WO2006035515A1 - 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 - Google Patents
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 Download PDFInfo
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- 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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- superficial bladder
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2310/00—Structure or type of the nucleic acid
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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.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010017319A3 (en) * | 2008-08-05 | 2010-04-15 | Mdrna, Inc. | Nucleic acid compounds for inhibiting plk1 gene expression and uses thereof |
JP2011507534A (ja) * | 2007-12-27 | 2011-03-10 | プロチバ バイオセラピューティクス インコーポレイティッド | 干渉rnaを使用したポロ様キナーゼ発現のサイレンシング方法 |
DE102013003869A1 (de) * | 2013-02-27 | 2014-08-28 | Friedrich-Schiller-Universität Jena | Verfahren zur gezielten Abtötung von Zellen durch zur mRNA-Anbindung ausgerichtete Nukleotid-Moleküle sowie Nukleotid-Moleküle und Applikationskit für solche Verwendung |
US9878042B2 (en) | 2009-07-01 | 2018-01-30 | Protiva Biotherapeutics, Inc. | Lipid formulations for delivery of therapeutic agents to solid tumors |
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Cited By (10)
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JP2011507534A (ja) * | 2007-12-27 | 2011-03-10 | プロチバ バイオセラピューティクス インコーポレイティッド | 干渉rnaを使用したポロ様キナーゼ発現のサイレンシング方法 |
US9006191B2 (en) | 2007-12-27 | 2015-04-14 | Protiva Biotherapeutics, Inc. | Silencing of polo-like kinase expression using interfering RNA |
WO2010017319A3 (en) * | 2008-08-05 | 2010-04-15 | Mdrna, Inc. | Nucleic acid compounds for inhibiting plk1 gene expression and uses thereof |
US9878042B2 (en) | 2009-07-01 | 2018-01-30 | Protiva Biotherapeutics, Inc. | Lipid formulations for delivery of therapeutic agents to solid tumors |
US11446383B2 (en) | 2009-07-01 | 2022-09-20 | Arbutus Biopharma Corporation | Lipid formulations for delivery of therapeutic agents |
US11786598B2 (en) | 2009-07-01 | 2023-10-17 | Arbutus Biopharma Corporation | Lipid formulations for delivery of therapeutic agents |
US12016929B2 (en) | 2009-07-01 | 2024-06-25 | Arbutus Biopharma Corporation | Lipid formulations for delivery of therapeutic agents |
DE102013003869A1 (de) * | 2013-02-27 | 2014-08-28 | Friedrich-Schiller-Universität Jena | Verfahren zur gezielten Abtötung von Zellen durch zur mRNA-Anbindung ausgerichtete Nukleotid-Moleküle sowie Nukleotid-Moleküle und Applikationskit für solche Verwendung |
WO2014131773A2 (de) | 2013-02-27 | 2014-09-04 | Friedrich-Schiller-Universität Jena | Verfahren zur gezielten abtötung von zellen durch zur mrna-anbindung ausgerichtete nukleotid-moleküle sowie nukleotid-moleküle und applikationskit für solche verwendung |
DE102013003869B4 (de) * | 2013-02-27 | 2016-11-24 | Friedrich-Schiller-Universität Jena | Verfahren zur gezielten Abtötung von Zellen durch zur mRNA-Anbindung ausgerichtete Nukleotid-Moleküle sowie Nukleotid-Moleküle und Applikationskit für solche Verwendung |
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