[go: up one dir, main page]

WO2017036398A1 - Petit arn interférant, composition pharmaceutique, et son application - Google Patents

Petit arn interférant, composition pharmaceutique, et son application Download PDF

Info

Publication number
WO2017036398A1
WO2017036398A1 PCT/CN2016/097560 CN2016097560W WO2017036398A1 WO 2017036398 A1 WO2017036398 A1 WO 2017036398A1 CN 2016097560 W CN2016097560 W CN 2016097560W WO 2017036398 A1 WO2017036398 A1 WO 2017036398A1
Authority
WO
WIPO (PCT)
Prior art keywords
sirna
pharmaceutical composition
group
cancer
sirrm2
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
Application number
PCT/CN2016/097560
Other languages
English (en)
Chinese (zh)
Inventor
张鸿雁
高山
黄渊余
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.)
Suzhou Ribo Life Science Co Ltd
Original Assignee
Suzhou Ribo Life Science 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 Suzhou Ribo Life Science Co Ltd filed Critical Suzhou Ribo Life Science Co Ltd
Priority to CN201680046055.3A priority Critical patent/CN108431224B/zh
Publication of WO2017036398A1 publication Critical patent/WO2017036398A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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

Definitions

  • the present invention relates to the field of biomedical technology, and in particular to an siRNA (small interfering nucleic acid), an RNA interference target nucleic acid of the siRNA, a pharmaceutical composition, and uses thereof.
  • siRNA small interfering nucleic acid
  • RNA interference target nucleic acid of the siRNA a pharmaceutical composition, and uses thereof.
  • pancreatic cancer the first choice for the treatment of pancreatic cancer is surgical resection, but only 15-20% of patients are diagnosed with surgery, and the 5-year survival rate of patients with pancreatic cancer is less than 5%.
  • Pancreatic cancer is not very sensitive to radiotherapy and chemotherapy, and most drugs are objectively less than 10% effective.
  • Gemcitabine is currently the first-line treatment for standard pancreatic cancer chemotherapy, but its efficacy in improving patient survival and quality of life is limited. In patients with advanced pancreatic cancer, the objective rate of gemcitabine is only about 12%, and the survival period is only slightly prolonged. In general, patients with pancreatic cancer still lack effective treatment.
  • ribonucleotide reductase is the only enzyme in vivo that catalyzes the reduction of four ribonucleotides to produce the corresponding deoxyribonucleotides.
  • the enzyme is a key enzyme and rate-limiting enzyme for DNA synthesis and repair, and plays a regulatory role in cell proliferation and differentiation.
  • Ribonucleotide Reductase (RR) consists of a large subunit M1 and a small subunit M2, both of which are dimeric structures, commonly referred to as RRM1 and RRM2.
  • RRM1 has a site that binds to a substrate and an allosteric effector, and contains a sulfur group directly supplying electrons.
  • the switch that controls the specificity and enzyme activity of the substrate is a tumor suppressor gene and is also gemcitabine. (Gemcitabine) molecular target.
  • RRM2 is an iron-sulfur protein which participates in a catalytic reaction by forming a specific free radical through the benzene ring of a tyrosine residue, which is both a catalytic region responsible for substrate conversion and a contact inhibition region.
  • RRM2 is a homodimer that forms two identical ferrous iron centers that stabilize a lysyl radical, which is important for triggering electron transport during the catalytic process.
  • RRM2 has been found to have high expression in tumors; although it is reported in the literature (CN200680018408.5) that siRNA pharmaceutical compositions containing RRM2 can be used to treat liver cancer, their siRNA activity is not high in vivo; The self-defect can only be administered locally in the tumor.
  • the dose of the cocktail composed of 2 siRNAs is still 2.5 mg/kg, and the frequency of administration is high, once a day, and 3 times for continuous administration.
  • This method of intrahepatic artery administration has a high frequency of administration and a large dose, which is difficult to meet clinical needs.
  • the object of the present invention is to provide a high-efficiency siRNA sequence against the RRM2 gene, and to provide a pharmaceutical composition comprising the siRNA sequence, which can effectively inhibit tumor cell proliferation, promote tumor cell apoptosis, thereby inhibiting tumor tissue growth. .
  • the present invention provides a new and high-efficiency siRNA and a pharmaceutical composition thereof, which can reduce the dosage of administration and prolong the administration interval by systematic administration, and has the reality of clinical research and commercial development.
  • the present invention provides an siRNA, wherein the sense strand base sequence of the siRNA is set forth in SEQ ID NO: 2, and the antisense strand base sequence of the siRNA is set forth in SEQ ID NO: 3; And the phosphate-sugar backbone of the siRNA has or does not have a modifying group, respectively.
  • the invention also provides an RNA interference target nucleic acid, wherein the RNA interferes with the target nucleic acid as set forth in SEQ ID NO:4.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA as described above and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier contains an organic amine, cholesterol and polyethylene a glycolated phospholipid; wherein the organic amine is a compound as shown in formula (1) and/or Pharmaceutically acceptable salt:
  • R 1 and R 2 are each independently a C 10 -C 20 linear alkyl group; n is an integer from 1 to 6; and R 3 , R 4 , R 5 and R 6 are each independently hydrogen or Rx; At least one of R 3 , R 4 , R 5 and R 6 is Rx;
  • Rx is or Ry is a linear alkyl group having a carbon number of C 12 - C 20 .
  • the invention provides the use of a siRNA as described above and/or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.
