WO2006035515A1 - Therapeutic or preventive pharmaceutical composition for superficial bladder cancer, and use thereof - Google Patents

Therapeutic or preventive pharmaceutical composition for superficial bladder cancer, and use thereof Download PDF

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Publication number
WO2006035515A1
WO2006035515A1 PCT/JP2004/017669 JP2004017669W WO2006035515A1 WO 2006035515 A1 WO2006035515 A1 WO 2006035515A1 JP 2004017669 W JP2004017669 W JP 2004017669W WO 2006035515 A1 WO2006035515 A1 WO 2006035515A1
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WIPO (PCT)
Prior art keywords
sirna
bladder cancer
ribosome
plk
superficial bladder
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PCT/JP2004/017669
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French (fr)
Japanese (ja)
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Taira Maekawa
Takeshi Yuasa
Shinya Kimura
Masaki Nogawa
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Univ Kyoto
Taira Maekawa
Takeshi Yuasa
Shinya Kimura
Masaki Nogawa
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Application filed by Univ Kyoto, Taira Maekawa, Takeshi Yuasa, Shinya Kimura, Masaki Nogawa filed Critical Univ Kyoto
Priority to JP2006537625A priority Critical patent/JPWO2006035515A1/en
Publication of WO2006035515A1 publication Critical patent/WO2006035515A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • composition for treatment or prevention of superficial bladder cancer and use thereof
  • the present invention provides a siRNA effective for the treatment of superficial bladder cancer, particularly for the treatment of small cancer lesions remaining after removal of superficial bladder cancer, and for the prevention of progression of precancerous lesions of bladder tissue to cancer
  • the present invention relates to a pharmaceutical composition for treating or preventing superficial bladder cancer using this siRNA, and a method for treating or preventing superficial bladder cancer using this pharmaceutical composition.
  • Superficial bladder cancer accounts for 70% of the initial diagnosis of bladder cancer. Superficial bladder cancer can be removed transurethrally, but some cancer cells may remain after surgery. There may also be precancerous cells nearby. In order to prevent the recurrence of bladder cancer due to the proliferation of such cells, the current clinical practice is to use Mycobacterium tuberculosis Bacillus Calmette Guerin (BCG) as an anticancer drug, mitomycin C, adriamycin. Intravesical infusion therapy has been used, and this treatment has hardly improved since the 1980s.
  • BCG Mycobacterium tuberculosis Bacillus Calmette Guerin
  • An object of the present invention is to provide a therapeutic or preventive agent for superficial bladder cancer having high selectivity for cancer cells, and a therapeutic or prophylactic method.
  • PLK-1 poly-like kinase-1
  • siRNA-encapsulated ribosome is transurethrally administered to a superficial bladder cancer model mouse, it penetrates into the cancer cell and effectively suppresses the growth of the cancer cell.
  • a liposome encapsulating each siRNA consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 can particularly effectively suppress the proliferation of bladder cancer cells.
  • the present invention has been completed based on the above findings, and provides the following siRNA, a pharmaceutical composition for treating or preventing superficial bladder cancer, and a method for treating or preventing superficial bladder cancer.
  • siRNA comprising the nucleotide sequence of SEQ ID NO: 1
  • siRNAo comprising a nucleotide sequence in which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 1 and specifically suppressing the expression of the human PLK-1 gene
  • siRNA comprising the nucleotide sequence of SEQ ID NO: 2
  • siRNAo comprising a nucleotide sequence to which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 2 and specifically suppressing the expression of the human PLK-1 gene
  • Item 3 A ribosome encapsulating the siRNA according to Item 1.
  • Item 4 A ribosome encapsulating the siRNA according to Item 2.
  • Item 5 A pharmaceutical composition comprising the ribosome according to Item 3.
  • Item 6 A pharmaceutical composition comprising the liposome according to Item 4.
  • Item 7. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the liposome according to Item 3.
  • Item 8 A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the ribosome according to Item 4.
  • Item 9 A pharmaceutical composition for treating or preventing superficial bladder cancer comprising a liposome encapsulating siRNA that inhibits expression of human PLK-1.
  • Item 10 The composition according to Item 9, wherein the siRNA concentration is 100 nM to lmM.
  • Item 1 1. The composition according to Item 9, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 ⁇ m.
  • Item 1 A method of treating or preventing superficial bladder cancer comprising administering the ribosome according to Item 3 transurethrally into a human bladder having a superficial bladder cancer or a precancerous lesion of the bladder.
  • Item 1 A method for treating or preventing bladder superficial cancer, wherein the ribosome according to Item 4 is administered transurethrally into a human bladder having a superficial bladder cancer or a precancerous lesion of the bladder.
  • Item 14 Superficial bladder administered with a liposome containing a siRNA that inhibits human PLK-1 expression transurethrally into a human bladder with superficial bladder cancer or precancerous lesion of the bladder A method for treating or preventing sex cancer.
  • Item 15 The method for treatment or prevention according to Item 14, wherein the single dose of the agent for treating or preventing superficial bladder cancer is an amount capable of administering 12 zg to 120 nig of siRNA.
  • Item 1 6 The treatment or prevention method according to Item 14, wherein the therapeutic or prophylactic agent for superficial bladder cancer is administered 5 to 10 times at intervals of 1 to 7 days.
  • Item 17 The treatment or prevention method according to Item 14, which is performed on a human having a minimal residual cancer lesion after excision of a superficial bladder cancer.
  • Item 18 Use of the ribosome according to Item 3 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
  • Item 19 Use of the ribosome according to Item 4 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
  • Liposome containing siRNA that inhibits expression of human PLK-1 Use as a pharmaceutical composition for treating or preventing superficial bladder cancer.
  • Item 21 The use according to Item 20, wherein the concentration of siRNA is from 100 nM to 1 mM.
  • Item 22. The use according to Item 20, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 m.
  • the superficial bladder cancer cells and bladder it penetrates into precancerous tissues and effectively suppresses the growth of bladder cancer cells.
  • ribosomes encapsulating various therapeutic agents including siRNA, intravenous administration, subcutaneous administration, intraperitoneal administration, etc. have been attempted, but they are encapsulated by administration of ribosomes into the bladder. It is completely unpredictable whether the therapeutic agent can be introduced into the superficial bladder cells.
  • siRNAs comprising the nucleotide sequences of SEQ ID NOs: 1 and 2 that particularly effectively suppress the expression of the PLK-1 gene are provided.
  • the designed siRNA has a low probability of actually suppressing the expression of the target gene, and it is generally difficult to find such an effective siRNA.
  • siRNAs of SEQ ID NOs: 1 and 2 are encapsulated in ribosomes and administered into the bladder to actually suppress the expression of PLK-1 gene in bladder cancer cells. Proliferation was effectively suppressed.
  • anticancer drugs such as mitomycin 7 doriamycin
  • the known bladder cancer therapeutic agent described above has a small difference between the effective dose and the maximum tolerated dose of about 10 times, and the usable dose range is narrow.
  • the pharmaceutical composition for treating or preventing superficial bladder cancer according to the present invention has no side effects.
  • the difference between the effective dose and the maximum tolerated dose is large and easy to use.
  • FIG. 1 is an immunohistological staining diagram showing the expression level of PLK-1 in a cancer tissue removed from a bladder cancer patient.
  • Figure 2 shows the results of Western blotting showing the expression level of PLK-1 protein in bladder cancer cell lines.
  • FIG. 3 shows the results of Western plotting showing that the siRNA of the present invention suppresses the expression of PLK-1 in cancer cell lines.
  • B shows the results of Western blotting showing that the siRNA of the present invention suppresses the expression of PLK-1 over time.
  • C shows the results of Western plotting showing that the siRNA of the present invention suppresses PLK-1 expression in a dose-dependent manner.
  • FIG. 4 is a diagram showing that the siRNA of the present invention suppresses spindle formation in bladder cancer cells.
  • FIG. 5 shows that (A) and (B) induce apoptosis of bladder cancer cells by contact with the siRNA-encapsulating ribosome of the present invention.
  • (C) is a graph showing that the survival number of bladder cancer cells is reduced by contact of ribosomes containing si ⁇ si of the present invention siRNA.
  • FIG. 6 shows that the bladder cancer cell line engrafts and grows in the mouse bladder.
  • B) and C) show that cancer cells penetrate into the mouse bladder when the siRNA of the present invention is administered transurethrally.
  • a pharmaceutical composition for treatment or prevention of superficial bladder cancer includes those in which siRNA specifically inhibiting or suppressing the expression of human PLK-1 is encapsulated in ribosomes.
  • This siRNA preferably contains a base sequence that can hybridize to about 15 to 30 bases, particularly about 19 to 23 bases, of the human PLK-1 gene (GenBank accession no. ⁇ 1 005030).
  • the base sequence capable of hybridizing to the human PLK-1 gene may contain up to about 3 base regions that do not hybridize to the human PLK-1 gene.
  • siRNA having a base sequence capable of hybridizing to about 15 to 30 bases of human PLK-1 gene, particularly about 19 to 23 bases is preferable.
  • Such siRNA can be designed, for example, by the method described in Biochem. Biophys. Res. Commun. (2004) Jun 18, 319 (1), 264-27. Whether the selected sequence inhibits the expression of only PLK-1 mRNA may be confirmed, for example, by a BLAST search.
  • the siRNA containing each of the base sequences of SEQ ID NOs: 1 to 4 is a double-stranded RNA in which an RNA having this base sequence is paired with an MA having a complementary or substantially complementary base sequence. Contains double-stranded RNA.
  • siRNA containing each base sequence of SEQ ID NOs: 1 to 4 contains each base sequence of SEQ ID NOs: 1 to 4, and has a maximum of about 50 bases.
  • siRNA containing each of the nucleotide sequences of SEQ ID NOs: 1 to 4 is preferable in that it has a strong inhibitory action on the expression of human PLK-1 gene.
  • siRNAs that suppress or inhibit PLK-1 expression in particular, siRNA comprising the nucleotide sequence of SEQ ID NO: 1 (preferably, siRNA comprising the nucleotide sequence of SEQ ID NO: 1), and siRNA comprising the nucleotide sequence of SEQ ID NO: 2 (preferably, the siRNA comprising the nucleotide sequence of SEQ ID NO: 2 is efficiently taken up into bladder cancer cells or cells in the precancerous state of the bladder and does not efficiently suppress the expression of the PLK-1 gene. It inhibits.
  • the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 each include a base sequence in which about 1 to 3, particularly about 1 to 2 nucleotides are added, deleted, or substituted. Even siRNA can be used as long as it specifically inhibits or suppresses the expression of the human PLK-1 gene.
  • the siRNA of the present invention may be a derivative of these nucleotides as long as it inhibits PLK-1 expression.
  • each base sequence of SEQ ID NOs: 1 and 2 whether or not siRNA containing a base sequence in which 1 to 3 nucleotides are deleted, added, or substituted suppresses the expression of the PLK-1 gene is, for example, As will be described later in Example 3, it may be confirmed by Western plotting using an anti-PLK-1 polyclonal antibody.
  • the siRNA of the present invention can be produced by a known chemical synthesis method.
  • the structure of the ribosome is not particularly limited, and a ribosome composed of a known ribosome material used for cancer gene therapy may be used.
  • Examples of such known ribosomes include cationized ribosomes. By using a cationized ribosome, permeability in cells can be improved.
  • lipids constituting the ribosome include natural or synthetic phospholipids such as phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardioribine, or the like. Hydrogenated in accordance with the law. These phospholipids may be used in combination with sterol. Lipids can be used singly or in combination of two or more. Ribosomes can be produced, for example, by dissolving lipids in a solvent such as t-butyl alcohol, cooling, and freeze-drying.
  • a solvent such as t-butyl alcohol, cooling, and freeze-drying.
  • ribosomes containing siRNA can be obtained simply by bringing the prepared ribosome into contact with the siRNA solution.
  • siRNA dissolved in lipid After adding the prepared liquid to swell and dispersing with ultrasonic waves, a liposome with entrapped siRNA directly can be obtained by adding polyethylene glycol phosphatidylethanolamine to the dispersion. .
  • the size of the ribosome is preferably 80% by weight or more, preferably a particle size of about 50 to 300 xm, more preferably a particle size of about 70 to 200 ⁇ m, and a particle size of about 70 to 100 m. It is even more preferable that it is in the range. In the above particle size range, a sufficient amount of siRNA can be encapsulated and no ribosome toxicity occurs.
  • physiological saline It only needs to contain a liquid component such as a culture solution for cells such as RPMI and a sugar solution.
  • the siRNA concentration in the ribosome is preferably about 100 nM to lmM, and more preferably about 100 nM to 6 ⁇ .
  • the therapeutic efficiency is good if the amount of siRNA enclosed is within the above range.
  • the amount of siRNA can actually be encapsulated.
  • the ribosome encapsulating siRNA is defined by the present invention because it is brought into contact with a solution in which siRNA is suspended and the ribosome.
