WO2020027640A1 - Composition pour l'inhibition de l'expression de ctgf - Google Patents

Composition pour l'inhibition de l'expression de ctgf Download PDF

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WO2020027640A1
WO2020027640A1 PCT/KR2019/095026 KR2019095026W WO2020027640A1 WO 2020027640 A1 WO2020027640 A1 WO 2020027640A1 KR 2019095026 W KR2019095026 W KR 2019095026W WO 2020027640 A1 WO2020027640 A1 WO 2020027640A1
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seq
sequence
strand
sirna
silica particles
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PCT/KR2019/095026
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English (en)
Korean (ko)
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원철희
민달희
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주식회사 레모넥스
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Priority to EP19843450.8A priority Critical patent/EP3831392A4/fr
Priority to CN201980050234.8A priority patent/CN112512532A/zh
Priority to AU2019314093A priority patent/AU2019314093B2/en
Priority to JP2021505193A priority patent/JP7152072B2/ja
Priority claimed from KR1020190065618A external-priority patent/KR20200014684A/ko
Publication of WO2020027640A1 publication Critical patent/WO2020027640A1/fr
Priority to US17/162,045 priority patent/US20210230602A1/en

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    • 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
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a composition which inhibits CTGF expression with high efficiency and has excellent prophylactic or therapeutic effect of fibroproliferative disease.
  • Tissue remodeling is the reconstruction of existing tissue in response to physiological or pathological stress.
  • Tissue remodeling in pathophysiology is characterized by connective tissue growth factor (CTGF), myofiber differentiation and activation, deposition of extracellular matrix (ECM) and overexpression of fibrosis.
  • CTGF connective tissue growth factor
  • ECM extracellular matrix
  • CTGF has been considered a pivotal mediator in tissue remodeling.
  • CTGF is involved in a variety of signal transduction pathways, resulting in cell adhesion and migration, ECM remodeling and alteration of organ structure.
  • Tissue remodeling and fibrosis are associated with numerous fibrotic disorders such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, diabetic retinopathy, and skin fibrosis (keloids and hypertrophic scars).
  • the wound healing process in the skin is very complex and consists of overlapping steps of inflammation, cell differentiation and proliferation, tissue remodeling (including collagen production).
  • Some cytokines and growth factors, especially TGF- ⁇ play an important role in the early and late stages of wound healing.
  • TGF- ⁇ mediates leukocyte migration, angiogenesis, fibroblast migration and ECM components (collagen and fibronectin) production through CTGF upregulation. Since overexpression of CTGF has been observed in hypertrophic scars and keloid patients, suppression of CTGF expression is an attractive strategy to modulate fibrosis mechanisms that can inhibit or reverse the fibrosis process.
  • siRNA is one of the promising candidates for CTGF inhibition.
  • RNA interference induced in siRNA is mediated by highly specific and efficient gene silencing machinery that sequencely recognize and cleave target mRNAs.
  • RNA interference induced in siRNA is mediated by highly specific and efficient gene silencing machinery that sequencely recognize and cleave target mRNAs.
  • several limitations such as 1) rapid degradation by nucleases in biological systems, 2) maintenance of effective siRNA doses, 3) difficulty in efficient delivery across biological barriers, and There is a barrier.
  • cationic polymers lipid nanoparticles (LNPs), viruses, and various nanomaterials have been developed for the delivery of siRNAs.
  • LNPs lipid nanoparticles
  • the clinical application of cationic polymers and LNPs should be prudent due to the toxicity and / or instability of the structures in vivo, and viral gene transfer poses mutagenesis in addition to low packaging capacity.
  • Chemical modifications of siRNA backbones can increase stability and cell uptake, but still suffer from disadvantages such as high cost, labor intensive, time consuming processing, and high amounts of siRNA administration for satisfactory efficacy in target cells.
  • An object of the present invention is to provide a composition having high efficiency of inhibiting CTGF expression and having an excellent prophylactic or therapeutic effect of fibroproliferative diseases.
  • a composition for inhibiting CTGF gene expression comprising: a nucleic acid molecule consisting of a sequence of SEQ ID NO: 1 and a sequence complementary to 10 nucleotides or more.
  • composition according to the above wherein the nucleic acid molecule consisting of a sequence of at least 16 nucleotides complementary to the sequence of SEQ ID NO: 1;
  • composition of 1 above wherein the nucleic acid molecule consisting of a sequence complementary to the entire sequence of SEQ ID NO: 1;
  • siRNA consisting of the sense RNA consisting of the sequence of SEQ ID NO: 1 and the antisense RNA consisting of the sequence of SEQ ID NO: 2, the strand consisting of the sequence of SEQ ID NO: 3 and dsRNA consisting of the strands complementary thereto
  • SEQ ID NO: 52 SiRNA consisting of a sense RNA consisting of a sequence of and an antisense RNA consisting of a sequence of SEQ ID NO: 53, a dsRNA consisting of a strand consisting of the sequence of SEQ ID NO: 54 and a complementary strand thereof, a sense RNA consisting of the sequence of SEQ ID NO: 55, and SEQ ID NO: 56 SiRNA consisting of an antisense RNA consisting of a sequence of DNA, a strand consisting of a sequence of SEQ ID NO: 57 and a dsRNA consisting of a complementary strand thereof, a siRNA consisting of a sense RNA consisting of a sequence of SEQ
  • composition of 5 above further comprising a sequence of UU or dTdT at the 3 'end of the sense RNA and antisense RNA sequence.
  • composition of 4 above comprising at least one PNA consisting of one sequence selected from the group consisting of SEQ ID NOs: 87-99.
  • a pharmaceutical composition for the prevention or treatment of fibroproliferative diseases comprising the composition of any one of 1 to 8.
  • the fibrotic disease is hypertrophic scar, keloid, fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, cystic fibrosis, myelofibrosis, peritoneal fibrosis, scleroderma, diabetic retinopathy , Duchenne muscular dystrophy, radiation-induced fibrosis, myocardial fibrosis, diabetic kidney disease, chronic renal failure, chronic viral hepatitis, biliary fibrosis, fatty hepatitis, alcoholic fatty hepatitis, nonalcoholic steatohepatitis, proliferative vitreoretinopathy, musculoskeletal tumor, osteosarcoma , Rhabdomyosarcoma, glioblastoma, lung cancer, ovarian cancer, esophageal cancer, colon cancer, pancreatic cancer, kidney sclerosis, sarcoidosis,
  • the porous silica particles react with silica particles having pores less than 5 nm in diameter at 120 ° C. to 180 ° C. for 24 to 96 hours to expand the pores less than 5 nm in diameter; And calcining the pores of expanded silica particles at a temperature of 400 ° C. or higher for at least 3 hours.
  • the average diameter of the porous silica particles is 100 nm to 1000 nm, the BET surface area is 200m 2 / g to 700m 2 / g, the volume per g is 0.7ml to 2.2ml,
  • the porous silica particles, the ratio of the absorbance of the following formula 1 is t is 24 or more, CTGF gene expression inhibition composition:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • composition of 11 above, wherein the nucleic acid molecule is at least 10 nucleotides complementary to the sequence of SEQ ID NO: 1.
  • composition of 11 above, wherein the nucleic acid molecule is at least 16 nucleotides complementary to the sequence of SEQ ID NO: 1.
  • siRNA consisting of the sense RNA consisting of the sequence of SEQ ID NO: 1 and the antisense RNA consisting of the sequence of SEQ ID NO: 2, the strand consisting of the sequence of SEQ ID NO: 3 and dsRNA consisting of strands complementary thereto, SEQ ID NO: 52 SiRNA consisting of a sense RNA consisting of a sequence of and an antisense RNA consisting of a sequence of SEQ ID NO: 53, a dsRNA consisting of a strand consisting of the sequence of SEQ ID NO: 54 and a complementary strand thereof, a sense RNA consisting of the sequence of SEQ ID NO: 55, and SEQ ID NO: 56 SiRNA consisting of an antisense RNA consisting of a sequence of DNA, a strand consisting of a sequence of SEQ ID NO: 57 and a dsRNA consisting of a complementary strand thereof, a siRNA consisting of a sense RNA consisting of a sequence of a sequence of a sequence of
  • composition of 18 above further comprising a sequence of UU or dTdT at the 3 'end of the sense RNA and antisense RNA sequences.
  • composition of 17, wherein the composition comprises at least one PNA consisting of one sequence selected from the group consisting of SEQ ID NOs: 87 to 99.
  • a pharmaceutical composition for the prevention or treatment of fibroproliferative diseases comprising the composition of any one of 11 to 21 above.
  • fibrotic disease is hypertrophic scar, keloid, fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, cystic fibrosis, myelofibrosis, peritoneal fibrosis, scleroderma, diabetic retinopathy , Duchenne muscular dystrophy, radiation-induced fibrosis, myocardial fibrosis, diabetic kidney disease, chronic renal failure, chronic viral hepatitis, biliary fibrosis, fatty hepatitis, alcoholic fatty hepatitis, nonalcoholic steatohepatitis, proliferative vitreoretinopathy, musculoskeletal tumor, osteosarcoma , Rhabdomyosarcoma, glioblastoma, lung cancer, ovarian cancer, esophageal cancer, colon cancer, pancreatic cancer, kidney sclerosis, s
  • composition of the present invention contains a nucleic acid molecule capable of effectively inhibiting the expression of CTGF and collagen, it is possible to prevent or treat a variety of fibrotic diseases due to overexpression of CTGF or collagen.
  • FIG. 1 schematically illustrates the mechanism of inhibition of hypertrophic scars and keloids due to overexpression of CTGF induced by pathological pathways and the effect of inhibiting effective CTGF expression of LEM-S401.