  • the present invention provides a method of treating cancer comprising administering an siRNA as described above and/or a pharmaceutical composition as described above to a patient in need thereof.
  • the invention provides a method of inhibiting expression of a RRM2 gene in a cell, the method comprising introducing an siRNA as described above and/or a pharmaceutical composition as described above into the cell.
  • the present invention provides a new and effective treatment method for cancer, which can be administered by system, reducing the dose and lengthening the administration interval.
  • the siRNA of the present invention can be administered intravenously at a dose of 1 mg/kg and the administration interval is twice a week, and a tumor inhibition rate of up to 82% can be obtained in vivo, which is significantly superior to the literature.
  • Paraneoplastic administration continuous administration 3 times in 3 days, 2.5 mg/kg of treatment per administration per day; meanwhile, the siRNA in the literature was modified by the modification method similar to the present invention. Under the mode of administration, 68% of the tumor inhibition rate was far less than the 85% inhibition efficiency achieved by the siRNA of the present invention.
  • a specific pharmaceutical composition formed from a pharmaceutically acceptable carrier formed of an organic amine, a helper lipid, or a pegylated phospholipid, and a siRNA of the present invention, and a pharmaceutical composition formed by the other conventional carrier and the siRNA of the present invention It has a very high biological activity and its mRNA inhibition efficiency is 2-20 times that of other conventional carrier-formed pharmaceutical compositions.
  • Figure 1 is a graph showing the results of inhibition of PANC-1 cell colony assembly by siRRM2-M and irrelevant siRNA (siNC).
  • Figure 2 is a graph showing tumor volume results of inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.
  • Figure 3 is a graph showing tumor weight results of inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.
  • Figure 4 is a photograph showing the results of tumor block photographs showing inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.
  • Figure 5 is a graph showing tumor weight results showing inhibition of hepatocarcinoma tumor tissue growth by siRRM2-M delivered by RBP131 vector.
  • RRM2 refers to a cDNA sequence such as the gene shown in Genebank Registry No.: NM_001034.3, and the RNA sequence thereof is shown in SEQ ID NO: 1.
  • the present invention provides an siRNA, wherein the sense strand base sequence of the siRNA is set forth in SEQ ID NO: 2, and the antisense strand base sequence of the siRNA is set forth in SEQ ID NO: 3; And the phosphate-sugar backbone of the siRNA has or does not have a modifying group, respectively.
  • the siRNA of the present invention contains a phosphate-sugar skeleton and a base.
  • the siRNA of the present invention contains a modifying group which does not cause a significant weakening or loss of the function of the siRNA to inhibit expression of the RRM2 gene.
  • a modifying group which does not cause a significant weakening or loss of the function of the siRNA to inhibit expression of the RRM2 gene.
  • the modifying group is an optionally substituted sugar group and an optionally substituted ester group, but is not limited thereto.
  • the phosphate-sugar backbone of the siRNA has the following modifying groups: the glycosyl group at the 1, 6, 14, 16 and 18 positions of the sense strand is a 2'-methoxyribosyl group; the antisense strand is 2 The glycosyl group at the position is a 2'-methoxyribosyl group, and the glycosyl group at the third position of the antisense strand is a 2'-fluororibosyl group.
  • 2'-methoxyribosyl refers to a group formed by substitution of a 2'-OH of a ribose group with a methoxy group
  • 2'-fluororibose group refers to a group formed by substitution of a 2'-OH of a ribose group with fluorine Group.
  • the ester group between the 20th and 21st positions of the sense strand and/or the antisense strand of the siRNA is a phosphorothioate group.
  • the phosphorothioate group refers to a group formed by replacing one oxygen atom in the phosphodiester group with a sulfur atom, as shown in the formula (5):
  • the invention also provides an RNA interference target nucleic acid, wherein the RNA interferes with the target nucleic acid as set forth in SEQ ID NO:4.
  • the RNA interference target nucleic acid refers to RRM2 mRNA In the fragment which hybridizes to the nucleic acids of positions 1 to 19 in the antisense strand as shown in SEQ ID NO: 3.
  • the invention provides a pharmaceutical composition comprising an siRNA as described above and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as Fe 3 O 4 , Fe 2 O 3 ), carbon nanotubes, Mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, polylysine (poly (L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly D or L Type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA) and poly(methacrylic acid) -N,N-dimethylaminoethyl ester (poly(2-dimethylaminoethyl methacrylate),
  • the pharmaceutical composition of the present invention may further comprise other excipients which are pharmaceutically acceptable, and the excipients may be one or more of various preparations or compounds conventionally employed in the art.
  • the pharmaceutically acceptable other excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.
  • the pH buffer may be a trishydroxymethylaminomethane hydrochloride buffer having a pH of 7.5-8.5 and/or a phosphate buffer having a pH of 5.5-8.5, preferably a phosphate having a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.
  • the protective agent may be included in an amount of from 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.
  • the osmotic pressure adjusting agent may be sodium chloride and/or Potassium chloride.
