WO2016206626A1 - 一种siRNA、含有该siRNA的药物组合物和缀合物及它们的应用 - Google Patents

一种siRNA、含有该siRNA的药物组合物和缀合物及它们的应用 Download PDF

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WO2016206626A1
WO2016206626A1 PCT/CN2016/087034 CN2016087034W WO2016206626A1 WO 2016206626 A1 WO2016206626 A1 WO 2016206626A1 CN 2016087034 W CN2016087034 W CN 2016087034W WO 2016206626 A1 WO2016206626 A1 WO 2016206626A1
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sirna
group
pharmaceutically acceptable
amine
pharmaceutical composition
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PCT/CN2016/087034
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English (en)
French (fr)
Chinese (zh)
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张鸿雁
高山
黄渊余
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苏州瑞博生物技术有限公司
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Priority to CN201680036513.5A priority Critical patent/CN107849567A/zh
Priority to US15/738,006 priority patent/US20190062749A1/en
Priority to EP16813742.0A priority patent/EP3315608B1/en
Priority to JP2018518771A priority patent/JP6715325B2/ja
Publication of WO2016206626A1 publication Critical patent/WO2016206626A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to an siRNA, a pharmaceutical composition and a conjugate comprising the same, and uses thereof. Specifically, the present invention relates to an siRNA for inhibiting expression of a hepatitis B virus (HBV) gene, a pharmaceutical composition and a conjugate comprising the siRNA as an active ingredient, and the siRNA, a pharmaceutical composition And use of the conjugate in the manufacture of a medicament for the prevention and/or treatment of hepatitis B.
  • HBV hepatitis B virus
  • Hepatitis B (also known as hepatitis B or hepatitis B) is a serious type of infectious disease that threatens the world, especially China.
  • hepatitis B prevention and treatment drugs that are recognized globally as interferon and nucleoside analogues. Disadvantages. Therefore, if the generation and replication of HBV can be inhibited from the gene level, thereby fundamentally reducing viral metabolism and infection of liver cells, it will undoubtedly be the most ideal treatment for hepatitis B.
  • small interfering RNA can inhibit or block any gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi). For example, the expression of a gene that causes a disease such as cancer, thereby achieving the purpose of treating a disease.
  • RNAi RNA interference
  • siRNA itself is less stable and is easily degraded by nucleases in the body (in particular, after systemic administration in vivo, siRNA first enters the blood circulation system in the body, and the blood is rich in endogenous Nuclease).
  • One way to overcome this obstacle is to chemically modify the siRNA to improve the stability of the siRNA in the blood.
  • modification sites is still an empirical process that must be verified by repeated experiments. Therefore, in order to obtain sufficient stability, a large number (even may be referred to as blind, redundant) chemical modification has to be introduced into the siRNA.
  • the stability of the siRNA may be improved to some extent by modification, the introduction of excessive modification or inappropriate modification will inevitably bring about an increase in cost, potential cytotoxicity of the modified siRNA, and modification.
  • the biological activity and specificity of the post-siRNA are affected.
  • siRNA which is stable in blood, has good biological activity, and has low cytotoxicity and inhibits expression of HBV gene has become an urgent problem to be solved.
  • siRNA CA1 which specifically inhibits the HBV gene
  • sense strand is 5'-CCUUGAGGCAUACUUCAAA-dTdT-3' (SEQ ID NO: 1); the antisense strand is 5'-UUUGAAGUAUGCCUCAAGG
  • SEQ ID NO: 2 Various chemical modification strategies for -dTdT-3' (SEQ ID NO: 2)) were investigated. The study found that different modification strategies have very different effects on siRNA stability, biological activity and cytotoxicity, for example, up to 20 chemically modified siRNA CA2 compared to unmodified naked siRNA CA1. Not only showed poor cell-level activity, high cytotoxicity, but also failed to reach a steady state in the blood.
  • siRNA A4 maintained blood stability while maintaining siRNA CA1. Basically equivalent inhibitory activity. Nevertheless, from the aspects of synthesis cost and synthesis efficiency, process convenience of synthesis process, siRNA stability, siRNA activity, etc., it is still necessary to further simplify and optimize the modification site of siRNA CA1 in order to obtain better siRNA. . Considering that previous studies in the field have shown that different chemical modifications to siRNA are likely to "make the whole body", that is, taking a different modification strategy at only one site will fundamentally To improve the stability of siRNA, it is not easy to accomplish the above-mentioned streamlining and optimization.
  • siRNAs designed to prevent hepatitis B it would be advantageous to be able to confer or enhance liver targeting or a conjugated molecule, which would greatly increase the efficiency of siRNA inhibition of HBV gene expression and reduce potential side effects.
  • the siRNA after the introduction of a liver-targeting vector or a conjugated molecule, the siRNA also needs to be able to function at the target site, ie, the encapsulation/conjugation of the vector or the conjugate molecule does not affect the activity of the siRNA itself (eg, does not affect siRNA)
  • the RNAi machine loaded into the cell, the RISC complex There is also an urgent need in the art for such pharmaceutically acceptable carriers or conjugated molecules capable of increasing the clinical application potential of siRNA, and siRNA pharmaceutical compositions or siRNA conjugates prepared using such vectors or conjugated molecules. demand.
  • the invention expands the selection space of the deprotection reagent after the synthesis is completed, shortens the reaction time, improves the synthesis efficiency, and maintains low cytotoxicity.
  • the reduced nucleotide-modified siRNA obtained by the novel modification of the present invention has better blood (plasma) stability in vitro than the siRNA A4 disclosed in the applicant's prior application CN102140461B.
  • the biological activity in animals is also better than siRNA A4, further enhancing its clinical application potential.
  • the inventors studied various pharmaceutically acceptable carriers for siRNA administration, the type and ratio of carrier molecules (including the ratio between the internal components of the carrier, and the carrier and the siRNA of the present invention). The ratio, physicochemical properties, drug loading efficiency, administration method, dosage, and drug treatment time were studied in detail.
  • the inventors have also studied various types of siRNA conjugates, including the selection of pharmaceutically acceptable conjugation molecules (selection of targeting molecules, linkers between targeting molecules and siRNAs of the invention). The selection), the development and establishment of synthetic routes, and the study of biological activity in vivo.
  • the inventors have discovered that the siRNAs of the invention can be combined with a variety of pharmaceutically acceptable carriers or with a variety of pharmaceutically acceptable conjugating molecules to achieve superior in vivo effects.
  • a lipid mixture formed of the three components containing an amine-containing compound, a helper lipid, and a pegylated lipid as a pharmaceutically acceptable carrier to form a pharmaceutical composition with the siRNA of the present invention.
