Modified nucleic acid having improved therapeutic efficacy and anticancer pharmaceutical composition comprising the same
阅读说明：本技术 具有改善治疗功效的修饰核酸和含有其的抗癌药物组合物 (Modified nucleic acid having improved therapeutic efficacy and anticancer pharmaceutical composition comprising the same ) 是由 李仲桓 李钟旭 于 2019-10-21 设计创作，主要内容包括：本发明涉及寡核苷酸变体,并且更具体地涉及具有下式1的结构的寡核苷酸变体和包含所述寡核苷酸变体或其药学上可接受的盐的药物组合物,所述药物组合物可以展现出优异的体内稳定性和抗癌作用：[式1](N)-x-[TGG]-m[TTG][TGG]-n-(M)-y(其中,N和M独立地为脱氧尿苷(dU)、脱氧胞苷(dC)、尿苷(U)或胞苷(C),其中卤素或羟基结合至N和M的5-位或2’-位；x和y独立地为0至10的整数(x和y同时为0的情况除外)；n为1至10的整数；并且m为1至10的整数)。(The present invention relates to an oligonucleotide variant, and more particularly, to an oligonucleotide variant having the structure of the following formula 1 and a pharmaceutical composition comprising the same, which can exhibit excellent in vivo stability and anticancer effect: [ formula 1](N) x ‑[TGG] m [TTG][TGG] n ‑(M) y (wherein, N and M are independently deoxyuridine (dU), deoxyuridine (dU)Cytidine (dC), uridine (U), or cytidine (C), wherein a halogen or hydroxyl group is bound to the 5-or 2' -position of N and M; x and y are independently integers from 0 to 10 (except where x and y are both 0); n is an integer of 1 to 10; and m is an integer of 1 to 10).)
1. An oligonucleotide variant having the structure of formula 1:
[ 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 hydroxyl group is bound to the 5-or 2' -position of N and M; x and y are independently integers from 0 to 10 (except where x and y are both 0); n is an integer of 1 to 10; and m is an integer of 1 to 10.
2. The oligonucleotide variant of claim 1, 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-iodocytidine, cytosine arabinoside, 2' -difluorodeoxycytidine, capecitabine, and bromovinyldeoxyuridine.
3. The oligonucleotide variant according to claim 1, wherein the structure of formula 1 is any one of the following formulae 2 to 34:
[ formula 2]
(N)2-[TGG]1[TTG][TGG]1，
[ formula 3]
(N)2-[TGG]1[TTG][TGG]2，
[ formula 4]
(N)2-[TGG]2[TTG][TGG]1，
[ formula 5]
(N)2-[TGG]2[TTG][TGG]2，
[ formula 6]
(N)2-[TGG]2[TTG][TGG]3，
[ formula 7]
(N)2-[TGG]3[TTG][TGG]2，
[ formula 8]
(N)2-[TGG]3[TTG][TGG]3
[ formula 9]
(N)2-[TGG]3[TTG][TGG]4，
[ formula 10]
(N)2-[TGG]4[TTG][TGG]3，
[ 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，
[ formula 28]
[TGG]4[TTG][TGG]4-(M)10，
[ formula 29]
(N)1-[TGG]4[TTG][TGG]4-(M)1，
[ formula 30]
(N)3-[TGG]4[TTG][TGG]4-(M)3，
[ formula 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，
[ formula 34]
(N)5-[TGG]4[TTG][TGG]5-(M)5。
4. The oligonucleotide variant according to claim 1, wherein n is an integer from 1 to 5 and m is an integer from 1 to 5.
5. The oligonucleotide variant according to claim 1, wherein x and y are independently integers from 0 to 5 (except for the case where x and y are both 0).
6. A pharmaceutical composition for preventing or treating cancer, comprising the oligonucleotide variant according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.
7. The pharmaceutical composition of claim 6, 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, stomach adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer.
8. The pharmaceutical composition of claim 6, wherein the composition is a atelopeptide collagen dispersion formulation.
9. The pharmaceutical composition of claim 8, wherein the atelocollagen dispersion contains 0.5g to 5.5g atelocollagen per 100ml of pbs solution.
10. The pharmaceutical composition of claim 6, wherein the composition is in the form of a sol-gel or patch.
11. The pharmaceutical composition of claim 8, wherein the atelocollagen is prepared by a process comprising: 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, and salting out the extracted substance by adding a neutral salt to the obtained filtrate, followed by second filtering; c) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; d) lyophilizing the filtrate obtained after the third filtration, and recovering the lyophilized powder; and e) dissolving the lyophilized powder in dilute hydrochloric acid (dil-HCl) of pH 4 to pH 8, dilute acetic acid or phosphate buffer and concentrating to prepare a atelopeptide collagen solution, injecting the prepared atelopeptide collagen solution into a column packed with polymer beads at 5-20 vol% of a bed volume, and developing the column in dilute hydrochloric acid, dilute acetic acid or phosphate buffer of pH 4 to pH 8, thereby recovering atelopeptide collagen.
Technical Field
The present invention relates to nucleic acid variants with improved therapeutic efficacy and pharmaceutical compositions for anticancer use comprising said nucleic acid variants.
Background
Since the 80's of the 20 th century, research has been actively conducted to develop oligonucleotides as therapeutic agents. Guanosine-rich oligonucleotides are known to have a cytostatic effect on a wide range of cancer cells, and these oligonucleotides may have a 4-strand structure by intramolecular or intermolecular bonds. Instead of forming a double helix structure by hydrogen bonding between adenosine and thiamine or guanosine and cytidine, four (4) guanosine groups are located on one plane to form hydrogen bonding in the form of a mustine (hoogsten) to form a G-quadruplex. In addition, it is known that G-quadruplexes-forming oligonucleotides have a stable structure due to their structural characteristics, thereby exhibiting relatively high blood stability and cell permeability. Various studies and developments are being conducted to further stabilize the G-quadruplex and increase the anticancer effect by introducing a modified nucleic acid for therapeutic use into an oligonucleotide having different functions as described above.
Disclosure of Invention
[ problems to be solved by the invention ]
It is an object of the present invention to provide nucleic acid variants with therapeutic efficacy and pharmaceutical compositions for anticancer use comprising said nucleic acid variants.
It is another object of the present invention to provide a pharmaceutical composition having excellent in vivo stability and anticancer effect.
[ means for solving the problems ]
In order to achieve the above object, the present invention adopts the following technical means.
1. An oligonucleotide variant having the structure of formula 1:
[ 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), to which a halogen or hydroxyl group is bound at the 5-or 2' -position; x and y are independently integers from 0 to 10 (except where x and y are both 0), n is an integer from 1 to 10; and m is an integer of 1 to 10.
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-iodocytidine, cytosine arabinoside, 2' -difluorodeoxycytidine, capecitabine (capecitabine), and bromovinyldeoxyuridine.
3. The oligonucleotide variant according to the above 1, wherein the structure of formula 1 is any one of the following formulae 2 to 34:
[ formula 2]
(N)2-[TGG]1[TTG][TGG]1，
[ formula 3]
(N)2-[TGG]1[TTG][TGG]2，
[ formula 4]
(N)2-[TGG]2[TTG][TGG]1，
[ formula 5]
(N)2-[TGG]2[TTG][TGG]2，
[ formula 6]
(N)2-[TGG]2[TTG][TGG]3，
[ formula 7]
(N)2-[TGG]3[TTG][TGG]2，
[ formula 8]
(N)2-[TGG]3[TTG][TGG]3
[ formula 9]
(N)2-[TGG]3[TTG][TGG]4，
[ formula 10]
(N)2-[TGG]4[TTG][TGG]3，
[ 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，
[ formula 28]
[TGG]4[TTG][TGG]4-(M)10，
[ formula 29]
(N)1-[TGG]4[TTG][TGG]4-(M)1，
[ formula 30]
(N)3-[TGG]4[TTG][TGG]4-(M)3，
[ formula 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，
[ formula 34]
(N)5-[TGG]4[TTG][TGG]5-(M)5，
4. The oligonucleotide variant according to 1 above, wherein n is an integer of 1 to 5, and m is an integer of 1 to 5.
5. The oligonucleotide variant according to 1 above, wherein x and y are independently an integer of 0 to 5 (except for the case where x and y are both 0).
6. A pharmaceutical composition for preventing or treating cancer, comprising an oligonucleotide variant according to any one of above 1 to 5 or a pharmaceutically acceptable salt thereof.
7. The pharmaceutical composition of 6 above, 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, stomach adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer.
8. The pharmaceutical composition of 6 above, wherein the composition is a atelocollagen dispersion formulation.
9. The pharmaceutical composition of 8 above, wherein the atelocollagen dispersion contains 0.5g to 5.5g atelocollagen per 100ml of PBS solution.
10. The pharmaceutical composition according to 6 above, wherein the composition is in the form of a sol-gel or a patch.
11. The pharmaceutical composition according to 8 above, wherein the atelocollagen is prepared by a process comprising: 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 and salting out the obtained filtrate by adding neutral salt thereto, followed by second filtering; c) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; d) lyophilizing 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 solution of pH 4 to pH 8 and concentrating to prepare a atelocollagen solution, injecting the prepared atelocollagen solution into a column packed with polymer beads at 5-20 vol% of a bed volume, and developing the column in dilute hydrochloric acid, dilute acetic acid or phosphate buffer solution of pH 4 to pH 8, thereby recovering atelocollagen.
