WO1999028452A1 - Matrilysin antisense compounds - Google Patents
Matrilysin antisense compounds Download PDFInfo
- Publication number
- WO1999028452A1 WO1999028452A1 PCT/JP1998/002327 JP9802327W WO9928452A1 WO 1999028452 A1 WO1999028452 A1 WO 1999028452A1 JP 9802327 W JP9802327 W JP 9802327W WO 9928452 A1 WO9928452 A1 WO 9928452A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antisense compound
- seq
- oligonucleotide
- matrilysin
- antisense
- Prior art date
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- PTMHPRAIXMAOOB-UHFFFAOYSA-N phosphoramidic acid Chemical group NP(O)(O)=O PTMHPRAIXMAOOB-UHFFFAOYSA-N 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 1
- 229950004616 tribromoethanol Drugs 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to antisense compounds that hybridize to a part of the gene encoding human tomato relysin or contain a sequence complementary thereto. Further, the present invention relates to a pharmaceutical composition comprising the antisense compound and a pharmaceutically acceptable carrier.
- Matrix 'meta-oral protease is a generic name for a group of metal-requiring proteases that use extracellular matrix proteins as the main substrate.
- a typical MMP is a group of collagenases such as stromal collagenase ( MMP-1), Neutrophil Collagenase (MMP-8), Collagenase 3 (MM P-13), Gelatinase A (MM P-2), Gelatinase B (MMP-9), Stromlysin , Stromlysin 1 (MM P-3), Stromlysin 2 (MM P-10), MT- MMP group as MT 1-MM P (MM P-14), MT 2-MM P (MMP-15) ), MT 3 -MM P (MM P— 16).
- MT 4 -MM P MM P- 17
- other matri lysine MM P-7
- stromlysin 3 MMP-11
- metalloesterase MMP-12
- a chromone derivative having a matrix-meta-oral protease inhibitory activity exhibited an inhibitory effect on the growth of black species of mice.
- the molecular weight of matrilysin is 28 K, which is smaller than other MMPs.
- matrilysin does not have the hemopoxin-like C-terminal domain of other MMPs.
- Matrilysin has protease activity against various ECMs such as fibronectin, laminin and type IV collagen.
- the native enzyme of matrilysin was first purified from the culture of human rectal cancer cells under the name of matrine (Miyazaki, K., Hattori, Y., Umenishi, F., Yasumitsu, H., , Cancer Res. 50, 7758-7764 (1990)).
- matrilysin is expressed in colorectal cancer, prostate cancer, brain tumors, and lymphoma cancerous tissues, and in particular, colorectal cancer is almost exclusively expressed. Furthermore, while stromelysins and collagenases are upregulated in stromal cells around cancer tissues, matrilysin is specifically expressed in cancer tissues themselves (Kamiya Miyazaki et al., Biochemistry, 6 8, 1 7 9 1 — 1 807 pages, (1 996)) o
- the human matrilysin cDNA sequence is described in Muller, D., Breathnach, R. et a, Biochem. J. 253, 187-192 (1988), and Marti, H-P., MaI co in , D. Lovett, DH et a and in Biochem. J. 285, 899-905 (1992) under the name pu mp-1. Also, Gaire,., Magbanua, Z., McDonne I, S., et aI., J. Biol. Chem., 269, 2032-2040 (1994) disclose the sequence of the genome.
- a vector was constructed in which the full-length cDNA of matrilysin was integrated in the opposite direction, and this vector was introduced into a colorectal cancer cell line to examine the role of matrilysin in cancer metastasis and invasion.
- In vitro studies of colon cancer cells incorporating the vector by in vitro studies showed no clear association between matrilysin expression and invasion rate (invasion).
- colorectal cancer cells incorporating the vector were inoculated into nude mice cecum and observed for liver metastasis. The introduction of the vector reduced the tumor-forming ability, and decreased tumor-forming ability also reduced metastasis to the liver. did. However, it has not been concluded that the introduction of Vector-1 directly suppresses the metastasis of tumors (Witty, JP, Matrisian, and M. et a, Cancer Res., 54, 4805-4812. (1994)).
- the present inventor has conducted various researches using antisense technology to specifically inhibit the expression of matrilysin and to find a substance having an activity of inhibiting metastasis to cancer in vivo. It was completed.
- an object of the present invention is to provide an antisense compound of matrilysin capable of suppressing cancer metastasis, particularly in vivo, and a pharmaceutical composition containing the same.
- the present invention relates to an antisense compound that hybridizes with at least a part of a gene encoding human trilysin.
- the present invention also relates to an antisense compound comprising a sequence complementary to at least a part of the gene encoding human tomato relysin.
- the present invention preferably relates to an antisense compound that hybridizes with at least a part of SEQ ID NO: 1 (Gore region), SEQ ID NO: 2 (Capping Site), or SEQ ID NO: 3 (near AUG).
- the present invention particularly relates to an antisense compound including a sequence complementary to at least a part of the partial sequence among such antisense compounds.
- an antisense compound having a cancer metastasis inhibitory effect in an in vivo experiment is preferable.
- the present invention further preferably relates to an antisense compound comprising a sequence complementary to at least a part of SEQ ID NO: 1, which is a partial sequence of a gene encoding human tomato relysin, and particularly preferably SEQ ID NO: 1.
- the present invention relates to the antisense compound represented by SEQ ID NO: 4 which is complementary to the underlined sequence in the above sequence.
- the antisense compound of the present invention can suppress the transcription or translation of the human matrilysin gene, and can suppress the expression of human matrilysin. Metastasis can be significantly suppressed.
- the present invention is preferably an antisense compound in which at least one of the internucleotide linking groups contains an ⁇ atom.
- the antisense compound of the present invention includes oligonucleotides, oligonucleotide derivatives, and substances other than oligonucleotide derivatives represented by peptide nucleic acids.
- the term “gene encoding human tomato relysin” means chromosomal DNA or its transcript (mRNA and its precursor), and defines the amino acid sequence of human tomato relysin. In addition to the structural genes It also includes an intervening sequence (intron) existing in the middle of the structural gene, a base sequence upstream of the structural gene (such as a promoter or an operator), a base sequence downstream of the structural gene, etc., involved in the expression of human tomato lysin. Examples of the partial sequence of such a gene include those represented by SEQ ID NOs: 1 to 3.
- hybridize refers to the formation of specific binding to chromosomal DNA or mRNA.
- the hybridization strength is 0.15 M phosphate buffer with a Tm value of 35 ° C or more, as long as it has a Tm value of 45 ° C or more. Those having a Tm value of 55 ° C. or more are more preferable.
- the binding observed during hybridization is mainly complementary binding, but may be any binding form that forms specific binding to the target sequence.
- the antisense compound of the present invention does not necessarily need to have a complete complementary sequence to the target sequence as described below, and may contain a universal base represented by inosine or 3-2-tropyrrole. However, some of them may contain non-complementary bases or sequences.
- the antisense compound of the present invention may form a Watson-Crick-type or a Hoogsteen-type or both double or triple chains in any portion of the gene encoding human tomato relysin.
- the antisense compound of the present invention is preferably an oligonucleotide containing a sequence complementary to a part of the gene encoding human tomato relysin.
- Complementary sequence '' refers to a base specific to the base sequence of DNA or RNA A base pair that forms an effective complementary base pair.
- complementary base pairs form between C (cytosine) and G (guanine), between T (thymine) and A (adenine), and between U (peracyl) and A (adenine). Is performed.
- nucleotide sequence containing 10 or more nucleotides is considered a specific sequence. Therefore, the oligonucleotide of the present invention is expected to specifically hybridize to the gene encoding human tomato relysin, as long as it contains a nucleotide sequence consisting of 10 or more bases.
- the oligonucleotide of the present invention may be of any length, but a length of 50 bases or less is suitable for taking up the oligonucleotide of the present invention into cells and effectively suppressing human matrilysin expression. It is.
- the oligonucleotide of the present invention hybridizes to a gene encoding human tomato relysin, preferably has a nucleotide of 10 to 50 bases, more preferably 10 to 30 bases, and particularly preferably 15 bases. Those having a base number of at least 25 and no more than 25 bases are preferred.
- the oligonucleotide of the present invention includes all kinds of derivatives, including those having a base, sugar, phosphate, and backbone structure, which do not exist in nature.
- the oligonucleotide derivatives included in the present invention include a backbone structure having a phosphodiester bond, a phosphorothioate bond, or a methylphosphonate bond in whole or in part thereof. (Methyl phosphonate) binding, phosphoramido
- DNG deoxyribonucleotide guanidine
- 2 'of sugar examples include those substituted at other positions with other atoms or substituents, or modified sugar moieties such as ⁇ -ribose (Bertrand JR. Biochem. ⁇ ophys. Res. Commun., O 4 Vol., 311 1, (1989).
- those in which the sugar moiety has been replaced with another substance those in which some bases have been replaced with inosine or universal bases (bases that bind to any of A, D, C, and G), and oligonucleotide 5 Cleavage of cholesterol acridine, poly-L-lysine, psoralen, long-chain alkyl, etc. at the 'or 3' end or inside (G. Degols et al., Nucleic Acid Research. Vol. 17, 934 1 A. McConna ghie et al., J. Med. Chem. 38, 3488, (1993); G. Godard et al., Eu J. Biochem. Oligonucleotide derivatives such as p. 404, (1995)) are also included in the present invention.
- the antisense compound of the present invention satisfies the requirements described in the claims. Any substance can be used as long as it is useful.
- the antisense compound is preferably an oligonucleotide derivative having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability, and avidity.
- an oligonucleotide derivative in which at least one of the bonding groups between nucleotides contains an iodo atom is preferable, and an oligonucleotide derivative having a phosphorothioate bond as a backbone structure is more preferable.
- oligonucleotides and derivatives thereof can be obtained from S. Agra a Protocol for Oligonucleotides and Ana logs. Method in Molecular Biology). Series 20, Volume 4, Humana Press, S. Agrawal, etc.Antisense Research and Development 4, Volume 1, 185, (1994)
- the oligonucleotide of the present invention can be obtained by a PCR method using the gene encoding human tomato relysin as a type I.
- the methylphosphonate type, the phosphorothioate type, and the like include a chemical synthesizer.
- the obtained synthetic product be cowpea to be purified by H P L C method using a reverse-phase chroma Togurafi first class, it is possible to obtain a Origonukure Ochido derivative of interest.
- the antisense compounds of the present invention are labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like according to a known method.
- DNA or mRNA is prepared from cells of a patient whose expression of matrilysin in humans is to be examined by a known method, and a test substance is used as a test substance. Remove the labeled probe of the reaction. If the test substance contains human trilysin DNA or RNA, the labeled probe binds to them.
- the presence or absence of bond formation can be determined by using a labeled enzyme, a fluorescent substance, a luminescent substance, a radioisotope, or the like as an indicator of luminescence, fluorescence, radioactivity, or the like.
- the antisense compound of the present invention can be used as a diagnostic probe for diagnosis of a disease mediated by matrilysin, specifically, metastasis of cancer. It can also be used for diagnosis to determine the degree of cancer and treatment.
- the present invention further provides a pharmaceutical composition containing such an antisense compound, particularly a drug as a cancer metastasis inhibitor. Related to the composition.
- the subject to which the medicament of the present invention is administered is not particularly limited as long as it is a cancer.
- Examples include colon cancer, prostate cancer, brain tumor, and lymphoma, and more preferably, they can be used for the prevention or treatment of colon cancer, particularly liver metastasis thereof.
- the medicament of the present invention When the medicament of the present invention is used, it is preferable to use a medicament of a purity suitable for use as a medicament in a pharmacologically acceptable use method.
- the antisense compounds of the present invention may be used by directly dissolving or suspending them in an appropriate solvent, or may be used by encapsulating them in ribosomes or by incorporating them in an appropriate vector. Is also good.
- a pharmaceutically acceptable carrier is added to the antisense compound of the present invention, and injections, tablets, capsules, eye drops, creams, suppositories, sprays, patches, etc. May be used in an appropriate dosage form.
- Pharmaceutically acceptable carriers include solvents, bases, stabilizers, preservatives, solubilizers, excipients, and buffers well known to those skilled in the art.
- the antisense compound of the present invention When the antisense compound of the present invention is in the above-mentioned dosage form, its administration method and dosage can be set and used according to the patient's age, sex, disease type, degree, etc. . That is, an amount suitable for suppressing the expression of matrilysin and improving the condition is orally or parenterally administered. For example, continuously or divided into one or several times for each statement, 0.00 ⁇ ! ⁇ 200 mgZkg is administered. In the case of intravenous injection, 0.01 to 10 O mgZkg is preferable, and 0.1 to 5 OmgZkg is particularly preferable. Further, the antisense compound of the present invention is sufficiently safe at the above dose.
- Oral administration includes sublingual administration.
- Parenteral administration is appropriate from inhalation, transdermal administration, ophthalmic administration, intravaginal administration, intraarticular administration, rectal administration, intraarterial administration, intravenous administration, local administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, etc. What is necessary is to choose and administer the appropriate method.
