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WO1999028452A1 - Composes non codants de matrilysine - Google Patents

Composes non codants de matrilysine Download PDF

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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
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WIPO (PCT)
Prior art keywords
antisense compound
seq
oligonucleotide
matrilysin
antisense
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP1998/002327
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English (en)
Japanese (ja)
Inventor
Kaoru Miyazaki
Hiroshi Shimada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mochida Pharmaceutical Co Ltd
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Mochida Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mochida Pharmaceutical Co Ltd filed Critical Mochida Pharmaceutical Co Ltd
Publication of WO1999028452A1 publication Critical patent/WO1999028452A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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.
  • Sequence type nucleic acid
  • Organism name h um a n
  • Sequence type nucleic acid
  • Organism name h um a n
  • Sequence type nucleic acid
  • Organism name h um a n
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

On décrit des substances qui sont capables d'inhiber de manière spécifique l'expression de la matrilysine pour inhiber ainsi la métastase, ces substances étant préparées à l'aide d'une technique non codante. On décrit également des composés non codants pouvant s'hybrider avec au moins une partie d'une séquence partielle d'un gène codant la matrilysine humaine; des compositions médicinales et plus particulièrement des compositions ayant pour effet d'inhiber la métastase, qui contiennent ces composés non codants.
PCT/JP1998/002327 1997-11-28 1998-05-27 Composes non codants de matrilysine Ceased WO1999028452A1 (fr)

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Application Number Priority Date Filing Date Title
JP9/343784 1997-11-28
JP34378497 1997-11-28

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WO1999028452A1 true WO1999028452A1 (fr) 1999-06-10

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2802945A1 (fr) * 1999-12-28 2001-06-29 Pf Medicament Nouvelle metalloprotease matricielle mmp-25 homologue de la matrilysine
WO2001019853A3 (fr) * 1999-09-11 2001-11-15 Univ Sheffield Transfection cellulaire
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001019853A3 (fr) * 1999-09-11 2001-11-15 Univ Sheffield Transfection cellulaire
FR2802945A1 (fr) * 1999-12-28 2001-06-29 Pf Medicament Nouvelle metalloprotease matricielle mmp-25 homologue de la matrilysine
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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