Nucleic acid delivery enhancer

Abstract

To provide a novel anti-tumor agent capable of delivering siRNA or shRNA specifically to pancreatic cancer cells and suppressing tumor growth, invasion, and metastasis of pancreatic cancer.SOLUTION: The present invention provides a nucleic acid delivery enhancer for delivering siRNA or shRNA into cells, which comprises a folic acid-cationic oligopeptide complex. The present invention also provides an anti-tumor agent comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells to inhibit its expression, and a folic acid-cationic oligopeptide complex. The siRNA is capable of binding to RNA selected from the group consisting of SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to nucleic acid delivery enhancers for delivering nucleic acid molecules such as siRNA into cells. The invention also relates to agents that can be used for the treatment of diseases using RNA interference (RNAi). In particular, this invention relates to nucleic acid preparations and pharmaceutical compositions containing the same, which have an antitumor effect against pancreatic cancer and can effectively suppress tumor growth, invasion, and metastasis of pancreatic cancer. [Background technology]

[0002] Pancreatic cancer is said to have the worst prognosis of all cancers. This is because, in addition to the difficulty of early detection due to the pancreas being a retroperitoneal organ, pancreatic cancer cells have extremely high motility, making them highly prone to peritoneal invasion and metastasis to blood vessels, the digestive tract, nerves, and other organs.

[0003] The present inventors have previously investigated the mechanisms of invasion and metastasis in pancreatic cancer and discovered that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), which is normally found in the nucleolus and is known to bind to the 5' untranslated region of insulin-like growth factor II mRNA and inhibit the translation of insulin-like growth factor II, is present in the cell membrane protrusions of pancreatic cancer cells, and that various mRNAs bind to this IGF2BP3 and accumulate in the cell membrane protrusions. They have also reported that inhibiting these mRNAs by RNA interference (RNAi) effectively suppresses the invasion and metastasis of pancreatic cancer cells (Patent Document 1).

[0004] Small interfering RNA (siRNA), which is representative of nucleic acid pharmaceuticals using RNA interference (RNAi) and has attracted attention as a new therapeutic agent in recent years, is generally a small double-stranded RNA consisting of 21 to 23 base pairs. However, due to its high anionic nature and low cell membrane permeability from its structure, siRNA has a problem in terms of delivery into cells. To address this problem, the inventors previously prepared an siRNA-folic acid-polyethylene glycol (PEG)-chitosan oligosaccharide lactate (COL) nanoparticle complex and confirmed the promoting effect of this nanoparticle on the uptake of siRNA into cells. It has also been reported that siRNA against mRNA having IGF2BP3 binding ability taken up into pancreatic cancer cells suppresses the invasion and metastasis of pancreatic cancer (Non-Patent Document 1).

[0005] On the other hand, the inventors have found that a cationic oligopeptide having an amino group or a guanidino group in the side chain is useful for the delivery of double-stranded RNA into cells (Patent Document 2, Non-Patent Document 2). In addition, the use of a complex containing vitamin E and a cationic sugar for the delivery of RNAi molecules is also being studied (Non-Patent Documents 3 and 4).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

Chemical formula

[0011] 2. The delivery promoter according to item 1 above, wherein the cationic oligopeptide moiety consists of 8 to 12 amino acids. 3. The delivery promoter according to item 1 or 2 above, wherein the cationic oligopeptide moiety is a homopolymer of L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), L-2-amino-4-guanidinobutyric acid (Agb), or L-arginine (Arg).

[0012] [[ID=四十二]]4. The delivery promoter according to any one of items 1 to 3 above, wherein the cationic oligopeptide has an octamer of diaminobutyric acid having the following structure as a partial structure.

Chemical formula

[0013] 5. A delivery accelerator according to any one of 1 to 4 above, wherein folic acid is linked to the N-terminus, C-terminus, or side chain of a cationic oligopeptide, with or without a linker. 6. The delivery accelerator according to item 5, wherein a folic acid-cationic oligopeptide complex is linked via a linker, and the linker is a peptide linker. 7. The delivery accelerator according to item 6 above, wherein the peptide linker is a peptide consisting of 1 to 4 glycine residues.

[0014] 8. The delivery accelerator according to item 4 above, wherein the folic acid-cationic oligopeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure. [ka] [ka]

[0015] 9. An antitumor agent comprising an siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and a delivery enhancer as described in any of items 1 to 8 above. 10. The antitumor agent described in item 9 above, wherein the mRNA or snoRNA expressed in pancreatic cancer cells binds to insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). 11. The antitumor agent according to 9 or 10 above, wherein the mRNA or snoRNA expressed in pancreatic cancer cells is selected from the group consisting of SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA. 12. An antitumor agent according to any one of the above 9 to 11, comprising 0.5 to 10 equivalents of a folic acid-cationic oligopeptide complex relative to siRNA or shRNA. 13. An antitumor agent according to any of the above 9-12, wherein the siRNA is RNA / RNA double-stranded. 14. An antitumor agent according to any one of 9 to 13 above, wherein the siRNA or shRNA comprises a modified base, a modified sugar, and / or a modified nucleoside bond. 15. The antitumor agent according to 14 above, wherein the modification of the modified sugar is a 2'-OMe modification. 16. The antitumor agent according to 14 or 15 above, wherein the modified nucleoside bond is a phosphorothioate bond. 17. A pharmaceutical composition containing any of the antitumor agents described in 9 to 16 above.

[0016] 18. The following: (a) Preparations comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and (b) A formulation comprising a folic acid-cationic oligopeptide complex comprising a cationic oligopeptide moiety consisting of 8 to 40 amino acids, the moiety comprising a portion of at least two consecutive amino acid residues of formula (I) below, and one other amino acid residue that is not consecutive except for the portion of amino acid residues of formula (I) below: [ka] [In equation (I), R 1 is based on H3N + -CH2-, or the group represented by formula (II), R 2 R 1 is based on H3N + In the case of -CH2-, it does not exist, or it is an alkylene group with 1 to 3 carbon atoms, R 1 In the case of the group represented by formula (II), it is an alkylene group having 1 to 4 carbon atoms. In one cationic oligopeptide, R 1 and R 2 They are all identical. [ka] [In formula (II), R 3 , R 4 and R 5 These are either the same or different hydrogen atoms or methyl groups. A combination formulation containing the above.

[0017] 19. The following: (a) siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and (b) A folate-cationic oligopeptide complex comprising a cationic oligopeptide moiety consisting of 8 to 40 amino acids, the moiety comprising at least two consecutive amino acid residues of formula (I) below, and one other amino acid residue that is not consecutive except for the consecutive amino acid residues of formula (I): [ka] [In equation (I), R 1 is based on H3N + -CH2-, or the group represented by formula (II), R 2 R 1 is based on H3N + In the case of -CH2-, it does not exist, or it is an alkylene group with 1 to 3 carbon atoms, R 1 In the case of the group represented by formula (II), it is an alkylene group having 1 to 4 carbon atoms. In one cationic oligopeptide, R 1 and R 2 They are all identical. [ka] [In formula (II), R 3 , R 4 and R 5 These are either the same or different hydrogen atoms or methyl groups. A medical kit for the treatment of pancreatic cancer, including [specific components / items]. This specification includes the disclosures of Japanese Patent Application No. 2019-194646, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0018] The present invention provides a nucleic acid preparation that can effectively deliver siRNA or shRNA specifically to pancreatic cancer cells and suppress tumor growth, invasion, and metastasis of pancreatic cancer. [Brief explanation of the drawing]

