WO2019022586A2 - Traitements pharmaceutiques pour prévenir ou traiter le cancer du foie - Google Patents

Traitements pharmaceutiques pour prévenir ou traiter le cancer du foie Download PDF

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WO2019022586A2
WO2019022586A2 PCT/KR2018/008611 KR2018008611W WO2019022586A2 WO 2019022586 A2 WO2019022586 A2 WO 2019022586A2 KR 2018008611 W KR2018008611 W KR 2018008611W WO 2019022586 A2 WO2019022586 A2 WO 2019022586A2
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group
dsrna
sequence
porous silica
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PCT/KR2018/008611
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WO2019022586A9 (fr
WO2019022586A3 (fr
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원철희
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주식회사 레모넥스
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Priority to CN202310211629.XA priority Critical patent/CN116236501A/zh
Priority to CN201880062438.9A priority patent/CN111132682B/zh
Priority to AU2018306411A priority patent/AU2018306411B2/en
Priority to JP2020504312A priority patent/JP6967810B2/ja
Priority to EP18838318.6A priority patent/EP3689355A4/fr
Priority to CN202310211626.6A priority patent/CN116898870A/zh
Priority to US16/634,675 priority patent/US20210093654A1/en
Application filed by 주식회사 레모넥스 filed Critical 주식회사 레모넥스
Priority claimed from KR1020180088375A external-priority patent/KR20190013635A/ko
Publication of WO2019022586A2 publication Critical patent/WO2019022586A2/fr
Publication of WO2019022586A3 publication Critical patent/WO2019022586A3/fr
Publication of WO2019022586A9 publication Critical patent/WO2019022586A9/fr
Priority to US17/536,880 priority patent/US20220072027A1/en
Priority to AU2022201756A priority patent/AU2022201756A1/en
Priority to AU2022201755A priority patent/AU2022201755A1/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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • 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 pharmaceutical composition for preventing or treating liver cancer.
  • HCC Hepatocellular carcinoma
  • the primary treatment for hepatocellular carcinoma is surgical resection, and few patients are eligible for curative treatment at the initial treatment stage.
  • Resection and percutaneous ablation have a recurrence rate of 70% after 5 years and are closely related to the survival rate.
  • HCC is characterized by multiple tumor progression.
  • the damaged liver tissue evolves in the early stages to small nodular hypercellular lesions called dysplastic nodules (DNs).
  • DNs small nodular hypercellular lesions
  • eHCC early hepatocarcinoma
  • eHCC early hepatocarcinoma
  • liver cancer diagnostic markers BANF1, PLOD3 and SF3B contribute to early malignant transformation of hepatocytes in liver tumor formation and are promising targets for molecular therapy of liver malignancy.
  • siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 157 and an antisense RNA consisting of the sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 158 to 310;
  • a prophylactic or therapeutic agent for liver cancer comprising at least one sequence selected from the group consisting of SEQ ID NOs: 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310 A pharmaceutical composition.
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 and an antisense RNA comprising a sequence complementary thereto;
  • a dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181;
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 and an antisense RNA comprising a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208;
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 and an antisense RNA consisting of a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273.
  • composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 121 to 157 and antisense RNA having a sequence complementary thereto; or
  • a pharmaceutical composition for preventing or treating liver cancer comprising a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310.
  • the siRNA or dsRNA may be selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, , Gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butylcyanoacrylate, gel foam, gelatin, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, poly Polyglycolic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and polyglycolic acid, and mixtures thereof.
  • the carrier is supported on at least one support selected from the group consisting of hydrogels.
  • the support is porous silica particles having a t of 20 or more, wherein the ratio of absorbance of the following formula (1) is 1/2:
  • 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 and A t is the absorbance of the porous silica particles measured after passage of time from the measurement of A 0 ).
  • composition of claim 8 wherein t is greater than or equal to 40.
  • the siRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 28, 119, and 136, and an antisense RNA comprising a sequence complementary thereto,
  • the dsRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 181, 272, and 289.
  • porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
  • the porous silica particles may be in the form of particles of an aliphatic group having an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, , A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aryl group, A substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, in the presence of at least one hydrophilic substituent selected from the group consisting of a substituted or unsubsti
  • porous silica particles have a positive or negative charge at an external surface or pore interior at neutral pH.
  • porous silica particles are positively charged at neutral pH at the outer surface and inside the pores.
  • porous silica particles have an average diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
  • the pharmaceutical composition of the present invention provides a preventive and therapeutic effect of liver cancer by specifically knocking down the genes expressed in early liver cancer cells to prevent the development of liver cancer and inhibiting the metastasis and proliferation of liver cancer cells.
  • Fig. 1 shows siRNAs comprising the sense RNA having the sequence of Table 11 and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell strains of Example 1, , And then the amount of expression of corresponding indicator factors of each siRNA was measured by Western blotting.
  • FIG. 2 shows the results of in vitro transfection of the SNU-449 cell line of Example 1-1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA of the complementary sequence according to the method of Example 1-2 , Migration and invasion responses of the corresponding surface factors of each siRNA were analyzed according to the methods of Examples 1-5, and the results of analysis of the scratch wound healing ability according to the method of Example 1-6 are shown.
  • FIG. 3 (A) shows the results of in vitro transfection of SNU-449 cell line of Example 1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA consisting of the complementary sequence according to the method of Example 1-2
  • FIG. 3 (B) is a graph showing the results of analysis of the expression level of the EMT regulatory proteins and the expression level of the corresponding indicator factors of the respective siRNAs according to the method of Example 1-9, In a subcutaneous tissue of athymic nude mice, and then analyzed the size of the liver tumor and the survival rate of the mouse.
  • FIG. 4 (A) shows siRNAs consisting of sense RNA having the sequence of Table 13 and antisense RNA consisting of the complementary sequence thereof in vivo according to the method of Example 1-8, and the process, ultrasound image
  • FIG. 4 (B) is a graph showing the results of analysis of inhibitory levels of the expression levels of the respective indicator genes of the siRNAs carried on the porous nanoparticles by the method of Example 1-9.
  • 5 is a photomicrograph of a porous silica particle.
  • FIG. 7 is a microphotograph of a small pore particle during the production of porous silica particles.
  • 10 is a tube having a cylindrical permeable membrane.
  • 11 is a graph showing the results of absorbance reduction with time of the porous silica particles.
  • FIG. 12 is a graph showing the results of absorbance reduction by particle size of porous silica particles over time.
  • 13 is a graph showing the results of absorbance reduction of pore diameters with time of porous silica particles.
  • 15 is a graph showing the results of absorbance reduction with time of the porous silica particles.
  • 16 is a tube for confirming release of physiologically active substance from porous silica particles.
  • 17 is a graph showing the release of the physiologically active substance carried on the porous silica particles with time.
  • 18 is a photomicrograph showing the release of siRNA in mice by loading siRNA onto porous silica particles.
  • siRNA means a nucleic acid molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method.
  • the siRNA molecule may have a structure in which the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) are located on opposite sides to form a double strand.
  • siRNA molecules may have a single stranded structure with self-complementary sense and antisense strands.
  • the siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included.
  • the siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by RNA interference (RNAi) effect.