[ advantageous effects ]
The oligonucleotide variants of the invention can effectively target nucleolin present on the surface, cytoplasm or nucleus of cancer cells.
The oligonucleotide variants of the invention can inhibit the growth of or kill cancer cells.
The oligonucleotide variants of the invention can bind a modified nucleic acid (N) to an oligonucleotide having a specific sequence, thereby reducing the rate at which the modified nucleic acid is degraded by an enzyme in vivo.
Drawings
Fig. 1 shows calibration curves for dFdC and dFdU for determining the concentration of gemcitabine (dFdC) and gemcitabine inactive metabolites, i.e. 2, 2' -difluorodeoxyuridine (dFdU), in plasma.
FIG. 2 shows (Gem)2-[TGG]4[TTG][TGG]4(IO101) evaluation results of in vitro anticancer efficacy of pancreatic cancer cell lines.
Fig. 3 shows the confirmation result of apoptosis of the pancreatic cancer cell line BXPC treated according to IO 101.
FIG. 4 shows the confirmation result of apoptosis of the pancreatic cancer cell line mia-paca-2 treated according to IO 101.
FIG. 5 shows the confirmation of apoptosis of pancreatic cancer cell line Panc-1 treated according to IO 101.
FIG. 6 shows the confirmation result of apoptosis of the pancreatic cancer cell line Capan-1 treated according to IO 101.
FIG. 7 shows the evaluation (Gem)2-[TGG]4[TTG][TGG]5(IO101L) results of in vitro anticancer efficacy against pancreatic cancer cell lines.
Fig. 8 shows the change in plasma concentration of gemcitabine after administration of gemcitabine (●, n-4) or IO101L (o, n-4) to rats.
Fig. 9 shows the plasma concentrations of dFdU after gemcitabine (●, n-4) or IO101L (o, n-4) was administered to rats.
FIG. 10 shows the results of determination of the size of a tumor 30 days after injecting sol-gel type IO101-0.5mg/AC (atelopeptide collagen), IO101-1.0mg/AC, IO101-1.5mg/AC and IO101-2.0mg/AC, respectively, into a mouse with subcutaneous pancreatic cancer.
FIG. 11 shows the relative change in tumor size within 30 days after injection of sol-gel type IO101-1.0mg/AC and IO101-2.0mg/AC, respectively, into mice with subcutaneous pancreatic cancer.
FIG. 12 shows the confirmation of the effect of anticancer therapy by varying the concentration of atelopeptide collagen while fixing the dose of IO101 at 2 mg.
FIG. 13 shows a graph of the change in tumor size of pancreatic cancer in mice after implantation of IO101/AC discs.
FIG. 14 shows pancreatic cancer tumor size of mice after implantation of IO101/AC discs.
FIG. 15 shows the measurement of pancreatic cancer tumor size over time after implantation of IO101/AC disks.
FIG. 16 shows a graph comparing tumor size before and after implantation of the IO101/AC disc and the Gem/AC disc, respectively.
FIG. 17 shows observations and comparisons between IO101/AC discs and Gem/AC discs with respect to tumor inhibition and intraperitoneal metastasis, respectively, of pancreatic cancer.
FIG. 18 shows the effect of inhibiting intraperitoneal metastasis of pancreatic cancer after implantation of IO101/AC and IO101L/AC discs, respectively.
FIG. 19 shows the change in survival of mice after implantation of IO101L/AC discs.
FIG. 20 shows tumor suppression or metastasis suppression after removal of residual tumor and implantation of IO101L/AC sol-gel type or insertion of discs in a pancreatic cancer in situ mouse model.
FIG. 21 shows a process for making a mouse model of the Capan-1 pancreatic cancer.
FIG. 22 shows the effect of inhibiting peritoneal metastasis 1 month after implantation of the IO101L/AC disc into the Capan-1 pancreatic cancer mouse model.
FIG. 23 shows the results of metastasis in the liver, diaphragm and kidney 1 month after implantation of a atelocollagen disc (control) into the Capan-1 pancreatic cancer mouse model.
Figure 24 shows histologically similar features of primary tumors compared to a patient-derived xenograft (PDX) pancreatic cancer model using patient-derived pancreatic cancer cells.
FIG. 25 shows tumor suppression after implantation of control (untreated), Gem-IP, IO101/AC disks (2.0 mg/3.0%), IO101-Con/AC disks (2.0 mg/3.0%), and Gem/atelocollagen disks (0.12 mg/3.0%) into a PDX mouse model, respectively.
Figure 26 shows the results of apoptosis analysis by TUNEL staining in pancreatic cancer tumors.
FIG. 27 shows the results demonstrating no side effects in other organs after implantation of the IO101/AC discs in the PDX model.
Fig. 28 shows results regarding cell proliferation inhibitory efficacy of BxPC3 (pancreatic cancer), MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer), and Mv4-11 (AML).
Fig. 29 shows results regarding 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
The present invention provides oligonucleotide variants in which an oligonucleotide and a modified nucleic acid are linked.
The oligonucleotide of the present invention has [ TGG ]]m[TTG][TGG]nThe sequence of (a).
Herein, n may be an integer from 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. Further, n can be an integer from 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.
m may be an integer from 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. Further, m may be an integer from 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.
The oligonucleotide of the present invention may be one of the sequences listed in table 1 below, but is not limited thereto.
[ Table 1]
The oligonucleotides of the invention are rich in guanosine to form a G-quadruplex structure and are aptamers specific for nucleolin.
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 to the cell surface. Nucleolin has multiple functions in cells and can be involved in ribosome production, cell growth and DNA replication.
The oligonucleotides of the invention can bind more selectively to cancer cells and inhibit the growth of cancer cells by various mechanisms in the cell.
One or more modified nucleic acids may be incorporated into the oligonucleotides of the invention to stabilize the modified nucleic acids or to prevent inactivation of the modified nucleic acids in vivo.
Furthermore, when one or more modified nucleic acids are bound to the 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.
The term "modified nucleic acid" as used herein may be a chemically modified nucleoside or nucleotide.
The modified nucleic acid may be deoxyuridine (dU), deoxycytidine (dC), uridine (U), or cytidine (C), wherein at least one halogen or hydroxyl group is bound to its 5-or 2' -position. For example, the modified nucleic acid may 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' -difluorodeoxycytidine, capecitabine, and bromovinyldeoxyuridine, or a derivative thereof.
The modified nucleic acid may be ligated in at least one of the 5' and 3' directions of the oligonucleotide, and preferably in the 5' direction of the oligonucleotide.
The modified nucleic acid may be attached to at least one of the 5 'and 3' directions of the oligonucleotide by a linker.
The joint may be [ - (CH)2)a-]、[-(CH2CH2O)b-]And [ butanamido methyl-1- (2-nitrophenyl) -ethyl]-2-cyanoethyl-][1', 2' -dideoxyribose-]Or (PEG)y. Here, 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 from 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6. y may be an integer from 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.
When the modified nucleic acid is ligated in the 3' direction of the oligonucleotide, idT, LNA, PEG or 2 ' OMeNu may be further ligated in the 3' direction of the modified nucleic acid.
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 nuclease attack, thereby reducing the rate of degradation of the oligonucleotide variant in vivo. Thus, the modified nucleic acids can be bound to the oligonucleotides for a longer period of time, thereby increasing the anti-cancer efficacy of the modified nucleic acids.
Specifically, the oligonucleotide variant of the present invention may be a compound having the structure of formula 1:
[ formula 1]
(N)x-[TGG]m[TTG][TGG]n-(M)y
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. Details of the modified nucleic acid are the same as those described above.
In formula 1, x and y may be independently integers of 0 to 10, except for the case where x and y are simultaneously 0.
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.
In formula 1, n may be an integer of 1 to 10, and m may be an integer of 1 to 10.
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. Further, 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.
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 from 1 to 10, 2 to 9, 3 to 8, 4 to 7, or 5 to 6.
For example, the oligonucleotide variant having the structure of formula 1 may be a compound having the structure of formula listed in table 2 below.
[ Table 2]
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 in at least one of the 5 'and 3' directions of an oligonucleotide comprising the sequence shown in SEQ ID NO: 10. For example, an oligonucleotide variant of the 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 incorporated in the 5' direction of an oligonucleotide comprising the sequence shown in SEQ ID NO 10.
According to another embodiment, the oligonucleotide variant of the present invention may be one in which at least one gemcitabine is attached in the 5 'or 3' direction of an oligonucleotide comprising the sequence as set forth in SEQ ID NO 11. For example, an oligonucleotide variant of the 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 incorporated in the 5' direction of an oligonucleotide comprising the sequence shown in SEQ ID NO 11.
A method for linking an oligonucleotide and a modified nucleic acid may include a step of binding the modified nucleic acid to at least one of the 5 'end and the 3' end of the oligonucleotide.
Another method for ligating oligonucleotides and modified nucleic acids may comprise: linking a linker to at least one of the 5 'end and the 3' end of the oligonucleotide by chemical synthesis; then, the modified nucleic acid is bound to the above linker.
The binding of the modified nucleic acid for preparing the oligonucleotide variant may comprise the use of a solid phase reactor.
In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-described oligonucleotide variant or a pharmaceutically acceptable salt thereof.
The oligonucleotide variants are the same as described above.
