Methods and lipid compositions for delivery of therapeutic agents

Abstract

# imgabs0. The purpose of the present invention is to provide: a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, said method being capable of achieving excellent delivery efficiency to organs other than the liver; and a composition comprising the therapeutic agent and the lipid nanoparticles, which is capable of achieving excellent delivery efficiency to organs other than the liver. The present invention provides a method of delivering a therapeutic agent to an endothelial cell, a mesenchymal cell or a cancer cell, comprising administering to an individual a lipid composition wherein the lipid composition comprises the therapeutic agent and a lipid nanoparticle, and wherein the lipid nanoparticle comprises an ionizable lipid and a compound represented by Formula (1) or a salt thereof. Wherein G1 represents-C (O)-,-OC (O)-,-O (CO) O-or-C (O) O-, LY represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms. And X represents a basic functional group.

Description

Technical Field

[0001] The present invention relates to methods of delivering therapeutic agents to endothelial cells, mesenchymal cells, or cancer cells, and lipid compositions comprising a therapeutic agent and lipid nanoparticles (LNPs). Background Art

[0002] LNPs are materials capable of delivering therapeutic agents such as nucleic acids to the liver. An example of an LNP is the recently U.S. Food and Drug Administration-approved short interfering RNA (siRNA) LNP therapy for transthyretin-mediated amyloidosis, called Onpattro TM . Despite these advancements, it is currently not possible to predict and rationally design nanoparticles for delivery to targeted tissues other than the liver.

[0003] Generally, effective intracellular delivery materials rely on an optimal balance of ionizable amines (pKa of 6.0 - 6.5) that bind and release RNA and nanoparticle-stabilizing hydrophobicity. This exhaustive focus on ionizable cationic lipids has yielded highly efficient carriers for hepatocytes in the liver, but has not yielded effective carriers capable of reaching other organs.

[0004] Lipid nanoparticles (LNPs) are self-assembled nanostructures capable of encapsulating, protecting, and delivering nucleic acids. Conventional LNPs are composed of ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. Screening and designing new ionizable lipids has yielded highly efficient RNA delivery carriers for hepatocytes in the liver and mRNA vaccines. However, these efforts have not yielded effective carriers capable of reaching extrahepatic organs.

[0005] The inventors have demonstrated that by incorporating permanent cationic lipids (Non-Patent Documents 1 - 3) or by surface-modifying LNPs with ligands, RNA can be delivered to endothelial cells in the lung (Non-Patent Documents 4 - 7). However, cationic lipids are known to be toxic, and ligand modification of LNPs is labor-intensive and may be heterogeneous. Moreover, even with these techniques, RNA delivery is still limited to lung endothelial cells. Thus, there is a need for lipid nanoparticle compositions that do not use any constitutive cationic lipids or ligands.

[0006] There have been several studies on using cholesterol analogs to replace cholesterol in LNPs. For example, oxidized cholesterol and cholesterol esters have been used to improve the RNA delivery efficiency to liver endothelial cells and all cell types in the liver (Non-Patent Documents 8 and 9), respectively. Additionally, naturally occurring cholesterol analogs such as phytosterols have been tested in vitro for improved endosomal escape efficiency (Non-Patent Document 10).

[0007] Several synthetic cholesterol analogues with basic functional groups have been developed for use as nucleic acid delivery vectors. DC-cholesterol is a derivative of cholesterol with a tertiary amino group and a pKa value of 7.8 (Non-Patent Literatures 11 and 12). Generally, DC-cholesterol is formulated with the phospholipid DOPE to encapsulate nucleic acids in liposomes. Although our group has tested the combination of non-biodegradable ionizable lipids (C12-200 and cKK-E12) and DC-cholesterol in subcutaneous mRNA vaccines to enhance the delivery of mRNA to dendritic cells in lymph nodes, it did not show any advantage over cholesterol and even reduced the delivery efficiency (Non-Patent Literature 13).

[0008] List of Citations

[0009] [Non-Patent Literature]

[0010] [NPL 1]

[0011] Q.Cheng,T.Wei,L.Farbiak,L.T.Johnson,S.A.Dilliard,and D.J.Siegwart,“Selective organ targeting(SORT)nanoparticles for tissue-specific mRNAdelivery and CRISPR-Cas gene editing,”Nat.Nanotechnol.,vol.15,no.April,2020.

[0012] [NPL 2]

[0013] L.M.Kranz et al.,“Systemic RNAdelivery to dendritic cells exploitsantiviral defence for cancer immunotherapy,”Nature,vol.534,no.7607,pp.396-401,2016.

[0014] [NPL 3]

[0015] Q.CHENG,T.Wei,and J.SIEGWART,Daniel,“COMPOSITIONS AND METHODS FORORGAN SPECIFIC DELIVERY OF NUCLEIC ACIDS,”WO 2020 / 051220,2018.

[0016] [NPL 4]

[0017] Q. Li et al., “Engineering Caveolae-Targeted Lipid Nanoparticles To Deliver mRNA to the Lungs,” ACS Chem. Biol., 2020.

[0018] [NPL 5]

[0019] H. Parhiz et al., “PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake,” J. Control. Release, vol. 291, no. August, pp. 106 - 115, 2018.

[0020] [NPL 6]

[0021] Y. Sakurai, T. Hada, A. Kato, Y. Hagino, W. Mizumura, and H. Harashima, “Effective Therapy Using a Liposomal siRNA that Targets the Tumor Vasculature in a Model Murine Breast Cancer with Lung Metastasis,” Mol. Ther.-Oncolytics, vol. 11, no. December, pp. 102 - 108, 2018.

[0022] [NPL 7]

[0023] K. Kusumoto et al., “Lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic siRNA delivery to the pulmonary endothelium,” ACS Nano, vol. 7, no. 9, pp. 7534 - 7541, 2013.

[0024] [NPL 8]

[0025] K. Paunovska et al., “Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses,” vol. 1807748, pp. 1 - 7, 2019.

[0026] [NPL 9]

[0027] K. Paunovska et al., “Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery,” ACS Nano, vol. 12, no. 8, pp. 8341 - 8349, 2018.

[0028] [NPL 10]

[0029] S. Patel et al., “Naturally - occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA,” Nat. Commun., vol. 11, no. 1, pp. 1 - 13, 2020.

[0030] [NPL 11]

[0031] X. Gao and L. Huang, “A NOVEL CATIONIC LIPOSOME REAGENT FOR EFFICIENT TRANSFECTION OF MAMMALIAN CELLS,” Biochem. Biophys. Res. Commun., vol. 179, no. 1, pp. 280 - 285, 1991.

[0032] [NPL 12]

[0033] D. Pozzi et al., “Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition,” Biochim. Biophys. Acta - Biomembr., vol. 1838, no. 3, pp. 957 - 967, 2014.

[0034] [NPL 13]

[0035] M. A. Oberli et al., “Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy,” Nano Lett., vol. 17, no. 3, pp. 1326 - 1335, 2017. Summary of the Invention

[0037] Technical Problem

[0038] In view of the above objects in the background art, an object of the present invention is to provide a lipid composition capable of delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells in organs other than the liver.

[0039] Another object to be solved by the present invention is to provide a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, which can achieve excellent delivery efficiency to organs other than the liver. In addition, an object to be solved by the present invention is to provide a composition comprising a therapeutic agent and lipid nanoparticles, which can achieve excellent delivery efficiency to organs other than the liver.

[0040] Technical Solution

[0041] The inventors of the present invention conducted in - depth research to solve the above objects and found that by using ionizable lipids and cholesterol derivatives with specific structures, excellent delivery efficiency of a therapeutic agent to organs other than the liver can be achieved. Based on the above findings, the present invention was completed. According to the present invention, the following inventions are provided.

[0042] <1>A method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, which comprises administering a lipid composition to an individual,

[0043] wherein the lipid composition comprises the therapeutic agent and lipid nanoparticles,

[0044] and wherein the lipid nanoparticle comprises an ionizable lipid and a compound represented by formula (1) or a salt thereof,

[0045] [Chemical Formula 1]

[0046]

[0047] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-;

[0048] L Y represents a single bond, an alkylene having 1 - 14 carbon atoms, a substituted alkylene having 1 - 14 carbon atoms, a heteroalkylene having 1 - 14 carbon atoms, and a substituted heteroalkylene having 1 - 14 carbon atoms,

[0049] X represents a basic functional group.

[0050] <2>The method according to <1>, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidyl group, a 5 - or 6 - membered heterocycloalkyl group, or a 5 - or 6 - membered heteroaryl group.

[0051] <3>The method according to <1> or <2>, wherein the compound represented by formula (1) is a compound represented by formula (2)

[0052] [Chemical Formula 2]

[0053]

[0054] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-;

[0055] L Y represents a single bond, an alkylene having 1 - 14 carbon atoms, a substituted alkylene having 1 - 14 carbon atoms, a heteroalkylene having 1 - 14 carbon atoms, and a substituted heteroalkylene having 1 - 14 carbon atoms,

[0056] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 - 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and one of R 2 、R 3 and R 4 may be absent.

[0057] <4>The method according to <3>, wherein the compound represented by formula (2) is a compound represented by formula (3)

[0058] [Chemical Formula 3]

[0059]

[0060] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0061] L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms

[0062] R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms

[0063] G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0064] L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group

[0065] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and

[0066] R 2 、R 3 and R 4 one of them may be absent

[0067] <5> The method according to <4>, wherein the compound represented by formula (3) is a compound represented by formula (3A):

[0068] [Chemical formula 4]

[0069]

[0070] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0071] L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms

[0072] R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms

[0073] G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0074] L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group, and

[0075] R 2 and R3 Each independently is a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms that may be substituted with a hydroxyl group, or -C(NH2)=NH2.

[0076] <6>The method according to any one of <1> to <5>, wherein G1 represents -C(O)- or -C(O)O-.

[0077] <7>The method according to <5>, wherein R1 represents a hydrogen atom or an aminoalkyl group having 1 to 4 carbon atoms.

[0078] <8>The method according to <5>, wherein R1 represents a hydrogen atom.

[0079] <9>The method according to <5>, wherein G2 represents a single bond or -C(O).

[0080] <10>The method according to <5>, wherein G2 represents a single bond.

[0081] <11>The method according to <5>, wherein L2 represents an alkylene group having 1 to 3 carbon atoms, and R 2 and R 3 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms that may be substituted with a hydroxyl group.

[0082] <12>The method according to <1> or <2>, wherein the compound represented by the formula (1) or a salt thereof is any one of the following:

[0083] [Chemical formula 5]

[0084]

[0085] [Chemical formula 6]

[0086]

[0087] [Chemical formula 7]

[0088]

[0089] [Chemical formula 8]

[0090]

[0091] [Chemical formula 9]

[0092]

[0093] [Chemical formula 10]

[0094]

[0095] [Chemical formula 11]

[0096]

[0097] [Chemical formula 12]

[0098]

[0099] <13>The method according to any one of <1>-<12>, wherein based on the total lipid, the content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol%.

[0100] <14>The method according to any one of <1>-<13>, wherein the therapeutic agent is a nucleic acid.

[0101] <15>The method according to any one of <1>-<14>, wherein the therapeutic agent is DNA or RNA.

[0102] <16>The method according to any one of <1>-<15>, wherein the therapeutic agent is mRNA or siRNA.

[0103] <17>The method according to any one of <1>-<16>, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and wherein the biodegradable group is represented by -O(CO)O-, -O(CO)- or -(CO)O-.

[0104] <18>The method according to any one of <1>-<17>, wherein the ionizable lipid is a compound represented by formula (4):

[0105] [Chemical formula 13]

[0106]

[0107] wherein X represents NR 1 - or -O-,

[0108] R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 21 -L 1 -R 22 -, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0109] [Chemical formula 14]

[0110]

[0111] R 22is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0112] R 2 and R 3 each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms or a group represented by R 31 -L 2 -R 32 -, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0113] [Chemical formula 15]

[0114]

[0115] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0116] R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms,

[0117] R 4 and R 5 、R 10 and R 5 、R 5 and R 12 、R 4 and R 6 、R 5 and R 6 、R 6 and R 7 、R 6 and R 10 、R 12 and R 7 and R 7 and R 8 any one or more groups of which may be linked together to form a 4- to 7-membered ring which may contain an O atom,

[0118] the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms represents a hydroxyl group, a carboxyl group, by NR 45 R 46An amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0119] The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group represent alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 OR 44 represents a group, and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0120] a, b, c and d each independently represent an integer of 0 to 3, wherein a+b is 1 or more, and c+d is 1 or more.

[0121] <19> like <1> - <17> The method according to any one of the preceding claims, wherein the ionizable lipid is a compound represented by formula (1):

[0122] [Chemical Formula 16]

[0123]

[0124] In the formula,

[0125] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by the present invention may be selected from one or more of -OH, COOH, -NR 51 R52 、 -OC(O)O-R 53 、 -C(O)O-R 54 、 -OC(O)-R 55 and -O-R 56 are substituted by substituents of

[0126] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0127] R 5 and R 6 each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , excluding the case where both R 5 and R 6 are hydrocarbon groups having 1 to 8 carbon atoms,

[0128] R 7 represents -R 10 -L 2 -R 11 -L 3 -R 12 ,

[0129] R 51 and R 52 each independently represent a hydrocarbon group having 1 to 8 carbon atoms,

[0130] R 53 、R 54 、R 55 and R 56 each independently represent a hydrocarbon group having 1 to 24 carbon atoms,

[0131] The hydrocarbon groups represented by R 53 、R 54 、R 55 and R 56 can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 substituted,

[0132] The above-mentioned aryl group having 6 to 20 carbon atoms can be substituted by -OH, COOH, -NR 51 R 52 、 -OC(O)O-R 53 、 -C(O)O-R 54 、 -OC(O)-R 55 、 -O-R 56 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 substituted,

[0133] R 58represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0134] R 57 represents -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 or -O-R 66 ,

[0135] R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0136] R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0137] The hydrocarbon group represented by R 63 , R 64 , R 65 and R 66 can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 68 .

[0138] The above-mentioned aryl group having 6 to 20 carbon atoms can be substituted by -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 , -O-R 66 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 .

[0139] R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0140] L 1 , L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-,

[0141] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0142] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0143] R10 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0144] R 11 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0145] R 12 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0146] The hydrocarbon group represented by R 9 and R 12 may be substituted by an aryl group, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 wherein R 53 , R 54 , R 55 and R 58 are as defined above, and

[0147] The hydrocarbon group represented by R 11 may be substituted by -OC(O)O-R 53 , -C(O)O-R 54 or -OC(O)-R 55 wherein R 53 , R 54 and R 55 are as defined above.

[0148] <20>The method according to any one of <1> to <17>, wherein the ionizable lipid is a compound represented by the following formula (5):

[0149] [Chemical formula 17]

[0150]

[0151] wherein R 51 and R 52 each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A,

[0152] The substituent A represents a hydroxyl group, or a group represented by -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ) or -G 20 -R 60 ;

[0153] G 20 represents -O(CO)- or -(CO)O-,

[0154] R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0155] R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have substituent B,

[0156] The said substituent B is -N(R 61 )(R 62 ),

[0157] R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,

[0158] R 60 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0159] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0160] G 30 represents -S-(CO)-NR 64 ,

[0161] R 64 represents a group represented by -L 30 -G 20 -CH(R 55 )(R 56 ),

[0162] a represents 0 or 1,

[0163] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0164] G 10 represents -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 ),

[0165] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0166] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0167] b represents 0 or 1,

[0168] R 53 、R 54 and R 57Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C,

[0169] wherein the substituent C represents a group represented by -(CO)OR 65 or -O(CO)-R 65 represents the group,

[0170] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by -L 40 -CH(R 66 )(R 67 ) represents the group,

[0171] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0172] R 66 and R 67 represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.

[0173] <21>The method according to any one of <1> - <20>, wherein the lipid nanoparticle further comprises a lipid having a non-ionic hydrophilic polymer.

[0174] <22>The method according to any one of <1> - <21>, wherein the lipid nanoparticle further comprises a phospholipid.

[0175] <23>The method according to any one of <1> - <22>, wherein the lipid composition is administered to an individual by intravenous or intramuscular injection.

[0176] <24>The method according to any one of <1> - <23>, wherein the mesenchymal cell is a myocyte.

[0177] <25>The method according to any one of <1> - <23>, wherein the mesenchymal cell is an extracellular matrix-producing cell.

[0178] <26>The method according to any one of <1> - <23>, wherein the extracellular matrix cell is a stellate cell or a fibroblast.