  • RNAi RNA interference
  • the sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.
  • siRNA molecules may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence To form a hairpin structure, which in turn forms a stem-and-loop structure.
  • This stem-and-loop structure is processed in vitro or in vivo to produce siRNA molecules that are capable of mediating RNAi.
  • the "dsRNA” is a siRNA precursor molecule that meets the RISC complex containing the DICER enzyme of the target cell (Ribonuclease III) and is cleaved into siRNA, which in turn generates RNAi.
  • the dsRNA has a sequence that is several nucleotides longer than the siRNA, and the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) As shown in FIG.
  • Nucleic acid is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.
  • Gene means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression.
  • the gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein.
  • the nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.
  • 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 the cell.
  • gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide.
  • gene expression refers to a process in which a precursor siRNA is produced from a gene.
  • this process is referred to as transcription, although the transcription product of the siRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on the protein coding gene. Nevertheless, the generation of mature siRNAs 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 modulation using methods and compositions of the subject matter disclosed herein.
  • the target gene comprises a nucleic acid sequence whose expression level is down-regulated by the siRNA to an mRNA or polypeptide level.
  • target RNA or " target mRNA” refers to a transcript of a target gene that will bind siRNA and induce modulation of expression of the target gene.
  • transcription refers to a cellular process involving the interaction of an RNA polymerase with a gene that induces expression as RNA of structural information present in the coding sequence of the gene.
  • down-regulation refers to the expression of a specific gene into mRNA or the expression level of a protein by an intracellular transcription or gene translation in an activated cell, .
  • Treatment means an approach to obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction
  • treatment may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented.
  • compositions herein may prevent early onset symptoms, or related disorders when administered prior to appearance.
  • the present invention provides a siRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 5 to 157 and an antisense RNA comprising a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 310.
  • the present invention also provides a pharmaceutical composition for preventing or treating liver cancer.
  • siRNA or dsRNA of the present invention can be used in an animal including humans, such as monkeys, pigs, horses, cows, sheeps, dogs, cats, Mice, rabbits, and the like, and preferably may be derived from humans.
  • the siRNA or dsRNA of the present invention may be modified by deletion, substitution or insertion of a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the base sequence of the siRNA or dsRNA of the present invention , And variants capable of functionally functioning with the siRNA or dsRNA of the present invention.
  • siRNA or dsRNA of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available.
  • the composition of the present invention includes siRNA or dsRNA itself of the present invention, as well as other substances capable of increasing the expression rate of the siRNA or dsRNA of the present invention in a cell, for example, a compound, a natural product, .
  • siRNA or dsRNA of the present invention can be provided as a vector for intracellular expression.
  • the siRNA or dsRNA of the present invention can be introduced into cells using various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and nuclear protein, a complex of DNA and lipid, etc.
  • the dsRNA may be in a form that is contained within a carrier that enables efficient introduction into the cell.
  • the carrier is preferably a vector, and both viral vectors and non-viral vectors are usable.
  • the viral vector for example, lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector can be used, Preferably a lentiviral vector, but is not limited thereto.
  • Lentiviruses are a type of retrovirus that is not only a mitotic cell but also a mitotic cell due to the nucleophilicity of a nucleopore or a pre-integrated complex (a virus "shell") that allows active incorporation into a complete nuclear membrane There is a feature that can be made.
  • the vector comprising siRNA or dsRNA of the present invention preferably further comprises a selection marker.
  • the " selection marker" is intended to facilitate screening of cells into which siRNA or dsRNA of the present invention has been introduced.
  • the selectable marker used in the vector is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of a vector.
  • the selectable marker is selected from the group consisting of resistance to drugs, tolerance to cytotoxic agents, Such as GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidine dehydrogenase Histidinol dehydrogenase gene hisD) and guanine phosphosribosyltransferase (Gpt).
  • GFP green fluorescent protein
  • puromycin marker can be used.
  • the present invention relates to a composition
  • a composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 below.
  • SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA consisting of the complementary sequence thereof; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 targets the variant 1 sequence (SEQ ID NO: 1) of the human BANF1 gene, and the expression of the human BANF1 gene variant 1 RNAi, thereby preventing or treating liver cancer.
  • Target sequence 5 5'-CAA GAA GCT GGA GGA AAG-3 '(Position in gene sequence: 601) GC content: 45.0% SEQ ID NO: 5 Sense strand: 5'-CAA GAA GCU GGA GGA AAG UU-3 ' SEQ ID NO: 326 Antisense strand: 5'- CUU UCC UCC AGC UUC UUG UU-3 ' SEQ ID NO: 158 dsRNA: 5'-CAA GAA GCU GGA GGA AAG UU UCU AAA G-3 ' Target sequence 6: 5'-GAA AGA TGA AGA CCT CTT-3 '(Position in gene sequence: 667) GC content: 40.9% SEQ ID NO: 6 Sense strand: 5'-GAA AGA UGA AGA CCU CUU CCU U-3 ' SEQ ID NO: 327 Antisense strand: 5'-GGA AGA GGU CUU CAU CU UCU U-3 ' SEQ ID NO:
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208 in Table 1 below.
  • siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and antisense RNAs comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 182 to 208 shown in Table 2 below targets the variant 2 sequence (SEQ ID NO: 2) of the human BANF1 gene and the expression of the human BANF1 gene variant 2 RNAi, thereby preventing or treating liver cancer.
  • Target sequence 29 5'-ATG ACA ACC TCC CAA AAGCA-3 '(Position in gene sequence: 452) GC content: 40.9% SEQ ID NO: 29 Sense strand: 5'- AUGACAACCUCCCAAAAGCA UU-3 ' SEQ ID NO: 350 Antisense strand: 5'- UGCUUUUGGGAGGUUGUCUCUU UU-3 ' SEQ ID NO: 182 dsRNA: 5'- AUGACAACCUCCCAAAAGCA UU UCU AAA G-3 ' Target sequence 30: 5'-CCG AGA CTT CGT GGC AGA-3 '(Position in gene sequence: 472) GC content: 40.9% SEQ ID NO: 30 Sense strand: 5'- CCGAGACUUCGUGGCAGA UU-3 ' SEQ ID NO: 351 Antisense strand: 5'-UCUGCCACGAAGUCUCGG UU-3 ' SEQ ID NO: 183 dsRNA: 5'-CCGAGA
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 56-120 of Table 3 and an antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3 below.
  • SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 of Table 3 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3, targets the sequence of the human PLOD3 gene (SEQ ID NO: 3) and inhibits the expression of the human PLOD3 gene through RNAi Thereby preventing or treating liver cancer.
  • Target sequence 56 5'-CCA GAG AAG CTG CTG GTG AT-3 '(Position in gene sequence: 562) GC content: 50.0% SEQ ID NO: 56 Sense strand: 5'- CCAGAGAAGCUGCUGGUGAU UU-3 ' SEQ ID NO: 377 Antisense strand: 5'- AUCACCAGCAGCUUCUCUGG UU-3 ' SEQ ID NO: 209 dsRNA: 5'- CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3 ' Target sequence 57: 5'-CCA CAG CTG AAA CCG AGG-3 '(Position in gene sequence: 590) GC content: 55.0% SEQ ID NO: 57 Sense strand: 5'- CCACAGCUGAAACCGAGG UU-3 ' SEQ ID NO: 378 Antisense strand: 5'- CCUCGGUUUCAGCUGUGG UU-3 ' SEQ ID NO: 210 dsRNA: 5'-
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 shown in Table 4 below and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below.
  • siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 of Table 4 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below, targets the sequence of the human SF3B4 gene (SEQ ID NO: 4) and inhibits the expression of the human SF3B4 gene through RNAi Thereby preventing or treating liver cancer.
  • Target sequence 121 5'-AAT CAG GAT GCC ACT GTG TA-3 '(Position in gene sequence: 521) GC content: 40.9% SEQ ID NO: 121 Sense strand: 5'- AAUCAGGAUGCCACUGUGUA UU-3 ' SEQ ID NO: 442 Antisense strand: 5'- UACACAGUGGCAUCCUGAUU UU-3 ' SEQ ID NO: 274 dsRNA: 5'- AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3 ' Target sequence 122: 5'-CTG GAT GAG AAG GTT AGT GA-3 '(Position in gene sequence: 551) GC content: 40.9% SEQ ID NO: 122 Sense strand: 5'- CUGGAUGAGAAGGUUAGUGA UU-3 ' SEQ ID NO: 443 Antisense strand: 5'- UCACUAACCUUCUCAUCCAG UU-3 ' SEQ ID NO: 275 d
  • the siRNA or dsRNA of the present invention may be one that is carried on a carrier and is capable of carrying RNA molecules in the kind of the carrier.
  • the siRNA or dsRNA is not particularly limited as long as it is known in the art.
  • the siRNA or dsRNA of the present invention may be carried on a porous silica particle, and the particle is a particle of silica (SiO 2 ) and has a nano-sized particle size.
  • the porous silica particles are porous particles having nano-sized pores and can carry physiologically active substances such as siRNA or dsRNA of the present invention on the surface and / or pores thereof.
  • the porous silica particles are biodegradable particles, and when physiologically active substances are supported on the body, they can be biodegraded in the body and release physiologically active substances. That is, biodegradation of the porous silica particles results in the release of the physiologically active substance.
  • the porous silica particles according to the present invention may be slowly decomposed in the body, so that the supported physiologically active substance may have sustained release properties. For example, t at which the ratio of the absorbance of the following formula (1) is 1/2 is 20 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 was 7.4,
  • a t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
  • Equation (1) means that the porous silica particles are decomposed at a certain rate in an environment similar to the inside of the body.
  • the absorbances A 0 and A t in Equation (1) may be measured by, for example, placing the porous silica particles and suspension in a cylindrical permeable membrane and putting the same suspension in the outside of the permeable membrane.
  • the porous silica particles are biodegradable and can be slowly decomposed in the suspension.
  • the porous silica particles having a diameter of 50 kDa corresponding to about 5 nm can pass through the permeable membrane having a diameter of 50 kDa and the cylindrical permeable membrane can be permeated under a condition of 60 rpm Therefore, the suspension may be uniformly mixed, and the decomposed porous silica particles may come out of the permeable membrane.
  • the absorbance in Equation (1) may be measured under an environment in which, for example, the suspension outside the permeable membrane is replaced with a new suspension.
  • the suspension may be constantly being replaced, replaced at regular intervals, and the period may be a periodic or irregular period. For example, in the range of 1 hour to 1 week, an interval of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 7 Day interval, etc., but is not limited thereto
  • the absorbance becomes half of the initial absorbance after t time, meaning that about half of the porous silica particles are decomposed.
  • the suspension may be a buffer solution, and may be at least one selected from the group consisting of, for example, PBS (phosphate buffered saline) and SBF (simulated body fluid), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • T may be 20 or more, for example, t may be 20 to 120, for example, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65, and the like.
  • the porous silica particles may have a ratio t of 1/5 of the absorbance of the formula (1), for example, 70 to 140, and may be, for example, in the range of 80 to 140, 80 to 120, 80 to 110, To 140, 70 to 120, 70 to 110, and the like.
  • the porous silica particles may have a ratio t of 1/20 of the absorbance of the formula (1), for example, 130 to 220, and for example, within the range of 130 to 200, 140 to 200, 140 to 180, 150 To 180, and the like.
  • the porous silica particles may have a t of, for example, not less than 250, such as not less than 300, not less than 350, not less than 400, not less than 500, not less than 1000, and the like, , But is not limited thereto.
  • the ratio of the absorbance of Equation 1 to t has 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 represents the degree of decomposition of the porous silica particles at a certain rate in an environment similar to that of the body. This means that the surface area, the particle size, the pore diameter, the surface of the porous silica particles, and / The substituent, the degree of compactness of the surface, and the like.
  • the surface area of a particle can be increased to decrease t, or the surface area can be decreased to increase t.
  • the surface area can be controlled by controlling the diameter of the particles and the diameter of the pores. It is also possible to increase the t by placing substituents in the surface and / or pores to reduce the direct exposure of the porous silica particles to the environment (solvent, etc.). Also, it is possible to increase the affinity between the physiologically active substance and the porous silica particles by supporting the physiologically active substance on the porous silica particles, and to reduce the direct exposure of the porous silica silica particles to the environment, thereby increasing t. It is also possible to increase the t by fabricating the surface more densely during the production of the particles. While various examples have been described above for adjusting t in Equation 1, the present invention is not limited thereto.
  • the porous silica particles may be, for example, spherical particles, but are not limited thereto.
  • the average diameter of the porous silica particles may be, for example, 100 nm to 1000 nm, and may be within the above range, for example, 100 nm to 800 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, , But is not limited thereto.
  • the porous silica particles may have an average pore diameter of, for example, 1 nm to 100 nm, and may be within the above range, for example, 4 nm to 100 nm, 4 nm to 50 nm, 4 nm to 30 nm, 10 nm to 30 nm, It is not. It is possible to carry a large amount of the physiologically active substance and to carry the physiologically active substance having a large size.
  • the porous silica particles 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 700m 2 / g.
  • 200m 2 / g to 650m 2 / g 250m 2 / g to 650m 2 / g
  • 300m 2 / g to 700m 2 / g 300m 2 / g to 650 m 2 / g
  • 300 m 2 / g to 550 m 2 / g 300 m 2 / g to 500 m 2 / g, 300 m 2 / g to 450 m 2 /
  • the porous silica particles may have a volume per gram of, for example, 0.7 ml to 2.2 ml. For example, it may be within the range of 0.7 ml to 2.0 ml, 0.8 ml to 2.2 ml, 0.8 ml to 2.0 ml, 0.9 ml to 2.0 ml, 1.0 ml to 2.0 ml, and the like. If the volume per gram is too small, the rate of decomposition may become too high, and excessively large particles may be difficult to manufacture or may not have a perfect shape.
  • the porous silica particles may have a hydrophilic substituent and / or a hydrophobic substituent on the outer surface and / or inside the pore.
  • a hydrophilic substituent and / or a hydrophobic substituent on the outer surface and / or inside the pore.
