Application of ATR / Chk1 signal channel inhibitor in promoting erythrocyte denucleation

Abstract

The invention provides application of an ATR / Chk1 signal channel inhibitor in promoting erythrocyte denucleation, and experiments prove that the ATR / Chk1 signal channel inhibitor can promote denucleation of an erythroid in-vitro differentiation system derived from fetal liver and an erythroid in-vitro differentiation system derived from peripheral blood and promote denucleation of erythroblasts in bone marrow. The ATR / Chk1 signaling pathway inhibitor can also promote erythrocyte denucleation in bone marrow and spleen of a thalassemia subject, and has a good application prospect in treatment of erythrocyte denucleation abnormality related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of an ATR / Chk1 signaling pathway inhibitor in promoting erythrocyte enucleation. Background Art

[0002] Red blood cell diseases are the most common diseases in humans. Currently, about 1 billion people in the world suffer from various types of red blood cell developmental disorders, including thalassemia, congenital pure red cell aplasia, myelodysplastic syndrome, congenital erythroid hypoplasia, anemia caused by malarial parasite infection, and anemia caused by insufficient hematopoietic raw materials.

[0003] Blood transfusion is an indispensable treatment method in modern clinical medicine. For patients with anemia, timely and sufficient blood transfusion may improve symptoms, improve quality of life, and even save patients' lives. Since the current source of blood products is mainly donated by volunteers, this causes an imbalance between the amount of blood used and the amount of blood donated. In addition, donated blood products may also be contaminated with pathogenic microorganisms, which can easily cause cross-infection in patients due to blood transfusions, increasing the risk of disease caused by blood transfusions. Mature red blood cells have no nucleus and contain only a very small amount of genetic material, and there is no risk of pathogenic microorganism contamination in in vitro cultured mature red blood cells. Studies in recent years have shown that it is possible to induce differentiation in vitro to produce mature red blood cells and ultimately apply them to clinical blood transfusions, which has broad prospects.

[0004] However, the current technology for inducing the generation of mature red blood cells in vitro is still far from the level of clinical application, and the low rate of red blood cell denucleation is a key issue that needs to be urgently addressed. Summary of the invention

[0005] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0006] The first aspect of the present invention provides any of the following applications: (1) Application of ATR / Chk1 signaling pathway inhibitors in promoting erythrocyte enucleation / preparation of mature erythrocytes; (2) Use of ATR / Chk1 signaling pathway inhibitors in the preparation of drugs for treating and / or preventing diseases related to abnormal red blood cell denucleation.

[0007] In the present invention, the type of ATR / Chk1 signaling pathway inhibitor is not limited, and it can be an ATR gene expression inhibitor, an ATR protein activity inhibitor, a Chk1 gene expression inhibitor, or a Chk1 protein activity inhibitor.

[0008] The ATR / Chk1 gene expression inhibitor may include any material known in the art, as long as it inhibits the mRNA of the gene encoding the ATR / Chk1 protein, and may be prepared by various methods, such as chemical synthesis or in vitro transcription synthesis. The expression inhibitor may include, but is not limited to, antisense nucleotides, siRNA, shRNA or ribozymes that complementarily bind to the polynucleotides constituting the ATR / Chk1 gene.

[0009] In the present invention, antisense nucleotide refers to a sequence that interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to a complementary nucleotide sequence of DNA, immature mRNA or mature mRNA. In addition, since antisense nucleotides are long chains of monomeric units, antisense nucleotides of target RNA sequences can be easily synthesized.

[0010] In the present invention, the siRNA refers to a short double-stranded RNA that can induce RNA interference by cutting specific mRNA. In addition, siRNA is not limited to the region where the double-stranded RNA is fully paired, but can include regions where the chains are not paired due to mismatches (the corresponding nucleotides are not complementary) or protrusions (there are no nucleotides corresponding to one chain). The siRNA terminal structure can have a flat end or an overhanging end, as long as the expression of the target gene can be inhibited by the RNA interference effect, and the adhesion end structure can be a 3' terminal overhanging structure and a 5' terminal overhanging structure.

[0011] In the present invention, the shRNA refers to an RNA sequence that produces a strong hairpin bend, which can be used to silence gene expression by RNA interference. In addition, shRNA can be delivered to cells using a vector for cell introduction, and this vector is always delivered to daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is degraded into siRNA by an intracellular mechanism and binds to an RNA-induced silencing complex that binds to the mRNA corresponding to the siRNA to degrade it.