[0179] <27>The method according to any one of <1> - <23>, wherein the stellate cell is a hepatic stellate cell, a pancreatic stellate cell or a colonic stellate cell.

[0180] <28>A lipid composition comprising a therapeutic agent and a lipid nanoparticle,

[0181] wherein the lipid nanoparticle comprises a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group,

[0182] [Chemical Formula 18]

[0183]

[0184] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0185] L Y represents a single bond, an alkylene group having 1 - 14 carbon atoms, a substituted alkylene group having 1 - 14 carbon atoms, a heteroalkylene group having 1 - 14 carbon atoms, and a substituted heteroalkylene group having 1 - 14 carbon atoms

[0186] X represents a basic functional group.

[0187] <29>The lipid composition as described in <28>, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5 - 6 - membered heterocycloalkyl group or a 5 - 6 - membered heteroaryl group.

[0188] <30>The lipid composition as described in <28> or <29>, wherein the compound represented by formula (1) is the compound represented by formula (2)

[0189] [Chemical Formula 19]

[0190]

[0191] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0192] L Y represents a single bond, an alkylene group having 1 - 14 carbon atoms, a substituted alkylene group having 1 - 14 carbon atoms, a heteroalkylene group having 1 - 14 carbon atoms, and a substituted heteroalkylene group having 1 - 14 carbon atoms

[0193] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 - 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and one of R 2 、R 3 and R 4 may be absent.

[0194] <31>The lipid composition as described in <30>, wherein the compound represented by formula (2) is the compound represented by formula (3)

[0195] [Chemical Formula 20]

[0196]

[0197] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0198] L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms

[0199] R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms

[0200] G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-

[0201] L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group

[0202] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and

[0203] R 2 、R 3 and R 4 one of them may be absent

[0204] <32>The lipid composition according to any one of <28>-<31>, wherein the ionizable lipid having a biodegradable group is a compound represented by formula (4)

[0205] [Chemical formula 21]

[0206]

[0207] wherein X represents NR 1 - or -O-

[0208] R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0209] [Chemical formula 22]

[0210]

[0211] R22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0212] R 2 and R 3 each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms or a group represented by R 31 -L 2 -R 32 -, where R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or

[0213] [Chemical formula 23]

[0214]

[0215] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms,

[0216] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms,

[0217] R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 and R 7 and R 8 any one or more groups of which can be joined together to form a 4- to 7-membered ring which may contain an O atom,

[0218] the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms represents a hydroxyl group, a carboxyl group, or by NR 45 R 46An amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 , where R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0219] The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group represent an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 , and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0220] a, b, c, and d each independently represent an integer from 0 to 3, where a + b is 1 or more, and c + d is 1 or more.

[0221] <33>The lipid composition according to any one of <28>-<31>, wherein the ionizable lipid is a compound represented by formula (1):

[0222] [Chemical formula 24]

[0223]

[0224] In the said formula,

[0225] R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R 1 , R 2 and R 3 can be substituted with one or more selected from -OH, COOH, -NR 51 R52 、 -OC(O)O-R 53 、 -C(O)O-R 54 、 -OC(O)-R 55 and -O-R 56 are substituted by substituents of

[0226] R 4 represents a hydrocarbon group having 1 - 8 carbon atoms,

[0227] R 5 and R 6 each independently represent a hydrocarbon group having 1 - 8 carbon atoms or -R 8 -L 1 -R 9 , excluding the case where both R 5 and R 6 are hydrocarbon groups having 1 - 8 carbon atoms,

[0228] R 7 represents -R 10 -L 2 -R 11 -L 3 -R 12 ,

[0229] R 51 and R 52 each independently represent a hydrocarbon group having 1 - 8 carbon atoms,

[0230] R 53 、R 54 、R 55 and R 56 each independently represent a hydrocarbon group having 1 - 24 carbon atoms,

[0231] The hydrocarbon groups represented by R 53 、R 54 、R 55 and R 56 can be substituted by an aryl group having 6 - 20 carbon atoms or -S-R 58 substituents,

[0232] The above-mentioned aryl group having 6 - 20 carbon atoms can be substituted by -OH, COOH, -NR 51 R 52 、 -OC(O)O-R 53 、 -C(O)O-R 54 、 -OC(O)-R 55 、 -O-R 56 or -(hydrocarbon group having 1 - 12 carbon atoms)-R 57 substituents,

[0233] R 58represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0234] R 57 represents -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 or -O-R 66 ,

[0235] R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0236] R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0237] The hydrocarbon groups represented by R 63 , R 64 , R 65 and R 66 can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 68 .

[0238] The above-mentioned aryl group having 6 to 20 carbon atoms can be substituted by -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 , -O-R 66 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 .

[0239] R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0240] L 1 , L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-,

[0241] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0242] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0243] R10 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0244] R 11 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0245] R 12 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0246] The hydrocarbon group represented by R 9 and R 12 can be substituted by an aryl group, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 wherein R 53 , R 54 , R 55 and R 58 are as defined above, and

[0247] The hydrocarbon group represented by R 11 can be substituted by -OC(O)O-R 53 , -C(O)O-R 54 or -OC(O)-R 55 wherein R 53 , R 54 and R 55 are as defined above.

[0248] <34>The lipid composition according to any one of <28>-<31>, wherein the ionizable lipid having a biodegradable group is a compound represented by formula (5):

[0249] [Chemical formula 25]

[0250]

[0251] wherein R 51 and R 52 each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A,

[0252] The substituent A represents a hydroxyl group, or a group represented by -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ) or -G 20 -R 60 ,

[0253] G 20 represents -O(CO)- or -(CO)O-,

[0254] R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0255] R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have substituent B,

[0256] The said substituent B is -N(R 61 )(R 62 ),

[0257] R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,

[0258] R 60 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0259] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0260] G 30 represents -S-(CO)-NR 64 ,

[0261] R 64 is represented by the group -L 30 -G 20 -CH(R 55 )(R 56 ),

[0262] a represents 0 or 1,

[0263] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0264] G 10 represents -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 ),

[0265] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0266] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0267] b represents 0 or 1,

[0268] R 53 、R 54 and R 57Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C,

[0269] The substituent C represents a group represented by -(CO)OR 65 or -O(CO)-R 65 represents a group,

[0270] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by -L 40 -CH(R 66 )(R 67 ) represents a group,

[0271] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0272] R 66 and R 67 represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.

[0273] <35> The lipid composition according to any one of <28> to <34>, wherein the content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipid.

[0274] <36> The lipid composition according to any one of <28> to <35>, wherein the therapeutic agent is a nucleic acid.

[0275] <37> The lipid composition according to any one of <28> to <36>, wherein the therapeutic agent is DNA or RNA.

[0276] <38> The lipid composition according to any one of <28> to <37>, wherein the therapeutic agent is mRNA or siRNA.

[0277] Advantages of the invention

[0278] According to the method and composition of the present invention, excellent delivery efficiency of a therapeutic agent can be achieved even for organs other than the liver. Brief description of the drawings

[0279] Figure 1

[0280] Figure 1 Shows the results of measuring the uptake of the lipid composition in various tissues.

[0281] Figure 2

[0282] Figure 2 Shows the results of measuring the uptake of the lipid composition in various tissues. ​​​​

[0283] Figure 3

[0284] Figure 3 Shows the results of measuring the uptake of lipid compositions in various tissues.

[0285] Figure 4

[0286] Figure 4 Shows the results of measuring the uptake of lipid compositions in various tissues.

[0287] Figure 5

[0288] Figure 5 Shows the results of measuring the uptake of lipid compositions in various tissues.

[0289] Figure 6

[0290] Figure 6 Shows the results of measuring the uptake of lipid compositions in various tissues.

[0291] Figure 7

[0292] Figure 7 Shows the results of in vivo endothelial cell RNA delivery with various ionizable lipids.

[0293] Figure 8

[0294] Figure 8 Shows the results of in vivo endothelial cell RNA delivery with various ionizable cholesterol ratios (1) HAPC-cholesterol.

[0295] Figure 9

[0296] Figure 9 Shows the results of in vivo endothelial cell RNA delivery with various ionizable cholesterol ratios (2) DC-cholesterol.

[0297] Figure 10

[0298] Figure 10 Shows the results of in vivo endothelial cell RNA delivery with various ionizable cholesterol ratios (3) HAPC-cholesterol.

[0299] Figure 11

[0300] Figure 11 Shows the results of serum protein-independent RNA delivery to endothelial cells.

[0301] ​​​​​​​​​​​​​​​​​​​Figure 12 ]

[0302] Figure 12 Shown are the results of an in vitro LNP-mediated endothelial cell injury assay.

[0303] [ Figure 13 ]

[0304] Figure 13 Shown are the results of in vitro hemocompatibility analysis using primary human erythrocytes.

[0305] [ Figure 14 ]

[0306] Figure 14 Shown are the results of in vivo hepatic stellate cell delivery.

[0307] [ Figure 15 ]

[0308] Figure 15 Results are shown for in vivo RNA delivery to extrahepatic extracellular matrix-producing cells.

[0309] [ Figure 16 ]

[0310] Figure 16 Results are shown for in vivo RNA delivery to extrahepatic extracellular matrix-producing cells.

[0311] [ Figure 17 ]

[0312] Figure 17 Results are shown for intramuscular administration and delivery to myocytes and endothelial cells.

[0313] [ Figure 18 ]

[0314] Figure 18 Results are shown for intramuscular administration and delivery to myocytes and endothelial cells.

[0315] [ Figure 19 ]

[0316] Figure 19 Results are shown for in vitro delivery to cancer cells.

[0317] Detailed description of implementation plan

[0318] Hereinafter, the present invention will be described in detail.

[0319] In the present specification, “to” represents a range including numerical values described before and after it as a minimum value and a maximum value, respectively.

[0320] The present invention relates to a method of delivering a therapeutic agent to an endothelial cell, a mesenchymal cell, or a cancer cell, comprising administering to a subject a lipid composition,

[0321] wherein the lipid composition comprises the therapeutic agent and lipid nanoparticles,

[0322] and wherein the lipid nanoparticles comprise an ionizable lipid and a compound represented by formula (1) or a salt thereof.

[0323] The present invention relates to a lipid composition comprising a therapeutic agent and lipid nanoparticles, wherein the lipid nanoparticles comprise a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group,

[0324] In the present invention, it has been found that by replacing cholesterol with a cholesterol analogue, the cell type and tissue type tropism can be transformed from hepatocytes in the liver to endothelial cells, mesenchymal cells or cancer cells in various organs. The combination of an ionizable lipid and a cholesterol analogue as the compound represented by formula (1) can be used for the safe and effective delivery of therapeutic agents for systemic and local delivery to endothelial cells, mesenchymal cells or cancer cells.

[0325] Examples of mesenchymal cells include osteocytes (osteoblasts), chondrocytes (cartilage cells or chondrocytes), muscle cells (myocytes, skeletal muscle cells, cardiomyocytes), connective tissue cells (fibroblasts, myofibroblasts, stellate cells), bone marrow stromal cells, tendon cells and adipocytes (lipocytes).

[0326] Fibroblasts are a ubiquitous type of mesenchymal cell that are commonly present in the stroma of many tissues.

[0327] Myofibroblasts are an activated form of fibroblasts that are capable of contraction due to the presence of cytoskeletal proteins (especially α-smooth muscle actin) that are normally found in smooth muscle cells.

[0328] Stellate cells are a type of fibroblast that stores retinoids and are found in various organs such as the liver, pancreas, lung, kidney, intestine, spleen, adrenal gland, vas deferens and vocal cords.

[0329] <Compound represented by formula (1), formula (2) or formula (3)>

[0330] In the present invention, the following compound represented by formula (1) or a salt thereof is used.

[0331] [Chemical formula 26]

[0332]

[0333] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0334] L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms.

[0335] X represents a basic functional group.

[0336] Preferably, G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-.

[0337] The alkylene group having 1 to 14 carbon atoms represented by L Y can be straight-chain or branched, can be chain-like or cyclic, and can have 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and further preferably 2 to 10 carbon atoms. Specific examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, undecamethylene, dodecamethylene, etc.

[0338] In the substituted alkylene group having 1 to 14 carbon atoms represented by L Y the examples of the alkylene group having 1 to 14 carbon atoms are as described above. Examples of the substituents contained in the alkylene group having 1 to 14 carbon atoms include a hydrocarbon group having 1 to 4 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, and an amino group.

[0339] The heteroalkylene group having 1 to 14 carbon atoms represented by L Y is a group in which one or more heteroatoms selected from an oxygen atom, a nitrogen atom, or a sulfur atom are attached to the alkylene group having 1 to 14 carbon atoms, or a group in which one or more heteroatoms selected from an oxygen atom, a nitrogen atom, or a sulfur atom are present in the alkyl chain of the alkylene group having 1 to 14 carbon atoms. As examples thereof, the heteroalkylene group contains -O-, -S-, -NH-, -NR-, -C(O)-, -CN-, -NR-C(O)-, -C(O)O-, -OC(O)-, -OC(O)O- in the alkyl, etc., but the examples are not limited thereto. In addition, two heteroatoms can be consecutive, such as -S-S-.

[0340] In the substituted heteroalkylene group having 1 to 14 carbon atoms represented by L Y the examples of the substituents on the alkylene group having 1 to 14 carbon atoms include a hydrocarbon group having 1 to 4 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, and an amino group.

[0341] L YPreferably a heteroalkylene group having 1 to 14 carbon atoms or a substituted heteroalkylene group having 1 to 14 carbon atoms.

[0342] Examples of the basic functional group represented by X include an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered heteroalkyl group or a 5- or 6-membered heteroaryl group. Examples of the substituent on the substituted amino group include an alkyl group having 1 to 4 carbon atoms which may be substituted, -C(NH2)=NH2, etc.

[0343] Examples of the heteroalkyl group include a cyclic functional group containing one or more heteroatoms selected from an oxygen atom, a nitrogen atom or a sulfur atom and carbon atoms. Examples include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, imidazolinyl, piperidinyl, piperazinyl and morpholinyl.

[0344] Examples of the heteroaryl group include an aromatic ring containing one or more heteroatoms selected from an oxygen atom, a nitrogen atom or a sulfur atom and carbon atoms. Examples include, but are not limited to, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, tetrazinyl, pentazinyl, etc.

[0345] The compound represented by the formula (1) may preferably be a compound represented by the formula (2).

[0346] [Chemical formula 27]

[0347]

[0348] Wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0349] L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms.

[0350] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and one of R 2 and R 3 and R 4 may be absent.

[0351] The definitions of G1 and L Y in the formula (2) are synonymous with the definitions in the formula (1).

[0352] In R 2 、R3 or R 4 Among the hydrocarbyl groups having 1 to 4 carbon atoms which may be substituted by a hydroxyl group represented by, examples of the hydrocarbyl groups having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.

[0353] R 2 、R 3 and R 4 are preferably a hydrogen atom, a methyl group, 1-hydroxyethyl or -C(NH2)=NH2.

[0354] The compound represented by formula (2) may preferably be the compound represented by formula (3).

[0355] [Chemical formula 28]

[0356]

[0357] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0358] L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms,

[0359] R1 represents a hydrogen atom, a hydrocarbyl group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms,

[0360] G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0361] L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group,

[0362] R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbyl group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group or -C(NH2)=NH2, and

[0363] R 2 、R 3 and R 4 one of may be absent.

[0364] G in formula (3) 1 、R 2 、R 3 and R 4 are synonymous with the definitions in formula (1) and formula (2).

[0365] Examples of the alkylene group having 1 to 6 carbon atoms represented by L1 include methylene, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, etc. L1 is preferably a single bond or hexane-1,6-diyl.

[0366] The compound represented by formula (3) may preferably be the compound represented by formula (3A).

[0367] [Chemical formula 29]

[0368]

[0369] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0370] L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms,

[0371] R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms,

[0372] G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-,

[0373] L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group, and

[0374] R 2 and R 3 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2.

[0375] Examples of the hydrocarbon group having 1 to 4 carbon atoms represented by R 1 include methyl, ethyl, propyl, butyl, etc.

[0376] Examples of the aminoalkyl group having 1 to 4 carbon atoms represented by R 1 include aminomethyl, aminoethyl, aminopropyl, aminobutyl, etc.

[0377] R 1 is preferably a hydrogen atom or an aminoalkyl group having 1 to 4 carbon atoms.

[0378] G2 preferably represents a single bond or -C(O).

[0379] Among the alkylene groups having 1 to 6 carbon atoms which may have an amino group represented by L2, examples of the alkylene group having 1 to 6 carbon atoms include methylene, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, etc.