  • hydrophilic substituents may be present on both the surface and the pores, only hydrophobic substituents may be present, hydrophilic substituents may be present only on the surface or pores, hydrophobic substituents may be present, hydrophilic substituents may be present on the surface, Or vice versa, and vice versa.
  • the release of the physiologically active substance carried on the porous silica particles is mainly performed by the decomposition of the particles, and the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by the control of the substituent,
  • the rate of release of the physiologically active substance can be controlled by adjusting the rate of the physiologically active substance, and the physiologically active substance can be diffused and released from the particles. Can be adjusted.
  • a hydrophobic substituent is present inside the pore, and a hydrophilic substituent may be present on the surface of the particle in view of ease of use and formulation Do.
  • the hydrophilic substituent may be, for example, an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, An unsubstituted C 3 to C 30 cycloalkyl group, a substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, and the hydrophobic substituent includes, for example, a substituted or unsub
  • the 'substituted' functional group in the 'substituted or unsubstituted' may be an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, At least one member selected from the group consisting of an alkyl group, an alkoxy group, an alkoxy group, an ammonium group, a sulfhydryl group, a phosphate group, an ester group, an imide group, a thioimide group, a keto group, an ether group, an indene group, a sulfonyl group, a methylphosphonate group and a polyethylene glycol group But is not limited thereto.
  • porous silica particles may be those in which the outer surface and / or the inside of the pores are positively charged and / or negatively charged.
  • both the surface and the pores can be positively charged, negatively charged, only positively charged on the surface or pore, negatively charged, surface can be positively charged, negatively charged inside the pore , And vice versa.
  • the charging may be performed, for example, by the presence of a cationic substituent or an anionic substituent.
  • the cationic substituent may be, for example, an amino group or other nitrogen-containing group as a basic group, and the anionic substituent may be, for example, a carboxyl group (-COOH), a sulfonic acid group (-SO 3 H) SH), and the like.
  • the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by controlling the substituent, so that the rate of decomposition of the particles themselves can be controlled to control the release rate of the physiologically active substance, May be diffused and released from the particles.
  • the substituent By controlling the substituent, the binding force of the physiologically active substance to the particles can be controlled and the release of the physiologically active substance can be controlled.
  • porous silica particles may be coated on the surface and / or inside of the pores by carrying a physiologically active substance, transferring a physiologically active substance to a target cell, carrying a substance for other purposes, And may further include an antibody, a ligand, a cell permeable peptide, an umbilical cord or the like coupled thereto.
  • Substituents, charges, bonding substances, etc. in the surface and / or pores described above can be added, for example, by surface modification.
  • the surface modification can be performed, for example, by reacting a compound having a substituent to be introduced with a particle, and the compound may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.
  • the alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
  • the porous silica particles may be prepared by, for example, preparing fine pore particles and a pore expansion process, and may be manufactured by further performing a calcination process, a surface modification process or the like, if necessary. When both the calcination and the surface modification process are performed, the surface modification may be performed after the calcination.
  • the particles of the small pores 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 to a solvent and stirring and homogenizing the same.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • water and the organic solvent may be used in a volume ratio of 1: 0.7 to 1.5, for example, 1: 0.8 to 1.3 by volume, It is not.
  • the surfactant may be, for example, CTAB (cetyltrimethylammonium bromide), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added in an amount of, for example, 1 g to 10 g per liter of solvent, for example, in the range of 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, and the like.
  • the silica precursor may be added after stirring and adding a surfactant to the solvent.
  • the silica precursor may be, for example, TMOS (Tetramethyl orthosilicate), but is not limited thereto.
  • the stirring may be carried out, for example, for 10 minutes to 30 minutes, but is not limited thereto.
  • the silica precursor may be added, for example, in an amount of 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, But may be further added with sodium hydroxide as a catalyst as needed, which may be added with stirring before addition of the surfactant to the solvent, followed by addition of the silica precursor.
  • the sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example 1 ml of sodium hydroxide solution, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, But is not limited thereto.
  • the solution can be reacted with stirring.
  • the stirring may be carried out for example for 2 hours to 15 hours, for example, within the above range for 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.
  • the aging can be carried out, for example, for 8 hours to 24 hours, for example within the above range, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 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 the unreacted material may be preceded by washing, for example, by separating the supernatant liquid by centrifugation.
  • the centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the washing may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times alternately. Alternatively, water or an organic solvent may be used once or several times It can be washed several times. The number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • ethers such as 1,4-dioxane (particularly, cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane
  • Acetone methyl isobutyl ketone
  • the washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the drying may be performed at, for example, 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, which can be carried out using a pore-expanding agent.
  • pore-expanding agent for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene and the like can be used. Specifically, trimethylbenzene can be used. But is not limited to.
  • the pore-expanding agent may be, for example, N, N-dimethylhexadecylamine (DMHA), but is not limited thereto.
  • DMHA N, N-dimethylhexadecylamine
  • the pore expansion can be performed, for example, by mixing the porous silica particles in a solvent with a pore-expanding agent, and heating and reacting.
  • the solvent can be, for example, water and / or organic solvents
  • the organic solvent can be, for example, ethers (especially cyclic ethers) such as 1,4-dioxane
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane
  • Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone
  • Carbon-based aromatic compounds 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 butano
  • the porous silica particles may be present in an amount of from 10 g to 200 g per liter of solvent, for example from 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, But the present invention is not limited thereto.
  • the porous silica particles may be uniformly dispersed in a solvent, for example, the porous silica particles may be added to a solvent and ultrasonically dispersed.
  • the second solvent may be added after dispersing the porous silica particles in the first solvent.
  • the pore-expanding agent may be, for example, 10 to 200 parts of skin to 100 parts of skin, 10 to 150 parts of skin, 10 to 100 parts of skin, 10 to 80 parts of skin, 30 to 80 parts of skin, 70 parts skin, and the like, but the present invention is not limited thereto.
  • the reaction can be carried out, for example, at 120 ° C to 180 ° C.
  • 120 ° C to 180 ° C For example, within the above-mentioned range, it is possible to use a temperature of 120 to 170 ° C, 120 to 160 ° C, 120 to 150 ° C, 130 to 180 ° C, 130 to 170 ° C, 130 to 160 ° C, 130 to 150 ° C, But is not limited thereto.
  • the reaction can be carried out, for example, for 24 hours to 96 hours.
  • 24 hours to 96 hours For example, within the above range from 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 48 hours to 48 hours, 48 hours to 80 hours, 48 hours to 72 hours, and the like, for example, from 1 hour to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, But is not limited thereto.
  • the reaction time may be increased when the reaction temperature is lowered, or the reaction time may be shortened when the reaction temperature is lowered. If the reaction is insufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to over-expansion of the pores.
  • the reaction can be carried out, for example, by raising the temperature stepwise. Specifically, it can be performed by raising the temperature from room temperature to the temperature stepwise at a rate of 0.5 ° C / min to 15 ° C / min, for example, within a range of 1 ° C / min to 15 ° C / Min to 15 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
  • the reaction solution may be gradually cooled, for example, it may be cooled stepwise. Specifically, it may be performed by gradually warming the temperature to room temperature at a rate of 0.5 ° C / minute to 20 ° C / minute.