  • FIG. 2 shows A549 (FIG. 2) and HaCaT (FIG. 3) cells each treated for 6 hours with various doses of LEM-S401 (12.5, 25, 50, 100 nM) for 6 hours and CTGF expression was 12 ng.
  • TGF-ß of / mL, CTGF mRNA expression level was compared with the control (untreated, siCTGF only, DegradaBALL only, scrambled siRNA treatment) using RT-PCR ( * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.005).
  • FIG. 4 and 5 show that A549 (FIG. 4) and HaCaT (FIG. 5) cells were treated with lipoid nanoparticles (LiP nanoparticles (LNP)) carrying LEM-S401 and siCTGF, respectively, and the cells were 72 and 96 hours. And TGF-ß was treated 12 hours before harvesting ( * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.005).
  • LNP lipoid nanoparticles
  • FIG. 6 is a subcutaneous injection of LEM-S401 into mouse skin, and fluorescence images of the excised mouse skin were taken at different time points to measure skin retention time of siCTGF and DegradaBALL at the LEM-S401 injection site (1,3,5). 1) (left, Scale bar: 25mm), after the DAPI staining was taken fluorescence image of the treated skin section (right, Scale bar: 100 ⁇ m).
  • FIG. 7 shows fluorescence images of resected skin subcutaneously injected with FITC-conjugated siCTGF unsupported in DegradaBALL at different time points (left), and after fluorescence imaging of fluorescence images of skin sections treated in the same manner.
  • Scale bar 100 ⁇ m.
  • FIGS. 8 to 15 show the results of subcutaneous injection of LEM-S401 (1 nmol) around the mouse skin wound on day 0,4,8,12, Figures 8 to 10 mRNA expression of CTGF and collagen type 1,3 Levels were measured at day 16 using RT-PCR, FIG. 11 shows images of wounded mouse skin treated with skin irregularities and softness between LEM-S401 treated group and control (buffer, DegradaBALL only, siCTGF only).
  • 12 is a fluorescent image of skin sections treated with LEM-S401, free siCTGF, DegradaBALL only and buffer after tissue staining with antibodies that recognize CTGF and collagen types 1,3, respectively. Secondary antibodies were treated to skin sections (Scale bar: 100 ⁇ m), and FIGS. 13 to 15 quantitatively analyze immunohistochemical data based on the image of FIG. 12 ( * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.005).
  • Figure 16 shows the toxicity in A549 and HaCaT cells with increasing concentrations of DegradaBALL was measured by CCK-8 analysis.
  • FIG. 17 shows the results of treatment of A549 and HaCaT cells with 2 ng / mL of TGF-ß for 24 hours. Cells were obtained at different time points, and CTGF expression levels were analyzed by RT-PCR.
  • FIGS. 18 to 24 show the results of subcutaneous injection of LEM-S401 into the wound site 10, 14, 18 and 22 days after wound formation, and FIGS. 18 to 20 show mRNA expression levels of CTGF and collagen type 1,3 at the injection site. Measured by RT-PCR, FIG. 21 is a fluorescence image of CTGF and collagen types 1,3 of the obtained mouse skin was measured by immunohistochemistry (Scale bar: 100 ⁇ m), and FIGS. 22 to 24 are immune tissues. Chemical data were quantified ( * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.005).
  • Fig. 25 shows comparison results of detection of fluorescent images in the body when siRNA was injected subcutaneously with DDV (porous silica particles, DegradaBALL) and when mouse was injected subcutaneously with unsupported free-siRNA.
  • DDV porous silica particles, DegradaBALL
  • siRNA (# 1) consisting of a sense RNA consisting of a sequence of SEQ ID NO: 1 and an antisense RNA consisting of a sequence of SEQ ID NO: 2
  • SiRNA (# 2) consisting of a sense RNA consisting of a sequence of SEQ ID NO: 4 and an antisense RNA consisting of a sequence of SEQ ID NO: 5
  • SiRNA (# 3) consisting of sense RNA consisting of the sequence of SEQ ID NO: 7 and antisense RNA consisting of the sequence of SEQ ID NO: 8 confirmed the inhibitory ability of CTGF expression levels in A549 and HaCaT cells.
  • FIG. 26 shows TGF- 12 hours after siRNA treatment. ⁇ was treated, and FIG. 27 was treated with siRNA 24 hours after TGF-ß treatment.
  • 29 is a micrograph of porous silica particles according to one embodiment of the present invention.
  • FIG. 30 is a micrograph of the small pore particles in the manufacturing process of the porous silica particles according to an embodiment of the present invention.
  • Figure 31 is a micrograph of the small pore particles according to an embodiment of the present invention.
  • DDV Delivery Vehicle
  • the number in parenthesis means the diameter of the particle
  • the number of subscripts means the pore diameter.
  • DDV 200 10 refers to a particle of an embodiment having a particle diameter of 200 nm and a pore diameter of 10 nm.
  • Figure 33 is a micrograph to confirm the biodegradability of the porous silica particles according to an embodiment of the present invention.
  • 34 is a tube with a cylindrical permeable membrane according to one example.
  • 35 is a result of decreasing absorbance over time of porous silica particles according to an embodiment of the present invention.
  • 40 is a tube confirming siRNA or dsRNA release according to one example.
  • Figure 41 is the degree of release over time of the siRNA supported on the porous silica particles according to an embodiment of the present invention.
  • siRNA refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing. siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of the target gene.
  • the siRNA® molecule may have a double-stranded structure in which the sense strand (corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (sequence complementary to the mRNA sequence of the target gene) are positioned opposite to each other.
  • siRNA® molecules may have a single chain structure with self-complementary sense and antisense strands.
  • siRNAs are not limited to the complete pairing of double-stranded RNA portions paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included.
  • the siRNA® terminal structure can be either blunt or cohesive, as long as the expression of the target gene can be suppressed by RNAi (RNA interference) effects.
  • the cohesive end structure can be both a 3'-end protrusion structure and a 5'-end protrusion structure.
  • siRNA molecules may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted between self-complementary sense and antisense strands, in which case the siRNA molecules formed by expression of the nucleotide sequence are intramolecular hybridization.
  • a short nucleotide sequence eg, about 5-15 nt
  • the siRNA molecules formed by expression of the nucleotide sequence are intramolecular hybridization.
  • the hairpin structure is formed, and as a whole, the stem-and-loop structure is formed.
  • This stem-and-loop structure is processed in vitro or in vivo to produce active “siRNA” molecules that can mediate RNAi.
  • dsRNA is a precursor molecule of siRNA, meets the RISC complex containing the target cell's DICER enzyme (Ribonuclease III) and is cleaved into siRNA, in which RNAi occurs.
  • dsRNA has a sequence that is several nucleotides longer than siRNA, and the double stranded strand of the sense strand (corresponding to the target gene) and the antisense strand (sequence complementary to the mRNA sequence of the target gene) It may have a structure forming a.
  • PNA is a synthetic polymer that has a structure similar to DNA or RNA, but unlike DNA or RNA, and is designed to have no charge, and has a strong binding force, wherein the DNA and RNA are deoxyribose or ribose sugar backbones ( backbones, respectively, while the backbone of the PNA has a structure in which repeating N- (2-aminoethyl) -glycine ((N- (2-aminoethyl) -glycine) units are linked by peptide bonds.
  • methylene methylene
  • -C O-
  • Nucleic acid is meant to include any PNA, DNA or RNA, eg, chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in tissue samples.
  • PNA protein-binding nucleic acid
  • DNA or RNA DNA or RNA
  • chromosomes chromosomes
  • mitochondria mitochondria
  • viruses and / or bacterial nucleic acids present in tissue samples.
  • Gene means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression.
  • the gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein.
  • Nucleic acid sequences can include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.
  • gene expression generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in a cell.
  • gene expression includes not only transcriptional and translational processes, but also post-transcriptional and posttranslational processes that can affect the biological activity of a gene or gene product.
  • the processes include, but are not limited to, RNA synthesis, processing and transport, as well as post-translational modifications of the polypeptide synthesis, transport and polypeptide.
  • siRNA genes the term “gene expression” refers to the process by which precursor siRNAs are produced from a gene.
  • this process is referred to as transcription, although unlike transcription induced by RNA polymerase II for a protein coding gene, the transcription product of the siRNA gene is not translated to produce a protein. Nevertheless, generation of mature siRNA from siRNA genes is encompassed by the term "gene expression" as that term is used herein.
  • target gene refers to a gene that is targeted for regulation using the methods and compositions of the subject matter disclosed herein. Therefore, the target gene comprises a nucleic acid sequence whose expression level is down regulated by siRNA at the mRNA or polypeptide level.
  • target RNA or “target mRNA” refers to a transcript of a target gene to which siRNA binds to induce regulation of expression of the target gene.
  • transcription refers to a cellular process that involves the interaction of an RNA polymerase with a gene that drives expression as RNA of structural information present in the coding sequence of the gene.
  • down-regulation refers to the expression of specific genes in mRNA or the expression of proteins in activated cells by intracellular transcription or translation in activated cells compared to normal tissue cells. Means reduced.
  • Treatment means an approach to obtain beneficial or desirable clinical results.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected.
  • Treatment may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders.
  • Prevention means any action that inhibits or delays the development of a related disease. It will be apparent to those skilled in the art that the compositions herein can prevent the initial symptoms, or related diseases, if administered before they appear.
  • the present invention provides a composition for inhibiting CTGF gene expression comprising; a nucleic acid molecule consisting of a sequence of SEQ ID NO: 1 and a sequence complementary to 10 nucleotides or more.
  • nucleotides comprising at least 10 nucleotides (nt), at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, Or at least 17 nucleotides or all 18 nucleotides.
