Modified oligonucleotide-drug conjugates and their uses

Abstract

The present invention relates to a modified oligonucleotide-drug conjugate, comprising a modified oligonucleotide comprising at least one modified nucleic acid and a drug conjugated to the modified oligonucleotide. The modified oligonucleotide-drug conjugate of the present invention exhibits high stability in the body and can exhibit excellent anticancer effects.

Description

[Technical Field]

[0001] The present invention relates to modified oligonucleotide-drug conjugates and uses thereof. [Background technology]

[0002] Currently, many therapeutic agents, including anticancer drugs, have been developed and clinically tested. However, continued research is needed to more selectively and effectively deliver therapeutic substances to the desired site of onset, such as targeted anticancer drugs. Specifically, anticancer drugs have drawbacks, such as a low therapeutic index and therapeutic window due to non-tumor-specific systemic toxicity and cytotoxicity, and the possibility of developing resistance to anticancer drugs over long-term treatment. Therefore, there is a pressing need for new and improved therapies that precisely deliver drugs to cancer cells and kill them.

[0003] On the other hand, guanosine-rich oligonucleotides can have special structures due to intramolecular or intermolecular bonds in addition to triple hydrogen bonds between guanine and cytosine. Instead of forming a double helix structure through the typical hydrogen bonds between adenine and thymine or guanine and cytosine, four guanines are positioned in a single plane and form Hoogsteen hydrogen bonds, forming a square planar structure called a guanine tetrad (G-tetrad). Two or more guanine tetrads (G-tetrads) stack together to form a quadruple helix structure called a G-quadruplex. These G-quadruplex oligonucleotides are known to have excellent cell permeability due to their structural stability.

[0004] However, oligonucleotides that form G-quadruplexes primarily induce apoptosis through cell growth inhibition, but because the apoptosis rate is relatively low, they must be administered continuously via infusion for a certain period of time, typically 4 to 7 days. This poses the problem of administering more drugs than necessary for a long period of time. Therefore, extensive research is being conducted to develop new modified nucleic acid-containing substances that exhibit stable, high cell permeability in the body and excellent efficacy. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide modified oligonucleotide-drug conjugates.

[0006] An object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer, which comprises a modified oligonucleotide-drug conjugate. [Means for solving the problem]

[0007] 1. A modified oligonucleotide-drug conjugate comprising a modified oligonucleotide represented by the following Chemical Formula 1 and a drug conjugated to deoxyuridine (dU) contained in the modified oligonucleotide: [ka] (In the above Chemical Formula 1, X1 to X3 and X7 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: Y1 is deoxyguanosine (dG) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: X4 to X6 each independently represent thymidine (T), deoxyuridine (dU), or a modified nucleic acid represented by the following chemical formula 3 or 4, and at least one of X4 to X6 is deoxyuridine (dU), M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, provided that when all of X1 to X9 are thymidine (T) and Y1 is deoxyguanosine (dG), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0008] 2. A modified oligonucleotide-drug conjugate in the above item 1, wherein the modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4 is selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodocytidine, cytosine arabinoside, 2',2'-difluoro-2'-deoxycytidine, and bromovinyldeoxyuridine.

[0009] 3. In the above item 1, the drug is paclitaxel, monomethyl auristatin E (MMAE), monomethyl auristatin F, monomethyl auristatin D, cytarabine, gemcitabine, maytansine, DM1 (mertansine), DM4, calicheamicin and its derivatives, doxorubicin, duocarmycin and its derivatives, pyrrolobenzodiazepine (PBD), SN-38, α-amanitin, tubulysin analogues analog, cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine ), camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel, docetaxel, ixabepilone, vinblastine, vincristine, vindesine, vinorelbine, estramustine, maytansine, auristatin E, auristatin F, and nemorubicin.

[0010] 4. In the above item 1, the drug and deoxyuridine (dU) contained in the modified oligonucleotide are conjugated via a linker (L) connecting L1 and L2; L1 is selected from the group consisting of 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serinol, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2 dT, amino-modifier C6 dT, S-Bz-thiol-modifier C6-dT, phosphodiester bond, and nucleotide, and is selected from acrylamide-C2-NH2, C12-NH2, C3-NH2, acrylamide-C6-propanamide-SH, acrylamide-C6-NH2, C6-NH2, C6-SH obtained by a deprotection process; L2 is a modified oligonucleotide-drug conjugate selected from the group consisting of maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinic acid, hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproyl, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), a phosphodiester bond, and a nucleotide.

[0011] 5. A modified oligonucleotide-drug conjugate according to item 1, wherein the modified oligonucleotide consists of a sequence selected from SEQ ID NO: 4 to SEQ ID NO: 10.

[0012] 6. A pharmaceutical composition for preventing or treating cancer, comprising the modified oligonucleotide-drug conjugate according to any one of items 1 to 5 above.

[0013] 7. A pharmaceutical composition for preventing or treating cancer according to item 6, wherein the cancer is selected from the group consisting of leukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, cervical carcinoma, glioma cancer, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer.

[0014] 8. A modified oligonucleotide-drug conjugate comprising a modified oligonucleotide represented by the following Chemical Formula 2 and a drug conjugated to at least one of the 5' end or the 3' end of the modified oligonucleotide: [ka] (In the above Chemical Formula 2, X1 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, and when X1 to X9 are all thymidine (T), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0015] 9. A modified oligonucleotide-drug conjugate according to item 8, wherein the modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4 is selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodocytidine, cytosine arabinoside, 2',2'-difluoro-2'-deoxycytidine, and bromovinyldeoxyuridine.

[0016] 10. In the above item 8, the drug is paclitaxel, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D (MMAD), cytarabine, gemcitabine, maytansine, DM1 (mertansine), DM4, calicheamicin and its derivatives, doxorubicin, duocarmycin and its derivatives, pyrrolobenzodiazepine (PBD), SN-38, α-amanitin, tubulysin analogues analog, cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine ), camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel, docetaxel, ixabepilone, vinblastine, vincristine, vindesine, vinorelbine, estramustine, maytansine, auristatin E, auristatin F, and nemorubicin.

[0017] 11. In the above item 8, the drug and at least one of the 5' end and the 3' end of the modified oligonucleotide represented by Chemical Formula 2 are conjugated with a linker (L) connecting L1 and L2; L1 is selected from the group consisting of 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serinol, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2 dT, amino-modifier C6 dT, S-Bz-thiol-modifier C6-dT, phosphodiester bond, and nucleotide, and is selected from acrylamide-C2-NH2, C12-NH2, C3-NH2, acrylamide-C6-propanamide-SH, acrylamide-C6-NH2, C6-NH2, C6-SH obtained by a deprotection process; L2 is a modified oligonucleotide-drug conjugate selected from the group consisting of maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinic acid, hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproyl, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), a phosphodiester bond, and a nucleotide.

[0018] 12. The modified oligonucleotide-drug conjugate according to item 8, wherein the modified oligonucleotide consists of a sequence selected from SEQ ID NO: 11 and SEQ ID NO: 12.

[0019] 13. A pharmaceutical composition for preventing or treating cancer, comprising the modified oligonucleotide-drug conjugate according to any one of items 8 to 12. [Effects of the Invention]

[0020] The "modified oligonucleotide-drug conjugate" of the present invention comprises an oligonucleotide containing a modified nucleic acid (hereinafter referred to as "modified oligonucleotide") and a drug conjugated thereto, and is therefore highly stable in the body and can exhibit excellent anticancer effects.

[0021] The "modified oligonucleotide-drug conjugate" of the present invention can exhibit a superior anti-cancer effect to when the "modified oligonucleotide" is used alone or when the "drug" is administered alone.

[0022] The "modified oligonucleotide-drug conjugate" of the present invention has a longer half-life in the body than the "modified oligonucleotide" in the same amount as the modified oligonucleotide contained in the conjugate. The "modified oligonucleotide-drug conjugate" of the present invention has a lower toxicity than the "drug" in the same amount as the drug contained in the conjugate.

[0023] The "modified oligonucleotide-drug conjugate" of the present invention can exhibit superior anticancer effects compared to "oligonucleotide-drug conjugates" in which only a drug is conjugated to an oligonucleotide that does not contain a modified nucleic acid. [Brief explanation of the drawings]