  • the osmotic pressure adjusting agent is present in an amount such that the osmotic pressure of the pharmaceutical composition is from 200 to 700 milliosmoles per kilogram.
  • the content of the osmotic pressure adjusting agent can be easily determined by those skilled in the art depending on the desired osmotic pressure.
  • the pharmaceutical composition may be a liquid preparation, such as an injection solution, or may be a lyophilized powder injection, which is mixed with a liquid adjuvant when administered, and formulated into a liquid preparation.
  • the liquid formulation can be, but is not limited to, for subcutaneous, intramuscular or intravenous administration, and can be, but is not limited to, administered to the lungs by spraying, or administered to other organ tissues (such as the liver) via the lungs by spraying.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid and/or a poly Ethylene glycolated phospholipids.
  • the organic amine, helper lipid, and pegylated phospholipid are each selected from the group consisting of amine-containing transfection compounds described in CN201180060664.1 (hermby incorporated by reference herein in its entirety)
  • a salt or a derivative, a helper lipid, and a pegylated phospholipid One or more of a salt or a derivative, a helper lipid, and a pegylated phospholipid.
  • the pharmaceutically acceptable carrier contains an organic amine, cholesterol and a pegylated phospholipid; wherein the organic amine is as shown in formula (1) a compound and/or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are each independently a C 10 -C 20 linear alkyl group; n is an integer from 1 to 6; and R 3 , R 4 , R 5 and R 6 are each independently hydrogen or Rx; At least one of R 3 , R 4 , R 5 and R 6 is Rx;
  • Rx is or Ry is a linear alkyl group having a carbon number of C 12 - C 20 .
  • the organic amine is an organic amine as shown in formula (2) and/or an organic amine as shown in formula (3):
  • the pegylated phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
  • the molar ratio between the organic amine, cholesterol and the pegylated phospholipid may be (19.7-80): (19.7-80): (0.3-50).
  • the molar ratio between the organic amine, cholesterol and the PEGylated phospholipid is (50-70) in the pharmaceutical composition: (20) -40): (3-20).
  • the liposome particles formed from the siRNA of the present invention and the above carrier have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more typically, the liposome particles have an average diameter of from about 50 nm to about 120 nm, From about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
  • the weight ratio (weight/weight ratio) of the siRNA of the invention to all lipids is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1 :17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1:6 to about 1:10,
  • the weight ratio of the siRNA of the present invention to the total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 , 1:14, 1:15, 1:16, 1:17 or 1:18.
  • the pharmaceutical composition further comprises at least one additional anti-cancer chemotherapeutic agent that inhibits cancer cells in an additive or synergistic manner with the nucleic acid.
  • the nucleic acid and the chemotherapeutic agent can be prepared in advance as a combined preparation, or can be prepared independently and selected for appropriate time and dosage to achieve the combined effect.
  • the anti-cancer chemotherapeutic agent is doxorubicin.
  • the siRNA of the present invention and doxorubicin are capable of exerting an excellent synergistic effect.
  • doxorubicin can be stored separately.
  • siRNA can be administered simultaneously with doxorubicin, and siRNA can also be administered sequentially with doxorubicin.
  • the invention provides the use of a siRNA as described above and/or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.
  • the cancer includes, but is not limited to, leukemia, lymphoma, multiple myeloma, brain tumor, breast cancer, adrenal adenoma, thyroid cancer, pancreatic cancer, pituitary cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer. , rectal cancer, liver cancer, gallbladder cancer, lung cancer, testicular cancer, prostate cancer, head and neck cancer Including oral cancer, etc.), skin cancer, kidney cancer.
  • the siRNA as described above and/or the pharmaceutical composition as described above is particularly suitable for the treatment of liver cancer and/or pancreatic cancer.
  • the present invention provides a method of treating cancer comprising administering an siRNA as described above and/or a pharmaceutical composition as described above to a patient in need thereof.
  • Routes of administration suitable for the methods of the invention include topical administration and systemic administration.
  • Administration can be administered to a subject by any suitable route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal.
  • Drug airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration).
  • Dosages suitable for the methods of the invention may be those conventional in the art, which may be determined based on various parameters, particularly the age, weight and sex of the subject.
  • the range of human doses can be derived based on data obtained from cell culture assays and animal studies.
  • the invention provides a method of inhibiting expression of a RRM2 gene in a cell, the method comprising introducing an siRNA as described above and/or a pharmaceutical composition as described above into the cell.
  • the cells include, but are not limited to, human hepatoma cell line HepG2, human cervical cancer cell line Hela, human pancreatic cancer cell line PANC-1, and human breast cancer cell line MDA-MB-231.
  • the sequence of the siRNA is shown in Table 1.
  • One single strand of the siRNA has the sequence represented by SEQ ID NO: 2, which is identical to the corresponding RNA interference target nucleic acid (shown as SEQ ID NO: 4) in the mRNA sequence of RRM2.
  • the other single strand has the sequence represented by SEQ ID NO: 3, which is complementary to the corresponding RNA interference target nucleic acid in the mRNA sequence of RRM2.
