Nucleic acid delivery promoter

Abstract

Provided is a novel anti-tumor agent capable of delivering siRNA or shRNA specifically to pancreatic cancer cells and suppressing tumor enlargement, infiltration, and metastasis of pancreatic cancer.　The present invention provides a nucleic acid delivery enhancer that is for delivering siRNA or shRNA into cells and that comprises a folate-cationic oligopeptide complex. The present invention also provides an anti-tumor agent that contains a folate-cationic oligopeptide complex and siRNA or shRNA capable of binding to mRNA or snoRNA expressed in pancreatic cancer cells to inhibit the expression. Said siRNA is capable of binding to RNA selected from the group consisting of, for example, SNORA18 snoRNA, NUP85 mRNA, WASF2 mRNA, and SNORA22 snoRNA.

Description

Technical Field

[0001] The present invention relates to a nucleic acid delivery promoter for delivering nucleic acid molecules such as siRNA into cells. The present invention also relates to a drug that can be used for treating diseases and the like by utilizing RNA interference (RNAi). In particular, the present invention relates to a nucleic acid preparation having an antitumor effect against pancreatic cancer and capable of effectively suppressing tumor growth, invasion, and metastasis of pancreatic cancer, and a pharmaceutical composition containing the same.

Background Art

[0002] Pancreatic cancer is said to have the worst prognosis among cancers. As its causes, in addition to the difficulty of early detection because the pancreas is a retroperitoneal organ, the motility of pancreatic cancer cells is extremely high, so there is a strong tendency to metastasize to peritoneal invasion, blood vessels, the digestive tract, nerves, etc.

[0003] The present inventors have previously found, while examining the mechanism of invasion and metastasis of pancreatic cancer, that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), which is normally found in nucleosomes and binds to the 5'untranslated region of the mRNA of insulin-like growth factor II to inhibit the translation of insulin-like growth factor II, is present in cell membrane protrusions in pancreatic cancer cells, and various mRNAs bind to this IGF2BP3 and accumulate in cell membrane protrusions. And it has been reported that the invasion and metastasis of pancreatic cancer cells are effectively suppressed by inhibiting these mRNAs by RNA interference (RNAi) (Patent Document 1).

[0004] Small interfering RNA (siRNA), which is typical of nucleic acid pharmaceuticals using RNA interference (RNAi) and has attracted attention as a new therapeutic agent in recent years, is generally a low-molecular-weight double-stranded RNA consisting of 21 to 23 base pairs. However, due to its highly anionic nature and low cell membrane permeability resulting from its structure, siRNA has a problem in terms of delivery into cells. In response to this problem, the present 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 by pancreatic cancer cells suppresses the invasion and metastasis of pancreatic cancer (Non-Patent Document 1).

[0005] On the other hand, the present 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 investigated (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

[0008] As described above, the delivery of RNAi molecules such as siRNA to cells as nucleic acid drugs is a very important issue in enhancing the practicality of nucleic acid drugs. There are still many problems to be achieved, such as not only simply delivering but also being specifically delivered to target cells and not inhibiting the effect of nucleic acid drugs after being taken up into cells. Means for Solving the Problems

[0009] As a result of repeated studies in view of the above problems, the present inventors have found that siRNA and shRNA can be specifically delivered into cells having folate receptors by using a complex of folic acid and a cationic oligopeptide. In addition, by using this method, targeting pancreatic cancer cells, effectively incorporating siRNA or shRNA into the targeted pancreatic cancer cells, and knocking down the expression of a specific RNA, a new treatment strategy capable of suppressing tumor growth, invasion and metastasis against pancreatic cancer has been found, and the present invention has been completed.

[0010] That is, the present invention provides the following. 1. A delivery promoter for siRNA or shRNA comprising a folic acid-cationic oligopeptide complex, wherein the cationic oligopeptide contains at least 2 consecutive portions of amino acid residues of the following formula (I), and is 1 other amino acid residue that is not consecutive other than the consecutive portions of amino acid residues of the following formula (I), and the above delivery promoter containing a cationic oligopeptide moiety consisting of 8 to 40 amino acids. Chemical Formula [In formula (I), R 1 is the group H3N + -CH2-, or a group represented by formula (II), and R 2 is R 1 when the group is H3N + -CH2-, it does not exist, or is an alkylene group having 1 to 3 carbon atoms, and R 1 when the group is represented by formula (II), it is an alkylene group having 1 to 4 carbon atoms. In one cationic oligopeptide, R 1 and R 2 are all the same.] [Chemical formula] [In formula (II), R 3 , R 4 and R 5 are the same or different and are a hydrogen atom or a methyl group.]

[0011] 2. The delivery promoter according to 1 above, wherein the cationic oligopeptide moiety consists of 8 to 12 amino acids. 3. The delivery promoter 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).

[0012] 4. The delivery promoter 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. [Chemical formula]

[0013] 5. The delivery promoter 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, either directly or via a linker. 6. The delivery promoter according to 5 above, wherein the folic acid-cationic oligopeptide complex is linked via a linker, and the linker is a peptide linker. 7. The delivery promoter according to 6 above, wherein the peptide linker is a peptide consisting of 1 to 4 glycine residues.

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

Chemical formula

Chemical formula

[0015] 9. An antitumor agent comprising siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression, and the delivery promoter according to any one of 1 to 8 above. 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). 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. The antitumor agent according to any one of 9 to 11 above, comprising 0.5 to 10 equivalents of the folic acid-cationic oligopeptide complex with respect to siRNA or shRNA. 13. The antitumor agent according to any one of 9 to 12 above, wherein the siRNA is an RNA / RNA double strand. 14. An antitumor agent according to any one of 9 to 13 above, wherein the siRNA or shRNA contains 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 the antitumor agent according to any one of 9 to 16 above.