[0019] [Figure 1] The HPLC analysis results of the crude product containing Fol-Dab8A and Fol-Dab8B are shown. [Figure 2] The effects of 1-3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B on siRNA RNase A resistance are shown by changes in fluorescence intensity induced by fluorescence resonance energy transfer (FRET). [Figure 3] A: Confocal microscopy image showing the uptake of Alexa488-labeled siRNA into S2-013 pancreatic cancer cells in the presence of a folate-cationic oligopeptide complex. B: Confocal microscopy image showing the uptake of Alexa488-labeled siRNA into S2-013 pancreatic cancer cells in the absence of a folate-cationic oligopeptide complex. C: Shows the uptake efficiency of siRNA into S2-013 pancreatic cancer cells in the presence of a cationic oligopeptide or a folate-cationic oligopeptide complex. Results are shown as mean and standard error. Dab8-1 indicates the addition of 1 equivalent of cationic oligopeptide Dab8, and Fol-Dab8B-3 indicates the addition of 3 equivalents of the folate-cationic oligopeptide complex Dab8B. [Figure 4] A: Shows the inhibitory effect of combining scrambled control siRNA or SNORA18 siRNA with a cationic oligopeptide or folate-cationic oligopeptide complex on SNORA18 expression. The data are shown as relative intensity for bands amplified by RT-PCR, with the band intensity for the combination of control siRNA and Dab8 set to 1. B: Shows the number of infiltrating cells in a Matrigel assay using the combination of scrambled control siRNA or SNORA18 siRNA with a folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B). [Figure 5]A: Shows the inhibitory effect of NUP85 expression by combining scrambled control siRNA or NUP85 siRNA with a cationic oligopeptide or folate-cationic oligopeptide complex. The data are shown as relative intensity for bands amplified by RT-PCR, with the band intensity for the combination of control siRNA and Dab8 set to 1. B: Shows the number of infiltrating cells in a Matrigel assay using scrambled control siRNA or NUP85 siRNA combined with a folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B). [Figure 6] A: Shows the inhibitory effect of WASF2 expression by combining scrambled control siRNA or WASF2 siRNA with a cationic oligopeptide or folate-cationic oligopeptide complex. The data are shown as relative intensity for bands amplified by RT-PCR, with the band intensity for the combination of control siRNA and Dab8 set to 1. B: Shows the number of infiltrating cells in a Matrigel assay using scrambled control siRNA or WASF2 siRNA combined with a folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B). [Figure 7] A: Shows the inhibitory effect of combining scrambled control siRNA or SNORA22 siRNA with a cationic oligopeptide or folate-cationic oligopeptide complex on SNORA22 expression. The data are shown as relative intensity for bands amplified by RT-PCR, with the band intensity for the combination of control siRNA and Dab8 set to 1. B: Shows the number of infiltrating cells in a Matrigel assay using the combination of scrambled control siRNA or SNORA22 siRNA with a folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B). [Figure 8] The number of infiltrating cells in a Matrigel assay using a scrambled control siRNA (A), SNORA18 siRNA (B), NUP85 siRNA (C), WASF2 siRNA (D), or SNORA22 siRNA (E) in combination with a cationic oligopeptide (Dab8) or folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B) is shown. [Figure 9]These are confocal microscope images showing the staining of folate receptor (FOLR1) and SNORA22 siRNA in S2-013 pancreatic cancer cells (A) and HPNE normal pancreatic ductal epithelial cells (B) after adding Alexa488-labeled SNORA22 siRNA and a folate-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents) to the culture medium and incubating overnight. [Figure 10] This shows the %) efficiency (%) of introducing SNORA22 siRNA into S2-013 pancreatic cancer cells and HPNE normal pancreatic ductal epithelial cells in the presence of a folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents). Results are shown as mean and standard error. * indicates a statistically significant difference (P<0.05). [Figure 11] These are confocal microscope images showing the staining of lysosomes (LysoTracker) and SNORA22 siRNA in S2-013 pancreatic cancer cells incubated overnight with Alexa488-labeled SNORA22 siRNA and a folate-cationic oligopeptide complex (Fol-Dab8A(A) or Fol-Dab8B(B), 2 equivalents) added to the culture medium. Merge / DAPI images show the combined staining of Alexa488 and DAPI. [Figure 12] The stability of SNORA22 siRNA immediately after (0), 3 hours (3), or 6 hours (6) after mixing unmodified SNORA22 siRNA (A) or chemically modified SNORA22 siRNA (B) with a cationic oligopeptide (Dab8) or a folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B) and adding it to serum (10% FCS / PBS) is shown by SDS-PAGE results using a non-reducing gel. -: No FCS (PBS only), Control: Each SNORA22 siRNA added alone. [Figure 13]The images show the results of in vivo imager imaging of the delivery of chemically modified SNORA22 siRNA to pancreatic cancer tissue from mice carrying human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells. AC: SNORA22 siRNA with 1-3 equivalents of Dab8 added; DF: SNORA22 siRNA with 1-3 equivalents of Fol-Dab8A added; GI: SNORA22 siRNA with 1-3 equivalents of Fol-Dab8B added. [Figure 14] This study demonstrates the antitumor effect of co-administration of SNORA22 siRNA and a folic acid-cationic oligopeptide complex against pancreatic cancer tumors in mice carrying human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells. The group includes: Control (no administration), SNORA22-Dab8 (SNORA22 siRNA administered with the folic acid-cationic oligopeptide complex), Scr-Fol-Dab8B (scrambled control siRNA), and SNORA22-Fol-Dab8B (SNORA22 siRNA administered with the folic acid-cationic oligopeptide complex). * indicates a statistically significant difference (P<0.05). [Figure 15] The images show the results of in vivo imager imaging of the delivery of chemically modified SNORA18 siRNA to pancreatic cancer tissue from mice carrying human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells. AC: SNORA18 siRNA with 1-3 equivalents of Fol-Dab8A added, DF: SNORA18 siRNA with 1-3 equivalents of Fol-Dab8B added. [Figure 16] The following images show the delivery of chemically modified WASF2 siRNA to pancreatic cancer tissue from mice carrying human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells, as captured by in vivo imager. AC: WASF2 siRNA with 1-3 equivalents of Fol-Dab8A added; DF: WASF2 siRNA with 1-3 equivalents of Fol-Dab8B added. [Modes for carrying out the invention]

[0020] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0021] <Nucleic acid delivery enhancer> The present invention provides a nucleic acid delivery enhancer comprising a folic acid-cationic oligopeptide complex.

[0022] <Folic acid> Folic acid is a water-soluble vitamin B and has the structure shown below. Folic acid receptors have been reported to be highly expressed on the surface of tumor cells. The inventors previously reported that folic acid receptors are expressed in the pancreatic cancer cell line S2-013, and that intracellular delivery of siRNA using a folic acid-containing complex was achieved (Oncotarget, 2019, Vol.10, No.30, pp.2869-2886).

[0023] [ka]

[0024] The folic acid used to create a complex with a cationic oligopeptide in the present invention may be folic acid having the above structure, or it may be a folate or folic acid derivative that retains binding affinity to cationic oligopeptides and binding affinity to folic acid receptors.

[0025] For example, as can be understood from the structure described above, the folic acid molecule contains two carboxyl groups, an amino group, an imino group, etc. Therefore, substituents known in this field can be added to these functional groups that do not participate in binding with cationic oligopeptides, and labeling compounds can also be attached to folic acid.

[0026] <Cationic oligopeptide> Cationic oligonucleotides usable in the present invention may be those disclosed in International Publication WO2014 / 148620 and Bioorganic & Medicinal Chemistry 21 (2013) 1717-1723, as appropriate. Specifically, the cationic oligonucleotide used in the present invention may contain eight or more amino acid residues having an amino group or a guanidino group.

[0027] More specifically, the cationic oligopeptide usable in the present invention may be an oligopeptide consisting of 8 to 40 amino acids, which includes a portion comprising at least two consecutive amino acid residues of formula (I) below, and a cationic oligopeptide moiety which is one other amino acid residue that is not consecutive except for the portion comprising the consecutive amino acid residues of formula (I) below.

[0028] [ka] [In equation (I), R 1 is based on H3N + -CH2-, or the group represented by formula (II), R 2 R 1 is based on H3N + In the case of -CH2-, it does not exist, or it is an alkylene group with 1 to 3 carbon atoms, R 1 In the case of the group represented by formula (II), it is an alkylene group having 1 to 4 carbon atoms. In one cationic oligopeptide, R 1 and R 2 They are all identical.

[0029] [ka] [In formula (II), R 3 , R 4 and R 5 These are either the same or different hydrogen atoms or methyl groups.

[0030] The "amino acid residue of formula (I)" mentioned above is not limited to any specific example, but could include L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), L-2-amino-4-guanidinobutyric acid (Agb), or L-arginine (Arg).

[0031] The "other amino acid residues" mentioned above are not particularly limited and can be selected as appropriate. For example, in addition to glycine, L-alanine, and L-proline, amino acids with a proline backbone such as L-aminoproline and L-guanidinoproline can be listed, but are not limited to these.

[0032] In one embodiment, the cationic oligopeptide moiety may be a heteropolymer containing the above-mentioned "other amino acid residues." In another embodiment, the cationic oligopeptide moiety may be a homopolymer that does not contain the above-mentioned "other amino acid residues." Considering the ease of synthesis of the cationic oligopeptide and the stabilizing effect on siRNA and shRNA confirmed by the present inventors, it is preferable that the cationic oligopeptide moiety be a homopolymer composed of a single amino acid.

[0033] In this case, the amino acids as monomers may be either natural or unnatural. Furthermore, the amino acids may be either L-form or D-form, and both forms may be present within the oligopeptide molecule.

[0034] Therefore, cationic oligopeptides may have homopolymers of, for example, L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), L-ornithine (Orn), L-lysine (Lys), L-2-amino-3-guanidinopropionic acid (Agp), L-2-amino-4-guanidinobutyric acid (Agb), or L-arginine (Arg) as partial structures.