The cancer may be a nucleolin-associated cancer, such as solid cancer and leukemia. For example, the cancer may be selected from the group consisting of: leukemia, lymphoma, myeloproliferative diseases, solid tissue carcinoma, sarcoma, melanoma, adenoma, hypoxic tumors, oral squamous cell carcinoma, squamous cell carcinoma of the throat, squamous cell carcinoma of the larynx, squamous cell carcinoma of the lung, uterine cancer, bladder cancer, hematopoietic cancer, head and neck cancer, nervous system cancer, and papilloma, but are not limited thereto. Furthermore, the nucleolin-associated cancer may be 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, hepatoma, stomach adenocarcinoma, uterine cancer, bladder cancer, thyroid cancer, ovarian cancer, melanoma, and cervical cancer, but is not limited thereto.
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 the severity of disease of a subject.
The atelocollagen dispersion may contain 0.5g to 5.5g, 1g to 4.5g, or 2g to 3.5g of atelocollagen per 100ml 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%.
Depending on the concentration of atelocollagen contained in the pharmaceutical composition of the present invention, differences may occur in the duration of the drug or the therapeutic effect. Therefore, the composition should contain a suitable concentration of atelocollagen.
The atelocollagen contained in the pharmaceutical composition of the invention may be present in the form surrounding the oligonucleotide variants contained in the composition.
The atelocollagen of the present invention may be prepared by a process comprising the steps of: a) swelling fresh pigskin in acetic acid, removing a fat layer, crushing and suspending a dermal portion in the acetic acid, and then adding at least one of alkaline protease, catalase, pepsin and papain to the suspension to extract a substance; b) first filtering the extracted substance and salting out the obtained filtrate by adding a neutral salt thereto, 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) lyophilizing the filtrate obtained by the third filtration to obtain high-purity atelopeptide collagen.
The atelocollagen of the present invention may be prepared by a process comprising the steps of: 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 material; c) adding a neutral salt to the filtrate obtained by the first filtration and salting out the extracted substance, followed by the second filtration; d) dissolving the collagen salt obtained by the second filtration to adsorb fat, followed by a third filtration; e) lyophilizing the filtrate obtained by the third filtering, and recovering the lyophilized powder; and f) dissolving the lyophilized powder in dilute hydrochloric acid (dil-HCl) of pH 4 to pH 8, dilute acetic acid or phosphate buffer and concentrating to prepare a atelocollagen solution, injecting the prepared atelocollagen solution into a column packed with polymer beads, and developing the column in dilute hydrochloric acid, dilute acetic acid or phosphate buffer of pH 4 to pH 8, thereby recovering atelocollagen by molecular weight.
The neutral salt used in the process for preparing atelopeptide collagen may be a sodium chloride solution.
In the method for preparing atelopeptide collagen, the lyophilized powder may be dissolved in dilute acetic acid at a concentration of 10mM or in a phosphate buffer solution at a concentration of 10 mM.
In a process for preparing atelopeptide collagen, lyophilized powder can be concentrated with MWCO 100K Dalton.
In the method for preparing atelopeptide collagen, the polymer beads injected with the atelopeptide collagen solution may be Sephadex G-200 Sephacryl.
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 bed volume.
In the method for preparing atelopeptide collagen, a atelopeptide collagen solution may be injected into a column packed with polymer beads, and then developed with dilute acetic acid having a concentration of 10mM or a phosphate buffer solution having a concentration of 10mM to recover the atelopeptide collagen.
The pharmaceutical composition of the present invention can be used in various formulations.
For example, the formulation 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 is not limited thereto. The pharmaceutical composition of the invention can be a telopeptide collagen dispersion preparation.
The pharmaceutical compositions of the present invention can be administered to a subject in need of treatment and/or inhibition of aberrantly proliferating cells.
The pharmaceutical composition may be administered as a sole therapeutic agent or in combination with other therapeutic agents. Furthermore, when administered in combination with other therapeutic agents, they may be administered sequentially or simultaneously, and may be administered in a single dose or multiple doses. When administering the pharmaceutical composition of the present invention, it is important to administer an amount that can ensure the maximum effect in the minimum amount without side effects, which can be easily determined by a person skilled in the art.
Administration may be oral or parenteral, and may include, for example, intravenous, subcutaneous, or intramuscular injection, but is not limited thereto.
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.
In addition, the present invention provides a method for preparing the pharmaceutical composition for preventing or treating cancer as described above.
The method for producing the pharmaceutical composition according to the present invention may comprise preparing a dispersion in which the above-described oligonucleotide variants are dispersed.
Dispersions in which the oligonucleotide variants are dispersed can be prepared by mixing the oligonucleotide variants with PBS.
The method for producing a pharmaceutical composition according to the present invention may further comprise the step of mixing a dispersion in which the above-described oligonucleotide variants are dispersed with a atelopeptide collagen dispersion.
Dispersions in which the oligonucleotide variants are dispersed can be prepared by mixing the oligonucleotide variants with PBS.
A atelopeptide collagen dispersion can be prepared by adding atelopeptide collagen to NaOAc/HAc (acetic acid) buffer solution.
The atelopeptide collagen dispersion may be prepared by adding 0.5g to 5.5g, 1g to 4.5g, or 2g to 3.5g of atelopeptide collagen per 100ml of the buffer solution.
The buffer solution may be at 0.3M NaOAc and 45% HAC.
The atelopeptide collagen added to the buffer solution can be lyophilized atelopeptide collagen.
The step of mixing the dispersion in which the oligonucleotide variant is dispersed with the atelopeptide collagen dispersion may include mixing 0.1mg to 3mg of the dispersion in which the oligonucleotide variant is dispersed per 400 μ L of the atelopeptide collagen dispersion.
For example, the dispersion in which the oligonucleotide variant is dispersed may be mixed in an amount of 0.1mg to 3mg, 0.5mg to 2.5mg, or 1mg to 2mg per 400 μ L of the atelopeptide collagen dispersion.
Hereinafter, the embodiments 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.
Example 1 preparation of oligonucleotide variants
Example 1-1 Synthesis of Gemcitabine-containing oligonucleotides
28 types of oligonucleotides were designed and prepared. In addition, gemcitabine is combined with each of the prepared oligonucleotides to produce oligonucleotide variants. The 28 types of oligonucleotide sequences and 28 types of oligonucleotide variants designed (in which modified nucleic acids were combined with oligonucleotides) are listed in table 3 below. In the following description, gemcitabine may also be referred to as Gem.
[ Table 3]
"Gemcitabine-containing oligonucleotides" (oligonucleotide variants) were synthesized by solid-phase phosphoramidite chemistry using a Mermade 12 DNA synthesizer (BioAutomation Manufacturing, Irging, TX).
Desalting was performed using Biotage MPLC, C18 cartidge. After dissolving the compound in d.w., the solution was isolated and purified on an Xbridge Oligonucleotide BEH C18130A, 1.7 μ M, 2.1x50mm column at 50 ℃ column oven temperature, mobile phase a solvent (0.1M TEAA), B solvent (100ACN) using Waters Acquity UPLC H-Class at a flow rate of 0.3 mL/min. Molecular weights were confirmed by Waters G2-XS Q-TOF mass spectrometer. The synthesis of all oligonucleotides was performed according to an internal protocol.
1-2 Synthesis of different types of oligonucleotide variants
Synthesis of ((N) according to the same procedure as described in 1-1 abovex-[TGG]4[TTG][TGG]4 or 5)、 (([TGG]4[TTG][TGG]4 or 5-(N)x) And ((N)x-[TGG]4[TTG][TGG]4 or 5-(N)y) (see table 4 below).
[ Table 4]
Example 2 confirmation of in vitro stability of oligonucleotide variants
Gemcitabine (2,2 '-difluorodeoxycytidine, dFdC) is converted by cytidine deaminase in plasma into the inactive metabolite, 2' -difluorodeoxyuridine (dFdU), and in this case, reduces the anticancer efficacy in vivo. To confirm the inhibition of degradation of gemcitabine in vivo by cytidine deaminase when it is bound to an oligonucleotide, the concentrations of dFdC and dFdU in plasma were determined.
2-1 liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for determining plasma concentrations of dFdC and dFdU
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 the active metabolite of gemcitabine in mouse plasma. From the chromatograms obtained by the established LC-MS/MS determination method, peak area ratios of the peak areas of dFdC, dFdU and dFdCTP to the internal standard substance were estimated, after which the concentration in the plasma and the concentration in the tissue of the mice were calculated from the previously created calibration curve.
2-1-1 preparation of Tetrauridine (THU) -treated blood
1mL of mouse blood was collected and placed in a 1.5mL eppendorf tube containing 10. mu.L (in DW) of 10mg/mL Tetrahydrouridine (THU). After centrifugation using a centrifuge at 14,000rpm and 4 ℃ for 2 minutes, only plasma was removed and transferred to another vial and stored at-80 ℃. After transferring 50. mu.L of plasma to a 1.5mL amber eppendorf tube, 150. mu.L of internal standard substance (1.29. mu.g/mL melt in ACN) was added and vortexed for 10 minutes. Centrifugation was carried out using a centrifuge at 14,000rpm at 4 ℃ for 20 minutes. Transfer 150. mu.L of the sample from the centrifuge tube to another 1.5mL amber eppendorf tube. The organic solvent was dried using a GeneVac EZ-2 automatic evaporation system (GeneVac Ltd., Ipswich, UK). (GeneVac HPLC mode, maximum temperature 35 ℃, 1 hour). The organic solvent was reconstituted by adding 125 μ L of DW to a 1.5mL dry amber eppendorf tube. After vortexing for 5 minutes, centrifugation was performed using a centrifuge at 14,000rpm at 4 ℃ for 5 minutes. The supernatant of the centrifuged solution was added to each vial for LC-MS/MS in an amount of 100 μ L using a PTFE (hydrophilic) 0.2 μm syringe filter (Toyo Toshi, Japan). Specifically, 5 μ L of the supernatant was injected into each LC-MS/MS.