[0016] 18. The following: (a) A preparation containing an siRNA or shRNA that can bind to an mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression, and (b) A preparation containing a folic acid-cationic oligopeptide complex containing a cationic oligopeptide site consisting of 8 to 40 amino acids, wherein at least 2 consecutive amino acid residues of the following formula (I) are included, and the other amino acid residues outside the consecutive part of the amino acid residues of the following formula (I) are non-consecutive:

Chemical formula

Chemical formula

[0017] 19. The following: (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 oligopeptide complex comprising a cationic oligopeptide moiety consisting of 8 to 40 amino acids, wherein at least 2 consecutive amino acid residues of the following formula (I) are included, and the other amino acid residues other than the consecutive portion of the amino acid residues of the following formula (I) are not consecutive:

Chemical formula

Chemical formula

Advantages of the Invention

[0018] According to the present invention, there is provided a nucleic acid preparation that can effectively deliver siRNA or shRNA specifically to pancreatic cancer cells and inhibit tumor growth, invasion, and metastasis of pancreatic cancer.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

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

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

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

[0023]

Chemical formula

[0024] In the present invention, the folic acid used to prepare a complex with a cationic oligopeptide may be folic acid having the above structure, or a folate or folic acid derivative that retains the binding property with a cationic oligopeptide and the binding property with a folic acid receptor.

[0025] For example, as understood from the above structure, since there are two carboxyl groups, amino groups, imino groups, etc. in the folic acid molecule, substituents known in the art can be added to these functional groups that do not participate in the binding with the cationic oligopeptide, and a labeling compound can also be bound to the folic acid.

[0026] <Cationic oligopeptide> As the cationic oligonucleotides that can be used in the present invention, those disclosed in International Publication WO2014 / 148620 and Bioorganic & Medicinal Chemistry 21 (2013) 1717-1723 can be appropriately used. Specifically, the cationic oligonucleotide used in the present invention may contain 8 or more amino acid residues having an amino group or a guanidino group.

[0027] More specifically, the cationic oligopeptide that can be used in the present invention contains at least 2 consecutive amino acid residues of the following formula (I), and is an oligopeptide consisting of 8 to 40 amino acids containing a cationic oligopeptide moiety that is 1 other non-consecutive amino acid residue other than the consecutive portion of the amino acid residues of the following formula (I).

[0028] [Chemical formula] [In formula (I), R 1 is the group H3N + -CH2-, or a group represented by formula (II), and R 2 is non-existent when R 1 is the group H3N + -CH2-, or is an alkylene group having 1 to 3 carbon atoms, and when R 1 is a 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 are all the same.]

[0029] [Chemical formula] [In formula (II), R 3 , R 4 and R 5 are the same or different and are a hydrogen atom or a methyl group.]

[0030] Examples of the "amino acid residue of formula (I)" include, but are not limited to, 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).

[0031] The above-mentioned "other amino acid residues" are not particularly limited and can be appropriately selected. For example, in addition to glycine, L-alanine, and L-proline, examples of amino acids having a proline skeleton include L-aminoproline and L-guanidinoproline, but are not limited thereto.

[0032] In one embodiment, the cationic oligopeptide moiety can be a heteromultimer containing the above-mentioned "other amino acid residues". In another embodiment, the cationic oligopeptide moiety can be a homomultimer that does not contain the above-mentioned "other amino acid residues". Considering the simplicity of the synthesis of the cationic oligopeptide and the stabilization effect of siRNA and shRNA confirmed by the present inventors, etc., the cationic oligopeptide moiety is preferably a homomultimer composed of a single amino acid.

[0033] In this case, the amino acid as a monomer may be a natural amino acid or a non-natural amino acid. Also, the amino acid may be of the L-form or the D-form, and they may be mixed in the oligopeptide molecule.

[0034] Therefore, the cationic oligopeptide can be, for example, one having a homomultimer 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.

[0035] The cationic oligopeptide may be in the form of a salt, and suitable salts that can be preferably used include hydrochloride, acetate, trifluoroacetate, etc., but are not particularly limited.

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

[0037] The number of amino acid residues constituting the cationic oligopeptide may generally be 8 - 12 in order to improve the stability of siRNA consisting of 21 - 23 bases and shRNA that gives rise to such siRNA.

[0038] As a specific embodiment, as actually studied in the following examples and resulting in favorable outcomes, the cationic oligopeptide moiety can be an octamer of diaminobutyric acid having the following structure.

[0039]

Chemical formula

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

[0041] As the linker, those commonly used in the art for the preparation of conjugates can be appropriately used, and are not particularly limited. For example, it can be a peptide linker composed of amino acids such as glycine and serine. For example, the peptide linker can be a peptide composed of 1 to 4 glycine residues. For example, as a result of actual consideration in the following examples and bringing favorable results, a linker composed of 3 glycine residues can be mentioned.

[0042] In the folic acid-cationic oligopeptide complex, the ratio of folic acid to the cationic oligopeptide can be 1:1 in consideration of the interaction with the folate receptor and the interaction with double-stranded nucleic acid, but is not particularly limited.

[0043] As described above, since there are two carboxyl groups in the folic acid molecule, when Dab8 having the above structure is bound via the reaction with the carboxyl group of folic acid, the following two isomers can occur. In this specification, for convenience, these compounds are denoted as Fol-Dab8A and Fol-Dab8B.

[0044]

Chemical formula

[0045]

Chemical formula

[0046] In the above Fol-Dab8A and Fol-Dab8B, 3 glycines are used as the linker, but the length of the linker can be appropriately changed. Depending on the type of cationic oligopeptide used for complex formation, the folic acid-cationic oligopeptide complex may be obtained in the form of a salt. Suitable salts that can be used include hydrochloride, acetate, trifluoroacetate, etc., but are not particularly limited. By forming a folic acid-cationic oligopeptide complex, the stability of siRNA and shRNA can be enhanced, and targeted delivery to cells having a folic acid receptor becomes possible.

[0047] In the Examples, as a control compound showing the effectiveness of Fol-Dab8A and Fol-Dab8B, a cationic oligopeptide Dab8 having no folic acid is used, which has tyrosine (the N-terminus is protected with an acetyl group) and three glycines at the N-terminus to enable UV detection and quantification, and this is referred to as Dab8 for convenience in the Examples.

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

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

[0050] [Antitumor agent] The present invention also provides a nucleic acid preparation comprising an siRNA or shRNA that can bind to mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression, and the above-described delivery promoter of the present invention. The nucleic acid preparation of the present invention can effectively suppress the tumor growth, invasion, and metastasis of pancreatic cancer. Therefore, the nucleic acid preparation of the present invention has an action as a tumor growth inhibitor, an invasion / metastasis inhibitor, and an antitumor agent against pancreatic cancer.

