A GalNAc compound-coupled nucleic acid lipid nanoparticle containing a ribose ring structure and its preparation method and application

Abstract

The present invention provides a GalNAc compound-coupled nucleic acid lipid nanoparticle containing a ribose ring structure, and a preparation method and application thereof. The components of the lipid nanoparticle include: nucleic acid, lipid and anchor compound of GalNAc containing a ribose ring; the lipid includes: cationic lipid, neutral lipid, auxiliary lipid and long-circulating lipid; the anchor compound of GalNAc containing a ribose ring includes: a GalNAc part containing a ribose ring and a lipid chain group; the structural formula of the anchor compound is shown in Formula IX. The conjugate provided in the present invention can achieve efficient liver-targeted delivery and improve the efficacy of the drug.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a GalNAc compound-coupled nucleic acid lipid nanoparticle containing a ribose ring structure, and a preparation method and application thereof. Background Art

[0002] Currently, there is a rapid increase in oligonucleotide-based therapies that aim to alter the expression of specific genes and proteins for various cancers, cardiovascular diseases, neurological diseases, and other diseases. Although this type of therapy is becoming increasingly popular, nucleic acids are rapidly degraded in the body, especially for "unmodified" nucleic acid drugs. Although adenovirus and lentivirus as vectors have been widely explored in the field of gene therapy, they are known to stimulate unwanted immune responses in addition to high manufacturing costs and ethical issues.

[0003] Therefore, non-viral vectors are more suitable for nucleic acid drugs. Currently, the delivery platform technologies commonly used in mainstream nucleic acid therapies include: GalNAc (N-acetylgalactosamine) ligand-modified short interfering RNA (siRNA) conjugates and lipid nanoparticles (LNPs). The above delivery technologies all have important applications in the field of nucleic acid drug delivery.

[0004] GalNAc and ASGPR (Asialoglycoprotein receptor, also known as Asialoglycoprotein Receptor, or ASGP-R) play an important role in liver metabolism. GalNAc is a ligand that binds to ASGPR. ASGPR is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, some progress has been made in the liver-targeted delivery of nucleic acid drugs using GalNAc, a high-affinity ligand of ASGPR, as a targeting molecule.

[0005] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a glycoprotein composed of 692 amino acids. It is the ninth member of the proprotein convertase (PCs) family and a secretory serine protease. It is mainly expressed in tissues such as the liver and intestines and then secreted into the blood. After entering the blood circulation, PCSK9 can specifically bind to the epidermal growth factor-like domain of the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes, guide it into the hepatocytes to reach the lysosomes, and degrade LDL-R in the lysosomes, thereby reducing LDL-R on the surface of hepatocytes, thereby reducing the liver's ability to bind and clear LDL-C, and ultimately leading to an increase in LDL-C levels in the blood. Therefore, hypercholesterolemia can be treated by inhibiting PCSK9.

[0006] For liver-targeted drug delivery, oligonucleotide drugs can be delivered in liver-targeted manner using LNP in addition to the GalNAc / ASGPR combination. It is well known that nucleic acid drugs have difficulty entering cells and face the problem of degradation by nucleases. Due to their double-stranded structure (such as siRNA), they may also form unstable structures in the body, further affecting their stability. Therefore, nucleic acid drugs are encapsulated in LNP, which is beneficial to improve their stability and bioavailability in the body. This method utilizes the characteristics of lipid polymorphism to enable cationic lipids to bind to RNA to form complexes. These complexes can pass through the cell membrane and enter the interior of the cell to achieve the purpose of drug delivery, thereby preventing nucleic acid drugs from being degraded by nucleases and immune substances in the serum.

[0007] Most intravenously administered LNPs can spontaneously aggregate in the liver. Surface-modified LNPs can also achieve targeted delivery through ApoE / LDL-R combined targeted delivery, specifically combining apolipoprotein (ApoE) on the surface of low-density lipoprotein (LDL) in plasma with LDL-R on the surface of hepatocytes. Therefore, LNP is also regarded as a carrier with liver targeting. In addition, LNP is composed of biocompatible and biodegradable lipid molecules, which is beneficial to the body's tolerance. At the same time, the sustained release expression of LNP can prolong the effect. Although the purpose of coupling nucleic acid drugs to GalNAc is also to increase bioavailability, it is more to achieve this goal by utilizing its efficient targeting.

[0008] The LNP delivery system promotes the intracellular uptake of oligonucleotides by actively fusing lipid particles with the cell membrane. In contrast, the GalNAc delivery system relies on binding to specific receptors and being internalized by cells through receptor-mediated endocytosis. The use of LNP may reduce certain specific side effects. Although the GalNAc delivery system is highly targeted, it may cause higher local side effects or toxicity to the liver due to its high degree of targeting to the liver.

[0009] Combining the above characteristics of GalNAc and LNP delivery, the method of coupling LNP-encapsulated oligonucleotide drugs with GalNAc compounds should have better targeting and stability. Therefore, providing a new LNP-GalNAc-coupled drug that improves the liver targeting of nucleic acid drugs, enhances the efficacy and reduces side effects has important application prospects. Summary of the invention

[0010] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a GalNAc compound containing a ribose ring structure coupled with nucleic acid lipid nanoparticles and a preparation method and application thereof. The present invention couples GalNAc containing a ribose ring with LNP through a one-step preparation method, thereby improving the liver targeting of oligonucleotide drugs, enhancing the efficacy and reducing side effects.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a GalNAc compound containing a ribose ring structure coupled to a nucleic acid lipid nanoparticle, wherein the components of the lipid nanoparticle include: a nucleic acid, a lipid, and an anchoring compound of GalNAc containing a ribose ring;

[0013] The lipids include: cationic lipids, neutral lipids, auxiliary lipids and long-circulating lipids;

[0014] The anchor compound of the GalNAc containing a ribose ring comprises: a GalNAc part containing a ribose ring and a lipid chain group; the structural formula of the anchor compound is shown in Formula IX:

[0015] ;

[0016] Wherein, R1 is oxygen or sulfur;

[0017] R2 is hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy or halogen;

[0018] R3 is hydrogen or a hydroxyl protecting group;

[0019] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; b is an integer from 1 to 7, for example, 1, 2, 3, 4, 5, 6 or 7; c is an integer from 1 to 7, for example, 1, 2, 3, 4, 5, 6 or 7; d is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5;

[0020] B is -(CH2) e -, wherein e is an integer from 0 to 7, for example, 0, 1, 2, 3, 4, 5, 6 or 7; when e is 0, B is a single bond;

[0021] L1 is -CONH- or -NHCO-;

[0022] G is ;

[0023] wherein T is N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine or N-isobutyryl-galactosamine, wherein the hydroxyl group is fully protected with an acyl group or unprotected, and wherein the acyl group is an acetyl group or a benzoyl group;

[0024] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, wherein f is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5;

[0025] X2 is -(CH2) g -, wherein g is an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6;

[0026] Y1 is 0 or 1;

[0027] Y2 is 0, 1 or 2;

[0028] Y3 is 1, 2 or 3;

[0029] m is an integer from 0 to 4, for example, 1, 2, 3 or 4; when m is 0, -OR3 is directly connected to the ring;

[0030] n is an integer from 0 to 4, for example, 1, 2, 3 or 4; when n is 0, the ring is a five-membered ring;

[0031] L2 is -P(=O)(OH)-O-, -P(=S)(OH)-O- or -C(=O)-;

[0032] L3 and L5 are each independently -(CH2CH2O) j -CH2-CH2-, -(CH2CH2O) j -、-(OCH2CH2) j -、-(CH2) k -、-((CH2) q NHCO) r -or-((CH2) q CONH) r -, wherein j is selected from an integer of 0-200, for example, 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200, etc., k is an integer of 1-15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, q is an integer of 1-7, for example, 1, 2, 3, 4, 5, 6 or 7, and r is an integer of 1-5, for example, 1, 2, 3, 4 or 5;

[0033] L4 and L6 are independent of each other. , -CONH- or -NHCO-;

[0034] L7 is a lipid chain group selected from: , or Any one of .

