Modified nucleic acid with improved therapeutic efficacy and anticancer drug composition containing the same

Abstract

The present invention relates to oligonucleotide variants, and more specifically to oligonucleotide variants having a structure of the following formula 1 and pharmaceutical compositions comprising the oligonucleotide variants or pharmaceutically acceptable salts thereof, wherein the pharmaceutical compositions can exhibit excellent in vivo stability and anticancer effects: [Formula 1] (N) x ‑[TGG] m [TTG][TGG] n ‑(M) y (wherein, N and M are independently deoxyuridine (dU), deoxycytidine (dC), uridine (U) or cytidine (C), wherein a halogen or a hydroxyl group is bound to the 5-position or the 2'-position of N and M; x and y are independently integers from 0 to 10 (except for the case where x and y are both 0); n is an integer from 1 to 10; and m is an integer from 1 to 10).

Description

Technical Field

[0001] The present invention relates to nucleic acid variants with improved therapeutic efficacy and pharmaceutical compositions for anti-cancer use comprising the nucleic acid variants. Background Art

[0002] Since the 1980s, research has been actively conducted to develop oligonucleotides as therapeutic agents. It is known that oligonucleotides rich in guanosine have a cell growth inhibitory effect on a wide range of cancer cells, and these oligonucleotides can have a structure of 4 chains through intramolecular bonds or intermolecular bonds. Instead of forming a double helix structure through hydrogen bonds between adenosine and thiamine or guanosine and cytidine, four (4) guanosines are located on a plane to form hydrogen bonds in the form of Hoogsten, thereby forming a G-quadruplex. In addition, it is known that the oligonucleotides forming G-quadruplexes have a stable structure due to their structural characteristics, thereby showing relatively high blood stability and cell permeability. Various research and development are being conducted to further stabilize the G-quadruplex and increase the anticancer effect by introducing modified nucleic acids for therapeutic use into oligonucleotides with different functions as described above. Summary of the invention

[0003] [Problems to be solved by the present invention]

[0004] The object of the present invention is to provide a nucleic acid variant having therapeutic efficacy and a pharmaceutical composition for anti-cancer use comprising the nucleic acid variant.

[0005] Another object of the present invention is to provide a pharmaceutical composition having excellent in vivo stability and anticancer effect.

[0006] [Methods used to solve the problem]

[0007] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present invention.

[0008] 1. An oligonucleotide variant having the structure of the following formula 1:

[0009] [Formula 1]

[0010] (N) x -[TGG] m [TTG][TGG] n -(M) y

[0011] wherein N and M are independently deoxyuridine (dU), deoxycytidine (dC), uridine (U) or cytidine (C), wherein a halogen or a hydroxyl group is bound to the 5-position or 2'-position thereof; x and y are independently integers of 0 to 10 (except for the case where x and y are both 0), n is an integer of 1 to 10; and m is an integer of 1 to 10.

[0012] 2. The oligonucleotide variant according to 1 above, wherein N and M are independently selected from the group consisting of 5-fluorodeoxyuridine, 5-fluorouridine, 5-fluorodeoxycytidine, 5-fluorocytidine, 5-iododeoxyuridine, 5-iodouridine, 5-iododeoxycytidine, 5-iodine, cytosine arabinoside, 2',2'-difluorodeoxycytidine, capecitabine and bromovinyl deoxyuridine.

[0013] 3. The oligonucleotide variant according to 1 above, wherein the structure of Formula 1 is any one of the following Formulas 2 to 34:

[0014] [Formula 2]

[0015] (N) 2 -[TGG] 1 [TTG][TGG] 1 ,

[0016] [Formula 3]

[0017] (N) 2 -[TGG] 1 [TTG][TGG] 2 ,

[0018] [Formula 4]

[0019] (N) 2 -[TGG] 2 [TTG][TGG] 1 ,

[0020] [Formula 5]

[0021] (N) 2 -[TGG] 2 [TTG][TGG] 2 ,

[0022] [Formula 6]

[0023] (N) 2 -[TGG] 2 [TTG][TGG] 3 ,

[0024] [Formula 7]

[0025] (N) 2 -[TGG] 3 [TTG][TGG] 2 ,

[0026] [Formula 8]

[0027] (N) 2 -[TGG]3 [TTG][TGG] 3

[0028] [Formula 9]

[0029] (N) 2 -[TGG] 3 [TTG][TGG] 4 ,

[0030] [Formula 10]

[0031] (N) 2 -[TGG] 4 [TTG][TGG] 3 ,

[0032] [Formula 11]

[0033] (N) 2 -[TGG] 4 [TTG][TGG] 4 ,

[0034] [Formula 12]

[0035] (N) 2 -[TGG] 4 [TTG][TGG] 5 ,

[0036] [Formula 13]

[0037] (N) 2 -[TGG] 5 [TTG][TGG] 4 ,

[0038] [Formula 14]

[0039] (N) 2 -[TGG] 5 [TTG][TGG] 5 ,

[0040] [Formula 15]

[0041] (N) 2 -[TGG] 5 [TTG][TGG] 6 ,

[0042] [Formula 16]

[0043] (N) 2 -[TGG] 6 [TTG][TGG] 5 ,

[0044] [Formula 17]

[0045] (N) 2 -[TGG] 6 [TTG][TGG] 6 ,

[0046] [Formula 18]

[0047] [TGG] 4 [TTG][TGG] 4 -(M) 1 ,

[0048] [Formula 19]

[0049] [TGG] 4 [TTG][TGG] 4 -(M) 2 ,

[0050] [Formula 20]

[0051] [TGG] 4 [TTG][TGG] 4 -(M) 3 ,

[0052] [Formula 21]

[0053] [TGG] 4 [TTG][TGG] 4 -(M) 4 ,

[0054] [Formula 22]

[0055] [TGG] 4 [TTG][TGG] 4 -(M) 5

[0056] [Formula 23]

[0057] [TGG] 4 [TTG][TGG] 5 -(M) 1 ,

[0058] [Formula 24]

[0059] [TGG] 4 [TTG][TGG] 5 -(M) 2 ,

[0060] [Formula 25]

[0061] [TGG] 4 [TTG][TGG] 5 -(M)3 ,

[0062] [Formula 26]

[0063] [TGG] 4 [TTG][TGG] 5 -(M) 4 ,

[0064] [Formula 27]

[0065] [TGG] 4 [TTG][TGG] 5 -(M) 5 ,

[0066] [Formula 28]

[0067] [TGG] 4 [TTG][TGG] 4 -(M) 10 ,

[0068] [Formula 29]

[0069] (N) 1 -[TGG] 4 [TTG][TGG] 4 -(M) 1 ,

[0070] [Formula 30]

[0071] (N) 3 -[TGG] 4 [TTG][TGG] 4 -(M) 3 ,

[0072] [Formula 31]

[0073] (N) 5 -[TGG] 4 [TTG][TGG] 4 -(M) 5 ,

[0074] [Formula 32]

[0075] (N) 1 -[TGG] 4 [TTG][TGG] 5 -(M) 1 ,

[0076] [Formula 33]

[0077] (N) 3 -[TGG] 4 [TTG][TGG]5 -(M) 3 ,

[0078] [Equation 34]

[0079] (N) 5 -[TGG] 4 [TTG][TGG] 5 -(M) 5 ,

[0080] 4. The oligonucleotide variant according to 1 above, wherein n is an integer from 1 to 5, and m is an integer from 1 to 5.

[0081] 5. The oligonucleotide variant according to 1 above, wherein x and y are independently integers from 0 to 5 (except for the case where x and y are both 0).

[0082] 6. A pharmaceutical composition for preventing or treating cancer, comprising the oligonucleotide variant according to any one of 1 to 5 above or a pharmaceutically acceptable salt thereof.

[0083] 7. The pharmaceutical composition according to 6 above, wherein the cancer is selected from the group consisting of leukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, cervical cancer, glioma cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma and cervical cancer.

[0084] 8. The pharmaceutical composition according to 6 above, wherein the composition is an atelocollagen dispersion preparation.

[0085] 9. The pharmaceutical composition according to 8 above, wherein the atelocollagen dispersion contains 0.5 g to 5.5 g of atelocollagen per 100 ml of PBS solution.

[0086] 10. The pharmaceutical composition according to 6 above, wherein the composition is in the form of a sol-gel or a patch.

[0087] 11. A pharmaceutical composition according to 8 above, wherein the atelocollagen is prepared by a method comprising: a) treating a collagen-containing animal tissue with at least one of alkaline protease, catalase, pepsin and papain to extract a substance; b) first filtering the extracted substance and salting it out by adding a neutral salt to the obtained filtrate, followed by a second filtration; c) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; d) freeze-drying the filtrate obtained after the third filtration, and recovering the lyophilized powder; and e) dissolving the lyophilized powder in dilute hydrochloric acid (dil-HCl), dilute acetic acid or phosphate buffer at pH 4 to pH 8 and concentrating to prepare an atelocollagen solution, injecting the prepared atelocollagen solution into a column filled with polymer beads at 5% to 20% by volume of the column bed volume, and developing the column in dilute hydrochloric acid, dilute acetic acid or phosphate buffer at pH 4 to pH 8, thereby recovering the atelocollagen.

[0088] [Beneficial Effects]

[0089] The oligonucleotide variants of the present invention can effectively target nucleolin present on the surface, cytoplasm or nucleus of cancer cells.

[0090] The oligonucleotide variants of the present invention can inhibit the growth of cancer cells or kill cancer cells.

[0091] The oligonucleotide variant of the present invention can combine the modified nucleic acid (N) with an oligonucleotide having a specific sequence, thereby reducing the rate at which the modified nucleic acid is degraded by enzymes in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Shown are calibration curves for dFdC and dFdU used to determine the concentrations of gemcitabine (dFdC) and the inactive metabolite of gemcitabine, 2,2'-difluorodeoxyuridine (dFdU), in plasma.

[0093] Figure 2 Shown (Gem) 2 -[TGG] 4 [TTG][TGG] 4 (IO101) Evaluation results of in vitro anticancer efficacy on pancreatic cancer cell lines.

[0094] Figure 3 The results of confirming apoptosis of the pancreatic cancer cell line BXPC treated with IO101 are shown.

[0095] Figure 4 The results of confirming apoptosis of the pancreatic cancer cell line mia-paca-2 treated with IO101 are shown.

[0096] Figure 5 The results of confirming apoptosis of the pancreatic cancer cell line Panc-1 treated with IO101 are shown.

[0097] Figure 6 The results of confirming apoptosis of the pancreatic cancer cell line Capan-1 treated with IO101 are shown.

[0098] Figure 7 Shows evaluation (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The results of in vitro anticancer efficacy of (IO101L) on pancreatic cancer cell lines.

[0099] Figure 8 Shown are changes in plasma concentrations of gemcitabine after administration of gemcitabine (●, n=4) or IO101L (○, n=4) to rats.

[0100] Fig. 9 Shown are the plasma concentrations of dFdU after administration of gemcitabine (●, n=4) or IO101L (○, n=4) to rats.