  • the temperature may be 1 ° C / minute to 20 ° C / 20 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
  • reaction product may be washed and dried to obtain pore-expanded porous silica particles, and if necessary, separation of the unreacted material may be preceded by separation of the supernatant by, for example, centrifugation .
  • the centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the washing may be performed using water and / or an organic solvent.
  • water and an organic solvent may be used once or several times alternately.
  • water or an organic solvent may be used once or several times It can be washed several times.
  • the number of times may be, for example, two times or more and ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds 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., and specifically ethanol, more specifically ethanol, may be used, but the present invention
  • the washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
  • the obtained particles can be calcined.
  • the calcination is a process for heating the particles to have a more dense structure on the surface and inside thereof, and removing organic substances that fill the pores.
  • calcination is performed at 400 to 700 ° C for 3 hours To 8 hours, more specifically, from 500 ° C to 600 ° C for 4 hours to 5 hours, but the present invention is not limited thereto.
  • porous silica particles can be surface-modified.
  • the surface modification can be performed inside the surface and / or pores.
  • the surface of the particle and the inside of the pore may be surface-modified in the same manner or may be surface-modified differently.
  • the surface modification may allow the particles to be charged or have hydrophilic and / or hydrophobic properties.
  • the surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, and anionic to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group But is not limited thereto.
  • the alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
  • an alkoxysilane When the alkoxysilane is reacted with the porous silicon particles, an alkoxysilane can be bonded to the surface and / or pores of the porous silicon particles by forming a covalent bond between the silicon atom and the oxygen atom, and the alkoxysilane has a substituent
  • the substituent may be introduced into the surface and / or pores of the porous silicon particles.
  • the above reaction can be carried out by reacting the porous silica particles dispersed in a solvent with an alkoxysilane.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • the charging 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 or an aminoalkyl group.
  • an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group or an aminoalkyl group.
  • Specific examples thereof include N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, N- [3- propyl] silane, 3- (2-Aminoethylamino) propyldimethoxymethylsilane, and the like, but the present invention is not limited thereto.
  • the charging of the negative charge can be carried out, for example, by reacting with an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group or a thiol group.
  • an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group or a thiol group.
  • 3-Mercaptopropyl) trimethoxysilane can be used, but is not limited thereto.
  • the hydrophilic property may be a hydrophilic property such as a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, a sulfhydryl group, a phosphate group, a thiol group, an ammonium group, an ester group, an imide group, a thioimide group, a keto group, A polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyethylene glycol group and the like.
  • the hydrophobic property may be a hydrophobic substituent, for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted A C2 to C30 heteroaryl group, a halogen group, an ester group of C1 to C30, a halogen-containing group, and the like.
  • a hydrophobic substituent for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted A C2 to C30 heteroaryl group, a halogen group, an ester group of C1 to C30
  • trimethoxy (octadecyl) silane trimethoxy n-octylsilane, trimethoxy propyl silane
  • isobutyl trimethoxy silane trimethoxy (7-octen- silane, trimethoxy (2-phenylethyl) silane, vinyltrimethoxysilane, cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate and (3-Bromopropyl) trimethoxysilane.
  • hydrophobic substituent in the pores for enhancing the bonding strength with the poorly soluble (hydrophobic) physiologically active substance through surface modification, and the hydrophobic substituent is present on the surface of the particle in terms of ease of use and formulation And a substituent for binding another substance other than the physiologically active substance may be present on the surface.
  • the surface modification may be performed in combination.
  • two or more surface modification may be performed on the outer surface or inside the pores.
  • a compound containing a carboxyl group may be bonded to an amide-introduced silica particle with an amide bond to change positively charged particles to have different surface characteristics, but the present invention is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating.
  • the heating may be performed at a temperature of, for example, 80 to 180 DEG C, for example, 80 to 160 DEG C, 80 to 150 DEG C, 100 to 160 DEG C, 100 to 150 DEG C, 110 to 150 DEG C, But is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane may be carried out, for example, for 4 to 20 hours, for example 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, and the like.
  • the reaction temperature, time, and the amount of the compound used for surface modification can be selected depending on the desired degree of surface modification.
  • the reaction conditions vary depending on the hydrophilicity, hydrophobicity, and electric charge of the physiologically active substance,
  • the hydrophilic property, the hydrophobicity, and the electric charge level of the physiologically active substance can be controlled. For example, when a physiologically active substance has a strong negative charge at a neutral pH, the reaction temperature can be increased or the reaction time can be lengthened and the compound throughput can be increased in order to allow the porous silica particles to have a strong positive charge , But is not limited thereto.
  • the porous silica particles may be prepared by, for example, preparing pores of small pores, expanding pores, modifying the surface, or modifying pores inside.
  • the particle preparation and the pore expansion process of the small pores can be performed by the processes described above, and the cleaning and drying processes can be performed after the particle preparation of the small pores and after the pore expansion process.
  • the separation of the unreacted material may be preceded by washing before washing, for example, by separating the supernatant by centrifugation.
  • the centrifugation may be performed at, for example, 6,000 to 10,000 rpm, for example, 3 minutes to 60 minutes, specifically 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the cleaning after the particle preparation of the small pores may be carried out by any method / condition within the range exemplified above, but is not limited thereto.
  • the purging after the pore expansion can be performed under more relaxed conditions than in the previous examples. For example, washing may be performed within 3 times, but is not limited thereto.
  • the surface modification and the internal reforming of the pores can be performed by the processes described above, and the processes can be performed in the order of the surface modification and the internal pore modification, and the particle washing process is further performed between the two processes .
  • the inside of the pores are filled with the reaction liquid such as the particles used for the preparation of the particles and the pore expansion, Only the surface can be modified. The particles may then be washed to remove the reaction liquid inside the pores.
  • the washing of the particles between the surface modification and the pore interior modification process may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times, Water or an organic solvent can be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
  • the washing may be carried out under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically for 3 to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
  • the drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
  • the physiologically active substance such as siRNA or dsRNA of the present invention can be carried on the surface and / or inside the pores of the porous silica particles.
  • the support may be carried out, for example, by mixing the porous silica particles in the solvent and the physiologically active substance.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds 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. may be used.
  • PBS phosphate buffered saline solution
  • SBF Simulated Body Fluid
  • borate-buffered saline borate-buffered saline
  • Tris-buffered saline Tris-buffered saline, etc. may be used as the solvent.
  • the ratio of the porous silica particles to the physiologically active substance is not particularly limited and may be, for example, a weight ratio of 1: 0.05 to 0.8, such as 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
  • the physiologically active substance such as siRNA or dsRNA of the present invention carried on the porous silica particles can be gradually released over an extended period of time. Such slow release may be continuous or non-continuous, linear or non-linear, and may vary due to the characteristics of the porous silica particles and / or their interaction with the bioactive material.
  • the physiologically active substance supported on the porous silica particles is released while the porous silica particles are biodegraded.
  • the porous silica particles according to the present invention may be slowly decomposed to release the supported physiologically active substance slowly. This can be controlled, for example, by controlling the surface area, particle size, pore diameter, surface area and / or porosity of the porous silica particles, the degree of compactness of the surface, and the like.