[0024] [Figure 1] Figures 1 to 5 show the results of IVIS imaging to evaluate the efficacy of modified oligonucleotide-drug conjugates in an orthotopic pancreatic cancer transplant animal model (Figure 1: Untreated, Figure 2: Gemcitabine-loaded collagen patch-treated group, Figure 3: IO101L-loaded collagen patch-treated group, Figure 4: IO176-loaded collagen patch-treated group, Figure 5: IO142-loaded collagen patch-treated group). [Figure 2]Figures 1 to 5 show the results of IVIS imaging to evaluate the efficacy of modified oligonucleotide-drug conjugates in an orthotopic pancreatic cancer transplant animal model (Figure 1: Untreated, Figure 2: Gemcitabine-loaded collagen patch-treated group, Figure 3: IO101L-loaded collagen patch-treated group, Figure 4: IO176-loaded collagen patch-treated group, Figure 5: IO142-loaded collagen patch-treated group). [Figure 3] Figures 1 to 5 show the results of IVIS imaging to evaluate the efficacy of modified oligonucleotide-drug conjugates in an orthotopic pancreatic cancer transplant animal model (Figure 1: Untreated, Figure 2: Gemcitabine-loaded collagen patch-treated group, Figure 3: IO101L-loaded collagen patch-treated group, Figure 4: IO176-loaded collagen patch-treated group, Figure 5: IO142-loaded collagen patch-treated group). [Figure 4] Figures 1 to 5 show the results of IVIS imaging to evaluate the efficacy of modified oligonucleotide-drug conjugates in an orthotopic pancreatic cancer transplant animal model (Figure 1: Untreated, Figure 2: Gemcitabine-loaded collagen patch-treated group, Figure 3: IO101L-loaded collagen patch-treated group, Figure 4: IO176-loaded collagen patch-treated group, Figure 5: IO142-loaded collagen patch-treated group). [Figure 5] Figures 1 to 5 show the results of IVIS imaging to evaluate the efficacy of modified oligonucleotide-drug conjugates in an orthotopic pancreatic cancer transplant animal model (Figure 1: Untreated, Figure 2: Gemcitabine-loaded collagen patch-treated group, Figure 3: IO101L-loaded collagen patch-treated group, Figure 4: IO176-loaded collagen patch-treated group, Figure 5: IO142-loaded collagen patch-treated group). [Figure 6] FIG. 6 shows the results of examining changes in body weight in an animal model of orthotopic pancreatic cancer transplantation treated with a collagen patch carrying a modified oligonucleotide-drug conjugate. [Figure 7a]Figures 7a to 7e show the results of examining organ and tumor size in an orthotopic pancreatic cancer animal model treated with collagen patches carrying modified oligonucleotide-drug conjugates (Figure 7a: untreated; Figure 7b: gemcitabine-loaded collagen patch-treated group; Figure 7c: IO101L-loaded collagen patch-treated group; Figure 7d: IO176-loaded collagen patch-treated group; Figure 7e: IO142-loaded collagen patch-treated group). [Figure 7b] Figures 7a to 7e show the results of examining organ and tumor size in an orthotopic pancreatic cancer animal model treated with collagen patches carrying modified oligonucleotide-drug conjugates (Figure 7a: untreated; Figure 7b: gemcitabine-loaded collagen patch-treated group; Figure 7c: IO101L-loaded collagen patch-treated group; Figure 7d: IO176-loaded collagen patch-treated group; Figure 7e: IO142-loaded collagen patch-treated group). [Figure 7c] Figures 7a to 7e show the results of examining organ and tumor size in an orthotopic pancreatic cancer animal model treated with collagen patches carrying modified oligonucleotide-drug conjugates (Figure 7a: untreated; Figure 7b: gemcitabine-loaded collagen patch-treated group; Figure 7c: IO101L-loaded collagen patch-treated group; Figure 7d: IO176-loaded collagen patch-treated group; Figure 7e: IO142-loaded collagen patch-treated group). [Figure 7d] Figures 7a to 7e show the results of examining organ and tumor size in an orthotopic pancreatic cancer animal model treated with collagen patches carrying modified oligonucleotide-drug conjugates (Figure 7a: untreated; Figure 7b: gemcitabine-loaded collagen patch-treated group; Figure 7c: IO101L-loaded collagen patch-treated group; Figure 7d: IO176-loaded collagen patch-treated group; Figure 7e: IO142-loaded collagen patch-treated group). [Figure 7e]Figures 7a to 7e show the results of examining organ and tumor size in an orthotopic pancreatic cancer animal model treated with collagen patches carrying modified oligonucleotide-drug conjugates (Figure 7a: untreated; Figure 7b: gemcitabine-loaded collagen patch-treated group; Figure 7c: IO101L-loaded collagen patch-treated group; Figure 7d: IO176-loaded collagen patch-treated group; Figure 7e: IO142-loaded collagen patch-treated group). [Figure 8a] 8a and 8b show the results of examining organ and tumor weights in an orthotopic pancreatic cancer animal model treated with collagen patches loaded with modified oligonucleotide-drug conjugates. [Figure 8b] 8a and 8b show the results of examining organ and tumor weights in an orthotopic pancreatic cancer animal model treated with collagen patches loaded with modified oligonucleotide-drug conjugates. [Figure 9] 9 is a graph showing the plasma MMAE concentration after administration of a modified oligonucleotide-drug conjugate (IO176) according to one embodiment. The small graph within the graph in FIG. 9 shows the plasma MMAE concentration from immediately before drug administration (0 minutes) to 120 minutes after drug administration. [Figure 10] FIG. 10 is a graph showing plasma concentrations of a modified oligonucleotide-drug conjugate (IO176) according to one embodiment, and gemcitabine and its metabolite (dFdU). [Figure 11] FIG. 11 is a reaction scheme showing the conjugation of MMAE to a modified oligonucleotide in which an internal, but not terminal, nucleotide is substituted with a modified nucleic acid. [Figure 12] FIG. 12 is a reaction scheme showing the conjugation of paclitaxel to a modified oligonucleotide in which an internal, but not terminal, nucleotide is substituted with a modified nucleic acid. [Figure 13] FIG. 13 is a reaction scheme showing the conjugation of DM1 with a modified oligonucleotide in which an internal, but not terminal, nucleotide is substituted with a modified nucleic acid. [Figure 14]FIG. 14 is a reaction scheme showing the process of conjugating a modified oligonucleotide having a modified nucleic acid attached to either the 5′ or 3′ end with MMAE. [Figure 15] FIG. 15 is a reaction scheme showing the process of conjugating paclitaxel with a modified oligonucleotide having a modified nucleic acid attached to either the 5′ or 3′ end. [Figure 16] FIG. 16 is a reaction scheme showing the process of conjugating a modified oligonucleotide having a modified nucleic acid attached to either the 5′ or 3′ end with DM1. [Figure 17] FIG. 17 shows the reaction scheme for producing Comparative Example 2. [Figure 18] FIG. 18 shows the reaction scheme for preparing Example 8. [Figure 19] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. [Figure 20] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. [Figure 21] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. [Figure 22] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. [Figure 23] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. [Figure 24] 19 to 27 show the evaluation results and graphs of IC50 for several examples and comparative examples. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be described in further detail.

[0026] The present invention provides a modified oligonucleotide-drug conjugate comprising a modified oligonucleotide comprising at least one modified nucleic acid, and a drug conjugated to the modified oligonucleotide.

[0027] Modified Oligonucleotides The modified oligonucleotide can comprise at least one modified nucleic acid.

[0028] The modified oligonucleotide may contain a modified nucleic acid and form a G-quadruplex structure.

[0029] The guanosine (G) that contributes to the G-quadruplex structure of the oligonucleotide may be one or more selected from 2'-deoxyguanosine, guanosine, 2'-O-methyl-guanosine, 2'-fluoro-guanosine (2'-F-guanosine), LNA (Locked Nucleic Acid)-guanosine, D-deoxyguanosine, and D-guanosine, and can be synthesized to include modified nucleic acids with therapeutic effects.

[0030] The modified oligonucleotide can be produced by solid-phase synthesis using a DNA synthesizer so that the above-mentioned modified nucleic acid is contained in a guanosine-rich oligonucleotide.

[0031] Modified oligonucleotides can be produced by substituting a modified nucleic acid for a predetermined nucleotide in a known oligonucleotide sequence, or by introducing a modified nucleic acid at least at one of the 3' or 5' ends of a known oligonucleotide sequence.

[0032] The modified oligonucleotides may be aptamers that have binding strength or specificity for a target protein.

[0033] The sequence of the modified oligonucleotide and the position of the modified nucleic acid may affect the binding strength or specificity to the target protein, etc. Therefore, the sequence and the position of the modified nucleic acid must be appropriately selected taking into consideration the binding strength or specificity to the target protein, etc.

[0034] The usefulness of the modified oligonucleotide-drug conjugates of the present invention can be determined by the physiological effects they exhibit, which depend on factors such as binding strength or specificity to the target protein.

[0035] The modified nucleic acid may have a therapeutic effect, for example, but not limited to, a sugar- or base-modified guanosine, thymidine, cytidine, or uridine. The modified nucleic acid may be pyrimidine-based. Specifically, the modified nucleic acid may be derived from uridine or cytidine.

[0036] The modified nucleic acid can be selected from compounds represented by the following formula 3 or 4:

[0037] [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0038] R in Chemical Formula 3 1 may be hydrogen, halogen or a hydroxy group, and specifically may be hydrogen.

[0039] R in Chemical Formula 3 2 may be hydrogen, halogen or a hydroxy group, and specifically may be hydrogen.

[0040] R in Chemical Formula 3 3 may be hydrogen, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, or C2-C6 haloalkenyl, specifically halogen, C1-C6 haloalkyl, or C2-C6 haloalkenyl. According to one embodiment, Chemical Formula 3 may be 5-fluorodeoxyuridine.

[0041] R in Chemical Formula 4 1 may be hydrogen, halogen or a hydroxy group, specifically hydrogen or halogen (eg, fluoro, chloro, bromo, or iodo).

[0042] R in Chemical Formula 4 2 may be hydrogen, halogen or a hydroxy group, specifically hydrogen or halogen (eg, fluoro, chloro, bromo, or iodo).

[0043] R in Chemical Formula 4 3 may be hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group, and specifically may be hydrogen or a C1-C6 alkyl group.

[0044] R in Chemical Formula 4 1 and R 2 is a halogen and R 3 may be hydrogen or a C1-C3 alkyl group. According to one embodiment, Chemical Formula 4 may be gemcitabine.

