New use of known eIF5B inhibitors in treatment of acute myelogenous leukemia (AML)

Abstract

The invention discloses a novel application of a known eIF5B inhibitor in treating acute myelogenous leukemia (AML). It is found for the first time that the eIF5B inhibitor has high toxicity to various AML cells, can inhibit proliferation of the various AML cells and induce differentiation of the AML cells, has no significant influence on normal hematopoietic stem / progenitor cells, and can be used for developing drugs for treating AML. The invention provides a theoretical basis for research and development of anti-AML drugs, opens up a new application of the eIF5B inhibitor, provides a brand new method for treating AML, and has a wide application prospect in the technical field of AML treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a new use of a known eIF5B inhibitor in treating acute myeloid leukemia (AML). BACKGROUND

[0002] Acute myeloid leukemia (AML) is a hematological malignancy characterized by the excessive proliferation and differentiation block of immature myeloid hematopoietic stem / progenitor cells, which shows high heterogeneity in pathogenesis, genetic background and prognosis. At present, the treatment of AML mainly relies on chemotherapy, targeted therapy and hematopoietic stem cell transplantation, but still has the following limitations: 1) poor selectivity of existing targeted drugs: many AML targeted drugs (such as FLT3 inhibitors, IDH inhibitors) are only effective for specific mutant subtypes, and may affect normal hematopoietic stem cells (HSPC), leading to bone marrow suppression, infection, bleeding and other side effects; 2) lack of effective means to regulate cell differentiation: existing therapies mainly kill tumor cells directly, while drugs that promote AML cell differentiation (such as ATRA) are only suitable for specific subtypes (APL) and have limited scope of application.

[0003] As a member of eukaryotic translation initiation factors, eIF5B is involved in the last step of the translation initiation process, promoting the binding of 40S and 60S ribosomal subunits to form an 80S initiation complex with elongation ability. In addition, the interaction of eIF5B with eIF5, eIF2A and eIF1A, as well as its ability to bind and deliver initiator tRNA, is crucial for mRNA translation. So far, no study has confirmed the link between eIF5B and leukemia.

[0004] 1282NJ0069 was first reported in 2024, and its structure is 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone, which can be used as an inhibitor of in vitro protein synthesis and significantly inhibits the growth of pathogenic fungi. After verification of drug target experiments, it was confirmed that eIF5B in Saccharomyces cerevisiae is a potential antifungal target of 1282NJ0069, and 1282NJ0069 was identified as a translation initiation inhibitor. However, no study has converted 1282NJ0069 from the antifungal field to the treatment of hematological tumors, and its potential use needs to be explored. SUMMARY

[0005] To make up for the shortcomings of the prior art, the present application provides a new use of a known eIF5B inhibitor in treating acute myeloid leukemia (AML), providing a safe and effective drug for the treatment of AML to overcome the technical problem that there is an urgent need for an effective and universal drug to inhibit the proliferation of AML cells and induce the differentiation of AML cells in the field.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The first aspect of the present invention provides the use of an eIF5B inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hematologic malignancies.

[0008] Furthermore, the hematologic malignancies include leukemia, myeloma, and lymphoma.

[0009] Furthermore, the leukemia includes acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphoblastic leukemia.

[0010] Furthermore, the leukemia mentioned is acute myeloid leukemia.

[0011] Furthermore, the eIF5B inhibitor includes nucleic acid inhibitors and protein inhibitors.

[0012] Furthermore, the nucleic acid inhibitors include siRNA, shRNA, ribozymes, and antisense oligonucleotides.

[0013] In some embodiments, the siRNA refers to a short double-stranded RNA capable of inducing RNA interference by cleaving specific mRNAs. Furthermore, the siRNA is not limited to regions where the double-stranded RNA is perfectly paired, but may include regions where the strands are not paired due to mismatch (corresponding nucleotides are not complementary) or protrusion (absence of a nucleotide corresponding to one strand). The siRNA terminal structure may have blunt or protruding ends, as long as the expression of the target gene can be suppressed by the RNA interference effect, and the adhesive end structure may be a 3' protrusion or a 5' protrusion.

[0014] In some embodiments, the shRNA refers to an RNA sequence that produces a strong hairpin bend, which can be used to silence gene expression via RNA interference. Furthermore, the shRNA can be delivered into cells using a vector for cell introduction, and this vector is always delivered to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure degrades into siRNA via intracellular mechanisms and binds to an RNA-induced silencing complex, which binds to the corresponding mRNA to degrade it.