[0101] Fig.10 Shown are the results of determining the size of tumors 30 days after injection of sol-gel type IO101-0.5 mg / AC (atelopeptide collagen), IO101-1.0 mg / AC, IO101-1.5 mg / AC, and IO101-2.0 mg / AC into mice with subcutaneous pancreatic cancer, respectively.

[0102] Fig.11 The relative changes in tumor size within 30 days after injection of sol-gel forms of IO101-1.0 mg / AC and IO101-2.0 mg / AC, respectively, into mice bearing subcutaneous pancreatic cancer are shown.

[0103] Fig.12 The results of confirming the anticancer therapeutic effect produced by changing the concentration of atelocollagen while keeping the IO101 dose fixed at 2 mg are shown.

[0104] Fig.13 A graph showing changes in pancreatic cancer tumor size in mice after implantation of IO101 / AC discs.

[0105] Fig.14 Shown are pancreatic cancer tumor sizes in mice after implantation of IO101 / AC discs.

[0106] Fig.15Shown are measurements of pancreatic cancer tumor size over time following implantation of the IO101 / AC disc.

[0107] Fig.16 Shown are graphs comparing tumor size before and after implantation of IO101 / AC discs and Gem / AC discs, respectively.

[0108] Fig.17 Shown are the observation results and comparison between IO101 / AC disc and Gem / AC disc regarding tumor inhibition and intraperitoneal metastasis of pancreatic cancer, respectively.

[0109] Fig.18 The effect of inhibiting intraperitoneal metastasis of pancreatic cancer after implantation of IO101 / AC and IO101L / AC discs, respectively, is shown.

[0110] Fig.19 Shown are changes in the survival rate of mice after implantation of IO101L / AC discs.

[0111] Fig. 20 Tumor inhibition or metastasis inhibition after removal of residual tumors followed by implantation of IO101L / AC sol-gel or insert discs in an orthotopic mouse model of pancreatic cancer was shown.

[0112] Fig.21 Demonstrates the process used to generate the Capan-1 pancreatic cancer mouse model.

[0113] Fig. 22 The effect of suppressing intraperitoneal metastasis 1 month after implantation of IO101L / AC discs in the Capan-1 pancreatic cancer mouse model is shown.

[0114] Fig.23 Shown are the results of metastases in the liver, diaphragm, and kidney 1 month after implantation of atelocollagen disks (control) in the Capan-1 pancreatic cancer mouse model.

[0115] Fig.24 Shown are histologically similar features of primary tumors compared to patient-derived xenograft (PDX) pancreatic cancer models using patient-derived pancreatic cancer cells.

[0116] Fig.25 The tumor inhibition effect after implantation of control (untreated), Gem-IP, IO101-IP, IO101 / AC disc (2.0 mg / 3.0%), IO101-Con / AC disc (2.0 mg / 3.0%) and Gem / atelocollagen disc (0.12 mg / 3.0%) into the PDX mouse model is shown.

[0117] Fig.26 Shown are the results of apoptosis analysis by TUNEL staining in pancreatic cancer tumors.

[0118] Fig. 27 Results demonstrating the absence of adverse effects in other organs following implantation of IO101 / AC discs in a PDX model are shown.

[0119] Fig.28 Results are shown for cell proliferation inhibitory efficacy of BxPC3 (pancreatic cancer), MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer), and Mv4-11 (AML).

[0120] Fig.29 Results are shown for cell proliferation inhibitory efficacy of BxPC3 (pancreatic cancer), MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer), and Mv4-11 (AML). DETAILED DESCRIPTION

[0121] The present invention provides oligonucleotide variants in which the oligonucleotide and the modified nucleic acid are linked.

[0122] The oligonucleotide of the present invention has [TGG] m [TTG][TGG] n sequence.

[0123] Herein, n may be an integer of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In addition, n may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.

[0124] m may be an integer of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In addition, m may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.

[0125] The oligonucleotide of the present invention may be one of the sequences listed in Table 1 below, but is not limited thereto.

[0126] [Table 1]

[0127]

[0128]

[0129] The oligonucleotide of the present invention is rich in guanosine to form a G-quadruplex structure, and is an aptamer specific for nucleolin.

[0130] The term "nucleolin" as used herein is a protein expressed at high levels in transformed cells, and it is known that most tumor cells express nucleolin in their cytoplasm and nucleus, as well as exposing nucleolin on the cell surface. Nucleolin has multiple functions in cells and can be involved in ribosome production, cell growth, and DNA replication.

[0131] The oligonucleotides of the present invention can more selectively bind to cancer cells and inhibit the growth of cancer cells through various mechanisms in cells.

[0132] One or more modified nucleic acids may be bound to the oligonucleotides of the present invention to stabilize the modified nucleic acids or prevent the modified nucleic acids from being inactivated in vivo.

[0133] Furthermore, when one or more modified nucleic acids are bound to an oligonucleotide, not only the cell growth inhibitory effect of the oligonucleotide itself but also the cell growth inhibitory effect of the modified nucleic acid can be exhibited, thereby improving the anticancer effect.

[0134] The term "modified nucleic acid" as used herein may be a chemically modified nucleoside or nucleotide.

[0135] The modified nucleic acid can be deoxyuridine (dU), deoxycytidine (dC), uridine (U) or cytidine (C), wherein at least one halogen or hydroxyl group is bound to the 5-position or 2'-position thereof. For example, 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'-difluorodeoxycytidine, capecitabine and bromovinyl deoxyuridine, or a derivative thereof.

[0136] The modified nucleic acid may be attached in at least one of the 5' and 3' directions of the oligonucleotide, and preferably in the 5' direction of the oligonucleotide.

[0137] The modified nucleic acid may be linked via a linker in at least one of the 5' and 3' directions of the oligonucleotide.

[0138] The linker can be [-(CH 2 ) a -]、[-(CH 2 CH 2 O) b -], [butylamidomethyl-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-], [1',2'-dideoxyribose-] or (PEG) yHere, a may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6. b may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6. y may be an integer of 1 to 20, 2 to 19, 3 to 18, 4 to 17, 5 to 16, 6 to 15, 7 to 14, 8 to 13, 9 to 12, or 10 to 11.

[0139] When the modified nucleic acid is linked in the 3' direction of the oligonucleotide, idT, LNA, PEG or 2'OMeNu may be further linked in the 3' direction of the modified nucleic acid.

[0140] By further linking idT, LNA, PEG or 2'OMeNu in the 3' direction of the modified nucleic acid, the 3' end of the oligonucleotide variant can be protected from attack by nucleases, thereby reducing the in vivo degradation rate of the oligonucleotide variant. Therefore, the modified nucleic acid can be bound to the oligonucleotide for a longer time, thereby increasing the anti-cancer efficacy of the modified nucleic acid.

[0141] Specifically, the oligonucleotide variant of the present invention may be a compound having a structure of Formula 1:

[0142] [Formula 1]

[0143] (N) x -[TGG] m [TTG][TGG] n -(M) y

[0144] In Formula 1, N and M are modified nucleic acids, and N and M may be the same or different types of modified nucleic acids. The details of the modified nucleic acid are the same as described above.

[0145] In Formula 1, x and y may independently be an integer of 0 to 10, except for the case where x and y are both 0.

[0146] For example, in Formula 1, x may be an integer of 0 to 10, 1 to 9, 2 to 8, 3 to 7, or 4 to 6, and y may be an integer of 0 to 10, 1 to 9, 2 to 8, 3 to 7, or 4 to 6. However, the case where x and y are both 0 is excluded.

[0147] In Formula 1, n may be an integer of 1 to 10, and m may be an integer of 1 to 10.

[0148] In Formula 1, n may be an integer of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In addition, in Formula 1, n may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.

[0149] In Formula 1, m may be an integer of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. For another example, m in Formula 1 may be an integer of 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.

[0150] For example, the oligonucleotide variant having the structure of Formula 1 may be a compound having the structure of the formula listed in Table 2 below.

[0151] [Table 2]

[0152]

[0153]

[0154]

[0155] According to one embodiment, the oligonucleotide variant of the present invention may be one in which gemcitabine (which may be represented as 2',2'-difluorodeoxycytidine) is bound to at least one of the 5' and 3' directions of the oligonucleotide comprising the sequence as shown in SEQ ID NO: 10. For example, the oligonucleotide variant of the present invention may be one in which 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less gemcitabine is bound to the 5' direction of the oligonucleotide comprising the sequence as shown in SEQ ID NO: 10.

[0156] According to another embodiment, the oligonucleotide variant of the present invention may be one in which at least one gemcitabine is linked in the 5' or 3' direction of the oligonucleotide comprising the sequence as shown in SEQ ID NO: 11. For example, the oligonucleotide variant of the present invention may be one in which 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less gemcitabines are bound in the 5' direction of the oligonucleotide comprising the sequence as shown in SEQ ID NO: 11.

[0157] A method for ligating an oligonucleotide and a modified nucleic acid may include the step of binding the modified nucleic acid to at least one of the 5' end and the 3' end of the oligonucleotide.

[0158] Another method for linking an oligonucleotide and a modified nucleic acid may include: linking a linker to at least one of the 5' end and the 3' end of the above-mentioned oligonucleotide by chemical synthesis; and then, combining the modified nucleic acid with the above-mentioned linker.

[0159] The incorporation of modified nucleic acids for the preparation of oligonucleotide variants may involve the use of a solid phase reactor.

[0160] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above oligonucleotide variant or a pharmaceutically acceptable salt thereof.

[0161] Oligonucleotide variants are the same as above.

[0162] The above-mentioned cancer can be a nucleolin-related cancer, such as solid cancer and leukemia. For example, the cancer can be selected from the group consisting of: leukemia, lymphoma, myeloproliferative disease, solid tissue cancer, sarcoma, melanoma, adenoma, hypoxic tumor, oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, lung squamous cell carcinoma, uterine cancer, bladder cancer, hematopoietic system cancer, head and neck cancer, nervous system cancer and papilloma, but not limited to this. In addition, the nucleolin-related cancer can be selected from the group consisting of: leukemia, lymphoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, cervical cancer, glioma cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, hepatoma, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma and cervical cancer, but not limited to this.

[0163] The content of the oligonucleotide variant or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition for preventing or treating cancer may be an amount capable of exhibiting an effect on preventing or treating cancer, and may be appropriately adjusted according to the condition and / or disease severity of the subject.

[0164] The atelocollagen dispersion may contain 0.5 g to 5.5 g, 1 g to 4.5 g, or 2 g to 3.5 g of atelocollagen per 100 ml of PBS solution. The atelocollagen concentration of the atelocollagen dispersion may be 0.5% to 5.5%, 1% to 4.5%, or 2% to 3.5%.

[0165] Depending on the concentration of atelocollagen contained in the pharmaceutical composition of the present invention, a difference may occur in the duration or therapeutic effect of the drug. Therefore, an appropriate concentration of atelocollagen should be contained in the composition.

[0166] The atelocollagen contained in the pharmaceutical composition of the present invention may be present in the form of surrounding the oligonucleotide variant contained in the composition.