- Figure 1 shows the structure of the mRNA of matrilysin, as well as SEQ ID NO: 1 and the sequence 2 shows the nucleotide sequences of SEQ ID NOS: 2 and 3, and the nucleotide sequence of SEQ ID NO: 4 which is the antisense oligonucleotide of the present invention.
- FIG. 2 shows the invasion-suppressing effect of the antisense oligonucleotide AS-1 of the present invention in an in vitro system. The mean value is shown together with the standard deviation.
- FIG. 3 shows the effect of the antisense oligonucleotide AS-1 of the present invention on metastasis to the liver in an in vivo system. Mean values are shown with standard deviations. The symbols in the figure are control PBS ( ⁇ ), control oligonucleotide CL-11 ( ⁇ ), and antisense oligonucleotide AS-1 ( ⁇ ).
- FIG. 4 shows the dose dependence of the anti-metastatic effect on the liver by the antisense oligonucleotide AS-1 of the present invention in an in vivo system. Mean values are shown with standard deviation. BEST MODE FOR CARRYING OUT THE INVENTION
- the antisense and control oligonucleotides specific for matrilysin are provided by the computer program HYB simulator (Advanced Gene Computing Technologies, Irvine, CA). Measured.
- 15mer oligonucleotide AS-1 (SEQ ID NO: 4) hybridizing to the core region of matrilysin mRNA was selected as a sequence having high specificity and low secondary structure-forming ability.
- a S-1 GTATATGATACGATC
- a 15mer oligonucleotide CL-1 obtained by randomly substituting adenine and thymine residues so as to have the same length and the same GC content as the above antisense sequence was used.
- C L-1 GTATTAGTATCGAAC (SEQ ID NO: 5)
- oligonucleotides were synthesized using an automatic DNA synthesizer (Applied Biosystems Model 380B, Foster City CA).
- Human rectal cancer cell lines C aR-1 and SW480 are JCRB cell (Tokyo). These cell lines were cultured in DMEM medium (Gibco BRI_) supplemented with 10% fetal calf serum (Gibeo BRL, Gaitherburg, A), 100 units I penicillin G, and 0.1 mg Zml streptomycin sulfate. ° C, and cultured in a humid condition of 5% C0 2 and 950/0 air. Subcultured every 3-4 days at an initial cell concentration 2 X 1 0 5 pieces ZML. The culture plate and culture dish used were those manufactured by Sumitomo BeiClient (Tokyo).
- oligonucleotides prepared above were labeled with [r- 32 P] ATP (300 OCiZmmol) using Pacteriophage T4 polynucleotide kinase (Gibco BRL).
- Radiolabeled oligonucleotides were purified by separating free of [r one 32 P] with liquid chromatography scratch.
- the above cell line was inoculated in a serum-free DMEM medium in a 96-well plate, and 1 ng of each radiolabeled oligonucleotide was added to each well. After each time period, cells are washed and placed on glass fiber filters (Glass Fiber Strips 240-1: Cambridge Technology, Watertown, Mass.) Using a semi-automated cell harvester (PHD model 290: Cambridge Technology). Collected. The amount of incorporated 32 P-labeled oligonucleotide was measured using a liquid scintillation counter (LS 6000 ic) (Beckman Industries, Fulerton, Calif.).
- Phosphorothioate oligonucleotides AS-1 and CL-11 were purified using fluoroscein isothiocyanate (FITC) (Leonetti J P. et al., Proc Natl Acad Sc USA USA 88: 2702-2706, (1991))
- the cell line (C aR-1) was inoculated in a serum-free DMEM medium in a 24-well plate and incubated with 10 M fluorescein-labeled oligonucleotide for 24 hours. The cells were thoroughly washed to remove the oligonucleotide outside the cells, and observed under a fluorescent confocal microscope (Nikon). As a result, both AS-1 and CL-1 were found to be distributed in the nucleus and cytoplasm of the cells after 24 hours.
- cDNA was randomly primed using M-M and V reverse transcriptase (Gibco BRl_), and then subjected to PCR amplification treatment (Perkin-Elmer / Centus, Norwalk, CT).
- the primer pairs used are as follows.
- Matrilysin sense primer 5 '-GTGACGGCGAGTTTTCAAAGC-3' (SEQ ID NO: 6)
- Matrilysin antisense primer 5 '-CGTTGCGGGACTGGATTATCA G-3' (SEQ ID NO: 7)
- One actin sense primer 5'-CTTCGGGGGGGACGATGC-3 '(SEQ ID NO: 8)
- ⁇ —Actin antisense primer 5'-CGTACATGGCTGGGGTGTTG-3 '(SEQ ID NO: 9)
- PCR amplification involves cycles of denaturation at 94 ° C (1 minute), annealing at 55 ° C (1.5 minutes), and polymerase reaction at 72 ° C (4 minutes). Cycles (matrilysin) and 25 cycles (actin) were repeated. The resulting PCR product was electrophoresed in a 1.2% agarose gel and stained with ethidium bromide.
- the C aR-1 cell line showed a marked decrease in its expression level under normal conditions when treated with the AS-1 oligonucleotide, which has the ability to express endogenous matrix lysin mRNA. did.
- AS-1 suppressed 92% of the expression of matrilysin mRNA.
- the growth medium obtained after the above incubation (48 hours) was centrifuged at 15,000 X g for 30 minutes. To this was added 80% saturated ammonium sulfate to precipitate the protein, which was recovered by centrifugation at 15,000 xg for 30 minutes. The resulting protein was subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules. GA).
- This membrane was blocked with 3% gelatin ZTBS (20 m Tris-HC and ⁇ 7.6, 0.9 ⁇ / ⁇ sodium chloride) for 10 minutes at room temperature. After that, the mixture was allowed to react with a heron anti-human trimeric triclonal antibody (Miyazaki K, et al. Cancer Res 50, 7758-7765 (1990)) diluted 1:10 with 1% gelatin for 2 hours. The cells were allowed to react for 1 hour with similarly diluted Alphosphite phosphatase-conjugated goat anti-Peacock IgG antibody (Sigma).
- a color reaction was carried out using an alkaline phosphatase binding substrate kit (Bio-Rad) according to the product protocol.
- WiDR Human colon cancer cell line WiDR has a very high level of matrilysin. It is known to produce in bells, and it has been confirmed by the present inventors that none of gelatinase, stromal collagenase, and stromlysin secrete detectable amounts. WiDR was obtained from JC RB Cell Bank (Tokyo).
- This cell line was cultured in DMEMZF12 medium (Life Technologies, Inc., Gaithersburg, USA) supplemented with 10% fetal calf serum (Gibco BR), 100 units Zml penicillin G, and 1 mg Zml streptomycin sulfate. MD, USA) 0. 24-well plates containing 5 m I in each ⁇ E Le of (Sumitomo base one Cry preparative (Tokyo) Ltd.) 5 X 1 0 or implantation, 37 ° C, 5% C0 2 and 95 The cells were cultured for 6 hours in a humid state of% air. After washing twice, the cells were cultured for 2 hours in a serum-free medium.
- the cells were cultured for 2 days while adding PBS 5I containing no ribonucleotide every 8 hours. Thereafter, the growth media from each of the two wells were combined and subjected to Western blotting according to the procedure described in Example 1 below.
- the WiDr cell line was added to 10% fetal calf serum (Gibco BRL), 100 units ml ⁇ nicillin G, and 1 mgZml streptomycin sulfate in DMEF 12 medium (Life Technologies, Inc., Gaithersburg, D.). , USA) at 37 ° C, 5% CO 2 and 95% air. This was treated with trypsin (0.25% trypsin 0.02% EDTA) and suspended in the same medium to a final concentration of 1 ⁇ 10 8 ml.
- trypsin 0.25% trypsin 0.02% EDTA
- BALB / c nu / nu nude mice are anesthetized with 2.5% avertin solution, aseptically incised in the left flank to temporarily expose the spleen outside the body, and use a 1 ml syringe (27 gauge needle). was injected to 5 1 0 6 W i D r the suspension solution 50 / I containing the spleen was this exposed.
- PBS containing the 15-mer antisense oligonucleotide AS-1 (120 ⁇ g) or the 15-mer control port-oligonucleotide CL-11 (120-S) prepared in Example 1 and 0.25 ml of each of these oligonucleotide-free PBSs was intraperitoneally injected daily from 1 day before the spleen treatment to 10 days after the spleen treatment.
- the number of tumors in the liver metastatic foci of the mice injected with the antisense oligonucleotide AS-1 of the present invention was 11, 28 and 42, respectively, 13%, 23 and 29%.
- the spleen was removed from the mouse into which the WiDr cells had been injected into the spleen in the same manner as in Example 3, 24 hours after the injection into the spleen, and the 15-mer antisense oligonucleotide prepared in Example 1 was removed.
- 10 ⁇ g of PBS containing nucleotides AS-1 or 15mer control oligonucleotide CL-11, and PBS containing no such oligonucleotides, and PBS containing no such oligonucleotides were injected intraperitoneally for 10 consecutive days from the following: The mice were sacrificed 4 weeks later, and liver metastases were observed.
- liver metastasis nodules was 7 ⁇ 0.56 in the PBS group and 16.3 ⁇ 4.8 in the control oligonucleotide group, but was 0 in the antisense oligonucleotide group.
- the transfer inhibition effect of the sense oligonucleotide was observed.
- the antisense compound of the present invention has an effect of actually significantly suppressing liver metastasis of colorectal cancer cells in an in vivo system using experimental animals, and is useful as a pharmaceutical. Was confirmed.
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Abstract
Substances capable of specifically inhibiting the expression of matrilysin to thereby inhibit metastasis and prepared by utilizing the antisense technique. Antisense compounds hybridizable with at least a part of a partial sequence of a gene encoding human matrilysin; and medicinal compositions, in particular ones having the effect of inhibiting metastasis, containing these antisense compounds.
Description
明細書 マトリライシンアンチセンス化合物 技術分野 Description Matrilysin antisense compound Technical field
本発明は、 ヒ トマトリライシンをコードする遺伝子の一部とハイプリ ダイズするか、 又はそれに相補的な配列を含むアンチセンス化合物に関 する。 さらに、 該アンチセンス化合物および薬学的に許容しうる担体を 含む医薬組成物に関する。 背景技術 The present invention relates to antisense compounds that hybridize to a part of the gene encoding human tomato relysin or contain a sequence complementary thereto. Further, the present invention relates to a pharmaceutical composition comprising the antisense compound and a pharmaceutically acceptable carrier. Background art
宮崎香 等、 生化学、 第 68巻、 1 791 — 1 807頁、 （1 996 ) には、 「マトリックス ' メタロプロテアーゼの構造と機能一特にがん の浸潤 '転移における役割」 と題して、 マトリックス ' メタ口プロテア ーゼに関する総説が記載されている。 Kazu Miyazaki et al., Biochemistry, 68, 1791-1780, (1996), entitled "Matrix 'Structure and Function of Metalloproteases, Especially Roles in Cancer Invasion' Metastasis" 'Contains a review on meta-oral proteases.
マトリックス ' メタ口プロテアーゼ （MM P) は細胞外マ卜リックス タンパク質を主要な基質とする一群の金属要求性プロテアーゼの総称で あり、 代表的な MMPとしては、 コラゲナーゼ群として間質型コラゲナ ーゼ （MMP— 1 ) 、 好中球コラゲナーゼ （MMP— 8) 、 コラゲナー ゼー3 (MM P- 1 3) 、 ゼラチナーゼ群としてゼラチナーゼ A (MM P— 2) 、 ゼラチナーゼ B (MMP— 9) 、 ストロムライシン群として 、 ストロムライシン 1 (MM P— 3) 、 ストロムライシン 2 (MM P- 1 0) 、 MT— MMP群として MT 1 —MM P (MM P - 1 4) 、 M T 2 -MM P (MMP— 1 5) 、 M T 3 -MM P (MM P— 1 6 ) . M T
4 -MM P (MM P- 1 7 ) 、 その他の MM Pとしてマ トリライシン （ MM P- 7 ) 、 ストロムライシン 3 (MMP— 1 1 ) 、 メタロェラスタ ーゼ （MMP— 1 2) が知られている。 Matrix 'meta-oral protease (MMP) is a generic name for a group of metal-requiring proteases that use extracellular matrix proteins as the main substrate. A typical MMP is a group of collagenases such as stromal collagenase ( MMP-1), Neutrophil Collagenase (MMP-8), Collagenase 3 (MM P-13), Gelatinase A (MM P-2), Gelatinase B (MMP-9), Stromlysin , Stromlysin 1 (MM P-3), Stromlysin 2 (MM P-10), MT- MMP group as MT 1-MM P (MM P-14), MT 2-MM P (MMP-15) ), MT 3 -MM P (MM P— 16). MT 4 -MM P (MM P- 17), other matri lysine (MM P-7), stromlysin 3 (MMP-11) and metalloesterase (MMP-12) are known. .
これまでの研究によれば、 種々のがん細胞における転移性や浸潤能と MM P産生能の間には相関関係が見られ、 がんの浸潤■転移における M M Pの役割を示唆している。 Previous studies have shown a correlation between metastatic and invasive potential in various cancer cells and MMP-producing ability, suggesting a role for MMPs in cancer invasion-metastasis.