  • the nucleic acid molecule may be one strand of siRNA, dsRNA, PNA or miRNA, in which case the siRNA, dsRNA, PNA or miRNA is expressed in the CTGF gene by RNAi (RNA interference). It may be to suppress the, and more specifically, in the mRNA sequence which is a transcript of the CTGF gene, it may be to complementarily bind to at least a portion of the region consisting of the sequence of SEQ ID NO: 1 to inhibit the expression of the CTGF gene.
  • RNAi RNA interference
  • the sequence conforms to the conditions to be considered in the design of the siRNA, dsRNA, PNA or miRNA at the level of those skilled in the art. It is obvious that it is designed in this case, in this case, it may be designed to avoid the degradation of RNAi efficiency caused by the hairpin shape (crosspin), cross-shaped (hairpin shape) so as not to have a palindrome sequence.
  • siRNA or dsRNA of the present invention may be designed not to include the sequence of SEQ ID NO: 100 (5'-AACUUGAACU-3 '), for example, PNA of the present invention, both ends of the CNA It may be designed to avoid having a complementary relationship with each and G, it can be designed to have a stable RNAi efficiency by maintaining the entire length of the nucleic acid molecule more than the appropriate length.
  • the total sequence length of the nucleic acid molecule is 10 to 30, 11 to 29, 12 to 28, 13 to 27, 14 to 26, 15 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21 or 16 to It may be 20 nucleotides in length, but is not necessarily limited thereto, but preferably 16 to 16 in order to maintain the efficiency of RNAi by maintaining an appropriate length or more, but to maintain the efficiency in vivo by maintaining the appropriate length or less. It may be 20 nucleotides in length.
  • the nucleic acid molecule may be an antisense RNA (sequence of SEQ ID NO: 2) that forms a siRNA by complementarily binding with a sense RNA consisting of the sequence of SEQ ID NO: 1, a strand consisting of the sequence of SEQ ID NO: 3 It may be a strand complementary to the dsRNA to bind to, and the antisense RNA (sequence of SEQ ID NO: 53) that complementarily binds to the sense RNA consisting of the sequence of SEQ ID NO: 1 to form a siRNA, SEQ ID NO: 54 It may be a strand forming a dsRNA by binding complementarily to the strand consisting of a strand, and may be an antisense RNA (sequence of SEQ ID NO: 56) that complementarily binds to a sense RNA consisting of the sequence of SEQ ID NO: 55 to form an siRNA , Strand may form a dsRNA by complementarily binding to a ds
  • antisense RNA which complementarily binds to the sense RNA consisting of the sequence of SEQ ID NO: 67 to form an siRNA. It may be a strand consisting of dsRNA by binding complementarily to the strand consisting of a strand, and may be an antisense RNA (sequence of SEQ ID NO: 71) that complementarily binds to a sense RNA consisting of the sequence of SEQ ID NO: 70 to form an siRNA It may be a strand forming a dsRNA by binding complementary to the strand consisting of the sequence of SEQ ID NO: 72.
  • the nucleic acid molecule may be a PNA consisting of one sequence selected from the group consisting of SEQ ID NOs: 87 to 99.
  • Nucleic acid molecules of the present invention are animals including humans, for example monkeys, pigs, horses, cows, sheep, dogs, cats, mice (mice), rabbits (rabbits) and the like, preferably may be of human origin.
  • the nucleic acid molecule of the present invention has been modified by deletion, substitution or insertion of functional equivalents of the nucleic acid molecule constituting the same, for example, some nucleotide sequences of the nucleic acid molecule of the present invention. It is a concept that includes a variant (variants) that can function functionally the same as the nucleic acid molecule of the invention.
  • the nucleic acid molecule of the present invention when it forms a sense RNA or antisense RNA of siRNA, it may further include a sequence of UU or dTdT at the 3 'end of the sense RNA and antisense RNA sequence, which is a nucleic acid
  • the siRNA or dsRNA can be given to the siRNA or the dsRNA by increasing the structural stability of the siRNA or dsRNA by increasing the resistance to the enzyme, and increasing the RNAi efficiency of the siRNA or the dsRNA through the induction of a stable RISC.
  • nucleic acid molecule of the present invention forms a PNA
  • at least one peptide consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 101 to 113; Or mPEG 5000 may be further bound, which may be for the purpose of increasing the solubility or penetration in the composition of the PNA, at the C-terminus at both ends (N-terminus or C-terminus). Bonding is more preferred in that it does not require a separate linker.
  • Nucleic acid molecules of the present invention may be isolated or prepared using standard molecular biology techniques, such as chemical synthesis or recombinant methods, or may be commercially available.
  • the composition of the present invention may contain not only the nucleic acid molecule of the present invention, but also other substances capable of increasing the expression rate of the nucleic acid molecule of the present invention in cells, for example, compounds, natural products, novel proteins, and the like. .
  • nucleic acid molecule of the present invention can be provided included in the vector for expression in the cell.
  • Nucleic acid molecules of the present invention can be introduced into cells using a variety of transformation techniques, such as complexes of DNA and DEAE-dextran, complexes of DNA and nuclear proteins, complexes of DNA and lipids, for this purpose nucleic acid molecules of the present invention Can be in a form contained within a carrier that allows for efficient introduction into a cell.
  • the carrier is preferably a vector, and both viral and non-viral vectors can be used.
  • a viral vector for example, lentiviruses, retroviruses, adenoviruses, adenoviruses, herpesviruses, and abipoxvirus vectors may be used.
  • lentiviral vector is a lentiviral vector, but is not limited thereto.
  • Lentiviruses are a type of retrovirus that infects dividing as well as dividing cells due to the nucleophilicity of a pre-integrated complex (virus "shell") that enables active introduction into the nucleopore or the complete nuclear membrane. There are features that can be made.
  • the vector containing the nucleic acid molecule of the present invention preferably further comprises a selection marker.
  • the "selection marker” is intended to facilitate selection of cells into which the nucleic acid molecule of the present invention has been introduced.
  • the selectable markers that can be used in the vector are not particularly limited as long as they are genes capable of easily detecting or measuring the introduction of the vector, but typically, drug resistance, nutritional requirements, resistance to cytotoxic agents, or surface proteins.
  • Markers that confer a selectable phenotype such as expression, for example GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol dihydro Genase (histidinol dehydrogenase gene: hisD) and guanine phosphosribosyltransferase (Gpt), and the like, and preferably GFP (green fluorescent protein) and puromycin markers can be used.
  • GFP green fluorescent protein
  • puromycin puromycin
  • Neo neomycin
  • Hyg hygromycin
  • histidinol dihydro Genase histidinol dehydrogenase gene: hisD
  • Gpt guanine phosphosribosyltransferase
  • the present invention provides a pharmaceutical composition for the prevention or treatment of fibroproliferative diseases comprising the composition described above.
  • the pharmaceutical composition of the present invention has a prophylactic or therapeutic effect of fibrotic disease, which may be an effect achieved by inhibiting the expression of the CTGF gene of the nucleic acid molecule of the present invention.
  • fibrotic diseases which are diseases to be prevented or treated for the pharmaceutical composition of the present invention, include hypertrophic scars, keloids, fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, cystic fibrosis, myelofibrosis, and post-peritoneal Fibrosis, scleroderma, diabetic retinopathy, Duken's muscular dystrophy, radiation-induced fibrosis, myocardial fibrosis, diabetic kidney disease, chronic renal failure, chronic viral hepatitis, biliary fibrosis, fatty hepatitis, alcoholic fatty hepatitis, nonalcoholic steatohepatitis, proliferation Vitreoretinopathy, Musculoskeletal Tumor, Osteosarcoma, Rhabdomyosarcoma, Glioblastoma, Lung Cancer, Ovarian Cancer, Esophageal Cancer, Colon Cancer, Pancre
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • a pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention is applicable to any formulation containing the nucleic acid molecule of the present invention as an active ingredient, and can be prepared in oral or parenteral formulations.
  • Pharmaceutical formulations of the present invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels depend on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other factors well known in the medical field. Can be determined.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
  • the dosage of the pharmaceutical composition of the present invention varies widely depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and the appropriate dosage is, for example, Depending on the amount of drug accumulated in the patient's body and / or the specific efficacy of the nucleic acid molecules of the invention used. It can be calculated on the basis of EC50, which is generally determined to be effective in in vivo animal models and in vitro, for example from 0.01 ⁇ g to 1 g per kg of body weight, in unit periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition can be administered for relapse.
  • the pharmaceutical composition of the present invention may further contain a compound which maintains / increases the solubility and / or absorption of at least one active ingredient or the active ingredient having the same or similar function in the treatment of fibroproliferative diseases. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • composition of the present invention may be carried on a carrier having a nucleic acid molecule that complementarily binds to at least a portion of a transcript of a CTGF gene, and is known in the art as capable of supporting a nucleic acid molecule in a kind of the carrier. If there is no particular limitation, for example, it may be at least one selected from the group consisting of liposomes, lipofectamine, dendrimers, micelles, porous silica particles, aminoclays and hydrogels, but preferably high nucleic acid molecule loading rate, sustained release It may be porous silica particles having advantages such as property, biodegradability and the like.
  • the present invention provides a composition for inhibiting CTGF gene expression, comprising; porous silica particles carrying nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene.
  • the porous silica particles are particles of silica (SiO 2 ) material, and have a particle size of nano size.
  • Porous silica nanoparticles of the present invention is a porous particle, having a nano-sized pores, can carry a nucleic acid molecule that complementarily binds to at least a portion of the transcript of the CTGF gene on the surface and / or inside the pores.