[0045] The modified nucleic acid may be contained in the modified oligonucleotide in the form of a compound represented by Chemical Formula 3 or Chemical Formula 4, to which a phosphate group is bound. Specifically, the modified oligonucleotide may be contained in the modified oligonucleotide in the form of a phosphoramidite. Nucleotide phosphoramidites can be purchased from Glen Research, Berry and Associates, Okeanos Tech, Chemgene, Proligo, etc., or can be prepared by known methods (Oligonucleotides and Analogues: A Practical Approach, Fritz Eckstein et al. 1991, IRL Press: Oxford).

[0046] The modified nucleic acid can be selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodocytidine, cytosine arabinoside, 2',2'-difluoro-2'-deoxycytidine, and bromovinyldeoxyuridine. The modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4 can be selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodocytidine, cytosine arabinoside, 2',2'-difluoro-2'-deoxycytidine, and bromovinyldeoxyuridine. The 2',2'-difluoro-2'-deoxycytidine may be gemcitabine.

[0047] For example, the modified oligonucleotide may have a sequence represented by Chemical Formula 1 below.

[0048] [ka]

[0049] Formula 1 is shown from the left in the 5' to 3' direction.

[0050] As used herein, G shown in Chemical Formula 1 may be deoxyguanosine (dG).

[0051] X1 to X3 and X7 to X9 in Chemical Formula 1 may each independently represent thymidine (T) or a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0052] Y1 in Chemical Formula 1 may be deoxyguanosine (dG) or a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0053] X4 to X6 in Chemical Formula 1 may each independently be thymidine (T), deoxyuridine (dU), or a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0054] At least one of X4 to X6 in Chemical Formula 1 may be deoxyuridine (dU).

[0055] M1 and M2 in Chemical Formula 1 may each independently be a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0056] N1 and N2 in Chemical Formula 1 may each independently be thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG).

[0057] In the above chemical formula 1, a and d may each independently be an integer of 0 to 10.

[0058] In Chemical Formula 1, when X1 to X9 are all thymidine (T) and Y1 is deoxyguanosine (dG), a and d do not have to be simultaneously 0. That is, the modified oligonucleotide represented by Chemical Formula 1 contains at least one modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0059] In the above chemical formula 1, b and c may each independently be an integer of 0 to 10.

[0060] A drug can be conjugated to the deoxyuridine (dU) of the modified oligonucleotide of Chemical Formula 1.

[0061] Specifically, the modified oligonucleotide of the present invention can be represented by the following chemical formula 1.

[0062] [ka] (In the above Chemical Formula 1, X1 to X3 and X7 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: Y1 is deoxyguanosine (dG) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: X4 to X6 each independently represent thymidine (T), deoxyuridine (dU), or a modified nucleic acid represented by the following chemical formula 3 or 4, and at least one of X4 to X6 is deoxyuridine (dU), M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, provided that when all of X1 to X9 are thymidine (T) and Y1 is deoxyguanosine (dG), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0063] According to some embodiments, the modified oligonucleotide may consist of a sequence selected from SEQ ID NO:4 to SEQ ID NO:10.

[0064] In another example, the modified oligonucleotide may have a sequence represented by Chemical Formula 2 below.

[0065] [ka]

[0066] The formula 2 is shown from the left in the 5' to 3' direction.

[0067] As used herein, G shown in Chemical Formula 2 may be deoxyguanosine (dG).

[0068] X1 to X9 in Chemical Formula 2 may each independently be thymidine (T), or a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4.

[0069] M1 and M2 in Chemical Formula 2 may each independently be a modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4 below.

[0070] N1 and N2 in Chemical Formula 2 may each independently be thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG).

[0071] In the above chemical formula 2, a and d may each independently be an integer of 0 to 10.

[0072] When X1 to X9 in Chemical Formula 2 are all thymidine (T), a and d do not have to be 0 at the same time.

[0073] In the above chemical formula 2, b and c may each independently be an integer of 0 to 10.

[0074] A drug can be conjugated to at least one of the 5' end and the 3' end of the modified oligonucleotide represented by Chemical Formula 1.

[0075] Specifically, the modified oligonucleotide of the present invention can be represented by the following chemical formula 2.

[0076] [ka] (In the above Chemical Formula 2, X1 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, and when X1 to X9 are all thymidine (T), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0077] According to some embodiments, the modified oligonucleotide may consist of a sequence selected from SEQ ID NO:11 or SEQ ID NO:12.

[0078] drugs The drug is not limited to a specific type as long as it has a structure that allows it to be conjugated to the modified oligonucleotide.

[0079] The drug may have, but is not limited to, a functional group (e.g., a hydroxy group, a carboxy group, or an amino group) that can be directly conjugated to the modified oligonucleotide or that can be conjugated to a linker.

[0080] For example, drugs include paclitaxel, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D (MMAD), cytarabine, gemcitabine, maytansine, DM1 (mertansine), DM4, calicheamicin and its derivatives, doxorubicin, duocarmycin and its derivatives, pyrrolobenzodiazepines (PBD), SN-38, α-amanitin, tubulysin analogs, analog, cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine The anti-cancer agent may be selected from the group consisting of fluticasone, fluoxetine, fluoxetine-3, fluoxetine-4, fluoxetine-5, fluoxetine-6, fluoxetine-7, fluoxetine-8, fluoxetine-9, fluoxetine-10, fluoxetine-11, fluoxetine-12, fluoxetine-13, fluoxetine-14, fluoxetine-15, fluoxetine-16, fluoxetine-17, fluoxetine-18, fluoxetine-19, fluoxetine-20, fluoxetine-21, fluoxetine-22, fluoxetine-23, fluoxetine-24, fluoxetine-25, fluoxetine-26, fluoxetine-27, fluoxetine-28, fluoxetine-29, fluoxetine-30, fluoxetine-31, fluoxetine-32, fluoxetine-33, fluoxetine-34, fluoxetine-35, fluoxetine-36, fluoxetine-37, fluoxetine-38, fluoxetine-39, fluoxetine-40, fluoxetine-41, fluoxetine-42, fluoxetine-43, fluoxetine-44, fluoxetine-45, fluoxetine-46, fluoxetine-47, fluoxetine-48, fluoxetine-49, fluoxetine-50, fluoxetine-51, fluoxetine-52, fluoxetine-53, fluoxetine-54, fluoxetine-55, fluoxetine-55, fluoxetine-

[0081] The drug can be conjugated to the modified oligonucleotide directly or indirectly, for example, the drug can be conjugated to the modified oligonucleotide directly or via a linker.

[0082] The drug can be conjugated to at least one base contained within the modified oligonucleotide. Specifically, the drug can be conjugated to a uridine or deoxyuridine (dU) contained within the modified oligonucleotide, but is not limited thereto.

[0083] The drug can be conjugated to a base located at the end of the modified oligonucleotide. Specifically, the drug can be conjugated to at least one of the 5' or 3' end of the modified oligonucleotide.

[0084] The modified oligonucleotides are as described above, and a detailed description thereof will be omitted.

[0085] Linker The modified oligonucleotide and the drug can be linked via a linker (L). The type of linker (L) is not limited as long as it can link the modified oligonucleotide and the drug.

[0086] The linker (L) may be a combination of L1 and L2.

[0087] L1 and L2 can be independently conjugated to a modified oligonucleotide and a drug. L1 and L2 can be independently conjugated to a modified oligonucleotide and a drug, and the modified oligonucleotide and the drug can be conjugated by the reaction of L1 and L2.

[0088] According to one embodiment, L1 can be linked to the modified oligonucleotide, and L2 can be linked to the drug. For example, L1 can be linked to a nucleotide (e.g., dU) contained within the modified oligonucleotide, and L2 can be linked to the drug. In another example, L1 can be linked to at least one of the 5' or 3' end of the modified oligonucleotide, and L2 can be linked to the drug.

[0089] It is sufficient for L1 to contain a functional group that can bond to L2 through a chemical reaction, and there are no limitations on the type of L2. It is sufficient for L2 to contain a functional group that can bond to L1 through a chemical reaction, and there are no limitations on the type of L2.

[0090] For example, L1 may include an -SH functionality and L2 may include a maleimide functionality. In another example, L1 may include an -NH2 functionality and L2 may include a -COOH functionality.

[0091] L1 can be selected from the group consisting of, but not limited to, 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serinol, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2 dT, amino-modifier C6 dT, S-Bz-thiol-modifier C6-dT, a phosphodiester bond, and a nucleotide. L1 may be a deprotected form of the foregoing examples, such as, but not limited to, acrylamide-C2-NH2, C12-NH2, C3-NH2, acrylamide-C6-propanamide-SH, acrylamide-C6-NH2, C6-NH2, or C6-SH.

[0092] L2 can be selected from the group consisting of, but not limited to, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinic acid, hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproyl, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), a phosphodiester bond, and a nucleotide.

[0093] Some examples of methods for conjugating modified oligonucleotides and drugs using linkers (L) are as follows:

[0094] For example, the method includes the steps of: preparing a modified oligonucleotide in which a predetermined nucleotide is substituted with a modified nucleic acid based on a known oligonucleotide (e.g., an aptamer having the sequence of Comparative Example 1); and binding L1 to one end of the modified oligonucleotide. conjugating L2 to a drug; The modified oligonucleotide-drug conjugate can be prepared by a method comprising the steps of reacting a modified oligonucleotide having L1 attached thereto with a drug having L2 attached thereto (see Figures 11 to 13).

[0095] In another example, a modified oligonucleotide is prepared by adding at least one modified nucleic acid to at least one of both ends (5' end or 3' end) of a known oligonucleotide (e.g., an aptamer having the sequence of Comparative Example 1), and binding L1 to one end of the modified oligonucleotide. conjugating L2 to a drug; The modified oligonucleotide-drug conjugate can be produced by a method comprising the steps of reacting a modified oligonucleotide having L1 attached thereto with a drug having L2 attached thereto (see Figures 14 to 16).

