Application of ATR / Chk1 signaling pathway inhibitor in promoting enucleation of erythrocytes

Abstract

The present invention provides the use of ATR / Chk1 signaling pathway inhibitors in promoting enucleation of red blood cells, and experiments prove that ATR / Chk1 signaling pathway inhibitors can promote enucleation in the in vitro differentiation system of erythroid cells derived from fetal liver and the in vitro differentiation system of erythroid cells derived from peripheral blood, and can promote enucleation of erythroblasts in bone marrow. ATR / Chk1 signaling pathway inhibitors can also promote enucleation of erythroblasts in the bone marrow and spleen of thalassemia subjects, and have good application prospects in the treatment of diseases related to abnormal enucleation of red blood cells.

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 worldwide 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 the patient's life. Because the current source of blood products is mainly donated by volunteers, this has caused an imbalance between the amount of blood used and the amount of blood donated. In addition, donated blood products may be contaminated with pathogenic microorganisms, which can easily cause cross-infection in patients due to blood transfusion, increasing the risk of transfusion-related diseases. Mature red blood cells have no nucleus and contain only a very small amount of genetic material. In addition, mature red blood cells cultured in vitro do not have the risk of pathogenic microbial contamination. Recent studies have shown that it is possible to induce differentiation of mature red blood cells in vitro and ultimately use them in clinical blood transfusion, 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:

[0007] (1) Application of ATR / Chk1 signaling pathway inhibitors in promoting erythrocyte enucleation / preparation of mature erythrocytes;

[0008] (2) The 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.

[0009] In the present invention, the type of ATR / Chk1 signaling pathway inhibitor is not limited, and can be an ATR gene expression inhibitor, an ATR protein activity inhibitor, a Chk1 gene expression inhibitor, or a Chk1 protein activity inhibitor.

[0010] 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 can 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 polynucleotide constituting the ATR / Chk1 gene.

[0011] In the present invention, an antisense nucleotide refers to a sequence that interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to the complementary nucleotide sequence of DNA, immature mRNA, or mature mRNA. In addition, since an antisense nucleotide is a long chain of monomer units, an antisense nucleotide of a target RNA sequence can be easily synthesized.

[0012] In the present invention, the siRNA refers to a short double-stranded RNA that can induce RNA interference by cleaving a specific mRNA. In addition, siRNA is not limited to the region where the double-stranded RNA is completely paired, and may include regions where the strands are unpaired due to mismatches (corresponding nucleotides are not complementary) or bulges (there are no nucleotides corresponding to one strand). The siRNA terminal structure may have blunt ends or overhangs as long as the expression of the target gene can be inhibited by the RNA interference effect, and the sticky end structures can be 3'-end overhang structures and 5'-end overhang structures.

[0013] In the present invention, the shRNA refers to an RNA sequence that produces a strong hairpin bend and can be used to silence gene expression by RNA interference. In addition, the shRNA can be delivered into cells using a vector for cell introduction, and such a vector is always delivered into daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is degraded into siRNA by an intracellular mechanism and binds to the RNA-induced silencing complex, which binds to the mRNA corresponding to the siRNA to degrade it.

[0014] In the present invention, the ribozyme refers to an enzymatic RNA molecule capable of catalyzing specific RNA cleavage. Specific hybridization of the molecular sequence of the ribozyme and the complementary target RNA can induce endonucleolytic cleavage. The ribozyme may include known sequences responsible for cleaving one or more sequences complementary to the target RNA or functionally equivalent sequences. In addition, the ribozyme may be a hammerhead ribozyme or a Cech-type ribozyme, i.e., an endoribonuclease RNA, and may be formed from modified oligonucleotides to improve safety and targeting. Meanwhile, the ribozyme can be distributed in cells expressing the target gene in vivo. A DNA construct encoding the ribozyme can be used under the control of a strong constitutive polymerase III or polymerase II promoter, such that transfected cells can disrupt endogenous target messengers and produce sufficient amounts of ribozyme to inhibit translation. Since the ribozyme has catalytic activity, unlike other antisense molecules, it may have to be maintained at a low concentration in cells.