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

[0052] <RNAi molecule> siRNA and shRNA are known to target specific mRNAs and block their 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 protrude by 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 protrude by 1 to 4 bases, and the 3'-protruding end may be composed of DNA. shRNA is degraded in cells 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 purpose of the present invention is an siRNA or shRNA that can bind to the mRNA or snoRNA expressed in pancreatic cancer cells and inhibit its expression.

[0056] The "mRNA or snoRNA expressed in pancreatic cancer cells" described herein is not particularly limited, but may be one that can 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 cell membrane protrusions, various mRNAs bind to this IGF2BP3, and accumulate in cell membrane protrusions. By inhibiting the mRNAs bound to IGF2BP3 in these cell membrane protrusions by RNA interference, tumor growth, invasion, and metastasis in pancreatic cancer can be effectively suppressed.

[0058] Examples of the mRNA expressed in pancreatic cancer cells for which the present inventors confirmed the antitumor effect on pancreatic cancer using RNA interference include the mRNAs of NUP85, WASF2, ARHGEF4, CCDC88A, LAMTOR2, and mTOR.

[0059] <nup85> NUP85 (nucleoporin 85) is a protein belonging to the nucleoporin protein family, which is a component of the nuclear pore complex that forms an entrance / exit regulating the movement of macromolecules between the cell nucleus and the cytoplasm. Information such as the amino acid sequence of human NUP85 and the nucleotide sequence of the mRNA encoding the same is registered in databases such as NCBI under Gene ID: 79902, NCBI reference sequence: NM_024844, etc.

[0060] <wasf2> Wiskott-Aldrich syndrome, a type of primary immunodeficiency disorder, is a disease characterized by thrombocytopenia with a decrease in size, eczema, and susceptibility to infection. Wiskott-Aldrich syndrome protein family member 2 (WASF2) belongs to the Wiskott-Aldrich syndrome protein family that forms a multi-protein complex connecting receptor kinase and actin. Information such as the amino acid sequence of human WASF2 and the nucleotide sequence of the mRNA encoding the same is registered in databases such as NCBI as Gene ID: 10163, NCBI reference sequence: NM_006990, etc.

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

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

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

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

[0065] As is well known in the art, mRNA is an RNA transcribed from a gene (DNA) and contains information encoding a protein. Usually double-stranded siRNA is known to form a complex called RISC (RNA-Induced-Silencing-Complex) with a specific protein after its double-stranded dissociation and one of its strands (antisense strand). RISC recognizes and binds to an mRNA having a homologous sequence with the nucleotide sequence of the sense strand of siRNA, and cleaves the mRNA by RNaseIII-like enzyme activity. On the other hand, shRNA can generate siRNA after processing in the delivered cell and then function similarly.

[0066] In contrast, snoRNA is a non-coding RNA (small nuclear RNA) present in the nucleolus, and it has been reported that it is a group of RNA molecules having functions such as leading to chemical modifications such as methylation and pseudouridylation of ribosomal RNA and other RNAs (for example, Mol. Biol. Cell, 2004, 15: 281-293; J. Biol. Chem, 2015, 290: 11741-11748).

[0067] The present inventors have recently found that SNORA18 and SNORA22 belonging to such snoRNA have the ability to bind to IGF2BP3 and are involved in the motility or invasion of pancreatic cancer cells. It has also been found that snoRNA binds to KH-type splicing regulatory protein (KHSRP) and localizes in the cytoplasmic P-body (Oncotarget, 2020, Vol. 11, No. 2, pp: 131-147). The inventors have found that siRNA or shRNA against these snoRNAs can also recognize and bind to snoRNAs and knockdown them, thereby suppressing the tumor growth, invasion, and metastasis of pancreatic cancer.

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

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

[0070] Therefore, although not particularly limited, examples of the 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 to which siRNA or shRNA can hybridize may include 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or other nucleic acid regions.

[0072] The siRNA and shRNA used in the present invention can knockdown mRNA or snoRNA and inhibit its function. Therefore, 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 1 to 2 mismatches with respect to the target nucleotide sequence. Those skilled in the art, when the nucleotide sequence of the target mRNA or snoRNA has been obtained, can select a region suitable for inhibiting / knocking down the expression of the target sequence without homology to the nucleotide sequence of nucleic acids other than the target sequence, and design and synthesize appropriate siRNA and shRNA with high specificity.

[0073] For example, siRNA has 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 When 4 or more of the 7 bases at the 5'-terminal portion of the antisense strand are A or U, it is known that the RNA interference effect is high. Therefore, the siRNA used in the present invention may have such a nucleotide sequence, but is not limited thereto. Similarly, the shRNA used in the present invention may generate such siRNA after processing in cells, but is not limited thereto. The determination of effective siRNA sequences and shRNA sequences for a certain target sequence can also be carried out using programs available via the Internet.

[0074] In this specification, inhibition / suppression of expression or knockdown refers to degradation of target mRNA or snoRNA and inhibition / suppression of translation into the encoded protein. Suppression includes reducing the amount of target mRNA / snoRNA in cells or cell populations (pancreatic cancer tissues) 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 the control. The inhibition / suppression of expression or knockdown can be determined by any method known in the art, for example, by a method based on RT-PCR.

[0075] siRNA and shRNA may be composed of only natural (unmodified) nucleotides, or some or all of the nucleotides may 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 for the intended activity of siRNA and shRNA to be reduced by the modification. For example, it is understood that modifications that interfere with the intracellular processing of shRNA are not used. Modifications as non-natural nucleotides can be modifications of sugars and / or bases.

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

[0077] As base modifications, for example, but not limited to, 5-methylation, 5-fluorination, 5-bromination, 5-iodination, N4-methylation of cytosine, N6-methylation, 8-bromination of adenine, N2-methylation, 8-bromination of guanine, 5-fluorination, 5-bromination, 5-iodination, 5-hydroxylation of uracil and the like can be mentioned.

[0078] The same or different nucleotides in siRNA and shRNA can have the above-described sugar and base modifications. siRNA and shRNA can also include modified internucleoside linkages.