[0015] In some embodiments, the ribozyme refers to an enzyme RNA molecule capable of catalyzing the cleavage of specific RNA. Specific hybridization of the ribozyme's molecular sequence with complementary target RNA can induce endonuclease cleavage. The ribozyme may include a known sequence responsible for cleaving one or more sequences complementary to or functionally equivalent to the target RNA. Furthermore, the ribozyme may be a hammerhead ribozyme or a Cech-type ribozyme, i.e., a ribonuclease RNA, and may be formed from modified oligonucleotides to improve safety and targeting. Simultaneously, the ribozyme can be distributed in cells expressing the target gene in vivo. DNA constructs encoding the ribozyme under the control of a strong constitutive polymerase III or polymerase II promoter can be used, allowing transfected cells to disrupt endogenous target messengers and produce sufficient amounts of the ribozyme to inhibit translation. Due to its catalytic activity, unlike other antisense molecules, the ribozyme may need to be maintained at low concentrations in the cell.

[0016] In some embodiments, an antisense nucleotide is 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. Furthermore, because antisense nucleotides are long chains of monomeric units, antisense nucleotides of target RNA sequences can be readily synthesized.

[0017] Furthermore, the protein inhibitors include protein expression inhibitors and protein activity inhibitors.

[0018] Furthermore, the protein expression inhibitor refers to a drug or substance that can inhibit protein translation, protein modification, or promote protein degradation in an organism, including but not limited to proteolytic enzymes and transcription factors.

[0019] Furthermore, the protein activity inhibitor refers to a drug or substance that inhibits the functional activity of a protein, including but not limited to protein-binding molecules, phosphatase inhibitors, etc.

[0020] Furthermore, the eIF5B inhibitor is 1282NJ0069, and its structural formula is shown in formula (I):

[0021]

[0022] Formula (I).

[0023] Furthermore, the eIF5B inhibitor has the effect of inhibiting AML cell proliferation or inducing its differentiation.

[0024] Furthermore, the drug also contains pharmaceutically acceptable excipients and / or excipients.

[0025] Furthermore, the drug can be administered orally, parenterally, or locally.

[0026] Furthermore, the dosage form of the drug is oral solution, syrup, tablet, capsule, granule, powder, pill, emulsion, suspension, injection, aerosol, powder spray, spray, suppository, patch, implant, external solution, lotion, liniment, ointment, or gel.

[0027] Furthermore, the application concentration of the eIF5B inhibitor or its pharmaceutically acceptable salt is 0.1 μg / mL - 10 μg / mL.

[0028] Furthermore, the application concentration of the eIF5B inhibitor or its pharmaceutically acceptable salt is 0.15 μg / mL - 5 μg / mL.

[0029] Furthermore, the application concentration of the eIF5B inhibitor or its pharmaceutically acceptable salt is 1 μg / mL to 4 μg / mL.

[0030] In this invention, the eIF5B inhibitor is 1282NJ0069, a fluorophenyl oxazole derivative identified by in vitro fungal translation assays. Its specific structure is 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)acetonequinolol, as shown in formula (I) above. 1282NJ0069 exerts its translational inhibitory effect by weakening the functional contact between eIF5B and the ribosome complex in fungi. Currently, there are no reports or studies on the treatment of hematological malignancies with 1282NJ0069. This invention does not impose any particular restrictions on the specific source of the eIF5B inhibitor 1282NJ0069, which can be obtained by those skilled in the art through conventional means.

[0031] In some embodiments, the hydrates, enantiomers, diastereomers, solvates, crystalline forms, or derivatives thereof corresponding to the eIF5B inhibitor are also within the scope of protection of this invention. That is, the use of the hydrates, enantiomers, diastereomers, solvates, crystalline forms, or derivatives thereof corresponding to the eIF5B inhibitor in the preparation of drugs for treating AML also falls within the scope of protection of this invention.

[0032] In some implementations, the pharmaceutically acceptable salt refers to a salt that is pharmaceutically acceptable for the eIF5B inhibitor. Specifically, a pharmaceutically acceptable salt for the eIF5B inhibitor refers to a salt form suitable for pharmaceutical formulation and clinical application obtained by salting the eIF5B inhibitor. The salts used in the salting modification process include, but are not limited to: inorganic acid salts (e.g., hydrochloride, sulfate, phosphate, nitrate, hydrogen sulfate, sulfite, pyrosulfate, pyrophosphate, metaphosphate, bromide, monohydrogen phosphate, dihydrogen phosphate, etc.) and organic acid salts (e.g., citrate, maleate, tartrate, acetate, sebate, caprylate, isobutyrate, malonate, oxalate, propionate, succinate, mandelate, caprylate, fumarate, benzoate, etc.). These salts are suitable for patient contact within the limits of reliable medical judgment and will not produce inappropriate toxicity, irritation, or allergic reactions.

[0033] In some embodiments, examples of pharmaceutically acceptable salts of the eIF5B inhibitor include, but are not limited to, those having an alkali metal ion, such as Na+, as an anti-counterion. + Li + or K + Salts, or those containing alkaline earth metal ions such as Ca 2+ or Mg 2+ Salts, or those containing any other pharmaceutically acceptable metal ions such as Zn 2+ Or Al 3+ Salts; or pharmaceutically acceptable salts formed with organic bases such as diethanolamine, ethanolamine, N-methylglucosamine, triethanolamine or tromethamine.