2-1-2.LC-MS/MS analysis conditions
For mass spectrometry, the AB SCIEX QTRAP 5500 electrospray ionization mode (SCIEX, Framingham, MA, USA) was used. The quantitative conditions are listed in table 5 below.
[ Table 5]
Chromatography was performed using an Agilent 1200 series separation module (Agilent Technologies, Waldbronn, Germany). Furthermore, Hypersil gold C18, 1.9 μm, 100X2.1mm was used2Columns (ThermoScientific, matrices, Oslo, Norway). The injection volume was 5 μ L, 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 20ng/mL to 5,000 ng/mL. The chromatographic conditions are listed in table 6 below.
[ Table 6]
Fig. 1 shows a dFdC calibration curve and a dFdU calibration curve. In fig. 1(a), x is gemcitabine concentration and y is area in HPLC. Further, in fig. 1(b), x is the dFdU concentration, and y is the area in HPLC.
2-1-3.LC-MS/MS analysis results
Each sample was treated according to 2-1-1 and 2-1-2 above, and with respect to (Gem)x-[TGG]m[TTG][TGG]nThe compound type (d) measures the dFdC/dFdU ratio in rat plasma. The measurement results are listed in table 7 below (Gem ═ gemcitabine).
[ Table 7]
Selection of several Compounds with high dFdC/dFdU ratios (Compound 10, also hereinafterReferred to as IO 101; and compound 11, hereinafter also referred to as IO101L), followed by additional experiments to determine the effect of the oligonucleotide variants. Furthermore, to compare the effect of IO101 and IO101L with other oligonucleotide variants, experiments were performed using oligonucleotide variants of different sequences such as the following, and gemcitabine that was not bound to the oligonucleotide: [ (Gem)2-[TCC]4[TTG][TCC]4；IO101-Con(CRO)， (Gem)2-GGTGGTGGTGGTTGTGGTGGTGGTGGTGG; IO100 sum (Gem)2-CCTCCTCCTCCTTCTCCTCCTCCTCCTCC；IO100-Con((Gem)2-CRO))。
Example 3 evaluation of oligonucleotide variants for in vitro efficacy against cancer cell lines
Confirmation of anticancer efficacy of IO101 on pancreatic cancer cell lines
BxPC3, PANC-1, Miapaca-2 and Capan-1 were each cultured as pancreatic cancer cell lines. When cells were stable after several subcultures, 1 × 103Individual cells were seeded in 96-well plates to verify drug effects. The next day, IO101, IO100-Con, IO101-Con and Gem at maximum concentration of 100. mu.M were diluted in 1/10 per well and prepared at 4-point concentration of maximum 100 nM. Then, cells seeded in 96-well plates were treated with the variants prepared above. After 48 hours, 10. mu.l of WST-1 solution was added to the wells and incubated in an incubator at 37 ℃ for 1 hour. Subsequently, each sample was measured at 440nm in a microplate reader (VERSA Max). After the measurements were calculated, a plot of cell viability was plotted for each treatment concentration.
In all four pancreatic cancer cell lines, (Gem) was observed compared to IO100, IO100-Con, IO101-Con and Gem2-[TGG]4[TTG][TGG]4(IO101) is effective in inhibiting pancreatic cancer cell growth in proportion to drug concentration (see FIG. 2).
At 1x10 respectively4After 4 types of pancreatic cancer cell lines were seeded per well and then treated with IO101, the treated cell lines were labeled with a red probe and imaged within 48 hours to observe apoptosis of the cancer cells. FIGS. 3 to 6Confirmation of apoptosis of 4 types of pancreatic cancer cells treated according to IO101 is shown. Intensity values are dead cell indices and are indicated as fold changes.
Confirmation of anticancer Effect of IO101L on pancreatic cancer cell lines
Validated by cell viability assay (Gem) in the same manner as the efficacy assessment in 3-1 above2-[TGG]4[TTG][TGG]5(IO101L) efficacy on pancreatic cancer cell lines. As a result, in all four pancreatic cancer cell lines, pancreatic cancer cell growth was effectively inhibited by IO101L compared to the control (see fig. 7).
Example 4 confirmation of in vivo stability of oligonucleotide variants
Gemcitabine (8mg/kg) was injected intravenously in rats, or as a novel agent prepared by combining gemcitabine with an oligonucleotide (Gem)x-[TGG]m[TTG][TGG]n(160mg/kg, which corresponds to 8mg/kg based on gemcitabine), to compare pharmacokinetic changes between gemcitabine and its metabolite, i.e., 2', 2-difluorodeoxyuridine (dFdU) anthocyanin-3-glucoside.
4-1. intravenous injection of oligonucleotide variants or gemcitabine in rats
Sprague-Dawley male rats were subjected to induction anesthesia with isoflurane as an inhalation anesthetic, and then Polyethylene (PE) tubes (Clay Adams, Becton Dickinson, NJ, USA) were inserted into the carotid artery (for blood collection) and sutured with sutures, after which the ends of the sutures were fixed at the back of the neck. During surgery, anesthesia was maintained with ether and about 0.5mL of heparin (20 units/mL) in saline was injected to prevent blood from clotting in the cannula. After the end of the surgery, the rats were placed in metabolic cages and allowed to recover completely from the anaesthesia state (4 to 5 hours), respectively. Thereafter, the animals were divided into (Gem)2-[TGG]4[TTG][TGG]5Administration group (N ═ 4) and gemcitabine administration group (N ═ 4), and the corresponding drugs were administered to these groups. On an electronic balance (CP224S, Sar)torius, GER) by combining gemcitabine with (Gem)2-[TGG]4[TTG][TGG]5After weighing to a certain dose (8mg/kg and 160mg/kg), each drug was dissolved in 0.9% sterile physiological saline and prepared. 1ml/kg and 2ml/kg of drug were administered intravenously. Application in clinical settings (Gem)2-[TGG]4[TTG][TGG]5And 0.3mL of blood was collected via the jugular vein before gemcitabine (0min) and after 1, 5, 15, 30, 45, 60, 120, 360 and 720 minutes post-administration, respectively. In addition, in order to prevent gemcitabine in blood from being metabolized by cytidine deaminase, the collected blood was put into an eppendorf tube previously placed with 10. mu.L of distilled water containing Tetrahydrouridine (THU) dissolved at a concentration of 10mg/mL per 1mL of blood as a cytidine deaminase inhibitor. Then, the blood in the eppendorf tube was immediately centrifuged, after which the plasma was stored. Since gemcitabine is known to be photodegradable, all plasma samples were placed in brown eppendorf tubes at 50 μ L per tube and stored at-80 ℃ until LC-MS/MS analysis was performed.
4-2 analysis of plasma concentrations of Gemcitabine and its metabolite dFdU
After pre-treatment by plasma protein precipitation using pre-established acetonitrile plasma, sample treatment and analysis of plasma concentrations of gemcitabine and dFdU was performed by LC-MS/MS in AB SCIEX QTRAP 5500 electrospray ionization mode (SCIEX, Framingham, MA, USA) using an Agilent 1200 series analyzer (Agilent Technologies). All analytical procedures were carried out 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, 100X2.1mm2(Thermo Scientific, matrices, Oslo, Norway); and mobile phase conditions: distilled water (a) and acetonitrile (B) containing 0.1% formic acid (see table 6 above).
4-3 creation of plasma calibration curves
Gemcitabine and dFdU are respectively dissolved in distilled water to prepare a storage solution of 1mg/mL, stored frozen and diluted with three-stage distilled water to prepare a working solution having gemcitabine concentrations of 200, 400, 800, 2000, 10000, 20000 and 100000ng/mL and dFdU concentrations of 200, 400, 800, 2000, 10000, 20000 and 50000ng/mL, and then stored in a refrigerator. Metformin was dissolved in acetonitrile as an internal standard substance (IS) to prepare 645ng/mL of another working solution. Gemcitabine and dFdU working solutions were added to empty plasma of rats, respectively, and standard plasma samples were prepared so that the plasma concentrations of gemcitabine and dFdU were 10, 20, 40, 100, 500, 1000, and 5000ng/mL, and 10, 20, 40, 100, 500, 1000, and 2500ng/mL, respectively.
4-4. method for processing plasma samples
mu.L of internal standard (i.e., metformin) was added to 50. mu.L of plasma (645 ng/mL in acetonitrile) and vortexed for 10 minutes, followed by centrifugation at 15,000rpm at-4 ℃ for 20 minutes. After that, 150. mu.L of the supernatant was taken and transferred to another eppendorf tube, and then the organic solvent was dried under a nitrogen stream. Then, 125. mu.L of tertiary distilled water was added to the dried eppendorf tube and redissolved. The solution was then vortexed for 5 minutes and centrifuged at 15,000rpm at-4 ℃ for 5 minutes. Thereafter, 100. mu.L of the supernatant was transferred to a vial for LC-MS/MS, 5. mu.L of the supernatant was injected and analyzed by LC-MS/MS.