  • Porous silica particles of the present invention are biodegradable particles, which carry a nucleic acid molecule that complementarily binds to at least a portion of the transcript of the CTGF gene, and are complemented to at least a portion of the transcript of the CTGF gene while being biodegraded in the body when administered to the body.
  • the porous silica particles of the present invention may be slowly degraded in the body to allow sustained release of nucleic acid molecules that complementarily bind to at least a portion of the transcript of the supported CTGF gene.
  • t which is the ratio of the absorbance of the following formula 1 to 1/2, is 24 or more:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • Equation 1 means that the rate at which the porous silica particles are degraded in an environment similar to the body.
  • Absorbance A 0 , A t in Equation 1 may be measured by putting porous silica particles and a suspension in a cylindrical permeable membrane and putting the same suspension outside the permeable membrane, as illustrated in FIG. 34, for example.
  • the porous silica particles of the present invention are biodegradable, and can be slowly decomposed in suspension, 50 kDa in diameter corresponds to about 5 nm, and biodegradable porous silica particles can pass through a permeable membrane of 50 kDa in diameter, and a cylindrical permeable membrane is 60 rpm horizontal. Under stirring, the suspension can be mixed evenly and the degraded porous silica particles can come out of the permeable membrane.
  • the absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the permeable membrane is replaced with a new suspension.
  • the suspension can be one that is constantly replaced, one that can be replaced every period, and the period can be periodic or irregular. For example, within the range of 1 hour to 1 week, 1 hour interval, 2 hours interval, 3 hours interval, 6 hours interval, 12 hours interval, 24 hours interval, 2 days interval, 3 days interval, 4 days interval, 7 It may be replaced at day intervals, but is not limited thereto.
  • the ratio of the absorbance to 1/2 means that the absorbance is half of the initial absorbance after t hours, which means that approximately half of the porous silica particles are decomposed.
  • the suspension may be a buffer solution, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • T of the absorbance ratio of Equation 1 of the present invention is 1/2 or more, for example, t may be 24 to 120, for example, 24 to 96, 24 to 72, 30 within the above range To 70, 40 to 70, 50 to 65 and the like, but is not limited thereto.
  • t for example, the absorbance ratio of Equation 1 is 1/5 may be, for example, 70 to 140, for example, 80 to 140, 80 to 120, and 80 to 110 within the above range. , 70 to 140, 70 to 120, 70 to 110, and the like, but is not limited thereto.
  • t may be 130 to 220, for example, wherein the ratio of absorbance of Equation 1 is 1/20, for example, 130 to 200, 140 to 200, 140 to 180 within the above range. , 150 to 180, and the like, but is not limited thereto.
  • the porous silica particles of the present invention may have a measured absorbance of 0.01 or less, for example, 250 or more, for example, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, and the upper limit thereof is 2000 days. May be, but is not limited thereto.
  • the ratio of the absorbance of Formula 1 and t have a high positive correlation.
  • the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more. .
  • T in Equation 1 means how fast the porous silica particles decompose in an environment similar to the body, for example, the surface area, particle diameter, pore diameter, surface and / or inside the pores of the porous silica particles. It can be controlled by controlling the substituent, the degree of compactness of the surface, and the like.
  • the surface area of the particles can be increased to reduce t, or the surface area can be reduced to increase t.
  • the surface area can be adjusted by adjusting the diameter of the particles and the diameter of the pores.
  • substituents on the surface and / or within the pores it is possible to increase t by reducing the direct exposure of porous silica particles to the environment (such as solvents).
  • the porous silica particles carry nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene, and increase the affinity between the nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene and the porous silica particles.
  • Porous silica particles of the present invention may be, for example, spherical particles, but is not limited thereto.
  • the porous silica particles of the present invention may have an average diameter of, for example, 150 nm to 1000 nm, for example, within the above range, for example, 150 nm to 800 nm, 150 nm to 500 nm, 150 nm to 400 nm, 150 nm to 300 nm, and 150 nm to 200 nm. May be, but is not limited thereto.
  • the porous silica particles of the present invention may have an average pore diameter of, for example, 1 nm to 100 nm, for example, within the above range, for example, 5 nm to 100 nm, 7 nm to 100 nm, 7 nm to 50 nm, 10 nm to 50 nm, 10 nm to 30 nm. , 7 nm to 30 nm, but is not limited thereto.
  • a nucleic acid molecule having a large diameter as described above may carry a nucleic acid molecule that complementarily binds to at least a portion of a transcript of a large amount of CTGF gene, and complementarily binds to at least a portion of a transcript of a large CTGF gene. It can also be supported.
  • the porous silica particles of the present invention may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g.
  • a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g, 200 m 2 / g to 650 m 2 / g, 250 m 2 / g to 650 m 2 / g, 300 m 2 / g to 700 m 2 / g, 300 m 2 / g to 650m 2 / g, 300m 2 / g to 600m 2 / g, 300m 2 / g to 550m 2 / g, 300m 2 / g to 500m 2 / g, 300m 2 / g to 450m 2 / g, etc. It is not limited to this.
  • the porous silica nanoparticles of the present invention may have a volume per g, for example, 0.7 ml to 2.2 ml.
  • a volume per g for example, 0.7 ml to 2.2 ml.
  • within the above range may be 0.7ml to 2.0ml, 0.8ml to 2.2ml, 0,8ml to 2.0ml, 0.9ml to 2.0ml, 1.0ml to 2.0ml and the like, but is not limited thereto. If the volume per gram is too small, the rate of decomposition may be too high, and excessively large particles may be difficult to manufacture or may not have an intact shape.
  • the porous silica particles of the present invention may have hydrophilic substituents and / or hydrophobic substituents on the outer surface and / or inside the pores.
  • hydrophilic substituents may exist on both the surface and inside of the pores, or only hydrophobic substituents may exist, hydrophilic substituents may exist on the surface or inside of the pores, hydrophobic substituents may exist on the surface, hydrophilic substituents on the surface, and hydrophobic substituents inside the pores. It may be present and vice versa.
  • the release of nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene supported on the porous silica particles of the present invention is mainly performed by the decomposition of the nanoparticles.
  • the interaction of the porous silica particles with the nucleic acid molecule release environment that complementarily binds to at least a portion of the carcass is regulated so that the rate of degradation of the nanoparticles is controlled to complementally bind to at least a portion of the transcript of the CTGF gene.
  • the release rate may be regulated, and nucleic acid molecules complementarily binding to at least a portion of the transcript of the CTGF gene may be diffused and released from the nanoparticles, and at least a portion of the transcript of the CTGF gene is controlled by the control of the substituents.
  • the binding force of the nucleic acid molecules that complementarily bind to the nanoparticles is controlled to at least the transcript of the CTGF gene. Release of nucleic acid molecules that complementarily bind to some can be controlled.
  • hydrophobic substituents are present inside the pores to enhance the binding ability of the poorly soluble (hydrophobic) CTGF gene to at least a portion of the transcript of the transcript, and in view of ease of use and formulation.
  • the surface of the particles may also be treated such that a hydrophilic substituent is present.
  • Hydrophilic substituents are, for example, hydroxyl groups, carboxy groups, amino groups, carbonyl groups, sulfhydryl groups, phosphate groups, thiol groups, ammonium groups, ester groups, imide groups, thiimide groups, keto groups, ether groups, indene groups, sulfonyl groups, polyethylene Glycol groups and the like
  • the hydrophobic substituent is, for example, substituted or unsubstituted C1 to C30 alkyl group, substituted or unsubstituted C3 to C30 cycloalkyl group, substituted or unsubstituted C6 to C30 aryl group, substituted Or an unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, a halogen-containing group, and the like.
  • the porous silica particles of the present invention may be one in which the outer surface and / or the inside of the pores are positively charged, negatively charged and / or uncharged.
  • both the surface and the inside of the pore may be positively charged, or may be negatively charged, only the surface or the inside of the pore may be positively charged, or may be negatively charged, the surface may be positively charged, and the interior of the pore may be negatively charged. The reverse is also possible, and vice versa.
  • the charging may be, for example, by the presence of a nonionic substituent, a cationic substituent or an anionic substituent.
  • the cationic substituent may be, for example, an amino group, another nitrogen-containing group, or the like as a basic group, and specifically, a heterocyclic aromatic compound group including a amino group, an aminoalkyl group, an alkylamino group, and a nitrogen atom, a cyan group, and a guanidine group. At least one functional group selected from the group consisting of, but is not limited thereto.
  • the anionic substituent may be, for example, a carboxy group (-COOH), a sulfonic acid group (-SO 3 H), a thiol group (-SH), etc., as an acidic group, but is not limited thereto.
  • the interaction of the porous silica particles with the nucleic acid molecule release environment that complementarily binds to at least a part of the transcript of the CTGF gene is regulated by controlling the substituent, thereby controlling the rate of degradation of the nanoparticles themselves.
  • the rate of release of nucleic acid molecules complementarily binding to at least a portion of the transcript may be controlled, and the nucleic acid molecules complementarily binding to at least a portion of the transcript of the CTGF gene may be diffused and released from the nanoparticles.
  • the binding force of the nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene is controlled to the nanoparticles, thereby controlling the release of the nucleic acid molecules that complementarily bind to the transcript of the CTGF gene.
  • the porous silica particles of the present invention support nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene in addition to the surface and / or the pores thereof, and complementarily to at least a portion of the transcript of the CTGF gene.
  • Substituents may be used to transfer the binding nucleic acid molecules to target cells, to carry a substance for other purposes, or to bind other additional substituents, and may further include antibodies, ligands, cell permeable peptides, or aptamers bound thereto. It may include.
  • Substituents, charges, binders and the like within the aforementioned surfaces and / or pores may be added, for example, by surface modification.