[0035] Cationic oligopeptides may also be in the form of salts, and suitable salts include hydrochloride salts, acetate salts, trifluoroacetate salts, etc., but are not particularly limited.

[0036] Double-stranded nucleic acids have two distinct helical structures, type A and type B. DNA / DNA double strands have a type B double helix structure with a major groove width of 13-18 Å, while RNA / RNA double strands and DNA / RNA strands have a type A double helix structure with major groove widths of 7-14 Å and 8-15 Å, respectively. Since this invention aims to improve the stability of siRNA, it is necessary to use a cationic oligopeptide that can bind to an RNA / RNA strand with a type A double helix structure.

[0037] The number of amino acid residues that make up a cationic oligopeptide is generally 8 to 12 to improve the stability of siRNA, which consists of 21 to 23 bases, and the shRNA that gives rise to such siRNA.

[0038] As a specific embodiment, in the following examples, it was actually investigated and a favorable result was obtained that the cationic oligopeptide moiety can be an octamer of diaminobutyric acid having the following structure.

[0039] [ka]

[0040] <Folic acid-cationic oligopeptide complex> The bond between folic acid and cationic oligopeptide is preferably a covalent bond. The covalent bond can be a direct bond to the N-terminus, C-terminus, or side chain of the cationic oligopeptide, or a bond via a linker.

[0041] As a linker, any linker commonly used in this field for the preparation of conjugates can be used as appropriate, and is not particularly limited; for example, a peptide linker consisting of amino acids such as glycine or serine can be used. For example, the peptide linker may be a peptide consisting of 1 to 4 glycine residues. For example, in the examples below, a linker consisting of 3 glycine residues was actually investigated and yielded favorable results.

[0042] In the folate-cationic oligopeptide complex, the ratio of folate to cationic oligopeptide can be 1:1, taking into account the interaction with the folate receptor and the interaction with double-stranded nucleic acids, but this is not particularly limited.

[0043] As described above, since the folic acid molecule has two carboxyl groups, when Dab8 of the above structure is bonded to the carboxyl group of folic acid via a reaction, the following two isomers can be formed. For convenience, these compounds will be referred to as Fol-Dab8A and Fol-Dab8B in this specification.

[0044] [ka]

[0045] [ka]

[0046] In the Fol-Dab8A and Fol-Dab8B described above, three glycine molecules are used as linkers, but the length of the linkers can be changed as needed. Depending on the type of cationic oligopeptide used for complex formation, the folate-cationic oligopeptide complex may be obtained in the form of a salt. Suitable salts include, but are not limited to, hydrochloride, acetate, and trifluoroacetate. By forming a folate-cationic oligopeptide complex, the stability of siRNA and shRNA is enhanced, and targeted delivery to cells possessing folate receptors becomes possible.

[0047] In the examples, a cationic oligopeptide Dab8 without folic acid is used as a control compound to demonstrate the effectiveness of Fol-Dab8A and Fol-Dab8B. However, to enable UV detection and quantification, it has tyrosine (protected by an acetyl group at the N-terminus) and three glycine molecules at the N-terminus, and in the examples, this is referred to as Dab8 for convenience.

[0048] [ka] [In the formula, Ac-YGGG represents N-acetyl-L-tyrosine-glycine-glycine-glycine]

[0049] As demonstrated in the examples, the nucleic acid delivery enhancer of the present invention can promote the delivery of siRNA or shRNA to cells in vitro and in vivo. The nucleic acid delivery enhancer of the present invention can specifically promote delivery to cells that highly express folate receptors, particularly cancer cells.

[0050] <Anticonomastic agents> The present invention also provides a nucleic acid formulation comprising an siRNA or shRNA capable of binding to and inhibiting the expression of mRNA or snoRNA expressed in pancreatic cancer cells, and the delivery enhancer of the present invention. The nucleic acid formulation of the present invention can effectively suppress tumor growth, invasion, and metastasis of pancreatic cancer. Therefore, the nucleic acid formulation of the present invention has effects as a tumor growth inhibitor, an invasion and metastasis inhibitor, and an antitumor agent against pancreatic cancer.

[0051] In the present invention, siRNA or shRNA and the folic acid-cationic oligopeptide complex can be bound via an additional covalent bond. However, even without a covalent bond, due to the interaction brought about by the negative charge of siRNA or shRNA and the positive charge of the cationic oligopeptide, the folic acid-cationic oligopeptide complex can partially enter the major groove of siRNA or shRNA to have a stable structure, but it is not intended to be restricted by the mechanism of this stabilization effect.

[0052] <RNAi molecule> siRNA and shRNA are known to target specific mRNA and block its translation (expression) by a mechanism called RNA interference. The number of bases in the target sequence is not particularly limited and can be selected in the range of 15 to 500 bases. siRNA is a short double-stranded RNA molecule, and shRNA is a hairpin-type RNA that can be processed by Dicer in vivo to generate siRNA. In this specification, there may be cases where "RNAi molecule" is described including siRNA and shRNA.

[0053] siRNA is a double-stranded RNA in which a sense strand homologous to a partial base sequence of the target RNA hybridizes with an antisense strand capable of hybridizing with the sense strand. Usually, the 3' end remains OH while the 5' end is phosphorylated, and the 3' end side may have a protrusion of 1 to 4 bases.

[0054] On the other hand, shRNA consisting of single-stranded RNA has a region of a sense strand homologous to a partial base sequence of the target mRNA and a region of an antisense strand capable of hybridizing with the sense strand linked by a linker region, and has a hairpin-like structure as a whole. The 3' end of shRNA may have a protrusion of 1 to 4 bases, and the 3' protruding end may be composed of DNA. shRNA is degraded intracellularly as described above and causes RNA interference in the same manner as siRNA. Therefore, shRNA may be used instead of siRNA.

[0055] The siRNA or shRNA used for the purposes of the present invention is an siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit their expression.

[0056] The "mRNA or snoRNA expressed in pancreatic cancer cells" as described herein may be, but are not limited to, those that bind to insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3).

[0057] As described above, in pancreatic cancer cells, human IGF2BP3 is present in the cell membrane protrusions, and various mRNAs bind to this IGF2BP3, accumulating in the cell membrane protrusions. By inhibiting the mRNAs bound to IGF2BP3 in these cell membrane protrusions through RNA interference, tumor growth, invasion, and metastasis in pancreatic cancer can be effectively suppressed.

[0058] Examples of mRNAs expressed in pancreatic cancer cells that the inventors have confirmed to have an antitumor effect against pancreatic cancer using RNA interference include NUP85, WASF2, ARHGEF4, CCDC88A, LAMTOR2, and mTOR mRNA.

[0059] <nup85> NUP85 (nucleoporin 85) is a protein belonging to the nucleoporin protein family, which is a component of the nuclear pore complex, a complex that forms entry and exit points that regulate the movement of macromolecules between the cell nucleus and cytoplasm. The amino acid sequence of human NUP85 and the base sequence of the mRNA encoding it are listed in databases such as NCBI as Gene ID: 79902, NCBI reference sequence: NM_024844, etc.

[0060] <wasf2> Wiscott-Aldrich syndrome, a type of primary immunodeficiency disorder, is characterized by thrombocytopenia with reduced size, eczema, and increased susceptibility to infection. Wiscott-Aldrich syndrome protein family member 2 (WASF2) belongs to the Wiscott-Aldrich syndrome protein family, which forms a multiprotein complex linking receptor kinase and actin. Information on the amino acid sequence of human WASF2 and the base sequence of the mRNA encoding it is listed in databases such as NCBI as Gene ID: 10163, NCBI reference sequence: NM_006990, etc.

[0061] <arhgef4> ARHGEF4 (Rho guanine nucleotide exchange factor 4) is a protein involved in intracellular processes initiated by stimuli that function via G protein-coupled states. Information on the amino acid sequence of human ARHGEF4 and the base sequence of the mRNA encoding it is listed in databases such as NCBI as Gene ID: 50649, NCBI reference sequence: NM_015320, etc.

[0062] <ccdc88a> CCDC88A (88A containing a coiled-coil domain) is a gene that encodes the actin-binding protein Girdin. Information on the mRNA sequence and other details of human CCDC88A is listed in databases such as NCBI as Gene ID: 55704, NCBI reference sequence: NM_001135597, etc.

[0063] <lamtor2> LAMTOR2 (late endosomal / lysosomal adaptor, MAPK and mTOR activator 2) is a homeostatic regulator in Langerhans cells and has been reported to be involved in signal transduction and the mTOR cascade. Information on the amino acid sequence of human LAMTOR2 and the base sequence of the mRNA encoding it is listed in databases such as NCBI as Gene ID: 28956, NCBI reference sequence: NM_014017, etc.

[0064] <mtor> mTOR (mammalian target of rapamycin kinase) is a type of protein kinase involved in intracellular signal transduction in mammals and other organisms. Information on the amino acid sequence of human mTOR and the base sequence of the mRNA encoding it is listed in databases such as NCBI as Gene ID: 2475, NCBI reference sequence: NM_004958, etc.