  • the siRNA of the present invention is specifically delivered to the liver and exhibits superior HBV mRNA inhibition while greatly reducing the dose administered.
  • the inventors completed the present invention.
  • the invention provides a specific ability to target as a HBV gene
  • the siRNA of the X gene region of one of the four open reading frames, the target mRNA sequence of the siRNA is shown in SEQ ID NO: 3, wherein the sequence of the siRNA is as follows:
  • the capital letters C, G, U, A, and T represent the base composition of the nucleotide;
  • the lowercase letter d indicates that one nucleotide to the right of the letter d is a deoxyribonucleotide;
  • the lowercase letter m indicates the letter m.
  • the 2'-hydroxyl group of the ribose group of one nucleotide on the left is substituted with a methoxy group;
  • the lowercase letter f indicates that the 2'-hydroxyl group of the ribose group of one nucleotide to the left of the letter f is substituted with fluorine.
  • Cm consisting of the first capital letter C at the 5' end of the sense strand and m on the right side thereof indicates a nucleoside in which the base is a cytosine, and the 2'-hydroxy group of the ribose group is substituted with a methoxy group.
  • An acid; a "Uf” consisting of the first uppercase letter U at the 5' end of the antisense strand and the f on the right side thereof indicates a nucleotide in which the base of the uracil or ribose group is substituted with fluorine. .
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA of the first aspect as an active ingredient and a pharmaceutically acceptable carrier.
  • the invention provides an siRNA conjugate obtained by conjugating an siRNA as described in the first aspect to a pharmaceutically acceptable conjugated molecule.
  • the invention provides a kit comprising the siRNA of the first aspect and/or the pharmaceutical composition of the second aspect and/or the siRNA of the third aspect Conjugate.
  • the present invention provides the siRNA according to the first aspect and/or the pharmaceutical composition according to the second aspect and/or the siRNA conjugate according to the third aspect, in preparation for prevention and / or the application of drugs for the treatment of hepatitis B.
  • the invention provides a method of treating hepatitis B, the method comprising the siRNA of the first aspect and/or the pharmaceutical composition of the second aspect and/or the third aspect
  • the siRNA conjugate is administered to a patient in need thereof.
  • the present invention provides a method of inhibiting expression of a HBV gene in a hepatitis cell infected with chronic HBV, the method comprising the siRNA according to the first aspect and/or the pharmaceutical composition according to the second aspect And/or the siRNA conjugate as described in the third aspect is introduced into the hepatitis cell infected with chronic HBV.
  • the siRNA of the present invention further reduces the number of modified nucleotides, but still achieves similar or even superior pharmacodynamics (the HBV mRNA expression inhibitory activity in cell experiments is similar; in animal experiments) HBV mRNA expression inhibitory activity, hepatitis B surface antigen HBsAg inhibition is stronger); more importantly, after reducing the number of modified nucleotides, it is also expected to improve blood stability (full length siRNA fragment in vitro) It is able to maintain higher stability in plasma for a long time); at the same time, due to the reduction of the amount of nucleotide modification, the siRNA of the invention has lower synthesis cost, wider technical solution and higher production efficiency than siRNA A4. This provides a more clinically useful siRNA.
  • the pharmaceutical composition and siRNA conjugate formed by the siRNA of the present invention exhibit excellent HBV mRNA inhibition efficiency in hepatitis B model mice, and can effectively reduce surface antigen expression, showing good treatment for chronic hepatitis B. effect.
  • a lipid combination formed by using the three components of an amine-containing compound, a helper lipid, and a PEGylated lipid as a pharmaceutically acceptable carrier forms a pharmaceutical combination with the siRNA of the present invention.
  • the siRNA of the present invention can be specifically delivered to the liver, and exhibits superior HBV mRNA inhibition while greatly reducing the dose administered (in hepatitis B model mice, the dose is only given by other conventional In the case of 1/5 of the pharmaceutical composition formed by the carrier, the HBV mRNA inhibition efficiency of the pharmaceutical composition is nearly twice as high as that of the conventionally formed carrier, and can even reach 10 times or more.
  • Figure 1a - Figure 1c show: Figure 1a, (GalNAc) 3 -X2M2 double-stranded Ultra Performance Liquid Chromatography (UPLC) spectrum results (after melting); Figure 1b, (GalNAc) 3 MS mass spectrometry results for the -X2M2 sense strand; and MS mass spectrometry results for the (GalNAc) 3 -X2M2 antisense strand of Figure 1c.
  • Figure 1a Figure 1a - Figure 1c show: Figure 1a, (GalNAc) 3 -X2M2 double-stranded Ultra Performance Liquid Chromatography (UPLC) spectrum results (after melting); Figure 1b, (GalNAc) 3 MS mass spectrometry results for the -X2M2 sense strand; and MS mass spectrometry results for the (GalNAc) 3 -X2M2 antisense strand of Figure 1c.
  • UPLC Ultra Performance Liquid Chromatography
  • Figure 2 shows the results of detection of HBV mRNA expression inhibitory activity in HepG2.2.15 cells by the test siRNAs listed in Table 1 and control siRNAs.
  • Figure 3 shows the results of the stability test of the test siRNA and the control siRNA listed in Table 1 in human plasma in vitro.
  • Figure 4 shows the results of detection of HBV mRNA expression inhibitory activity in C57BL/6J-Tg (AlblHBV) 44 Bri/J transgenic mice, in which RBP131/siRNA liposome preparations containing the test siRNAs listed in Table 1 were loaded.
  • Fig. 5 shows the results of detecting the inhibitory activity of HBsAg expression in the serum of M-TgHBV transgenic mice at different time points in each test group.
  • Fig. 6 shows the results of detection of HBsAg expression inhibitory activity in the liver of pHBV mice (based on high-pressure injection of HBV-transfected mice) on the 7th and 28th day of each test group.
  • Figure 7 shows the results of detection of HBV mRNA expression inhibitory activity in liver of pHBV mice (HBV-transfected mice based on high pressure injection) of each test group.
  • Fig. 8 shows the results of detection of HBsAg expression inhibitory activity in serum of each group of test samples against pHBV mice (based on high-pressure injection of HBV-transfected mice).
  • Figure 9 shows the results of detection of HBsAg expression inhibitory activity in serum of pHBV mice (based on high-pressure injection of HBV-transfected mice) at various time points of each test group.
  • siRNA described in the first aspect of the invention may also be referred to as X2M2.
  • siRNA of the present invention is a siRNA of the present invention.