[0034] In some embodiments, the pharmaceutically acceptable base addition salt is formed with a metal or amine, such as an alkali metal hydroxide with an alkaline earth metal hydroxide or an organic amine. Examples of metals used as cations include magnesium, potassium, sodium, and calcium. Examples of suitable amines include N,N'-dibenzylethylenediamine, diethanolamine, chloroprocaine, choline, ethylenediamine, N-methylglucosamine, or procaine.

[0035] In some embodiments, the base addition salt of the acidic compound can be prepared by contacting the free acid in a conventional manner with a sufficient amount of the required base to form a salt. Contacting the salt in a conventional manner with the acid, followed by separation of the free acid, can regenerate the free acid.

[0036] In some embodiments, pharmaceutically acceptable salts may include alkali metal and alkaline earth metal-based cations, such as sodium, potassium, calcium, lithium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to: ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, etc. Salts of amino acids are also included, such as gluconate, arginine, galacturonate, etc.

[0037] In a specific embodiment of the present invention, cell experiments have demonstrated that eIF5B inhibitors are highly toxic to a variety of AML cells, inhibiting their in vitro proliferation and inducing their differentiation, while having no significant effect on normal hematopoietic stem / progenitor cells. This confirms that eIF5B inhibitors or their pharmaceutically acceptable salts can be effectively used to prepare drugs for the treatment of AML.

[0038] In some embodiments, the pharmaceutically acceptable excipients and / or excipients include, but are not limited to: stabilizers, preservatives, solubilizers, wetting agents, emulsifiers, buffers, sweeteners, colorants, osmotic pressure regulating salts, flavoring agents, masking agents, or antioxidants. Additionally, by adding ingredients capable of delaying the absorption of the active ingredient (i.e., the eIF5B inhibitor or its pharmaceutically acceptable salt as described above), the absorption of the active ingredient in the drug can be prolonged.

[0039] The term "treatment" as used in this invention includes the injection or other internal administration of the drug of the invention to a patient, or the application or administration of the drug of the invention to the patient's cells or tissues for the purpose of treating, curing, alleviating, reducing, altering, compensating, improving, modifying, or influencing the disease or condition, the symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. The term "treatment" also refers to any sign of success in the treatment or relief of an injury, lesion, or condition, including any objective or subjective parameters such as the reduction, relief, weakening of symptoms, or making the injury, lesion, or condition more tolerable for the patient; slowing the rate of degeneration or decline; making the final point of degeneration less weak; or improving the patient's physical or mental condition.

[0040] A second aspect of the present invention provides a pharmaceutical composition for treating hematologic malignancies, the pharmaceutical composition comprising the eIF5B inhibitor described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0041] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the eIF5B inhibitor or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention. In other embodiments, the main active ingredient of the pharmaceutical composition is the eIF5B inhibitor or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention, and in addition to the eIF5B inhibitor or a pharmaceutically acceptable salt thereof, the pharmaceutical composition may also comprise other drugs that can be used to treat or as adjunctive therapy for AML.

[0042] In some implementations, a therapeutically effective amount refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response sought by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, or human. The therapeutically or pharmaceutically effective amount of the compound to be administered will be determined by such considerations and is the minimum amount necessary to improve, cure, or treat a disease or condition or one or more of its symptoms. The pharmaceutical composition of the invention will be formulated, administered, and applied in a manner consistent with good medical practice, i.e., the dosage, concentration, regimen, process, medium, and route of administration. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery, the method of administration, the administration regimen, and other factors known to a medical professional, such as the individual patient's age, weight, and response.

[0043] In some embodiments, the other drugs that can be used to treat or as adjunctive therapy for AML include, but are not limited to, chemotherapy drugs (e.g., cytarabine), targeted drugs (e.g., venetoclax), and immunotherapy drugs (e.g., PD-1 / PD-L1 inhibitors). In this invention, the other drugs that can be used to treat or as adjunctive therapy for AML are not limited to the specific drugs listed above; any drug that may be used to treat or as adjunctive therapy for AML falls within the scope of protection of this invention.

[0044] It should be noted that, given that this invention has clearly demonstrated through detailed cell experiments that the eIF5B inhibitor has the effect of inhibiting AML cell proliferation and inducing AML cell differentiation, those skilled in the art, based on common knowledge, will know that the eIF5B inhibitor or its pharmaceutically acceptable salt described above can be used alone to achieve this invention. Therefore, the use of the eIF5B inhibitor or its pharmaceutically acceptable salt described above, either alone or in combination with other drugs, will fall within the protection scope of this invention.

[0045] A third aspect of the present invention provides a pharmaceutical preparation for treating hematologic malignancies, the pharmaceutical preparation comprising the pharmaceutical composition described in the second aspect of the present invention.