[0035] After the GalNAc compound containing a ribose ring of the present invention is coupled with a nucleic acid drug (e.g., a PCSK9 inhibitor), the binding of PCSK9 to the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes can be inhibited by utilizing the principle of GalNAc binding to ASGPR, so that LDL-R takes up more LDL-C, thereby improving the clearance of LDL-C in plasma. Although the coupling with GalNAc-nucleic acid drugs has been widely used, the coupling of the GalNAc compound containing a ribose ring with the siRNA nucleic acid drug has stronger liver targeting compared with the conventional GalNAc-nucleic acid drug coupling. This is because ASGPR is a heterogeneous multimeric receptor complex composed of multiple subunits with carbohydrate recognition domains. Increasing the number of sugars that bind to multiple receptor subunits at the same time will increase the affinity exponentially. And the spatial arrangement between the sugar and the ligand (GalNAc) determines its specificity and efficiency of binding to ASGPR.

[0036] The present invention combines the advantages of GalNAc and LNP delivery, combines the oligonucleotide drug encapsulated in LNP with the coupling method of GalNAc compound, and the obtained nanoparticle drug has better targeting and stability. Although there are a few studies on the coupling of LNP with GalNAc, the method of coupling LNP with GalNAc containing ribose ring described in the present invention has not been reported. The present invention is to couple the GalNAc compound containing ribose ring with LNP through a one-step preparation method. Thereby improving the liver targeting of oligonucleotide drugs, enhancing the efficacy while reducing side effects.

[0037] Preferably, in formula IX, R1 is oxygen.

[0038] Preferably, in formula IX, R3 is hydrogen.

[0039] Preferably, in Formula IX, A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d-, wherein a is an integer from 3 to 13, b is an integer from 2 to 5, c is an integer from 2 to 6, and d is an integer from 1 to 3.

[0040] Preferably, in Formula IX, B is -(CH2) e -, where e is an integer from 1 to 5.

[0041] Preferably, in Formula IX, G is , wherein T is N-acetyl-galactosamine with unprotected hydroxyl group.

[0042] Preferably, in Formula IX, m is 0, 1 or 2.

[0043] Preferably, in Formula IX, n is 0, 1 or 2.

[0044] In one embodiment, A is -(CH2) 10 -, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 11 -、-(CH2) 12 -, -(CH2CH2O)3-, -(CH2)4NHCO- or -(CH2)6NHCO-.

[0045] In one embodiment, B is -CH2-, -(CH2)2-, -(CH2)4-, or -(CH2)3-.

[0046] In one embodiment, R3 is a hydroxy protecting group, preferably a trityl group, a monomethoxytrityl group or a 4,4'-dimethoxytrityl group, and more preferably a 4,4'-dimethoxytrityl group.

[0047] In one embodiment, m is 1.

[0048] In one embodiment, n is 0.

[0049] In one embodiment, G is ,

[0050] or .

[0051] In the present invention, the above compound or a pharmaceutically acceptable salt thereof can bind to asialoglycoprotein receptor (ASGPR).

[0052] Preferably, the cationic lipid is selected from one or more of the following compounds:

[0053] (1) A compound of formula I, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein G1 is C 1-6 Alkylene; G2 is C2-8 Alkylene; G3 is C 1-3 Alkylene; L8 is C 6-15 Straight chain alkyl; L9 is C 12-25 Branched chain alkyl;

[0054] ;

[0055] (2) A compound of formula II, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein G4 is C 2-8 Alkylene; G5 is C 2-8 Alkylene; L 10 is -C(O)O- or -OC(O)-; L 11 is -C(O)O- or -OC(O)-; R4 is C 6-25 Straight or branched alkyl; R5 is C 6-25 Straight or branched alkyl; G6 is HO(CH2)2- or HO(CH2)3-; G7 is HO(CH2)2- or HO(CH2)3-; L 12 is -(CH2)2- or -(CH2)3- or -(CH2)4-;

[0056] ;

[0057] (3) A compound of formula III, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein G8 is C 1-6 Alkylene; G9 is C 2-8 Alkylene; R6 is C 6-20 Straight or branched alkyl; R7 is C 12-25 Branched alkyl; G 10 are: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2) 2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-;

[0058] ;

[0059] (4) A compound of formula IV, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein G 11 C 1-8 Alkylene; G 12 C 2-8Alkylene; R9 is C 6-25 Straight or branched alkyl; R 10 C 12-25 Straight or branched alkyl; G 13 is: HO(CH2)2N(R m )CH2CH(OH)CH2-, where R m is -CH3, -CH2CH3 or -CH2CH2OH;

[0060] ;

[0061] (5) A compound of formula V, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein G 1 and G 2 Each is independently unsubstituted C 6-10 Alkylene; G 3 For unsubstituted C 1-12 Alkylene; R 1 and R 2 Each independently is C 6-24 Alkyl or C 6-24 Alkenyl; R 3 OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; Among them, R 4 C 1-12 A hydrocarbon group; and R 5 H or C 1-6 Hydrocarbon;

[0062] ;

[0063] (6) A compound of formula VI, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; wherein R 11 Selected from -(CH2) t Q or -(CH2) t CHQR; where R 11 Each Q in is independently selected from the group consisting of: -OR, -OH, -O(CH2) twherein: -N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycle; t is 1, 2 or 3; each R is independently selected from the group consisting of: C 1-3 , C 2-3 Alkenyl, (CH2) p OR 6 or H, and p is independently selected from 1, 2 or 3; each R 6 Independently selected from C 1-12 Alkyl and / or C 2-12 each X is independently selected from the group consisting of: F, Cl, Br or I;

[0064] ;

[0065] (7) A compound of formula VII, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof;

[0066] .

[0067] Preferably, the cationic lipid comprises:

[0068] , , , or Any one or a combination of at least two.

[0069] Preferably, the neutral lipid comprises any one of DSPC, DPPC, DMPC, POPC or DOPE, or a combination of at least two thereof.

[0070] Preferably, the auxiliary lipid comprises: any one or a combination of at least two of cholesterol, vitamin E, DC-cholesterol or their derivatives.

[0071] Preferably, the long-circulating lipid comprises any one or a combination of at least two of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) or methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0072] Preferably, the molar ratio of the cationic lipid, neutral lipid, auxiliary lipid, long-circulating lipid, and ribose ring-containing GalNAc anchor compound is (30-60):(5-15):(30-50):(0.5-5):(0.1-10).

[0073] Among them, 30-60 can be, for example, 30, 35, 40, 45, 50, 55 or 60, etc.; 5-15 can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, etc.; 30-50 can be, for example, 30, 35, 40, 45 or 50, etc.; 0.5-5 can be, for example, 0.5, 1, 2, 3, 4 or 5, etc.; 0.1-10 can be, for example, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0074] Preferably, the nucleic acid is selected from any one or a combination of at least two of small interfering nucleotides (siRNA), DNA, micro RNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA) or antisense nucleotides (ASO).

[0075] Preferably, the mass ratio of the nucleic acid to the cationic lipid is 1:(10-12), for example, it can be 1:10, 1:11 or 1:12, etc.

[0076] Preferably, the components of the lipid nanoparticles further include: buffer salts and excipients.

[0077] Preferably, the buffer salt comprises: any one of Tris-HCl / Tris buffer salt, DPBS buffer salt or phosphate buffer salt, or a combination of at least two thereof.

[0078] Preferably, the pH of the buffer salt is 4-8, for example, 4, 5, 6, 7 or 8.

[0079] Preferably, the excipient comprises: any one of sucrose, trehalose or maltose, or a combination of at least two of them.