4-5. determination of analytical suitability
To determine the suitability of the analytical process, the samples used to create the calibration curve were analyzed for each batch of analytical samples during sample processing, and then the samples for analytical suitability were analyzed twice (for gemcitabine, lowest limit of quantitation: LLoQ, 10ng/mL, low concentration: LoQC, 30ng/mL, medium concentration: MiQC, 900ng/mL, high concentration: HiQC, 4000 ng/mL; for dFdU, lowest limit of quantitation: LLoQ, 10ng/mL, low concentration: LoQC, 30ng/mL, medium concentration: MiQC, 900ng/mL, high concentration: HiQC, 2000 ng/mL). At least 67% of 6 suitable samples (e.g., 4 out of 6 samples) should be within 15% of theoretical, and whether 50% or more is within 15% of theoretical at the same concentration was investigated.
4-6. the medicine acts as a substituteMechanical parameter calculation and statistical processing
Pharmacokinetic parameters of gemcitabine were obtained by the Winnonlin Professional program (Pharsight, Mountain View, CA, USA). Plasma concentration-time Curve (AUC)t) The area values below are calculated from the plasma concentration-time curve from the time after administration of the drug to the final quantification using a log-linear trapezoidal equation (specifically, the linear-trapezoidal equation is used in the portion where the plasma concentration increases, and the trapezoidal equation is used by logarithmically transforming the concentration values in the other portion where the plasma concentration decreases). Following oral administration, the maximum plasma concentration (C) is determined from the plasma concentration-time curvemax) And the time to reach maximum plasma concentration (T)max). The area under plasma concentration (AUC) up to an infinite time-time curve was determined using the following equationinf). Determination of the terminal elimination rate constant (. gamma.Z) and half-life (t) from the slope of elimination of plasma concentration1/2)。
AUinf＝AUCt+Ct/γZ(Ct: final quantitative concentration, γ Z: terminal elimination rate constant)
To compare pharmacokinetic parameters between the two groups, a separate sample t-test was performed using SPSS (19 th edition, Chicago, IL, USA).
4-7 comparison of in vivo pharmacokinetic Properties between Gemcitabine and dFdU
As to gemcitabine (8mg/kg) or (Gem)2-[TGG]4[TTG][TGG]5(160mg/kg, which corresponds to 8mg/kg based on gemcitabine) the plasma concentrations of gemcitabine are shown in figure 8, while the plasma concentrations of dFdU concentrations are shown in figure 9. In addition, in gemcitabine or (Gem)2-[TGG]4[TTG][TGG]5The values of the pharmacokinetic parameters of gemcitabine and dFdU are listed in table 8 as mean ± standard deviation in intravenous injections. In Table 8 below, AUCtIs the area under the curve from 0min to the time of the last blood collection, t1/2Is terminal half-life (plasma elimination half-life), CL is systemic clearance, VdssIs in vivoAnd MRT is the mean residence time of the drug in the body, TmaxIs median (range) and metabolic conversion value is determined by using dFdU AUCtValue divided by gemcitabine AUCtValue, or a calculated value.
[ Table 8]
As a result of the experiment, the gemcitabine administration group reached the highest plasma concentration immediately after administration, as in the general pharmacokinetic pattern after intravenous injection of the drug. However, (Gem)2-[TGG]4[TTG][TGG]5The administration group reached C5 to 30 minutes after intravenous injectionmax. Intravenous injection (Gem)2-[TGG]4[TTG][TGG]5The plasma gemcitabine concentration thereafter refers to the in vivo nucleic acid degrading enzyme ("nuclease") from the oligonucleotide, i.e., [ TGG ]]4[TTG][TGG]5Concentration of released gemcitabine. As a result of preliminary experiments, it was confirmed that (Gem) was generated during the plasma sample treatment (organic solvent such as acetonitrile or methanol)2-[TGG]4[TTG][TGG]5Will not be released in the form of gemcitabine. In addition, because gemcitabine is a drug with a very low plasma protein binding rate, it is injected intravenously (Gem) in rats2-[TGG]4[TTG][TGG]5The gemcitabine concentration measured later in the plasma is indicative of gemcitabine free concentration produced by nuclease in vivo. (Gem)2-[TGG]4[TTG][TGG]5The administration group showed significantly lower gemcitabine plasma concentration up to 120 minutes than the gemcitabine administration group. However, after 120 minutes (360 minutes, 720 minutes), (Gem)2-[TGG]4[TTG][TGG]5The administration group showed a higher gemcitabine concentration than the gemcitabine administration group (see fig. 8). This means [ TGG]4[TTG][TGG]5Is metabolized into gemcitabine by nucleases in the body, which in turn is slowly released into the plasma. Results, (Gem)2-[TGG]4[TTG][TGG]5Plasma elimination half-life (t) of gemcitabine in administration group1/2) Statistically significantly increased by about two-fold (177 ± 28.5 minutes compared to 360 ± 54.8 minutes; see table 8 above). Furthermore, it is understood that (Gem)2-[TGG]4[TTG][TGG]5Vd of gemcitabine in administration groupssThe volume distributed in vivo, which indicates the affinity between the drug and the tissues in vivo, increased significantly by more than two-fold. (1150. + -. 223mL/kg compared to 2670. + -. 139 mL/kg; see Table 7 above). That is, it can predict (Gem)2-[TGG]4[TTG][TGG]5Gemcitabine of the administration group had a higher distribution in the in vivo tissue, and thus distributed much more into the tissue than the gemcitabine administration group. AUC of Gemcitabine in both groupstAnd AUCinfThe values were similar and showed no statistical significance. In (Gem)2-[TGG]4[TTG][TGG]5In the case of the administration group, plasma concentrations of dFdU detected as an inactive metabolite of gemcitabine were lower than those of the gemcitabine administration group until 360 minutes after administration (see fig. 9). Due to binding to [ TGG ]]4[TTG][TGG]5Gemcitabine is not metabolized to dFdU, so the measured dFdU concentration in plasma means intravenous injection (Gem)2-[TGG]4[TTG][TGG]5Thereafter, when gemcitabine is converted from [ TGG ]]4[TTG][TGG]5The concentration of dFdU produced by cytidine deaminase in the plasma upon release. After 360 minutes (Gem)2-[TGG]4[TTG][TGG]The 5 administration group showed higher plasma concentrations of dFdU, however, no further confirmation was possible due to the relatively short sampling time limit. (Gem)2-[TGG]4[TTG][TGG]5C of dFdU in administration groupmaxValues showed a trend of decrease (364. + -. 52.3ng/mL vs 477. + -. 87.9 ng/mL), but did not show statistical significance between the two groups (see Table 8 above). To compare the ratio of dFdU produced by cytidine deaminase in the two groups, the dFdU AUC was determined by comparingtValue divided by gemcitabine AUCtThe value is used to calculate the metabolic conversion rate. From the results, both groups showed similar values (0.190 ± 0.019 compared to 0.181 ± 0.0124) with no statistical significance. According to the principleMeasuring, at (Gem)2-[TGG]4[TTG][TGG]5In the case of [ TGG ]]4[TTG][TGG]5The gemcitabine of (a) is slowly released into plasma and distributed to tissues in large quantities, but the released gemcitabine does not affect the dFdU produced by the cytidine deaminase present in plasma and tissues.
Table 9 below shows the results of gemcitabine and (Gem)2-[TGG]4[TTG][TGG]5Followed by plasma gemcitabine concentrations over time.
[ Table 9]
Table 10 below shows the results of gemcitabine and (Gem)2-[TGG]4[TTG][TGG]5Followed by plasma concentrations of dFdU over time. BLLoQ means a value below a minimum quantitation limit, and ND means an undetected value.
[ Table 10]
Example 5 preparation of atelocollagen for medical use
5-1. preparation of pigskin
The pigskin was washed three times with tap water and three times with primary purified water, divided into 3kg (20cm x 20 cm) and stored in a freezer at-20 ℃. The frozen pigskin was allowed to stand at 4 ℃ for 2 hours, thawed and finely cut to a size of 1.5cm x 8 cm. Thereafter, 7.5L of 0.5M acetic acid was added to the pigskin, followed by standing overnight and observing the swelling of the pigskin.
5-2. fat removal step
The swollen pigskin was removed, cut to a size of 1.5cm x 1.5cm, and 7.5L of 0.5M fresh acetic acid was added again. Then, after standing for several hours, only the dermis was filtered through a sieve. The dermis was washed with 10L of purified water. The washing process was repeated five times in total. After adding 20L of ethanol to the washed dermis, the mixture was stirred at 4 ℃ overnight. After recovering the dermis only from the overnight stirred sample, 20L of ethanol was added, followed by stirring again for 1 hour at 4 ℃. The dermis was filtered using a sieve and left for about 1 hour to remove ethanol. The dermis, from which fat has been removed, is subdivided into portions of appropriate weight (500g) and stored in a cryofreezer at-80 ℃.