[0046] Furthermore, the administration methods of the drug formulation include, but are not limited to: oral administration, parenteral administration, and topical administration.

[0047] In some implementations, oral administration refers to the absorption of a drug in the gastrointestinal tract after oral administration to exert its therapeutic effect. Dosage forms for oral administration include, but are not limited to: tablets, capsules, granules, powders, pills, syrups, oral solutions, suspensions, and emulsions.

[0048] Specifically, the tablets are tablet-shaped preparations made by mixing drugs and excipients and then compressing them. They have advantages such as accurate dosage, good stability, high production efficiency, and convenient administration. They can be divided into various types, including ordinary tablets, coated tablets, chewable tablets, and dispersible tablets.

[0049] Specifically, the capsules are divided into hard capsules and soft capsules. Hard capsules are made by filling a hollow hard capsule with drug powder or granules; soft capsules are made by dissolving or suspending the drug in a suitable oily or non-oily liquid medium and then using compression or dripping methods. Capsules can mask unpleasant odors of drugs, improve drug stability, and are convenient to take.

[0050] Specifically, the granules are dry granular preparations made by combining drugs with suitable excipients to form granules of a certain particle size. They can be classified into soluble granules, suspension granules, effervescent granules, etc. Granules are easy to dissolve, absorb quickly, and are convenient to carry and take.

[0051] Specifically, the powder is a dry powder preparation made by pulverizing and uniformly mixing a drug with suitable excipients. Powders have small particle size, large specific surface area, are easily dispersed, and have a rapid onset of action, but their stability is relatively poor.

[0052] Specifically, the pills are spherical or near-spherical preparations made by combining fine powder or extracts of medicine with suitable binders or other excipients. Depending on the excipients used and the preparation method, they can be classified as honey pills, water pills, water-honey pills, paste pills, wax pills, etc.

[0053] Specifically, the syrup refers to a concentrated sucrose aqueous solution containing the drug. The sucrose in the syrup can mask the bitterness of the drug and also has a certain preservative effect. It has a pleasant taste and is especially suitable for children.

[0054] Specifically, the oral solution is a clear liquid preparation for oral administration, made by dissolving the drug in a suitable solvent. The drug is uniformly dispersed in the solution, absorbed rapidly, and has high bioavailability.

[0055] Specifically, the suspension is a non-homogeneous liquid formulation formed by dispersing poorly soluble solid drugs in a dispersion medium in a particulate state. To ensure the stability and homogeneity of the drug, excipients such as suspending agents and wetting agents are often added.

[0056] Specifically, the emulsion is a heterogeneous liquid formulation formed by emulsifying two immiscible liquids, dispersing one liquid in droplets within the other. Oral emulsions can improve the taste and stability of drugs and enhance drug bioavailability.

[0057] In some implementations, parenteral administration refers to the delivery of drugs into the body via routes other than oral administration. The dosage forms for parenteral administration include, but are not limited to: injections, aerosols, powder inhalers, sprays, suppositories, patches, and implants.

[0058] Specifically, the aforementioned injectable preparations are the most common parenteral dosage forms, including solution-type injections, suspension-type injections, emulsion-type injections, and sterile powders for injection. They can be administered via intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection. Intravenous injection allows the drug to rapidly enter the bloodstream, resulting in a fast-acting effect, suitable for emergency situations and drugs requiring rapid onset of action; intramuscular and subcutaneous injections have relatively slower absorption rates, but their effects last longer.

[0059] Specifically, the aerosol is a formulation made by encapsulating a drug and a suitable propellant in a pressure-resistant container with a special valve system. In use, the drug is atomized using the pressure of the propellant and inhaled through the respiratory tract or sprayed directly onto the mucous membranes, skin, or other sites to exert its effect. Aerosols have rapid and targeted effects, uniform drug distribution, reduced irritation from local administration, and are convenient to use.

[0060] Specifically, the powder inhalers include inhaled powder inhalers, non-inhaled powder inhalers, and topical powder inhalers. Inhaled powder inhalers are preparations in which the patient actively inhales the nebulized medication into the lungs; non-inhaled powder inhalers are preparations in which drug powder is sprayed onto the mucous membranes of body cavities using a specially designed dry powder delivery device; topical powder inhalers are preparations in which drug powder is sprayed onto the skin or mucous membrane surface using a special device. Powder inhalers have advantages such as being propellant-free, having good drug stability, and providing accurate dosage.

[0061] Specifically, the spray refers to a formulation containing a drug solution, emulsion, or suspension filled in a specially designed device, which is used to release the contents as a mist using the pressure of a manual pump, high-pressure gas, ultrasonic vibration, or other methods. It can be used for pulmonary inhalation, nasal mucosal administration, or skin administration.

