Nucleic acid delivery enhancers

Abstract

A novel antitumor agent is provided, which can inhibit the tumor growth, infiltration and metastasis of pancreatic cancer by specifically delivering siRNA or shRNA to pancreatic cancer cells. The present invention provides a nucleic acid delivery facilitator, which is composed of a folate-cationic oligopeptide complex, and is used to deliver siRNA or shRNA into cells. The present invention also provides an antitumor agent, comprising siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and hinder its expression, and a folate-cationic oligopeptide complex. siRNA can bind to RNA selected from the group consisting of, for example, SNORA18snoRNA, NUP85mRNA, WASF2mRNA, and SNORA22snoRNA.

Description

Technical Field

[0001] The present invention relates to a nucleic acid delivery facilitator for delivering nucleic acid molecules such as siRNA into cells. The present invention also relates to a drug that can use RNA interference (RNAi) to treat diseases. In particular, the present invention relates to a nucleic acid preparation having an anti-tumor effect on pancreatic cancer and being able to effectively inhibit the tumor growth, infiltration and metastasis of pancreatic cancer, and a pharmaceutical composition containing the same. Background Art

[0002] It is generally believed that pancreatic cancer has the worst prognosis among all cancers. This is because the pancreas is a retroperitoneal organ, making it difficult to detect early, and pancreatic cancer cells are highly motile, making it more likely to infiltrate the peritoneum and metastasize to blood vessels, the digestive tract, and nerves.

[0003] At present, the inventors have studied the invasion and metastasis mechanism of pancreatic cancer and found that insulin-like growth factor 2mRNA-binding protein 3 (IGF2BP3) exists in the cell membrane protrusions of pancreatic cancer cells. A variety of mRNAs are bound to IGF2BP3, resulting in the accumulation of these mRNAs in the cell membrane protrusions. Insulin-like growth factor 2mRNA-binding protein 3 is usually found in nucleosomes. It is known that it binds to the 5' untranslated region of insulin-like growth factor II mRNA to inhibit the translation of insulin-like growth factor II. It has also been reported that by using RNA interference (RNAi) to block these mRNAs, the invasion and metastasis of pancreatic cancer cells can be effectively inhibited (Patent Document 1).

[0004] In recent years, nucleic acid drugs using RNA interference (RNAi) have attracted much attention as new therapeutic drugs, and siRNA (small interfering RNA) is a representative of them, which is usually a low-molecular double-stranded RNA composed of 21 to 23 base pairs. However, due to its structure, siRNA has high anionicity and low cell membrane permeability, so there are technical problems in delivering it into cells. In response to this technical problem, the inventors prepared siRNA-folic acid-polyethylene glycol (PEG)-chitosan oligosaccharide lactic acid (COL) nanoparticle complexes and confirmed that the nanoparticles have the effect of promoting cell uptake of siRNA. It has also been reported that siRNA against mRNA with IGF2BP3 binding ability taken into pancreatic cancer cells will inhibit the infiltration and metastasis of pancreatic cancer (Non-patent Document 1).

[0005] On the other hand, the present inventors have also found that cationic oligopeptides having amino or guanidine groups in the side chains can be used to deliver double-stranded RNA into cells (Patent Document 2, Non-Patent Document 2). In addition, in order to deliver RNAi molecules, the application of complexes comprising vitamin E and cationic sugars has been investigated (Non-Patent Documents 3 and 4).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. WO2016 / 002844

[0009] Patent Document 2: International Publication No. WO2014 / 148620

[0010] Non-patent literature

[0011] Non-patent literature 1: Oncotarget, 2019, Vol. 10, No. 30, pp. 2869-2886

[0012] Non-patent document 2: Bioorganic & Medicinal Chemistry 21 (2013) 1717-1723

[0013] Non-patent document 3: Bioorganic & Medicinal Chemistry 22 (2014) 1394-1403

[0014] Non-patent document 4: Bioorganic & Medicinal Chemistry Letters 25 (2015) 815-819 Summary of the invention

[0015] As mentioned above, in terms of improving the practicality of nucleic acid drugs, the delivery of RNAi molecules such as siRNA as nucleic acid drugs to cells is a very important technical issue. There are still many technical problems to be solved, such as not only delivery but also specific delivery to target cells; and not hindering the effect of nucleic acid drugs after being taken into cells.

[0016] The present inventors have repeatedly studied the above technical problems and found that by using a complex of folic acid and a cationic oligopeptide, siRNA and shRNA can be specifically delivered to cells with folic acid receptors. In addition, a new treatment strategy has been discovered, that is, by using this method, pancreatic cancer cells are targeted, siRNA or shRNA is effectively taken into the targeted pancreatic cancer cells, and the expression of specific RNA is knocked down, which can inhibit the tumor growth, infiltration and metastasis of pancreatic cancer, thereby completing the present invention.

[0017] That is, the present invention provides the following solutions.

[0018] 1. A delivery facilitator for siRNA or shRNA, comprising a folic acid-cationic oligopeptide complex, wherein the cationic oligopeptide comprises a cationic oligopeptide site composed of 8 to 40 amino acids, wherein the cationic oligopeptide site comprises at least two consecutive amino acid residues of the following formula (I), and other than the consecutive amino acid residues of the following formula (I), there is one other discontinuous amino acid residue.

[0019] [Chemical formula 1]

[0020]

[0021] In formula (I), R 1 For group H 3 N + -CH 2 - or a group represented by formula (II), in R 1 For group H 3 N + -CH 2 -In the case of R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group having 1 to 4 carbon atoms; in a cationic oligopeptide, R 1 and R 2 All the same;

[0022] [Chemical formula 2]

[0023]

[0024] In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different.

[0025] 2. The delivery-enhancing agent according to item 1 above, wherein the cationic oligopeptide moiety consists of 8 to 12 amino acids.

[0026] 3. The delivery enhancer according to 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).

[0027] 4. The delivery-enhancing agent according to any one of 1 to 3 above, wherein the cationic oligopeptide has an octamer of diaminobutyric acid having the following structure as a partial structure.

[0028] [Chemical formula 3]

[0029]

[0030] 5. The delivery-enhancing agent according to any one of 1 to 4 above, wherein folic acid is linked to the N-terminus, C-terminus or side chain of the cationic oligopeptide via or without a linker.

[0031] 6. The delivery-enhancing agent according to 5 above, wherein the folic acid-cationic oligopeptide complex is connected via a linker, and the linker is a peptide linker.

[0032] 7. The delivery-enhancing agent according to 6 above, wherein the peptide linker is a peptide consisting of 1 to 4 glycine residues.

[0033] 8. The delivery-enhancing agent according to 4 above, wherein the folic acid-cationic oligopeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure.

[0034] [Chemical formula 4]

[0035]

[0036] [Chemical formula 5]

[0037]

[0038] 9. An antitumor agent comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells to inhibit the expression thereof, and the delivery enhancing agent according to any one of 1 to 8 above.

[0039] 10. The antitumor agent according to 9 above, wherein the mRNA or snoRNA expressed in pancreatic cancer cells binds to insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3).

[0040] 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.

[0041] 12. The antitumor agent according to any one of 9 to 11 above, comprising 0.5 to 10 equivalents of a folic acid-cationic oligopeptide complex relative to siRNA or shRNA.

[0042] 13. The antitumor agent according to any one of 9 to 12 above, wherein the siRNA is an RNA / RNA double strand.

[0043] 14. The 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 an altered nucleoside bond.

[0044] 15. The antitumor agent according to 14 above, wherein the modification of the modified sugar is 2'-OMe modification.

[0045] 16. The antitumor agent according to 14 or 15 above, wherein the modified nucleoside bond is a phosphorothioate bond.

[0046] 17. A pharmaceutical composition comprising the antitumor agent according to any one of 9 to 16 above.

[0047] 18. A combined preparation comprising:

[0048] (a) a preparation comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting the expression thereof; and

[0049] (b) a preparation comprising a folic acid-cationic oligopeptide complex, wherein the folic acid-cationic oligopeptide complex comprises a cationic oligopeptide site composed of 8 to 40 amino acids, wherein the cationic oligopeptide site comprises at least two consecutive amino acid residues of the following formula (I), and one other discontinuous amino acid residue other than the consecutive amino acid residues of the following formula (I),

[0050] [Chemical formula 6]

[0051]

[0052] In formula (I), R 1 For group H 3 N + -CH 2 - or a group represented by formula (II), in R 1 For group H 3 N + -CH 2 -In the case of R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group having 1 to 4 carbon atoms; in a cationic oligopeptide, R 1 and R 2 All the same;

[0053] [Chemical formula 7]

[0054]

[0055] In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different.

[0056] 19. A drug kit for treating pancreatic cancer, comprising:

[0057] (a) siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression; and

[0058] (b) a folic acid-cationic oligopeptide complex comprising a cationic oligopeptide site consisting of 8 to 40 amino acids, wherein the cationic oligopeptide site comprises at least two consecutive amino acid residues of the following formula (I), and one other discontinuous amino acid residue other than the consecutive amino acid residues of the following formula (I).

[0059] [Chemical formula 8]

[0060]

[0061] In formula (I), R 1 For group H 3 N + -CH 2 - or a group represented by formula (II), in R 1 For group H 3 N + -CH 2 -In the case of R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group having 1 to 4 carbon atoms; in a cationic oligopeptide, R 1 and R 2 All the same;

[0062] [Chemical formula 9]

[0063]

[0064] In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different.

[0065] This specification includes the disclosure of Japanese Patent Application No. 2019-194646 which is the basis of priority of this application.

[0066] According to the present invention, a nucleic acid preparation is provided, which can effectively deliver siRNA or shRNA specifically to pancreatic cancer cells, thereby inhibiting tumor growth, infiltration and metastasis of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The results of HPLC analysis of the crude product containing Fol-Dab8A and Fol-Dab8B are shown.

[0068] Figure 2 The effect of 1 to 3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B on the RNase A resistance of siRNA was shown by changes in fluorescence intensity obtained by fluorescence resonance energy transfer (FRET).

