treatment group.
  • Figure 5C shows the results of red blood cell-specific colony formation on day 14 detected under a bright-field microscope using a 5 ⁇ objective lens. There were significant differences in the colony area and the number of cells per colony between the treatment group and the control group. Scale bar, 500 ⁇ m.
  • Figure 5D shows that BRAF inhibitors promote erythropoiesis and lineage development of cord blood-derived CD34+ cells (myeloid medium).
  • the CD34+ cells were revived one day before the experiment, and kept overnight in the hematopoietic stem cell maintenance medium.
  • 200 CD34+ cells per well were inoculated in methylcellulose containing cytokines required for each lineage of myeloid development
  • Methocult H4435 StemCell, Vancouver, BC, CA, Cat H4435
  • the medicines of the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed at 14 days using a Leica inverted phase contrast microscope.
  • BRAF inhibitors did not result in a significant hematopoietic lineage shift (Panel D).
  • A At the beginning (Day 0), 100 CD34+ cells were inoculated on the culture medium of the treatment group and the control group, and then eluted with PBS on the fourteenth day and counted for each well.
  • B Number of red blood cells (Non CD11b+) in each well.
  • C The proportion of granulocytes (CD11b+) in each well.
  • D On Methocult H4435, UCB-hCD34+ was administered, and the number of colonies grown from GDC and the control group was counted.
  • Example 6 The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
  • Example 6-1 Small molecule test results for the same target
  • CD34+ cells were pre-cultured in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100 (StemCell Technologies, Vancouver, BC, CA, Cat 09690) for 3 days.
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. A total of 9 days were administered, and no treatment was administered after the 9th day.
  • Figure 6A shows the results of detection of other small molecules targeting BRAF with the same target (the concentration of small molecules not specifically marked is 1 ⁇ M, treated for 9 days).
  • the combined treatment group of 1 ⁇ M Encorafenib and Dabrafenib had more than 3 times the number of red blood cell expansion on the 10th day.
  • Example 6-2 The effect of small molecules targeting BRAF on promoting the proliferation of erythroid precursor cells
  • the CD34+ cells were revived one day before the experiment, and overnight in the differentiation medium.
  • 300 CD34+ cells per well (22mm) were inoculated in EPO-only methylcellulose medium Methocult H4330 (StemCell Technologies, Vancouver) , BC, CA, Cat H4330), the drugs in the administration group were premixed evenly, and cultured in the incubator for 14 days. Colonies were photographed and counted using a Leica inverted phase-contrast microscope at 14 days.
  • Figure 6B shows the results of erythrocyte-specific colony formation on day 14 in EPO-only methylcellulose medium with other inhibitors of BRAF.
  • 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2 replicates per treatment group.
  • Gal treated with 2 ⁇ M Galunisertib
  • Enco treated with 5 ⁇ M Encorafenib
  • Dabr treated with 5 ⁇ M Dabrafenib
  • GDC treated with 5 ⁇ M GDC-0879
  • SB treated with 5 ⁇ M SB-590885
  • Ctrl control (DMSO).
  • Figure 6C shows the results of erythrocyte-specific colony formation at day 14 for other combinations containing BRAF inhibitors in EPO-only methylcellulose media.
  • 300 CD34+ cells were pre-cultured overnight in SFEM II (StemCell Technologies, Vancouver, BC, CA, Cat 09655) supplemented with CC100, and then placed in 35mm wells the next day, and cultured on the 14th day base photo. 2/3 replicates per treatment group.
  • GDC Gal treated with 2 ⁇ M galunisertib and 5 ⁇ M GDC-0879
  • SB Gal treated with 2 ⁇ M galunisertib and 5 ⁇ M SB-590885
  • Ctrl control.
  • Figure 6D shows the colony formation results under the light field microscope, and the erythrocyte-specific colony formation results on day 14 were detected under the 5 ⁇ objective lens. Under the microscope, the largest colony of erythrocytes in Figures 6B and 6C is shown in Figure D. The colony area and the number of cells per colony were significantly different between the treatment group and the control group.