  • “Intraribosomal siRNA concentration” is the concentration of siRNA in the solution that comes into contact with the ribosome during ribosome production. In the present invention, this concentration is regarded as the siRNA concentration in the liposome.
  • the method for treating or preventing superficial bladder cancer according to the present invention comprises the above-described siRNA-encapsulating ribosome of the present invention in the human bladder having a superficial bladder cancer or a precancerous lesion of the bladder transurethrally (this book).
  • the pharmaceutical composition for treatment or prevention of bladder cancer of the invention comprises the above-described siRNA-encapsulating ribosome of the present invention in the human bladder having a superficial bladder cancer or a precancerous lesion of the bladder transurethrally (this book).
  • Patients subject to the method of the present invention include humans with untreated bladder superficial cancers, humans after removal of superficial bladder cancers, humans whose bladder cancer has been reduced by administration of anticancer agents, intravesical A human having a precancerous lesion on the surface. Since bladder cancer cells are stacked in multiple layers, even if the superficial bladder cancer is removed, Usually, a few cancer cells remain. Humans having such minimal residual lesions are also subject to the method of the present invention.
  • the siRNA-encapsulating ribosome of the present invention By administering the siRNA-encapsulating ribosome of the present invention to a patient in such a state, the growth of cancer cells can be suppressed, and the progression of precancerous tissue to cancer can be suppressed.
  • the subject who can expect the highest therapeutic effect is a human who has a minimal residual cancer lesion on the inner surface of the bladder after the removal of the bladder cancer.
  • the siRNA-encapsulated ribosome may be administered into the bladder through the urethra from the urethral orifice, usually using a catheter, in a state suspended in, for example, PBS, physiological saline, cell culture medium, sugar solution, or the like. As a result, siRNA is introduced into bladder cancer cells.
  • the single dose of siRNA-encapsulated ribosome is preferably about 12 x g to 120 nig, more preferably about 50 g to 10 mg, in terms of the amount of siRNA. Within the above dose range, a sufficient therapeutic or prophylactic effect can be obtained, and non-specific effects do not occur.
  • this amount is preferable to administer this amount divided into about 3 to 10 times. If the number of doses is within the above range, the single dose will not cause side effects and the burden on the patient is small.
  • the administration interval is preferably about 1 to 7 days. With the above administration interval, no infection is induced and the burden on the patient is small. In addition, the effective siRNA concentration can be maintained in the patient body within the above administration interval.
  • siRNA-encapsulating ribosome of the present invention described above can be used as a pharmaceutical composition for prevention or treatment of superficial bladder cancer.
  • the preferred mode of use is as described above.
  • FIG. 1 A, B, and C are bladder cancers with high grade undifferentiated muscle layer infiltration. E and F are well-differentiated and superficial bladder cancers of low grade. It can be seen that PLK-1 is highly expressed in higher grade cancer tissues. D is a tissue with lymphatic invasion, and PLK-1 is also highly expressed in this tissue.
  • Table 1 shows the relationship between the clinicopathological characteristics of bladder cancer and the PLK-1 expression level for 58 specimens.
  • FIG. 2 shows that PLK-1 is very strongly expressed in bladder cancer cell lines compared to normal cells.
  • PLK-1 plays an important role in the invasion / malignancy of bladder cancer. It can also be seen that PLK-1 is suitable as a molecular therapy target for bladder cancer.
  • siRNAs of SEQ ID NOs: 1 to 4 for PLK-1 were chemically synthesized (Nippon- Shinyaku Co., Kyoto, Japan). The nucleotide sequences of these siRNAs are as follows.
  • PLK-1 siRNA 1345 5, -GACAGCCUGCAGUACAUAGdTdT-3 '(SEQ ID NO: 1)
  • PLK-1 siRNA 1412 5,-CCUUGAUGAAGA AGAUCACdTdT- 3' (SEQ ID NO: 2)
  • PLK-1 siRNA 183 5 '-GGGCGGCUUUGCCAAGUGCdTdT-3' (SEQ ID NO: 3)
  • PLK-1 siRNA 1418 5, -GAAGAAGAUCAC CCUCCUUdTdT-3 ′ (SEQ ID NO: 4) Cationic liposome containing a cationic lipid analog (Cancer Res. 1999 Sep 1; 59 (17): 4325_33.
  • siRNA was encapsulated by contact. More than 80% by weight of the ribosome has a particle size of 70-80 / zm. These ribosomes are filled with 10% aqueous maltose solution and contain siRNA. This siRNA concentration is the siRNA concentration in the siRNA solution used in the preparation of the siRNA-encapsulated ribosome.
  • siRNA was introduced into cells by adding the siRNA-encapsulated ribosome to each cell culture. Proteins were prepared from the cells after siRNA introduction, and Western blotting using anti-human PLK-1 polyclonal antibody was performed in the same manner as in Example 2.
  • Fig. 3 (A) The results are shown in Fig. 3 (A).
  • untreated cells represent cells that do not allow siRNA-encapsulating liposomes to act, and the constrictor contains siRNA having a nonsense sequence (5'-UUCUCCGAACGUGUCACGUdTdT-3 '(SEQ ID NO: 5)). Cells that have been allowed to act are shown.
  • Fig. 3 four siRNAs suppressed the expression of PLK-1 in cancer cells.
  • PLK-1 1412 and: PLK-1 1418 strongly suppressed the expression of PLK-1, especially PLK-1. It can be seen that 1412 most effectively suppresses the expression of PLK-1.
  • the ribosome encapsulating the siRNA of the present invention acts on bladder cancer cells and suppresses the expression of PLK-1.
  • PLK-1 protein plays an important role in cell cycle control and is thought to be involved in the degradation of cyclin B1.
  • PLK-1 1412 we investigated the effects of cyclin B1 protein on bladder cancer cell lines UM-UC-3 and 376376 into which ribosomes encapsulating each of the above concentrations of siRNA were introduced.
  • the expression level was examined by Western plotting using a rabbit anti-cyclin B1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
  • Figure 3 (C) shows that PLK-1 1412 suppresses the degradation of cyclin B1 in a concentration-dependent manner.
  • siRNA The influence of siRNA on spindle formation, which is considered to be an important role of PL -1 protein, was examined as follows.
  • PLK-1 siRNA 1412-encapsulated ribosome was allowed to act on the above cells in the bladder cancer cell line UM-UC-3 (ATCC; American Type Culture Collection). Introduced siRNA but not bladder cancer cells and controls ]-Immunocytostaining with antibodies specific for tubulin and tubulin (Sigma, ST. Louis, M0) and Hoechs t 33342 DNA staining (Molecular Probes, Eugene, OR) Were carried out according to the manual attached to them.
  • the UM-UC-3 bladder cancer cell line was stained with propidium iodide (PI) and the cell cycle was examined by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Fig. 5 (A) The results are shown in Fig. 5 (A). From Fig. 5 (A), apoptosis was not induced in the control bladder cancer cells not introduced with PLK-1 siRNA 1412, but in the bladder cancer cells introduced with PLK-1 siRNA 1412, the cell cycle was at G2 / M phase. It can be seen that it has been stopped and apoptosis has been induced.
  • a UM-UC-3 bladder cancer cell line that does not introduce PLK-1 siRNA 1412, MEBSTAIN apoptosis Kit II (MBL, Nagoya, Japan) for UM-UC-3 bladder cancer cells transfected with control siRNA (SEQ ID NO: 5) and UM-UC-3 bladder cancer cells transfected with PLK-1 siRNA 1412 Annexin V staining was carried out using this.
  • the siRNA concentration in the ribosome was 100 nM, and the contact time with the cells was 36 hours.
  • FIG. 3 (B) The results are shown in Fig. 3 (B).
  • a in Fig. 3 (B) is a stained photograph, and B is the number of viable cells. From FIG. 3 (B), the percentage of Annexin V positive cells was high in UM-UC-3 bladder cancer cells treated with PLK-1 siRNA 1412, confirming the induction of apoptosis.
  • Example 6 which will be described later, the liposome encapsulating PLK-1 siRNA 1412 was found to have an inhibitory effect on cancer cell proliferation in a concentration range of 150 nM to 6 / z M in vivo. IC 5 . The in effect was observed at a concentration 3 to 120 times higher than that of.
  • a liposome containing PLK-1 siRNA 1412 at a concentration of ⁇ ⁇ was applied in the same manner as in Example 3 for 0 hour. The number of viable cells after 1, 2 and 3 hours was counted. The relative cell viability of each cell treated with PLK-1 siRNA 1412 is 0.187 ⁇ 0.0191 and 0.0891 ⁇ 0.0290 for UM-UC-3 cells, assuming that the initial viable cell number of control cells is 1.0. 0.75J for 253J cells 0.0342 and 0.399 ⁇ 0.0693, and 0.466 soil 0.108 and 0.243 0.0717 for KU-7 cells.
  • Figure 5 (C) shows that when bladder cancer cells were treated with a relatively high concentration of PLK-1 siRNA 1412 ( ⁇ ), the number of viable cells decreased in a short time of 1 to 3 hours. .
  • PGL3 Promega, Madison, WI
  • pSV2 neovector-1 ATCC
  • the 6 LUC-labeled bladder cancer cells thus obtained were transplanted into 1 ⁇ 10 6 mouse mice using a 24G angio 'catheter. Mice were observed using the Xenogen in vivo imaging system (Xenogen, Alameda, CA) 10 minutes, 1 day, 1 week, 3 weeks, and 4 weeks after transplantation.
  • FIG. 6 A shows 10 minutes later, B 1 day later, C 1 week later, D 3 weeks later, E 4 weeks later. It is observed that bladder cancer cells are growing in the mouse bladder.
  • a ribosome encapsulating siRNA at a concentration of 100 nM was prepared. Furthermore, this ribosome was labeled with FITC (fluorescent with a peak wavelength of 530 nm). After urinating from the bladder of a mouse transplanted with LUC-labeled bladder cancer cells using a catheter, 200 ⁇ 1 of the above-mentioned liposo was injected into the urinary bladder from the urethral orifice. Twenty-four hours after ribosome injection, mouse bladders were removed and observed using a fluorescence microscope. The results are shown in Fig. 6 (B). In Fig.
  • FIG. 6 (B) A and B are administered with ribosome, C and D are not administered with ribosome, A and C are observed with a fluorescence microscope, and B and D are serial sections of hematoxylin HE) Shows the dyed result.
  • Fig. 6 (B) A, when ribosome is administered, fluorescence is observed in the bladder. This indicates that administration of siRNA-encapsulating ribosomes transurethrally penetrates the bladder cancer tissue.
  • the resected bladder cancer tissue was also subjected to HE staining.
  • FIG. 6 The results are shown in Fig. 6 (0. A and B in Fig. 6 (C) are mouse tissues treated with siRNA-encapsulated ribosomes, and D is the tissue of mice that do not receive this treatment.
  • Figure 6 (C) shows the expression of PLK-1 by transurethrally administration of siMA-encapsulated ribosome, using anti-PLK-1 monoclonal antibody and B, D by HE staining. It can be seen that is suppressed.
  • mice transplanted with the UM-UC-3 cell line into which the luciferase gene had been introduced into the bladder were divided into 4 groups (7 mice per group), reared for 21 days, and PLK-1 siRNA 1412-encapsulating liposome or control siRNA (SEQ ID NO: 5) was administered from the 5th day to the 9th day. Administration was performed once a day by administering ribosomes into the bladder at once, tying the urethral opening with surgical thread, and opening it 4 hours later.
  • These 4 groups are the non-administration group, concentration 6
  • concentration 6 are a group administered with ribosomes containing urn control siRNA, a group administered with PLK-1 s iRNA 141 2 at a concentration of 200 nM, and a group administered with PLK-ls iRNA 141 2 at a concentration of 6 ra.
  • Fig. 6 (D) The proliferation of bladder cancer cells was measured for each group of mice. The results are shown in Fig. 6 (D).
  • the horizontal axis of the graph in Fig. 6 (D) shows the breeding period, and the vertical axis shows the photon count.
  • PLK-ls iRNA 1412 was shown to have a therapeutic effect on superficial bladder cancer micro-recurrence and minimal residual disease by transurethral injection into the bladder. Sufficient experimental results were obtained to provide a rationale for this. Industrial applicability
  • the pharmaceutical composition for prevention or treatment of superficial bladder cancer of the present invention penetrates into superficial bladder cancer cells and precancerous tissues and effectively suppresses the proliferation of bladder cancer cells when administered into the bladder. To do.
  • it can be suitably used as a pharmaceutical composition for the treatment of humans having minimal residual cancer lesions after excision of bladder cancer.

Abstract

Liposomes having, packed therein, siRNA capable of inhibiting the expression of human PLK-1 gene are useful as a therapeutic or preventive pharmaceutical composition for superficial bladder cancer. In particular, siRNA containing the base sequence of SEQ ID NO: 1 and siRNA containing the base sequence of SEQ ID NO: 2 are preferred. Per urethra administration of this therapeutic or preventive pharmaceutical composition for superficial bladder cancer into the bladder of patient with superficial bladder cancer or precancerous lesion of the bladder realizes inhibition of cancerous cell proliferation or progression to cancer.

