Double-stranded nucleic acid-galactosamine-cholesterol compound and its application

Abstract

The present invention discloses a double-stranded nucleic acid-galactosamine-cholesterol compound and its application, and belongs to the technical field of nucleic acid drugs. The double-stranded nucleic acid-galactosamine-cholesterol compound is composed of a double-stranded nucleic acid, galactosamine and cholesterol connected in sequence; wherein, the double-stranded nucleic acid is a double-stranded nucleic acid with the function of inhibiting the transcription level and / or expression level of the target gene, and is composed of an antisense nucleic acid chain and a complementary chain; the galactosamine is connected to the 5' end of the complementary chain of the double-stranded nucleic acid. The double-stranded nucleic acid-galactosamine-cholesterol compound is used in the preparation of a drug that has the effect of inhibiting the transcription level and / or expression level of the target gene and has a protective effect on the kidney. The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention can penetrate the blood-brain barrier and can significantly reduce the burden on the kidney, providing a technical platform for the treatment of refractory diseases of the nervous system such as Alzheimer's disease and Parkinson's disease.

Description

Technical Field

[0001] The invention relates to a double-stranded nucleic acid-galactosamine-cholesterol compound and application thereof, belonging to the technical field of nucleic acid drugs. Background Art

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic characteristics and pharmacological activity. They are an important class of drugs that can be used to treat tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. A huge challenge in the application of nucleic acid drugs is the specificity and effectiveness of delivery to target cells in vivo. Chemical modification, nanoparticle encapsulation and ligand coupling of oligonucleotides can improve their in vivo stability and are strategies to improve drug pharmacokinetics and targeting specificity. Many studies have shown that targeted delivery technology may drive the further development of oligonucleotide drug therapy.

[0003] Chinese invention patent CN 104126010 A discloses a double-stranded nucleic acid complex that inhibits the expression of a target gene by means of an antisense effect, comprising a first nucleic acid chain renatured with a second nucleic acid chain, wherein the first nucleic acid chain (i) comprises nucleotides and optionally nucleotide analogs, and the total number of nucleotides and nucleotide analogs in the first nucleic acid chain is 8 to 100, (ii) comprises at least 4 consecutive nucleotides recognized by RNase H when the first nucleic acid chain hybridizes with a transcription product, and (iii) the first nucleic acid chain hybridizes with the transcription product; and the second nucleic acid chain comprises nucleotides and optionally nucleotide analogs. Antisense nucleic acids can be delivered in double-stranded complexes and can selectively and very effectively inhibit the expression of target genes or the level of transcription products by antisense nucleic acids. The double-stranded complex can be delivered to the target site with high specificity and high efficiency by associating a delivery portion with the complex. However, double-stranded nucleic acid complexes will increase the burden on the kidneys when administered in large doses or multiple times (one of the reasons is that the double-stranded nucleic acid complexes contain phosphorothioate nucleotides or phosphorothioate nucleotide analogs; if phosphorothioate is not performed, the stability of the double-stranded nucleic acid is extremely poor), which greatly limits the population that can benefit from the drug.

[0004] In addition, double-stranded nucleic acid complexes cannot effectively cross the blood-brain barrier, and how to deliver sufficient levels of double-stranded nucleic acid complexes to the site of administration is a difficult problem. According to relevant reports, researchers from Tokyo Medical and Dental University (TMDU), Ionis Pharmaceuticals, and Takeda have collaborated to discover a new method for delivering subcutaneously or intravenously injected ASOs across the blood-brain barrier to the central nervous system: cholesterol (CHS) is coupled to heteroduplex oligonucleotides (HDOs) composed of DNA and RNA. In experiments with mice or rats, it can effectively knock down the expression of target genes in the brain through subcutaneous or intravenous injection. Experimental results show that this cholesterol-coupled HDOs can be widely distributed in the brain, spine, and peripheral tissues, and can inhibit the expression levels of four target genes evaluated in the central nervous system (CNS) by up to 90%. Moreover, the research team further demonstrated that the ability of this HDOs to cross the blood-brain barrier did not cause a decrease in the integrity of the blood-brain barrier itself.

[0005] N-acetylgalactosamine (GalNAc), also known as 2-acetylamino-2-deoxy-D-galactose, D-GalNAc, N-acetylgalactosamine, has a chemical formula of C8H 15 N06. N-acetylgalactosamine is a commonly used liver-targeting carrier in the prior art. N-acetylated galactosamine coupling modification is the most commonly used small nucleic acid drug delivery system: GalNAc is covalently conjugated to the 3' end of the positive chain of DNA or RNA of different sequences in a monovalent or trivalent state to form polysaccharide DNA and polysaccharide RNA conjugates, thereby achieving specific delivery to hepatocytes. GalNAc is a ligand of the asialoglycoprotein receptor (ASGPR) and can bind to ASGPR with high affinity at the nM level. Subsequently, the receptor takes up GalNAc and nucleic acids into the cell under clathrin-mediated endocytosis. The advantage of GalNAc coupling modification is that the coupled nucleic acid has a small molecular weight and the duration of action is as long as several months. However, since ASGPR is only specifically and highly expressed in hepatocytes, there are tissue limitations. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a double-stranded nucleic acid-galactosamine-cholesterol compound and its application. The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention can pass through the blood-brain barrier and can significantly reduce the burden on the kidneys. The compound can be used to prepare a drug that has the effect of inhibiting the transcription level and / or expression level of the target gene and has a protective effect on the kidneys. It has great application potential and broad application prospects.

[0007] The present invention is achieved through the following technical solutions:

[0008] A double-stranded nucleic acid-galactosamine-cholesterol compound, which is composed of a double-stranded nucleic acid, galactosamine and cholesterol connected in sequence; wherein the double-stranded nucleic acid is a double-stranded nucleic acid that has the ability to inhibit the transcription level and / or expression level of a target gene, and is composed of an antisense nucleic acid strand (also known as a sense strand, template strand, parent strand, main strand) and a complementary strand (also known as a side strand); the galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid.