  • Gal treated with 2 ⁇ M Galunisertib
  • Enco treated with 5 ⁇ M Encorafenib
  • Dabr treated with 5 ⁇ M Dabrafenib
  • GDC treated with 5 ⁇ M GDC-0879
  • SB treated with 5 ⁇ M SB-590885
  • +SB 2 ⁇ M Galunisertib and 5 ⁇ M SB-590885 treatment
  • Ctrl control.
  • Scale bar 500 ⁇ m.
  • Gal treated with 2 ⁇ M Galunisertib; Enco: treated with 5 ⁇ M Encorafenib; Dabr: treated with 5 ⁇ M Dabrafenib;
  • GDC treated with 5 ⁇ M GDC-0879; SB: treated with 5 ⁇ M SB-590885;
  • Gal+GDC treated with 2 ⁇ M Galunisertib and 5 ⁇ M GDC-0879; : 2 ⁇ M Galunisertib and 5 ⁇ M SB-590885 treatment;
  • Ctrl control (DMSO).
  • BRAF inhibitors generally have paradoxical activation effects, and have the potential to promote erythroid proliferation synergistically with other small molecules in the colony formation ability.
  • Embodiment 7 effective dosing cycle test
  • Embodiment 7-1 Effective administration cycle test result 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Different administration time groups were administered for 0-3 days, 3-6 days, 6-9 days and 9-12 days respectively.
  • Figure 7A shows the test results of GDC-0879 administered in different cycles from the 0th day to the 12th day, and the final cell count was counted on the 15th day, with 3 days as the administration period, to explore the drug dependence of continuous administration effect.
  • Figure 7B shows the comparison of the number of cells in the 0-3 day treatment group and the control group on day 13. In the different cycles of red blood cells treated in the differentiation medium, the expansion ability of the 6-9 day treatment group was the strongest. This figure demonstrates the persistent effect of GDC-0879 expansion. The number of cells in the single-dose treatment group also increased significantly, indicating that GDC-0879 expanded erythroid precursor cells at all stages.
  • Embodiment 7-2 Effective administration cycle test result 2
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed. Dosing for 3 days, after the 3rd day, no more dosing treatment.
  • Fig. 7C shows the FACS results of the experimental group and the control group on day 13 in the differentiation medium.
  • the treatment group was administered from the 0th to the 3rd day. It can be seen from the FACS graph that the differentiation rate of the 1 ⁇ M GDC-0879 treatment group for 0-3 days was almost the same as that of the control group on the 13th day, and even had a higher enucleation rate.
  • 1 ⁇ M GDC-0879 was administered only from day 1 to day 3. This indicated that early drug administration still promoted proliferation, but did not affect differentiation at all, and even promoted terminal maturation and denucleation.
  • Embodiment 7-3 Effective administration cycle test result 3
  • the cells were counted at the beginning of Day 0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day 0, and the cell density was kept below 100,000/ml when changing the medium.
  • the medium and small molecules were changed every 3 days, and the treatment group was administered at the same time as the medium was changed.
  • Different administration time groups were administered for 0-3 days, 3-6 days, 0-6 days, 3-9 days and 6-9 days respectively.
  • Figure 7D shows statistics on the enucleation rate of each group administered for 3 days from day 13 to day 16 (treatment group, 1 ⁇ M GDC-0879 treatment group). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes.
  • the vertical axis is the percentage of cells.
  • Figure 7E shows statistics on the enucleation rate of each group in the two administration periods (6 consecutive days) from day 13 to day 16 (treatment group: 1 ⁇ M GDC-0879 treatment). It can be seen from the figure that GDC-0879 does not directly affect the process of denucleation and terminal maturation of erythrocytes.
  • the vertical axis is the percentage of cells.
  • Example 8 Expansion of erythroblasts in PBMCs in vitro
  • Example 8-1 In vitro expansion of erythroblasts from healthy donor PBMCs in serum-free differentiation medium
  • the cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced. After the ninth day of administration, the different administration groups were centrifuged and the cell pellets were photographed.
