RNAi agent for inhibiting AGT expression as well as preparation method and application of RNAi agent

Abstract

The invention belongs to the technical field of biological medicines, relates to an RNAi (Ribonucleic Acid Interference) agent of a targeted angiotensin (AGT) gene, a preparation method and application of the RNAi agent, and further relates to a pharmaceutical composition of the RNAi agent and application of the pharmaceutical composition containing the RNAi agent in prevention or treatment of AGT-related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to an RNAi agent targeting angiotensinogen (AGT) gene, a preparation method and uses thereof, and further relates to a pharmaceutical composition of the RNAi agent and uses of the pharmaceutical composition comprising the RNAi agent for preventing or treating AGT-related diseases. BACKGROUND

[0002] Angiotensinogen (AGT) is a member of the serpin family and a component of the renin-angiotensin-aldosterone system (RAAS). It is mainly produced in the liver and released into the circulation, where it is converted to angiotensin I by renin. Angiotensin I is then converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates the secretion of the hormone aldosterone from the adrenal cortex. Aldosterone causes the kidneys to increase the reabsorption of sodium and water, leading to an increase in the volume of fluid in the body, which in turn can increase blood pressure.

[0003] The present application aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can inhibit the expression of AGT gene in the liver, so as to achieve the purpose of disease treatment. SUMMARY

[0004] In view of the problems existing in the prior art, the present application aims to provide an RNAi agent for inhibiting AGT gene expression, a preparation method and uses thereof.

[0005] Specifically, the present application provides an RNAi agent for inhibiting the expression of angiotensinogen (AGT) in cells, the RNAi agent comprising a sense strand and an antisense strand, the sense strand and the antisense strand forming a duplex region, wherein the sense strand and the antisense strand are selected from the sequences shown in Table 1.

[0006] Further, the RNAi agent is connected with a targeting ligand.

[0007] Further, the targeting ligand is connected with the 3' or 5' end of the sense strand.

[0008] Further, the targeting ligand comprises an N-acetyl-galactosamine (GalNac) moiety.

[0009] Further, the targeting ligand can be selected from the following structures:

[0010]

[0011] Further, the targeting ligand is selected from:

[0012]

[0013] Further, the targeting ligand is linked to the 3' end of the sense strand.

[0014] Further, the RNAi agent is selected from the sequences shown in Table 3.

[0015] Further, the present application also provides a pharmaceutical composition comprising the RNAi agent of any of the preceding items, and one or more than one pharmaceutically acceptable excipient and / or carrier.

[0016] Further, the present application also provides a use of the RNAi agent or the pharmaceutical composition of any of the preceding items in the manufacture of a medicament for preventing or treating a disease associated with AGT; the disease associated with AGT is selected from hypertension.

[0017] The term "AGT" includes human AGT, the amino acid and the entire coding sequence of which can be found, for example, in GenBank Accession No. GI: 188595658 (NM_000029.3); cynomolgus monkey AGT, the amino acid and the entire coding sequence of which can be found, for example, in GenBank Accession No. GI: 90075391 (AB170313.1); mouse (Mus musculus) AGT, the amino acid and the entire coding sequence of which can be found, for example, in GenBank Accession No. GI: 113461997 (NM_007428.3); and rat (Rattus norvegicus) AGT, the amino acid and the entire coding sequence of which can be found, for example, in GenBank Accession No. GI: 51036672 (NM_134432).