  • Surface modification can be carried out, for example, by reacting a compound having a substituent to be introduced with the particles, which may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the porous silica particles of the present invention may be manufactured through a small pore particle preparation and a pore expansion process, and may be manufactured through a calcination process, a surface modification process, and the like, as necessary. If both the calcination and the surface modification process has gone through may be surface modified after calcination.
  • the small pore particles may be, for example, particles having an average pore diameter of 1 nm to 5 nm.
  • the small pore particles can be obtained by adding a surfactant and a silica precursor in a solvent, stirring and homogenizing.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the ratio may be, for example, water and the organic solvent in a volume ratio of 1: 0.7 to 1.5, for example, 1: 1: 0.8 to 1.3, but is not limited thereto.
  • the surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added, for example, in an amount of 1 g to 10 g, for example, 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, etc., per liter of solvent, but is not limited thereto.
  • the silica precursor may be added after stirring with the addition of a surfactant to the solvent.
  • the silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but is not limited thereto.
  • the stirring may be performed, for example, for 10 minutes to 30 minutes, but is not limited thereto.
  • the silica precursor may be added, for example, 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, etc. within the above range, but is not limited thereto. It doesn't happen.
  • sodium hydroxide may further be used as a catalyst, which may be added with stirring after adding the surfactant to the solvent and before adding the silica precursor.
  • the sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, 2 ml to 3 ml, etc., based on 1 M aqueous sodium hydroxide solution. However, it is not limited thereto.
  • the solution can be reacted with stirring.
  • the stirring may be performed for example, for 2 hours to 15 hours, for example, within the above range, for example, 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours. , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.
  • the solution may be aged. Aging may be performed for example, from 8 hours to 24 hours, for example, within the range of 8 hours to 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 16 hours. , 10 hours to 14 hours, and the like, but is not limited thereto.
  • reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the pores of the obtained porous silica particles are expanded, and the pore expansion may be performed using a pore swelling agent.
  • the pore swelling agent may be trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, and the like, and specifically, trimethylbenzene may be used. It is not limited.
  • the pore swelling agent may use, for example, N, N-dimethylhexadecylamine (N, N-dimethylhexadecylamine, DMHA), but is not limited thereto.
  • the pore expansion may be carried out, for example, by mixing porous silica particles in a solvent with a pore swelling agent and heating to react.
  • the solvent may be, for example, water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like
  • the porous silica particles are, for example, 10 g to 200 g per liter of solvent, for example, 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, 60 g to 80 g, etc., within the above range. It may be added in a ratio of, but is not limited thereto.
  • the porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed.
  • the second solvent may be added after the porous silica particles are dispersed in the first solvent.
  • the pore swelling agent is for example 10 to 200 parts by volume, 100 to 150 parts by volume, 10 to 100 parts by volume, 10 to 80 parts by volume, 30 to 80 parts by volume, 30 to 80 parts by volume based on 100 parts by volume of solvent. 70 parts by volume may be added, but is not limited thereto.
  • the reaction can be carried out, for example, at 120 ° C to 190 ° C.
  • 120 ° C to 190 ° C For example, within the range of 120 °C to 190 °C, 120 °C to 180 °C, 120 °C to 170 °C, 130 °C to 170 °C, 130 °C to 160 °C, 130 °C to 150 °C, 130 °C to 140 °C It may be performed, but is not limited thereto.
  • the reaction may be performed, for example, for 6 hours to 96 hours.
  • 6 hours to 96 hours within the range of 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 36 hours to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, 48 hours to 96 hours, 48 hours to 88 hours, 48 hours to 80 hours, 48 hours to 72 hours, 6 hours to 96 hours, 7 hours to 96 hours, 8 hours to 80 hours, 9 hours to 72 hours, 9 hours to 80 hours, 6 hours to 72 hours, 9 hours to 96 hours, 10 hours to 80 hours, 10 hours to 72 hours , 12 hours to 66 hours, 13 hours to 60 hours, 14 hours to 96 hours, 15 hours to 88 hours, 16 hours to 80 hours, 17 hours to 72 hours, and the like, but is not limited thereto.
  • the time and temperature can be adjusted within the ranges exemplified above so that the reaction can be carried out sufficiently without excess. For example, when the reaction temperature is lowered, the reaction time may be increased, or when the reaction temperature is lowered, the reaction time may be shortened. If the reaction is not sufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.
  • the reaction can be carried out, for example, by gradually raising the temperature. Specifically, it may be carried out by gradually raising the temperature at a rate of 0.5 °C / min to 15 °C / min from the room temperature to the above temperature, for example, 1 °C / min to 15 °C / min, 3 °C / min within the above range To 15 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but are not limited thereto.
  • the reaction can be carried out under stirring. For example, it may be stirred at a speed of 100 rpm or more, and specifically, may be performed at a speed of 100 rpm to 1000 rpm, but is not limited thereto.
  • the reaction solution can be cooled slowly, for example, it can be cooled by gradually reducing the temperature. Specifically, it may be carried out by gradually decreasing the temperature at a rate of 0.5 °C / min to 20 °C / min from the temperature to room temperature, for example, 1 °C / min to 20 °C / min, 3 °C / min to within the above range 20 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but is not limited thereto.
  • reaction product After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more times, ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like, and specifically, alcohol, more specifically ethanol can be used, but is not limited
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the obtained particles may be calcined, which is a process of heating the particles to remove silanol groups on the surface and inside thereof to lower the reactivity of the particles, to have a more compact structure, and to remove organic substances filling the pores.
  • it may be heated to a temperature of 400 °C or more.
  • the upper limit thereof is not particularly limited, and may be, for example, 1000 ° C, 900 ° C, 800 ° C, 700 ° C, or the like. Heating can be carried out for example for 3 hours or more.
  • the upper limit is not particularly limited and may be, for example, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or the like. More specifically, it may be performed for 3 hours to 8 hours at 400 ° C to 700 ° C, specifically 4 hours to 5 hours at 500 ° C to 600 ° C, but is not limited thereto.
  • the porous silica particles obtained can then be surface modified, and the surface modification can be carried out on the surface and / or inside the pores.
  • the particle surface and the inside of the pore may be surface modified identically, or may be surface modified differently.
  • the surface modification can cause the particles to charge or to have hydrophilic and / or hydrophobic properties.
  • a heterocyclic aromatic compound group including an amino group, an aminoalkyl group, an alkylamino group, a nitrogen atom, a cyan group and a guanidine group
  • Surface modification of the porous silica particles may be performed by having at least one substituent selected from the group consisting of.
  • Surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, anionic and the like to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group. However, it is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the alkoxysilane reacts with the porous silicon particles, a covalent bond is formed between the silicon atom and the oxygen atom so that the alkoxysilane may be bonded to the surface and / or the inside of the pores of the porous silicon particle, and the alkoxysilane has a substituent to be introduced.
  • the corresponding substituents may be introduced into the surface of the porous silicon particles and / or within the pores.
  • the reaction may be carried out by reacting porous silica particles dispersed in a solvent with an alkoxysilane.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the charge to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • Charging to the negative charge may be carried out by reacting with an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • 3-Mercaptopropyl) trimethoxysilane may be used, but is not limited thereto.
  • the charge to the non-charge (not positive or negative charge, non-charged state) can be carried out by reacting with an alkoxysilane having a common functional group having no charge, a combination of charging to the positive charge and negative charge appropriately By doing so, it is possible to charge with no charge through the offset of positive and negative charge, but is not limited thereto.
  • the hydrophilic property is a hydrophilic group such as hydroxy group, carboxy group, amino group, carbonyl group, sulfhydryl group, phosphate group, thiol group, ammonium group, ester group, imide group, thiimide group, keto group, ether group, indene group, sulfo It may be made to react with the alkoxysilane which has a silyl group, a polyethyleneglycol group, etc.
  • the hydrophobic nature may include hydrophobic substituents such as substituted or unsubstituted C1 to C30 alkyl groups, substituted or unsubstituted C3 to C30 cycloalkyl groups, substituted or unsubstituted C6 to C30 aryl groups, substituted or unsubstituted It can be made to react with the alkoxysilane which has a C2-C30 heteroaryl group, a halogen group, C1-C30 ester group, a halogen containing group, etc.
  • Trimethoxy (octadecyl) silane, Trimethoxy-n-octylsilane, Trimethoxy (propyl) silane, Isobutyl (trimethoxy) silane, Trimethoxy (7-octen-1-yl) silane, Trimethoxy (3,3,3-trifluoropropyl) Silane, Trimethoxy (2-phenylethyl) silane, Vinyltrimethoxysilane, Cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate, (3-Bromopropyl) trimethoxysilane, etc. may be used, but is not limited thereto.
  • hydrophobic substituents are present in the pores to enhance the binding force with nucleic acid molecules or substances complementarily bound to at least a part of the transcript of the poorly soluble (hydrophobic) CTGF gene through the surface modification.
  • the surface of the particle may be treated such as to have a hydrophilic substituent, and a substituent may be present on the surface to bind a nucleic acid molecule or a substance complementarily to at least a portion of a transcript of another CTGF gene. It may be.
  • the surface modification may be carried out in combination.
  • two or more surface modifications may be performed on the outer surface or inside the pores.
  • a compound including a carboxyl group may be bonded to silica particles into which amino groups are introduced by amide bonds to change the positively charged particles to have different surface properties, but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating, and the heating is for example from 80 ° C. to 180 ° C., for example from 80 ° C. to 160 ° C., from 80 ° C. to 150 ° C. within the above range. , 100 ° C. to 160 ° C., 100 ° C. to 150 ° C., 110 ° C. to 150 ° C., etc., but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane is, for example, 4 hours to 20 hours, for example, 4 hours to 18 hours, 4 hours to 16 hours, 6 hours to 18 hours, 6 hours to 16 hours within the above range. , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, etc., but is not limited thereto.