[0167] The atelocollagen of the present invention can be prepared by a method comprising the following steps: a) swelling fresh pig skin in acetic acid, removing the fat layer, crushing the dermis part and suspending it in acetic acid, and then adding at least one of alkaline protease, catalase, pepsin and papain to the suspension to extract the substance; b) first filtering the extracted substance and salting it out by adding a neutral salt to the obtained filtrate, followed by a second filtration; c) dissolving the residue obtained by the second filtration to adsorb fat using fumed silica, followed by a third filtration; and d) freeze-drying the filtrate obtained by the third filtration to obtain high-purity atelocollagen.

[0168] The atelocollagen of the present invention can be prepared by a method comprising the following steps: a) treating collagen-containing animal tissue with at least one of alkaline protease, catalase, pepsin and papain to extract a substance; b) first filtering the extracted substance; c) adding a neutral salt to the filtrate obtained by the first filtration and salting out the extracted substance, followed by a second filtration; d) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; e) freeze-drying the filtrate obtained by the third filtration and recovering the freeze-dried powder; and f) dissolving the freeze-dried powder in dilute hydrochloric acid (dil-HCl), dilute acetic acid or phosphate buffer at pH 4 to pH 8 and concentrating to prepare an atelocollagen solution, injecting the prepared atelocollagen solution into a column filled with polymer beads, and developing the column in dilute hydrochloric acid, dilute acetic acid or phosphate buffer at pH 4 to pH 8, thereby recovering the atelocollagen according to molecular weight.

[0169] The neutral salt used in the method for preparing atelocollagen may be a sodium chloride solution.

[0170] In the method for preparing atelocollagen, the lyophilized powder may be dissolved in dilute acetic acid at a concentration of 10 mM or in a phosphate buffer solution at a concentration of 10 mM.

[0171] In the method for preparing atelocollagen, the lyophilized powder can be concentrated with MWCO 100K Dalton.

[0172] In the method for preparing atelocollagen, the polymer beads injected with the atelocollagen solution may be Sephadex G-200 Sephacryl.

[0173] In the method for preparing atelocollagen, the volume of the atelocollagen solution injected into the column filled with polymer beads may be 5% to 20% of the column bed volume.

[0174] In the method for preparing atelocollagen, the atelocollagen solution may be injected into a column filled with polymer beads and then developed with 10 mM dilute acetic acid or 10 mM phosphate buffer to recover the atelocollagen.

[0175] The pharmaceutical composition of the present invention can be used in various preparations.

[0176] For example, the preparation may include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders, sol-gel forms, stent forms or patch forms (disk forms), but are not limited thereto. The pharmaceutical composition of the present invention may be a telopeptide collagen dispersion preparation.

[0177] The pharmaceutical composition of the present invention can be administered to a subject in need of treating and / or inhibiting abnormally proliferating cells.

[0178] The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other therapeutic agents. In addition, when administered in combination with other therapeutic agents, they can be administered sequentially or simultaneously, and can be administered in single or multiple doses. When administering the pharmaceutical composition of the present invention, it is important to administer an amount that can ensure maximum effect without side effects in a minimum amount, which amount can be easily determined by those skilled in the art.

[0179] Administration may be oral or parenteral, and may include, for example, intravenous injection, subcutaneous injection, or intramuscular injection, but is not limited thereto.

[0180] The subject to be administered may be a mammal such as a primate, mouse, rat, hamster, rabbit, horse, cow, dog or cat, but is not limited thereto.

[0181] Furthermore, the present invention provides a method for preparing the pharmaceutical composition for preventing or treating cancer as described above.

[0182] The method for producing a pharmaceutical composition according to the present invention may comprise preparing a dispersion in which the above-mentioned oligonucleotide variant is dispersed.

[0183] The dispersion liquid in which the oligonucleotide variant is dispersed can be prepared by mixing the oligonucleotide variant with PBS.

[0184] The method for producing the pharmaceutical composition according to the present invention may further comprise the step of mixing the dispersion in which the above-mentioned oligonucleotide variant is dispersed with the atelocollagen dispersion.

[0185] The dispersion liquid in which the oligonucleotide variant is dispersed can be prepared by mixing the oligonucleotide variant with PBS.

[0186] The atelocollagen dispersion can be prepared by adding atelocollagen to a NaOAc / HAc (acetic acid) buffer solution.

[0187] The atelocollagen dispersion can be prepared by adding 0.5 g to 5.5 g, 1 g to 4.5 g, or 2 g to 3.5 g of atelocollagen per 100 ml of the buffer solution.

[0188] The buffer solution may be under the condition of 0.3 M NaOAc and 45% HAC.

[0189] The atelocollagen added to the buffer solution may be lyophilized atelocollagen.

[0190] The step of mixing the dispersion in which the oligonucleotide variant is dispersed with the atelocollagen dispersion may include mixing 0.1 mg to 3 mg of the dispersion in which the oligonucleotide variant is dispersed per 400 μL of the atelocollagen dispersion.

[0191] For example, the dispersion in which the oligonucleotide variant is dispersed may be mixed in an amount of 0.1 mg to 3 mg, 0.5 mg to 2.5 mg, or 1 mg to 2 mg per 400 μL of the atelocollagen dispersion.

[0192] Hereinafter, examples will be described in detail to specifically describe the present invention. However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the examples.

[0193] Example 1, Preparation of oligonucleotide variants

[0194] Example 1-1. Synthesis of Gemcitabine-Containing Oligonucleotides

[0195] Designed and prepared 28 types of oligonucleotide. In addition, gemcitabine is combined with each in the prepared oligonucleotide to produce oligonucleotide variants. Designed 28 types of oligonucleotide sequences and 28 types of oligonucleotide variants (wherein modified nucleic acid is combined with oligonucleotide) are listed in the following table 3. In the following description, gemcitabine may also be referred to as Gem.

[0196] [Table 3]

[0197]

[0198]

[0199]

[0200] "Gemcitabine-containing oligonucleotides" (oligonucleotide variants) were synthesized by solid phase phosphoramidite chemistry using a Mermade 12 DNA synthesizer (BioAutomation Manufacturing, Irging, TX).

[0201] Desalting was performed using Biotage MPLC, C18 Cartridge. After the compound was dissolved in DW, under the conditions of Xbridge Oligonucleotide BEH C18 130A, 1.7 μm, 2.1x50mm column, the solution was separated and purified using Waters Acquity UPLC H-Class at a flow rate of 0.3 mL / min at 50°C column oven temperature, mobile phase A solvent (0.1 M TEAA), B solvent (100 ACN). Molecular weight was confirmed by Waters G2-XS Q-TOF mass spectrometer. All oligonucleotides were synthesized according to internal protocols.

[0202] 1-2. Synthesis of different types of oligonucleotide variants

[0203] According to the same procedure as described in 1-1 above, ((N) x -[TGG] 4 [TTG][TGG] 4或5 )、(([TGG] 4 [TTG][TGG] 4或5 -(N) x ) and ((N) x -[TGG] 4 [TTG][TGG] 4或5 -(N) y )(See Table 4 below).

[0204] [Table 4]

[0205]

[0206]

[0207]

[0208] Example 2. Confirmation of in vitro stability of oligonucleotide variants

[0209] Gemcitabine (2,2'-difluorodeoxycytidine, dFdC) is converted into an inactive metabolite, 2,2'-difluorodeoxyuridine (dFdU), by cytidine deaminase in plasma, and in this case, the anticancer efficacy in vivo is reduced. In order to confirm the inhibitory effect of cytidine deaminase on the degradation of gemcitabine in vivo when gemcitabine is conjugated to oligonucleotides, the concentrations of dFdC and dFdU in plasma were measured.

[0210] 2-1. Liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis for determination of plasma concentrations of dFdC and dFdU

[0211] Mouse plasma and tissue assays were established by modifying the liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay to confirm the concentrations of gemcitabine triphosphate (dFdCTP) and dFdC, dFdU, as active metabolites of gemcitabine in mouse plasma. From the chromatograms obtained by the established LC-MS / MS assay method, the peak area ratios of dFdC, dFdU, and dFdCTP to the peak area of ​​the internal standard were estimated, and then the concentrations in mouse plasma and tissues were calculated based on the calibration curves previously created.

[0212] 2-1-1. Preparation of blood treated with tetrahydrouridine (THU)

[0213] 1mL mouse blood is collected and placed in a 1.5mL eppendorf tube containing 10 μL (in DW) 10mg / mL tetrahydrouridine (THU). After centrifugation for 2 minutes at 14,000rpm and 4°C using a centrifuge, only plasma is taken out and transferred to another vial and stored at -80°C. After 50 μL plasma is transferred to a 1.5mL amber eppendorf tube, 150 μL internal standard substances (melt of 1.29 μg / mL in ACN) are added and vortexed for 10 minutes. Centrifuge is used to centrifuge for 20 minutes at 14,000rpm and 4°C. 150 μL samples are transferred from a centrifuge tube to another 1.5mL amber eppendorf tube. GeneVac EZ-2 automatic evaporation system (GeneVac Ltd., Ipswich, UK) is used to dry the organic solvent. (GeneVac HPLC mode, maximum temperature 35°C, 1 hour). Organic solvent was reconstituted by adding 125 μL DW to a 1.5 mL dried amber eppendorf tube. After vortexing for 5 minutes, centrifuge was used to centrifuge for 5 minutes at 14,000 rpm and 4 ° C. Using a PTFE (hydrophilic) 0.2 μm syringe filter (Toyo Toshi, Japan), the supernatant of the centrifuged solution was added to each vial for LC-MS / MS in an amount of 100 μL. Specifically, 5 μL supernatant was injected into each LC-MS / MS.

[0214] 2-1-2. LC-MS / MS analysis conditions

[0215] For mass spectrometry, an AB SCIEX QTRAP 5500 electrospray ionization mode (Sciex, Framingham, MA, USA) was used. The quantification conditions are listed in Table 5 below.

[0216] [Table 5]

[0217]

[0218] Chromatography was performed using an Agilent 1200 series separation module (Agilent Technologies, Waldbronn, Germany). In addition, a Hypersil gold C18, 1.9 μm, 100 x 2.1 mm 2 Column (ThermoScientific, Matriks, Oslo, Norway). The injection volume was 5 μL, the mobile phase was a gradient eluent system [A: 100% ACN, B: 0.1% formic acid in DW], and the total run time was 20 minutes. The calibration range was set to 20 ng / mL to 5,000 ng / mL. The chromatographic conditions are listed in Table 6 below.

[0219] [Table 6]

[0220]

[0221] Figure 1 A dFdC calibration curve and a dFdU calibration curve are shown. Figure 1 In (a), x is the gemcitabine concentration, and y is the area in HPLC. Figure 1 In (b), x is the dFdU concentration, and y is the area in HPLC.

[0222] 2-1-3. LC-MS / MS analysis results

[0223] Each sample was processed according to 2-1-1 and 2-1-2 above, and about (Gem) x -[TGG] m [TTG][TGG] n The dFdC / dFdU ratio in rat plasma was measured for the compound type. The measurement results are listed in Table 7 below (Gem = gemcitabine).