[0009] Furthermore, the type of the antisense nucleic acid chain is selected from any one or a combination of two or more of circular RNA, small activating RNA (SaRNA), antisense RNA (asRNA), antisense DNA (asDNA) and micro RNA (miRNA).

[0010] Furthermore, the antisense nucleic acid chain is composed of nucleotides and / or nucleotide analogs, with a length of 7 to 50 bp, 2 to 3 bridging nucleotides or nucleotide analogs at the 3' end; 2 to 3 bridging nucleotides or nucleotide analogs at the 5' end; the number of bridging nucleotides or nucleotide analogs accounts for 10% to 70% of the total number of nucleotides in the chain; the bridging nucleotides are selected from LNA, cEt-BNA, amide BNA (AmNA), cMOE-BNA or any other type of bridging nucleotides; the nucleotides and nucleotide analogs are all phosphorylated.

[0011] Furthermore, the nucleic acid type of the complementary chain is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and / or peptide nucleic acid (PNA).

[0012] Furthermore, the complementary chain is composed of nucleotides and / or nucleotide analogs, with 2 to 3 methylated (2-O-methylated) nucleotides or nucleotide analogs at the 3' end; and 2 to 3 methylated nucleotides or nucleotide analogs at the 5' end; the nucleotides and nucleotide analogs are connected by phosphodiester bonds.

[0013] Furthermore, the galactosamine is selected from N-acetylgalactosamine or N-acetylglucosamine (GlcNAc).

[0014] Furthermore, the connection method of galactosamine and cholesterol can be TEG-link (a conventional connection method in the prior art, cholesterol is connected to TEG-link through an amide bond, and TEG and GalNAc are connected through a phosphodiester bond), PEG-link or directly connected through a phosphodiester bond, or other connection methods known in the prior art.

[0015] The double-stranded nucleic acid-galactosamine-cholesterol compound is used as or in the preparation of a drug that has the efficacy of inhibiting the transcription level and / or expression level of a target gene and has a protective effect on the kidney.

[0016] A drug that has the efficacy of inhibiting the transcription level and / or expression level of a target gene and has a protective effect on the kidneys, wherein the active ingredient is the above-mentioned double-stranded nucleic acid-galactosamine-cholesterol compound, and may also include excipients commonly used in pharmacy, such as solvents, fillers, etc.

[0017] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention is administered to mammals including humans. The administration route for animal in vivo experiments or clinical administration is intravenous administration, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intraventricular injection and other parenteral routes or gastrointestinal administration (oral administration, gastrointestinal nutrition tube, anal plug, etc.), wherein the dosage is 0.01-100 mg / kg / day, and the administration can be repeated.

[0018] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention is composed of double-stranded nucleic acid, galactosamine and cholesterol, wherein cholesterol carries double-stranded nucleic acid through the blood-brain barrier into neurons, and double-stranded nucleic acid has the effect of inhibiting the transcription level and / or expression level of target genes, and galactosamine can bind to ASGPR on the surface of liver parenchymal cells, thereby bringing excess double-stranded nucleic acid into liver cells for metabolism, reducing the burden on the kidneys. The present invention solves the following technical problems and achieves the following technical effects:

[0019] (1) Solved the problem that double-stranded nucleic acids cannot penetrate the blood-brain barrier. The present invention has found through research that in vivo animal experiments (mice) after the compound of the present invention is injected into the tail vein, it can significantly increase the amount of double-stranded nucleic acids that penetrate the blood-brain barrier and enter the brain neuronal cells, which provides a technical platform for the treatment of Alzheimer's disease, Parkinson's disease and other refractory diseases of the nervous system.

[0020] (2) The problem of acute and chronic renal damage after increasing the dose or multiple administrations of double-stranded nucleic acids is solved. At present, in order to improve the delivery effectiveness and stability of double-stranded nucleic acids, it is usually necessary to combine multiple modification technologies and carriers. The kidney is the metabolic organ of some drugs, and some patients will increase the burden on the kidney after long-term medication. After the N-acetylgalactosamine binds to the ASGPR on the surface of liver parenchymal cells, it enters the cell through endocytosis to form endosomes, thereby bringing the excess double-stranded nucleic acids into the liver cells for decomposition; therefore, multiple injections can be performed without affecting the effectiveness of the drug and at the same time have a significant protective effect on the kidneys, expanding the population that can benefit from double-stranded nucleic acids (including people with kidney damage).

[0021] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention has the following action mechanisms: ① antisense DNA combines with genomic DNA in the form of complementary base pairing in the cell nucleus to form a triple-stranded nucleic acid structure or combines with single-stranded DNA to form a double-stranded structure, thereby preventing the replication and transcription of the target gene; ② antisense RNA combines with mRNA to form a complementary double-stranded structure, thereby blocking the binding of ribosomes with mRNA and inhibiting the process of ribosome-mediated translation of mRNA into protein; ③ after the antisense nucleic acid combines with mRNA in the cell nucleus, it can inhibit the nuclear export of mRNA; ④ antisense nucleic acid combines with mRNA to form a hybrid double-stranded molecule, thereby inducing RNase H to degrade RNA, thereby shortening the half-life of mRNA and reducing the translation of target protein.