[0069] Figure 3 In the figure, A: shows a confocal microscopy image, which shows that siRNA labeled with Alexa488 is taken up into S2-013 pancreatic cancer cells in the presence of a folic acid-cationic oligopeptide complex. B: shows a confocal microscopy image, which shows that siRNA labeled with Alexa488 is taken up into S2-013 pancreatic cancer cells in the absence of a folic acid-cationic oligopeptide complex. C: shows the efficiency of siRNA uptake into S2-013 pancreatic cancer cells in the presence of a cationic oligopeptide or a folic acid-cationic oligopeptide complex. The results show the mean and standard error. Dab8-1 means that the cationic oligopeptide Dab8 is added in 1 equivalent, and Fol-Dab8B-3 means that the folic acid-cationic oligopeptide complex Dab8B is added in 3 equivalents.

[0070] Figure 4 In the figure, A: shows the inhibitory effect of scrambled control siRNA or SNORA18 siRNA and cationic oligopeptide or folate-cationic oligopeptide complex on SNORA18 expression. For the bands amplified by RT-PCR, the data are displayed with the relative intensity when the band intensity when the control siRNA and Dab8 are combined is set to 1. B: shows the number of infiltrating cells in the matrigel assay when scrambled control siRNA or SNORA18 siRNA and folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B) are combined.

[0071] Figure 5A: shows the inhibitory effect of scrambled control siRNA or NUP85 siRNA in combination with cationic oligopeptide or folate-cationic oligopeptide complex on NUP85 expression. Data are displayed by relative intensity when the band intensity when the control siRNA and Dab8 combination is set to 1 for the band amplified by RT-PCR. B: shows the number of infiltrating cells in the matrigel assay when scrambled control siRNA or NUP85 siRNA and folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B) are used in combination.

[0072] Figure 6 In the figure, A: shows the inhibitory effect of scrambled control siRNA or WASF2 siRNA on WASF2 expression when used in combination with cationic oligopeptide or folic acid-cationic oligopeptide complex. For the bands amplified by RT-PCR, the data are displayed using the relative intensity when the band intensity when the control siRNA and Dab8 are combined is set to 1. B: shows the number of infiltrating cells in the matrigel assay when scrambled control siRNA or WASF2 siRNA and folic acid-cationic peptide complexes (Fol-Dab8A and Fol-Dab8B) are used in combination.

[0073] Figure 7 In the figure, A: shows the inhibitory effect of scrambled control siRNA or SNORA22 siRNA on SNORA22 expression when used in combination with cationic oligopeptide or folate-cationic oligopeptide complex. For the bands amplified by RT-PCR, the data are displayed using the relative intensity when the band intensity when the control siRNA and Dab8 are combined is set to 1. B: shows the number of infiltrating cells in the matrigel assay when scrambled control siRNA or SNORA22 siRNA and folate-cationic peptide complex (Fol-Dab8A and Fol-Dab8B) are used in combination.

[0074] Figure 8 The numbers of infiltrating cells in the Matrigel assay when scrambled control siRNA (A), SNORA18 siRNA (B), NUP85 siRNA (C), WASF2 siRNA (D), or SNORA22 siRNA (E) were combined with a cationic oligopeptide (Dab8) or folate-cationic peptide complexes (Fol-Dab8A and Fol-Dab8B) are shown.

[0075] Fig. 9The confocal microscopy images show the staining of folate receptor (FOLR1) and SNORA22 siRNA in S2-013 pancreatic cancer cells (A) and HPNE normal pancreatic duct epithelial cells (B) after adding Alexa488-labeled SNORA22 siRNA and folate-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents) to the culture medium and incubating overnight.

[0076] Fig.10 The figure shows the efficiency (%) of SNORA22 siRNA introduction into S2-013 pancreatic cancer cells and HPNE normal pancreatic duct epithelial cells in the presence of folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents). The results are the mean and standard error. * indicates significant difference, P<0.05.

[0077] Fig.11 This is a confocal microscope image showing lysosomes (LysoTracker) and SNORA22 siRNA staining in S2-013 pancreatic cancer cells after adding Alexa488-labeled SNORA22 siRNA and folate-cationic oligopeptide complexes (Fol-Dab8A (A) or Fol-Dab8B (B), 2 equivalents) to the culture medium and incubating overnight. Merge / DAPI is an image in which Alexa488 staining and DAPI staining are merged.

[0078] Fig.12 In the present invention, unmodified SNORA22 siRNA (A) or chemically modified SNORA22 siRNA (B) was mixed with a cationic oligopeptide (Dab8) or a folate-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B), and added to serum (10% FCS / PBS). The results of SDS-PAGE using a non-reducing gel showed the stability of siRNA immediately after addition (0), 3 hours after addition (3), or 6 hours after addition (6). -: no FCS (PBS only), control: each SNORA22 siRNA was added alone.

[0079] Fig.13 The results of in vivo imager imaging of the delivery of chemically modified SNORA22 siRNA to pancreatic cancer tissues of mice loaded with human pancreatic cancer organoids from S2-013 pancreatic cancer cells are shown. AC: 1-3 equivalents of Dab8 were added to SNORA22 siRNA; DF: 1-3 equivalents of Fol-Dab8A were added to SNORA22 siRNA; GI: 1-3 equivalents of Fol-Dab8B were added to SNORA22 siRNA.

[0080] Fig.14 The anti-tumor effect of co-administration of SNORA22 siRNA and folate-cationic oligopeptide complex on pancreatic cancer tumors in mice loaded with human pancreatic cancer organoids from S2-013 pancreatic cancer cells is shown. Control: non-administered control group, SNORA22-Dab8: group co-administered with SNORA22 siRNA and folate-cationic oligopeptide complex, Scr-Fol-Dab8B: group co-administered with scrambled control siRNA, SNORA22-Fol-Dab8B: group co-administered with SNORA22 siRNA and folate-cationic oligopeptide complex. * indicates significant difference, P<0.05.

[0081] Fig.15 The results of in vivo imaging of the delivery of chemically modified SNORA18 siRNA to pancreatic cancer tissues of mice loaded with human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells are shown. AC: 1-3 equivalents of Fol-Dab8A were added to SNORA18 siRNA, DF: 1-3 equivalents of Fol-Dab8B were added to SNORA18 siRNA.

[0082] Fig.16 The results are shown by in vivo imager of the delivery of chemically modified WASF2 siRNA to pancreatic cancer tissues of mice loaded with human pancreatic cancer organoids derived from S2-013 pancreatic cancer cells. AC: 1-3 equivalents of Fol-Dab8A were added to WASF2 siRNA, DF: 1-3 equivalents of Fol-Dab8B were added to WASF2 siRNA. DETAILED DESCRIPTION

[0083] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0084] <Nucleic Acid Delivery Enhancer>

[0085] The present invention provides a nucleic acid delivery accelerator composed of a folic acid-cationic oligopeptide complex.

[0086] <Folic acid>

[0087] Folic acid is a water-soluble vitamin B group with the following structure. It has been reported that folate receptors are expressed at high concentrations on the surface of tumor cells. Since folate receptors are expressed in pancreatic cancer cell line S2-013, the inventors first reported the use of a complex containing folate to deliver siRNA into cells (Oncotarget, 2019, Vol. 10, No. 30, pp. 2869-2886).

[0088] [Chemical formula 10]

[0089]

[0090] In the present invention, the folic acid used to prepare a complex with a cationic oligopeptide may be the folic acid having the above structure, or may be a folate or a folic acid derivative that retains the binding properties to the cationic oligopeptide and the binding properties to the folate receptor.

[0091] For example, according to the above structure, there are two carboxyl groups, an amino group, an imine group, etc. on the folic acid molecule. Therefore, substituents well known in the art can be added to these functional groups that do not participate in the binding with the cationic oligopeptide. In addition, a labeled compound can also be bound to folic acid.

[0092] <Cationic Oligopeptide>

[0093] As the cationic oligonucleotide that can be used in the present invention, nucleotides disclosed in International Publication No. WO2014 / 148620 and Bioorganic & Medicinal Chemistry 21 (2013) 1717-1723 can be appropriately used.

[0094] Specifically, the cationic oligonucleotide used in the present invention may have 8 or more amino acid residues, and the amino acid residues may have an amino group or a guanidine group.

[0095] More specifically, the cationic oligopeptide that can be used in the present invention can be an oligopeptide composed of 8 to 40 amino acids, which includes a cationic oligopeptide site, wherein the cationic oligopeptide site includes at least two consecutive portions of amino acid residues of the following formula (I), and in addition to the consecutive portions of amino acid residues of the following formula (I), there is one other discontinuous amino acid residue.

[0096] [Chemical formula 11]

[0097]

[0098] In formula (I), R 1 For group H 3 N + -CH 2 - or a group represented by formula (II), in R1 For group H 3 N + -CH 2 -In the case of R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group having 1 to 4 carbon atoms; in a cationic oligopeptide, R 1 and R 2 All the same.

[0099] [Chemical formula 12]

[0100]

[0101] In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different.

[0102] The above-mentioned "amino acid residue of formula (I)" is not limited, and specifically, for example, it can be listed: 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).

[0103] The "other amino acid residues" are not particularly limited and can be appropriately selected, and examples thereof include, but are not limited to, glycine, L-alanine, L-proline, and amino acids having a proline skeleton such as L-aminoproline and L-guanidinoproline.

[0104] In one embodiment, the cationic oligopeptide site may be a heteropolymer comprising the above-mentioned "other amino acid residues". In another embodiment, the cationic oligopeptide site may be a homopolymer not comprising the above-mentioned "other amino acid residues". Considering the simplicity of synthesis of cationic oligopeptides, and the stabilization effect of siRNA and shRNA confirmed by the present inventors, etc., the cationic oligopeptide site preferably adopts a homopolymer composed of a single amino acid.

[0105] In this case, the amino acid as a monomer may be a natural amino acid or a non-natural amino acid. In addition, the amino acid may be an L-type or a D-type, and these may be mixed in the oligopeptide molecule.