  • the reaction temperature, time, and the amount of the compound used for surface modification may be selected according to the degree to be surface modified, and the reaction conditions vary depending on the degree of hydrophilicity, hydrophobicity, and charge of the nucleic acid molecules or materials of the present invention.
  • the degree of hydrophilicity, hydrophobicity, and charge of the porous silica particles By controlling the degree of hydrophilicity, hydrophobicity, and charge of the porous silica particles, the release rate of nucleic acid molecules or substances that complementarily bind to at least a portion of the transcript of the CTGF gene can be controlled. For example, if nucleic acid molecules or substances that complementarily bind to at least a portion of the transcript of the CTGF gene have a strong negative charge at neutral pH, the reaction temperature is increased to make the porous silica particles have a strong positive charge. Or increase the reaction time and increase the compound throughput, but are not limited thereto.
  • porous silica particles of the present invention may be produced through, for example, the preparation of small pores, pore expansion, surface modification, and internal pore modification.
  • the small pore particle production and pore expansion process may be based on the above-described process, and the washing and drying process may be performed after the small pore particle production and after the pore expansion process.
  • separation of the unreacted material may be preceded before washing, and separation of the unreacted material may be performed by separating the supernatant, for example, by centrifugation.
  • the centrifugation may be performed, for example, at 6,000 to 10,000 rpm, and the time may be, for example, 3 to 60 minutes, specifically, 3 to 30 minutes, 3 to 30 minutes, and 5 minutes within the above range. To 30 minutes, etc., but is not limited thereto.
  • the washing after the preparation of the particles of the small pores may be performed by a method / condition within the above-described range, but is not limited thereto.
  • the washing after the pore expansion may be performed under more relaxed conditions than the above example.
  • washing may be performed within three times, but is not limited thereto.
  • the surface modification and internal pore modification may be by the processes described above, respectively, the process may be performed in the order of surface modification and internal pore modification, and the washing process of the particles may be further performed between the two processes. Can be.
  • the reaction solution such as a surfactant used for particle production and pore expansion is filled in the pores so that the inside of the pores is not modified during surface modification. Only the surface can be modified. Then, washing the particles may remove the reaction solution in the pores.
  • Particle washing between the surface modification and the internal pore reforming process may be water and / or an organic solvent, and in particular, water and an organic solvent may be alternately used once or several times because different materials may be dissolved for each solvent.
  • Water or organic solvents alone may be washed once or several times. The number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, specifically 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • Nucleic acid molecules complementarily bound to at least a portion of the transcript of the CTGF gene may be supported on the surface and / or within the pores of the porous silica particles, and the supported nucleic acid may be, for example, a porous silica particle in a solvent and a transcript of the CTGF gene. It may be performed by mixing a nucleic acid molecule that binds to at least a portion complementarily.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. can be used.
  • PBS phosphate buffered saline solution
  • SBF Simulated Body Fluid
  • Borate-buffered saline Borate-buffered saline
  • Tris-buffered saline may be used as the solvent.
  • the ratio of the porous silica particles and the nucleic acid molecule of the present invention is not particularly limited, for example, the weight ratio is 1: 0.05 to 0.8, for example, within the above range 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.1 to 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
  • Nucleic acid molecules complementarily binding to at least a portion of the transcript of the CTGF gene supported on the porous silica particles may be gradually released over an extended time. Such slow release may be continuous or discontinuous, linear or nonlinear, due to the characteristics of the porous silica particles and / or their interaction with nucleic acid molecules that complementarily bind to at least a portion of the transcript of the CTGF gene. Can vary.
  • a nucleic acid molecule that complementarily binds to at least a portion of the transcript of the CTGF gene supported on the porous silica particles is released as the porous silica particles are biodegraded, and the porous silica particles according to the present invention are slowly degraded to transfer the supported CTGF gene.
  • Nucleic acid molecules that complementarily bind to at least a portion of the corpse can be released in a sustained manner. This may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface and / or pores, degree of compactness of the porous silica particles, and the like, but are not limited thereto.
  • nucleic acid molecules complementarily bound to at least a portion of the transcript of the CTGF gene supported on the porous silica particles may be released while being separated from the porous silica particles and diffused, and thus, the transcripts of the porous silica particles and the CTGF gene may be released. It is influenced by the relationship with the nucleic acid molecule complementarily binding to at least a portion, the nucleic acid molecule release environment complementary to at least a portion of the transcript of the CTGF gene, thereby controlling at least the transcript of the CTGF gene
  • the release of nucleic acid molecules that complementarily bind to some can be regulated. For example, it can be controlled by enhancing or weakening the binding strength of the porous silica particles to nucleic acid molecules that complementarily bind to at least a part of the transcript of the CTGF gene of the porous silica particles.
  • the surface of the particle and / or the inside of the pore have a hydrophobic substituent so that the porous silica
  • the binding force between the particle and the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript of the CTGF gene may be increased, whereby the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript of the CTGF gene This can be released slowly.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” means to be insoluble (practically insoluble) or only slightly soluble (with respect to water), which means “Pharmaceutical Science” 18 th Edition ( USP, Remington, Mack Publishing Company).
  • the poorly water-soluble material may be, for example, water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 1 atmosphere and 25 ° C., but is not limited thereto.
  • the surface of the particle and / or the inside of the pore have a hydrophilic substituent, so that the porous silica particles and the transcript of the CTGF gene
  • the binding force with a nucleic acid molecule or substance complementarily binding to at least a portion of the may be increased, whereby the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript of the CTGF gene may be released in a sustained manner.
  • This may be, for example, the surface of the porous silica particles modified with an alkoxysilane having a hydrophilic substituent.
  • the water-soluble substance may have a water solubility of 10 g / L or more at 1 atmosphere and 25 ° C., but is not limited thereto.
  • nucleic acid molecule or substance complementary to at least a portion of the transcript of a supported CTGF gene is charged, the surface of the particle and / or the inside of the pore are charged with opposite charges, thereby The binding force with the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript may be increased, whereby the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript of the CTGF gene is released slowly.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having an acidic group or a basic group.
  • the surface of the particle and / or the inside of the pore may be negatively charged at neutral pH.
  • the binding force between the porous silica particles and the nucleic acid molecule or substance complementarily binding to at least a portion of the transcript of the CTGF gene is increased, and the nucleic acid molecule or substance complementarily binds to at least a portion of the transcript of the CTGF gene. This can be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) and a sulfonic acid group (-SO 3 H).
  • the surface of the particle and / or the inside of the pore may be positively charged, whereby Sustained release of nucleic acid molecules or substances that complementarily bind to at least a portion of the transcript of the CTGF gene by increasing the binding force between the silica particles and at least a portion of the transcript of the CTGF gene Can be.
  • the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or another nitrogen-containing group.
  • Nucleic acid molecules or substances that complementarily bind to at least a portion of the transcript of the CTGF gene may be released for a period of, for example, 7 days to 1 year or more, depending on the type of treatment required, the release environment, and the porous silica particles used. have.
  • the porous silica particles of the present invention are 100% biodegradable, the nucleic acid molecules or substances complementarily binding to at least a part of the transcript of the CTGF gene supported thereon may be 100% released.
  • the nucleic acid molecule that complementarily binds to at least a portion of the transcript of the CTGF gene has a complementarity with the sequence of SEQ ID NO: 10 nucleotides (nt), 11 nucleotides, 12 nucleotides, 13 It may be at least nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, or at least 18 nucleotides in total, and the related detailed description is as described above.
  • the present invention provides a pharmaceutical composition for preventing or treating fibroproliferative diseases, including; a composition for inhibiting CTGF gene expression comprising porous silica particles carrying nucleic acid molecules complementarily bound to at least a portion of a transcript of the CTGF gene described above. To provide a composition.
  • nucleic acid molecule porous silica particles, suppression of CTGF gene expression, fibrotic disease, various formulations of the pharmaceutical composition and the like are as described above.
  • siRNA used in the present invention may be abbreviated as 'siCTGF', porous silica particles of the present invention as 'DegradaBALL or DDV', and DegradaBALL carrying siCTGF may be abbreviated as 'LEM-S401'.
  • DegradaBALL combined with DegradaBALL and TAMRA is Lemonex, Inc. (Cell counting kit-8) is provided by Dojindo molecular technologies, Inc. (Maryland, USA).
  • TGF-ß was purchased from Peprotech (New Jersey, USA), 10% Phosphate Buffered Saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Roswell Park Memorial Laboratory 1640 (RPMI 1640) , Penicillin-streptomycin and 0.05% trypsin-EDTA were purchased from WelGene (South Korea).
  • nucleic acid molecules were synthesized by Lemonex (Seoul, Korea), and their sequences and nucleic acid molecule sequences used throughout the present specification are shown in Table 1 below. All PCR primers were purchased from Cosmogenetech (Seoul, Korea). Anti-mouse CTGF antibodies were purchased from Abcam (Cambridge, UK) and anti-mouse collagen 1, 3 antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Trizol cell lysis solution was purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA) and all PCR reagents were obtained from TaKaRa Bio Inc. Purchased from (Shiga, Japan). All chemicals were used as received.
  • Target sequence 1 5'-CTC ATT AGA CTG GAA CTT -3 '(Position in gene sequence: 1280)
  • Target sequence 2 5'-G GAA CTT GAA CTG ATT CA-3 '(Position in gene sequence: 1291)
  • A549 and HaCaT cells were seeded in 96-well culture plates with 100 ⁇ l of growth medium (50-70% confluency) at a density of 10,000 cells per well.