5-3 homogenizing and grinding dermis
7.5L of 0.5M acetic acid was added to 3kg of frozen dermis thawed by standing at 4 ℃ and then left to stand for 30 minutes. After removing acetic acid by filtration using a sieve, the dermis was subdivided into 250g portions. 250g of dermis and 2L of purified water were put into a mixer and then ground for 2 minutes. Then, 2L of purified water was further added, followed by grinding again for 2 minutes. 4L of 0.73M acetic acid was added to the ground tissue. The tissue was again ground using the homogenizer for 3 minutes. The grinding process was repeated four times to grind and blend 1kg of frozen dermis. Further, 18L of 0.73M acetic acid was added thereto, thereby adjusting the final acetic acid concentration to 0.5M. Subsequently, a pH of about 2.5 to 4 was confirmed. The mixture was stirred at low speed for 3 hours using a stirrer.
5-4 pepsin treatment
Each kg of dermis 15X 107Units of pepsin were added to the finished dermal sample, followed by gentle stirring for 24 hours using a stirrer. To the pepsin treated samples 10M NaOH was added and stirred to reach pH 8 to 9 and further stirred for 10 minutes to inactivate the pepsin. After inactivating pepsin by alkaline 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 ℃ for 10 minutes using a centrifuge, the fat on the surface of the supernatant was removed, and the remaining supernatant was collected and stored.
5-5 salting out and Generation of atelopeptide collagen intermediates
5M NaCl was slowly added to 1L of the supernatant prepared from dermis at a rate of 163ml, stirred for 15 minutes, and then allowed to stand overnight at 4 ℃ to perform salting out. After salting out, the supernatant was removed by aspiration, and the precipitate was centrifuged (7800rpm, 4 ℃, 10 minutes) to completely remove the supernatant. To the precipitate was added 30L of ethanol, and the mixture was washed while stirring overnight at 4 ℃. After centrifugation (7800rpm, 4 ℃, 10 minutes), 30L of ethanol was again added to the precipitate, followed by stirring at 4 ℃ for 6 hours for a second wash. After centrifugation (7800rpm, 4 ℃, 10 minutes), the weight of the precipitated atelocollagen intermediate was measured and subdivided and stored in a low temperature freezer at-80 ℃.
5-6 production of atelocollagen for medical use
After 2.8L of 0.02M urea was added to 200g of atelocollagen intermediate, the mixture was stirred overnight. Diafiltration of the atelocollagen was performed using Centramate (tangential flow filtration system) and the recovered solution was stirred in a stirrer while adding 0.5M NaOH to reach pH 7. The prepared neutral atelopeptide collagen was thinly and flatly subdivided, after which each subdivided portion was put into a zipper bag and stored in a low-temperature freezer at-80 ℃. After primary freezing (-40 ℃) in a lyophilizer for at least 1 hour, the atelopeptide collagen stored in the cryofreezer is transferred to a lyophilizer and lyophilized. The lyophilized medical atelopeptide collagen was cut into an appropriate size and vacuum-packed, followed by refrigeration.
5-7 preparation of atelocollagen solution with an identified pH neutral by substitution with buffer solution
5-7-1. step of dissolving lyophilized atelopeptide collagen in sodium acetate buffer solution (0.3M sodium acetate (NaOAC), 45% acetic acid)
To prepare 0.3M NaOAC, a 45% acetic acid buffer solution, 2.4g of CH3CO2Na was dissolved in 55 ml of tertiary sterile water and 45ml of an acetic acid solution having a purity of 99% or more. Then, the solution was titrated with acetic acid using a pH meter to reach pH 3.0. 3g of lyophilized atelopeptide collagen was finely cut using sterile forceps and scissors, and the finely cut pieces were dissolved in 0.3M NaOAC, 45% acetic acid solution. Specifically, finely cut atelocollagen was slowly added for complete dissolution while it was mixed with the solution by a stir bar.
5-7-2. diafiltration Using Tangential Flow Filter (TFF) System and replacement of atelocollagen solution with PBS
As shown, a 3% atelopeptide collagen solution was pumped into the prepared TFF system (via TFF 100K) using a pump, and the filtered sodium acetate buffer solution was transferred to waste for disposal, while the remaining solution was returned to the storage vessel via the retentate tube. 1x PBS buffer solution was added to the storage vessel to maintain the 3.0% atelopeptide collagen solution at a predetermined level while continuing the dialysis filtration. Specifically, the dialysis filtration was continued using a PBS solution having a volume corresponding to 10 times that of the telopeptide collagen-sodium acetate buffer solution initially prepared, thereby performing the buffer exchange. When the buffer exchange is complete, completion of the buffer exchange is confirmed by monitoring the pH of the osmotic solution to determine if a neutral pH is detected. After dialysis and filtration, 3% atelopeptide collagen was aliquoted into sterile tubes in 10ml and stored in a freezer at 4 ℃.
Example 6 Generation of oligonucleotide variant/atelopeptide collagen compositions
6-1 preparation of Atelocollagen (AC) Dispersion
0.5%, 1.0%, 1.5%, 2.0% and 3.0% of high purity medical atelocollagen (0.5 g, 1.0g, 1.5g, 2.0g and 3.0g atelocollagen per 100ml buffer, respectively) was placed in a NaOAc/HAc solution (0.3M sodium acetate, 45% acetic acid), maintained at pH3.0 and completely dissolved while stirring. The solution was diafiltered using Tangential Flow Filtration (TFF) and then the atelopeptide collagen solution was diafiltered with 10 volumes of 1x PBS solution to prepare a dispersion of medical collagen in PBS solution.
Generation of IO 101/atelocollagen Sol-gel type and IO 101L/atelocollagen Sol-gel type
In general (Gem)2-[TGG]4[TTG][TGG]4(IO101) Or (Gem)2-[TGG]4[TTG][TGG]5(IO101L) after being placed in PBS and completely dissolved by a mixer at room temperature, a solution of IO101 or IO101L mixed in PBS was added to the atelocollagen dispersion in the following amounts: 0.5mg, 1.0mg, 1.5mg, 2.0mg, 4.0mg or 8.0mg (0.5%, 1.0%, 1.5%, 2.0%, 3.0%, respectively) per 400. mu.l of atelocollagen dispersion.
The above solution was mixed at room temperature for 30 minutes by a rotating sleeve device to prepare a mixture. In the liquid state (Gem)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 be cured (gelled) at 37 ℃ when injected directly into tumors in vivo. In addition, because of the surround (Gem)2-[TGG]4[TTG][TGG]4(IO101) or (Gem)2-[TGG]4[TTG][TGG]5The atelocollagen of (IO101L) is slowly dissolved, so that the drug is gradually released, thereby enabling effective tumor treatment.
Generation of IO 101/atelocollagen disks and IO 101L/atelocollagen disks
The high-concentration collagen dispersion prepared above and (Gem) were used in the same manner as the sol-gel type manufacturing method2-[TGG]4[TTG][TGG]4(IO101) or (Gem)2-[TGG]4[TTG][TGG]5(IO101L) preparation of the mixture.
The prepared mixture was placed in a cylindrical silicone mold having a diameter of 1cm and diffused to form a uniform film of 0.5 mm, followed by lyophilization at-80 ℃. The completely lyophilized sample was again lyophilized in a lyophilizer maintained at-70 ℃ for 30 hours to form a porous thin film. Hereinafter, the film prepared as described above is referred to as a wafer or a patch.
Example 7 evaluation of the stability of oligonucleotide variant/atelopeptide collagen compositions in plasma
For gemcitabine and (Gem) in rat plasmax-[TGG]m[TTG][TGG]nAnd concentration analysis of gemcitabine and gemcitabine inactive metabolites (i.e., dFdU) released from the high purity atelocollagen formulation. Sample processing, calibration curve creation and LC-MS/MS analysis conditions were the same as described above.
7-1 evaluation of stability of rat plasma (IO 101L)/atelocollagen discs
To evaluate the stability of gemcitabine/atelocollagen discs in plasma, experiments were performed with 5 groups classified as listed in table 12 below.
Gemcitabine/atelocollagen discs from groups 2, 3, 4 and 5 were added to 2mL rat plasma to achieve a plasma concentration of 60 μ g/mL before CO2Incubate at 37 ℃ for 2 hours in an incubator. Sample processing was performed as follows. After 2 hours, to remove the collagen attached to the gemcitabine/atelocollagen disc in the plasma, 500 μ L of plasma containing gemcitabine was added to Ultracell-3K, followed by centrifugation at 15,000rpm and 20 ℃ for 30 minutes. Thereafter, the supernatant was taken and then centrifuged again under the same conditions as above. Then, 5 μ L of the concentrate was taken and diluted 10-fold with the same rat plasma to measure the concentration in plasma ("plasma concentration"). The concentration of gemcitabine concentrated in the upper portion of Ultracell-3K was measured, as well as another concentration of gemcitabine in the lower portion of filtered plasma. In addition, the concentration of gemcitabine eluted from the disc after 2 hours of incubation was also measured. The above sample treatment was performed in the same manner as the calibration curve creation, followed by sample analysis under the same LC-MS/MS conditions.
Results of analysis of gemcitabine (dFdC) and its metabolites (i.e., dFdU) in rat plasma with a minimum quantitation limit of 10 ng/mL. In addition, because the crosstalk phenomenon occurs, the retention time of gemcitabine (dFdC) is different from that of dFdU, in which a dFdU peak is generated, and thus in the gemcitabine (dFdC) analysis, measurement is performed while a gradient is given to a mobile phase to prevent each peak from being affected. Thus, the retention times of gemcitabine and dFdU were measured as 2.1 minutes and 4.23 minutes, respectively. As a result of analyzing the standard plasma samples, all samples were determined to have an accuracy within ± 15%, so that the sample concentrations determined as above were reliable (see table 11 below).