[0380] L2 is preferably ethylene, propane-1,3-diyl or butane-1,4-diyl having an amino group.

[0381] Examples of salts of the compounds represented by formula (1), formula (2), formula (3) or formula (3A) include salts in basic groups and include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0382] Specific examples of the compound represented by formula (1) or a salt thereof include the following compounds.

[0383] [Chemical formula 30]

[0384] DC cholesterol

[0385] [Chemical formula 31]

[0386] GL67: N4-cholesteryl-spermine

[0387] [Chemical formula 32]

[0388] DMPAC-Chol

[0389]

[0390] [Chemical formula 33]

[0391] OH-Chol

[0392]

[0393] [Chemical formula 34]

[0394] HAPC-Chol

[0395]

[0396] [Chemical formula 35]

[0397] MHAPC-Chol

[0398]

[0399] [Chemical formula 36]

[0400] DMHAPC-Chol

[0401]

[0402] [Chemical Formula 37]

[0403] Arg-Chol

[0404]

[0405] Among them, the preferred examples are

[0406] [Chemical Formula 38]

[0407] DC cholesterol

[0408] [Chemical Formula 39]

[0409] OH-Chol

[0410]

[0411] [Chemical Formula 40]

[0412] HAPC-Chol

[0413]

[0414] [Chemical Formula 41]

[0415] MHAPC-Chol

[0416]

[0417] [Chemical Formula 42]

[0418] Arg-Chol

[0419]

[0420] Particularly preferred examples are

[0421] [Chemical Formula 43]

[0422] DC cholesterol

[0423] [Chemical Formula 44]

[0424] OH-Chol

[0425]

[0426] [Chemical Formula 45]

[0427] HAPC-Chol

[0428]

[0429] [Chemical Formula 46]

[0430] MHAPC-Chol

[0431]

[0432] The compound represented by the above formula (1) can be obtained as a commodity from, for example, Cayman chemicals and Avantipolar lipids.

[0433] The content of the compound represented by formula (1) is preferably 5-80 mol%, more preferably 10-80 mol%, still more preferably 10-60 mol%, and further preferably 30-50 mol% relative to the total lipids.

[0434] <Ionizable lipid>

[0435] In the present invention, an ionizable lipid is used. The ionizable lipid may be a lipid having at least one biodegradable group. The ionizable lipid may be a lipid having at least one ionizable amino group and at least one biodegradable group. Examples of the above biodegradable groups include groups represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

[0436] <<Lipid represented by formula (4) or its salt>>

[0437] For example, the lipid represented by formula (4) or its salt can be used as an ionizable lipid.

[0438] [Chemical formula 47]

[0439]

[0440] In the formula, X represents -NR 1 - or -O-,

[0441] R 1 represents a hydrogen atom, a hydrocarbon group having 6-24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, where R 21 represents a hydrocarbon group having 1-24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula,

[0442] [Chemical formula 48]

[0443]

[0444] And R 22 represents a divalent hydrocarbon linking group having 1-18 carbon atoms,

[0445] R 2 and R 3 each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31 -L 2 -R 32 -, where R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula,

[0446] [Chemical formula 49]

[0447]

[0448] and R 32 represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,

[0449] R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted,

[0450] R 4 and R 5 、R 10 and R 5 、R 5 and R 12 、R 4 and R 6 、R 5 and R 6 、R 6 and R 7 、R 6 and R 10 、R 12 and R 7 、R 7 and R 8 any one or more pairs of the groups in can be connected to each other to form a 4- to 7-membered ring which may contain an O atom,

[0451] the substituents on the alkyl group having 1 to 18 carbon atoms which may be substituted are a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 -, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or -O(CO)O-R 41, -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 a group represented by, wherein R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0452] The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 represented by, wherein R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and

[0453] a, b, c and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more.

[0454] As a hydrocarbon group having 6 to 24 carbon atoms represented by R 1 and a hydrocarbon group represented by R 2 , R 3A hydrocarbon group having 3 to 24 carbon atoms, preferably an alkyl group, an alkenyl group or an alkynyl group, more preferably an alkyl group or an alkenyl group. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and the alkyl group having 3 to 24 carbon atoms is more preferably an alkyl group having 6 to 20 carbon atoms. Specifically, examples thereof include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, nonadecyl, eicosyl, etc. The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms. Specifically, examples thereof include hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadec-9,12-dienyl), nonadecenyl, eicosenyl (preferably (Z)-eicos-11-enyl), eicosadienyl (preferably (11Z,14Z)-eicos-11,14-dienyl), etc. The alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms. Specifically, examples thereof include hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0455] represented by R 21 and R 31The hydrocarbon group having 1 to 24 carbon atoms represented is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. The alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms. Specifically, examples thereof include decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, 2-butyloctyl, 1-pentylhexyl, 2-pentylheptyl, 3-pentyl octyl, 1-hexylheptyl, 1-hexylnonyl, 2-hexyl octyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylnonyl, 2-heptylundecyl, 3-heptyldecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl, etc. The alkenyl group having 10 to 24 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. Specifically, examples thereof include decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadec-8-enyl), hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadec-9,12-dienyl), etc. The alkynyl group having 10 to 24 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. Specifically, examples thereof include decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0456] by R 22 and R 32The divalent hydrocarbon linking group having 1 to 18 carbon atoms represented is preferably an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. The number of carbon atoms in the alkylene group is preferably 1 to 12, more preferably 1 to 10, and further preferably 2 to 10. Specifically, examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, undecamethylene, dodecamethylene, etc. The alkenylene group having 2 to 18 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic. The number of carbon atoms in the alkenylene group is preferably 1 to 12, more preferably 2 to 10.

[0457] -O(CO)O-, -O(CO)-, and -(CO)O- are within the preferred range of L 1 and -O(CO)- and -(CO)O- are within the more preferred range of L 1 in the range.

[0458] -O(CO)O-, -O(CO)-, and -(CO)O- are within the preferred range of L 2 and -O(CO)- and -(CO)O- are within the more preferred range of L 2 in the range.

[0459] The optionally substituted alkyl group having 1 to 18 carbon atoms represented by R 4 , R 6 , R 9 , R 10 , R 11 and R 12 can be straight-chain or branched-chain, or can be chain-like or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. When the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 is preferred, and a group represented by -O(CO)-R 42 or -(CO)O-R 43 is more preferred.

[0460] The optionally substituted alkyl group having 1 to 18 carbon atoms represented by R 5 , R 7 and R 8The replaceable alkyl group having 1 to 18 carbon atoms may be straight-chain or branched-chain, and may also be chain-like or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12, more preferably 1 to 8. Specifically, examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. When the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 is preferred, and a group represented by -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 is more preferred.

[0461] Examples of the 4- to 7-membered ring that may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. The 4- to 7-membered ring is preferably a 6-membered ring, and is preferably a piperidine ring or a morpholine ring.

[0462] When the replaceable alkyl group having 1 to 18 carbon atoms represented by R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 has a substituted or unsubstituted aryl group as a substituent, the number of carbon atoms in the aryl group is preferably 6 to 22, more preferably 6 to 18, and further preferably 6 to 10. Specifically, examples of the aryl group include phenyl, naphthyl, anthryl, phenanthryl, etc. As the substituent on the aryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 , or -O-R 44 is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specifically, examples of the substituted aryl group include hydroxyphenyl, carboxyphenyl, etc.

[0463] When the group represented by R 4 , R 5 , R 6 , R 7 , R 8 , R9 , R 10 , R 11 and R 12 When the optionally substituted alkyl group having 1 - 18 carbon atoms represented by has a substituted or unsubstituted heteroaryl group as a substituent, the number of carbon atoms in the heteroaryl group is preferably 1 - 12, more preferably 1 - 6. Specifically, examples of the heteroaryl group include pyridyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, oxazolyl, etc. As substituents on the heteroaryl group, an alkyl group having 1 - 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 , or -O-R 44 are preferred, and a hydroxyl group or a carboxyl group is more preferred. Specifically, examples of the substituted or unsubstituted heteroaryl group include hydroxypyridyl, carboxypyridyl, pyridone, etc.

[0464] As represented by R 41 , R 42 , R 43 , R 44 , R 45 and R 46A hydrocarbon group having 1 to 18 carbon atoms, preferably an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms or an alkynyl group having 2 to 18 carbon atoms, more preferably an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be straight-chain or branched-chain, and may also be chain-like or cyclic. The number of carbon atoms in the alkyl group is preferably 3 to 18, more preferably 5 to 18. Specifically, examples thereof include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc. The alkenyl group having 2 to 18 carbon atoms may be straight-chain or branched-chain, and may also be chain-like or cyclic. The number of carbon atoms in the alkenyl group is preferably 3 to 18, more preferably 5 to 18. Specifically, examples thereof include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadec-8-enyl), hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadec-9,12-dienyl), etc. The alkynyl group having 2 to 18 carbon atoms may be straight-chain or branched-chain, and may also be chain-like or cyclic. The number of carbon atoms in the alkynyl group is preferably 3 to 18, more preferably 5 to 18. Specifically, examples thereof include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc.

[0465] When X represents -NR 1 -, R 1 preferably represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 21 -L 1 -R 22 -. In this case, preferably one of R 2 and R 3 represents a hydrogen atom, and the other represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 31 -L2 -R 32 -represented group.

[0466] When X represents -O-, preferably R 2 and R 3 each independently represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 31 -L 2 -R 32 -represented group.

[0467] Preferably R 4 、R 6 、R 9 、R 10 、R 11 and R 12 each represents a hydrogen atom.

[0468] R 5 is preferably a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted by -O(CO)-R 42 or -(CO)O-R 43 substituted alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted by an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted by a hydroxyl group. When R 5 is an alkyl group, R 5 can be connected to R 4 、R 6 、R 10 and R 12 to form a ring which may contain an O atom. In particular, R 5 is preferably an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted by -O(CO)-R 42 or -(CO)O-R 43 substituted alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 12 carbon atoms which may be substituted by an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted by a hydroxyl group, more preferably an alkyl group having 1 to 18 carbon atoms, or an alkyl group having 1 to 18 carbon atoms which may be substituted by -O(CO)-R 42 or -(CO)O-R 43 substituted alkyl group having 1 to 18 carbon atoms.

[0469] R 7 and R 8 are preferably each independently a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted by -O(CO)-R 42 or -(CO)O-R 43 substituted alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 8 carbon atoms which may be substituted by an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted by a hydroxyl group. Or, preferably R7 and R 8 are connected to each other to form a 4- to 7-membered ring which may contain an O atom.

[0470] R 5 is not connected to R 7 or R 8 and is not connected to R 7 or R 8 to form a ring.

[0471] a + b is preferably 1 or 2, more preferably 1. c + d is preferably 1 or 2, more preferably 1.

[0472] The compound represented by formula (4) is preferably the compound represented by formula (21).

[0473] [Chemical formula 50]

[0474]

[0475] In the formula, R 2 and R 3 each independently represent a hydrocarbon group having 1 or more unsaturated bonds and 3 - 24 carbon atoms, or R 2 and R 3 each independently represent a group represented by R 31 -L 2 -R 32 -, or one of R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, and the other represents a hydrocarbon group having 3 - 24 carbon atoms.

[0476] R 31 represents a hydrocarbon group having 1 - 24 carbon atoms.

[0477] L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula

[0478] [Chemical formula 51]

[0479]

[0480] And R 32 represents a divalent hydrocarbon linking group having 1 - 18 carbon atoms.

[0481] R 5 represents a group which may be substituted by -O(CO)-R 42 or -(CO)O-R 43A substituted alkyl group having 1 to 18 carbon atoms, wherein R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms,

[0482] R 7 and R 8 each independently represent an alkyl group having 1 to 4 carbon atoms

[0483] e represents 2 or 3.

[0484] In formula (21), preferably one of R 2 and R 3 is a group represented by R 31 -L 2 -R 32 -, and the other is a hydrocarbon group having 3 to 24 carbon atoms. In formula (21), L 2 preferably represents -O(CO)-- or -(CO)O-.

[0485] The compound represented by formula (4) can form salts.

[0486] Examples of salts in basic groups include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0487] Examples of salts in acidic groups include salts formed with alkali metals such as sodium and potassium; salts formed with alkaline earth metals such as calcium and magnesium; ammonium salts; salts formed with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, etc.

[0488] Among the above salts, for example, pharmaceutically acceptable salts are preferred.

[0489] The lipid represented by formula (4) and its production method are described in WO2019 / 235635A and WO2021 / 095876A.

[0490] <<The lipid represented by formula (1) or its salt>>

[0491] As another example, the lipid represented by formula (1) or its salt can be used as an ionizable lipid.

[0492] [Chemical formula 52]

[0493]

[0494] In the formula,

[0495] R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein the hydrocarbon groups represented by R 1 , R 2 and R 3 can be substituted by one or more substituents selected from -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 and -O-R 56 .

[0496] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0497] R 5 and R 6 each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , excluding the case where both R 5 and R 6 are hydrocarbon groups having 1 to 8 carbon atoms.

[0498] R 7 represents -R 10 -L 2 -R 11 -L 3 -R 12 .

[0499] R 51 and R 52 each independently represent a hydrocarbon group having 1 to 8 carbon atoms,

[0500] R 53 , R 54 , R 55 and R 56 each independently represent a hydrocarbon group having 1 to 24 carbon atoms,

[0501] The hydrocarbon groups represented by R 53 , R 54 , R 55 and R 56 can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58Substituted,

[0502] The above aryl group having 6 to 20 carbon atoms may be substituted by -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 , -O-R 56 or -(hydrocarbyl group having 1 to 12 carbon atoms)-R 57 Substituted,

[0503] R 58 represents a hydrocarbyl group having 1 to 12 carbon atoms, and

[0504] R 57 represents -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 or -O-R 66 .

[0505] R 61 and R 62 each independently represents a hydrocarbyl group having 1 to 8 carbon atoms,

[0506] R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbyl group having 1 to 24 carbon atoms,

[0507] The hydrocarbyl group represented by R 63 , R 64 , R 65 and R 66 may be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 68 Substituted,

[0508] The above aryl group having 6 to 20 carbon atoms may be substituted by -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 , -O-R 66 or -(hydrocarbyl group having 1 to 12 carbon atoms)-R 67 Substituted,

[0509] R 68 represents a hydrocarbyl group having 1 to 12 carbon atoms, and

[0510] L 1 , L 2 and L 3 Each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-.

[0511] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0512] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0513] R 10 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0514] R 11 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0515] R 12 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0516] By R 9 and R 12 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 or -SR 58 Substituted, where R 53 , R 54 , R 55 and R 58 is defined above, and

[0517] By R 11 The hydrocarbon group represented by -OC(O)OR 53 、-C(O)OR 54 or -OC(O)-R 55 Substituted, where R 53 , R 54 and R 55 The definition of is as above.

[0518] The hydrocarbyl group having 1 to 24 carbon atoms, the hydrocarbyl group having 1 to 18 carbon atoms, the hydrocarbyl group having 1 to 12 carbon atoms, the hydrocarbyl group having 2 to 8 carbon atoms and the hydrocarbyl group having 1 to 8 carbon atoms are preferably an alkyl group, an alkenyl group or an alkynyl group.

[0519] The alkyl group can be straight-chain or branched-chain, and can also be chain-like or cyclic. Specifically, examples of the alkyl group include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, 2-butyloctyl, 1-pentylhexyl, 2-pentylheptyl, 3-pentyl octyl, 1-hexylheptyl, 1-hexylnonyl, 2-hexyl octyl, 2-hexyldecyl, 3-hexylnonyl, 1-heptyloctyl, 2-heptylundecyl, 3-heptyldecyl, 1-octylnonyl, 2-octyldecyl, 2-octyldodecyl, 3-octylundecyl, 2-nonylundecyl, 3-nonyldodecyl, 2-decyldodecyl, 2-decyltetradecyl, 3-decyltridecyl, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl, etc.

[0520] The alkenyl group can be straight-chain or branched-chain, and can also be chain-like or cyclic. Specifically, examples of the alkenyl group include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadec-8-enyl), hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadec-9,12-dienyl), etc.

[0521] The alkynyl group can be straight-chain or branched-chain, and can also be chain-like or cyclic. Specifically, examples of the alkynyl group include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc.