[0224] [Table 7]

[0225]

[0226]

[0227] Several compounds with high dFdC / dFdU ratios were selected (compound 10, also referred to as IO101 hereinafter; and compound 11, also referred to as IO101L hereinafter) and then additional experiments were performed to determine the effects of the oligonucleotide variants. In addition, in order to compare the effects of IO101 and IO101L with other oligonucleotide variants, experiments were performed using oligonucleotide variants of different sequences such as the following and gemcitabine not bound to the oligonucleotides: [(Gem) 2 -[TCC] 4 [TTG][TCC] 4 IO101-Con(CRO), (Gem) 2 -GGTGGTGGTGGTTGTGGTGGTGGTGGTGG; IO100 and (Gem) 2 -CCTCCTCCTCCTTCTCCTCCTCCCTCTCC;IO100-Con((Gem) 2 -CRO)).

[0228] Example 3. Evaluation of in vitro efficacy of oligonucleotide variants against cancer cell lines

[0229] 3-1. Confirmation of the anticancer efficacy of IO101 on pancreatic cancer cell lines

[0230] BxPC3, PANC-1, Miapaca-2, and Capan-1 were cultured as pancreatic cancer cell lines. When the cells became stable after several subcultures, 1×10 3 Cells were seeded in 96-well plates to verify the drug effects. The next day, IO101, IO100, IO100-Con, IO101-Con and Gem with a maximum concentration of 100 μM were diluted 1 / 10 per well and prepared with a 4-point concentration of up to 100 nM. Then, the cells seeded in 96-well plates were treated with the variants prepared above. After 48 hours, 10 μl of WST-1 solution was added to the wells and incubated in a 37°C incubator for 1 hour. Subsequently, each sample was measured at 440 nm in a microplate reader (VERSAMax). After calculating the measured values, a cell viability graph was drawn for each treatment concentration.

[0231] In all four pancreatic cancer cell lines, (Gem) was observed compared with IO100, IO100-Con, IO101-Con, and Gem. 2 -[TGG] 4 [TTG][TGG] 4 (IO101) effectively inhibits the growth of pancreatic cancer cells in proportion to drug concentration (see Figure 2 ).

[0232] In 1x10 4 Four types of pancreatic cancer cell lines were seeded at 10 cells / well and then treated with 10101. The treated cell lines were labeled with a red probe and imaged within 48 hours to observe apoptosis of cancer cells. Figures 3 to 6 The results of confirming apoptosis of 4 types of pancreatic cancer cells treated with IO101 are shown. The intensity value is the death cell index and is indicated as a fold change.

[0233] 3-2. Confirmation of the anticancer effect of IO101L on pancreatic cancer cell lines

[0234] Verification by cell viability assay in the same manner as the efficacy evaluation in 3-1 above (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The results showed that the growth of pancreatic cancer cells was effectively inhibited by IO101L in all four pancreatic cancer cell lines compared with the control (see Figure 7 ).

[0235] Example 4. Confirmation of in vivo stability of oligonucleotide variants

[0236] Gemcitabine (8 mg / kg) was injected intravenously in rats, or (Gem) which is a new agent prepared by combining gemcitabine with oligonucleotides was injected intravenously. x -[TGG] m [TTG][TGG] n (160 mg / kg, which is equivalent to 8 mg / kg based on gemcitabine) to compare the pharmacokinetic changes between gemcitabine and its metabolite (i.e., 2',2-difluorodeoxyuridine (dFdU) cyanidin-3-glucoside).

[0237] 4-1. Intravenous injection of oligonucleotide variants or gemcitabine in rats

[0238] Sprague-Dawley male rats were anesthetized by induction with isoflurane as an inhalation anesthetic, and then a polyethylene (PE) tube (Clay Adams, Becton Dickinson, NJ, USA) was inserted into the carotid artery (for blood collection) and sutured with sutures, after which the ends of the sutures were fixed to the back of the neck. During the operation, ether was used to maintain anesthesia, and approximately 0.5 mL of normal saline containing heparin (20 units / mL) was injected to prevent blood from clotting in the cannula. After the operation, the rats were placed in metabolic cages and allowed to fully recover from anesthesia (4 to 5 hours). Thereafter, the animals were divided into (Gem) 2 -[TGG]4 [TTG][TGG] 5 The gemcitabine and gemcitabine were administered to the 4 groups (N=4) and the corresponding drugs were administered to these groups. Gemcitabine and gemcitabine were measured on an electronic balance (CP224S, Sartorius, GER). 2 -[TGG] 4 [TTG][TGG] 5 After weighing to a certain dose (8 mg / kg and 160 mg / kg), each drug was dissolved and prepared in 0.9% sterile saline. 1 ml / kg and 2 ml / kg of the drug were administered intravenously. 2 -[TGG] 4 [TTG][TGG] 5 Before (0min) and gemcitabine and after 1,5,15,30,45,60,120,360 and 720 minutes of administration, 0.3mL blood was collected by jugular vein respectively.In addition, in order to prevent the gemcitabine in the blood from being metabolized by cytidine deaminase, the blood collected was put into the eppendorf tube previously placed with 10 μL distilled water, wherein the distilled water contained tetrahydrouridine (THU) as cytidine deaminase inhibitor dissolved with the concentration of 10mg / mL of every 1mL blood.Then, the blood in the centrifugal eppendorf tube was stored with plasma afterwards.Due to known gemcitabine can be degraded by light, all plasma samples were placed in brown eppendorf tubes with 50 μL of every tube, and stored at-80 ℃, until LC-MS / MS analysis was performed.

[0239] 4-2. Analysis of plasma concentrations of gemcitabine and its metabolite dFdU

[0240] After pretreatment by plasma protein precipitation using pre-established acetonitrile plasma, sample processing and analysis of plasma concentrations of gemcitabine and dFdU were performed by LC-MS / MS using an Agilent 1200 series analyzer (Agilent Technologies) in AB SCIEX QTRAP 5500 electrospray ionization mode (Sciex, Framingham, MA, USA). All analytical procedures were implemented under shielded tube conditions. That is, all procedures were performed under the following conditions: quantitative conditions: SRM (selected reaction monitoring) mode (see Table 5 above); stationary phase conditions: Hypersil gold C18, 1.9 μm, 100x2.1 mm 2 (Thermo Scientific, Matriks, Oslo, Norway); and mobile phase conditions: distilled water (A) and acetonitrile (B) containing 0.1% formic acid (see Table 6 above).

[0241] 4-3. Creation of plasma calibration curve

[0242] Gemcitabine and dFdU were dissolved in distilled water to prepare a 1 mg / mL stock solution, stored frozen and diluted with tertiary distilled water to prepare working solutions with gemcitabine concentrations of 200, 400, 800, 2000, 10000, 20000 and 100000 ng / mL and dFdU concentrations of 200, 400, 800, 2000, 10000, 20000 and 50000 ng / mL, and then stored in a refrigerator. Metformin was dissolved in acetonitrile as an internal standard (IS) to prepare another working solution of 645 ng / mL. Gemcitabine and dFdU working solutions were added to empty plasma of rats, and standard plasma samples were prepared so that the plasma concentrations of gemcitabine and dFdU were 10, 20, 40, 100, 500, 1000 and 5000 ng / mL and 10, 20, 40, 100, 500, 1000 and 2500 ng / mL, respectively.

[0243] 4-4. Method for processing plasma samples

[0244] 150 μL of internal standard (i.e., metformin) was added to 50 μL of plasma (645 ng / mL in acetonitrile) and vortexed for 10 minutes, followed by centrifugation at 15,000 rpm and -4°C for 20 minutes. Thereafter, 150 μL of supernatant was taken and transferred to another eppendorf tube, and the organic solvent was then dried under a stream of nitrogen. Then, 125 μL of tertiary distilled water was added to the dried eppendorf tube to redissolve it. Then, the solution was vortexed for 5 minutes and centrifuged at 15,000 rpm and -4°C for 5 minutes. Thereafter, 100 μL of supernatant was transferred to a vial for LC-MS / MS, 5 μL of supernatant was injected and analyzed by LC-MS / MS.

[0245] 4-5. Determination of analytical suitability

[0246] To determine the suitability of the analytical process, samples used to create calibration curves were analyzed for each batch of analytical samples during sample processing, and then samples for analytical suitability were analyzed twice (for gemcitabine, lower limit of quantification: LLoQ, 10 ng / mL, low concentration: LoQC, 30 ng / mL, medium concentration: MiQC, 900 ng / mL, high concentration: HiQC, 4000 ng / mL; for dFdU, lower limit of quantification: LLoQ, 10 ng / mL, low concentration: LoQC, 30 ng / mL, medium concentration: MiQC, 900 ng / mL, high concentration: HiQC, 2000 ng / mL). At least 67% of the 6 suitable samples (e.g., 4 of the 6 samples) should be within ±15% of the theoretical value, and whether 50% or more at the same concentration are within ±15% of the theoretical value was investigated.

[0247] 4-6. Calculation and statistical processing of pharmacokinetic parameters

[0248] The pharmacokinetic parameters of gemcitabine were obtained by Winnonlin Professional program (Pharsight, Mountain View, CA, USA). Plasma concentration-time curves (AUC t ) was calculated from the plasma concentration-time curve from the administration of the drug to the final quantitative time using a logarithmic-linear trapezoidal equation (specifically, the linear-trapezoidal equation was used in the portion where the plasma concentration increased, and the trapezoidal equation was used by logarithmically transforming the concentration values ​​in the other portion where the plasma concentration decreased). After oral administration, the maximum plasma concentration (C max ) and the time to reach the maximum plasma concentration (T max The area under the plasma concentration (AUC) of the time-time curve up to infinity was determined using the following equation: inf The terminal elimination rate constant (γZ) and half-life (t 1 / 2 ).

[0249] AU inf =AUC t +C t / γZ(C t : final quantitative concentration, γZ: terminal elimination rate constant)

[0250] To compare the pharmacokinetic parameters between the two groups, independent sample t-test was performed using SPSS (version 19, Chicago, IL, USA).

[0251] 4-7. Comparison of in vivo pharmacokinetic properties between gemcitabine and dFdU

[0252] About Gemcitabine (8mg / kg) or (Gem) 2 -[TGG] 4 [TTG][TGG] 5 (160 mg / kg, which is equivalent to 8 mg / kg based on gemcitabine) intravenous injection, the plasma concentration of gemcitabine is shown in Figure 8 The plasma concentrations of dFdU are shown in Fig. 9 In addition, in gemcitabine or (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The pharmacokinetic parameter values ​​of gemcitabine and dFdU in the intravenous injection of are listed in Table 8 as mean ± standard deviation. In the following Table 8, AUC t is the area under the curve from 0 min to the last blood collection time, t 1 / 2 is the terminal half-life (plasma elimination half-life), CL is the systemic clearance, Vd ss is the volume of distribution in the body, and MRT is the mean residence time of the drug in the body, T max is the median (range), and metabolic conversion rate values ​​are calculated by using dFdU AUC t Divide the value by the gemcitabine AUC t value calculated based on the value.

