New application of adapalene and vinca in preparation of medicine for treating acute myelogenous leukemia by combining adapalene and vinca

Abstract

The invention discloses a novel application of adapalene and vinca in preparation of a medicine for treating acute myelogenous leukemia by combination, and finds that the adapalene and the vinca have a synergistic inhibition effect on proliferation of an acute myelogenous leukemia cell line, namely the adapalene and the vinca have a remarkable synergistic effect by combination for the first time, so that the application of the adapalene and the vinca in preparation of the medicine for treating the acute myelogenous leukemia is realized; the invention provides an effective drug combination strategy for treatment of acute myelogenous leukemia, and has good clinical application prospects and important transformation significance in the technical field of development of drugs for treating acute myelogenous leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a new use of adapalene and venetoclax in combination in the preparation of a drug for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a common type of acute leukemia in adults, with a median age of onset between 67 and 70 years. The disease is prone to relapse, and patients often develop resistance to chemotherapy drugs after relapse. In terms of existing treatment options, the "7+3" regimen (7 days of continuous intravenous infusion of cytarabine combined with 3 days of intravenous injection of anthracyclines such as daunorubicin) is one of the main treatment options. The complete remission rate (CR) in young patients can reach 60%-80%, but in elderly patients (>60 years old) due to poor tolerance, the CR rate drops to 40%-50%. Although high-dose cytarabine (HiDAC) can reduce the risk of relapse, it is very likely to cause bone marrow suppression, with the incidence of grade III / IV neutropenia exceeding 90%.

[0003] Venetoclax (ABT-199) is a novel Bcl-2 selective inhibitor. The U.S. FDA approved venetoclax for the treatment of elderly AML patients who are newly diagnosed and cannot tolerate high-intensity conventional chemotherapy. The drug needs to be used in combination with azacitidine, decitabine or low-dose cytarabine. Although venetoclax has shown good clinical anti-AML activity, some AML patients will still relapse, become drug-resistant and eventually die after receiving venetoclax treatment. Natural or acquired resistance to venetoclax is a huge obstacle to the clinical use of venetoclax in the treatment of AML. Therefore, finding new treatment strategies to enhance the anti-AML activity of venetoclax is of great significance for improving the cure rate of AML.

[0004] Current treatments for AML have obvious defects in efficacy, safety, and monitoring. There is an urgent need to develop new effective treatment strategies and drugs, which are expected to bring new breakthroughs in the treatment of AML. Summary of the Invention

[0005] In the clinical treatment of AML, the inherent drug resistance of venetoclax and the secondary drug resistance generated during the treatment process seriously limit its efficacy. To address this treatment bottleneck, the present invention proposes for the first time an innovative treatment strategy for the combined treatment of acute myeloid leukemia with adapalene and venetoclax, which improves the treatment response rate of venetoclax through a synergistic enhancement mechanism, aiming to break through the existing treatment dilemma and provide AML patients with a more effective survival benefit plan.

[0006] The present invention discovered for the first time that adapalene can effectively induce apoptosis in leukemia cell lines and leukemia patient cells, and can enhance the anti-AML activity of venetoclax. By using adapalene and venetoclax in combination, the apoptosis pathway is activated to overcome the problem of drug resistance. The small molecule drug adapalene is expected to overcome existing treatment difficulties, open up new paths for the treatment of leukemia, especially AML, significantly improve treatment effects, and prolong patients' long-term survival rates.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0008] The first aspect of the present invention provides a combined pharmaceutical composition for treating leukemia.

[0009] Furthermore, the pharmaceutical composition comprises adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof.

[0010] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0011] Furthermore, the concentration ratio of adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (1 μM-30 μM): (0.01-5 μM).

[0012] Furthermore, the concentration ratio of adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is any one of 5 μM:0.05 μM, 5 μM:0.1 μM, 5 μM:0.1 μM, and 10 μM:0.1 μM.