[0080] In a second aspect, the present invention provides a method for preparing the GalNAc compound-coupled nucleic acid lipid nanoparticles containing a ribose ring structure as described in the first aspect, the preparation method comprising:

[0081] (a) preparing an aqueous phase containing nucleic acid and an organic phase containing lipids and an anchoring compound containing a ribose ring-containing GalNAc, and encapsulating the nucleic acid using a microfluidic device to obtain a nucleic acid-lipid nanoparticle solution;

[0082] (b) Nucleic acid-lipid nanoparticle solution was dialyzed against buffer solution.

[0083] In a third aspect, the present invention provides use of the GalNAc compound-containing ribose ring structure-coupled nucleic acid lipid nanoparticles described in the first aspect in the preparation of a drug for treating and / or preventing pathological conditions or diseases caused by the expression of specific genes in hepatocytes.

[0084] The specific gene is selected from: any one of the hepatitis B virus gene, the angiopoietin protein 3 gene or the apolipoprotein C3 gene, or a combination of at least two of them.

[0085] Preferably, the disease comprises any one of chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease or dyslipidemia, or a combination of at least two thereof.

[0086] Preferably, the dyslipidemia includes any one of hypercholesterolemia, hypertriglyceridemia or atherosclerosis, or a combination of at least two thereof.

[0087] In a fourth aspect, the present invention provides a ribose ring-containing GalNAc anchor compound, wherein the ribose ring-containing GalNAc anchor compound comprises: a ribose ring-containing GalNAc portion and a lipid chain group; the structural formula of the anchor compound is shown in Formula IX;

[0088] ;

[0089] Wherein, R1 is oxygen or sulfur, preferably oxygen;

[0090] R2 is hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy or halogen;

[0091] R3 is hydrogen or a hydroxyl protecting group, preferably hydrogen;

[0092] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer of 1-15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably an integer of 3-13, b is an integer of 1-7, for example, 1, 2, 3, 4, 5, 6 or 7, preferably an integer of 2-5, c is an integer of 1-7, for example, 1, 2, 3, 4, 5, 6 or 7, preferably an integer of 2-6, d is an integer of 1-5, for example, 1, 2, 3, 4 or 5, preferably an integer of 1-3;

[0093] B is -(CH2)e -, wherein e is an integer from 0 to 7, for example, 0, 1, 2, 3, 4, 5, 6 or 7; when e is 0, B is a single bond; preferably an integer from 1 to 5;

[0094] L1 is -CONH- or -NHCO-;

[0095] G is , wherein T is N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine or N-isobutyryl-galactosamine, wherein the hydroxyl group is fully protected or unprotected with an acyl group, wherein the acyl group is an acetyl group or a benzoyl group, the acyl group is preferably an acetyl group, and T is preferably N-acetyl-galactosamine, which is an unprotected hydroxyl group;

[0096] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, wherein f is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5;

[0097] X2 is -(CH2) g -, wherein g is an integer from 1 to 6, for example, 1, 2, 3, 4, 5 or 6;

[0098] Y1 is 0 or 1;

[0099] Y2 is 0, 1 or 2;

[0100] Y3 is 1, 2 or 3;

[0101] m is an integer from 0 to 4, for example, 1, 2, 3 or 4; when m is 0, -OR3 is directly connected to the ring; preferably 0, 1 or 2;

[0102] n is an integer of 0-4, for example, 1, 2, 3 or 4; when n is 0, the ring is a five-membered ring; preferably 0, 1 or 2;

[0103] L2 is -P(=O)(OH)-O-, -P(=S)(OH)-O- or -C(=O)-;

[0104] L3 and L5 are each independently -(CH2CH2O) j -CH2-CH2-, -(CH2CH2O) j -、-(OCH2CH2) j -、-(CH2) k -、-((CH2) q NHCO) r -or-((CH2) q CONH) r-, wherein j is selected from an integer of 0-200, for example, 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200, etc., k is an integer of 1-15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, q is an integer of 1-7, for example, 1, 2, 3, 4, 5, 6 or 7, and r is an integer of 1-5, for example, 1, 2, 3, 4 or 5;

[0105] L4 and L6 are independent of each other. , -CONH- or -NHCO-;

[0106] L7 is a lipid chain group selected from:

[0107] , or Any one of .

[0108] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the GalNAc compound containing a ribose ring structure coupled to nucleic acid lipid nanoparticles as described in the first aspect and a pharmaceutically acceptable excipient.

[0109] The present invention also provides a method for inhibiting the expression of specific genes in hepatocytes, comprising contacting an effective amount of the GalNAc compound containing a ribose ring structure described in the first aspect above with nucleic acid lipid nanoparticles, or the pharmaceutical composition of the fifth aspect or the pharmaceutical composition of the third aspect with the hepatocytes.

[0110] In the present invention, the specific gene may be, for example, any one of the following genes: proprotein convertase subtilisin / kexin type 9 gene (PCSK9), ApoB, ApoC, ANGPTL3, SCD1, FVII, p53, HBV or HCV.

[0111] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0112] Compared with the prior art, the present invention has the following beneficial effects:

[0113] The present invention couples a GalNAc compound containing a ribose ring with LNP through a one-step preparation method, thereby improving the liver targeting and stability of the nucleic acid drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 The inhibition rates of PCSK9 protein expression in mouse serum of the samples of Example 1, Comparative Examples 1 and 2 at 7 days and 14 days.

[0115] Figure 2 The effects of the samples of Example 1, Comparative Examples 1 and 2 on the LDL-C levels in mouse serum at 7 days and 14 days.

[0116] Figure 3 The inhibition rates of PCSK9 protein expression in mouse serum of the samples of Example 1 and Comparative Examples 3, 4 and 7 at 7 and 14 days.

[0117] Figure 4 The effects of the samples of Example 1 and Comparative Examples 3, 4 and 7 on the LDL-C levels in mouse serum at 7 days and 14 days.

[0118] Figure 5 The inhibition rates of PCSK9 protein expression in mouse serum of the samples of comparative examples 1, 5 and 6 at 7 and 14 days.

[0119] Figure 6 The effects of the samples of comparative examples 1, 5 and 6 on the LDL-C levels in the serum of mice at 7 and 14 days. DETAILED DESCRIPTION

[0120] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0121] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0122] The term "pharmaceutically acceptable" in the present invention means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the formulation and / or with humans or mammals for preventing or treating diseases or disorders.

[0123] The term "pharmaceutically acceptable salt" in the present invention refers to a relatively non-toxic, inorganic acid or organic acid addition salt of the compound of the present invention. For example, see S. M. Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19.

[0124] The term "alkyl" in the present invention refers to a branched and straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. 1-4The term "alkyl" includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0125] The term "alkoxy" herein refers to the formula -OR, wherein R is an alkyl group as defined herein. A non-limiting list of alkoxy groups is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, phenoxy, and benzoyloxy. In some cases, the alkoxy group may be -OR, wherein R is an unsubstituted C 1-4 Alkyl. Alkoxy may be substituted or unsubstituted.

[0126] The term "halogen" in the present invention refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), preferably fluorine (F) and chlorine (Cl). In one embodiment, the halogen is fluorine.

[0127] The term "protecting group" in the present invention refers to any atom or atomic group introduced into a molecule to prevent an existing group in the molecule from undergoing undesired chemical reactions, which can be removed to leave an unprotected group.

[0128] The hydroxyl protecting group may be, for example, a protecting group commonly used to protect the hydroxyl group of a ribose structure in the synthesis of RNA or its derivatives, or may be a reference to the protecting groups described in Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc. by Green et al. For example: acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4,4'-dimethoxytrityl (DMTr), monomethoxytrityl (MMTr, monomethoxytrityl), 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, etc., preferably 4,4'-dimethoxytrityl (DMTr, 4,4'-dimethoxytrityl).

[0129] The term "nucleotide" in the present invention includes naturally occurring nucleotides and chemically modified nucleotides. Chemically modified nucleotides are non-naturally occurring nucleotides, also referred to herein as "nucleotide analogs".

[0130] The “hydroxyl groups are fully protected with acyl groups” described for “T” in the present invention means that all hydroxyl groups in the galactose structure except the hydroxyl group used for connecting to X1 are protected with acyl groups, wherein the acyl group is, for example, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl or benzoyl.

