Ionizable cationic lipid compound for nucleic acid delivery and composition, and use thereof

Abstract

The present invention provides an ionizable cationic lipid compound having a structure as represented by formula (I). The compound can be used for preparing lipid nanoparticles (LNPs) for delivery of a therapeutic agent and / or a preventive agent. The LNPs prepared using the ionizable cationic lipid compound of the present invention have high stability and transfection efficiency, and can efficiently and stably deliver biologically active substances (including a nucleic acid, such as mRNA) to target cells or organs, thereby inducing a highly specific antibody response in vivo.

Description

Ionizable cationic lipid compound and composition for nucleic acid delivery and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an ionizable cationic lipid compound and composition for nucleic acid delivery and applications thereof. Background Art

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic characteristics and pharmacological activity. They can be used to treat tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. After nucleic acid drugs are injected into the human body, an efficient and safe drug delivery system is required to deliver them to the lesion site. The drug delivery system needs to stay for a sufficient time to accurately target the lesion site while avoiding damage to normal cells.

[0003] Current delivery systems can be divided into viral vectors and non-viral vectors. Viral vectors are less used in nucleic acid drugs due to their immunogenicity, tumorigenicity, and limited drug loading capacity; non-viral vectors, such as polymers and lipids (liposomes or LNPs), can bind nucleic acid drugs to specific ligands to enable them to target specific cells, and are widely used in current nucleic acid drugs. LNP is one of the most widely used delivery systems for nucleic acid drugs. The LNP delivery system can safely and effectively deliver nucleic acids. It has advantages such as high nucleic acid encapsulation rate, ability to effectively transfect cells, strong tissue penetration, low cytotoxicity and immunogenicity, which are conducive to drug delivery. Compared with other delivery systems, it has huge advantages. Therefore, the lipid nanoparticle delivery system has broad development and application prospects.

[0004] In the prior art, LNP delivery systems are often composed of four components: ionizable lipids (cationic lipids), steroids, neutral lipids and PEG-lipids. For example, patent document AU2020325221A1 discloses a composition for delivering LNPs to target cells, including (i) ionizable lipids; (ii) sterols or other structural lipids; (iii) non-cationic auxiliary lipids or phospholipids; (iv) PEG lipids and (v) agents encapsulated in and / or associated with LNPs (such as nucleic acid molecules). These four components are used in a specific ratio to achieve the encapsulation and delivery of nucleic acid drugs. Traditional ionizable lipids (or cationic lipids) are basically composed of branched or linear fatty chains. Cationized cholesterol compounds, such as DC-Chol, can also be used to deliver nucleic acids. Compared with linear fatty chains, this type of structure has less structural variability. Patent US7514099B2 and CN112424214A Two types of ionizable cholesterol-modified amino lipid compounds were disclosed. Studies have shown that although this asymmetric cholesterol-amino lipid compound can improve the orderliness of lipid nanoparticles, this type of compound still requires the use of more than four or even five lipid components to construct a nanodelivery carrier, and its construction method is still relatively complicated.

[0005] The present invention introduces a sterol structure into a cationic lipid compound through a piperazine or piperazine dione structure to form a new type of steroid-cationic lipid compound. The three-component lipid nanoparticles constructed with the steroid-cationic lipid compound, auxiliary lipids and PEG lipids can achieve efficient encapsulation and delivery of nucleic acids without the participation of additional cholesterol. Its construction is simpler than that of traditional four-component lipid nanoparticles. Since this type of compound has better endosomal escape ability, its delivery efficiency of nucleic acids is higher. Summary of the Invention

[0006] In one aspect, the present invention provides a lipid compound represented by formula (I),

[0007] or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein,

[0008] L1 and L2 are each independently selected from a bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 Alkynylidene, optionally substituted C1-C 20 acyl, optionally substituted carbocyclylene, optionally substituted arylene;