Description

明 細 書 膀胱表在性癌の治療又は予防用医薬組成物、及びその利用 技術分野  Description Pharmaceutical composition for treatment or prevention of superficial bladder cancer, and use thereof
本発明は、 膀胱表在性癌の治療、 特に膀胱表在性癌の摘出後に残存す る微小癌病変の治療や、 膀胱組織の前癌病変の癌への進行の予防などに 有効な s i RNA、 この s i RNAを用いた膀胱表在性癌の治療又は予防用医薬 組成物、 及び、 この医薬組成物を用いた膀胱表在性癌の治療又は予防方 法に関する。 背景技術  The present invention provides a siRNA effective for the treatment of superficial bladder cancer, particularly for the treatment of small cancer lesions remaining after removal of superficial bladder cancer, and for the prevention of progression of precancerous lesions of bladder tissue to cancer The present invention relates to a pharmaceutical composition for treating or preventing superficial bladder cancer using this siRNA, and a method for treating or preventing superficial bladder cancer using this pharmaceutical composition. Background art
膀胱癌の初診断時にはその 70 %を膀胱表在性癌が占める。膀胱表在性 癌は経尿道的に切除可能であるが、 術後に癌細胞が僅かに残存する場合 がある。 また、 周囲に前癌状態の細胞が存在する場合もある。 このよう な細胞の増殖により膀胱癌が再発することを予防するために、 現在の臨 床では、 結核菌である Bacillus Calmette Guerin (BCG)ゃ抗癌剤のマイト マイシン C (mitomycin C)、 アドリアマイシン（adriamycin)などの膀胱内 注入療法がされており、 この治療法は 1980 年代からほとんど改善され ていない。  Superficial bladder cancer accounts for 70% of the initial diagnosis of bladder cancer. Superficial bladder cancer can be removed transurethrally, but some cancer cells may remain after surgery. There may also be precancerous cells nearby. In order to prevent the recurrence of bladder cancer due to the proliferation of such cells, the current clinical practice is to use Mycobacterium tuberculosis Bacillus Calmette Guerin (BCG) as an anticancer drug, mitomycin C, adriamycin. Intravesical infusion therapy has been used, and this treatment has hardly improved since the 1980s.
しかし、 これらの薬剤は再発予防効果は認めるものの十分とは言いが たく約半数は再発し、 その内 20%— 30%は浸潤癌となる。 このような進 行期膀胱癌では膀胱全摘手術を施行せざるをえず、 術後に排尿および性 機能に関し QOL を著しく損なう。また、 BCGは生細菌であるため、 BCG の使用により膀胱炎ゃ菌血症になる恐れがある。 また、 マイトマイシン Cゃァドリァマイシン等の抗癌剤は、 癌細胞に対する選択性が十分では なく正常細胞にも傷害を与える恐れがある。 発明の開示 本発明は、 癌細胞に対する選択性が高い表在性膀胱癌の治療又は予防 剤、及び治療又は予防方法を提供することを課題とする。 However, although these drugs have a recurrence-preventing effect, they are not sufficient, but about half of them relapse, of which 20% -30% have invasive cancer. In advanced stage bladder cancer, total cystectomy must be performed, and postoperatively, QOL is significantly impaired in terms of urination and sexual function. Since BCG is a live bacterium, the use of BCG may cause cystitis or bacteremia. In addition, anticancer drugs such as mitomycin C adadrimycin are not sufficiently selective for cancer cells and may damage normal cells. Disclosure of the invention An object of the present invention is to provide a therapeutic or preventive agent for superficial bladder cancer having high selectivity for cancer cells, and a therapeutic or prophylactic method.
上記課題を解決するために本発明者らは研究を重ね、 以下の知見を得 た。  In order to solve the above-mentioned problems, the present inventors have conducted research and obtained the following knowledge.
(i) 細胞の分裂及び増殖に重要な役割を担っている PLK-1 (polo- like kinase- 1)は、 癌細胞の中でも膀胱癌細胞において特に強く発現してい る。  (i) PLK-1 (polo-like kinase-1), which plays an important role in cell division and proliferation, is particularly strongly expressed in bladder cancer cells among cancer cells.
(ii) PLK-1 の発現を特異的に阻害する siRNAを封入したリボソームを 表在性膀胱癌モデルマウスに経尿道的に投与すると癌細胞に浸透し、 癌 細胞の増殖が効果的に抑制される。  (ii) Specific inhibition of PLK-1 expression When siRNA-encapsulated ribosome is transurethrally administered to a superficial bladder cancer model mouse, it penetrates into the cancer cell and effectively suppresses the growth of the cancer cell. The
(iii) 配列番号 1及び 2の塩基配列からなる各 siRNAを封入したリポ ゾームは、 膀胱癌細胞の増殖を特に効果的に抑制できる。  (iii) A liposome encapsulating each siRNA consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 can particularly effectively suppress the proliferation of bladder cancer cells.
本発明は上記知見に基づき完成されたものであり、 以下の siRNA、 表 在性膀胱癌治療又は予防用医薬組成物、 及び表在性膀胱癌の治療又は予 防方法を提供する。  The present invention has been completed based on the above findings, and provides the following siRNA, a pharmaceutical composition for treating or preventing superficial bladder cancer, and a method for treating or preventing superficial bladder cancer.
項 1. 以下の（a)又は（b)の siRNA  Item 1. siRNA of (a) or (b) below
(a) 配列番号 1の塩基配列を含む siRNA  (a) siRNA comprising the nucleotide sequence of SEQ ID NO: 1
(b) 配列番号 1において 1〜 3個の塩基が付加、 欠失、 又は置換され た塩基配列を含み、 かつヒト PLK- 1 遺伝子の発現を特異的に抑制する siRNAo  (b) siRNAo comprising a nucleotide sequence in which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 1 and specifically suppressing the expression of the human PLK-1 gene
項 2. 以下の（c)又は（d)の siRNA  Item 2. siRNA of (c) or (d) below
(c) 配列番号 2の塩基配列を含む siRNA  (c) siRNA comprising the nucleotide sequence of SEQ ID NO: 2
(d) 配列番号 2において 1〜 3個の塩基が付加、 欠失、 又は置換され た塩基配列を含み、 かつヒト PLK-1 遺伝子の発現を特異的に抑制する siRNAo  (d) siRNAo comprising a nucleotide sequence to which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 2 and specifically suppressing the expression of the human PLK-1 gene
項 3. 項 1に記載の siRNAが内包されたリボソーム。  Item 3. A ribosome encapsulating the siRNA according to Item 1.
項 4. 項 2に記載の siRNAが内包されたリボソーム。  Item 4. A ribosome encapsulating the siRNA according to Item 2.
項 5. 項 3に記載のリボソームを含む医薬組成物。  Item 5. A pharmaceutical composition comprising the ribosome according to Item 3.
項 6. 項 4に記載のリポソ一ムを含む医薬組成物。 項 7. 項 3に記載めリポソームを含む膀胱表在性癌の治療又は予防 用医薬組成物。 Item 6. A pharmaceutical composition comprising the liposome according to Item 4. Item 7. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the liposome according to Item 3.
項 8. 項 4に記載のリボソームを含む膀胱表在性癌の治療又は予防 用医薬組成物。  Item 8. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the ribosome according to Item 4.
項 9. ヒト PLK- 1の発現を阻害する siRNAが内包されたリポソ一ム を含む膀胱表在性癌の治療又は予防用医薬組成物。  Item 9. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising a liposome encapsulating siRNA that inhibits expression of human PLK-1.
項 1 0. siRNA濃度が 100nM〜lmMである項 9に記載の組成物。  Item 10. The composition according to Item 9, wherein the siRNA concentration is 100 nM to lmM.
項 1 1. リボソームの 80重量％以上が 50〜300 ^mの粒径を有する ものである項 9に記載の組成物。  Item 1 1. The composition according to Item 9, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 ^ m.
項 1 2. 膀胱表在性癌又は膀胱の前癌病変を有するヒトの膀胱内に、 経尿道的に、 項 3に記載のリボソームを投与する膀胱表在性癌の治療又 は予防方法。  Item 1 2. A method of treating or preventing superficial bladder cancer comprising administering the ribosome according to Item 3 transurethrally into a human bladder having a superficial bladder cancer or a precancerous lesion of the bladder.
項 1 3. 膀胱表在性癌又は膀胱の前癌病変を有するヒトの膀胱内に、 経尿道的に、 項 4に記載のリボソームを投与する膀胱表在性癌の治療又 は予防方法。  Item 1 3. A method for treating or preventing bladder superficial cancer, wherein the ribosome according to Item 4 is administered transurethrally into a human bladder having a superficial bladder cancer or a precancerous lesion of the bladder.
項 14. 膀胱表在性癌又は膀胱の前癌病変を有するヒトの膀胱内に、 経尿道的に、 ヒト PLK-1の発現を阻害する siRNAが内包されたリポソ一 ムを投与する膀胱表在性癌の治療又は予防方法。  Item 14. Superficial bladder administered with a liposome containing a siRNA that inhibits human PLK-1 expression transurethrally into a human bladder with superficial bladder cancer or precancerous lesion of the bladder A method for treating or preventing sex cancer.
項 1 5. 膀胱表在性癌の治療又は予防剤の 1 回投与量が、 siRNAを 12 zg〜120nig投与できる量である項 1 4に記載の治療又は予防方法。 項 1 6. 膀胱表在性癌の治療又は予防剤を 1〜7日間間隔で 5〜10 回投与する項 14に記載の治療又は予防方法。  Item 15. The method for treatment or prevention according to Item 14, wherein the single dose of the agent for treating or preventing superficial bladder cancer is an amount capable of administering 12 zg to 120 nig of siRNA. Item 1 6. The treatment or prevention method according to Item 14, wherein the therapeutic or prophylactic agent for superficial bladder cancer is administered 5 to 10 times at intervals of 1 to 7 days.
項 1 7. 膀胱表在性癌を切除後に微小残存癌病変を有するヒトに対 して行われるものである項 1 4に記載の治療又は予防方法。  Item 17. The treatment or prevention method according to Item 14, which is performed on a human having a minimal residual cancer lesion after excision of a superficial bladder cancer.
項 1 8. 項 3に記載のリボソームの膀胱表在性癌の治療又は予防用 医薬組成物としての使用。  Item 18. Use of the ribosome according to Item 3 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
項 1 9. 項 4に記載のリボソームの膀胱表在性癌の治療又は予防用 医薬組成物としての使用。  Item 19. Use of the ribosome according to Item 4 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
項 2 0. ヒト PLK-1の発現を阻害する siRNAが内包されたリポソ一 ムの膀胱表在性癌の治療又は予防用医薬組成物としての使用。 Item 20. Liposome containing siRNA that inhibits expression of human PLK-1 Use as a pharmaceutical composition for treating or preventing superficial bladder cancer.
項 2 1 . s i RNAの濃度が 1 00nM〜l mMである項 2 0に記載の使用。 項 2 2 . リボソームの 80重量％以上が 50〜 300 mの粒径を有する ものである項 2 0に記載の使用。  Item 21. The use according to Item 20, wherein the concentration of siRNA is from 100 nM to 1 mM. Item 22. The use according to Item 20, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 m.
本発明の治療又は予防方法によれば、 PLK- 1 遺伝子の発現を抑制でき る s i RNAを封入したリボソームを尿道を経由して膀胱内に投与すること により、 膀胱表在性癌細胞や膀胱の前癌組織に浸透し、 膀胱癌細胞の増 殖を効果的に抑制する。  According to the treatment or prevention method of the present invention, by administering a ribosome encapsulating siRNA capable of suppressing the expression of the PLK-1 gene into the bladder via the urethra, the superficial bladder cancer cells and bladder It penetrates into precancerous tissues and effectively suppresses the growth of bladder cancer cells.
s i RNA を始めとする各種治療剤を封入したリボソームの投与方法とし ては、 静脈内投与、 皮下投与、 腹腔内投与などの方法が試みられている が、 膀胱内へのリボソームの投与により内包された治療剤を膀胱表在性 の細胞に導入できるか否かは全く予測できないことである。  As methods for administering ribosomes encapsulating various therapeutic agents including siRNA, intravenous administration, subcutaneous administration, intraperitoneal administration, etc. have been attempted, but they are encapsulated by administration of ribosomes into the bladder. It is completely unpredictable whether the therapeutic agent can be introduced into the superficial bladder cells.
