Pharmaceutical compositions and uses thereof

Abstract

A pharmaceutical composition comprising a first RNAi agent and a second RNAi agent, wherein the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA complexes capable of suppressing APOC3 mRNA, and the second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes capable of suppressing PCSK9 mRNA, and the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 10:1 to 1:10 in terms of the total amount of siRNAs and siRNA groups. The pharmaceutical composition and treatment method provided by the present disclosure can effectively treat and / or prevent diseases or symptoms associated with hyperlipidemia.

Description

[Technical Field]

[0001] The present disclosure relates to a pharmaceutical composition capable of treating and / or preventing a disease or condition associated with hyperlipidemia. The present disclosure also relates to uses of the pharmaceutical composition and methods for treating and / or preventing a disease or condition associated with hyperlipidemia. [Background technology]

[0002] Dyslipidemia, also known as hyperlipidemia, is a systemic disease in which plasma lipids are elevated above normal levels due to abnormalities in lipid metabolism or function. Persistently elevated low-density lipoprotein cholesterol (LDL-C) is a significant risk factor that induces and accelerates the development and progression of atherosclerosis, and is closely related to ischemic heart disease and cerebrovascular disease, posing a serious threat to the health of patients worldwide.

[0003] Research has shown that apolipoprotein C3 (APOC3) plays an important role in lipid metabolism, and that individuals with APOC3 mutations have a 46% reduction in circulating APOC3 expression and a 39% reduction in plasma triglyceride levels compared to the general population. The proprotein convertase subtilisin / kexin type 9 (PCSK9) gene plays an important role in lipid metabolism, particularly cholesterol metabolism, and suppressing the expression of the PCSK9 gene and / or APOC3 gene can effectively lower plasma total cholesterol (TC) levels.

[0004] Therefore, blocking APOC3 and PCSK9 by silencing gene expression at the gene level using small interfering RNA (siRNA) would undoubtedly be an ideal means of treating dyslipidemia. However, current research has shown that when siRNA inhibiting APOC3 or PCSK9 is used alone, it is difficult to reduce LDL-C to very low levels, even with increasing dosage. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present disclosure have unexpectedly discovered that when an RNAi agent capable of suppressing APOC3 mRNA and an RNAi agent capable of suppressing PCSK9 mRNA are used in combination at a specific ratio or dose to prevent and / or treat a disease or symptom associated with hyperlipidemia, a highly synergistic effect is exhibited, and not only can the TC level in plasma be reduced, but the plasma LDL-C level can also be stably and significantly reduced over a long period of time, thereby demonstrating excellent therapeutic effects. [Means for solving the problem]

[0006] In one aspect, the present disclosure provides a pharmaceutical composition comprising a first RNAi agent and a second RNAi agent, wherein the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA complexes, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of the first siRNA complexes comprises an siRNA group formed from one of the first siRNAs and a complex group complexed thereto; the second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each second siRNA being independently an siRNA capable of suppressing PCSK9 mRNA, and each second siRNA complex comprises an siRNA group formed from one of the second siRNAs and a complex group complexed thereto; The weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively.

[0007] In another aspect, the present disclosure further provides a use of a pharmaceutical composition of the present disclosure in the preparation of a medicament for treating and / or preventing a disease or condition associated with hyperlipidemia.

[0008] In another aspect, the present disclosure further provides a method for treating and / or preventing a disease or condition associated with hyperlipidemia, the method comprising administering a first RNAi agent and a second RNAi agent to a subject in need thereof, wherein the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA complexes, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of the first siRNA complexes comprises an siRNA group formed from one of the first siRNAs and a complex group conjugated thereto; the second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each second siRNA being an siRNA capable of suppressing PCSK9 mRNA, and each second siRNA complex comprising an siRNA group formed from one of the second siRNAs and a complex group complexed thereto; The weight ratio of the first RNAi agent to the second RNAi agent administered to the subject is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively.

[0009] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Effects of the Invention]

[0010] Compared with the single administration of an RNAi agent that inhibits APOC3 mRNA or an RNAi agent that inhibits PCSK9 mRNA, the composition and treatment method provided by the present disclosure have significantly superior effects in reducing both TC levels and LDL-C levels, demonstrating clear synergistic effects. Particularly noteworthy is the strong synergistic effect shown in the reduction of LDL-C levels, which unexpectedly breaks through the limitations of LDL-C level reduction achieved by the single administration of the first or second RNAi agent. The pharmaceutical composition and treatment method of the present disclosure have excellent potential for application in the prevention and / or treatment of symptoms or diseases associated with hyperlipidemia.

[0011] Regarding changes in TC levels, on the one hand, under the condition that the total dose was 6 mg / kg in both cases, mice administered 6 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone showed almost no decrease in TC, whereas mice administered the pharmaceutical compositions of the present disclosure (3 mg / kg each of an RNAi agent that inhibits APOC3 mRNA and an RNAi agent that inhibits PCSK9 mRNA) showed a TC reduction rate of 45% or more (TC reduction rate = 100% - normalized value of TC content), and even when the TC reduction rate in the mice of the group administered with an RNAi agent that inhibits APOC3 mRNA and the group administered with an RNAi agent that inhibits PCSK9 mRNA were added together, the TC reduction rate was less than 45%. Under the condition that the total dose was 4 mg / kg in both cases, the combined TC reduction rate in the mice of the group administered 1 mg / kg of an RNAi agent that inhibits APOC3 mRNA alone and the group administered 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone was a maximum of 34.9%, whereas the TC reduction rate in the mice administered the pharmaceutical composition of the present disclosure (1 mg / kg of an RNAi agent that inhibits APOC3 mRNA, 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA) reached 37.2%. On the other hand, taking the change in TC levels on day 8 after administration as an example, under the condition that the total dose was 6 mg / kg in all cases, no decrease in TC was observed in the mice of the group administered 6 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone, whereas the group administered the pharmaceutical composition of the present disclosure (3 mg / kg each of an RNAi agent that inhibits APOC3 mRNA and an RNAi agent that inhibits PCSK9 mRNA) showed a 45.6% decrease in TC, and the sum of the TC decrease rates in the group administered the RNAi agent that inhibits APOC3 mRNA (3 mg / kg) and the group administered the RNAi agent that inhibits PCSK9 mRNA (3 mg / kg) was only 33.3%.Under the condition that the total dose was 4 mg / kg in both cases, the combined TC reduction rate of Group 1, which received 1 mg / kg of an RNAi agent that inhibits APOC3 mRNA alone, and the combined TC reduction rate of the group that received 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone was a maximum of 34.9%, while the TC reduction rate of the group that received the pharmaceutical composition of the present disclosure (1 mg / kg of an RNAi agent that inhibits APOC3 mRNA, 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA) reached 37.0%.

[0012] Regarding changes in LDL-C levels, on the one hand, under the condition that the total dose was 6 mg / kg, in the bodies of mice administered with an RNAi agent inhibiting PCSK9 mRNA alone, a maximum LDL-C reduction rate of 43.3% (LDL-C reduction rate = 100% - normalized value of LDL-C content) was shown, while in the bodies of mice administered with the pharmaceutical composition of the present disclosure, an LDL-C reduction rate of 72.8% was shown. On the other hand, neither the administration of a high dose of an RNAi agent inhibiting APOC3 mRNA alone nor the administration of a high dose of an RNAi agent inhibiting PCSK9 mRNA alone was able to reduce plasma LDL-C levels to 50% or less, while in the bodies of mice administered with a low dose (4 mg / kg) of the pharmaceutical composition of the present disclosure, an LDL-C reduction rate of 67% was already shown. Taking the change in LDL-C levels on the 8th day after administration as an example, under the condition that the total dose was 6 mg / kg in both cases, the group administered only the RNAi agent that inhibits PCSK9 mRNA showed a 41.4% reduction in LDL-C, whereas the group administered the pharmaceutical composition of the present disclosure (3 mg / kg each of the RNAi agent that inhibits APOC3 mRNA and the RNAi agent that inhibits PCSK9 mRNA) showed a 72.8% reduction in LDL-C. DETAILED DESCRIPTION OF THE INVENTION

[0013] Specific embodiments of the present disclosure will be described in detail below. It should be understood that the specific embodiments described herein are merely for the purpose of illustrating and interpreting the present disclosure, and are not intended to limit the present disclosure.

[0014] In this disclosure, unless otherwise specified, APOC mRNA or "mRNA expressed by the APOC3 gene" refers to the mRNA having the sequence set forth in Genbank accession number NM_000040.3, the APOC3 gene refers to the gene that transcribes the above-mentioned APOC3 mRNA, PCSK9 mRNA or "mRNA expressed by the PCSK9 gene" refers to the mRNA having the sequence set forth in Genbank accession number NM_174936.3, the PCSK9 gene refers to the gene that transcribes the above-mentioned PCSK9 mRNA, ANGPTL3 mRNA or "mRNA expressed by the ANGPTL3 gene" refers to the mRNA having the sequence set forth in Genbank accession number NM_014495.4, and the ANGPTL3 gene refers to the gene that transcribes the above-mentioned ANGPTL3 mRNA.

[0015] definition Unless otherwise specified in the context, capital letters C, G, U, and A represent the base composition of a nucleotide, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letter s represents that two nucleotides adjacent to the left and right of the letter s are bonded by a thiophosphate ester, and P1 represents that one nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. In some embodiments, P1 is VP, Ps, or P, where the modification is specified, and the combination VP indicates that one nucleotide adjacent to the right of the combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide, the combination Ps indicates that one nucleotide adjacent to the right of the combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that one nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide, and the capital letter P is underlined. S is the character Srepresents a stabilizing modified nucleotide. Each U and T base in all sequences of the present disclosure can be substituted for another without appreciably affecting the activity or off-target effects of the siRNA of the present disclosure.

[0016] In this context, the term "fluoro-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl of the ribose of the nucleotide is substituted with fluorine, and the term "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the 2'-hydroxyl of the ribose of the nucleotide is substituted with a non-fluorine group. These nucleotides in which the 2'-hydroxyl of the ribose is substituted with a non-fluorine group are well known to those skilled in the art, and these nucleotides may be one selected from 2'-alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides. In some embodiments, the 2'-alkoxy-modified nucleotide may be a methoxy-modified nucleotide. The term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl of the ribose is substituted with methoxy. "Nucleotide analog" refers to a group that can substitute for a nucleotide in a nucleic acid but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide, such as an isonucleotide, bridged nucleotide (abbreviated as BNA), or acyclic nucleotide.

[0017] In some embodiments, the "non-fluoro-modified nucleotide" may be a stabilizing modified nucleotide, where the stabilizing modified nucleotide refers to a nucleotide in which the 2'-hydroxyl of the ribose of the nucleotide is substituted with a stabilizing modified group, and the siRNA containing the stabilizing modified nucleotide has increased thermal stability compared to an siRNA in which the nucleotide in the corresponding position is an unmodified nucleotide. The stabilizing modified nucleotide refers to a nucleotide in which the 2'-hydroxyl of the ribose of the nucleotide is substituted with a stabilizing modified group, and the siRNA containing the stabilizing modified nucleotide has increased thermal stability compared to an siRNA in which the nucleotide in the corresponding position is an unmodified nucleotide. In some embodiments, the stabilizing modified group is one or more selected from 2'-O-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-O-2-N-methylamino-2-oxiylideneethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl, and 2'-O-2,4-dinitrophenyl.

[0018] In the context of this specification, the terms "complementary" and "reverse complementary" may be used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, bases on one strand pair complementary to bases on the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, the strands are considered complementary and the sequence of the strand can be deduced from the sequence of the complementary strand. Accordingly, in the art, the term "mismatch" means that bases at corresponding positions in a double-stranded nucleic acid are not paired complementary to each other.

