Pharmaceutical composition for preventing or treating bcl-2 family inhibitor-resistant acute myeloid leukemia

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Bcl-2 family inhibitor-resistant acute myeloid leukemia. More specifically, the present invention may provide a pharmaceutical composition for preventing or treating cancer that has acquired resistance to a Bcl-2 family inhibitor, which is used as a conventional therapeutic agent for resistant acute myeloid leukemia, through inhibition of the expression of nucleolin and Mcl-1.

Description

Pharmaceutical composition for the prevention or treatment of BCL-2 inhibitor-resistant acute myeloid leukemia

[0001] The present invention relates to a compound for preventing or treating cancer that has acquired resistance to Bcl-2 class inhibitors currently used as treatments for acute myeloid leukemia, and to the use of a pharmaceutical composition containing the same to exhibit a therapeutic effect.

[0002] Acute myeloid leukemia (AML) is a rapidly growing cancer characterized by abnormal white blood cells accompanied by various genetic mutations. While cytarabine (Ara-C) and daunorubicin are standard first-line chemotherapy regimens for AML, there is a problem in that approximately 30% of patients are refractory, and more than 50% of them relapse due to resistance. Furthermore, FLT3 inhibitors, the only targeted therapies for AML, are effective in only 20% of patients, and resistance develops within a few months.

[0003] Oligonucleotides forming G-quadroplexes are known to inhibit cell growth by binding to specific proteins that are highly expressed on the surface of cancer cells, as described in U.S. Patent No. 7314926 and U.S. Patent Publication No. 2007-105805, and then infiltrating cancer cells via endocytosis and binding to proteins involved in apoptosis, and have been reported to induce apoptosis.

[0004] Recently, venetoclax, a targeted anticancer drug that inhibits the anti-apoptotic protein Bcl-2 (B-cell lymphoma-2), has been widely used in the treatment of acute myeloid leukemia (AML); however, the development of drug resistance in a significant number of patients acts as a major inhibiting factor in treatment. This resistance is known to be induced mainly by the compensatory activation of another anti-apoptotic protein, Mcl-1 (Myeloid cell leukemia 1).

[0005] In particular, since cancer cells that have acquired resistance to venetoclax are highly likely to exhibit extensive cross-resistance to other Bcl-2 inhibitors such as lisaftoclax and navitoclax, there is an urgent need to develop therapeutic agents with new mechanisms to effectively control this.

[0006] Currently, no prior art has disclosed that the compound of the present invention has such an excellent anticancer therapeutic effect in cancers resistant to Bcl-2 series inhibitors.

[0007] Accordingly, the inventors confirmed that the compound exhibits an excellent anticancer effect in resistant cancer cells that do not respond to a wide range of existing Bcl-2 inhibitors. In particular, the invention was completed by confirming the anticancer therapeutic effect, in which the compound of the present invention showed a significant improvement in survival rate when administered alone, even in animal models exhibiting resistance to Bcl-2 inhibitors.

[0008] Accordingly, the objective of the present invention is to provide a pharmaceutical composition for preventing or treating cancer that has acquired resistance to Bcl-2 class inhibitors currently used as treatments for acute myeloid leukemia.

[0009] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become more apparent from the following description and will be realized by the means and combinations thereof described in the claims.

[0010] To achieve the above objective, a pharmaceutical composition for the prevention or treatment of Bcl-2 series inhibitor-resistant acute myeloid leukemia is provided, comprising as active ingredients an oligonucleotide modified nucleic acid having the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof.

[0011] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0012] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0013] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0014] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0015] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0016] The above guanosine or guanosine derivative may be one or more selected from 2'-deoxyguanosine, guanosine, 2'-O-methylguanosine, 2'-fluoroguanosine, LNA (Locked Nucleic Acid)-guanosine, D-deoxyguanosine, and D-guanosine.

[0017] The above Bcl-2 inhibitor may be one or more selected from the group consisting of venetoclax, lisaptoclax, nabitoclax, obatoclax, and felcitoclax.

[0018] The above composition can inhibit the expression of one or more proteins selected from the group consisting of nucleolin, Mcl-1, Bcl-2, and DNMT1.