[0065] As is well known in this field, mRNA is RNA transcribed from genes (DNA) and contains information that codes for proteins. siRNA, which is normally double-stranded, is known to form a complex called RISC (RNA-Induced-Silencing-Complex) after the two strands dissociate, with one strand (the antisense strand) forming a complex with a specific protein. RISC recognizes and binds to mRNA having a homologous sequence to the sense strand of the siRNA, and cleaves the mRNA through RNase III-like enzymatic activity. On the other hand, shRNA can produce siRNA after processing in the delivered cell and subsequently function similarly.

[0066] In contrast, snoRNAs are non-coding RNAs (small nuclear RNAs) present in the nucleolus, and have been reported to be a group of RNA molecules that perform functions such as inducing chemical modifications of ribosomal RNA and other RNAs, such as methylation and pseudouridine (e.g., Mol. Biol. Cell, 2004, 15:281-293; J. Biol. Chem, 2015, 290:11741-11748).

[0067] The inventors have recently discovered that SNORA18 and SNORA22, which belong to this group of snoRNAs, have the ability to bind to IGF2BP3 and are involved in the motility or invasion of pancreatic cancer cells. Furthermore, it has been found that snoRNAs bind to KH-type splicing regulatory proteins (KHSRPs) and are localized in cytoplasmic P-bodies (Oncotarget, 2020, Vol.11, No.2, pp:131-147). The inventors have discovered that siRNA or shRNA targeting these snoRNAs can also recognize and bind to snoRNAs, thereby suppressing tumor growth, invasion, and metastasis of pancreatic cancer by knocking them down.

[0068] <snora18> Micronucleolar RNA SNORA18 has been reported as a member of the RNA group that guides the modification site from uridine to pseudouridine. Information on the nucleotide sequence and other details of human SNORA18 is listed in databases such as NCBI as Gene ID: 677805, NCBI reference sequence: NR_002959, etc.

[0069] <snora22> Micronucleolar RNA SNORA22 has also been reported as a member of the RNA group that guides the modification site from uridine to pseudouridine. Information on the nucleotide sequence of human SNORA22 is listed in databases such as NCBI as Gene ID: 677807, NCBI reference sequence: NR_002961, etc.

[0070] Therefore, although not particularly limited, examples of mRNA or snoRNA expressed in pancreatic cancer cells that can be targeted by siRNA or shRNA in the present invention include those selected from the group consisting of SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA.

[0071] When targeting mRNA, the target mRNA that siRNA or shRNA can hybridize may include the 3'UTR, 5'UTR, exons, introns, coding regions, translation initiation regions, translation termination regions, or other nucleic acid regions.

[0072] The siRNA and shRNA used in the present invention are capable of knocking down mRNA or snoRNA and inhibiting its function. More specifically, the siRNA and shRNA of the present invention consist of a nucleotide sequence substantially complementary to a specific nucleotide sequence of the target RNA. However, the siRNA and shRNA may have one or two mismatches with the target nucleotide sequence. Those skilled in the art can, if the nucleotide sequence of the target mRNA or snoRNA is obtained, select a region that is not homologous to the nucleotide sequences of nucleic acids other than the target sequence and is suitable for inhibiting / knockdown of the expression of the target sequence, and design and synthesize appropriate siRNA and shRNA with high specificity.

[0073] For example, siRNA is subject to the following conditions: (1) The 5' end of the antisense strand is A or U, (2) The 5' end of the sense strand is G or C, and (3) At least four of the seven bases at the 5' end of the antisense strand are A or U. It is known that RNA interference is highly effective when these conditions are met. Therefore, the siRNA used in the present invention may have such a base sequence, but is not limited thereto. Similarly, the shRNA used in the present invention may produce such siRNA after intracellular processing, but is not limited thereto. The determination of effective siRNA and shRNA sequences for a given target sequence can also be performed using programs available via the internet.

[0074] In this specification, inhibition / suppression or knockdown of expression means the degradation of target mRNA or snoRNA, or the inhibition / suppression of its translation into the encoded protein. Suppression includes reducing the amount of target mRNA / snoRNA in cells or cell populations (pancreatic cancer tissue) by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to a control. Inhibition / suppression or knockdown of expression may be determined by any method known in the art, for example, by a method based on RT-PCR.

[0075] siRNA and shRNA may consist solely of natural (unmodified) nucleotides, or some or all of their nucleotides may be modified. The chemical stability of siRNA and shRNA can be increased by using any of the modifications known in this field. However, it is undesirable for modifications to reduce the intended activity of siRNA and shRNA. For example, it is understood that modifications that interfere with the intracellular processing of shRNA should not be used. Modifications as non-natural nucleotides may include sugar and / or base modifications.

[0076] Examples of sugar modifications include, but are not limited to, bicyclic sugars, 5'-vinyl, 5'-methyl, 4'-S, 2'-F, 2'-OCH3(2'-OMe), and 2'-O(CH2)2OCH3 substituents. Bicyclic sugars are generally referred to as bridged nucleic acids (BNA), and examples, but are not limited to, LNA (Locked Nucleic Acid®), 2,4-BNA, and ENA (ethyleneoxy(4-(CH2)2-O-2)BNA). Alternatively, the sugar may contain deoxyribose in part instead of ribose, in which case DNA and RNA may be mixed in one or both oligonucleotide chains of the siRNA, or in the oligonucleotide chain of the shRNA. Preferably, the siRNA is an RNA / RNA double-stranded molecule. Also, the shRNA is preferably an RNA chain. As a sugar modification, for example, 2'-OMe modification can be suitably used.

[0077] Examples of base modifications include, for instance, (but are not limited to) Cytosine 5-methylation, 5-fluoration, 5-bromination, 5-iodation, N4-methylation, N6-methylation and 8-bromination of adenine, N2-methylation and 8-bromination of guanine, 5-fluorinated, 5-brominated, 5-iodinated, and 5-hydroxylated uracil These are some examples.

[0078] Identical or different nucleotides in siRNA and shRNA can have the sugar and base modifications described above. siRNA and shRNA may also contain modified nucleoside bindings.

[0079] Modified nucleoside bonds may be those that replace naturally occurring phosphodiester bonds, such as phosphorothioate bonds, phosphorodithioate bonds, boranophosphate bonds, phosphorodiamidate bonds, and phosphoramidate bonds.

[0080] At least one of the nucleoside bonds of siRNA and shRNA may be a modified nucleoside bond. Alternatively, at least two, three, four, or more of the nucleoside bonds of siRNA and shRNA may be modified nucleoside bonds. The modified nucleoside bonds are preferably phosphorothioate bonds.

[0081] In siRNA and shRNA, different nucleotides within the same strand can undergo independently different modifications. Furthermore, the same nucleotide may possess modified nucleoside bonds (e.g., phosphorothioate bonds) and modified sugars (e.g., bicyclic sugars). The same nucleotide may also possess modified nucleic acid bases (e.g., 5-methylcytosine) and modified sugars (e.g., 2'-OMe modifications, bicyclic sugars, etc.).

[0082] The siRNA and shRNA of the present invention can be produced by methods known in the art. For example, siRNA and shRNA can be produced by synthesizing them using a commercially available automated nucleic acid synthesizer and then purifying them using an ion exchange column or a reversed-phase column. Alternatively, siRNA and shRNA can be ordered from a manufacturer (e.g., Gene Design Co., Ltd.) by specifying the nucleic acid base sequence, modification site, and type.

[0083] Furthermore, functional molecules such as labeling compounds (fluorescent proteins, luciferase, etc.) and purification compounds (biotin, avidin, His-tagged peptides, GST-tagged peptides, FLAG-tagged peptides, etc.) may be bound to the siRNA and shRNA. The binding may be direct or indirect via other substances, but direct binding by covalent bonds or the like is preferred.

[0084] The antitumor agent of the present invention may contain, but is not limited to, 0.5 to 10 equivalents, preferably 1 to 5 equivalents, more preferably 1 to 3 equivalents, of a folic acid-cationic oligopeptide complex per siRNA or shRNA molecule.

[0085] As demonstrated in the examples, the combined use of siRNA or shRNA with a folic acid-cationic oligopeptide complex can significantly suppress tumor growth and invasion in pancreatic cancer. Since the folic acid-cationic oligopeptide complex of the present invention exhibits sufficient effect with only a small amount (approximately 1-3 equivalents) added to siRNA or shRNA, it is expected to be an extremely useful treatment method for pancreatic cancer.

[0086] The antitumor agent according to the present invention is not particularly limited as long as the active ingredient is delivered to the target site, pancreatic cancer tissue, and can be, for example, an injectable, a liquid, or a sustained-release agent. Water is preferred as the solvent for these formulations, but it is preferable to use physiological saline, PBS, serum albumin solution, etc., so that the formulation ultimately becomes an isotonic or nearly isotonic solution.