[0009] G1 and G2 are each independently selected from a bond, -O-, -S-, -N(R a )-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-、-N(R a )C(=O)-, -S(=O)-, -S(=O)2-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-、-SP(=O)(OR a )O-、-OP(=S)(OR a )O-、-OP(=O)(SR a )O-、-P(=O)(OR a )(OR a )-、-P(=S)(ORa )(OR a )-、-P(=O)(SR a )(OR a )-、-N(R a )C(=O)O-、-OC(=O)N(R a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-;

[0010] R1 and R2 are each independently selected from a steroid group, an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl; wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 One or more -CH2- in the alkynyl group may be optionally replaced by -O-, -S-, -NR a -, carbocyclyl, aryl, heteroaryl, and / or heterocyclyl replacement;

[0011] R3 is selected from C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, carbocyclic, aryl, aralkyl, halogen, C1-C 20 Alkoxy, C1-C 20 Alkylthio, NR4R4', R4-C(O)-, R4-S(O)-, R4-S(O)2-, R4-C(O)O-, R4-OC(O)-, R4-NHC(O)-, R4-C(O)NH-, oxo;

[0012] R4 and R4' are each independently selected from H, Cl-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and / or heterocyclylalkyl;

[0013] R a and R b are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20Alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl; wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 One or more -CH2- in the alkynyl group may be optionally replaced by -O-, -S-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NH-, -NHC(=O)-, -S(=O)-, -S(=O)2-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, or -C(=S)S-;

[0014] m, n, p and q are each independently selected from 1, 2 or 3;

[0015] r is selected from 0, 1, 2, 3 or 4;

[0016] Provided that at least one of R1 and R2 is selected from a steroid group.

[0017] On the other hand, the present invention also provides a lipid nanoparticle comprising a lipid compound represented by formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

[0018] In another aspect, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

[0019] In another aspect, the present invention also provides a method of delivering a therapeutic and / or prophylactic agent, comprising administering the pharmaceutical composition described herein to a subject in need thereof.

[0020] On the other hand, the present invention also provides the use of the lipid compound represented by formula (I) described herein or its stereoisomers, tautomers, and pharmaceutically acceptable salts in the preparation of a therapeutic and / or preventive agent delivery system. Beneficial effects:

[0021] The three-component lipid nanoparticles prepared using the lipid compounds of the present invention, or their stereoisomers, tautomers, or pharmaceutically acceptable salts, have a simple process and exhibit excellent stability and transfection efficiency. The lipid nanoparticles can be used to deliver nucleic acids (e.g., mRNA) efficiently and stably to target cells or organs, eliciting high specific antibody and cellular immune responses in experimental animals with good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 shows the expression level of firefly luciferase in Hep3B cells transfected with Luc-mRNA-LNP complexes.

[0023] Figure 2 shows the serum antibody titer of mice after immunization with SARS-CoV-2 mRNA-LNP. DETAILED DESCRIPTION

[0024] definition

[0025] As used in this specification, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0026] As used herein, the term "lipid nanoparticle," or "LNP," refers to a particle having a nanometer size, eg, 1 nm to 1,000 nm, which comprises one or more types of lipid molecules.

[0027] As used herein, the term "gene medicine" generally consists of a vector or delivery system containing an engineered gene construct, the active ingredient of which may be DNA, RNA, genetically modified viruses, bacteria or cells. By introducing exogenous genes into target cells or tissues, it replaces, compensates, blocks or corrects specific genes to achieve the purpose of treating and preventing diseases.

[0028] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA, and hybrids thereof.

[0029] As used herein, the term "lipid compound" refers to a group of organic compounds, which include but are not limited to esters of fatty acids and are generally characterized by being poorly soluble in water but soluble in many organic solvents. The organic solvents of the present invention include but are not limited to: benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, ether, ethyl acetate, acetone, carbon tetrachloride.