[0079] Modified internucleoside linkages are those that exist instead of the naturally occurring phosphodiester bond, and can be, for example, phosphorothioate bonds, phosphorodithioate bonds, boranophosphate bonds, phosphorodiamidate bonds, and phosphoramidate bonds.

[0080] At least one of the internucleoside linkages of the siRNA and shRNA can be a modified internucleoside linkage. Alternatively, at least two, three, four, or more of the internucleoside linkages of the siRNA and shRNA can be modified internucleoside linkages. The modified internucleoside linkage is preferably a phosphorothioate linkage.

[0081] In siRNA and shRNA, different nucleotides in the same strand can independently undergo different modifications. Also, the same nucleotide can have a modified internucleoside linkage (e.g., a phosphorothioate linkage) and further can have a modified sugar (e.g., a bicyclic sugar). The same nucleotide can also have a modified nucleobase (e.g., 5-methylcytosine) and further can have a modified sugar (e.g., 2'-OMe modification, bicyclic sugar, etc.).

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

[0083] Also, functional molecules such as a labeling compound (fluorescent protein, luciferase, etc.), a purification compound (biotin, avidin, His-tag peptide, GST-tag peptide, FLAG-tag peptide, etc.) may be bound to the siRNA and shRNA. The binding may be a direct binding or an indirect binding via another substance, but it is preferably directly bound by a covalent bond or the like.

[0084] The anti-tumor agent of the present invention can contain a folic acid-cationic oligopeptide complex in an amount of 0.5 to 10 equivalents, preferably 1 to 5 equivalents, more preferably 1 to 3 equivalents, relative to 1 molecule of siRNA or shRNA, although not limited thereto.

[0085] As demonstrated in the examples, by using siRNA or shRNA in combination with a folic acid-cationic oligopeptide complex, it is possible to significantly suppress the tumor growth and invasion of pancreatic cancer. Since the folic acid-cationic oligopeptide complex of the present invention can exert a sufficient effect by adding only a small amount of about 1 to 3 equivalents relative to siRNA or shRNA, it is expected to be an extremely useful treatment means for pancreatic cancer.

[0086] The anti-tumor agent according to the present invention is not particularly limited as long as the active ingredient is delivered to pancreatic cancer tissue, which is the target site. For example, it can be an injection, a solution, or a sustained-release agent. Water is preferably used as the solvent for these preparations, but it is preferable to use physiological saline, PBS, a serum albumin solution, etc. so that the preparation finally becomes an isotonic solution or a substantially isotonic solution.

[0087] <Pharmaceutical composition> The present invention also provides a pharmaceutical composition containing one or more of the anti-tumor agents of the present invention. The target site of the anti-tumor 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. Also, in order to more surely deliver the active ingredient to the target site, an injection is preferably used as the dosage form.

[0088] The pharmaceutical composition can include carriers, excipients, stabilizers, disintegrants, surfactants, binders, lubricants, emulsifiers, suspending agents, antioxidants, odor correctors, fillers, solubilizing agents, coating agents, coloring agents, flavoring agents, preservatives, buffers, etc. commonly used in the pharmaceutical field. Specifically, 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, gum arabic, xanthan gum, casein, gelatin, agar, propylene glycol, polyethylene glycol, petrolatum, paraffin, glycerin, stearyl alcohol, stearic acid, sorbitol, etc. can be mentioned.

[0089] The pharmaceutical composition can be administered to a subject orally or parenterally. Parenteral administration includes, but is not limited to, injection or infusion subcutaneously, intravenously, intraperitoneally, intratumorally, etc., and administration during procedures via an endoscope or laparoscope.

[0090] The dosage and dosing frequency of the pharmaceutical composition of the present invention can be appropriately adjusted according to the respective dosage forms, the age, sex, weight of the patient, the severity of the disease, etc. For example, the pharmaceutical composition can be in an amount such that the siRNA or shRNA is 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 of body weight / day to 0.1 mg / kg / day, and the amount of the folic acid-cationic oligopeptide complex can be determined to be 0.5 to 10 equivalents thereto for administration.

[0091] The pharmaceutical composition can be administered as a single dose or multiple doses, and can be administered, 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] Subjects to which the pharmaceutical composition is administered can be mammals, such as primates including humans and monkeys, and non-primates such as cows, pigs, sheep, horses, cats, dogs, guinea pigs, rats and mice, but more preferably humans. Also, the subject may be a pancreatic cancer model animal, such as a pancreatic cancer model animal transplanted with human pancreatic cancer cells, which can be used for evaluating the efficacy on humans.

[0093] Although not particularly limited, as the pancreatic cancer model animal, for example, a non-human animal transplanted with the cancer organoid described in JP 2018-110575 can be used. Here, the "organoid" is an organ produced three-dimensionally in vitro and refers to an aggregate of cells specific to a particular organ. In the art, organoids of various organs have already been produced. The "cancer organoid" described in JP 2018-110575 is an organoid that reproduces the microenvironment of cancer tissue. A research group including the inventors of the present application has modified the method of JP 2018-110575 to produce a human pancreatic cancer mouse model useful for determining the effect of a pancreatic cancer therapeutic agent (Japanese Patent Application No. 2020-078771), and this mouse model can also be suitably used for evaluating the efficacy of the antitumor agent and pharmaceutical composition of the present invention on humans.

[0094] <Combined preparation> The present invention also provides a combined preparation for suppressing tumor growth, invasion and metastasis of pancreatic cancer, which comprises siRNA or shRNA that can bind to mRNA or snoRNA expressed in the above pancreatic cancer cells and inhibit its expression, and the above folic acid-cationic oligopeptide complex.

[0095] As understood from the description herein, siRNA or shRNA and the folic acid-cationic oligopeptide complex are not covalently bound and are not restricted by a mechanism, but it is intended that the folic acid-cationic oligopeptide complex is partially inserted into the major groove of siRNA or shRNA to stabilize siRNA or shRNA.

[0096] Therefore, siRNA or shRNA and the folic acid-cationic oligopeptide complex do not necessarily have to be contained in the same composition in advance. That is, a formulation containing siRNA or shRNA and a formulation containing the folic acid-cationic oligopeptide complex may be prepared separately and combined prior to administration.