[0087] <Pharmaceutical composition> The present invention also provides a pharmaceutical composition comprising one or more antitumor agents of the present invention. The target site of the antitumor agent according to the present invention may be not only the pancreas, but also lymph nodes or other organs to which pancreatic cancer cells have metastasized. Furthermore, in order to more reliably deliver the active ingredient to the target site, an injectable formulation is preferred.

[0088] Pharmaceutical compositions may include carriers, excipients, stabilizers, disintegrants, surfactants, binders, lubricants, emulsifiers, suspending agents, antioxidants, deodorizers, fillers, solubilizers, coatings, colorants, flavoring agents, preservatives, buffers, etc., commonly used in the pharmaceutical field. Specifically, examples include water, physiological saline, other aqueous solvents, pharmaceutically acceptable organic solvents, mannitol, lactose, starch, microcrystalline cellulose, glucose, calcium, polyvinyl alcohol, collagen, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, acacia gum, xanthan gum, casein, gelatin, agar, propylene glycol, polyethylene glycol, petrolatum, paraffin, glycerin, stearyl alcohol, stearic acid, sorbitol, and the like.

[0089] The pharmaceutical composition can be administered to the subject orally or parenterally. Parenteral administration includes, but is not limited to, injection or infusion into the subcutaneous, intravenous, intraperitoneal, or tumor, and administration during procedures via endoscope or laparoscope.

[0090] The dosage and frequency of administration of the pharmaceutical composition of the present invention may be appropriately adjusted depending on the dosage form, the patient's age, sex, weight, and the severity of the disease. For example, the pharmaceutical composition may be administered in an amount of siRNA or shRNA of 0.001 mg / kg / day to 1 mg / kg / day, 0.005 mg / kg / day to 0.5 mg / kg / day, or 0.01 mg / kg / body weight to 0.1 mg / kg / day, with the amount of folic acid-cationic oligopeptide complex determined to be 0.5 to 10 equivalents in relation to this.

[0091] The pharmaceutical composition can be administered as a single dose or multiple doses, for example, at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, etc.

[0092] The subjects to whom the pharmaceutical composition is administered may be mammals, such as humans and primates, and non-primates, such as cattle, pigs, sheep, horses, cats, dogs, guinea pigs, rats, and mice, but are more preferably humans. The subjects may also be animal models of pancreatic cancer, such as those transplanted with human pancreatic cancer cells, which are suitable for evaluating efficacy in humans.

[0093] While there are no particular limitations on the pancreatic cancer model animal, for example, a non-human animal transplanted with cancer organoids as described in Japanese Patent Application Publication No. 2018-110575 can be used. Here, "organoid" refers to an organ fabricated three-dimensionally in vitro, which is an aggregate of cells specific to a particular organ. Various organ organoids have already been fabricated in this field. The "cancer organoid" described in Japanese Patent Application Publication No. 2018-110575 is an organoid that reproduces the microenvironment of cancer tissue. The research group including the inventors of this application has modified the method of Japanese Patent Application Publication No. 2018-110575 to create a human pancreatic cancer mouse model useful for determining the efficacy of pancreatic cancer therapeutics (Japanese Patent Application No. 2020-078771), and this mouse model can also be suitably used to evaluate the efficacy of the antitumor drug and pharmaceutical composition of the present invention in humans.

[0094] <Combination formulations> The present invention also provides a combination formulation for suppressing tumor growth, invasion, and metastasis of pancreatic cancer, comprising an siRNA or shRNA capable of binding to and inhibiting the expression of mRNA or snoRNA expressed in the pancreatic cancer cells, and the folate-cationic oligopeptide complex described above.

[0095] As can be understood from the description herein, the siRNA or shRNA and the folate-cationic oligopeptide complex are not covalently bound and are not constrained by any particular mechanism, but the intention is that the folate-cationic oligopeptide complex will be partially inserted into the main groove of the siRNA or shRNA, thereby stabilizing the siRNA or shRNA.

[0096] Therefore, the siRNA or shRNA and the folic acid-cationic oligopeptide complex do not necessarily have to be included in the same composition beforehand. In other words, the preparation containing the siRNA or shRNA and the preparation containing the folic acid-cationic oligopeptide complex may be prepared separately and combined prior to administration.

[0097] Alternatively, the two formulations described above can be administered separately. However, considering the stabilization of siRNA or shRNA by the folate-cationic oligopeptide complex, it is preferable to administer these two formulations simultaneously (or consecutively) and via the same route of administration, rather than administering them at different times or via different routes of administration.

[0098] <Kit> The present invention also provides a pharmaceutical kit for the treatment of pancreatic cancer, comprising an siRNA or shRNA capable of binding to and inhibiting the expression of mRNA or snoRNA expressed in the pancreatic cancer cells described above, and the folate-cationic oligopeptide complex described above. In addition to the siRNA or shRNA and the folate-cationic oligopeptide complex, the kit may include instructions regarding other drugs that can be administered simultaneously or are appropriate for administration, a carrier, a method of administration, etc. [Examples]

[0099] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments.

[0100] [Example 1: Synthesis of Folic Acid-Cationic Oligopeptide Complex] The folic acid-cationic oligopeptide complex was synthesized as shown below. The reagents and analytical instruments used in this example are as follows.

[0101] <Reagents> The resin used as a solid-phase synthesis support for Fmoc amino acid derivatives and peptides was purchased from Watanabe Chemical Industry Co., Ltd., and folic acid was purchased from Tokyo Chemical Industry Co., Ltd. and used as is. Each peptide chain was synthesized using the Fmoc solid-phase synthesis method with Fmoc-NH-SAL-PEG resin as the solid-phase support. Fmoc-Dab(Boc)-OH, Fmoc-Gly-OH, and Fmoc-Tyr(t-Bu)-OH were used as Fmoc-AA-OH reagents.

[0102] <ESI MS> Varian 910-MS (JASCO) <UV-visible spectrophotometer> V-550 (JASCO) <Temperature-Variable UV-Vis Spectrophotometer> UV-1650PC (SIMADZU) <Spectrofluorometer> FP-6500 (JASCO) <hplc> Pump: PU-2080i plus (JASCO) Detector:UV-2075i plus(JASCO) Low-pressure gradient unit: LG-2080-02 (JASCO) Degassa: DG-2080-53 (JASCO) Reverse-phase columns: μ-Bondasphere 150×3.9mm C18 5μm 100Å (Waters), SunFire C18 OBD 5μm 19×150mm (Waters)

[0103] <Step 1: Coupling operation> First, an octamer (Dab8) of L-2,4-diaminobutyric acid was synthesized by Fmoc solid-phase synthesis. A solid support was added to PetiSyzer (Hypep Institute) so that 13 μmol of amino groups were introduced. After washing five times with 1.3 mL of dimethylformamide (DMF), 1.3 mL of DMF was added and the mixture was allowed to stand for more than one hour to swell the support.

[0104] (i) After swelling, the mixture was washed five times with 1.3 mL of DMF, then 1.3 mL of 25% piperidine / DMF solution was added and the mixture was reacted for 5 minutes to remove the Fmoc groups. The mixture was stirred in several stages using a vortex mixer.

[0105] (ii) Next, after washing five times with 1.3 mL of DMF, 5 equivalents of Fmoc-amino acid (Fmoc-AA-OH), 5 equivalents of N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan aminiumhexafluorophosphate-N-oxide (HATU·H2O, 5 equivalents) as condensation reagents, 10 equivalents of diisopropylethylamine (DIPEA), and 1.3 mL of DMF as the reaction solvent were added to the amino groups on the resin, and the condensation reaction was carried out for 15 minutes. During this time, the mixture was stirred in several stages using a vortex mixer.

[0106] After repeating steps (i) and (ii) until the N-terminal amino acid, step (i) was performed again to remove the Fmoc group, thereby synthesizing NH2-GGG-Dab8 on a solid support. Subsequently, after washing five times with 1.3 mL of DMF, 1.3 mL of a solution containing 2 equivalents of folic acid and 2 equivalents of N,N'-dicyclohexylcarbodiimide (DCC) dissolved in 1.5 mL of a DMF-dimethyl sulfoxide (DMSO) mixed solvent (1:1, v / v), which had been allowed to stand for 6 hours, was added to the amino groups on the resin, and the condensation reaction was carried out for 14 hours.