例えば、 今井一志 蛋白核酸酵素、 第 42巻、 1 694— 1 700頁 、 ( 1 997) には、 がん細胞の浸潤■転移に伴う ECM (細胞外マト リックス） 破壊には、 細胞周囲の EC M破壊が必要であり、 なかでも細 胞膜貫通型 MM P (M T -MM P) と MMP— 2 (ゼラチナーゼ A) の 果たす役割が大きいことが述べられている。 For example, Kazushi Imai, Protein Nucleic Acid Enzyme, Vol. 42, pp. 1694–1700, (1997) states that ECM (extracellular matrix) destruction associated with invasion and metastasis of cancer cells involves EC surrounding cells. M-destruction is required, and it is stated that transmembrane MMPs (MT-MMP) and MMP-2 (gelatinase A) play a particularly important role.
又、 岡田明子等、 蛋白核酸酵素、 第 42巻、 2386— 2392頁、 (1 997) によれば、 ヒ ドロキサメ一ト誘導体のバチマスタッ トゃマ リマスタッ トはマトリックス ' メタロプロテアーゼに対してスぺク トラ ムの広い強力なインヒビターであり、 マウスの移植大腸がんなどの増殖 を抑制し転移も抑制すると示されている。 Also, according to Akada Okada et al., Protein Nucleic Acid Enzyme, Vol. 42, pp. 2386-2392, (1997), the batimastat-mammariat of a hydroxamethate derivative is more sensitive to matrix metalloproteases. It is a powerful inhibitor with a wide range of trum and has been shown to suppress the growth of transplanted colorectal cancer in mice and to suppress metastasis.
さらに、 マトリックス■ メタ口プロテアーゼ阻害活性を有するクロモ ン誘導体がマウス黒色種の増殖抑制作用を示したことも開示されている Furthermore, it has been disclosed that a chromone derivative having a matrix-meta-oral protease inhibitory activity exhibited an inhibitory effect on the growth of black species of mice.
(特開平 9一 1 1 0864) 。 (Japanese Unexamined Patent Publication No. 9-111864).
さて、 MM Pの一種であるマトリライシンの分子量は 28 Kと他の M MPに比べ小さい。 又、 他の MM Pが持つへモぺキシン様 C末端ドメィ ンをマトリライシンは持っていない。 Now, the molecular weight of matrilysin, a kind of MMP, is 28 K, which is smaller than other MMPs. In addition, matrilysin does not have the hemopoxin-like C-terminal domain of other MMPs.
マトリライシンはフイブロネクチン、 ラミニン及び I V型コラーゲン 等の種々の EC Mに対してプロテアーゼ活性を有している。
マトリライシンの天然型酵素はヒ 卜直腸がん細胞の培養液からマトリ ンの名称で初めて精製された （Miyazaki, K. , Hattor i, Y.， Umen i sh i , F.， Yasumitsu, H. , & Umeda, . , Cancer Res. 50, 7758-7764(19 90)) 。 Matrilysin has protease activity against various ECMs such as fibronectin, laminin and type IV collagen. The native enzyme of matrilysin was first purified from the culture of human rectal cancer cells under the name of matrine (Miyazaki, K., Hattori, Y., Umenishi, F., Yasumitsu, H., , Cancer Res. 50, 7758-7764 (1990)).
マトリライシンは大腸がん、 前立腺がん、 脳腫瘍、 リンフォーマのが ん組織で発現していることがこれまでの研究で確認されており、 特に、 大腸がんではほとんど例外なしに発現が見られる。 更に、 ストロムライ シン類ゃコラゲナーゼ類はがん組織周辺の間質細胞で発現が亢進するの に対して、 マトリライシンはがん組織自身に特異的な発現が見られる （ 宮崎香 等、 生化学、 第 6 8巻、 1 7 9 1 — 1 8 0 7頁、 （ 1 9 96 ) ) o Previous studies have confirmed that matrilysin is expressed in colorectal cancer, prostate cancer, brain tumors, and lymphoma cancerous tissues, and in particular, colorectal cancer is almost exclusively expressed. Furthermore, while stromelysins and collagenases are upregulated in stromal cells around cancer tissues, matrilysin is specifically expressed in cancer tissues themselves (Kamiya Miyazaki et al., Biochemistry, 6 8, 1 7 9 1 — 1 807 pages, (1 996)) o
又、 マ卜リライシン c D N A構築物のヒ 卜前立腺がん細胞株 D U— 1 45に於ける過剰発現によって、 マウスに於ける該細胞の浸潤能が増大 したことが報告されている （Powel l WC, Knox JD, Narve M et al. , C ancer Res. 53, 417-722 (1993)) 。 It has also been reported that overexpression of the matrilysin cDNA construct in the human prostate cancer cell line DU-145 increased the invasiveness of the cells in mice (Powell WC, Knox JD, Narve M et al., Cancer Res. 53, 417-722 (1993)).
更に、 A p c遺伝子に変異が入っているため小腸に腫瘍が多数出現す るコンジエニックマウスのマトリライシンを相同組み換えで欠損させた ものは、 その腫瘍の数が著しく減少したという報告がなされた （Wi lson , C. L. et al. , Proc. Natl. Acad. Scに USA, 94, 1402-1407(1997 In addition, it has been reported that the congenic mice, in which a large number of tumors appear in the small intestine due to mutations in the Apc gene, were deleted by homologous recombination in matrilysin, the number of tumors was significantly reduced ( Wilson, CL et al., Proc. Natl. Acad. Sc in USA, 94, 1402-1407 (1997
))o )) o
尚、 ヒ トのマトリライシン c D N A配列は、 Mul ler, D. , Breathnach ， R. et aに Biochem. J. 253, 187—192(1988) 、 および、 Marti, H -P. , Ma I co in, D. Lovett, D. H. et aに， Biochem. J. 285, 899 - 90 5(1992) に p u mp— 1の名称で開示されている。
また、 Ga i re, . , Magbanua, Z. , McDonne I , S. , et a I . , J. Biol . Chem. , 269, 2032-2040(1994) にそのゲノムの配列が開示されてい る。 The human matrilysin cDNA sequence is described in Muller, D., Breathnach, R. et a, Biochem. J. 253, 187-192 (1988), and Marti, H-P., MaI co in , D. Lovett, DH et a and in Biochem. J. 285, 899-905 (1992) under the name pu mp-1. Also, Gaire,., Magbanua, Z., McDonne I, S., et aI., J. Biol. Chem., 269, 2032-2040 (1994) disclose the sequence of the genome.
一方、 マトリライシンの c D N A全長を逆方向に組み込んだベクター を作成し、 これを大腸がん細胞株に導入して、 がんの転移と浸潤におけ るマ卜リライシンの役割について検討された。 インビトロ試験によるべ クタ一を組み込んだ大腸がん細胞の浸潤能試験では、 マトリライシンの 発現と進入速度 （浸潤） に明確な関連が認められていない。 インビボ試 験においてベクターを組み込んだ大腸がん細胞をヌードマウス盲腸へ接 種し肝転移を観察したところ、 ベクターの導入により腫瘍形成能が減少 し、 腫瘍形成能の減少によって肝への転移も減少した。 しかし、 ベクタ 一の導入が直接的に腫瘍の転移抑制をしているとは結論されていない （ Witty, J. P.， Matr isian, し. M. et aに， Cancer Res. , 54, 4805 - 4 812(1994))。 On the other hand, a vector was constructed in which the full-length cDNA of matrilysin was integrated in the opposite direction, and this vector was introduced into a colorectal cancer cell line to examine the role of matrilysin in cancer metastasis and invasion. In vitro studies of colon cancer cells incorporating the vector by in vitro studies showed no clear association between matrilysin expression and invasion rate (invasion). In an in vivo test, colorectal cancer cells incorporating the vector were inoculated into nude mice cecum and observed for liver metastasis.The introduction of the vector reduced the tumor-forming ability, and decreased tumor-forming ability also reduced metastasis to the liver. did. However, it has not been concluded that the introduction of Vector-1 directly suppresses the metastasis of tumors (Witty, JP, Matrisian, and M. et a, Cancer Res., 54, 4805-4812. (1994)).
更に、 別のグループにより同様に c D N A全長を逆方向に組み込んだ ベクターを作成し、 これを大腸がん細胞株に導入して、 がんの浸潤にお けるマトリライシンの役割について検討された。 インビトロ試験におい てはベクターを組み込んだ大腸がん細胞はベクターの導入により、 その 浸潤能が約 1 2に減少したが、 転移について明確な記載がない （Yama moto H. , Imai Κ.， et al. , Int. J. Cancer, 61, 218 - 222 (1995))。 又、 インビ卜口の実験系に於いては、 マ卜リライシン発現ヒ 卜直腸が ん細胞株 C a R- 1の浸潤が A U G開始コ ドンを含む領域を認識するマ トリライシンアンチセンスオリゴヌクレオチドによって濃度依存性の抑 制を受けることが示されてはいるが （嶋田紘ら、 曰消外会誌、 2 9、 8
7 8 - 8 8 3 ( 1 996) ) 、 インビポにおいてはマトリライシンアン チセンスオリゴヌクレオチドが転移抑制効果を有するか否かについては 明らかにされていない。 In addition, another group similarly created a vector in which the full-length cDNA was integrated in the opposite direction, introduced this vector into a colorectal cancer cell line, and examined the role of matrilysin in cancer invasion. In an in vitro test, the invasive ability of colorectal cancer cells incorporating the vector was reduced to about 12 by the introduction of the vector, but metastasis was not clearly described (Yamamoto H., Imai III., Et al. , Int. J. Cancer, 61, 218-222 (1995)). In addition, in the experimental system at the mouth, the invasion of the mouse rectocrine cell line CaR-1 expressing matrilysin was induced by matrilysin antisense oligonucleotide which recognizes the region containing the AUG initiation codon. Although it has been shown to be concentration-dependent, see Hiroshi Shimada et al. 78-8883 (1996)), it has not been clarified whether or not matrilysin antisense oligonucleotide has a metastasis inhibitory effect in in vivo.
更に、 インビトロでの浸潤アツセィとインビボでの転移能との間には 関連が見られないという報告もなされた （Simon, N. et al. , Invasio η Metastasis, 12. 156-167(1992)) 。 Furthermore, it has been reported that there is no association between invasive invasion in vitro and metastatic potential in vivo (Simon, N. et al., Invasio η Metastasis, 12.156-167 (1992)). .
発明の開示 Disclosure of the invention
そこで、 本発明者は、 アンチセンス技術を利用して、 マトリライシン の発現を特異的に阻害し、 インビボにおいて、 がんに対する転移抑制活 性を有するような物質を見出すべく研究を重ね、 本発明を完成するに至 つたのである。 Therefore, the present inventor has conducted various researches using antisense technology to specifically inhibit the expression of matrilysin and to find a substance having an activity of inhibiting metastasis to cancer in vivo. It was completed.
即ち、 本発明の課題は、 特にインビボにおいて、 がんの転移を抑制し 得るマトリライシンのアンチセンス化合物およびこれを含む医薬組成物 を提供することである。 That is, an object of the present invention is to provide an antisense compound of matrilysin capable of suppressing cancer metastasis, particularly in vivo, and a pharmaceutical composition containing the same.
本発明は、 ヒ トマ卜リライシンをコードする遺伝子の少なくとも一部 とハイブリダィズするアンチセンス化合物に係わる。 The present invention relates to an antisense compound that hybridizes with at least a part of a gene encoding human trilysin.
本発明は又、 ヒ トマトリライシンをコードする遺伝子の少なくとも一 部に相補的な配列を含むアンチセンス化合物に係わる。 The present invention also relates to an antisense compound comprising a sequence complementary to at least a part of the gene encoding human tomato relysin.
本発明は好ましくは、 配列番号 1 (Gore領域） 、 配列番号 2 (Cappin g Site), 又は、 配列番号 3 (A U G近傍） のいずれかの少なくとも一 部とハイブリダィズするアンチセンス化合物に係わる。 The present invention preferably relates to an antisense compound that hybridizes with at least a part of SEQ ID NO: 1 (Gore region), SEQ ID NO: 2 (Capping Site), or SEQ ID NO: 3 (near AUG).
本発明は特に、 かかるアンチセンス化合物の中でも、 上記部分配列の 少なくとも一部に相補的な配列を含むアンチセンス化合物に係わる。
前述のように、 嶋田紘らの A U G近傍の領域の遺伝子に対するアンチ センス化合物はインビトロの実験では株化癌細胞の浸潤を抑制する効果 を示したが、 癌転移モデルを用いたインビポの実験では明確な効果を示 すことができなかった。 The present invention particularly relates to an antisense compound including a sequence complementary to at least a part of the partial sequence among such antisense compounds. As mentioned above, antisense compounds against genes in the region near AUG by Hiroshi Shimada showed an effect of suppressing invasion of established cancer cells in in vitro experiments, but were clear in in vivo experiments using cancer metastasis models. Could not show any significant effect.
そこで、 本発明のアンチセンス化合物としては、 インビボの実験で癌 転移抑制効果を有するアンチセンス化合物が好ましい。 Thus, as the antisense compound of the present invention, an antisense compound having a cancer metastasis inhibitory effect in an in vivo experiment is preferable.