[0013] Furthermore, the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, juvenile myelomonocytic leukemia, mixed phenotype acute leukemia and / or plasma cell leukemia.

[0014] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia.

[0015] Furthermore, the pharmaceutically acceptable adjuvant and / or excipient is a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and / or a disintegrant.

[0016] In the present invention, adapalene is a third-generation retinoid drug primarily used to treat acne vulgaris. It is a first-line topical medication for acne treatment, particularly suitable for mild to moderate acne and for preventing recurrence. Its mechanism of action includes regulating keratinocyte differentiation, improving hyperkeratinization of the hair follicle opening, and preventing pore clogging; inhibiting the growth of Propionibacterium acnes; and providing anti-inflammatory effects, alleviating acne redness and papule reactions. Currently, adapalene has not been used in the treatment of acute myeloid leukemia.

[0017] In some embodiments, the adapalene or a pharmaceutically acceptable salt thereof, or venetoclax or a pharmaceutically acceptable salt thereof refers to salt forms obtained by salt modification of adapalene or venetoclax, suitable for use in pharmaceutical formulations and clinical applications. Salts used in the salt modification process include, but are not limited to, inorganic acid salts (e.g., hydrochlorides, sulfates, phosphates) and organic acid salts (e.g., citrates, maleates, tartrates). These salts are suitable for contact with patients within the scope of sound medical judgment and do not produce undue toxicity, irritation, or allergic reactions.

[0018] In some embodiments, examples of pharmaceutically acceptable salts include, but are not limited to, salts with (as counter ions) alkali metal ions such as Na + 、Li + or K + or salts with alkaline earth metal ions such as Ca 2+ or Mg 2+ or any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+ or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0019] In some embodiments, pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include magnesium, potassium, sodium, calcium, and the like. Examples of suitable amines include N,N'-dibenzylethylenediamine, diethanolamine, chloroprocaine, choline, ethylenediamine, N-methylglucamine, or procaine.

[0020] In some embodiments, base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.

[0021] In some embodiments, pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, potassium, calcium, lithium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids, such as gluconate, arginate, galacturonate, and the like are also contemplated.

[0022] In some embodiments, the pharmaceutically acceptable excipients and / or vehicles are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used to help stabilize the drug or help improve the activity of the active ingredient (i.e., adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof as described above in the present invention). The substances include but are not limited to: diluents, surfactants, wetting agents, binders, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, and pH control agents. The pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed.

[0023] In some embodiments, the pharmaceutically acceptable adjuvants and / or excipients may additionally contain liquids such as water, saline, glycerol, and ethanol.

[0024] In some embodiments, the pharmaceutical composition is a single compound preparation or a combination of two separate single preparations. The compound preparation is a compound preparation comprising adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof; and the combination of single preparations is a combination of a single preparation comprising adapalene or a pharmaceutically acceptable salt thereof and a single preparation comprising venetoclax or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the administration of the two single-ingredient preparations in the combination of single-ingredient preparations includes: simultaneous administration and sequential administration. When the administration of the two single-ingredient preparations in the combination of single-ingredient preparations is sequential administration, the administration includes: first administering the single-ingredient preparation containing adapalene or a pharmaceutically acceptable salt thereof, and then administering the single-ingredient preparation containing venetoclax or a pharmaceutically acceptable salt thereof; first administering the single-ingredient preparation containing venetoclax or a pharmaceutically acceptable salt thereof, and then administering the single-ingredient preparation containing adapalene or a pharmaceutically acceptable salt thereof.

[0026] A second aspect of the present invention provides a pharmaceutical preparation.

[0027] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the first aspect of the present invention.

[0028] Furthermore, the dosage form of the pharmaceutical preparation includes an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form or a skin dosage form.

[0029] In some embodiments, the dosage form of the pharmaceutical preparation refers to a dosage form prepared by conventional methods known in the art that is convenient for administration to a subject, including but not limited to: parenteral dosage forms, gastrointestinal dosage forms, specific examples include but are not limited to: aqueous solution injection, powder injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release agents and controlled-release agents, etc.