[0022] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention has been found through experimental studies that the double-stranded nucleic acid can only exert its inhibitory effect on the target gene when galactosamine-cholesterol is connected to the 5' end of the complementary chain of the double-stranded nucleic acid. When galactosamine-cholesterol is connected to other positions (3' end of the sense chain, 3' end of the complementary chain, 5' end of the sense chain), the double-stranded nucleic acid does not show an inhibitory effect on the target gene. Moreover, the present invention has also found through experimental studies that the connection order of the double-stranded nucleic acid, galactosamine, and cholesterol will also have a significant effect on the effect of the compound. Only when the double-stranded nucleic acid is connected in the order of double-stranded nucleic acid-galactosamine-cholesterol can the double-stranded nucleic acid play a significant inhibitory effect. When the connection order is changed to double-stranded-cholesterol-galactosamine, the double-stranded nucleic acid does not show an inhibitory effect on the target gene. In addition, it is generally believed in the prior art that the smaller the molecular weight or volume of the vector is, the easier it is to be absorbed by cells. However, the present invention has shown through experimental studies that when the nucleic acid chain is less than 100 bp, the vector extension (TEG-link), the increase in molecular weight (double or multiple vectors) or the increase in volume have no obvious negative impact on its inhibitory effect.

[0023] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : A simplified structural diagram of a double-stranded nucleic acid-galactosamine-cholesterol compound.

[0025] Figure 2 : Schematic diagram of the connection between cholesterol and galactosamine.

[0026] Figure 3 : Mass spectrum of galactosamine-cholesterol.

[0027] Figure 4 : Schematic diagram of the relative SNCA mRNA levels in the mouse cerebral cortex in Example 2, wherein the mRNA level of the control group is taken as 1.

[0028] Figure 5 : Schematic diagram of the relative MALAT1 mRNA levels in the mouse cerebral cortex in Example 3, wherein the mRNA level of the control group is taken as 1.

[0029] Figure 6 : Schematic diagram of the relative MALAT1 mRNA levels in the mouse cerebral cortex in Example 4, wherein the mRNA level of the control group is taken as 1.

[0030] Figure 7 : Schematic diagram of the relative SRB1 mRNA levels in the substantia nigra of mice in Example 5, wherein the mRNA level of the control group is 1

[0031] Figure 8 : Schematic diagram of the relative MALAT1 mRNA levels in the mouse cerebral cortex in Example 6, wherein the mRNA level of the control group is taken as 1.

[0032] Fig. 9 : Schematic diagram of the relative MALAT1 mRNA levels in the mouse hippocampus in Example 6, wherein the mRNA level of the control group is taken as 1.

[0033] Fig.10 : Schematic diagram of the relative MALAT1 mRNA levels in the mouse striatum in Example 6, wherein the mRNA level of the control group is taken as 1.

[0034] Fig.11 : Schematic diagram of the relative OAT3 mRNA levels in mouse vascular endothelial cells in Example 7, wherein the mRNA level of the control group is taken as 1. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0036] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0037] The double-stranded nucleic acid of the present invention can be completely or partially synthesized by chemical synthesis methods (currently mainly using automated synthesizers) or by enzymatic reaction processes (including but not limited to polymerase, ligase or restriction reactions).

[0038] The present invention does not particularly limit the chain length of the sense nucleic acid chain (template chain), but the chain length is generally 7 to 50 bases. The chain length can be up to 120 bases. The choice of length generally depends on the balance between the effect of the antisense action and the specificity of the target nucleic acid chain, and other factors such as cost, synthesis yield, etc. also have an impact.

[0039] The DNA mentioned in the present invention means naturally occurring DNA deoxynucleotides, or DNA deoxynucleotides partially having modified base, sugar or phosphate bond subunits. The so-called modified base, sugar or phosphate bond subunits mainly refer to those in which a single substituent has been added or substituted in the subunit, such as 5-methylation, phosphorylation, 2'-O-methylation, etc. The main connection mode between DNA deoxynucleotides is phosphorothioate.

[0040] The locked nucleic acid LNA mentioned in the present invention is a kind of oligonucleotide derivative, in which the 2'-O and 4'-C positions of β-D-ribofuranose are connected by hydration to form a rigid structure.

[0041] The preparation of the nucleotide chain of the present invention adopts the conventional phosphoramidite triester synthesis method, which is the most widely used oligonucleotide synthesis method and is also the synthesis method adopted by the mainstream synthesizer at home and abroad. The method has the characteristics of high efficiency, rapid coupling and relatively stable starting reactants. First, RNA monomers and DNA monomers are synthesized by conventional methods. The RNA methylation, DNA thiolation, locked nucleic acid (bridging nucleic acid) and the like used in the present invention are all conventional methods well known to those skilled in the art. RNA monomers, DNA monomers or locked nucleic acids are fixed on a solid phase carrier to complete the synthesis of RNA or DNA chains, and the synthesis is extended from the 3' end to the 5' end of the primer to be synthesized, and adjacent nucleotides are connected by 3'→5' phosphodiester bonds. The steps include four-step cycles of deprotection, coupling, capping and oxidation. According to the predetermined base sequence, the corresponding phosphoramidite synthesis monomers and other necessary chemical reagents are added to the solid phase carrier with surface modification in advance in sequence through the liquid path system to complete the synthesis of the specified oligonucleotide sequence. After the synthesis is completed, the product is cut off and collected from the solid phase carrier by ammonia or other alkaline conditions, and the oligonucleotide of the target base sequence is obtained. The specific operations can be as follows:

[0042] (i) Synthesis: The synthesis of crude products is carried out by a synthesizer, which mainly includes deprotection, condensation connection, sealing, oxidation and other processes.