[0522] All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0523] in -(hydrocarbon group having 1-12 carbon atoms)-R 67Among them, the hydrocarbon group having 1 to 12 carbon atoms is preferably an alkylene group having 1 to 12 carbon atoms or an alkenylene group having 2 to 12 carbon atoms. The alkylene group having 1 to 12 carbon atoms and the alkenylene group having 2 to 12 carbon atoms can be straight-chain or branched-chain, and can also be chain-like or cyclic.

[0524] Specifically, examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, undecamethylene, etc.

[0525] The aryl group preferably has 6 to 20 carbon atoms, more preferably has 6 to 18 carbon atoms, and even more preferably has 6 to 10 carbon atoms. Specifically, examples of the aryl group include phenyl, naphthyl, anthryl, phenanthryl, etc.

[0526] R 1 and R 2 each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and further preferably represents a hydrocarbon group having 1 to 3 carbon atoms.

[0527] R 3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0528] The hydrocarbon group represented by R 1 、R 2 and R 3 can preferably be substituted by -OH.

[0529] L 1 and L 3 each independently preferably represents -C(O)O- or -OC(O)-.

[0530] L 2 preferably represents -OC(O)O-, -C(O)O- or -OC(O)-.

[0531] R 8 preferably represents a hydrocarbon group having 1 to 10 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0532] R 9 preferably represents a hydrocarbon group having 1 to 20 carbon atoms, more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.

[0533] R 11 preferably represents a hydrocarbon group having 1 to 16 carbon atoms, more preferably represents a hydrocarbon group having 1 to 9 carbon atoms.

[0534] R 12Preferably represents a hydrocarbon group having 1 to 20 carbon atoms, more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.

[0535] The hydrocarbon group represented by R 9 and R 12 can preferably be substituted by an aryl group or -S-R 58 In this text, R 58 preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0536] The hydrocarbon group represented by R 11 can preferably be substituted by -C(O)O-R 55 or -OC(O)-R 56 wherein R 55 and R 56 each independently represent a hydrocarbon group having 1 to 16 carbon atoms.

[0537] The hydrocarbon group represented by R 55 and R 56 can preferably be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 where the definition of R 58 is as described above.

[0538] As a first example, the compound represented by formula (1) is preferably the compound represented by formula (1-1).

[0539] [Chemical formula 53]

[0540]

[0541] In the formula,

[0542] R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein the hydrocarbon groups represented by R 1 , R 2 and R 3 can be substituted by -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -O-R 56 substituted,

[0543] R 4 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0544] R 5 and R 6Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , excluding R 5 and R 6 both being hydrocarbon groups having 1 to 8 carbon atoms,

[0545] L 1 represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-,

[0546] R 8 represents a hydrocarbon group having 1 to 12 carbon atoms,

[0547] R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, wherein the hydrocarbon group represented by R 9 can be substituted by an aryl group, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 substituted,

[0548] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0549] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms,

[0550] The hydrocarbon groups represented by R 53 , R 54 , R 55 and R 56 can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 substituted,

[0551] The above-mentioned aryl group having 6 to 20 carbon atoms can be substituted by -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 , -O-R 56 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 substituted,

[0552] R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, and

[0553] R 57 represents -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -O-R 56 ,

[0554] R 13 represents a hydrocarbon group having 1 to 8 carbon atoms,

[0555] R 14 represents -R 15 -L 5 -R 16 , where R 15 represents a hydrocarbon group having 1 to 24 carbon atoms, L 5 represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-, and R 16 represents a hydrocarbon group having 1 to 24 carbon atoms,

[0556] The hydrocarbon group having 1 to 24 carbon atoms represented by R 15 can be substituted by -OC(O)O-R 53 , -C(O)O-R 54 or -OC(O)-R 55 , where R 53 , R 54 and R 55 are defined as above, and

[0557] The hydrocarbon group having 1 to 24 carbon atoms represented by R 16 can be substituted by an aryl group having 6 to 20 carbon atoms, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 , where R 53 , R 54 , R 55 and R 58 are defined as above.

[0558] In formula (1-1), R 1 and R 2 each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and further preferably represents a hydrocarbon group having 1 to 3 carbon atoms.

[0559] R 3Preferably represents a hydrocarbon group having 2 to 6 carbon atoms, more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0560] Represented by R 1 , R 2 and R 3 The represented hydrocarbon group may preferably be substituted by -OH.

[0561] L 1 Preferably represents -C(O)O- or -OC(O)-.

[0562] R 8 Preferably represents a hydrocarbon group having 1 to 10 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0563] R 9 Preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and the hydrocarbon group represented by R 9 May be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 Substituted.

[0564] R 14 Preferably represents -R 15 -L 5 -R 16 , where R 15 Represents a hydrocarbon group having 1 to 18 carbon atoms, L 5 Represents -OC(O)O-, and R 16 Represents a hydrocarbon group having 1 to 18 carbon atoms.

[0565] The hydrocarbon group having 1 to 18 carbon atoms represented by R 15 May preferably be substituted by -C(O)O-R 55 Or -OC(O)-R 56 Substituted. R 55 And R 56 Each independently represents a hydrocarbon group having 1 to 16 carbon atoms, and the hydrocarbon groups represented by R 55 And R 56 May be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 Substituted, where R 58 Is defined as above.

[0566] The hydrocarbon group having 1 to 18 carbon atoms represented by R 16 May preferably be substituted by an aryl group or -S-R 58 Substituted, where R 58 Is defined as above.

[0567] As a second example, the compound represented by formula (1) is preferably the compound represented by formula (1-2).

[0568] [Chemical Formula 54]

[0569]

[0570] In the formula,

[0571] R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein the hydrocarbon groups represented by R 1 , R 2 and R 3 can be substituted by -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -O-R 56 .

[0572] R 4 and R 8 each independently represent a hydrocarbon group having 1 to 8 carbon atoms,

[0573] R 21 and R 22 each independently represent a hydrocarbon group having 1 to 18 carbon atoms,

[0574] R 23 and R 24 each independently represent a hydrocarbon group having 1 to 12 carbon atoms,

[0575] R 25 and R 26 each independently represent a hydrocarbon group having 1 to 24 carbon atoms,

[0576] L 21 and L 22 each independently represent -OC(O)O-, -C(O)O-, -OC(O)- or -O-,

[0577] The hydrocarbon groups represented by R 25 and R 26 can be substituted by an aryl group having 6 to 20 carbon atoms, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 .

[0578] R 51 and R 52each independently represents a hydrocarbon group having 1 to 8 carbon atoms

[0579] R 53 、R 54 、R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms

[0580] The aryl group having 6 to 20 carbon atoms described above may be substituted by OH, COOH, -NR 51 R 52 、-OC(O)O-R 53 、-C(O)O-R 54 、-OC(O)-R 55 、-O-R 56 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 and

[0581] R 57 represents -OH, COOH, -NR 51 R 52 、-OC(O)O-R 53 、-C(O)O-R 54 、-OC(O)-R 55 or -O-R 56 。

[0582] R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0583] In formula (1-2), R 1 and R 2 each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 6 carbon atoms, and still more preferably represents a hydrocarbon group having 1 to 3 carbon atoms. The hydrocarbon group represented by R 1 and R 2 may preferably be substituted by -OH, but is more preferably a hydrocarbon group without substituents.

[0584] R 3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0585] R 21 and R 22 each independently preferably represents a hydrocarbon group having 1 to 12 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms, and still more preferably represents a hydrocarbon group having 1 to 6 carbon atoms.

[0586] R 23 and R 24Each independently preferably represents a hydrocarbon group having 1 to 10 carbon atoms, more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0587] R 25 and R 26 Each independently preferably represents a hydrocarbon group having 1 to 20 carbon atoms, more preferably represents a hydrocarbon group having 1 to 16 carbon atoms, and still more preferably represents a hydrocarbon group having 1 to 12 carbon atoms.

[0588] L 21 and L 22 Each independently preferably represents -C(O)O- or -OC(O)-.

[0589] As a third example, the compound represented by formula (1) is preferably the compound represented by formula (1-3).

[0590] [Chemical formula 55]

[0591]

[0592] In the formula,

[0593] R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein the hydrocarbon groups represented by R 1 、R 2 and R 3 can be substituted by -OH, COOH, -NR 51 R 52 、-OC(O)O-R 53 、-C(O)O-R 54 、-OC(O)-R 55 or -O-R 56 .

[0594] R 4 and R 8 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms.

[0595] R 31 、R 32 、R 33 and R 34 Each independently represents a hydrocarbon group having 1 to 12 carbon atoms.

[0596] R 35 、R 36 、R 37 and R 38 Each independently represents a hydrocarbon group having 1 to 24 carbon atoms.

[0597] L 31 , L 32 , L 33 and L 34 Each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-,

[0598] By R 35 , R 36 , R 37 and R 38 The hydrocarbon group represented by may be replaced by an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 or SR 58 replace,

[0599] R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms,

[0600] R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0601] The above aromatic groups having 6 to 20 carbon atoms may be substituted with OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 Replace, and

[0602] R 57 Indicates -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 OR 56 ,

[0603] R 58 represents a hydrocarbon group having 1 to 12 carbon atoms.

[0604] In formula (1-3), R 1 and R 2Each independently represents preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and further preferably a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented by may preferably be substituted with -OH, but is more preferably a hydrocarbon group having no substituent.

[0605] R 3 Preferably, it represents a hydrocarbon group having 2 to 6 carbon atoms, and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0606] R 31 , R 32 , R 33 and R 34 Each independently represents preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and further preferably a hydrocarbon group having 1 to 3 carbon atoms.

[0607] R 35 , R 36 , R 37 and R 38 Each independently represents preferably a hydrocarbon group having 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and further preferably a hydrocarbon group having 1 to 12 carbon atoms. 35 , R 36 , R 37 and R 38 The hydrocarbon group represented by may preferably be replaced by an aryl group having 6 to 20 carbon atoms or an SR 58 More preferably, these may be replaced by -SR 58 replace.

[0608] R 35 , R 36 , R 37 and R 38 Each independently particularly preferably represents -SR 58 A substituted hydrocarbon group having 1 to 12 carbon atoms, or a hydrocarbon group having 1 to 12 carbon atoms.

[0609] L 31 , L 32 , L 33 and L 34 Each independently preferably represents -C(O)O- or -OC(O)-.

[0610] R 58 It preferably represents a hydrocarbon group having 1 to 10 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0611] The compounds according to the embodiments of the present invention can form salts.

[0612] Examples of salts in basic groups include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0613] Examples of salts in acidic groups include salts formed with alkali metals such as sodium and potassium; salts formed with alkaline earth metals such as calcium and magnesium; ammonium salts; salts formed with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine; and so on.

[0614] Among the above salts, for example, pharmaceutically acceptable salts are preferred.

[0615] The lipid represented by formula (1) and its preparation method are described in WO2022 / 230964A, the entire content of which is incorporated herein by reference.

[0616] <<The lipid represented by formula (5) or a salt thereof>>

[0617] For example, the lipid represented by formula (5) or a salt thereof can be used as an ionizable lipid.

[0618] [Chemical formula 56]

[0619]

[0620] Wherein R 51 and R 52 each independently represent a hydrocarbon group having 1-21 carbon atoms which may have substituent A,

[0621] The substituent A represents a hydroxyl group, or a group represented by -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 represented group,

[0622] G 20 represents -O(CO)- or -(CO)O-,

[0623] R 55 and R 56Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0624] R 58 and R 59 Each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have substituent B, and substituent B is -N(R 61 )(R 62 ),

[0625] R 61 and R 62 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,

[0626] R 60 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0627] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0628] G 30 represents -S-(CO)-NR 64 ,

[0629] R 64 represents a group represented by -L 30 -G 20 -CH(R 55 )(R 56 ),

[0630] a represents 0 or 1,

[0631] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0632] G 10 represents -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 ),

[0633] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0634] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0635] b represents 0 or 1,

[0636] R 53 、R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have substituent C, and the said substituent C is represented by -(CO)OR 65 or -O(CO)-R65 The group represented by

[0637] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a -L 40 -CH(R 66 )(R 67 ),

[0638] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0639] R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms.

[0640] The compound represented by formula (5) may be a compound represented by formula (5A):

[0641] [Chemical Formula 57]

[0642]

[0643] Where R 51 and R 52 Each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, wherein the substituent A represents a hydroxyl group or a -G 20 -CH(R 55 )(R 56 ),

[0644] G 20 represents -O(CO)- or -(CO)O-,

[0645] R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms,

[0646] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0647] G 10 represents -O(CO)- or -(CO)O-,

[0648] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0649] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0650] The compound represented by formula (5) may be a compound represented by formula (5B):

[0651] [Chemical Formula 58]

[0652]

[0653] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0654] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0655] G 10 represents -O(CO)O-,

[0656] L 20 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0657] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, wherein the substituent C represents -O(CO)-R 65 The group represented by

[0658] R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a -L 40 -CH(R 66 )(R 67 ),

[0659] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0660] R 66 and R 67 represents an alkoxy group having 1 to 10 carbon atoms.

[0661] The compound represented by formula (5) may be a compound represented by formula (5C):

[0662] [Chemical Formula 59]

[0663]

[0664] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0665] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0666] G 10 Indicates -N(C(O)R63 )-,

[0667] R 63 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0668] R 53 , R 54 and R 57 Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, wherein the substituent C represents -(CO)OR 65 The group represented by

[0669] R 65 Indicated by -L 40 -CH(R 66 )(R 67 ),

[0670] L 40 represents a hydrocarbon group having 1 to 6 carbon atoms,

[0671] R 66 and R 67 represents a hydrocarbon group having 1 to 10 carbon atoms.

[0672] The compound represented by formula (5) may be a compound represented by formula (5D):

[0673] [Chemical Formula 60]

[0674]

[0675] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms,

[0676] L 10 represents a hydrocarbon group having 1 to 18 carbon atoms,

[0677] G 30 Represents -S-(CO)-NR 64 ,

[0678] R 64 Indicated by -L 30 -G 20 -CH(R 55 )(R 56 ),

[0679] L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,

[0680] G 20 represents -(CO)O-,

[0681] R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,

[0682] G 10 represents -(CO)O-,

[0683] R 53 、R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0684] The hydrocarbon group having 1 to 21 carbon atoms is preferably an alkyl group having 1 to 21 carbon atoms, an alkenyl group having 2 to 21 carbon atoms, or an alkynyl group having 2 to 21 carbon atoms, more preferably an alkyl group having 1 to 21 carbon atoms or an alkenyl group having 2 to 21 carbon atoms. The alkyl group having 1 to 21 carbon atoms can be straight-chain or branched-chain and can be linear or cyclic. The number of carbon atoms is preferably 3 to 21, more preferably 5 to 21 carbon atoms. Examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. The alkenyl group having 2 to 18 carbon atoms can be straight-chain or branched-chain and can be linear or cyclic. The number of carbon atoms is preferably 3 to 18, more preferably 5 to 18. Examples include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably (Z)-tridec-8-enyl), tetradecenyl (preferably tetradec-9-enyl), pentadecenyl (preferably (Z)-pentadec-8-enyl), hexadecenyl (preferably (Z)-hexadec-9-enyl), hexadecadienyl, heptadecenyl (preferably (Z)-heptadec-8-enyl), heptadecadienyl (preferably (8Z,11Z)-heptadec-8,11-dienyl), octadecenyl (preferably (Z)-octadec-9-enyl), octadecadienyl (preferably (9Z,12Z)-octadec-9,12-dienyl), etc. The alkynyl group having 2 to 21 carbon atoms can be straight-chain or branched-chain and can be linear or cyclic. The number of carbon atoms is preferably 3 to 21, more preferably 5 to 21 carbon atoms. Examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, etc. Examples of the hydrocarbon group having 1 to 18 carbon atoms include those of the hydrocarbon group having 1 to 21 carbon atoms that have 1 to 18 carbon atoms.

[0685] As the cyclic hydrocarbon group, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, a cycloalkynyl group having 3 to 10 carbon atoms, and an aryl group having 6 to 10 carbon atoms are preferred.

[0686] The hydrocarbon group having 1 to 6 carbon atoms is preferably an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms or an alkynyl group having 2 to 6 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. Specific examples thereof include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl and hexyl. The alkenyl group having 2 to 6 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. Specific examples thereof include allyl, isoprenyl, pentenyl and hexenyl. The alkynyl group having 2 to 6 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. Specific examples thereof include propargyl, butynyl, pentynyl and hexynyl.

[0687] The hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and is preferably an alkyl group having 1 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10 carbon atoms. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl and decyl. The alkenyl group having 2 to 10 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include allyl, isoprenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), and decenyl. The alkynyl group having 2 to 10 carbon atoms can be straight-chain or branched-chain, and can be chain-like or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10 carbon atoms. Specific examples thereof include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.