[0107] <Step 2: Deprotection, de-resinization, and purification> The resin was washed five times each with DMF and CHCl3, and dried under reduced pressure in a desiccator. The obtained resin was deprotected and deresinated by stirring for 1 hour and 30 minutes in a mixed solvent of trifluoroacetic acid (TFA)-triisopyrropyrsilane-H2O (96.5 / 1.0 / 2.5, v / v / v) at room temperature. The resin was filtered off, and the solvent was vaporized under an argon stream. Et2O was then added to precipitate the peptide. The supernatant was removed by centrifugation, and this process was repeated three times. After that, Et2O was vaporized under an argon stream to obtain the crude product (folic acid-cationic oligopeptide). The obtained crude product was dissolved in 1 mL of Otsuka distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then purified by reverse-phase HPLC under the following conditions.

[0108] Gradient cycle: Ratio of solvent B (CH3CN-0.05% TFA) in solvent A (H2O-0.05% TFA). Linear gradient: 0% to 5% in 5 minutes; 5% to 25% in 40 minutes; 25% to 100% in 5 minutes. Measurement temperature: 30℃ Flow rate: 0.5mL / min

[0109] Figure 1 shows the HPLC analysis results of the crude Fol-Dab8 product. Due to the presence of two carboxyl groups in folic acid, the Dab8 with folic acid introduced (referred to as Fol-Dab8) eluted as two separate peaks. The peak that eluted first in the HPLC analysis was designated Fol-Dab8A, and the peak that eluted second was designated Fol-Dab8B. The chemical structures of each are shown below.

[0110] [ka]

[0111] [ka]

[0112] The solution separated by HPLC was freeze-dried to obtain a yellow powder. Purified Fol-Dab8A or Fol-Dab8B was dissolved in Otsuka distilled water, and the molar extinction coefficient of folic acid was calculated in advance (molar extinction coefficient at a wavelength of 368 nm: 7967 L / mol·cm). The yield was calculated from the UV absorption of folic acid. Dab8 was obtained by synthesizing NH2-GGG-Dab8 on a solid support, then coupling it with tyrosine protected by an acetyl group at the N-terminus, performing the procedure in <Step 2>, and purifying it by HPLC. The yield was calculated from the UV absorption of Tyr. Each peptide was identified by mass spectrometry (ESI-MS) ([M+H] of Dab8). + m / z Calculated value: 1194.681, Measured value: 1194.682; Fol-Dab8A [M+H] + m / z Calculated value: 1412.737, Measured value: 1412.738; Fol-Dab8B [M+H] + (m / z calculated value: 1412.737, measured value: 1412.739).

[0113] [Example 2: Melting Temperature (Tm) Analysis] The melting temperatures of nucleic acid double helix structures were measured in the presence and absence of the cationic oligopeptide or folate-cationic oligopeptide complex synthesized in Example 1.

[0114] In this example, a cationic oligopeptide (Dab8) or a folate-cationic oligopeptide (Fol-Dab8A or Fol-Dab8B) was added to the annealed RNA double strand, and the melting temperature (Tm value) was measured. As the RNA double strand, an oligonucleotide pair consisting of the following sequence was used as siRNA. 5'-r(GUCAUCACACUGAAUACCA)dTdT-3'(Sequence ID 1) 5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-3'(Sequence ID 2)

[0115] 144 μL of a 50 μM nucleic acid aqueous solution was prepared and kept at 95°C for 5 minutes, then slowly cooled to 4°C at -0.5°C / min. This was added to a mixed solution of a pH 7.0 buffer containing 200 mM NaCl and 20 mM Na2HPO4-NaH2PO4, Otsuka distilled water, and a 0.1 mM peptide aqueous solution. The sample was then adjusted to a final concentration of 10 mM Na2HPO4-NaH2PO4, 100 mM NaCl, 4 μM nucleic acid double helix, and 0, 4, 8, 12, 16, and 20 μM peptides (0, 1, 2, 3, 4, and 5 equivalents, respectively).

[0116] The absorbance at 260 nm was measured while the temperature was increased from 20°C to 95°C at a rate of 0.5°C / min, and the melting curve was determined. To remove background noise, the absorbance at 320 nm was measured, and the melting curve was created by subtracting the absorbance at 320 nm from the absorbance at 260 nm. The Tm value was then determined using the median line method.

[0117] As a result, as shown in Table 1, the Tm value was 72.3°C when no cationic oligopeptide was added to the RNA duplex, while the Tm value increased when one equivalent or more of Dab8, Fol-Dab8A, or Fol-Dab8B was added. This suggests that Fol-Dab8A and B, like Dab8, bind to the RNA duplex and improve its thermodynamic stability.

[0118] [Table 1]

[0119] [Example 3: Evaluation of RNaseA resistance] We performed RNA-degrading enzyme resistance tests on double-stranded nucleic acids in the presence of cationic oligopeptides. In this example, a peptide was added to the annealed RNA double helix to form a complex, and the rate of RNA degradation was measured.

[0120] Otsuka distilled water, 100 mM Tris-HCl, and 1 M NaCl buffer (pH 7.3) were added to a PCR tube. Then, equal volumes of 0.1 mM aqueous solution of 6-FAM modified single-stranded RNA (5'-FAM-r(GUCAUCACACUGAAUACCA)dTdT-3', SEQ ID NO: 1) with a fluorescent 5' end, and 0.1 mM aqueous solution of Dabcyl modified single-stranded RNA (5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-dabcyl-3', SEQ ID NO: 2) with a quenching group at the 3' end were mixed together to prepare a final concentration of 10 mM Tris-HCl, 100 mM NaCl, and 10 μM siRNA. The mixture was then maintained at 95°C for 5 minutes, followed by slow cooling to 4°C at -0.5°C / min.

[0121] A 10 mM Tris-HCl, 100 mM NaCl, 10 μM siRNA aqueous solution, and 0.1 mM cationic oligopeptide aqueous solution were added to a quartz cell to prepare a 3 mL solution with final concentrations of 10 mM Tris-HCl, 100 mM NaCl, 10 nM siRNA, and 0, 10, 20, and 30 nM peptides (0, 1, 2, and 3 equivalents, respectively). Subsequently, 15 μL of 100 μg / mL bovine pancreas-derived RNase A (Roche) was added while stirring at 37°C, and fluorescence intensity was measured and tracked over time (excitation wavelength: 490 nm; measurement wavelength: 520 nm; measurement time: 60 minutes).

[0122] The results are shown in Figure 2. When the RNA double helix dissociates, the distance between the fluorescent group and the quenching group increases, resulting in higher fluorescence intensity. Therefore, when one equivalent or more of Dab8, Fol-Dab8A, and Fol-Dab8B were added, the rate of increase in fluorescence intensity slowed down. This suggests that, similar to Dab8, Fol-Dab8A and B improved the resistance of RNA double helix to degrading enzymes.

[0123] [Example 4: Confirmation of siRNA uptake by pancreatic cancer cells] Because flow cytometry makes it difficult to distinguish between siRNAs that have been taken up by cells and those that have not, we observed the uptake of siRNA into pancreatic cancer cells using a confocal microscope.

[0124] Pancreatic cancer cell line S2-013 (Medical Cell Resource Center, Cell Bank, Institute of Development, Aging and Cancer, Tohoku University, ID: TKG0709) 2 × 10 4 Cells were seeded at a cell density of 1 cell / well in 4-well chamber slides (Thermo Fisher Scientific). Scrambled control siRNAs labeled with Alexa647 (Thermo Fisher Scientific) (SEQ ID NOs: 3 and 4) were then mixed with 1 to 3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B, and cultured for 48 hours. 25 μL of siRNA was used at a concentration of 8.28 μg / mL, while the concentrations of Dab8, Fol-Dab8A, and Fol-Dab8B were obtained by diluting the stock solutions (Dab8: 119.3 μg / mL, Fol-Dab8A, Fol-Dab8B: 141.2 μg / mL) 25-fold. The amounts corresponding to 1, 2, and 3 equivalents of siRNA were 3.9 μL, 7.8 μL, and 11.7 μL, respectively.

[0125] The following day, the cells were fixed with 4% paraformaldehyde, mounted with a DAPI-containing mounting medium, and observed using an all-in-one fluorescence microscope (BZ-X800, keyence). As shown in Figure 3A, the uptake of siRNA into cells was confirmed by the fluorescence of Alexa647. On the other hand, when only siRNA was added to the culture medium of pancreatic cancer cell line S2-013 and cultured, fluorescence by Alexa647 confirmed that the siRNA was hardly taken up by the cells (Figure 3B).

[0126] To quantify uptake, the hybrid cell counting function of the keyence analysis software BZ-X800 Analyzer was used to count cells that had taken up siRNA in the presence of Dab8, Fol-Dab8A, and Fol-Dab8B. Cells with more than one-third of the nucleus stained intensely, or those with dense staining around the periphery of the nucleus, were measured.

[0127] As a result, as shown in Figure 3C, when using cationic oligopeptide (Dab8) and folate-cationic oligopeptide complexes (Fol-Dab8A, Fol-Dab8B), 20-40% of the added siRNA was observed to be taken up into cells. Furthermore, the highest transfusion efficiency was observed with 3 equivalents for Fol-Dab8A and 1 equivalent for Fol-Dab8B, which was better than the transfusion efficiency of Dab8 with 1-3 equivalents added.