[0015] In the present invention, the ATR / Chk1 protein may include a polypeptide composed 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 a range that does not affect the protein function. Amino acid exchanges in proteins or peptides that do not alter the overall activity of the molecule are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc.

[0016] Furthermore, the ATR / Chk1 protein activity inhibitors include, but are not limited to, small molecule compounds, peptides, peptidomimetics, matrix analogs, aptamers, or antibodies.

[0017] In the present invention, the 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.

[0018] Furthermore, the small molecule compounds include, but are not limited to, bezosertib, elimusertib, M1774, RP-3500, AZ20, VE-821, Ceralasertib, and / or Rabusertib. Among them, bezosertib, elimusertib, M1774, RP-3500, AZ20, VE-821, Ceralasertib are ATR inhibitors, and Rabusertib is a Chk1 inhibitor.

[0019] Furthermore, the small molecule compounds are selected from AZ20, VE-821, Ceralasertib, Rabusertib.

[0020] In the present invention, peptides and peptidomimetics can inhibit the activity of ATR / Chk1 proteins by inhibiting the binding of ATR / Chk1 proteins to other proteins. Peptidomimetics can be peptides or non-peptides and can be composed of amino acids linked by non-peptide bonds such as psi bonds. Non-hydrolyzable peptidomimetics can be prepared using a β-turn dipeptide core, keto methylene pseudopeptides, azepine, benzodiazepine, β-amino alcohols, or substituted γ-lactam rings as the main residues.

[0021] In the present invention, the aptamer refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure by itself and can bind to a target molecule with high affinity and specificity. Since aptamers can bind to organic compounds, peptides, membrane proteins, etc. and block their functions, they 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 or biological molecules with high affinity and selectivity using an oligonucleotide library called SELEX (Systematic Evolution of Ligands by Exponential Enrichment).

[0022] In the present invention, the antibody can be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. If the sequence of a 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 the hybridoma method or phage antibody library technology. Generally, hybridoma cells secreting monoclonal antibodies can be prepared by fusing immune cells isolated from an immunologically suitable host animal (such as a mouse) injected with the 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 antibody-producing cells can be proliferated by standard culture methods. After obtaining a homogeneous cell population by subcloning using the limiting dilution method, hybridoma cells capable of producing antigen-specific antibodies can be prepared by culturing them in large quantities in vitro or in vivo. The antibodies prepared by the above methods can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

[0023] 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, rat, sheep, and rabbit. When the immunogen is administered by intramuscular injection, intraperitoneal injection, or subcutaneous injection, it can be administered together with an adjuvant to increase antigenicity. Thereafter, blood is regularly collected from the external host to obtain serum showing an increased titer and specificity against the antigen, and the antibody can be separated and purified therefrom.

[0024] Further, the diseases related to abnormal erythrocyte enucleation include, but are not limited to, ineffective hematopoiesis diseases, where ineffective hematopoiesis can be caused by increased differentiation of erythroid progenitor cells or maturation arrest of nucleated erythrocytes. In the present invention, ineffective hematopoiesis diseases include, but are not limited to, myelodysplastic syndromes (MDS), α-thalassemia, β-thalassemia, hereditary sideroblastic anemia, and congenital dyserythropoietic anemia.

[0025] In the present invention, the diseases related to abnormal erythrocyte enucleation also include other types of thalassemia except α-thalassemia and β-thalassemia, such as γ-thalassemia, δβ-thalassemia, γδβ-thalassemia, and δ-thalassemia.

[0026] In the present invention, thalassemia shows the characterization of abnormal erythrocyte morphology due to abnormal enucleation.

[0027] Further, the diseases related to abnormal erythrocyte enucleation are selected from β-thalassemia.

[0028] Further, the erythrocytes include erythrocytes in an in vitro hematopoiesis system and nucleated erythrocytes in vivo.