[0018] In some embodiments, the pharmaceutical compositions of the present application can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., through a transdermal patch), pulmonary (e.g., through inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal), percutaneous, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, subcutaneous, intravenous, intraarterial, intraperitoneal or intramuscular injection or infusion; subcutaneous (via an implanted device), intracranial, intra-parenchymal, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0019] In some embodiments, the gene expression level and / or mRNA level of AGT in subjects administered the AGT RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to subjects before administration of the AGT RNAi agent or subjects who did not receive the AGT RNAi agent. The gene expression level and / or mRNA level in the subjects may be reduced in the subjects' cells, cell populations, and / or tissues. In some embodiments, the protein level of AGT in subjects administered the AGT RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to subjects before administration of the AGT RNAi agent or those who did not receive the AGT RNAi agent. The protein level in the subject may be reduced in the subject's cells, cell populations, tissues, blood, and / or other fluids. The reduction in gene expression, mRNA, or protein levels can be assessed by any method known in the art. The reduction or decrease in AGT mRNA and / or protein levels is collectively referred to herein as a reduction or decrease in AGT, or inhibition or reduction of AGT expression.

[0020] The present invention also provides a method for preparing the nucleic acid described in any of the preceding claims, wherein the sense and antisense strands of the nucleic acid can be conveniently and routinely prepared using known solid-phase synthesis techniques. Other methods known in the art for such synthesis, such as liquid-phase synthesis or fermentation, may also be used, either alternatively or as an alternative. The preparation of other oligonucleotides (such as phosphate thioides and alkylated derivatives) using similar techniques is also known.

[0021] The term “including” as used herein refers to the phrase “including (but not limited to)” and is used interchangeably with it, unless the context clearly indicates otherwise.

[0022] The terms “silence,” “reduction,” “suppression,” “downregulation,” or “knockdown of gene expression” when referring to the AGT gene mean that when cells, cell populations, or tissues are treated with the AGT RNAi agent, the expression of the gene (measured, for example, by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA) is reduced compared to the same cells, cell populations, or tissues before the application of the AGT RNAi agent.

[0023] The term "targeting ligand" can include naturally occurring substances such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, styrax, chitosan, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylglucosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include the following: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, dendritic polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0024] Targeting ligands can also be cell or tissue-targeting agents that bind to specific cell types, such as kidney cells, for example, lectins, glycoproteins, lipids, or proteins, such as antibodies. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics.

[0025] Targeting ligands can also be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for co-ligands, or antibodies, such as antibodies that bind to a specific cell type, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38MAP kinase, or activators of NF-κB.

[0026] Targeting ligands can be substances that can increase the uptake of RNAi agents into cells, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments), such as drugs. Drugs can be, for example, taxon, vincristine, vinblastine, pinocembrin, nocodazole, iaplakinolide, red sea sponge A, phalloidin, swinholide A, indanocine, or myoservin.

[0027] The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to the target sequence. When used herein, the term "complementary region" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0028] The term "sense strand" generally refers to a strand of an RNAi agent that includes regions substantially complementary to the region referred to herein as the antisense strand. The "sense" strand is sometimes called the "positive" strand, the "passenger" strand, or the "antiguide" strand. With their sequence, the antisense strand targets the desired mRNA, while the positive strand targets a different target. Therefore, if the antisense strand is incorporated into a RISC, the correct target is targeted. Incorporation of the positive strand can lead to off-target effects. These off-target effects can be limited by using modifications on the positive strand or by using a 5' cap.

[0029] In this invention, "complementary" has the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0030] The term "complementary" when used to describe the first nucleotide sequence (e.g., the positive strand of an RNAi agent or AGT mRNA) in relation to the second nucleotide sequence (e.g., the antisense strand of an RNAi agent), refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize (form base pair hydrogen bonds) with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions, forming a double helix or double-stranded structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include native or modified nucleotides or nucleotide mimics, provided that the above requirements regarding their hybridization ability are met. "Complementarity" does not necessarily require nucleobase complementarity on every nucleotide. Instead, some mismatches are tolerated.

[0031] The term "fully complementary" generally means that all (100%) bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The sequential sequence may comprise all or a portion of the first or second nucleotide sequence. As used herein, "partially complementary" generally means that in the hybridized nucleobase sequence pair, at least about 70% of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. As used herein, "substantially complementary" generally means that in the hybridized nucleobase sequence pair, at least about 90% of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used in relation to base matching between the sense and antisense strands of the RNAi agent or between the antisense strand of the RNAi agent and the sequence of AGT mRNA. Sequence identity or complementarity is independent of modification. For the purpose of determining identity or complementarity, for example, a and Af are complementary to U (or T) and are identical to A.