[0131] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below.

[0132] Example 1

[0133] This embodiment provides a method for preparing GalNAc-coupled nucleic acid-lipid nanoparticles, the method comprising the following steps:

[0134] 1. Preparation of siRNA

[0135] The synthesized siRNA sequences are as follows:

[0136] Sense strand: 5'-Cms-Ums-Am-Gm-Am-Cm-Cf-Um-Gf-Um-dT-Um-Um-Gm-Cm- Um-Um-Um-Um-Gm-Um-3' (SEQ ID NO: 1);

[0137] Antisense strand: 5'-Ams-Cfs-Am-Af-Af-Af-Gm-Cf-Am-Af-Am-Af-Cm-Af-Gm-Gf-Um-Cf-Um-Am-Gms-Ams-Am-3' (SEQ ID NO: 2).

[0138] The corresponding basic sequence is as follows:

[0139] Sense strand SEQ ID NO: 1: 5′-CUAGACCUGUUUUGCUUUUGU-3′;

[0140] Antisense strand SEQ ID NO: 2: 5'-ACAAAAGCAAAACAGGUCUAGAA-3'.

[0141] Among them, A, U, C, and G represent the base composition of the nucleotide; dT represents deoxythymidine nucleotide; m represents that the nucleotide adjacent to the left of m is 2'-OMe modified; f represents that the nucleotide adjacent to the left of m is 2'-F modified; s represents that the two adjacent nucleotides on the left and right of s are connected by thiophosphate groups.

[0142] The above sequence comes from a public sequence and is the oligonucleotide sequence of the siRNA drug inclisiran that has been marketed overseas.

[0143] The inclisiran sequence is a synthetic, chemically modified double-stranded small interfering RNA (siRNA) that targets and binds to the mRNA encoding the PCSK9 protein, inhibiting the production of the PCSK9 protein through the RNA interference mechanism, thereby regulating the recycling and reuse of the LDL receptor and enhancing its binding with LDL, thereby achieving the purpose of lowering LDL in the blood.

[0144] siRNA sense and antisense strands were synthesized on a universal CPG solid support using phosphoramidite chemistry.

[0145] (1) Preparation of reagents and monomers

[0146] The monomer solution in acetonitrile (1 / 20, w / v), 0.25 M 5-benzylthiotetrazolyl solution in acetonitrile was used as an activating agent, 0.2 M hydroxanthin solution in acetonitrile / pyridine (1 / 4, v / v) was used as a thiolation agent, 0.05 M iodine solution in water / pyridine (1 / 9, v / v) was used as an oxidizing agent, 20% acetic anhydride in acetonitrile (v / v) was used as a capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) was used as a capping agent B, 20% diethylamine in acetonitrile (v / v) was used as a decyanoethylation agent, and 3% dichloroacetic acid in toluene (v / v) was used as a de-DMT agent. The reagents were loaded into the designated reagent position of the 192 P DNA / RNA automatic synthesizer.

[0147] (2) Crude product synthesis

[0148] Enter the designated oligonucleotide sequence and set the synthesis program. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The monomer coupling time is about 1 minute, of which the oxygen generation time is about 30-45 seconds and the thio generation time is about 2 minutes. After the cycle is completed, the solid phase synthesis of the oligonucleotide is completed.

[0149] (3) Deprotection

[0150] After the synthesis is completed, the solid phase carrier is transferred to a reactor, and the oligonucleotide is cleaved from the solid phase carrier with concentrated ammonia (25-28%) at 50-60°C for 16-24 hours. The system is cooled to room temperature and then filtered, rinsed with a mixed solution of purified water and ethanol, the filtrates are combined, and the filtrates are concentrated at low temperature to obtain a crude residue.

[0151] (4) Purification

[0152] The crude residue after deprotection was dissolved in purified water and purified by HPLC. The product peak solution was collected and the content was measured by an enzyme marker, and the molecular weight was confirmed by ESI MS.

[0153] (5) Annealing

[0154] The siRNA sense strand and the complementary antisense strand were mixed in a ratio of 1:1 according to the UV absorption content, heated to 95°C, and cooled to room temperature after 3 minutes to form a double strand. The obtained double-stranded solution was characterized by HPLC, and the content was determined by an ELISA instrument after the product purity was qualified, and lyophilized to obtain a solid powder for storage.

[0155] 2. Preparation of GalNAc-Lipids (anchored lipids) containing ribose rings

[0156] The method for preparing the GalNAc compound containing a ribose ring refers to CN116854754A.

[0157] (1) Synthesis of GL1

[0158] The synthetic route is as follows:

[0159]

[0160] Step 1: Synthesis of GL1-3.

[0161] Pyridine (2.68 g, 33.84 mmol, 4.0 eq.) was added to a solution of compound GL1-1 (5.05 g, 8.46 mmol, 1.0 eq.) and GL1-2 (6.82 g, 33.84 mmol, 4.0 eq.) in dichloromethane (50 mL) at room temperature and stirred for 4 h. The reaction mixture was concentrated to dryness and purified by preparative chromatography to obtain compound GL1-3 (5.22 g, 6.85 mmol, yield: 81%), MS (ESI) m / z [M+H] + =763.5.

[0162] Step 2: Synthesis of GL1-5.

[0163] At room temperature, pyridine (0.72 g, 9.16 mmol, 2.0 eq.) was added to a dichloromethane solution (40 mL) of compound GL1-3 (3.49 g, 4.58 mmol, 1.0 eq.) and GL1-4 (11.27 g, 6.87 mmol, 1.5 eq.) and stirred overnight. The reaction mixture was diluted with water. The product was extracted into dichloromethane and concentrated to dryness to obtain a crude product. The crude product was further separated and purified by preparative chromatography to obtain compound GL1-5 (8.61 g, 3.80 mmol, yield: 83%), MS (ESI-TOF) m / z[M+H] + =2264.7.

[0164] Step 3: Synthesis of GL1-8.

[0165] At room temperature, dicyanoimidazole (0.39 g, 3.28 mmol, 2.0 eq.) was added to a solution (50 mL) of GL1-6 (4.30 g, 1.64 mmol, 1.0 eq., prepared according to the preparation method on page 60 of patent CN116854754B) and GL1-7 (0.54 g, 2.46 mmol, 1.5 eq.) in acetonitrile, stirred for 2 h, then tert-butyl hydroperoxide (0.74 g, 8.20 mmol, 5.0 eq.) was added and stirred for 30 min, and then trifluoroacetic acid (1.87 g, 16.4 mmol, 10.0 eq.) was added and stirred for 2 h. The reaction mixture was quenched with aqueous sodium bicarbonate solution. The product was extracted into dichloromethane and concentrated to dryness to obtain a crude product. The crude product was further separated and purified by preparative chromatography to obtain compound GL1-8 (3.42 g, 1.39 mmol, yield: 85%), MS (ESI-TOF) m / z [M+H] + =2459.3.

[0166] Step 4: Synthesis of GL1-9.

[0167] Aqueous ammonia (12 mL) was added to a mixture of compound GL1-8 (3.42 g, 1.39 mmol) and ethanol (6 mL) at room temperature, and the resulting reaction mixture was heated at 45 °C for 20 h. The reaction mixture was concentrated under reduced pressure at 40 °C. The product was extracted into dichloromethane and concentrated to dryness to obtain a crude product. Compound GL1-9 (2.54 g, 1.25 mmol, yield: 90%) was obtained, MS (ESI-TOF) m / z [M+H] + =2028.6.

[0168] Step 5: Synthesis of GL1.