[0062] Specifically, the suppository is a solid preparation of a specific shape, made by combining a drug with a suitable matrix, for administration through body cavities. Rectal suppositories are commonly used. After melting or dissolving within the cavity, the suppository allows the drug to be absorbed through the mucosa into the bloodstream, avoiding the first-pass effect of the liver, making it suitable for patients who cannot take oral medication.

[0063] Specifically, the patches include transdermal patches and mucosal patches. Transdermal patches are plasters made by uniformly dispersing or dissolving drugs in an adhesive matrix and coating it onto a backing material. The drugs are slowly released through the skin into the bloodstream to exert a systemic therapeutic effect. Mucosal patches are preparations applied to the mucous membranes of the mouth, nose, conjunctiva, etc. The drugs are absorbed through the mucous membrane to exert local or systemic effects.

[0064] Specifically, the implant is a sterile solid preparation made of drugs and excipients for implantation into the body. It is typically inserted into subcutaneous or intramuscular tissues using a specialized syringe. The drug is released slowly, maintaining an effective blood drug concentration for a long period, thus providing a long-lasting therapeutic effect.

[0065] In some implementations, local administration refers to the application of a drug to local tissues to achieve a local therapeutic effect. The local administration dosage forms include, but are not limited to: topical solutions, lotions, liniments, ointments, and gels.

[0066] Specifically, the topical solution is a clear liquid preparation for external use made by dissolving the drug in a suitable solvent.

[0067] Specifically, the lotion is a solution, emulsion, or suspension containing a drug, and is a preparation for washing or applying to unbroken skin.

[0068] Specifically, the liniment refers to a liquid preparation made of ethanol, oil or a suitable solvent for rubbing on unbroken skin.

[0069] Specifically, the ointment is a homogeneous semi-solid topical preparation made by mixing a drug with an oily or water-soluble base.

[0070] Specifically, the gel is a homogeneous, suspension, or emulsion-type viscous liquid or semi-solid preparation made from a drug and excipients capable of forming a gel. Topical gels are commonly used on the skin or mucous membranes.

[0071] The fourth aspect of the present invention provides a method for inhibiting the proliferation of AML cells or inducing their differentiation in vitro, the method comprising the eIF5B inhibitor described in the first aspect of the present invention or a system thereof that can be treated with pharmaceutically acceptable salts as needed.

[0072] Furthermore, the AML cells include MOLM13, HL60, primary AML cells, Kasumi-1, and THP1.

[0073] Furthermore, the method described is not for therapeutic purposes.

[0074] In some embodiments, the present invention does not particularly limit the desired system. Exemplarily, the system includes the desired cellular system, subcellular system, tissue system, or organ system.

[0075] In some implementations, the desired cell system, subcellular system, tissue system, or organ system is a system that needs to inhibit AML cell proliferation or induce AML cell differentiation.

[0076] In some implementation schemes, the specific dosage of the eIF5B inhibitor or its pharmaceutically acceptable salt can be conventionally selected and adjusted by those skilled in the art based on the actual situation.

[0077] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0078] This invention is the first to discover that eIF5B inhibitors are highly toxic to a variety of AML cells, can inhibit the proliferation of various AML cells, and can also induce their differentiation, while having no significant effect on normal hematopoietic stem / progenitor cells. They can be used in the development of drugs for the treatment of AML. This invention provides a theoretical basis for the research and development of drugs for the treatment of AML, opens up a new use for eIF5B inhibitors, and provides a brand-new method for the treatment of AML. It has broad application prospects in the field of AML treatment technology. Attached Figure Description

[0079] Figure 1 This figure shows the cytotoxicity results of eIF5B inhibitors against different AML cell lines, primary AML cells, and non-AML cells. A: MOLM13; B: THP1; C: HL60; D: Kasumi-1; E: K562; F: primary AML; G: primary HSPC.

[0080] Figure 2 This figure shows the experimental results of eIF5B inhibitors inhibiting the proliferation of different AML cell lines, primary AML cells, and normal hematopoietic stem cells. A: HL60; B: MOLM13; C: primary AML; D: primary HSPC.

[0081] Figure 3 This figure shows the results of differentiation induction experiments of eIF5B inhibitors on different AML cell lines, primary AML cells, and normal hematopoietic stem cells. A: MOLM13; B: HL60; C: primary AML; D: primary HSPC. Detailed Implementation

[0082] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0083] Example 1: Toxicity study of eIF5B inhibitors on AML cells

[0084] I. Experimental Methods

[0085] 1. Experimental Materials

[0086] (1) Cells / samples:

[0087] AML cell lines (MOLM13, THP1, HL60, Kasumi-1) were obtained from the Cell Center of Peking Union Medical College.

[0088] Other cell lines: (CML-K562) from the Cell Center of Peking Union Medical College.

[0089] Primary AML patient sample: bone marrow from AML patients at Chongqing Xinqiao Hospital.

[0090] Normal sample: Umbilical cord blood from a healthy donor at Haidian District Maternal and Child Health Hospital, Beijing.