[0029] Further, the in vitro hematopoiesis system includes, but is not limited to, an erythroid in vitro differentiation system derived from fetal liver, an erythroid in vitro differentiation system derived from umbilical cord blood, an erythroid in vitro differentiation system derived from pluripotent stem cells, and an erythroid in vitro differentiation system derived from peripheral blood.

[0030] Further, the in vitro hematopoiesis system is selected from an erythroid in vitro differentiation system derived from fetal liver and an erythroid in vitro differentiation system derived from peripheral blood.

[0031] In the present invention, the source of the in vitro hematopoiesis system is not restricted and can be derived from, including but not limited to, mice, humans, pigs, rabbits, monkeys, etc.

[0032] Further, the source of the in vitro hematopoiesis system is selected from humans and mice.

[0033] Further, the nucleated erythrocytes in vivo are derived from thalassemia subjects and non-thalassemia subjects.

[0034] In the present invention, the subjects include humans, mammals (such as cats, dogs, horses, etc.), living cells and other living organisms. The living organisms can be as simple as a single eukaryotic cell or as complex as a mammal. A typical patient is a mammal, especially a primate, particularly a human. For veterinary applications, a variety of subjects will be suitable, such as livestock like cows, sheep, goats, cattle, pigs, etc.; poultry like chickens, ducks, geese, turkeys, etc.; and domestic animals, especially 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.

[0035] Further, the subject is selected from humans and mice.

[0036] In the present invention, the term "treatment and / or prevention" may refer to a therapeutic treatment or a preventive measure, where the goal is to prevent or slow down (mitigate) an undesired physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms; reducing the degree of a condition, disorder, or disease; stabilizing (i.e., not worsening) the state of a condition, disorder, or disease; delaying the onset or slowing the progression of a condition, disorder, or disease; improving the state of a condition, disorder, or disease; and relieving (whether partially or completely) (whether detectable or undetectable) or improving or ameliorating a condition, disorder, or disease. Treatment may include eliciting a clinically significant response without excessive side effects. Treatment also includes a survival period that is extended compared to the expected survival period without treatment.

[0037] A second aspect of the present invention provides a pharmaceutical composition for promoting enucleation of red blood cells, the pharmaceutical composition comprising an ATR / Chk1 signaling pathway inhibitor selected from those described in the first aspect of the present invention.

[0038] Further, the red blood cells include red blood cells in an in vitro hematopoietic system and nucleated red blood cells in vivo.

[0039] Further, the in vitro hematopoietic system includes, but is not limited to, an erythroid in vitro differentiation system derived from fetal liver, an erythroid in vitro differentiation system derived from umbilical cord blood, an erythroid in vitro differentiation system derived from pluripotent stem cells, and an erythroid in vitro differentiation system derived from peripheral blood.

[0040] Further, the in vitro hematopoietic system is selected from an erythroid in vitro differentiation system derived from fetal liver and an erythroid in vitro differentiation system derived from peripheral blood.

[0041] 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.

[0042] Further, the source of the in vitro hematopoietic system is selected from humans and mice.

[0043] Further, the enucleated erythrocytes in the body are derived from thalassemia subjects and non-thalassemia subjects.

[0044] In the present invention, the pharmaceutical composition refers to a composition comprising at least one ATR / Chk1 signaling pathway inhibitor, which may comprise one ATR / Chk1 signaling pathway inhibitor or multiple ATR / Chk1 signaling pathway inhibitors.

[0045] Further, the pharmaceutical composition provided by the present invention can be used in combination with other drugs that promote enucleation of erythrocytes.

[0046] Further, other drugs that promote enucleation of erythrocytes include, but are not limited to, N-aryl cinnamide, Bcl-2 inhibitor, Vacuolin-1, zkscan3 inhibitor, RO8191, and AS2863619.

[0047] Further, other drugs that promote enucleation of erythrocytes are selected from N-aryl cinnamide, Bcl-2 inhibitor, or Vacuolin-1.