  • the physiologically active substance carried on the porous silica particles can be released while diffusing from the porous silica particles, which is affected by the relationship between the porous silica particles, the physiologically active substance, and the environment for releasing the physiologically active substance , And it is possible to control the release of the biologically active substance by controlling this. For example, by strengthening or weakening the binding force of the porous silica particles with the physiologically active substance by surface modification.
  • the surface of the particle and / or the inside of the pore may have a hydrophobic substituent to increase the binding force between the porous silica particle and the physiologically active substance, Whereby the physiologically active substance can be released in a sustained manner.
  • the porous silica particles are surface-modified with an alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” is a meant to include (for water) in that the insoluble (insoluble), substantially insoluble (practically insoluble) or very slightly soluble (only slightly soluble) This "Pharmaceutical Science,” 18 th Edition ( USP, Remington, published by Mack Publishing Company).
  • the water-insoluble physiologically active substance may have a water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 25 ° C under 1 atm, but is not limited thereto.
  • the surface and / or the pores of the particles may have a hydrophilic substituent and the binding strength between the porous silica particles and the physiologically active substance may be increased, It can be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having a hydrophilic substituent.
  • the water-soluble physiologically active substance may have a water solubility of 10 g / L or more at 25 ° C and 1 atm, but is not limited thereto.
  • the surface and / or pore of the particle may be charged with opposite charge to increase the binding force between the porous silica particle and the physiologically active substance, Material may be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having an acidic group or a basic group.
  • the surface and / or pore interior of the particle may be negatively charged at a neutral pH, whereby the binding force between the porous silica particle and the physiologically active substance Is increased, so that the physiologically active substance 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) or a sulfonic acid group (-SO 3 H).
  • the surface and / or pores of the particles may be positively charged, thereby increasing the binding force between the porous silica particles and the physiologically active substance, Material may be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or other nitrogen-containing groups.
  • the physiologically active substance 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.
  • the porous silica particles can be decomposed 100% as biodegradable, the supported physiologically active substance can be 100% released.
  • the pharmaceutical composition for preventing or treating liver cancer comprising siRNA or dsRNA of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with a carrier.
  • a pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.
  • the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention can be applied to any formulation containing the siRNA or dsRNA of the present invention as an active ingredient, and can be manufactured into oral or parenteral formulations.
  • the pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.
  • composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels will depend on factors well known in the art and other medical disciplines including the type of disease, severity of the patient, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, Can be determined.
  • the composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
  • the dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease and the like.
  • the amount of the drug accumulated in the body, and / or the degree of specific activity of the siRNA or dsRNA of the present invention to be used. May be calculated on the basis of the EC50 generally measured as effective in the in vivo animal model and in vitro, for example from 0.01 [mu] g to 1 g per kg of body weight and may be divided into daily, weekly, monthly, May be administered once or several times per unit period, or may be continuously administered for a long period using an infusion pump.
  • the number of repeated administrations is determined in consideration of the duration of the drug in the body, the drug concentration in the body, and the like.
  • the composition may be administered for recurrence, even after treatment according to the course of the disease treatment.
  • composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions with respect to the treatment of liver cancer, or a compound which maintains / increases the solubility and / or absorbency of the active ingredient. Also optionally, it may further comprise a chemotherapeutic agent, an anti-inflammatory agent, an antiviral agent and / or an immunomodulator.
  • compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.
  • Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7 liver cancer cell line were obtained from Korean Cell Line Bank (Seoul, Korea). All cell lines were cultured in EMEM (American Type) supplemented with 10% fetal bovine serum (FBS, Lonza) and 100 units / mL penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37 ° C and 5% CO 2 in a humidified incubator Culture Collection, Manassas, Va.), RPMI-1640 or DMEM medium (Lonza, Walkersville, Md.).
  • EMEM American Type
  • FBS fetal bovine serum
  • penicillin-streptomycin Invitrogen, Carlsbad, CA
  • siRNA and dsRNA used in this experiment were synthesized by Lemonex (Seoul, Korea) and the gene ORF sequence (BANF1: NM_003860, PLOD3: NM_001084, SF3B4: NM_005850) in the pcDNA3.1 + / C- (K) Human BANF1, PLOD3, and SF3B4 expression plasmids were purchased from Genscript TM (Piscataway, NJ, USA). Transfection was performed using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's manual.
  • the cell line was supplied to a 12-well plate in 30% confluence. After transfusion or inhibition treatment, the cells were incubated with 0.5 mg / mL of (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) at 37 ° C for 1 hour every 24 hours .
  • the formazan crystal was dissolved in DMSO and the absorbance at 570 nm was read using a VICTOR3 TM multilabel plate reader (PerkinElmer, Boston, Mass.).
  • cell proliferation assay cell lines were fed to 24 well plates in 30% confluence. After transfection, cells were treated with 5-bromo-2'-deoxyuridine (BrdU) reagent for 2 h and fixed at room temperature for 30 min. Cells were incubated with anti-BrdU antibody for 1 hour at room temperature. Unbound antibody was removed by washing buffer. Horseradish peroxidase-conjugated secondary antibody was added to each well. The substrate solution was added and the reaction was stopped after 30 minutes in the stop solution. The final product was quantified at 490 nm by a VICTOR3 TM multilabel plate reader (PerkinElmer).
  • RhdU 5-bromo-2'-deoxyuridine
  • Transwell plates and cell culture inserts were used for in vitro cell motility and invasion analysis.
  • (BD Biosciences) was diluted to 0.3 mg / ml with coating buffer (0.01 M Tris, 0.7% NaCl, pH 8.0) for coating of the invasion assays and 100 ⁇ l of Macri Gel was applied to the upper compartment of the cell culture insert Lt; / RTI > After incubation at 37 ° C for 1 hour, the cell culture insert was ready for seeding.
  • the cells 5% FBS a serum present in chemoattractant- were properly seeded into the cell culture insert of the pre-culture medium (0.5 ⁇ 10 5 cells / well for the motility assay, 1 ⁇ 10 5 cells / well for the invasion assay).
  • the migrated or invaded cells were stained with a Diff-Quik staining kit (Sysmex, Japan). Cells were photographed at 200x magnification on an Axiovert 200 inverted microscope (Zeiss, Jena, Germany). Cells were enumerated in three randomized clocks.
  • Transfected cells were fed into wells of 6 well plates. At 100% confluence, a scratch was made on the same layer using a micropipette tip. Photographs of the same area of the wound were taken at 0 h and 24 h using an IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).
  • xenograft tumorigenicity evaluation 1 x 10 7 transduced cells were mixed in 0.2 ml PBS (pH 7.4) and 30% (v / v) Matrigel Matrix (BD Biosciences). Cell suspensions were subcutaneously injected into Balb / c-nude mice at 6 weeks of age. Rats were examined twice a week for confirmation of tumor formation at the injection site. Tumor volume was calculated as 0.5 x length (L) x width 2 (W 2 ). Each experimental group consisted of 10 rats, and tumor growth was quantified by caliper measurements in three orthogonal directions. Results were expressed as mean tumor volume and 95% confidence interval. The H-ras12V activated homotypic transgenic mice were provided by Dr.