また、 本発明によれば、 PLK-1 遺伝子の発現を特に効果的に抑制する 配列番号 1及び 2の塩基配列からなる各 s i RNAが提供された。 設計した s i RNAが実際に標的遺伝子の発現を抑制する確率は低く、 このように効 果的な s i RNAを見出すことは一般に困難である。このような状況の下で、 配列番号 1及び 2の s i RNAはリボソームに封入して膀胱内に投与するこ とにより実際に膀胱癌細胞における PLK- 1遺伝子の発現を抑制し、 膀胱 癌細胞の増殖を効果的に抑制した。  In addition, according to the present invention, siRNAs comprising the nucleotide sequences of SEQ ID NOs: 1 and 2 that particularly effectively suppress the expression of the PLK-1 gene are provided. The designed siRNA has a low probability of actually suppressing the expression of the target gene, and it is generally difficult to find such an effective siRNA. Under such circumstances, siRNAs of SEQ ID NOs: 1 and 2 are encapsulated in ribosomes and administered into the bladder to actually suppress the expression of PLK-1 gene in bladder cancer cells. Proliferation was effectively suppressed.
また、従来膀胱癌の治療に使用されている抗癌剤（マイトマイシン 7ドリアマイシン等) は活発に増殖する癌細胞に選択性を示すが、 その 作用機序は夕ンパク質合成や核酸合成の阻害であることから、 ある程度 正常細胞に対しても作用し、 副作用を引き起こす。 これに対して本発明 の膀胱表在性癌の治療又は予防用組成物は、 癌細胞、 中でも膀胱癌細胞 に特異的に強く発現している PLK- 1遺伝子の発現を抑制する s i RNAを有 効成分とするため、 癌細胞に対する選択性が極めて高い。  In addition, anticancer drugs (such as mitomycin 7 doriamycin) that have been used in the treatment of bladder cancer are selective for actively proliferating cancer cells, but their mechanism of action is inhibition of protein synthesis and nucleic acid synthesis. Therefore, it acts on normal cells to some extent and causes side effects. In contrast, the composition for the treatment or prevention of superficial bladder cancer of the present invention has siRNA that suppresses the expression of PLK-1 gene that is specifically and strongly expressed in cancer cells, in particular, bladder cancer cells. Because it is an active ingredient, it has extremely high selectivity for cancer cells.
また、 上記の公知の膀胱癌治療剤は、 有効投与量と最大耐容量との差 がせいぜい 1 0 倍程度と小さく、 使用可能な用量範囲が狭い。 これに対 して本発明の膀胱表在性癌の治療又は予防用医薬組成物は、 副作用が認 められない又は殆ど認められないため、 有効投与量と最大耐容量との差 が大きく、 使用し易い。 図面の簡単な説明 In addition, the known bladder cancer therapeutic agent described above has a small difference between the effective dose and the maximum tolerated dose of about 10 times, and the usable dose range is narrow. On the other hand, the pharmaceutical composition for treating or preventing superficial bladder cancer according to the present invention has no side effects. The difference between the effective dose and the maximum tolerated dose is large and easy to use. Brief Description of Drawings
図 1は、 膀胱癌患者から摘除された癌組織における PLK- 1発現量を示 す免疫組織染色図である。  FIG. 1 is an immunohistological staining diagram showing the expression level of PLK-1 in a cancer tissue removed from a bladder cancer patient.
図 2は、 膀胱癌細胞株における PLK-1タンパク質の発現量を示すゥェ スタンプロッティングの結果である。  Figure 2 shows the results of Western blotting showing the expression level of PLK-1 protein in bladder cancer cell lines.
図 3は、 （A)は、 癌細胞株において本発明の siRNAが PLK- 1 の発現を 抑制することを示すウェスタンプロティングの結果である。 （B)は、 本 発明の siRNAが経時的に PLK- 1の発現を抑制することを示すウェスタン ブロッテイングの結果である。 （C)は、 本発明の siRNA が用量依存的に PLK-1 の発現を抑制することを示すウエスタンプロッティングの結果で ある。  FIG. 3 (A) shows the results of Western plotting showing that the siRNA of the present invention suppresses the expression of PLK-1 in cancer cell lines. (B) shows the results of Western blotting showing that the siRNA of the present invention suppresses the expression of PLK-1 over time. (C) shows the results of Western plotting showing that the siRNA of the present invention suppresses PLK-1 expression in a dose-dependent manner.
図 4は、 本発明の siRNAが膀胱癌細胞において紡錐体形成を抑制する ことを示す図である。  FIG. 4 is a diagram showing that the siRNA of the present invention suppresses spindle formation in bladder cancer cells.
図 5は、 （A)及び（B)は、 本発明の siRNA内包リボソームの接触により 膀胱癌細胞のアポト一シスが誘導されることを示す図である。 （C)は、 ΙΟΟηΜの本発明の siRNAを内包するリボソームの接触により膀胱癌細胞 の生存数が減少することを示す図である。  FIG. 5 shows that (A) and (B) induce apoptosis of bladder cancer cells by contact with the siRNA-encapsulating ribosome of the present invention. (C) is a graph showing that the survival number of bladder cancer cells is reduced by contact of ribosomes containing siη si of the present invention siRNA.
図 6は、 （A)は膀胱癌細胞株がマウスの膀胱に生着し、 増殖すること を示す図である。 （B)及び（C)は、 マウスの膀胱内に経尿道的に本発明の siRNA を投与した場合に癌細胞に浸透することを示す図である。 （D)は、 に本発明の siRNAを経尿道的に膀胱内投与した膀胱癌細胞移植マウスに おいて、 膀胱癌細胞の増殖が抑制されることを示す図である。 発明の詳細な記述  FIG. 6 (A) shows that the bladder cancer cell line engrafts and grows in the mouse bladder. (B) and (C) show that cancer cells penetrate into the mouse bladder when the siRNA of the present invention is administered transurethrally. (D) shows that the proliferation of bladder cancer cells is suppressed in mice transplanted with bladder cancer cells in which the siRNA of the present invention was intraurethrally administered into the bladder. Detailed description of the invention
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
(I)膀胱表在性癌の治療又は予防用医薬組成物 本発明の膀胱表在性癌の治療又は予防用医薬組成物は、 ヒト PLK-1 の 発現を特異的に阻害ないしは抑制する siRNAがリボソームに内包された ものを含む。 (I) A pharmaceutical composition for treatment or prevention of superficial bladder cancer The pharmaceutical composition for the treatment or prevention of superficial bladder cancer of the present invention includes those in which siRNA specifically inhibiting or suppressing the expression of human PLK-1 is encapsulated in ribosomes.
siRNA siRNA
この siRNAは、 ヒト PLK- 1遺伝子 （GenBank access i on no.匪一 005030) の連続する 15〜30塩基程度、 特に 19〜23塩基程度にハイブリダィズで きる塩基配列を含むことが好ましい。 ヒト PLK-1遺伝子にハイブリダィ ズできる塩基配列には、 ヒ卜 PLK- 1遺伝子にハイブリダィズしない塩基 領域が 3塩基程度までなら含まれていてもよい。 中でも、 ヒト PLK- 1遺 伝子の連続する 15〜30塩基程度、 特に 19〜23塩基程度にハイブリダィ ズできる塩基配列からなる siRNAが好ましい。 このような siRNAの設計 は、 例えば、 Biochem. Biophys.Res. Commun. ( 2004) Jun 18, 319(1), 264-27 に記載の方法により行うことができる。 選択した配列が PLK- 1 の mRNAのみ発現阻害するか否かは、 例えば BLASTサーチで確認すれば よい。  This siRNA preferably contains a base sequence that can hybridize to about 15 to 30 bases, particularly about 19 to 23 bases, of the human PLK-1 gene (GenBank accession no. 匪 1 005030). The base sequence capable of hybridizing to the human PLK-1 gene may contain up to about 3 base regions that do not hybridize to the human PLK-1 gene. Among them, siRNA having a base sequence capable of hybridizing to about 15 to 30 bases of human PLK-1 gene, particularly about 19 to 23 bases is preferable. Such siRNA can be designed, for example, by the method described in Biochem. Biophys. Res. Commun. (2004) Jun 18, 319 (1), 264-27. Whether the selected sequence inhibits the expression of only PLK-1 mRNA may be confirmed, for example, by a BLAST search.
このような siRNAとして、 例えば配列番号 1〜 4のいずれかの塩基配 列を含む siRNAが挙げられる。 本発明において、 配列番号 1〜4の各塩 基配列を含む siRNAとは、 この塩基配列の RNAとそれに相補的又は略相 補的な塩基配列の MAとが対になった 2本鎖 RNAを含む 2本鎖 RNAを指 す。  Examples of such siRNA include siRNA containing any one of the nucleotide sequences of SEQ ID NOs: 1 to 4. In the present invention, the siRNA containing each of the base sequences of SEQ ID NOs: 1 to 4 is a double-stranded RNA in which an RNA having this base sequence is paired with an MA having a complementary or substantially complementary base sequence. Contains double-stranded RNA.
配列番号 1〜4の各塩基配列を含む siRNAは、 配列番号 1〜4の各塩 基配列を含み、 最大 50塩基程度のものである。 特に、 ヒト PLK-1 遺伝 子の発現阻害作用が強い点で、 配列番号 1〜 4の各塩基配列を含む siRNAが好ましい。  The siRNA containing each base sequence of SEQ ID NOs: 1 to 4 contains each base sequence of SEQ ID NOs: 1 to 4, and has a maximum of about 50 bases. In particular, siRNA containing each of the nucleotide sequences of SEQ ID NOs: 1 to 4 is preferable in that it has a strong inhibitory action on the expression of human PLK-1 gene.
PLK-1 の発現を抑制又は阻害する siRNAの中でも、 特に配列番号 1の 塩基配列を含む siRNA (好ましくは、 配列番号 1の塩基配列からなる siRNA) 、及び配列番号 2の塩基配列を含む siRNA (好ましくは、 配列番 号 2の塩基配列からなる siRNA) は、 膀胱癌細胞又は膀胱の前癌状態の 細胞に効率よく取り込まれて、 PLK- 1 遺伝子の発現を効率的に抑制ない しは阻害する。 Among siRNAs that suppress or inhibit PLK-1 expression, in particular, siRNA comprising the nucleotide sequence of SEQ ID NO: 1 (preferably, siRNA comprising the nucleotide sequence of SEQ ID NO: 1), and siRNA comprising the nucleotide sequence of SEQ ID NO: 2 ( Preferably, the siRNA comprising the nucleotide sequence of SEQ ID NO: 2 is efficiently taken up into bladder cancer cells or cells in the precancerous state of the bladder and does not efficiently suppress the expression of the PLK-1 gene. It inhibits.
また、 配列番号 1の塩基配列、及び配列番号 2の塩基配列において、 ぞれぞれ 1〜 3個程、 特に 1〜 2個程度のヌクレオチドが付加、 欠失、 又は置換された塩基配列を含む s i RNAであっても、 ヒト PLK-1遺伝子の 発現を特異的に阻害又は抑制する限り使用することができる。  In addition, the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 each include a base sequence in which about 1 to 3, particularly about 1 to 2 nucleotides are added, deleted, or substituted. Even siRNA can be used as long as it specifically inhibits or suppresses the expression of the human PLK-1 gene.
また、 本発明の s i RNAは、 PLK- 1 の発現を阻害する限り、 これらのヌ クレオチドの誘導体であってもよい。  The siRNA of the present invention may be a derivative of these nucleotides as long as it inhibits PLK-1 expression.
配列番号 1及び 2の各塩基配列において、 1〜 3個のヌクレオチドが 欠失、 付加、 又は置換された塩基配列を含む s i RNAが PLK-1遺伝子の発 現を抑制するか否かは、 例えば後述する実施例 3で説明するように、 抗 PLK- 1 ポリク口一ナル抗体を用いたウエスタンプロッティングなどによ り確かめればよい。  In each base sequence of SEQ ID NOs: 1 and 2, whether or not siRNA containing a base sequence in which 1 to 3 nucleotides are deleted, added, or substituted suppresses the expression of the PLK-1 gene is, for example, As will be described later in Example 3, it may be confirmed by Western plotting using an anti-PLK-1 polyclonal antibody.
本発明の s i RNAは、 公知の化学合成法により製造できる。  The siRNA of the present invention can be produced by a known chemical synthesis method.
リボソーム Ribosome
リボソームの構成は特に限定されず、 癌の遺伝子治療に使用される公 知のリボソーム材料からなるものを使用すればよい。 このような公知の リボソームとしては、 例えば、 カチオン化されたリボソームが挙げられ る。 カチオン化されたリボソームを用いることにより、 細胞内での浸透 性を向上させることができる。  The structure of the ribosome is not particularly limited, and a ribosome composed of a known ribosome material used for cancer gene therapy may be used. Examples of such known ribosomes include cationized ribosomes. By using a cationized ribosome, permeability in cells can be improved.