[0019] The above-mentioned resistant acute myeloid leukemia may be relapsed or refractory.

[0020] The above-mentioned resistant acute myeloid leukemia may be due to the overexpression of nucleolin, Mcl-1, and Bcl-2 resulting from the acquisition of resistance to Bcl-2 series inhibitors.

[0021] In addition, the present invention may provide a method for treating Bcl-2 inhibitor-resistant acute myeloid leukemia, comprising the step of administering a therapeutically effective amount of an oligonucleotide modified nucleic acid having the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0022] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0023] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0024] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0025] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0026] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0027] The expression of nucleolin is inhibited by the administration of the above-mentioned oligonucleotide modified nucleic acid, and the expression of Mcl-1 and Bcl-2 may be simultaneously reduced by the inhibited nucleolin.

[0028] A decrease in the expression of the above Mcl-1 and Bcl-2 may increase the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bax, and Bak, thereby inducing apoptosis.

[0029] In addition, the present invention may provide an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of Bcl-2 series inhibitor-resistant acute myeloid leukemia.

[0030] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0031] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0032] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0033] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0034] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0035] In addition, the present invention may provide a method for preventing or treating acute myeloid leukemia resistant to Bcl-2 inhibitors, comprising the steps of: inhibiting nucleolin in a patient with acute myeloid leukemia resistant to Bcl-2 inhibitors; inhibiting Mcl-1 expression; and inhibiting Bcl-2 expression.

[0036] The step of inhibiting the above Nucleolin may be achieved by administering to a patient an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof.

[0037] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0038] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0039] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0040] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0041] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0042] The present invention relates to a pharmaceutical composition for the prevention or treatment of acute myeloid leukemia resistant to Bcl-2 inhibitors, and more specifically, can provide a pharmaceutical composition for the prevention or treatment of cancer that has acquired resistance to Bcl-2 inhibitors currently used as treatments for acute myeloid leukemia.

[0043] The effects of the present invention are not limited to those mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

[0044] Figure 1 is the result of confirming the inhibitory effect on cancer cell proliferation in venetoclax-resistant AML cancer cell lines.

[0045] Figure 2 is a graph showing the results of confirming the inhibitory effect on cancer cell proliferation in venetoclax-resistant AML cancer cell lines.

[0046] Figure 3 shows the changes in expression of Nucleolin, Mcl-1, and Bcl-2 when compound 1 was treated in venetoclax-resistant MOLM-13.

[0047] Figure 4 shows the results confirming that Nucleolin, Mcl-1, and Bcl-2 expression were increased in venetoclax-resistant MOLM-13, and that NCL-MCL-1-BCL2-mediated apoptosis in venetoclax-resistant MOLM-13 is the key mechanism of the anticancer effect.

[0048] Figure 5 shows the results of confirming the antitumor effect following the administration of compound 1 in a venetoclax-resistant AML mouse allogeneic model.

[0049] Figure 6 shows the results of confirming the change in Mcl-1 expression following the administration of compound 1 in bone marrow cells of a venetoclax-resistant AML mouse allogeneic transplant model.

[0050] Figure 7 shows the results of confirming the change in Bcl-2 expression following the administration of compound 1 in bone marrow cells of a venetoclax-resistant AML mouse allogeneic transplant model.

[0051] Figure 8 shows the results of confirming the change in Bax expression following the administration of compound 1 in bone marrow cells of a venetoclax-resistant AML mouse allogeneic transplant model.

[0052] The above objects, other objects, features, and advantages of the present invention will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0053] In describing each drawing, similar reference numerals have been used for similar components. In the attached drawings, the dimensions of the structures are depicted enlarged compared to their actual size for the clarity of the invention.

[0054] Unless otherwise specified, all numbers, values, and / or expressions used herein to represent amounts of ingredients, reaction conditions, polymer compositions, and formulations should be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among other things. Furthermore, where numerical ranges are disclosed herein, such ranges are continuous and, unless otherwise indicated, include all values ​​from the minimum value of such range to the maximum value including said maximum value. Moreover, where such ranges refer to integers, they include all integers from the minimum value to said maximum value including said maximum value, unless otherwise indicated.