[0043] (1) First step of deprotection: the chemically active group pre-attached to the solid phase support is placed in the synthesizer to react with TCA added to the synthesizer from the reagent bottle pipeline;

[0044] (2) Second step: Tetrazolium is added from a reagent bottle into the synthesizer through a pipe, mixed with the monomer dissolved in acetonitrile to obtain an activated intermediate, thereby causing a condensation reaction;

[0045] (3) The third step is sealing: the reagents CapA and CapB are pumped from the reagent bottles into the synthesizer to terminate the condensation reaction and allow the minority of substances that have not participated in the reaction to proceed with the subsequent reaction;

[0046] (4) The fourth step is oxidation: using a pump to pump iodine-added tetrahydrofuran reagent from a reagent bottle to react with the condensed group to oxidize the phosphite to form a more stable phosphotriester;

[0047] (5) Repeat the above four steps until all the required groups are connected to obtain the crude product.

[0048] (ii) Ammonolysis: First, the pre-treatment of ammonolysis is carried out to remove impurities generated during the synthesis process, and then the crude product is washed to further purify it. After the pre-treatment is completed, the crude product is transferred to the ammonolysis chamber and added to the ammonolysis instrument for ammonolysis deprotection, and finally the sample to be eluted is obtained.

[0049] (iii) HPLC purification: The sample after aminolysis is purified using an HPLC purifier.

[0050] The mice used in the experiments mentioned in the present invention were all 4- to 6-week-old C57 / BL mice (male and female), weighing about 20 g. Unless otherwise specified, the number of mice used in each group was 3 or 4.

[0051] Example 1 Preparation of double-stranded nucleic acid-galactosamine-cholesterol compound

[0052] A double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound S in the present invention) was prepared for the target gene α-synuclein (SNCA) gene.

[0053] Compound S is composed of double-stranded nucleic acid, galactosamine and cholesterol connected in sequence. The structural diagram is as follows Figure 1 As shown; wherein, galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid; galactosamine and cholesterol are connected via TEG-link (cholesterol is connected to TEG-link via an amide bond, and TEG and GalNAc are connected via a phosphodiester bond), as shown Figure 2 shown.

[0054] Here are the steps:

[0055] (1) Synthesis of single-stranded and double-stranded nucleic acids

[0056] A double-stranded nucleic acid targeting α-synuclein was designed, wherein the main chain of the initial design was DNA, and the nucleotide sequence was: 5'-aatgacattctta-3', as shown in SEQ ID NO.1.

[0057] The initially designed side chain is RNA, and the nucleotide sequence is: 5'-TAAGAATGTCATT-3', as shown in SEQ ID NO.2.

[0058] Then, without changing the nucleotide sequence, some bases are modified (the purpose of the modification is to enhance the therapeutic effect or make it more stable or play other positive roles), and the modification method is selected from any one or more of locked nucleic acid modification, methylation modification (including conventional 2' methylation modification and 5' methylation modification of cytosine), phosphorothioate diester bond modification, DNA nucleic acid chain chimeric RNA modification, etc.

[0059] The modified main chain is as follows: 5'-A(+)^A(+)^t^g^a^c^a^t^t^c^T(+)^T(+)^A(+)-3'.

[0060] The modified side chain is as follows: 5'-T(M)^A(M)^A(M)^GAATGTCA^T(M)^T(M)-3'.

[0061] Among them, capital letters represent RNA; lowercase letters represent DNA; capital letters (+) represent locked nucleic acid modification (2'-O and 4' carbon on ribose are linked together); (M) represents methylation modification (2'-O-methylation); ^ represents phosphorothioate diester bond modification.

[0062] After synthesizing the modified main chain and side chain by conventional methods, the main chain and side chain were mixed in the same amount of substances, heated at 95°C for 5 minutes, kept at a constant temperature of 37°C for 1 hour to allow the chains to renature, and stored in a 4°C environment.

[0063] (2) Preparation of double-stranded nucleic acid-galactosamine-cholesterol compounds

[0064] (a) Cholesterol is linked to TEG-linker. The hydroxyl group on cholesterol is acylated by a condensing agent and reacts with the activated lipid of TEG to form an amide bond (this method is a conventional technical means). The specific method is as follows: 300 g of cholesterol and 350 g of TEG-linker are dissolved in 50 ml of acetone, 0.5 g of condensing agent acylase is added, and the mixture is placed in an air bath constant temperature oscillator for oscillation (23°C, 250 r·min -1 ) react for 24 hours; after the reaction is completed, filter out the enzyme, recover the solvent, and obtain cholesterol-TEG;

[0065] (b) Synthesis of galactosamine-cholesterol by enzymatic method (this method is a conventional technical means), the specific method is as follows: 100 g hydrogenated soybean phospholipids (HSPC), 300 g cholesterol, 20 g sodium distearoylphosphatidylglycerol (DSPG-Na), 330 g cholesterol-TEG and 300 g N-acetylgalactosamine were mixed, dissolved in 500 ml chloroform, placed in a 37°C water bath rotary evaporator, and the chloroform was evaporated until a lipid film was formed on the inner wall of the bottle, and dried in a vacuum desiccator for 12 hours, and then 500 ml ultrapure water was added for hydration, slowly stirred at 55°C, incubated in the dark for 2 hours, and then passed through 0.1 μm and 0.05 μm polycarbonate membranes 10 times each using a high-pressure extruder to obtain galactosamine-cholesterol. The product was purified by rapid silica gel column chromatography, and the purified product was structurally identified by ESI, HNMR and CNMR. The results are shown as follows: Figure 3 As shown, the structure of the product is determined to be Figure 2 The structure shown has three N-acetylgalactosamines attached to a cholesterol molecule.