[0128] [Example 5: Knockdown effect by folic acid-cationic peptide-added siRNA] The knockdown effects of siRNA against SNORA18, NUP85, WASF2, and SNORA22 in the presence of cationic oligopeptides or folate-cationic oligopeptide complexes were investigated. Table 2 shows the sequences of the siRNAs used in this example. Each siRNA was prepared using a double-stranded configuration of sense strands and antisense strands, as is commonly practiced in this field, as shown in Table 2.

[0129] [Table 2]

[0130] Scrambled control siRNAs (SEQ ID NOs. 3 and 4), SNORA18 siRNA (SEQ ID NOs. 5 and 6), NUP85 siRNA (SEQ ID NOs. 7 and 8), WASF2 siRNA (SEQ ID NOs. 9 and 10), and SNORA22 siRNA (SEQ ID NOs. 11 and 12), each containing 1 equivalent of a cationic oligopeptide or folate-cationic peptide attached to a Dab8-siRNA complex, 3 equivalents of a Fol-Dab8A-siRNA complex, and 1 equivalent of a cationic oligopeptide or folate-cationic peptide attached to a 6-well plate (Thermo Fisher Scientific), were added to a culture medium (1.0 × 10⁶). 5 The solution was added to cells per well, and cells were harvested after 48 hours. Semi-quantitative RT-PCR was performed using RNA from the recovered cells to confirm the knockdown effects of SNORA18, NUP85, WASF2, and SNORA22 in the cells.

[0131] Specifically, total RNA obtained from S2-013 cells was reverse transcribed using StrataScript reverse transcriptase (Agilent) and random primers. Appropriate dilutions of each single-stranded cDNA were prepared for subsequent PCR amplification. GAPDH mRNA was used as an internal quantitative control. The primer sequences used to amplify SNORA18, NUP85, WASF2, and SNORA22 are listed in Table 3 below.

[0132] [Table 3]

[0133] PCR reactions were performed on a TaKaRa PCR Thermal Cycler Dice Gradient for initial denaturation at 94°C for 2 minutes, followed by 21 cycles (for GAPDH) or 25 cycles (for SNORA18, NUP85, WASF2, and SNORA22) at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute. Scanning and densitometry analysis for band intensity measurement were performed using a Quantity One analysis system (Bio-Rad).

[0134] As shown in Figures 4A, 5A, 6A, and 7A, respectively, no knockdown effect was observed for SNORA18, NUP85, WASF2, and SNORA22 when the control siRNA was combined with Dab8, Fol-Dab8A, or Fol-Dab8B. However, when SNORA18 siRNA, SNORA22 siRNA, NUP85 siRNA, and WASF2 siRNA were added in combination with Fol-Dab8A or Fol-Dab8B, a significant knockdown effect was observed for all of them. This knockdown effect was not observed with Dab8 that did not contain folic acid.

[0135] [Example 6: Cell invasion inhibitory effect of folic acid-cationic oligopeptide complex + siRNA] Scrambled control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA (all used in Example 5), each of which was attached to a folic acid-cationic oligopeptide complex, was added to the culture medium of S2-013 cells, and a Matrigel infiltration assay was performed after 48 hours.

[0136] 4.0 × 10⁶ suspended in serum-free medium 4 Cells were seeded in the upper chamber of a Matrigel Invasion Chamber (24-well plate, pore size: 8 μm, Becton Dickinson). A solvent containing 5% fetal bovine serum was added to the lower chamber. After incubating the cells in the upper chamber for 20 hours, three independent regions were observed under a microscope, and the cells that had infiltrated the lower chamber were counted. The same experiment was repeated three times, and the cell invasion ability of S2-013 cells incorporating each siRNA was compared.

[0137] Figures 4B, 5B, 6B, and 7B show the number of cells that migrated from the upper chamber to the lower chamber in assays in which cells were treated with either scrambled control siRNA or one of the following: SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA, along with a folate-cationic oligopeptide complex. An asterisk (*) indicates a statistically significant difference compared to the control (P<0.05) in the t-test.

[0138] As a result, S2-013 cells that incorporated SNORA18siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA showed significantly suppressed cell invasion compared to S2-013 cells that incorporated control siRNA.

[0139] Based on these results, we were able to confirm that siRNA added to the culture medium of cultured cells in combination with a folate-cationic oligopeptide complex was taken up into S2-013 cells and inhibited the expression of snoRNA and mRNA involved in cell invasion.

[0140] [Example 7: Effects on chemically modified siRNA] Chemically modified siRNAs were prepared to enhance their stability, and the effects of folic acid-cationic oligopeptide complexes were investigated. Table 4 shows the sequences of the sense and antisense strands of the siRNA used in this example. Each strand contains chemically modified bases and a phosphorothioate bond at its 3' end.

[0141] [Table 4]

[0142] The uptake into cells was investigated using scrambled control siRNAs (SEQ ID NOs: 3 and 4) and the above-mentioned siRNAs, in the same manner as in Example 4.

[0143] Culture medium of pancreatic cancer cell line S2-013 in culture (2 × 10 4 Each well contained 2.5 μg / mL of siRNA labeled with Alexa488 (Thermo Fisher Scientific), to which 1, 2, and 3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B were added.

[0144] Next, the effect of chemically modified siRNA on pancreatic cancer cell invasion was investigated in the presence or absence of the folate-cationic oligopeptide complex, in the same manner as in Example 5. Scrambled control siRNA (SEQ ID NOs: 3 and 4), SNORA18 siRNA (SEQ ID NOs: 21 and 22), NUP85 siRNA (SEQ ID NOs: 23 and 24), WASF2 siRNA (SEQ ID NOs: 25 and 26), and SNORA22 siRNA (SEQ ID NOs: 27 and 28) were each added to the culture medium of the pancreatic cancer cell line S2-013 along with 2 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B. As a control, the control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA were each added individually to the culture medium of S2-013 cells.

[0145] After 48 hours of culture, a Matrigel infiltration assay was performed. The number of cells that migrated from the upper chamber to the lower chamber in the Matrigel infiltration assay is shown. "*" indicates a statistically significant difference compared to the control in the t-test (P<0.05).

[0146] As a result, as shown in Figure 8, in the presence of Fol-Dab8A and Fol-Dab8B, S2-013 cell invasion was significantly suppressed when using SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA, compared to S2-013 cells treated with scrambled control siRNA in the presence of Fol-Dab8A and Fol-Dab8B, and when siRNA was treated alone. In the presence of folic acid-free Dab8, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA were observed to suppress S2-013 cell invasion, but the suppressive effect was weaker compared to when Fol-Dab8A and Fol-Dab8B were added.

[0147] [Example 8: Confirmation of uptake of chemically modified siRNA into pancreatic cancer cells 1] The chemically modified SNORA22 siRNAs (SEQ ID NOs. 27 and 28) prepared in Example 7 were labeled with Alexa488 (Thermo Fisher Scientific) and combined with a folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents) in a 4-well chamber (Thermo Fisher Scientific) containing culture medium (5 × 10⁻¹⁰) of S2-013 pancreatic cancer cells or HPNE normal pancreatic ductal epithelial cells (ATCC). 4 After adding the solution to individual cells / well and incubating overnight at 37°C, staining of DNA (DAPI), folic acid receptor (FOLR1), and siRNA (Alexa488) was observed.

[0148] As a result, as shown in Figure 9, S2-013 pancreatic cancer cells showed higher staining intensity for folate receptors compared to HPNE normal pancreatic ductal epithelial cells, and simultaneously, stronger staining intensity for SNORA22 siRNA taken up into the cells. This indicates that pancreatic cancer cells that express more folate receptors take up more SNORA22 siRNA.

[0149] Figure 10 shows the efficiency (%) of SNORA22 siRNA introduction into S2-013 pancreatic cancer cells and HPNE normal pancreatic ductal epithelial cells in the presence of a folate-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents). The uptake of SNORA22 siRNA into S2-013 pancreatic cancer cells was nearly three times higher than in normal cells.

[0150] [Example 9 Confirmation of uptake of chemically modified siRNA into pancreatic cancer cells 2] It is known that siRNA taken up into cells by endocytosis is taken up into endosomes and fuses with lysosomes. To verify whether the siRNA complex of the present invention and Fol-Dab8A or Fol-Dab8B is taken up via endocytosis, confocal microscopy images of stained lysosomes and siRNA were obtained.