[ Table 11]
7-2.(IO 101L)/evaluation of in vivo stability of atelocollagen discs
To evaluate the stability of the IO 101L/atelocollagen disc formulation in rat plasma, experiments were performed with 5 groups (1-5 groups) (see table 12 below). Normal collagen (SK Co.) was used for comparison with atelocollagen.
[ Table 12]
In CO2As a result of incubation in the incubator for 2 hours, the gemcitabine concentration in the gemcitabine/atelocollagen discs of groups 2 and 3 was lower than that of group 1 in the stock state. As (Gem)2-[TGG]4[TTG][TGG]5Groups 4 and 5 of the/atelocollagen disc formulations were determined to have lower concentrations than groups 2 and 3 containing collagen alone. As a result of preliminary experiments, it can be seen that the release rate of the IO 101L/atelocollagen disc formulation in rat plasma is stable and sustained. In addition, the formulations conjugated to atelopeptide collagen showed a rather low metabolic rate of gemcitabine to 2 ', 2' -difluorodeoxyuridine, an inactive metabolite of gemcitabine (see table 13 below).
[ Table 13]
Example 8 evaluation of anti-cancer efficacy of oligonucleotide variant/atelopeptide collagen compositions
8-1 anticancer efficacy of IO 101/atelopeptide collagen (Sol-gel type) using subcutaneous pancreatic cancer animal models
The pancreatic cancer therapeutic effect of IO 101/atelocollagen (sol-gel type) was demonstrated using subcutaneous pancreatic cancer animal models implanted with pancreatic cancer cell lines.
8-1-1 evaluation of anticancer efficacy against IO101 content
It was confirmed that IO 101/atelopeptide collagen (sol-gel type) is superior to gemcitabine/atelopeptide collagen (sol-gel type) in terms of pancreatic cancer inhibitory efficacy. To cope with different clinical situations, a sol type drug for local injection therapy is used. After injection, the therapeutic effect was verified by determining changes in tumor size and histochemical changes.
In other words, after culturing the pancreatic cancer cell line Capan-1 cells in the recommended medium (RPMI, 10% FBX, 1% AA), 1X10 was added6Individual cells were injected subcutaneously into nude mice. Tumor size was measured with calipers, and mice with tumor size up to 0.5cm diameter were selected as treatment targets. For statistical analysis, medical efficacy was evaluated from 5 or more mice per group. After injecting IO 101/atelopeptide collagen (sol-gel type) directly into the tumor, tumor size was measured to determine therapeutic effect. At the end of 30 days of treatment, animals were sacrificed and tumors were removed to determine the effect of treatment.
IO 101/atelocollagen (sol-gel type) with different doses (IO101-0.5mg/AC, IO101-1.0mg/AC, IO101-1.5mg/AC and IO101-2.0mg/AC) was injected subcutaneously into mice with pancreatic cancer by mixing 0.5mg, 1.0mg, 1.5mg and 2.0mg of IO101 dispersion with atelocollagen dispersion per 400. mu.L, respectively, after 30 days tumor was extracted and compared in size. It was confirmed that tumors were best inhibited at IO101-2 mg/AC (sol-gel type) (see FIGS. 10 and 11).
8-1-2 evaluation of anticancer efficacy against atelopeptide collagen concentration
Animals were evaluated for weight loss and toxicity following subcutaneous injection of IO 101/atelocollagen (sol-gel type), with the dose of IO101 set at 2mg/400 μ L of atelocollagen dispersion. Since the duration of the drug and its therapeutic effect may vary depending on the concentration of Atelocollagen (AC), comparative experiments were performed with respect to atelocollagen concentrations of 0.5%, 1.0% and 1.5% (g per 100ml of buffer), respectively. It was confirmed that the therapeutic effect was highest in mice with subcutaneous pancreatic cancer when 1.5% concentration (g amount per 100ml buffer) of atelopeptide collagen was used (see fig. 12).
8-2 in vivo anticancer efficacy of IO 101/atelocollagen (disc) in subcutaneous pancreatic cancer cell line transplanted animals
Pancreatic cancer cell line BXPC3 at 2x106The individual cells were transplanted subcutaneously in BALB/C nude mice. Three days later, 3 discs were implanted on the tumor per group. After transplantation, tumor volume was measured twice weekly for 30 days using a 2x0.5 (major axis x minor axis) caliper and body weight was measured twice weekly. It can be seen that the IO101/AC disc inhibited pancreatic cancer better than the other groups. After disc implantation, tumor size was observed in POD30 mouse images. It can be visually confirmed that the tumor volume of the IO101/AC disc is smaller than the other groups. In fact, the tumor volume of the IO101/AC discs was found to be rather small, and in particular the IO101-2 mg/1.5% to 3.0% AC group had the smallest tumor volume (see fig. 13 and 14).
8-3 pancreatic cancer of IO 101/atelocollagen (discs) in mouse model of orthotopic xenograft pancreatic cancer Therapeutic efficacy
5x10 which will express luciferase by replacement of specific vectors5One BxPC3 cancer cell line was injected into the pancreas (male, 6 weeks old, 30 animals) of Balb/c-nude mice with saline. At week 2 of the construction of the pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. To reduce autofluorescence, non-fluorescent feed was provided 1 week prior to imaging. Intraperitoneal insertion (intraperitoneal insertion using surgery) IO101/AC disc and comparative drug. Immediately after modeling, luciferase imaging was performed by in vivo IVIS spectroscopy to measure tumors and tumor size. Luciferase imaging was performed using the IVIS spectrometer with time (days 6, 18, 21, 23, 25, 28, 31 and 35). After the last in vivo IVIS spectral imaging, animals were sacrificed for ex vivo measurements of each organ (including spleen, liver, heart, lung, kidney and pancreas of tumors). The size of the extracted tumor was measured and compared by caliper. Immediately after insertion of disc drug into experimental animals, changes in tumor size were monitored over time by luciferase imaging. In addition, changes in luciferase imaging were determined using IVIS spectroscopy as described below (see fig. 15). After sacrifice of experimental animals, the actual size of the tumor was measured ex vivo. Table 14 below lists the actual size variation of the extracted tumors, which is graphically shown in fig. 16.
[ Table 14]
Further, with respect to the group implanted with only AC discs, it was confirmed that tumors had a larger size than the other groups, and most progressed to intraperitoneal metastatic cancer. The liver, spleen and kidney showed the most metastases and 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 inhibited, while the Gem/AC disc and AC disc implants were observed to be mostly translocated to the intraperitoneal liver and septum. However, the IO101/AC discs did not show intraperitoneal cancer metastasis (see fig. 17).
8-4 IO 101/atelopeptide collagen (discs) and IO 101L/Deplasia in mouse model of orthotopic xenograft pancreatic cancer Pancreatic cancer metastasis inhibition efficacy of telopeptide collagen (discs)
5x10 which will express luciferase by replacement of specific vectors5One BxPC3 cancer cell line was injected into the pancreas (male, 6 weeks old, 30 animals) of Balb/c-nude mice with saline. At week 2 of the construction of the pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. To reduce autofluorescence, non-fluorescent feed was provided 1 week prior to imaging. Intraperitoneal insertion (intraperitoneal insertion using surgery) of IO101/AC disc or IO101L/AC disc and comparative drug. Immediately after modeling, luciferase imaging was performed by in vivo IVIS spectroscopy to measure tumors and tumor size. Luciferase imaging was performed using the IVIS spectrometer with time (days 6, 18, 21, 23, 25, 28, 31 and 35). After the last in vivo IVIS spectral imaging, animals were sacrificed for ex vivo measurements of each organ (including spleen, liver, heart, lung, kidney and pancreas of tumors). The organs were observed for intraperitoneal metastases. In particular, the AC discs were observed to migrate primarily to the liver, kidneys and/or septum within the abdominal cavity. However, no intraperitoneal cancer metastasis was observed in the IO101/AC disc and the IO101L/AC disc (see FIG. 18).
8-5 survival Change after Implantation of IO 101L/atelocollagen (discs)
Survival by concentration after implantation of IO101L/AC discs was compared in an in situ mouse model of pancreatic cancer. After transplantation of AC discs alone, 2 of 5 animals survived 32 days later. Furthermore, 4 of 5 animals survived for 32 days with IO101L-2mg (mix IO101L 2mg per 400 μ L atelocollagen dispersion)/AC disc implantation. Furthermore, all 5 animals died on day 6 with IO101L-4mg (IO101L 4mg per 400. mu.L atelocollagen dispersion)/AC disc and IO101L-8mg (IO101L 8mg per 400. mu.L atelocollagen dispersion)/AC disc (see FIG. 19).