  • Figure 8A shows the cells of the treated and control groups at day 9.
  • PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were cultured in serum-free erythrocyte expansion medium (SFEM II (StemCell Technologies, Vancouver, BC, Canada, Cat 09655)) for 9 days, and 3 IU/ ml erythropoietin (Amgen, Thousand Oaks, CA, Cat 55513-144-10), 50ng/ml human stem cell factor (StemCell, Vancouver, BC, CA, Cat 78062), 10ng/ml interleukin (IL)- 3 (StemCell, Vancouver BC CA Cat 78042) and insulin-like growth factor (IGF)-1 (40ng/mL, PeproTech, Rocky Hill, NJ, USA, Cat AF-100-11)).
  • SFEM II serum-free erythrocyte expansion medium
  • IGF insulin-like growth factor
  • Example 8-2 In vitro expansion of erythroblasts in PBMC Result 1
  • the cells were counted at the start of Day 0, and 500,000 PBMC cells from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) were inoculated in each well of serum-free differentiation medium, and at Day 0, according to the corresponding concentration for administration.
  • the culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the number of cells was counted on the ninth day of administration.
  • Figure 8B shows the change in the number of erythroblasts in the PBMCs of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) expanded in vitro on day 9.
  • GDC GDC-0879; SB: SB-590885. Data are presented as mean ⁇ SD.
  • BRAF inhibitors can efficiently expand the erythroid precursor cells therein, and a large number of erythrocytes can be obtained without any separation means for erythroid precursor cells.
  • Example 8-3 In vitro expansion of erythroblasts in PBMC results 2
  • the cells were counted at the beginning of Day 0, and 500,000 PBMC cells from healthy donors were inoculated in each well of serum-free differentiation medium, and administered at the corresponding concentration on Day 0, and the cell density was kept at Below 1000000/ml.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
  • Figure 8C shows the FACS results of PBMCs from healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) cultured in serum-free erythrocyte expansion medium on day 9.
  • Treatment group 1 ⁇ M SB-590885.
  • Figure 8D shows the statistics of the FACS results of the PBMC of healthy donors (Healthy Donor 1-Healthy Donor 5, HD1-HD5) on day 10 of the cell population ratio.
  • the vertical axis is the percentage of cells. Double negative: CD71 and CD235a double negative; Double positive: CD71 and CD235a double positive; SB: SB-590885; GDC: GDC-0879.
  • Example 8-4 In vitro expansion of erythroblasts in PBMC results 3
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry on the ninth day of administration.
  • Figure 8F shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 ⁇ M SB-590885.
  • Figure 8G shows the FACS results of PBMC from healthy donors (Donor1-Donor5) cultured in serum-free erythrocyte expansion medium. Treatment group: 1 ⁇ M SB-590885.
  • BRAF inhibitors can greatly expand the proportion of erythroid precursor cells in PBMC, and better promote the proliferation of erythroid precursor cells in PBMC.
  • Example 8-5 In vitro expansion of erythroblasts in PBMC Result 4
  • Dex glucocorticoid dexamethasone
  • Example 8-6 In vitro expansion of erythroblasts in PBMC Result 5 (SB-590885)
  • Figure 8I Whole-body clonogenic images and representative erythroid monoclonal colony images of SB and controls administered to PBMCs on Methocult H4435.
  • Figure 8K Statistics of the number of erythrocyte colonies (left) and the area of individual erythrocyte colonies in different groups (right) in different Methocult H4435 administration groups and control groups.
  • Embodiment 9 Therapeutic effect of medicine under low EPO condition
  • Embodiment 9-1 Therapeutic effect of medicine under low EPO condition 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • Figure 9A shows the effect of GDC-0879 in different concentrations of erythropoietin (EPO) differentiation medium.
  • EPO erythropoietin
  • Embodiment 9-2 Therapeutic effect of medicine under low EPO condition 2
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, for a total of six days, and no further administration for 4 days after six days.