  • the siRNA of the present invention contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group, and a base.
  • the siRNA of the present invention contains a modified nucleotide group that does not cause a significant weakening or loss of the function of the siRNA to inhibit HBV gene expression.
  • backbone modifications such as phosphate group modification
  • ribose group modifications and base modifications
  • Watts, JK, GF Deleavey, and MJDamha Chemically modified siRNA: Tools and applications. Drug Discov Today, 2008. 13 (19-20): p. 842-55.
  • the modified nucleotide group is a nucleotide group in which a ribose group and an optional phosphate group are modified, but is not limited thereto.
  • the phosphate group modification refers to a phosphorothioate modification, that is, replacing a non-bridged oxygen atom in a phosphodiester bond with a sulfur atom, thereby using a phosphorothioate
  • the diester bond replaces the phosphodiester bond. This modification stabilizes the structure of the siRNA, maintaining high specificity and high affinity for base pairing.
  • ribose group modification means that the 2'-hydroxyl group of the ribose group is substituted with methoxy or fluoro.
  • the introduction of, for example, a methoxy group or a fluorine at the 2'-hydroxyl position of the ribose group makes it difficult for the ribonuclease in the blood to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. performance.
  • the 3' end of the sense strand and the antisense strand are linked to an overhang dTdT.
  • the phosphodiester bond between the 3'-hanging end dTdT of the sense strand and/or the antisense strand is subjected to a phosphorothioate modification.
  • an siRNA in which a phosphodiester bond between a sense strand and an antisense strand 3' hanging end dTdT is phosphorothioate-modified is referred to as X2M2 (S++); the sense strand and the antisense strand can be 3'
  • the siRNA that does not undergo phosphorothioate modification between the dtdT at the end dTdT is called X2M2(S--); only the sense chain 3' can be suspended.
  • the phospho-modified siRNA of the phosphodiester bond between the dTdT is called X2M2(S+-); and the phosphodiester bond between the dSdT of the antisense strand only 3' can be thiophosphoric acid
  • the modified siRNA is referred to as X2M2 (S-+).
  • siRNA X2M2 it is intended to cover X2M2 (S++), X2M2 (S--), X2M2 (S+-), and X2M2 (S-+) listed herein.
  • siRNAs of the present invention can be obtained by conventional siRNA preparation methods (e.g., solid phase synthesis and liquid phase synthesis), such as X2M2 (S++), X2M2 (S--), X2M2. (S+-) and X2M2 (S-+).
  • solid phase synthesis has commercialized customized services, and Suzhou Ruibo Biotechnology Co., Ltd. also has such solid phase synthesis capabilities.
  • a method of preparing a nucleotide monomer having a corresponding modification and a modified nucleotide group by introducing a modified nucleotide group having a corresponding modification into a siRNA of the present invention Methods of introducing siRNA into a group are also well known to those skilled in the art.
  • the siRNA of the present invention and a pharmaceutically acceptable carrier are contained.
  • the pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as Fe 3 O 4 , Fe 2 O 3 ), carbon nanotubes, Mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-lysine (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly-D Or L-type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA) and poly (poly(2-dimethylaminoethyl me
  • the pharmaceutical composition of the present invention may further comprise other excipients which are pharmaceutically acceptable, and the excipients may be one or more of various preparations or compounds conventionally employed in the art.
  • the pharmaceutically acceptable other excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.
  • the pH buffer may be a trishydroxymethylaminomethane hydrochloride buffer having a pH of 7.5-8.5 and/or a phosphate buffer having a pH of 5.5-8.5, preferably a phosphate having a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.
  • the protective agent may be included in an amount of from 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.
  • the osmotic pressure adjusting agent may be sodium chloride and/or potassium chloride.
  • the osmotic pressure adjusting agent is present in an amount such that the osmotic pressure of the pharmaceutical composition is from 200 to 700 milliosmoles per kilogram.
  • the content of the osmotic pressure adjusting agent can be easily determined by those skilled in the art depending on the desired osmotic pressure.
  • the pharmaceutical composition may be a liquid preparation, such as an injection solution, or may be a lyophilized powder injection, which is mixed with a liquid adjuvant when administered, and formulated into a liquid preparation.
  • the liquid preparation may be, but not limited to, for subcutaneous, intramuscular or intravenous administration, and may be, but is not limited to, It is administered by spray to the lungs or by spraying through the lungs to other organ tissues (such as the liver).
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an amine-containing compound), a helper lipid, and/or Pegylated lipids.
  • the amine-containing compound, the helper lipid, and the PEGylated lipid are each selected from the group consisting of the amine-containing transfection compound described in CN201180060664.1 (hereby incorporated by reference in its entirety)
  • One or more of an acceptable salt or derivative, helper lipid, and pegylated lipid One or more of an acceptable salt or derivative, helper lipid, and pegylated lipid.
  • the amine-containing compound may be a compound of formula I, or a pharmaceutically acceptable salt thereof, as described in CN201180060664.1:
  • X 1 and X 2 are each independently O, S, NA or CA, wherein A is hydrogen or a C 1 - C 20 hydrocarbon chain;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic radical a cyclic, acyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted or not Substituted, branched or straight-chain aryl, substituted or unsubstituted, branched or straight-chain heteroaryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • HCC represents a hydrocarbon chain
  • * represents a nitrogen atom in formula I.
  • the amine-containing compound may be an amine-containing compound 72 or an amine-containing compound 87 as described in CN201180060664.1, as follows:
  • the helper lipid may be cholesterol or an analog, derivative or the like thereof.
  • the PEGylated lipid may be 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000], ie 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000].
  • the pharmaceutically acceptable carrier used simultaneously comprises the three groups of the amine-containing compound, the helper lipid, and the PEGylated lipid described above. These three components form a lipid mixture.
  • the molar percentage of the amine-containing compound, the helper lipid, and the PEGylated lipid in the pharmaceutically acceptable carrier may be: 19.7%-80% of the amine-containing compound, the auxiliary lipid The quality is 19.7%-80%, and the PEGylated lipid is 0.3%-50%.
  • the molar percentage of the amine-containing compound, the helper lipid, and the PEGylated lipid in the pharmaceutically acceptable carrier may be: 50%-70% of the amine-containing compound, 20% of the helper lipid- 40%, PEGylated lipid 3%-20%.
  • a method of preparing a pharmaceutical composition in the form of a functional liposome preparation using the above-described amine-containing compound, helper lipid, PEGylated lipid, and siRNA can also be found in CN201180060664.1.