[0012] In the present invention, the ribozyme refers to an enzyme RNA molecule that can catalyze specific RNA cutting. The specific hybridization of the molecular sequence of the ribozyme and the complementary target RNA can induce endonucleolytic cleavage. The ribozyme may include a known sequence responsible for cutting one or more sequences complementary to the target RNA or a functionally equivalent sequence. In addition, the ribozyme may be a hammerhead ribozyme or a Cech-type ribozyme, i.e., an endoribonuclease RNA, and may be formed by modified oligonucleotides to improve safety and targeting. At the same time, the ribozyme may be distributed in cells expressing the target gene in vivo. A DNA construct encoding a ribozyme under the control of a strong constitutive polymerase III or polymerase II promoter may be used so that the transfected cells can destroy the endogenous target messenger and produce a sufficient amount of ribozyme to inhibit translation. Because the ribozyme has catalytic activity, unlike other antisense molecules, it may have to be kept at a low concentration in the cell.

[0013] In the present invention, the ATR / Chk1 protein may include a polypeptide consisting of any sequence known in the art. The polypeptide may be an amino acid variant having a different sequence by deletion, insertion, substitution or a combination thereof of amino acid residues within the range that does not affect the function of the protein. Amino acid exchanges in proteins or peptides that do not change the overall activity of the molecule are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc.

[0014] Furthermore, inhibitors of ATR / Chk1 protein activity include, but are not limited to, small molecule compounds, peptides, peptide mimetics, matrix analogs, aptamers or antibodies.

[0015] In the present invention, small molecule compounds include but are not limited to compounds that can bind to the ATR / Chk1 kinase domain, compounds that can prevent ATP from binding to ATR / Chk1, and compounds that directly inhibit the activity of ATR / Chk1.

[0016] Further, the small molecule compound includes but is not limited to berzosertib, elimusertib, M1774, RP-3500, AZ20, VE-821, Ceralasertib and / or Rabusertib. Among them, berzosertib, elimusertib, M1774, RP-3500, AZ20, VE-821, Ceralasertib are ATR inhibitors, and Rabusertib is a Chk1 inhibitor.

[0017] Furthermore, the small molecule compound is selected from AZ20, VE-821, Ceralasertib, and Rabusertib.

[0018] In the present invention, peptides and peptide mimetics can inhibit the activity of ATR / Chk1 protein by inhibiting the binding of ATR / Chk1 protein to other proteins. Peptide mimetics can be peptides or non-peptides, and can be composed of amino acids bound by non-peptide bonds such as psi bonds. Non-hydrolyzable peptide mimetics can be prepared using β-turn dipeptide core, keto methylene pseudopeptides, azepine, benzodiazepine, β-amino alcohol or substituted γ-lactam ring as the main residue.

[0019] In the present invention, the aptamer refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid), which itself has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. Because the aptamer can bind to organic matter, peptides, membrane proteins, etc., and block their functions, it can be regarded as a chemical antibody that replaces a single antibody. In addition, aptamers can be obtained by separating oligomers that bind to specific chemical molecules or biomolecules with high affinity and selectivity using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment).

[0020] In the present invention, the antibody can be a monoclonal antibody, a polyclonal antibody or a recombinant antibody. If the sequence of the protein is known, antibodies against a specific protein can be easily prepared using techniques well known in the art. In particular, antibodies can be prepared using hybridoma methods or phage antibody library technology. Typically, hybridoma cells secreting monoclonal antibodies can be prepared by fusing immune cells separated from an immunologically suitable host animal (such as a mouse) injected with an antigen protein with a cancer cell line. The fusion of these two groups of cells can be carried out using polyethylene glycol, etc., and the cells producing antibodies can be propagated by standard culture methods. After obtaining a homogeneous cell population by using a limiting dilution method subclone, hybridoma cells capable of producing antigen-specific antibodies can be prepared by mass culture in vitro or in vivo. The antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography and affinity chromatography.

[0021] Polyclonal antibodies can be prepared by injecting a biomarker protein or a fragment thereof as an immunogen into an external host. The external host can be a mammal, such as a mouse, a rat, a sheep, and a rabbit. When the immunogen is injected intramuscularly, intraperitoneally, or subcutaneously, it can be administered with an adjuvant to increase antigenicity. Thereafter, blood is collected regularly from the external host to obtain serum that shows an increased titer and specificity for the antigen, and antibodies can be isolated and purified therefrom.