  • Oligonucleotide single strands of siRNA are chemically synthesized according to methods well known in the art. For the synthesis, two deoxythymidine dTdTs are added to the 3' end of the single strand of the oligonucleotide.
  • the complementary oligonucleotides are single-stranded to form a double strand, and the two ends of the double strand have a 3' overhang of dTdT, respectively.
  • the sequences of the synthesized oligonucleotides are shown in Table 1.
  • This example was used to examine the inhibitory efficiency of siRNA against RRM2 mRNA expression levels in vitro.
  • the human hepatoma cell line HepG2 was inoculated into a 24-well plate with DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, and the cell density was 4 ⁇ 10 5 . /well, 0.5 mL per well, incubated overnight at 37 °C.
  • Transfected specific steps are as follows: 100ng diluted in Preparation Example 1 siRNA in serum-free medium 50 ⁇ L DEME while 1 ⁇ L Lipofectamine TM 2000 (Invitrogen) was diluted in serum-free medium 50 ⁇ L DEME, the above two The solution was incubated after 5 minutes at room temperature. After the mixed solution was allowed to stand at room temperature for 20 minutes, 100 ⁇ L of the above mixed solution was added to a 24-well plate inoculated with PANC-1 cells. The final concentration of siRNA was approximately 10 nM.
  • the cells were cultured at 37 ° C for 4 hours, and then 1 mL of DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin was added, and then cultured at 37 ° C. hour.
  • RNA concentration ( ⁇ g/ ⁇ L) 0.04 ⁇ OD260 ⁇ dilution factor.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara Company, Catalog No.
  • inhibition rate [1-(experimental RRM2 mRNA expression level / experimental hole ⁇ -Actin mRNA expression level) / (negative control well RRM2 mRNA expression level) / Negative control well ⁇ -Actin mRNA expression level)] ⁇ 100%.
  • inhibition rate [1-(experimental RRM2 mRNA expression level / experimental hole ⁇ -Actin mRNA expression level) / (negative control well RRM2 mRNA expression level) / Negative control well ⁇ -Actin mRNA expression level)] ⁇ 100%.
  • the results are shown in Table 3.
  • siRNA mRNA inhibition rate (%) siNC 0 siPC1 65.7 siPC2 82.4 siRRM2 91.3
  • the activity of the siRNA of the present invention is significantly higher than that of the literature (CN200680018408.5)
  • the disclosed sequence can inhibit the expression of the target gene RRM2 more efficiently.
  • the oligonucleotides listed in Table 4 were synthesized.
  • the oligonucleotides in Table 4 contain modified nucleotide residues, and the complementary oligonucleotide strands anneal to form modified siRNAs, designated siRRM2-M, siPC1-M, and siPC2-M, respectively.
  • (OMe) represents a pentose group in the nucleotide residue to the left of which is a 2'-methoxyribosyl group
  • (F) represents a pentose group in the nucleotide residue to the left of which is 2'- The fluororibose group
  • (S) represents an ester group between the deoxyribonucleotides dTdT on both sides thereof is a phosphorothioate group.
  • the nucleotide sequences before the modification of these siRNAs corresponded to siRRM2, siPC1 and siPC2 in Preparation Example 1, respectively.
  • This example was used to examine the effect of chemical modification on siRNA serum stability.
  • siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2, and siRRM2, siPC1 and siPC2 obtained in Preparation Example 1 were measured in a serum environment. Specific steps are as follows.
  • 10 ⁇ L of the above modified and unmodified siRNA (20 ⁇ M) were mixed with 90 ⁇ L of 50% human plasma (Human plasma, HP, PBS diluted), respectively, and incubated at 37 ° C for 0, 2, 4, 8, 24, 48 and 72 in vitro.
  • the treated sample was obtained after an hour.
  • the sample was sampled and sampled at 10 ⁇ L, and immediately frozen in liquid nitrogen, and stored at -80 ° C for use.
  • 72 hours degradation rate [1-( 72 hours of electrophoresis main strip gray scale / 0 hour electrophoresis main strip gray scale)] ⁇ 100%.
  • the stability of the modified siRNA was significantly enhanced in the human serum environment compared to the unmodified siRNA.
  • This example was used to detect the inhibition efficiency of siRNA on the expression level of RRM2 mRNA in vitro before and after chemical modification.
  • the inhibitory efficiency of the expression levels of RRM2 mRNA was determined by siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2, and siRRM2, siPC1 and siPC2 obtained in Preparation Example 1, respectively, in the same manner as in Example 1. .
  • the specific steps of transfection and fluorescence quantitative real-time PCR are as described in Example 1.
  • the above siRNAs were transfected with a gradient dose to a final concentration of 0.5 nM, 1 nM and 10 nM, respectively.
  • siRNA (siNC) as in Example 1 was used.
  • inhibition rate [1-(experimental RRM2 mRNA expression level / experimental hole ⁇ -Actin mRNA expression level) / (negative control well RRM2 mRNA expression level) / Negative control well ⁇ -Actin mRNA expression level)] ⁇ 100%. Result As shown in Table 6.
  • siRRM2-M has a stronger inhibitory efficiency against siPC1-M and siPC2-M, and siRRM2-M has a similar inhibitory effect on siRRM2 as the expression of RRM2 mRNA at doses of 1 nM and 10 nM.
  • siRRM2-M had a more superior inhibitory effect (46% vs. 0%) than the corresponding siRRM2. This may be due to the fact that the modification enhances the stability of the siRNA and thereby increases the retention time of the siRNA in the cell, thereby increasing the inhibition of the activity of the siRNA.
  • This example was used to detect the inhibitory effect of the modified siRNA on the growth of liver cancer tumor cells, and specifically, the growth inhibitory effect of siRRM2-M on HepG2 cells was examined using MTT.