リボソームを構成する脂質としては、 例えば、 ホスファチジルコリン (レシチン）、 ホスファチジルェタノ一ルァミン、 ホスファチジルセリ ン、 ホスファチジルイノシト一ル、 スフインゴミエリン、 カルジオリビ ン等の天然または合成のリン脂質、 又はこれらを常法に従って水素添加 したものが挙げられる。 また、 これらのリン脂質にはステロ一ルを併用 してもよい。脂質は、 1種を単独で又は 2種以上を混合して使用できる。 リボソームは、 例えば、 脂質を t —ブチルアルコールなどの溶媒に溶 解させ、冷却後、凍結乾燥することにより作製することができる。また、 作製したリボソームと siRNA溶液とを接触させるだけでも siRNAを内 包したリボソームが得られる。 さらに、 例えば、 脂質に siRNA を溶解 させた液体を加えて膨潤させ、 超音波で分散させた後、 分散体にポリェ チレンダリコール一フォスファチジルエタノールアミンを添加するこ とにより直接 siRNAが封入されたリポソ一ムを得ることができる。 Examples of lipids constituting the ribosome include natural or synthetic phospholipids such as phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardioribine, or the like. Hydrogenated in accordance with the law. These phospholipids may be used in combination with sterol. Lipids can be used singly or in combination of two or more. Ribosomes can be produced, for example, by dissolving lipids in a solvent such as t-butyl alcohol, cooling, and freeze-drying. In addition, ribosomes containing siRNA can be obtained simply by bringing the prepared ribosome into contact with the siRNA solution. For example, siRNA dissolved in lipid After adding the prepared liquid to swell and dispersing with ultrasonic waves, a liposome with entrapped siRNA directly can be obtained by adding polyethylene glycol phosphatidylethanolamine to the dispersion. .
リボソームの大きさは、 その 80重量％以上が、 粒径 50~300 xm程度 であることが好ましく、 粒径 70〜200^m 程度の範囲であることがより 好ましく、 粒径 70〜100 m 程度の範囲であることがさらにより好まし い。 上記の粒径範囲であれば、 十分量の siRNA を封入できるとともに、 リボソームによる毒性が生じない。  The size of the ribosome is preferably 80% by weight or more, preferably a particle size of about 50 to 300 xm, more preferably a particle size of about 70 to 200 ^ m, and a particle size of about 70 to 100 m. It is even more preferable that it is in the range. In the above particle size range, a sufficient amount of siRNA can be encapsulated and no ribosome toxicity occurs.
また、 リボソーム内には、その形態及び機能を維持するために、通常、 siRNA の他にリン酸緩衝液-生理食塩水 （PBS； pH=7〜7.4 程度） のよ うな緩衝液、 生理食塩水、 RPMIのような細胞用培養液、 糖溶液などの液 体成分が含まれていればよい。  In addition, in order to maintain the form and function of the ribosome, in addition to siRNA, a buffer solution such as phosphate buffer-saline (PBS; pH = 7 to 7.4), physiological saline It only needs to contain a liquid component such as a culture solution for cells such as RPMI and a sugar solution.
リボソーム内の siRNAの濃度は、 100nM〜lmM程度であることが好まし く、 100nM〜 6 ζΜ程度であることがより好ましい。 上記の siRNAの封入 量の範囲であれば治療効率が良い。 また、 現実に siRNAを封入できる範 囲の量である。 なお、 siRNA を内包するリボソームは、 siRNA を懸濁し た溶液とリボソームとの接触により行うことから、 本発明に規定する The siRNA concentration in the ribosome is preferably about 100 nM to lmM, and more preferably about 100 nM to 6 ζΜ. The therapeutic efficiency is good if the amount of siRNA enclosed is within the above range. In addition, the amount of siRNA can actually be encapsulated. The ribosome encapsulating siRNA is defined by the present invention because it is brought into contact with a solution in which siRNA is suspended and the ribosome.
「リボソーム内 siRNA濃度」 は、 リボソーム作製の際にリボソームと接 触させる溶液中の siRNA濃度である。 本発明においては、 この濃度をリ ポソ一ム内 siRNA濃度とみなす。 “Intraribosomal siRNA concentration” is the concentration of siRNA in the solution that comes into contact with the ribosome during ribosome production. In the present invention, this concentration is regarded as the siRNA concentration in the liposome.
(II)膀胱表在性癌の治療又は予防方法  (II) Treatment or prevention of superficial bladder cancer
本発明の膀胱表在性癌の治療又は予防方法は、 膀胱表在性癌又は膀胱 の前癌病変を有するヒトの膀胱内に、 経尿道的に、 上記説明した本発明 の siRNA内包リボソーム（本発明の膀胱癌の治療又は予防用医薬組成物） を投与する方法である。  The method for treating or preventing superficial bladder cancer according to the present invention comprises the above-described siRNA-encapsulating ribosome of the present invention in the human bladder having a superficial bladder cancer or a precancerous lesion of the bladder transurethrally (this book). The pharmaceutical composition for treatment or prevention of bladder cancer of the invention).
本発明方法の対象となる患者は、 未処置の膀胱表在性癌を有するヒト、 膀胱表在性癌を摘除後のヒト、 抗癌剤を投与したことにより膀胱癌が小 さくなったヒト、 膀胱内表面に前癌状態の病変を有するヒトなどである。 膀胱癌細胞は複数層重なっているため、 膀胱表在性癌を摘除しても、 通 常はわずかに癌細胞が残存する。 このような微小残存病変を有するヒト も本発明方法の対象となる。 このような状態の患者に本発明の s i RNA内 包リボソームを投与することにより、 癌細胞の増殖を抑制することがで き、 また前癌組織の癌への進行を抑制することができる。 最も高い治療 効果を期待できる対象は、 膀胱癌を摘除後に、 膀胱内表面に微小残存癌 病変を有するヒトである。 Patients subject to the method of the present invention include humans with untreated bladder superficial cancers, humans after removal of superficial bladder cancers, humans whose bladder cancer has been reduced by administration of anticancer agents, intravesical A human having a precancerous lesion on the surface. Since bladder cancer cells are stacked in multiple layers, even if the superficial bladder cancer is removed, Usually, a few cancer cells remain. Humans having such minimal residual lesions are also subject to the method of the present invention. By administering the siRNA-encapsulating ribosome of the present invention to a patient in such a state, the growth of cancer cells can be suppressed, and the progression of precancerous tissue to cancer can be suppressed. The subject who can expect the highest therapeutic effect is a human who has a minimal residual cancer lesion on the inner surface of the bladder after the removal of the bladder cancer.
s i RNA 内包リボソームは、 例えば PBS、 生理食塩水、 細胞用培養液、 糖溶液等に懸濁した状態で、 尿道口から、 通常カテーテルを用いて尿道 を通して膀胱内に投与すればよい。 これにより、 膀胱癌細胞内に s iRNA が導入される。  The siRNA-encapsulated ribosome may be administered into the bladder through the urethra from the urethral orifice, usually using a catheter, in a state suspended in, for example, PBS, physiological saline, cell culture medium, sugar solution, or the like. As a result, siRNA is introduced into bladder cancer cells.
s i RNA 内包リボソームの 1回投与量は、 s iRNA 量に換算して 12 x g〜 120nig 程度が好ましく、 50 g〜10mg 程度がより好ましい。 上記の投与 量の範囲であれば、 十分に治療又は予防効果が得られるとともに、 非特 異的な作用が生じることもない。  The single dose of siRNA-encapsulated ribosome is preferably about 12 x g to 120 nig, more preferably about 50 g to 10 mg, in terms of the amount of siRNA. Within the above dose range, a sufficient therapeutic or prophylactic effect can be obtained, and non-specific effects do not occur.
また、 この量を、 3〜10回程度に分けて投与することが好ましい。 上 記投与回数の範囲であれば、 副作用の出るような 1回投与量になること がなく、 かつ患者の負担が小さい。  In addition, it is preferable to administer this amount divided into about 3 to 10 times. If the number of doses is within the above range, the single dose will not cause side effects and the burden on the patient is small.
また、投与間隔は 1〜 7日間程度が好ましい。上記投与間隔であれば、 感染症を誘発せず、患者の負担が小さい。また、上記投与間隔であれば、 患者体内で有効 s iRNA濃度を保つことができる。  The administration interval is preferably about 1 to 7 days. With the above administration interval, no infection is induced and the burden on the patient is small. In addition, the effective siRNA concentration can be maintained in the patient body within the above administration interval.
(I I I)膀胱表在性癌の予防又は治療用医薬組成物としての使用 (I I I) Use as a pharmaceutical composition for prevention or treatment of superficial bladder cancer
上記説明した本発明の s iRNA内包リボソームは、 膀胱表在性癌の予防 又は治療用医薬組成物として使用できる。 使用の好ましい態様は、 前述 した通りである。 実施例  The siRNA-encapsulating ribosome of the present invention described above can be used as a pharmaceutical composition for prevention or treatment of superficial bladder cancer. The preferred mode of use is as described above. Example
以下、 本発明を実施例を示してより詳細に説明するが、 本発明はこれ らに限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例 1 膀胱癌細胞における PLK- 1の発現 Example 1 Expression of PLK-1 in bladder cancer cells
滋賀医科大学にて根治的膀胱全摘が施行され Helsinki 宣言に基づい てィンフォームドコンセントを得た 58 例の膀胱癌患者から摘除された 癌組織の標本を用意した。 一次抗体として、 100 倍希釈の抗ヒト PLK-1 モノクロ一ナ レ抗体（Transduct ion Laborator ies, Lexington, KY) を 用い、 周知のアビジン一ピオチン—パーォキシダーゼ複合体法 ( ABC-El i te, Vector Laboratories, Burl ingame, CA ； Jpn. J. Cancer Res.93, 523-531) により免疫組織染色を行った。  We prepared specimens of cancer tissue removed from 58 patients with bladder cancer who underwent radical total cystectomy at Shiga Medical University and obtained informed consent based on the Helsinki declaration. As a primary antibody, a 100-fold diluted anti-human PLK-1 monoclonal antibody (Transduct ion Laboratories, Lexington, KY) was used, and the well-known avidin-piotin-peroxidase complex method (ABC-El ite, Vector Laboratories, Burl ingame, CA; Jpn. J. Cancer Res. 93, 523-531).
結果を図 1に示す。 図 1の A， B, Cは、 グレードの高い未分化な筋層 浸潤を認める膀胱癌である。 E， Fはグレードの低い高分化で表在性の膀 胱癌である。 グレードの高い癌組織の方が、 PLK- 1が高発現しているこ とが分かる。 また、 Dはリンパ管浸潤が認められる組織であるが、 この 組織にも PLK-1は高発現していることが分かる。  The results are shown in Figure 1. In Fig. 1, A, B, and C are bladder cancers with high grade undifferentiated muscle layer infiltration. E and F are well-differentiated and superficial bladder cancers of low grade. It can be seen that PLK-1 is highly expressed in higher grade cancer tissues. D is a tissue with lymphatic invasion, and PLK-1 is also highly expressed in this tissue.
また、 58標本について、 膀胱癌の臨床病理学的特徴と PLK-1 発現レ ベルとの関係を以下の表 1に示す。 Table 1 shows the relationship between the clinicopathological characteristics of bladder cancer and the PLK-1 expression level for 58 specimens.
表 1 table 1
Figure imgf000012_0001
Figure imgf000012_0001
表 1から、 腫瘍の筋層浸潤の有無、 リンパ管浸潤の有無、 及び悪性度 と PLK-1 の発現との間には正の相関性が認められた。 即ち、 グレードの 高い膀胱癌組織ではグレードの低い膀胱癌組織より PLK-1 が高発現し ている。 また、 表在性の癌より侵襲性の癌の方が PLK-1 が高発現して いる。 さらに、 リンパ管浸潤が認められる癌では PLK-1が高発現してい る。 このように、 PLK-1は膀胱癌において特に高発現していることが分か る。 From Table 1, a positive correlation was observed between the presence or absence of muscle invasion in the tumor, the presence or absence of lymphatic vessel invasion, and the degree of malignancy and the expression of PLK-1. That is, PLK-1 is highly expressed in bladder cancer tissue of higher grade than in bladder cancer tissue of lower grade. Also, PLK-1 is highly expressed in invasive cancers than in superficial cancers. Furthermore, PLK-1 is highly expressed in cancers with lymphatic vessel invasion. Thus, it can be seen that PLK-1 is particularly highly expressed in bladder cancer.
実施例 2 Example 2
膀胱癌細胞株における PLK- 1 の発現 Expression of PLK-1 in bladder cancer cell lines
次に、 膀胱癌の樹立細胞株における PLK-1 の発現量を検討した。 ヒト 膀胱癌細胞株の 253J, 5637, HT1197, HT1376, J82, RT4, RT112, SCaBER, TCCSUP, KU-7, 及び 醫- UC- 3； マウス膀胱癌細胞株の MBT-2； ヒト正 常肝細胞 Hepatocyte；並びにヒト正常線維芽細胞 HF， NHDFを American Type Culture collection (Rockville, MD) から購入した。 これらの細 胞から調製したタンパク質について、 抗ヒト PLK- 1ポリクローナル抗体 (rabbit; Upstate Biotechnology, Waltham, MA) を用いてウェスタン · ブロッティングを行った。  Next, the expression level of PLK-1 in an established cell line of bladder cancer was examined. Human bladder cancer cell lines 253J, 5637, HT1197, HT1376, J82, RT4, RT112, SCaBER, TCCSUP, KU-7, and 醫 -UC-3; Mouse bladder cancer cell line MBT-2; Human normal hepatocytes Hepatocyte; and normal human fibroblasts HF and NHDF were purchased from American Type Culture collection (Rockville, MD). Proteins prepared from these cells were subjected to Western blotting using an anti-human PLK-1 polyclonal antibody (rabbit; Upstate Biotechnology, Waltham, MA).