[0096] According to some embodiments, the linker (L) is -[acrylamide-C6-NH-(CO)-(CH2)2-(CO)O]-, -[C6-NH-(CO)-(CH2)2-(CO)O]-, -[C6-S-MC-Val-Cit-PAB]-, -[C6-NH-CO-A]-, -[acrylamide-C6-NH-CO-A]-, or (wherein A is [ka] ) may be used, but is not limited to these.

[0097] Modified Oligonucleotide-Drug Conjugates According to some embodiments, the modified oligonucleotide-drug conjugate of the present invention may comprise a modified oligonucleotide represented by Chemical Formula 1 below and a drug conjugated to deoxyuridine (dU) contained in the modified oligonucleotide.

[0098] [ka] (In the above Chemical Formula 1, X1 to X3 and X7 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: Y1 is deoxyguanosine (dG) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: X4 to X6 each independently represent thymidine (T), deoxyuridine (dU), or a modified nucleic acid represented by the following chemical formula 3 or 4, and at least one of X4 to X6 is deoxyuridine (dU), M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, provided that when all of X1 to X9 are thymidine (T) and Y1 is deoxyguanosine (dG), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0099] The drug and deoxyuridine (dU) can be linked via a linker (L). The linker may be a combination of the above-mentioned L1 and L2.

[0100] L1 and L2 can each independently bind to deoxyuridine (dU) in the modified oligonucleotide and the drug.

[0101] Specifically, the modified oligonucleotide may consist of a sequence selected from SEQ ID NO:4 to SEQ ID NO:10.

[0102] The modified nucleic acid, the drug, L1 and L2, etc. are as described above, and therefore a detailed explanation will be omitted.

[0103] According to some embodiments, the modified oligonucleotide-drug conjugate of the present invention can include a modified oligonucleotide represented by Chemical Formula 2 and a drug conjugated to at least one of the 5' end or the 3' end of the modified oligonucleotide.

[0104] [ka] (In the above Chemical Formula 2, X1 to X9 each independently represent thymidine (T) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: M1 and M2 are each independently a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4: N1 and N2 are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d each independently represent an integer of 0 to 10, and when X1 to X9 are all thymidine (T), a and d are not simultaneously 0, b and c are each independently an integer of 0 to 10. [ka] [ka] (In the above Chemical Formula 3 or Chemical Formula 4, R 1 is hydrogen, halogen or a hydroxy group, R 2 is hydrogen, halogen or a hydroxy group, R 3 is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein the R 1 is hydrogen or a hydroxy group, and the R 2 is hydrogen, and the R 3 Except when is hydrogen or methyl.

[0105] The drug and at least one of the 5' end and the 3' end of the modified oligonucleotide can be linked by a linker (L). The linker may be a combination of the above-mentioned L1 and L2.

[0106] L1 and L2 can each independently bind to at least one of the 5' end or the 3' end of the modified oligonucleotide and the drug.

[0107] In particular, the modified oligonucleotide may consist of a sequence selected from SEQ ID NO:11 or SEQ ID NO:12.

[0108] The modified nucleic acid, the drug, L1 and L2, etc. are as described above, and therefore a detailed explanation will be omitted.

[0109] Uses of Modified Oligonucleotide-Drug Conjugates The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned modified oligonucleotide-drug conjugate.

[0110] The cancer can be selected from the group consisting of, but not limited to, leukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, cervical carcinoma, glioma cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer.

[0111] The present invention can also provide a method for preventing or treating cancer, which comprises the step of administering the above-described modified oligonucleotide-drug conjugate to an individual.

[0112] The individual may be a mammal, including but not limited to a human, cow, horse, dog, rabbit, cat, goat, and rat.

[0113] The modified oligonucleotide-drug conjugate and cancer have been described above, and therefore a detailed explanation will be omitted.

[0114] The present invention will be described in more detail below with reference to examples. However, the following examples are provided for illustrative purposes to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0115] The inventors of the present invention have successfully conjugated drugs to guanosine-rich modified oligonucleotides containing modified nucleic acids to synthesize modified oligonucleotide-drug conjugates, and have confirmed that the synthesized modified oligonucleotide-drug conjugates are non-toxic, stable, and exhibit excellent cancer therapeutic effects.

[0116] 1. Preparation of Modified Oligonucleotide-Drug Conjugates (ApDDC; Aptamer-Double Drug-Conjugate) The examples (modified oligonucleotide-drug conjugates) and comparative examples in Table 1 were designed and produced by the following method. In Table 1 below, M is gemcitabine, N is 5-FdU (5-fluorodeoxyuridine), G is deoxyguanosine (dG), T is thymidine (T), and U is deoxyuridine (dU). That is, in the description of the present invention, G is deoxyguanosine (dG), T is thymidine (T), and U is deoxyuridine (dU) in SEQ ID NOS: 1 to 12 and conjugates produced therefrom, SEQ ID NOS: 13 to 33.

[0117] [Table 1-1] [Table 1-2]

[0118] (1) Preparation of Comparative Example 1 Comparative Example 1 [:GGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 1)] was produced by a general DNA synthesis method.

[0119] Specifically, oligonucleotides were synthesized using Mermade 12 or Mermade 48 (BioAutomation, USA). Each cycle consisted of four steps: deblocking, coupling, capping, and oxidation. The oligonucleotides were synthesized one nucleotide at a time, sequentially from the 3' end to the 5' end. Specifically, the four steps of deblocking, coupling, capping, and oxidation were as follows:

[0120] A. Deprotection: TCA Deblock was added to remove the 4,4-dimethoxytriyl group (DMT) of the nucleotide supported on CPG (Controlled Pore Glass) to generate a 5'-hydroxyl group so that the next nucleotide could be bound.

[0121] B. Coupling: Nucleotide phosphoramidite and ETT activator were added to activate the nucleotide, which was then coupled to the 5'-hydroxyl group of the nucleotide supported by the CPG.

[0122] C. Inactivation (Capping): Cap A and Cap B were added to inactivate the 5'-hydroxyl group of the (n-1)th nucleotide that did not participate in the reaction.

[0123] D. Oxidation: An oxidizer was added to oxidize the phosphate bonds between nucleotides.

[0124] The synthesized oligonucleotide was transferred to a solution (aqueous ammonia, 55°C) to separate the CPG support and the protecting groups were removed.

[0125] Purification and analysis of the deprotected oligonucleotides were performed using a Waters Prep150 (Waters, USA) and a Waters ACQUITY UPLC H-Class PLUS Bio System (Waters, USA) with a reversed-phase C18 column, and mass spectrometry of the purified nucleic acid ligands was performed using a Waters Xevo G2-XS Q-TOF System (Waters, USA).

[0126] (2) Preparation of Comparative Example 2 Comparative Example 2 [an oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in GGTGGTGGTGGTTGUGGTGGTGGTGG (SEQ ID NO: 2)] was prepared in two steps: a conventional DNA synthesis method and drug conjugation (see Figure 17).

[0127] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0128] 2) Drug conjugation [ka] and paclitaxel-succinic acid (PTX-SA 98%, MedKoo Biosciences, Cat#620101) to synthesize the oligonucleotide-drug conjugate of Comparative Example 2. The linker (L) linking U in the sequence of SEQ ID NO: 2 to paclitaxel in the oligonucleotide-drug conjugate of Comparative Example 2 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety linked to U in SEQ ID NO: 2, and succinic acid is the moiety linked to paclitaxel).

[0129] Specifically, paclitaxel-succinic acid was dissolved in DMSO, and EDC (-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide) dissolved in ultrapure water were added and reacted at room temperature (RT) for 1 hour. The sequence of SEQ ID NO: 13 was dissolved in a pH 8.4 buffer, and the activated paclitaxel mixture was added. The reaction was allowed to proceed at RT for 1 hour, and the progress of the reaction was monitored using a Waters ACQUITY UPLC H-Class PLUS Bio System (Waters, USA).

[0130] Purification and analysis of the sequence of Comparative Example 2 (oligonucleotide-drug conjugate) were performed using a Waters Prep150 (Waters, USA) and a Waters ACQUITY UPLC H-Class PLUS Bio System (Waters, USA) with a reversed-phase C18 column. Mass spectrometry of the purified nucleic acid ligand was also performed using a Waters Xevo G2-XS Q-TOF System (Waters, USA).

[0131] (3) Preparation of Comparative Example 3 Comparative Example 3 [an oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in GGTGGTGGTGGTUGTGGTGGTGGTGG (SEQ ID NO: 3)] was prepared.

[0132] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0133] 2) Drug conjugation The oligonucleotide-drug conjugate of Comparative Example 3 was synthesized in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO: 14 was used instead of the sequence of SEQ ID NO: 13. The linker (L) connecting U in the sequence of SEQ ID NO: 3 to paclitaxel in the oligonucleotide-drug conjugate of Comparative Example 3 was "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety linked to U in SEQ ID NO: 3, and succinic acid is the moiety linked to paclitaxel).

[0134] (4) Preparation of Example 1 Example 1 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGUMGTGGTGGTGGTGG (SEQ ID NO: 4), where M=gemcitabine] was prepared.

[0135] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0136] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 1 was produced in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO: 15 was used instead of the sequence of SEQ ID NO: 13. The linker (L) linking U in the sequence of SEQ ID NO: 4 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 1 was "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety linked to U in SEQ ID NO: 4, and succinic acid is the moiety linked to paclitaxel).