[0030] In some embodiments, the pharmaceutical preparation contains pharmaceutical excipients, which can be conventionally used in various preparations, including but not limited to: isotonicity agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants and lubricants; or selected for compatibility with the substance, including but not limited to: emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants. Such excipients can effectively improve the stability and solubility of the active ingredients contained in the composition or change the release rate and absorption rate of the active ingredients, thereby improving the metabolism of various active ingredients in the organism and enhancing the administration effect of the composition. In addition, excipients used to achieve specific administration purposes or methods, such as sustained-release administration, controlled-release administration and pulse administration, include but are not limited to: gelatin, albumin, chitosan, polyether and polyester polymer materials (for example: polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc.). The main manifestations of the advantages of administration include: improving therapeutic effect, improving bioavailability, reducing toxic side effects and improving patient compliance.

[0031] In some embodiments, when the pharmaceutical preparation is an injectable preparation, the solvent is one or more of water for injection, physiological saline, and 5% glucose injection, and excipients such as pH adjusters, osmotic pressure regulators, and antioxidants may be added as needed. The pH adjusters are selected from one or more of hydrochloric acid, sodium hydroxide, and citric acid-sodium citrate buffer; the osmotic pressure regulators are selected from one or more of sodium chloride, glucose, and mannitol; and the antioxidants are selected from one or more of sodium sulfite, sodium metabisulfite, and sodium thiosulfate.

[0032] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention may also be used in combination with other therapeutic compounds that can be used for the treatment or adjuvant treatment of leukemia, and the other therapeutic compounds can be administered simultaneously with the main active ingredient in the pharmaceutical composition or pharmaceutical preparation of the present invention, or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the course of treatment, the dosage of the pharmaceutical composition or pharmaceutical preparation of the present invention can be adjusted according to the severity of the symptoms, the frequency of relapses, and the physiological response of the treatment regimen.

[0033] In some embodiments, when the pharmaceutical composition or pharmaceutical preparation provided by the present invention is actually used, its administration regimen and dosage regimen can be selected according to a variety of factors, including the type, species, age, weight, sex and type of disease being treated of the subject; the severity of the disease being treated; the route of administration; the patient's renal and liver function; and the specific compound used or other forms of the compound. A dosing and / or dosage regimen can be used, for example, to prevent a disease, inhibit (completely or partially inhibit) a disease, or stop the development of the disease. In a specific embodiment of the present invention, the disease refers to leukemia, especially acute myeloid leukemia.

[0034] Those skilled in the art will understand that, although the pharmaceutical composition or pharmaceutical preparation mentioned above in the present invention may further contain pharmaceutically acceptable excipients and / or vehicles, when the adapalene or its pharmaceutically acceptable salt; and venetoclax or its pharmaceutically acceptable salt are used as drugs for humans or animals, they may also be administered in their own form, that is, the present invention can be achieved without adding any of the above-mentioned pharmaceutically acceptable excipients and / or vehicles.

[0035] The third aspect of the present invention provides an in vitro non-therapeutic method for inhibiting the growth of leukemia cells and promoting the apoptosis of leukemia cells.

[0036] Furthermore, the method comprises: treating in vitro leukemia cells with adapalene or a pharmaceutically acceptable salt thereof and venetoclax or a pharmaceutically acceptable salt thereof;

[0037] Optionally, the leukemia cells are acute myeloid leukemia cells, acute lymphocytic leukemia cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells, hairy cell leukemia cells, juvenile myelomonocytic leukemia cells, mixed phenotype acute leukemia cells and / or plasma cell leukemia cells.

[0038] In a specific embodiment of the present invention, the present invention has experimentally discovered for the first time that the combination of adapalene and venetoclax can produce a synergistic effect in the treatment of acute myeloid leukemia, significantly inhibiting the growth of acute myeloid leukemia cells and promoting their apoptosis. Therefore, the combined pharmaceutical composition can be used as an inhibitor to inhibit the growth and proliferation of leukemia (especially acute myeloid leukemia) cells for non-therapeutic purposes, and can be used in scientific research, such as to further study the growth and metabolic mechanisms or behaviors of leukemia cells, and to screen for potential drugs for the treatment of leukemia.