[0066] (c) synthesizing the complementary chain by conventional methods (synthesizing from 3' to 5', removing the 5' end protecting group DMT after synthesis to expose the hydroxyl group); placing the complementary chain and galactosamine-cholesterol in a molar ratio of 1:3 in L96 CPG, synthesizing the L96 carrier containing the oligonucleotide sequence with the protecting group by solid phase synthesis technology, and then cutting and deprotecting from the solid phase carrier (3% DCA, 0.3M BMT, 0.5% NMI in ACN) to obtain the crude oligonucleotide, coupling the galactosamine-cholesterol with the exposed hydroxyl group, and conjugating to the 5' end of the complementary chain through oxidation to form a phosphodiester bond to form a complementary chain-galactosamine-cholesterol;

[0067] (d) The main chain and the complementary chain - galactosamine-cholesterol are mixed in equal amounts, the mixture is heated at 95°C for 5 minutes, and allowed to stand at a constant temperature of 37°C for 1 hour to allow the chains to renature, thereby obtaining a double-stranded nucleic acid-galactosamine-cholesterol compound, which is then stored at 4°C.

[0068] The sources of raw materials used are as follows:

[0069] Cholesterol (Japan Seika Co., Ltd., batch number: A90719, mass fraction > 98.5%);

[0070] N-acetylgalactosamine (Shanghai Aladdin Biochemical Technology Co., Ltd., mass fraction > 98.0%);

[0071] TEG-linker (Shanghai Zhaowei Technology Development Co., Ltd., CB96327030);

[0072] Lipozyme TLIM lipase (Novozymes, Denmark, enzyme activity > 250 IUN·g -1 );

[0073] Ultrafiltration centrifuge tube (PALL, USA, 1 mL, 100 KD);

[0074] MD34 dialysis bag (Shanghai Baisai Biotechnology Co., Ltd., cut-off molecular weight 0.8×10 4 ~1.4×10 4 );

[0075] Other reagents were commercially available of analytical grade.

[0076] Example 2 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 1

[0077] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Excessive aggregation of α-synuclein in cells is a characteristic pathological change of Parkinson's disease. Its early pathological changes are limited to dopaminergic neurons in the substantia nigra, so local gene delivery is likely to be an effective treatment measure.

[0078] The mice were randomly divided into 5 groups: control group, single chain experimental group 1, single chain experimental group 2, compound experimental group 1, and compound experimental group 2.

[0079] Compound Experiment Group 1 and Compound Experiment Group 2 were both injected into the tail vein with the double-stranded nucleic acid-galactosamine-cholesterol compound prepared in Example 1, which was diluted with PBS buffer to a concentration of 1200 μM. The dose of Compound Experiment Group 1 (referring to the dose of the compound) was 50 mg / kg, and the dose of Compound Experiment Group 2 was 25 mg / kg, and the injection was once a week for 4 consecutive weeks, for a total of 4 times.

[0080] Single-stranded nucleic acid (i.e., the main chain of Example 1) was injected into the tail vein of single-stranded experimental group 1 and single-stranded experimental group 2, which was diluted with PBS buffer to a concentration of 1200 μM before injection. The dose of single-stranded experimental group 1 (referring to the dose of single-stranded nucleic acid) was 50 mg / kg, and the dose of single-stranded experimental group 2 was 25 mg / kg, and the injection was once a week for 4 consecutive weeks, for a total of 4 times.

[0081] The control group was injected with an equal volume of PBS buffer only.

[0082] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0083] Figure 4Relative SNCA mRNA levels in the mouse cerebral cortex are shown by Figure 4 It can be seen that after the tail vein injection of single-stranded nucleic acid and double-stranded nucleic acid-galactosamine-cholesterol compounds, the SNCA mRNA levels decreased significantly, and the double-stranded nucleic acid-galactosamine-cholesterol compound had a better effect, which was significantly different from the single-stranded experimental group; at the same time, high doses also had better therapeutic effects than low doses, but high doses also had greater side effects.

[0084] Table 1 shows the biochemical indexes of mouse serum, and the lower the value, the better (indicating less side effects). As can be seen from Table 1, the liver function (AST, ALT) and kidney function (BUN, Cre) damage in the compound experimental group were significantly reduced compared with the single-chain experimental group, and the double-stranded nucleic acid-galactosamine-cholesterol compound had no obvious side effects.

[0085] Table 1 Biochemical parameters of mouse serum

[0086] T-Bil (mg / dl) BUN (mg / dl) Cre (mg / dl) AST (U / l) ALT (U / l) LD(U / l) ALP (U / l) PBS 0.067±0.009 33.033±1.25 0.110±0.0058 101±15 37±11 312±62 427±35 Single-chain experimental group 2 0.963±0.111 28.133±1.43 0.113±0.0120 933±76 821±19 802±43 949±7 Single-chain experimental group 1 1.953±0.120 31.500±1.95 0.110±0.0058 1519±76 1967±90 1998±122 1971±296 Compound experimental group 2 0.803±0.093 24.767±3.11 0.103±0.0088 183±28 135±3 343±16 515±25 Compound experimental group 1 1.007±0.038 18.300±2.95 0.090±0.0100 317±25 287±15 329±47 736±9

[0087] Example 3 Application 2 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0088] In this example, single-stranded nucleic acid and double-stranded nucleic acid were designed and prepared for the target gene MALAT1, and galactosamine, cholesterol, and galactosamine-cholesterol were connected to the 5' end of the side chain of the double-stranded nucleic acid (the connection method is a conventional technical means) to prepare a double-stranded nucleic acid-galactosamine compound, a double-stranded nucleic acid-cholesterol compound, and a double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound MA in the present invention).

[0089] Specifically, the double-stranded nucleic acid is composed of a sense strand (main strand) and a complementary strand (side strand), wherein the initially designed main strand is DNA, and the nucleotide sequence is: 5'-gaactcaaaatatatt-3', as shown in SEQ ID NO.3.

[0090] The initially designed side chain is RNA, and the nucleotide sequence is: 5'-AATATATTTTGAGTTC-3', as shown in SEQ ID NO.4.

[0091] Then, without changing the nucleotide sequence, some bases were modified and the modified main chain was as follows: 5'- G(+)^A(+)^A(+)^c^t^c^a^a^a^a^t^a^t^A(+)^T(+)^T(+)-3'.