[0055] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0056] Specifically, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as the base for suppositories.

[0057] According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner through intravenous, intra-arterial, intra-abdominal, intramuscular, intrasternal, transdermal, nasal, inhalation, local, rectal, oral, ocular, or intradermal routes.

[0058] The dosage of the active ingredient according to the present invention may vary depending on the condition and body weight of the subject, the type and severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.1 mg / kg to 5000 mg / kg, preferably 1 mg / kg to 3000 mg / kg, more preferably 10 mg / kg to 2000 mg / kg. The administration may be performed once a day or divided into several doses, and the scope of the present invention is not limited by this.

[0059]

[0060] Conventional technological attempts to overcome drug resistance in acute myeloid leukemia (AML) have primarily focused on genomic-level mechanisms, such as defects in DNA replication processes or epigenetic mutations. In particular, resistance to DNA synthesis inhibitors such as cytarabine or DNA methyltransferase (DNMT) inhibitors such as decitabine and azacitidine has been identified as a key factor causing AML relapse and treatment failure, and the mechanisms of action for responding to this have also been limited to inducing cytotoxicity through interference with DNA synthesis or methylation regulation.

[0061] However, with the recent widespread use of Bcl-2 inhibitors, including venetoclax, as standard therapy, a novel resistance caused by the "compensatory activation of apoptosis-regulating proteins"—which operates via biological pathways distinct from conventional genomic metabolism-related resistance—is emerging as a serious clinical limitation. This mechanism involves cancer cells becoming highly dependent on anti-apoptotic factors such as Mcl-1 for survival, and there are clear technical barriers to fundamentally controlling this apoptosis-evasion resistance using only existing approaches aimed at inhibiting DNA replication.

[0062] To overcome the limitations of the prior art, the inventors provide a unique mechanism of action that induces nucleolin targeting and the direct inhibition of Mcl-1 / Bcl-2 protein expression. It was confirmed that inhibiting Mcl-1 exhibits an excellent anticancer effect in resistant cancer cells that do not respond to a wide range of existing Bcl-2 inhibitors. This represents a unique pharmacological action that could not have been predicted at all by biological mechanism from existing technologies for overcoming resistance to DNA synthesis inhibitors or methyltransferase inhibitors, and is characterized by exhibiting a significantly enhanced therapeutic effect on relapsed and refractory AML that has acquired broad cross-resistance to Bcl-2 inhibitors. In particular, the present invention was completed by confirming the anticancer therapeutic effect of the compound of the present invention, which showed a significant improvement in survival rate when administered alone even in animal models exhibiting resistance to Bcl-2 inhibitors.

[0063]

[0064] The present invention may provide a pharmaceutical composition for the prevention or treatment of Bcl-2 series inhibitor-resistant acute myeloid leukemia comprising, as an active ingredient, an oligonucleotide modified nucleic acid having the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof.

[0065] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0066] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0067] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0068] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0069] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0070] The above guanosine or guanosine derivative may be one or more selected from 2'-deoxyguanosine, guanosine, 2'-O-methylguanosine, 2'-fluoroguanosine, LNA (Locked Nucleic Acid)-guanosine, D-deoxyguanosine, and D-guanosine, but is not limited thereto.

[0071] The above Bcl-2 inhibitors may be one or more selected from the group consisting of venetoclax, lisaptoclax, nabitoclax, obatoclax, and felcitoclax, but are not limited thereto.

[0072] The above composition may inhibit the expression of one or more proteins selected from the group consisting of Mcl-1, Bcl-2, nucleolin, and DNMT1, but is not limited thereto.

[0073] The above composition can simultaneously reduce the expression of Mcl-1 and Bcl-2, which are located in the nucleolin downstream signaling pathway, by inhibiting the expression of nucleolin.

[0074] A decrease in the expression of the above Mcl-1 and Bcl-2 can increase the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bax, and Bak, which are cell death induction markers, thereby inducing cell death.

[0075] The above composition may increase the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bak, and Bax, which are cell death-inducing markers, but is not limited thereto.