  • HepG2 cells were taken at 5 ⁇ 10 3 /mL, 100 ⁇ L/well were seeded in 96-well microplates, and after 24 hours of culture, siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2 were transfected, each siRNA. The final concentrations were in turn 50 nM, 100 nM and 200 nM, respectively. There is also a cell-free zeroing hole. Tumor cells were cultured for 48 hours at 37 ° C, 5% CO 2 , and then added to MTT (Sigma, Cat. No.
  • the IC 50 of siRRM2-M was 65.0 nmol/L
  • the IC 50 of siPC1-M was 143.7 nmol/L
  • the IC 50 of siPC2-M was 129.2 nmol/L. It can be seen that the siRRM2-M provided by the present invention has an effect of significantly inhibiting the growth of liver cancer cells relative to siPC1-M and siPC2-M.
  • the experimental results on the pancreatic cancer cell line PANC-1 were similar (results not shown).
  • This example was used to detect the blocking effect of the modified siRNA on the cell cycle of pancreatic cancer cells, and specifically, the effect of siRRM2-M on the cell cycle arrest of PANC-1 was detected by flow cytometry.
  • siRNA was transfected with Lipofectamine 2000 to give a final concentration of siNC of 50 nM, a final concentration of siRRM2-M of 25 nM and 50 nM; after 4 hours, 2 mL of DMEM medium containing 10% FBS was added.
  • the cells were collected at 1000 rpm/min, centrifuged for 5 min, and the supernatant was discarded; washed once with pre-cooled PBS, centrifuged at 1000 rpm/min for 5 min, and the supernatant was discarded; then uniformly sprayed with 500 ⁇ L of pre-cooled PBS.
  • Each sample was further fixed with 3 mL of 70% pre-chilled ethanol, gently pipetted several times, and frozen at -20 ° C for 3 hours.
  • siRNA S phase cell ratio (%) No processing group 24.74 siNC (50nM) 27.99 siRRM2-M (25nM) 57.42 siRRM2-M (50nM) 63.46
  • This example was used to detect the inhibitory effect of modified siRNA on colony assembly of pancreatic cancer tumor cells.
  • the exponential growth phase cells were taken and the cells were collected by subculture.
  • the cells were resuspended in 10 mL of 10% FBS, 1% penicillin/streptomycin in DMEM, and cell counts were performed.
  • the plate was plated with a 6-well plate, and the number of cells per well was 300.
  • siRRM2-M was transfected with Lipofectamine 2000 and an unrelated siRNA control (siNC) at a transfection concentration of 50 nM, and 3 replicate wells per siRNA.
  • siRRM2-M was transfected with Lipofectamine 2000 and an unrelated siRNA control (siNC) at a transfection concentration of 50 nM, and 3 replicate wells per siRNA.
  • the medium was replaced with fresh medium (10% FBS, 1% penicillin/streptomycin in DMEM).
  • the culture was continued for 10 days after changing the solution, and the fresh medium was changed every three days.
  • Figure 1 shows the inhibitory effect of siRRM2-M on PANC-1 cell colony assembly.
  • the results showed that the number of colony formation of cells treated with siRRM2-M was significantly reduced compared to cells treated with irrelevant siRNA (siNC), demonstrating that inhibition of RRM2 expression inhibits the growth of PANC-1 cells.
  • siNC irrelevant siRNA
  • Parallel, in the liver The experimental results on the cancer cell line HepG2 were similar (results not shown).
  • This preparation was used to prepare siRNA pharmaceutical compositions RBP131/siRNA and RBP130/siRNA.
  • Three dry powder lipid compounds ie, organic amines (such as those shown in formula (2) or formula (3), the preparation method thereof, see compound 87 or 72 in CN201180060664.1), cholesterol, PEGylated lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000]))) suspended in ethanol at a molar ratio of 59:29:12 And mixed, the total mass concentration of the three lipid compounds was about 8.85 mg/ml.
  • organic amines such as those shown in formula (2) or formula (3), the preparation method thereof, see compound 87 or 72 in CN201180060664.1
  • PEGylated lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000]
  • siRNA to be tested (siNC, siRRM2 in Preparation Example 1 and siRRM2-M, siPC1-M, siPC2-M in Preparation Example 2) was dissolved in a 200 mM sodium acetate (pH 5.2) solution to have a siRNA concentration of 0.2 mg. /ml.
  • the obtained lipid ethanol solution and siRNA aqueous sodium acetate solution were quickly mixed at a volume ratio of 1:3.
  • the specific composition of the liposome preparation obtained after mixing is described in Table 8.
  • the liposome preparation obtained after mixing (i.e., a combination of an organic amine, cholesterol, PEGylated lipid and siRNA) was incubated at about 50 ° C for 10 minutes. After incubation, use The phase-cut flow system, the hollow fiber column 100KDa ultrafiltration, the ultrafiltration exchange solution was pH 7.4 PBS. The preparation can be concentrated or diluted to the desired siRNA concentration while ultrafiltration. The ultrafiltered preparation was sterilized by filtration on a 0.22 ⁇ m filter.
  • the mixture is called RBP131, an organic amine represented by formula (3), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)-2000
  • the composition of the lipid mixture is called RBP130.
  • the obtained RBP131/siRNA or RBP130/siRNA liposome preparations were stored at 4 ° C before use, and the relevant physical and chemical properties were examined. The physical and chemical parameters of the RBP131/siRNA and RBP130/siRNA liposome preparations were similar, and the test results are shown in Table 9.
  • This example was used to detect the synergistic effect of the modified siRNA in combination with the broad-spectrum anticancer drug doxorubicin on inhibiting the growth of pancreatic cancer tumor tissue.
  • compositions RBP131/siNC and RBP131/siRRM2-M were prepared according to the method described in Preparation 3.
  • mice Animals were randomly assigned to 5 groups of 8 mice each. The five groups of animals were treated as follows: (1) 1 ⁇ PBS group; (2) doxorubicin (ADM, purchased from Klamal klamar, article number 25316-40-9) single use group, agent The dose was 1 mg/kg; (3) the combination of doxorubicin and unrelated siRNA (siNC), wherein the dose of doxorubicin was 1 mg/kg, the dose of siNC was 5 ⁇ g/head; (4) the combination of doxorubicin and siRRM2-M In the group, the dose of doxorubicin was 1 mg/kg, and the dose of siRRM2-M was 2 ⁇ g/head; (5) the combination of doxorubicin and siRRM2-M, wherein the dose of doxorubicin was 1 mg/kg, and the dose of siRRM2-M It is 5 ⁇ g/only.