[0092] The modified side chain is as follows: 5'-A(M)^A(M)^T(M)^ATATTTTGAGT(M)^T(M)^C(M)-3'.

[0093] The single-stranded nucleic acid refers to the sense strand mentioned above.

[0094] The effects of these three compounds were compared, and the experimental process was as follows:

[0095] The mice were randomly divided into 5 groups: control group, single-chain experimental group, double-chain experimental group, galactosamine double-chain experimental group, cholesterol double-chain experimental group, and cholesterol-galactosamine double-chain experimental group. The single-chain experimental group was injected with the above single-chain nucleic acid, the double-chain experimental group was injected with the above double-chain nucleic acid, the galactosamine double-chain experimental group was injected with the above double-chain nucleic acid-galactosamine compound, the cholesterol double-chain experimental group was injected with the above double-chain nucleic acid-cholesterol compound, and the cholesterol-galactosamine double-chain experimental group was injected with the above compound MA. The control group was injected with an equal amount of PBS buffer only. The PBS buffer was diluted to a concentration of 1200 µM and then injected. The dose was 50 mg / kg, once a week, for 4 consecutive weeks, for a total of 4 times.

[0096] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0097] Figure 5 Relative MALAT1 mRNA levels in the mouse cerebral cortex are shown. Figure 5 It can be seen that after tail vein injection of single-stranded nucleic acid, double-stranded nucleic acid, double-stranded nucleic acid-galactosamine compound, double-stranded nucleic acid-cholesterol compound, and compound MA, the MALAT1 mRNA level decreased. After injection of compound MA, the decrease was significant, which was significantly better than double-stranded nucleic acid-galactosamine compound and double-stranded nucleic acid-cholesterol compound. This shows that only connecting galactosamine or cholesterol to double-stranded nucleic acid cannot play an ideal inhibitory role; when both galactosamine and cholesterol are connected to double-stranded nucleic acid, they can play a significant inhibitory role.

[0098] Example 4 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 3

[0099] In this example, a double-stranded nucleic acid (same as Example 3) was prepared for the target gene MALAT1, and galactosamine-cholesterol was connected to the 3' end of the sense chain, the 3' end of the complementary chain, the 5' end of the sense chain, and the 5' end of the complementary chain, respectively, through conventional coupling and condensation reactions, to prepare four compounds, which were respectively referred to as: sense chain 3' compound A, complementary chain 3' compound A, sense chain 5' compound A, and complementary chain 5' compound A (this is compound MA of the present invention).

[0100] The effects of these four compounds were compared, and the experimental process was as follows:

[0101] The mice were randomly divided into 5 groups: control group, compound main chain 3' end A group, compound main chain 5' end A group, compound side chain 3' end A group, and compound side chain 5' end A group. Among them, the compound main chain 3' end A group was injected with the above-mentioned sense chain 3' compound A, the compound main chain 5' end A group was injected with the above-mentioned sense chain 5' compound A, the compound side chain 3' end A group was injected with the above-mentioned complementary chain 3' compound A, and the compound side chain 5' end A group was injected with the above-mentioned compound MA. The control group was injected with an equal amount of PBS buffer only. After diluting with PBS buffer to a concentration of 1200 µM, the dose was 12.5 mg / kg, once a week, for 4 consecutive weeks, for a total of 4 times.

[0102] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0103] Figure 6 Relative MALAT1 mRNA levels in the mouse cerebral cortex are shown by Figure 6 It can be seen that after the tail vein injection of compound A at the 3' end of the sense chain and compound A at the 5' end of the sense chain, the MALAT1 mRNA level increased significantly; after the injection of compound A at the 3' end of the complementary chain, the MALAT1 mRNA level did not change significantly; after the injection of compound MA, the MALAT1 mRNA level decreased significantly. This shows that only compound MA can achieve an inhibitory effect, that is, when galactosamine-cholesterol is connected to the 5' end of the complementary chain of the double-stranded nucleic acid, it can play a significant inhibitory role. When connected at other positions, the double-stranded nucleic acid cannot play the preset role.

[0104] Example 5 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 4

[0105] In this example, a double-stranded nucleic acid is designed and prepared for the target gene SRB1, and galactosamine-cholesterol is connected to the 3' end of the sense strand, the 3' end of the complementary strand, the 5' end of the sense strand, and the 5' end of the complementary strand, respectively, through conventional coupling and condensation reactions, to prepare four compounds, which are respectively referred to as: sense strand 3' compound B, complementary strand 3' compound B, sense strand 5' compound B, and complementary strand 5' compound B (this is the double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention, referred to as compound SR).

[0106] Specifically, the double-stranded nucleic acid is composed of a sense strand (main strand) and a complementary strand (side strand), wherein the initially designed main strand is DNA, and the nucleotide sequence is: 5'-ttcagtcatgact-3', as shown in SEQ ID NO.5.

[0107] The initially designed side chain is RNA, and the nucleotide sequence is: 5'-AGTCATGACTGAA-3', as shown in SEQ ID NO.6.

[0108] Then, without changing the nucleotide sequence, some bases were modified and the modified main chain was as follows: 5'- T(+)^T(+)^c^a^g^t^c^a^t^g^A(+)^C(+)(5M) ^T(+)-3', where 5M represents the 5' methylation modification of cytosine (5'-O-methylation).

[0109] The nucleotide sequence of the modified side chain is as follows: 5'-A(M)^G(M)^T(M)^CATGACTGA(M)^A(M)-3'.