  • Example 12- (1) -2) The porous silica nanoparticles of Example 12- (1) -2) -2, 80 ⁇ l of InViVojection TM RNAi-nano reagent, which is specific for BANF1, PLOD3 and SF3B4, No. DHMSN-vivo RNA; Lemonex Inc., Seoul, Korea) and prepared in 200 [mu] l PBS.
  • a mixture of siRNA or dsRNA and nanoparticles was injected into the H-ras transgenic HCC mouse model by weekly intravenous injection from week 9 to week 23. Ultrasonography was taken at 17, 19, and 21 weeks with an ultrasound machine (Affiniti 50, Philips, Seoul, Korea).
  • reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
  • TMB trimethyl benzene
  • the reaction was carried out by starting at 25 ° C and heating at a rate of 10 ° C / min and then slowly cooling down at a rate of 1 to 10 ° C / min in an autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
  • the porous silica particles prepared in 2) were placed in a glass vial and heated at 550 ° C for 5 hours. After completion of the reaction, the particles were gradually cooled to room temperature to prepare particles.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11, 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 item (1) of Example 11, except that 5-fold larger vessels were used and all the materials were used in a 5-fold capacity.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 920 ml of distilled water and 850 ml of methanol were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 800 ml of distilled water, 1010 ml of methanol and 10.6 g of CTAB were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in Example 11- (1), except that 620 ml of distilled water, 1380 ml of methanol and 7.88 g of CTAB were used in the preparation of the small pore particles.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 2.5 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 4.5 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 11 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 12.5 mL of TMB was used at the time of pore expansion.
  • Example 11- (1) -2 The small pore particles were reacted with TMB in the same manner as in Example 11- (1) -2), cooled, and centrifuged to remove the supernatant. Thereafter, the mixture was centrifuged under the same conditions as in Example 11- (1) -2), washed three times with ethanol and distilled water alternately and then dried under the same conditions as in Example 11- (1) -2) Silica particles (pore diameter 10 to 15 nm, particle diameter 200 nm) were obtained.
  • the reaction solution in the previous step remains in the pores, and the inside of the pores are 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 DEG C, and washed five times with ethanol and distilled water alternately.
  • Example 11- (4) The porous silica particles of Example 11- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • porous silica particles were dispersed in 10 mL of toluene in a 100 mL round bottom flask with a bath sonicator. Then, 1 mL of APTES was added, and the mixture was stirred at 400 rpm and stirred at 130 DEG C for 12 hours.
  • the mixture was slowly cooled to room temperature, centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed 5 times with ethanol and distilled water.
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with (3-aminopropyl) triethoxysilane (APTES) to positively charge, except that 0.4 ml of APTES was added and the reaction time was changed to 3 hours Was modified in the same manner as in Example 12- (1) -1).
  • APTES (3-aminopropyl) triethoxysilane
  • Example 11- (9) The porous silica particles of Example 11- (9) were reacted with (3-Aminopropyl) triethoxysilane (APTES) and positively charged.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 11- (10) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with trimethoxy (propyl) silane to introduce a propyl group into the surface and pores, except that 0.35 ml of trimethoxy (propyl) silane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with trimethoxy-n-octylsilane to introduce a propyl group into the surface and pores, except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with succinic anhydride to negatively charge.
  • DMSO dimethyl sulfoxide
  • 80 mg of succinic anhydride was added instead of APTES
  • the reaction was carried out in the same manner as in Example 12- (1) -1) except that DMSO was used instead of distilled water at the time of washing.
  • Example 12- (3) -2 100 mg of the porous silica nanoparticles of Example 12- (3) -2) was dispersed in 1 mL of a 1 M aqueous sulfuric acid solution and 20 mL of 30% aqueous hydrogen peroxide and stirred at room temperature to induce an oxidation reaction to oxidize the thiol group with a sulfonic acid group. Thereafter, it was washed and dried in the same manner as in Example 12- (1) -1).
  • GFP green fluorescent protein
  • Example 2 Analysis of inhibitory rate of indicator gene expression of siRNA or dsRNA of the present invention
  • SiRNA, dsRNA validation test for inhibition of human BANF1, transcript variant 1, mRNA (Gene Bank number: NM_003860.3) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 5 87.73 11 73.18 17 84.11 23 76.24 6 79.64 12 85.44 18 88.36 24 87.7 7 82.3 13 69.57 19 87.83 25 62.57 8 76.21 14 77.3 20 67.72 26 72.92 9 89.6 15 82.92 21 82.29 27 65.58 10 83.42 16 91.38 22 63.23 28 72.91
  • SiRNA, dsRNA validity test for inhibition of human BANF1, transcript variant 2, mRNA (Gene Bank number: NM_001143985.1) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 29 92.55 37 81.64 45 83.22 53 84.31 30 91.49 38 68.4 46 78.16 54 64.9 31 86.44 39 79.72 47 73.48 55 74.72 32 77.1 40 91.6 48 68.3 33 73.82 41 87.37 49 85.27 34 76.6 42 53.77 50 88.74 35 88.33 43 86.39 51 92.32 36 82.53 44 68.63 52 74.8
  • SiRNA, dsRNA validity test for expression suppression of human human PLOD3 gene sequence (Gene Bank number: NM_001084.4) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 56 87.62 73 76.8 90 72.7 107 74.5 57 78.13 74 68.27 91 83.69 108 86.25 58 92.72 75 77.44 92 85.3 109 83.7 59 83.49 76 86.26 93 76.62 110 74.13 60 86.8 77 84.3 94 82.11 111 76.29 61 64.29 78 81.52 95 83.46 112 73.52 62 73.33 79 79.35 96 71.25 113 82.86 63 85.83 80 76.63 97 72.73 114 73.52
  • SiRNA for the inhibition of the expression of the human SF3B4 gene sequence (human SF3B4 gene sequence (Gene Bank number: NM_005850.4), dsRNA validity test Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 121 83.71 131 74.32 141 78.33 151 73.12 122 81.83 132 92.19 142 72.45 152 78.66 123 87.62 133 84.72 143 76.72 153 82.5 124 86.39 134 81.3 144 81.36 154 76.63 125 78.64 135 83.4 145 83.2 155 62.95 126 82.7 136 88.63 146 72.41 156 89.6 127 84.25 137 78.25 147 73.64 157 77.2 128 74.11 138 85.1 148
  • SiRNAs comprising the sense RNA having the sequence shown in Table 11 below and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell lines of Example 1 were subjected to the methods of Examples 1-2 or 1-8, respectively After in vitro transfection, the expression levels of the corresponding indicator factors of each siRNA were measured by Western blotting, and the results are shown in Fig.
  • SEQ ID NO: Sense RNA sequence The naming in Figure 1 Target gene SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUU-3 ' Banf1 Mouse BANF1 gene SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3 Mouse PLOD3 gene SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4 Mouse SF3B4 gene SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3 ' Control - SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1 Human BANF1 gene SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3 Human PLOD3 gene SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4 Human SF3B4
  • SiRNAs comprising the sense RNA having the sequence shown in Table 12 below and the antisense RNA consisting of the complementary sequence of the SNU-449 cell line of Example 1-1 were in vitro transfected according to the method of Example 1-2, The migration and invasion responses of the corresponding surface factors of the siRNAs were analyzed according to the methods of Examples 1-5, and the scratch wound healing ability was analyzed according to the method of Examples 1-6, and the results are shown in FIG.