  • Cells were treated with the appropriate concentration of DegradaBALL in serum containing medium and incubated at 37 ° C. for 24 hours. After incubation, the cells were washed twice with 1 x PBS, and then 100 ⁇ l of serum-free medium containing 10 ⁇ l of CCK-8 was added, followed by further incubation for 1 hour.
  • the optical density of each well in the culture plate was measured at 450 nm wavelength. Mean and standard deviation of the deviation of triplicates were calculated and plotted.
  • A549 and HaCaT cells were treated with 500 ⁇ l of LEM-S401 (50 nM siCTGF) and siCTGF (50 nM) containing LNP in serum-free medium.
  • LEM-S401 50 nM siCTGF
  • siCTGF 50 nM
  • serum-free medium was replaced.
  • TGF-ß 2 ng / mL
  • RNAs extracted in vitro and in vivo were used for thermal cycling using the following reaction conditions.
  • cDNA synthesis 5 minutes at 65 ° C., 2 minutes at 42 ° C., 50 minutes at 42 ° C., 15 minutes at 70 ° C., 1 cycle of inactivation, amplification: 30 seconds at 95 ° C., 60 seconds at 55 ° C., 72 ° C. 30 seconds 30 cycles.
  • LEM-S401 33 mM
  • FITC-conjugated siCTGF and TAMRA-binding DegradaBALL were injected into the mouse skin at seven different sites.
  • fluorescence images of the excised mouse skin were taken using an FOBI in vivo imaging device (NeoScience Co., Ltd., Seoul, Korea).
  • the obtained skin sample was placed in 4% PFA solution.
  • the sample was inserted into paraffin and cut to 10 ⁇ m thickness. After dehydration, the sections were stained with DAPI. Samples were observed with a BX71 microscope equipped with a 20x objective (Olympus, Tokyo, Japan).
  • LEM-S401 can inhibit CTGF expression during tissue remodeling.
  • a hole was cut in the mouse skin, wounded and wrapped with a band using a biopsy punch (4 mm). After the wound was completely closed, 30 ⁇ l of LEM-S401 (33 mM) in 1 ⁇ PBS was injected subcutaneously into the wound site at 4 different sites every 4 days (10 days, 14 days, 18 days, 22 days), and mice were Sacrifice was made on day 26.
  • Mouse skin samples were incubated in 4% PFA solution at 4 ° C. for 24 hours. The sample was then inserted into paraffin and sections were made 10 ⁇ m thick. The sectioned samples were dehydrated and incubated twice for 10 minutes each in permeate solution (0.2% tween 20 in 1 ⁇ PBS). The samples were then incubated for 45 minutes in humidified atmospheric blocking solution (5% normal goat serum, 0.2% tween 20 in 1 ⁇ PBS). Samples were incubated for 3 hours at room temperature with a primary antibody solution containing 0.2% tween 20 with a 1: 100 dilution of 2% normal goat serum and antibody in PBS in a humidification chamber.
  • Samples were rinsed three times for 10 minutes each in permeate solution and incubated with a secondary antibody dilution solution containing 2% normal goat serum and 0.2% tween 20 in 1 ⁇ PBS for 2 hours at room temperature. Samples were washed with permeate solution and stained with DAPI. Samples were observed under a BX71 microscope equipped with a 20x objective (Olympus, Tokyo, Japan).
  • Porous Silica Particles DDV or DegradaBALL
  • reaction solution was then centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C., and washed five times with alternating ethanol and distilled water.
  • the reaction was carried out starting at 25 ° C. and warming up at a rate of 10 ° C./min, then slowly cooling at a rate of 1-10 ° C./min in the autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, and centrifuged at 8000 rpm for 10 minutes at 25 ° C. and washed five times with ethanol and distilled water.
  • the porous silica particles prepared in 2) were put in a glass vial, heated at 550 ° C. for 5 hours, and cooled slowly to room temperature after completion of the reaction to prepare particles.
  • Porous silica particles were prepared in the same manner as in 9-1- (1), except that the reaction conditions at the time of pore expansion were changed to 140 ° C. and 72 hours.
  • Porous silica particles were prepared in the same manner as in Example 9-1- (1), except that a 5 times larger container was used and each material was used in a 5 times capacity.
  • Porous silica particles were prepared by the same method as 9-1- (1), except that 920 ml of distilled water and 850 ml of methanol were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 9-1- (1), except that 800 ml of distilled water, 1010 ml of methanol, and 10.6 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 9-1- (1), except that 620 ml of distilled water, 1380 ml of methanol, and 7.88 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as 9-1- (1), except that 2.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as 9-1- (1), except that 4.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as 9-1- (1), except that 11 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as 9-1- (1), except that 12.5 mL of TMB was used for pore expansion.
  • Example 9-1- (1) -2 In the same manner as in Example 9-1- (1) -2), the small pore particles were reacted with TMB, cooled and centrifuged to remove the supernatant. Thereafter, centrifuged under the same conditions as in Example 9-1- (1) -2), washed three times with alternating ethanol and distilled water, and then dried under the same conditions as in Example 9-1- (1) -2). Powdery porous silica particles (pore diameter 10-15 nm, particle diameter 200 nm) were obtained.
  • N-Hydroxysuccinimide 200 mg was dispersed in 30 mL of PBS and allowed to react for 12 hours while stirring at room temperature. The product is then washed and dried.
  • reaction solution of the previous step remains inside the pore, so that the inside of the pore is not modified.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed five times with alternating ethanol and distilled water.
  • Example 9-1- (4) The porous silica particles of Example 9-1- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to charge with a positive charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • porous silica particles were dispersed in a 10 mL toluene in a 100 mL round bottom flask with a bath sonicator. Then 1 mL of APTES was added and stirred at 400 rpm and stirred at 130 ° C. for 12 hours.
  • Example 9-1- (1) The porous silica particles of Example 9-1- (1) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), except that 0.4 ml of APTES was added and the reaction time was 3 hours.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 9-1- (9) The porous silica particles of Example 9-1- (9) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and the other method was the method of 9-2- (1) -1). Modified in the same manner as
  • Example 9-1- (10) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and were modified in the same manner as in the method of 9-2- (1) -1). It was.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 9-1- (1) The porous silica particles of Example 9-1- (1) were reacted with Trimethoxy (propyl) silane to introduce propyl groups into the surface and the pores, and 0.35ml of Trimethoxy (propyl) silane was added instead of APTES, followed by 12 hours of reaction. Modification was carried out in the same manner as in Example 9-2- (1) except for the above.
  • Example 9-1- (1) The porous silica particles of Example 9-1- (1) were reacted with Trimethoxy-n-octylsilane to introduce propyl groups on the surface and inside of the pores, and 0.5 ml of Trimethoxy-n-octylsilane was added instead of APTES, and reacted for 12 hours. Modification was carried out in the same manner as in Example 9-2- (1) except for the above.
  • Example 9 except that DMSO (dimethyl sulfoxide) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES, stirred at room temperature for 24 hours, and DMSO was used instead of distilled water. The modification was carried out in the same manner as in the method of -2- (1) -1).
  • DMSO dimethyl sulfoxide
  • Example 9-2- (1) -1) It was modified in the same manner as in Example 9-2- (1) -1) except that 1.1 mL of MPTES was used instead of APTES.
  • Example 9-2- (3) -2 100 mg of the porous silica nanoparticles of Example 9-2- (3) -2) were dispersed in 1 mL of 1 M aqueous sulfuric acid solution and 20 mL of 30% hydrogen peroxide solution, and stirred at room temperature to induce an oxidation reaction. Oxidized to a group. After the same washing and drying in the same manner as in Example 9-2- (1) -1).
  • the inhibition rate of CTGF expression of the prepared nucleic acid molecules is shown in Tables 2 and 3 below.
  • a nucleic acid molecule comprising a strand having complementarity with at least 10 nucleotides complementarity with the sequence of SEQ ID NO: 1 (sense RNA consisting of the sequence of SEQ ID NO: 1 and antisense RNA consisting of the sequence of SEQ ID NO: 2)
  • a dsRNA consisting of a strand and its complementary strand a siRNA consisting of a sense RNA consisting of a sequence of SEQ ID NO: 55 and an antisense RNA consisting of a sequence of SEQ ID NO: 56, a strand consisting of
  • nucleic acid molecule comprising a strand having complementarity of only 7 nucleotides (sense RNA consisting of the sequence of SEQ ID NO: 4 and antisense RNA consisting of the sequence of SEQ ID NO: 5; strand consisting of the sequence of SEQ ID NO: 6 and complementary thereto
  • sense RNA consisting of the sequence of SEQ ID NO: 4
  • antisense RNA consisting of the sequence of SEQ ID NO: 5
  • strand consisting of the sequence of SEQ ID NO: 6 and complementary thereto
  • the expression rate is significantly lower than about 60%, suggesting that the technical significance of the complementarity with the sequence of SEQ ID NO: 1 is 10 nucleotides or more.
  • siRNA consisting of a sense RNA consisting of a sequence of SEQ ID NO: 1 and an antisense RNA consisting of a sequence of SEQ ID NO: 2, including the strand having the highest expression inhibition and comprising a strand complementary to the sequence of SEQ ID NO: 1 and all 18 nucleotides;
  • siRNA consisting of a sense RNA consisting of a sequence of SEQ ID NO: 1 and an antisense RNA consisting of a sequence of SEQ ID NO: 2
  • the following experiment was performed by selecting a strand consisting of a strand consisting of the sequence of SEQ ID NO: 3 and a strand complementary thereto.