[0688] The compound represented by formula (5) can form salts.

[0689] Examples of salts in the basic group include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts formed with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts formed with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.

[0690] Among the above salts, for example, pharmaceutically acceptable salts are preferred.

[0691] Examples of Ionizable Lipids

[0692] Examples of ionizable lipids include the following lipids. Note that cKK-E12 (MD-1) and C12-200 are compounds not included in the above formula (5).

[0693] [Chemical Formula 61]

[0694] cKK-E12 (MD-1)

[0695]

[0696] [Chemical Formula 62]

[0697] C12-200

[0698]

[0699] [Chemical Formula 63] FL-A

[0700]

[0701] [Chemical Formula 64]

[0702] FL-B

[0703]

[0704] [Chemical Formula 65]

[0705] FL-C

[0706]

[0707] [Chemical Formula 66]

[0708] MC3

[0709]

[0710] [Chemical Formula 67]

[0711] L-319

[0712]

[0713] [Chemical Formula 68]

[0714] 13-B43

[0715]

[0716] [Chemical Formula 69]

[0717] Lipid 9

[0718]

[0719] [Chemical formula 70]

[0720] Lipid 19

[0721]

[0722] [Chemical formula 71]

[0723] TCL053

[0724]

[0725] [Chemical formula 72]

[0726] TCL065

[0727]

[0728] [Chemical formula 73]ALC-0315

[0729]

[0730] [Chemical formula 74]SM-102

[0731]

[0732] [Chemical formula 75]Lipid 5

[0733]

[0734] [Chemical formula 76]Lipid 29

[0735]

[0736] [Chemical formula 77]

[0737] ATX-100

[0738]

[0739] [Chemical formula 78]

[0740] Lipid A9

[0741]

[0742] [Chemical formula 79]

[0743] Lp01

[0744]

[0745] [Chemical formula 80]

[0746] FL-D

[0747]

[0748] [Chemical formula 81] FL-E

[0749]

[0750] [Chemical formula 82] FL-F

[0751]

[0752] [Chemical formula 83] FL-G

[0753]

[0754] [Chemical formula 84]

[0755] FL-H

[0756]

[0757] In the lipid composition of the present invention, the content of the ionizable lipid or its salt is preferably 10 mol% - 80 mol%, more preferably 20 mol% - 80 mol%, further preferably 30 mol% - 70 mol%, and further preferably 40 mol% - 60 mol% based on the total lipid.

[0758] <Neutral lipid>

[0759] The lipid particles according to the present invention may contain neutral lipids.

[0760] The neutral lipid is preferably an amphiphilic lipid.

[0761] As the amphiphilic lipid, phospholipids are preferred. Examples thereof include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, etc. As the phospholipid, phospholipids having choline groups such as phosphatidylcholine are preferred. The amphiphilic lipid can be used alone or in combination with a variety of different neutral lipids.

[0762] There is no particular limitation on phosphatidylcholine, and examples thereof include soybean phosphatidylcholine (SPC), hydrogenated soybean phosphatidylcholine (HSPC), egg yolk phosphatidylcholine (EPC), hydrogenated egg yolk phosphatidylcholine (HEPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dilauroyl phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), etc. Among them, dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), and dilauroyl phosphatidylcholine (DLPC) are preferred. Distearoyl phosphatidylcholine (DSPC) is particularly preferred.

[0763] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0764] [Chemical formula 85]

[0765]

[0766] There is no particular limitation on phosphatidylethanolamine, and examples thereof include dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylethanolamine (DLoPE), diphytanoyl phosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), bis(tetradecyl) phosphatidylethanolamine, bis(hexadecyl) phosphatidylethanolamine, bis(octadecyl) phosphatidylethanolamine, diphytanoyl ethanolamine, etc.

[0767] There is no particular limitation on sphingomyelin, and examples thereof include sphingomyelin derived from egg yolk, sphingomyelin derived from milk, etc.

[0768] In the lipid composition of the present invention, the amount of neutral lipid mixed is preferably 1 - 30 mol%, more preferably 5 - 25 mol%, and still more preferably 7 - 23 mol% relative to the total amount of lipid components.

[0769] <Lipid having a nonionic hydrophilic polymer>

[0770] The lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer. The lipid having a nonionic hydrophilic polymer preferably contains an acyl group, and the carbon chain length of the acyl group is preferably 8 - 26.

[0771] There is no particular limitation on the nonionic hydrophilic polymer, and examples thereof include nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers containing two or more of these polymers as constituent units, etc.

[0772] Among these nonionic hydrophilic polymers, nonionic polyethers, nonionic polyesters, nonionic polyamino acids or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are still more preferred, and polyethylene glycol (hereinafter, polyethylene glycol is also referred to as PEG) is particularly preferred.

[0773] There is no particular limitation on the lipid having a nonionic hydrophilic polymer, and examples thereof include PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, ceramide PEG derivatives, and the like. Among them, monoacylglycerol PEG and diacylglycerol PEG are preferred.

[0774] The alkyl chain of the lipid having a nonionic hydrophilic polymer preferably has 8 to 26 carbon atoms, and more preferably has 10 to 22 carbon atoms.

[0775] The weight-average molecular weight of the nonionic hydrophilic polymer is preferably 100 to 10,000, more preferably 500 to 5,000, and further preferably 750 to 3,000.

[0776] The nonionic hydrophilic polymer chain may be branched or may have substituents such as hydroxymethyl.

[0777] Preferred examples of the lipid having a nonionic hydrophilic polymer include the following lipids.

[0778] DMG-mPEG2000: 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0779] DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0780] DSG-mPEG2000: 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0781] [Chemical formula 86]

[0782] DMG-mPEG2000

[0783] DPG-mPEG2000

[0784] DSG-mPEG2000

[0785] In the lipid composition of the present invention, the amount of the lipid having a nonionic hydrophilic polymer is preferably 0.1 mol% to 10 mol%, more preferably 0.3 mol% to 8 mol%, further preferably 0.5 mol% to 5 mol%, and even more preferably 1 mol% to 3 mol% with respect to the total amount of the lipid.

[0786] <Nucleic acid>

[0787] The lipid composition of the present invention contains a therapeutic agent. As the therapeutic agent, a nucleic acid is preferred. The nucleic acid can be DNA or RNA, and can be a plasmid, single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (microRNA), mRNA, antisense oligonucleotide (also known as ASO), ribozyme, aptamer, decoy nucleic acid, gRNA for genome editing, etc. It can also contain modified nucleic acids.

[0788] In the lipid composition of the present invention, the weight ratio of lipid to therapeutic agent is preferably 5-100, more preferably 5-70, further preferably 5-40, and particularly preferably 5-35.

[0789] <Preparation method of the composition>

[0790] The preparation method of the lipid composition of the present invention will be described.

[0791] There is no limitation on the preparation method of the lipid composition. For example, the lipid composition can be prepared by dissolving all the constituent components of the lipid particles or a part of the oil-soluble components of the lipid particles in an organic solvent, etc. to form an oil phase, dissolving the water-soluble components of the lipid particles in water to form a water phase, and mixing the oil phase and the water phase together. Mixing can be carried out using a micro mixer, or emulsification can be carried out using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, etc.

[0792] Alternatively, the lipid composition can also be prepared by the following method: evaporating the lipid-containing solution to dryness using an evaporator under reduced pressure, or spray-drying the lipid-containing solution using a spray dryer to prepare a dry lipid-containing mixture, adding this mixture to an aqueous solvent, and further emulsifying using the above emulsifier, etc.

[0793] An example of the preparation method of lipid particles containing nucleic acids is a method including the following steps:

[0794] Step (a): Dissolving the constituent components of the lipid particles containing the compound of the embodiment of the present invention in an organic solvent to obtain an oil phase;

[0795] Step (b): Mixing the oil phase obtained in step (a) with an aqueous phase containing nucleic acid;

[0796] Step (c): Diluting the mixed solution containing the oil phase and the water phase obtained in step (b) to obtain a dispersion of the nucleic acid-containing lipid composition; and

[0797] Step (d): Removing the organic solvent from the dispersion of the nucleic acid lipid composition obtained in step (c).

[0798] In step (a), the lipid component is dissolved in an organic solvent (such as an alcohol like ethanol, an ester, etc.). The total lipid concentration is not particularly limited, but is generally from 1 mmol / L to 100 mmol / L, preferably from 3 mmol / L to 50 mmol / L, and more preferably from 5 mmol / L to 30 mmol / L.

[0799] In step (b), an aqueous phase can be obtained by dissolving a nucleic acid (such as siRNA, antisense nucleic acid, mRNA, etc.) in water or a buffer. If necessary, components such as an antioxidant can be added. The mixing ratio (volume ratio) of the aqueous phase:oil phase is preferably from 5:1 to 1:1, more preferably from 4:1 to 2:1.

[0800] In step (b), the mixed solution can be diluted with water or a buffer (such as phosphate buffered saline (PBS), etc.).

[0801] In step (c), as a method for removing the organic solvent from the dispersion of the lipid composition, general methods can be used without particular limitation. For example, the organic solvent can be removed by dialyzing the dispersion with phosphate buffered saline.

[0802] If necessary, sizing can be performed on the lipid composition. The sizing method is not particularly limited, and an extruder or the like can be used to reduce the particle size.

[0803] <Composition>

[0804] The composition of the present invention can be lipid particles. Lipid particles refer to particles composed of lipids and include compositions having any structure selected from the following: lipid aggregates formed by lipids (such as lipid nanoparticles), micelles, and liposomes. However, as long as the composition contains lipids, the structure of the lipid particles is not limited to these.

[0805] The morphology of the lipid particles can be confirmed by an electron microscope, structural analysis using X-rays, etc. For example, by the method using a cryogenic transmission electron microscope (CryoTEM method), it can be confirmed whether lipid particles such as liposomes have a structure composed of a bimolecular lipid membrane structure (lamellar structure) and an internal water layer, or a structure composed of a core with a high electron density and filled with constituent components including lipids. Small angle X-ray scattering (SAXS) analysis can also confirm whether the lipid particles have a bimolecular lipid membrane structure (lamellar structure).

[0806] When the lipid composition of the present invention is in the form of particles, the particle size is not particularly limited, but is preferably from 10 - 1000 nm, more preferably from 30 - 500 nm, and further preferably from 50 - 250 nm. The particle size of the lipid particles can be measured by general methods (such as dynamic light scattering method, laser diffraction method, etc.).

[0807] When the lipid composition of the present invention is a particle, the zeta potential of the particle is not particularly limited, but is preferably -20 to +20 mV, more preferably -10 to 10 mV. The zeta potential in the present invention is a value measured by electrophoresis by diluting the lipid composition in a phosphate buffer solution, but the method is not limited thereto.

[0808] The pKa of the lipid composition of the present invention is not particularly limited, but is preferably 9 to 4, more preferably 8 to 5, and even more preferably 7.5 to 6. The pKa of the lipid composition of the present invention is a value measured by the TNS assay, but is not limited thereto.

[0809] <Applications of lipid compositions>

[0810] As an example of the purposes of the lipid composition in the present invention, therapeutic agent (such as nucleic acid) can be imported into cell by the lipid composition containing nucleic acid.That is, the lipid composition of the present invention can be used as the composition for nucleic acid to be imported into cell.

[0811] Furthermore, the lipid compositions of the present invention can be used as pharmaceutical compositions for in vivo nucleic acid delivery.

[0812] In the present invention, in particular, therapeutic agent can be delivered to endothelial cells, mesenchymal cells or cancer cells. Therefore, therapeutic agent can be delivered to organs other than the liver. Organs other than the liver include spleen, kidney, lung, heart, muscle and brain. The lipid composition of the present invention delivers the therapeutic agent to organs other than the liver, preferably spleen, kidney, lung, heart, muscle and brain, more preferably kidney, lung, heart, muscle and brain, further preferably lung and heart.

[0813] In addition, when the lipid composition of the present invention contains the nucleic acid with pharmaceutical use, the lipid composition can be used as a nucleic acid drug to an organism. When the lipid composition of the present invention is used as a nucleic acid drug, the lipid composition of the present invention can be administered separately to an organism, or the lipid composition can be mixed with a pharmaceutically acceptable carrier (for example, an administration medium such as saline or phosphate buffer) and administered to an organism. That is, the lipid composition of the present invention can further include a pharmaceutically acceptable carrier.

[0814] In the mixture mixed with a pharmaceutically acceptable carrier, the concentration of the lipid composition is not particularly limited and can generally be 0.05% to 90% by weight. In addition, other pharmaceutically acceptable additives such as pH regulating buffers, osmotic pressure regulators, etc. can also be added to the nucleic acid drug containing the lipid composition of the present invention.

[0815] The route of administration for administering the lipid composition of the present invention is not particularly limited. The lipid composition can be administered by any method. Examples of administration methods include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intraperitoneal administration, intramuscular administration, vaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, sublingual administration, nasal administration, intracisternal administration, inhalation, etc.). Among them, parenteral administration is preferred. As the administration method, intravenous injection, subcutaneous injection, intradermal injection or intramuscular injection is preferred. Intravenous injection or intramuscular injection is particularly preferred. As the administration, nucleic acid delivery can also be carried out by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection into the diseased site.

[0816] The dosage form of the lipid particles of the embodiments of the present invention is not particularly limited. For oral administration, the lipid composition of the present invention can be combined with a suitable excipient and used in the form of tablets, lozenges, capsules, pills, suspensions, syrups, etc. In addition, additives such as antioxidants, buffers, bacteriostatic agents, isotonic sterile injectables, suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. can be appropriately incorporated into the preparations suitable for parenteral administration.

[0817] <Use of lipid nanoparticles as nucleic acid delivery carriers>

[0818] The lipid particles of the present invention can retain nucleic acids with a high encapsulation rate. Therefore, the lipid particles are extremely useful as nucleic acid delivery carriers. According to the use of the nucleic acid delivery carrier of the present invention, for example, by mixing the obtained lipid particles with nucleic acids, etc. and performing transfection in vitro or in vivo, nucleic acids, etc. can be introduced into cells. In addition, the nucleic acid delivery carrier of the present invention can also be used as a nucleic acid delivery carrier in nucleic acid drugs. That is, the lipid particles according to the embodiments of the present invention can be used as a composition for in vitro or in vivo (preferably in vivo) delivery of nucleic acids.

[0819] Next, the present invention will be described based on examples, but the present invention is not limited thereto. Examples

[0820] <Materials and methods>

[0821] <sirna>

[0822] The following custom siRNAs are manufactured by Horizon.

[0823] siRNAs targeting murine VECadherin (siVEcad, siCdh5)

[0824] Sense: 5'-mCmCAAAAGAGAGAmCmUGGAmUmUdTsdT-3'

[0825] Antisense: 5'-AAUCmCAGUCUCUCUUUUGGdTsdT-3'

[0826] Abbreviations

[0827] A Adenosine-3'-phosphate

[0828] C Cytidine-3'-phosphate

[0829] G Guanosine-3'-phosphate

[0830] U Uridine-3'-phosphate

[0831] mA 2'-O-methyladenosine-3'-phosphate

[0832] mC 2'-O-methylcytidine-3'-phosphate

[0833] mG 2'-O-methylguanosine-3'-phosphate

[0834] mU 2'-O-methyluridine-3'-phosphate

[0835] dT 2'-deoxythymidine-3'-phosphate

[0836] dTs 2'-deoxythymidine-5'-phosphate-thiophosphate

[0837] Cadherin 5 (Cdh5), also known as vascular endothelial cadherin (VE-cadherin), is a junction protein whose expression is restricted to endothelial cells. The delivery efficiency to endothelial cells can be evaluated by quantifying the remaining Cdh5 mRNA after administration of siCdh5.

[0838] <Lipid nanoparticle formulation>

[0839] All chemicals obtained from commercial sources were stored according to the manufacturer's instructions and used without further purification.

[0840] Lipid nanoparticles were synthesized using the microfluidic chip device as described previously. Lipid nanoparticles were formed by mixing the lipid-containing ethanol phase with the siRNA-containing aqueous phase and pumped through the microfluidic channels in a PDMS (polydimethylsiloxane) chip.

[0841] Dissolve the ionizable lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, NOF), cholesterol (Sigma), and 1,2-dimyristoyl-rac-glycero-3-methylpolyethylene glycol (DMG-mPEG2000, NOF) in ethanol and mix them in a predetermined molar ratio.