[0076] The above-mentioned resistant acute myeloid leukemia may be relapsed or refractory.

[0077] The above-mentioned resistant acute myeloid leukemia may be due to the overexpression of nucleolin, Mcl-1, and Bcl-2 resulting from the acquisition of resistance to Bcl-2 series inhibitors.

[0078] In addition, the present invention may provide a method for treating Bcl-2 inhibitor-resistant acute myeloid leukemia, comprising the step of administering a therapeutically effective amount of an oligonucleotide modified nucleic acid having the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0079] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0080] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0081] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0082] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0083] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0084] The expression of nucleolin is inhibited by the administration of the above-mentioned oligonucleotide modified nucleic acid, and the expression of Mcl-1 and Bcl-2 may be simultaneously reduced by the inhibited nucleolin.

[0085] A decrease in the expression of the above Mcl-1 and Bcl-2 may increase the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bax, and Bak, thereby inducing apoptosis.

[0086] The subject may be a group of patients with acute myeloid leukemia resistant to Bcl-2 inhibitors in which nucleolin, Mcl-1, and Bcl-2 are overexpressed due to the acquisition of resistance to Bcl-2 inhibitors.

[0087] In addition, the present invention may provide an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of Bcl-2 series inhibitor-resistant acute myeloid leukemia.

[0088] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0089] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0090] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0091] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0092] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0093] The expression of nucleolin is inhibited by the administration of the above-mentioned oligonucleotide modified nucleic acid, and the expression of Mcl-1 and Bcl-2 may be simultaneously reduced by the inhibited nucleolin.

[0094] A decrease in the expression of the above Mcl-1 and Bcl-2 may increase the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bax, and Bak, thereby inducing apoptosis.

[0095] In addition, the present invention may provide a method for preventing or treating acute myeloid leukemia resistant to Bcl-2 inhibitors, comprising the steps of: inhibiting nucleolin in a patient with acute myeloid leukemia resistant to Bcl-2 inhibitors; inhibiting Mcl-1 expression; and inhibiting Bcl-2 expression.

[0096] Patients with acute myeloid leukemia resistant to Bcl-2 inhibitors may have overexpression of nucleolin, Mcl-1, and Bcl-2 due to the acquisition of resistance to Bcl-2 inhibitors.

[0097] The step of inhibiting the above Nucleolin may be achieved by administering to a patient an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof.

[0098] Sequence 1) GGGTGGTGGTNNTGGTGGTGG;

[0099] Sequence 2) GGGTGGTGGTTNTGGTGGTGG;

[0100] Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or

[0101] Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG;

[0102] In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

[0103]

[0104] The present invention will be explained in detail below through examples and comparative examples. However, the following examples and comparative examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0105] <Example 1> Synthesis of a Novel Oligonucleotide-Modified Nucleic Acid

[0106] An oligonucleotide modified nucleic acid was prepared as described in Example 1 of Korean Registered Patent No. 10-2097519, and an oligonucleotide modified nucleic acid preparation was prepared as described in Example 2 of Korean Registered Patent No. 10-2097519, and the experiment was conducted.

[0107] Compound Number N (Modified Nucleic Acid) G (Guanosine Derivative) T (Thymine Derivative) Sequence LC / MS Compound 11-β-D-Arabinofuranosylcytosine 2'-Deoxyguanosine Thymidine GGTGGTGGTNNTGGTGGTGG6324.1 Compound 21-β-D-Arabinofuranosylcytosine 2'-Deoxyguanosine Thymidine GGTGGTGGTTNTGGTGGTGG6323.1 Compound 31-β-D-Arabinofuranosylcytosine 2'-Deoxyguanosine Thymidine NGGTGGTGGTTGTGGTGGTGG6652.3 Compound 41-β-D-Arabinofuranosylcytosine 2'-Deoxyguanosine Thymidine NNGGTGGTGGTTGTGGTGGTGG6957.5

[0108] <Example 2> Confirmation of anticancer activity and drug resistance overcoming effect in general AML cancer cell lines and venetoclax-resistant AML cancer cell lines (MTTassay)