[0019] Unless otherwise specified in context, "essentially reverse complementary" refers to the presence of no more than three base mismatches between two related nucleotide sequences, "substantially reverse complementary" refers to the presence of no more than one base mismatch between two nucleotide sequences, and "fully reverse complementary" refers to the absence of any base mismatches between two nucleotide sequences.

[0020] In the context of the present disclosure, particularly when describing the methods for preparing siRNA, pharmaceutical compositions, or siRNA complexes, unless otherwise specified, the term "nucleoside monomer" refers to modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites; RNA phosphoramidites are sometimes referred to as nucleoside phosphoramidites) used in solid-phase phosphoramidite synthesis, depending on the type and order of nucleotides in the siRNA or siRNA complex to be prepared. Solid-phase phosphoramidite synthesis is a method used for RNA synthesis known to those skilled in the art. All nucleoside monomers used in the present disclosure are commercially available.

[0021] It will be understood by those skilled in the art that with respect to any group that contains one or more substituents, it is not intended that these groups introduce any substitutions or substitution patterns that are sterically impractical, synthetically impractical, and / or inherently unstable.

[0022] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl is a straight and branched chain alkyl containing 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbon atoms, it is intended to include all branched and straight chain forms having that number of carbon atoms. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue similar to alkyl but having two points of attachment.

[0023] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl having at least one carbon-carbon double bond, which is obtained by removing one hydrogen molecule from adjacent carbon atoms of a parent alkyl. The group may be in the cis or trans configuration about the double bond. Typical alkenyl groups include, but are not limited to, vinyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl), butenyl (e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl), and the like. In some embodiments, alkenyl groups have from 2 to 20 carbon atoms, and in others, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to the same residues as alkenyl but with two points of attachment.

[0024] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl having at least one carbon-carbon triple bond, where the carbon-carbon triple bond is obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl (e.g., prop-1-yn-1-yl, prop-2-yn-1-yl), butynyl (e.g., but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl), and the like. In some embodiments, alkynyl has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to a residue similar to alkynyl but with two points of attachment.

[0025] As used herein, "alkoxy" refers to an alkyl having the specified number of carbon atoms attached through an oxygen bridge, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. Alkoxy typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through the oxygen bridge.

[0026] As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms from 6 to 18 carbon atoms, and at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl includes, but is not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to a residue similar to aryl but with two points of attachment.

[0027] "Heteroaryl" refers to a group derived from a 5- to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, in which at least one ring is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system according to Hückel theory. Heteroaryl includes fused or bridged ring systems. In some embodiments, a heteroatom in a heteroaryl is an oxidized heteroatom. In some embodiments, a heteroaryl contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in a heteroaryl is a quaternized nitrogen atom. A heteroaryl is bonded to the remainder of the molecule through any atom in the ring. Illustrative examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzindole, 1,3-benzodioxazolyl, benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzo Pyrronyl, benzofuryl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,[2-c]pyridazinyl, dibenzofuryl, dibenzothiophenyl, furyl, furanonyl, furo[3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridyl, isothiazolyl, imidazolyl, indazolyl, indole, isoindole, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, oxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolidinyl Allyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo Examples include, but are not limited to, [4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl / thienyl.

[0028] A variety of hydroxy protecting groups can be used in the present disclosure. Generally, a protecting group can render a chemical functionality insensitive to certain reaction conditions and can be added to or removed from that functionality in a molecule without substantially damaging the remainder of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive illustrative examples of hydroxy protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0029] The term "subject," as used herein, refers to any animal, e.g., a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0030] Pharmaceutical Compositions of the Present Disclosure In one aspect, the present disclosure provides pharmaceutical compositions capable of treating and / or preventing diseases or conditions associated with hyperlipidemia. As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic effect. A "therapeutic effect" refers to eradicating or ameliorating the underlying disorder being treated. A therapeutic effect is also achieved by observing an improvement in a subject by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the subject may still suffer from the underlying disorder.

[0031] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including, but not limited to, a prophylactic effect. To achieve a "prophylactic effect," a pharmaceutical composition of the present disclosure can be administered to a subject at risk of developing a specific disease, or to a subject who has been reported to have one or more physiological symptoms of the disease, even if the subject has not been diagnosed with the disease. The term "preventing a disease or symptom associated with hyperlipidemia" refers to preventing the rise of lipids in the subject's body to abnormal levels by administering a pharmaceutical composition of the present disclosure to the subject when the subject begins to show a tendency toward elevated lipids. The abnormal levels refer to a total cholesterol (TC) level exceeding 6.2 mmol / L, a triglyceride (TG) level exceeding 2.3 mmol / L, and / or a low-density lipoprotein cholesterol (LDL-C) level exceeding 4.1 mmol / L in the body of a human subject.

[0032] The pharmaceutical composition of the present disclosure comprises a first RNAi agent and a second RNAi agent, wherein the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA complexes, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of the first siRNA complexes comprises an siRNA group formed from one of the first siRNAs and a complex group complexed thereto.

[0033] The second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each of which is independently an siRNA capable of suppressing PCSK9 mRNA, and each of which comprises an siRNA group formed from one of the second siRNAs and a complex group complexed thereto.

[0034] In the context of the present disclosure, unless otherwise specified, "conjugate" refers to the covalent bonding between two or more chemical moieties, each having a specific function, and accordingly, "conjugate" refers to a compound formed by the covalent bonding between the respective chemical moieties. Furthermore, "siRNA complex" refers to a compound formed by covalently bonding one or more chemical moieties having a specific function to an siRNA. Depending on the context, the siRNA complex should be understood as a general term for multiple siRNA complexes or an siRNA complex represented by a certain chemical formula. In the context of the present disclosure, a "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by reaction to ultimately form an siRNA complex. As used in the present disclosure, the "siRNA complex" includes an siRNA group and a conjugate group portion, and the siRNA group refers to a chemical moiety formed by removing one or more atoms from an siRNA molecule. It will be understood by those skilled in the art that the removal of one or more atoms does not impair the inhibitory activity or stability of the siRNA against the target mRNA, and that, for example, the siRNA group may be a chemical moiety formed by removing a hydrogen atom from a phosphate ester bond from an siRNA, a chemical moiety formed by removing a hydrogen atom from the 5' hydroxyl of the 5'-terminal nucleotide of the sense strand or antisense strand from an siRNA, or a chemical moiety formed by removing a hydrogen atom from the 3' hydroxyl of the 3'-terminal nucleotide of the sense strand or antisense strand from an siRNA.

[0035] The weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition, based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively, is 100:1 to 1:100, and may be, for example, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:100, 1:50, 1:25, 1:15, 1:10, 1:5, 1:3, 1:4, or 1:2. In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is preferably 10:1 to 1:10, 5:1 to 1:5, or 2:1 to 1:5. In some embodiments, to achieve a better therapeutic / prophylactic effect, the weight of the second RNAi agent is equal to or greater than the weight of the first RNAi agent, and the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 1:1 to 1:10, 1:1 to 1:5, or 1:1 to 1:3.

[0036] In some embodiments, the weight of the first RNAi agent in the pharmaceutical composition is 1 mg to 1000 mg, e.g., 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg, based on the total amount of siRNA and siRNA group contained in the first and second RNAi agents, respectively. In some embodiments, the weight of the first RNAi agent is 5 mg to 500 mg.

[0037] In some embodiments, the weight of the second RNAi agent is 1 mg to 1000 mg, e.g., 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg. In some embodiments, the weight of the first RNAi agent in the pharmaceutical composition is 5 mg to 500 mg, and in some embodiments, the weight of the second RNAi agent is 5 mg to 800 mg.

[0038] In some embodiments, the pharmaceutical composition further comprises a third RNAi agent, the third RNAi agent comprising one or more third siRNAs and / or one or more third siRNA complexes, each of the third siRNAs independently being an siRNA capable of suppressing expression of ANGPTL3 mRNA, and each of the third siRNA complexes comprising a third siRNA group formed from one of the third siRNAs and a complex group conjugated thereto. In some embodiments, the third RNAi agent, the first RNAi agent, and / or the second RNAi agent can further enhance the therapeutic and / or preventive effect of the pharmaceutical composition of the present disclosure on diseases and / or symptoms associated with hyperlipidemia.

[0039] In some embodiments, the weight ratio of the third RNAi agent to the second RNAi agent in the pharmaceutical composition, based on the total amount of siRNA and siRNA groups contained in the third RNAi agent and the second RNAi agent, respectively, is 100:1 to 1:100, for example, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:100, 1:50, 1:25, 1:15, 1:10, 1:5, 1:3, 1:4, or 1:2. In some embodiments, the weight ratio of the third RNAi agent to the second RNAi agent in the pharmaceutical composition is preferably 10:1 to 1:10, 5:1 to 1:5, or 2:1 to 1:5.

[0040] In some embodiments, the weight of the third RNAi agent is 1 mg to 1000 mg, for example, 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg. In some embodiments, the weight of the third RNAi agent in the pharmaceutical composition is 5 mg to 500 mg, and in some embodiments, the weight of the third RNAi agent is 5 mg to 800 mg.

[0041] First RNAi agent As described above, the pharmaceutical composition of the present disclosure includes a first RNAi agent, which includes one or more first siRNAs and / or one or more first siRNA complexes, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of which includes an siRNA group formed from one of the first siRNAs and a complex group conjugated thereto.

[0042] The siRNA capable of suppressing APOC3 mRNA may be any siRNA capable of suppressing APOC3 mRNA. In some embodiments, each first siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently modified or unmodified, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the first siRNA comprises at least one siRNA, and the sense strand of the first siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 1, or the sense strand has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 2. 5'-UGGCCUCCCAAUAAAGCUGGACAAGAAGCU-3' (SEQ ID NO: 1) 5'-UGCUUAAAAGGGACAGUAUUCUCAGUGCUC-3' (SEQ ID NO: 2)

[0043] In some embodiments, the sense strand has at least 15, 16, 17, 18, or 19 contiguous nucleotides that match, for example, the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, in the 5'-3' direction, the sense strand comprises a contiguous nucleotide sequence that matches nucleotides 9-27 in the nucleotide sequence set forth in SEQ ID NO: 1, or a contiguous nucleotide sequence that matches nucleotides 4-22 in the nucleotide sequence set forth in SEQ ID NO: 2.

[0044] The length of the sense strand and the length of the antisense strand of the first siRNA may be the same or different. In some embodiments, the ratio of the lengths of the sense strand and the antisense strand of the first siRNA is 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 20 / 28, 20 / 29, 20 / 30, 20 / 31, 20 / 32, 20 / 33, 20 / 34, 20 / 35, 20 / 36, 20 / 37, 20 / 38, 20 / 39, 20 / 40, 2 For example, a length ratio of "19 / 19" indicates that the sense strand and antisense strand are both 19 nucleotides long, a length ratio of "19 / 21" indicates that the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long, and so on. In some embodiments, the length ratio of the sense strand to the antisense strand of the first siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0045] In some embodiments, in the first siRNA, some nucleotides in the sense strand and antisense strand are modified nucleotides, and some nucleotides are unmodified nucleotides. In some embodiments, in the first siRNA, all nucleotides in the sense strand and antisense strand are modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 7 to 9 of the sense strand are 2'-fluoro-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the other nucleotides in the antisense strand are each independently one of non-fluoro-modified nucleotides.

[0046] In some embodiments, the first siRNA may be, for example, any one of the siRNAs shown in Table 1a and Table 1b, and siRNA complexes containing these siRNAs or siRNA groups formed from these siRNAs exhibit high APOC3 mRNA suppression activity. To achieve a better suppression effect, in some embodiments, the first siRNA has the sequence shown in, for example, siAPOC3b1-M1SP1 and / or siAPOC3b3-M1S.