  • the molar percentage of the amine-containing compound, the auxiliary lipid, and the PEGylated lipid in the pharmaceutically acceptable carrier in the pharmaceutical composition may be: 19.7%-80% of the amine-containing compound, auxiliary The lipid is 19.7%-80%, and the PEGylated lipid is 0.3%-50%.
  • the molar percentage of the amine-containing compound, the auxiliary lipid, and the PEGylated lipid in the pharmaceutically acceptable carrier in the pharmaceutical composition is: 50%-70% of the amine-containing compound, the auxiliary lipid
  • the quality is 20%-40%, and the PEGylated lipid is 3%-20%.
  • the liposome particles formed from the siRNA of the present invention and the above lipid mixture have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more typically, the liposome particles have an average diameter of from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
  • the weight ratio (weight/weight ratio) of the siRNA of the invention to all lipids is in From about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1:6 to about 1:10
  • the weight ratio of the siRNA of the present invention to the total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1: 13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • the siRNA conjugates of the invention are obtained by conjugating an siRNA of the invention to a pharmaceutically acceptable conjugate molecule comprising a pharmaceutically acceptable targeting molecule and an optional linker.
  • the siRNA may be non-covalently conjugated to the conjugate molecule, but is preferably covalently conjugated to the conjugate molecule.
  • the conjugation site of the siRNA to the conjugate molecule can be at the 3' or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand.
  • the conjugate site of the siRNA and the conjugate molecule can be in addition to the 3' or 5' terminus The internal sequence of the siRNA.
  • the siRNA and the conjugated molecule can be linked by acid-labile or reducible chemical bonds, and in the acidic environment of the cell endosomes, these chemical bonds can be degraded, thereby making the siRNA free.
  • the conjugated molecule can be ligated to the sense strand of the siRNA to minimize the effect of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting molecule may be a targeting molecule conventionally used in the field of siRNA administration, such as but not limited to one or more of the following targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, Bile acids, vitamins (eg vitamin E), lipid molecules of different chain lengths; polymers such as polyethylene glycol; polypeptides such as transmembrane peptides; aptamers; antibodies; quantum dots; saccharides such as lactose, poly Lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; or receptor ligands expressed by hepatocytes, such as asialoglycoprotein, asialoglycohol residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin and so on.
  • lipophilic molecules such as cholesterol, Bile acids, vitamins (
  • the pharmaceutically acceptable targeting molecule may be selected from one or more of the following molecules: a asialoglycoprotein, such as asialoic acid Serum mucin (ASOR); a ligand for the asialoglycoprotein receptor (ASGPR), such as lactose, polylactose, mannose, galactose, and N-acetylgalactosamine.
  • ASOR asialoic acid Serum mucin
  • ASGPR ligand for the asialoglycoprotein receptor
  • the pharmaceutically acceptable targeting molecule may be galactose or N-acetylgalactosamine, wherein galactose or N-acetylgalactosamine
  • the molecules can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent terms described herein refer to the formation of siRNA conjugates by siRNA molecules and conjugated molecules containing galactose or N-acetylgalactosamine molecules as targeting molecules, respectively.
  • the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3, and 1:4. More preferably, the pharmaceutically acceptable targeting molecule is N-acetylgalactosamine. Even more preferably, when the siRNA of the present invention is conjugated to a conjugated molecule containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. Even more preferably, when the siRNA of the present invention is conjugated to a conjugated molecule containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
  • the conjugated molecule can be linked to the siRNA molecule via a suitable linker, and one skilled in the art can select a suitable linker depending on the particular type of target molecule.
  • a suitable linker can be of the structure: -(L A ) n L C -L B -, wherein
  • n is an integer from 1 to 3;
  • L A is a chain moiety containing an amide bond, and is bonded to the N-acetylgalactosamine molecule and the L C moiety via an ether bond, and its structure is as follows:
  • L B is a chain moiety containing an amide bond, and is linked to the L C moiety via an amide bond, and is linked to the siRNA moiety via a phosphate bond, and its structure is as follows:
  • L C is a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trishydroxymethylaminomethane, and one end of L C can pass an ether bond via an oxygen atom and 1-3 L A moieties. They are linked so as to be linked to 1-3 N-acetylgalactosamine molecules via the L A moiety; the other end of L C is linked to the L B moiety via an amide bond via a nitrogen atom.
  • L C is a tetramethylolaminomethane-based tetravalent linking group, by -(L A ) 3 trishydroxymethylaminomethane-L B as a linker -
  • the structure of the siRNA conjugate formed by linking the N-acetylgalactosamine molecule and the siRNA molecule is as follows:
  • the conjugation site of the siRNA to the conjugated molecule can be at the 3' or 5' end of the siRNA sense strand, also at the 5' end of the antisense strand, and also within the internal sequence of the siRNA.
  • the 3' terminus of the sense strand of the siRNA of the invention is covalently linked to three N-acetylgalactosamine (GalNAc) molecules via a linker-(L A ) 3 trishydroxymethylaminomethane-L B - Conjugation, an siRNA conjugate having a molar ratio of siRNA molecule to GalNAc molecule of 1: 3 is obtained, which may also be referred to as (GalNAc) 3 -X2M2 hereinafter, and its structure is as follows:
  • siRNA conjugates of the invention may also be combined with other pharmaceutically acceptable excipients, which may be one or more of the various formulations or compounds conventionally employed in the art, as described above. Description of the pharmaceutical composition of the invention.
  • one container can be used to provide siRNA, and at least another container can be used to provide a pharmaceutically acceptable carrier and/or adjuvant.
  • a pharmaceutically acceptable carrier and/or adjuvant In addition to the siRNA and the pharmaceutically acceptable carrier and/or adjuvant, other components such as stabilizers or preservatives and the like may be included in the kit.
  • the other ingredients may be included in the kit, but are present in a different container than the container providing the siRNA and the pharmaceutically acceptable carrier and/or adjuvant.
  • the kit can include instructions for mixing the siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients.
  • the siRNA conjugate can be stored in a container; with or without at least one other container, with or without pharmaceutically acceptable excipients.
  • other ingredients such as stabilizers or preservatives and the like may be included in the kit.
  • the other ingredients may be included in the kit, but are present in a different container than the container providing the siRNA conjugate and optionally the pharmaceutically acceptable excipient.
  • the kit can comprise instructions for mixing the siRNA conjugate with a pharmaceutically acceptable excipient (for excipients) or other ingredients.
  • the siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the siRNA conjugate and optionally the pharmaceutically acceptable excipient may be provided in any form, such as a liquid form, a dry form Or lyophilized form.