[0030] As used herein, the term "alkyl" refers to a monovalent group having a straight or branched saturated hydrocarbon chain of 1 to 20 carbon atoms, more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, and the like.

[0031] As used herein, the term "alkylene" refers to a divalent group having a straight or branched saturated hydrocarbon chain of 1 to 20 carbon atoms, more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. The term is exemplified by groups such as methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like.

[0032] As used herein, the term "alkenyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond can be present at any stable point along the chain. The term is exemplified by groups such as vinyl (i.e., -CH=CH2), propen-1-yl (i.e., -CH=CHCH3), propen-3-yl (or allyl, i.e., -CH2CH=CH2), propen-2-yl (i.e., -C(CH3)=CH2), butadienyl (including 1,2-butadienyl and 1,3-butadienyl), and the like.

[0033] As used herein, the term "alkenylene" refers to a divalent group of a straight or branched unsaturated hydrocarbon chain having 2 to 20 carbon atoms (more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms) and having carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond can be present at any stable point along the chain. The term is exemplified by groups such as ethenylene, propenylene, butenylene, pentenylene, hexenylene, and the like.

[0034] As used herein, the term "alkynyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds). The term is exemplified by groups such as ethynyl (i.e., -C≡CH), propargyl (i.e., -CH2C≡CH), propynyl (i.e., -C≡CCH3), and the like.

[0035] As used herein, the term "alkynylene" refers to a divalent group of a straight or branched unsaturated hydrocarbon chain having 2 to 20 carbon atoms (more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms) and having carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds). The unsaturated carbon-carbon triple bond can exist at any stable point along the chain. The term is exemplified by groups such as ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.

[0036] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0037] As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. This term is exemplified by groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0038] As used herein, the term "acyl" refers to "alkyl-C(=O)-", "alkenyl-C(=O)-", "alkynyl-C(=O)-", "aryl-C(=O)-", "heteroaryl-C(=O)-", "carbocyclyl-C(=O)-", "heterocyclyl-C(=O)-" groups, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, and heterocyclyl are as defined herein. This term is exemplified by groups such as formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, n-hexanoyl, acryloyloxy, benzoyl, cyclopropylacyl, and the like.

[0039] As used herein, the term "acyloxy" refers to "alkyl-C(=O)O-", "alkenyl-C(=O)O-", "alkynyl-C(=O)O-", "aryl-C(=O)O-", "heteroaryl-C(=O)O-", "carbocyclyl-C(=O)O-", "heterocyclyl-C(=O)-" groups, wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, and heterocyclyl are as defined herein. This term is exemplified by groups such as formyloxy, acetyloxy, propionyloxy, n-butyryloxy, isobutyryloxy, n-pentanoyloxy, n-hexanoyloxy, and the like.

[0040] As used herein, the term "aryl" refers to an aromatic carbocyclic group of 6 to 14 carbon atoms (more typically 6 to 10 carbon atoms, or 6 carbon atoms) having a single ring (e.g., phenyl) or multiple rings (e.g., biphenyl) or multiple condensed (fused) rings (e.g., naphthyl, fluorenyl, and anthracenyl). The term is exemplified by groups such as phenyl, fluorenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthalene (if the point of attachment is through the aryl group), and the like.

[0041] As used herein, term " carbocyclic radical " refers to have 3 to 14 carbon atoms (more typically have 3 to 8 carbon atoms, or 3 to 6 carbon atoms) as the monocycle of annular atoms or the monoradical saturation or part unsaturated group of a plurality of thick (condensed) rings or bridged rings or spirocycles.Carbocyclic ring or carbocyclic radical can be saturated or partly unsaturated, and can be condensed with another saturated, partly unsaturated or aromatic ring, and condition is that the annular atoms being connected with target molecule is not aromatic carbon.The example of carbocyclic ring or carbocyclic radical includes, but is not limited to cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene etc.