  • doxorubicin ADM, purchased from Klamal klamar, article number 25316-40-9 single use
  • Doxorubicin was administered by intraperitoneal injection, and the drug was administered three times a week; the siRNAs were all RBP131-encapsulated preparations (ie, RBP131/siNC and RBP131/siRRM2-M), administered by intratumoral injection, with an injection volume of 30 ⁇ L, two doses per week. Times.
  • the tumor size was measured with a digital vernier caliper during the administration; the mice were weighed on the 25th day of administration, the mice were weighed, anesthetized and photographed, and then the mice were sacrificed to isolate the liver, spleen and tumor tissue, and the liver and spleen. Tumor tissue was weighed.
  • Figures 2, 3 and 4 show the inhibition of tumor growth by the combination of doxorubicin and RPR131 vector delivered siRRM2-M.
  • the results showed that the group (4) and the group (5) during the administration period, that is, the RBP131/siRRM2-M (2 ⁇ g or 5 ⁇ g) group in which doxorubicin was used was slower in growth rate than the other groups.
  • the combination of doxorubicin and RBP131/siRRM2-M (5 ⁇ g) showed a significant decrease in tumor weight compared with the combination of doxorubicin and RBP131/siNC (5 ⁇ g). No abnormalities such as behavior, spirit, and feces were found during the administration.
  • the ratio of liver to body and spleen were normal, and there was no significant change in body weight.
  • This example was used to detect the inhibitory effect of modified siRNA on the growth of liver cancer tumor tissue.
  • siRNA pharmaceutical composition The pharmaceutical compositions RBP131/siNC, RBP131/siRRM2 and RBP131/siRRM2-M were prepared according to the method described in Preparation 3.
  • Preparation and treatment of subcutaneous xenografts Human hepatoma cells HepG2 were subcultured, and cells in logarithmic growth phase were collected into single cell suspensions, which were inoculated subcutaneously into the back of 4 nude mice. Each nude mouse was injected with 0.2 mL, containing cells. The number was 2 ⁇ 10 6 , and the conventional feeding was continued to observe the subcutaneous tumor formation in the back of the nude mice. When the tumor volume reached 1000 mm 3 , the nude mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.7 mL/100 g). The tumor was removed under aseptic conditions, necrotic tissue was removed, and the tumor tissue was cut into 1 mm ⁇ 1 mm ⁇ 1 mm tumor tissue with ophthalmic scissors, and placed in serum-free DMEM medium for use.
  • 5% chloral hydrate 0.7 mL/100 g
  • Nude mice were randomly divided into 5 groups of 10 animals. The 5 groups of animals were treated as follows: (1) PBS control group; (2) RBP131/siNC-independent control group; (3) RBP131/siRRM2 sample group; (4) RBP131/siRRM2-M sample group; (5) 5-Fu positive control group.
  • 5-Fu fluorouracil is one of the commonly used anti-tumor compounds of chemotherapy, purchased from Tianjin Jinyao Amino Acid Co., Ltd. The above five groups of animals were each administered by tail vein injection.
  • the siRNA was administered in an amount of 1 mg/kg, the administration volume was 10 mL/kg, and the administration was performed twice a week for 5 times; the 5-Fu dose was 5 mg/kg, and the administration volume was 10 mL/kg every two days.
  • the drug was administered once for a total of 8 times.
  • B-ultrasound was performed on tumor size after administration to determine tumor growth. On the 20th day after the first administration, the mice were weighed, anesthetized and photographed, and then the mice were sacrificed, and the liver, spleen and tumor tissues were separated, and the liver, spleen and tumor tissues were weighed.
  • Figure 5 shows inhibition of liver tumor growth by siRRM2-M siRNA delivered by RBP131 vector.
  • the results showed that the pharmaceutical composition administered siRRM2-M by the tail vein injection system can effectively inhibit tumor tissue growth compared with the PBS control group, and the tumor weight is reduced by 82%, which is close to the level of the chemotherapeutic agent 5-Fu; The siRRM2 tumor weight was only reduced by 45%.
  • This example was used to compare the inhibition efficiency of different siRNAs on the growth of liver cancer tumor tissues.
  • siRNA pharmaceutical compositions RBP130/siRRM2-M, RBP130/siRRM2-M2, RBP130/siPC1-M, RBP130/siPC2-M and RBP130/si501/842 were prepared according to the method described in Preparation Example 3; The method of preparing a nude mouse liver orthotopic transplantation tumor model.
  • siRRM2-M2 is another modified form of siRRM2, and its antisense strand is modified differently than siRRM2-M;
  • si501/842 is modified by the method disclosed in US8946176B2, specific oligonucleotide sequence See Table 10.
  • the oligonucleotides in Table 10 contain modified nucleotide residues that are annealed to form modified siRNA.
  • OMe represents a pentose group in the nucleotide residue to the left of which is a 2'-methoxyribosyl group
  • F represents a pentose group in the nucleotide residue to the left of which is 2'- Fluororibosyl
  • S represents the ester group between the deoxyribonucleotides dTdT on both sides thereof is a phosphorothioate group
  • p represents the 5'-terminal linked phosphate group in the first nucleotide residue (in the sequence) Without p, it means no 5'-phosphate group.
  • Nude mice were randomly divided into 6 groups of 6 animals.
  • the 6 groups of animals were treated as follows: (1) PBS control group; (2) RBP130/siRRM2-M sample group; (3) RBP130/siRRM2-M2 sample group; (4) RBP130/siPC1-M sample set; (5) RBP130/siPC2-M sample set; (6) RBP130/si501/842 sample set.