[0097] Alternatively, the above two formulations can also be administered separately. However, considering the stabilization of siRNA or shRNA by the folic acid-cationic oligopeptide complex, it is preferable that these two formulations are administered simultaneously (or continuously) and via the same administration route, rather than at different times or via different administration routes.

[0098] <Kit> The present invention also provides a pharmaceutical kit for treating pancreatic cancer, which comprises siRNA or shRNA capable of binding to mRNA or snoRNA expressed in the above pancreatic cancer cells and inhibiting their expression, and the above folic acid-cationic oligopeptide complex. In addition to siRNA or shRNA and the folic acid-cationic oligopeptide complex, the kit can include instructions for other drugs, carriers, administration methods, etc. that can be administered simultaneously or are appropriate for administration.

Example

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

[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 shown below.

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

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

[0103] <Step 1: Coupling operation> First, an octamer of L-2,4-diaminobutyric acid (Dab8) was synthesized by Fmoc solid-phase synthesis. A solid-phase support was added to PetiSyzer (Hyperpep Research Institute) so that the introduced amino groups reached 13 μmol, and after washing 5 times with 1.3 mL of dimethylformamide (DMF), 1.3 mL of DMF was added and left standing for 1 hour or more to swell the support.

[0104] (i) After swelling, it was washed 5 times with 1.3 mL of DMF, then 1.3 mL of 25% piperidine / DMF solution was added and reacted for 5 minutes to remove the Fmoc group. During this time, it was stirred several times with a vortex mixer.

[0105] (ii) Subsequently, after washing 5 times with 1.3 mL of DMF, 5 equivalents of Fmoc-amino acid (Fmoc-AA-OH) relative to the amino groups on the resin, as a condensation reagent, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate-N-oxide (HATU·H2O, 5 equivalents), diisopropylethylamine (DIPEA, 10 equivalents), and DMF as a reaction solvent were added to a total of 1.3 mL and subjected to a condensation reaction for 15 minutes. During this time, it was stirred several times with a vortex mixer.

[0106] After repeating the operations (i) and (ii) up to the N-terminal amino acid and then performing operation (i) again to remove the Fmoc group, NH2-GGG-Dab8 was synthesized on the solid support. Subsequently, after washing 5 times with 1.3 mL of DMF, 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) for the amino groups on the resin, and 1.3 mL of the solution that had been allowed to stand for 6 hours was added, followed by a condensation reaction for 14 hours.

[0107] <Step 2: Deprotection, resin removal, and purification> The resin was washed 5 times each with DMF and CHCl3 and dried under reduced pressure in a desiccator. The obtained resin was stirred in a trifluoroacetic acid (TFA)-triisopropylsilane-H2O mixed solvent (96.5 / 1.0 / 2.5, v / v / v) at room temperature for 1 hour and 30 minutes to perform deprotection and resin removal. The resin was filtered off, the solvent was evaporated under an argon stream, and then Et2O was added to precipitate the peptide. After repeating the operation of centrifuging to remove the supernatant 3 times, Et2O was evaporated 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 Factory) and then purified by reverse-phase HPLC using the following conditions.

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

[0109] Figure 1 shows the HPLC analysis results of the Fol-Dab8 crude product. Due to folic acid containing two carboxyl groups, Dab8 with folic acid introduced (referred to as Fol-Dab8) eluted in two peaks. The one eluted first in the HPLC analysis was designated as Fol-Dab8A, and the one eluted later was designated as Fol-Dab8B. Their respective chemical structural formulas are shown below.

[0110] [Chemical formula]

[0111] [Chemical formula]

[0112] The solution fractionated by HPLC was lyophilized to obtain a yellow powder. The purified Fol-Dab8A or Fol-Dab8B was dissolved in Otsuka distilled water. First, the molar absorption coefficient of folic acid was calculated (molar absorption coefficient at a wavelength of 368 nm: 7967 L / mol·cm), and the yield was calculated from the UV absorption of folic acid. For Dab8, after synthesizing NH2-GGG-Dab8 on a solid-phase carrier, a coupling operation with tyrosine whose N-terminus was protected with an acetyl group was performed. After performing the operation of <Step 2>, it was purified by HPLC to obtain a white powder, and the yield was calculated from the UV absorption of Tyr. Each peptide was identified by mass spectrometry (ESI-MS) ([M+H] of Dab8 + Calculated m / z value: 1194.681, measured value: 1194.682; [M+H] of Fol-Dab8A + Calculated m / z value: 1412.737, measured value: 1412.738; [M+H] of Fol-Dab8B + Calculated m / z value: 1412.737, measured value: 1412.739).

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

[0114] In this example, a cationic oligopeptide (Dab8) or folic acid-cationic oligopeptide (Fol-Dab8A or Fol-Dab8B) was added to the annealed RNA duplex, and the melting temperature (Tm value) was measured. As the RNA duplex, an oligonucleotide pair consisting of the following sequences was used as siRNA. 5'-r(GUCAUCACACUGAAUACCA)dTdT-3' (SEQ ID NO: 1) 5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-3' (SEQ ID NO: 2)

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

[0116] The temperature was raised from 20°C to 95°C at 0.5°C / min to measure the absorbance at 260 nm, and a melting curve was obtained. To remove noise due to the background, the absorbance at 320 nm was measured, and a melting curve was created using the value obtained by subtracting the absorbance at 320 nm from the absorbance at 260 nm, and the Tm value was determined by the midpoint method.

[0117] As a result, as shown in Table 1, when no cationic oligopeptide was added to the RNA duplex, the Tm value was 72.3°C, whereas when 1 equivalent or more of Dab8, Fol-Dab8A, or Fol-Dab8B was added, the Tm value increased in all cases. That is, it was suggested that both Fol-Dab8A and B, like Dab8, bind to the RNA duplex and improve its thermodynamic stability.

[0118]

Table 1

[0119] [Example 3 Evaluation of RNase A Resistance] An RNase resistance test of a nucleic acid duplex in the presence of a cationic oligopeptide was performed. In this example, a peptide was added to the annealed RNA duplex to form a complex, and the rate of RNA degradation was measured.