[0109] 1. Cell line and drug preparation

[0110] In this experiment, C1498, a mouse-derived AML cell line resistant to venetoclax, and MOLM-13, a human-derived AML cell line (parental and venetoclax resistant), were used. The C1498 cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium, HyClone™, Cat. No. SH30243.01) containing 10% FBS (Fetal bovine serum, Gibco, Cat. No. 16000044), and the MOLM-13 cell line was cultured in RPMI1640 (Cytiva, Cat. No. SH30027.01) containing 20% ​​FBS (Fetal bovine serum, Gibco, Cat. No. 16000044) at 37°C in a 5% CO2 incubator. Bcl-2 inhibitors venetoclax, lisaptoclax, and nabitoclax were used as control drugs.

[0111] 2. Measurement of cell viability and calculation of IC50

[0112] Each cancer cell line was seeded into a 96-well plate, and after 24 hours, the compound of the present invention (Compound 1) and control drugs were treated at various concentrations. After incubation for a set period (72 to 120 hours), 20 μl of 5 mg / ml MTT solution (Invitrogen, Cat. No. 6494) was added per well (final treatment concentration: 0.5 mg / ml). After incubation for 2 hours in a 37 ℃, 5% CO2 incubator, 200 μl / well culture medium was removed, and 200 μl / well DMSO was added to dissolve formazan. The absorbance (SYNERGY HTX (BioTek)) was measured at a wavelength of 570 nm, and the half-maximum inhibitory concentration (IC10) for cell activity was determined using GraphPad Prism software. 50 ) was produced.

[0113] Cell line compound 1 (IC50,μM)Venetoclax (IC50,μM)Lisaftoclax (IC50,μM)Navitoclax (IC50,μM)C14980.164.6418.974.91

[0114] According to Table 2 above, in C1498, a mouse-derived AML cell line resistant to venetoclax, Bcl-2 inhibitors such as venetoclax, lisaptoclax, and nabitoclax did not exhibit effective anticancer efficacy, whereas compound 1 showed excellent anticancer efficacy.

[0115] Cell line Venetoclax (IC50,μM) Compound 1 (IC50,μM) MOLM-13 (Parental) 0.015 0.025 Venetoclax-resistant MOLM-13 3.87 0.072

[0116] According to Table 3 above, in MOLM-13 (Venetoclax-resistant MOLM-13), a human-derived AML cell line engineered to be resistant to venetoclax, venetoclax did not exhibit effective anticancer efficacy, whereas compound 1 showed excellent anticancer efficacy.

[0117] In other words, while Bcl-2 inhibitors exhibit very poor anticancer efficacy in cell lines possessing strong resistance to the existing Bcl-2 inhibitor venetoclax, Compound 1 has a very low IC50 50 It was confirmed that it exhibits excellent anticancer activity by displaying a value.

[0118]

[0119] <Example 3> Confirmation of cancer cell proliferation inhibitory effect in venetoclax-resistant AML cancer cell lines (Clonogenicassay)

[0120] Colony formation ability was evaluated in venetoclax-resistant MOLM-13 cells following drug treatment. Venetoclax-resistant MOLM-13 cell lines were pretreated with venetoclax (50 nM), Compound 1 (50 nM), and lisaptoclax (50 nM), respectively, for 24 hours. After counting the pretreated cells, 1 x 10⁶ cells were placed in a tube. 4 The cells were suspended at a concentration of cells / ml, and each drug was treated again at the same final treatment concentration. Cells were mixed with StemMACS™ HSC-CFU complete with Epo, human (Miltenyi Biotec, cat, No. 130-091-280) medium at a ratio of 1:10 and inoculated into 35 mm culture dishes. After 6 days, colony counts and colony photographs were taken, and graphs were created using GraphPad Prism software.

[0121] As a result, in Figures 1 and 2, venetoclax and lisaptoclax did not differ significantly from the number of colonies in the control group in venetoclax-resistant MOLM-13, whereas the number and size of colonies in the compound 1 treatment group were significantly reduced, confirming that compound 1 exhibited excellent cell proliferation inhibitory efficacy (Figure 2, *p<0.05).