[0047] Table 1a. Sequence of the first siRNA [Table 1] TIFF2026507286000002.tif244170 TIFF2026507286000003.tif173170

[0048] Table 1b. Sequence of the first siRNA [Table 2]

[0049] Here, the capital letters C, G, U, and A represent the base composition of the nucleotide, the lower case letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, and the underlined capital letters S is the character S represents that the adjacent nucleotide on the left side of P1 is a stabilizing modified nucleotide, the lowercase letter s represents that the two nucleotides on the left and right sides of the letter s are linked by a phosphorothioate, and P1 represents that the adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. S is a specific stabilizing modification, e.g. tired and the underlined combination letters tired is the combination character tired represents that the nucleotide adjacent to the left of is a nucleotide having a 2'-O-methoxyethyl modification. In some embodiments, P1 represents VP, Ps, or P, with a specified modification; the combination letter VP represents that the nucleotide adjacent to the right of the combination letter VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; the combination letter Ps represents that the nucleotide adjacent to the right of the combination letter Ps is a phosphorothioate-modified nucleotide; and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide. Furthermore, each U in the sequences shown in Tables 1a to 1c above can be arbitrarily substituted with T without any apparent effect on the activity or off-target effects of the siRNA.

[0050] In some embodiments, the conjugate group in each of the first siRNA complexes comprises a linker and a pharmaceutically acceptable targeting group and / or delivery-assisting group, wherein the first siRNA group, the linker, the targeting group, or the delivery-assisting group are covalently or non-covalently linked in that order, each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery-assisting group is selected from groups capable of improving the biocompatibility of the first siRNA complex in the target organ or tissue.

[0051] Typically, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, with the siRNA group, the linker, and the targeting group being bound in that order. In some embodiments, the number of targeting groups is 1 to 6. In some embodiments, the number of targeting groups is 2 to 4. The siRNA group may be conjugated to the conjugated group non-covalently or covalently, for example, covalently. The conjugation site between the siRNA group and the conjugated group may be at the 3' or 5' end of the sense strand of the siRNA group, the 5' end of the antisense strand, or an internal sequence of the siRNA group. In some embodiments, the conjugation site between the siRNA group and the conjugated group is at the 3' end of the sense strand.

[0052] In some embodiments, the conjugated group may be attached to the phosphate group, 2'-hydroxyl, or base of the siRNA group. In some embodiments, the conjugated group may be attached to the 3'-hydroxyl, in which case the nucleotides are linked via a 2'-5' phosphodiester bond. When attached to the end of the siRNA group, the conjugated group is typically attached to the phosphate group of the nucleotide, and when attached to the internal sequence of the siRNA group, the conjugated group is typically attached to the ribose sugar ring or base. For various linking methods, see Muthiah Manoharan et al., "siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes." ACS Chemical Biology, 2015, 10 (5): 1181-7.

[0053] In some embodiments, the siRNA group and the conjugation group can be linked by an acid-labile or reducible chemical bond, and in the acidic environment of cell endosome, these chemical bonds can be degraded, so that the siRNA group can be released, for example, the siRNA group can form the siRNA molecule again.For non-degradable conjugation methods, the conjugation group can be bound to the sense strand of the siRNA group, so that the influence of conjugation on the silencing activity of the siRNA group can be minimized.

[0054] Targeting group can be linked to siRNA group via suitable linker, and those skilled in the art can select suitable linker according to the specific type of targeting group.For these linkers, types of targeting group and methods of linking to siRNA group, refer to the disclosure of WO2015006740A2, the contents of which are incorporated herein by reference in their entirety.

[0055] In some embodiments, the targeting group may be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the disclosure of which is incorporated herein by reference in its entirety.

[0056] In some embodiments, the or each targeting group is selected from a ligand capable of binding to a cell surface receptor expressing the gene of interest.

[0057] In some embodiments, at least one or each of the targeting groups is selected from ligands capable of binding to receptors on the surface of mammalian liver parenchymal cells. In some embodiments, each of the targeting groups is independently a ligand with affinity for an asialoglycoprotein receptor on the surface of mammalian liver cells. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each said targeting group is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, and Nn-butyrylgalactosamine , N-Isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formylamino-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranoside The hydroxybenzoate is one selected from the group consisting of methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.In some embodiments, at least one or each said targeting group is galactose or N-acetylgalactosamine.

[0058] In some embodiments, the linker in the siRNA complex has the structure shown in formula (301):

[0059] [ka] Formula (301) In the formula, k is an integer of 1 to 3, L A has a structure containing an amide bond represented by formula (302), and L B has a structure containing an N-acylpyrrolidine represented by formula (303), contains a carbonyl and an oxygen atom, and L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane.

[0060] [ka] Formula (302) TIFF2026507286000007.tif121111 Formula (303) In the formula, n 302 , q 302 and p 302 are each independently an integer from 2 to 6, and optionally, n 302 , q 302 and p 302 are each independently 2 or 3, and n 303 is an integer between 4 and 16, and optionally, n 303 is an integer between 8 and 12, TIFF2026507286000008.tif1779 represents the site to which the group is covalently attached.

[0061] In the linker, LA are each bonded to one of the targeting groups by an ether bond, and L C The oxygen atom of the hydroxyl in the moiety L C is bonded to the L moiety by forming an ether bond, B is expressed by the carbonyl in formula (303) as L C The siRNA group is bonded to the siRNA via the oxygen atom in formula (303) by forming an amide bond with the nitrogen atom of the amino group in the siRNA group, and is bonded to the siRNA group via the oxygen atom in formula (303) by forming a phosphate bond or a thiophosphate bond with the oxygen atom in formula (303).

[0062] In some embodiments, the siRNA complex has the structure shown in formula (305).

[0063] [ka] Formula (305) In the formula, Nu represents the first siRNA group, the second siRNA group, or the third siRNA group.

[0064] In some embodiments, the linker in the siRNA complex has the structure shown in formula (306):

[0065] [ka] Formula (306) In the formula, n 306 is an integer from 0 to 3, and each p 306 are independently an integer from 1 to 6, TIFF2026507286000011.tif24112 represents a site to which a group is covalently attached, said linking group being *The linking group is linked to the targeting group by forming an ether bond through the oxygen atom indicated by #, and the linking group is linked to the siRNA group by forming a phosphate bond or a thiophosphate bond through at least one of the oxygen atoms indicated by #, and the remaining oxygen atom indicated by # is linked to a hydrogen atom to form a hydroxy or a C1-C3 alkyl to form a C1-C3 alkoxy.

[0066] In some embodiments, the siRNA complex has the structure shown in formula (307):

[0067] [ka] Formula (307) In the formula, Nu represents the first siRNA group, the second siRNA group, or the third siRNA group.

[0068] In some embodiments, the siRNA complex has the structure shown in formula (308):

[0069] [ka] Formula (308) In the formula, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4. m1, m2, and m3 each independently represent an integer selected from 2 to 10, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H or C1 to C 10 Alkyl, C1-C 10 Alkyl halides and C1-C 10 alkoxy; R3 has the structure shown in formula A59.

[0070] [ka] (A59) wherein E1 is OH, SH, or BH2, and Nu represents the first siRNA group, the second siRNA group, or the third siRNA group; R2 is a straight chain alkylene of 1 to 20 carbon atoms in length, where one or more of the carbon atoms is C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 R2 is optionally substituted with one or more groups selected from the group consisting of C1 to C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Alkyl halides, -OC1~C 10 Alkyl, -OC1~C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1~C 10 Alkyl halides, -SC1~C 10 Alkyl, -SC1~C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1~C 10 Alkyl halides, halogen substituents, -OH, -SH, -NH, -C 10 Alkyl-NH2, -N(C1-C 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -COH, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 Alkyl) (C1-C 10alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 Alkyl)C(O)(phenyl), -C(O)C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 Alkyl halides), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 and optionally one or more substituents from the group consisting of: alkyl halide; Each L1 is independently a straight chain alkylene of 1 to 70 carbon atoms in length, wherein one or more of the carbon atoms is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 L1 is optionally substituted with one or more groups selected from the group consisting of C1 to C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Alkyl halides, -OC1~C 10 Alkyl, -OC1~C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1~C 10 Alkyl halides, -SC1~C 10 Alkyl, -SC1~C10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1~C 10 Alkyl halides, halogen substituents, -OH, -SH, -NH, -C 10 Alkyl-NH2, -N(C1-C 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -COH, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 Alkyl) (C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 Alkyl)C(O)(phenyl), -C(O)C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 Alkyl halides), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 and optionally one or more substituents from the group consisting of: alkyl halide; TIFF2026507286000015.tif726 represents the site to which the group is covalently attached; M1 represents a targeting group, and its definition and selectable range are the same as those described above. In some embodiments, each M1 is independently selected from ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes.

[0071] For convenience, L1 is defined as a linear alkyl, but one of skill in the art will understand that, for example, amino or alkenyl resulting from the above-described replacement and / or substitution may not be a linear group or may have a different name. For purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, a ring (e.g., heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene is considered to be one atom.

[0072] When M1 is a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes, in some embodiments, n1 may be an integer from 1 to 3, and n3 may be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, whereby the number of M1 ligands is at least 3, which facilitates binding of the M1 ligand to the asialoglycoprotein receptor on the surface of the liver and promotes cellular uptake of the conjugate. Experiments have shown that using more than 3 M1 ligands does not significantly improve the ease of binding of the M1 ligand to the asialoglycoprotein receptor on the surface of the liver. Therefore, taking into consideration various aspects such as ease of synthesis, structure / processing costs, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, and n3 is an integer from 0 to 1, and n1 + n3 = 2 to 3.

[0073] In some embodiments, the spatial positioning between multiple M1 ligands can be optimized for binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface when m1, m2, and m3 are independently integers selected from 2 to 10. To simplify, facilitate synthesis, and / or reduce costs of the conjugates provided by the present disclosure, in some embodiments, m1, m2, and m3 are each independently an integer from 2 to 5, and in some embodiments, m1 = m2 = m3.

[0074] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are independently H, C1 to C 10 Alkyl, C1-C 10 Alkyl halides and C1-C 10 It will be understood by those skilled in the art that when R is one selected from alkoxy, any of them can achieve the objectives of the present disclosure without changing the properties of the conjugates of the present disclosure. 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, methyl, and ethyl. In some embodiments, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are both H.

[0075] In some embodiments, R3 is a group having the structure shown in formula A59, wherein E1 is OH, SH, or BH2, and in some embodiments, E1 is OH or SH in consideration of the availability of preparation raw materials.

[0076] In some embodiments, R2 is selected to provide a bond between N on the nitrogen-containing backbone and A59. In the context of this disclosure, a "nitrogen-containing backbone" refers to R 10 , R 11 , R 12 , R 13 , R 14 and R 15 R2 refers to a chain structure in which the carbon atom to which A59 is attached and N are bonded to each other. Therefore, R2 may be any linking group capable of appropriately linking the group of A59 to the N on the nitrogen-containing backbone. In some embodiments, when the siRNA complex is prepared by a solid-phase synthesis process, the R2 group must contain both a binding site bound to the N on the nitrogen-containing backbone and a binding site bound to P in R3. In some embodiments, the site bound to N on the nitrogen-containing backbone in R2 forms an amide bond with N, and the site bound to P on R3 forms a phosphate ester bond with P. In some embodiments, the length of R2 is 4 to 15 atoms or 6 to 10 atoms. In some embodiments, R2 is B5, B6, B5', or B6'.

[0077] [ka] , (B5) TIFF2026507286000017.tif76168, (B6) TIFF2026507286000018.tif8577, (B5') TIFF2026507286000019.tif73162 (B6') During the ceremony, TIFF2026507286000020.tif27127 represents the site to which the group is covalently attached.

[0078] The value of q2 may range from 1 to 10, and in some embodiments, q2 is an integer from 1 to 5.