本発明はさらに好まし〈は、 ヒ トマトリライシンをコードする遺伝子 の部分配列である、 配列番号 1の少なくとも一部に相補的な配列を含む アンチセンス化合物に係わり、 特に好ましくは、 配列番号 1の配列中の 下線部の配列に相補的な配列番号 4で示されるアンチセンス化合物に係 わる。 The present invention further preferably relates to an antisense compound comprising a sequence complementary to at least a part of SEQ ID NO: 1, which is a partial sequence of a gene encoding human tomato relysin, and particularly preferably SEQ ID NO: 1. The present invention relates to the antisense compound represented by SEQ ID NO: 4 which is complementary to the underlined sequence in the above sequence.
本発明のアンチセンス化合物は、 ヒ 卜マトリライシン遺伝子の転写ま たは翻訳を抑制し、 ヒ トマ卜リライシンの発現を抑制しうるものである さらに本発明のアンチセンス化合物は、 インビボ系において、 がんの 転移を有意に抑制し得るものである。 The antisense compound of the present invention can suppress the transcription or translation of the human matrilysin gene, and can suppress the expression of human matrilysin. Metastasis can be significantly suppressed.
本発明は好ましくは、 ヌクレオチド間の結合基の少なくとも 1 つがィ ォゥ原子を含むアンチセンス化合物である。 The present invention is preferably an antisense compound in which at least one of the internucleotide linking groups contains an ゥ atom.
本発明のアンチセンス化合物には、 オリゴヌクレオチド、 オリゴヌク レオチド誘導体、 及びべプチド核酸に代表されるようなオリゴヌクレオ チド誘導体以外の物質が含まれる。 The antisense compound of the present invention includes oligonucleotides, oligonucleotide derivatives, and substances other than oligonucleotide derivatives represented by peptide nucleic acids.
本明細書中で、 Γヒ トマトリライシンをコードする遺伝子」 とは、 染 色体 D N Aまたはその転写物 （m R N Aおよびその前駆体） を意味し、 ヒ トマ卜リライシンのアミノ酸配列を規定している構造遺伝子に加えて
、 構造遺伝子の途中に存在する介在配列 （イントロン） 、 ヒ トマトリラ イシンの発現に関与する、 構造遺伝子上流の塩基配列 （プロモーターや オペレーターなど） 及び構造遺伝子下流の塩基配列等も包含するもので ある。 かかる遺伝子の部分配列としては、 例えば配列番号 1 〜 3で表さ れるものが挙げられる。 In the present specification, the term “gene encoding human tomato relysin” means chromosomal DNA or its transcript (mRNA and its precursor), and defines the amino acid sequence of human tomato relysin. In addition to the structural genes It also includes an intervening sequence (intron) existing in the middle of the structural gene, a base sequence upstream of the structural gene (such as a promoter or an operator), a base sequence downstream of the structural gene, etc., involved in the expression of human tomato lysin. Examples of the partial sequence of such a gene include those represented by SEQ ID NOs: 1 to 3.
又、 本明細書中で 「ハイブリダィズする」 とは、 染色体 D N Aもしく は m R N Aに対する特異的結合を形成することをいう。 ハイブリダイズ の強さとしては 0 . 1 5 Mリン酸緩衝液で、 3 5 °C以上の T m値を持つ ものであればよく、 4 5 °C以上の T m値を持つものであれば好ましく、 さらに 5 5 °C以上の T m値を持つものがよリ好ましい。 The term "hybridize" as used herein refers to the formation of specific binding to chromosomal DNA or mRNA. The hybridization strength is 0.15 M phosphate buffer with a Tm value of 35 ° C or more, as long as it has a Tm value of 45 ° C or more. Those having a Tm value of 55 ° C. or more are more preferable.
ハイプリダイズする際に見られる結合は主として相補的結合であるが 、 標的配列に対する特異的結合を形成するものであればいずれの結合様 式であってもよい。 つまり、 本発明のアンチセンス化合物は必ずしも標 的配列に対し、 以下に述べるような相補的配列を完全に持つ必要はなく 、 イノシンや 3—二トロピロールに代表されるユニバーサルベースを含 んでいてもよいし、 その一部に相補的でない塩基もしくは配列を含んで いてもよい。 The binding observed during hybridization is mainly complementary binding, but may be any binding form that forms specific binding to the target sequence. In other words, the antisense compound of the present invention does not necessarily need to have a complete complementary sequence to the target sequence as described below, and may contain a universal base represented by inosine or 3-2-tropyrrole. However, some of them may contain non-complementary bases or sequences.
また、 本発明のアンチセンス化合物はヒ トマトリライシンをコードす る遺伝子のいかなる部分に Watson Cr i ck型、 もしくは Hoogsteen 型また はその両方の二重鎖もしくは三重鎖を形成してもよい。 In addition, the antisense compound of the present invention may form a Watson-Crick-type or a Hoogsteen-type or both double or triple chains in any portion of the gene encoding human tomato relysin.
本発明のアンチセンス化合物は、 ヒ トマトリライシンをコードする遺 伝子の一部に相補的な配列を含むオリゴヌクレオチドであるのが好まし い。 The antisense compound of the present invention is preferably an oligonucleotide containing a sequence complementary to a part of the gene encoding human tomato relysin.
Γ相補的な配列」 とは、 D N Aや R N Aの塩基配列に対して塩基特異
的な相補的塩基対を形成するような塩基対をいう。 一般的には C (シト シン） と G (グァニン） の間、 T (チミン） と A (アデニン） の間、 お よび U (ゥラシル） と A (アデニン） との間で相補的塩基対が形成され る。 `` Complementary sequence '' refers to a base specific to the base sequence of DNA or RNA A base pair that forms an effective complementary base pair. In general, complementary base pairs form between C (cytosine) and G (guanine), between T (thymine) and A (adenine), and between U (peracyl) and A (adenine). Is performed.
一般的には、 1 0塩基以上の塩基を含む塩基配列であれば、 特異的配 列と考えられている。 したがって、 本発明のオリゴヌクレオチドは 1 0 塩基以上からなる塩基配列を含むものであれば、 ヒ トマトリライシンを コードする遺伝子に特異的にハイプリダイズすることが期待される。 Generally, a nucleotide sequence containing 10 or more nucleotides is considered a specific sequence. Therefore, the oligonucleotide of the present invention is expected to specifically hybridize to the gene encoding human tomato relysin, as long as it contains a nucleotide sequence consisting of 10 or more bases.
—方、 オリゴヌクレオチドを細胞内に取り込ませるには、 その長さが 余り長すぎても不適当である。 本発明のオリゴヌクレオチドはいかなる 長さのものでもよいが、 本発明のオリゴヌクレオチドを細胞内に取り込 ませ、 ヒトマ卜リライシンの発現を有効に抑制する為には 5 0塩基以下 の長さが適当である。 On the other hand, it is inappropriate for the oligonucleotide to be taken up into the cell, even if its length is too long. The oligonucleotide of the present invention may be of any length, but a length of 50 bases or less is suitable for taking up the oligonucleotide of the present invention into cells and effectively suppressing human matrilysin expression. It is.
従って本発明のオリゴヌクレオチドは、 ヒ トマトリライシンをコード する遺伝子にハイブリダィズし、 1 0塩基以上 5 0塩基以下のものが好 ましく、 更に、 1 0塩基以上 3 0塩基以下、 特に 1 5塩基以上 2 5塩基 以下の塩基数からなるものが好ましい。 Accordingly, the oligonucleotide of the present invention hybridizes to a gene encoding human tomato relysin, preferably has a nucleotide of 10 to 50 bases, more preferably 10 to 30 bases, and particularly preferably 15 bases. Those having a base number of at least 25 and no more than 25 bases are preferred.
アンチセンス技術の進歩とともに、 オリゴヌクレオチドの効果を高め ることを目的として様々な誘導体が見いだされてきた。 現在、 目的の D N Aや m R N Aとの結合力、 組織選択性、 細胞透過性、 ヌクレアーゼ耐 性、 細胞内安定性の高い様々なオリゴヌクレオチド誘導体が得られてい る。 従って、 本発明のオリゴヌクレオチドには、 天然には存在しないよ うな、 塩基、 糖、 リン酸、 バックボーン構造からなるものも含めて、 あ らゆる種類の誘導体が含まれる。
本発明に含まれるオリゴヌクレオチド誘導体の例としては、 バックボ ーン構造として、 その全部または一部にフォスフォジエステル （phosph odi ester) 結合、 フォスフォロチォエート （phosphoroth i oate) 結合、 メチルフォスフォネート （methyl phosphonate)結合、 フォスフォロアミWith the advance of antisense technology, various derivatives have been found for the purpose of enhancing the effect of oligonucleotides. At present, various oligonucleotide derivatives having high binding strength to target DNA or mRNA, tissue selectivity, cell permeability, nuclease resistance and intracellular stability have been obtained. Therefore, the oligonucleotide of the present invention includes all kinds of derivatives, including those having a base, sugar, phosphate, and backbone structure, which do not exist in nature. Examples of the oligonucleotide derivatives included in the present invention include a backbone structure having a phosphodiester bond, a phosphorothioate bond, or a methylphosphonate bond in whole or in part thereof. (Methyl phosphonate) binding, phosphoramido
7 卜 (phosphoroami date) 結合、 フォスフォロンチォェ一卜 (phosph orodithioate) 結合、 モルホリノ基を有するもの等 （東海林洋子 等、 癌と化学療法、 2 0巻、 1 8 99— 1 90 7頁、 （ 1 9 93年） ） が挙 げられる。 7 (phosphoroamidate) bond, phosphorodithioate bond, morpholino group, etc. (Yoko Tokai Hayashi, etc., Cancer and Chemotherapy, Vol. 20, pp. 1899-1907, ( 1 1993)).
又、 デォキシリボヌクレオチドグァニジン （D N G) (Robert P等、 Proc. Natl. Acad. Sc i . USA、 92巻、 6 0 9 7頁、 （ 1 9 9 5年） ) 、 糖の 2 ' 位が他の原子あるいは置換基に置換されたもの、 或いは α 一リボース等の糖部分を修飾したものも誘導体の例として挙げられる （ Bertrand JR. Biochem. Β ι ophys. Res. Commun. ， o 4卷、 3 1 1 ¾ 、 ( 1 98 9年） ） 。 Also, deoxyribonucleotide guanidine (DNG) (Robert P et al., Proc. Natl. Acad. Sci. USA, 92, 6097, (1995)), 2 'of sugar Examples of derivatives include those substituted at other positions with other atoms or substituents, or modified sugar moieties such as α-ribose (Bertrand JR. Biochem. Βι ophys. Res. Commun., O 4 Vol., 311 1, (1989).
更に、 糖部分が他の物質に置換されたもの、 一部の塩基がイノシンや ユニバーサル塩基 （A、 丁、 C、 Gのいずれにでも結合する塩基） に置 換されたもの、 オリゴヌクレオチドの 5 ' 末端もしくは 3 ' 末端もしく は内部にコレステロールゃァクリジン、 ポリ一 L—リジン、 ソラレン （ psoralen) 、 長鎖アルキル等が結合したもの （G. Degols 等、 Nucleic Acid Research. 1 7卷、 934 1頁、 （ 1 98 9年） ； A. McConna ghie等、 J. Med. Chem. 3 8巻、 348 8頁、 （ 1 993年） ； G. Go dard 等 Eu J. Biochem. 2 3 2巻、 404頁、 （ 1 9 95年） ） 等 のオリゴヌクレオチド誘導体も本発明に含まれる。 In addition, those in which the sugar moiety has been replaced with another substance, those in which some bases have been replaced with inosine or universal bases (bases that bind to any of A, D, C, and G), and oligonucleotide 5 Cleavage of cholesterol acridine, poly-L-lysine, psoralen, long-chain alkyl, etc. at the 'or 3' end or inside (G. Degols et al., Nucleic Acid Research. Vol. 17, 934 1 A. McConna ghie et al., J. Med. Chem. 38, 3488, (1993); G. Godard et al., Eu J. Biochem. Oligonucleotide derivatives such as p. 404, (1995)) are also included in the present invention.
本発明のアンチセンス化合物は、 特許請求の範囲に記載した要件を満
たすものであれば、 例示したオリゴヌクレオチド誘導体をはじめとして いかなる物質であってもよい。 しかしながら、 アンチセンス化合物は、 ヌクレアーゼ耐性、 組織選択性、 細胞透過性、 結合力の少なくとも 1 つが 高められたオリゴヌクレオチド誘導体であることが好ましい。 特に、 ヌ クレオチド間の結合基の少なくとも 1 つがィォゥ原子を含むオリゴヌク レオチド誘導体が好ましく、 フォスフォロチォエート結合をバックボー ン構造として有するオリゴヌクレオチド誘導体がより好ましい。 The antisense compound of the present invention satisfies the requirements described in the claims. Any substance can be used as long as it is useful. However, the antisense compound is preferably an oligonucleotide derivative having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability, and avidity. In particular, an oligonucleotide derivative in which at least one of the bonding groups between nucleotides contains an iodo atom is preferable, and an oligonucleotide derivative having a phosphorothioate bond as a backbone structure is more preferable.