[0122]

[0123] <Example 4> Confirmation of changes in cell death-related protein expression (Western Blot)

[0124] 1. Protein changes in venetoclax-resistant cell lines following treatment with Compound 1

[0125] To confirm the mechanism of Compound 1 in overcoming venetoclax resistance, venetoclax-resistant MOLM-13 cell lines were treated with Compound 1 and venetoclax at various concentrations (0.1, 0.5, 1, 5 μM), and the cells were lysed with cell lysis buffer to perform western blot analysis. Samples were loaded using 10% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad, Cat. No. #4561036), and transfer was performed using the Trans-Blot® Turbo™ Transfer System and Trans-Blot® Turbo™ Transfer Pack (Bio-Rad, Cat. No. #1704156). Subsequently, primary blocking was completed using 5% skim milk in T-TBST, followed by overnight shaking at 4°C using the antibodies listed below, and thorough washing with T-TBST the next day. Protein expression of each secondary antibody was confirmed after incubation at room temperature for 1 hour using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, REF 34580) and an Amersham ImageQuant™ 800 imaging system. The conditions for the primary antibodies to detect Mcl-1, Bcl-2, Bak, Bax, Cleaved Caspase-3, and Nucleolin were described as follows.

[0126] Nucleolin (Cell signaling, #14574): 1st 1:3000, 2nd 1:5000 (Rabbit)

[0127] Mcl-1 (Cell signaling, #5453): 1st 1:2000, 2nd 1:5000 (Rabbit)

[0128] Bcl-2 (Cell signaling, #3498): 1st 1:1000, 2nd 1:5000 (Rabbit)

[0129] Bak (Cell signaling, #12105): 1st 1:1000, 2nd 1:5000 (Rabbit)

[0130] Bax (Cell signaling, #2772): 1st 1:1000, 2nd 1:5000 (Rabbit)

[0131] Cleaved Caspase-3 (Cell signaling, #9664): 1st 1:1000, 2nd 1:5000 (Rabbit)

[0132] β-actin (Santacruz, #sc-47778): 1st 1:5000, 2nd 1:5000 (Mouse)

[0133] As a result, according to Figure 3, compound 1 inhibited the expression of Mcl-1 and Bcl-2, which are known as major causative proteins of venetoclax resistance, in a concentration-dependent manner, and effectively increased the expression of Cleaved Caspase-3, Bak, and Bax, which are downstream cell death-inducing markers. On the other hand, venetoclax did not induce the same changes in protein expression in the cell lines when treated under the same conditions.

[0134]

[0135] 2. Confirmation of Protein Changes and Anticancer Mechanisms in Venetoclax-Resistant Cell Lines

[0136] To compare changes in protein expression between MOLM-13 progenitor cells and venetoclax-resistant MOLM-13 cell lines, both untreated cells were lysed with cell lysis buffer and western blots were performed. The conditions for the western blots were as described above.

[0137] As a result, according to Figure 4, it was confirmed that the expression of Nucleolin, Mcl-1, and Bcl-2 was increased in venetoclax-resistant MOLM-13 cell lines compared to MOLM-13 progenitor cells.

[0138] Furthermore, to confirm that the mechanism of overcoming venetoclax resistance by compound 1 is a mechanism by the inhibition of nucleolin expression, venetoclax-resistant MOLM-13 cell lines were treated with 200 nM Nucleoilon siRNA (Sense: GGUCGUCAUACCUCAGAAG, Antisense: CUUCUGAGGUAUGACGACC [Sequence (5'-3')]) (Bioneer) + Lipofector-pMAX Regaent (Aptabio, Cat#AB-LF-M100), and then the cells were lysed with cell lysis buffer to perform western blot, and the process was described above.

[0139] As a result, according to Figure 4, the expression of Mcl-1 and Bcl-2, which are associated with resistance, was effectively suppressed in the Nucleolin siRNA-treated group, and the expression of downstream cell death-inducing markers Cleaved Caspase-3, Bax, and Bak was increased, indicating that NCL-MCL-1-BCL2-mediated cell death in venetoclax-resistant MOLM-13 is the key mechanism of the anticancer effect.