[0253] [Table 8]

[0254]

[0255] The results of the experiment showed that the gemcitabine administration group reached the highest plasma concentration immediately after administration, as is the general pharmacokinetic pattern after intravenous injection of the drug. 2 -[TGG] 4 [TTG][TGG] 5 The administration group reached C 5 to 30 minutes after intravenous injection. max . Intravenous injection (Gem) 2 -[TGG] 4 [TTG][TGG] 5 Plasma gemcitabine concentrations are determined by the degradation of oligonucleotides by nucleases ("nucleases") in vivo, namely [TGG] 4 [TTG][TGG] 5 The results of preliminary experiments confirmed that during the plasma sample treatment (organic solvents such as acetonitrile or methanol), (Gem) 2 -[TGG] 4 [TTG][TGG] 5Since gemcitabine is a drug with very low plasma protein binding, it is not released in the form of gemcitabine when injected intravenously in rats (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The gemcitabine concentration measured in plasma afterwards indicates the free concentration of gemcitabine generated by nucleases in vivo. (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The administration group showed significantly lower gemcitabine plasma concentration than the gemcitabine administration group until 120 minutes. However, after 120 minutes (360 minutes, 720 minutes), (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The administration group showed a higher gemcitabine concentration than the gemcitabine administration group (see Figure 8 ). This means [TGG] 4 [TTG][TGG] 5 It is metabolized to gemcitabine by nucleases in the body, and gemcitabine is slowly released into the plasma. As a result, (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The plasma elimination half-life of gemcitabine in the administration group (t 1 / 2 ) was statistically significantly increased by about two times (177 ± 28.5 minutes compared to 360 ± 54.8 minutes; see Table 8 above). In addition, it can be understood that (Gem) 2 -[TGG] 4 [TTG][TGG] 5 Vd of gemcitabine in the administration group ss (Volume of distribution in vivo), which indicates the affinity between the drug and the tissues in the body, increased significantly by more than two times. (1150±223 mL / kg vs. 2670±139 mL / kg; see Table 7 above). That is, it can be predicted that (Gem) 2 -[TGG] 4 [TTG][TGG] 5 The gemcitabine in the administration group had a higher distribution in the tissues in the body, and thus was distributed to the tissues much more than the gemcitabine administration group. In both groups, the AUC of gemcitabine t and AUC inf The values ​​were similar and did not show statistical significance. 2 -[TGG] 4 [TTG][TGG] 5In the case of the gemcitabine administration group, the plasma concentration of dFdU, an inactive metabolite of gemcitabine, was detected until 360 minutes after administration, which was lower than that of the gemcitabine administration group (see Fig. 9 ). Due to the binding to [TGG] 4 [TTG][TGG] 5 Gemcitabine is not metabolized to dFdU, so the dFdU concentration measured in plasma means the concentration of gemcitabine in the blood after intravenous injection (Gem) 2 -[TGG] 4 [TTG][TGG] 5 Later, when gemcitabine was taken from [TGG] 4 [TTG][TGG] 5 The concentration of dFdU generated by cytidine deaminase in plasma during release. After 360 minutes, (Gem) 2 -[TGG] 4 The [TTG][TGG]5 administration group showed higher dFdU plasma concentrations, however, this could not be further confirmed due to the relatively short sampling time limit. (Gem) 2 -[TGG] 4 [TTG][TGG] 5 C of dFdU in the administration group max The values ​​showed a decreasing trend (364 ± 52.3 ng / mL vs. 477 ± 87.9 ng / mL), but no statistical significance was shown between the two groups (see Table 8 above). In order to compare the ratio of dFdU generated by cytidine deaminase in the two groups, the dFdU AUC t Divide the value by the gemcitabine AUC t The metabolic conversion rate was calculated using the values ​​of (Gem) and the results showed that the two groups showed similar values ​​(0.190 ± 0.019 vs. 0.181 ± 0.0124), which was not statistically significant. 2 -[TGG] 4 [TTG][TGG] 5 In the case of binding to [TGG] 4 [TTG][TGG] 5 The gemcitabine is slowly released into plasma and distributed to tissues in large quantities, but the released gemcitabine does not affect the dFdU produced by cytidine deaminase present in plasma and tissues.

[0256] Table 9 below shows the effects of gemcitabine and (Gem) 2 -[TGG] 4 [TTG][TGG] 5 Gemcitabine plasma concentrations over time.

[0257] [Table 9]

[0258]

[0259] Table 10 below shows the effects of gemcitabine and (Gem) 2 -[TGG] 4 [TTG][TGG] 5 BLLoQ means value below the minimum limit of quantitation, and ND means value not detected.

[0260] [Table 10]

[0261]

[0262] Example 5. Preparation of medical atelocollagen

[0263] 5-1. Pigskin preparation steps

[0264] The pigskin was washed three times with tap water, three times with primary purified water, divided into 3 kg (20 cm x 20 cm), and stored in a freezer at -20°C. The frozen pigskin was left to stand at 4°C for 2 hours, thawed, and finely cut into a size of 1.5 cm x 8 cm. Thereafter, 7.5 L of 0.5 M acetic acid was added to the pigskin, which was then left overnight and the swelling of the pigskin was observed.

[0265] 5-2. Fat removal steps

[0266] Take out the swollen pig skin, cut it into a size of 1.5cm x 1.5cm, and add 7.5L of 0.5M fresh acetic acid again. Then, after standing for several hours, filter only the dermis through a sieve. Wash the dermis with 10L of purified water. Repeat the washing process a total of five times. After adding 20L of ethanol to the washed dermis, stir the mixture overnight at 4°C. After recovering the dermis only from the sample stirred overnight, add 20L of ethanol, then stir again at 4°C for 1 hour. Filter the dermis using a sieve and stand for about 1 hour to remove ethanol. The dermis from which fat has been removed is subdivided into parts with appropriate weight (500g) and stored in a low-temperature freezer at -80°C.

[0267] 5-3. Dermis homogenization and grinding

[0268] 7.5L 0.5M acetic acid was added to 3kg of frozen dermis thawed by placing at 4°C, and then left to stand for 30 minutes. After removing the acetic acid by filtering using a sieve, the dermis was subdivided into 250g portions. 250g of dermis and 2L of purified water were placed in a mixer and then ground for 2 minutes. Then, 2L of purified water was further added and then ground again for 2 minutes. 4L 0.73M acetic acid was added to the ground tissue. The tissue was ground again using a homogenizer for 3 minutes. The grinding process was repeated four times to grind and blend 1kg of frozen dermis. In addition, 18L 0.73M acetic acid was added thereto to adjust the final acetic acid concentration to 0.5M. Subsequently, it was confirmed that the pH was about 2.5 to 4. The mixture was stirred at a low speed for 3 hours using an agitator.

[0269] 5-4. Pepsin treatment

[0270] 15×10 per kg of leather 7 Unit of pepsin was added to the finished dermis sample, followed by gentle stirring for 24 hours using a stirrer. 10M NaOH was added to the pepsin-treated sample and stirred to reach pH 8 to 9, and further stirred for 10 minutes to inactivate pepsin. After inactivating pepsin by alkali treatment, 4M HCl was added and stirred to reach pH 3.4, followed by further stirring for 10 minutes. After centrifuging the sample at 7800rpm and 4°C for 10 minutes using a centrifuge, the fat on the surface of the supernatant was removed, and the remaining supernatant was collected and stored.

[0271] 5-5. Salting out and production of atelocollagen intermediates

[0272] 5M NaCl was slowly added to 1L of the supernatant prepared from the dermis at a rate of 163ml, stirred for 15 minutes, and then allowed to stand overnight at 4°C for salting out. After salting out, the supernatant was removed by suction, and the precipitate was centrifuged (7800rpm, 4°C, 10 minutes) to completely remove the supernatant. 30L of ethanol was added to the precipitate, and the mixture was washed while stirring overnight at 4°C. After centrifugation (7800rpm, 4°C, 10 minutes), 30L of ethanol was added to the precipitate again, followed by stirring at 4°C for 6 hours for a second wash. After centrifugation (7800rpm, 4°C, 10 minutes), the weight of the precipitated atelocollagen intermediate was measured, and it was subdivided and stored in a -80°C cryogenic freezer.

[0273] 5-6. Production of medical atelocollagen

[0274] After 2.8L 0.02M urea is added to 200g atelocollagen intermediate, the mixture is stirred overnight.Use Centramate (tangential flow filtration system) to carry out the diafiltration of atelocollagen, and in agitator, add 0.5M NaOH to reach pH 7 while the solution of recovery is stirred.Prepared neutral atelocollagen is thinly and evenly subdivided, and each subdivided part is put into zipper bag afterwards, and is stored in -80 ℃ low temperature freezer.After carrying out preliminary freezing (-40 ℃) at least 1 hour in freeze dryer, the atelocollagen stored in the low temperature freezer is moved to freeze dryer and lyophilized.Freeze-dried medical atelocollagen is cut into suitable size and vacuum packed, refrigerated afterwards.

[0275] 5-7. Preparation of atelocollagen solution having neutral pH identified by substitution with buffer solution

[0276] 5-7-1. Step of dissolving freeze-dried atelocollagen in sodium acetate buffer solution (0.3 M sodium acetate (NaOAC), 45% acetic acid)

[0277] To prepare a 0.3M NaOAC, 45% acetic acid buffer solution, 2.4g CH3CO2Na was dissolved in 55ml of tertiary sterile water and 45ml of acetic acid solution with a purity of 99% or higher. Then, the solution was titrated with acetic acid using a pH meter to reach pH 3.0. 3g of freeze-dried atelocollagen was finely cut using sterile tweezers and scissors, and the finely cut pieces were dissolved in a 0.3M NaOAC, 45% acetic acid solution. Specifically, the finely cut atelocollagen was slowly added so as to be completely dissolved while being mixed with the solution by a stirring rod.

[0278] 5-7-2. Diafiltration using a tangential flow filter (TFF) system and replacement of the atelocollagen solution with PBS

[0279] As shown in the figure, 3% atelocollagen solution is pumped into the prepared TFF system (by TFF100K) using a pump, and the filtered sodium acetate buffer solution is transferred to waste and discarded, while the remaining solution is returned to the storage container by the retentate tube. 1 x PBS buffer solution is added to the storage container to maintain 3.0% atelocollagen solution at a predetermined level while continuing to perform diafiltration. Specifically, 10 times of PBS solution equivalent to the initially prepared atelocollagen-sodium acetate buffer solution is used to continue diafiltration, thereby performing buffer exchange. When buffer exchange is completed, the completion of buffer exchange is confirmed by monitoring the pH of the permeate solution to determine whether neutral pH is detected. After dialysis and filtration, 3% atelocollagen is divided into 10ml aliquots in sterilized tubes and stored in a refrigerator at 4°C.