[0079] L1 serves to link the M1 ligand to the N on the nitrogen-containing backbone and provide targeting functionality to the siRNA complex. In some embodiments, L1 is a combination of one or more bonds selected from the groups of formulae A1-A26. In some embodiments, L1 is a combination of one or more bonds selected from A1, A4, A5, A6, A8, A10, A11, and A13; in some embodiments, L1 is a combination of at least two bonds selected from A1, A4, A8, A10, and A11; and in some embodiments, L1 is a combination of at least two bonds selected from A1, A8, and A10.

[0080] In some embodiments, L1 can be 3 to 25 atoms in length, 3 to 20 atoms in length, 4 to 15 atoms in length, or 5 to 12 atoms in length, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms in length.

[0081] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is C1-C4 alkyl, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is C1-C5 alkyl, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, and Rb in Formulae A1-A26 are each selected to realize bonding between the M1 ligand and N on the nitrogen-containing backbone, and to provide a spatial position between the M1 ligands that is more suitable for binding between the M1 ligand and the asialoglycoprotein receptor on the liver surface.

[0082] In some embodiments, the siRNA complex has a structure as shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422).

[0083] [ka] Expression (403) TIFF2026507286000022.tif101141 Expression (404) TIFF2026507286000023.tif116148 Formula (405) TIFF2026507286000024.tif97150 Formula (406) TIFF2026507286000025.tif130166 Equation (407) TIFF2026507286000026.tif106134 Equation (408) TIFF2026507286000027.tif121161 Equation (409) TIFF2026507286000028.tif123168 Equation (410) TIFF2026507286000029.tif94139 Equation (411) TIFF2026507286000030.tif95139 Equation (412) TIFF2026507286000031.tif107168 Equation (413) TIFF2026507286000032.tif98131 Equation (414) TIFF2026507286000033.tif125143 Equation (415) TIFF2026507286000034.tif118136 Equation (416) TIFF2026507286000035.tif122134 Equation (417) TIFF2026507286000036.tif112141 Equation (418) TIFF2026507286000037.tif105139 Equation (419) TIFF2026507286000038.tif96135 formula(420) TIFF2026507286000039.tif149164 Formula (421) TIFF2026507286000040.tif198167 Formula (422)

[0084] In some embodiments, P in formula A59 may be bound to any available position of the siRNA group. For example, P in formula A59 may be bound to any one nucleotide of the sense strand or antisense strand of the siRNA group. In some embodiments, P in formula A59 is bound to any one nucleotide of the sense strand. In some embodiments, P in formula A59 is bound to the end of the sense strand or antisense strand. In some embodiments, P in formula A59 is bound to the end of the sense strand. The end refers to the first four nucleotides from one end of the sense strand or the antisense strand. In some embodiments, P in formula A59 is bound to the end of the sense strand or the antisense strand of the siRNA group. In some embodiments, P in formula A59 is bound to the 3' end of the sense strand. When bound to the above position of the sense strand, the siRNA complex can release the antisense strand of the single siRNA when unwound after entering a cell, thereby regulating the expression of a target gene.

[0085] P in Formula A59 may be attached to any available position on a nucleotide in the siRNA group, for example, the 5' position, the 2' position, the 3' position, or the base of the nucleotide. In some embodiments, P in Formula A59 may be attached to the 2', 3', or 5' position of a nucleotide in the siRNA group by forming a phosphodiester bond. In some embodiments, P in Formula A59 is attached to the oxygen atom formed by dehydrogenating the 3' hydroxyl of the 3'-terminal nucleotide of the sense strand, or P in Formula A59 is attached to a nucleotide by replacing the hydrogen in the 2'-hydroxyl of one nucleotide in the sense strand, or P in Formula A59 is attached to a nucleotide by replacing the hydrogen in the 5'-hydroxyl of the 5'-terminal nucleotide of the sense strand.

[0086] As those skilled in the art will be aware, modified nucleotide group can be introduced into the siRNA described in the present disclosure by using the nucleoside monomer with corresponding modification.The method of preparing the nucleoside monomer with corresponding modification and the method of introducing modified nucleotide group into siRNA are also well known to those skilled in the art.All modified nucleoside monomers can be purchased commercially or can be prepared by known methods.

[0087] The siRNA complex of the present disclosure can be prepared by any reasonable synthetic route.For example, in the case of the complex molecule that comprises targeting group and the active reactive group that can react with phosphoramidite to form covalent bond, first, the active group in this complex molecule is protected with protecting agent, and then is bound to solid support; then, by using phosphoramidite solid phase synthesis method, according to the type and order of the nucleotide of the sense strand and antisense strand of siRNA, nucleoside monomer is bound one by one from 3' to 5', and the binding of each nucleoside monomer comprises the four reactions of deprotection, coupling, capping, oxidation or sulfurization; then, the sense strand and antisense strand of siRNA are isolated and annealed, thereby obtaining the siRNA complex of the present disclosure.

[0088] Furthermore, the preparation of siRNA complexes can also be carried out by referring to the disclosures of existing documents. For example, WO2019010274A1 describes in Example 1 a method for reacting a linking group having a specific structure with a targeting ligand and sequentially binding it to siRNA. The disclosure is incorporated herein by reference in its entirety. Also, for example, WO2015006740A2 describes in detail methods for preparing multiple siRNA complexes. For example, WO2014025805A1 describes a method for preparing a structure represented by formula (305), and Rajeev et al. describe a method for preparing a structure represented by formula (307) in ChemBioChem 2015, 16, 903-908. Chinese Patent Application CN110959011A also discloses in detail a method for preparing an siRNA complex represented by formula (308). The above-mentioned documents are incorporated herein by reference in their entirety.

[0089] The above description of the siRNA complex also applies to the second and third siRNA complexes described below.

[0090] In the siRNA, pharmaceutical composition, or siRNA complex described herein, the siRNA or siRNA complex may exist in a salted or unsalted form. In the siRNA, pharmaceutical composition, or siRNA complex described herein, the siRNA or siRNA complex may exist partially or entirely in the form of a water-soluble salt. The water-soluble salt may be an amine salt or alkali metal salt thereof. In some embodiments, the alkali metal salt may be a potassium salt or a sodium salt. In some embodiments, the amine salt may be an ammonium salt, a methylamine salt, or a triethylamine salt. In the first siRNA and / or first siRNA complex, the second siRNA and / or second siRNA complex, and the third siRNA and / or third siRNA complex described in the context, adjacent nucleotides are linked by a phosphodiester bond or a phosphorothioate bond, and the non-bridging oxygen or sulfur atom in the phosphodiester or phosphorothioate bond may be negatively charged and exist in the form of a hydroxyl or sulfhydryl, and the hydrogen ions in the hydroxyl or sulfhydryl may be partially or entirely replaced by a cation. The cation may be any cation, for example, a metal cation, an ammonium ion NH4 + , or one of organic ammonium cations. The organic ammonium cation may be a cation formed from methylamine, a cation formed from triethylamine, or a quaternary ammonium cation. In consideration of improving solubility, in one embodiment, the cation is one or more selected from alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations. The alkali metal ion is K + and / or Na +The cation formed from the tertiary amine may be an ammonium ion formed from triethylamine and / or an ammonium ion formed from N,N-diisopropylethylamine. Thus, the siRNA or siRNA complex described in the present disclosure may exist at least partially in the form of a salt. In one embodiment, at least a portion of the non-bridging oxygen or sulfur atoms in the phosphodiester bond or thiophosphodiester bond is bound to sodium ions, i.e., the siRNA or siRNA complex exists in the form of a sodium salt or partial sodium salt.

[0091] In some embodiments, the first RNAi agent contains two to four first siRNAs and / or first siRNA complexes, and the content of each first siRNA and / or first siRNA complex may be equal or unequal. In some embodiments, the first RNAi agent contains two first siRNAs and / or first siRNA complexes, and the ratio between the two may be 0.1 to 10, for example, 0.4 to 5.

[0092] In some embodiments, for ease of preparation, the first RNAi agent contains only one first siRNA or first siRNA complex, and in some embodiments, for better delivery efficiency, the first RNAi agent contains only one first siRNA complex.

[0093] Second RNAi Agents in the Pharmaceutical Compositions of the Disclosure In the context of the present disclosure, the second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each of which is independently an siRNA capable of suppressing PCSK9 mRNA, and each of which includes an siRNA group formed from one of the second siRNAs and a complex group complexed thereto.

[0094] The siRNA capable of suppressing PCSK9 mRNA may be any siRNA capable of suppressing PCSK9 mRNA, and in some embodiments, each second siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently modified or unmodified, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the second siRNA comprises at least one siRNA, and the sense strand of the second siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:3. 5'-UUCUAGACCUGUUUUGCUUUUGUAACUUGA-3' (SEQ ID NO: 3)

[0095] In some embodiments, the sense strand of the siRNA has at least 15, 16, 17, 18, or 19 contiguous nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, in the 5'-3' direction, the sense strand comprises a contiguous nucleotide sequence that matches nucleotides 9 to 27 in the nucleotide sequence set forth in SEQ ID NO: 3.

[0096] The length of the sense strand and the length of the antisense strand of the second siRNA may be the same or different. In some embodiments, the ratio of the length of the sense strand to the antisense strand of the second siRNA is 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 20, 20 / 21, 20 / 2 ...4, 20 / 25, 20 / 26, 20 / 20, 20 / 21 In some embodiments, the ratio of the lengths of the sense strand and the antisense strand of the second siRNA may be 19 / 21, 21 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0097] In some embodiments, in the second siRNA, some nucleotides in the sense strand and the antisense strand are modified nucleotides, and some nucleotides are unmodified nucleotides. In some embodiments, in the second siRNA, all nucleotides in the sense strand and the antisense strand are modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 7 to 9 of the sense strand are 2'-fluoro-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the other nucleotides in the antisense strand are each independently one of non-fluoro-modified nucleotides.

[0098] In some embodiments, the second siRNA may be, for example, any one of the siRNAs shown in Table 2. An siRNA complex containing these siRNAs or an siRNA group formed from these siRNAs exhibits high PCSK9 mRNA suppression activity. To achieve a better suppression effect, in some embodiments, the second siRNA has, for example, the sequence shown in siPCSKd1-M1S.

[0099] Table 2. Sequence of the second siRNA [Table 3] TIFF2026507286000042.tif246170

[0100] Here, capital letters C, G, U, and A represent the base composition of the nucleotide, lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letter s represents that the two nucleotides on the left and right of the letter s are linked by a phosphorothioate, and P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. In some embodiments, P1 represents VP, Ps, or P, with a specified modification, the combination letter VP represents that the nucleotide adjacent to the right of the combination letter VP is a vinyl phosphate (5'-(E)-vinylphosphonate (E-VP))-modified nucleotide, the combination letter Ps represents that the nucleotide adjacent to the right of the combination letter Ps is a phosphorothioate-modified nucleotide, and capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide. Furthermore, each U in the sequences shown in Table 2 above can be optionally substituted with T without any apparent effect on the activity or off-target effects of the siRNA.

[0101] In some embodiments, the conjugate group in each of the second siRNA complexes comprises a linker and a pharmaceutically acceptable targeting group and / or delivery-assisting group, wherein the second siRNA group, the linker, the targeting group, or the delivery-assisting group are sequentially bound covalently or non-covalently, and the definition and selection of the targeting group are the same as those of the first siRNA complex described above. In some embodiments, the second siRNA complex has a structure shown in formula (403).

[0102] In some embodiments, the second RNAi agent comprises two to four second siRNAs and / or second siRNA complexes, and the content of each second siRNA and / or second siRNA complex may be equal or unequal. In some embodiments, the second RNAi agent comprises two second siRNAs and / or second siRNA complexes, and the ratio between the two may be 0.1 to 10, for example, 0.4 to 5.