[0140]

[0141] <Example 5> Confirmation of increased survival time and antitumor effect in an AML mouse allogeneic model (Syngeneic model)

[0142] 1. Preparation of animal model and drug administration

[0143] To confirm the antitumor effect in an AML mouse allograft model, 6-week-old female C57BL / 6N mice (Orientbio) were purchased and used in the experiment after an acclimatization period of 1 week.

[0144] C1498 (ATCC, #TIB-49), a venetoclax-resistant C57BL / 6 mouse-derived blood cancer cell, was proliferated at 37°C in a 5% CO2 incubator. The medium used was DMEM (Dulbecco's Modified Eagle's Medium, HyClone™, Cat. No. SH30243.01) containing 10% FBS (Fetal bovine serum, Gibco, Cat. No. 16000044).

[0145] C1498 cells were suspended in PBS and administered 200 µl per mouse to 1x10 C57BL / 6 mice. 6 An AML mouse allogeneic transplantation model was established by intravenously injecting cells.

[0146] As the administered drug, Compound 1 was prepared by dissolving it in injectable physiological saline at a concentration of 150 mg / 10 ml / kg and administered subcutaneously. Venetoclax, a Bcl-2 inhibitor, was prepared as a mixture of 5% DMSO, 40% PEG300, 5% Tween-80, and 50% Saline in that order; to ensure complete dissolution of the drug, it was subjected to ultrasonic treatment and heating to 49°C to be prepared at a concentration of 5 mg / mL, and was prepared for oral administration at a concentration of 100 mg / 20 ml / kg. Both drugs were administered from the day after cancer cell transplantation until the time of death.

[0147] 2. Confirmation of antitumor efficacy and survival rate

[0148] The body weight of mice was measured at intervals of 2 to 3 days starting from the start of drug administration to check for drug toxicity. No significant changes in body weight or abnormal signs were observed in any of the populations until the end of the experiment. The survival period for each group was recorded to construct Kaplan-Meier survival curves, and statistical significance was examined.

[0149] As a result of the experiment, the venetoclax administration group showed no anticancer efficacy, exhibiting a survival curve similar to the control group, whereas the survival period of the compound 1 administration group was effectively extended (Fig. 4, ****p<0.0001).

[0150] 3. Collection of Bone Marrow-Derived Blood Cells and Protein Expression Analysis (Western Blot)

[0151] After the drug administration was completed, mice in each experimental group were euthanized, and femurs were aseptically excised. After cutting off both ends of the excised femurs, the inside of the bone marrow cavity was flushed with cold PBS solution using a 26G syringe to collect bone marrow cells. The collected cell suspension was treated with Red Blood Cell Lysis Buffer to remove red blood cells. The finally recovered blood cells were washed twice with PBS and used for analysis. Lysis Buffer containing a protease inhibitor cocktail was added to the isolated blood cells and reacted at 4°C for 30 minutes, after which the mixture was centrifuged at 13,000 rpm for 20 minutes to collect only the supernatant. The extracted proteins were quantified to equal concentrations using the BCA Protein Assay, and the subsequent western blot procedure was the same as described in Example 3.

[0152] As a result of the analysis experiment, it was confirmed that the group administered Compound 1 significantly inhibited the expression of Mcl-1 compared to the Vehicle control group in AML mouse allogeneic transplantation model bone marrow cells, whereas the venetoclax group showed no significant change (Fig. 5, *p<0.05). Bcl-2 was significantly increased in the venetoclax group compared to the Vehicle control group, whereas no significant change was observed in the group administered Compound 1 (Fig. 6, **p<0.01). Furthermore, the expression of Bax, a cell death-promoting factor, increased in the group administered Compound 1 compared to the Vehicle control group, demonstrating that the cell death mechanism induced by Compound 1 operates effectively in in vivo bone marrow tissue (Fig. 7, **p<0.01).