[0039] In a specific embodiment of the present invention, the present invention further verifies the synergistic effect of the combination of adapalene and venetoclax by the CI index of the combination. The CI (combination index, CI) index of the combination is a method for evaluating the combined effect of drugs, which is based on the dose-effect curve of the drug, by calculating the ratio of the actual effect to the theoretical effect during the combination (the specific calculation method is a method for calculating the combination index CI using CalcuSyn software known to those skilled in the art. CalcuSyn software calculates the CI index based on the Chou-Talalay theory by fitting the dose-effect curves of the single drug and the combination group). It is judged whether the combination has a synergistic effect, an additive effect or an antagonistic effect. A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect.

[0040] A fourth aspect of the present invention provides the application of any of the following aspects:

[0041] (1) Use of adapalene or a pharmaceutically acceptable salt thereof and venetoclax or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating leukemia;

[0042] (2) Use of adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical preparation for treating leukemia;

[0043] (3) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the anti-leukemia activity of venetoclax;

[0044] (4) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating leukemia;

[0045] (5) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical preparation for treating leukemia.

[0046] Furthermore, the concentration ratio of adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof is (1 μM-30 μM): (0.01-5 μM);

[0047] Optionally, the concentration ratio of adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof is any one of 5 μM:0.05 μM, 5 μM:0.1 μM, 5 μM:0.1 μM, and 10 μM:0.1 μM;

[0048] Optionally, the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, juvenile myelomonocytic leukemia, mixed phenotype acute leukemia and / or plasma cell leukemia.

[0049] In addition, the present invention also provides a method for treating leukemia, comprising: administering a therapeutically effective amount of the pharmaceutical composition of the first aspect of the present invention or the pharmaceutical preparation of the second aspect of the present invention to a subject in need.

[0050] In some embodiments, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. In certain embodiments, the subject is preferably a human. In a specific embodiment of the present invention, the subject is a leukemia patient, particularly an acute myeloid leukemia patient.

[0051] In some embodiments, the term "treating" refers to the complete or partial improvement or alleviation of a disease or condition or disorder, or a symptom, adverse effect or consequence, or a phenotype associated therewith. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease condition, and alleviating or improving prognosis. The term does not imply a complete cure of a disease or complete elimination of any symptoms or an effect on all symptoms or consequences.

[0052] In some embodiments, the therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments used concurrently.

[0053] In some embodiments, the pharmaceutical composition or formulation may have a formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Furthermore, administration may be single or multiple. In this case, the pharmaceutical composition or formulation is administered in the form of a liquid formulation, powder, aerosol, capsule, or suppository. Routes of administration may include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration. For oral administration, the active ingredient in the pharmaceutical composition or formulation may be formulated to protect it from degradation in the stomach. Furthermore, the active ingredient may be administered via any device capable of transporting the active ingredient to target cells. In specific embodiments, the pharmaceutical composition or formulation provided herein may be formulated into various dosage forms according to actual needs, and the dosage may be determined by the clinician based on factors such as the type, age, weight, general condition, and route of administration of the patient to be administered. The administration method can be, for example, injection or any other appropriate administration method known to those skilled in the art.

[0054] In some embodiments, the specific dosage and frequency (single or multiple doses) of the pharmaceutical composition or pharmaceutical preparation administered to the subject may vary according to a variety of factors, such as whether the mammal suffers from another disease and its route of administration; the subject's age, sex, health status, weight, body mass index, and diet; the nature and extent of the symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of concurrent treatment, complications caused by the disease being treated, or other health-related issues. Other treatment regimens or agents may be used in conjunction with the pharmaceutical composition or pharmaceutical preparation and treatment methods described herein. Adjustment and manipulation of the established dosage (e.g., frequency and duration) are well within the capabilities of those skilled in the art.