[0103] In some embodiments, for ease of preparation, the second RNAi agent contains only one second siRNA or second siRNA complex, and in some embodiments, for better delivery efficiency, the second RNAi agent contains only one second siRNA complex.

[0104] In some embodiments, in the pharmaceutical composition provided by the present disclosure, the first RNAi agent comprises only one first siRNA complex, and / or the second RNAi agent comprises only one second siRNA complex, and the sense strand of the first siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand of the first siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 1; or the sense strand of the second siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 2, and the sense strand of the second siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 3. 5'-UGGCCUCCCAAUAAAGCUGGACAAGAAGCU-3' (SEQ ID NO: 1) 5'-UGCUUAAAAGGGACAGUAUUCUCAGUGCUC-3' (SEQ ID NO: 2) 5'-UUCUAGACCUGUUUUGCUUUUGUAACUUGA-3' (SEQ ID NO: 3)

[0105] Third RNAi Agents in the Pharmaceutical Compositions of the Disclosure In some embodiments, the pharmaceutical composition of the present disclosure may further comprise a third RNAi agent, the third RNAi agent comprising one or more third siRNAs and / or one or more third siRNA complexes, each of which is independently an siRNA capable of silencing ANGPTL3 mRNA, and each of which comprises an siRNA group formed from one of the third siRNAs and a complex group conjugated thereto. In some embodiments, the third RNAi agent has a synergistic effect with the first RNAi agent and / or the second RNAi agent, thereby further enhancing the therapeutic and / or preventive effect of the pharmaceutical composition of the present disclosure on diseases and / or symptoms associated with hyperlipidemia.

[0106] The siRNA capable of suppressing ANGPTL3 mRNA may be any siRNA capable of suppressing ANGPTL3 mRNA, and in some embodiments, each third siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently modified or unmodified, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the third siRNA comprises at least one siRNA, and the sense strand of the third siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:4. 5'-AGCCAAAAUCAAGAUUUGCUAUGUUAGACG-3' (SEQ ID NO: 4)

[0107] In some embodiments, the sense strand of the siRNA has at least 15, 16, 17, 18, or 19 contiguous nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, in the 5'-3' direction, the sense strand comprises a contiguous nucleotide sequence that matches nucleotides 9-27 in the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence that differs by no more than one nucleotide from nucleotides 9-27 in the nucleotide sequence set forth in SEQ ID NO: 3.

[0108] The length of the sense strand and the length of the antisense strand of the third siRNA may be the same or different. In some embodiments, the ratio of the length of the sense strand to the antisense strand of the third siRNA is 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 20 / 27, 20 / 28, 20 / 29, 20 / 30, 20 / 31, 20 / 32, 20 / 33, 20 / 34, 20 / 35, 20 / 36, 20 / 37, 20 / 38, 20 / 39, 20 / 40, 2 In some embodiments, the ratio of the lengths of the sense strand and the antisense strand of the second siRNA may be 19 / 21, 21 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0109] In some embodiments, in the third siRNA, some nucleotides in the sense strand and the antisense strand are modified nucleotides, and some nucleotides are unmodified nucleotides. In some embodiments, in the third siRNA, all nucleotides in the sense strand and the antisense strand are modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 7 to 9 of the sense strand are 2'-fluoro-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the other nucleotides in the antisense strand are each independently one of non-fluoro-modified nucleotides.

[0110] In some embodiments, the third siRNA may be, for example, any one of the siRNAs shown in Table 3. An siRNA complex containing these siRNAs or an siRNA group formed from these siRNAs exhibits high ANGPTL3 mRNA suppression activity. To achieve a better suppression effect, in some embodiments, the third siRNA has, for example, the sequence shown in siANGa1-M1S or siANGa1-M2S.

[0111] Table 3. Sequence of the third siRNA [Table 4] TIFF2026507286000044.tif247170 TIFF2026507286000045.tif248170 TIFF2026507286000046.tif246170 TIFF2026507286000047.tif190170

[0112] Here, the capital letters C, G, U, and A represent the base composition of the nucleotide, the lower case letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, and the underlined capital letters S is the character S represents that the adjacent nucleotide on the left side of P1 is a stabilizing modified nucleotide, the lowercase letter s represents that the two nucleotides on the left and right sides of the letter s are linked by a phosphorothioate, and P1 represents that the adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. S is a specific stabilizing modification, e.g. tired and the underlined combination letters tired is the combination character tiredrepresents that the nucleotide adjacent to the left of is a nucleotide having a 2'-O-methoxyethyl modification. In some embodiments, P1 represents VP, Ps, or P, with a specified modification, and the combination letter VP represents that the nucleotide adjacent to the right of the combination letter VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide, the combination letter Ps represents that the nucleotide adjacent to the right of the combination letter Ps is a phosphorothioate-modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide. Furthermore, each U in the sequences shown in Table 3 above can be arbitrarily substituted with T without any apparent effect on the activity or off-target effects of the siRNA.

[0113] In some embodiments, the conjugate group in each of the third siRNA complexes comprises a linker and a pharmaceutically acceptable targeting group and / or delivery-assisting group, wherein the third siRNA group, the linker, the targeting group, or the delivery-assisting group are sequentially bound covalently or non-covalently, and the definition and selection of the targeting group are the same as those of the first siRNA complex described above. In some embodiments, the third siRNA complex has a structure shown in formula (403).

[0114] In some embodiments, the third RNAi agent comprises two to four third siRNAs and / or third siRNA complexes, and the content of each third siRNA and / or third siRNA complex may be equal or unequal. In some embodiments, the second RNAi agent comprises two third siRNAs and / or third siRNA complexes, and the ratio between the two may be 0.1 to 10, for example, 0.4 to 5.

[0115] In some embodiments, for ease of preparation, the third RNAi agent contains only one third siRNA or third siRNA complex, and in some embodiments, for better delivery efficiency, the third RNAi agent contains only one third siRNA complex.

[0116] Solvents, Pharmaceutically Acceptable Carriers and Excipients In some embodiments, for convenience of use, the first, second, and third RNAi agents are present in the form of various RNAi formulations commonly used in the art.

[0117] In some embodiments, at least one or each of the first, second, and third RNAi agents further comprises one or more of a solvent, a pharmaceutically acceptable carrier, and an excipient. The following definitions and selection ranges of solvent, pharmaceutically acceptable carrier, and excipient apply to the first, second, and third RNAi agents.

[0118] In some embodiments, the RNAi formulation may be a liquid formulation, such as an injectable solution. The liquid formulation may be an injectable solution for subcutaneous injection, intramuscular injection, or intravenous injection. In some embodiments, the solvent may include various common solvents, such as water, deionized water, ethanol, and pH buffer solutions. The pH buffer solution may be Tris-hydrochloride buffer solution of pH 7.5 to 8.5 and / or phosphate buffer solution of pH 5.5 to 8.5, for example, phosphate buffer solution of pH 5.5 to 8.5.

[0119] The amount of the solvent used is adjusted depending on the concentration of the required injection solution. For siRNA or siRNA base, the solution is prepared by dissolving siRNA or siRNA to a concentration of 0.01 mg / mL to 200 mg / mL siRNA, 20 mg / mL to 200 mg / mL, 50 mg / mL to 100 mg / mL, 0.01 mg / mL to 5 mg / mL siRNA, 0.1 mg / mL to 5 mg / mL, or 0.5 mg / mL to 3 mg / mL. Those skilled in the art can easily determine the amount of the solvent used depending on the required concentration.

[0120] In some embodiments, the first RNAi agent, the second RNAi agent, and the third RNAi agent are all present in the form of a subcutaneous injection preparation to achieve a better suppressive effect.

[0121] In some embodiments, for ease of transportation and / or storage, all of the siRNAs and siRNA complexes in the first RNAi agent and / or second RNAi agent and / or third RNAi agent are present in powder form, for example, as a lyophilized powder injection, and when administration is performed, the lyophilized powder injection is mixed with a solvent to prepare a liquid formulation.

[0122] In some embodiments, the first RNAi agent, the second RNAi agent, and the third RNAi agent may be drug formulations administered by a method such as a spray formulation administered to the lungs by aerosol or to other organ tissues (e.g., the liver) through the lungs by aerosol, an inhalation formulation inhaled through the oral cavity, or nasal administration. Thus, the first component comprises at least one of an siRNA and an siRNA complex, and a pharmaceutically acceptable carrier and / or excipient, and the type and content of the carrier and / or excipient in the drug formulations administered by a method such as a subcutaneous injection, an intramuscular injection, an intravenous injection, a spray formulation administered to the lungs by aerosol or to other organ tissues (e.g., the liver) through the lungs by aerosol, an inhalation formulation inhaled through the oral cavity, or nasal administration are known to those skilled in the art.

[0123] The pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration, such as magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(aminoethyl ethylene phosphate ester) (poly(2-aminoethyl ethylene phosphate ester)), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate ester) (poly(2-aminoethyl ethylene phosphate ester) ... Examples of suitable polyimide polymers include, but are not limited to, poly(N,N-dimethylaminoethyl methacrylate) (PPEEA) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0124] The contents of the siRNA, siRNA complex, and pharmaceutically acceptable carrier in the first component are the usual contents known to those skilled in the art. In some embodiments, the weight ratio of the total amount of the siRNA and siRNA complex to the pharmaceutically acceptable carrier in the siRNA or siRNA group may be 1:1 to 1:1000, 1:1 to 1:500, 1:1 to 1:200, 1:1 to 1:100, or 1:1 to 1:50.

[0125] The pharmaceutically acceptable excipients are one or more of a variety of ingredients commonly used in the art, such as diluents, binders, disintegrants, lubricants, encapsulating agents, protective agents, osmotic agents, etc., but do not include the aforementioned solvents.

[0126] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose, and the content of the protective agent may be 0.01 to 30% of the total weight of the first component.

[0127] The osmolality adjusting agent may be sodium chloride and / or potassium chloride. The content of the osmolality adjusting agent is determined so that the osmolality of the injection solution is 200 to 700 milliosmoles per kilogram (mOsm / kg). Those skilled in the art can easily determine the content of the osmolality adjusting agent depending on the required osmolality. In some embodiments, the dose of the first component during administration is adjusted depending on the administration route.

[0128] In some embodiments, the first siRNA, second siRNA, and / or third siRNA are present in the form of liposomes. In some embodiments, the pharmaceutically acceptable carrier used in the liposomes comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid, and / or a PEGylated lipid. The organic amine, auxiliary lipid, and PEGylated lipid may be one or more selected from the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, auxiliary lipids, and PEGylated lipids described in Chinese Patent Application CN103380113A (incorporated herein by reference in its entirety), respectively.

[0129] In some embodiments, the organic amine may be a compound represented by formula (201) or a pharmaceutically acceptable salt thereof, as described in Chinese Patent Application CN103380113A.

[0130] [ka] Formula (201) During the ceremony, X 101 and X 102 are each independently O, S, NA, or CA, where A is hydrogen or C1-C 20 is a hydrocarbon chain, Y 101 and Z 101 are each independently C=O, C=S, S=O, CH—OH, or SO; R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl, a substituted or unsubstituted, branched or straight-chain aryl, or a substituted or unsubstituted, branched or straight-chain heteroaryl; x is an integer from 1 to 10, n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1. When m=p=0, R 102 is hydrogen, When at least one of n and m is 2, R 103 and the nitrogen in formula (201) form a structure shown in formula (202) or formula (203).

[0131] [ka] Formula (202), JPEG2026507286000050.jpg116137 Formula (203) In the formula, g, e, and f are each independently an integer of 1 to 6, and "HCC" represents a hydrocarbon chain. * N represents a nitrogen atom in formula (201).