結果を図 2に示す。 図 2から、 膀胱癌細胞株では正常細胞に比べて PLK-1が非常に強く発現していることが分かる。  The result is shown in figure 2. Figure 2 shows that PLK-1 is very strongly expressed in bladder cancer cell lines compared to normal cells.
実施例 1及び 2より、 膀胱癌の浸潤 ·悪性化に PLK- 1が重要な役割を 果たしていることが示唆された。 また、 PLK- 1 が膀胱癌の分子治療の標 的として好適であることが分かる。  From Examples 1 and 2, it was suggested that PLK-1 plays an important role in the invasion / malignancy of bladder cancer. It can also be seen that PLK-1 is suitable as a molecular therapy target for bladder cancer.
実施例 3 Example 3
本発明の siRNA内包リポソ一ムを用いた膀胱癌細胞株における PLK- 1発 現の抑制 Inhibition of PLK-1 expression in bladder cancer cell lines using siRNA-encapsulating liposomes of the present invention
(A) PLK- 1 に対する配列番号 1〜4の 4種類の siRNAを化学合成し た （Nippon- Shinyaku Co., Kyoto, Japan) 。 これらの siRNA の塩基配 列は以下の通りである。  (A) Four types of siRNAs of SEQ ID NOs: 1 to 4 for PLK-1 were chemically synthesized (Nippon- Shinyaku Co., Kyoto, Japan). The nucleotide sequences of these siRNAs are as follows.
PLK-1 siRNA 1345: 5， -GACAGCCUGCAGUACAUAGdTdT-3 ' (配列番号 1 ) PLK-1 siRNA 1412: 5， - CCUUGAUGAAGA AGAUCACdTdT- 3 ' (配列番号 2 ) PLK-1 siRNA 183: 5' -GGGCGGCUUUGCCAAGUGCdTdT-3 ' (配列番号 3 ) PLK-1 siRNA 1418: 5， -GAAGAAGAUCAC CCUCCUUdTdT-3 ' (配列番号 4) カチオン性脂質アナログを含むカチオン製リポソ一ム （Cancer Res. 1999 Sep 1;59(17):4325_33.に記載のリボソーム） に、 それぞれ上記 4 種の siRNAを接触により封入した。 このリボソームは、 その 80重量％ 以上が粒径 70〜80/zmである。 これらのリボソーム内には、 10%マルト ース水溶液が満たされており、 siRNAが ΙΟΟηΜ含まれている。 この siRNA 濃度は、 siRNA封入リボソームの作製時に使用した siRNA溶液中の siRNA 濃度である。 PLK-1 siRNA 1345: 5, -GACAGCCUGCAGUACAUAGdTdT-3 '(SEQ ID NO: 1) PLK-1 siRNA 1412: 5,-CCUUGAUGAAGA AGAUCACdTdT- 3' (SEQ ID NO: 2) PLK-1 siRNA 183: 5 '-GGGCGGCUUUGCCAAGUGCdTdT-3' (SEQ ID NO: 3) PLK-1 siRNA 1418: 5, -GAAGAAGAUCAC CCUCCUUdTdT-3 ′ (SEQ ID NO: 4) Cationic liposome containing a cationic lipid analog (Cancer Res. 1999 Sep 1; 59 (17): 4325_33. In each of the above 4) Species siRNA was encapsulated by contact. More than 80% by weight of the ribosome has a particle size of 70-80 / zm. These ribosomes are filled with 10% aqueous maltose solution and contain siRNA. This siRNA concentration is the siRNA concentration in the siRNA solution used in the preparation of the siRNA-encapsulated ribosome.
ヒト膀胱癌細胞株 HT1376、 マウス膀胱癌細胞株 MBT-2、 及びヒト子宮 顏癌細胞株 HeLa における PLK- 1 タンパク質の発現に与える上記 4種の siRNA 内包リボソームの影響を以下のようにして調べた。 即ち、 上記各 細胞培養に対して上記 siRNA封入リボソームを添加することにより細胞 に siRNAを導入した。 siRNA導入後の細胞からタンパク質を調製し、 実 施例 2と同様にして、 抗ヒト PLK- 1ポリクロ一ナル抗体を用いたウェス タン · ブロッテイングを行った。  The effects of the above four siRNA-encapsulating ribosomes on the expression of PLK-1 protein in human bladder cancer cell line HT1376, mouse bladder cancer cell line MBT-2, and human uterine uterine cancer cell line HeLa were examined as follows. . That is, siRNA was introduced into cells by adding the siRNA-encapsulated ribosome to each cell culture. Proteins were prepared from the cells after siRNA introduction, and Western blotting using anti-human PLK-1 polyclonal antibody was performed in the same manner as in Example 2.
結果を図 3 (A)に示す。 図 3 (A)中、 非処理は siRNA内包リポソ一ムを 作用させない細胞を示し、 コ ン ト 口一ルは、 ナンセンス配列 ( 5 ' -UUCUCCGAACGUGUCACGUdTdT-3 ' (配列番号 5 ))を有する siRNA を作用させた細胞を示す。 図 3から、 4種の siRNA は癌細胞における PLK- 1 の発現を抑制しているが、 中でも PLK- 1 1412及び: PLK- 1 1418が PLK-1 の発現を強く抑制し、 特に PLK-1 1412が最も効果的に PLK- 1の発 現を抑制していることが分かる。  The results are shown in Fig. 3 (A). In Fig. 3 (A), untreated cells represent cells that do not allow siRNA-encapsulating liposomes to act, and the constrictor contains siRNA having a nonsense sequence (5'-UUCUCCGAACGUGUCACGUdTdT-3 '(SEQ ID NO: 5)). Cells that have been allowed to act are shown. From Fig. 3, four siRNAs suppressed the expression of PLK-1 in cancer cells. Among them, PLK-1 1412 and: PLK-1 1418 strongly suppressed the expression of PLK-1, especially PLK-1. It can be seen that 1412 most effectively suppresses the expression of PLK-1.
(B) また、 PLK-1 1412の膀胱癌細胞株 UM- UC- 3における siMA発現 抑制を経時的に観察した。 即ち、 siRNA 内包リボソームと細胞との接触 時間を 0、 2、及び 4時間として、各場合の UM-UC- 3細胞における PLK-1 タンパク質の発現量を、 実施例 2と同様にして、 抗ヒト PLK-1ポリクロ ーナル抗体を用いたウエスタンプロッティングで調べた。 結果を図 3 (B)に示す。 図 3 (B)より、 PLK-1 1412は PLK- 1の発現を時間依存的に抑 制することが分かる。 (B) In addition, inhibition of siMA expression in PLK-1 1412 in the bladder cancer cell line UM-UC-3 was observed over time. That is, the contact time between the siRNA-encapsulated ribosome and the cells was set to 0, 2, and 4 hours, and the expression level of PLK-1 protein in UM-UC-3 cells in each case was determined in the same manner as in Example 2. It was examined by Western plotting using PLK-1 polyclonal antibody. The results are shown in Fig. 3 (B). From FIG. 3 (B), it can be seen that PLK-1 1412 suppresses PLK-1 expression in a time-dependent manner.
(C) また、 PLK- 1 1412の使用量と膀胱癌細胞株 UM- UC- 3及び HT1376 における siRNA発現抑制効果との関係を同様にして調べた。 即ち、 膀胱 癌細胞株 UM- UC- 3 については、 リボソーム内の PLK-1 1412 濃度 （リポ ゾームと接触させる siRNA溶液中の siRNA濃度）を 1ηΜ、 ΙΟηΜ、及び ΙΟΟηΜ に変化させ、 膀胱癌細胞株 ΗΊ 376 については、 リボソーム内 siRNA濃 度（リボソームと接触させる siRNA溶液中の siRNA濃度） を 3nM、 10nM、 30ηΜ、 ΙΟΟηΜに変化させて、 各場合の膀胱癌細胞における PLK- 1 タンパ ク質の発現量を、 実施例 2と同様にして、 抗ヒト PLK- 1ポリクローナル 抗体を用いたウエスタンプロッティングにより調べた。 (C) Also, the amount of PLK-1 1412 used and bladder cancer cell lines UM-UC-3 and HT1376 In the same manner, the relationship with the siRNA expression inhibitory effect was investigated. That is, for the bladder cancer cell line UM-UC-3, the PLK-1 1412 concentration in the ribosome (siRNA concentration in the siRNA solution in contact with the liposome) was changed to 1ηΜ, ΙΟηΜ, and ΙΟΟηΜ, and the bladder cancer cell lineに つ い て For 376, change the siRNA concentration in the ribosome (siRNA concentration in the siRNA solution in contact with the ribosome) to 3nM, 10nM, 30ηΜ, and ΙΟΟηΜ to express PLK-1 protein in bladder cancer cells in each case The amount was examined in the same manner as in Example 2 by Western plotting using an anti-human PLK-1 polyclonal antibody.
結果を図 3 (C)に示す。 いずれの膀胱癌細胞を用いた場合も、 PLK - 1 1412は用量依存的に PLK- 1の発現を抑制している。  The results are shown in Fig. 3 (C). Regardless of which bladder cancer cell is used, PLK-1 1412 suppresses the expression of PLK-1 in a dose-dependent manner.
以上より、 本発明の siRNAを内包するリボソームは、 膀胱癌細胞に作 用して PLK- 1 の発現を抑制することが分かる。  From the above, it can be seen that the ribosome encapsulating the siRNA of the present invention acts on bladder cancer cells and suppresses the expression of PLK-1.
(D) PLK-1 タンパク質は細胞周期の制御に重要な役割を果たしてお り、 サイクリン B1 の分解に関与するとされている。 サイクリン B1 の分 解に及ぼす PLK-1 1412 の影響を調べるために、 上記各濃度の siRNAを 内包するリボソームを導入した膀胱癌細胞株 UM- UC-3及び ΗΠ 376 につ いて、 サイクリン B1タンパク質の発現量を、 ゥサギ抗サイクリン B1ポ リク口一ナル抗体 （Santa Cruz Biotechnology, Santa Cruz, CA) を用い たウェスタンプロッティングにより調べた。 (D) PLK-1 protein plays an important role in cell cycle control and is thought to be involved in the degradation of cyclin B1. In order to investigate the effect of PLK-1 1412 on the degradation of cyclin B1, we investigated the effects of cyclin B1 protein on bladder cancer cell lines UM-UC-3 and 376376 into which ribosomes encapsulating each of the above concentrations of siRNA were introduced. The expression level was examined by Western plotting using a rabbit anti-cyclin B1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
結果を図 3(C)に示す。 図 3 (C)から、 PLK-1 1412は濃度依存的にサイ クリン B1の分解を抑制していることが分かる。  The results are shown in Fig. 3 (C). Figure 3 (C) shows that PLK-1 1412 suppresses the degradation of cyclin B1 in a concentration-dependent manner.
実施例 4 Example 4
本発明の siRNAによる紡錐体形成の抑制 Inhibition of spindle formation by siRNA of the present invention
PL -1 タンパク質の重要な役割とされている紡錐体形成に及ぼす siRNAの影響を以下のようにして調べた。  The influence of siRNA on spindle formation, which is considered to be an important role of PL -1 protein, was examined as follows.
膀胱癌細胞株 UM-UC-3(ATCC; American Type Culture Collection)に、 PLK-1 siRNA 1412内包リボソームを実施例 3と同様にして上記細胞に作 用させた。 siRNA を導入したが膀胱癌細胞及び導入しないコントロール の膀胱癌細胞について、 ]3—チューブリン及びァ一チューブリンに特異 的な抗体 (Sigma, ST.Louis,M0) 及び Hoechs t 33342 DNA染色 (Molecular Probes, Eugene, OR)を用いた免疫細胞染色を、 これらに添付されたマ ニュアルに従って行った。 In the same manner as in Example 3, PLK-1 siRNA 1412-encapsulated ribosome was allowed to act on the above cells in the bladder cancer cell line UM-UC-3 (ATCC; American Type Culture Collection). Introduced siRNA but not bladder cancer cells and controls ]-Immunocytostaining with antibodies specific for tubulin and tubulin (Sigma, ST. Louis, M0) and Hoechs t 33342 DNA staining (Molecular Probes, Eugene, OR) Were carried out according to the manual attached to them.