[0137] (5) Preparation of Example 2 Example 2 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGUTMTGGTGGTGGTGG (SEQ ID NO: 5), where M=gemcitabine] was prepared.

[0138] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0139] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 2 was produced in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO: 17 was used instead of the sequence of SEQ ID NO: 13.

[0140] The linker (L) linking U in sequence number 5 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 2 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety linked to U in sequence number 5, and succinic acid is the moiety linked to paclitaxel).

[0141] (6) Preparation of Example 3 Example 3 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGTUMTGGTGGTGGTGG (SEQ ID NO: 6), where M=gemcitabine] was prepared.

[0142] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0143] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 3 was prepared in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO: 18 was used instead of the sequence of SEQ ID NO: 13.

[0144] The linker (L) connecting U in the sequence of SEQ ID NO: 6 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 3 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety connected to U in SEQ ID NO: 6, and succinic acid is the moiety connected to paclitaxel).

[0145] (7) Preparation of Example 4 Example 4 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGTUGMGGTGGTGGTGG (SEQ ID NO: 7), where M=gemcitabine] was prepared.

[0146] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0147] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 4 was prepared in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO: 19 was used instead of the sequence of SEQ ID NO: 13.

[0148] The linker (L) connecting U in the sequence of SEQ ID NO: 7 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 4 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety connected to U in SEQ ID NO: 7, and succinic acid is the moiety connected to paclitaxel).

[0149] (8) Preparation of Example 5 Example 5 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGMUGMGGTGGTGGTGG (SEQ ID NO: 8), where M=gemcitabine] was prepared.

[0150] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0151] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 5 was prepared in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO:20 was used instead of the sequence of SEQ ID NO:13.

[0152] The linker (L) connecting U in the sequence of SEQ ID NO: 8 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 5 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety connected to U in SEQ ID NO: 8, and succinic acid is the moiety connected to paclitaxel).

[0153] (9) Preparation of Example 6 Example 6 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGTUGTGGTGGTGGMGG (SEQ ID NO: 9), where M=gemcitabine] was prepared.

[0154] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0155] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 6 was produced in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO:21 was used instead of the sequence of SEQ ID NO:13.

[0156] The linker (L) connecting U in the sequence of SEQ ID NO: 9 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 6 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety connected to U in SEQ ID NO: 9, and succinic acid is the moiety connected to paclitaxel).

[0157] (10) Preparation of Example 7 Example 7 [a modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to U in:GGTGGTGGTGGTTGUGGTGGTGGMGG (SEQ ID NO: 10), where M=gemcitabine] was prepared.

[0158] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0159] 2) Drug conjugation The modified oligonucleotide-drug conjugate of Example 7 was produced in the same manner as the drug conjugation method of Comparative Example 2, except that the sequence of SEQ ID NO:22 was used instead of the sequence of SEQ ID NO:13.

[0160] The linker (L) connecting U in the sequence of SEQ ID NO: 10 to paclitaxel in the modified oligonucleotide-drug conjugate of Example 7 is "-acrylamide-C6-NH-succinic acid-" (acrylamide is the moiety connected to U in SEQ ID NO: 10, and succinic acid is the moiety connected to paclitaxel).

[0161] (11) Reference example 1 Manufacturing By the following method, Reference example 1 A modified oligonucleotide-drug conjugate in which paclitaxel was conjugated to the 5' end (i.e., nucleotide 1) of [:GGTGGTGGTGGTTGTGGTGGTGGTGGMM (SEQ ID NO: 11), where M = gemcitabine] was prepared (see Figure 18).

[0162] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0163] 2) Drug conjugation The same drug conjugation method as in Comparative Example 2 was carried out, except that the sequence of SEQ ID NO: 23 was used instead of SEQ ID NO: 13. Reference example 1 modified oligonucleotide-drug conjugates were prepared.

[0164] Reference example 1 In the modified oligonucleotide-drug conjugate, the linker (L) connecting the first nucleotide (G) at the 5' end of the sequence of SEQ ID NO: 11 to paclitaxel is "-C6-NH-succinic acid-" (C6 is the moiety linked to the 5' end of SEQ ID NO: 11, and succinic acid is the moiety linked to paclitaxel).

[0165] (12) Preparation of Comparative Example 4 Comparative Example 4 [an oligonucleotide-drug conjugate in which MMAE was conjugated to the 5' end (i.e., the first nucleotide) of GGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 1)] was prepared in two steps: a conventional DNA synthesis method and drug conjugation.

[0166] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0167] 2) Drug conjugation Z of SEQ ID NO:24 was reduced with dithiothreitol (DTT), [ka] The oligonucleotide-drug conjugate of Comparative Example 4 was prepared by conjugating L1 of SEQ ID NO: 25 with MC (maleimide-caproic acid)-Val-Cit-PAB (para-aminobenzyl carbamate)-MMAE (Monomethyl auristatin E) (VcMMAE 96%, MedKoo Biosciences, Cat# 407406). In the oligonucleotide-drug conjugate of Comparative Example 4, the linker (L) linking the first nucleotide (G) at the 5' end of SEQ ID NO: 1 to MMAE was "-C6-S-MC-Val-Cit-PAB-" (C6 is the moiety linked to the 5' end of SEQ ID NO: 1, and PAB is the moiety linked to MMAE).

[0168] Specifically, DTT dissolved in a pH 8.5 buffer was [ka] The reaction was carried out at RT, followed by centrifugation at 4°C to remove the remaining DTT. [ka] was dissolved in a pH 6.0 buffer, and then VcMMAE dissolved in DMSO was added and reacted at room temperature (RT) for 1 hour. The progress of the reaction was monitored using a Waters ACQUITY UPLC H-Class PLUS Bio System (Waters, USA).

[0169] Purification and analysis of the prepared Comparative Example 4 were performed using a Waters Prep150 (Waters, USA) and a Waters ACQUITY UPLC H-Class PLUS Bio System (Waters, USA) with a reversed-phase C18 column. Mass spectrometry of the purified nucleic acid ligand was also performed using a Waters Xevo G2-XS Q-TOF System (Waters, USA).

[0170] (13) Preparation of Example 9 Example 9 [a modified oligonucleotide-drug conjugate in which MMAE was conjugated to the 5' end (i.e., first nucleotide) of:GGTGGTGGTGGTTGTGGTGGTGGTGGMM (SEQ ID NO: 11), where M = gemcitabine] was prepared.

[0171] 1) Oligonucleotide synthesis By the same method as in Comparative Example 1, [ka] was synthesized.

[0172] 2) Drug conjugation The same drug conjugation method as in Comparative Example 4 was carried out, except that the sequence of SEQ ID NO: 26 was reduced with DTT instead of the sequence of SEQ ID NO: 24. [ka] The compound of Example 9 was prepared by conjugating L1 of SEQ ID NO: 27 with VcMMAE.

[0173] In the modified oligonucleotide-drug conjugate of Example 9, the linker (L) connecting the first nucleotide (G) at the 5' end of SEQ ID NO: 11 to MMAE is "-C6-S-MC-Val-Cit-PAB-" (C6 is the moiety linked to the 5' end of SEQ ID NO: 11, and PAB is the moiety linked to MMAE).

[0196] 2. Confirmation of the anti-cancer effect of modified oligonucleotide-drug conjugates - in vitro test The in vitro anticancer effects of modified oligonucleotide-drug conjugates were examined using a pancreatic cancer cell line. The pancreatic cancer cell line (BxPC-3) was treated with the above-mentioned Examples and Comparative Examples at concentrations of 0, 0.01, 0.1, 1, 10, 100, 500, and 1000 μM. The specific IC50 measurement method and results are as follows:

[0197] (1) Cell culture All cell lines were cultured at 37°C in a 5% CO2 incubator. The BxPC-3 pancreatic cancer cell line was cultured in ATCC-modified RPMI-1640 (Thermo Scientific, USA) medium supplemented with 10% FBS and 1% antibiotics. The medium was changed every 2–3 days, and cells were subcultured when they filled approximately 70–90% of the culture dish. First, cells were washed with PBS, then added with 0.05% trypsin-EDTA and incubated at 37°C for 2 minutes. After that, fresh medium was added to inactivate the trypsin. Cells were separated from the trypsin and medium using a centrifuge and then subcultured at a 1:4 ratio in fresh medium. All cells were periodically checked for mycoplasma contamination using a kit (Intron, Korea), and only cells without detectable contamination were used for in vitro efficacy evaluation experiments.

[0198] (2) Confirmation of the inhibitory effect on pancreatic cancer cell proliferation WST reagent (DonginLS, Korea) was used to confirm the inhibitory effect of drug treatment on pancreatic cancer cell growth and measure IC50. Mitochondrial dehydrogenase in the cells produces an orange coloring substance called formazan in WST, which is only effective in living cells. The specific test method is as follows:

[0199] The cells were cultured as described above and plated in a 96-well culture vessel at 2 × 10 4 The cells were then aliquoted into individual wells and cultured for 24 hours. The medium for the cells attached to the culture vessel was replaced with ATCC-modified RPMI-1640 medium containing 5% FBS, and the cells were treated with the modified oligonucleotide-drug conjugate samples (Examples 1-7, 9-12, and Comparative Examples 1-7) at concentrations of 0, 0.01, 0.1, 1, 10, 100, 500, and 1000 μM. After 72 hours, 10 μl of WST reagent was added to each well and allowed to react for 2 hours. The formazan produced was measured at an absorbance of 450 nm using a Glomax plate reader (Promega, USA), and the IC50 was evaluated according to the instrument's protocol. IC 50 The evaluation results and graphs are shown in the following Table 2 and Figures 19 to 27 (in Table 2, M=gemcitabine, N=5-FdU).