[0032] In this invention, the modified nucleotides include, but are not limited to: 5'-thiophosphate nucleotides, 5'-methylated cytosine nucleotides, 5'-methylphosphate nucleotides, 2'-O-methyl modified nucleotides, reverse 2'-O-methyl nucleotides, 3'-O-methyl nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, alkyl nucleotides, 5'-C-methylphosphate nucleotides, 2'-F-arabinonucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNA), 2'-fluorinated modified nucleotides, and 3'-nitrogen-substituted modified nucleotides. Nucleotides, 2'-deoxy-2'-fluorinated nucleotides, 5'-methyl-2'-fluorinated nucleotides, 2'-deoxy nucleotides, vinylphosphonate nucleotides, thiophosphate nucleotides, dithiophosphate nucleotides, locked nucleic acids (LNA), debased nucleotides, abasic ribose (Ab), reverse deoxyribonucleotides (3'-3' linked nucleotides), ethylene phosphate deoxyribonucleotides, reverse abasic nucleotides, deoxythymidine, reverse deoxythymidine, 2'-amino modified nucleotides, morpholino oligonucleotides (PMO), polypeptide nucleotides, aminophosphates, or non-natural base nucleotides.

[0033] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, have the following structure: 2'-Ethylnucleotide, with the following structure: The nucleotide modified with 2'-methoxy group has the following structure: 2'-Methoxyethyl nucleotide, with the following structure: 2'-O-methoxyethyl nucleotide, with the following structure:

[0034] 2'-Fluoronucleotide, with the following structure: Vinylphosphonate nucleotide (VP), with the following structure: Phosphophosphate nucleotide (S), with the following structure: Phosphonucleotide (p), structure: 2'-Deoxyribonucleotide, with the following structure: The reverse debasing deoxyribose residue (invAb) has the following structure:

[0035] Glycol nucleic acids (GNAs), including (S)-glycol nucleic acids ((S)-GNAs), have the following structures: And (R)-ethylene glycol nucleic acid ((R)-GNA), the structure is: The TNA structure is as follows:

[0036] 3'-OMe-2',5'-PS, structure is

[0037] Where Base represents a base, Me represents a methyl group, and Et represents an ethyl group.

[0038] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2′ and 4′ carbons. This structure effectively "locks" the ribose in a 3′-endonucleotide conformation. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) MolCancTher 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0039] Representative U.S. patents for the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125 and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0040] In some embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholine sugar moieties and their use in oligomeric compounds have been reported (see, for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0041] The term "morpholino" refers to a sugar substitute having the following formula:

[0042]

[0043] In some embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the morpholino structure described above. Such sugar substitutes are referred to herein as "modified morpholino groups".

[0044] In this invention, unless otherwise specified, uppercase letters C, G, U, and A represent the base composition of nucleotides. Lowercase letters c, g, u, and a respectively indicate that the nucleotide represented by the corresponding uppercase letter is modified with a methoxy group; underscores indicate that the nucleotide represented by the uppercase letter is modified with fluorination; the spacer "·" indicates that the two nucleotide residues adjacent to the spacer "·" are linked by a thiophosphate group; VP indicates that the nucleotide to the right of the letter VP is a (E)-vinylphosphonate modified nucleotide. For example, "a·g" indicates that the a and g residues are linked by a thiophosphate group.

[0045] It should be emphasized that the “modification” of nucleotides described in this disclosure includes, but is not limited to, the examples above. Nucleotides may also be replaced with other nucleotides, such as (S)-glycerol nucleic acids.