[0106] Thus, the cationic oligopeptide may have, for example, 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) as a partial structure.

[0107] The cationic oligopeptide may be in the form of a salt. Examples of the salt that can be suitably used include hydrochloride, acetate, trifluoroacetate, etc., but are not particularly limited.

[0108] Double-stranded nucleic acids have different helical structures, type A and type B. In the DNA / DNA double-stranded nucleic acid, the main groove width is The B-type double helix structure, while in RNA / RNA double helix and DNA / RNA chain, the main groove width is and The present invention aims to improve the stability of siRNA, and therefore, it is necessary to use a cationic oligopeptide that can bind to the RNA / RNA chain of the A-type double helix structure.

[0109] Generally, in order to improve the stability of siRNA composed of 21 to 23 bases and shRNA that will produce such siRNA, the number of amino acid residues constituting the cationic oligopeptide may be 8 to 12.

[0110] As a specific embodiment, in the following examples, it was found that a substance that brought about a preferred result after actual study was conducted. In the cationic oligopeptide portion, an octamer of diaminobutyric acid having the following structure can be used.

[0111] [Chemical formula 13]

[0112]

[0113] <Folic acid-cationic oligopeptide complex>

[0114] The folic acid and the cationic oligopeptide are preferably bound by covalent bonding, which can be directly or via a linker to the N-terminus, C-terminus or side chain of the cationic oligopeptide.

[0115] As a linker, a substance commonly used in the art for preparing a conjugate can be appropriately used, without particular limitation, and a peptide linker composed of amino acids such as glycine and serine can be used. For example, the peptide linker can be a peptide composed of 1 to 4 glycine residues. For example, in the following examples, after actual research, it was found that a preferred result was obtained, and a linker composed of three glycine residues can be cited.

[0116] In the folic acid-cationic oligopeptide complex, the ratio of folic acid to cationic oligopeptide may be 1:1 in consideration of the interaction with the folate receptor and the interaction with the double-stranded nucleic acid, but is not particularly limited.

[0117] As described above, since there are two carboxyl groups in the folic acid molecule, when Dab8 of the above structure is bonded via a reaction with the carboxyl group of folic acid, the following two isomers are generated. In this specification, for convenience, these compounds are referred to as Fol-Dab8A and Fol-Dab8B.

[0118] [Chemical formula 14]

[0119]

[0120] [Chemical formula 15]

[0121]

[0122] In the above-mentioned Fol-Dab8A and Fol-Dab8B, three glycines are used as the linker, but the length of the linker can be changed appropriately.

[0123] Depending on the type of cationic oligopeptide used to form the complex, the folic acid-cationic oligopeptide complex can also be obtained in the form of a salt. Examples of the salt that can be suitably used include hydrochloride, acetate, trifluoroacetate, etc., but are not particularly limited.

[0124] By making a folate-cationic oligopeptide complex, the stability of siRNA and shRNA can be improved, while targeted delivery to cells with folate receptors can be achieved.

[0125] It should be noted that although the cationic oligopeptide Dab8 without folic acid was used as a control compound for demonstrating the effectiveness of Fol-Dab8A and Fol-Dab8B in the examples, it was made to have a tyrosine (the N-terminus was protected by an acetyl group) and three glycines at the N-terminus that allow UV detection and quantification, and in the examples, it is referred to as Dab8 for convenience.

[0126] [Chemical formula 16]

[0127]

[0128] In the formula, Ac-YGGG represents N-acetyl-L-tyrosine-glycine-glycine-glycine.

[0129] As confirmed in the examples, the nucleic acid delivery promoter of the present invention can promote the delivery of siRNA or shRNA into cells in vitro and in vivo. The nucleic acid delivery promoter of the present invention can specifically promote the delivery into cells that highly express folate receptors, particularly cancer cells.

[0130] <Antitumor agent>

[0131] The present invention also provides a nucleic acid preparation comprising siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit their expression, and the above-described delivery promoter of the present invention. The nucleic acid preparation of the present invention can effectively inhibit the tumor growth, invasion, and metastasis of pancreatic cancer. Therefore, the nucleic acid preparation of the present invention has the functions of a tumor growth inhibitor, an invasion-metastasis inhibitor, and an antitumor agent against pancreatic cancer.

[0132] In the present invention, the siRNA or shRNA and the folate-cationic oligopeptide complex may also be bound via another covalent bond, but they may not be bound via a covalent bond. Instead, due to the interaction between the negative charge of the siRNA or shRNA and the positive charge of the cationic oligopeptide, a part of the folate-cationic oligopeptide complex enters the major groove of the siRNA or shRNA, thereby having a stable structure. The mechanism of this stabilization effect is not intended to be limited.

[0133] <RNAi molecule>

[0134] It is known that siRNA and shRNA target specific mRNA and inhibit its translation (expression) through a mechanism called RNA interference. The number of bases in the target sequence is not particularly limited and can be selected within 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 enzyme in vivo to generate siRNA. In this specification, siRNA and shRNA are sometimes collectively referred to as "RNAi molecule".

[0135] siRNA is a double-stranded RNA formed by hybridization of a sense strand homologous to a partial base sequence of the target RNA and an antisense strand that can hybridize with the sense strand. Usually, the 3' end is kept as OH, the 5' end is phosphorylated, and the 3' end side can protrude by 1 base or more and 4 bases or less.

[0136] In contrast, the shRNA consisting of single-stranded RNA is formed by combining the region of the sense strand homologous to the partial base sequence of the target mRNA and the region of the antisense strand that can hybridize with the sense strand through the joint region, and the overall structure has a hairpin shape. The 3' end of the shRNA protrudes more than 1 base and less than 4 bases, and in addition, the 3' protruding end can also be composed of DNA. The shRNA is decomposed in the cell as above, and causes RNA interference in the same way as siRNA. Therefore, shRNA can also be used to replace siRNA.

[0137] The siRNA or shRNA used to achieve the purpose of the present invention is a siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit the expression thereof.

[0138] The "mRNA or snoRNA expressed in pancreatic cancer cells" described in the present specification is not particularly limited and may be a substance that binds to insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3).

[0139] As mentioned above, human IGF2BP3 is present in the cell membrane protrusions of pancreatic cancer cells, and various mRNAs bind to this IGF2BP3 and accumulate in the cell membrane protrusions. By using RNA interference to block these mRNAs that bind to IGF2BP3 in the cell membrane protrusions, the growth, invasion, and metastasis of pancreatic cancer tumors can be effectively inhibited.

[0140] Among the mRNAs expressed in pancreatic cancer cells, examples of mRNAs for which the present inventors have confirmed antitumor effects against pancreatic cancer using RNA interference include mRNAs of NUP85, WASF2, ARHGEF4, CCDC88A, LAMTOR2, and mTOR.

[0141] <nup85>

[0142] NUP85 (nucleoporin 85) is a protein belonging to the nucleoporin protein family. Nucleoporins are components of the nuclear membrane pore complex, which forms an entrance and exit for regulating the movement of macromolecules between the cell nucleus and the cytoplasm. The amino acid sequence of human NUP85 and the base sequence of the mRNA encoding it are included in the NCBI database as Gene ID: 79902, NCBI reference sequence: NM_024844, etc.

[0143] <wasf2>

[0144] Wiskott-Aldrich syndrome is a primary immunodeficiency characterized by thrombocytopenia with reduced platelet size, eczema, and susceptibility to infection. Wiskott-Aldrich syndrome protein family member 2 (WASF2) belongs to the Wiskott-Aldrich syndrome protein family, which forms a multiprotein complex that connects receptor kinases and actin. The amino acid sequence of human WASF2 and the base sequence of the mRNA encoding it are included in databases such as NCBI with Gene ID: 10163 and NCBI reference sequence: NM_006990.

[0145] <arhgef4>

[0146] ARHGEF4 (Rho guanine nucleotide exchange factor 4) is a protein involved in an intracellular process that is activated by stimulation, wherein the stimulation acts via the G protein conjugation state. The amino acid sequence of human ARHGEF4 and the base sequence of the mRNA encoding it are included in databases such as NCBI with Gene ID: 50649 and NCBI reference sequence: NM_015320.

[0147] <ccdc88a>

[0148] CCDC88A (coiled-coil domain-containing 88A) is a gene encoding a Girdin protein that is an actin-binding protein. The base sequence and other information of the mRNA of human CCDC88A are stored in databases such as NCBI as Gene ID: 55704, NCBI reference sequence: NM_001135597, etc.

[0149] <lamtor2>

[0150] LAMTOR2 (late endosomal / lysosomal adaptor, MAPK and mTOR activator 2) is a regulator of Langerhans cell homeostasis and is reported to be involved in signal transduction and mTOR cascade. The amino acid sequence of human LAMTOR2 and the base sequence of mRNA encoding it are included in NCBI and other databases as Gene ID: 28956 and NCBI reference sequence: NM_014017.

[0151] <mtor>

[0152] mTOR (mammalian target of rapamycin kinase) is a protein kinase involved in intracellular signal transduction in mammals, etc. The amino acid sequence of human mTOR and the base sequence of mRNA encoding it are included in databases such as NCBI with Gene ID: 2475 and NCBI reference sequence: NM_004958.

[0153] In the art, it is well known that mRNA is an RNA transcribed from a gene (DNA) and contains information encoding proteins. It is known that after the double strands of siRNA, which is usually double-stranded, are dissociated, one strand (the antisense strand) forms a complex called RISC (RNA-Induced-Silencing-Complex) together with a specific protein. RISC recognizes and binds to mRNA with a homologous sequence to the base sequence of the sense strand of siRNA, and cuts the mRNA through RNaseIII-like enzyme activity. On the other hand, shRNA is processed in the cells to which it is delivered to produce siRNA, which can then play the same role.