[0151] Chemically modified SNORA22 siRNA labeled with Alexa488 (SEQ ID NOs. 27 and 28) and folate-cationic oligopeptide complexes (Fol-Dab8A or Fol-Dab8B, 2 equivalents) were added to the culture medium of S2-013 pancreatic cancer cells in a 4-well chamber (Thermo Fisher Scientific) (2 × 10⁻¹⁶). 4 The sample was added to individual cells / wells and incubated overnight. The staining of lysosomes (stained using LysoTracker, Thermo Fisher Scientific) and siRNA (Alexa488) was observed.

[0152] As a result, as shown in Figure 11, SNORA22 siRNA was taken up by S2-013 pancreatic cancer cells and localized to lysosomes, suggesting that the siRNA and folate-cationic oligopeptide complex were taken up by endocytosis.

[0153] [Example 10: Confirmation of improved siRNA stability through chemical modification] In this example, we investigated whether the stability of siRNA in serum can be improved by chemical modification.

[0154] Unmodified SNORA22 siRNA (SEQ ID NOs. 11 and 12) or chemically modified SNORA22 siRNA (SEQ ID NOs. 27 and 28) were mixed with a cationic oligopeptide (Dab8) or a folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B). After standing at room temperature for 15 minutes, 1 μL of each mixture was added to PBS or 10% FCS / PBS and mixed (final 20 μM). As a control, each SNORA22 siRNA was added alone.

[0155] Samples were taken immediately after mixing, and again after 3 or 6 hours. The samples were then frozen in liquid nitrogen and stored at -80°C. After all samples were obtained, SDS-PAGE was performed using a non-reducing gel, and detection was performed using SYBR GOLD (Thermo Fisher Scientific).

[0156] As a result, as shown in Figure 12, unmodified siRNA was shown to be rapidly degraded even when cationic oligopeptides or folate-cationic oligopeptide complexes were present. However, chemical modification of siRNA improved its stability in serum and suggested that degradation by RNase could be almost completely suppressed.

[0157] [Example 11: siRNA uptake in vivo 1] Human pancreatic cancer organoids were created using S2-013 pancreatic cancer cells by modifying the method described in Japanese Patent Application Publication No. 2018-110575. Specifically, S2-013 pancreatic cancer cells (20 × 10 4 (individual), human mesenchymal stem cell MSC (LONZA, 40 x 10) 4 (14 × 10¹), and human umbilical vein endothelial cells (HUVECs) (LONZA, 14 × 10¹⁴). 4 The samples were added to a DMEM / Matrigel mixed solution in a 48-well plate (Thermo Fisher Scientific) and incubated in a 37°C CO2 incubator for 30 minutes. Subsequently, 300 μL / well of DMEM / EGM mixed solution was added, and the mixture was incubated again at 37°C for 24 hours to produce one pancreatic cancer organoid in each well.

[0158] Next, the skin of the flanks of nude mice (6-week-old BALB / cSlc-nu / nu (Pathogen-free female athymic nude mice), SLC Japan Co., Ltd.) was incised, and the pancreatic cancer organoids obtained above were transplanted subcutaneously (2 mice per group).

[0159] Six weeks post-transplant, chemically modified SNORA22 siRNA labeled with Alexa-594 (SEQ ID NOs. 27 and 28, 5 μg) was administered via tail vein injection along with one, two, or three equivalents of a cationic oligopeptide (Dab8) or a folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B). Images were taken 24 hours later using an in vivo imager.

[0160] Measurement equipment: Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA) Measurement conditions: kexc 640nm, kemi 680nm

[0161] As a result, as shown in Figure 13, delivery of SNORA22 siRNA to pancreatic cancer tissue was enhanced when using Fol-Dab8A (DF) and Fol-Dab8B (GI) compared to when using Dab8 (AC), and particularly when 3 equivalents of Fol-Dab8A were added (F) and 2 equivalents of Fol-Dab8B were added (H), resulting in high concentrations of delivery. Furthermore, this experiment confirmed that siRNA was hardly transferred to or accumulated in the liver, and was instead excreted by the kidneys.

[0162] [Example 12: Confirmation of antitumor effect] One week after transplanting the human pancreatic cancer organoids prepared in Example 11 subcutaneously into nude mice, chemically modified SNORA22 siRNA (SEQ ID NOs. 27 and 28, 5 μg) was administered weekly via tail vein injection with cationic oligopeptide (Dab8, 2 equivalents) or folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents), and tumor volume was measured weekly using calipers (n=8 in each group). As a control, scrambled control siRNA (SEQ ID NOs. 3 and 4) was administered with folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents).

[0163] As a result, as shown in Figure 14, measurements taken from the start of administration up to 9 weeks later showed that the group administered SNORA22 siRNA with a folic acid-cationic oligopeptide complex (SNORA22-Fol-Dab8B) exhibited a significant tumor growth suppression effect from week 8 onwards, compared to the control group (no administration), the group administered scrambled control siRNA (Scr-Fol-Dab8B), and the group administered with a cationic oligopeptide (SNORA22-Dab8).

[0164] [Example 13: siRNA uptake in vivo 2] In the same manner as in Example 11, the delivery of chemically modified SNORA18 siRNAs (SEQ ID NOs. 21 and 22) to pancreatic cancer-carrying mice when administered together with a folic acid-cationic oligopeptide conjugate (Fol-Dab8A or Fol-Dab8B) was imaged in vivo using an imager.

[0165] As a result, as shown in Figure 15, both Fol-Dab8A and Fol-Dab8B were confirmed to promote the delivery of SNORA18 siRNA to pancreatic cancer tissue, and in particular, when Fol-Dab8A was added at 1 equivalent (A) or 3 equivalents (C), it was delivered to a high concentration.

[0166] [Example 14: siRNA uptake in vivo 3] In the same manner as in Examples 11 and 13, the delivery of chemically modified WASF2 siRNAs (SEQ ID NOs. 25 and 26) to pancreatic cancer-carrying mice when administered together with a folic acid-cationic oligopeptide conjugate (Fol-Dab8A or Fol-Dab8B) was imaged in vivo using an imager.

[0167] As a result, as shown in Figure 16, both Fol-Dab8A and Fol-Dab8B were confirmed to promote the delivery of WASF2 siRNA to pancreatic cancer tissue, and in particular, when Fol-Dab8B was added at 1 equivalent (D) or 3 equivalents (F), it was delivered to a high concentration. [Industrial applicability]

[0168] The present invention provides a therapeutic method that can effectively suppress tumor growth, invasion, and metastasis in pancreatic cancer, which is said to have the worst prognosis among cancers. The antitumor agent of this application is effectively delivered specifically to pancreatic cancer cells, and when combined with other anticancer agents and / or anticancer treatments, it is possible to dramatically improve the therapeutic effect of pancreatic cancer. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.< / hplc> < / mtor>

Claims

1. A siRNA or shRNA delivery enhancer comprising a folic acid-cationic oligopeptide complex, The delivery enhancer is a cationic oligopeptide homopolymer of L-2,4-diaminobutyric acid (Dab) and consists of 8 to 12 amino acid residues.

2. The delivery accelerator according to claim 1, wherein the cationic oligopeptide is in the form of a salt.

3. The delivery accelerator according to claim 1 or 2, wherein folic acid is linked to the N-terminus, C-terminus, or side chain of a cationic oligopeptide, with or without a linker.

4. The delivery accelerator according to claim 3, wherein folic acid is linked to a cationic oligopeptide via a linker, and the linker is a peptide linker.

5. The delivery promoter according to claim 4, wherein the peptide linker is a peptide consisting of 1 to 4 glycine residues.

6. An antitumor agent comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and a delivery promoter according to any one of claims 1 to 5.

7. The antitumor agent according to claim 6, wherein the mRNA or snoRNA expressed in pancreatic cancer cells binds to insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3).

8. The antitumor agent according to claim 6 or 7, wherein the mRNA or snoRNA expressed in pancreatic cancer cells is selected from the group consisting of SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA.

9. An antitumor agent according to any one of claims 6 to 8, comprising 0.5 to 10 equivalents of a folic acid-cationic oligopeptide complex relative to siRNA or shRNA.

10. The antitumor agent according to any one of claims 6 to 9, wherein the siRNA is RNA / RNA double-stranded.

11. The antitumor agent according to any one of claims 6 to 10, wherein the siRNA or shRNA comprises a modified base, a modified sugar, and / or a modified nucleoside bond.

12. The antitumor agent according to claim 11, wherein the modification of the modified sugar is a 2'-OMe modification.

13. The antitumor agent according to claim 11 or 12, wherein the modified nucleoside bond is a phosphorothioate bond.

14. A pharmaceutical composition containing the antitumor agent according to any one of claims 6 to 13.

15. below: (a) A preparation comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and (b) A formulation comprising a delivery accelerator according to any one of claims 1 to 5 A combination formulation containing the above.

16. below: (a) siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and (b) Delivery accelerator according to any one of claims 1 to 5 A medical kit for the treatment of pancreatic cancer, including [specific components / items].

Images