以下に、 本発明のアンチセンス化合物の製造方法を説明する。 Hereinafter, a method for producing the antisense compound of the present invention will be described.
本発明のアンチセンス化合物は当業者であれば公知方法で容易に製造 することができる （例えば、 オリゴヌクレオチドやその誘導体は、 S. A gra a I Protocol for ol igonucleotides and Ana logs. Method in Molecular Biology Series 2 0巻、 Humana Press, S. Agrawal等 Ant i sense Research and Development 4巻、 1 8 5頁、 ( 1 9 9 4年) 天然の D N Aや R N Aであれば、 化学合成機を使用して合成したリ、 ヒ トマトリライシンをコードする遺伝子を錶型として P C R法により本 発明のオリゴヌクレオチドを得ることができる。 また、 メチルフォスフ ォネート型やフォスフォロチォエー卜型等の中には、 化学合成機 （たと えば、 パーキンエルマ一ジャパン （株） 製、 3 9 4型） を使用して合成 できるものもある。 この場合には、 化学合成機に添付されたマニュアル に従って操作を行い、 得られた合成産物を逆相クロマ トグラフィ一等を 用いた H P L C法により精製することによつても、 目的のオリゴヌクレ ォチド誘導体を得ることができる。 Those skilled in the art can easily produce the antisense compound of the present invention by a known method (for example, oligonucleotides and derivatives thereof can be obtained from S. Agra a Protocol for Oligonucleotides and Ana logs. Method in Molecular Biology). Series 20, Volume 4, Humana Press, S. Agrawal, etc.Antisense Research and Development 4, Volume 1, 185, (1994) For natural DNA and RNA, synthesize using chemical synthesizer The oligonucleotide of the present invention can be obtained by a PCR method using the gene encoding human tomato relysin as a type I. In addition, the methylphosphonate type, the phosphorothioate type, and the like include a chemical synthesizer. (For example, PerkinElmer Japan, Inc., Model 3394), which can be synthesized by following the manual attached to the chemical synthesizer. The obtained synthetic product be cowpea to be purified by H P L C method using a reverse-phase chroma Togurafi first class, it is possible to obtain a Origonukure Ochido derivative of interest.
次に本発明のアンチセンス化合物の利用方法について説明する。
本発明のアンチセンス化合物を診断用のプローブとして使用する場合 には、 それらを、 公知の方法に従い、 ラジオアイソ トープや、 酵素、 蛍 光物質、 発光物質等で標識する。 次に、 ヒ 卜におけるマトリライシンの 発現を調べたい患者の細胞から D N Aもしくは m R N Aを公知方法で調 製し、 これを被検物質として、 標識プローブを加えて反応させた後、 洗 浄して未反応の標識プローブを除去する。 被検物質中にヒ トマ卜リライ シン D N Aもしくは R N Aが含まれていれば、 標識プローブはそれらと 結合する。 結合形成の有無は、 標識した酵素、 蛍光物質、 発光物質、 あ るいは放射性同位元素等により発光、 蛍光、 放射能等を指標として知る ことができる。 Next, a method of using the antisense compound of the present invention will be described. When the antisense compounds of the present invention are used as probes for diagnosis, they are labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like according to a known method. Next, DNA or mRNA is prepared from cells of a patient whose expression of matrilysin in humans is to be examined by a known method, and a test substance is used as a test substance. Remove the labeled probe of the reaction. If the test substance contains human trilysin DNA or RNA, the labeled probe binds to them. The presence or absence of bond formation can be determined by using a labeled enzyme, a fluorescent substance, a luminescent substance, a radioisotope, or the like as an indicator of luminescence, fluorescence, radioactivity, or the like.
したがって、 本発明のアンチセンス化合物は診断用プローブとして、 マトリライシンを介する疾患、 具体的にはがんの転移の診断に利用する ことができる。 また、 がんの程度や治療方法を決定するための診断にも 使用することができる。 Therefore, the antisense compound of the present invention can be used as a diagnostic probe for diagnosis of a disease mediated by matrilysin, specifically, metastasis of cancer. It can also be used for diagnosis to determine the degree of cancer and treatment.
本発明のアンチセンス化合物はインビボでのがんの転移を抑制する効 果を有しているので、 本発明は更に、 かかるアンチセンス化合物を含む 医薬組成物、 特にがん転移抑制剤としての医薬組成物に係わる。 Since the antisense compound of the present invention has an effect of suppressing metastasis of cancer in vivo, the present invention further provides a pharmaceutical composition containing such an antisense compound, particularly a drug as a cancer metastasis inhibitor. Related to the composition.
本発明の医薬品の投与する対象はがんであれば特に限定しない。 例え ば大腸がん、 前立腺がん、 脳腫瘍、 及びリンフォーマ等であり、 より好 ましくは大腸がん、 特にその肝転移の予防あるいは治療に用いることが できる。 The subject to which the medicament of the present invention is administered is not particularly limited as long as it is a cancer. Examples include colon cancer, prostate cancer, brain tumor, and lymphoma, and more preferably, they can be used for the prevention or treatment of colon cancer, particularly liver metastasis thereof.
本発明の医薬品を使用する場合には、 医薬品として使用するのに適し た純度のものを、 薬理学的に許容されうる使用方法で使用することが好 ましい。
本発明のアンチセンス化合物は、 それらを直接適当な溶媒に溶解もし くは懸濁して使用してもよいし、 リボソーム中に封入したり、 適当なベ クタ一に組み込んだ形にして使用してもよい。 また、 必要に応じて、 本 発明のアンチセンス化合物に薬学的に許容され得る担体を添加し、 注射 剤、 錠剤、 カプセル剤、 点眼剤、 クリーム剤、 座剤、 噴霧剤、 及びパッ プ剤等の適当な剤型にして使用してもよい。 薬学的に許容し得る担体に は、 当業者には周知の溶媒、 基剤、 安定化剤、 防腐剤、 溶解剤、 賦形剤 、 及び緩衝剤等が含まれる。 When the medicament of the present invention is used, it is preferable to use a medicament of a purity suitable for use as a medicament in a pharmacologically acceptable use method. The antisense compounds of the present invention may be used by directly dissolving or suspending them in an appropriate solvent, or may be used by encapsulating them in ribosomes or by incorporating them in an appropriate vector. Is also good. Also, if necessary, a pharmaceutically acceptable carrier is added to the antisense compound of the present invention, and injections, tablets, capsules, eye drops, creams, suppositories, sprays, patches, etc. May be used in an appropriate dosage form. Pharmaceutically acceptable carriers include solvents, bases, stabilizers, preservatives, solubilizers, excipients, and buffers well known to those skilled in the art.
本発明のアンチセンス化合物は、 上述のような剤型とした場合、 患者 の年齢、 性別、 疾患の種類、 程度等に応じて、 その投与方法、 その投与 量を設定して使用することができる。 すなわち、 マトリライシンの発現 を抑制し、 病態を改善するのに適した量を、 経口投与または非経口投与 する。 例えば、 連続的にあるいは 1 曰あたり 1回若しくは数回に分けて 、 0 . 0 0 ·！ 〜 2 0 0 O mgZkgが投与される。 静脈注射の場合は 0 . 0 1 〜 1 0 O mgZkgが好ましく、 0 . 1 〜 5 O mgZkgが特に好ましい。 ま た、 本発明のアンチセンス化合物は上記投与量においては十分に安全で る。 When the antisense compound of the present invention is in the above-mentioned dosage form, its administration method and dosage can be set and used according to the patient's age, sex, disease type, degree, etc. . That is, an amount suitable for suppressing the expression of matrilysin and improving the condition is orally or parenterally administered. For example, continuously or divided into one or several times for each statement, 0.00 ·! ~ 200 mgZkg is administered. In the case of intravenous injection, 0.01 to 10 O mgZkg is preferable, and 0.1 to 5 OmgZkg is particularly preferable. Further, the antisense compound of the present invention is sufficiently safe at the above dose.
経口投与には舌下投与を含む。 非経口投与は、 吸入、 経皮投与、 点眼 、 膣内投与、 関節内投与、 直腸投与、 動脈内投与、 静脈内投与、 局所投 与、 筋肉内投与、 皮下投与、 及び腹腔内投与等から適当な方法を選んで 投与すればよい。 図面の簡単な説明 Oral administration includes sublingual administration. Parenteral administration is appropriate from inhalation, transdermal administration, ophthalmic administration, intravaginal administration, intraarticular administration, rectal administration, intraarterial administration, intravenous administration, local administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, etc. What is necessary is to choose and administer the appropriate method. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マトリライシンの m R N Aの構造、 並びに配列番号 1 、 配列
番号 2、 及び配列番号 3の塩基配列、 並びに本発明のアンチセンスオリ ゴヌクレオチドである配列番号 4の塩基配列を示す。 Figure 1 shows the structure of the mRNA of matrilysin, as well as SEQ ID NO: 1 and the sequence 2 shows the nucleotide sequences of SEQ ID NOS: 2 and 3, and the nucleotide sequence of SEQ ID NO: 4 which is the antisense oligonucleotide of the present invention.
図 2は、 インビトロ系に於ける本発明のアンチセンスオリゴヌクレオ チド AS— 1 による浸潤抑制効果を表す。 平均値を標準偏差と共に示す 図 3は、 インビボ系に於ける本発明のアンチセンスオリゴヌクレオチ ド AS— 1 による肝臓への転移抑制効果を表す。 平均値を標準偏差と共 に示す。 尚、 図中の記号は、 コントロール PB S (□) 、 コントロール オリゴヌクレオチド C L一 1 (△) 及びアンチセンスオリゴヌクレ才チ ド A S— 1 (〇） である。 FIG. 2 shows the invasion-suppressing effect of the antisense oligonucleotide AS-1 of the present invention in an in vitro system. The mean value is shown together with the standard deviation. FIG. 3 shows the effect of the antisense oligonucleotide AS-1 of the present invention on metastasis to the liver in an in vivo system. Mean values are shown with standard deviations. The symbols in the figure are control PBS (□), control oligonucleotide CL-11 (△), and antisense oligonucleotide AS-1 (〇).
図 4は、 インビボ系に於ける本発明のアンチセンスオリゴヌクレオチ ド AS— 1 による肝臓への転移抑制効果の用量依存性を表す。 平均値を 標準偏差と共に示す。 発明を実施するための最良の形態 FIG. 4 shows the dose dependence of the anti-metastatic effect on the liver by the antisense oligonucleotide AS-1 of the present invention in an in vivo system. Mean values are shown with standard deviation. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 実施例を参照しながら本発明を詳細に説明するが、 該実施例は 本発明の範囲を何等限定するものではない。 実施例 1 Hereinafter, the present invention will be described in detail with reference to examples, but the examples do not limit the scope of the present invention in any way. Example 1
(1 ) アンチセンスフォスフォロチォェ一卜オリゴヌクレオチドの製 造 (1) Production of antisense phosphorothioate oligonucleotide
マ卜リライシンに特異的なアンチセンスオリゴヌクレオチドとコント ロールオリゴヌクレオチドは、 コンピュータープログラム HYB simulato r (Advanced Gene Computing Technologies, Irvine, CA) ¾:Ηΐΐ て設
計した。 The antisense and control oligonucleotides specific for matrilysin are provided by the computer program HYB simulator (Advanced Gene Computing Technologies, Irvine, CA). Measured.
かかるオリゴヌクレオチドの設計に際しては、 融点として 3 5°Cを有 する可能性のある全ての配列を、 目標となるマトリライシン mR N Aの あらゆる部位から抽出した。 In designing such oligonucleotides, all sequences with a potential melting point of 35 ° C. were extracted from every site of the target matrilysin mRNA.
その後、 このようにして抽出した各配列の特異性 （GenBank データべ ースにある全配列に対するクロス一ハイプリダイゼーシヨンの度合） 及 び該ヌクレオチド配列とマトリライシン mR N A両者に於ける 2次構造 形成能をコンピュータで分析した （Mitsuhashi M. et al. , Nature 36 7: 759-761, (1994)； Hyndman D. et aに， B i otechn i ques 20: 1090- 1097, (1996))。 Thereafter, the specificity of each sequence extracted in this manner (the degree of cross-hybridization for all sequences in the GenBank database) and the formation of secondary structures in both the nucleotide sequence and matrilysin mRNA Noh was analyzed by computer (Mitsuhashi M. et al., Nature 366: 759-761, (1994); Hyndman D. et a, Biotechniques 20: 1090-1097, (1996)).
その結果、 特異性が高く 2次構造形成能が低い配列として、 マト リライシン mR N Aのコア （Core) 領域にハイブリダィズする 1 5m e rオリゴヌクレオチド A S— 1 (配列番号 4) が選ばれた。 As a result, 15mer oligonucleotide AS-1 (SEQ ID NO: 4) hybridizing to the core region of matrilysin mRNA was selected as a sequence having high specificity and low secondary structure-forming ability.
A S - 1 ： GTATATGATACGATC A S-1: GTATATGATACGATC
コントロール配列としては、 上記アンチセンス配列と同一の長さと同 一の G C含量を有するようにアデニン及びチミン残基でランダムに置換 して得られた 1 5me rオリゴヌクレオチド C L— 1 を使用した。 As a control sequence, a 15mer oligonucleotide CL-1 obtained by randomly substituting adenine and thymine residues so as to have the same length and the same GC content as the above antisense sequence was used.