[0055] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0056] The present invention discovers for the first time that the combined use of adapalene and venetoclax exerts a synergistic inhibitory effect on the proliferation of acute myeloid leukemia cell lines, that is, the combination of the two has a significant synergistic enhancing effect. The inhibitory effect of the combined pharmaceutical composition on acute myeloid leukemia cell lines is significantly better than that of single adapalene or venetoclax. The present invention provides an effective drug combination strategy for the treatment of acute myeloid leukemia, and has good clinical application prospects and important translational significance in the technical field of development of therapeutic drugs for acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1:Untreated cell lines (H-60, SKNO-1, MOLM-13, MV4-11, and Kasumi-1) were treated with different concentrations of Adapalene (0.1 μM-50 μM) for 72 hours, and the proportion of viable cells was detected by MTT assay.

[0058] Figure 2 : Effects of different concentrations of Adapalene or the same concentration of Adapalene treated for different time periods on cell death of MOLM-13 cells or AML clinical samples, wherein, Figure A: Effects of different concentrations of Adapalene (0μM, 2.5μM, 5.0μM, 10.0μM) treated for 24h on MOLM-13 cell death; Figure B: Effects of 10.0μM Adapalene treated for different time periods (0h, 12h, 24h, 48h) on MOLM-13 cell death; Figure C: Effects of different concentrations of Adapalene (0μM, 2.5μM, 5.0μM, 10.0μM) treated for 48h on cell death of AML patients; Figure D: Effects of different concentrations of Adapalene (0μM, 2.5μM, 5.0μM, 10.0μM) treated for 48h on CD34 + the effects of cell death;

[0059] Figure 3 : AML cell lines MOLM-13 and Kasumi-1 cells were treated with different concentrations of adapalene (Ada) and venetoclax (Ven) alone or in combination for 12 h and 24 h, respectively. Cell death was detected by Annexin V-FITC / PI double staining and flow cytometry, and the synergistic coefficient (CI) (CI < 1 indicates a synergistic effect of the two drugs) was calculated using calcusyn software. AB figures: representative flow cytometry figures; CD figures: statistical figures of cell apoptosis detected by flow cytometry, **** represents p < 0.0001. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to specific embodiments. The following specific embodiments are intended only to illustrate the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0061] The drugs, reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0062] Example 1 Effect of Adapalene on Cell Viability of Acute Myeloid Leukemia (AML) Cell Lines

[0063] 1. Experimental Materials

[0064] AML cell lines: HL-60, SKNO-1, MOLM-13, MV4-11, and Kasumi-1;

[0065] Drug: Adapalene.

[0066] 2. Experimental methods

[0067] Based on drug solubility, adapalene is serially diluted with DMSO or other solvents, with each drug concentration reaching 10 times the final concentration. Add 10 μL of each drug to the corresponding well of a 96-well plate. Add 10 μL of the corresponding solvent to the single-drug wells and 20 μL of the corresponding solvent to the control wells. Add 80 μL of cell suspension to each well. All other steps are identical to those described above.

[0068] Adapalene drugs at different concentrations (0.1 μM-50 μM) were used to act on untreated cell lines (HL-60, SKNO-1, MOLM-13, MV4-11 and Kasumi-1) for 72 h. The proportion of living cells was detected by MTT assay. The absorbance of each well at a specific wavelength was measured by microplate reader. IC was calculated using GraphPad Prism. 50 The values were calculated and a standard isobologram was made to reflect the cell viability and to determine the half-maximal inhibitory concentration (IC50) of the drug on different cell lines. 50 ).