  • the siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the siRNA conjugate and optionally the pharmaceutically acceptable excipient are substantially pure and/or sterile.
  • Sterile water can optionally be provided in the kit of the invention.
  • the siRNA and/or pharmaceutical compositions and/or siRNA conjugates of the invention are useful for the prevention and/or treatment of hepatitis B, or for the preparation of a medicament for the prevention and/or treatment of hepatitis B.
  • administering/administering refers to a method or route by which a siRNA or pharmaceutical composition or siRNA conjugate is at least partially localized to a desired site to produce a desired effect, siRNA or pharmaceutical composition. Or the siRNA conjugate is placed into the subject.
  • Routes of administration suitable for the methods of the invention include topical administration and systemic administration. In general, topical administration results in delivery of more siRNA or pharmaceutical composition or siRNA conjugate to a particular site than the subject's entire body; whereas systemic administration results in the siRNA or pharmaceutical composition or siRNA.
  • the conjugate is delivered to the substantially entire body of the subject.
  • a mode of administration capable of delivering a drug to the liver is preferred.
  • Administration can be administered to a subject by any suitable route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal. Drug, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration).
  • oral or parenteral routes including intravenous, intramuscular, subcutaneous, transdermal.
  • Drug airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration).
  • the dose of the siRNA or pharmaceutical composition or siRNA conjugate of the present invention may be a dose conventional in the art, which may be determined according to various parameters, particularly the age, weight and sex of the subject. It can be determined Toxicity and therapeutic efficacy by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determination of LD 50 (50% of the population the dose lethal) and the ED 50 (the reaction finger in an amount to cause dose lethal to 50% of the maximum intensity of reaction, and in The qualitative reaction refers to the dose that causes 50% of the subjects to have a positive reaction).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD 50 /ED 50 .
  • siRNA or pharmaceutical compositions or siRNA conjugates that exhibit a high therapeutic index are preferred.
  • the range of human doses can be derived based on data obtained from cell culture assays and animal studies.
  • the drug combination When administering the pharmaceutical composition or siRNA conjugate of the present invention, for example, for a male or female, 6-12 week old, 18-25 g body weight C57BL/6J or C3H/HeNCrlVr mouse, the drug combination
  • the amount of siRNA in the siRNA or siRNA conjugate (i) a pharmaceutical composition for siRNA and a pharmaceutically acceptable carrier, which may be used in an amount of 0.001 to 50 mg/kg body weight, preferably 0.01 to 10 mg/kg.
  • the body weight is more preferably 0.05-5 mg/kg body weight, most preferably 0.1-3 mg/kg body weight; (ii) for the siRNA conjugate formed by the siRNA and the pharmaceutically acceptable conjugate molecule, the siRNA amount may be 0.001- 100 mg/kg body weight, preferably 0.01-50 mg/kg body weight, more preferably 0.05-20 mg/kg body weight, most preferably 0.1-10 mg/kg body weight.
  • the siRNA of the present invention is administered, the above amounts can be referred to.
  • the expression of HBV gene in hepatitis cells infected with chronic HBV can also be achieved by the mechanism of RNA interference.
  • the cell is a HepG2.2.15 cell.
  • the invention will be described in detail below by way of examples. Unless otherwise specified, the reagents and culture media used in the following examples are commercially available, and the nucleic acid electrophoresis, real-time PCR and the like used are carried out according to a conventional scheme. For example, it can carry out as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
  • HepG2.2.15 cells were purchased from ATCC, and cells were cultured in DMEM complete medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco) and 380 ⁇ g/ml G418 at 37 °C. Incubate in an incubator containing 5% CO 2 /95% air.
  • siRNA sequences in Table 1 below were screened by combining evaluation and analysis of various modification methods and modification sites.
  • NC-pos, NC-bas and NC are negative controls that have no inhibitory effect on HBV gene, which are also referred to as control siRNA hereinafter; the remaining siRNAs are X-based specifically targeting HBV.
  • the siRNA of the region is also referred to below as a test siRNA.
  • siRNAs listed in Table 1 were obtained by Suzhou Ruibo Biotechnology Co., Ltd. by conventional solid phase synthesis. An equimolar mixture of sense and antisense strands is solubilized with an annealing salt solution followed by conventional annealing to form an siRNA duplex.
  • the capital letters C, G, U, A and T indicate the base composition of the nucleotide; the lowercase d indicates that one nucleotide to the right of the letter d is a deoxyribonucleotide; the lowercase letter m indicates the letter m.
  • the 2'-hydroxyl group of the ribose group of one nucleotide on the left is substituted with a methoxy group;
  • the lowercase letter f indicates that the 2'-hydroxyl group of the ribose group of one nucleotide to the left of the letter f is substituted by fluorine;
  • lowercase The letter s indicates that the phosphodiester bond between the 3' hanging end dTdT is replaced by a phosphorothioate diester bond.
  • lipid compounds ie, amine-containing compound, helper lipid, PEGylated lipid
  • the synthesized siRNA listed in Table 1 was dissolved in a 200 mM sodium acetate (pH 5.2) solution to have a siRNA concentration of 0.2 mg/ml.
  • the obtained lipid ethanol solution and siRNA aqueous sodium acetate solution were mixed very quickly in a volume ratio of 1:3.
  • the lipids obtained after mixing are described in Table 2.
  • the liposome preparation obtained after mixing (i.e., the composition containing the amine compound, cholesterol, PEGylated lipid and siRNA) was incubated at about 50 ° C for 10 minutes. After incubation, use The phase-cut flow system, the hollow fiber column 100KDa ultrafiltration, the ultrafiltration exchange solution was pH 7.4 PBS. The preparation can be concentrated or diluted to the desired siRNA concentration while ultrafiltration. The ultrafiltered preparation was sterilized by filtration on a 0.22 ⁇ m filter.
  • RBP131 A lipid mixture composed of an amine-containing compound 87, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] is referred to as RBP131.
  • the obtained RBP131/siRNA liposome preparation was stored at 4 ° C before use, and the relevant physical and chemical properties were examined. The test results are shown in Table 3.
  • the liposome preparation RBP130/siRNA was prepared in the same manner as described above except that the amine-containing compound was changed to the compound 72 in CN201180060664.1.
  • the physicochemical parameters of the liposome preparation RBP130/siRNA were similar to those shown in Table 3.