[0169] Compound GL1-5 (2.60 g, 1.15 mmol, 1.75 eq.) was added to a solution of compound GL1-9 (1.33 g, 0.66 mmol, 1.00 eq.) in dimethyl sulfoxide (15 mL). Sodium ascorbate (0.65 g, 3.28 mmol, 5.00 eq.) was added to the above mixture. Tris(3-hydroxypropyltriazolemethyl)amine (0.57 g, 1.31 mmol, 2.00 eq.) and copper sulfate (0.32 g, 1.96 mmol, 3.00 eq.) were dissolved in pure water (3 mL) and added to the reaction mixture. The reaction mixture was stirred at 45°C for 16 h. The reaction solution was diluted with dichloromethane (15 mL) and washed twice with water (150 mL×2). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was further separated and purified by preparative chromatography to obtain compound GL1 (2.49 g, 0.58 mmol, yield: 88%).

[0170] 1 HNMR (400 MHz, DMSO-d6) δ ppm δ8.18 (d, J =6.2Hz, 1H), 7.84 - 7.72 (m, 3H), 7.30 (m, 7H), 6.44 (m, 1H), 5.83 (m, 1H), 5.26 (d, J = 3.5 Hz, 4H), 4.22 - 4.00 (m, 10H), 3.87 (dt, J = 13.4, 5.5 Hz, 6H), 3.77 (m, 6H), 3.70 (m, 3H), 3.58 - 3.32 (m, 14H), 3.29 - 3.03 (m, 186H), 2.95 - 2.80 (m, 6H), 2.24 - 2.09 (m, 14H), 1.75 (s, 8H), 1.64 - 1.57 (m, 15H), 1.53 - 1.37 (m, 26H), 1.24 (s, 66H), 0.89 - 0.81 (m, 6H). MS(ESI-TOF) m / z [M+H] + =4292.5.

[0171] 2. Synthesis of GL2

[0172] The synthetic route is as follows:

[0173]

[0174] Step 1: Synthesis of GL2-2.

[0175] Pyridine (0.21 g, 2.60 mmol, 2.00 eq.) was added to a dichloromethane solution of GL2-1 (0.58 g, 1.30 mmol, 1.00 eq.) and compound GL1-4 (2.13 g, 1.30 mmol, 1.00 eq.) at room temperature, and then stirred for 2 h. The reaction mixture was concentrated to dryness and purified by preparative chromatography to obtain compound GL2-2 (2.47 g, 1.20 mmol, yield: 92%). MS (ESI-TOF) m / z [M+H] + =2054.1.

[0176] Step 2: Synthesis of GL2.

[0177] Compound GL2-2 (1.81 g, 0.88 mmol, 1.75 eq.) was added to a solution of compound GL1-9 (1.01 g, 0.50 mmol, 1.00 eq.) in dimethyl sulfoxide (10 mL). Sodium ascorbate (0.50 g, 2.50 mmol, 5.00 eq.) was added to the above mixture. Tris(3-hydroxypropyltriazolemethyl)amine (0.43 g, 1.00 mmol, 2.00 eq.) and copper sulfate (0.24 g, 1.50 mmol, 3.00 eq.) were dissolved in pure water (2 mL) and added to the reaction mixture. The reaction mixture was stirred at 45°C for 16 h. The reaction solution was diluted with dichloromethane (10 mL) and washed twice with water (10 mL×2). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was further separated and purified by preparative chromatography to obtain compound GL2 (1.81 g, 0.45 mmol, yield: 90%).

[0178] 1HNMR (400 MHz, DMSO-d6) δ ppm δ8.17 (d, J =6.1Hz, 1H), 7.82 - 7.72 (m, 3H), 7.28 (m, 7H), 6.43 (m, 1H), 5.86 (m, 1H), 5.25 (d, J = 3.5 Hz, 4H), 4.22 - 4.03 (m, 9H), 3.87 (dt, J = 13.2, 5.6 Hz, 5H), 3.77 (m, 6H), 3.69 (m, 3H), 3.51 - 3.30 (m, 14H), 3.28 - 3.01 (m, 182H), 2.96 - 2.80 (m, 6H), 2.29 - 2.11 (m, 20H), 1.74 (s, 8H), 1.63 - 1.54 (m, 15H), 1.50 - 1.34 (m, 24H), 1.25 (s, 26H), 0.88 - 0.81 (m, 12H). MS(ESI-TOF) m / z [M+H] + =4067.5.

[0179] 3. Synthesis of GL3

[0180] The synthetic route is as follows:

[0181]

[0182] Step 1: Synthesis of GL3-2.

[0183] At room temperature, dicyanoimidazole (0.31 g, 2.60 mmol, 2.0 eq.) was added to a solution of compound GL1-6 (3.42 g, 1.30 mmol, 1.0 eq.) and GL3-1 (3.32 g, 1.95 mmol, 1.5 eq.) in acetonitrile (50 mL), stirred for 2 h, then tert-butyl hydroperoxide (0.59 g, 6.50 mmol, 5.0 eq.) was added and stirred for 30 min, and then trifluoroacetic acid (1.48 g, 13.0 mmol, 10.0 eq.) was added and stirred for 2 h. The reaction mixture was quenched with aqueous sodium bicarbonate solution. The product was extracted into dichloromethane and concentrated to dryness to obtain a crude product. The crude product was further separated and purified by preparative chromatography to obtain compound GL3-2 (4.11 g, 1.07 mmol, yield: 82%), MS (ESI-TOF) m / z [M+H] + =3843.0.

[0184] Step 2: Synthesis of GL3-3.

[0185] Ammonia water (12 mL) was added to a mixture of compound GL3-2 (3.23 g, 0.84 mmol) and ethanol (6 mL) at room temperature, and the resulting reaction mixture was heated at 45 °C for 20 h. The reaction mixture was concentrated under reduced pressure at 40 °C. The product was extracted into dichloromethane and concentrated to dryness to obtain a crude product. Compound GL3-3 (2.69 g, 0.77 mmol, yield: 92%) was obtained, MS (ESI-TOF) m / z [M+H] + =3411.4.

[0186] Step 3: Synthesis of GL3.

[0187] GL3-4 (147 mg, 0.47 mmol, 1.0 eq.), diisopropylethylamine (121 mg, 0.94 mmol, 1.0 eq.) and O-benzotriazole-tetramethyluronium hexafluorophosphate (269 mg, 0.71 mmol, 1.5 eq.) were dissolved in N,N-dimethylformyl (10 mL), and GL3-3 (1.65 g, 0.47 mmol, 1.0 eq.) was added, and stirred at 15 ° C for 1 h under nitrogen protection. The reaction mixture was concentrated to dryness and purified by preparative chromatography to obtain compound GL3 (1.37 g, 0.37 mmol, yield: 78%).

[0188] 1 HNMR (400 MHz, DMSO-d6) δ ppm δ8.18 (d, J =6.1Hz, 1H), 7.82 - 7.73 (m, 4H), 7.31 (m, 7H), 5.25 (d, J = 3.5 Hz, 4H), 4.21 - 4.00 (m, 9H), 3.84 (dt, J = 13.2, 5.6 Hz, 6H), 3.74 (m, 4H), 3.67 (m, 3H), 3.56 - 3.31 (m, 8H), 3.28 - 3.01 (m, 178H), 2.99 - 2.80 (m, 6H), 2.24 - 2.09 (m, 14H), 1.77 (s, 10H), 1.66 - 1.53 (m, 15H), 1.50- 1.35 (m, 20H), 1.25 (s, 32H), 0.87 - 0.82 (m, 3H). MS (ESI-TOF) m / z [M+H] + =3706.5.

[0189] 3. Water phase preparation

[0190] The aqueous phase was prepared by dissolving siRNA in pH 4.0 citrate buffer.

[0191] 4. Organic phase preparation

[0192] Cationic lipid (YK009), neutral lipid (DSPC), auxiliary lipid (chol), long-circulating lipid (mPEG-DMG) and anchoring lipid (GL3) were dissolved in anhydrous ethanol at a ratio of 49:10:39:2 to prepare an organic phase.

[0193] 5. Encapsulation

[0194] The organic phase and the aqueous phase were quickly mixed in a microfluidic device (manufacturer: Precision NanoSystems) for encapsulation. The encapsulation method was that the ratio of the organic phase and the aqueous phase inflow rate was 1:3, the total inflow flow rate was 12 mL / min, and the chip model was Ignite NxGen.