[0022] Furthermore, the diseases related to abnormal erythrocyte denucleation include but are not limited to ineffective hematopoiesis, wherein ineffective hematopoiesis may be caused by increased differentiation of erythroid precursor cells or blockage of nucleated erythrocyte maturation. In the present invention, ineffective hematopoietic diseases include but are not limited to myelodysplastic syndrome (MDS), α-thalassemia, β-thalassemia, hereditary sideroblastic anemia, and congenital dyserythropoietic anemia.

[0023] In the present invention, the diseases associated with abnormal red blood cell denucleation also include other types of thalassemia except α-thalassemia and β-thalassemia, such as γ-thalassemia, δβ-thalassemia, γδβ-thalassemia, and δ-thalassemia.

[0024] In the present invention, thalassemia may show abnormalities in the morphology of red blood cells due to abnormal denucleation.

[0025] Furthermore, the disease associated with abnormal red blood cell denucleation is selected from β-thalassemia.

[0026] Furthermore, the red blood cells include red blood cells of an in vitro hematopoietic system and in vivo nucleated red blood cells.

[0027] Furthermore, the in vitro hematopoietic system includes, but is not limited to, an in vitro erythroid differentiation system derived from fetal liver, an in vitro erythroid differentiation system derived from umbilical cord blood, an in vitro erythroid differentiation system derived from pluripotent stem cells, and an in vitro erythroid differentiation system derived from peripheral blood.

[0028] Furthermore, the in vitro hematopoietic system is selected from a fetal liver-derived erythroid in vitro differentiation system and a peripheral blood-derived erythroid in vitro differentiation system.

[0029] In the present invention, the source of the in vitro hematopoietic system is not limited, and can be derived from, including but not limited to, mice, humans, pigs, rabbits, monkeys, etc.

[0030] Furthermore, the source of the in vitro hematopoietic system is selected from humans and mice.

[0031] Furthermore, the in vivo nucleated red blood cells are derived from thalassemia subjects and non-thalassemia subjects.

[0032] In the present invention, subjects include humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and other living organisms. Living organisms can be as simple as, for example, a single eukaryotic cell or as complex as a mammal. Typical patients are mammals, particularly primates, and especially humans. For veterinary applications, a variety of subjects will be suitable, such as livestock such as cattle, sheep, goats, cows, pigs, etc.; poultry such as chickens, ducks, geese, turkeys, etc.; and domestic animals, particularly pets such as dogs and cats. For research applications, suitable subjects will be a variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs, etc.

[0033] Furthermore, the subject is selected from humans and mice.

[0034] In the present invention, the term "treatment and / or prevention" may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, relieving symptoms; alleviating the extent of a condition, disorder or disease; stabilizing (i.e., not worsening) the state of a condition, disorder or disease; delaying the onset of a condition, disorder or disease or slowing its progression; improving a condition, disorder or disease state; and alleviating (whether partial or complete) (whether detectable or undetectable) or improving or ameliorating a condition, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a prolonged survival period compared to the expected survival period when not receiving treatment.

[0035] The second aspect of the present invention provides a pharmaceutical composition for promoting erythrocyte enucleation, wherein the pharmaceutical composition comprises an ATR / Chk1 signaling pathway inhibitor selected from the group consisting of the inhibitors described in the first aspect of the present invention.

[0036] Furthermore, the red blood cells include red blood cells of an in vitro hematopoietic system and in vivo nucleated red blood cells.

[0037] Furthermore, the in vitro hematopoietic system includes, but is not limited to, an in vitro erythroid differentiation system derived from fetal liver, an in vitro erythroid differentiation system derived from umbilical cord blood, an in vitro erythroid differentiation system derived from pluripotent stem cells, and an in vitro erythroid differentiation system derived from peripheral blood.

[0038] Furthermore, the in vitro hematopoietic system is selected from a fetal liver-derived erythroid in vitro differentiation system and a peripheral blood-derived erythroid in vitro differentiation system.

[0039] In the present invention, the source of the in vitro hematopoietic system is not limited, and can be derived from, including but not limited to, mice, humans, pigs, rabbits, monkeys, etc.