結果を図 4に示す。 β—チューブリン及びァ—チューブリン染色によ り、 PLK-1 siMA 1412を導入しないコントロール細胞では、 正常に形成 された M-期の双極性の紡錐体が観察された。 また、 Hoechst33342 DNA 染色により、 染色体 DNA が細胞の中心に整列しているのが観察された。 これに対して、 PLK- 1 siRNA 1412を導入した膀胱癌細胞では、 M-期の円 形の細胞が増加し、 双極性の紡錐体の形成が強く抑制されている。 サ イクリン B1の分解が抑制され、また紡錐体形成が抑制されることから、 本発明の siRNAを内包するリボソームは、 膀胱癌細胞に作用して PLK - 1 の機能を効果的に抑制することが分かる。  The results are shown in Fig. 4. By β-tubulin and a-tubulin staining, normally formed M-phase bipolar spindles were observed in control cells not transfected with PLK-1 siMA 1412. Hoechst33342 DNA staining showed that chromosomal DNA was aligned in the center of the cell. In contrast, in bladder cancer cells into which PLK-1 siRNA 1412 was introduced, the number of M-phase circular cells increased, and the formation of bipolar spindles was strongly suppressed. Since the degradation of cyclin B1 is suppressed and the formation of spindles is also suppressed, the ribosome encapsulating the siRNA of the present invention acts on bladder cancer cells and effectively suppresses the function of PLK-1. I understand.
実施例 5 Example 5
本発明の siRNA内包リボソームのアポトーシス誘導に対する影響 Effect of siRNA-encapsulating ribosome of the present invention on apoptosis induction
PLK-1 siRNA 1412を内包するリボソームが膀胱癌細胞の細胞周期に及 ぼす影響を以下のようにして調べた。  The effect of ribosomes encapsulating PLK-1 siRNA 1412 on the cell cycle of bladder cancer cells was examined as follows.
(A) UM-UC-3 膀胱癌細胞株について propidium iodide (PI)の単染 色を行い、 Fluorescence- Activated Cell Sorting (FACS) で細胞周期 を調べた。 また、 実施例 3と同様にして ΙΟΟηΜ濃度の PLK-1 siRNA 1412 を内包するリボソームを UM- UC- 3細胞に作用させて 24時間培養した細 胞についても、 同様にして PI の単染色を行い、 FACS により細胞周期を 調べた。  (A) The UM-UC-3 bladder cancer cell line was stained with propidium iodide (PI) and the cell cycle was examined by fluorescence-activated cell sorting (FACS). In the same manner as in Example 3, ribosomes containing PLK-1 siRNA 1412 at a concentration of ΙΟΟηΜ were allowed to act on UM-UC-3 cells and cultured for 24 hours in the same manner. The cell cycle was examined by FACS.
結果を図 5 (A)に示す。 図 5 (A)より、 PLK-1 siRNA 1412を導入しない コントロール膀胱癌細胞ではアポトーシスは誘導されていないが、 PLK-1 siRNA 1412 を導入した膀胱癌細胞では、 G2/M 期にて細胞周期が 停止され、 アポト一シスが誘導されていることが分かる。  The results are shown in Fig. 5 (A). From Fig. 5 (A), apoptosis was not induced in the control bladder cancer cells not introduced with PLK-1 siRNA 1412, but in the bladder cancer cells introduced with PLK-1 siRNA 1412, the cell cycle was at G2 / M phase. It can be seen that it has been stopped and apoptosis has been induced.
(B) 同様に、 PLK-1 siRNA 1412を導入しない UM- UC- 3膀胱癌細胞株、 コントロール siRNA (配列番号 5 ) を導入した UM- UC- 3膀胱癌細胞、 及 び PLK- 1 siRNA 1412を導入した UM- UC- 3膀胱癌細胞について、 MEBSTAIN apoptosis Kit II (MBL, Nagoya, Japan)を用レ て Annexin V 染色を行 つた。 この場合、 リボソーム内 siRNA濃度は 100nM、 細胞との接触時間 は 36時間とした。 (B) Similarly, a UM-UC-3 bladder cancer cell line that does not introduce PLK-1 siRNA 1412, MEBSTAIN apoptosis Kit II (MBL, Nagoya, Japan) for UM-UC-3 bladder cancer cells transfected with control siRNA (SEQ ID NO: 5) and UM-UC-3 bladder cancer cells transfected with PLK-1 siRNA 1412 Annexin V staining was carried out using this. In this case, the siRNA concentration in the ribosome was 100 nM, and the contact time with the cells was 36 hours.
結果を図 3(B)に示す。 図 3(B)の Aは染色写真であり、 Bは生存細胞数 である。 図 3(B)から、 PLK- 1 siRNA 1412 を作用させた UM- UC-3膀胱癌 細胞では Annexin Vポジティブである細胞の比率が高く、 アポトーシス の誘導が確認された。  The results are shown in Fig. 3 (B). A in Fig. 3 (B) is a stained photograph, and B is the number of viable cells. From FIG. 3 (B), the percentage of Annexin V positive cells was high in UM-UC-3 bladder cancer cells treated with PLK-1 siRNA 1412, confirming the induction of apoptosis.
(C) さらに、 実施例 3と同様にして、 膀胱癌細胞株濯- UC- 3, 253J, 及び KU- 7に、 ΙΟΟηΜの PLK-1 siRNA 1412を内包するリボソームを 1, 3， 6， 及び 72 時間作用させた後の生存細胞数をカウントした。 PLK-1 siRNA 1412による上記各細胞に対する増殖抑制効果の IC5。はそれぞれ、 46.1ηΜ， 94.5nM, 及び 60.4 Μであった。 (C) Further, in the same manner as in Example 3, the ribosome encapsulating PLK-1 siRNA 1412 of KUη を was added to the bladder cancer cell line Rinse-UC-3, 253J, and KU-7 in the same manner as in Example 3, The number of viable cells after counting for 72 hours was counted. IC 5 of the growth inhibitory effect of PLK-1 siRNA 1412 on the above cells. Were 46.1 ηΜ, 94.5 nM, and 60.4 そ れ ぞ れ, respectively.
後述する実施例 6において、 PLK- 1 siRNA 1412を内包するリポソ一ム は、 in vivoで 150nM〜 6 /z Mの濃度範囲でがん細胞増殖抑制効果が認め られたことから、 in vitroでの IC5。の 3〜120倍の濃度で in 効果 が認められたことになる。 In Example 6, which will be described later, the liposome encapsulating PLK-1 siRNA 1412 was found to have an inhibitory effect on cancer cell proliferation in a concentration range of 150 nM to 6 / z M in vivo. IC 5 . The in effect was observed at a concentration 3 to 120 times higher than that of.
なお、 A431膀胱癌細胞株 （AKC) を用い、 siRNA内包リボソームとし てそれぞれ PLK- 1 siRNA 246, 1345, 1412,及び 246を内包するリボソーム を用い、 同様にして IC5。を求めたところ、 それぞれ 221nM、 357nM、 2nM、 13nMであった。 Incidentally, using the A431 bladder cancer cell lines (AKC), respectively PLK- 1 siRNA 246 as the siRNA contained ribosomes, 1345, 1412, and 246 using the ribosomes containing the, IC 5 in the same manner. Was found to be 221 nM, 357 nM, 2 nM, and 13 nM, respectively.
また、 膀胱癌細胞株 UM-UC- 3, 253J,及び KU-7について、 実施例 3と同 様にして、 濃度 ΙΟΟηΜの PLK-1 siRNA 1412 を内包するリポソ一ムを作 用させ、 0 時間、 1時間、 2時間、 及び 3時間後の生存細胞数をカウン トした。 PLK-1 siRNA 1412を作用させた各細胞の相対細胞生存数は、 コ ントロ一ル細胞の当初生存細胞数を 1.0 とした場合、 UM- UC- 3細胞では 0.187±0.0191 及び 0.0891 ±0.0290 であり、 253J 細胞では 0.705土 0.0342 及び 0.399 ±0.0693 であり、 KU- 7 細胞では 0.466土 0.108 及び 0.243士 0.0717であった。 In addition, for the bladder cancer cell lines UM-UC-3, 253J and KU-7, a liposome containing PLK-1 siRNA 1412 at a concentration of 作 ηΙΟΟ was applied in the same manner as in Example 3 for 0 hour. The number of viable cells after 1, 2 and 3 hours was counted. The relative cell viability of each cell treated with PLK-1 siRNA 1412 is 0.187 ± 0.0191 and 0.0891 ± 0.0290 for UM-UC-3 cells, assuming that the initial viable cell number of control cells is 1.0. 0.75J for 253J cells 0.0342 and 0.399 ± 0.0693, and 0.466 soil 0.108 and 0.243 0.0717 for KU-7 cells.
結果を図 5(C)に示す。図 5(C)より、 ΙΟΟηΜという比較的高濃度の PLK-1 siRNA 1412を内包するリボソームで膀胱癌細胞を処理した場合は、 1〜3 時間という短時間で生存細胞数が減少したことが分かる。  The results are shown in Fig. 5 (C). Figure 5 (C) shows that when bladder cancer cells were treated with a relatively high concentration of PLK-1 siRNA 1412 (ΙΟΟηΜ), the number of viable cells decreased in a short time of 1 to 3 hours. .
これは、 PLK- 1 siRNA 1412によって PLK-1タンパク質の発現が阻害さ れた結果、 膀胱癌細胞の増殖が抑制されたものと考えられる。  This is thought to be because the proliferation of bladder cancer cells was suppressed as a result of the inhibition of PLK-1 protein expression by PLK-1 siRNA 1412.
実施例 6 Example 6
マウス正所性モデルを用いた膀胱癌増殖抑制効果 Bladder cancer growth inhibitory effect using mouse orthotopic model
(A) in vivoにおいて膀胱癌に対する PLK- 1 siRNA 1412の抗腫瘍効 果を観察するために、 ルシフェラ一ゼ遺伝子を導入した膀胱癌細胞株を 用いた正所性膀胱癌マウスモデルを確立した。  (A) In order to observe the antitumor effect of PLK-1 siRNA 1412 against bladder cancer in vivo, an orthotopic bladder cancer mouse model using a bladder cancer cell line into which a luciferase gene was introduced was established.
リポフエクタミン 2000 (Invitrogen, Carlsbad, CA) を用いて、 マウス 膀胱癌細胞株 UM- UC- 3に pGL3 (Promega, Madison, WI)及び pSV2ネオべ クタ一 （ATCC)を導入した。 このようにして得られた LUC -ラベルされた 膀胱癌細胞 1 X106個を、 マウス膀胱へ 24Gのアンギオ'カテーテルを用 いて移植した。 移植して 10 分後、 1日後、 1週間後、 3週間後、 及び 4 週間後に、 Xenogen 社の in vivo イ メ ージ ングシス テム (Xenogen, Alameda, CA) を用いてマウスを観察した。 PGL3 (Promega, Madison, WI) and pSV2 neovector-1 (ATCC) were introduced into the mouse bladder cancer cell line UM-UC-3 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The 6 LUC-labeled bladder cancer cells thus obtained were transplanted into 1 × 10 6 mouse mice using a 24G angio 'catheter. Mice were observed using the Xenogen in vivo imaging system (Xenogen, Alameda, CA) 10 minutes, 1 day, 1 week, 3 weeks, and 4 weeks after transplantation.
結果を図 6 (A)に示す。 Aは 10分後、 Bは 1 日後、 Cは 1週間後、 Dは 3週間後、 E は 4週間後の様子を示す。 膀胱癌細胞がマウス膀胱で増殖 していることが観察される。  The results are shown in Fig. 6 (A). A shows 10 minutes later, B 1 day later, C 1 week later, D 3 weeks later, E 4 weeks later. It is observed that bladder cancer cells are growing in the mouse bladder.
このことから、 上記システムにより、 非観血的に反復的に、 さらに定 量的に膀胱癌細胞の増殖を観察できることが分かる。  From this, it can be seen that the above system can observe the proliferation of bladder cancer cells non-invasively and quantitatively, and further quantitatively.
(B) 次いで、 実施例 3と同様にして、 濃度 100nMの siRNAを内包す るリボソームを作製した。 さらに、 このリボソームを FITC (ピーク波長 530nm の蛍光を発する） でラベルした。 LUC-ラベルされた膀胱癌細胞を 移植したマウスの膀胱からカテーテルを用いて排尿させた後、 力テーテ ルを用いて尿道口から膀胱内に上記リポソ一厶を 200 x 1注入した。 リボソーム注入の 24 時間後に、 マウスの膀胱を摘出し、 蛍光顕微鏡 を用いて観察した。 結果を図 6 (B)に示す。 図 6(B)の A，Bはリボソーム を投与した場合、 C，D はリボソームを投与しない場合、 A，C は蛍光顕微 鏡で観察した場合、 B，D は連続切片をへマトキシリン 'ェォジン（HE)染 色した結果を示す。 図 6(B)Aに示すように、 リボソームを投与した場合 は、 膀胱部分に蛍光が観察される。 このことから、 経尿道的に siRNA内 包リボソームを投与することにより、 膀胱癌組織に浸透することが分か る。 (B) Next, in the same manner as in Example 3, a ribosome encapsulating siRNA at a concentration of 100 nM was prepared. Furthermore, this ribosome was labeled with FITC (fluorescent with a peak wavelength of 530 nm). After urinating from the bladder of a mouse transplanted with LUC-labeled bladder cancer cells using a catheter, 200 × 1 of the above-mentioned liposo was injected into the urinary bladder from the urethral orifice. Twenty-four hours after ribosome injection, mouse bladders were removed and observed using a fluorescence microscope. The results are shown in Fig. 6 (B). In Fig. 6 (B), A and B are administered with ribosome, C and D are not administered with ribosome, A and C are observed with a fluorescence microscope, and B and D are serial sections of hematoxylin HE) Shows the dyed result. As shown in Fig. 6 (B) A, when ribosome is administered, fluorescence is observed in the bladder. This indicates that administration of siRNA-encapsulating ribosomes transurethrally penetrates the bladder cancer tissue.
