Medicine for treating leukemia

Abstract

The invention discloses a medicine for treating leukemia. It is found for the first time that CPI-0610 can effectively promote apoptosis of leukemia cell lines and leukemia patient cells and enhance the activity of imatinib, existing treatment problems are expected to be solved, a new path is developed for treatment of leukemia, the treatment effect is remarkably improved, and the life of a patient is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a drug for treating leukemia. Background Technology

[0002] Leukemia is the most common malignant clonal disease of hematopoietic stem cells, characterized by the massive accumulation and proliferation of clonal leukemic cells in the bone marrow and other hematopoietic tissues. The mechanisms include uncontrolled proliferation, impaired differentiation, and inhibited apoptosis. Leukemic cells not only infiltrate other non-hematopoietic tissues and organs but also suppress normal hematopoietic function. High morbidity, high mortality, and high relapse rates are the main challenges in leukemia treatment. Therefore, there is an urgent need to develop new therapeutic drugs to alleviate the difficulties in treating leukemia. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a drug for treating leukemia.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention provides the use of CPI-0610 and / or imatinib in the preparation of a medicament for treating leukemia.

[0006] Furthermore, the leukemia described is Ph-positive leukemia.

[0007] A second aspect of the invention provides the use of CPI-0610 in the preparation of a medicament that enhances the anti-leukemia activity of imatinib.

[0008] Furthermore, the leukemia described is Ph-positive leukemia.

[0009] Furthermore, the drug also includes other drugs for treating leukemia.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0012] Furthermore, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0013] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0014] A third aspect of the present invention provides a medicament for treating leukemia, the medicament comprising CPI-0610 and / or imatinib.

[0015] Furthermore, the drug is a single compound preparation or a combination of two single preparations.

[0016] Furthermore, the concentration ratio of CPI-0610 to imatinib is (0.5-2):(1.25-2.5).

[0017] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0018] A fourth aspect of the present invention provides a method for promoting apoptosis of leukemia cells in vitro, the method comprising administering CPI-0610 and / or imatinib.

[0019] Furthermore, the leukemia cells include one or both of K562 and BV173 cells.

[0020] Furthermore, the method described is not for therapeutic purposes.

[0021] A fifth aspect of the present invention provides a method for treating leukemia, the method comprising administering CPI-0610 and / or imatinib.

[0022] Advantages and beneficial effects of the present invention:

[0023] This application is the first to discover that CPI-0610 can effectively induce apoptosis in leukemia cell lines and leukemia patients, and enhance the activity of imatinib. This discovery is expected to overcome existing treatment challenges, open up new avenues for the treatment of leukemia, significantly improve treatment efficacy, and prolong patient life. Attached Figure Description

[0024] Figure 1 This is a graph showing the effect of CPI-0610 on the cell viability of leukemia cell lines;

[0025] Figure 2 The graphs show the effects of different concentrations of CPI-0610 and imatinib, alone or in combination, on cells in CML patients. 2A is a flow cytometry representation graph, and 2B is a flow cytometry-based statistical graph of cell apoptosis.

[0026] Figure 3 The graphs show the effects of different concentrations of CPI-0610 and imatinib, alone or in combination, on cells of Ph-positive leukemia patients. 3A is a flow cytometry representation graph, and 3B is a flow cytometry statistical graph showing the proliferation and apoptosis of resting stem cells. Detailed Implementation

[0027] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] This invention provides the use of CPI-0610 and / or imatinib in the preparation of medicaments for treating leukemia.