[0048] Further, when used in combination, the order of administration of the pharmaceutical composition provided by the present invention and other drugs that promote enucleation of erythrocytes is not restricted. The pharmaceutical composition provided by the present invention can be administered first, followed by other drugs that promote enucleation of erythrocytes; or other drugs that promote enucleation of erythrocytes can be administered first, followed by the pharmaceutical composition provided by the present invention; or the pharmaceutical composition provided by the present invention and other drugs that promote enucleation of erythrocytes can be administered simultaneously.

[0049] Further, the pharmaceutical composition includes its pharmaceutically acceptable carrier and / or excipient.

[0050] The pharmaceutically acceptable carrier and / or excipient described in the present invention includes any substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or excipient is used as needed to help the stability of the formulation or to enhance its activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration. The pharmaceutical composition thus formulated can be administered by any suitable administration method known to those skilled in the art as needed. When using the pharmaceutical composition, a safe and suitable dosage of the pharmaceutical composition described in the present invention is administered to the subject.

[0051] Furthermore, the pharmaceutically acceptable carrier and / or excipient include diluents, binders, surfactants, wetting agents, adsorbent carriers, lubricants and / or disintegrants. Among them, diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, water; binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose; surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, cetyl alcohol; wetting agents include, but are not limited to, glycerol, starch; adsorbent carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, saponite; lubricants include, but are not limited to, zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium dodecyl magnesium sulfate.

[0052] Furthermore, the pharmaceutical composition can be administered in any convenient pharmaceutical dosage form.

[0053] Furthermore, the dosage forms include, but are not limited to, dosage forms for gastrointestinal administration and dosage forms for non-gastrointestinal administration.

[0054] Furthermore, the dosage forms for gastrointestinal administration include, but are not limited to, solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, dripping pills, suppositories, aerosols, sprays, powder inhalations, patches, ointments or creams.

[0055] Furthermore, the dosage forms for non-gastrointestinal administration include, but are not limited to, dosage forms for injection, dosage forms for respiratory tract, dosage forms for body cavities, dosage forms for mucous membranes, dosage forms for skin.

[0056] Furthermore, the dosage forms for injection include, but are not limited to, various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the dosage forms for respiratory tract include, but are not limited to, sprays, aerosols, powder inhalations, etc.; the dosage forms for body cavities include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the dosage forms for mucous membranes include, but are not limited to, eye drops, nose drops, eye ointments, gargles, sublingual tablets, adhesive tablets, film dressings, etc.; the dosage forms for skin include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0057] In the present invention, the pharmaceutical composition is administered to a patient at a pharmaceutically effective dose. A "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, and the age and weight of the patient, and different doses may be required. The dose administration can be carried out by a single administration in the form of individual dose units (otherwise, several smaller dose units) and also by multiple administrations of divided doses at specific time intervals.

[0058] Furthermore, the administration dose of the ATR / Chk1 signaling pathway inhibitor is 5.5 - 11 mg / kg.

[0059] 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.

[0060] Furthermore, the erythrocytes include erythrocytes of the in vitro hematopoietic system and nucleated erythrocytes in vivo.

[0061] 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.

[0062] Furthermore, the in vitro hematopoietic system includes but is not limited to an erythroid in vitro differentiation system derived from fetal liver, an erythroid in vitro differentiation system derived from umbilical cord blood, an erythroid in vitro differentiation system derived from pluripotent stem cells, and an erythroid in vitro differentiation system derived from peripheral blood.

[0063] Furthermore, the in vitro hematopoietic system is selected from an erythroid in vitro differentiation system derived from fetal liver and an erythroid in vitro differentiation system derived from peripheral blood.

[0064] Furthermore, the in vitro hematopoietic system is selected from an erythroid in vitro differentiation system derived from mouse fetal liver and an erythroid in vitro differentiation system derived from human peripheral blood.