  • Compound 30 was synthesized according to the preparation method described in WO2014025805A1, ie, containing the linker-(L A ) 3 trishydroxymethylaminomethane-L B - as described above and the N-acetylgalactosamine molecule as a targeting molecule ( Wherein, each L A may be linked to one N-acetylgalactosamine molecule, such that one linker may be linked to three N-acetylgalactosamine molecules) conjugating molecules, which may also be referred to herein as (GalNAc) 3
  • the molecular structure of the compound 30 is as follows:
  • nucleic acid solid phase synthesizer (ABI, USA, model: ABI394 synthesizer), automatic cycle completion of nucleic acid extension from 3' to 5' sequence, each cycle including deprotection, coupling, capping, oxidation four-step reaction .
  • All conventional nucleoside monomers, methoxy monomers, and fluoromonomers used for the synthesis of (GalNAc) 3 -X2M2 are formulated into a 0.1 M acetonitrile solution and deprotected and coupled using standard methods in solid phase synthesis. , caps, oxidizing reagents. The coupling time of the monomers was 12 min.
  • the conjugated molecule is ligated to the siRNA via a phosphodiester bond, and the resulting (GalNAc) 3 -X2M2 conjugate has a molar ratio of siRNA molecule to N-acetylgalactosamine molecule of 1:3.
  • the (GalNac) 3 conjugation molecule can be conjugated to the 3' end of the siRNA sense strand by covalent attachment by initiating a cycle of attachment of a (GalNAc) 3 conjugated molecule.
  • Purification and Desalting Purification of nucleic acids was accomplished by preparative ion chromatography purification column (Source 15Q) eluting with a gradient of NaBr. The purity-qualified products were collected and piped, and subjected to desalting using a reverse phase chromatography purification column.
  • Detection Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by LC-MS.
  • Annealing The S chain and the AS chain were mixed in an equimolar ratio, heated to 95 ° C, and cooled at room temperature to form a double-stranded structure by hydrogen bonding.
  • Test Example 1 This test example was used to test the cellular level toxicity and animal level toxicity of the siRNA X2M2 of the present invention.
  • the cytotoxicity of the siRNA X2M2 of the present invention was examined by MTT method on HepG2.2.15 cells and rat primary hepatocytes using conventional methods in the art, and no cytotoxicity was observed at a concentration of 500 nM (100 times the concentration of the drug).
  • Reaction in C57BL/6J mice, RB131/X2M2 was administered intravenously at a single dose of 10 mg/kg (33 times the onset dose) for 14 consecutive days. No animal death occurred and no adverse drug reactions were observed.
  • Clinical symptoms; in C57BL/6J mice, (GalNAc) 3 -X2M2 conjugate was administered subcutaneously at a dose of 10 mg/kg for 14 consecutive days without animal death and no clinically relevant adverse drug reactions were observed. Symptoms indicate that the siRNAX2M2 of the present invention and its pharmaceutical composition or conjugate have lower cytotoxicity and higher safety, and have clinical application possibilities.
  • Experimental Example 1 This experimental example was used to examine the invitro inhibition efficiency of the siRNA obtained in Preparation Example 1 for the expression level of HBV mRNA.
  • HepG2.2.15 cells were transfected in vitro with the siRNA obtained in Preparation Example 1, three in each group, and the experiment was repeated at least three times.
  • the final concentrations of siRNA were 50 nM, 10 nM, and 1 nM, respectively.
  • the expression level of HBV mRNA in the above harvested cells was determined by real-time fluorescent qPCR, specifically: using the RNeasy Mini Kit (QIAGEN, Cat. No. 74106) kit, The instructions were used to extract total RNA, and the extracted total RNA was reverse transcribed into cDNA, and then the inhibition efficiency of siRNA on HBV mRNA expression of HepG2.2.15 cells was detected by real-time PCR.
  • the GAPDH gene was used as an internal reference gene, and HBV and GAPDH were detected using primers for HBV and primers for GAPDH, respectively.
  • the primer sequences are shown in Table 5 below:
  • siRNA inhibitory activity is expressed by the remaining amount of HBV gene expression, and is calculated as follows:
  • Remaining amount of HBV gene expression (copy number of HBV gene in test group / copy number of GAPDH in test group) / (copy number of HBV gene in mock group / copy number of GAPDH in mock group) ⁇ 100%.
  • each test group is HepG2.2.15 cells treated with the siRNA listed in Table 1, respectively, the siRNA includes siRNA specifically targeting the X gene region of HBV and three control NC siRNA; the mock group is without any siRNA-treated HepG2.2.15 cells.
  • Figure 2 shows the results of detection of HBV mRNA expression inhibitory activity in HepG2.2.15 cells by the test siRNAs listed in Table 1 and control siRNAs.
  • the HBV mRNA inhibitory activity of each test siRNA was similar to that of the naked sequence siRNA CA1 except for siRNA 3d and siRNA CA2.
  • siRNA 3d is an siRNA which reduces the nucleotide modification at the 5' end of the 5' end of the siRNA A4 antisense strand
  • siRNA CA2 is a siRNA which performs nucleotide spacer modification on the naked siRNA CA1.
  • siRNA X2M2 of the present invention showed excellent activity at the cellular level.
  • Experimental Example 2 This experimental example was used to detect the siRNA obtained in Preparation Example 1 in vivo. Stability in the plasma of outsiders.
  • siRNA (20 ⁇ M, 10 ⁇ l) obtained in Preparation Example 1 was incubated with 50% human plasma (Human plasma, HP) at 37 ° C for a certain period of time, and then sampled, specifically, at 0, 2, 4, 6, respectively. 10 ⁇ l of the sample was taken out at 8, 24, and 48 hours, and immediately frozen in liquid nitrogen, followed by freezing at -80 ° C for use.
  • human plasma Human plasma, HP
  • Figure 3 shows the results of the stability test of the test siRNA and the control siRNA listed in Table 1 in human plasma in vitro, wherein Marker at both ends represents an equal amount of siRNA not treated with human plasma to indicate the treated siRNA. Size changes. By quantitatively reading the gray value, the ratio of the longest fragment remaining after incubation of these test siRNAs and control siRNAs to human plasma and the longest fragment of siRNA not treated with human plasma (ie, Marker shown in Figure 3) was calculated (ratio) Of the longest fragments, RL), the results are shown in Table 6.
  • siRNA X2M2 exhibits superior plasma stability than siRNA A4, in comparison with human After incubated for 48 hours, the RL was guaranteed to be above 90%. Considering this issue The siRNA X2M2 also reduced the modification of one nucleotide based on siRNA A4, which is very surprising.
  • siRNA A4 was able to maintain 81.2% RL after 48 h incubation with human plasma, it was observed that its bands were significantly down-shifted (other specificity)
  • siRNA targeting the X gene region of HBV that is, a certain degree of degradation still occurs (this may be because the base at its end is cleaved by RNase), while the siRNA X2M2 of the present invention is always maintained. Full length status.