[0029] In some embodiments, CPI-0610 or imatinib also includes pharmaceutically acceptable salts, solvates, or stereoisomers thereof, or combinations thereof. A pharmaceutically acceptable salt is any acid addition salt or base addition salt that is safe and effective for topical application in mammals and has the desired biological activity. The main components of pharmaceutically acceptable salts are well known in the art. If pharmaceutically acceptable salts of CPI-0610 and / or imatinib of this application are used in these medicaments, those salts are preferably derived from inorganic acids or organic acids and bases. This class of acid salts includes, but is not limited to, the following: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, diglucurons, dodecyl sulfates, ethanesulfonates, fumarates, luchoptanoates, glycerophosphates, hemisulfates, heptanates, hexanoates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, and 2-naphthalenesulfonic acid. Salts, nicotinates, oxalates, pyrates, pectates, persulfates, 3-phenyl-propionates, picrates, pentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, hydrohalides (e.g., hydrochlorides and hydrobromates), sulfates, phosphates, nitrates, aminosulfonates, malonates, salicylates, methylene-bis-β-hydroxynaphthylcarboxylate, gentianates, hydroxyethylsulfonates, di-p-toluyl tartrate, ethanesulfonates, cyclohexylaminosulfonates, quinates, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts containing organic bases (such as dicyclohexylamine salts, N-methyl-D-glucosamine), and salts containing amino acids such as arginine and lysine.

[0030] In some embodiments, the drug also includes other adjunctive therapies for leukemia, including anti-apoptotic agents, mitotic inhibitors (anti-microtubule drugs, such as vincristine (VCR) and paclitaxel (PTX), which block cell division by interfering with microtubule function), immunomodulators (including glucocorticoids (such as prednisone) and monoclonal antibodies (such as rituximab), which enhance anti-leukemia effects by modulating immune responses or targeting antigens such as CD20), alkylating agents (such as cyclophosphamide (CTX) and busulfan, which inhibit leukemia cell proliferation by disrupting DNA structure and are used in various leukemia chemotherapy regimens), antimetabolites (such as methotrexate (MTX) and cytarabine (Ara-C), which interfere with nucleic acid metabolism and are widely used in induction and consolidation therapy for leukemia), and topoisomerase inhibitors (such as etoposide (…)). VP-16), irinotecan (which kills leukemia cells by inhibiting the activity of DNA replication-related enzymes), antibiotics (anthracyclines, such as doxorubicin (ADM) and daunorubicin (DNR), which embed into the DNA double helix to prevent replication and are the core drugs in chemotherapy for acute leukemia), platinum compounds (such as carboplatin and oxaliplatin, which exert their anti-leukemic effect by forming DNA cross-linking damage), hormonal drugs (glucocorticoids (dexamethasone) used for ALL induction therapy, which can enhance chemotherapy sensitivity and inhibit lymphocyte proliferation), targeted drugs (non-TKIs, such as FLT3 inhibitors (midotolamine) and CD33 monoclonal antibodies (gestuzumab), which target specific molecular targets to treat relapsed / refractory AML), and chemotherapy protectants (such as leucovorin calcium (methotrexate antidote) and dexrazoxane (which reduces the cardiotoxicity of anthracyclines) used to reduce the side effects of chemotherapy).

[0031] The drug also includes pharmaceutically acceptable excipients.

[0032] In some embodiments, pharmaceutically acceptable excipients include all solvents, diluents, buffers (e.g., neutral buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, solvents, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aromatizers, thickeners, substances that provide storage effects, coating agents, antifungal agents, preservatives (e.g., Thimerosal™, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, extenders (e.g., lactose, mannitol), etc. The use of such media and reagents for formulating pharmaceuticals is well known in the art. Their use in pharmaceuticals may be considered unless any conventional media or reagent is incompatible with the active ingredient.

[0033] The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0034] In some embodiments, the gastrointestinal dosage form includes tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release formulations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0035] In some embodiments, the non-gastrointestinal dosage form includes injectable dosage form, respiratory dosage form, cavity dosage form, mucosal dosage form, and skin dosage form.

[0036] In some embodiments, the injectable dosage forms include, but are not limited to, various injectables such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0037] The drug is a single compound preparation or a combination of two single preparations.

[0038] In some embodiments, the compound formulation refers to a formulation made of two or more active pharmaceutical ingredients. In this application, the compound formulation is a compound formulation containing CPI-0610 and imatinib. The single-ingredient formulation refers to a formulation made of a single active pharmaceutical ingredient. In this application, the combination of single-ingredient formulations is a combination of a single-ingredient formulation containing CPI-0610 and a single-ingredient formulation containing imatinib. The two single-ingredient formulations in the combination are administered simultaneously or sequentially.