[0200] [Table 2-1] [Table 2-2]

[0201] Control aptamer, Comparative Example 1 IC 50 On the other hand, the modified oligonucleotide-drug conjugate example has an IC value several to several tens of times lower than that of the control aptamer, Comparative Example 1. 50 The values ​​shown were:

[0202] In addition, in Examples (e.g., Examples 1 to 12) in which a drug is conjugated to some amino acids in the sequence of Comparative Example 1 and other amino acids are simultaneously substituted with modified nucleic acids or modified nucleic acids are further included, the IC was at least 2-fold, and often about 40-fold, lower than in Comparative Examples (Comparative Examples 2 to 7) in which only a drug was conjugated to some amino acids without substitution with modified nucleic acids. 50 It was confirmed that it had a value.

[0203] This means that the modified oligonucleotide-drug conjugates of the present invention can exhibit superior anti-cancer effects compared to the control aptamer.

[0204] 3. Confirmation of the anti-cancer effect of modified oligonucleotide-drug conjugates - Animal experiments (in vivo) I C 50 Example 5 (IO142) and Example 9 (IO176) which showed excellent measurement results were selected and subjected to animal experiments.

[0205] (1) Preparation of a "patch-type collagen drug delivery vehicle" carrying a modified oligonucleotide-drug conjugate To deliver modified oligonucleotide-drug conjugates to animal models, a 1.0% dispersion of high-purity collagen (COLTRIX® Tendoregen, Ubiosis) was mixed with the test sample (e.g., modified oligonucleotide-drug conjugate, gemcitabine, etc.), poured into a 1-cm-diameter circular silicone mold, and freeze-dried to produce a "collagen patch-type drug delivery device" in which the sample was supported on collagen. The amount of sample supported per collagen patch-type drug delivery device is shown in Table 3 below (in Table 3, SEQ ID NO: 16 is 5'-(Gem)(Gem)[TGG]4[TTG][TGG]5-3', where Gem = gemcitabine).

[0206] [Table 3]

[0207] Specifically, the test samples (gemcitabine, IO101L (SEQ ID NO: 16) and modified oligonucleotide-drug conjugates (Examples 5 and 9)) were added to highly purified collagen and mixed uniformly at 4°C for 1 hour using a multimixer (SLRM-3, MYLAB). A predetermined amount of the collagen-drug mixture was then dispensed into a cylindrical silicone mold with a diameter of 1 cm. The cylindrical silicone mold containing the collagen-drug mixture was subjected to primary freezing at -20°C for at least 4 hours. After primary freezing, the mixture was separated from the cylindrical silicone mold and transferred to a sterile dish or plate, followed by secondary freezing at -80°C for at least 2 hours. The mixture was pre-frozen so that the temperature of the cold trap of the freeze dryer was -80°C, and then placed in the freeze dryer and freeze-dried for at least 16 hours. The freeze-dried mixture was pressed into a patch mold using an acrylic plate, then placed in an aluminum pouch and sealed. The packaged collagen drug delivery patch was refrigerated at 4°C.

[0208] Hereinafter, the patch-type collagen drug delivery device prepared by the above-mentioned method is referred to as a patch or a drug-loaded collagen patch.

[0209] (2) Evaluation of the stability and efficacy of a "patch-type collagen drug delivery system" carrying modified oligonucleotide-drug conjugates in animal models 1) Construction of an orthotopic pancreatic cancer model and implantation of a collagen patch-type drug delivery system Frozen cancer cell line (BxPC-3-Luc) (Asan Medical Center) was thawed and then cultured in RPMI 1640 medium (Biowest, Cat#: S1480) supplemented with 10% fetal bovine serum (Biowest, Cat#: S1480) and 1% antibiotic (Gibco, Cat#: 15240062) at 37°C and 5% CO2. The thawed cells were cultured for one week, and the morphology, viability, and doubling time were confirmed to be normal.

[0210] Cancer cells with a cell viability of 95% or more were prepared for transplantation into mice. The cells were harvested using trypsin, suspended in RPMI 1640 medium, and then cultured at a concentration of 5 × 10 5 Six-week-old BALB / c-nude male mice (Jabio, Korea) that had been stabilized for one week were anesthetized intraperitoneally, and the pancreas was removed and then cultured at 5 × 10 5 Cells were transplanted at a rate of 25 μl per 1000 cells. Two weeks later, tumor formation was confirmed by luciferase imaging. The mice were then divided into groups of five, as shown in Table 4 below. The collagen patch-type drug delivery device prepared in (1) above was inserted into the tumor site of each group of mice (intra-abdominal cavity insertion using surgery). All animal experiments included in this study were conducted in accordance with the animal experiment protocol [2022-14-058] approved by the Institutional Animal Care and Use Committee (IACUC) of Asan Life Science Institute, Korea.

[0211] [Table 4]

[0212] 2) Evaluation of efficacy using IVIS imaging After insertion of the collagen patch, changes in tumor size and body weight were monitored three times a week for four weeks using an IVIS imaging system (Xenogen IVIS spectrum system; Caliper Life Science, Inc., Hopkinton, MA). Luciferin was intraperitoneally injected at a concentration of 150 mg / kg per mouse, and after a 7-minute incubation, IVIS imaging was performed. After implantation, IVIS imaging was performed on day 28. After weight measurement, the mice were sacrificed and their vital organs (heart, lungs, spleen, kidneys, and liver), pancreas, and tumor were removed. The organs were weighed and photographed. Because mice needed time to recover on the day of implantation, IVIS imaging was performed the day after implantation.

[0213] 2-1) IVIS whole body image In the drug-untreated mice group (Group 1) and the mice group with the 2.0 mg IO101L-loaded collagen patch inserted (Group 3), some mice died, and luminescence increased over time. In the mice group with the 0.2 mg IO176-loaded collagen patch inserted (Group 4) from Example 9 and the 2.0 mg IO142-loaded collagen patch inserted (Group 5) from Example 5, the fluorescence change decreased. An increase in luminescence indicates cancer growth, while a decrease in luminescence indicates tumor suppression (see Figures 1 to 5).

[0214] 2-2) Weight change After implantation of the collagen patch, the mice's weights were measured three times a week for four weeks. The weights on day 0 were separated from the daily measurements, and the percentage change in mouse weight compared to day 0 was observed. All mouse groups (groups 2 to 5) that received the collagen patch showed weight loss for two to six days after implantation of the test substance, but gradually recovered (see Figure 6).

[0215] 2-3) Photographing and weighing the excised organs The mice were sacrificed 28 days after the insertion of the collagen patch-type drug delivery device, and the major organs (heart, lungs, spleen, kidneys, liver), pancreas, and tumor were removed and photographed, and the organ weights were measured.

[0216] Similar to the experimental results in "2-2," the group of mice implanted with a 0.2 mg-loaded collagen patch of Example 9 (IO176) (Group 4) had the smallest tumor size, and no metastatic tumors were observed. Furthermore, the group of mice implanted with a 0.2 mg-loaded collagen patch of Example 9 (IO176) (Group 4) had the lowest tumor weight, followed by the group of mice implanted with a 2.0 mg-loaded collagen patch of Example 5 (IO142) (Group 5) and the group of mice implanted with a 2.0 mg-loaded collagen patch of IO101L (Group 3) (see Figures 7A to 7E and 8).

[0217] Based on the results of IVIS whole-body imaging and tumor size and weight measurements, excellent tumor suppression effects were confirmed, with no mice dying in the group of mice implanted with a 0.2 mg-loaded collagen patch of Example 9 (IO176) (Group 4) and the group of mice implanted with a 2.0 mg-loaded collagen patch of Example 5 (IO142) (Group 5). Furthermore, when mice in the test groups were dissected, no drug-loaded collagen patches were observed, indicating that they had all dissolved and been absorbed.

[0218] 4. Pharmacokinetic Evaluation of Modified Oligonucleotide-Drug Conjugates—Use in Rats Rats were administered 40 mg / kg of Example 9 (IO176), 4 mg / kg of MMAE (MedChemExpress, Cat# HY-15162), the same amount as monomethyl auristatin E (hereinafter referred to as "MMAE") contained in Example 9, and 2 mg / kg of gemcitabine (BetaPharma (shanghai) Co., Ltd., Cat# 86-39157), the same amount as gemcitabine contained in Example 9, via the jugular vein. Then, i) the pharmacokinetic index of MMAE and ii) the pharmacokinetic index of gemcitabine and dFdU, an inactive metabolite of gemcitabine, were calculated and compared.