8-6 IO 101L/atelocollagen after removal of residual tumor in orthotopic xenograft pancreatic cancer mouse model (Sol-gel)Glue type) and IO 101L/atelocollagen (disc) tumor and metastasis inhibition
8-6-1.BxPC3 pancreas cancer transplantation mouse experiment
After tumor removal by surgical procedure in situ pancreatic cancer mice, IO101L/AC sol-gel type or IO101L/AC disc drugs were inserted, followed by observation to confirm whether residual tumor was inhibited and whether metastasis of residual tumor to other organs in the abdominal cavity was inhibited. Specifically, 5x10 expressing luciferase by substituting a specific vector was prepared in Balb/c nude mice (male, 6 weeks old, 21 animals)5One BxPC3 cancer cell line (25 μ L). The pancreas was removed by cutting the abdominal region of the intraperitoneally anesthetized mouse, and then BxPC-3-Luc cells were injected into the prepared pancreas. At week 2 of the construction of the pancreatic cancer mouse model, luciferase imaging was performed to confirm the tumor. The tumor cell line used here was BxPC-3-Luc cells expressing luciferase by substituting a specific vector in BxPC3 cells, and tumors were measured using luciferin. To reduce autofluorescence, non-fluorescent feed was provided 1 week prior to imaging. After removal of pancreatic tumors by incision of the abdominal region of intraperitoneal anesthetized mice, IO101L/AC sol-gel type, IO101L/AC disc, Gem/atelocollagen sol-gel or Gem/atelocollagen disc was transplanted to the remaining tumor site (intra-abdominal insertion using surgery). Tumor size and metastasis were confirmed by luciferase imaging, and the results thereof are shown in fig. 20.
Experiment in mice transplanted with Capan-1 pancreatic cancer 8-6-2
When mice ordered for the generation of an in situ mouse model of pancreatic cancer were offered, a 1 week period was maintained to accommodate the environment. During the adaptation period, at 1 × 106The capan-1 cells were prepared per 100. mu.l. The mouse skin was opened 2mm, the spleen was folded up and the above cells were directly injected into the pancreas using a syringe. After 4 weeks, the tumors were confirmed by MRI to be 5mm to 6mm in diameter. When the cancer cell diameter is determined to be 5mm to 6mm, the abdomen is opened and then pancreatic tumors are removed as much as possible. After removal, IO101L/AC disc and atelocollagen disc (control) were inserted into the remainingThe tumor portion was sealed (see fig. 21).
After 1 month, tumor size was monitored by MRI. After all images are taken, the CO is used in the operating room2Mice were euthanized in the room. Intraperitoneal tumor metastasis was confirmed in each group, and each tumor tissue was obtained and fixed with 10% formalin. By using H&E and the corresponding antibodies were immunostained to confirm the histopathology. No metastasis occurred in the abdominal cavity with the IO101L/AC disc inserted, whereas metastasis to the spleen and liver in the abdominal cavity was found in the atelocollagen disc (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).
8-7 in a patient-derived xenograft (PDX) pancreatic cancer mouse model using patient-derived pancreatic cancer cells Anticancer effect of
8-7-1 construction of PDX pancreatic cancer mouse model Using patient-derived pancreatic cancer cells
The PDX model was successfully established using pancreatic tumors excised in 65 year old female patients undergoing laparoscopic pancreatic amniotic resection. Pathological examination revealed ductal adenocarcinoma of the pancreas with a size of 3.2cm and showing frequent lymphatic infiltrates. One metastatic lymph node was found among seven lymph nodes searched (AJCC version 8T 2N1M0, IIB). When the histological features of PDX and primary tumors were compared by H & E and nucleolar (nucleoline) immunostaining, overall histological similarity was found between primary tumors and PDX tumors (see figure 24).
Male diabetic/Severe Combined immunodeficiency mice, i.e., NOD/Shi-scid, IL-2R γ KO 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, the primary tumor will be preserved in the patient (institutional review)Examination committee No. 4-2017-. Then, after matrigel-coating the above fragments on a petri dish, 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), followed by suturing with 5-0 suture (VCP490G, ETHICON). When the growing tumor size reached 1500mm, the donor mouse (F1) tumor, as well as the same tumor and the rest of the tumors, were then stored in liquid nitrogen in frozen vials containing 5% dimethyl sulfide/95% fetal bovine serum, which were then implanted into the contemporary group of mice (F2). Subcutaneously grown tumors (1500 mm) were excised from F1 mice3) And subcultured to the next generation of a synchronized group of mice (F2). About 150-200mm of xenografts from the same patient in each compartment3Tumors take about 50 days to reach a touchable tumor. A pair of primary tumor (F0) and F1 (passage 1) and F2 (passage 2) samples were used in 2 patients. When the tumor reached a palpable size (average size 266.5 ± 58.0 mm)3) When 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 μ g) suspended in PBS was administered intraperitoneally to mice every 4 weeks; group 3, in which IO101(100 μ g) suspended in PBS was administered intraperitoneally to mice every 4 weeks; and groups 4, 5 and 6, in which three types of patches were implanted. Untreated control mice were also included for comparison.
After tumor tissues obtained from patients with pancreatic cancer were transplanted into NSG (NOD/SCID/IL-2Rg KO) mice, animal models that had successfully grown were transplanted for multiple generations and individually expanded by bulb formation in order to prepare PDX models that could be used to evaluate the therapeutic effect. For the unmanipulated subcutaneous infusion surgery, tumor size, tumor markers (CA 19-9, CFB) and weight were determined at 1 week post-surgery, 2 weeks post-surgery, 3 weeks post-surgery, 4 weeks post-surgery, 5 weeks post-surgery and 6 weeks post-surgery, after which histological examination was performed and survival was confirmed.
Evaluation of tumor suppression Effect of IO101/AC disks in a mouse model of PDX pancreatic cancer Using patient-derived pancreatic cancer cells
After making skin incisions in PDX mice, controls (untreated), Gem-IP (sol-gel type), IO101/AC disks (2.0 mg/3.0%), IO101-Con/AC (2.0 mg/3.0%), and Gem/atelocollagen disks (0.12 mg/3.0%), were implanted locally between the skin and the tumor by subcutaneous anesthetic dissection, respectively. Tumor size was measured by caliper measurement (Mitutoyo, Absolute AOS Digmatic, Kawasaki, Japan) three times a week 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). The statistical significance of the data is determined byStatistical version 23 calculated. All results are expressed as mean ± standard deviation, 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).
1 month after disc implantation, mice were sacrificed and tissue samples and blood were collected. The extracted tumors were weighed on a balancer and recorded with a Nikon digital camera (Japan). For hematology measurements, the blood sample was prepared from a sample containing K2E (K)2EDTA) was collected for anticoagulation and toxicity testing in a serum collection BD Microtainer chemical tube SST (BD, USA). The anti-cancer effect of the IO101/AC discs was verified by TUNEL analysis. No tumor necrosis or cell death was found in the control. In the Gem-IP group, the 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 set showed a significant apoptotic process, but TUNNEL positive cancer cells were found along the surface layer of the tumor surface (see fig. 26).
8-7-3 evaluation of side effects in other organs after implantation of IO101 disks in a PDX pancreatic cancer mouse model Using patient-derived pancreatic cancer cells
Microscopic examination confirmed that there was no evidence of potential toxicity (such as inflammatory or necrotic changes) in liver, lung, kidney and spleen tissues (see figure 27). No leukopenia, anemia and neutropenia were observed in the IO101/AC disc set. On the other hand, leukopenia (WBC) (3.2 ± 2.9 compared to 5.4 ± 2.9, P ═ 0.028), low hemoglobin level (HB) (10.3 ± 4.6 compared to 18.5 ± 11.9) and neutropenia (0.76 ± 0.71 compared to 2.69 ± 2.66, P ═ 0.010) in the IP-GEM group associated with systemic effects of gemcitabine were demonstrated. Table 15 below shows the blood test results.
[ Table 15]
1) Mann-Whitney U between control and Patch I; 2) Mann-Whitney U between IP-GEM and Patch I
Example 9 screening for in vitro efficacy of oligonucleotide variants
To (N)x-[TGG]4[TTG][TGG]4 or 5、[TGG]4[TTG][TGG]4 or 5-(N)xAnd (N)x-[TGG]4[TTG][TGG]4 or 5-(N)yThe cell proliferation inhibition efficacy of BxPC3 (pancreatic cancer), MD-MBA 231 (breast cancer), Uuh-7 (liver cancer), HT29 (colon cancer) and Mv4-11(AML) cell lines was examined.
2.5x105To 5.0x105Individual cells/well, which is the cell number defined by the cell assay used to determine the appropriate cell concentration in pancreatic cancer cell line BxPC3 cells (ATCC, IMDM + 10% PBS), were seeded in 96-well plates and incubated for one day. Heating each sample at 95 deg.C for 5 minutesThereafter, the temperature was slowly lowered to room temperature, and then the samples were immediately treated for each well by concentration. Treated BxPC3 cells in 5% CO2The incubation was performed in an incubator for 3 days, treated with 20 μ L of reagent solution to perform MTT assay (Cell Proliferation kit ii, Roche), and then incubated in hours (10 minutes, 30 minutes, 60 minutes), after which the absorbance at 490nm was measured by an ELISA reader (see fig. 28 and 29).
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 described above. The cell viability of each cell line is listed in table 16 below.
[ Table 16]
<110> Neteorelbu Co
<120> modified nucleic acid having improved therapeutic efficacy and anticancer pharmaceutical composition comprising 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> 21
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _7
<400> 7
tggtggtggt tgtggtggtg g 21
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _8
<400> 8
tggtggtggt tgtggtggtg gtgg 24
<210> 9
<211> 24
<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> 21
<211> 48
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _21
<400> 21
tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtgg 48
<210> 22
<211> 51
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _22
<400> 22
tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtggtg g 51
<210> 23
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _23
<400> 23
tggtggtggt ggtggtggtg gtggttgtgg tggtggtggt ggtggtggtg gtgg 54
<210> 24
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> oligonucleotide _24
<400> 24
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