[0153]

[0154] Compound 1, the core compound of the present invention, exhibits a significant IC50-fold or greater reduction in AML cell lines showing strong cross-resistance to existing Bcl-2 inhibitors. 50 The efficacy of improvement has been proven. In particular, by directly and simultaneously inhibiting the expression of Mcl-1 and Bcl-2 proteins, which are the root causes of resistance, and potently reactivating apoptotic pathways (Bak, Bax, and Cleaved Caspase-3), it induces the complete death of resistant cancer cells beyond mere proliferation inhibition; this demonstrates remarkable effects in actual allogeneic mouse models, including the inhibition of lethal tumor proliferation and a significant extension of survival time.

[0155]

[0156] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0157] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of Bcl-2 inhibitor-resistant acute myeloid leukemia comprising, as an active ingredient, an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof: Sequence 1) GGGTGGTGGTNNTGGTGGTGG; Sequence 2) GGGTGGTGGTTNTGGTGGTGG; Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG; In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

2. In Claim 1, A pharmaceutical composition characterized in that the above guanosine or guanosine derivative is one or more selected from 2'-deoxyguanosine, guanosine, 2'-O-methylguanosine, 2'-fluoroguanosine, LNA (Locked Nucleic Acid)-guanosine, D-deoxyguanosine, and D-guanosine.

3. In Claim 1, A pharmaceutical composition characterized in that the above Bcl-2 series inhibitor is one or more selected from the group consisting of venetoclax, lisaptoclax, nabitoclax, obatoclax, and felcitoclax.

4. In Claim 1, A pharmaceutical composition characterized by inhibiting the expression of one or more proteins selected from the group consisting of nucleolin, Mcl-1, Bcl-2, and DNMT1.

5. In Claim 1, A pharmaceutical composition characterized in that the above-mentioned resistant acute myeloid leukemia is relapsed or refractory.

6. In Claim 1, A pharmaceutical composition characterized by the overexpression of nucleolin, Mcl-1, and Bcl-2 due to the acquisition of resistance to Bcl-2 series inhibitors in the above-mentioned resistant acute myeloid leukemia.

7. A method for treating Bcl-2 inhibitor-resistant acute myeloid leukemia, comprising the step of administering a therapeutically effective amount of an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof, to a subject in need thereof: Sequence 1) GGGTGGTGGTNNTGGTGGTGG; Sequence 2) GGGTGGTGGTTNTGGTGGTGG; Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG; In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

8. In Claim 7, A treatment method characterized by the suppression of nucleolin expression by the administration of the above-mentioned oligonucleotide modified nucleic acid, and the simultaneous reduction of Mcl-1 and Bcl-2 expression by the suppressed nucleolin.

9. In Claim 8, A treatment method characterized by inducing apoptosis by increasing the expression of one or more proteins selected from the group consisting of Cleaved Caspase-3, Bax, and Bak through a decrease in the expression of Mcl-1 and Bcl-2.

10. Use of an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure and a pharmaceutically acceptable salt thereof for the prevention or treatment of Bcl-2 series inhibitor-resistant acute myeloid leukemia: Sequence 1) GGGTGGTGGTNNTGGTGGTGG; Sequence 2) GGGTGGTGGTTNTGGTGGTGG; Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG; In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

11. Step of inhibiting nucleolin in patients with Bcl-2 inhibitor-resistant acute myeloid leukemia; A step of inhibiting Mcl-1 expression; and A method for the prevention or treatment of acute myeloid leukemia resistant to Bcl-2 inhibitors, comprising a step of inhibiting Bcl-2 expression.

12. In Claim 11, A method for prevention or treatment characterized by the step of inhibiting the above-mentioned Nucleolin being performed by administering to a patient an oligonucleotide modified nucleic acid comprising the following sequence and forming a G-quadroplex structure, and a pharmaceutically acceptable salt thereof: Sequence 1) GGGTGGTGGTNNTGGTGGTGG; Sequence 2) GGGTGGTGGTTNTGGTGGTGG; Sequence 3) NGGTGGTGGTTGTGGTGGTGG ; or Sequence 4) NNNGGTGGTGGTTGTGGTGGTGG; In the above sequence, G is guanosine or a guanosine derivative, T is thymine or a thymine derivative, and N is 1-β-D-arabinofuranosylcytosine as a modified nucleic acid.

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