[0195] 6. Dialysis

[0196] After encapsulation, the sample was placed in a dialysis bag (purchased from Shanghai Yuanye, 50 KD) and dialyzed overnight in a 25 mM buffer with a pH value of 7.6 to obtain a siRNA lipid nanoparticle (siRNA-LNP) sample.

[0197] Comparative Example 1

[0198] This comparative example provides a GalNAc-siRNA conjugate containing a ribose ring, and the preparation method of the conjugate is detailed in Examples 2 and 3 of Patent CN116854754A. Among them, the siRNA is inclisiran.

[0199] Comparative Example 2

[0200] This comparative example provides a LNP encapsulated siRNA, wherein the siRNA uses inclisiran. The preparation method thereof comprises: directly encapsulating the siRNA in the LNP through a microfluidic device. The preparation method is detailed in Example 1 in patent CN116672316B. Among them, the aqueous phase preparation, organic phase preparation (without anchoring lipid), encapsulation and dialysis steps are the same as those in Example 1.

[0201] Comparative Example 3

[0202] This comparative example provides a GalNAc-LNP conjugate, which is prepared according to the patented prescription of Onpattro.

[0203] This method is similar to Example 1, which is to first connect GalNAc to PEG, and then prepare it into LNP together with siRNA and other lipids through a microfluidic device, thereby achieving the coupling of GalNAc and LNP. The anchoring lipid structure is shown in patent CN102625696A, and the structure is shown below.

[0204]

[0205] The steps of aqueous phase preparation, organic phase preparation, encapsulation and dialysis are the same as those in Example 1.

[0206] Comparative Example 4

[0207] This comparative example uses other patented prescriptions to prepare GalNAc-LNP conjugates, wherein inclisiran is used as siRNA.

[0208] This method is similar to Example 1 and Comparative Example 3, which is to first connect GalNAc to cholesterol, and then prepare it together with siRNA and other lipids into LNP through a microfluidic device, thereby achieving the coupling of GalNAc and LNP. The anchored lipid structure is detailed in patent WO2023212622A1 Part IIA and FIG.11, and the structure is shown below.

[0209]

[0210] The steps of aqueous phase preparation, organic phase preparation, encapsulation and dialysis are the same as those in Example 1.

[0211] Comparative Example 5

[0212] This comparative example prepares a GalNAc compound with a similar structure containing a ribose ring. (siRNA uses inclisiran)

[0213] This method is similar to Comparative Example 1, and also directly chemically couples the GalNAc containing a ribose ring with the siRNA to form a conjugate. The difference is that the structure of the GalNAc compound containing a ribose ring is different from that of Comparative Example 1, as shown below, see Example 31 of patent WO 2013033230A for details.

[0214]

[0215] Comparative Example 6

[0216] Preparation of siRNA conjugates using GalNAc ligand L96

[0217] This method is similar to Comparative Examples 1 and 5, and also directly chemically couples GalNAc to siRNA to form a conjugate. The difference is that the structure of GalNAc is different from that of Comparative Examples 1 and 5, as shown below. The preparation method is detailed in Example 2 of patent CN116854754A (GalNAc ligand L96 compound is disclosed in US10465194B2).

[0218]

[0219] Comparative Example 7

[0220] This comparative example uses Verve Therapeutics patented formula to prepare GalNAc-LNP conjugates (siRNA uses inclisiran).

[0221] This method is similar to Example 1, in that GalNAc is first connected to a lipid chain as an anchoring compound, and then it is prepared into LNP together with siRNA and other lipids through a microfluidic device, thereby achieving the coupling of GalNAc and LNP. However, its GalNAc structure is different from that of Example 1 and does not have a ribose ring. The preparation method of the anchoring lipid is detailed in the publication Kasiewicz et al., in Nature Communications (doi.org / 10.1038 / s41467-023-37465-1). The aqueous phase preparation, organic phase preparation, encapsulation and dialysis steps are the same as those of Example 1.

[0222] Test Example 1

[0223] This test example compares the effects of GalNAc-LNP (encapsulated siRNA) coupling (GL1 in Example 1 is the anchoring lipid), GalNAc-siRNA conjugate containing a ribose ring (Comparative Example 1), and LNP encapsulated siRNA (LNP-siRNA) (Comparative Example 2), and examines the inhibition rate of PCSK9 protein expression and LDL-C levels in mouse serum using the three methods.

[0224] 1. Experiment on inhibition rate of PCSK9 protein expression in mouse serum

[0225] Experimental process:

[0226] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and the serum was separated by centrifugation at 4000 r / min for 10 min at 4°C. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological Company).

[0227] Experimental results:

[0228] The inhibition rate of PCSK9 protein in serum of negative control group and each test drug group 7 days and 14 days after administration was as follows Figure 1 As shown, Figure 1 The inhibition rates of PCSK9 protein expression in mouse serum of the samples of Example 1, Comparative Examples 1 and 2 at 7 days and 14 days were shown in Table 1. The data were statistically analyzed using GraphPad Prism software.

[0229] 2. Experiment on LDL-C level in mouse serum

[0230] Experimental process:

[0231] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at 4000 r / min for 10 min at 4°C to separate serum, and the level of serum LDL-C was detected.

[0232] Experimental results:

[0233] Serum LDL-C (Mean±SD) was statistically analyzed and graphed using GraphPad Prism software. Specific results are shown in Figure 2 , Figure 2 The effects of the samples of Example 1, Comparative Examples 1 and 2 on the LDL-C levels in mouse serum at 7 days and 14 days.

[0234] Summary: The experimental results show that the LNP conjugate prepared by GalNAc-lipid containing a ribose ring has a better inhibition rate of serum PCSK9 protein expression and LDL-C level after subcutaneous administration to mice than the sample directly conjugated with GalNAc containing a ribose ring and siRNA, and is better than the delivery form of directly encapsulating siRNA in LNP.

[0235] Test Example 2

[0236] Comparison of different LNP formulations.

[0237] In October 2018, the world's first siRNA drug patisiran (trade name: onpattro) was approved by the FDA for marketing. This product uses GalNAc-LNP as a delivery carrier. However, it is different from the LNP prescription and anchoring component used in the present invention. Therefore, this test example prepared LNP (all siRNA drugs are inclisiran) using the prescription of the present invention (Example 1), the patented prescription using onpattro (Comparative Example 3), another patented method using GalNAc-cholesterol as an anchoring component (Comparative Example 4), and the patented method of Verve Pharmaceuticals using GalNAc-lipid as an anchoring component (Comparative Example 7). Experimental comparisons were also conducted on the inhibition rate of PCSK9 protein expression and LDL-C levels in mouse serum.

[0238] Table 1 is a comparison of the LNP lipid formulations in Example 1, Comparative Examples 3, 4, and 7.

[0239] Table 1

[0240]

[0241] Note: YK009 is as follows:

[0242]

[0243] MC3 looks like this:

[0244]

[0245] 1. Experiment on inhibition rate of PCSK9 protein expression in mouse serum

[0246] Experimental process:

[0247] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and the serum was separated by centrifugation at 4000 r / min for 10 min at 4°C. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological Company).

[0248] Experimental results:

[0249] The inhibition rate of PCSK9 protein in serum of negative control group and each test drug group 7 days and 14 days after administration was as follows Figure 3 As shown, Figure 3 The inhibition rates of PCSK9 protein expression in mouse serum of the samples of Example 1, Comparative Examples 3, 4, and 7 at 7 days and 14 days were shown in Table 1. The data were statistically analyzed using GraphPad Prism software.

[0250] 2. Experiment on LDL-C level in mouse serum

[0251] Experimental process:

[0252] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at 4000 r / min for 10 minutes at 4°C to separate serum, and the level of serum LDL-C was detected.