  • Flow cytometry detection was performed on the 14th day of culture.
  • Figure 9B shows the FACS results on day 14 after 5-10 days of culture in the absence of EPO.
  • Left 1 ⁇ M GDC-0879 treatment from day 5 to day 10; right: control.
  • the treatment group used complete erythropoietin differentiation medium (3IU/ml erythropoietin, EPO) for the first 4 days, and used erythropoietin (EPO)-free differentiation medium for 5 to 10 days.
  • EPO erythropoietin
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0. Culture in only 1/3 EPO concentration (i.e. 1IU EPO). The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the treatment group was only administered for 9 days. A total of 15 days were cultured in this 1/3 EPO concentration differentiation medium.
  • Figure 9C shows the FACS results on the 15th day after culturing for 1-15 days under the condition of 1/3 concentration of EPO. From day 1 to day 14, 1/3 EPO differentiation medium (1IU/ml EPO) was used. In the treatment group, 1 ⁇ M GDC-0879 was added to the culture medium from day 1 to day 9.
  • Embodiment 9-4 Therapeutic effect of medicine under low EPO condition 4
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • the culture medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the administration group was administered for 12 days.
  • a total of 19 days were cultured in the differentiation medium of these two EPO concentrations.
  • Fig. 9D shows the statistics of the denucleation rate of red blood cells under the conditions of various EPO concentrations from day 13 to day 19. Most of the cells in the control group were apoptotic by day 15, therefore no further data were available after day 15.
  • the treatment group was treated with 1 ⁇ M GDC-0879 from day 1 to day 12.
  • 3 IU/ml erythropoietin was added to the differentiation medium; in the 1/3 EPO group, 1.5 IU/ml EPO was added to the differentiation medium.
  • BRAF inhibitors can promote normal erythroid development, promote erythroid proliferation, and maintain erythroid denucleation ratio.
  • Example 9-5 Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration Conditions
  • Example 9-5a Therapeutic effect of drugs under low EPO and SCF conditions 1
  • the cells were counted at the beginning of Day0, and 10,000 CD34+ cells were inoculated in the differentiation medium of each well, and administered at the corresponding concentration on Day0.
  • Differentiation medium with EPO concentration and SCF concentration were changed in each group (3 IU for Full Ctrl, 0.15 IU for 5% EPO, no SCF for NO SCF).
  • Figure 9E is a growth curve of UCB CD34+ cells treated with 2 ⁇ M GDC-0879 administration in erythroid medium with low cytokine concentrations of 0% SCF or 5% EPO. In all conditions, the number of cells in the administration group was an order of magnitude higher than that in the control group.
  • Figure 9F is the flow cytometry of 0% SCF (left) and 5% EPO (right) low concentrations of cytokines differentiated (UCB CD34+ cells) on day 9 in the control group (top) and GDC-0879 treatment group (bottom) Representative images of cell surface markers. Compared with the control group, the erythroid development process of the administration group was normal, and the proportion of the undeveloped group was lower.
  • Example 9-7 Effect of BRAF Inhibitor Treatment and Promotion of Erythroid Differentiation under Low Cytokine Concentration-2
  • Figures 9G and 9H Expression of typical erythrocyte surface markers (CD117, CD71 and CD235) of erythroblasts in different erythropoietic conditioned media of GDC treatment group and control group on day 6.
  • Figures 9I and 9J Expression of erythrocyte surface markers (CD117, CD71 and CD235) in different erythropoietic conditioned media in the GDC treatment group and control group on day 14.
  • Figure 9J shows the difference in the expression of CD235 between GDC and the control group under different stress conditions.
  • Figure 9K 5% E.: Typical photographs of red blood cells of samples cultured under different stress conditions after centrifugation of 5% EPO-conditioned medium for 14 days (the number of cells in each EP tube is equal); S.Free: cultured without SCF base. Apart from the cytokine differences mentioned, the other components in the conditioned medium were the same as in the erythropoietic medium.