[0154] SnoRNA is a non-coding RNA (small nuclear RNA) present in nucleosomes. It is reported that snoRNA is a group of RNA molecules that have the function of guiding the chemical modification of methylation and pseudouridylation of ribosomal RNA and other RNAs (e.g. Mol. Biol. Cell, 2004, 15: 281-293; J. Biol. Chem, 2015, 290: 11741-11748).

[0155] The present inventors recently discovered that SNORA18 and SNORA22, which belong to the above-mentioned snoRNA, have the ability to bind to IGF2BP3 and are involved in the movement or infiltration of pancreatic cancer cells. In addition, it was found that snoRNA binds to KH-type splicing regulatory protein (KHSRP) and is concentrated in the P-body in the cytoplasm (Oncotarget, 2020, Vol.11, No.2, pp: 131-147).

[0156] The present inventors have found that siRNA or shRNA targeting the above snoRNA can also inhibit tumor growth, invasion and metastasis of pancreatic cancer by recognizing, binding and knocking down snoRNA.

[0157] <snora18>

[0158] Small nucleosomal RNA SNORA18 is reported to be a member of RNA that guides the modification site from uridine to pseudouridine. The base sequence and other information of human SNORA18 are included in databases such as NCBI with Gene ID: 677805 and NCBI reference sequence: NR_002959.

[0159] <snora22>

[0160] Small nucleosomal RNA SNORA22 is also reported to act on a member of RNA that guides the modification site from uridine to pseudouridine. The base sequence and other information of human SNORA22 are included in databases such as NCBI with Gene ID: 677807 and NCBI reference sequence: NR_002961.

[0161] Therefore, in the present invention, examples of mRNA or snoRNA expressed in pancreatic cancer cells that can be targeted by siRNA or shRNA include, but are not particularly limited to, substances selected from the group consisting of SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA.

[0162] In the case of targeting mRNA, the target mRNA to which the siRNA or shRNA can hybridize may include a 3'UTR, a 5'UTR, an exon, an intron, a coding region, a translation start region, a translation end region, or other nucleic acid regions.

[0163] The siRNA and shRNA used in the present invention can knock down mRNA or snoRNA to hinder its function. Therefore, more specifically, the siRNA and shRNA of the present invention are composed of a base sequence that is substantially complementary to a specific base sequence of the target RNA. However, siRNA and shRNA may have 1 to 2 mismatches relative to the target base sequence. In the case of obtaining the base sequence of the target mRNA or snoRNA, those skilled in the art can select a region that has no homology with the base sequence of the nucleic acid other than the target sequence and is suitable for hindering / knocking down the expression of the target sequence, thereby designing and synthesizing suitable high-specificity siRNA and shRNA.

[0164] For example, it is known that siRNA has good RNA interference effect when the following conditions are met:

[0165] (1) The 5' end of the antisense strand is A or U;

[0166] (2) The 5' end of the sense strand is G or C; and

[0167] (3) Four or more bases out of the seven bases at the 5' end of the antisense strand are A or U. Therefore, the siRNA used in the present invention may be an siRNA having the above base sequence, but is not limited thereto. Similarly, the shRNA used in the present invention may be an shRNA that produces the above siRNA after being processed in cells, but is not limited thereto.

[0168] It is also possible to use a program available on the Internet to determine siRNA sequences and shRNA sequences that are effective for a certain target sequence.

[0169] In this specification, blocking / inhibiting or knocking down expression means blocking / inhibiting the degradation of target mRNA or snoRNA and translating it into the encoded protein. Inhibition includes reducing the amount of target mRNA / snoRNA in cells or cell groups (pancreatic cancer tissue) by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% compared to the control. The blocking / inhibition or knocking down of expression can be determined by any known method in the art, for example, by a method based on RT-PCR.

[0170] siRNA and shRNA can be composed of only natural (non-modified) nucleotides, or some or all of the nucleotides can be modified. By using any modification known in the art, the chemical stability of siRNA and shRNA can be increased. However, it is not desirable to reduce the activity of the required siRNA and shRNA due to modification. Therefore, it is understandable that modifications such as those that hinder shRNA intracellular processing can not be used. The modification as non-natural nucleotides can be the modification of sugar and / or base.

[0171] Examples of sugar modifications include bicyclic sugars, 5'-vinyl, 5'-methyl, 4'-S, 2'-F, 2'-OCH 3 (2'-OMe), and 2'-O(CH 2 ) 2 OCH 3 Substituents, etc., but are not limited thereto. Bicyclic sugars are generally referred to as bridged nucleic acids (BNA), and examples thereof include LNA (locked nucleic acid (registered trademark)), 2,4-BNA, ENA (vinyloxy (4-(CH 2 ) 2 -O-2) BNA), etc., but not limited thereto. In addition, the sugar may also partially contain deoxyribose instead of ribose, in which case DNA and RNA may be mixed in one or two oligonucleotide chains of siRNA or oligonucleotide chains of shRNA. siRNA is preferably RNA / RNA double-stranded. In addition, shRNA is preferably RNA chain. As a modification of the sugar, for example, 2'-OMe modification can be appropriately used.

[0172] Examples of base modifications include:

[0173] 5-methylation, 5-fluorination, 5-bromination, 5-iodination, N4-methylation of cytosine,

[0174] N6-methylation, 8-bromination,

[0175] N2-methylation, 8-bromination,

[0176] Examples of the uracil include 5-fluorination, 5-bromination, 5-iodination, and 5-hydroxylation, but are not limited thereto.

[0177] The same nucleotide or different nucleotides in siRNA and shRNA may have the above-mentioned modifications of sugar and base.

[0178] siRNA and shRNA may also contain engineered internucleoside linkages.

[0179] The engineered internucleoside linkages may be, for example, phosphorothioate linkages, phosphorodithioate linkages, boranophosphate linkages, phosphodiamidate linkages, and phosphoamidate linkages, which exist instead of the naturally occurring phosphodiester linkages.

[0180] At least one of the internucleoside bonds of the siRNA and shRNA can be a modified internucleoside bond. Alternatively, at least two, three, or more than four of the internucleoside bonds of the siRNA and shRNA can be modified internucleoside bonds. The modified internucleoside bond is preferably a phosphorothioate bond.

[0181] In siRNA and shRNA, different nucleotides in the same chain can be independently modified differently. In addition, the same nucleotide can have a modified internucleoside bond (e.g., a phosphorothioate bond) and further have a modified sugar (e.g., a bicyclic sugar). The same nucleotide can also have a modified nucleic acid base (e.g., 5-methylcytosine) and further have a modified sugar (e.g., 2'-OMe modification, bicyclic sugar, etc.).

[0182] The siRNA and shRNA of the present invention can be manufactured by methods known in the art. For example, siRNA and shRNA can be synthesized using a commercially available automatic nucleic acid synthesis device and then purified using an ion exchange column or a reverse phase column. Alternatively, siRNA and shRNA can also be ordered from a manufacturer (e.g., Gene Design Co., Ltd.) after specifying the nucleic acid base sequence and the modification site and type.

[0183] In addition, siRNA and shRNA may also be bound to functional molecules such as labeling compounds (fluorescent protein, luciferase, etc.), purification compounds (biotin, avidin, His tag peptide, GST tag peptide, FLAG tag peptide, etc.). The binding may be direct binding or indirect binding via other substances, preferably direct binding via covalent bonds, etc.

[0184] The anti-tumor agent of the present invention may contain 0.5 to 10 equivalents of folic acid-cationic oligopeptide complex relative to siRNA or shRNA1 molecule, preferably 1 to 5 equivalents of folic acid-cationic oligopeptide complex, and more preferably 1 to 3 equivalents of folic acid-cationic oligopeptide complex, but is not limited thereto.

[0185] As demonstrated in the examples, the combination of siRNA or shRNA and a folic acid-cationic oligopeptide complex can significantly inhibit the tumor growth and infiltration of pancreatic cancer. The folic acid-cationic oligopeptide complex of the present invention can exert a sufficient effect by adding only a small amount, such as adding about 1 to 3 equivalents relative to siRNA or shRNA, and is therefore expected to become a very useful method for treating pancreatic cancer.

[0186] The antitumor agent of the present invention is not particularly limited as long as its active ingredient is delivered to pancreatic cancer tissue as the target site, and can be prepared, for example, as an injection, a liquid, or a sustained-release agent. The solvent of these preparations is preferably water, but it is preferred that the preparation is finally prepared into an isotonic solution or a quasi-isotonic solution by using a physiological saline, PBS, a serum albumin solution, or the like.

[0187] <Pharmaceutical Composition>

[0188] The present invention also provides a pharmaceutical composition comprising one or more antitumor agents of the present invention.

[0189] The target site of the antitumor agent of the present invention may be not only the pancreas but also lymph nodes and other organs to which pancreatic cancer cells metastasize. In addition, in order to more reliably deliver the active ingredient to the target site, the dosage form is preferably an injection.

[0190] The pharmaceutical composition can contain carriers, excipients, stabilizers, disintegrants, surfactants, coupling agents, lubricants, emulsifiers, suspending agents, antioxidants, flavoring agents, fillers, solubilizers, coating agents, colorants, taste masking agents, preservatives, buffers, etc. commonly used in the field of pharmaceutical preparations. Specifically, examples include: water, physiological saline, other aqueous solvents, pharmaceutically acceptable organic solvents, mannitol, lactose, starch, microcrystalline cellulose, glucose, calcium, polyvinyl alcohol, collagen, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, gum arabic, xanthan gum, casein, gelatin, agar, propylene glycol, polyethylene glycol, vaseline, paraffin, glycerol, stearyl alcohol, stearic acid, sorbitol, etc.

[0191] The pharmaceutical composition can be administered to the subject orally or parenterally. Examples of parenteral administration include, but are not limited to, injection or infusion into the subcutaneous, intravenous, intraperitoneal, or tumor cavity, and administration during endoscopic or laparoscopic procedures.

[0192] The dosage and frequency of the pharmaceutical composition of the present invention can be appropriately adjusted according to different dosage forms, the patient's age, sex, body weight, severity of the disease, etc. For example, when administering the pharmaceutical composition, the amount of siRNA or shRNA can be used to ensure that it reaches 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 / body weight kg / day to 0.1 mg / kg / day, and the amount of folic acid-cationic oligopeptide complex can be determined so that it reaches 0.5 to 10 equivalents relative to siRNA or shRNA.