[0253] Experimental results:

[0254] Serum LDL-C (Mean±SD) was statistically analyzed using GraphPad Prism software for plotting and data analysis. The specific results are shown in Figure 4 , Figure 4 The effects of the samples of Example 1 and Comparative Examples 3, 4 and 7 on the LDL-C levels in mouse serum at 7 days and 14 days.

[0255] Summary: The experimental results show that although Example 1, Comparative Examples 3, 4, and 7 all use GalNAc and lipid to prepare anchoring components, and then mix the anchoring components with other lipid components to prepare LNP conjugates. However, the results of mouse serum PCSK9 protein expression inhibition rate and LDL-C level show that the GalNAc-lipid conjugate containing a ribose ring used in the present invention (Example 1) is better than the GalNAc-PEG conjugation method used in Comparative Example 3, better than the GalNAc-cholesterol conjugation method used in Comparative Example 4, and better than the GalNAc-lipid conjugation method used in Comparative Example 7.

[0256] Test Example 3

[0257] Comparative Examples 1, 5, and 6 are comparisons of three different methods of direct connection of GalNAc to siRNA.

[0258] The GalNAc compound containing a ribose ring described in the present invention (Comparative Example 1) is similar in structure to the GalNAc compounds of Comparative Examples 5 and 6, and both use a method of direct coupling of GalNAc with siRNA. Therefore, the samples of Comparative Examples 1, 5, and 6 were also compared in terms of mouse serum PCSK9 protein expression inhibition rate and LDL-C level.

[0259] Comparative Example 5 also uses a GalNAc containing a ribose ring to directly connect with the siRNA drug, thereby achieving a targeted drug delivery effect.

[0260] Comparative Example 6 is the first siRNA drug Inclisiran injection (Lecoway®) approved for marketing by the National Medical Products Administration (NMPA). As a siRNA drug, Inclisiran is directly coupled with GalNAc ligand L96 (see the formula below) to reduce LDL-C by inhibiting the synthesis of protein convertase PCSK9 in stem cells. All the GalNAc-siRNA conjugates currently on the market use L96 as a carrier.

[0261]

[0262] The products of the above three methods were used to compare the PCSK9 protein expression inhibition rate and LDL-C level in mouse serum.

[0263] 1. Experiment on inhibition rate of PCSK9 protein expression in mouse serum

[0264] Experimental process:

[0265] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and the serum was separated by centrifugation at 4000 r / min for 10 min at 4°C. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological Company).

[0266] Experimental results:

[0267] The inhibition rate of PCSK9 protein in serum of negative control group and each test drug group 7 days and 14 days after administration was as follows Figure 5 As shown, Figure 5 The inhibition rates of PCSK9 protein expression in mouse serum of samples of comparative examples 1, 5 and 6 on days 7 and 14 were shown in Table 1. GraphPad Prism software was used for data statistics and analysis.

[0268] 2. Experiment on LDL-C level in mouse serum

[0269] Experimental process:

[0270] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at 4000 r / min for 10 minutes at 4°C to separate serum, and the level of serum LDL-C was detected.

[0271] Experimental results:

[0272] Serum LDL-C (Mean±SD) was statistically analyzed using GraphPad Prism software for plotting and data analysis. The specific results are shown in Figure 6 , Figure 6 The effects of the samples of comparative examples 1, 5 and 6 on the LDL-C levels in the serum of mice at 7 and 14 days.

[0273] Summary: The experimental results show that although Comparative Examples 1, 5, and 6 all use chemically modified GalNAc to couple with siRNA, the results of mouse serum PCSK9 protein expression inhibition rate and LDL-C level show that the structure and site containing ribose ring used in the present invention (Comparative Example 2) are superior to the GalNAc modification methods of Comparative Examples 5 and 6.

[0274] Test Example 4

[0275] After the LNP samples described in Example 1 and Comparative Examples 2, 3, 4, and 7 were stored at room temperature for 3 months, the particle size, dispersion, encapsulation efficiency, and integrity of each sample were measured and compared with the 0-day data, which helped to evaluate the effect of each coupling method on stability.

[0276] 1. Particle size and dispersion

[0277] The particle size and particle size distribution of liposomes are related to their encapsulation efficiency and stability, directly affecting the behavior of liposomes in body tissues, and are important parameters for evaluating their biochemistry, biophysics and drug delivery systems, affecting the product's in vivo biodistribution and drug pharmacokinetics. Therefore, particle size and particle size distribution are important characterization indicators of LNP product stability. Dynamic light scattering is used to detect particle size and dispersion.

[0278] 2. Encapsulation rate

[0279] The encapsulation efficiency is a key quality attribute of liposomes. It refers to the percentage of drug content encapsulated in the lipid bilayer to the total dosage. The higher the encapsulation efficiency, the greater the proportion of drug encapsulated by the liposomes. The encapsulation efficiency is an important indicator of the stability of LNP products.

[0280] The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK). The specific method is as follows:

[0281] The sample was diluted to 2.8 μg / mL, one part was mixed with an equal volume (50 μL) of Triton X-100, and the emulsion was fully broken for the determination of total RNA concentration, and the other part was mixed with an equal volume (50 μL) of TE (Tris-EDTA) for the determination of free unencapsulated RNA concentration. After the samples with TE (Tris-EDTA) and Triton X-100 were incubated at 37°C for a certain period of time, 100 μL of Ribogreen reagent diluted 200 times was added, and after centrifugation to remove bubbles, the fluorescence value was measured using a multifunctional microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the plate was read.

[0282] 3. Purity

[0283] 3.1 Non-denaturing IP-RP-HPLC method

[0284] Determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0285] Test solution: Accurately measure an appropriate amount of the product and dilute it with 1×PBS (0.01 mol / L phosphate buffered saline) to make a solution containing approximately 1 mg of INCLISIRAN per 1 mL.

[0286] System suitability solution: Take appropriate amount of INCLISIRAN control substance (or typical test substance) and antisense strand control substance (or typical test substance), add 1× PBS to dilute to make a solution containing approximately 1 mg of INCLISIRAN and 0.1 mg of antisense strand per 1 mL.

[0287] Sensitivity solution: Accurately pipette an appropriate amount of system suitability solution and dilute with 1× PBS to make a solution containing approximately 0.002 mg of INCLISIRAN per 1 mL.

[0288] Chromatographic conditions: chromatographic column with octadecyl bonded silica as filler (Xbridge Oligonucleotide BEH C18, 130Å, 2.5 μm, 4.6×50mm or equivalent); mobile phase A: aqueous solution containing 120 mmol hexafluoroisopropanol, 15 mmol dibutylamine, and 1 mmol disodium ethylenediaminetetraacetate (EDTA·2Na) per 1 L; mobile phase B: acetonitrile-methanol (30:70), gradient elution according to Table 2. Flow rate: 0.30 mL per minute; detection wavelength: 260 nm; column temperature: 10°C; injection volume: 3 μL; sample tray temperature: 5°C.

[0289] Table 2

[0290]

[0291] System suitability requirements: In the system suitability solution chromatogram, the separation between the INCLISIRAN main peak and the antisense strand peak should be greater than 3.0; the relative standard deviation of the main peak area of ​​the 6-pin system suitability solution should be no greater than 2.0%, and the relative standard deviation of the retention time should be no greater than 1.0%. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10.

[0292] Determination method: Accurately measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0293] Limit: If there are impurity peaks in the chromatogram of the test solution, the purity should be no less than 90.0% calculated by the area normalization method.

[0294] 3.2 Denaturing IP-RP-HPLC method

[0295] Determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0296] Test solution: Accurately measure an appropriate amount of this product and dilute with water to make a solution containing approximately 1.5 mg of INCLISIRAN per 1 mL.

[0297] System suitability solution: Take an appropriate amount of INCLISIRAN reference substance (or typical test substance) and add water to make a solution containing approximately 1.5 mg of INCLISIRAN per 1 mL.

[0298] Sensitivity solution: Accurately pipette an appropriate amount of system suitability solution and dilute with water to make a solution containing approximately 0.003 mg of INCLISIRAN per 1 mL.