[0219] Specifically, male Sprague-Dawley rats (8 weeks old, YOUNG BIO) were acclimated for one week in an animal room on the Sungshin campus of The Catholic University of Korea before undergoing pharmacokinetic studies. The rats were anesthetized with the inhalation anesthetic isoflurane, and PE50 (polyethylene tubing, Clay Adams, Becton Dickinson, NJ, USA) tubes were inserted into the carotid artery (for blood sampling) and jugular vein (for drug administration), sutured, and secured to the back of the neck. Anesthesia was maintained during surgery with ether, and approximately 0.5 mL of saline containing heparin (20 units / mL) was injected to prevent blood clotting in the cannula. After surgery, rats were placed in metabolic cages and allowed to fully recover from the anesthesia (4-5 hours). Then, they were divided into three groups: Example 9 (IO176) alone (n=6), MMAE alone (n=7), and gemcitabine alone (n=6). Each group was administered with each test (Example 9, MMAE alone, gemcitabine alone). Example 9, MMAE, and gemcitabine were all dissolved in saline at 2 mL per kg of body weight and administered intrajugularly at doses of 40 mg / kg, 4 mg / kg, and 2 mg / kg, respectively. To prevent gemcitabine metabolites from being converted by cytidine deaminase, 0.2 mL of blood was collected in a light-tight Eppendorf tube containing 2 μL of a 10 mg / mL solution of tetrahydrouridine (THU), a cytidine deaminase inhibitor, dissolved in distilled water. From each of the three groups, 0.2 mL of blood was collected via the carotid artery into a pre-frozen, light-tight Eppendorf tube immediately before (0 min), 1, 5, 10, 15, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 20 h, 24 h, and 30 h after administration of each drug. Immediately after blood collection, plasma stored in an ice bath was centrifuged at 4°C, and 100 μL of plasma was dispensed into a light-tight Eppendorf tube. The aliquots of plasma were immediately stored in an ice bath and kept at -80°C until LC-MS / MS analysis.After carotid blood sampling, rats were euthanized in a CO2 gas chamber.

[0220] (1) Plasma concentration analysis method 1) Method for analyzing MMAE concentration After pretreatment of the samples by protein precipitation, the plasma concentrations of MMAE were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) established in the Pharmacology / Pharmacokinetics Laboratory at the Catholic University of Korea. All analytical procedures were performed under light-protected conditions.

[0221] B. LC-MS / MS conditions -Analytical instruments: Agilent 1290 series HPLC, Qtrap 5500 LC-MS / MS -Detector: Tandem mass spectrometer (triple quadruple type) Ion source: Electrospray ionization (positive ion mode) Desolvation temperature: 500℃ Nebulizing gas: Nitrogen, Collision gas: Nitrogen Quantitation: MRM (multiple reaction monitoring) mode -Stationary phase: Luna C18 (2.0×100mm, 3μm, Phenomenex, California, USA) -Stationary phase temperature: 40℃ Mobile phase: 0.1% formic acid in distilled water (A) / 0.1% formic acid in acetonitrile (B) (50:50, v / v) (see Table 5 below) -Flow rate: 0.3mL / min Mass parameters: See Table 6 below (CE: Collision energy)

[0222] [Table 5]

[0223] [Table 6]

[0224] B. Preparation of calibration curve The MMAE standard was dissolved in acetonitrile to prepare a 1 mg / mL stock solution, which was then stored frozen. The stock solution was then diluted with acetonitrile to prepare working solutions at concentrations of 0.250, 0.500, 2.50, 5.00, 12.5, and 50.0 μg / mL, which were then stored in a refrigerator. The internal standard (IS), verapamil, was dissolved in acetonitrile to prepare a 200 ng / mL working solution. The MMAE working solution was spiked into rat co-plasma containing THU to prepare standard plasma samples with MMAE concentrations of 10, 20, 100, 200, 500, and 2000 ng / mL.

[0225] C. Plasma sample processing method 200 μL of the internal standard verapamil (200 ng / mL) dissolved in acetonitrile was added to 25 μL of rat plasma, vortexed for 10 minutes, and then centrifuged at 14,000 rpm for 5 minutes at 4° C. 190 μL of the supernatant was then collected and transferred to an LC-MS / MS vial via a 0.2 μm syringe filter, after which 2 μL was injected and analyzed by LC-MS / MS.

[0226] D. Determining the suitability of the analysis To determine the suitability of the analytical process, after analyzing the calibration curve samples for each batch of analytical samples during sample processing, analytically suitable samples (MMAE lowest limit of quantitation: LLoQ, 10 ng / mL; low concentration: LoQC, 25 ng / mL; medium concentration: MiQC, 250 ng / mL; high concentration: HiQC, 1500 ng / mL) were analyzed in duplicate. It was confirmed that at least 67% of the six suitable samples (e.g., 4 out of 6) were within ±15% of the theoretical value, and that at least 50% of the same concentrations were within ±15% of the theoretical value.

[0227] E. Calculation of plasma drug concentration The peak area ratio of MMAE to the peak area of ​​the internal standard was determined from the obtained chromatogram, and the plasma MMAE concentration was calculated from a calibration curve prepared in advance.

[0228] 2) Method for analyzing gemcitabine and dFdU concentrations B. LC-MS / MS conditions -Analytical instruments: Agilent 1290 series HPLC, Qtrap 5500 LC-MS / MS -Detector: Tandem mass spectrometer (triple quadruple type) Ion source: Electrospray ionization (positive ion mode) Desolvation temperature: 500℃ Nebulizing gas: Nitrogen, Collision gas: Nitrogen Quantitation :MRM (multiple reaction monitoring) mode -Stationary phase: Hypersil gold C18 column (2.1×100mm, 1.9μm, Thermo Fisher Scientific Inc., Boston, USA) -Stationary phase temperature: 40℃ Mobile phase: distilled water (A) and acetonitrile (B) containing 0.1% formic acid (95:5, v / v) (see Table 7 below) -Flow rate: 0.22mL / min Mass parameters: See Table 8 below (CE: Collision energy)

[0229] [Table 7]

[0230] [Table 8]

[0231] B. Preparation of calibration curve Gemcitabine and dFdU standards were dissolved in distilled water to prepare stock solutions of 400 μg / mL and 700 μg / mL, which were then stored frozen. The stock solutions were diluted with triple-distilled water to prepare working solutions of 0.200, 0.400, 0.800, 2.00, 10.0, and 40.0 μg / mL, which were then stored in a refrigerator. The internal standard (IS), 5'-deoxy-5-fluorocytidine, was dissolved in acetonitrile to prepare a 50 ng / mL working solution. Standard plasma samples were prepared by adding the gemcitabine and dFdU working solutions to rat co-plasma containing THU, resulting in gemcitabine and dFdU plasma concentrations of 10, 20, 40, 100, 500, and 2000 ng / mL, respectively.

[0232] C. Plasma sample processing method 20 μL of rat plasma was mixed with 150 μL of the internal standard, 5'-deoxy-5-fluorocytidine (50 ng / mL) dissolved in acetonitrile. The mixture was vortexed for 10 minutes and then centrifuged at 14,000 rpm at 4°C for 5 minutes. 150 μL of the supernatant was then transferred to another Eppendorf tube, and the organic solvent was removed under a stream of nitrogen. 100 μL of triple-distilled water was added to the dried Eppendorf tube for reconstitution. The mixture was then vortexed for 10 minutes and centrifuged at 14,000 rpm at -4°C for 5 minutes. 90 μL of the supernatant was then transferred to another Eppendorf tube and centrifuged at 14,000 rpm at -4°C for 5 minutes. 80 μL of the supernatant was transferred to an LC-MS / MS vial, and 5 μL was injected for LC-MS / MS analysis.

[0233] 2. Determining the suitability of the analysis To determine the suitability of the analytical process, after analyzing the calibration curve samples for each batch of analytical samples during sample processing, analytically acceptable samples (lowest limit of quantitation for gemcitabine and dFdU, respectively: LLoQ, 10 ng / mL; low concentration: LoQC, 30 ng / mL; medium concentration: MiQC, 300 ng / mL; high concentration: HiQC, 900 ng / mL) were analyzed in duplicate. It was confirmed that at least 67% of the acceptable samples (e.g., 4 out of 6) were within ±15% of the theoretical value, and at least 50% of the acceptable samples at the same concentrations were within ±15% of the theoretical value.

[0234] E. Calculation of plasma drug concentration From the obtained chromatogram, the peak area ratios of gemcitabine and dFdU relative to the peak area of ​​the internal standard were determined, and the concentrations of gemcitabine and dFdU in plasma were calculated from a calibration curve prepared in advance.

[0235] 3) Calculation method and statistical processing of pharmacokinetic parameters The pharmacokinetic parameters of MMAE, gemcitabine, and dFdU were analyzed by non-compartmental analysis using the Phoenix Winnonlin™ (Version 6.4, Certara) program. The area under the plasma concentration-time curve (AUCt) was calculated from the plasma concentration-time curve up to the final measurement time point after drug administration using the log-linear trapezoidal method. The area under the plasma concentration-time curve up to infinity (AUCinf) was calculated using the following mathematical formula 1. The terminal elimination rate constant (γZ) and half-life (t1 / 2) were calculated from the slope of the elimination phase of the plasma concentration profile.

[0236] [Mathematical formula 1] AUCinf=AUCt+Ct / γZ (Ct: final quantitative concentration, γZ: terminal disappearance rate constant)

[0237] Statistical analysis for comparing the pharmacokinetic parameters of each of the two groups was performed using the t-test.

[0238] (2) Results of plasma concentration analysis 1) Results of MMAE concentration analysis When Example 9 (IO176) was administered intravenously at 40 mg / kg and the same amount of MMAE alone at 4 mg / kg was administered intravenously to rats, i) the plasma concentration of MMAE is shown in Figure 9, and ii) the pharmacokinetic coefficient of MMAE is summarized in Table 9 below.