  • SEQ ID NO: Sense RNA sequence The naming in Figure 2 Target gene SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3 ' Control - SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1 Human BANF1 gene SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3 Human PLOD3 gene SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4 Human SF3B4 gene
  • N-cadherin, Fibronectin, Snail, and Slug which are representative epithelial-mesenchymal transition (EMT) regulatory proteins related to the metastasis of liver cancer cells, can be inhibited by inhibiting the expression of each marker by the siRNA or dsRNA of the present invention.
  • SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were in vitro transfected in the SNU-449 cell line of Example 1 according to the method of Example 1-2 Then, the amount of expression of the corresponding indicator factors and the expression level of the EMT regulatory proteins of each siRNA were analyzed according to the method of Examples 1-9, and the results are shown in FIG. 3 (A).
  • SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were transfected in vitro in the SNU-449 cell line of Example 1 according to the method of Example 1-2, and transfected cells After injection into athymic nude mice, the size of the liver tumor and the survival rate of the mice were analyzed and the results are shown in FIG. 3 (B).
  • knockdown of the marker factors via the siRNA or dsRNA of the present invention can increase the overall tumor growth rate And the average tumor volume is decreased.
  • the tumor-free survival rate is significantly higher than that of the control group. More specifically, when 50 days have elapsed after subcutaneous transfection of the transfected cells, In the control group, tumors were found in 6 rats of 10 rats, but in the experimental group, tumors were found in 1 to 2 rats among 10 rats, indicating that the siRNA or dsRNA of the present invention can effectively inhibit the growth of liver tumors Able to know.
  • SiRNAs consisting of sense RNA having the sequence of Table 13 below and antisense RNA consisting of the complementary sequence were transfected in vivo according to the method of Examples 1-8 and the number of tumors 4 (A).
  • the level of inhibition of the expression of the respective indicator genes of the siRNAs carried on the porous nanoparticles was analyzed by the method of Example 1-9 and shown in FIG. 4 (B).
  • SEQ ID NO: Sense RNA sequence The naming in Figure 1 Target gene SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUU-3 ' Banf1 Mouse BANF1 gene SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3 Mouse PLOD3 gene SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4 Mouse SF3B4 gene
  • FIG. 5 is a photograph of the porous silica particles of 11- (1)
  • FIG. 6 is a photograph of the porous silica particles of 11- (2)
  • 7 is a photograph of small pore particles of 11- (1)
  • FIG. 8 is a comparative photograph of small pore particles of 11- (1) and 11- (3) have.
  • porous silica particles were almost completely decomposed after 360 hours of biodegradation.
  • 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,
  • a t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
  • porous silica particle powder 5 mg was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in the permeable membrane having the pore diameter of 50 kDa shown in Fig. 15 ml of SBF was added to the outer membrane, and SBF of the outer membrane was replaced every 12 hours.
  • the decomposition of the porous silica particles was carried out at 37 ° C with 60 rpm of horizontal stirring.
  • porous silica particles of the examples have significantly larger t than the control.
  • t which is the ratio of the absorbance of the positively charged particles to 1/2, was 24 or more.
  • the release solvent Prior to 24 hours, the release solvent was withdrawn at 0.5, 1, 2, 4, 8, 12, 24 hours, then every 24 hours thereafter, 0.5 ml of the release solvent was recovered for fluorescence measurement, SBF was added.
  • the time at which 50% of the siRNA was released was about 40 hours or more.
  • Balb / c nude male (5 weeks old) was purchased from Orient Bio Inc., and 3 million HeLa cells (cervical cancer cells) were dispersed in sterilized 1x PBS.
  • Xenograft tumors were subcutaneously injected into mice to grow 70 mm when the solidification of the 3 tumor size is confirmed, PBS, FITC- porous silica particles (example 12- (1) -2) -2 of porous silica particles), porous FITC- loaded with siRNA of example 1-13 (Silica particles of Example 12- (1) -2) -2) were injected into each mouse tumor, and the fluorescence intensity and distribution were measured immediately before, immediately after, and after 48 hours by FOBI fluorescence in vivo imaging system (Neo science, Korea).
  • the FITC label was prepared by dispersing 50 mg of silica particles in 1 mL of DMSO and adding 25 ⁇ g of FITC-NHS solution (2.5 mg / mL) (10 ⁇ l) The reaction mixture was reacted for 18 hours, and the reaction product was purified by centrifugation (8500 rpm, 10 minutes). The supernatant was discarded and the precipitated particles were collected and dispersed evenly in ethanol. This was repeated 3-4 times with ethanol- distilled water, And purified until no color was observed.
  • the control was administered alone in PBS, the siRNA of Example 1-13 alone, the siRNA alone of Example 1-13, the FITC-DDV of FITC alone, the complex
  • the siRNA transferred into the body to be loaded into the body has a longer period of maintaining the activity, After staying longer, we can confirm that the fluorescence is strong even after 48 hours.

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Abstract

La présente invention concerne un ARNsi ou un ARNdb, qui peut inhiber efficacement l'expression de trois marqueurs hautement exprimés dans le cancer du foie, et une composition pharmaceutique le comprenant pouvant assurer un excellent effet de prévention ou de traitement du cancer du foie par l'intermédiaire de l'ARNi.
PCT/KR2018/008611 2017-07-28 2018-07-30 Traitements pharmaceutiques pour prévenir ou traiter le cancer du foie WO2019022586A2 (fr)

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CN202310211626.6A CN116898870A (zh) 2017-07-28 2018-07-30 用于预防或治疗肝癌的药物组合物
CN201880062438.9A CN111132682B (zh) 2017-07-28 2018-07-30 用于预防或治疗肝癌的药物组合物
AU2018306411A AU2018306411B2 (en) 2017-07-28 2018-07-30 Pharmaceutical composition for preventing or treating liver cancer
JP2020504312A JP6967810B2 (ja) 2017-07-28 2018-07-30 肝癌の予防または治療用の薬学的組成物
EP18838318.6A EP3689355A4 (fr) 2017-07-28 2018-07-30 Traitements pharmaceutiques pour prévenir ou traiter le cancer du foie
CN202310211629.XA CN116236501A (zh) 2017-07-28 2018-07-30 用于预防或治疗肝癌的药物组合物
US16/634,675 US20210093654A1 (en) 2017-07-28 2018-07-30 Pharmaceutical composition for preventing or treating liver cancer
US17/536,880 US20220072027A1 (en) 2017-07-28 2021-11-29 Pharmaceutical composition for preventing or treating liver cancer
AU2022201755A AU2022201755A1 (en) 2017-07-28 2022-03-14 Pharmaceutical composition for preventing or treating liver cancer
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JP2022520467A (ja) * 2019-02-22 2022-03-30 レモネックス インコーポレイテッド 免疫活性もしくは癌の予防または治療用の医薬組成物
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WO2024065649A1 (fr) * 2022-09-30 2024-04-04 谛邈生物科技（新加坡）有限公司 Procédé de chargement efficace d'adn dans un exosome

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