  • Example 10 In vitro therapeutic effect of anemia caused by pure red blood cell regeneration defects after hematopoietic stem cell transplantation
  • the cells were counted at the start of Day0, and 500,000 PBMC cells from patients with erythroid dysplasia after hematopoietic stem cell transplantation were inoculated in serum-free differentiation medium per well, and administered at the corresponding concentration on Day0. When changing the medium, keep the cell density below 1,000,000/ml. The medium and small molecules were replaced every 3 days, and the treatment group was administered at the same time as the medium was replaced, and the cells were detected by flow cytometry and cell number on the 10th day of administration.
  • FIG. 10A shows that GDC-0879 may promote the development and self-renewal of erythroid progenitors in erythroid failure or pure erythroid aplasia after hematopoietic stem cell transplantation.
  • the patient had a history of leukemia and had a low red blood cell count after a hematopoietic stem cell transplant.
  • After 10 days of treatment of this patient's PBMC with GDC-0879 in differentiation medium not only the cell number expanded to about 6-fold (Fig. 10A), but also FACS showed a low proportion of CD71/CD235a negative cell population and good erythroid development outcome.
  • FIG. 10B Media and small molecules were changed every 3 days. Data are expressed as mean ⁇ SD, and statistical results are calculated from three independent biological replicates.
  • FIG. 10B Growth curves of bone marrow mononuclear cells (BMMNCs) from patients with defective erythroid regeneration after hematopoietic stem cell transplantation (EFAHSCT). *Represents pairwise comparisons between different treatment and control groups.
  • GDC GDC-0879; SB: SB-590885; ***: P ⁇ 0.001; ****: P ⁇ 0.0001.
  • Figure 10C Red blood cell surface markers (CD71, CD235, and Hoechst 33342) in SB-treated (bottom) and control (top) erythrocyte development of BMMNCs from EFAHSCT at days 6 (left) and 16 (right).
  • Figure 10F The number of colonies of different lineages cultured from 50,000 EFAHSCT BMMNCs in H4435 myeloid methylcellulose medium.
  • ns no significant difference; ***: p ⁇ 0.001. Data are shown as mean ⁇ SD. Statistical significance of two-group comparisons was assessed using an unpaired Student's t-test.
  • BRAF inhibitors can rescue erythroid developmental failure in patients with EFAHSCT, greatly promote the differentiation and development of erythroid, and expand the number of cells at the same time.
  • Example 11 SB-590885 effectively expands erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
  • PBMC cells derived from DBA patients were revived one day before the experiment, and overnight in the differentiation medium.
  • 300,000 PBMC cells per well were inoculated in the methyl group containing EPO.
  • Cellulose medium Methocult H4330 (StemCell, Vancouver, BC, CA, Cat H4330), supplemented to 10ng/ml IL3 (StemCell, Vancouver, BC, CA, Cat 78042) and 50ng/ml hSCF (StemCell, Vancouver, BC, CA, Cat 78062), the medicines of the administration group were premixed evenly, and cultivated in the incubator for 14 days. At 14 days, the colonies were photographed using a Leica inverted phase-contrast microscope and the number of erythroid colonies in each well was counted.
  • Figure 11 shows that in the methylcellulose medium containing EPO, IL3, and SCF, 1 ⁇ M SB-590885 treated peripheral mononuclear cells of different types of DBA patients, and inoculated 300,000 PBMCs per well; the figure shows the results of erythrocyte colony formation at 14 days. Erythrocyte colony formation results of SB-590885 cultured in methylcellulose medium for erythroid cell culture for 14 days. The results of erythrocyte-specific colony formation on day 14 were detected with a 5 ⁇ objective lens. Under the microscope, typical erythrocyte colonies formed by in vitro culture of PBMC from each DBA patient. The colony area of the treatment group was significantly different from that of the control group.
  • RPL11Mut refers to DBA patients with RPL11 mutation, and there are two patient samples of DBA1 and DBA2 in this batch;
  • RPL5Mut refers to DBA patients with RPL5 mutation.
  • Ctrl treated without administration; Treated: only initially administered 1 ⁇ M SB-590885.