[0065] Furthermore, the nucleated erythrocytes in vivo are derived from thalassemia subjects and non-thalassemia subjects.

[0066] Furthermore, the subject is selected from mice.

[0067] Furthermore, the method includes a method for promoting erythrocyte enucleation of an erythroid in vitro differentiation system derived from mouse fetal liver, the method comprising the following steps: amplifying the erythroid in vitro differentiation system derived from mouse fetal liver using a proliferation medium, then differentiating and culturing using a differentiation medium, and when the positive rate of Ter119 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.

[0068] Further, the amplification time is 0 to 7 days.

[0069] Further, the amplification time is 3 to 5 days.

[0070] Further, the differentiation time is 1 to 5 days.

[0071] Further, the differentiation time is 2 to 4 days.

[0072] Further, add 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 for 6 h to 24 h, and observe the enucleation rate of Ter119-positive cells.

[0073] Further, the method includes amplifying and culturing an in vitro differentiation system of erythroid cells derived from mouse fetal liver using a proliferation medium for 3 to 5 days, then changing to a differentiation medium for culturing for 2 to 4 days. When the positive rate of Ter119 reaches about 50% or more, add 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 observe the enucleation efficiency of Ter119-positive cells for 6 h to 24 h.

[0074] Further, the method includes a method for promoting enucleation of erythroid cells in an in vitro differentiation system derived from human peripheral blood. The method includes the following steps: amplifying the in vitro differentiation system of erythroid cells derived from human peripheral blood using a proliferation medium, then differentiating and culturing using a differentiation medium, 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.

[0075] Further, the amplification time is 0 to 10 days.

[0076] Further, the amplification time is 7 days.

[0077] Further, the differentiation time is 0 to 21 days.

[0078] Further, the differentiation time is 6 days.

[0079] Further, add 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 for 6 h to 24 h on the 6th day of differentiation, and observe the enucleation rate of CD235a-positive cells.

[0080] Further, the method includes culturing the erythroid in vitro differentiation system derived from human peripheral blood in a proliferation medium for 7 days, then replacing it with 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 enucleation efficiency of CD235a-positive cells for 6 h to 24 h.

[0081] Further, the method also includes a method for promoting enucleation of erythrocytes in mouse bone marrow, and the method includes 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 abdominal cavity of the mouse.

[0082] Further, the mice include thalassemia mice and wild-type mice.

[0083] Further, the dosage of the ATR / Chk1 signaling pathway inhibitor used in mice is 50 - 100 mg / kg.

[0084] Advantages and beneficial effects of the present invention:

[0085] The present invention provides the application of an ATR / Chk1 signaling pathway inhibitor in promoting erythrocyte enucleation, and experiments prove that the ATR / Chk1 signaling pathway inhibitor can promote enucleation of the erythroid in vitro differentiation system derived from fetal liver and the erythroid in vitro differentiation system derived from peripheral blood, promote enucleation of erythroblasts in bone marrow, and the ATR / Chk1 signaling pathway inhibitor can also promote enucleation 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 enucleation. Description of the drawings

[0086] Figure 1 It is a diagram of the culture medium formula of the erythroid in vitro differentiation system derived from mouse fetal liver.

[0087] Figure 2 It is a diagram of the enucleation efficiency results of erythroblasts (Ter119 + cells) after treatment with an ATR inhibitor (AZ20).

[0088] Figure 3 It is a diagram of the Wright-Giemsa staining results of the erythroid in vitro differentiation system derived from mouse fetal liver after treatment with AZ20.

[0089] Figure 4 It is a diagram of the enucleation rate results of the erythroid in vitro differentiation system derived from mouse fetal liver after treatment with AZ20 for 12 h.