[0091] (2) eIF5B inhibitors:

[0092] 1282NJ0069 was synthesized by Shanghai Ruizhi Pharmaceutical Technology Co., Ltd. (solvent: DMSO, final concentration ≤0.1%).

[0093] (3) Culture medium:

[0094] Cell line: RPMI-1640 + 10% fetal bovine serum FBS.

[0095] Primary cell culture medium: 70% IMDM (Gibco) + 30% fetal bovine serum (Gibco) + 10% BSA (Beijing Solarbio Science & Technology Co., Ltd.) + recombinant human cytokines IL-3 (50 ng / ml), SCF (100 ng / ml) (PeproTech Biotechnology Co., Ltd., China) + β-mercaptoethanol (0.1 mM) (Shanghai Zeye Biotechnology Co., Ltd.).

[0096] (4) Reagents: CCK-8 (Beyotime Biotechnology Co., Ltd.), CD34 antibody (Medlan), FBS (Gibco), EDTA (Thermo Fisher Scientific), bispecific antibody (Thermo Fisher Scientific), hydroxyethyl starch (HES) solution (Sigma), and blocking agent (Medlan).

[0097] 2. Experimental Procedure

[0098] (1) Extraction of bone marrow mononuclear cells

[0099] Prepare the sorting buffer: 10 mL of FBS (2%); 2 mL of 0.5 M EDTA (0.4%); 5 mL of bispecific antibody (1%).

[0100] Collect bone marrow blood into an anticoagulant tube, spray with 75% alcohol, and place in a sterile operating table. Proceed with the following steps.

[0101] Open the anticoagulant tube and slowly pour the contents into a 15 mL sterile tube. Add HES solution at a ratio of bone marrow blood to HES of 4:1. Tighten the cap, invert the tube to mix, and place it on a tube rack to stand for about 1 hour to allow the red blood cells to settle fully.

[0102] After 30 minutes, take a new 15 mL sterile tube and add half the volume of human peripheral blood lymphocyte separation medium. Slowly add the clear upper layer of the solution after the first step of settling and separation to the 15 mL centrifuge tube, being careful not to disrupt the interface between the two solutions. After tightening the cap, adjust the centrifuge speed to the lowest setting and centrifuge at 680 g for 20 minutes.

[0103] After centrifugation, the white film layer and the liquid above and below it were aspirated into a new 15 mL tube. The separation buffer was added to 15 mL, the tube cap was tightened, and the tube was centrifuged at 1600 r / min for 10 min at normal speed.

[0104] After centrifugation, discard the supernatant in the 15 mL tube, add 15 mL of sorting buffer to resuspend the cells, transfer 10 µL of the cell suspension to a 1.5 mL EP tube, add 190 µL of sorting buffer to dilute the cell suspension 20-fold, and then count the cells. Centrifuge the remaining cells at 1400 r / min for 10 min, discard the supernatant after centrifugation, and obtain bone marrow mononuclear cells.

[0105] (2) Extraction of mononuclear cells from umbilical cord blood

[0106] Prepare the sorting buffer: 10 mL of FBS (2%); 2 mL of 0.5 M EDTA (0.4%); 5 mL of bispecific antibody (1%).

[0107] Collect umbilical cord blood into a disposable plastic blood bag, spray the blood bag with 75% alcohol, and then place it in a sterile operating table. Proceed with the following steps.

[0108] Cut open the blood bag and slowly pour the blood into a 50 mL sterile tube. Each tube should contain a maximum of 40 mL of cord blood. Add HES solution at a ratio of cord blood to HES of 4:1. Tighten the cap, invert the tube to mix, and place it on a tube rack to stand for about 1 hour to allow the red blood cells to settle completely.

[0109] After 30 minutes, take several 15 mL sterile tubes and add 5 mL of human peripheral blood lymphocyte separation medium to each tube. Slowly add the clear upper layer of the solution after the first step of settling and separation to the 15 mL centrifuge tubes, being careful not to disrupt the interface between the two solutions. After tightening the tube caps, adjust the centrifuge speed to the lowest setting and centrifuge at 680 g for 20 minutes.

[0110] After centrifugation, the white film layer and the liquid above and below it were aspirated into a new 50 mL tube. The separation buffer was added to 50 mL, the tube cap was tightened, and the tube was centrifuged at 1600 r / min for 10 min at normal speed.

[0111] After centrifugation, discard the supernatant in the 50 mL tube, add 40 mL of sorting buffer to resuspend the cells, transfer 10 µL of the cell suspension to a 1.5 mL EP tube, add 190 µL of sorting buffer to dilute the cell suspension 20-fold, and then count the cells. Centrifuge the remaining cells at 1400 r / min for 10 min, discard the supernatant after centrifugation, and obtain umbilical cord blood mononuclear cells.