[0193] The pharmaceutical composition can be administered once or multiple times, 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.

[0194] The test subject to which the pharmaceutical composition is administered may be a mammal, such as a primate such as a human or a monkey, and a non-primate such as a cow, pig, sheep, horse, cat, dog, guinea pig, rat, and mouse, more preferably a human. In addition, the test subject may also be, for example, a pancreatic cancer model animal transplanted with human pancreatic cancer cells, which model animal may be used to evaluate the effectiveness for humans.

[0195] Pancreatic cancer model animals can use non-human animals transplanted with cancer organoids described in, for example, Japanese Patent Application No. 2018-110575, but are not particularly limited. Here, "organoid" refers to an organ made in vitro, which is a collection of cells that are specific to a particular organ. In the art, organoids of various organs have been made. The "cancer organoids" recorded in Japanese Patent Application No. 2018-110575 are organoids that reproduce the microenvironment of cancer tissue. A research team including the inventors of the present application improved the method of Japanese Patent Application No. 2018-110575 and made a human pancreatic cancer mouse model (Japanese Patent Application No. 2020-078771) that can be used to judge the effect of pancreatic cancer treatment drugs. The mouse model can also be appropriately used to evaluate the effectiveness of the anti-tumor drugs and pharmaceutical compositions of the present invention on humans.

[0196] <Combination preparations>

[0197] The present invention also provides a combined preparation comprising the above-mentioned siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting their expression, and the above-mentioned folic acid-cationic oligopeptide complex, for inhibiting tumor growth, infiltration and metastasis of pancreatic cancer.

[0198] According to the description of this specification, siRNA or shRNA and folate-cationic oligopeptide complex are not covalently bound, but the folate-cationic oligopeptide complex is intentionally partially inserted into the major groove of siRNA or shRNA to stabilize siRNA or shRNA, but the mechanism is not limited thereto.

[0199] Therefore, siRNA or shRNA and folate-cationic oligopeptide complex may not be included in the same composition in advance. That is, a preparation containing siRNA or shRNA and a preparation containing folate-cationic oligopeptide complex may be prepared separately and combined before administration.

[0200] In addition, the above two preparations can also be administered separately. However, considering that the siRNA or shRNA is stabilized by the folic acid-cationic oligopeptide complex, the two preparations are preferably administered simultaneously (or continuously) and by the same administration route, rather than at different times or by different administration routes.

[0201] <Kit>

[0202] The present invention also provides a drug kit for preparing pancreatic cancer, comprising the above-mentioned siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting its expression, and the above-mentioned folic acid-cationic oligopeptide complex. In addition to the siRNA or shRNA and the folic acid-cationic oligopeptide complex, the kit also includes instructions for use of other drugs that can be administered simultaneously or are suitable for administration, carriers, and administration methods.

[0203] Example

[0204] The present invention is specifically described by the following examples, but the present invention is not limited to these examples.

[0205] [Example 1 Synthesis of folic acid-cationic oligopeptide complex]

[0206] The folic acid-cationic oligopeptide complex was synthesized as follows. It should be noted that the reagents and analytical equipment used in this example are as follows.

[0207] <Reagents>

[0208] Fmoc amino acid derivatives and resins used as peptide solid phase synthesis supports were purchased from Watanabe Chemical Industry Co., Ltd., and folic acid was purchased from Tokyo Chemical Industry Co., Ltd., and all of them were used directly. Each peptide chain was synthesized using the Fmoc solid phase synthesis method with Fmoc-NH-SAL-PEG resin as a solid phase support. Fmoc-AA-OH reagents used were Fmoc-Dab(Boc)-OH, Fmoc-Gly-OH, and Fmoc-Tyr(t-Bu)-OH.

[0209] <ESI MS>

[0210] Varian 910-MS (JASCO)

[0211] <Ultraviolet-visible spectrophotometer>

[0212] V-550 (JASCO)

[0213] <Variable-temperature ultraviolet-visible spectrophotometer>

[0214] UV-1650PC (SIMADZU)

[0215] <Fluorescence spectrophotometer>

[0216] FP-6500 (JASCO)

[0217] <hplc>

[0218] Pump: PU-2080i plus (JASCO)

[0219] Detector: UV-2075i plus (JASCO)

[0220] Low pressure gradient assembly: LG-2080-02 (JASCO)

[0221] Degassing device: DG-2080-53 (JASCO)

[0222] Reversed phase column: μ-Bondasphere 150×3.9mm C18 5μm (Waters),SunFire C18 OBD5μm 19×150mm(Waters)

[0223] <Step 1: Coupling operation>

[0224] First, an octamer of L-2,4-diaminobutyric acid (Dab8) was synthesized by Fmoc solid phase synthesis. A solid phase carrier was added to PetiSyzer (hipep Institute) so that the introduced amino group was 13 μmol, and after washing five times with 1.3 mL of dimethylformamide (DMF), 1.3 mL of DMF was added and allowed to stand for more than 1 hour to allow the carrier to swell.

[0225] (i) After swelling, the mixture was washed five times with 1.3 mL of DMF, and then 1.3 mL of a 25% piperidine / DMF solution was added and allowed to react for 5 minutes to remove the Fmoc group. At this time, the mixture was stirred several times with a vortex mixer.

[0226] (ii) After washing with 1.3 mL of DMF five times, 5 equivalents of Fmoc-amino acid (Fmoc-AA-OH) and condensation reagent N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan aminiumhexafluorophosphate-N-oxide (HATU·H) were added to the resin. 2 The mixture was stirred several times with a vortex mixer.

[0227] The steps (i) and (ii) are repeated until the amino acid at the N-terminus is reached, and then the step (i) is repeated to remove the Fmoc group, thereby synthesizing NH 2 -GGG-Dab8. Then, after washing with 1.3 mL of DMF five times, 2 equivalents of folic acid and 2 equivalents of N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in 1.5 mL of a DMF-dimethyl sulfoxide (DMSO) mixed solvent (1:1, v / v), and after standing for 6 hours, 1.3 mL was added to allow the condensation reaction to proceed for 14 hours.

[0228] <Step 2: Deprotection / Resin Removal and Purification>

[0229] Use DMF and CHCl 3 The resin was washed 5 times and dried in a desiccator under reduced pressure. 2 O mixed solvent (96.5 / 1.0 / 2.5, v / v / v) was stirred for 1 hour and 30 minutes to perform deprotection / resin removal. The resin was removed by filtration, the solvent was vaporized under an argon stream, and then Et 2 O to precipitate the peptide. Add the supernatant to the centrifuge and repeat this process 3 times. Then, add Et 2 O was evaporated under an argon stream to obtain a crude product (folate-cationic oligopeptide).

[0230] 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. The reverse phase HPLC used the following conditions.

[0231] Gradient cycle: Solvent A (H 2 Solvent B (CH 3 The ratio of CN-0.05% TFA) was measured by linear gradient, 0% to 5% at 5 minutes; 5% to 25% at 40 minutes; 25% to 100% at 5 minutes

[0232] Measurement temperature: 30℃

[0233] Flow rate: 0.5mL / min

[0234] Figure 1 The results of HPLC analysis of crude Fol-Dab8 are shown. Since folic acid contains two carboxyl groups, two peaks will be eluted separately in Dab8 (denoted as Fol-Dab8) after the introduction of folic acid. During HPLC analysis, the peak that eluted first was Fol-Dab8A, and the peak that eluted later was Fol-Dab8B. The chemical structural formulas of each are shown below.

[0235] [Chemical formula 17]

[0236]

[0237] [Chemical formula 18]

[0238]

[0239] The solution prepared in HPLC was lyophilized to obtain a yellow powder. The purified Fol-Dab8A or Fol-Dab8B was dissolved in Otsuka distilled water, the molar extinction coefficient of folic acid was calculated in advance (molar extinction coefficient at a wavelength of 368 nm: 7967 L / mol·cm), and the yield was calculated based on the UV absorption of folic acid. 2 -GGG-Dab8, and then coupled with tyrosine protected by an acetyl group at the N-terminus, and after the operation of <Step 2>, purified by HPLC, thereby obtaining Dab8 as a white powder, and the yield was calculated based on the UV absorption of Tyr. Each peptide was identified by mass spectrometry (ESI-MS) (Dab8 [M+H] + m / z calculated: 1194.681, found: 1194.682; [M+H] + m / z calculated: 1412.737, found: 1412.738; [M+H] of Fol-Dab8B + m / z calculated: 1412.737, found: 1412.739).

[0240] [Example 2 Melting Temperature (Tm) Analysis]

[0241] The melting temperatures of double-stranded nucleic acids in the presence and absence of the cationic oligopeptide synthesized in Example 1 or the folic acid-cationic oligopeptide complex were measured.

[0242] In this example, cationic oligopeptide (Dab8) or folate-cationic oligopeptide (Fol-Dab8A or Fol-Dab8B) was added to the annealed RNA double strands, and the melting temperature (Tm value) was measured. As RNA double strands, an oligonucleotide pair consisting of the following sequence was used as siRNA.

[0243] 5'-r(GUCAUCACACUGAAUACCA)dTdT-3'(SEQ ID NO: 1)

[0244] 5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-3'(SEQ ID NO: 2)

[0245] Prepare 144 μL of a 50 μM nucleic acid aqueous solution, keep it at 95°C for 5 minutes, and then slowly cool it to 4°C at -0.5°C / min. Add 200 mM NaCl, 20 mM Na 2 HPO 4 -NaH 2 PO 4 The final concentration was 10 mM Na 2 HPO 4 -NaH 2 PO 4 , 100 mM NaCl, 4 μM nucleic acid double-stranded, 0, 4, 8, 12, 16, 20 μM peptide (0, 1, 2, 3, 4, 5 equivalents, respectively), thereby preparing the samples.