[0280] Example 6. Generation of oligonucleotide variant / atelopeptide collagen compositions

[0281] 6-1. Preparation of atelocollagen (AC) dispersion

[0282] 0.5%, 1.0%, 1.5%, 2.0% and 3.0% of high-purity medical atelocollagen (0.5g, 1.0g, 1.5g, 2.0g and 3.0g atelocollagen per 100ml buffer) were placed in a NaOAc / HAc solution (0.3M sodium acetate, 45% acetic acid), maintained at pH 3.0 and completely dissolved while stirring. The solution was diafiltered using tangential flow filtration (TFF), and then the atelocollagen solution was diafiltered with 10 times the volume of 1 x PBS solution to prepare a medical collagen dispersion in PBS solution.

[0283] 6-2. Production of IO101 / atelopeptide collagen sol-gel type and IO101L / atelopeptide collagen sol-gel type

[0284] In Gem 2 -[TGG] 4 [TTG][TGG] 4 (IO101) or (Gem) 2 -[TGG] 4 [TTG][TGG] 5 After (IO101L) is placed in PBS and completely dissolved by a mixer at room temperature, a solution of IO101 or IO101L mixed in PBS is added to the atelocollagen dispersion in the following amounts: 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 4.0 mg or 8.0 mg (0.5%, 1.0%, 1.5%, 2.0%, 3.0%, respectively) per 400 μl of atelocollagen dispersion.

[0285] The above solution was mixed by a rotary hood apparatus at room temperature for 30 minutes to prepare a mixture. 2 -[TGG] 4 [TTG][TGG] 4 (IO101) / AC(Sol-Gel type) or (Gem) 2 -[TGG] 4 [TTG][TGG] 5 (IO101L) / AC (sol-gel type) can solidify (gel) at 37°C when injected directly into a tumor in vivo. In addition, because the surrounding (Gem) 2 -[TGG] 4 [TTG][TGG] 4 (IO101) or (Gem) 2 -[TGG] 4 [TTG][TGG]5 (IO101L) atelocollagen dissolves slowly, so the drug is gradually released, thus enabling effective tumor treatment.

[0286] 6-3. Production of IO101 / atelopeptide collagen discs and IO101L / atelopeptide collagen discs

[0287] The high concentration collagen dispersion prepared above and (Gem) were used in the same manner as in the sol-gel type production method. 2 -[TGG] 4 [TTG][TGG] 4 (IO101) or (Gem) 2 -[TGG] 4 [TTG][TGG] 5 (10101L) Prepare a mixture.

[0288] The prepared mixture was placed in a cylindrical silicone mold with a diameter of 1 cm and spread to form a uniform film of 0.5 mm, followed by freeze drying at -80°C. The completely freeze-dried sample was freeze-dried again for 30 hours in a freeze dryer maintained at -70°C to form a porous film. Hereinafter, the film prepared as described above is represented as a disc or patch.

[0289] Example 7. Evaluation of the stability of oligonucleotide variant / atelopeptide collagen compositions in plasma

[0290] Effect of gemcitabine and (Gem) in rat plasma x -[TGG] m [TTG][TGG] n The concentration of gemcitabine and gemcitabine inactive metabolite (i.e., dFdU) released from the high-purity atelocollagen preparation was analyzed. Sample processing, calibration curve creation, and LC-MS / MS analysis conditions were the same as above.

[0291] 7-1. Evaluation of the stability of (10101L) / atelopeptide collagen discs in rat plasma

[0292] To evaluate the stability of gemcitabine / atelopeptide collagen discs in plasma, experiments were performed with 5 groups categorized as listed in Table 12 below.

[0293] The gemcitabine / atelopeptide collagen discs of groups 2, 3, 4, and 5 were added to 2 mL of rat plasma to achieve a plasma concentration of 60 μg / ml, and then incubated under CO 2Incubator at 37 ° C for 2 hours. Sample processing is performed as follows. After 2 hours, in order to remove the collagen attached to the gemcitabine / atelocollagen disc in the plasma, 500 μL of plasma containing gemcitabine is added to Ultracell-3K, and then centrifuged at 15,000rpm and 20 ° C for 30 minutes. Thereafter, the supernatant is taken and then centrifuged again under the same conditions as above. Then, 5 μL of concentrate is taken and diluted 10 times with the same rat plasma to measure the concentration in plasma ("plasma concentration"). The concentration of gemcitabine concentrated in the upper part of Ultracell-3K and another concentration of gemcitabine in the plasma filtered in the lower part are measured. In addition, the concentration of gemcitabine eluted from the disc after incubation for 2 hours is also measured. The above sample processing is performed in the same manner as the calibration curve creation, and then the sample analysis is performed under the same LC-MS / MS conditions.

[0294] The result of gemcitabine (dFdC) and its metabolite (i.e. dFdU) in analysis rat plasma, minimum limit of quantification is 10ng / mL. In addition, due to the crosstalk phenomenon, the retention time of gemcitabine (dFdC) is different from dFdU, wherein a dFdU peak is produced, so in gemcitabine (dFdC) analysis, measurement is performed while giving mobile phase gradient to prevent each peak from being affected. Therefore, the retention time of gemcitabine and dFdU is measured as 2.1 minutes and 4.23 minutes respectively. The result of analyzing standard plasma samples is that all samples are determined to have an accuracy within ± 15%, so that the sample concentration determined as above is reliable (see Table 11 below).

[0295] [Table 11]

[0296]

[0297] 7-2. Evaluation of in vivo stability of (IO101L) / atelopeptide collagen discs

[0298] To evaluate the stability of the 10101L / atelopeptide collagen disc preparation in rat plasma, experiments were conducted using 5 groups (Groups 1-5) (see Table 12 below). Normal collagen (SK Co.) was used for comparison with atelopeptide collagen.

[0299] [Table 12]

[0300]

[0301] In CO 2 As a result of incubation in the incubator for 2 hours, the concentration of gemcitabine in the gemcitabine / atelopeptide collagen discs in groups 2 and 3 was lower than that in group 1 under the reserve state. 2 -[TGG] 4 [TTG][TGG]5 The 4th and 5th groups of the 1010L / atelocollagen disc preparations were determined to have lower concentrations than the 2nd and 3rd groups containing only collagen. As a result of the preliminary experiment, it can be seen that the release rate of the 1010L / atelocollagen disc preparations in rat plasma is stable and sustained. In addition, the preparations combined with atelocollagen showed that the metabolic rate of gemcitabine to 2',2'-difluorodeoxyuridine, which is an inactive metabolite of gemcitabine, is quite low (see Table 13 below).

[0302] [Table 13]

[0303]

[0304]

[0305] Example 8. Evaluation of the anticancer efficacy of oligonucleotide variants / atelopeptide collagen compositions

[0306] 8-1. Anticancer efficacy of IO101 / atelopeptide collagen (sol-gel type) using a subcutaneous pancreatic cancer animal model

[0307] The pancreatic cancer therapeutic effect of IO101 / atelopeptide collagen (sol-gel type) was verified using a subcutaneous pancreatic cancer animal model implanted with pancreatic cancer cell lines.

[0308] 8-1-1. Evaluation of the anticancer effect of IO101 content

[0309] It was confirmed that IO101 / atelocollagen (sol-gel type) is superior to gemcitabine / atelocollagen (sol-gel type) in terms of pancreatic cancer inhibitory efficacy. In order to cope with different clinical situations, sol-type drugs for local injection treatment were used. After injection, the therapeutic effect was verified by determining changes in tumor size and histochemical changes.

[0310] In other words, after culturing pancreatic cancer cell line Capan-1 cells in the recommended culture medium (RPMI, 10% FBX, 1% AA), 1x10 6 The cells were injected subcutaneously into nude mice. Tumor size was measured with a caliper, and mice with a tumor size reaching 0.5 cm in diameter were selected as treatment targets. For statistical analysis, the medical efficacy was evaluated from 5 or more mice per group. After 10101 / atelocollagen (sol-gel type) was directly injected into the tumor, the tumor size was measured to determine the therapeutic effect. At the end of 30 days of treatment, the animals were killed and the tumor was removed to determine the therapeutic effect.

[0311] By mixing 0.5 mg, 1.0 mg, 1.5 mg and 2.0 mg of IO101 dispersion with atelocollagen dispersion (400 μL of atelocollagen dispersion), IO101 / atelocollagen (sol-gel type) with different doses (IO101-0.5 mg / AC, IO101-1.0 mg / AC, IO101-1.5 mg / AC and IO101-2.0 mg / AC) were subcutaneously injected into mice with pancreatic cancer, and then the tumors were extracted 30 days later and the sizes were compared. It was confirmed that the tumor was best inhibited under IO101-2 mg / AC (sol-gel type) (see Fig.10 and Fig.11 ).

[0312] 8-1-2. Evaluation of anticancer efficacy in relation to atelocollagen concentration

[0313] Animals were evaluated for weight loss and toxicity after subcutaneous injection of IO101 / atelopeptide collagen (sol-gel type), while the dose of IO101 was set to 2 mg / 400 μL of atelopeptide collagen dispersion. Since the duration of the drug and its therapeutic effect may vary depending on the concentration of atelopeptide collagen (AC), comparative experiments were conducted on atelopeptide collagen at concentrations of 0.5%, 1.0%, and 1.5% (g per 100 ml of buffer). It was confirmed that the therapeutic effect was highest in mice with subcutaneous pancreatic cancer when 1.5% concentration (g per 100 ml of buffer) of atelopeptide collagen was used (see Fig.12 ).

[0314] 8-2. In vivo anticancer efficacy of IO101 / atelopeptide collagen (disk) in animals with subcutaneous pancreatic cancer cell line transplantation

[0315] Pancreatic cancer cell line BXPC3 was cultured at 2x10 6 The cells were subcutaneously transplanted in BALB / C nude mice. Three days later, 3 discs were implanted on the tumor in each group. After transplantation, the tumor volume of the mice was measured twice a week for 30 days using a 2x0.5 (long axis x short axis) caliper, and the body weight was measured twice a week. It can be seen that the IO101 / AC discs inhibited pancreatic cancer better than the other groups. After the discs were implanted, the tumor size was observed in the POD30 mouse images. It can be visually confirmed that the tumor volume of the IO101 / AC discs is smaller than that of the other groups. In fact, it was found that the tumor volume of the IO101 / AC discs was quite small, and in particular the IO101-2 mg / 1.5% to 3.0% AC group had the smallest tumor volume (see Fig.13 and Fig.14 ).

[0316] 8-3. Pancreatic cancer in an orthotopic xenograft pancreatic cancer mouse model with IO101 / atelopeptide collagen (disk) Therapeutic effects

[0317] 5x10 of luciferase will be expressed by replacing a specific vector 5 BxPC3 cancer cell lines and saline were injected into the pancreas of Balb / c-nude mice (male, 6 weeks old, 30 animals). In the 2nd week of building a pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. In order to reduce autofluorescence, a non-fluorescent feed was provided 1 week before imaging. IO101 / AC disks and comparative drugs were inserted intraperitoneally (using surgery to insert intraperitoneally). After building the model, luciferase imaging was performed immediately by an in vivo IVIS spectrometer to measure tumors and tumor size. Using an IVIS spectrometer, luciferase imaging was performed by time (the 6th, 18th, 21st, 23rd, 25th, 28th, 31st and 35th day). After the last in vivo IVIS spectrometer imaging, animals were killed to measure each organ (including the spleen, liver, heart, lungs, kidneys and pancreas of the tumor) in vitro. The size of the extracted tumor was measured and compared by calipers. After the disk drug was inserted into the experimental animal, the change of tumor size was monitored over time by luciferase imaging immediately. In addition, changes in luciferase imaging were determined using IVIS Spectrum as described below (see Fig.15 After the experimental animals were sacrificed, the actual size of the tumor was measured in vitro. Table 14 below lists the actual size changes of the extracted tumors, which are shown in a graphical form. Fig.16 middle.