[0039] In some embodiments, CPI-0610 and imatinib in the drug can be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. This differs from simultaneous or sequential administration, where the interval between administering one drug and another is significant, meaning that when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0040] This application directly verified the synergistic effect of the combination of CPI-0610 and imatinib using the combination drug CI index, with a CI value less than 1. The combination drug CI (CI) index is a method for evaluating the combined effects of drugs. Based on the dose-response curve of the drugs, this method calculates the corresponding drug combination index CI for each treatment group's cellular effect score (Fraction-affected, Fa, i.e., the cell growth inhibition rate after drug treatment), and plots a Fa-CI curve to determine whether the combined drug treatment has a synergistic, additive, or antagonistic effect. When CI < 1, it indicates a synergistic effect between the two drugs; when CI = 1, it indicates an additive effect; and when CI > 1, it indicates an antagonistic effect.

[0041] In some embodiments, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one identifiable symptom thereof. In some embodiments, treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated. In other embodiments, treatment refers to inhibiting or slowing the progression of a disease or condition, which may be physical, such as stabilizing identifiable symptoms, or physiological, such as stabilizing physiological parameters, or both. In still other embodiments, treatment refers to delaying the onset of a disease or condition.

[0042] In some embodiments, the dosage of the drug may vary depending on the patient's condition and weight, the severity of the disease, the drug formulation, the route of administration, and the duration of administration, but may be appropriately selected by those skilled in the art. The dosage may be in the range of approximately 0.001 mg / kg to approximately 100 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, or approximately 0.1 mg / kg to approximately 1 mg / kg per adult. It may be administered once daily, several times daily, twice or three times weekly, once to four times monthly, or once or twelve times annually.

[0043] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0044] Example 1

[0045] 1. Experimental Materials

[0046] Ph-positive leukemia cell lines (K562 and BV173) were purchased from ATCC.

[0047] Drug: CPI-0610 (Pelabresib).

[0048] 2. Experimental Methods

[0049] CPI-0610 was serially diluted with DMSO or other solvents according to the drug's solubility, with each drug gradient being 10 times the final concentration. 10 μL of each different drug was added to the corresponding well of a 96-well plate, with 10 μL of the corresponding solvent added to each single-drug well and 20 μL to each control well. 80 μL of cell suspension was added to each well, and the remaining steps were exactly the same as described above.

[0050] Untreated cells (K562 and BV173) were cultured at different concentrations (0.08 μM-40 μM) of CPI-0610 for 72 h. The proportion of viable cells was detected by the MTT assay, and the absorbance of each well at a specific wavelength was detected by a microplate reader. The IC50 value was calculated using GraphPad Prism and a standard equivalent line graph was plotted to reflect cell viability and thus determine the half-maximal inhibitory concentration (IC50) of the drug for different cell lines.

[0051] 3. Experimental Results

[0052] The effects of CPI-0610 (Pelabresib) on the viability of K562 and BV173 cell lines were investigated. The results showed that the viability of all cell lines decreased as the CPI-0610 concentration increased from 0.08 μM to 40 μM. Different cell lines showed varying sensitivities to CPI-0610, with K562 being the most sensitive (IC50) at 1.127 μM; BV173 showed an IC50 of 2.763 μM. Figure 1 ).

[0053] Example 2

[0054] 1. Experimental Materials

[0055] Sample: Cells from Ph-positive leukemia patients

[0056] Medications: CPI-0610 (Pelabresib) and imatinib

[0057] 2. Experimental Methods

[0058] Annexin V / PI staining and flow cytometry were used to detect cell apoptosis, as detailed below:

[0059] Ph-positive leukemia patient cells were adjusted to a cell count of 1 × 10^6 / well. After treatment with the drug for 24 h or 48 h, cells were collected, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cell pellet was washed with 1 mL of pre-chilled PBS. The pellet was then transferred to a 1.5 mL EP tube, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded as much as possible. 50 μL of binding buffer was added to the 1.5 mL EP tube, followed by 5 μL of Annexin-FITC and mixed well. The mixture was stained at room temperature in the dark for 15 min. 10 μL of propidium iodide (PI) was added and mixed well. The mixture was stained at room temperature in the dark for 5 min. 450 μL of binding buffer was added to stop the reaction. The sample was filtered through a double-layer 200-mesh nylon mesh and analyzed by flow cytometry within 1 hour. Data files were saved, images were generated using Cyt Expert, and data processing and statistical analysis were performed using GraphPad Prism software.