  • Example 12 Statistics of SB-590885 effectively amplifying erythroid precursor cells in peripheral mononuclear cells of patients with DBA anemia
  • Figure 12 shows the statistics for the results of Figure 11. Take photos of the erythroid colonies grown on methylcellulose medium in Figure 11, and use FIJI software to count the area of a single erythroid colony, and calculate the individual and average erythroid colonies in two replicate wells area. Statistical data were subjected to unpaired Student's t-test and statistical significance was calculated. Data are presented as mean ⁇ SD; *: p-value ⁇ 0.05; **: p-value ⁇ 0.01.
  • Example 13 BRAF inhibitors effectively promote erythroid differentiation and erythroid proliferation of MDS patient cells
  • MDS Myelodysplastic Syndrome. It is mainly due to the abnormal development (cancerous) of the blood progenitor cells of the patient, or the blood progenitor cells die in the bone marrow for various reasons, or after entering the circulation. In turn, the number of immature or defective cells is lower than that of healthy cells, and the number of healthy red blood cells, healthy white blood cells, and platelets is too small, but the number of pre-cancerous blood progenitor cells is abnormally increased.
  • Figure 13C Area statistics of the erythroid colonies in Figure 13A.
  • Figure 13E Erythrocyte surface markers (CD71, CD235 and Hoechst 33342) in the erythroid development process of BMMNC of MDS patients treated with GDC (lower) and control group (upper) on day 6 (left) and day 9 (right) .
  • Example 13 proves that BRAF inhibitors can rescue erythroid dysplasia in MDS patients, greatly promote the differentiation and development of erythroid, and simultaneously expand the number of cells.
  • BRAF inhibitors have an expansion effect on early erythroid precursor cells
  • the source of BRAF on erythroid progenitor cells that is, the drug's expansion effect on CD34+ cells
  • a and B are not from the same batch of CD34+.
  • Example 15 BRAF inhibitors promote hematopoiesis in mice 1 (normal conditions)
  • A Schematic representation of in vivo administration of BRAF inhibitors.
  • RBC red blood cells
  • HGB hemoglobin
  • MCV mean corpuscular volume
  • Example 16 BRAF inhibitors promote hematopoiesis in mice 2 (acute hemolysis model 1)
  • PHZ phenylhydrazine
  • PHZ can induce hemolytic anemia of red blood cells.
  • acute hemolytic anemia can be induced in mice, which can be used as an acute anemia model to verify the stress hematopoiesis promotion model of BRAF inhibitors.
  • FIG 16A Schematic representation of administration of BRAF inhibitors in the phenylhydrazine (PHZ)-induced hemolytic anemia model.
  • C57BL/6N mice were pretreated with vehicle (control group), GDC-0879 (30 mg/kg) or Encorafenib (25 mg/kg) for 10 days at 6–8 weeks prior to injection of phenylhydrazine (PHZ) on day 0 ( P.O., B.D., Mon-Fri 3 weeks). Blood samples were collected and examined on Day 0, Days 2-5, and Days 7-11. Mice were sacrificed on day 12, when bone marrow and spleen samples were collected. Red arrows indicate the days on which blood samples were collected. Yellow arrows indicate days on which spleen samples were collected.
  • Figure 16B Determination of red blood cells (RBC) (left panel) and red blood cell distribution width CV (RDW-CV) (middle panel) on day 0, days 2-5, and days 7-11. Spleens were weighed on day 12 (right panel). Error bars represent mean ⁇ standard deviation of 4 or 5 biological replicates. The two groups (ns: P>0.05; *: P ⁇ 0.05, **: P ⁇ 0.01) were statistically analyzed by unpaired two-tailed Student's t test.
  • FIG. 16C Hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), platelet count (PLT), white blood cell count (WBC) and PHZ-induced hemolytic anemia in the in vivo experiment in Example 16 Reticulocyte ratio.
  • HGB Hemoglobin
  • HCT hematocrit
  • MCH mean corpuscular hemoglobin
  • PHT platelet count
  • WBC white blood cell count
  • PHZ-induced hemolytic anemia in the in vivo experiment in Example 16 Reticulocyte ratio.