  • Sequence number (/ means pair between sense strand and antisense strand) Expression inhibition rate (%) Sequence number Expression inhibition rate (%) 1/2 or 3 93.7 37/38 or 39 74.6 4/5 or 6 62.4 40/41 or 42 71.8 7/8 or 9 67.2 43/44 or 45 63.5 10/11 or 12 72.6 46/47 or 48 74.2 13/14 or 15 74.1 49/50 or 51 71.6 16/17 or 18 61.3 52/53 or 54 90.3 19/20 or 21 74.7 55/56 or 57 90.1 22/23 or 24 72.3 58/59 or 60 91.9 25/26 or 27 67.8 61/62 or 63 92.2 28/29 or 30 63.5 64/65 or 66 91.5 31/32 or 33 71.6 67/68 or 69 92.8 34/35 or 36 72.9 70/71 or 72 91.6
  • PNA sequence number Expression inhibition rate (%) PNA sequence number Expression inhibition rate (%) 87 90.3 94 92.1 88 90.1 95 90.5 89 91.2 96 91.7 90 90.7 97 90.4 91 91.5 98 90.2 92 90.2 99 91.4 93 91.3
  • Porous Silica Particles DDV or DegradaBALL
  • FIG. 28 is a photograph of porous silica particles of 9-1- (1)
  • FIG. 29 is a photograph of porous silica particles of 9-1- (2), confirming that spherical porous silica particles having sufficiently expanded pores are formed evenly.
  • Fig. 30 is a photograph of small pore particles of 9-1- (1)
  • Fig. 31 is a comparative photograph of small pore particles of 9-1- (1) and 9-1- (3). You can see that is generated evenly.
  • Example 9-1- (1) The surface area and pore volume of the small pore particles of Example 9-1- (1) and the porous silica particles of Examples 9-1- (1), (7), (8) and (10) were calculated.
  • the surface area was calculated by Brunauer-Emmett-Teller (BET) method, and the pore size distribution was calculated by Barrett-Joyner-Halenda (BJH) method.
  • BET Brunauer-Emmett-Teller
  • BJH Barrett-Joyner-Halenda
  • Example 9-1- (1) 2.1 1337 0.69
  • Example 9-1- (7) 4.3 630 0.72
  • Example 9-1- (8) 6.9 521 0.79
  • Example 9-1- (1) 10.4 486 0.82
  • Example 9-1- (10) 23 395 0.97
  • porous silica particles are biodegraded and nearly decomposed after 360 hours.
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • porous silica particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a permeable membrane having pores having a diameter of 50 kDa shown in FIG. 34. 15 ml of SBF was added to the outer membrane, and the SBF of the outer membrane was replaced every 12 hours. Decomposition of the porous silica particles was performed at 37 ° C. with 60 rpm horizontal stirring.
  • porous silica particles of the example have a significantly larger t than the control.
  • t which has a ratio of absorbance 1/2 of the positively charged particles, was 24 or more.
  • the solvent was recovered at 0.5, 1, 2, 4, 8, 12, and 24 hours elapsed, and thereafter, at 24 hours, 0.5 ml of the solvent was recovered for fluorescence measurement. SBF was added.
  • the time of siRNA release by 50% is about 48 hours.
  • A549 (human lung cancer non-small cell) cells and HaCaT (human keratinocyte cells) cells were treated with DegradaBALL (LEM-S401) carrying siCTGF. Because both cell lines have functionally active CTGF transcription pathways, A549 and HaCaT cells are widely used as in vitro model cells in fibrosis studies.
  • A549 cells were treated with various concentrations of LEM-S401 (12.5, 25, 50 and 100 nM) and then incubated with 2 ng / mL TGF-ß to induce CTGF expression (FIG. 1).
  • LEM-S401 maintained a longer CTGF knockdown effect than LNP in A549 and HaCaT cells.
  • A549 and HaCaT cells were treated with siCTGF supported on LEM-S401 (50 nM) and LNP (50 nM), and then CTGF expression was induced by TGF-ß treatment.
  • Downregulation of CTGF in A549 cells lasted up to 96 hours after LEM-S401 treatment (CTGF expression level, 72 hours: 26.3%, 96 hours: 26.9%).
  • CTGF expression level, 72 hours: 46.4%, 96 hours: 58.6% was not high at both 72 and 96 hours.
  • LEM-S401 did not downregulate CTGF expression in vivo.
  • C57BL / 6 mice were injected with fluorescent label LEM-S401 consisting of FITC-conjugated siCTGF loaded on TAMRA-conjugated DegradaBALL and unsupported (free) FITC-conjugated
  • LEM-S401 consisting of FITC-conjugated siCTGF loaded on TAMRA-conjugated DegradaBALL and unsupported (free) FITC-conjugated
  • Fluorescence of TAMRA-DegradaBALL carrying FITC-siCTGF showed strong luminescence at the injection site on day 1.
  • the fluorescence decreased slowly with time, but the fluorescence was maintained at the injection site until 5 days after the injection (FIG. 6).
  • the tendency for fluorescence at the injection site to decrease with this time was in accordance with the tendency of the skin section slide (FIG. 6).
  • no fluorescence signal was observed in the excised skin or fragmented skin slides from mice injected with only unsupported free FITC-siCTGF, which was a small fragment that caused the free siCTGF to disperse rapidly in the body or to induce very rapid diffusion. Implying degradation (FIG. 7).
  • the data show that, compared to free siCTGF, LEM-S401 can maintain significantly higher concentration levels of siCTGF in the skin, at least 3 days after infusion.
  • LEM-S401 can induce CTGF gene knockdown and reduce collagen overproduction.
  • silicone splints were sutured around the wound for management and observation.
  • LEM-S401 was injected subcutaneously around the wound at 0,4,8,12 days. Mice were sacrificed on day 16, and expression levels of CTGF in the skin were analyzed by RT-PCR. The expression level of CTGF was significantly decreased in the LEM-S401 treated group, whereas no change was observed in the group treated with siCTGF or DegradaBALL alone.
  • collagen type 1,3 expression levels were also significantly downregulated in the LEM-S401 treated group (FIGS. 8-10).
  • Collagen types 1,3 known to be induced by CTGF are the major components of hypertrophic scars and keloids.
  • LEM-S401 was injected after the epidermis was recovered. Specifically, a biopsy punch was used to puncture the mouse skin (day 0), and after 10 days, 14, 18 and 22 days of epidermal recovery, LEM-S401 was injected into the wound site. Typically, in the mouse wound model, 10 days was sufficient to fully restore the epidermis after wound formation. After sacrifice on day 26, expression levels of CTGF and collagen type 1,3 were analyzed by RT-PCR.
  • CTGF and collagen in the LEM-S401 treated group were significantly lower than those in the control group (FIGS. 18 to 20).
  • Immunohistochemical analysis showed that CTGF and collagen type 1,3 protein expression levels were significantly reduced in the LEM-S401 treated group (FIGS. 21-24).

Abstract

La présente invention concerne une composition pour inhiber l'expression de CTGF, la composition permettant l'inhibition du niveau d'expression de CTGF au moyen d'interférence ARN (ARNi), et la présente invention concerne une composition permettant la prévention ou le traitement, avec une excellente efficacité, de diverses maladies fibroblastiques telles que des cicatrices hypertrophiques et des chéloïdes.
PCT/KR2019/095026 2018-07-31 2019-06-03 Composition pour l'inhibition de l'expression de ctgf WO2020027640A1 (fr)

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EP19843450.8A EP3831392A4 (fr) 2018-07-31 2019-06-03 Composition pour l'inhibition de l'expression de ctgf
CN201980050234.8A CN112512532A (zh) 2018-07-31 2019-06-03 用于抑制ctgf表达的组合物
AU2019314093A AU2019314093B2 (en) 2018-07-31 2019-06-03 Composition for inhibiting CTGF expression
JP2021505193A JP7152072B2 (ja) 2018-07-31 2019-06-03 Ｃｔｇｆ発現抑制用組成物
US17/162,045 US20210230602A1 (en) 2018-07-31 2021-01-29 Composition for inhibiting ctgf expression

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100130595A1 (en) * 2008-08-25 2010-05-27 Dean Nicholas M Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
KR20130062917A (ko) * 2010-03-24 2013-06-13 알엑스아이 파마슈티칼스 코포레이션 진피 및 섬유증성 적응증에서의 ｒｎａ 간섭
KR20140010285A (ko) * 2012-07-16 2014-01-24 서울대학교산학협력단 약물전달용 조성물 및 이를 이용한 약물전달방법
KR20160011565A (ko) * 2014-07-22 2016-02-01 주식회사 레모넥스 생리활성 물질 또는 단백질 전달용 조성물 및 이의 용도
WO2017178883A2 (fr) * 2016-04-11 2017-10-19 Olix Pharmaceuticals, Inc. Traitement de la fibrose pulmonaire idiopathique à l'aide de complexes d'arn qui ciblent le facteur de croissance du tissu conjonctif

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100130595A1 (en) * 2008-08-25 2010-05-27 Dean Nicholas M Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
KR20130062917A (ko) * 2010-03-24 2013-06-13 알엑스아이 파마슈티칼스 코포레이션 진피 및 섬유증성 적응증에서의 ｒｎａ 간섭
KR20140010285A (ko) * 2012-07-16 2014-01-24 서울대학교산학협력단 약물전달용 조성물 및 이를 이용한 약물전달방법
KR20160011565A (ko) * 2014-07-22 2016-02-01 주식회사 레모넥스 생리활성 물질 또는 단백질 전달용 조성물 및 이의 용도
WO2017178883A2 (fr) * 2016-04-11 2017-10-19 Olix Pharmaceuticals, Inc. Traitement de la fibrose pulmonaire idiopathique à l'aide de complexes d'arn qui ciblent le facteur de croissance du tissu conjonctif

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
U.S.P.: "Pharmaceutical Science", REMINGTON, MACK PUBLISHING COMPANY

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