[0246] The temperature was raised from 20°C to 95°C at 0.5°C / min, and the absorbance at 260nm was measured to obtain a melting curve. In order to remove background noise, the absorbance at 320nm was measured, and the melting curve was prepared by subtracting the absorbance at 320nm from the absorbance at 260nm, and the Tm value was obtained by the midline method.

[0247] As shown in Table 1, when no cationic oligopeptide was added to the RNA double strand, the Tm value was 72.3°C, but when more than 1 equivalent of Dab8, Fol-Dab8A, or Fol-Dab8B was added, the Tm values ​​increased. This indicates that Fol-Dab8A and B, like Dab8, bind to the RNA double strand and improve its thermodynamic stability.

[0248] [Table 1]

[0249]

[0250] [Example 3 Evaluation of RNase A resistance]

[0251] The RNA degrading enzyme resistance test of double-stranded nucleic acids was carried out in the presence of cationic oligopeptides.

[0252] In this example, a peptide was added to the annealed RNA double strands to form a complex, and the degradation rate of the RNA was measured.

[0253] After adding Otsuka distilled water, 100mM Tris-HCl, and 1M NaCl buffer (pH 7.3) to a PCR tube, an equal amount of 0.1mM 6-FAM-modified single-stranded RNA (5'-FAM-r(GUCAUCACACUGAAUACCA)dTdT-3', sequence number 1) aqueous solution with a fluorescent group at the 5' end and 0.1mM Dabcyl-modified single-stranded RNA (5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-dabcyl-3', sequence number 2) aqueous solution with a quenching group at the 3' end were mixed to a final concentration of 10mM Tris-HCl, 100mM NaCl, and 10μM siRNA. Then, after keeping at 95°C for 5 minutes, the mixture was slowly cooled to 4°C at -0.5°C / min.

[0254] 10mM Tris-HCl, 100mM NaCl, 10μM siRNA aqueous solution, and 0.1mM cationic oligopeptide aqueous solution were added to a quartz dish to make 3mL, so that the final concentrations were: 10mM Tris-HCl, 100mM NaCl, 10nM siRNA, 0, 10, 20, 30nM peptide (0, 1, 2, 3 equivalents, respectively). Then, 15μL of 100μg / mL RNase A from bovine pancreas (Roche) was added while stirring at 37°C, and the fluorescence intensity was measured and the change over time was tracked (excitation wavelength: 490nm; measurement wavelength: 520nm; measurement time: 60 minutes).

[0255] The results are shown in Figure 2 After the RNA double-strand dissociates, the distance between the fluorescent group and the quenching group increases, and the fluorescence intensity increases. Therefore, if more than 1 equivalent of Dab8, Fol-Dab8A, and Fol-Dab8B are added, the increase rate of the fluorescence intensity slows down, indicating that Fol-Dab8A and B, like Dab8, also improve the RNA double-stranded enzyme resistance.

[0256] [Example 4 Confirmation of siRNA Uptake into Pancreatic Cancer Cells]

[0257] Since it is difficult to distinguish between labeled siRNA that has been incorporated into cells and labeled siRNA that has not been incorporated into cells by flow cytometry, confocal microscopy was used to observe the incorporation of siRNA into pancreatic cancer cells.

[0258] Pancreatic cancer cell line S2-013 (Medical Cell Resource / Cell Bank, Institute of Aging Medicine, Tohoku University, ID: TKG0709) was cultured at 2×10 4 The cells were seeded at a density of 100 cells / well in a 4-well chamber slide (Thermo Fisher Scientific), and 1 to 3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B were added to the slide relative to Alexa647 (Thermo Fisher Scientific) labeled scrambled control siRNA (sequence numbers 3 and 4), and cultured for 48 hours. The concentration of siRNA was 8.28 μg / mL, and 25 μL was taken as the concentration of Dab8, Fol-Dab8A, and Fol-Dab8B. The stock solution (Dab8: 119.3 μg / mL; Fol-Dab8A, Fol-Dab8B: 141.2 μg / mL) was diluted 25 times before use. The amount of 1, 2, and 3 equivalents relative to siRNA was 3.9 μL, 7.8 μL, and 11.7 μL, respectively.

[0259] The next day, the cells were fixed with 4% paraformaldehyde and blocked with a blocking agent containing DAPI, and then observed using an integrated fluorescence microscope (BZ-X800, Keyence). Figure 3 As shown in A, 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, it was confirmed based on the fluorescence of Alexa647 that siRNA was hardly taken into cells ( Figure 3 B).

[0260] To convert the uptake into numerical values, the hybrid cell counting function of the Keyence analysis software BZ-X800 Analyzer was used to count the cells that took up siRNA in the presence of Dab8, Fol-Dab8A, and Fol-Dab8B. For determination, cells with more than 1 / 3 of the cell nuclei stained darkly and cells with dark masses around the cell nuclei were measured.

[0261] Results, such as Figure 3 As shown in Figure C, when using a cationic oligopeptide (Dab8) and a folic acid-cationic oligopeptide complex (Fol-Dab8A, Fol-Dab8B), 20-40% of the added siRNA was confirmed to be taken up into the cells. In addition, when 3 equivalents of Fol-Dab8A and 1 equivalent of Fol-Dab8B were used, the introduction efficiency was the highest, and the introduction efficiency was better than that when 1-3 equivalents of Dab8 were added.

[0262] [Example 5 Knockdown effect of folic acid-cationic peptide plus siRNA]

[0263] The knockdown effects of siRNA on SNORA18, NUP85, WASF2, and SNORA22 were investigated in the presence of cationic oligopeptides or folate-cationic oligopeptide complexes.

[0264] The sequences of siRNAs used in this example are shown in Table 2. As is commonly done in the art, double-stranded siRNAs having a sense strand and an antisense strand shown in Table 2 were used as each siRNA.

[0265] [Table 2]

[0266]

[0267] Cationic oligopeptide or folate-cationic peptide scrambled control siRNA (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) to which 1 equivalent of the complex of Dab8 and siRNA, 3 equivalents of the complex of Fol-Dab8A and siRNA, and 1 equivalent of the complex of Fol-Dab8B and siRNA were added were added to the culture medium (1.0 × 10 5 The cells were collected 48 hours later.

[0268] 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.

[0269] 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 for amplification of SNORA18, NUP85, WASF2 and SNORA22 are recorded in Table 3 below.

[0270] [Table 3]

[0271]

[0272] In the PCR reaction, the initial denaturation was performed at 94°C for 2 minutes on a TaKaRa PCR Thermal Cycler Dice Gradient, followed by 21 cycles (GAPDH) or 25 cycles (SNORA18, NUP85, WASF2, and SNORA22) at 94°C, 30 seconds, 58°C, 30 seconds, and 72°C, 1 minute. The band intensity was determined by scanning and optical density analysis using the Quantity One Analysis System (Bio-Rad).

[0273] Results, such as Figure 4 As shown in A, 5A, 6A, and 7A, respectively, in the combination of control siRNA and Dab8, Fol-Dab8A, or Fol-Dab8B, no knockdown effect was observed for SNORA18, NUP85, WASF2, and SNORA22, but when SNORA18 siRNA, SNORA22 siRNA, NUP85 siRNA, and WASF2 siRNA were added in combination with Fol-Dab8A or Fol-Dab8B, significant knockdown effects were confirmed. This knockdown effect was not observed in Dab8 that did not contain folic acid.

[0274] [Example 6 Inhibitory effect of folic acid-cationic oligopeptide complex + siRNA on cell infiltration]

[0275] Scrambled control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA (all of which were used in Example 5) to which folic acid-cationic oligopeptide complexes were added were added to the culture medium of S2-013 cells, and Matrigel infiltration was measured 48 hours later.

[0276] 4.0×10 4 The cells were inoculated in the upper chamber of Matrigel Invasion Chamber (24-well plate, pore size: 8 μm, manufactured by Becton Dickinson). A solvent containing 5% fetal bovine serum was added to the lower chamber. After the cells were incubated in the upper chamber for 20 hours, three independent areas were observed under a microscope, and the cells infiltrated into the lower chamber were counted. The same experiment was repeated 3 times to compare the cell infiltration ability of each S2-013 cell that had taken in siRNA.

[0277] Figure 4 B, 5B, 6B, and 7B show the number of cells that moved from the upper chamber to the lower chamber when the scrambled control siRNA, or any one of SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA was co-added with the folic acid-cationic oligopeptide complex to the cells for measurement. "*" indicates a significant difference relative to the control in the t-test, P < 0.05.

[0278] As a result, compared with S2-013 cells taken up with control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA took up, cell infiltration was significantly suppressed in S2-013 cells taken up with SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA.

[0279] From the above results, it was confirmed that siRNA added to the culture medium of cultured cells after combining with the folic acid-cationic oligopeptide complex was taken up into S2-013 cells and inhibited the expression of snoRNA and mRNA involved in cell infiltration.

[0280] [Effects of Example 7 on chemically modified siRNA]

[0281] Chemically modified siRNA was prepared to improve stability, and the effect of folate-cationic oligopeptide complex on it was investigated.

[0282] The sequences of the sense strand and antisense strand of siRNA used in this example are shown in Table 4. Each of them contains a chemically modified base and a phosphorothioate bond at the 3' end.

[0283] [Table 4]

[0284]

[0285] The uptake into cells was investigated in the same manner as in Example 4 using scrambled control siRNA (SEQ ID NOs. 3 and 4) and the above siRNA.

[0286] The culture medium (2×10 4 Alexa488 (Thermo Fisher Scientific)-labeled siRNA (2.5 μg / mL) and 1, 2, or 3 equivalents of Dab8, Fol-Dab8A, or Fol-Dab8B relative to siRNA were added to each well.

[0287] Next, similarly to Example 5, the effect of chemically modified siRNA on the infiltration of pancreatic cancer cells was investigated in the presence or absence of a folate-cationic oligopeptide complex.