[0110] The effects of these four compounds were compared, and the experimental process was as follows:

[0111] Mice were randomly divided into 5 groups: control group, compound main chain 3' end B group, compound main chain 5' end B group, compound side chain 3' end B group, and compound side chain 5' end B group. Among them, compound main chain 3' end B group was injected with the above-mentioned sense chain 3' compound B, compound main chain 5' end B group was injected with the above-mentioned sense chain 5' compound B, compound side chain 3' end B group was injected with the above-mentioned complementary chain 3' compound B, and compound side chain 5' end B group was injected with the above-mentioned compound SR. Compounds were injected into the tail vein of each experimental group, diluted with PBS buffer to a concentration of 1200 µM before injection. The dose was 12.5 mg / kg. The control group was injected with an equal amount of PBS buffer only.

[0112] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0113] Figure 7 Relative SRB1 mRNA levels in the substantia nigra of mice are shown by Figure 7 It can be seen that after the tail vein injection of compound B at the 3' end of the sense strand, compound B at the 5' end of the sense strand, and compound B at the 3' end of the complementary strand, the SRB1 mRNA level did not change significantly; after the injection of compound SR, the SRB1 mRNA level decreased significantly. This shows that only compound SR can achieve an inhibitory effect, that is, when galactosamine-cholesterol is connected to the 5' end of the complementary strand of the double-stranded nucleic acid, it can play a significant inhibitory role. When connected to other positions, the double-stranded nucleic acid cannot play the preset role.

[0114] Example 6 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 5

[0115] In this example, for the target gene MALAT1, a double-stranded nucleic acid (same as in Example 3) was prepared, and cholesterol was connected to the oligonucleotide on the solid phase carrier through conventional coupling and condensation reactions, and galactosamine was connected through oxidation; the 5' end of the side chain was linked by a phosphate bond to obtain a double-stranded nucleic acid-cholesterol-galactosamine compound. Compared with the double-stranded nucleic acid-galactosamine-cholesterol compound of the present invention (this is the compound MA of the present invention), the experimental process is:

[0116] The mice were randomly divided into three groups: control group, double-stranded-cholesterol-galactosamine compound group, and double-stranded nucleic acid-galactosamine-cholesterol compound group. The double-stranded-cholesterol-galactosamine compound group was injected into the tail vein, and the double-stranded nucleic acid-galactosamine-cholesterol compound group was injected into the tail vein. The compound MA was diluted with PBS buffer to a concentration of 1200 µM before injection. The dose was 25 mg / kg. The control group was only injected with an equal amount of PBS buffer.

[0117] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0118] Figure 8 Relative MALAT1 mRNA levels in the mouse cerebral cortex are shown by Figure 7 It can be seen that after tail vein injection of double-stranded cholesterol-galactosamine compound, the MALAT1 mRNA level did not change significantly; after injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0119] Fig. 9 Relative MALAT1 mRNA levels in mouse hippocampus are shown by Figure 8 It can be seen that after tail vein injection of double-stranded cholesterol-galactosamine compound, the MALAT1 mRNA level did not change significantly; after injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0120] Fig.10 Relative MALAT1 mRNA levels in the mouse striatum are shown by Fig. 9 It can be seen that after the tail vein injection of double-stranded cholesterol-galactosamine compound, the MALAT1 mRNA level increased significantly; after the injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0121] The above results show that double-stranded nucleic acid can only play a significant inhibitory role when it is connected in the order of double-stranded nucleic acid-galactosamine-cholesterol. When the connection order is changed to double-stranded-cholesterol-galactosamine, double-stranded nucleic acid cannot play the expected role (it cannot effectively pass through the blood-brain barrier and cannot reduce the burden on the kidneys).

[0122] Example 7 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 6

[0123] In this example, single-stranded nucleic acid and double-stranded nucleic acid were designed and prepared for the target gene OAT3, and galactosamine, cholesterol, and galactosamine-cholesterol were connected to the 5' end of the side chain of the double-stranded nucleic acid (the connection method is a conventional technical means) to prepare a double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound M in the present invention).

[0124] Specifically, the double-stranded nucleic acid is composed of a sense strand (main strand) and a complementary strand (side strand), wherein the initially designed main strand is DNA, and the nucleotide sequence is: 5'-cggccaaacctgtc-3', as shown in SEQ ID NO.7.

[0125] The initially designed side chain is RNA, and the nucleotide sequence is: 5'-GACAGGTTTGGCCG-3', as shown in SEQ ID NO.8.

[0126] Then, without changing the nucleotide sequence, some bases were modified and transformed. There were 5 modified main chains, named M-4, M-6, M-8, M-10, and M-12, and the corresponding numbers of locked nucleic acids in the main chains were 4, 6, 8, 10, and 12, respectively, as shown in Table 2 (direction 5'-3').

[0127] Table 2

[0128] M-4 C(+)(5M)^G(+)^g^c^c^a^a^a^c^c^t^g^T(+)^C(+)(5M) M-6 C(+)(5M)^G(+)^G(+)^c^c^a^a^a^c^c^t^G(+)^T(+)^C(+)(5M) M-8 C(+)(5M)^G(+)^G(+)^C(+)(5M)^c^a^a^a^c^c^T(+)^G(+)^T(+)^C(+)(5M) M-10 C(+)(5M)^G(+)^G(+)^C(+)(5M)^C(+)(5M)^a^a^a^c^C(+)(5M)^T(+)^G(+)^T(+)^C(+)(5M) M-12 C(+)(5M)^G(+)^G(+)^C(+)(5M)^C(+)(5M)^A(+)^a^a^C(+)(5M)^C(+)(5M)^T(+)^G(+)^T(+)^C(+)(5M)

[0129] The modified side chain is as follows: 5'-G(M)^A(M)^C(M)^AGGTTTGGC(M)^C(M)^G(M)-3'.

[0130] Among them, capital letters represent RNA; lowercase letters represent DNA; capital letters (+) represent locked nucleic acid modification (2'-O and 4' carbon on ribose are linked together); (M) represents methylation modification (2'-O-methylation); ^ represents phosphorothioate diester bond modification, and (5M) represents 5' methylation modification of cytosine (5'-O-methylation).