C L - 1 ： GTATTAGTATCGAAC (配列番号 5 ) C L-1: GTATTAGTATCGAAC (SEQ ID NO: 5)
これらのオリゴヌクレオチドは、 自動 D N A合成装置 （Appl ied Bios ystems Model 380B, Foster City CA)を用しゝて合成した。 These oligonucleotides were synthesized using an automatic DNA synthesizer (Applied Biosystems Model 380B, Foster City CA).
これらの合成したオリゴヌクレオチドはその都度調製した Dulbecco' s リン酸塩緩衝溶液 （P B S) (Hyclone, Logan, UT) 中に溶解した。 These synthesized oligonucleotides were dissolved in each time prepared Dulbecco's phosphate buffer solution (PBS) (Hyclone, Logan, UT).
(2) 細胞株及び培養条件 (2) Cell lines and culture conditions
ヒ ト直腸がん細胞株 C a R - 1及び SW4 8 0は J C R B 細胞バン
ク （東京） から入手した。 これらの細胞株は、 1 0%牛胎児血清 （Gibe o BRL, Gaitherburg, A) 、 1 00単位 Iペニシリン G、 及び 0. 1 mgZml硫酸ストレプトマイシン添加の DM EM培地 （Gibco BRI_)中で 、 3 7°C、 5 %C02及び 950/0空気の湿気状態で培養した。 初期細胞 濃度 2 X 1 05個 Zmlで 3〜4日毎に植継いだ。 培養プレート及び培養 デイシュは住友べ一クライ 卜 （東京） 製を用いた。 Human rectal cancer cell lines C aR-1 and SW480 are JCRB cell (Tokyo). These cell lines were cultured in DMEM medium (Gibco BRI_) supplemented with 10% fetal calf serum (Gibeo BRL, Gaitherburg, A), 100 units I penicillin G, and 0.1 mg Zml streptomycin sulfate. ° C, and cultured in a humid condition of 5% C0 2 and 950/0 air. Subcultured every 3-4 days at an initial cell concentration 2 X 1 0 5 pieces ZML. The culture plate and culture dish used were those manufactured by Sumitomo BeiClient (Tokyo).
(3) 放射標識 （ラベル） オリゴヌクレオチドの細胞内取込み (3) Incorporation of radiolabeled oligonucleotide into cells
上記で作成した各オリゴヌクレオチドを、 パクテリオファージ T 4ポ リヌクレオチドキナーゼ （Gibco BRL)を使用して 〔r—32 P〕 A T P ( 300 OCiZmmol) でラベルした。 Each of the oligonucleotides prepared above was labeled with [r- 32 P] ATP (300 OCiZmmol) using Pacteriophage T4 polynucleotide kinase (Gibco BRL).
放射標識されたオリゴヌクレオチドは液体クロマトグラフィ一を用い てフリーの 〔r一32 P〕 を分離することによって精製した。 Radiolabeled oligonucleotides were purified by separating free of [r one 32 P] with liquid chromatography scratch.
上記細胞株を 96穴プレー卜中の血清無添加 D MEM培地に植え込み 、 各穴に放射標識した各オリゴヌクレオチド 1 ngを添加した。 それぞれ の時間経過後に、 細胞を洗浄し、 半自動細胞ハーべスター （PHD model 290: Cambridge Techno I ogy)を用いてガラス繊維フィルター （Glass F iber Strips 240-1： Cambridge Technology, Watertown, MA)上に採取 した。 取り込まれた32 P標識オリゴヌクレオチドの量は液体シンチレ一 シヨンカウンター （L S 6000 i c ) (Beckman Industries, Fu I lerto n, CA)を用いて測定した。 The above cell line was inoculated in a serum-free DMEM medium in a 96-well plate, and 1 ng of each radiolabeled oligonucleotide was added to each well. After each time period, cells are washed and placed on glass fiber filters (Glass Fiber Strips 240-1: Cambridge Technology, Watertown, Mass.) Using a semi-automated cell harvester (PHD model 290: Cambridge Technology). Collected. The amount of incorporated 32 P-labeled oligonucleotide was measured using a liquid scintillation counter (LS 6000 ic) (Beckman Industries, Fulerton, Calif.).
その結果、 取込み量は 2 4時間頃迄は直接的に増加し、 約 48時間経 過するとほぼ平衡状態に達することが判った。 As a result, it was found that the uptake amount increased directly until about 24 hours, and reached an almost equilibrium state after about 48 hours.
添加した量の 1 8 % ( 24時間後） 及び 2 1 % (48時間後） が細胞 に取り込まれていた。 また、 A S— 1及び C L _ 1の間に取込みの速度
及び効率に関して顕著な差異は見られなかった。 18% (after 24 hours) and 21% (after 48 hours) of the added amount were taken up by the cells. Also, the acquisition speed between AS-1 and CL_1 And no significant differences were seen in efficiency.
(4) オリゴヌクレオチドの細胞内蓄積 (4) Intracellular accumulation of oligonucleotides
フォスフォロチォエー卜オリゴヌクレオチド A S— 1及び C L一 1 を フルォロセインイソチオシァネート （F I T C) を用いて （Leonetti J P. et al. , Proc Natl Acad Sc i USA 88: 2702— 2706, (1991))ラベノレ した。 Phosphorothioate oligonucleotides AS-1 and CL-11 were purified using fluoroscein isothiocyanate (FITC) (Leonetti J P. et al., Proc Natl Acad Sc USA USA 88: 2702-2706, (1991))
細胞株 （C a R— 1 ) を 2 4穴プレート中の血清無添加 D M E M培地 に植え込み、 1 0 Mのフルォロセイン標識オリゴヌクレオチドと共に 2 4時間ィンキュベートした。 細胞を充分に洗浄して細胞外部の該オリ ゴヌクレオチドを除去し、 蛍光共焦点顕微鏡 （ニコン） 下で観察した。 その結果、 A S— 1及び C L - 1の両者ともに 2 4時間後には細胞の 核及び細胞質内に分布していることが判つた。 The cell line (C aR-1) was inoculated in a serum-free DMEM medium in a 24-well plate and incubated with 10 M fluorescein-labeled oligonucleotide for 24 hours. The cells were thoroughly washed to remove the oligonucleotide outside the cells, and observed under a fluorescent confocal microscope (Nikon). As a result, both AS-1 and CL-1 were found to be distributed in the nucleus and cytoplasm of the cells after 24 hours.
( 5 ) C a R - 1細胞株に於けるマ卜リライシン mR N Aの発現に対 するアンチセンスオリゴヌクレオチドの効果 (5) Effect of antisense oligonucleotide on expression of matrilysin mRNA in CaR-1 cell line
2 4穴プレー卜の各ゥエル中の血清無添加 D M EM培地、 A S— 1又 は C L— 1オリゴヌクレオチドを 1 0 μ Μ含む同培地の各々 1 mlに、 5 0， 000個 Z穴の割合で細胞株を植え込み、 2 4時間インキュベート して処理した。 2 Serum-free DMEM medium in each well of a 4-well plate, 10 μl of the same medium containing 10 μl of AS-1 or CL-1 oligonucleotides The cell line was inoculated with and treated for 24 hours.
次に、 酸グァニジゥムチオシァネートーフエノール一クロ口ホルム法 を用いて全細胞 R N Aを抽出した。 ビォチン化オリゴ一 d T及びストレ プトアビジン常磁性粒子 （Promega, Madison, WI) を用いて全 R N A を捕捉した。 こうして単離した mR N Aから M— Mし V逆転写酵素 （Gi bco BRl_)を用いてランダムにプライマー化した c D N Aを調製した後、 P C R増幅処理を施した （Perkin-Elmer/Centus, Norwalk， CT)。
用いたプライマーペア一は以下の通リである。 Next, total cellular RNA was extracted using the acid guanidium thiosinate-phenol monochromic method. Total RNA was captured using biotinylated oligo-dT and streptavidin paramagnetic particles (Promega, Madison, WI). From the mRNA isolated in this manner, cDNA was randomly primed using M-M and V reverse transcriptase (Gibco BRl_), and then subjected to PCR amplification treatment (Perkin-Elmer / Centus, Norwalk, CT). The primer pairs used are as follows.
マトリライシンセンスプライマー： 5' - GTGACGGCGAGTTTTCAAAGC- 3' ( 配列番号 6) Matrilysin sense primer: 5 '-GTGACGGCGAGTTTTCAAAGC-3' (SEQ ID NO: 6)
マトリライシンアンチセンスプライマ一： 5' - CGTTGCGGGACTGGATTATCA G - 3' (配列番号 7) Matrilysin antisense primer: 5 '-CGTTGCGGGACTGGATTATCA G-3' (SEQ ID NO: 7)
一ァクチンセンスプライマー： 5' - CTTCGGGGGGGACGATGC- 3' (配列番 号 8) One actin sense primer: 5'-CTTCGGGGGGGACGATGC-3 '(SEQ ID NO: 8)
^—ァクチンアンチセンスプライマー： 5' -CGTACATGGCTGGGGTGTTG- 3' (配列番号 9) ^ —Actin antisense primer: 5'-CGTACATGGCTGGGGTGTTG-3 '(SEQ ID NO: 9)
P C R増幅は、 94 °Cでの変性 （1分間） 、 5 5 °Cでのアニーリング ( 1 . 5分間） 及び 7 2 °Cでのポリメラ一ゼ反応 （4分間） のサイクル を、 それぞれ 3 5サイクル （マトリライシン） 及び 2 5サイクル （ ー ァクチン） 繰り返した。 得られた P C R産物を 1 . 2%ァガロースゲル 中で電気泳動し、 ェチジゥムブロマイ ドで染色した。 PCR amplification involves cycles of denaturation at 94 ° C (1 minute), annealing at 55 ° C (1.5 minutes), and polymerase reaction at 72 ° C (4 minutes). Cycles (matrilysin) and 25 cycles (actin) were repeated. The resulting PCR product was electrophoresed in a 1.2% agarose gel and stained with ethidium bromide.
その結果、 C a R— 1細胞株は通常の条件下では内因性マ卜リライシ ン mR N Aを発現する力 A S— 1オリゴヌクレオチドで処理した場合 にはその発現量が顕著に低下することが判明した。 As a result, it was found that the C aR-1 cell line showed a marked decrease in its expression level under normal conditions when treated with the AS-1 oligonucleotide, which has the ability to express endogenous matrix lysin mRNA. did.
これに対して、 C L一 1 ヌクレオチドで処理した場合には、 このよう な発現抑制効果は観察されなかった。 In contrast, when treated with CL-11 nucleotides, such an effect of suppressing expression was not observed.
デンシトメ 卜リー分析によれば、 A S— 1 によってマ トリライシン m R N Aの発現の 92%が抑制されていた。 According to densitometry analysis, AS-1 suppressed 92% of the expression of matrilysin mRNA.
尚、 A S— 1又は C L— 1のいずれのオリゴヌクレオチドも/ S—ァク チン mR N Aの発現レベルに影響は与えていなかった。 It should be noted that neither the AS-1 nor the CL-1 oligonucleotide had an effect on the expression level of / S-actin mRNA.
(6) C a R- 1細胞株に於けるマ卜リライシン蛋白質発現に対する
アンチセンスオリゴヌクレオチドの効果 (6) Inhibition of matrilysin protein expression in C a R-1 cell line Effect of antisense oligonucleotide
上記インキュベーション （48時間） 後に得られた増殖培地を 1 5， 000 X gで 30分間遠心分離した。 これに 80%飽和硫酸アンモニゥ ムを添加し、 蛋白質を沈殿させ、 これを 1 5， 000 x g、 30分間の 遠心分離で回収した。 得られた蛋白質を 1 0%S D S—ポリアクリルァ ミ ドゲル電気泳動にかけ、 その後ニトロセルロース膜 （Bio- Rad, Hercu les. GA)に写しとつた。 The growth medium obtained after the above incubation (48 hours) was centrifuged at 15,000 X g for 30 minutes. To this was added 80% saturated ammonium sulfate to precipitate the protein, which was recovered by centrifugation at 15,000 xg for 30 minutes. The resulting protein was subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules. GA).
この膜を 3%ゼラチン ZT B S ( 20 m T r i s— H C し ρ Η7· 6 、 0.9 ο/ο塩化ナトリウム） で 1 0分間、 室温にてブロック処理した。 その後、 1 %ゼラチン 丁 85で1 ： 1 0に希釈したゥサギ抗ヒ トマ トリライシンポリクローナル抗体 （Miyazaki K， et a I . Cancer Res 5 0, 7758- 7765(1990))と 2時間反応させ、 更に、 同様に希釈したアル力 リフォスファターゼ結合ャギ抗ゥサギ I g G抗体 （Sigma)と 1時間反応 させた。 This membrane was blocked with 3% gelatin ZTBS (20 m Tris-HC and ρΗ7.6, 0.9ο / ο sodium chloride) for 10 minutes at room temperature. After that, the mixture was allowed to react with a heron anti-human trimeric triclonal antibody (Miyazaki K, et al. Cancer Res 50, 7758-7765 (1990)) diluted 1:10 with 1% gelatin for 2 hours. The cells were allowed to react for 1 hour with similarly diluted Alphosphite phosphatase-conjugated goat anti-Peacock IgG antibody (Sigma).