  • the above 6 groups of animals were each administered by tail vein injection.
  • the amount of siRNA administered was 1 mg/kg, and the administration volume was 10 mL/kg, which was administered twice a week for a total of 5 times. B-ultrasound was performed on tumor size after administration to determine tumor growth.
  • mice On the 20th day after the first administration, the mice were weighed, anesthetized and photographed. The mice were sacrificed, and the liver, spleen and tumor tissues were isolated. The liver, spleen and tumor tissues were weighed, and the tumor weight inhibition rate was determined. See Table 11.
  • the pharmaceutical composition administered RBP130/siRRM2-M by the tail vein injection system can effectively inhibit tumor tissue growth, the tumor weight is reduced by 85%, and the drug composition with RBP130/si501/842 is on the tumor tissue.
  • the growth inhibition was comparable; in the treatment group administered with the RBP130/siPC1-M pharmaceutical composition, the tumor weight was reduced by 53%, and the tumor weight was decreased in the treatment group administered with the RBP130/siPC2-M pharmaceutical composition.
  • the tumor weight was only reduced by 46%.
  • siRRM2-M containing fewer modification sites can achieve in vivo activity comparable to si501/842 containing more modification sites, while at the same time, although siRRM2- The M2 modification site was also less than si501/842, but the activity in vivo was poor.
  • This experimental example was used to detect commercial siRNA in vivo delivery vectors produced by different manufacturers, including Invivo fectamine 2.0 (purchased from Life technology), invivo jetPEI (purchased from PolyPlus-transfection (PT)), and Entranster (purchased from Engreen Biosystem). And the RBP131 of the present invention, after carrying the siRRM2-M, inhibits the tumor size and the RRM2 mRNA expression level in the nude mouse orthotopic liver cancer model (see Example 8 for the construction method).
  • the siRRM2-M of the present invention was packaged into the above commercial carrier according to standard operating procedures provided by the manufacturer to prepare corresponding vector/siRNA compositions, which were labeled as IVF2.0/RRM2, invivo, respectively. jetPEI/RRM2, Entranster/RRM2. Meanwhile, the RBP131/RRM2 and RBP130/RRM2 compositions were prepared in accordance with the method of Preparation 3.
  • mice Thirty-six tumor-bearing nude mice (average tumor volume of approximately 50 mm 3 ) were randomly divided into 6 groups (6 in each group, all male), respectively, PBS control group, IVF2.0/RRM2, invivo jetPEI/RRM2, Entranster /RRM2, RBP131/RRM2 and RBP130/RRM2 sample sets. All animals were dosed according to body weight, administered intravenously in the tail vein twice a week for a total of 6 doses.
  • the IVF2.0/RRM2, invivo jetPEI/RRM2, and Entranster/RRM2 groups were administered at a dose of 2.5 mg/kg (siRNA), and the RBP131/RRM2 and RBP130/RRM2 groups were administered at a dose of 0.5 mg/kg (siRNA).
  • the dosing volume of all 6 groups was 10 mL/kg.
  • the animals were sacrificed on the third day after the last administration, and the animals were weighed, anesthetized and photographed before sacrifice. The animals were grossly dissected to observe whether the organs in the body had lesions, and 10% of the tissues were observed by the naked eye.
  • the forest was further preserved for pathological observation.
  • the liver, spleen and tumor tissues were collected and weighed. Some liver tissues and tumor tissues were preserved with RNA later (Sigma Aldrich); the tumor tissues were homogenized with a tissue homogenizer, and then total RNA was extracted with Trizol. Total RNA was extracted from standard extraction procedures.
  • Real-time quantitative PCR was used to detect the expression level of RRM2 mRNA in liver tissue.
  • the detection method was the same as that described in Example 1, and the detection primers were as described in Table 2.
  • the inhibition efficiency of tumors by different siRNA pharmaceutical compositions is expressed by the change in tumor weight in the same manner as in Example 8.
  • Table 12 shows the results of the detection of RRM2 mRNA expression in the tumor tissues of orthotopic liver cancer animals and the detection rate of tumor weight inhibition.
  • RBP131/RRM2 and RBP130/RRM2 had similar inhibitory effects on gene expression and tumor size.
  • the RBP2/RRM2 and RBP130/RRM2 groups also increased the inhibition rate of RRM2 mRNA in liver cancer model mice compared with the IVF2.0/RRM2 group which inhibited mRNA expression in the pharmaceutical composition formed by other commercial carriers.
  • the inhibitory activity of the RBP131/RRM2 and RBP130/RRM2 groups was twice that of the invivo jetPEI/RRM2 group and nearly 20 times that of the Entranster/RRM2 group.
  • the tumor weight inhibition rate results were similar to the mRNA inhibition rate.
  • RBP131/RRM2 and RBP130/RRM2 groups are displayed compared to the pharmaceutical compositions formed by other commercial carriers. Very high biological activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention décrit un pARNi qui inhibe l'expression du gène RRM2. Une séquence de base de brin sens du pARNi est telle que présentée en SEQ ID NO : 2, une séquence de base de brin antisens du pARNi est telle que présentée en SEQ ID no : 3, et un squelette de sucre phosphate du pARNi peut en outre posséder un groupe modifié ; est également décrite une composition pharmaceutique comprenant le pARNi, qui peut inhiber la prolifération de cellules tumorales et faciliter l'apoptose des cellules tumorales, inhibant de là la croissance des tissus tumoraux.
PCT/CN2016/097560 2015-09-01 2016-08-31 Petit arn interférant, composition pharmaceutique, et son application Ceased WO2017036398A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201680046055.3A CN108431224B (zh) 2015-09-01 2016-08-31 一种小干扰核酸和药物组合物及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510551827 2015-09-01
CN201510551827.6 2015-09-01