[0288] 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), SNORA22 siRNA (SEQ ID NOs. 27 and 28) were added to the culture medium of the pancreatic cancer cell line S2-013 in culture together with 2 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B. As a control, control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA were added to the culture medium of S2-013 cells alone.

[0289] After 48 hours of culture, the Matrigel infiltration assay was performed. In the Matrigel infiltration assay, the number of cells that moved from the upper chamber to the lower chamber is shown. "*" indicates a significant difference relative to the control in the t-test, P<0.05.

[0290] The results, such as Figure 8 As shown, when SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA was used in the presence of Fol-Dab8A or Fol-Dab8B, the infiltration of S2-013 cells was significantly inhibited, compared with the case of adding scrambled control siRNA to S2-013 cells in the presence of Fol-Dab8A or Fol-Dab8B, and the case of adding siRNA alone. In the presence of Dab8 without folic acid, the inhibitory effect of SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA on the infiltration of S2-013 cells was also found, but the inhibitory effect was weaker than that of adding Fol-Dab8A or Fol-Dab8B.

[0291] [Example 8 Confirmation of Uptake of Chemically Modified siRNA into Pancreatic Cancer Cells 1]

[0292] The chemically modified SNORA22 siRNA (SEQ ID NOs. 27 and 28) prepared in Example 7 was labeled with Alexa488 (ThermoFisher Scientific) and added together with a folate-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents) to the culture medium (5×10 4 After incubation at 37°C overnight, staining of DNA (DAPI), folate receptor (FOLR1), and siRNA (Alexa 488) was observed.

[0293] Results, such as Fig. 9 As shown in the figure, the staining intensity of folate receptors in S2-013 pancreatic cancer cells was higher than that in HPNE normal pancreatic duct epithelial cells, and the staining intensity of SNORA22 siRNA taken into cells was also stronger. This indicates that pancreatic cancer cells expressing more folate receptors took up more SNORA22 siRNA.

[0294] Fig.10 The figure shows the efficiency (%) of SNORA22 siRNA introduction into S2-013 pancreatic cancer cells and HPNE normal pancreatic duct epithelial cells in the presence of folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents). The uptake of SNORA22 siRNA into S2-013 pancreatic cancer cells showed a value nearly 3 times that of normal cells.

[0295] [Example 9 Confirmation of Uptake of Chemically Modified siRNA into Pancreatic Cancer Cells 2]

[0296] It is known that siRNA taken into cells by endocytosis will fuse with lysosomes taken into endosomes. In order to verify whether the complex of the siRNA of the present invention and Fol-Dab8A or Fol-Dab8B is taken into cells by endocytosis, confocal microscopic images of lysosome staining and siRNA staining were obtained.

[0297] Alexa488-labeled chemically modified SNORA22 siRNA (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 (2×10 4 The cells were incubated overnight and the lysosomes (LysoTracker, Thermo Fisher Scientific) and siRNA (Alexa488) were observed.

[0298] Results, such as Fig.11 As shown, SNORA22 siRNA was taken up into S2-013 pancreatic cancer cells and concentrated in lysosomes, indicating that the siRNA and folate-cationic oligopeptide complex was taken up via endocytosis.

[0299] [Example 10 Confirmation that chemical modification improves the stability of siRNA]

[0300] In this example, whether chemical modification improves the stability of siRNA in serum was investigated.

[0301] 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 folate-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B), and allowed to stand at room temperature for 15 minutes, and then 1 μL was added to PBS or 10% FCS / PBS, respectively, and mixed (final concentration: 20 μM). As a control, each SNORA22 siRNA was added alone.

[0302] The samples were collected immediately after mixing, 3 hours after mixing, or 6 hours after mixing, frozen with liquid nitrogen, and stored at -80° C. After all samples were obtained, SDS-PAGE was performed using a non-reducing gel and detected using SYBR GOLD (Thermo Fisher Scientific).

[0303] Results, such as Fig.12 As shown, siRNA without chemical modification will decompose rapidly even in the presence of cationic oligopeptide or folic acid-cationic oligopeptide complex, indicating that chemical modification improves the stability of siRNA in serum and can basically inhibit the decomposition of siRNA by RNase.

[0304] [Example 11 In vivo uptake of siRNA1]

[0305] The method described in Japanese Patent Application Laid-Open No. 2018-110575 was modified to produce human pancreatic cancer organoids using S2-013 pancreatic cancer cells.

[0306] Specifically, S2-013 pancreatic cancer cells (20×10 4 ), human mesenchymal stem cells (LONZA, 40×10 4 ), and human umbilical vein endothelial cells HUVEC (LONZA, 14×10 4 ) were added to the DMEM / Matrigel mixed solution in a 48-well plate (Thermo Fisher Scientific) and incubated at 37°C in CO 2 Incubate in the incubator for 30 minutes.

[0307] Then, 300 μL / well of a DMEM / EGM mixed solution was added, and the mixture was incubated at 37°C for 24 hours to prepare pancreatic cancer organoids one by one in each well.

[0308] Next, the skin of the flank of nude mice (6-week-old BALB / cSlc-nu / nu (Pathogen-free female athymic nude mice), Japan SLC Co., Ltd.) was incised and the pancreatic cancer organoids obtained above were transplanted subcutaneously (2 mice per group).

[0309] At 6 weeks after transplantation, chemically modified SNORA22 siRNA (SEQ ID NOs. 27 and 28, 5 μg) labeled with Alexa-594 was injected into the tail vein together with 1, 2 or 3 equivalents of cationic oligopeptide (Dab8) or folate-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B). After 24 hours, images were taken using an in vivo imager.

[0310] Measurement equipment: Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA)

[0311] Measurement conditions: kexc 640nm, kemi 680nm

[0312] Results, such as Fig.13 As shown, when Dab8 (AC), Fol-Dab8A (DF), and Fol-Dab8B (GI) were used, the delivery of SNORA22 siRNA to pancreatic cancer tissues was promoted, especially when 3 equivalents of Fol-Dab8A (F) and 2 equivalents of Fol-Dab8B (H) were added, high concentrations of siRNA were delivered.

[0313] It should be noted that in this experiment, almost no siRNA transfer and accumulation to the liver was observed, and it was confirmed that siRNA was excreted from the body by the kidneys.

[0314] [Example 12 Confirmation of anti-tumor effect]

[0315] One week after the human pancreatic cancer organoids prepared in Example 11 were transplanted into nude mice subcutaneously, chemically modified SNORA22 siRNA (SEQ ID NOs. 27 and 28, 5 μg) was administered by tail vein injection once a week together with cationic oligopeptide (Dab8, 2 equivalents) or folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents), and tumor volume was measured weekly using a caliper (n=8 per group). As a control, scrambled control siRNA (SEQ ID NOs. 3 and 4) was administered together with folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents).

[0316] Results, such as Fig.14 As shown, according to the measurement results from the start of administration to 9 weeks later, compared with the non-administered control group (Control), the scrambled control siRNA administration group (Scr-Fol-Dab8B), and the cationic oligopeptide co-administration group (SNORA22-Dab8), in the SNORA22 siRNA and folic acid-cationic oligopeptide complex co-administration group (SNORA22-Fol-Dab8B), a significant effect of inhibiting tumor growth was observed after the 8th week.

[0317] [Example 13 In vivo uptake of siRNA 2]

[0318] As in Example 11, the delivery of chemically modified SNORA18 siRNA (SEQ ID NOs. 21 and 22) to pancreatic cancer-bearing mice was imaged using an in vivo imager when it was co-administered with a folate-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B).

[0319] Results, such as Fig.15 As shown, it was confirmed that both Fol-Dab8A and Fol-Dab8B promoted the delivery of SNORA18 siRNA to pancreatic cancer tissues, and in particular, high-concentration delivery was achieved when 1 equivalent (A) or 3 equivalents (C) of Fol-Dab8A was added.

[0320] [Example 14 In vivo uptake of siRNA 3]

[0321] As in Examples 11 and 13, the delivery of chemically modified WASF2 siRNA (SEQ ID NOs. 25 and 26) to pancreatic cancer-bearing mice when administered together with a folate-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B) was imaged using an in vivo imager.

[0322] Results, such as Fig.16 As shown, it was confirmed that both Fol-Dab8A and Fol-Dab8B promoted the delivery of WASF2 siRNA to pancreatic cancer tissues, and in particular, high-concentration delivery was achieved when 1 equivalent (D) or 3 equivalents (F) of Fol-Dab8B was added.

[0323] Industrial Applicability

[0324] According to the present invention, a treatment method is provided that can effectively inhibit the tumor growth, infiltration and metastasis of pancreatic cancer, which is considered to have the worst prognosis among all cancers. The anti-tumor agent of the present application is effectively and specifically delivered to pancreatic cancer cells, and by using it in combination with other anti-cancer agents and / or anti-cancer treatments, the therapeutic effect of pancreatic cancer can be greatly improved.