[0069] 3. Experimental results

[0070] This example investigates the effect of Adapalene on the viability of HL-60, SKNO-1, MOLM-13, MV4-11, and Kasumi-1 cell lines. The results show that as the concentration of Adapalene increases, the viability of each cell line decreases. Different cell lines have different sensitivities to Adapalene, with the HL-60 cell line being the most sensitive, with a median inhibitory concentration (IC50) of 1. 50 ) was 1.115 μM; Kasumi-1 cell line was relatively insensitive, IC 50 8.017 μM ( Figure 1 ).

[0071] Example 2 Adapalene induces apoptosis in AML cell lines

[0072] 1. Experimental Materials

[0073] Cell lines: AML cell line (MOLM-13), AML clinical sample cells;

[0074] Drug: Adapalene.

[0075] 2. Experimental methods

[0076] Annexin V / PI staining and flow cytometry were used to detect cell apoptosis. The specific experimental methods are as follows:

[0077] AML cell line (MOLM-13) and AML patient cells were adjusted to a cell number of 1×10 6 / well. After 24h or 48h of drug treatment, cells were collected and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded and the cell pellet was washed with 1mL of pre-cooled PBS. The cell pellet was transferred to a 1.5mL EP tube and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded as much as possible. 50μL of binding buffer was added to the 1.5mL EP tube, followed by 5μL of Annexin-FITC and mixed. The cells were stained at room temperature in the dark for 15 minutes. 10μL of propidium iodide (PI) was added and mixed. The cells were stained at room temperature in the dark for 5 minutes. 450μL of binding buffer was added to terminate the reaction. The samples were filtered through a double layer of 200-mesh nylon mesh and analyzed by flow cytometry within 1 hour. The data were stored and images were generated using CytExpert. Data processing and statistical analysis were performed using GraphPad Prism software.

[0078] 3. Experimental results

[0079] This example studies the effect of Adapalene on MOLM-13 cell death. The results show that when treated with 10 μM Adapalene, the proportion of MOLM-13 cell death increased significantly over time (12-48 h) (P < 0.0001) ( Figure 2 B). After 24 hours of treatment, as the concentration of adapalene increased from 2.5 μM to 10 μM, the death rate of MOLM-13 cells increased significantly, and the difference was extremely significant compared with the control group (P<0.0001) ( Figure 2 A). The experimental results showed that adapalene induced apoptosis of leukemia cells in a time-dependent and dose-dependent manner.

[0080] This example further explored the effects of different concentrations of adapalene (Ada, 0μM, 2.5μM, 5.0μM, 10.0μM) on AML patient cells after 48 hours of treatment. The results showed that as the concentration of Ada increased, the proportion of cell death increased. The difference between 2.5μM and 0μM was not significant (ns), while the differences between 5.0μM and 10.0μM and 0μM were extremely significant (P<0.0001) ( Figure 2 C) As the concentration of Ada increases, CD34 + Annexin V + The cell ratio also showed an upward trend. There was no significant difference between 2.5 μM and 0 μM (ns), while there was a very significant difference between 5.0 μM and 10.0 μM (P < 0.0001) ( Figure 2 D) The experimental results show that the increase of Ada concentration will significantly increase the cell death and CD34 + Cell death.

[0081] Example 3 Adapalene and Venetoclax combined to inhibit AML have a synergistic effect

[0082] 1. Experimental Materials

[0083] AML cell lines: MOLM-13 and Kasumi-1;

[0084] Drugs: Adapalene, Venetoclax.

[0085] 2. Experimental methods

[0086] Annexin V / PI staining and flow cytometry were used to detect cell apoptosis. The specific experimental methods are as follows:

[0087] AML cells (1×10 6) After drug treatment, cells were collected and centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was washed with 1 mL of pre-cooled PBS, transferred to a 1.5 mL EP tube, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded as much as possible. 50 μL of binding buffer was added to the 1.5 mL EP tube, and then 5 μL of Annexin-FITC was added and mixed, and the cells were stained at room temperature in the dark for 15 minutes. 10 μL of propidium iodide (PI) was added and mixed, and the cells were stained at room temperature in the dark for 5 minutes. 450 μL of binding buffer was added to terminate the reaction, and the sample was filtered through a double layer of 200 mesh nylon mesh. The sample was detected by flow cytometry within 1 hour and the data file was saved. Images were made using FlowJo 7.6, and data were processed using GraphPad Prism software.