[0120] After adding Otsuka distilled water, 100 mM Tris-HCl, and 1 M NaCl buffer (pH 7.3) to a PCR tube, an aqueous solution of single-stranded RNA (5'-FAM-r(GUCAUCACACUGAAUACCA)dTdT-3', SEQ ID NO: 1) modified with 6-FAM with a 0.1 mM 5'-terminal fluorescent group and an aqueous solution of single-stranded RNA (5'-r(UGGUAUUCAGUGUGAUGAC)dTdT-dabcyl-3', SEQ ID NO: 2) modified with Dabcyl with a 0.1 mM 3'-terminal quenching group were mixed in equal amounts and prepared to have a final concentration of 10 mM Tris-HCl, 100 mM NaCl, and 10 μM siRNA. Subsequently, after maintaining at 95°C for 5 minutes, it was gradually cooled to 4°C at -0.5°C / min.

[0121] 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 and adjusted to 3 mL so that the final concentration was 10 mM Tris-HCl, 100 mM NaCl, 10 nM siRNA, 0, 10, 20, 30 nM peptide (0, 1, 2, 3 equivalents respectively). Then, while stirring at 37°C, 15 μL of 100 μg / mL RNase A from bovine pancreas (manufactured by Roche) was added, and the measurement of fluorescence intensity was started to track the change over time (excitation wavelength: 490 nm; measurement wavelength: 520 nm; measurement time: 60 minutes).

[0122] The results are shown in Fig. 2. When the RNA duplex dissociates, the distance between the fluorophore and the quencher increases, resulting in an increase in fluorescence intensity. Therefore, when any of Dab8, Fol-Dab8A, or Fol-Dab8B is added in an amount of 1 equivalent or more, the rate of increase in fluorescence intensity becomes gradual, suggesting that, like Dab8, Fol-Dab8A and B improve the resistance of the RNA duplex to enzymatic degradation.

[0123] [Example 4 Confirmation of Uptake of siRNA into Pancreatic Cancer Cells] In flow cytometry, it is difficult to distinguish between cells that have taken up the label and those that have not. Therefore, the uptake of siRNA into pancreatic cancer cells was observed using a confocal microscope.

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

[0125] The next day, the samples were fixed with 4% paraformaldehyde, mounted with a mounting medium containing DAPI, and then observed using an all-in-one fluorescence microscope (BZ-X800, Keyence). As a result, as shown in Fig. 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, it was observed by fluorescence with Alexa647 that almost no siRNA was taken up by the cells (Fig. 3B).

[0126] To quantify the uptake, the hybrid cell count function of Keyence analysis software BZ-X800 Analyzer was used to count the cells in which siRNA was taken up in the presence of Dab8, Fol-Dab8A, and Fol-Dab8B. The determination was made by measuring those in which more than one-third of the cell nucleus was darkly stained and those in which there was a dark mass around the cell nucleus.

[0127] As a result, as shown in Fig. 3C, when the cationic oligopeptide (Dab8) and the folic acid-cationic oligopeptide complexes (Fol-Dab8A, Fol-Dab8B) were used, 20-40% uptake of the added siRNA into cells was confirmed. Also, the introduction efficiency was the highest at 3 equivalents for Fol-Dab8A and 1 equivalent for Fol-Dab8B, and the introduction efficiency was better than that of Dab8 added with 1-3 equivalents.

[0128] [Example 5 Knockdown effect by folic acid-cationic peptide-added siRNA] The knockdown effects of siRNA against SNORA18, NUP85, WASF2, and SNORA22 were examined in the presence of the cationic oligopeptide or the folic acid-cationic oligopeptide complex. Table 2 shows the sequences of the siRNAs used in this example. Each siRNA was a double-stranded one formed by the sense strand and the antisense strand shown in Table 2, as is usually done in the art.

[0129]

Table 2

[0130] The complex of Dab8 and siRNA was 1 equivalent, the complex of Fol-Dab8A and siRNA was 3 equivalents, and the complex of Fol-Dab8B and siRNA was 1 equivalent of a cationic oligopeptide or folic acid-cationic peptide-added 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), SNORA22 siRNA (SEQ ID NOs: 11 and 12) was added to the culture medium of S2-013 cells (1.0×10 5 cells / well) in a 6-well plate (Thermo Fisher Scientific), and the cells were collected after 48 hours. Semi-quantitative RT-PCR was performed using the RNA of the collected 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 described in Table 3 below.

[0132]

Table 3

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

[0134] As a result, as shown in FIGS. 4A, 5A, 6A, and 7A, respectively, no knockdown effect was observed for any of SNORA18, NUP85, WASF2, and SNORA22 in the combination of control siRNA and Dab8, Fol-Dab8A, or Fol-Dab8B, whereas a significant knockdown effect was confirmed when SNORA18 siRNA, SNORA22 siRNA, NUP85 siRNA, and WASF2 siRNA were added in combination with Fol-Dab8A or Fol-Dab8B. This knockdown effect was not observed with Dab8 without folic acid.

[0135] [Example 6 Inhibitory Effect on Cell Infiltration by Folic Acid-Cationic Oligopeptide Complex + siRNA] Each of the scrambled control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA (all used in Example 5) to which a folic acid-cationic oligopeptide complex was added was added to the culture medium of S2-013 cells, and a Matrigel invasion assay was performed 48 hours later.

[0136] Suspended in serum-free medium at 4.0×10 4 Individual cells were seeded into the upper chamber of a 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 incubating the cells in the upper chamber for 20 hours, three independent regions were observed under a microscope, and the number of cells that invaded into the lower chamber was counted. The same experiment was repeated three times to compare the cell invasion ability of S2-013 cells incorporating each siRNA.

[0137] Figures 4B, 5B, 6B, and 7B show the number of cells that migrated from the upper chamber to the lower chamber in an assay in which scrambled control siRNA, or either SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA was added to cells together with a folic acid-cationic oligopeptide complex. "*" indicates a significant difference with P < 0.05 compared to the control in a t-test.

[0138] As a result, in S2-013 cells incorporating SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA, cell invasion was significantly suppressed compared to S2-013 cells incorporating control siRNA.

[0139] From the above results, it was confirmed that siRNA added in combination with a folic acid-cationic oligopeptide complex to the medium of cultured cells was taken up into S2-013 cells and inhibited the expression of snoRNA and mRNA involved in cell invasion.