[0318] [Table 14]

[0319]

[0320]

[0321] In addition, regarding the group implanted with only the AC disc, it was confirmed that the tumor had a larger size than the other groups, and most of them progressed to intraperitoneal metastatic cancer. The liver, spleen, and kidney showed the most metastases, and it was confirmed that the tumor spread throughout all organs in the abdominal cavity. Even in the group implanted with the Gem / AC disc, there was no significant difference in tumor size compared to the AC disc group, and intraperitoneal metastasis was confirmed. In the IO101 / AC disc, the increase in tumor size was suppressed, and it was observed that the Gem / AC disc and AC disc implants mostly metastasized to the intraperitoneal liver and diaphragm. However, the IO101 / AC disc did not show intraperitoneal cancer metastasis (see Fig.17 ).

[0322] 8-4. IO101 / atelopeptide collagen (disk) and IO101L / atelopeptide collagen (disk) in an orthotopic pancreatic cancer mouse model The inhibitory effect of telopeptide collagen (disk) on pancreatic cancer metastasis

[0323] 5x10 of luciferase will be expressed by replacing a specific vector 5BxPC3 cancer cell lines and saline were injected into the pancreas of Balb / c-nude mice (male, 6 weeks old, 30 animals). In the 2nd week of building a pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. In order to reduce autofluorescence, a fluorescence-free feed was provided 1 week before imaging. IO101 / AC discs or IO101L / AC discs and comparative drugs were inserted intraperitoneally (using surgery to insert intraperitoneally). After building the model, luciferase imaging was performed immediately by an in vivo IVIS spectrometer to measure tumor and tumor size. Using an IVIS spectrometer, luciferase imaging was performed by time (the 6th, 18th, 21st, 23rd, 25th, 28th, 31st and 35th day). After the last in vivo IVIS spectrometer imaging, animals were killed to measure each organ (including the spleen, liver, heart, lungs, kidneys and pancreas of the tumor) in vitro. Observe whether the organ has intraperitoneal metastasis. Specifically, it was observed that the AC discs mainly metastasized to the liver, kidneys, and / or diaphragm in the peritoneal cavity. However, no intraperitoneal cancer metastasis was observed in the IO101 / AC discs and IO101L / AC discs (see Fig.18 ).

[0324] 8-5. Changes in survival rate after implantation of 10101L / atelopeptide collagen (disk)

[0325] In an orthotopic mouse model of pancreatic cancer, the survival rate by concentration after implantation of IO101L / AC discs was compared. After implantation of a single AC disc, 2 of 5 animals survived after 32 days. In addition, in the case of IO101L-2mg (mixed with IO101L 2mg per 400μL of atelocollagen dispersion) / AC disc implantation, 4 of 5 animals survived to 32 days. In addition, in the case of IO101L-4mg (mixed with IO101L 4mg per 400μL of atelocollagen dispersion) / AC disc and IO101L-8mg (mixed with IO101L 8mg per 400μL of atelocollagen dispersion) / AC disc, all 5 animals died on the 6th day (see Fig.19 ).

[0326] 8-6. IO101L / atelopeptide collagen after removal of residual tumor in an orthotopic xenograft pancreatic cancer mouse model Tumor inhibitory and metastasis inhibitory effects of (sol-gel type) and IO101L / atelopeptide collagen (disk)

[0327] 8-6-1.BxPC3 pancreatic cancer transplantation mouse experiment

[0328] After the tumor was removed by surgical operation in mice with orthotopic pancreatic cancer, 10101L / AC sol-gel type or 10101L / AC disc drugs were inserted, and then observation was performed to confirm whether the residual tumor and the metastasis of the residual tumor to other organs in the abdominal cavity were suppressed. Specifically, 5×10 luciferase expressing vectors were prepared in Balb / c nude mice (male, 6 weeks old, 21 animals). 5 BxPC3 cancer cell line (25 μL). The pancreas was removed by cutting the abdominal area of ​​intraperitoneal anesthetized mice, and then BxPC-3-Luc cells were injected into the prepared pancreas. In the second week of constructing the pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. The tumor cell line used here is BxPC-3-Luc cells that express luciferase by replacing a specific vector in BxPC3 cells, and luciferin is used to measure the tumor. In order to reduce autofluorescence, non-fluorescent feed was provided 1 week before imaging. After removing the pancreatic tumor by cutting the abdominal area of ​​intraperitoneal anesthetized mice, IO101L / AC sol-gel type, IO101L / AC disc, Gem / endopeptide collagen sol-gel or Gem / endopeptide collagen disc was transplanted to the remaining tumor site (intraperitoneal insertion using surgery). Tumor size and metastasis were confirmed by luciferase imaging, and the results are shown in Fig. 20 middle.

[0329] 8-6-2. Capan-1 pancreatic cancer transplantation mouse experiment

[0330] When ordering mice for the generation of an orthotopic mouse model of pancreatic cancer, maintain a 1-week period for acclimatization. During the acclimatization period, feed 1 × 10 6 Capan-1 cells were prepared at 10 cells / 100 μl. The mouse skin was opened 2 mm, the spleen was folded up and the above cells were injected directly into the pancreas with a syringe. After 4 weeks, the diameter of the tumor was confirmed to be 5 mm to 6 mm by MRI. When the cancer cell diameter was determined to be 5 mm to 6 mm, the abdomen was opened and the pancreatic tumor was removed as much as possible. After removal, 10101L / AC discs and atelocollagen discs (control) were inserted into the remaining tumor part and sealed (see Fig.21 ).

[0331] After 1 month, tumor size was monitored by MRI. After all images were taken, CO 2The mice were euthanized in the 100 μg / ml room. Intraperitoneal tumor metastasis was confirmed in each group, and each tumor tissue was obtained and fixed with 10% formalin. Histopathology was confirmed by immunostaining using H&E and corresponding antibodies. No metastasis occurred in the peritoneal cavity when the 10101L / AC disk was inserted, while metastasis to the spleen and liver in the peritoneal cavity was found in the atelocollagen disk (control) (see Fig. 22 ). In addition, in the case of the atelocollagen disc (control), metastasis to the liver, diaphragm, kidney, etc. in the abdominal cavity was observed after 1 month (see Fig.23 ).

[0332] 8-7. In a patient-derived xenograft (PDX) pancreatic cancer mouse model using patient-derived pancreatic cancer cells Anti-cancer effects

[0333] 8-7-1. Construction of PDX pancreatic cancer mouse model using patient-derived pancreatic cancer cells

[0334] A PDX model was successfully established using a pancreatic tumor removed from a 65-year-old female patient undergoing laparoscopic pancreatic amniotomy. Pathological examination revealed that the ductal adenocarcinoma of the pancreas was 3.2 cm in size and showed frequent lymphatic infiltration. One metastatic lymph node was found in the seven lymph nodes searched (AJCC 8th Edition T2N1M0, IIB). When the histological features of the PDX and the initial primary tumor were compared by H&E and nucleolar immunostaining, overall histological similarities were found between the primary tumor and the PDX tumor (see Fig.24 ).

[0335] Male diabetic / severe combined immunodeficient mice, namely NOD / Shi-scid, IL-2RγKO mice (NOG ) (Central Lab Animal Inc., Saeronbio Inc., Seoul) and female nu / nu athymic mice (Orientbio) were maintained in a 12-hour light / 12-hour dark cycle under pathogen-free conditions. At the time of surgery, 10 new pancreatic tumor specimens preserved in the patient's primary tumor (Institutional Inspection Board No. 4-2017-0594) were placed in ice-cold RPMI medium, and the PDX SOP of the PDTX Cleanbench Department Laboratory Animal Resources (Yonsei Biomedical Research Institute, Seoul) was washed with cold PBS, and the non-necrotic fragments were cut into small pieces (2× 2×2 mm) to remove blood or lipid parts. Then, after the above fragments were coated with matrix gel on a petri dish, the single pieces were implanted into the right and left sides of 6-week-old mice by Precision Trochar 10 gauge (MP182, Innovative Research of America), and then sutured with 5-0 sutures (VCP490G, ETHICON). When the growing tumors reached a size of 1500 mm, the donor mouse (F1) tumor and the same tumor and the remaining tumors were then stored in liquid nitrogen in cryovials containing 5% dimethyl sulfide / 95% fetal bovine serum and implanted into the same cohort of mice (F2). 3 ) and sub-passaged to the next generation of mice (F2). In each compartment, approximately 150-200 mm xenografts from the same patient were collected. 3 Tumors took about 50 days to reach palpable size. A pair of primary tumors (F0) and F1 (generation 1) and F2 (generation 2) samples were used in 2 patients. When tumors reached palpable size (mean size = 266.5 ± 58.0 mm 3 ), mice (n=8 to 13 / n=4 / patient) were randomly divided into the following five (5) groups: Group 1 (untreated control); Group 2, in which gemcitabine (100 μg) suspended in PBS was intraperitoneally administered to mice once every 4 weeks; Group 3, in which IO101 (100 μg) suspended in PBS was intraperitoneally administered to mice once every 4 weeks; and Groups 4, 5, and 6, which were implanted with three types of patches. Untreated control mice were also included for comparison.

[0336] After transplanting tumor tissue obtained from patients with pancreatic cancer into NSG (NOD / SCID / IL-2Rg KO) mice, the animal models that have been successfully grown were transplanted for multiple generations and expanded individually by spheroid cell formation to prepare PDX models that can be used to evaluate the effects of treatment. For unmanipulated subcutaneous infusion surgery, tumor size, tumor markers (CA19-9, CFB) and weight were determined at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks after surgery, followed by histological examination and confirmation of survival rate.

[0337] 8-7-2. Evaluation of the tumor suppressive effect of IO101 / AC disc in the PDX pancreatic cancer mouse model using patient-derived pancreatic cancer cells

[0338] After making a skin incision in PDX mice, control (untreated), Gem-IP (sol-gel), IO101-IP (sol-gel), IO101 / AC discs (2.0 mg / 3.0%), IO101-Con / AC (2.0 mg / 3.0%), and Gem / atelocollagen discs (0.12 mg / 3.0%) were implanted topically between the skin and the tumor by subcutaneous dissection under anesthesia. Tumor size was measured three times a week by caliper measurement (Mitutoyo, Absolute AOS Digmatic, Kawasaki, Japan), and volume was calculated as described above. Tumor growth in drug-treated animals compared to vehicle-treated mice was expressed as tumor growth rate (tumor volume / initial tumor volume). Statistical significance of the data was determined by All results were expressed as mean ± SD, Mann-Whitney U was applied to compare continuous variables according to different groups, and P values ​​less than 0.05 were considered significant (see Fig.25 ).