[0040] Furthermore, the source of the in vitro hematopoietic system is selected from humans and mice.

[0041] Furthermore, the nucleated red blood cells in the body are derived from thalassemia subjects and non-thalassemia subjects.

[0042] In the present invention, the pharmaceutical composition refers to a pharmaceutical composition comprising at least one ATR / Chk1 signaling pathway inhibitor, that is, it may comprise one ATR / Chk1 signaling pathway inhibitor or may comprise multiple ATR / Chk1 signaling pathway inhibitors.

[0043] Furthermore, the pharmaceutical composition provided by the present invention can be used in combination with other drugs that promote erythrocyte enucleation.

[0044] Furthermore, other drugs that promote erythrocyte enucleation include but are not limited to N-arylcinamide, Bcl-2 inhibitor, Vacuolin-1, zkscan3 inhibitor, RO8191, and AS2863619.

[0045] Furthermore, other drugs that promote erythrocyte enucleation are selected from N-arylcinamide, Bcl-2 inhibitor or Vacuolin-1.

[0046] Furthermore, when used in combination, the order of administration of the pharmaceutical composition provided by the present invention and other drugs that promote erythrocyte denucleation is not limited. The pharmaceutical composition provided by the present invention can be administered first, and then other drugs that promote erythrocyte denucleation; other drugs that promote erythrocyte denucleation can be administered first, and then the pharmaceutical composition provided by the present invention can be administered; the pharmaceutical composition provided by the present invention and other drugs that promote erythrocyte denucleation can also be administered at the same time.

[0047] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

[0048] The pharmaceutically acceptable carrier and / or adjuvant of the present invention include any material suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, a material with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant are used as needed to help the stability of the formulation or to help improve its activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration. The pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed. When using the pharmaceutical composition, the pharmaceutical composition of the present invention at a safe and suitable dosage is administered to the subject.

[0049] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant and / or a disintegrant. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; adhesives include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and hexadecanol; humectants include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0050] Furthermore, the pharmaceutical composition may be administered in any convenient pharmaceutical dosage form.

[0051] Furthermore, the dosage form includes, but is not limited to, a dosage form for enteral administration and a dosage form for parenteral administration.

[0052] Furthermore, the gastrointestinal dosage forms include, but are not limited to, solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder mists, patches, ointments or creams.

[0053] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0054] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the respiratory tract administration dosage forms include but are not limited to sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops, pills, etc., which are used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0055] In the present invention, the pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose. "Pharmaceutically effective dose" means a dose sufficient to produce the desired effect for the condition for which it is administered. The exact dose depends on the activity of the compound, the mode of administration, the nature and severity of the disease, the age and weight of the patient, and different doses may be required. Dosage administration can be implemented by a single administration in the form of individual dosage units (otherwise, several smaller dosage units) and multiple administrations of subdivided doses at specific time intervals.

[0056] Furthermore, the dosage of the ATR / Chk1 signaling pathway inhibitor was 5.5-11 mg / kg.

[0057] The third aspect of the present invention provides a method for promoting erythrocyte enucleation for non-therapeutic purposes, the method comprising administering the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0058] Furthermore, the red blood cells include red blood cells of an in vitro hematopoietic system and in vivo nucleated red blood cells.

[0059] In the present invention, the source of the in vitro hematopoietic system is not limited, and can be derived from, including but not limited to, mice, humans, pigs, rabbits, monkeys, etc.

[0060] Furthermore, the in vitro hematopoietic system includes, but is not limited to, an in vitro erythroid differentiation system derived from fetal liver, an in vitro erythroid differentiation system derived from umbilical cord blood, an in vitro erythroid differentiation system derived from pluripotent stem cells, and an in vitro erythroid differentiation system derived from peripheral blood.

[0061] Furthermore, the in vitro hematopoietic system is selected from a fetal liver-derived erythroid in vitro differentiation system and a peripheral blood-derived erythroid in vitro differentiation system.

[0062] Furthermore, the in vitro hematopoietic system is selected from an in vitro erythroid differentiation system derived from mouse fetal liver and an in vitro erythroid differentiation system derived from human peripheral blood.

[0063] Furthermore, the nucleated red blood cells in the body are derived from thalassemia subjects and non-thalassemia subjects.

[0064] Furthermore, the subject is selected from mice.