[0046] The term "homological" or "homology" generally refers to the number of nucleotides in the subject nucleic acid sequence that have matched the same nucleotides in the reference nucleic acid sequence, usually determined by sequence analysis procedures (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877) or by visual inspection. As used herein, the term "complete homology" or "complete homology" generally refers to complete (100%) homology or "identity" between the reference sequence and the subject nucleic acid sequence. As used herein, the terms “substantially homologous” or “substantially homologous” generally mean that the subject sequence and the reference sequence share at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) homologous nucleotides at the same nucleotide positions.

[0047] The terms “inducing,” “inhibiting,” “enhancing,” “increasing,” “reducing,” and “lowering” typically describe quantitative differences between two states. For example, “the amount of AGT activity or expression effectively inhibited” means that the level of AGT activity or expression in the treated sample will be lower than the level of AGT activity or expression in the untreated sample. These terms apply, for example, to expression levels and activity levels. The terms “reducing” and “lowering” are used interchangeably and generally indicate any change less than the original. “Reducing” and “lowering” are relative terms and need to be compared between before and after measurement. “Reducing” and “lowering” include complete depletion.

[0048] The term "at least" includes the number adjacent to it, and all subsequent numbers or integers that are logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in that series or range.

[0049] The term "reduction" refers to an overall reduction, detectable by standard methods known in the art (such as those described herein), of the expression level / amount of a gene, gene product (e.g., protein), or biomarker in a first sample compared to the expression level / amount of the corresponding gene, gene product (e.g., protein), or biomarker in a second sample by approximately 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the term "reduction" refers to a reduction in the expression level / amount of a gene or biomarker in the first sample, wherein the reduction is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold in the expression level / amount of the corresponding gene or biomarker in the second sample. In some embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0050] The term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modifications (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0051] The term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations typically contain salts, excipients, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. These pharmaceutically acceptable formulations may also typically contain compatible solid or liquid fillers, diluents, or encapsulation materials suitable for human administration. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and these should not be excluded from the scope of this application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0052] The pharmaceutical compositions disclosed herein include those suitable for oral, nasal, topical, sublingual, sublingual, rectal, and / or parenteral administration. The formulations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier substance to prepare a single-dose form is generally the amount of the compound that produces the therapeutic effect. Generally, in percent units, this amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0053] The term "prevention and / or treatment" encompasses not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it and / or preventing a further increase in the severity of the disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effects that are generally beneficial to the patient being treated. The nucleic acid or pharmaceutical composition of this application, constituting a viable therapeutic agent, does not require the complete cure or eradication of any symptom or manifestation of the disease. As recognized in the relevant art, a drug used as a therapeutic agent may reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically constitutes a viable preventive agent and does not need to completely and effectively prevent the onset of the condition. It is sufficient to simply reduce the effects of the disease in the subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reduce the likelihood of the disease occurring or worsening.

[0054] The terms "disease" and "symptom" are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the condition of the body or certain organs that impairs or disrupts the performance of functions, and / or causes symptoms such as discomfort, dysfunction, pain, or even death in those who are ill or in contact with them. Disease or symptom may also be referred to as disorder, ailing, ailment, malady, disorder, sickness, illness, complaint, inderdisposion, or affectation.

[0055] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of the present invention, prepared by reacting a compound having specific substituents discovered in the present invention with a pharmaceutically acceptable acid or base.

[0056] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of this invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, and transdermal penetration enhancers. Their formulations are well known to those skilled in the art of cosmetics or topical pharmaceuticals. For further information on carriers, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0057] The term "excipient" generally refers to the carrier, diluent, and / or medium required to formulate an effective pharmaceutical composition. Excipients include (but are not limited to): absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextran, diluents, disintegrants, emulsifiers, swelling agents, fillers, flavoring agents, flow aids, wetting agents, oils, polymers, preservatives, brine, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tensioning agents, mediators, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents, and aroma agents.

[0058] For pharmaceuticals or pharmacologically active agents, the term "effective amount" or "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. For the oral dosage forms of this invention, the "effective amount" of one active substance in the composition refers to the quantity required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. A suitable effective amount in any given case can be determined by a person skilled in the art through routine testing.