  • Experimental Example 3 This experimental example was used to test the preparation of the RBP131/siRNA liposome preparation obtained in Example 2 in HBV transgenic mouse C57BL/6J-Tg(Alb1HBV)44Bri/J (purchased from Beijing Vitallihua experiment). Animal Technology Co., Ltd.) Inhibition efficiency of HBV mRNA expression.
  • test siRNA used included siRNA X2M2 (S++), A4, 3d, X2M2 (S--) listed in Table 1, and was negative using an irrelevant siRNA siFL867 specific for firefly Luciferase (Firefly Luciferase). Control, the sequence is as follows:
  • Antisense strand 5'-GCGAAGAAGGAGAAUAGGGdTdT-3' (SEQ ID NO: 11).
  • the tissues with lesions observed by the naked eye were preserved with 10% formalin for further pathological observation, and the liver was collected for RNA later. (Sigma Aldrich) preservation; homogenate the liver tissue with a tissue homogenizer, and extract total RNA using Trizol according to the standard procedure of total RNA extraction.
  • Expression levels using real-time PCR detection of HBV mRNA in liver tissue and in particular: the use ImProm-II TM reverse transcription kit (Promega Corporation) according to its specification the extracted total RNA as the cDNA reverse transcription, followed by quantitative PCR The kit (Beijing Kangwei Century Biotechnology Co., Ltd.) was used to detect the inhibition efficiency of siRNA on HBV mRNA expression in liver tissue.
  • HBV and ⁇ -actin were detected using a ⁇ -actin gene as an internal reference gene, and a primer against HBV and a primer against ⁇ -actin.
  • Table 7 shows the sequence of the primers
  • siRNA inhibitory activity is expressed by the remaining amount of HBV gene expression, and is calculated as follows:
  • Remaining amount of HBV gene expression (copy number of HBV gene in test group / copy number of ⁇ -actin in test group) / (copy number of HBV gene in control group / copy number of ⁇ -actin in control group) ⁇ 100%.
  • control group was a control group of mice administered with PBS in the experiment, and each test group was a group of mice administered with different pharmaceutical compositions.
  • the results are shown in Figure 4.
  • siRNA X2M2 is excellent in biological activity in disease model mice and is superior to the siRNAA4 previously filed by the applicant in CN102140461B.
  • RBP130/X2M2 (S++) and RBP130/X2M2 (S--) liposome preparations obtained in Preparation Example 2 were tested in the same manner, and these liposome preparations were shown to be associated with RBP131/X2M2 ( S++) Similar effects as RBP131/X2M2 (S--).
  • Experimental Example 4 This experimental example was used to examine the inhibitory effect of RBP131 on different doses of X2M2 on the expression of serum HBsAg in HBV transgenic mice at different time points.
  • the RBP131/siRNA liposome preparation was prepared in accordance with the method of Preparation Example 2.
  • Eighty HBV transgenic mice designated M-TgHBV, purchased from the Animal Department of Shanghai Public Health Center, and preparation methods of transgenic mice, such as Ren J., et al. J. Medical Virology. 2006, 78: 551-560
  • the rats were randomly divided into 8 groups according to the serum HBsAg content (10 in each group, including 4 males and 6 females), respectively, 1 ⁇ PBS control group, RBP131/NC control group (1 mg/kg), and IFN interferon control.
  • HBsAg expression remaining amount Calculated as follows:
  • the remaining amount of HBsAg expression (test group HBsAg content / PBS control group HBsAg content) ⁇ 100%.
  • the HBsAg content is expressed by how many nanograms of HBsAg is contained per gram of liver tissue protein.
  • the biological activity of the siRNA X2M2 of the present invention is superior to that of the siRNA A4, further enhancing its clinical application potential.
  • RBP130/X2M2 (S++) and RBP130/X2M2 (S--) liposome preparations obtained in Preparation Example 2 were tested in the same manner, and these liposome preparations were shown to be associated with RBP131/X2M2 ( S++) Similar effects as RBP131/X2M2 (S--).
  • Experimental Example 5 This experimental example was used to examine the inhibitory effect of RBP131 on different doses of X2M2 on the expression of HBsAg in liver in high-pressure injection-based HBV-transfected mice at different time points.
  • the RBP131/X2M2 complex was prepared in accordance with the method of Preparation Example 2. Seventy-two high-pressure injections of HBV plasmid in C3H/HeN mice (designated pHBV, purchased from the Animal Health Department of Shanghai Public Health Center, and high-pressure injection mice were prepared by Peng XH., et al. World J. Gastroenterol.
  • the PBS group and each RBP131/X2M2 group were administered once in a single vein, and the entecavir group was intragastrically administered daily for 7 days, that is, from day 0 to day 6, once daily. On the 7th and 28th day, the animals were killed in batches, and the expression level of HBsAg in liver tissue was detected. The detection method of HBsAg was the same as before.
  • RBP130/X2M2 (S++) and RBP130/X2M2 (S--) liposome preparations obtained in Preparation Example 2 were tested in the same manner, and these liposome preparations were shown to be associated with RBP131/X2M2 ( S++) Similar effects as RBP131/X2M2 (S--).
  • Experimental Example 6 This experimental example was used to detect commercially available siRNA in vivo carriers produced by different manufacturers, including Invivo fectamine 2.0 (purchased from Life technology), invivo jetPEI (purchased from PolyPlus-transfection (PT)), and Entranster (purchased). From Engreen Biosystem) and the RBP131 of the present invention, after carrying the siRNA X2M2, the HBV transgenic model mouse C57BL/6J-Tg(Alb1HBV)44Bri/J (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) The efficiency of inhibition of HBV mRNA expression.
  • the siRNAX2M2 (S++) of the present invention was packaged into the above commercial carrier according to standard operating procedures provided by the manufacturer to prepare corresponding vector/siRNA compositions, which were labeled as IVF2.0/X2M2, invivo jetPEI/X2M2, Entranster/X2M2, respectively. . Simultaneously.
  • the RBP131/X2M2 composition was prepared in accordance with the method of Preparation Example 2.
  • mice Thirty C57BL/6J-Tg(Alb1HBV)44Bri/J mice were randomly divided into 5 groups (6 in each group, male and female), respectively, which were PBS control group, IVF2.0/X2M2, invivo jetPEI/ X2M2, Entranster/X2M2, RBP131/X2M2 sample set. All animals were dosed according to body weight, single dose of tail vein, IVF2.0/X2M2, invivo jetPEI/X2M2, Entranster/X2M2 were administered at 2.5 mg/kg (siRNA), and RBP131/X2M2 group was given. The dose was 0.5 mg/kg (siRNA) and the dose of all 5 groups was 10 ml/kg.