Publications (1)

Publication Number Publication Date
WO2017036398A1 true WO2017036398A1 (fr) 2017-03-09

Family

ID=58186709

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/097560 Ceased WO2017036398A1 (fr) 2015-09-01 2016-08-31 Petit arn interférant, composition pharmaceutique, et son application

Country Status (2)

Country Link
CN (1) CN108431224B (fr)
WO (1) WO2017036398A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113082001A (zh) * 2021-05-25 2021-07-09 中国科学院广州生物医药与健康研究院 一种核酸递送系统及其制备方法和应用
CN120005885A (zh) * 2025-02-17 2025-05-16 中国人民解放军陆军军医大学第一附属医院 siRNA组合物和衍生物及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114196672B (zh) * 2021-10-25 2023-12-22 北京理工大学 特异性抑制PAK4基因表达的siRNA及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW201226566A (en) * 2010-10-18 2012-07-01 Hoffmann La Roche Compositions and methods for inhibiting expression of RRM2 genes
CN103380113A (zh) * 2010-11-15 2013-10-30 生命科技公司 含胺的转染试剂及其制备和使用方法
CN104136423A (zh) * 2012-02-01 2014-11-05 希望之城 核糖核苷酸还原酶抑制剂和使用方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW201226566A (en) * 2010-10-18 2012-07-01 Hoffmann La Roche Compositions and methods for inhibiting expression of RRM2 genes
CN103380113A (zh) * 2010-11-15 2013-10-30 生命科技公司 含胺的转染试剂及其制备和使用方法
CN104136423A (zh) * 2012-02-01 2014-11-05 希望之城 核糖核苷酸还原酶抑制剂和使用方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113082001A (zh) * 2021-05-25 2021-07-09 中国科学院广州生物医药与健康研究院 一种核酸递送系统及其制备方法和应用
CN113082001B (zh) * 2021-05-25 2023-05-26 中国科学院广州生物医药与健康研究院 一种核酸递送系统及其制备方法和应用
CN120005885A (zh) * 2025-02-17 2025-05-16 中国人民解放军陆军军医大学第一附属医院 siRNA组合物和衍生物及其应用

Also Published As

Publication number Publication date
CN108431224A (zh) 2018-08-21
CN108431224B (zh) 2020-01-17

Similar Documents

Publication Publication Date Title
US20150232846A1 (en) Pseudocircularization oligonucleotides for modulating rna
CN108265052B (zh) 一种小干扰核酸和药物组合物及其用途
CN108239644B (zh) 一种小干扰核酸和药物组合物及其用途
JP7003044B2 (ja) Ａｎｇｐｔ２およびｐｄｇｆｂを標的化するｒｎａ複合体を用いる血管新生関連疾患の処置
CN109477107B (zh) 一种抑制EGFR基因表达的siRNA及其前体和应用
KR102246814B1 (ko) 치료용 올리고뉴클레오타이드
JP7385847B2 (ja) ｓｎｏＲＮＡの発現抑制剤を有効成分とするがん増殖抑制剤
EP3532617B1 (fr) Oligonucléotides antisens
CN109563513B (zh) 一种抑制K-RAS基因表达的siRNA及其前体和应用
CN108431224B (zh) 一种小干扰核酸和药物组合物及其用途
CN106467915A (zh) 小干扰RNA及其应用和抑制plk1基因表达的方法
TW201038278A (en) Rnai molecule for thymidylate synthase and use thereof
EP1731609A1 (fr) INHIBITEUR DE L'EXPRESSION DE Rad51, MÉDICAMENT CONTENANT L'INHIBITEUR COMME INGRÉDIENT ACTIF ET UTILISATION DE CELUI-CI
CN106232124B (zh) 制备用于局部施用的脂质体复合物的新方法及使用该脂质体复合物的抗肿瘤剂
JP2011251912A (ja) マイクロｒｎａ―１４３誘導体を含有する医薬
US11149271B2 (en) Method and pharmaceutical composition for treating cancer
US20150010502A1 (en) Compositions and Methods for Suppressing Gene Expression of p53 and Clusterin
EP4023297A1 (fr) Médicament à base d'acide nucléique pour le ciblage d'une molécule de cancer gastrique
US9617538B2 (en) Heptamer-type small guide nucleic acids inducing apoptosis of human leukemia cells
CN103361347B (zh) 针对血管生成素2的微小rna
AU2018101287A4 (en) Method and pharmaceutical composition for treating cancer
CN105779452A (zh) 一种抑制肿瘤生长的寡聚核酸及其应用
CN107693534A (zh) miR‑491在制备用于治疗骨肉瘤的药物中的应用
CN120591261A (zh) Crept基因的aso抑制剂及其对肝癌的抑制
CN121343987A (zh) 一种植物来源的miRNA在制备抗肿瘤药物中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16840827

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16840827

Country of ref document: EP

Kind code of ref document: A1