  • Figure 16D Non-erythroid (CD71 and Terl 19 double negative) ratio in the spleen at day 12 after PHZ induction in the in vivo experiment of Example 16.
  • FIG. 16E CD117+ progenitor cell ratio in bone marrow on day 12 after PHZ induction in Example 16.
  • BRAF inhibitors can promote stress hematopoiesis in vivo, especially extramedullary hematopoiesis (splenic hematopoiesis) in an acute hemolysis model.
  • Example 17 BRAF inhibitors promote hematopoiesis in mice 3 (acute hemolysis model 2)
  • Figure 17A 21 doses of GDC-0879 (50 mg/kg, P.O.) and SB-590885 (10 mg/kg, IP) were administered in the PHZ-induced hemolytic anemia model (Monday to Wednesday Q.D. and Thursday to Friday B.I.D. 2 week) diagram.
  • Figure 17B Measurement of red blood cell (RBC), HGB, HCT and red blood cell distribution width CV (RDW-CV) on days 0-4 and 7-11. 3 mice per group.
  • RBC red blood cell
  • HGB red blood cell
  • RDW-CV red blood cell distribution width CV
  • Example 18 BRAF inhibitors induce paradoxical activation of the MAPK pathway during erythroid differentiation
  • BRAF inhibitors are ATP-competitive inhibitors that competitively bind to the ATP-binding pocket in the BRAF mutant protein, and then it locks the R-Spine ( ⁇ C-helix) and DFG domain residue positions, thereby blocking the R-Spine And the formation of the DFG domain and the active conformation of the BRAF protein, thus preventing the activation of the mutant BRAF protein and the activation of downstream MAPKs.
  • BRAFV600E mutation In melanoma with BRAFV600E mutation, BRAFV600E mutation directly mediates downstream ERK activation after activating MEK, and finally leads to tumorigenesis.
  • BRAF inhibitors In normal BRAF (wild type), the addition of BRAF inhibitors will cause BRAF to be in an activated conformation similar to ATP binding. At this time, BRAF loses its kinase activity, but the conformation becomes activated, which makes it easy to align with the R-Spine of CRAF.
  • the formation of the R506 salt bridge greatly promotes the efficiency of BRAF and CRAF to form heterodimers.
  • the formation of BRAF-CRAF dimers is the key to RAS-RAF-MEK signal transduction in the MAPK pathway.
  • SCF Stem Cell Factor
  • EPO Erythropoietin
  • BRAF inhibitors SB-590885, GDC-0879 and Encorafenib could all activate pMEK and pERK to achieve paradoxical activation of the MAPKs pathway (Figure 18). It is worth noting that SB-590885 and GDC-0879 have stronger contradictory activation effects, not only in the stronger phosphorylation of pMEK and pERK, but also in the activation of pCRAF S338. Stronger MAPKs paradoxically activate small molecules. In summary, it can be basically concluded that BRAF inhibitors activate the MAPK/ERK pathway through paradox.
  • Figure 18 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • Example 19 BRAF inhibitors paradoxically activate MAPK/ERK pathway and have concentration-dependent effects
  • Figure 19 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • Example 20 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors depends on both upstream cytokine signaling and downstream signal transduction
  • hematopoietic stem cells first need to be activated from a quiescent state to a dividing state, similar to a transition from a long-term hematopoietic stem cell to a short-term hematopoietic stem cell, which is accompanied by cell division and strong activation of proliferation, a process that relies on extracellular signals to stimulate its entry into division.
  • the development of blood cells from hematopoietic stem cells to terminal lineages also requires the stimulation of various lineage-related cytokines.
  • One of the more common cytokines is stem cell factor (SCF).
  • SCF stem cell factor receptor
  • KIT stem cell factor receptor
  • both SCF and EPO activate the RAS/MAPK pathway to varying degrees to promote the proliferation, survival and development of erythroid progenitor cells. Therefore, the activated RAS brought about by cytokines is the result of normal hematopoiesis and erythroid development, and this result is the basis of paradoxical activation.