(C) 次いで、 リボソームを投与したマウス及びリボソームを投与し ないコントロールのマウスの膀胱癌組織を摘出し、 実施例 2と同様にし て、 但し、 抗 PLK-1ポリクローナル抗体に代えて抗 PLK-1モノクローナ ル抗体を用いてウェスタンブロッティングにより PLK- 1の発現量を調べ た。 (C) Next, the bladder cancer tissues of the mice administered with ribosome and the control mice not administered with ribosome were excised and treated in the same manner as in Example 2 except that anti-PLK-1 polyclonal antibody was used instead of anti-PLK-1 polyclonal antibody. The expression level of PLK-1 was examined by Western blotting using a monoclonal antibody.
また、 摘出した膀胱癌組織について、 HE染色も行った。  The resected bladder cancer tissue was also subjected to HE staining.
結果を図 6 (0に示す。 図 6 (C)の A，Bは siRNA内包リボソームで治療 したマウスの組織であり、 D はこの治療を行わないマウスの組織であ る。 また、 A， Cは抗 PLK- 1モノクローナル抗体を用いた場合であり、 B,D は HE染色による場合である。 図 6(C)から、 siMA内包リボソームを経 尿道的に投与することにより、 PLK- 1 の発現が抑制されていることが分 かる。  The results are shown in Fig. 6 (0. A and B in Fig. 6 (C) are mouse tissues treated with siRNA-encapsulated ribosomes, and D is the tissue of mice that do not receive this treatment. Figure 6 (C) shows the expression of PLK-1 by transurethrally administration of siMA-encapsulated ribosome, using anti-PLK-1 monoclonal antibody and B, D by HE staining. It can be seen that is suppressed.
(D) 次いで、 ルシフェラ一ゼ遺伝子を導入した UM-UC- 3細胞株を膀 胱内に移植したマウスを 4群 （ 1群 7匹） に分けて、 21 日間にわたり飼 育し、 PLK- 1 siRNA 1412内包リポソ一ム又はコントロール siRNA (配列 番号 5 ) を 5 日目から 9 日目にわたって投与した。 投与は、 1日に 1回 一度に膀胱内にリボソームを投与して尿道口を手術用糸で縛り、 4時間 後に開放する方法で行った。 これら 4群は、 それぞれ非投与群、 濃度 6 u rn のコントロール s i RNA を内包するリボソームを投与した群、 濃度 200nMの PLK- 1 s iRNA 141 2を投与した群、濃度 6 raの PLK- l s iRNA 141 2 を投与した群である。 (D) Next, mice transplanted with the UM-UC-3 cell line into which the luciferase gene had been introduced into the bladder were divided into 4 groups (7 mice per group), reared for 21 days, and PLK-1 siRNA 1412-encapsulating liposome or control siRNA (SEQ ID NO: 5) was administered from the 5th day to the 9th day. Administration was performed once a day by administering ribosomes into the bladder at once, tying the urethral opening with surgical thread, and opening it 4 hours later. These 4 groups are the non-administration group, concentration 6 These are a group administered with ribosomes containing urn control siRNA, a group administered with PLK-1 s iRNA 141 2 at a concentration of 200 nM, and a group administered with PLK-ls iRNA 141 2 at a concentration of 6 ra.
各群のマウスについて、 膀胱癌細胞の増殖を測定した。 結果を図 6 (D) に示す。 図 6 (D)のグラフの横軸は飼育期間を示し、 縦軸は光子のカウン トを示す。  The proliferation of bladder cancer cells was measured for each group of mice. The results are shown in Fig. 6 (D). The horizontal axis of the graph in Fig. 6 (D) shows the breeding period, and the vertical axis shows the photon count.
図 6 (D)中、 · は濃度 200nMの PLK- 1 s iRNA 1412 を投与した群、 〇は 濃度 6 の PLK- 1 s i RNA 1412を投与した群、 口は無投与群、 騸はコン トロール s iRNA投与群である。 PLK- 1 s i RNA 1412内包リボソームを投与 することにより、 膀胱癌細胞の増殖が効果的に抑制されたことが分かる。  In Fig. 6 (D) iRNA administration group. It can be seen that administration of PLK-1 siRNA 1412-encapsulating ribosome effectively suppressed the proliferation of bladder cancer cells.
この 4群間の末梢血中赤血球数、 白血球数、 血小板数および血清 aspartate aminotransferase ( ASl，, 血清 alanine ammotransrerase (ALT), 血 清 lactate dehydrogenase (LDH), 血清 total protein (TP), 血清 creatinine (Cre), 及び血清 blood urea nitrogen (BUN)を測定したところ、 4群間に有 意差を認めなかった。 調べた範囲では、 PLK- 1 siRNA 1412には副作用は 認められなかった。  Peripheral blood erythrocyte count, leukocyte count, platelet count and serum aspartate aminotransferase (ASl, serum alanine ammotransrerase (ALT), serum lactate dehydrogenase (LDH), serum total protein (TP), serum creatinine (Cre ), And serum blood urea nitrogen (BUN), no significant differences were found between the four groups, and PLK-1 siRNA 1412 had no adverse effects.
以上のことから PLK- l s iRNA 1412 は膀胱内に経尿道的に注入するこ とで表在性膀胱癌の微小再発巣、 微小残存病変の治療効果の可能性が示 され、 即ち再発予防的投与の理論的根拠となる十分な実験結果が得られ た。 産業上の利用可能性  Based on the above, PLK-ls iRNA 1412 was shown to have a therapeutic effect on superficial bladder cancer micro-recurrence and minimal residual disease by transurethral injection into the bladder. Sufficient experimental results were obtained to provide a rationale for this. Industrial applicability
本発明の膀胱表在性癌の予防又は治療用医薬組成物は、 膀胱内に投与 することにより、 膀胱表在性癌細胞や前癌組織に浸透し、 膀胱癌細胞の 増殖を効果的に抑制する。 特に、 膀胱癌を切除後に微小残存癌病変を有 するヒトの治療用医薬組成物として好適に使用できる。  The pharmaceutical composition for prevention or treatment of superficial bladder cancer of the present invention penetrates into superficial bladder cancer cells and precancerous tissues and effectively suppresses the proliferation of bladder cancer cells when administered into the bladder. To do. In particular, it can be suitably used as a pharmaceutical composition for the treatment of humans having minimal residual cancer lesions after excision of bladder cancer.

Claims

請求の範囲 The scope of the claims
1. 以下の（a)又は（b)の siRNA 1. siRNA of (a) or (b) below
(a) 配列番号 1の塩基配列を含む siRNA  (a) siRNA comprising the nucleotide sequence of SEQ ID NO: 1
(b) 配列番号 1において 1〜 3個の塩基が付加、 欠失、 又は置換され た塩基配列を含み、 かつヒト PLK- 1 遺伝子の発現を特異的に抑制する siRNAo  (b) siRNAo comprising a nucleotide sequence in which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 1 and specifically suppressing the expression of the human PLK-1 gene
2. 以下の（c)又は（d)の siRNA  2. siRNA of (c) or (d) below
(c) 配列番号 2の塩基配列を含む siRNA  (c) siRNA comprising the nucleotide sequence of SEQ ID NO: 2
(d) 配列番号 2において 1〜 3個の塩基が付加、 欠失、 又は置換され た塩基配列を含み、 かつヒト PLK- 1 遺伝子の発現を特異的に抑制する siRNAo  (d) siRNAo comprising a nucleotide sequence to which 1 to 3 bases are added, deleted or substituted in SEQ ID NO: 2 and specifically suppressing the expression of the human PLK-1 gene
3. 請求項 1に記載の siRNAが内包されたリボソーム。  3. A ribosome encapsulating the siRNA according to claim 1.
4. 請求項 2に記載の siRNAが内包されたリボソーム。 4. A ribosome encapsulating the siRNA according to claim 2.
5. 請求項 3に記載のリボソームを含む医薬組成物。 5. A pharmaceutical composition comprising the ribosome according to claim 3.
6. 請求項 4に記載のリボソームを含む医薬組成物。  6. A pharmaceutical composition comprising the ribosome according to claim 4.
7. 請求項 3に記載のリボソームを含む膀胱表在性癌の治療又は予防 用医薬組成物。  7. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the ribosome according to claim 3.
8. 請求項 4に記載のリポソ一ムを含む膀胱表在性癌の治療又は予防 用医薬組成物。  8. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising the liposome according to claim 4.
9. ヒト PLK-1 の発現を阻害する siRNAが内包されたリボソームを含 む膀胱表在性癌の治療又は予防用医薬組成物。  9. A pharmaceutical composition for treating or preventing superficial bladder cancer comprising a ribosome encapsulating siRNA that inhibits human PLK-1 expression.
1 0. siRNA濃度が 100nM〜lmMである請求項 9に記載の組成物。 10. The composition according to claim 9, wherein the siRNA concentration is 100 nM to lmM.
1 1. リボソームの 80重量％以上が 50〜300 ^mの粒径を有するもの である請求項 9に記載の組成物。 1 1. The composition according to claim 9, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 ^ m.
1 2. 膀胱表在性癌又は膀胱の前癌病変を有するヒトの膀胱内に、 経 尿道的に、 請求項 3に記載のリボソームを投与する膀胱表在性癌の治療 又は予防方法。  1 2. A method for the treatment or prevention of a superficial bladder cancer comprising administering the ribosome according to claim 3 transurethrally into a human bladder having a superficial bladder cancer or a precancerous lesion of the bladder.
1 3. 膀胱表在性癌又は膀胱の前癌病変を有するヒトの膀胱内に、 経 尿道的に、 請求項 4に記載のリボソームを投与する膀胱表在性癌の治療 又は予防方法。 1 3. In human bladder with superficial bladder cancer or precancerous lesions of the bladder, A method for treating or preventing superficial bladder cancer, wherein the ribosome according to claim 4 is administered urethrally.
1 4. 膀胱表在性癌又は膀胱の前癌病変を有するヒ トの膀胱内に、 経 尿道的に、 ヒト PLK- 1 の発現を阻害する siRNAが内包されたリボソーム を投与する膀胱表在性癌の治療又は予防方法。  1 4. Superficial bladder administered with ribosome containing siRNA that inhibits human PLK-1 expression transurethrally in the bladder of humans with superficial bladder cancer or precancerous lesions of the bladder A method for treating or preventing cancer.
1 5. 膀胱表在性癌の治療又は予防剤の 1 回投与量が、 siRNAを 12 g〜120mg投与できる量である請求項 1 4に記載の治療又は予防方法。 1 5. The method for treatment or prevention according to claim 14, wherein the single dose of the agent for treating or preventing superficial bladder cancer is an amount capable of administering 12 g to 120 mg of siRNA.
1 6. 膀胱表在性癌の治療又は予防剤を 1〜 7日間間隔で 5〜10回投 与する請求項 1 4に記載の治療又は予防方法。 1 6. The treatment or prevention method according to claim 14, wherein the therapeutic or prophylactic agent for superficial bladder cancer is administered 5 to 10 times at intervals of 1 to 7 days.
1 7. 膀胱表在性癌を切除後に微小残存癌病変を有するヒトに対して 行われるものである請求項 1 4に記載の治療又は予防方法。  1 7. The treatment or prevention method according to claim 14, which is performed on a human having a minimal residual cancer lesion after excision of a superficial bladder cancer.
1 8. 請求項 3に記載のリボソームの膀胱表在性癌の治療又は予防用 医薬組成物としての使用。  1 8. Use of the ribosome according to claim 3 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
1 9. 請求項 4に記載のリボソームの膀胱表在性癌の治療又は予防用 医薬組成物としての使用。  1 9. Use of the ribosome according to claim 4 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
2 0. ヒト PLK-1の発現を阻害する siRNAが内包されたリボソームの 膀胱表在性癌の治療又は予防用医薬組成物としての使用。  20. Use of a ribosome encapsulating siRNA that inhibits expression of human PLK-1 as a pharmaceutical composition for treating or preventing superficial bladder cancer.
2 1. siRNAの濃度が 100nM〜lmMである請求項 2 0に記載の使用。 2 1. Use according to claim 20, wherein the concentration of siRNA is between 100 nM and lmM.
2 2. リボソームの 80重量％以上が 50〜300 ^mの粒径を有するもの である請求項 2 0に記載の使用。 2. The use according to claim 20, wherein 80% by weight or more of the ribosome has a particle size of 50 to 300 ^ m.
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