[0088] 3. Experimental results

[0089] AML cell lines MOLM-13 and Kasumi-1 were treated with different concentrations of adapalene (Ada) and venetoclax (Ven) alone or in combination for 12 h and 24 h, and cell death was detected by Annexin V-FITC / PI double staining and flow cytometry ( Figure 3 AB), and the synergistic coefficient (CI) was calculated using calcusyn software; CI < 1 indicated that the two drugs had a synergistic effect.

[0090] The results showed that 5 μM adapalene combined with 0.05 μM and 0.1 μM venetoclax had a synergistic effect (CI < 0.65) in MOLM-13 cells treated for 12 h. Figure 3 C). Under the condition of 24h treatment of Kasumi-1 cells, 5μM adapalene and 10μM adapalene respectively combined with 0.1μM venetoclax had a synergistic effect (CI<0.42) ( Figure 3 D) The experimental results showed that the combination of adapalene and venetoclax can synergistically inhibit the proliferation and growth of AML cells, and the combination of the two drugs has synergistic anti-AML activity.

Claims

1. A combined pharmaceutical composition for treating leukemia, characterized in that: The pharmaceutical composition comprises adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and / or excipients.

3. The pharmaceutical composition according to claim 1, characterized in that Adapalene or a pharmaceutically acceptable salt thereof in the pharmaceutical composition; The concentration ratio of venetoclax or its pharmaceutically acceptable salt is (1 μM-30 μM): (0.01-5 μM).

4. The pharmaceutical composition according to claim 3, characterized in that Adapalene or a pharmaceutically acceptable salt thereof in the pharmaceutical composition; The concentration ratio of vinblastine or a pharmaceutically acceptable salt thereof is any one of 5 μM:0.05 μM, 5 μM:0.1 μM, 5 μM:0.1 μM, and 10 μM:0.1 μM.

5. The pharmaceutical composition according to claim 1, characterized in that The leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, juvenile myelomonocytic leukemia, mixed phenotype acute leukemia and / or plasma cell leukemia.

6. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical preparation according to claim 6, characterized in that The dosage form of the pharmaceutical preparation includes an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form or a skin dosage form.

8. A method for inhibiting the growth of leukemia cells and promoting apoptosis of leukemia cells in vitro for non-therapeutic purposes, characterized in that: The method comprises: treating in vitro leukemia cells with adapalene or a pharmaceutically acceptable salt thereof and venetoclax or a pharmaceutically acceptable salt thereof; Optionally, the leukemia cells are acute myeloid leukemia cells, acute lymphocytic leukemia cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells, hairy cell leukemia cells, juvenile myelomonocytic leukemia cells, mixed phenotype acute leukemia cells and / or plasma cell leukemia cells.

9. Any of the following applications: (1) Use of adapalene or a pharmaceutically acceptable salt thereof and venetoclax or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating leukemia; (2) Adapalene or a pharmaceutically acceptable salt thereof; Use of vinblastine and venetoclax or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical preparation for treating leukemia; (3) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the anti-leukemia activity of venetoclax; (4) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating leukemia; (5) Use of adapalene or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical preparation for treating leukemia.

10. The use according to claim 9, characterized in that Said adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or its pharmaceutically acceptable salt in a concentration ratio of (1 μM-30 μM): (0.01-5 μM); Optionally, the adapalene or a pharmaceutically acceptable salt thereof; and venetoclax or a pharmaceutically acceptable salt thereof in a concentration ratio of 5 μM:0.05 μM, 5 μM:0.1 μM, 5 μM:0.1 μM, or 10 μM:0.1 μM; Optionally, the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, juvenile myelomonocytic leukemia, mixed phenotype acute leukemia and / or plasma cell leukemia.

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