[0065] Furthermore, the method includes a method for promoting the enucleation of erythrocytes in an in vitro differentiation system of erythroid cells derived from mouse fetal liver, the method comprising the following steps: using a proliferation medium to amplify the in vitro differentiation system of erythroid cells derived from mouse fetal liver, and then using a differentiation medium for differentiation culture, and when the Ter119 positivity rate reaches about 50% or more, adding the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0066] Furthermore, the amplification time is 0 to 7 days.

[0067] Furthermore, the amplification time is 3 to 5 days.

[0068] Furthermore, the differentiation time is 1 to 5 days.

[0069] Furthermore, the differentiation time is 2 to 4 days.

[0070] Furthermore, the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention was added for 6 h to 24 h, and the denucleation rate of Ter119-positive cells was observed.

[0071] Furthermore, the method comprises using a proliferation medium to expand and culture an in vitro erythroid differentiation system derived from mouse fetal liver for 3 to 5 days, then changing to a differentiation medium for 2 to 4 days of culture, and when the Ter119 positivity rate reaches about 50% or more, adding the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention, and observing the denucleation efficiency of Ter119-positive cells for 6 hours to 24 hours.

[0072] Furthermore, the method includes a method for promoting the enucleation of erythrocytes in an in vitro erythroid differentiation system derived from human peripheral blood, the method comprising the following steps: using a proliferation medium to amplify the in vitro erythroid differentiation system derived from human peripheral blood, then using a differentiation medium for differentiation culture, and adding the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0073] Furthermore, the amplification time is 0 to 10 days.

[0074] Furthermore, the amplification time is 7 days.

[0075] Furthermore, the differentiation time is 0 to 21 days.

[0076] Furthermore, the differentiation time is 6 days.

[0077] Furthermore, on the 6th day of differentiation, the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention was added for 6 h to 24 h, and the denucleation rate of CD235a-positive cells was observed.

[0078] Furthermore, the method comprises using a proliferation medium to expand and culture an in vitro erythroid differentiation system derived from human peripheral blood for 7 days, then changing to a differentiation medium for 6 days, adding the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention, and observing the denucleation efficiency of CD235a-positive cells for 6 hours to 24 hours.

[0079] Furthermore, the method also includes a method for promoting erythrocyte enucleation in mouse bone marrow, the method comprising the following steps: injecting the ATR / Chk1 signaling pathway inhibitor described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention into the peritoneal cavity of the mouse.

[0080] Furthermore, the mice include thalassemia mice and wild-type mice.

[0081] Furthermore, the dosage of the ATR / Chk1 signaling pathway inhibitor for mice is 50-100 mg / kg.

[0082] The present invention has the following advantages and beneficial effects: The present invention provides the use of an ATR / Chk1 signaling pathway inhibitor in promoting erythrocyte denucleation, and experiments prove that the ATR / Chk1 signaling pathway inhibitor can promote the denucleation of an in vitro erythroid differentiation system derived from fetal liver and an in vitro erythroid differentiation system derived from peripheral blood, and promote the denucleation of erythroblasts in the bone marrow. The ATR / Chk1 signaling pathway inhibitor can also promote the denucleation of erythroblasts in the bone marrow and spleen of thalassemia subjects, and has good application prospects in the treatment of diseases related to abnormal erythrocyte denucleation. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is the culture medium formula for the erythroid in vitro differentiation system derived from mouse fetal liver.

[0084] Figure 2 After treatment with ATR inhibitor (AZ20), erythroblasts (Ter119 + Cell) denucleation efficiency results.

[0085] Figure 3 This is the result of Wright-Giemsa staining after the erythroid in vitro differentiation system derived from mouse fetal liver was treated with AZ20.

[0086] Figure 4 This is a graph showing the denucleation rate of the erythroid in vitro differentiation system derived from mouse fetal liver after AZ20 treatment for 12 hours.

[0087] Figure 5 ATR inhibitors (AZ20, VE-821, Ceralasertib) and Chk1 inhibitors (Rabusertib) have a positive effect on Ter119 +Figure 2. Effect of cell enucleation efficiency on results.

[0088] Figure 6 This is a picture of bone marrow sedimentation after intraperitoneal injection of AZ20 in wild-type mice.