[0090] Figure 5are ATR inhibitors (AZ20, VE-821, Ceralasertib), and Chk1 inhibitor (Rabusertib) on Ter119 + Results graph of the effect of cell enucleation efficiency

[0091] Figure 6 Is the bone marrow precipitation graph of wild-type mice after intraperitoneal injection of AZ20

[0092] Figure 7 Is the results graph of the enucleation rate of erythroblasts in the bone marrow of wild-type mice after intraperitoneal injection of AZ20

[0093] Figure 8 Is the results graph of the enucleation rate of erythroblasts in the bone marrow of thalassemic mice after intraperitoneal injection of AZ20

[0094] Figure 9 Is the results graph of aniline combined with Giemsa staining in the bone marrow of thalassemic mice after treatment with AZ20

[0095] Figure 10 Is the results graph of aniline combined with Giemsa staining in the spleen of thalassemic mice after treatment with AZ20

[0096] Figure 11 Is the medium formulation graph of the in vitro erythroid differentiation system derived from human peripheral blood

[0097] Figure 12 Is the results graph of Wright-Giemsa staining after treating the in vitro erythroid differentiation system derived from human peripheral blood with AZ20 Detailed implementation mode

[0098] The above disclosure provides a general description of the present invention. A more complete understanding can be obtained by referring to the following specific embodiments. The purpose of describing these embodiments is merely illustrative and is not intended to limit the scope of the present invention. Formal transformations and equivalent substitutions are considered as circumstances may suggest or afford convenience. Although specific terms are used herein, these terms are for descriptive purposes and not for limitation.

[0099] Example 1 ATR / Chk1 signaling pathway inhibitors promote enucleation of the erythroid in vitro differentiation system derived from mouse fetal liver

[0100] 1. Experimental materials

[0101] The medium formulation of the erythroid in vitro differentiation system derived from mouse fetal liver is as Figure 1 shown.

[0102] 2. Experimental method

[0103] Magnetic bead sorting of E14.5 pregnant mouse fetal liver Ter119 -After the cells, they were cryopreserved using a stem cell cryopreservation solution (CELLBANKER2). After resuscitation, in vitro culture was carried out in two stages of proliferation / differentiation.

[0104] After 3 - 5 days of amplification culture using a proliferation medium, it was replaced with a differentiation medium for 2 - 4 days of differentiation culture. When the positive rate of the cell membrane surface marker Ter119 reached about 50% or more, it was treated with ATR / Chk1 small molecule inhibitors, and the enucleation efficiency of erythroblasts (i.e., Ter119 - positive cells) was observed within a short time (6h - 24h). Among them, ATR inhibitors were AZ20, VE - 821, Ceralasertib, etc. The Chk1 inhibitor was Rabusertib, etc.

[0105] The methods for detecting enucleation efficiency were: ① Flow cytometry was used to detect the membrane surface marker Ter119 and the nuclear dye Hoechst33342. ② After preparing a monolayer cell smear using a Cytospin™ 4 cytocentrifuge, Wright - Giemsa staining was performed.

[0106] 3. Experimental results

[0107] (1) The results were as Figure 2 shown. Under the action of ATR inhibitor (AZ20) at various treatment times, the enucleation rate of Ter119+ cells could be significantly increased, and the enucleation rate could be increased by about 1 time at most.

[0108] (2) Wright - Giemsa staining was used to detect the enucleation situation ( Figure 3 ). When treated with AZ20 for 4h - 6h, a significant increase in enucleating and anucleated cells could be observed (arrows indicate enucleating erythroblasts).

[0109] (3) Under the action of ATR inhibitor (AZ20) for 12h, the enucleation rate increased with the increase in the concentration of AZ20 ( Figure 4 ).

[0110] (4) ATR inhibitors (AZ20, VE - 821, Ceralasertib) and Chk1 inhibitor (Rabusertib) could all improve the enucleation efficiency of Ter119+ cells ( Figure 5 ).

[0111] Example 2 Promotion of enucleation of erythroblasts in the bone marrow of wild - type mice by ATR inhibitors

[0112] 1. Experimental methods

[0113] Wild-type mice were intraperitoneally injected with an ATR inhibitor (AZ20) at a dose of 50 - 100 mg / kg. After 12 h, the whole bone marrow of the mice was collected, the color of the cell precipitate was observed, and the cell surface markers Ter119 and the nuclear dye Hoechst 33342 were detected by flow cytometry.