アル力リフォスファターゼ結合基質キッ 卜 （Bio-Rad)を用いて製品プ ロ トコールに従い、 発色反応を行った。 A color reaction was carried out using an alkaline phosphatase binding substrate kit (Bio-Rad) according to the product protocol.
その結果、 マトリライシンの前駆体 （29 K) 、 中間型 （23 K) 及 び活性型 （20 K) の 3種類の蛋白質が、 未処理 C a R- 1細胞株の増 殖培地中に見出された。 As a result, three proteins, matrilysin precursor (29 K), intermediate (23 K) and activated (20 K), were found in the growth medium of the untreated CaR-1 cell line. Was done.
ところが、 A S— 1オリゴヌクレオチドで処理することにより、 マト リライシン活性型は現れなくなり、 その他の型の発現も顕著に減少する ことが判明した。 デンシトメ一ターによる分析でマ トリライシン蛋白質 全量の 64%が発現を抑制されたことが判明した。 一方、 C L— 1オリ ゴヌクレオチドによる処理ではこのような抑制結果は示されなかった。
インビトロ浸潤アツセィ However, it was found that treatment with the AS-1 oligonucleotide stopped the matrilysin-active form from appearing and significantly reduced the expression of other forms. Analysis by densitometry revealed that the expression of 64% of the total amount of matrilysin protein was suppressed. On the other hand, treatment with CL-1 oligonucleotide did not show such a suppression result. In vitro infiltration atsushi
C a R - 1細胞 （5 X 1 0 3個） を M l C Sチャンバ一内のマトリゲ ル （再構成基底膜） 被膜フィルタ一 （8 ;u m細孔） 上に植え込み、 4 8 時間インキュベーションした。 この際、 種々の濃度 （2 . 5及び 1 0 μ Μ ) のオリゴヌクレオチドを添加した。 C a R - 1 cells (5 X 1 0 3 pieces) the Matorige le in M l CS chamber one (reconstituted basement membrane) coating the filter one; implantation on (8 um pores), and 48 hours of incubation. At this time, various concentrations (2.5 and 10 μM) of the oligonucleotide were added.
フィルター内に完全に浸潤した細胞をメタノールで固定し、 ギムザ染 色し、 顕微鏡で観察し、 浸潤細胞数を計測した。 フィルターに浸潤した 細胞数を上部ゥエル上の細胞数で除して、 浸潤細胞の割合 （％) を算出 した。 Cells completely infiltrated into the filter were fixed with methanol, stained with Giemsa, observed under a microscope, and the number of invading cells was counted. The number of cells invading the filter was divided by the number of cells on the upper well to calculate the percentage of invading cells (%).
その結果、 1 0 μ Μの A S - 1 で処理した場合に最大の効果が認めら れ、 未処理の細胞と較べて、 浸潤能が半減した （図 2 ) 。 As a result, the maximum effect was observed when the cells were treated with 10 μΜ of AS-1, and the invasive ability was reduced by half compared to untreated cells (Fig. 2).
A S— 1 による効果は 2 Mから 1 0 μ Μの間で用量依存的であり、 —方、 C L— 1 は全ての濃度でこのような効果を示さなかった。 The effect of AS-1 was dose-dependent between 2 M and 10 μM, whereas CL-1 did not show such an effect at all concentrations.
また、 陰性コントロールとしてマトリライシンを生産しないヒ 卜直腸 がん細胞である S W 4 8 0を用いて同様のアツセィを行った。 この結果 、 A S— 1又は Cし一 1のいずれのヌクレオチドもこの細胞の浸潤能に 影響を及ぼすことはなかった。 In addition, a similar assay was performed using SW480, a human rectal cancer cell that does not produce matrilysin, as a negative control. As a result, neither the AS-1 nor the C-1 nucleotide had an effect on the invasive ability of this cell.
以上のアツセィに於ける培養期間中、 細胞増殖速度はオリゴヌクレオ チドの存在の有無にかかわらず、 ほぼ同一であった。 実施例 2 During the above culture period in Atsushi, the cell growth rate was almost the same regardless of the presence or absence of the oligonucleotide. Example 2
W i D r細胞株に於けるマ トリライシン蛋白質発現に対するアンチセ ンスォリゴヌクレオチドの効果 Effect of antisense oligonucleotides on matrilysin protein expression in the WiDr cell line
ヒ 卜大腸がん細胞株である W i D rはマトリライシンを非常に高いレ
ベルで生産することが知られており、 更に本発明者等によって、 ゼラチ ナーゼ、 間質型コラゲナーゼ及びストロムライシンのいずれも検出可能 な量は分泌していないことが確認されている。 W i D rは J C RB 細 胞バンク （東京） から入手した。 Human colon cancer cell line WiDR has a very high level of matrilysin. It is known to produce in bells, and it has been confirmed by the present inventors that none of gelatinase, stromal collagenase, and stromlysin secrete detectable amounts. WiDR was obtained from JC RB Cell Bank (Tokyo).
この細胞株を、 1 0%牛胎児血清 （Gibco BRし）、 1 00単位 Zmlぺニ シリン G、 及び 1 mgZml硫酸ストレプ卜マイシン添加の D M E M Z F 1 2培地 (Life Technologies, Inc. , Ga i hersburg, MD, USA) 0. 5 m Iを含有する 24穴プレート （住友べ一クライ ト （東京） 製） の各ゥェ ル中に 5 X 1 0 個植え込み、 37°C、 5%C02及び 95%空気の湿気 状態下で 6時間培養した。 その後二回洗浄し、 血清無添加培地中で 2曰 間培養した。 This cell line was cultured in DMEMZF12 medium (Life Technologies, Inc., Gaithersburg, USA) supplemented with 10% fetal calf serum (Gibco BR), 100 units Zml penicillin G, and 1 mg Zml streptomycin sulfate. MD, USA) 0. 24-well plates containing 5 m I in each © E Le of (Sumitomo base one Cry preparative (Tokyo) Ltd.) 5 X 1 0 or implantation, 37 ° C, 5% C0 2 and 95 The cells were cultured for 6 hours in a humid state of% air. After washing twice, the cells were cultured for 2 hours in a serum-free medium.
実施例 1 (1 ) で作成した 1 5me rアンチセンスオリゴヌクレオチ ド A S— 1 ( 1 mM) 又は 1 5 m e rコン卜ロールォリゴヌクレオチド C L - 1 ( 1 mM) を含む P B S各 5 μ I 並びにォリゴヌクレオチドを 含有しない PBS 5 I を、 8時間毎に添加しながら 2日間培養した。 その後、 それぞれ 2穴からの増殖培地を合わせ、 以下、 実施例 1 に記載 したような手順に従ってウェスタンブロッ ト処理にかけた。 PBS containing 15mer antisense oligonucleotide AS-1 (1mM) or 15mer control oligonucleotide CL-1 (1mM), prepared in Example 1 (1) The cells were cultured for 2 days while adding PBS 5I containing no ribonucleotide every 8 hours. Thereafter, the growth media from each of the two wells were combined and subjected to Western blotting according to the procedure described in Example 1 below.
デンシ卜メータ一による分析によって、 AS— 1オリゴヌクレオチド で処理することにより、 マトリライシン蛋白質全量の発現の約 40〜8 θο/οが抑制されたことが判明した。 一方、 C L— 1オリゴヌクレオチド による処理ではこのような抑制効果は示されなかった。 Analysis with a densitometer revealed that treatment with the AS-1 oligonucleotide suppressed about 40 to 8 θο / ο of the expression of the total amount of matrilysin protein. On the other hand, treatment with the CL-1 oligonucleotide did not show such an inhibitory effect.
又、 W i D rの増殖自体には AS— 1オリゴヌクレオチド又は C L - 1オリゴヌクレオチドのいずれも全く影響を与えることはなかった。
実施例 3 Neither the AS-1 oligonucleotide nor the CL-1 oligonucleotide had any effect on the proliferation of WiDR itself. Example 3
W i D r細胞株に対するアンチセンスオリゴヌクレオチドのインビボ での効果 In vivo effects of antisense oligonucleotides on W i Dr cell lines
W i D r細胞株を 1 0%牛胎児血清 （Gibco BRL)、 1 0 0単位 mlぺ ニシリン G、 及び 1 mgZml硫酸ストレプトマイシン添加の D M E F 1 2培地 (Life Technologies, Inc. , Ga i thersburg, D, USA) 中で 3 7°C、 5 %C 02及び 9 5 %空気の湿気状態下で培養した。 これをト リプシン （0. 2 5 %トリプシンノ 0. 0 2 %E D T A) 処理し、 最終 濃度 1 X 1 08個 mlとなるように同培地中に懸濁した。 The WiDr cell line was added to 10% fetal calf serum (Gibco BRL), 100 units ml ぺ nicillin G, and 1 mgZml streptomycin sulfate in DMEF 12 medium (Life Technologies, Inc., Gaithersburg, D.). , USA) at 37 ° C, 5% CO 2 and 95% air. This was treated with trypsin (0.25% trypsin 0.02% EDTA) and suspended in the same medium to a final concentration of 1 × 10 8 ml.
BALB/c nu/nuヌードマウスを 2. 5 %アベルチン溶液で麻酔し、 無菌 的に左脇腹を切開して脾臓を一時的に体外に露出させ、 1 mlの注射器 （ 2 7ゲージ針） を使用して 5 1 06個の W i D rを含有する上記懸濁 液 50 / I をこの露出させた脾臓に注入した。 BALB / c nu / nu nude mice are anesthetized with 2.5% avertin solution, aseptically incised in the left flank to temporarily expose the spleen outside the body, and use a 1 ml syringe (27 gauge needle). was injected to 5 1 0 6 W i D r the suspension solution 50 / I containing the spleen was this exposed.
又、 実施例 1 で作成した 1 5 m e rアンチセンスオリゴヌクレオチド A S— 1 ( 1 2 0 μ g ) 又は 1 5 m e rコン卜口一ルォリゴヌクレオチ ド C L一 1 ( 1 2 0 S ) を含む P B S並びにこれらのオリゴヌクレオ チドを含有しない P B Sの各 0. 2 5 m l を上記脾臓処理の 1 日前から 1 0曰後までの間毎日腹腔内に注入した。 In addition, PBS containing the 15-mer antisense oligonucleotide AS-1 (120 μg) or the 15-mer control port-oligonucleotide CL-11 (120-S) prepared in Example 1 and 0.25 ml of each of these oligonucleotide-free PBSs was intraperitoneally injected daily from 1 day before the spleen treatment to 10 days after the spleen treatment.
それぞれ 1 1 、 2 8及び 4 2日後にマウスを屠殺して肝臓と脾臓を摘 出し、 それぞれにできた腫瘤の数を計測した。 その結果を図 3に示す。 図 3に示された結果から明らかなように、 コントロール P B S又はコ ントロールオリゴヌクレオチド C L一 1 を注入したマウスの場合には、 1 0日後に肝転移巣の腫瘤が現れ、 4 2日後には顕著な増加が見られた
これに対して、 本発明のアンチセンスオリゴヌクレオチド AS— 1 を 注入したマウスの場合にはこれらのマウスと較べて実験の全期間にわた つて、 肝転移巣の腫瘤の数が顕著に減少していることが判る。 コント口 ール PB Sを注入した対象群に対して、 本発明のアンチセンスオリゴヌ クレオチド AS— 1 を注入したマウスの肝転移巣の腫瘤の数は、 1 1、 28及び 42曰後にそれぞれ、 1 3%、 23及び 29%である。 The mice were sacrificed after 11, 28 and 42 days, respectively, and the liver and spleen were removed, and the number of tumors formed in each was counted. Figure 3 shows the results. As is evident from the results shown in Fig. 3, in mice injected with control PBS or control oligonucleotide CL-11, a tumor of liver metastatic focus appeared after 10 days, and was remarkable after 42 days. Increased In contrast, in the mice injected with the antisense oligonucleotide AS-1 of the present invention, the number of hepatic metastatic tumor masses was significantly reduced over the entire period of the experiment as compared to these mice. It turns out that there is. Compared to the control group injected with PBS, the number of tumors in the liver metastatic foci of the mice injected with the antisense oligonucleotide AS-1 of the present invention was 11, 28 and 42, respectively, 13%, 23 and 29%.
次に、 本発明のアンチセンスオリゴヌクレオチド A S— 1 による上記 抑制効果の用量依存性を確認した。 Next, the dose dependence of the above-described inhibitory effect by the antisense oligonucleotide AS-1 of the present invention was confirmed.
各群 5匹のマウスを使用して、 上記と同様に各量のアンチセンスオリ ゴヌクレオチド A S— 1で処理し、 W ί D r細胞の注入 42日後に屠殺 して肝臓にできた腫瘤の数を計測した。 その結果を図 4に示す。 Using 5 mice in each group, treated with each amount of antisense oligonucleotide AS-1 in the same manner as described above, and killed 42 days after injection of WίDr cells. Was measured. Fig. 4 shows the results.