[0239] When 4 mg / kg of MMAE was administered intravenously, all seven rats died between 480 and 600 minutes after administration. In contrast, in the group receiving 40 mg / kg of Example 9 (IO176) intravenously, the plasma concentration of MMAE was 605 ± 165 ng / mL at 1 minute after administration, approximately 50 times lower than the group receiving 4 mg / kg of MMAE intravenously alone (29,640 ± 7,190 ng / mL). The plasma concentration of MMAE gradually increased from 60 to 120 minutes after administration and continued to increase until 1,800 minutes, the final blood sampling time. All rats survived (see Figure 9). The AUC value of MMAE up to 120 minutes after administration (initial time) was 71.7±9.61 μg min / mL in the group receiving MMAE 4 mg / kg intravenously alone, which was calculated to be approximately 9.70 times higher than that of the group receiving Example 9 (IO176) 40 mg / kg intravenously (7.39±1.05 μg min / mL). However, the AUC value up to 1800 minutes (final blood collection time) was approximately 1.55 times higher in the group receiving Example 9 (IO176) 40 mg / kg intravenously (see Table 9 below).

[0240] Because the release rate of MMAE conjugated to Example 9 (IO176) into plasma is very slow, Example 9 can show a statistically significantly lower plasma MMAE concentration, which is presumably due to the reduced cytotoxicity of MMAE and the absence of deaths among the rats tested.

[0241] [Table 9]

[0242] 2) Results of gemcitabine and dFdU concentration analysis When Example 9 (IO176) was administered intravenously at 40 mg / kg and when the same amount of gemcitabine alone was administered intravenously at 2 mg / kg, i) the plasma concentrations of gemcitabine and its metabolite dFdU are shown in Figure 10, and ii) the pharmacokinetic coefficients of gemcitabine and its metabolite dFdU are summarized in Table 10 below.

[0243] The analytical results for gemcitabine are as follows (see Table 10 below). The group administered with Example 9 (IO176) exhibited statistically significantly higher gemcitabine plasma concentrations than the group administered intravenously with the same dose of gemcitabine alone (left panel of Figure 10). As a result, the AUCt values ​​of gemcitabine were calculated to be 610 ± 52.2 μg min / mL and 438 ± 124 μg min / mL, respectively, which were approximately 1.39 times higher. Furthermore, the plasma half-life (t1 / 2) of gemcitabine was confirmed to be statistically significantly increased in the group administered with Example 9 (IO176). From this, it is presumed that the initial release rate of gemcitabine in the group administered with IO176 (Example 9) was high, resulting in a high and sustained plasma concentration of gemcitabine, and that this sustained release increased the plasma half-life.

[0244] The analytical results for dFdU, an inactive metabolite of gemcitabine, are as follows (see Table 10 below). Although the plasma concentration tended to be slightly higher in the group administered with Example 9 (IO176), the AUC values ​​were 111 ± 22.3 μg min / mL and 79.8 ± 44.2 μg min / mL, respectively, and no statistical significance was observed. The Cmax (maximum plasma concentration) of dFdU was statistically significantly higher in the group administered with Example 9 (IO176). However, in both groups, the metabolic conversion rates of inactive metabolites (AUCt, dFdU / AUCt, gemcitabine) were 0.182 ± 0.0263 and 0.170 ± 0.0621, respectively, and no statistical significance was observed. Therefore, Example 9 (IO176) indicates that gemcitabine released into plasma does not affect the conversion rate of inactive metabolites.

[0245] In Table 10 below, AUCt is the area under the curve from 0 minutes to the last blood sampling time, t1 / 2 is the plasma elimination half-life, CL is the total body clearance, Vdss is the volume of distribution in the body, MRT is the mean residence time of the drug, Tmax is the median (ranges), and the metabolic conversion ratio value is calculated by dividing the AUCt value of dFdU by the AUCt value of gemcitabine.

[0246] [Table 10]

[0247] [National research and development project that supported this invention] [Project unique number]1425162626 [Project number] S3004867 [Department name] Small and Medium-sized Venture Business Department [Name of issue management (specialized) organization] Small and Medium Enterprise Technology Information Promotion Agency [Research Project Name] Small and Medium Enterprise Technology Innovation Development [Research title] Development technology for new anticancer drugs using multi-drug binding aptamers [Contribution rate] 1 / 1 [Project Implementation Organization Name] Interoligo Corporation [Research period] 20200921~20220920

Claims

1. A modified oligonucleotide-drug conjugate comprising a modified oligonucleotide represented by the following Chemical Formula 1 and a drug conjugated to deoxyuridine (dU) contained in the modified oligonucleotide: 【Chemistry 1】 (In the above Chemical Formula 1, X １ ~X ３ and X ７ ~X ９ are each independently thymidine (T) or a modified nucleic acid represented by the following Chemical Formula 3 or Chemical Formula 4, Y １ is deoxyguanosine (dG) or a modified nucleic acid represented by the following chemical formula 3 or chemical formula 4, X ４ ~X ６ are each independently thymidine (T), deoxyuridine (dU), or a modified nucleic acid represented by the following Chemical Formula 3 or Chemical Formula 4, ４ ~X ６ at least one of is deoxyuridine (dU), M １ and M ２ are each independently a modified nucleic acid represented by the following Chemical Formula 3 or Chemical Formula 4, N １ and N ２ are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a and d are each independently an integer of 0 to 10, １ ~X ９ are both thymidine (T), and the Y １ is deoxyguanosine (dG), the a and d are not 0 at the same time, b and c are each independently an integer of 0 to 10. 【Chemistry 2】 【Transformation 3】 (In the above Chemical Formula 3 or Chemical Formula 4, R １ is hydrogen, halogen or a hydroxy group, R ２ is hydrogen, halogen or a hydroxy group, R ３ is hydrogen, halogen, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group (wherein １ is hydrogen or a hydroxy group, and the R ２ is hydrogen, and the R ３ Except when is hydrogen or methyl.

2. The modified oligonucleotide-drug conjugate of claim 1, wherein the modified nucleic acid represented by Chemical Formula 3 or Chemical Formula 4 is selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodocytidine, cytosine arabinoside, 2',2'-difluoro-2'-deoxycytidine, and bromovinyldeoxyuridine.

3. The drugs include paclitaxel, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D (MMAD), cytarabine, gemcitabine, maytansine, DM1 (mertansine), DM4, calicheamicin and its derivatives, doxorubicin, duocarmycin and its derivatives, pyrrolobenzodiazepines (PBD), SN-38, α-amanitin, tubulysin analogs, and the like. analog), cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan (b usulfan), dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate), 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine ), camptothecin (camptothecin), topotecan (topotecan), irinotecan (irinotecan), etoposide (etoposide), teniposide (teniposide), mitoxantrone (mitoxantrone), paclitaxel (paclitaxel), docetaxel (docetaxel), ixabepilone (izabepilone), vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), vinorelbine (vinorelbine), estramustine (estramustine), maytansine (maytansine), auristatin E (auristatin 2. The modified oligonucleotide-drug conjugate of claim 1, wherein the modified oligonucleotide-drug conjugate is selected from the group consisting of ribozyme A (RI), ribozyme B (RI), ribozyme E (RI), auristatin F (RI), and nemorubicin (RI).

4. The drug and the deoxyuridine (dU) contained in the modified oligonucleotide are １ and L ２ is conjugated with a linker (L) to which Said L １ is selected from the group consisting of 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serinol, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2 dT, amino-modifier C6 dT, S-Bz-thiol-modifier C6-dT, phosphodiester bond, and nucleotide, and obtained by a deprotection process, acrylamide-C2-NH ２ , C12-NH ２ , C3-NH ２ , acrylamide-C6-propanamide-SH, acrylamide-C6-NH ２ , C6-NH ２ , C6-SH; Said L ２ is selected from the group consisting of maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinic acid, hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproyl, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), a phosphodiester bond, and a nucleotide.

5. 2. The modified oligonucleotide-drug conjugate of claim 1, wherein the modified oligonucleotide consists of a sequence selected from SEQ ID NO: 4 to SEQ ID NO:

10.

6. A pharmaceutical composition for preventing or treating cancer, comprising the modified oligonucleotide-drug conjugate according to any one of claims 1 to 5.

7. 7. The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the cancer is selected from the group consisting of leukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, cervical carcinoma, glioma cancer, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer.

8. A modified oligonucleotide-drug conjugate comprising a modified oligonucleotide represented by the following Chemical Formula 2 and MMAE conjugated to the 5' end of the modified oligonucleotide: 【Chemistry 4】 (In the above Chemical Formula 2, X １ ~X ９ are thymidine (T), M １ and M ２ are gemcitabine, respectively; N １ and N ２ are each independently thymidine (T), deoxyuridine (dU), deoxycytidine (dC), or deoxyguanosine (dG); a is 0, d is 2, b and c are each 0.

9. The 5' end of the modified oligonucleotide represented by the formula 2 and the MMAE is L １ and L ２ is conjugated with a linker (L) to which Said L １ is selected from the group consisting of 5'-thiol-modifier C6, thiol-modifier C6S-S, dithiol serinol, PC amino-modifier, 5'-amino-modifier C3, 5'-amino-modifier C6, 5'-amino-modifier C12, 5'-amino-modifier TEG, amino-modifier C2 dT, amino-modifier C6 dT, S-Bz-thiol-modifier C6-dT, phosphodiester bond, and nucleotide, and obtained by a deprotection process, acrylamide-C2-NH ２ , C12-NH ２ , C3-NH ２ , acrylamide-C6-propanamide-SH, acrylamide-C6-NH ２ , C6-NH ２ , C6-SH; Said L ２ is selected from the group consisting of maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-Val-Cit-PAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinic acid, hydrazone, peptide, disulfide, thioether, valine-citrulline, N-maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl, mercaptoacetamidocaproyl, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPDB), a phosphodiester bond, and a nucleotide.

10. A pharmaceutical composition for preventing or treating cancer, comprising the modified oligonucleotide-drug conjugate of claim 8 or 9.

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