[0114] 2. Experimental results

[0115] (1) Figure 6 It was shown that after intraperitoneal injection of AZ20 in wild-type mice, the bone marrow precipitate turned red, indicating enhanced erythroid terminal differentiation.

[0116] (2) Figure 7 It was shown that after intraperitoneal injection of AZ20 in wild-type mice, the enucleation rate of bone marrow erythroblasts increased.

[0117] Example 3: ATR inhibitor promotes enucleation of erythroblasts in the bone marrow of thalassemia mice

[0118] 1. Experimental method

[0119] (1) After intraperitoneal injection of AZ20 in β-thalassemia mice, the enucleation rate of bone marrow erythroblasts was detected by Wright-Giemsa staining.

[0120] (2) Aniline combined with Giemsa staining was used to detect the effect of ATR inhibitor AZ20 on the production of anucleated erythrocytes in the bone marrow of β-thalassemia mice.

[0121] (3) Aniline combined with Giemsa staining was used to detect the effect of ATR inhibitor AZ20 on the production of anucleated erythrocytes in the spleen of β-thalassemia mice.

[0122] 2. Experimental results

[0123] (1) Figure 8 The results showed that after intraperitoneal injection of AZ20 in β-thalassemia mice, the enucleation rate of bone marrow erythroblasts increased.

[0124] (2) Figure 9 The results showed that aniline combined with Giemsa staining indicated that ATR inhibitor AZ20 increased the production of anucleated erythrocytes in the bone marrow of β-thalassemia mice.

[0125] (3) Figure 10 The results showed that aniline combined with Giemsa staining indicated that ATR inhibitor AZ20 increased the production of anucleated erythrocytes in the spleen of β-thalassemia mice.

[0126] Example 4: ATR inhibitor promotes enucleation of erythroblasts in the in vitro erythroid differentiation system derived from human peripheral blood CD34 +

[0127] 1. Experimental materials

[0128] CD34 derived from human peripheral blood + The culture medium formula for the in vitro erythroid differentiation system is as Figure 11 shown.

[0129] 2. Experimental methods

[0130] Magnetic beads were used to separate CD34 + cells in human umbilical cord blood / peripheral blood mononuclear cells, and they were cryopreserved using a stem cell cryopreservation solution (CELLBANKER2). After thawing, in vitro culture of proliferation / differentiation was carried out.

[0131] After 7 days of amplification culture using a proliferation medium, it was replaced with a differentiation medium for 0 - 14 days of differentiation culture. On the 6th day of differentiation, treatment with an ATR / Chk1 small molecule inhibitor was carried out to observe the enucleation efficiency of erythroblasts (i.e., CD235a positive cells) within a short period (12 h). Among them, the ATR inhibitor was AZ20, and the working concentration was 2 μM.

[0132] 3. Experimental results

[0133] Wright-Giemsa staining was used to detect enucleation ( Figure 12 ). When treated with AZ20 for 12 h, an increase in anucleated cells (arrow indicates anucleated cells) could be observed in the in vitro erythroid differentiation system of CD34+ derived from human peripheral blood.

[0134] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of an ATR / Chk1 signaling pathway inhibitor in the preparation of a medicament for treating and / or preventing diseases associated with abnormal erythrocyte enucleation, wherein the ATR / Chk1 signaling pathway inhibitor is selected from AZ20, VE-821, Ceralasertib, and / or Rabusertib, and the diseases associated with abnormal erythrocyte enucleation are β-thalassemia.

2. A method for promoting enucleation of red blood cells for non-therapeutic purposes, characterized in that, The method comprises administering an ATR / Chk1 signaling pathway inhibitor, wherein the ATR / Chk1 signaling pathway inhibitor is selected from AZ20, VE-821, Ceralasertib, and / or Rabusertib.

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