[0299] Chromatographic conditions: chromatographic column with octadecyl bonded silica gel as filler (Acquity Oligonucleotide BEH C18130Å, 2.1mm×150mm, 1.7 μm or equivalent); mobile phase A: aqueous solution containing 200mmol hexafluoroisopropanol and 25mmol hexylamine per 1L; mobile phase B: acetonitrile-methanol (50:50), gradient elution according to Table 3. Flow rate: 0.30 mL per minute; detection wavelength: 260 nm; column temperature: 70℃; injection volume: 2 μL; sample tray temperature: 5℃.

[0300] Table 3

[0301]

[0302] System suitability requirements: In the system suitability solution chromatogram, the relative standard deviation of the main peak area of ​​the sense and antisense strands of the 6-pin system suitability solution should be no more than 5.0%, and the relative standard deviation of the retention time should be no more than 1.0%. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10.

[0303] Determination method: Accurately measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0304] Limit: If there are impurity peaks in the chromatogram of the test solution, the purity (sum of two single chains) calculated by the area normalization method should not be less than 85.0%.

[0305] 3.3 Denaturing IEX-HPLC method

[0306] Determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0307] Test solution: Accurately measure an appropriate amount of this product and dilute with water to make a solution containing approximately 1.5 mg of INCLISIRAN per 1 mL.

[0308] System suitability solution: Take an appropriate amount of INCLISIRAN reference substance (or typical test substance) and add water to make a solution containing approximately 1.5 mg of INCLISIRAN per 1 mL.

[0309] Sensitivity solution: Accurately pipette an appropriate amount of system suitability solution and dilute with water to make a solution containing approximately 0.003 mg of INCLISIRAN per 1 mL.

[0310] Chromatographic conditions: an anion exchange column (Dionex DNA PacTM PA-200, 4×250 mm or a column with equivalent performance); mobile phase A: 800 mL of mixed salt solution (pH 8.5) (20 mmol disodium hydrogen phosphate, 7 mmol citric acid, 1 mmol disodium ethylenediaminetetraacetic acid, pH adjusted to 8.5 with sodium hydroxide solution) and 200 mL of acetonitrile per 1 L; mobile phase B: mobile phase A solution containing 1 mol / L sodium bromide, gradient elution according to Table 4. Flow rate is 1.0 mL per minute; detection wavelength is 260 nm; column temperature is 85°C; injection volume is 20 μL; sample tray temperature is 5°C.

[0311] Table 4

[0312]

[0313] System suitability requirements: In the system suitability solution chromatogram, the relative standard deviation of the main peak area of ​​the sense and antisense strands of the 6-pin system suitability solution should be no more than 5.0%, and the relative standard deviation of the retention time should be no more than 1.0%. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10.

[0314] Determination method: Accurately measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0315] Limit: If there are impurity peaks in the chromatogram of the test solution, the purity (sum of two single chains) calculated by the area normalization method should not be less than 90.0%.

[0316] 4. Stability Staking

[0317] The preparation products obtained after filling the samples in the above Example 1, Comparative Examples 2, 3, 4 and 7 into vials were placed at room temperature for 3 months respectively. The particle size, dispersion, encapsulation rate and purity of the preparation samples at 0 day and 3 months were tested respectively to measure their stability.

[0318] Table 5 shows the comparison of particle size (nm) and dispersion degree (PDI) after being placed at room temperature for 3 months.

[0319] Table 5

[0320]

[0321] Summary: The measurement results show that the particle sizes of Example 1 and Comparative Examples 2, 3, and 4 did not change significantly after being placed at room temperature for 3 months.

[0322] Table 6 shows the encapsulation efficiency (%) comparison after being placed at room temperature for 3 months.

[0323] Table 6

[0324]

[0325] Summary: The test results show that the encapsulation efficiency of Example 1 and Comparative Examples 2, 3, and 4 did not change significantly after being placed at room temperature for 3 months.

[0326] Table 7 shows the comparison of purity (%) after being placed at room temperature for 3 months.

[0327] Table 7

[0328]

[0329] Summary: The test results show that the purity of Example 1 and Comparative Examples 2, 3, 4, and 7 did not change significantly after being placed at room temperature for 3 months.

[0330] Test Example 5

[0331] Comparison of cytotoxicity of the products in Example 1 and Comparative Examples 1-7.

[0332] Cytotoxicity test method: add 1 μg of the test sample to the cell culture medium of the 96-well plate per well, continue to culture for 24 hours, add 10 μL of CCK-8 solution to each well, return the culture plate to the incubator and continue to incubate for 1 hour, and measure the absorbance at 450 nm by microplate reader. The above samples were added to the cell solution to examine the cytotoxicity, and the results are shown in Table 8, which is the cell survival rate results.

[0333] Table 8

[0334]

[0335] Summary: The results show that there is no significant difference in the cell survival rate of the samples prepared by the preparation methods in Example 1 and Comparative Examples 1-7.

[0336] In summary, the present invention provides a conjugate of a GalNAc compound containing a ribose ring and oligonucleotide lipid nanoparticles, and the conjugate can achieve efficient liver-targeted delivery and improve the efficacy of the drug.

[0337] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A GalNAc compound containing a ribose ring structure coupled to a nucleic acid lipid nanoparticle, characterized in that: The components of the lipid nanoparticles include: nucleic acid, lipid and a GalNAc anchoring compound containing a ribose ring; The nucleic acid is siRNA; The lipids include: cationic lipids, neutral lipids, auxiliary lipids and long-circulating lipids; The cationic lipid is YK-009; the neutral lipid is DSPC; the auxiliary lipid is cholesterol; the long-circulating lipid is dimyristoylglycerol-3-methoxypolyethylene glycol 2000; The anchor compound of the GalNAc containing a ribose ring comprises: a GalNAc part containing a ribose ring and a lipid chain group; the structural formula of the anchor compound is shown below: ； The molar ratio of the cationic lipid, the neutral lipid, the auxiliary lipid, the long-circulating lipid, and the anchor compound of the ribose ring-containing GalNAc is (30-60):(5-15):(30-50):(0.5-5):(0.1-10); The mass ratio of the nucleic acid to the cationic lipid is 1:(10-12).

2. The GalNAc compound-coupled nucleic acid lipid nanoparticle containing a ribose ring structure according to claim 1, characterized in that: The components of the lipid nanoparticles also include: buffer salts and excipients; The buffer salt comprises: any one or a combination of at least two of Tris-HCl / Tris buffer salt, DPBS buffer salt or phosphate buffer salt; The pH of the buffer salt is 4-8; The excipient includes: any one of sucrose, trehalose or maltose or a combination of at least two of them.

3. A method for preparing the GalNAc compound-coupled nucleic acid lipid nanoparticles containing a ribose ring structure according to claim 1 or 2, characterized in that: The preparation method comprises: (a) preparing an aqueous phase containing nucleic acid and an organic phase containing lipids and an anchoring compound containing a ribose ring-containing GalNAc, and encapsulating the nucleic acid using a microfluidic device to obtain a nucleic acid-lipid nanoparticle solution; (b) Nucleic acid-lipid nanoparticle solution was dialyzed against buffer solution.

4. Use of the GalNAc compound-coupled nucleic acid lipid nanoparticles containing a ribose ring structure according to claim 1 or 2 in the preparation of a drug for treating and / or preventing dyslipidemia, characterized in that: The sequence of the siRNA is the oligonucleotide sequence of the drug inclisiran, and the dyslipidemia disease is any one of hypercholesterolemia, hypertriglyceridemia or atherosclerosis or a combination of at least two thereof.

5. A GalNAc anchor compound containing a ribose ring, characterized in that The anchor compound of the GalNAc containing a ribose ring comprises: a GalNAc part containing a ribose ring and a lipid chain group; the structural formula of the anchor compound is shown below: 。 6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the GalNAc compound-containing ribose ring structure coupled nucleic acid lipid nanoparticles according to claim 1 or 2 and pharmaceutically acceptable excipients.

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