[0112] (3) CD34 + Cell sorting

[0113] The above-mentioned bone marrow mononuclear cells or umbilical cord blood mononuclear cells were divided into groups of 1×10. 8Add 100 µL of blocking agent, 300 µL of sorting buffer, and 100 µL of CD34 antibody to each cell. Mix thoroughly in a 1.5 mL EP tube, wrap the tube with aluminum foil to protect from light, and incubate at 4°C for 30 min, gently tapping the tube every 10 min. Place the tube in a magnetic rack on the work surface and sterilize under UV light.

[0114] After incubation, the cells were washed once with an appropriate amount of sorting buffer and centrifuged at 1500 r / min for 10 min.

[0115] Install the adsorption column, rinse the column with 5-10 mL of sorting buffer, and do not interrupt the flow during this process to avoid generating air bubbles. Transfer the waste liquid to a 15 mL centrifuge tube.

[0116] After centrifugation, discard the supernatant of the cells, resuspend the cells in 5 mL of sorting buffer, pass the cell suspension through the column, and finally add 3 mL of sorting buffer to wash the adsorption column.

[0117] Remove the adsorption column, place it on a new 15 mL centrifuge tube, add 2 mL of sorting buffer, install the stopcock, and quickly and forcefully push the stopcock down to elute CD34. + Cells. Repeat elution. After counting the eluted cells, add sorting buffer and centrifuge at 300 g for 10 min.

[0118] After centrifugation, discard the supernatant and resuspend the cells in prepared primary cell culture medium at a rate of 2 × 10⁻⁶. 6 Cells were cultured per dish for subsequent experiments, and CD34 cells were obtained from bone marrow mononuclear cell sorting. + The cells were primary AML cells; CD34 cells were obtained from umbilical cord blood mononuclear cell sorting. + The cells are primary normal hematopoietic stem / progenitor cells (primary HSPC cells).

[0119] (4) CCK8 detection

[0120] Cell plating: Spread cells at a density of 5 × 10⁻⁶ 3 ~1×10 4 Inoculate at a density of 100 μL of culture medium per well in a 96-well plate.

[0121] 1282NJ0069 Serial dilution: The eIF5B inhibitor was serially diluted with culture medium at the following concentrations: 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.02 μg / ml. 100 μL of drug-containing culture medium was added to each well (final volume 200 μL / well), n=3 replicates.

[0122] Culture conditions: AML cell lines are cultured at 37℃ and 5% CO2 for 48-72 hours. Primary AML / HSPC cells are cultured for 96-120 hours (primary cells proliferate more slowly).

[0123] Cell viability assay: Add CCK-8 reagent (10 μL / well), incubate for 2-4 h, and measure absorbance at 450-630 nm.

[0124] II. Experimental Results

[0125] The eIF5B inhibitor 1282NJ0069 has shown highly selective toxicity to AML cells. For example... Figure 1 As shown, it is effective against the AML cell line (MOLM13: Figure 1 A. THP1: Figure 1 B, HL60: Figure 1 C. Kasumi-1: Figure 1 The IC50 of D) was 0.54-2.50 μg / ml, but for the CML cell line K562 ( Figure 1 E) No significant effect. In primary AML cells (n=6), the IC50 distribution (1.08–2.29 μg / ml) was largely consistent with the results for cell lines. Figure 1 F). Notably, 1282NJ0069 had no significant effect on the activity of HSPC (n=2) at the highest concentration of 10 μg / ml. Figure 1 G). These data indicate that the eIF5B inhibitor 1282NJ0069 has significant targeting potential for AML and clinical translational potential.

[0126] Example 2: Study on the Inhibition of AML Cell Proliferation by eIF5B Inhibitors

[0127] I. Experimental Methods

[0128] 1. Experimental Materials

[0129] Same as Example 1.

[0130] 2. Experimental Procedure

[0131] The cell extraction and sorting steps are the same as in Example 1.

[0132] CCK8 assay: Cells were seeded into 96-well plates at a density of 6000 cells / 100 µL of complete culture medium. Different concentrations of eIF5B inhibitor 1282NJ0069 were added, with 3-5 replicates per sample. Five to six identical 96-well plates were seeded. The 96-well plates were incubated for 0, 2, 4, 6, 8, or 10 days. At each time point, one well of the 96-well plate was removed, and 10 µL of CCK8 reagent was added to each well, followed by incubation for 2 hours. The absorbance was measured at 450 nm–630 nm using a microplate reader. After measuring the absorbance at 5-6 time points, the cell proliferation level was statistically analyzed.

[0133] II. Experimental Results

[0134] The eIF5B inhibitor 1282NJ0069 has an inhibitory effect on the proliferation of various AML cell types. For example... Figure 2 As shown, within the concentration range of 1-4 μg / mL, with increasing concentration of 1282NJ0069, its effect on the AML cell line (HL60: Figure 2 A and MOLM13: Figure 2 B) The inhibition of proliferation gradually increased. This inhibitory effect was also verified in primary AML cells (n=3). Figure 2 C). Most importantly, even at the highest tested concentration (4 μg / mL), 1282NJ0069 had no significant effect on the proliferation activity of HSPCs (n=2). Figure 2 D). These results confirm that the eIF5B inhibitor 1282NJ0069 has a significant inhibitory effect on the proliferation of various AML cells, while having no significant effect on normal hematopoietic cells.