[0842] Prepare an aqueous phase in 10 mM citrate buffer (pH 3.0, fisher) with siRNA. As described previously, mix ethanol and the aqueous phase in a 3:1 ratio in a microfluidic chip device using an injection pump. The total flow rate is 1.2 mL / min.

[0843] Dialyze the lipid nanoparticles against 1x PBS in a 20,000 MWCO cassette (Invitrogen) overnight at 4 °C. Concentrate the formulation using an Amicon ultra centrifugal filter (Millipore Sigma), sterilize it through a 0.22 um filter (Millipore Sigma), and store it at 4 °C.

[0844] <Lipid Nanoparticle Characterization>

[0845] According to the method of Walsh C. et al., Methods Mol Biol. 2014;1141:109-20, use a modified QUANT-IT TM RIBOGREEN TM RNA Assay (Invitrogen Corporation Carlsbad, Calif.) to calculate the nucleic acid encapsulation efficiency of the lipid nanoparticles. Briefly, dilute the sample to a concentration of approximately 1.5 μg / ml in TE buffer. Transfer 50 μl of the diluted sample to a black U-bottom 96-well plate, and add 50 μl of TE buffer or 50 μl of TE buffer containing 2% Triton X-100 to the wells. Incubate the plate at 37 °C for 15 minutes. Dilute the RIBOGREEN TM reagent 1:200 in TE buffer and add 100 μl of this solution to each well. Measure the fluorescence intensity using a plate reader (Tecan 200Pro, Tecan) at an excitation wavelength of 485 nm and an emission wavelength of 515 nm. Subtract the fluorescence value of the reagent blank from the fluorescence value of each sample, and determine the percentage of free RNA by dividing the fluorescence intensity of the intact sample (without Triton X-100) by the fluorescence value of the disrupted sample (with Triton X-100).

[0846] The particle size, polydispersity index (PDI), and Zeta potential of the nanoparticle composition were measured using a Zetasizer Nano ZS (Malvern Instruments), where the particle size was measured in 1×PBS and the Zeta potential was measured in 0.1×PBS.

[0847] <Determination of pKa value by TNS assay>

[0848] According to the method of Heyes J. et al., Journal of Controlled Release 107 (2005) 276 - 287, the apparent pKa value of the lipid nanoparticles was determined using the TNS assay. Briefly, TNS was prepared as a 100 μM stock solution in DMSO. The lipid nanoparticles were diluted to 25 ng / mL ionizable lipid in 0.1xPBS. Additionally, this lipid nanoparticle solution was diluted 10 - fold in buffer solutions with pH ranging from approximately 2.5 to 9.0. 1 TNS stock solution was added to the lipid nanoparticle solutions with different pH values to a final concentration of 6 μM and mixed well in a black 384 - well plate. The fluorescence intensity was monitored using excitation and emission wavelengths of 320 nm and 465 nm in a Tecan Pro200 microplate reader. Using the obtained fluorescence values, a sigmoidal curve of fluorescence versus buffer pH was created, and the logarithm of the inflection point of this curve was the apparent pKa of the lipid nanoparticle formulation. The pKa value was obtained using Prism software.

[0849] <Animal experiments>

[0850] All animal studies were approved by the Massachusetts Institute of Technology Committee on Animal Care and Use (CAC) and complied with applicable local, state, and federal regulations. All experimental procedures were conducted ethically and approved according to the guidelines of the Massachusetts Institute of Technology Division of Comparative Medicine. Female C57BL / 6 mice (6 weeks old) were obtained from Jackson Laboratories, housed in the Massachusetts Institute of Technology animal facility, and acclimated for at least 3 days before the start of the study.

[0851] For intravenous administration, after warming the animals using a heat lamp, siRNA - lipid nanoparticles diluted in PBS were injected via the tail vein using a 29g, 3 / 10cc insulin syringe (BD Biosciences). For intramuscular administration, siRNA - lipid nanoparticles diluted in PBS were injected via the tail vein using a 29g, 3 / 10cc insulin syringe (BD Biosciences).

[0852] At 48 - 72 hours after injection, organs or tissues were collected, including the heart, liver, spleen, lung, kidney, muscle (quadriceps and diaphragm), brain, and tumor, and immersed in RNAlater solution at 4 °C for 12 - 48 hours, and stored at -20 °C after removing RNAlater.

[0853] <Quantifying tissue mRNA by qPCR method>

[0854] Total RNA was isolated from tissues using TRIzol. Briefly, tissue punch samples were placed in a deep well 96 - well plate with 4 - mm stainless steel beads and lysed with 350 μL TRIzol using a GenoGrinder2010. Then 300 μL of the lysate was transferred to a new deep well 96 - well plate. Total RNA was further purified using Direct - zol - 96 MagBead (Zymo Rsearche) according to the manufacturer's protocol.

[0855] Using Luna TM The Luna Universal Probe One - Step RT - qPCR Kit (NEB) and Taqman probes - Cdh5 Mm00486938_m1), Gusb (Mm01197698_m1), and B2m (Mm00437762_m1) were used to analyze gene expression in a one - step multiplex qPCR. Samples were amplified using a LightCycler480 qPCR machine (Roche). Cdh5 expression was normalized to B2m or Gusb.

[0856] Example 1: DC - cholesterol incorporation

[0857] To examine the potential of synthetic cholesterol analogs with basic functional groups, we first replaced cholesterol in a standard hepatocyte - targeting LNP with DC - cholesterol. DC - cholesterol was initially developed for nucleic acid - delivering liposomes and has an ionizable tertiary amine group with a pKa of 7.8 at the cholesterol C3 position (Non - Patent Documents 11 and 12). Although the combination of ionizable lipids and DC - cholesterol has been tested in subcutaneous mRNA vaccines for enhancing mRNA delivery to dendritic cells in lymph nodes, it did not show any advantage over cholesterol and even decreased the delivery efficiency.

[0858] LNP1 is a standard hepatocyte - targeting LNP formulation containing approximately 50 mol% ionizable lipid, approximately 10 mol% DSPC, approximately 38.5 mol% cholesterol, and approximately 1.5 mol% PEG - DMG. This type of formulation is used in Onpattro TM and Spikevax TM . LNP2 replaced cholesterol with DC - cholesterol at the same lipid ratio as LNP1.

[0859] [Table 1]

[0860] Table 1. Physicochemical properties of cholesterol and DC-cholesterol LNPs

[0861] Ionizable lipid Sterol Encapsulation efficiency (%) Z-average (nm) pKa Zeta potential (mV) LNP1 FL-A Cholesterol 77 98.4 6.72 -5.6 LNP2-1 FL-A DC-Cholesterol 83 87.4 6.86 1.3

[0862] To evaluate gene silencing in endothelial cells in various organs, we used siRNA against Cdh5, a cell adhesion molecule whose expression is restricted to endothelium. A formulation containing siRNA against Cdh5 was intravenously administered to mice at 0.3 mg / kg, and organs were harvested 48 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene B2m. A PBS control was also tested.

[0863] As Figure 1 shown, replacement of cholesterol in conventional LNPs with DC-cholesterol enabled silencing of endothelial genes in various organs of mice. Three days after injection, VE-cadherin mRNA expression was measured based on ΔΔCt calculation compared to PBS. (0.3 mg / kg siVEcad)

[0864] Example 2: GENERALIZATION of Other Ionizable Lipids

[0865] To test the generalization of the DC-cholesterol LNP formulation, we tested different types of ionizable lipids. Table 2 summarizes the physicochemical properties of the test formulations. FL-A, FL-B, and FL-C were developed by FUJIFILM.

[0866] [Chemical formula 87]

[0867] FL-A

[0868]

[0869] [Chemical formula 88]

[0870] FL-B

[0871]

[0872] [Chemical formula 89] FL-C

[0873]

[0874] [Chemical formula 90] MC3

[0875]

[0876] [Chemical formula 91] SM-102

[0877]

[0878] [Chemical Formula 92] cKK-E12

[0879]

[0880] [Chemical Formula 93] ALC-0315

[0881]

[0882] [Chemical Formula 94] Lipid 5

[0883]

[0884] [Table 2]

[0885] Table 2. Physicochemical properties of DC-cholesterol LNPs with different ionizable lipids

[0886]

[0887] A formulation containing siRNA against Cdh5 was intravenously administered to mice at 0.5 mg / kg, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene Gusb. A PBS control was also tested.

[0888] The results are as Figure 2 shown. From Figure 2 it was demonstrated by replacement with DC-cholesterol that silencing of endothelial genes with various ionizable lipids. Three days after injection, Cdh5 mRNA expression was measured based on ΔΔCt calculation compared to PBS. (0.5 mg / kg siCdh5)

[0889] Example 3: Evaluation of other cholesterol derivatives modified at the C3 position

[0890] To clarify the structural requirements of cholesterol derivatives, a series of cholesterol derivatives were formulated in LNPs. The lipid composition of the LNPs was fixed as FL-A:DSPC:sterol:DMG-mPEG2000 = 50:10:38.5:1.5.

[0891] [Chemical Formula 95]

[0892] DC cholesterol

[0893]

[0894] [Chemical Formula 96]

[0895] DMPAC-Chol

[0896]

[0897] [Chemical Formula 97]

[0898] OH-Chol

[0899]

[0900] [Chemical Formula 98]

[0901] HAPC-Chol

[0902]

[0903] [Chemical Formula 99]

[0904] MHAPC-Chol

[0905]

[0906] [Chemical Formula 100]

[0907] DMHAPC-Chol

[0908]

[0909] [Chemical Formula 101]

[0910] Arg-Chol

[0911]

[0912] [Chemical Formula 102]

[0913] GL67: N4-Cholesteryl-Spermine

[0914]

[0915] [Table 3]

[0916] Table 3: Physicochemical properties of 3'-modified cholesterol LNPs

[0917] Ionizable lipid Sterol Encapsulation efficiency (%) Z-average (nm) pKa Zeta potential (mV) LNP2-3 FL-A DC-Cholesterol 84 74.3 6.71 4.0 LNP11 FL-A DMPAC-Chol 80 69.3 6.78 6.2 LNP12 FL-A OH-Chol 89 61.5 6.66 8.0 LNP13 FL-A HAPC-Chol 92 65.8 6.90 7.2 LNP14 FL-A MHAPC-Chol 91 56.5 6.83 2.4 LNP15 FL-A DMHAPC-Chol 76 84.2 6.71 5.9 LNP16 FL-A Arg-Chol 92 58.3 6.36 8.9 LNP17 FL-A GL-67 100 72.1 7.80 13.0

[0918] A formulation containing siRNA against Cdh5 was administered intravenously to mice at 0.5 mg / kg, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene Gusb. A PBS control was also tested.

[0919] The results are as Figure 3 shown. As Figure 3 As shown, various 3'-modified cholesterol analogs exhibited silencing of endothelial genes in various organs of mice. Three days after injection, Cdh5 mRNA expression was measured based on ΔΔCt calculation compared to PBS. (0.5 mg / kg siCdh5)

[0920] Example 4: DC-Cholesterol Ratio (FL-B)

[0921] To optimize the DC-cholesterol ratio, we prepared LNP formulations with different DC-cholesterol ratios and tested them in vivo. In this example, FL-A was used for the ionizable lipid, and the DSPC and DMG-mPEG-2000 ratios were fixed at 10% and 1.5% respectively.

[0922] Table 4 summarizes the contents and properties of several formulations of lipid components that can be used in the nanoparticle compositions of the present invention.

[0923] [Table 4]

[0924] Table 4: Physicochemical Properties of DC-Cholesterol LNPs with Different Molar Ratios

[0925]

[0926] Formulations containing siRNA against Cdh5 were intravenously administered to mice at 0.5 mg / kg, and organs were harvested 72 hours after injection. After isolating and purifying total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene B2m. A PBS control was also tested.

[0927] The results are as Figure 4 shown. As Figure 4 shown, LNPs with different DC-cholesterol ratios exhibited strong silencing of endothelial genes in the lungs and hearts of mice. Three days after injection, Cdh5 mRNA expression was measured based on ΔΔCt calculation compared to PBS. (0.5 mg / kg siCdh5)

[0928] Example 5: DC-Cholesterol Ratio 2 (FL-A)

[0929] To optimize the DC-cholesterol ratio, we prepared LNP formulations with different DC-cholesterol ratios and tested them in vivo. In this example, FL-A was used for the ionizable lipid, and the DSPC and DMG-mPEG-2000 ratios were fixed at 10% and 1.5% respectively.

[0930] Table 5 summarizes the contents and properties of several formulations of lipid components that can be used in the nanoparticle compositions of the present invention.

[0931] [Table 5]

[0932] Table 5: Physicochemical properties of DC-cholesterol LNPs with different molar ratios

[0933]

[0934]

[0935] A formulation containing siRNA against Cdh5 was administered intravenously to mice at 0.5 mg / kg, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene Gusb. A PBS control was also tested.

[0936] The results are shown in Figure 5 as Figure 5 shown, LNPs with different DC-cholesterol ratios exhibited strong silencing of endothelial genes in the lungs and hearts of mice. Three days after injection, Cdh5 mRNA expression (0.5 mg / kg siCdh5) was measured based on ΔΔCt calculations compared to PBS.

[0937] Example 6: Universality of other ionizable lipids

[0938] To further test the universality of the DC-cholesterol LNP formulation, we tested different types of ionizable lipids. Table 6 summarizes the physicochemical properties of the test formulations.

[0939] [Chemical formula 103]

[0940] Lipid A9

[0941]

[0942] [Chemical formula 104]

[0943] Lipid 29

[0944]

[0945] [Chemical formula 105]

[0946] ATX-100

[0947]

[0948] [Chemical formula 106]

[0949] Lp01

[0950]

[0951] [Chemical formula 107]

[0952] TCL053

[0953]

[0954] [Table 6]

[0955] Table 6: Physicochemical properties of DC-cholesterol LNPs with different ionizable lipids

[0956]

[0957] A formulation containing siRNA against Cdh5 was intravenously administered to mice at 0.5 mg / kg, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene Gusb. A PBS control was also tested.

[0958] DC-cholesterol replacement would demonstrate the silencing of endothelial genes with various ionizable lipids. At 3 days after injection, Cdh5 mRNA expression was measured based on ΔΔCt calculation compared to PBS. (0.5 mg / kg siCdh5)

[0959] Example 7: In vivo endothelial cell RNA delivery using various ionizable lipids

[0960] To evaluate whether endothelial RNA delivery via ionizable cholesterol (iChol) LNPs could be generalized to various ionizable lipids, we prepared a library of iChol LNPs with different ionizable lipids (Table 7). In this experiment, we selected siRNA against the Cdh5 gene that is specifically expressed in endothelial cells. By using siCdh5, endothelial delivery efficiency could be evaluated without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Subsequently, at 48 - 72 hours after administration, individual organs were harvested, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression level of Cdh5 was quantified by real-time PCR.

[0961] The results are as Figure 7 shown. All iChol LNP formulations with different ionizable lipids showed significant gene silencing in the liver, kidney, lung, heart, and skeletal muscle (quadriceps femoris), demonstrating that the iChol LNP formulation can be generalized to various ionizable lipids.

[0962] Endothelial cells: siCdh5

[0963] 5’-ccAAAAGAGAGAcuGGAuudTsdT-3’

[0964] 5’-AAUCcAGUCUCUCUUUUGGdTsdT-3’

[0965] [Table 7]

[0966] Table 7: Physicochemical properties of DC-cholesterol LNPs with different ionizable lipids

[0967]

[0968] Figure 7 The figure shows that the iChol LNP formulation can be extended to various ionizable lipids. The figure shows the mRNA levels of Cdh5 relative to the PBS control group. Each symbol represents an individual animal. Data are represented as mean ± SD.

[0969] Example 8: In vivo endothelial cell RNA delivery with different ionizable cholesterol (1) HAPC-cholesterol ratios

[0970] To optimize the ionizable cholesterol ratio, we prepared LNP formulations with different HAPC-cholesterol ratios and tested them in vivo. In this example, FL-A was used as the ionizable lipid, and the ratios of FL-A, DSPC, and DMG-mPEG-2000 were fixed at 50%, 10%, and 1.5%, respectively (Table 8).

[0971] In this experiment, we selected siRNA against the Cdh5 gene specifically expressed in endothelial cells. By using siCdh5, the endothelial delivery efficiency can be evaluated without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Subsequently, at 48 - 72 hours after administration, individual organs were harvested, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression level of Cdh5 was quantified by real-time PCR.