  • PBS control group IVF2.0/X2M2, invivo jetPEI/ X2M2, Entranster/X2M2, RBP131/X2M2 sample set. All animals were dosed according to body weight, single dose of tail vein, IVF2.0/X2M2, in
  • siRNA inhibitory activity is calculated according to the following equation:
  • siRNA inhibitory activity [1 - (copy number of test group HBV gene / copy number of test group GAPDH) / (copy number of control group HBV gene / copy number of control group GAPDH)] ⁇ 100%.
  • control group was the control mice to which PBS was administered in the experiment, and each treatment group was a group of mice administered with different pharmaceutical compositions.
  • Table 8 shows the results of detection of HBV mRNA expression inhibitory activity in C57BL/6J-Tg (AlblHBV) 44 Bri/J transgenic mice of each test group.
  • This experimental example was used to detect siRNA in vivo administration of different literature sources and the RBP131 of the present invention carried siRNA carrying the apolipoprotein B (ApoB) gene (siApoB: sense strand (5' ⁇ 3') GUCAUCACACUGAAUACCAAUdTdT (SEQ ID NO: 16); antisense strand (5' ⁇ 3')AUUGGUAUUCAGUGUGAUGACACdTdT (SEQ ID NO: 17))
  • ApoB mRNA was expressed in normal C57BL/6J mice ( Or the inhibition rate of the expression level of blood lipids.
  • the main function of ApoB is to transport lipids from the liver into the blood circulatory system. When the expression of ApoB is inhibited, the lipids in the liver accumulate and the lipid levels in the blood decrease.
  • siRNA (containing the amount of siRNA required) was mixed with PCL-g-PDMAEMA nanoparticles (containing the desired amount of amino groups) and incubated for 15 minutes at room temperature to obtain a binary complex.
  • An additional amount of PGA-g-PEG (containing the desired amount of carboxyl groups) was added to the binary complex and incubation was continued for 15 minutes at room temperature to obtain a ternary complex.
  • PEA and PEAG complexes N/P ratio of polymer PEA, PEAG and siRNA The calculation is based on the number of moles of the amino group/mercapto group (N) on the polymer and the number of moles of the phosphate group (P) of the siRNA.
  • the RBP131/siApoB complex was prepared according to the method of Preparation Example 2. These complexes may be volume adjusted with 1 x PBS as needed prior to administration to suit the needs of the respective experiment.
  • mice Thirty-six normal C57BL/6J mice were randomly divided into 6 groups (6 in each group, half male and half female), which were PBS control group, Binary Complex 5:1 (binary complex) group, and Ternary Complex 5:1:2. (Ternary complex) group, PEA/siApoB 10:1 group, PEAG/siApoB 10:1 group, RBP131/siApoB group.
  • All animals were dosed according to body weight, single dose of tail vein, Binary Complexc 5:1 (binary complex) group, Ternary Complex 5:1:2 (ternary complex) group, PEA/siApoB 10:1 group
  • the dose of PEAG/siApoB 10:1 was 2.5 mg/kg (siRNA)
  • the dose of RBP131/siApoB was 0.5 mg/kg (siRNA)
  • the dose of all 6 groups was 10 ml/kg.
  • the animals were sacrificed, the blood was taken from the orbital venous plexus of the mice, and the serum was obtained by centrifugation.
  • RNA later Sigma Aldrich
  • Trizol total RNA was extracted by Trizol according to the standard procedure of total RNA extraction.
  • Real-time fluorescent qPCR was used to detect the expression level of ApoB mRNA in liver tissue. Specifically, the total RNA extracted was reverse transcribed into the target using the ImpProm-II TM reverse transcription kit (Promega). cDNA, followed by detection of the inhibition efficiency of siRNA on ApoB mRNA expression in liver tissue using a fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.).
  • siRNA inhibitory activity is calculated according to the following equation:
  • siRNA inhibitory activity [1 - (copy number of test group ApoB gene / copy number of test group ⁇ -actin) / (copy number of control group ApoB gene / copy number of control group ⁇ -actin)] ⁇ 100%.
  • control group was the control mice to which PBS was administered in the experiment, and each test group was a group of mice administered with different pharmaceutical compositions.
  • the level of serum lipids was directly measured using a PM4000/3 serum biochemical analyzer (SABA, Italy).
  • Table 10 shows the results of detection of ApoB mRNA expression inhibitory activity and blood lipid expression levels in each of the CHOBL/6J normal mice.
  • Experimental Example 8 This experimental example was used to examine the inhibitory effect of different doses of (GalNAc) 3 -X2M2 conjugate on the expression of HBV mRNA in the liver and the amount of HBsAg in serum in high-pressure injection-based HBV-transfected mice.
  • the PBS group, (GalNAc) 3 -NC group and each (GalNAc) 3 -X2M2 group were administered subcutaneously in a single dose, and the entecavir group was intragastrically administered for 14 days, ie, from day 0 to day 13, once daily. . All animals were sacrificed on the 14th day, and the expression levels of HBV mRNA and HBsAg in the liver were measured. The detection methods were the same as those in Experimental Example 3 and Experimental Example 4, respectively.
  • the (GalNAc) 3 -X2M2 conjugate of the present invention can exert a good biological activity and has potential for clinical application.
  • the (GalNAc) 3 -X2M2 of the present invention has a significant inhibitory effect on HBsAg relative to the clinical first-line anti-hepatitis B drug entecavir, further highlighting the function of the (GalNAc) 3 -siRNA conjugate against viral hepatitis B The possibility of sexual cure.
  • (GalNAc) 3 -X2M2 conjugate was prepared according to the method of Preparation Example 3.
  • 40 C3H/HeN mice injected with high pressure HBV plasmid designated as pHBV, purchased from the Animal Department of Shanghai Public Health Center, and prepared by high pressure injection of mice such as Peng XH., et al. World J. Gastroenterol. 2015, 21(12):3527-3536

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CN112423795A (zh) * 2018-12-28 2021-02-26 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
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CN113330117B (zh) * 2019-01-18 2024-05-28 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
CN113330117A (zh) * 2019-01-18 2021-08-31 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
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CN113663089A (zh) * 2021-06-25 2021-11-19 北京理工大学 可电离脂质纳米颗粒组合物、制备方法及应用
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