Λ . 2 μ 個体の投与では肝転移巣の腫瘤の形成に関して何等の影 響も見られない。 しかしながら、 1 2 gZ個体を投与した場合には抑 制効果が観察され、 1 20 gZ個体を投与すると肝転移巣の腫瘤の数 はコントロール PBSを注入した対象群に対して約 30%に減少した。 尚、 全てのマウスで W i D I"細胞が注入された脾原発巣に腫瘍の形成 が見られた。 その大きさには、 コントロール PB S、 コントロールオリ ゴヌクレオチド C L一 1、 及びアンチセンスオリゴヌクレオチド AS— 1の投与において、 有意な差は認められなかった。 このことより、 本発 明のアンチセンスオリゴヌクレオチド AS— 1 は腫瘍の増殖には影響を 与えないものと思われる。 Λ. The administration of 2μ individuals has no effect on the formation of liver metastatic lesions. However, 1 2 gZ If individual was administered suppression effect was observed in, 1 20 g Z number of mass of an individual liver metastases when administered decreased about 30% relative to the control group injected with control PBS did. In all mice, tumor formation was observed in the splenic primary lesions injected with WiDI "cells. The size of the tumors included control PBS, control oligonucleotide CL-11, and antisense oligonucleotide. No significant difference was observed in the administration of AS-1.This suggests that the antisense oligonucleotide AS-1 of the present invention does not affect tumor growth.
更に、 肝転移巣の腫瘤の大きさに関しても各群で有意な差異は見られ なかった。
実施例 4 Furthermore, there was no significant difference between the groups in the size of the liver metastasis mass. Example 4
術後投与モデルでのアンチセンスオリゴヌクレオチドの効果 Effect of antisense oligonucleotide on postoperative administration model
実施例 3と同様な手順で脾臓内に W i D r細胞を注入したマウスにつ いて、 脾臓への注入から 24時間後に脾臓摘出術を行い、 実施例 1 で作成 した 1 5m e rアンチセンスオリゴヌクレオチド A S— 1又は 1 5me rコントロールォリゴヌクレオチド C L一 1 を含む P B Sを 1 0 μ g Z マウス、 並びにこれらのオリゴヌクレオチドを含有しない P B Sを、 翌 曰から 1 0日間連続して腹腔内に注入し、 4週間後に屠殺したのち肝転 移巣を観察した。 The spleen was removed from the mouse into which the WiDr cells had been injected into the spleen in the same manner as in Example 3, 24 hours after the injection into the spleen, and the 15-mer antisense oligonucleotide prepared in Example 1 was removed. 10 μg of PBS containing nucleotides AS-1 or 15mer control oligonucleotide CL-11, and PBS containing no such oligonucleotides, and PBS containing no such oligonucleotides were injected intraperitoneally for 10 consecutive days from the following: The mice were sacrificed 4 weeks later, and liver metastases were observed.
4週間後の肝転移結節数は P B S群： 7 ±0. 5 6個、 コン トロール オリゴヌクレオチド群： 1 6. 3 ±4. 8個であつたがアンチセンスォ リゴヌクレオチド群： 0個であり、 アンチセンスオリゴヌクレオチドに よる転移抑制効果が認められた。 産業上の利用可能性 After 4 weeks, the number of liver metastasis nodules was 7 ± 0.56 in the PBS group and 16.3 ± 4.8 in the control oligonucleotide group, but was 0 in the antisense oligonucleotide group. The transfer inhibition effect of the sense oligonucleotide was observed. Industrial applicability
本発明のアンチセンス化合物は、 実験動物を用いたインビボ系に於い て、 実際に大腸がん細胞の肝転移を有意に抑制することが効果を有し、 医薬としての有用なものであることが確認された。
The antisense compound of the present invention has an effect of actually significantly suppressing liver metastasis of colorectal cancer cells in an in vivo system using experimental animals, and is useful as a pharmaceutical. Was confirmed.
配列表 Sequence listing
配列番号： 1 SEQ ID NO: 1
配列の長さ ： 5 5 Array length: 5 5
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー ： 直鎖状 Topology: linear
配列の種類： c DNA t o mRNA Sequence type: cDNA t o mRNA
起源 Origin
生物名 ： h um a n Organism name: h um a n
配列 Array
TCCAAAGTGG TCACCTACAG GATCGTATCA TATACTCGAG ACTTACCGCA TATTA 55 TCCAAAGTGG TCACCTACAG GATCGTATCA TATACTCGAG ACTTACCGCA TATTA 55
配列番号： 2 SEQ ID NO: 2
配列の長さ ： 2 1 Array length: 2 1
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー ： 直鎖状 Topology: linear
配列の種類： c DNA t o mRNA Sequence type: cDNA t o mRNA
起源 Origin
生物名 ： h um a n Organism name: h um a n
配列 Array
AAATCAACCA TAGGTCCAAG A 21 AAATCAACCA TAGGTCCAAG A 21
配列番号： 3 SEQ ID NO: 3
配列の長さ ： 36 Array length: 36
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 2本鎖 Number of chains: 2 chains
トポロジー ： 直鎖状
配列の種類： c DNA t o mRNA Topology: linear Sequence type: cDNA to mRNA
起源 Origin
生物名 ： h um a n Organism name: h um a n
配列 Array
TCTGGACGGC AGCTATGCGA CTCACCGTGC TGTGTG 36 TCTGGACGGC AGCTATGCGA CTCACCGTGC TGTGTG 36
配列番号： 4 SEQ ID NO: 4
配列の長さ ： 1 5 Array length: 1 5
配列の型 ：核酸 Sequence type: Nucleic acid
鎖の数： 1本鎖 Number of chains: 1 strand
トポロジー ： 直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
GTATATGATA CGATC 15 配列番号： 5 GTATATGATA CGATC 15 SEQ ID NO: 5
配列の長さ ： 1 5 Array length: 1 5
配列の型 ：核酸 Sequence type: Nucleic acid
鎖の数： 1本鎖 Number of chains: 1 strand
トポロジー ：直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA Sequence type: other nucleic acid synthetic DNA
配列 Array
GTATTAGTAT CGAAC 15 GTATTAGTAT CGAAC 15
配列番号： 6 SEQ ID NO: 6
配列の長さ ： 2 1 Array length: 2 1
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 1本鎖
トポロジー ： 直鎖状 Number of chains: 1 strand Topology: linear
配列の種類：他の核酸 合成 DNA 配列 Sequence type: Other nucleic acid Synthetic DNA sequence
GTGACCGCGA CTTTTCAAAG C 21 配列番号： 7 GTGACCGCGA CTTTTCAAAG C 21 SEQ ID NO: 7
配列の長さ ： 2 2 Array length: 2 2
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 1本鎖 Number of chains: 1 strand
トポロジー ： 直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA 配列 Sequence type: Other nucleic acid Synthetic DNA sequence
CGTTGCGGGA CTGGATTATC AG 22 CGTTGCGGGA CTGGATTATC AG 22
配列番号： 8 SEQ ID NO: 8
配列の長さ ： 1 8 Array length: 1 8
配列の型 ：核酸 Sequence type: Nucleic acid
鎖の数： 1本鎖 Number of chains: 1 strand
トポロジー ： 直鎖状 Topology: linear
配列の種類：他の核酸 合成 DNA 配列 Sequence type: Other nucleic acid Synthetic DNA sequence
CTTCGCGGGC GACGATGC 18 CTTCGCGGGC GACGATGC 18
配列番号： 9 SEQ ID NO: 9
配列の長さ ： 20 Array length: 20
配列の型：核酸 Sequence type: nucleic acid
鎖の数： 1本鎖 Number of chains: 1 strand
トポロジー ： 直鎖状
配列の種類：他の核酸 合成 DNA 配列 Topology: linear Sequence type: Other nucleic acid Synthetic DNA sequence
CGTACATGGC TGGGGTGTTG 20
CGTACATGGC TGGGGTGTTG 20
Claims
1. ヒ トマトリライシンをコードする遺伝子の部分配列である配列番 号 1 (Gore領域） 、 配列番号 2 (Capping Site), 又は、 配列番号 3 ( AUG近傍） のいずれかの少なくとも一部とハイブリダィズするアンチ センス化合物。 1. Hybridized with at least a part of SEQ ID NO: 1 (Gore region), SEQ ID NO: 2 (Capping Site), or SEQ ID NO: 3 (near AUG), which is a partial sequence of the gene encoding human tomato relysin Antisense compounds.
2. ヒ トマトリライシンをコードする遺伝子の部分配列である配列番 号 1 (Gore領域） 、 配列番号 2 (Capping Site), 又は、 配列番号 3 ( AUG近傍） のいずれかの少なくとも一部に相補的な配列を含むオリゴ ヌクレオチドである、 請求項 1 に記載のアンチセンス化合物。 2. Complementary to at least a part of SEQ ID NO: 1 (Gore region), SEQ ID NO: 2 (Capping Site), or SEQ ID NO: 3 (near AUG), which is a partial sequence of the gene encoding human tomato relysin The antisense compound according to claim 1, which is an oligonucleotide containing a specific sequence.
3. 配列番号 1の塩基配列の少なくとも一部に相補的な配列を含む請 求項 2に記載のアンチセンス化合物。 3. The antisense compound according to claim 2, which comprises a sequence complementary to at least a part of the nucleotide sequence of SEQ ID NO: 1.
4. ヒ トマトリライシンの発現を抑制しうる請求項 1 ~ 3のいずれか 一項に記載のアンチセンス化合物。 4. The antisense compound according to any one of claims 1 to 3, wherein the antisense compound is capable of suppressing expression of human tomato relysin.
5. 塩基数が 1 0ないし 50である請求項 1 〜 4のいずれか一項に記 載のアンチセンス化合物。 5. The antisense compound according to any one of claims 1 to 4, wherein the antisense compound has 10 to 50 bases.
6. 塩基数が 1 0ないし 30である請求項 5に記載のアンチセンス化 合物。 6. The antisense compound according to claim 5, wherein the number of bases is 10 to 30.
7. ヌクレオチド間の結合基の少なくとも 1 つがィォゥ原子を含む請 求項 1 〜6のいずれか一項に記載のアンチセンス化合物。 7. The antisense compound according to any one of claims 1 to 6, wherein at least one of the internucleotide linking groups contains an iodine atom.
8. 配列番号 4で示されるアンチセンス化合物。 8. An antisense compound represented by SEQ ID NO: 4.
9. 請求項 1 〜 8のいずれかに記載のアンチセンス化合物を含む医薬組 成物。 9. A pharmaceutical composition comprising the antisense compound according to any one of claims 1 to 8.
1 0. がん転移抑制剤である請求項 9に記載の医薬組成物。
10. The pharmaceutical composition according to claim 9, which is a cancer metastasis inhibitor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34378497 | 1997-11-28 | ||
| JP9/343784 | 1997-11-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999028452A1 true WO1999028452A1 (en) | 1999-06-10 |
Family
ID=18364220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1998/002327 WO1999028452A1 (en) | 1997-11-28 | 1998-05-27 | Matrilysin antisense compounds |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1999028452A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2802945A1 (en) * | 1999-12-28 | 2001-06-29 | Pf Medicament | New nucleic acid encoding matrix metalloprotease-25, useful for treatment and diagnosis of cancer, angiogenesis and inflammation |
| WO2001019853A3 (en) * | 1999-09-11 | 2001-11-15 | Univ Sheffield | Cell transfection |
| US7500272B2 (en) | 2000-08-04 | 2009-03-03 | First Data Corporation | Manufacturing unique devices that generate digital signatures |
| US7552333B2 (en) | 2000-08-04 | 2009-06-23 | First Data Corporation | Trusted authentication digital signature (tads) system |
-
1998
- 1998-05-27 WO PCT/JP1998/002327 patent/WO1999028452A1/en active Application Filing
Non-Patent Citations (3)
| Title |
|---|
| BIOCHEM. J., 253, (1988), DANIELE MULLER et al., "The Collagenase Gene Family in Humans Consists of at Least Four Members", p. 187-193. * |
| BIOCHEM. J., 285, (1992), HANS-PETER MARTI et al., "Molecular Characterization of a Low-Molecular-Mass Matrix Metallo-Proteinase Secreted by Glomerular Mesangial Cells as PUMP-1", p. 899-905. * |
| THE JOURNAL OF BIOLOGICAL CHEMISTRY, 269, (1994), MIREILLE GAIRE et al., "Structure and Expression of the Human Gene for the Matrix Metalloproteinase Matrilysin", p. 2032-2040. * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001019853A3 (en) * | 1999-09-11 | 2001-11-15 | Univ Sheffield | Cell transfection |
| FR2802945A1 (en) * | 1999-12-28 | 2001-06-29 | Pf Medicament | New nucleic acid encoding matrix metalloprotease-25, useful for treatment and diagnosis of cancer, angiogenesis and inflammation |
| US7500272B2 (en) | 2000-08-04 | 2009-03-03 | First Data Corporation | Manufacturing unique devices that generate digital signatures |
| US7552333B2 (en) | 2000-08-04 | 2009-06-23 | First Data Corporation | Trusted authentication digital signature (tads) system |
| US7784106B2 (en) | 2000-08-04 | 2010-08-24 | First Data Corporation | Manufacturing unique devices that generate digital signatures |
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