[0972] The results are as Figure 8 shown. All iChol LNPs with different HAPC-cholesterol ratios showed significant gene silencing in the liver, and iChol LNPs with 20% or more HAPC-cholesterol showed more efficient RNA delivery to extrahepatic organs.

[0973] Endothelial cells: siCdh5

[0974] 5’-ccAAAAGAGAGAcuGGAuudTsdT-3’

[0975] 5’-AAUCcAGUCUCUCUUUUGGdTsdT-3’

[0976] [Table 8]

[0977] Table 8

[0978]

[0979] Figure 8 Shown is that iChol LNP delivers RNA to endothelial cells with different HAPC-cholesterol ratios. The figure shows the mRNA levels of Cdh5 relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD.

[0980] Example 9: In Vivo Endothelial Cell RNA Delivery with Different Ionizable Cholesterol(2):DC-Cholesterol Ratios

[0981] To optimize the ionizable cholesterol ratio, we prepared LNP formulations with different DC-cholesterol ratios and tested them in vivo. In this example, FL-A was used as the ionizable lipid, and the ratios of FL-A, DSPC, and DMG-mPEG-2000 were fixed at 50%, 10%, and 1.5%, respectively (Table 9).

[0982] In this experiment, we selected siRNA against the Cdh5 gene that is specifically expressed in endothelial cells. By using siCdh5, the endothelial delivery efficiency can be evaluated without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Subsequently, at 48 - 72 hours after administration, each organ was harvested, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression level of Cdh5 was quantified by real-time PCR.

[0983] The results are as Figure 9 shown. All iChol LNPs with different DC-cholesterol ratios showed significant gene silencing in the liver, and iChol LNPs with 20% or more HAPC-cholesterol showed more efficient RNA delivery to extrahepatic organs.

[0984] [Table 9]

[0985] Table 9:

[0986]

[0987] Figure 9 Shown is that iChol LNPs with different DC-cholesterol ratios deliver RNA to endothelial cells. The figure shows the mRNA levels of Cdh5 relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD.

[0988] Example 10: In Vivo Endothelial Cell RNA Delivery with Different Ionizable Cholesterol (3)HAPC-Cholesterol Ratios

[0989] To optimize the ionizable cholesterol ratio, we prepared LNP formulations with different HAPC-cholesterol ratios and tested them in vivo. In this example, FL-A was used as the ionizable lipid, and the DMG-mPEG-2000 ratio was fixed at 1.5% (Table 10).

[0990] In this experiment, we selected siRNA against the Cdh5 gene specifically expressed in endothelial cells. By using siCdh5, the endothelial delivery efficiency could be evaluated without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Subsequently, at 48 - 72 hours after administration, each organ was harvested, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression level of Cdh5 was quantified by real-time PCR.

[0991] The results are as Figure 10 shown. All iChol LNPs with different DC-cholesterol ratios showed significant gene silencing in the liver, and iChol LNPs with 20% or more HAPC-cholesterol and 20% or more ionizable lipid showed more efficient RNA delivery to extrahepatic organs.

[0992] [Table 10]

[0993] Table 10:

[0994]

[0995]

[0996] Figure 10 Shows that iChol LNPs with different HAPC-cholesterol ratios deliver RNA to endothelial cells. The figure shows the mRNA levels of the Cdh5 gene relative to the PBS control group. Each symbol represents each animal. Data are presented as mean ± SD.

[0997] Example 11: Serum Protein-Independent RNA Delivery to Endothelial Cells

[0998] Apolipoprotein E (ApoE) is a protein present in the blood that participates in the transport of lipids and cholesterol throughout the body. Recent studies have shown that ApoE plays an important role in the uptake and distribution of hepatocyte LNP. Specifically, it has been found that LNP is taken up by cells in the liver through a process mediated by ApoE adsorbed on the surface of LNP. In the case of LNP containing constitutive cationic lipids, it has been reported that the cellular uptake mechanism is also serum protein-dependent. Among serum proteins, vitronectin has been reported to play a crucial role in the cellular uptake of these cationic LNP.

[0999] WO2020051220

[1000] WO2020051223

[1001] Dilliard S.A.Proc.Nat.Assoc.Sci.2022.https: / / doi.org / 10.1073 / pnas.2109256118

[1002] Dilliard S.A.J.Cont.Rel.2023.https: / / doi.org / 10.1016 / j.jconrel.2023.07.058.

[1003] To analyze the serum protein-dependence of ionizable cholesterol LNP uptake, in vitro transfection efficiency was evaluated in serum-containing and serum-free media. LNP formulations encapsulating siCdh5 were prepared as described in Materials and Methods (Table 11) and the resulting LNP were further tested in the bEND.3 murine brain endothelial cell line. bEND.3 cells were cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000 - 15,000 cells per well. bEND.3 endothelial cells were transfected with a siCdh5 concentration of 100 nM per well. After incubation for 2 hours, the cell culture medium containing LNP was removed, the cells were washed with serum-free medium, and then cultured in serum-containing medium. After overnight incubation, total RNA was extracted using the QuickExtract RNA Extraction Kit (LGC Biosearchtechnologies) and cDNA was synthesized by reverse transcription. Cdh5 expression levels were quantified by real-time PCR.

[1004] The results are as Figure 11 shown. Although hepatocyte LNP showed more efficient gene silencing in serum-containing medium, ionizable cholesterol LNP showed similar gene silencing efficiency in serum-free and serum-containing media, indicating that ionizable cholesterol LNP uptake is not dependent on serum proteins.

[1005] [Table 11]

[1006] Table 11:

[1007]

[1008] Figure 11 It shows that endothelial cells take up iChol LNP in a serum-independent manner.

[1009] Upper panel: Schematic diagram of the in vitro experimental design showing bEND.3 endothelial cells treated with siRNA-LNP in serum-containing and serum-free media.

[1010] Lower panel: This figure shows the mRNA levels of Cdh5 relative to the PBS control group. Each symbol represents each well. Data are expressed as mean ± SD.

[1011] Example 12: In Vitro Endothelial RNA Delivery Using Different LNP Formulations

[1012] To optimize the DC-cholesterol ratio, we prepared LNP formulations encapsulating firefly luciferase mRNA (TriLink) with different lipid ratios and further tested the resulting LNPs in the bEND.3 murine brain endothelial cell line. bEND.3 cells were cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000 - 15,000 cells per well. Before transfection, the cell medium was replaced with serum-free medium. bEND.3 endothelial cells were transfected at a dose of 1 μg per well. A total of 29 LNP formulations (including hepatocyte LNPs) were transfected into bEND.3 cells. After incubation for 2 hours, the luciferase expression level was quantified using the Steady-GloTM Luciferase Assay System (Promega).

[1013]

[1014] [Table 12]

[1015] Table 12: LNP Formulations with Different Lipid Ratios (Ionizable Lipid / RNA Ratio Fixed at 10)

[1016]

[1017]

[1018] Example 13: Analysis of LNP-Mediated In Vitro Endothelial Cell Injury

[1019] Endothelial cell injury in vivo may lead to internal hemorrhage ( Figure 12 )。To quantify the cytotoxicity of LNPs against endothelial cells, the murine endothelial cell line bEND.3 was used. 250 nM siRNA targeting Cdh5 encapsulated in LNPs was administered to 10,000 cells in a 96-well plate, and these cells were incubated for 24 hours after transfection and then analyzed, where cell viability was measured using a CCK-8 assay kit (Dojindo) according to the manufacturer's protocol and normalized to the PBS-treated group.

[1020] The results are as Figure 12 shown. These results indicate that LNPs containing ionizable cholesterol are more tolerable than LNPs containing the constitutive cationic lipid DOTAP.

[1021] [Table 13]

[1022] Table 13:

[1023]

[1024] The ionizable lipid / RNA ratio was fixed at 10

[1025] Figure 12 showing that iChol LNPs have lower endothelial cell cytotoxicity than cationic lipid-containing LNPs

[1026] Upper panel: Schematic of potential internal bleeding induced by LNP-mediated endothelial cell injury

[1027] Lower panel: This figure shows cell viability relative to the PBS control group. Each symbol represents each well. Data are presented as mean ± SD.

[1028] Example 14: In vitro blood compatibility analysis using human primary erythrocytes

[1029] To evaluate the blood compatibility of LNPs, a hemolysis assay was performed. In the hemolysis assay, human primary red blood cells (RBCs, Innovative Research) were washed three times with PBS, and 90 μL of a 4% vol / vol suspension of RBCs in PBS was transferred to a 96-well plate. Subsequently, 10 μL of a siRNA LNP solution containing 150 μg / mL total lipid was added to the RBC suspension. After incubation at 37 °C for 1 hour, the plate was centrifuged at 1,000 x g for 5 minutes, and 80 μL of the supernatant was transferred to a clear flat-bottom 96-well plate. Hemoglobin release was quantified by measuring the UV-vis absorbance at 490 nm. The cationic detergent Triton X-100, which completely lyses the RBC membrane, was used as a positive control, and hemoglobin release was normalized to the Triton X-100-treated group.

[1030] The results are as Figure 13 As shown, 50% DOTAP LNP showed significant hemolysis, while LNPs containing ionizable cholesterol such as DC-cholesterol and HAPC-cholesterol did not. These data demonstrate the higher blood compatibility of ionizable cholesterol LNPs.

[1031] [Table 14]

[1032] Table 14:

[1033]

[1034] The ionizable lipid / RNA ratio was fixed at 10

[1035] Figure 13 showing that iChol LNP has lower hemolytic activity than LNP containing cationic lipid

[1036] Upper panel: Schematic diagram of hemolysis assay

[1037] Lower panel: This figure shows % hemolysis normalized to the Triton X-100 positive control group. Each symbol represents each well. Data are expressed as mean ± SD.

[1038] Statistically significant differences were evaluated using one-way ANOVA and Dunnett's post-test. A corrected P-value less than 0.05 was considered statistically significant, *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001.

[1039] Example 15: In Vivo Hepatic Stellate Cell Delivery

[1040] To analyze the specific cell types in the liver that undergo transfection with ionizable cholesterol LNP, we performed a detailed assessment of the gene silencing efficiency within each cell population. To obtain cell type-specific information, we employed different siRNAs targeting genes characterized by cell type-specific expression patterns. We prepared LNPs encapsulating the following siRNAs (Table 15) and administered these LNPs intravenously to mice. Subsequently, at 48 - 72 hours after administration, each organ was harvested and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription and the gene expression levels of Cdh5, Reln, and Sirpa were quantified by real-time PCR. For Fvii gene silencing, serum factor VII protein levels were quantified using the BIOPHEN TM FVII assay kit (Aniara).

[1041] The results are as [[ID=1 As shown. Although hepatocyte LNPs exhibited gene silencing among all cell types evaluated in the liver, ionizable cholesterol LNPs showed more efficient and specific gene silencing in endothelial cells and hepatic stellate cells.

[1042] Hepatocytes: siFvii

[1043] 5’-GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT-3’

[1044] 5’-GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT-3’

[1045] Endothelial cells: siCdh5

[1046] 5’-ccAAAAGAGAGAcuGGAuudTsdT-3’

[1047] 5’-AAUCcAGUCUCUCUUUUGGdTsdT-3’

[1048] Hepatic stellate cells: siReln

[1049] 5’-GGmUmCmUmCAAGmCmCAmCmUmCGmUmUmUdTsdT-3’

[1050] 5’-AAACGAGUGGCUUGAGACCdTsdT-3’

[1051] Kupffer cells: siSirpa

[1052] 5’-mCmUAAmCAAmCmCAmCAmCAGAAmUAdTsdT-3’

[1053] 5’-mUAUUCUGUGUGGUUGUmUAGdTsdT-3’

[1054] [Table 15]

[1055] Table 15:

[1056]

[1057]

[1058] ​ The figure shows that iChol LNP delivers RNA to liver endothelial cells and hepatic stellate cells. These figures show the mRNA levels of the Cdh5 gene (endothelial cells), Sirpa gene (Kupffer cells), or Reln gene (hepatic stellate cells), or the serum Factor VII protein level (hepatocytes) relative to the PBS control group. siRNA-LNP was intravenously administered to mice at doses of siCdh5 0.3 mg / kg, siFvii 0.3 mg / kg, siSirpa 0.4 mg / kg, and siReln 0.2 mg / kg, respectively. Each symbol represents an individual animal. Data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA and Dunnett's post-test. A corrected P-value of less than 0.05 was considered statistically significant, *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

[1059] Example 16: In Vivo RNA Delivery to Extracellular Matrix-Producing Cells outside the Liver

[1060] Since stellate cells are also found in extrahepatic organs such as the pancreas, lung, kidney, intestine, spleen, adrenal gland, vas deferens, and vocal cords, we also evaluated the delivery of RNA to extrahepatic stellate cells using siRNA against Col1a1, which is specifically expressed in extracellular matrix-producing cells such as stellate cells and fibroblasts. We prepared LNP encapsulating siCol1a1 (Table 16) and intravenously administered these LNP to mice at a dose of 0.5 mg / kg siCola1. Subsequently, at 48 - 72 hours after administration, individual organs were harvested and total RNA was extracted using the Quick-RNA MagBead Kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the Col1a1 expression level was quantified by real-time PCR.

[1061] The results are shown in ​ and ​ As shown. Compared with hepatocyte LNP, DC-cholesterol LNP showed more efficient gene silencing in pancreatic and colonic stellate cells. In addition, gene silencing in extracellular matrix-producing cells of other organs was also confirmed.

[1062] siCol1a1

[1063] 5’-GmUmCmUAGAmCAmUGmUmUmCAGmCmUmUdTsdT-3’

[1064] 5’-AAGCUGAAmCAUGUCmUAGACdTsdT-3’

[1065] [Table 16]

[1066] Table 16:

[1067]

[1068]

[1069] ​ Shown is that iChol LNP delivers RNA to extrahepatic stellate cells. These graphs show the mRNA levels of the Col1a1 gene in the pancreas (left) and colon (right) relative to the PBS control group, respectively. Each symbol represents an individual animal. Data are presented as mean ± SD.

[1070] ​ Shown is that iChol LNP delivers RNA to extrahepatic extracellular matrix-producing cells. These graphs show the mRNA levels of the Col1a1 gene in the spleen, kidney, lung, and pancreas relative to the PBS control group, respectively. Each symbol represents an individual animal. Data are presented as mean ± SD.

[1071] Example 17: Intramuscular Administration and Delivery to Myocytes and Endothelial Cells

[1072] Lipid nanoparticles (LNPs) have proven to be highly versatile delivery vehicles, and their utility extends far beyond the well-known area of vaccine development. When considering intramuscular administration, LNPs offer several potential advantages in various therapeutic areas by localizing the therapeutic effect in the affected muscle group, reducing the likelihood of systemic side effects, and enhancing treatment specificity.

[1073] To evaluate whether the LNP effect is local, we administered iChol LNP intramuscularly into the quadriceps muscle and quantified gene silencing in the quadriceps muscle (injection side) and liver, as hepatocyte LNPs have been reported to deliver significant amounts of RNA to the liver upon intramuscular administration. We also evaluated gene silencing in skeletal muscle cells of the quadriceps muscle (injection side). To obtain cell type-specific information, we employed different siRNAs targeting genes characterized by cell type-specific expression patterns. We prepared LNPs encapsulating the following siRNAs (Table 17) and administered these LNPs intramuscularly to mice at a dose of 0.2 mg / kg siMstn. Subsequently, at 48 - 72 hours after administration, the quadriceps muscle and liver were harvested, and total RNA was extracted using the Quick-RNA MagBead Kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression levels of Cdh5 and Mstn were quantified by real-time PCR.

[1074] The results are shown in ​ and ​ As shown, DC-cholesterol LNP showed more efficient gene silencing in myoendothelial cells, while it did not show off-target delivery to liver endothelial cells. As for myocytes, hepatocyte LNP and DC-Chol LNP showed comparable gene silencing. When applied to local tissue regeneration therapy, this low off-target delivery property in local administration may be potentially beneficial.

[1075] Endothelial cells: siCdh5

[1076] 5’-ccAAAAGAGAGAcuGGAuudTsdT-3’

[1077] 5’-AAUCcAGUCUCUCUUUUGGdTsdT-3’

[1078] Myocytes: siMstn

[1079] 5’-AmUGGmCAAAGAAmCAAAmUAAmUdTsdT-3’

[1080] 5’-AUmUAUUUGUUCUUUGCmCAUdTsdT-3’

[1081] [Table 17]

[1082] Table 17

[1083]

[1084]

[1085] ​ Show that ionizable cholesterol LNP delivered intramuscularly delivers RNA to endothelial cells in skeletal muscle around the injection site, with minimized off-target delivery to liver endothelial cells.