[0325] Publications, patents and patent applications cited in this specification are directly incorporated into this specification by reference. Sequence Listing <110> National University Corporation Kochi University Tokyo University of Science <120> Nucleic acid delivery enhancers <130> PH-8588-PCT <150> JP 2019-194646 <151> 2019-10-25 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial <220> <223> siRNA Sense Strand <220> <223> DNA / RNA binding molecules <400> 1 gucaucacac ugaauaccat t 21 <210> 2 <211> twenty one <212> DNA <213> Artificial <220> <223> siRNA antisense strand <220> <223> DNA / RNA binding molecules <400> 2 ugguauucag ugugaugact t 21 <210> 3 <211> twenty one <212> RNA <213> Artificial <220> <223> Control siRNA <400> 3 uucuccgaac gugucacgua u 21 <210> 4 <211> twenty one <212> RNA <213> Artificial <220> <223> Control siRNA <400> 4 auacgugaca cguucggaga a 21 <210> 5 <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA18 siRNA positive strand <400> 5 uuuacuuuac ucacaggacu a 21 <210> 6 <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA18 siRNA Antisense <400> 6 uaguccugug aguaaaguaa a 21 <210> 7 <211> twenty one <212> RNA <213> Artificial <220> <223> NUP85 siRNA positive strand <400> 7 cagcggcaga ugacugaaca a 21 <210> 8 <211> twenty one <212> RNA <213> Artificial <220> <223> NUP85 siRNA Antisense <400> 8 uuguucaguc aucugccgcu g 21 <210> 9 <211> twenty one <212> RNA <213> Artificial <220> <223> WASF2 siRNA positive strand <400> 9 uaggauuaga ucauuagcuc a 21 <210> 10 <211> twenty one <212> RNA <213> Artificial <220> <223> WASF2 siRNA Antisense <400> 10 ugagcuaaug aucuaauccu a 21 <210> 11 <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA22 siRNA positive strand <400> 11 cuuggcuuug acccugugcu a 21 <210> 12 <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA22 siRNA Antisense <400> 12 uagcacaggg ucaaagccaa g 21 <210> 13 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 13 ccctcttggt agcttcgttc 20 <210> 14 <211> twenty three <212> DNA <213> Artificial <220> <223> Primers <400> 14 cgcaggtatg aaataagact gag 23 <210> 15 <211> twenty one <212> DNA <213> Artificial <220> <223> Primers <400> 15 gctcctccag tcacacaacc t 21 <210> 16 <211> twenty four <212> DNA <213> Artificial <220> <223> Primers <400> 16 ctcggggcag taatcaaagt aatc 24 <210> 17 <211> twenty one <212> DNA <213> Artificial <220> <223> Primers <400> 17 gtgccagctt ggacagattg a 21 <210> 18 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 18 ggacacggtg ggaatgctta 20 <210> 19 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 19 gtgcagggag aggaatcaat 20 <210> 20 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 20 gcatgtacga aagctccaga 20 <210> twenty one <211> twenty one <212> DNA <213> Artificial <220> <223> SNORA18 modified siRNA positive strand <220> <221> Modified_base <222> (1)..(1), (6)..(6), (18)..(18) <223> um <220> <221> Modified_base <222> (8)..(8), (10)..(10) <223> cm <220> <223> DNA / RNA binding molecules <400> twenty one uacuuuacuc acaggacuat t 21 <210> twenty two <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA18 modified siRNA antisense strand <220> <221> Modified_base <222> (1)..(1), (13)..(13), (18)..(18) <223> um <400> twenty two uaguccugug aguaaaguaa a 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial <220> <223> NUP85 modified siRNA positive strand <220> <221> Modified_base <222> (5)..(5), (17)..(17) <223> cm <220> <223> DNA / RNA binding molecules <400> twenty three gcggcagaug acugaacaat t 21 <210> twenty four <211> twenty one <212> RNA <213> Artificial <220> <223> NUP85 modified siRNA antisense strand <220> <221> Modified_base <222> (6)..(6), (10)..(10) <223> cm <400> twenty four uuguucaguc aucugccgcu g 21 <210> 25 <211> twenty one <212> DNA <213> Artificial <220> <223> WASF2 modified siRNA positive strand <220> <221> Modified_base <222> (5)..(5), (13)..(13) <223> um <220> <221> Modified_base <222> (10)..(10), (18)..(18) <223> cm <220> <223> DNA / RNA binding molecules <400> 25 ggauuagauc auuagcucat t 21 <210> 26 <211> twenty one <212> RNA <213> Artificial <220> <223> WASF2 modified siRNA antisense strand <220> <221> Modified_base <222> (6)..(6), (14)..(14), (20)..(20) <223> um <400> 26 ugagcuaaug aucuaauccu a 21 <210> 27 <211> twenty one <212> DNA <213> Artificial <220> <223> SNORA22 modified siRNA positive strand <220> <221> Modified_base <222> (18) <223> um <220> <223> DNA / RNA binding molecules <400> 27 uggcuuugac ccugugcuat t 21 <210> 28 <211> twenty one <212> RNA <213> Artificial <220> <223> SNORA22 modified siRNA antisense strand <220> <221> Modified_base <222> (1) <223> um <220> <221> Modified_base <222> (4)..(4), (6)..(6), (12)..(12), (18)..(18) <223> cm <400> 28 uagcacagggg uhaagccaa g 21< / hplc> < / mtor>

Claims

1. An anti-tumor agent comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting its expression, and a delivery enhancer for siRNA or shRNA consisting of a folic acid-cationic polypeptide complex, wherein the cationic polypeptide in the complex comprises a cationic polypeptide site consisting of 8 to 40 amino acids, wherein the cationic polypeptide site comprises a continuous portion of at least two amino acid residues of the following formula (I), and one other discontinuous amino acid residue other than the continuous portion of the amino acid residues of the following formula (I). In formula (I), R 1 For the group H3N + -CH2- or a group represented by formula (II), in R 1 For the group H3N + In the case of -CH2-, R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group with 1 to 4 carbon atoms; in a cationic polypeptide, R 1 All the same, R 2 All the same; In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different, The siRNA or shRNA contains 0.5 to 10 equivalents of folic acid-cationic polypeptide complex. The folic acid-cationic polypeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure:

2. A delivery facilitator for siRNA or shRNA, comprising a folic acid-cationic polypeptide complex, wherein the cationic polypeptide comprises a cationic polypeptide site composed of 8 to 40 amino acids, wherein the cationic polypeptide site comprises at least two consecutive amino acid residues of the following formula (I), and except for the consecutive amino acid residues of the following formula (I), there is one other discontinuous amino acid residue or no other amino acid residue. In formula (I), R 1 For the group H3N + -CH2- or a group represented by formula (II), in R 1 For the group H3N + In the case of -CH2-, R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group with 1 to 4 carbon atoms; in a cationic polypeptide, R 1 All the same, R 2 All the same; In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different, The siRNA or shRNA contains 0.5 to 10 equivalents of folic acid-cationic polypeptide complex. The folic acid-cationic polypeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure:

3. An antitumor agent comprising siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells and inhibiting the expression thereof, and the delivery enhancing agent according to claim 2.

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

5. The antitumor agent according to claim 3, 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.

6. The antitumor agent according to claim 3 or 4, wherein siRNA is a double-stranded RNA / RNA.

7. The antitumor agent according to claim 5, wherein siRNA is a double-stranded RNA / RNA.

8. The antitumor agent according to claim 3 or 4, wherein The siRNA or shRNA comprises modified bases, modified sugars, and / or engineered nucleoside bonds.

9. The antitumor agent according to claim 5, wherein The siRNA or shRNA comprises modified bases, modified sugars, and / or engineered nucleoside bonds.

10. The antitumor agent according to claim 6, wherein The siRNA or shRNA comprises modified bases, modified sugars, and / or engineered nucleoside bonds.

11. The antitumor agent according to claim 7, wherein The siRNA or shRNA comprises modified bases, modified sugars, and / or engineered nucleoside bonds.

12. The antitumor agent according to claim 8, wherein The modification of the modified sugar is 2'-OMe modification.

13. The antitumor agent according to claim 9, wherein The modification of the modified sugar is 2'-OMe modification.

14. The antitumor agent according to claim 10, wherein The modification of the modified sugar is 2'-OMe modification.

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

16. The antitumor agent according to claim 8, wherein The modified nucleoside bond is a phosphorothioate bond.

17. The antitumor agent according to claim 9, wherein The modified nucleoside bond is a phosphorothioate bond.

18. The antitumor agent according to claim 10, wherein The modified nucleoside bond is a phosphorothioate bond.

19. The antitumor agent according to claim 11, wherein The modified nucleoside bond is a phosphorothioate bond.

20. The antitumor agent according to claim 12, wherein The modified nucleoside bond is a phosphorothioate bond.

21. The antitumor agent according to claim 13, wherein The modified nucleoside bond is a phosphorothioate bond.

22. The antitumor agent according to claim 14, wherein The modified nucleoside bond is a phosphorothioate bond.

23. The antitumor agent according to claim 15, wherein The modified nucleoside bond is a phosphorothioate bond.

24. A pharmaceutical composition comprising the antitumor agent according to any one of claims 3 to 23.

25. A combined preparation comprising: (a) a preparation comprising siRNA or shRNA that binds to mRNA or snoRNA expressed in pancreatic cancer cells and inhibits the expression thereof; and (b) a preparation comprising a folic acid-cationic polypeptide complex, wherein the folic acid-cationic polypeptide complex comprises a cationic polypeptide site consisting of 8 to 40 amino acids, wherein the cationic polypeptide site comprises at least two consecutive amino acid residues of the following formula (I), and one other discontinuous amino acid residue other than the consecutive amino acid residues of the following formula (I), In formula (I), R 1 For the group H3N + -CH2- or a group represented by formula (II), in R 1 For the group H3N + In the case of -CH2-, R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group with 1 to 4 carbon atoms; in a cationic polypeptide, R 1 All the same, R 2 All the same; In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different, The siRNA or shRNA contains 0.5 to 10 equivalents of folic acid-cationic polypeptide complex. The folic acid-cationic polypeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure:

26. A drug kit for treating pancreatic cancer, comprising: (a) siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression; and (b) a folic acid-cationic polypeptide complex comprising a cationic polypeptide site consisting of 8 to 40 amino acids, wherein the cationic polypeptide site comprises at least two consecutive amino acid residues of the following formula (I), and one other discontinuous amino acid residue other than the consecutive amino acid residues of the following formula (I). In formula (I), R 1 For the group H3N + -CH2- or a group represented by formula (II), in R 1 For the group H3N + In the case of -CH2-, R 2 does not exist or is an alkylene group with 1 to 3 carbon atoms, in R 1 In the case of a group represented by formula (II), R 2 is an alkylene group with 1 to 4 carbon atoms; in a cationic polypeptide, R 1 All the same, R 2 All the same; In formula (II), R 3 , R 4 and R 5 is a hydrogen atom or a methyl group, which may be the same or different, and contains 0.5 to 10 equivalents of a folic acid-cationic polypeptide complex relative to the siRNA or shRNA, The folic acid-cationic polypeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure:

Images