[0140] [Example 7 Effect on Chemically Modified siRNA] Chemically modified siRNA was prepared to enhance stability, and the effect of a folic acid-cationic oligopeptide complex was examined. Table 4 shows the sequences of the sense strand and antisense strand of the siRNA used in this example. Each contains chemically modified bases and a phosphorothioate bond at the 3' end.

[0141]

Table 4

[0142] Using scrambled control siRNA (SEQ ID NOs: 3 and 4) and the above siRNA, uptake into cells was examined in the same manner as in Example 4.

[0143] In the culture medium of pancreatic cancer cell line S2-013 during culture (2×10 4 cells / well), siRNA labeled with Alexa488 (Thermo Fisher Scientific) (2.5 μg / mL) was added, and 1, 2, and 3 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B were added thereto.

[0144] Next, in the same manner as in Example 5, the effect of chemically modified siRNA on the invasion of pancreatic cancer cells was examined in the presence or absence of a folic acid-cationic oligopeptide complex. Into the culture medium of pancreatic cancer cell line S2-013 during culture, each of 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) was added together with 2 equivalents of Dab8, Fol-Dab8A, and Fol-Dab8B. As a control, each of control siRNA, SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, and SNORA22 siRNA was added alone into the culture medium of S2-013 cells.

[0145] After culturing for 48 hours, a Matrigel invasion assay was performed. In the Matrigel invasion assay, the number of cells that migrated from the upper chamber to the lower chamber is shown. "*" indicates a significant difference with P < 0.05 compared to the control in the t-test.

[0146] As a result, as shown in Fig. 8, in the presence of Fol-Dab8A and Fol-Dab8B, the invasion of S2-013 cells was significantly suppressed when any of SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA was used, as compared with S2-013 cells to which scrambled control siRNA was added in the presence of Fol-Dab8A and Fol-Dab8B, and with the case of adding siRNA alone. In the presence of Dab8 without folic acid, an inhibitory effect on the invasion of S2-013 cells by SNORA18 siRNA, NUP85 siRNA, WASF2 siRNA, or SNORA22 siRNA was observed, but the inhibitory effect was weaker than that in the case of adding Fol-Dab8A and Fol-Dab8B.

[0147] [Example 8 Confirmation of Uptake of Chemically Modified siRNA into Pancreatic Cancer Cells 1] The chemically modified SNORA22 siRNA (SEQ ID NOs: 27 and 28) prepared in Example 7 was labeled with Alexa488 (Thermo Fisher Scientific) and added together with folic acid-cationic oligopeptide complex (Fol-Dab8B, 2 equivalents) to the culture medium (5×10 4 cells / well) of S2-013 pancreatic cancer cells or HPNE normal pancreatic duct epithelial cells (ATCC) in a 4-well chamber (Thermo Fisher Scientific), and after 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 Fig. 9, in S2-013 pancreatic cancer cells, the staining intensity of the folic acid receptor was higher and the staining intensity of SNORA22 siRNA taken up into the cells was stronger, as compared with HPNE normal pancreatic duct epithelial cells. This indicates that more SNORA22 siRNA is taken up in pancreatic cancer cells that express more folic acid receptors.

[0149] Figure 10 shows the transfection efficiency (%) of SNORA22 siRNA 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 three times that of 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 complex of the siRNA of the present invention and Fol-Dab8A or Fol-Dab8B is taken up via endocytosis, confocal microscope images of lysosome staining and siRNA staining were obtained.

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

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

[0153] [Example 10 Confirmation of Improvement in Stability of siRNA by Chemical Modification] In this example, it was examined whether the stability of siRNA in serum was 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), was mixed with a cationic oligopeptide (Dab8) or a folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B), allowed to stand at room temperature for 15 minutes, and then added dropwise (1 μL each) into PBS or 10% FCS / PBS and mixed (final 20 μM). As a control, each SNORA22 siRNA was added alone.

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

[0156] As a result, as shown in Fig. 12, it was shown that in the case of unmodified siRNA, it can be rapidly degraded even in the presence of a cationic oligopeptide or a folic acid-cationic oligopeptide complex, but chemical modification of siRNA was suggested to improve its stability in serum and almost suppress degradation by RNase.

[0157] [Example 11 Uptake of siRNA in vivo 1] The method described in JP 2018-110575 was modified to prepare human pancreatic cancer organoids using S2-013 pancreatic cancer cells. Specifically, S2-013 pancreatic cancer cells (20×10 4 cells), human mesenchymal stem cells MSC (LONZA, 40×10 4 cells), and human umbilical vein endothelial cells HUVEC (LONZA, 14×10 4 cells) 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. Thereafter, 300 μL / well of DMEM / EGM mixed solution was added, and the mixture was incubated again at 37 °C for 24 hours. One pancreatic cancer organoid was prepared in each well.

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

[0159] At the 6th week after transplantation, chemically modified SNORA22 siRNA labeled with Alexa-594 (SEQ ID NOs: 27 and 28, 5 μg) was administered by tail vein injection together with 1 equivalent, 2 equivalents, or 3 equivalents of cationic oligopeptide (Dab8) or folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B). After 24 hours, imaging was performed using an in vivo imager.

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

[0161] As a result, as shown in Fig. 13, compared with the case of using Dab8 (A-C), the delivery of SNORA22 siRNA to pancreatic cancer tissues was promoted in the case of using Fol-Dab8A (D-F) and the case of using Fol-Dab8B (G-I). In particular, high concentrations were delivered when 3 equivalents of Fol-Dab8A were added (F) and when 2 equivalents of Fol-Dab8B were added (H). In addition, from this experiment, it was also confirmed that almost no migration and accumulation of siRNA in the liver were observed, and it was excreted from the kidneys.

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

[0163] As a result, as shown in Fig. 14, from the measurement results up to 9 weeks after the start of administration, compared with the non-administered control group (Control), the group administered with scrambled control siRNA (Scr-Fol-Dab8B), and the group administered with the cationic oligopeptide (SNORA22-Dab8), a significant tumor growth inhibitory effect was observed in the group administered with SNORA22 siRNA together with the folic acid-cationic oligopeptide complex (SNORA22-Fol-Dab8B) from the 8th week onwards.