[1086] (Left panel) Liver endothelial cells (Right panel) Endothelial cells in the muscle at the injection site. This figure shows the mRNA levels of Cdh5 in the liver (left panel) and quadriceps muscle (right panel) relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD. Statistical significance differences were evaluated using one-way ANOVA and Dunnett's post-test. A corrected P-value less than 0.05 was considered statistically significant, *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001.

[1087] ​ The figure shows that intramuscularly administered LNP delivers RNA to skeletal muscle cells around the injection site. The figure shows the Mstn mRNA levels in the quadriceps muscle relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA and Dunnett's post-test. A corrected P-value of less than 0.05 was considered statistically significant, *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

[1088] Example 18: Delivery to Cancer Cells in Vitro

[1089] To test the applicability to cancer cells, ionizable cholesterol LNP was tested in cancer model mice. We used the B16F10 murine melanoma lung metastasis model, in which cancer cells were administered intravenously.

[1090] We prepared LNP (Table 18) encapsulating siRNA against the CD47 gene encoding the CD47 protein. CD47 is a ubiquitously expressed membrane receptor, and the interaction between CD47 and signal regulatory protein α (SIRPα) expressed on macrophages transduces an inhibitory signal that suppresses macrophage phagocytic activity. It has been reported that the CD47 expression level is elevated in various cancer cells, leading to evasion of immune surveillance by the innate immune system and tumor progression.

[1091] The in vitro transfection efficiency was evaluated in serum-free medium. LNP formulations encapsulating siCD47 were prepared as described in Materials and Methods and further tested in the B16F10 murine melanoma cell line. B16F10 cells were cultured using standard culture conditions and transferred to 96-well plates at a density of 10,000 - 15,000 cells / well. B16F10 cells were transfected with siCD47 at a concentration of 30 nM per well. After incubation for 2 hours, the cell culture medium containing LNP was removed, the cells were washed with serum-free medium, and then cultured in serum-containing medium. After overnight incubation, total RNA was extracted using the QuickExtract RNA Extraction Kit (LGCBiosearch technologies), and cDNA was synthesized by reverse transcription. The Cd47 expression level was quantified by real-time PCR.

[1092] The results are as ​ shown. Ionizable cholesterol LNP exhibited more efficient gene silencing than hepatocyte LNP.

[1093] siCd47

[1094] 5’-mCmCGAAGAAAmUGmUmUmUGmUGAAdTsdT-3’

[1095] 5’-UUmCAmCAAAmCAUUUCUUCGGdTsdT-3’

[1096] [Table 18]

[1097] < / sirna>

Claims

1. A method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, which comprises administering a lipid composition to an individual, wherein the lipid composition comprises the therapeutic agent and lipid nanoparticles, and wherein the lipid nanoparticles comprise an ionizable lipid and a compound represented by formula (1) or a salt thereof, [Chemical formula 1] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms X represents a basic functional group.

2. The method according to claim 1, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered cycloalkyl group, or a 5- or 6-membered heteroaryl group.

3. The method according to claim 1 or 2, wherein the compound represented by formula (1) is a compound represented by formula (2) [Chemical formula 2] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms R 2 , R 3 and R 4 Each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and R 2 , R 3 and R 4 One of the may not exist.

4. The method according to claim 3, wherein the compound represented by formula (2) is a compound represented by formula (3) [Chemical formula 3] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L1 represents a single bond or an alkylene group having 1-6 carbon atoms, R1 represents a hydrogen atom, a hydrocarbon group having 1-4 carbon atoms, or an aminoalkyl group having 1-4 carbon atoms, G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L2 represents an alkylene group having 1-6 carbon atoms which may have an amino group, R 2 , R 3 and R 4 Each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and R 2 , R 3 and R 4 may not exist.

5. The method according to claim 4, wherein the compound represented by formula (3) is a compound represented by formula (3A): [Chemical formula 4] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L1 represents a single bond or an alkylene group having 1-6 carbon atoms, R1 represents a hydrogen atom, a hydrocarbon group having 1-4 carbon atoms, or an aminoalkyl group having 1-4 carbon atoms, G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L2 represents an alkylene group having 1-6 carbon atoms which may have an amino group, and R 2 and R 3 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2.

6. The method according to any one of claims 1 to 5, wherein G1 represents -C(O)- or -C(O)O-.

7. The method according to claim 5, wherein R1 represents a hydrogen atom or an aminoalkyl group having 1-4 carbon atoms.

8. The method according to claim 5, wherein R1 represents a hydrogen atom.

9. The method according to claim 5, wherein G2 represents a single bond or -C(O).

10. The method according to claim 5, wherein G2 represents a single bond.

11. The method according to claim 5, wherein L2 represents an alkylene group having 1 to 3 carbon atoms, and R 2 and R 3 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms which may be substituted by a hydroxyl group.

12. The method according to claim 1 or 2, wherein the compound represented by formula (1) or a salt thereof is any one of the following: [Chemical formula 5] [Chemical formula 6] [Chemical formula 7] [Chemical formula 8] [Chemical formula 9] [Chemical formula 10] [Chemical formula 11] [Chemical formula 12] 13. The method according to any one of claims 1 to 12, wherein the content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipid.

14. The method according to any one of claims 1 to 13, wherein the therapeutic agent is a nucleic acid.

15. The method according to any one of claims 1 to 14, wherein the therapeutic agent is DNA or RNA.

16. The method according to any one of claims 1 to 15, wherein the therapeutic agent is mRNA or siRNA.

17. The method according to any one of claims 1 to 16, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and wherein the biodegradable group is represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

18. The method according to any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by formula (4): [Chemical formula 13] wherein X represents NR 1 - or -O-, R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 - where R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 14] R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32 -represented group, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 Represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 15] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 each of which independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 and R 7 and R 8 Any one or more of the groups may be linked together to form a 4- to 7-membered ring which may contain an O atom, The substituents on the optionally substituted alkyl group having 1 to 18 carbon atoms represent hydroxyl, carboxyl, NR 45 R 46 An amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 OR 44 A group represented by 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group represent alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, -NR 45 R 46 An amino group represented by -O(CO)OR 41 、-O(CO)-R 42 、-(CO)OR 43 OR 44 represents a group, and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer from 0 to 3, where a + b is 1 or more, and c + d is 1 or more.

19. The method according to any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by formula (1): [Chemical formula 16] In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein the hydrocarbon groups represented by R 1 , R 2 and R 3 may be substituted by one or more substituents selected from -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 and -O-R 56 . R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 Each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , excluding R 5 and R 6 In the case of hydrocarbon groups having 1 to 8 carbon atoms, R 7 Indicates -R 10 -L 2 -R 11 -L 3 -R 12 , R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 、R 54 、R 55 and R 56 each independently represents a hydrocarbyl group having 1 to 24 carbon atoms, by R 53 、R 54 、R 55 and R 56 The hydrocarbon groups represented by can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 substituted The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 、-OR 56 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 57 replace, R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, and R 57 Indicates -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 OR 66 , R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 , R 64 , R 65 and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, by R 63 、R 64 、R 65 and R 66 The hydrocarbon groups represented by can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 68 substituted, The above aryl group having 6 to 20 carbon atoms may be substituted by -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 , -O-R 66 or -(hydrocarbyl group having 1 to 12 carbon atoms)-R 67 substituted R 68 represents a hydrocarbyl group having 1 to 12 carbon atoms, and L 1 , L 2 and L 3 Each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-, R 8 represents a hydrocarbyl group having 1 to 12 carbon atoms, R 9 represents a hydrocarbyl group having 1 to 24 carbon atoms, R 10 represents a hydrocarbon group having 1 to 8 carbon atoms, R 11 represents a hydrocarbyl group having 1 to 24 carbon atoms, R 12 represents a hydrocarbyl group having 1 to 24 carbon atoms, represented by R 9 and R 12 represents a hydrocarbon group which may be substituted by an aryl group, -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 or -S-R 58 , where R 53 , R 54 , R 55 and R 58 are as defined above, and The hydrocarbon group represented by R 11 may be substituted by -OC(O)O-R 53 , -C(O)O-R 54 or -OC(O)-R 55 , wherein R 53 , R 54 and R 55 are as defined above.

20. The method according to any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by the following formula (5): [Chemical formula 17] Where R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A represents a hydroxyl group, or a group represented by -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 wherein G 20 represents -O(CO)- or -(CO)O- R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have substituent B The substituent B is -N(R 61 )(R 62 ), R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbyl group having 1 to 18 carbon atoms, L 10 represents a hydrocarbyl group having 1 to 18 carbon atoms, G 30 Represents -S-(CO)-NR 64 , R 64 Indicated by -L 30 -G 20 -CH(R 55 )(R 56 ), a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms G 10 represents -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- R 63 represents a hydrocarbyl group having 1 to 18 carbon atoms, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R 53 、R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C represents a group represented by -(CO)OR 65 or -O(CO)-R 65 wherein the group is represented by R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by -L 40 -CH(R 66 )(R 67 ) L 40 represents a hydrocarbon group having 1 to 6 carbon atoms R 66 and R 67 represent a hydrocarbyl or alkoxy group having 1 to 10 carbon atoms.

21. The method according to any one of claims 1 to 20, wherein the lipid nanoparticle further comprises a lipid having a nonionic hydrophilic polymer.

22. The method according to any one of claims 1 to 21, wherein the lipid nanoparticle further comprises a phospholipid.

23. The method according to any one of claims 1 to 22, wherein the lipid composition is administered to an individual by intravenous or intramuscular injection.

24. The method according to any one of claims 1 to 23, wherein the mesenchymal cell is a myocyte.

25. The method according to any one of claims 1 to 23, wherein the mesenchymal cell is an extracellular matrix-producing cell.

26. The method according to any one of claims 1 to 23, wherein the extracellular matrix cell is a stellate cell or a fibroblast.

27. The method according to any one of claims 1 to 23, wherein the stellate cell is a hepatic stellate cell, a pancreatic stellate cell, or a colonic stellate cell.

28. A lipid composition comprising a therapeutic agent and a lipid nanoparticle, wherein the lipid nanoparticle comprises a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group, [Chemical formula 18] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms X represents a basic functional group.

29. The lipid composition according to claim 28, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5-6 membered heterocycloalkyl group, or a 5-6 membered heteroaryl group.

30. The lipid composition according to claim 28 or 29, wherein the compound represented by formula (1) is the compound represented by formula (2) [Chemical formula 19] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O- L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, and a substituted heteroalkylene group having 1 to 14 carbon atoms R 2 , R 3 and R 4 Each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and R 2 , R 3 and R 4 One of the may not exist.

31. The lipid composition according to claim 30, wherein the compound represented by formula (2) is the compound represented by formula (3) [Chemical formula 20] wherein G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O- L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms G2 represents a single bond, -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O- L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group R 2 、R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted by a hydroxyl group, or -C(NH2)=NH2, and R 2 , R 3 and R 4 One of the may not exist.

32. The lipid composition according to any one of claims 28 to 31, wherein the ionizable lipid having a biodegradable group is the compound represented by formula (4) [Chemical formula 21] wherein X represents NR 1 - or -O-, R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, where R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 22] R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 Each independently represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a 31 -L 2 -R 32 -represented group, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 Represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [Chemical formula 23] R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R 4 and R 5 、R 10 and R 5 、R 5 and R 12 、R 4 and R 6 、R 5 and R 6 、R 6 and R 7 、R 6 and R 10 、R 12 and R 7 and R 7 and R 8 Any one or more of the groups can be joined together to form a 4- to 7-membered ring that can contain an O atom. The substituents on the optionally substituted alkyl group having 1 to 18 carbon atoms represent hydroxyl, carboxyl, amino represented by NR 45 R 46 , substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 , where R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl and the substituted or unsubstituted heteroaryl represent an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 or a group represented by -O(CO)O-R 41 -O(CO)-R 42 -(CO)O-R 43 or -O-R 44 and R 41 R 42 R 43 R 44 R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms, a, b, c and d each independently represent an integer from 0 to 3, wherein a + b is 1 or more, and c + d is 1 or more.

33. The lipid composition according to any one of claims 28 to 31, wherein the ionizable lipid is the compound represented by formula (1): [Chemical formula 24] In the said formula, R 1 and R 2 Each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, wherein R 1 , R 2 and R 3 The hydrocarbon group represented by the present invention may be one or more selected from -OH, COOH, -NR 51 R 52 、-OC(O)OR 53 、-C(O)OR 54 、-OC(O)-R 55 AND-OR 56 Substituents substituted, R 4 represents a hydrocarbyl group having 1 to 8 carbon atoms, R 5 and R 6 each independently represents a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , excluding the case where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 represents -R 10 -L 2 -R 11 -L 3 -R 12 , R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 , R 54 , R 55 and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, by R 53 、R 54 、R 55 and R 56 The hydrocarbon groups represented by can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 58 substituted The above aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR 51 R 52 , -OC(O)O-R 53 , -C(O)O-R 54 , -OC(O)-R 55 , -O-R 56 or -(hydrocarbyl group having 1 to 12 carbon atoms)-R 57 substituted. R 58 represents a hydrocarbyl group having 1 to 12 carbon atoms, and R 57 represents -OH, COOH, -NR 61 R 62 , -OC(O)O-R 63 , -C(O)O-R 64 , -OC(O)-R 65 or -O-R 66 , R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 、R 64 、R 65 and R 66 each independently represents a hydrocarbyl group having 1 to 24 carbon atoms, by R 63 、R 64 、R 65 and R 66 The hydrocarbon groups represented by can be substituted by an aryl group having 6 to 20 carbon atoms or -S-R 68 substituted, The above aromatic groups having 6 to 20 carbon atoms may be replaced by -OH, COOH, -NR 61 R 62 、-OC(O)OR 63 、-C(O)OR 64 、-OC(O)-R 65 、-OR 66 or -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 replace, R 68 represents a hydrocarbyl group having 1 to 12 carbon atoms, and L 1 、L 2 and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)- or -O- R 8 represents a hydrocarbyl group having 1 to 12 carbon atoms, R 9 represents a hydrocarbyl group having 1 to 24 carbon atoms, R 10 represents a hydrocarbyl group having 1 to 8 carbon atoms, R 11 represents a hydrocarbon group having 1 to 24 carbon atoms, R 12 represents a hydrocarbyl group having 1 to 24 carbon atoms, By R 9 and R 12 The hydrocarbon group represented by 53 、-C(O)OR 54 、-OC(O)-R 55 or -SR 58 Substituted, where R 53 , R 54 , R 55 and R 58 is defined as above, and The hydrocarbon group represented by R 11 may be substituted by -OC(O)O-R 53 , -C(O)O-R 54 or -OC(O)-R 55 , where R 53 , R 54 and R 55 are as defined above.

34. The lipid composition according to any one of claims 28 to 31, wherein the ionizable lipid having a biodegradable group is the compound represented by formula (5): [Chemical formula 25] wherein R 51 and R 52 each independently represents a hydrocarbyl group having 1 to 21 carbon atoms which may have substituent A The substituent A represents a hydroxyl group, or a group represented by -G 20 -CH(R 55 )(R 56 )、-N(R 58 )(R 59 ), or -G 20 -R 60 wherein G 20 represents -O(CO)- or -(CO)O- R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 Each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, The substituent B is -N(R 61 )(R 62 ), R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbyl group having 1 to 18 carbon atoms, L 10 represents a hydrocarbyl group having 1 to 18 carbon atoms, G 30 represents -S-(CO)-NR 64 , R 64 represents a group represented by -L 30 -G 20 -CH(R 55 )(R 56 ). a represents 0 or 1 L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms G 10 represents -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20 represents a hydrocarbyl group having 1 to 6 carbon atoms b represents 0 or 1 R 53 、R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C. The substituent C represents -(CO)OR 65 or -O(CO)-R 65 The group represented by R 65 represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by -L 40 -CH(R 66 )(R 67 ) L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represent a hydrocarbyl or alkoxy group having 1 to 10 carbon atoms.

35. The lipid composition according to any one of claims 28 to 34, wherein the content of the compound represented by formula (1) or its salt is 5 to 80 mol% based on the total lipids.

36. The lipid composition according to any one of claims 28 to 35, wherein the therapeutic agent is a nucleic acid.

37. The lipid composition according to any one of claims 28 to 36, wherein the therapeutic agent is DNA or RNA.

38. The lipid composition according to any one of claims 28 to 37, wherein the therapeutic agent is mRNA or siRNA.

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