[0132] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, R in formula (201) 101 and R 102 are each independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl, said alkyl or alkenyl having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0133] In some embodiments, when each of n and m is independently 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0134] [ka] Formula (204), JPEG2026507286000052.jpg30140 Formula (205), JPEG2026507286000053.jpg88140 Formula (206), JPEG2026507286000054.jpg78140 Formula (207), JPEG2026507286000055.jpg17140 Formula (208), JPEG2026507286000056.jpg22140 Formula (209), JPEG2026507286000057.jpg42140 Formula (210), JPEG2026507286000058.jpg23140 Formula (211), JPEG2026507286000059.jpg119140 Equation (212) and JPEG2026507286000060.jpg72140 Formula (213) In formulas (204) to (213), g, e, and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each * is R 103 and the nitrogen atom in formula (201), and * Each H on position may be substituted to provide a bond with the nitrogen atom in formula (201).

[0135] Those skilled in the art can obtain the compound of formula (201) by any reasonable method. In some embodiments, the compound of formula (201) may be prepared according to the method described in Chinese Patent Application CN103380113A.

[0136] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0137] [ka] Formula (214), TIFF2026507286000062.tif62143 Formula (215)

[0138] the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0139] In some embodiments, the molar ratio of the organic amine, the colipid, and the polyethylene glycolated lipid in the pharmaceutical composition is (19.7 to 80):(19.7 to 80):(0.3 to 50), and may be, for example, (50 to 70):(20 to 40):(3 to 20).

[0140] In some embodiments, the siRNA liposome particles formed by the first siRNA and / or second siRNA and / or third siRNA and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0141] In some embodiments, in the siRNA liposome formed by the first siRNA and / or second siRNA and / or third siRNA and the amine-containing transfection reagent in the pharmaceutical composition of the present disclosure, the weight ratio (weight / weight ratio) of all siRNAs to all lipids (e.g., organic amine, auxiliary lipid, and / or polyethylene glycolated lipid) is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or about 1:6 to about 1:10, and may be, for example, about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0142] In some embodiments, the siRNA liposome may be prepared according to various known methods, and the siRNA may be used instead of conventional siRNA. In some embodiments, the siRNA liposome may be prepared according to the following method.

[0143] The organic amine, co-lipid, and polyethylene glycolated lipid are suspended in alcohol at the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol used is determined so that the total mass concentration of the resulting lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and may be, for example, ethanol.

[0144] The siRNA is dissolved in a buffer salt solution to obtain an siRNA aqueous solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, for example, 5.0 to 5.2. The amount of the buffer salt solution used is determined so that the siRNA concentration is 0.6 mg / mL or less, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, for example, sodium acetate and / or potassium acetate.

[0145] After mixing the lipid solution and the siRNA aqueous solution, the resulting product is incubated at 40-60°C for at least 2 minutes, e.g., 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), e.g., 1:4.

[0146] The incubated liposomes are concentrated or diluted to remove impurities and sterilized to obtain the liposomes containing siRNA, whose physicochemical parameters are pH 6.5 to 8, encapsulation efficiency of 80% or more, particle size of 40 to 200 nm, polydispersity index of 0.30 or less, and osmotic pressure of 250 to 400 mOsm / kg, and for example, the physicochemical parameters may be pH 7.2 to 7.6, encapsulation efficiency of 90% or more, particle size of 60 to 100 nm, polydispersity index of 0.20 or less, and osmotic pressure of 300 to 400 mOsm / kg.

[0147] Concentration or dilution may be performed before, after, or simultaneously with the removal of impurities. Various conventional methods may be used to remove impurities, such as ultrafiltration at 100 KDa using a tangential flow system or a hollow fiber column, and the ultrafiltration exchange solution may be converted into a phosphate buffer solution (PBS) at pH 7.4. Various conventional methods may be used to sterilize the solution, such as filtration through a 0.22 μm filter.

[0148] In some embodiments, the pharmaceutical composition comprises only the first RNAi agent and the second RNAi agent, the first RNAi agent comprises only one first siRNA complex, or the second RNAi agent comprises only one second siRNA complex, or the first RNAi agent comprises only one first siRNA complex and the second RNAi agent comprises only one second siRNA complex.

[0149] In some embodiments, the first RNAi agent and the second RNAi agent are each present in the form of a separate subcutaneous injection formulation, i.e., the first RNAi agent and the second RNAi agent are stored separately and can be used alone. In some embodiments, the first RNAi agent and the second RNAi agent are present in the form of a homogeneously mixed subcutaneous injection formulation.

[0150] In some embodiments, the pharmaceutical composition comprises a first RNAi agent, a second RNAi agent, and a third RNAi agent, wherein the first RNAi agent comprises only one first siRNA complex, or the second RNAi agent comprises only one second siRNA complex, or the third RNAi agent comprises only one third siRNA complex. In some embodiments, the first RNAi agent comprises only one first siRNA complex, the second RNAi agent comprises only one second siRNA complex, and the third RNAi agent comprises only one third siRNA complex. In some embodiments, the first RNAi agent, the second RNAi agent, and the third RNAi agent are all present individually, and in some embodiments, one of the first RNAi agent and the second RNAi agent and the third RNAi agent are present in the form of a homogeneously mixed preparation for subcutaneous injection, and in some embodiments, the first RNAi agent, the second RNAi agent, and the third RNAi agent are present in the form of a homogeneously mixed preparation for subcutaneous injection.

[0151] kit The pharmaceutical compositions provided by the present disclosure can be stored in a kit. In some embodiments, the kits described herein can provide the first, second, and third RNAi agents in the pharmaceutical compositions provided by the present disclosure in one or more containers. In some embodiments, the first and second RNAi agents in the kit are stored separately to facilitate use of the pharmaceutical compositions of the present disclosure. In some embodiments, the kit includes a container providing the first RNAi agent and a container providing the second RNAi agent, and optionally further includes a container providing the third component. In some embodiments, the kit may include other components, such as stabilizers or preservatives.

[0152] Uses of the Pharmaceutical Compositions of the Present Disclosure In another aspect, the present disclosure further discloses the use of a pharmaceutical composition of the present disclosure in the preparation of a medicament for treating and / or preventing a disease or condition associated with hyperlipidemia, wherein the disease or condition associated with hyperlipidemia is one or more selected from hypercholesterolemia, hypertriglyceridemia, and familial hypercholesterolemia.

[0153] In some embodiments, the first and second RNAi agents in the pharmaceutical composition are present as two separate formulations during preparation of the medicament. In some embodiments, the first and second RNAi agents in the pharmaceutical composition are present as a single formulation during preparation of the medicament.

[0154] Method for treating and / or preventing diseases associated with hyperlipidemia In another aspect, the present disclosure further provides a method for treating and / or preventing a disease associated with hyperlipidemia, the method comprising administering effective amounts of a first RNAi agent and a second RNAi agent to a subject in need thereof, wherein the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA complexes, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of the first siRNA complexes comprises an siRNA group formed from one of the first siRNAs and a conjugate group conjugated thereto; the second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each second siRNA being independently an siRNA capable of suppressing PCSK9 mRNA, and each second siRNA complex comprises an siRNA group formed from one of the second siRNAs and a complex group complexed thereto; The weight ratio of the first RNAi agent to the second RNAi agent administered to the subject is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively.

[0155] The disease or condition associated with hyperlipidemia is one or more selected from hypercholesterolemia, hypertriglyceridemia, and familial hypercholesterolemia.

[0156] The term "effective amount" refers to a dose that can reduce the TC or LDL-C level in a subject by at least 5%, 10%, or 20% compared to before administration.

[0157] In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent, based on the total amount of siRNA and siRNA groups contained in the first RNAi agent and the second RNAi agent, respectively, administered to a subject is 100:1 to 1:100, for example, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:100, 1:50, 1:25, 1:15, 1:10, 1:5, 1:3, 1:4, or 1:2. In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent administered to a subject is preferably 10:1 to 1:10, 5:1 to 1:5, or 2:1 to 1:5. In some embodiments, to achieve a better therapeutic / prophylactic effect, the weight of the second RNAi agent administered to the subject may be equal to or greater than the weight of the first RNAi agent, and the weight ratio of the first RNAi agent to the second RNAi agent administered to the subject may be 1:1 to 1:10, 1:1 to 1:5, or 1:1 to 1:3.

[0158] In some embodiments, the total amount of siRNA and siRNA groups contained in the first and second RNAi agents, respectively, of the first RNAi agent administered to the subject each time is 0.5 mg / kg to 10 mg / kg of the subject's body weight, and may be, for example, 0.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 3 mg / kg, 1 mg / kg to 3 mg / kg, or 1 mg / kg to 5 mg / kg of the subject's body weight. The total amount of the second RNAi agent administered to the subject each time is 0.5 mg / kg to 10 mg / kg of the subject's body weight, and may be, for example, 0.5 mg / kg to 9 mg / kg, 3 mg / kg to 10 mg / kg, 1 mg / kg to 9 mg / kg, 3 mg / kg to 9 mg / kg, 1 mg / kg to 6 mg / kg, or 3 mg / kg to 6 mg / kg.

[0159] In some embodiments, the total amount of siRNA and siRNA groups contained in the first RNAi agent administered to the subject each time is a fixed dose of 1 mg to 1000 mg, and may be, for example, 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg. In some embodiments, the dose of the first RNAi agent administered to the subject is 5 mg to 500 mg.

[0160] In some embodiments, the total amount of siRNA and siRNA groups contained in the second RNAi agent administered to the subject each time is a fixed dose of 1 mg to 1000 mg, and may be, for example, 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg. In some embodiments, the dose of the second RNAi agent administered to the subject is 5 mg to 800 mg.

[0161] To achieve better preventive / therapeutic effects, in some embodiments, the weight of the first RNAi agent administered to the subject is 5 mg to 500 mg, and the weight of the second RNAi agent administered to the subject is 5 mg to 800 mg.

[0162] In some embodiments, the method further comprises administering a third RNAi agent to the subject, wherein the third RNAi agent comprises one or more third siRNAs and / or one or more third siRNA complexes, each of the third siRNAs being independently an siRNA capable of suppressing expression of ANGPTL3 mRNA, and each of the third siRNA complexes comprising a third siRNA group formed from one of the third siRNAs and a complex group conjugated thereto.

[0163] In some embodiments, the weight ratio of the third RNAi agent to the second RNAi agent, based on the total amount of siRNA and siRNA groups contained in the third RNAi agent and the second RNAi agent, respectively, administered to the subject is 100:1 to 1:100, for example, 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:100, 1:50, 1:25, 1:15, 1:10, 1:5, 1:3, 1:4, or 1:2. In some embodiments, the weight ratio of the third RNAi agent to the second RNAi agent administered to the subject is preferably 10:1 to 1:10, 5:1 to 1:5, or 2:1 to 1:5.

[0164] In some embodiments, the total amount of siRNA and siRNA groups contained in the third RNAi agent, the dose of the third RNAi agent administered to the subject each time, is a fixed dose of 1 mg to 1000 mg, and may be, for example, 1 mg to 800 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 250 mg, or 1 mg to 100 mg.

[0165] In some embodiments, the dose of the third RNAi agent, which is the total amount of siRNA and siRNA groups respectively contained in the third RNAi agent, administered to the subject each time is 0.5 mg / kg to 10 mg / kg of the subject's body weight, and may be, for example, 0.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 3 mg / kg, 1 mg / kg to 3 mg / kg, or 1 mg / kg to 5 mg / kg.

[0166] The definitions and selection ranges of the first RNAi agent, second RNAi agent, and third RNAi agent are all the same as those of the first RNAi agent, second RNAi agent, and third RNAi agent in the pharmaceutical composition of the present disclosure described above.