[0089] Figure 7 This is a graph showing the denucleation rate of bone marrow erythroblasts in wild-type mice after intraperitoneal injection of AZ20.

[0090] Figure 8 This is a graph showing the denucleation rate of bone marrow erythroblasts after intraperitoneal injection of AZ20 in thalassemia mice.

[0091] Fig. 9 This is the result of anisidine combined with Giemsa staining in the bone marrow of thalassemia mice after AZ20 treatment.

[0092] Fig.10 This is the result of anisidine combined with Giemsa staining in the spleen of thalassemia mice after AZ20 treatment.

[0093] Fig.11 This is the culture medium formula for the in vitro erythroid differentiation system derived from human peripheral blood.

[0094] Fig.12 This is the Wright-Giemsa staining result after the in vitro erythroid differentiation system derived from human peripheral blood was treated with AZ20. DETAILED DESCRIPTION

[0095] The disclosure above generally describes the present invention. A more complete understanding can be obtained with reference to the following specific embodiments. The purpose of describing these embodiments is merely for illustration and is not intended to limit the scope of the present invention. Form changes and equivalent substitutions are considered to be circumstances that may suggest or confer convenience. Although specific terms are used in this article, the purpose of these terms is descriptive rather than limiting.

[0096] Example 1 ATR / Chk1 signaling pathway inhibitor promotes denucleation of mouse fetal liver-derived erythroid in vitro differentiation system 1. Experimental Materials The culture medium formula of the mouse fetal liver-derived erythroid in vitro differentiation system is as follows: Figure 1 shown.

[0097] 2. Experimental methods Magnetic bead sorting of Ter119 in E14.5 pregnant mouse fetal liver - After the cells were rehydrated, they were cryopreserved using stem cell cryopreservation medium (CELLBANKER2). After thawing, they were cultured in vitro in two stages: proliferation and differentiation.

[0098] After 3-5 days of expansion culture using proliferation medium, change to differentiation medium for 2-4 days of differentiation culture. When the cell membrane surface marker Ter119 positive rate reaches about 50% or more, use ATR / Chk1 small molecule inhibitors to observe the denucleation efficiency of erythroblasts (i.e. Ter119 positive cells) in a short period of time (6h~24h). Among them, ATR inhibitors are AZ20, VE-821, Ceralasertib, etc. Chk1 inhibitors are Rabusertib, etc.

[0099] The method for detecting the efficiency of denucleation is as follows: ① Flow cytometry detection of membrane surface marker Ter119 and nuclear dye Hoechst33342. ② Using Cytospin™ 4 cell smear machine to prepare monolayer cell smears, and then perform Wright-Giemsa staining.

[0100] 3. Experimental results (1) The results are as follows Figure 2 As shown in the figure, the ATR inhibitor (AZ20) can significantly increase the denuclearization rate of Ter119+ cells under the action of various treatment times, and the denuclearization rate can be increased by about 1 times at most.

[0101] (2) Wright-Giemsa staining to detect denucleation ( Figure 3 ). After 4h~6h of AZ20 treatment, a significant increase in the number of cells undergoing enucleation and anucleate cells can be observed (arrows indicate erythroblasts undergoing enucleation).

[0102] (3) After 12 h of treatment with the ATR inhibitor (AZ20), the denucleation rate increased with the increase of AZ20 concentration ( Figure 4 ).

[0103] (4) ATR inhibitors (AZ20, VE-821, Ceralasertib) and Chk1 inhibitors (Rabusertib) can improve the efficiency of Ter119+ cell denuclearization ( Figure 5 ).

[0104] Example 2 ATR inhibitor promotes denucleation of erythroblasts in the bone marrow of wild-type mice 1. Experimental methods Wild-type mice were intraperitoneally injected with ATR inhibitor (AZ20) at a dose of 50-100 mg / kg. After 12 hours, the whole bone marrow of the mice was obtained, the color of the cell pellet was observed, and the membrane surface marker Ter119 and the nuclear dye Hoechst 33342 were detected by flow cytometry.

[0105] 2. Experimental results (1) Figure 6 It was shown that after intraperitoneal injection of AZ20 in wild-type mice, the bone marrow pellet turned red, indicating enhanced erythroid terminal differentiation.

[0106] (2) Figure 7 The results showed that the rate of erythroblast denucleation increased after intraperitoneal injection of AZ20 in wild-type mice.