[0135] Example 3: Study on the induction of differentiation of AML cells by eIF5B inhibitors

[0136] I. Experimental Methods

[0137] 1. Experimental Materials

[0138] Flow cytometry antibodies: anti-human CD11b-FITC antibody (Elabscience), anti-human CD14-PE antibody (Elabscience).

[0139] The remaining materials are the same as in Example 1.

[0140] 2. Experimental Procedure

[0141] Cell plating: Placing cells at a density of 1×10⁶ 6 The density of cells / well was seeded in a 6-well plate.

[0142] Different concentrations of 1282NJ0069 were added to the cells, with gradient concentrations of 0, 1, 2, and 4 μg / mL, and the cells were incubated at 37℃ in a 5% CO2 incubator for 48 h.

[0143] Collect cells into a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0144] Gently resuspend the cells in 1× pre-cooled PBS (pH 7.4) and wash twice by centrifugation.

[0145] After the final wash, resuspend the cells in 100 µL PBS and adjust the cell density to 1×10⁻⁶. 6 per mL.

[0146] Add anti-human CD11b-FITC and anti-human CD14-PE antibodies (5 µL each per tube) and mix gently. Incubate at 4°C in the dark for 30-60 min.

[0147] After staining, wash once with 1 mL PBS and centrifuge at 1000 rpm for 5 min. Resuspend the cells in 300 µL PBS and filter through a 40 µm cell filter.

[0148] Immediately put it on the machine for testing.

[0149] II. Experimental Results

[0150] The eIF5B inhibitor 1282NJ0069 exhibited significant differentiation-inducing effects on various AML cell lines. Experimental data showed that, within a concentration range of 1-4 μg / mL, 1282NJ0069 promoted the differentiation of AML cell lines (HL60 and MOLM13) into mature myeloid cells. Specifically, after 48 hours of treatment with 1282NJ0069, CD11b levels in HL60 and MOLM13 cells decreased. + CD14 + The proportion of double-positive cells increased significantly. Figure 3 A, Figure 3 B). This differentiation-promoting effect was further validated in primary AML cells (n=3). Figure 3 C). Notably, under the same treatment conditions, the expression levels of differentiation markers in HSPC (n=2) remained stable. Figure 3 D). These data fully demonstrate that the eIF5B inhibitor 1282NJ0069 can selectively induce AML cell differentiation while having no significant interference with the maintenance of normal hematopoietic cell function.

Claims

1. The use of eIF5B inhibitors or their pharmaceutically acceptable salts in the preparation of drugs for treating hematologic malignancies.

2. The application according to claim 1, characterized in that, The hematologic malignancies include leukemia, myeloma, and lymphoma; Preferably, the leukemia includes acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphoblastic leukemia; Preferably, the leukemia is acute myeloid leukemia.

3. The application according to claim 1, characterized in that, The eIF5B inhibitors include nucleic acid inhibitors and protein inhibitors; Preferably, the nucleic acid inhibitor includes siRNA, shRNA, ribozymes, and antisense oligonucleotides; Preferably, the protein inhibitor includes a protein expression inhibitor and a protein activity inhibitor; Preferably, the eIF5B inhibitor is 1282NJ0069, and its structural formula is shown in formula (I): Formula (I).

4. The application according to claim 1, characterized in that, The eIF5B inhibitor has the effect of inhibiting the proliferation of AML cells or inducing their differentiation.

5. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients and / or excipients.

6. The application according to claim 1, characterized in that, The drug can be administered orally, parenterally, or locally.

7. The application according to claim 1, characterized in that, The eIF5B inhibitor or its pharmaceutically acceptable salt is administered at a concentration of 0.1 μg / mL - 10 μg / mL; Preferably, the application concentration of the eIF5B inhibitor or its pharmaceutically acceptable salt is 0.15 μg / mL - 5 μg / mL; Preferably, the application concentration of the eIF5B inhibitor or its pharmaceutically acceptable salt is 1 μg / mL to 4 μg / mL.

8. A pharmaceutical composition for treating hematologic malignancies, characterized in that, The pharmaceutical composition comprises the eIF5B inhibitor as described in any one of claims 1-7 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical preparation for treating hematologic malignancies, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition of claim 8.

10. A method for inhibiting the proliferation of AML cells or inducing their differentiation in vitro, characterized in that, The method includes using the eIF5B inhibitor of claim 1 or a pharmaceutically acceptable salt thereof to treat the desired system; Preferably, the AML cells include MOLM13, HL60, primary AML cells, Kasumi-1, and THP1.

Images