[0131] The single-stranded nucleic acid refers to the main chain mentioned above.

[0132] The effects of these five compounds were compared, and the experimental process was as follows:

[0133] The mice were randomly divided into 6 groups: control group, cholesterol-galactosamine double chain experimental group (compound M-4), cholesterol-galactosamine double chain experimental group (compound M-6), cholesterol-galactosamine double chain experimental group (compound M-8), cholesterol-galactosamine double chain experimental group (compound M-10), cholesterol-galactosamine double chain experimental group (compound M-12). Among them, the cholesterol-galactosamine double chain experimental group (compound M-4) was injected with the above-mentioned compound M-4 by tail vein, the cholesterol-galactosamine double chain experimental group (compound M-6) was injected with the above-mentioned compound M-6 by tail vein, the cholesterol-galactosamine double chain experimental group (compound M-8) was injected with the above-mentioned compound M-8 by tail vein, the cholesterol-galactosamine double chain experimental group (compound M-10) was injected with the above-mentioned compound M-10 by tail vein, and the cholesterol-galactosamine double chain experimental group (compound M-12) was injected with the above-mentioned compound M-12 by tail vein. The control group was injected with an equal amount of PBS buffer only. The drug was diluted with PBS buffer to a concentration of 1200 μM and then injected. The dose was 50 mg / kg.

[0134] Three days after the tail vein injection, blood was collected from the back of the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues were extracted by dissection and PBS perfusion.

[0135] Fig.11 Shows the relative OAT3 mRNA levels in mouse vascular endothelial cells. Fig.11 It can be seen that after the tail vein injection of cholesterol-galactosamine double-chain experimental group (compound M-4), cholesterol-galactosamine double-chain experimental group (compound M-6), cholesterol-galactosamine double-chain experimental group (compound M-8), cholesterol-galactosamine double-chain experimental group (compound M-10), and cholesterol-galactosamine double-chain experimental group (compound M-12), the OAT3 mRNA level decreased. After the injection of the compound cholesterol-galactosamine double-chain experimental group (compound M-6), the decrease was significant, which was significantly better than other compounds. This shows that the locked nucleic acid cannot play an ideal inhibitory role when the proportion of the main chain is too much or too little; when the locked nucleic acid accounts for 10% to 70% of the double chain, it can play a significant inhibitory role.

[0136] The above results show that when the locked nucleic acid accounts for too much or too little in the main chain, it cannot play an ideal inhibitory role; when the locked nucleic acid accounts for 10% to 70% of the double chain, it can play a significant inhibitory role.

[0137] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A double-stranded nucleic acid-galactosamine-cholesterol compound, characterized in that: It is composed of a double-stranded nucleic acid, galactosamine and cholesterol connected in sequence; wherein the double-stranded nucleic acid is a double-stranded nucleic acid that has the function of inhibiting the transcription level and / or expression level of a target gene, and is composed of an antisense nucleic acid strand and a complementary strand; the galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid; The galactosamine is selected from N-acetylgalactosamine; The connection mode between galactosamine and cholesterol is TEG-link and PEG-link; The target gene of the double-stranded nucleic acid is an α-synuclein gene. The double-stranded nucleic acid is modified by modifying the antisense nucleic acid chain and the complementary chain. The modified antisense nucleic acid chain is: 5'-A(+)^A(+)^t^g^a^c^a^t^t^c^T(+)^T(+)^A(+)-3'; the modified complementary chain is: 5'-T(M)^A(M)^A(M)^GAATGTCA^T(M)^T(M)-3'; Or: the target gene of the double-stranded nucleic acid is MALAT1, and the double-stranded nucleic acid is modified by modifying the antisense nucleic acid chain and the complementary chain, the modified antisense nucleic acid chain is: 5'-G(+)^A(+)^A(+)^c^t^c^a^a^a^a^t^a^t^A(+)^T(+)^T(+)-3'; the modified complementary chain is: 5'-A(M)^A(M)^T(M)^ATATTTTGAGT(M)^T(M)^C(M)-3'; Or: the target gene of the double-stranded nucleic acid is SRB1, and the double-stranded nucleic acid is modified by modifying the antisense nucleic acid chain and the complementary chain, and the modified antisense nucleic acid chain is: 5'-T(+)^T(+)^c^a^g^t^c^a^t^g^A(+)^C(+)(5M)^T(+)-3'; the modified complementary chain is: 5'-A(M)^G(M)^T(M)^CATGACTGA(M)^A(M)-3'; Or: the target gene of the double-stranded nucleic acid is OAT3, and the double-stranded nucleic acid is modified by modifying the antisense nucleic acid chain and the complementary chain, and the modified antisense nucleic acid chain is: 5'-C(+)(5M)^G(+)^G(+)^c^c^a^a^a^c^c^t^G(+)^T(+)^C(+)(5M)-3'; the modified complementary chain is: 5'-G(M)^A(M)^C(M)^AGGTTTGGC(M)^C(M)^G(M)-3'; Among them, capital letters represent RNA; lowercase letters represent DNA; capital letters (+) represent locked nucleic acid modification; (M) represents methylation modification; (5M) represents 5' methylation modification of cytosine; ^ represents phosphorothioate diester bond modification.

2. Use of the double-stranded nucleic acid-galactosamine-cholesterol compound according to claim 1 in the preparation of a drug that has the effect of inhibiting the transcription level and / or expression level of a target gene and has a protective effect on the kidney.

3. A drug that has the effect of inhibiting the transcription level and / or expression level of a target gene and has a protective effect on the kidney, characterized in that: The active ingredient is the double-stranded nucleic acid-galactosamine-cholesterol compound described in claim 1.

Images