[0339] One month after disc implantation, mice were sacrificed and tissue samples and blood were collected. The extracted tumors were weighed on a balance and recorded with a Nikon digital camera (Japan). For hematological measurements, the tumors were collected from the K2E (K 2Blood samples were collected in BD Microtainer tubes containing EDTA (EDTA) for anticoagulation and toxicity tests in serum collection BD Microtainer Chem Tubes SST (BD, USA). The anticancer effect of IO101 / AC discs was verified by TUNEL analysis. No tumor necrosis or cell death was found in the control. In the Gem-IP group, apoptotic process was confirmed, and TUNNEL-positive cancer cells in the tumor were found in the transplanted cancer tissue. On the other hand, the IO101 / AC disc group showed a significant apoptotic process, but TUNNEL-positive cancer cells were found along the superficial layer of the tumor surface (see Fig.26 ).

[0340] 8-7-3. Evaluation of side effects in other organs after implantation of IO101 discs in a PDX pancreatic cancer mouse model using patient-derived pancreatic cancer cells

[0341] Microscopic examination confirmed no evidence of potential toxicity (such as inflammatory or necrotic changes) in liver, lung, kidney, and spleen tissues (see Fig. 27 ). No leukopenia, anemia and neutropenia were observed in the IO101 / AC disc group. On the other hand, leukopenia (WBC) (3.2±2.9 vs. 5.4±2.9, P=0.028), low hemoglobin levels (HB) (10.3±4.6 vs. 18.5±11.9) and neutropenia (0.76±0.71 vs. 2.69±2.66, p=0.010) in the IP-GEM group associated with the systemic effects of gemcitabine were demonstrated. Table 15 below shows the blood test results.

[0342] [Table 15]

[0343]

[0344]

[0345] 1) Mann-Whitney U between control and patch I; 2) Mann-Whitney U between IP-GEM and patch I

[0346] Example 9. Screening of oligonucleotide variants for in vitro efficacy

[0347] Yes (N) x -[TGG] 4 [TTG][TGG] 4或5 , [TGG] 4 [TTG][TGG] 4或5 -(N) x Sum (N) x -[TGG]4 [TTG][TGG] 4或5 -(N) y The cell proliferation inhibitory efficacy was validated against BxPC3 (pancreatic cancer), MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer) and Mv4-11 (AML) cell lines.

[0348] 2.5x10 5 Up to 5.0x10 5 10 cells / well (which is the cell number defined by a cell test for determining the appropriate cell concentration in pancreatic cancer cell line BxPC3 cells (ATCC, IMDM + 10% PBS)) were seeded in a 96-well plate and incubated for one day. After heating each sample at 95°C for 5 minutes, the temperature was slowly lowered to room temperature, and then the samples were immediately treated for each well according to the concentration. The treated BxPC3 cells were incubated at 5% CO 2 The cells were incubated in an incubator for 3 days, treated with 20 μL of reagent solution for MTT assay (Cell Proliferation Kit II, Roche), and then incubated at different times (10 min, 30 min, 60 min), after which the absorbance at 490 nm was measured by an ELISA reader (see Fig.28 and Fig.29 ).

[0349] The cell proliferation inhibitory efficacy against other cell lines MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer) and Mv4-11 (AML) was also verified in the same manner as above. The cell viability of each cell line is listed in Table 16 below.

[0350] [Table 16]

[0351]

[0352]

[0353] <110> Inter Orient <120> Modified nucleic acid with improved therapeutic efficacy and anticancer drug composition containing the same <130> 18OP08013PCT <150> US 62 / 747,807 <151> 2018-10-19 <150> KR 2019 / 130669 <151> 2019-10-21 <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_1 <400> 1 tggttgtgg 9 <210> 2 <211> 12 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_2 <400> 2 tggttgtggt gg 12 <210> 3 <211> 12 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_3 <400> 3 tggtggttgt gg 12 <210> 4 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_4 <400> 4 tggtggttgt ggtgg 15 <210> 5 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_5 <400> 5 tggtggttgt ggtggtgg 18 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_6 <400> 6 tggtggtggt tgtggtgg 18 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_7 <400> 7 tggtggtggt tgtggtggtg g 21 <210> 8 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_8 <400> 8 tggtggtggt tgtggtggtg gtgg 24 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_9 <400> 9 tggtggtggt ggttgtggtg gtgg 24 <210> 10 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_10 <400> 10 tggtggtggt ggttgtggtg gtggtgg 27 <210> 11 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_11 <400> 11 tggtggtggt ggttgtggtg gtggtggtgg 30 <210> 12 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_12 <400> 12 tggtggtggt ggtggttgtg gtggtggtgg 30 <210> 13 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_13 <400> 13 tggtggtggt ggtggttgtg gtggtggtgg tgg 33 <210> 14 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_14 <400> 14 tggtggtggt ggtggttgtg gtggtggtgg tggtgg 36 <210> 15 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_15 <400> 15 tggtggtggt ggtggtggtt gtggtggtgg tggtgg 36 <210> 16 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_16 <400> 16 tggtggtggt ggtggtggtt gtggtggtgg tggtggtgg 39 <210> 17 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_17 <400> 17 tggtggtggt ggtggtggtt gtggtggtgg tggtggtggt gg 42 <210> 18 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_18 <400> 18 tggtggtggt ggtggtggtg gttgtggtgg tggtggtggt gg 42 <210> 19 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_19 <400> 19 tggtggtggt ggtggtggtg gttgtggtgg tggtggtggt ggtgg 45 <210> 20 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_20 <400> 20 tggtggtggt ggtggtggtg gttgtggtgg tggtggtggt ggtggtgg 48 <210> twenty one <211> 48 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_21 <400> twenty one tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtgg 48 <210> twenty two <211> 51 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_22 <400> twenty two tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtggtg g 51 <210> twenty three <211> 54 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_23 <400> twenty three tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtggtg gtgg 54 <210> twenty four <211> 54 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_24 <400> twenty four tggtggtggt ggtggtggtg gtggtggttg tggtggtggt ggtggtggtg gtgg 54 <210> 25 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide_25 <400> 25 tggtggtggt ggtggtggtg gtggtggttg tggtggtggt ggtggtggtg gtggtgg 57 <210> 26 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide_26 <400> 26 tggtggtggt ggtggtggtg gtggtggttg tggtggtggt ggtggtggtg gtggtggtgg 60 60 <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide_27 <400> 27 tggtggtggt ggtggtggtg gtggtggtgg ttgtggtggt ggtggtggtg gtggtggtgg 60 60 <210> 28 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide_28 <400> 28 tggtggtggt ggtggtggtg gtggtggtgg ttgtggtggt ggtggtggtg gtggtggtgg 60 tgg 63

Claims

1. An oligonucleotide variant having the structure of the following formula 1: [Formula 1] (N) x -[TGG] m [TTG][TGG] n -(M) y in, N and M are independently 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'-difluorodeoxycytidine, capecitabine and bromovinyl deoxyuridine; x and y are independently an integer from 0 to 10, wherein x and y are not simultaneously 0; n is an integer from 4 to 6; and m is an integer from 4 to 6.

2. The oligonucleotide variant according to claim 1, wherein the structure of Formula 1 is any one of the following Formulas 11 to 34: [Formula 11] (N)2-[TGG]4[TTG][TGG]4, [Formula 12] (N)2-[TGG]4[TTG][TGG]5, [Formula 13] (N)2-[TGG]5[TTG][TGG]4, [Formula 14] (N)2-[TGG]5[TTG][TGG]5, [Formula 15] (N)2-[TGG]5[TTG][TGG]6, [Formula 16] (N)2-[TGG]6[TTG][TGG]5, [Formula 17] (N)2-[TGG]6[TTG][TGG]6, [Formula 18] [TGG]4[TTG][TGG]4-(M)1,[Formula 19] [TGG]4[TTG][TGG]4-(M)2,[Formula 20] [TGG]4[TTG][TGG]4-(M)3,[Formula 21] [TGG]4[TTG][TGG]4-(M)4,[Formula 22] [TGG]4[TTG][TGG]4-(M)5[Formula 23] [TGG]4[TTG][TGG]5-(M)1,[Formula 24] [TGG]4[TTG][TGG]5-(M)2,[Formula 25] [TGG]4[TTG][TGG]5-(M)3,[Formula 26] [TGG]4[TTG][TGG]5-(M)4,[Formula 27] [TGG]4[TTG][TGG]5-(M)5, [Equation 28] [TGG]4[TTG][TGG]4-(M) 10 , [Equation 29] (N)1-[TGG]4[TTG][TGG]4-(M)1, [Formula 30] (N)3-[TGG]4[TTG][TGG]4-(M)3, [Equation 31] (N)5-[TGG]4[TTG][TGG]4-(M)5, [Formula 32] (N)1-[TGG]4[TTG][TGG]5-(M)1, [Formula 33] (N)3-[TGG]4[TTG][TGG]5-(M)3, [Equation 34] (N)5-[TGG]4[TTG][TGG]5-(M)5.

3. The oligonucleotide variant according to claim 1, wherein n is an integer from 4 to 5, and m is an integer from 4 to 5. The oligonucleotide variant according to claim 1 , wherein x and y are independently integers from 0 to 5, wherein x and y are not simultaneously 0.

5. A pharmaceutical composition for preventing or treating cancer, comprising the oligonucleotide variant according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition of claim 5, wherein the cancer is selected from the group consisting of leukemia, lymphoma, breast cancer, liver cancer, stomach cancer, ovarian cancer, cervical cancer, glioma cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, gastric adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma and cervical cancer.

7. The pharmaceutical composition according to claim 5, wherein the composition is an atelocollagen dispersion preparation.

8. The pharmaceutical composition according to claim 7, wherein the atelocollagen dispersion contains 0.5 g to 5.5 g of atelocollagen per 100 ml of PBS solution.

9. The pharmaceutical composition according to claim 5, wherein the composition is in the form of a sol-gel or a patch.

10. The pharmaceutical composition according to claim 7, wherein the atelocollagen is prepared by a method comprising: a) treating a collagen-containing animal tissue with at least one of alkaline protease, catalase, pepsin and papain to extract a substance; b) first filtering the extracted substance and salting out the extracted substance by adding a neutral salt to the obtained filtrate, followed by a second filtration; c) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; d) freeze-drying the filtrate obtained after the third filtration, and recovering the lyophilized powder; and e) dissolving the lyophilized powder in a dilute hydrochloric acid, dilute acetic acid or phosphate buffer having a pH of 4 to pH 8 and concentrating to prepare an atelocollagen solution, injecting the prepared atelocollagen solution into a column filled with polymer beads at 5% to 20% by volume of the column bed volume, and developing the column in a dilute hydrochloric acid, dilute acetic acid or phosphate buffer having a pH of 4 to pH 8, thereby recovering the atelocollagen.

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