  • BRAF inhibitors are drugs that potentially promote cell proliferation, so it is necessary to consider whether it will bring about a broad spectrum of abnormal proliferation or potential carcinogenesis.
  • Pharmacological inhibition of BRAF downstream MEK protein phosphorylation is a common approach for tumor therapy.
  • this project needs to prove that BRAF inhibitors will not cause RAS-independent proliferation and will not have significant carcinogenic effects; on the other hand, this project also needs to prove whether the direct inhibition of BRAF downstream MEK/ERK can directly Block paradox activation.
  • Figure 20 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • C From left to right, the drug was administered under the condition of containing SCF and EPO, containing only SCF, and containing only EPO.
  • Example 21 Paradoxical activation of MAPK/ERK pathway by BRAF inhibitors is mainly dependent on activation of CRAF
  • Figure 21 Cells in erythroid differentiation medium were harvested on Day 9 and analyzed for protein quantification by Western Blot.
  • A Protein expression of each knockdown group.
  • B Comparison of the doubling of the number of cells in the BRAF inhibitor administration group on Day 7 compared with the DMSO treatment group in the control group and the knockdown group. Three biological replicates were set for each condition, and the statistical data were mean ⁇ SD, ns: no significant difference, P>0.05; **: P ⁇ 0.01.
  • Example 22 BRAF inhibitors delay but only limitedly perturb transcriptional profiles of erythroid development
  • BRAF inhibitors are known to significantly delay the expression of surface differentiation marker proteins during erythroid differentiation; on the growth curve, after 6 days of exponential expansion of erythroid precursor cells, the cell number increased significantly slowing, which is characteristic of erythroid terminal differentiation, i.e., the balance of proliferation and differentiation is skewed toward differentiation during the final stages.
  • the administration of BRAF inhibitors did delay the time of slowing down the number of cell proliferation, so it can be confirmed that BRAF inhibitors lead to a significant delay in erythroid development in terms of cell surface markers and cell proliferation. After 72 hours of BRAF inhibitor treatment, it can also be determined that BRAF inhibitor can also delay the differentiation of erythrocytes at the transcriptional level.
  • Figure 22 Cells of erythroid precursor cells (CD71+, CD235-) were sorted, and transcriptional profiling was performed after 2 ⁇ M administration of GDC-0879 for 72 hours.
  • A Differences in the expression of specific genes between the treatment group (GDC) and the control group (DMSO).
  • B Volcano map of differential genes between the control group and the BRAF inhibitor-administered group (Foldchange>1.5; FDR ⁇ 0.1).
  • C GSEA enrichment analysis of downregulated genes (FDR ⁇ 0.1).
  • This example demonstrates that BRAF inhibitor treatment limitedly affected the transcriptional profile, delayed erythroid differentiation, and exhibited more early stem-progenitor characteristics. And it illustrates the synergistic effect of BRAF inhibitors and glucocorticoids from the perspective of transcriptional profiling.

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Abstract

L'invention concerne un procédé d'amplification de progéniteurs érythroïdes ou d'érythroblastes et de production finale de globules rouges dénucléés matures, ainsi qu'une application de celui-ci, comprenant spécifiquement l'utilisation d'un inhibiteur de kinase BRAF, et une combinaison de ceux-ci avec un inhibiteur de TGF-β et/ou un inhibiteur de SMAD2/3 et/ou un glucocorticoïde et/ou une érythropoïétine et/ou un facteur de cellule souche.
PCT/CN2022/122562 2021-09-30 2022-09-29 Procédé d'amplification de progéniteurs érythroïdes ou d'érythroblastes, et application associée Ceased WO2023051685A1 (fr)

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WO2025117266A3 (fr) * 2023-11-27 2025-08-21 Albert Einstein College Of Medicine Procédé de production de cellules érythroïdes à auto-renouvellement à partir de cellules progénitrices humaines

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