[0059] The terms “active ingredient,” “therapeutic agent,” “active substance,” or “active agent” refer to a chemical entity that can effectively treat a target disorder, disease, or symptom.

[0060] "Optional" or "optionally" means that the event or condition described below may occur but is not required to occur, and the description includes both the scenario in which said event or condition occurs and the scenario in which said event or condition does not occur.

[0061] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention. Attached Figure Description

[0062] Figure 1 A schematic diagram showing the changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugate of this invention. Detailed Implementation

[0063] The present application will be described in further detail below with reference to the embodiments, but the implementation of the present application is not limited thereto.

[0064] Example 1: Synthesis of the modified sequence of the RNAi agent

[0065] The modified RNAi agent was prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthetic route can be found in patent document CN112313335A. The sequence of the modified RNAi agent is shown in Table 1.

[0066] Table 1 shows the modified sequence list of RNAi agents.

[0067]

[0068] in,

[0069] A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; I = hypoxanthine-3'-phosphate; T = thymidine-3'-phosphate;

[0070] Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Im = 2'-O-methylhypoxanthine-3'-phosphate;

[0071] Am-s-=2'-O-methyladenosine-3'-thiophosphate; Um-s-=2'-O-methyluridine-3'-thiophosphate; Cm-s-=2'-O-methylcytidine-3'-thiophosphate; Gm-s-=2'-O-methylguanosine-3'-thiophosphate.

[0072] Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate;

[0073] Af-s-=2'-fluoroadenosine-3'-thiophosphate; Uf-s-=2'-fluorouridine-3'-thiophosphate; Cf-s-=2'-fluorocytidine-3'-thiophosphate; Gf-s-=2'-fluoroguanosine-3'-thiophosphate.

[0074] dA = 2′-deoxyadenosine-3′-phosphate; dC = 2′-deoxycytidine-3′-phosphate; dG = 2′-deoxyguanosine-3′-phosphate; dT = 2′-deoxythymidine-3′-phosphate;

[0075] m = 2'-O-methyl; f = 2'-fluorine; s = thiophosphate;

[0076] VP structure is The GNA structure is as follows: The TNA structure is as follows: The structure of 3Me-2-5 is Ribavirin (abbreviated as Ri) has the following structure: Where Base represents a base, including the bases A, U, G, C, T, and I.

[0077] Example 2: Detection of siRNA effect on AGT gene expression in HuH7 cells

[0078] The effect of siRNA on AGT gene expression in HuH7 cells was detected by q-PCR (SYBGREEN method).

[0079] To determine the inhibitory efficiency at the mRNA level, AGT siRNA was transfected into HuH7 cells, and q-PCR was performed to determine the expression level of AGT mRNA. HuH7 cells were cultured in DMEM medium (Gibco 11966025) containing 10% fetal bovine serum (Gibco A5669701), 1% glutamine (Gibco 35050061), 1% NEAA (Gibco 11140050), and 1% penicillin-streptomycin (Gibco 15140-122). HuH7 cells in logarithmic growth phase were cultured at a rate of 2 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1:1 in 96-well cell culture plates, and siRNA was simultaneously seeded using Lipofectamine. TM The lipo2000 reagent mixture (INVITROGEN11668030) was mixed to achieve final RNAi reagent concentrations of 1 nM, 0.1 nM, and 0.02 nM. The lipo2000 reagent mixture was then transfected into cells. Cells were incubated overnight at 37°C in a 5% CO2 incubator. A lipofectamine-containing solution was also provided. TM2000 cells were used as a control group without the chemical compound. 48 hours after transfection, the culture medium was removed, and total RNA was extracted and reverse transcribed. Target cDNA was detected using the SYBGREEN assay, and GAPDH cDNA was detected as an internal control in parallel.

[0080] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression level of the target gene was calculated using a 2-1T / T method. -ΔΔCt This is indicated by subtracting the internal reference gene from the CT value of the target gene in each sample.