[0060] 3. Experimental Results

[0061] CML patient cells were treated with different concentrations of CPI-0610 (Pelabresib) and imatinib, alone or in combination, for 48 h. Cell death was detected by Annexin V-FITC / PI double staining and flow cytometry. The synergistic coefficient (CI, CI < 1 indicates a synergistic effect of the two drugs) was calculated using Calcusyn software. The results showed that, under 48 h treatment, the combination of 1.0 μM CPI-0610 and 2.5 μM imatinib had a synergistic effect (CI < 0.86) in Ph-positive leukemia patient cells. Figure 2 ).

[0062] Example 3

[0063] 1. Experimental Materials

[0064] Sample: CD34+Ph positive leukemia stem cells, collected on July 4, 2024, at the Department of Hematology and Oncology, Shenzhen Second People's Hospital.

[0065] Medications: CPI-0610 (Pelabresib) and imatinib.

[0066] 2. Experimental Methods

[0067] Annexin V and CFSE staining and flow cytometry were used to detect the proliferation and apoptosis of CML CD34+ resting stem cells, as detailed below:

[0068] Ph-positive leukemia cells (1×10) 6 Centrifuge and discard the supernatant. Add 100 μL of premixed buffer containing CFSE (1 mL 2% FBS / PBS + 1 μL CFSE) to each well / tube and incubate at 37°C in the dark for 30 min. Add 500 μL of pre-chilled SFII medium to stop incubation, centrifuge, discard the supernatant, and wash once with pre-warmed medium at 37°C. Add the appropriate concentrations of CPI-0610 (Pelabresib) and imatinib (Imatinib) and treat for 72 hours. Discard the medium, add 5 μL of Annexin-V-APC antibody and binding buffer, incubate at room temperature for 15 min, and then add 450 μL of binding buffer to terminate the reaction. Filter the sample through a double-layer 200-mesh nylon mesh before analysis and data saving. GraphJo 7.6 is used to generate images, and GraphPad Prism software is used for data processing.

[0069] 3. Experimental Results

[0070] Ph-positive leukemia cells were treated with different concentrations of CPI-0610 (Pelabresib) and imatinib, alone or in combination, for 72 h. The proliferation and apoptosis of resting stem cells were labeled with Annexin V-APC and CFSE. The synergistic coefficient (CI, CI < 1 indicates a synergistic effect of the two drugs) was calculated using Calcusyn software. The results showed that, under 72 h treatment, the combination of 1.0 μM CPI-0610 and 2.5 μM imatinib had a synergistic effect (CI < 0.35). Figure 3 ).

[0071] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of CPI-0610 and / or imatinib in the preparation of drugs for treating leukemia; Preferably, the leukemia is Ph-positive leukemia.

2. Application of CPI-0610 in the preparation of drugs that enhance the anti-leukemia activity of imatinib; Preferably, the leukemia is Ph-positive leukemia.

3. The application according to claim 1 or 2, characterized in that, The medications also include other medications for treating leukemia.

4. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 1 or 2, characterized in that, The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

6. The application according to claim 5, characterized in that, The gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets. Preferably, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

7. A drug for treating leukemia, characterized in that, The drugs include CPI-0610 and / or imatinib.

8. The medicament according to claim 7, characterized in that, The drug is a single compound preparation or a combination of two single preparations.

9. The drug according to claim 7, characterized in that, The concentration ratio of CPI-0610 to imatinib is (0.5-2):(1.25-2.5). Preferably, the drug further includes pharmaceutically acceptable excipients.

10. A method for promoting apoptosis of leukemia cells in vitro, characterized in that, The method includes administering CPI-0610 and / or imatinib; Preferably, the leukemia cells include one or both of K562 and BV173 cells.

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