[0167] As used herein, the term "pharmaceutical administration" refers to the introduction of siRNA, pharmaceutical composition, and / or siRNA complex into the body of a subject by a method or route that at least partially localizes the siRNA, pharmaceutical composition, and / or siRNA complex to a desired site and produces a desired effect. Suitable administration routes for the methods of the present disclosure include local administration and systemic administration. Generally, local administration delivers more siRNA, pharmaceutical composition, and / or siRNA complex to a specific site than to the entire body of the subject, whereas systemic administration delivers the siRNA, pharmaceutical composition, and / or siRNA complex to nearly the entire body of the subject. Given that the present disclosure is intended to provide a means for preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, the administration method can deliver drugs to the liver.

[0168] Administration to a subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0169] In some embodiments, to achieve better preventive / therapeutic effects, all of the RNAi agents are administered to a subject by subcutaneous injection, for example, the first RNAi agent, the second RNAi agent, and the third RNAi agent are administered in the form of a homogeneously mixed subcutaneous injection solution.

[0170] In some embodiments, for a single administration, the subject is administered the first and second RNAi agents simultaneously, or the subject is administered the first and second RNAi agents singly, one after the other, within a 24-hour period.

[0171] In some embodiments, for a single administration, the method further comprises administering to the subject a third RNAi agent, wherein the third RNAi agent is administered to the subject simultaneously with or within 24 hours of the first RNAi agent and the second RNAi agent.

[0172] In some embodiments, the time interval between each administration of the first RNAi agent, the second RNAi agent, and the third RNAi agent is 7 days to 60 weeks, 14 days to 60 weeks, 14 days to 1 year, or 14 days to 2 years. In some embodiments, to achieve a better therapeutic effect, the time interval between each administration of the first RNAi agent, the second RNAi agent, and the third RNAi agent is 14 days to 1 year, 3 weeks to 1 year, 1 month to 1 year, 2 months to 1 year, 3 months to 1 year, or 6 months to 1 year. Specifically, in some embodiments, the time interval between each administration of the first RNAi agent, the second RNAi agent, and the third RNAi agent is 14 days, 21 days, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0173] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way. [Example]

[0174] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all procedures such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0175] Preparation Example 1 Preparation of a pharmaceutical composition provided by the present disclosure 1-1 Preparation of the first siRNA complex and the second siRNA complex in the pharmaceutical composition of the present disclosure The first and second siRNA complexes were prepared in the form of lyophilized powders using the method described in Preparation Example 13 of CN110959011A, except that the sense and antisense strands contained in the first and second siRNA complexes had the sequences shown in Table 4 below, respectively. Complexes 1 and 2 were diluted to a concentration of 0.2 mg / mL (based on the siRNA base) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ cm at 25°C), and then their molecular weights were detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The sense strand of the first siRNA complex had a theoretical molecular weight (MW) of 7581.42, and the actual molecular weight was 7581.22. The antisense strand had a theoretical molecular weight of 6948.55, and the actual molecular weight was 6948.05. The sense strand of the second siRNA complex had a theoretical molecular weight of 7426.26, and the actual molecular weight was 7425.55. The antisense strand had a theoretical molecular weight of 7084.88, and the actual molecular weight was 7084.14. The actual values ​​were consistent with the theoretical values, indicating that the synthesized first siRNA complex and second siRNA complex were the intended double-stranded nucleic acid sequences. The obtained first siRNA complex and second siRNA complex had the structure shown in formula (403).

[0176] Table 4. Sequences of the sense and antisense strands of the siRNA complexes [Table 5]

[0177] Here, the capital letters C, G, U, A, and T represent the base composition of the nucleotide, the lower case letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, and the underlined combination letters tiredindicates that the nucleotide adjacent to the left of the combination letter moe is a ribose 2'-O-methoxyethyl modified nucleotide, the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are linked by a thiophosphate ester, and P indicates that the nucleotide on the right of the letter P is a 5'-phosphate nucleotide.

[0178] 1-2 Preparation of the first and second RNAi agents in the pharmaceutical composition of the present disclosure The first siRNA complex powder prepared in 1-1 was dissolved in PBS to give solutions with concentrations of 0.2 mg / mL and 0.6 mg / mL, respectively, to give low-concentration and high-concentration first RNAi agents.

[0179] The second siRNA complex powder prepared in 1-1 was dissolved in PBS to give solutions with concentrations of 0.6 mg / mL and 1.2 mg / mL, respectively, to give low-concentration and high-concentration second RNAi agents.

[0180] 1-3 Preparation of the Pharmaceutical Composition of the Present Disclosure Pharmaceutical composition I was prepared by homogeneously mixing 0.9 mL of the low-concentration first RNAi agent and 0.9 mL of the low-concentration second RNAi agent, where the weight ratio of the first RNAi agent to the second RNAi agent was 1:3.

[0181] Pharmaceutical composition II was prepared by homogeneously mixing 0.9 mL of the high-concentration first RNAi agent and 0.9 mL of the low-concentration second RNAi agent, where the weight ratio of the first RNAi agent to the second RNAi agent was 1:1.

[0182] Pharmaceutical composition III was prepared by homogeneously mixing 0.9 mL of the high-concentration first RNAi agent and 0.9 mL of the high-concentration second RNAi agent, where the weight ratio of the first RNAi agent to the second RNAi agent was 1:2.

[0183] Experimental Example 1: Active effect of the pharmaceutical composition provided by the present disclosure in mice Thirty-five female hAPOC3 transgenic mice (species: (C57BL / 6J × CBA / J) F1, rank: SPF, purchased from Breisbach Co., Ltd.) were randomly divided into seven groups, with five mice per group, numbered Group 1 to Group 7. These mice had severe hypertriglyceridemia and significantly elevated cholesterol.

[0184] Mice in groups 1 to 7 were administered on the day of grouping according to the administration scheme in Table 5 below, with the administration day designated as day 1.

[0185] Table 5. Administration scheme for mice in groups 1 to 8 [Table 6]

[0186] All mice were bled once from the anterior orbit of their eyes before administration on Day 1 (D1), Day 8 (D8), and Day 15 (D15). They were fasted for 4-6 hours before blood collection, and 0.2 mL of blood was collected from the orbit using an EDTAK2 anticoagulant blood collection tube after anesthetizing with 70% CO2. The collected blood was centrifuged at 1800 g for 15 minutes at 2-8°C, and the supernatant was removed to separate the plasma, which was then stored in a -80°C refrigerator.

[0187] The obtained plasma samples were sent to Labcorp (Shanghai) for measurement of plasma TC and LDL-C contents. Normalized plasma TC and LDL-C values ​​= (content after administration (D8 or D15) / content before administration (D1)) × 100%. The results for each group are summarized in Table 6 below.

[0188] Table 6. Normalized values ​​of TC and LDL-C contents in the bodies of mice in Groups 1 to 8 [Table 7]

[0189] As can be seen from the results in Table 6, regarding changes in TC levels, on the one hand, under the condition that the total dose was 6 mg / kg in all cases, almost no reduction in TC was observed in the mice of Group 4 that were administered 6 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone, whereas in the mice of Group 6 that were administered the pharmaceutical composition of the present disclosure (3 mg / kg each of an RNAi agent that inhibits APOC3 mRNA and an RNAi agent that inhibits PCSK9 mRNA), a TC reduction rate of 45% or more (TC reduction rate = 100% - normalized value of TC content) was observed; even when the TC reduction rate in the mice of Group 2 that were administered an RNAi agent that inhibits APOC3 mRNA and the mice of Group 3 that were administered an RNAi agent that inhibits PCSK9 mRNA were added together, the TC reduction rate was less than 45%. Under the condition that the total dose was 4 mg / kg in both cases, the combined TC reduction rate in the mice of Group 1, which was administered 1 mg / kg of an RNAi agent that inhibits APOC3 mRNA alone, and the combined TC reduction rate in the mice of Group 3, which was administered 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone, was a maximum of 34.9%, whereas the TC reduction rate in the mice of Group 5, which was administered the pharmaceutical composition of the present disclosure (1 mg / kg of an RNAi agent that inhibits APOC3 mRNA and 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA), reached 37.2%. On the other hand, taking the change in TC levels on day 8 after administration as an example, under the condition that the total dose was 6 mg / kg in all cases, no decrease in TC was observed in the bodies of mice in Group 4 that were administered 6 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone, whereas Group 6 that was administered the pharmaceutical composition of the present disclosure (3 mg / kg each of an RNAi agent that inhibits APOC3 mRNA and an RNAi agent that inhibits PCSK9 mRNA) showed a 45.6% decrease in TC, and the combined TC decrease rates of Group 2 that were administered an RNAi agent that inhibits APOC3 mRNA (3 mg / kg) and Group 3 that were administered an RNAi agent that inhibits PCSK9 mRNA (3 mg / kg) was only 33.3%.Under the condition that the total dose was 4 mg / kg in both cases, the combined TC reduction rate of Group 1, which received 1 mg / kg of an RNAi agent that inhibits APOC3 mRNA alone, and Group 3, which received 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA alone, was a maximum of 34.9%, while the TC reduction rate of Group 5, which received the pharmaceutical composition of the present disclosure (1 mg / kg of an RNAi agent that inhibits APOC3 mRNA and 3 mg / kg of an RNAi agent that inhibits PCSK9 mRNA), reached 37.0%.

[0190] Regarding changes in LDL-C levels, on the one hand, under the condition that the total dose was 6 mg / kg, the mice in Group 4, which were administered with an RNAi agent inhibiting PCSK9 mRNA alone, showed a maximum LDL-C reduction rate of 43.3% (LDL-C reduction rate = 100% - normalized value of LDL-C content), whereas the mice in Group 6, which were administered with the pharmaceutical composition of the present disclosure, showed a 72.8% LDL-C reduction rate. On the other hand, neither the administration of a high dose of an RNAi agent inhibiting APOC3 mRNA alone nor the administration of a high dose of an RNAi agent inhibiting PCSK9 mRNA alone was able to reduce plasma LDL-C levels to 50% or less, whereas the mice in Group 5, which were administered with a low dose (4 mg / kg) of the pharmaceutical composition of the present disclosure, already showed a 67% LDL-C reduction rate. Taking the change in LDL-C levels on the 8th day after administration as an example, Group 4, which received only the RNAi agent that inhibits PCSK9 mRNA at a total dose of 6 mg / kg, showed a 41.4% reduction in LDL-C, while Group 6, which received the pharmaceutical composition of the present disclosure (3 mg / kg each of the RNAi agent that inhibits APOC3 mRNA and the RNAi agent that inhibits PCSK9 mRNA), showed a 72.8% reduction in LDL-C.

[0191] As can be seen from the above results, the composition and treatment method provided by the present disclosure have significantly superior effects on both TC level reduction and LDL-C level reduction compared to the administration of the first or second RNAi agent alone, demonstrating clear synergistic effects, and particularly noteworthy is the strong synergistic effect on LDL-C level reduction, unexpectedly breaking through the limitations of LDL-C level reduction achieved by the administration of the first or second RNAi agent alone. The pharmaceutical composition and treatment method of the present disclosure have excellent potential for application in the prevention and / or treatment of symptoms or diseases associated with hyperlipidemia.

[0192] Although several embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific details of the above embodiments, and within the scope of the technical idea of ​​the present disclosure, multiple simple modifications can be made to the technical solutions of the present disclosure, and all of these simple modifications fall within the protection scope of the present disclosure.

[0193] It should be noted that the specific technical features described in the above embodiments can be combined in any suitable manner if there is no contradiction, and in order to avoid unnecessary duplication, the present disclosure does not separately describe various possible combination methods.

[0194] Furthermore, various different embodiments of the present disclosure can be combined in any manner, and unless departing from the spirit of the present disclosure, such combinations should be considered to be the same as the contents disclosed in the present disclosure.