[0081] The ΔCT value was obtained by calculating the CT value of (GAPDH). Then, the ΔCT value of the control group (lipo2000 Control) containing only the transfection reagent was subtracted from the ΔCT value of the drug-treated group (sample) to obtain the ΔΔCT. Finally, the ΔΔCT was calculated using a 2-dimensional algorithm. -ΔΔCt Change

[0082] The relative expression level of AGT mRNA (value of sample) is then calculated.

[0083] Calculate the inhibition rate of the AGT gene mediated by siRNA analogs using the following formula.

[0084] AGT inhibition rate % = (1 - value of sample / Average value of lipo2000 Control) * 100.

[0085] The RNAi agent analogs of the present invention exhibit excellent inhibitory activity against AGT, as shown in Table 2:

[0086] Table 2

[0087]

[0088] Example 3: Modification sequence of RNAi agent + synthesis of ligand

[0089] The modified RNAi agent was prepared using an OligoMakerApS 192 RNA synthesizer (made in Denmark). The specific synthetic route can be found in patent document CN112313335A. The sequence of the modified RNAi agent is shown in Table 3.

[0090] Table 3

[0091]

[0092]

[0093] in,

[0094] A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; I = hypoxanthine-3'-phosphate; T = thymidine-3'-phosphate;

[0095] Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Im = 2'-O-methylhypoxanthine-3'-phosphate;

[0096] Am-s-=2'-O-methyladenosine-3'-thiophosphate; Um-s-=2'-O-methyluridine-3'-thiophosphate; Cm-s-=2'-O-methylcytidine-3'-thiophosphate; Gm-s-=2'-O-methylguanosine-3'-thiophosphate.

[0097] Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate;

[0098] Af-s-=2'-fluoroadenosine-3'-thiophosphate; Uf-s-=2'-fluorouridine-3'-thiophosphate; Cf-s-=2'-fluorocytidine-3'-thiophosphate; Gf-s-=2'-fluoroguanosine-3'-thiophosphate.

[0099] dA = 2′-deoxyadenosine-3′-phosphate; dC = 2′-deoxycytidine-3′-phosphate; dG = 2′-deoxyguanosine-3′-phosphate; dT = 2′-deoxythymidine-3′-phosphate;

[0100] m = 2'-O-methyl; f = 2'-fluorine; s = thiophosphate;

[0101] VP structure is The GNA structure is as follows: The TNA structure is as follows: The structure of 3Me-2-5 is Ribavirin (abbreviated as Ri) has the following structure:

[0102] Wherein, Base represents a base, including the bases A, U, G, C, T, and I;

[0103] L96 is:

[0104] Example 4: AGT Detection Test in Transgenic Mice

[0105] The in vivo efficacy of siRNA compounds was evaluated using 5-8 week old AGT humanized mice (purchased from Jicui Pharmaceutical). The specific procedures were as follows: On day -1 (one day before administration), plasma samples were obtained from mice before administration. AGT protein levels were detected using ELISA according to the experimental protocol provided by the supplier (Immuno-Biological Laboratories), and mice were randomly assigned to groups of four based on AGT protein levels. The siRNA compounds in each group were diluted with physiological saline, and then administered subcutaneously on day 0 according to the experimental group design at a dose of 1 mg / kg. The solvent group received an equal volume of physiological saline without siRNA compounds. Plasma samples were obtained from mice on days 7, 14, 21, and 28 after administration, and AGT protein expression was detected. The inhibition rate was calculated using the following formula to determine the AGT inhibition rate of a specific animal relative to the solvent, and then the average inhibition rate of plasma AGT in each group was calculated.