[0107] Example 3 ATR inhibitor promotes denucleation of erythroblasts in the bone marrow of thalassemia mice 1. Experimental methods (1) After intraperitoneal injection of AZ20 into β-thalassemia mice, the rate of erythroblast denucleation was detected by Wright-Giemsa staining.

[0108] (2) Anisidine combined with Giemsa staining was used to detect the effect of ATR inhibitor AZ20 on anucleated erythrocyte production in the bone marrow of β-thalassemia mice.

[0109] (3) Anisidine combined with Giemsa staining was used to detect the effect of ATR inhibitor AZ20 on anucleated erythrocyte production in the spleen of β-thalassemia mice.

[0110] 2. Experimental results (1) Figure 8 The results showed that after intraperitoneal injection of AZ20 in β-thalassemia mice, the rate of erythroblast denucleation increased.

[0111] (2) Fig. 9 The results showed that anisidine combined with Giemsa staining suggested that the ATR inhibitor AZ20 increased the production of anucleated erythrocytes in the bone marrow of β-thalassemia mice.

[0112] (3) Fig.10 The results showed that anisidine combined with Giemsa staining suggested that the ATR inhibitor AZ20 increased the production of anucleated erythrocytes in the spleen of β-thalassemia mice.

[0113] Example 4 ATR inhibitor promotes the expression of human peripheral blood derived CD34 + In vitro erythroid differentiation system erythroblast enucleation 1. Experimental Materials Human peripheral blood derived CD34 + In vitro erythroid differentiation system culture medium formula Fig.11 shown.

[0114] 2. Experimental methods Magnetic bead separation of CD34 in human umbilical cord blood / peripheral blood mononuclear cells + Cells were cryopreserved using stem cell cryopreservation medium (CELLBANKER2). After thawing, proliferation / differentiation in vitro culture was performed.

[0115] After 7 days of expansion culture using proliferation medium, the cells were replaced with differentiation medium for differentiation culture for 0-14 days. On the 6th day of differentiation, the ATR / Chk1 small molecule inhibitor was used to observe the denuclearization efficiency of erythroblasts (i.e., CD235a positive cells) in a short period of time (12 hours). Among them, the ATR inhibitor was AZ20, with an action concentration of 2uM.

[0116] 3. Experimental results Wright-Giemsa staining to detect nucleation ( Fig.12 ). After AZ20 treatment for 12 hours, anucleated cells were observed to increase in the CD34+ in vitro erythroid differentiation system derived from human peripheral blood (arrows indicate anucleated cells).

[0117] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Any of the following applications: (1) Application of ATR / Chk1 signaling pathway inhibitors in promoting erythrocyte enucleation / preparation of mature erythrocytes; (2) Use of ATR / Chk1 signaling pathway inhibitors in the preparation of drugs for treating and / or preventing diseases related to abnormal red blood cell denucleation.

2. The use according to claim 1, characterized in that: The ATR / Chk1 signaling pathway inhibitors include antisense nucleotides, siRNA, shRNA, ribozymes, small molecule compounds, peptides, peptide mimetics, matrix analogs, aptamers and / or antibodies.

3. The use according to claim 2, characterized in that: The ATR / Chk1 signaling pathway inhibitor is selected from small molecule compounds.

4. The use according to claim 3, characterized in that: The small molecule compound is selected from AZ20, VE-821, Ceralasertib and / or Rabusertib.

5. The use according to claim 1, characterized in that: The red blood cells include red blood cells of an in vitro hematopoietic system and in vivo nucleated red blood cells.

6. The use according to claim 1, characterized in that: The disease related to abnormal red blood cell denucleation is an ineffective hematopoietic disease.

7. The use according to claim 6, characterized in that: The ineffective hematopoietic diseases include myelodysplastic syndrome, α-thalassemia, β-thalassemia, hereditary sideroblastic anemia, and congenital dyserythropoietic anemia; Preferably, the ineffective hematopoietic disease is selected from β-thalassemia.

8. A pharmaceutical composition for promoting erythrocyte denucleation, characterized in that: The pharmaceutical composition comprises an ATR / Chk1 signaling pathway inhibitor selected from any one of claims 1-4.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

10. A method for promoting erythrocyte enucleation for non-therapeutic purposes, characterized in that: The method comprises administering the ATR / Chk1 signaling pathway inhibitor according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 8 to 9.

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