Claims

1. 1. A pharmaceutical composition comprising a first RNAi agent and a second RNAi agent, the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA conjugates, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of which comprises an siRNA group formed from one of the first siRNAs and a conjugate group conjugated thereto; The second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each second siRNA being independently an siRNA capable of suppressing PCSK9 mRNA, and each second siRNA complex comprises an siRNA group formed from one of the second siRNAs and a complex group conjugated thereto; A pharmaceutical composition, wherein the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively.

2. The pharmaceutical composition of claim 1, wherein the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 5:1 to 1:

5.

3. The pharmaceutical composition of claim 2, wherein the weight ratio of the first RNAi agent to the second RNAi agent in the pharmaceutical composition is 2:1 to 1:

5.

4. The pharmaceutical composition according to claim 1, wherein the total amount of the siRNA and the siRNA group contained in the first RNAi agent and the second RNAi agent, respectively, per portion of the pharmaceutical composition is 5 mg to 500 mg for the first RNAi agent and 5 mg to 800 mg for the second RNAi agent.

5. The pharmaceutical composition according to claim 4, wherein the content of the first RNAi agent is 25 mg to 250 mg and the content of the second RNAi agent is 50 mg to 500 mg per part of the pharmaceutical composition.

6. 10. The pharmaceutical composition of claim 1, wherein the first RNAi agent and / or the second RNAi agent further comprises one or more of a solvent, a pharmaceutically acceptable carrier, and an excipient.

7. 2. The pharmaceutical composition of claim 1, wherein each first siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, and the first siRNA comprises at least one siRNA, and the sense strand of the first siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 1, or the sense strand has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

2. 5'-UGGCCUCCCAAUAAAGCUGGACAAGAAGCU-3' (SEQ ID NO: 1) 5'-UGCUUAAAGGGACAGUAUUCUCAGUGCUC-3' (SEQ ID NO: 2)

8. 8. The pharmaceutical composition of claim 7, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides, the nucleotides at positions 7 to 9 of the sense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, and the nucleotides at other positions in the sense strand and the antisense strand are each independently one of non-fluoro-modified nucleotides.

9. The pharmaceutical composition of claim 8, wherein the first siRNA has a sequence shown in siAPOC3b1-M1SP1 or siAPOC3b3-M1S.

10. 2. The pharmaceutical composition of claim 1, wherein each second siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length, and the antisense strand is 18 to 26 nucleotides in length, the second siRNA comprises at least one siRNA, the sense strand of the siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand of the second siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

3. 5'-UUCUAGACCUGUUUUGCUUUGUAACUUGA-3' (SEQ ID NO: 3)

11. 11. The pharmaceutical composition of claim 10, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides, the nucleotides at positions 7 to 9 of the sense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, and the nucleotides at other positions in the sense strand and the antisense strand are each independently one of non-fluoro-modified nucleotides.

12. The pharmaceutical composition of claim 11, wherein the second siRNA has a sequence shown in siPCSKd1-M1S.

13. 2. The pharmaceutical composition of claim 1, wherein the conjugate group in each of the first siRNA conjugate and the second siRNA conjugate comprises a linker and a pharmaceutically acceptable targeting group, the first siRNA group or the second siRNA group, the linker and the targeting group are sequentially linked covalently or non-covalently, and each targeting group is selected from ligands capable of binding to a cell surface ASGPR.

14. The first siRNA conjugate and / or the second siRNA conjugate has a structure shown in formula (403): 【Chemistry 1】 Formula (403) 14. The pharmaceutical composition of claim 13, wherein Nu represents the first siRNA group or the second siRNA group.

15. 2. The pharmaceutical composition of claim 1, wherein the first RNAi agent comprises only one first siRNA complex, and / or the second RNAi agent comprises only one second siRNA complex, and the sense strand of the first siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand of the first siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 1, or the sense strand of the first siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 2, and the sense strand of the second siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand of the second siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

3. 5'-UGGCCUCCCAAUAAAGCUGGACAAGAAGCU-3' (SEQ ID NO: 1) 5'-UGCUUAAAGGGACAGUAUUCUCAGUGCUC-3' (SEQ ID NO: 2) 5'-UUCUAGACCUGUUUUGCUUUGUAACUUGA-3' (SEQ ID NO: 3)

16. 16. The pharmaceutical composition of claim 15, wherein the first RNAi agent comprises only one first siRNA conjugate and the second RNAi agent comprises only one second siRNA conjugate.

17. The pharmaceutical composition of claim 1, wherein the first RNAi agent and the second RNAi agent are each present in the form of a separate subcutaneous injection preparation, or the first RNAi agent and the second RNAi agent are present in a homogeneously mixed form for subcutaneous injection.

18. The pharmaceutical composition according to any one of claims 1 to 17, further comprising a third RNAi agent, the third RNAi agent comprising one or more third siRNAs and / or one or more third siRNA conjugates, each of the third siRNAs being independently an siRNA capable of suppressing expression of ANGPTL3 mRNA, and each of the third siRNA conjugates comprising a third siRNA group formed from one of the third siRNAs and a conjugate group conjugated thereto.

19. The pharmaceutical composition of claim 18, wherein the weight ratio of the third RNAi agent to the second RNAi agent in the pharmaceutical composition is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the third RNAi agent and the second RNAi agent, respectively.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein each of the third siRNAs comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length and the antisense strand is 18 to 26 nucleotides in length, the third siRNA comprises at least one siRNA, the sense strand of the siRNA is substantially reverse complementary or completely reverse complementary to the antisense strand, and the sense strand of the third siRNA has at least 17 consecutive nucleotides that correspond to the nucleotide sequence set forth in SEQ ID NO:

4. 5'-AGCCAAAAUCAAGAUUUGCUAUGUUAGACG-3' (SEQ ID NO: 4)

21. The pharmaceutical composition of claim 20, wherein the third siRNA has a sequence shown in siANGa1-M1S or siANGa1-M2S.

22. Use of a pharmaceutical composition according to any one of claims 1 to 21 in the preparation of a medicament for treating and / or preventing a disease or condition associated with hyperlipidemia.

23. 23. The use according to claim 22, wherein the disease or condition associated with hyperlipidemia is one or more selected from hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and familial hypercholesterolemia.

24. 1. A method for treating and / or preventing a disease or condition associated with hyperlipidemia, comprising: administering to a subject in need thereof effective amounts of a first RNAi agent and a second RNAi agent; the first RNAi agent comprises one or more first siRNAs and / or one or more first siRNA conjugates, each of which is independently an siRNA capable of suppressing APOC3 mRNA, and each of which comprises an siRNA group formed from one of the first siRNAs and a conjugate group conjugated thereto; The second RNAi agent comprises one or more second siRNAs and / or one or more second siRNA complexes, each second siRNA being independently an siRNA capable of suppressing PCSK9 mRNA, and each second siRNA complex comprises an siRNA group formed from one of the second siRNAs and a complex group conjugated thereto; The weight ratio of the first RNAi agent to the second RNAi agent administered to the subject is 10:1 to 1:10 based on the total amount of siRNA and siRNA group contained in the first RNAi agent and the second RNAi agent, respectively.

25. 25. The method of claim 24, wherein the weight ratio of the first RNAi agent to the second RNAi agent administered to the subject is from 5:1 to 1:

5.

26. 26. The method of claim 25, wherein the weight ratio of the first RNAi agent to the second RNAi agent administered to the subject is between 2:1 and 1:

5.

27. The method of claim 24, wherein the dose of the first RNAi agent administered to the subject each time is 0.5 mg / Kg to 5 mg / Kg of the subject's body weight, and the dose of the second RNAi agent administered to the subject each time is 0.5 mg / Kg to 9 mg / Kg of the subject's body weight, in terms of the total amount of siRNA and siRNA groups.

28. 28. The method of claim 27, wherein the dose of the first RNAi agent administered to the subject each time is 1 mg / Kg to 3 mg / Kg of the subject's body weight, and the dose of the second RNAi agent administered to the subject each time is 3 mg / Kg to 9 mg / Kg of the subject's body weight.

29. 25. The method of claim 24, wherein the dose of the first RNAi agent administered to the subject each time is a fixed dose of 5 mg to 500 mg, and the dose of the second RNAi agent administered to the subject each time is a fixed dose of 5 mg to 800 mg.

30. 30. The method of claim 29, wherein the dose of the first RNAi agent administered to the subject each time is a fixed dose of 25 mg to 250 mg, and the dose of the second RNAi agent administered to the subject each time is a fixed dose of 50 mg to 500 mg.

31. 25. The method of claim 24, wherein each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the first siRNA comprises at least one siRNA, and the sense strand of the first siRNA has at least 15 contiguous nucleotides that match the nucleotide sequence set forth in SEQ ID NO: 1, or the sense strand has at least 15 contiguous nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

2. 5'-UGGCCUCCCAAUAAAGCUGGACAAGAAGCU-3' (SEQ ID NO: 1) 5'-UGCUUAAAGGGACAGUAUUCUCAGUGCUC-3' (SEQ ID NO: 2)

32. 32. The method of claim 31, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides, the nucleotides at positions 7 to 9 of the sense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, and each other nucleotide in the antisense strand is independently one of the non-fluoro-modified nucleotides.

33. The method of claim 32, wherein the first siRNA has a sequence shown in siAPOC3b1-M1SP1 or siAPOC3b3-M1S.

34. 25. The method of claim 24, wherein each second siRNA comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the second siRNA comprises at least one siRNA, and the sense strand of the second siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

3. 5'-UUCUAGACCUGUUUUGCUUUGUAACUUGA-3' (SEQ ID NO: 3)

35. 35. The method of claim 34, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides, the nucleotides at positions 7 to 9 of the sense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, from the 5' to the 3' end, are 2'-fluoro-modified nucleotides, and the nucleotides at other positions in the sense strand and the antisense strand are each independently one of non-fluoro-modified nucleotides.

36. The method of claim 35, wherein the second siRNA has a sequence shown in siPCSKd1-M1S.

37. 25. The method of claim 24, further comprising administering a third RNAi agent to the subject, wherein the third RNAi agent comprises one or more third siRNAs and / or one or more third siRNA conjugates, each of which is independently an siRNA capable of suppressing expression of ANGPTL3 mRNA, and each of the third siRNA conjugates comprises a third siRNA group formed from one of the third siRNAs and a conjugate group conjugated thereto.

38. The method of claim 37, wherein the weight ratio of the third RNAi agent to the second RNAi agent administered to the subject each time is 10:1 to 1:10, based on the total amount of siRNA and siRNA group contained in the third RNAi agent and the second RNAi agent, respectively.

39. 38. The method of claim 37, wherein each of the third siRNAs comprises a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide, the sense strand is 18 to 23 nucleotides in length, the antisense strand is 18 to 26 nucleotides in length, the sense strand is substantially reverse complementary or completely reverse complementary to the antisense strand, the third siRNA comprises at least one siRNA, and the sense strand of the third siRNA has at least 15 consecutive nucleotides that match the nucleotide sequence set forth in SEQ ID NO:

4. 5'-AGCCAAAAUCAAGAUUUGCUAUGUUAGACG-3' (SEQ ID NO: 4)

40. The method of claim 39, wherein the third siRNA has the sequence shown in siANGa1-M1S or siANGa1-M2S.

41. 25. The method of claim 24, wherein the first and second RNAi agents are administered to the subject simultaneously, or the first and second RNAi agents are administered to the subject separately, one after the other, within a 24-hour period.

42. 38. The method of claim 37, further comprising administering to the subject an effective amount of a third RNAi agent, wherein the third RNAi agent is administered to the subject simultaneously with or within 24 hours of the first RNAi agent and the second RNAi agent.

43. 38. The method of claim 24 or 37, wherein all RNAi agents are administered to the subject by subcutaneous injection.

44. 44. The method of any one of claims 24 to 43, wherein the disease or condition is one or more selected from hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and familial hypercholesterolemia.

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