[0106] The inhibition rate of plasma AGT protein relative to the solvent group (%) = (1 - plasma AGT protein expression level of a specific mouse / mean plasma AGT protein expression level of mice in the solvent group) * 100, as shown in Table 4:

[0107] Table 4

[0108]

[0109] Example 5: AGT Detection Test in Transgenic Mice

[0110] The in vivo efficacy of siRNA compounds was evaluated using 5-8 week old AGT humanized mice (purchased from Jicui Pharmaceutical). The specific procedures were as follows: On day -1 (one day before administration), plasma samples were obtained from mice before administration. AGT protein levels were detected using ELISA according to the experimental protocol provided by the supplier (Immuno-Biological Laboratories), and mice were randomly assigned to groups of four based on AGT protein levels. The siRNA compounds in each group were diluted with physiological saline, and then administered subcutaneously on day 0 according to the experimental design, at a dose of 1 mg / kg. The solvent group received an equal volume of physiological saline without siRNA compounds. Plasma samples were obtained from mice on days 7, 14, 21, 28, 35, and 42 after administration, and AGT protein expression was detected. The inhibition rate was calculated using the following formula to determine the AGT inhibition rate of a specific animal relative to the solvent, and then the average inhibition rate of plasma AGT in each group was calculated.

[0111] The inhibition rate of AGT protein in mouse plasma before drug administration (%) = (Plasma AGT protein expression level of a specific mouse / Plasma AGT protein expression level of a specific mouse before drug administration - 1) * 100, as shown in Table 5:

[0112] Table 5

[0113]

[0114] Example 6: Drug Efficacy Test in Crab-Eating Monkeys

[0115] Twenty male cynomolgus macaques had their serum AGT protein levels measured three days prior to drug administration. Based on the test results and animal weight, they were randomly divided into five groups (n=4 per group) and administered a single subcutaneous injection of S2L, S9L, S15L, and AD-85481 at a dose of 1 mg / kg and a volume of 0.3 mL / kg.

[0116] The first administration date was designated as day 0 (D0). Blood samples were collected on days 3 before administration (D-3) and on days 7 (D7), 14 (D14), 21 (D21), 28 (D28), 35 (D35), 42 (D42), 49 (D49), 56 (D56), and 70 (D70) after administration. AGT protein expression was measured using a human AGT ELISA kit (IBL, 27412), and the change in AGT level relative to baseline (D-3) was calculated.

[0117] The results showed that all siRNA conjugates of the present invention significantly downregulated the concentration of AGT protein in cynomolgus monkey serum. Among them, S9L showed the most significant reduction in AGT protein levels in cynomolgus monkey serum, reaching steady state on day 14 after a single dose, and maintaining an AGT protein inhibition rate of over 90% until day 70 (day 70) after administration. Figure 1 As shown. Furthermore, S2L and S15L showed superior efficacy compared to AD-85481 at 7 and 14 days post-administration.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An RNAi agent for inhibiting the expression of angiotensinogen (AGT) in cells, said RNAi agent comprising a sense strand and an antisense strand, said sense strand and antisense strand forming a double-stranded region, wherein, The sense and antisense chains are selected from the sequences shown in Table 1.

2. The RNAi agent according to claim 1, characterized in that, The RNAi agent is linked to a target ligand.

3. The RNAi agent according to claim 2, characterized in that, The targeting ligand is attached to the 3' or 5' end of the sense chain.

4. The RNAi agent according to claim 2, characterized in that, The targeting ligand contains the N-acetyl-galactosamine (GalNAc) moiety.

5. The RNAi agent according to claim 2, characterized in that, The targeted ligand is selected from:

6. The RNAi agent according to claim 5, characterized in that, The targeting ligand is attached to the 3' end of the sense chain.

7. The RNAi agent according to claim 1, characterized in that, The RNAi agent is selected from the sequences shown in Table 3.

8. A pharmaceutical composition, characterized in that, It comprises the RNAi agent according to any one of claims 1-7, and one or more pharmaceutically acceptable excipients and / or carriers.

9. Use of the RNAi agent according to any one of claims 1-7 or the pharmaceutical composition according to claim 8 in the preparation for the prevention or treatment of diseases related to AGT.

10. The use according to claim 9, characterized in that, The AGT-related diseases mentioned are selected from hypertension.

Images