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

[0165] As a result, as shown in Fig. 15, it was confirmed that both Fol-Dab8A and Fol-Dab8B promoted the delivery of SNORA18 siRNA to pancreatic cancer tissues, and particularly high concentrations were delivered when 1 equivalent (A) or 3 equivalents (C) of Fol-Dab8A were added.

[0166] [Example 14 Uptake of siRNA in vivo 3] In the same manner 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 folic acid-cationic oligopeptide complex (Fol-Dab8A or Fol-Dab8B) was photographed using an in vivo imager.

[0167] As a result, as shown in FIG. 16, it was confirmed that both Fol-Dab8A and Fol-Dab8B promoted the delivery of WASF2 siRNA to pancreatic cancer tissue, and particularly high concentrations were delivered when 1 equivalent (D) or 3 equivalents (F) of Fol-Dab8B were added.

Industrial Applicability

[0168] According to the present invention, there is provided a therapeutic means capable of effectively suppressing tumor growth, invasion, and metastasis in pancreatic cancer, which is said to have the worst prognosis among cancers. The anti-tumor agent of the present application is specifically and effectively delivered to pancreatic cancer cells, and by combining it with other anti-cancer agents and / or anti-cancer treatments, it is possible to dramatically enhance the therapeutic effect of pancreatic cancer. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.< / hplc> < / mtor>

Claims

**Claim 1** A delivery promoter for siRNA or shRNA, comprising a folic acid-cationic oligopeptide complex, wherein the cationic oligopeptide contains at least two consecutive amino acid residues of the following formula (I): 【Chemical 1】 [In formula (I), R １ is a group H ３ N ＋ -CH ２ -, and R ２ is an alkylene group having 1 carbon atom. In one cationic oligopeptide, R １ and R ２ are all the same. ] and contains a cationic oligopeptide moiety consisting of 8 to 40 amino acids, which is one other non-consecutive amino acid residue except for the consecutive portion of the amino acid residues of formula (I), and the portion where at least two amino acid residues of formula (I) are consecutive has the following structure: 【Chemical Formula 2】 has an octamer of L-2,4-diaminobutyric acid (Dab) having the following as a partial structure, or is an octamer of the diaminobutyric acid, the above delivery promoter. **Claim 2** The delivery promoter according to claim 1, wherein the cationic oligopeptide consists of 8 to 12 amino acids. **Claim 3** The delivery promoter according to claim 1 or 2, wherein folic acid is linked to the N-terminus, C-terminus or side chain of the cationic oligopeptide, either via a linker or directly. **Claim 4** The delivery promoter according to claim 3, wherein folic acid is linked to the cationic oligopeptide via a linker, and the linker is a peptide linker. **Claim 5** The delivery promoter according to claim 4, wherein the peptide linker is a peptide consisting of 1 to 4 glycine residues. **Claim 6** The delivery promoter according to claim 1, wherein the folic acid-cationic oligopeptide complex is Fol-Dab8A and / or Fol-Dab8B having the following structure. [Chemical Formula 3] 【Chemical Formula 4】 **Claim 7** An antitumor agent comprising siRNA or shRNA that can bind to SNORA18 snoRNA and SNORA22 snoRNA expressed in pancreatic cancer cells and inhibit their expression, and the delivery promoter according to any one of claims 1 to 6. **Claim 8** The antitumor agent according to claim 7, comprising 0.5 to 10 equivalents of the folic acid-cationic oligopeptide complex relative to the siRNA or shRNA. **Claim 9** The antitumor agent according to claim 7 or 8, wherein the siRNA is an RNA / RNA double strand. **Claim 10** The antitumor agent according to any one of claims 7 to 9, wherein the siRNA or shRNA contains a modified base, a modified sugar, and / or a modified nucleoside bond. **Claim 11** The antitumor agent according to claim 10, wherein the modification of the modified sugar is a 2'-OMe modification. **Claim 12** The antitumor agent according to claim 10 or 11, wherein the modified nucleoside bond is a phosphorothioate bond. **Claim 13** A pharmaceutical composition containing the anti-tumor agent according to any one of claims 7 to 12.

14. The following: (a) A preparation containing siRNA or shRNA that can bind to SNORA18 snoRNA and SNORA22 snoRNA expressed in pancreatic cancer cells and inhibit their expression, and (b) An amino acid residue of the following formula (I): 【Chemical Formula 5】 [In formula (I), R １ is the group H ３ N ＋ -CH ２ -, and R ２ is an alkylene group having 1 carbon atom. In one cationic oligopeptide, R １ and R ２ are all the same.] A cationic oligopeptide site consisting of 8 to 40 amino acids, including at least two consecutive portions of the amino acid residue of formula (I), and the portion other than the consecutive portion of the amino acid residue of formula (I) is one other non-consecutive amino acid residue, and the portion where the amino acid residue of formula (I) has at least two consecutive portions has the following structure: 【Chemical Formula 6】 A preparation containing a folic acid-cationic oligopeptide complex having an octamer of L-2,4-diaminobutyric acid (Dab) having the following structure as a partial structure or being an octamer of the diaminobutyric acid A combined preparation.

15. The following: (a) siRNA or shRNA that can bind to SNORA18 snoRNA and SNORA22 snoRNA expressed in pancreatic cancer cells and inhibit their expression, and (b) An amino acid residue of the following formula (I): 【Chemical Formula 7】 [In formula (I), R １ is the group H ３ N ＋ -CH ２ -, and R ２ is an alkylene group having 1 carbon atom. In one cationic oligopeptide, R １ and R ２ are all the same. ] A cationic oligopeptide site consisting of 8 to 40 amino acids, including at least two consecutive portions of the amino acid residue of formula (I), and the portion other than the consecutive portion of the amino acid residue of formula (I) is one other non-consecutive amino acid residue, and the portion where the amino acid residue of formula (I) has at least two consecutive portions has the following structure: 【Chemical Formula 8】 A folic acid-cationic oligopeptide complex having an octamer of L-2,4-diaminobutyric acid (Dab) having the following structure as a partial structure or being an octamer of the diaminobutyric acid A pharmaceutical kit for the treatment of pancreatic cancer.

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