[0042] As used herein, the term "heteroaryl" refers to an aromatic ring group comprising a monocyclic or polycyclic fused ring (e.g., comprising 2 or 3 rings) having 5 to 14 ring atoms (more typically 5 to 10 ring atoms, or 5 to 6 ring atoms) in the ring, wherein in addition to carbon atoms, the ring atoms further comprise at least one heteroatom selected from oxygen, nitrogen and / or sulfur. If the ring is aromatic, the sulfur and nitrogen atoms may also exist in oxidized form. The polycyclic fused heteroaryl is a monocyclic heteroaryl as defined above fused with one or more rings selected from the following to form a polycyclic fused ring system: heteroaryl (to form, for example, naphthyridinyl, such as 1,8-naphthyridinyl), heterocycle (to form, for example, 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycle (to form, for example, 5,6,7,8-tetrahydroquinolinyl) and aryl (to form, for example, indazolyl). It will be understood that the point of attachment of the heteroaryl group can be at any suitable atom of the heteroaryl group, including carbon atoms and heteroatoms (eg, nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopenta[1,2-c]pyrazolyl.

[0043] As used herein, the term "heterocyclyl" refers to a monocyclic or multiple condensed (fused) ring or bridged ring or spirocyclic ring having 3 to 14 ring atoms (more typically 3 to 10 ring atoms, or 3 to 6 ring atoms) in the ring, wherein the ring atoms also include at least one or more nitrogen atoms in addition to carbon atoms. The example of a heterocyclyl group includes, but is not limited to, an aziridine ring, an azetidine ring, a tetrahydropyrrole ring, a piperidine ring, an azepane ring, an azooctane ring, a tetrahydroimidazole ring, a tetrahydropyrazole ring, a tetrahydrooxazole ring, a tetrahydroisoxazole ring, a tetrahydrothiazole ring, a tetrahydroisothiazole ring, a piperazine ring, a morpholine ring, a dihydropyridyl, 4,5,6,7-tetrahydro-1H-benzo [d] imidazole, 4,5,6,7-tetrahydro-1H-imidazo [4,5-c] pyridine, etc. The nitrogen heterocyclic group in the present invention is a heterocyclic group containing a nitrogen atom in its structure, including but not limited to substituted or unsubstituted: aziridinyl, azetidinyl, β-propiolactam, pyrrolyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, caprolactam, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, piperazinyl, indolyl, benzimidazolyl, carbazolyl, quinolyl, isoquinolyl, pteridinyl, acridinyl, 7H-purinyl, phenazinyl, phenothiazinyl or 1H-azepinyl.

[0044] As used herein, the term "optionally substituted" means unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halogen, hydroxy, cyano, nitro, amino, C3-C6 cycloalkyl, and oxo.

[0045] As used herein, the term "steroid" is an organic compound having a four-ring carbon skeleton structure as shown below.

[0046] Steroids include naturally occurring or synthetic steroids and their analogs. Steroids or their analogs include sterols or their analogs derived from plants and / or animals. Examples of steroids described herein include, but are not limited to, avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucoxosterol, hexahydroluminosterol, hydroxycholesterol, luminosterol, algaesterol, sitostanol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, choletenone, 3p-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Ch ol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cyclohexyl alcohol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumiesterol, cetocalciferol, calcipotriol, coprostol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroautocalciferol, tomatine, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, etc.

[0047] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to effect treatment, as defined below, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending on the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art.

[0048] As used herein, the term "stereoisomer" refers to a compound that has the same chemical composition and connectivity, but whose atoms have different orientations in space that cannot be interchanged by rotation about a single bond. "Stereoisomer" includes "diastereomers" and "enantiomers." "Diastereomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics, and reactivity. Diastereomeric mixtures can be separated under high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography. "Enantiomers" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other.

[0049] As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ only in the position and electron distribution of one (or more) mobile atoms, such as keto-enol tautomers.

[0050] As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a given compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be acid addition salts and / or base addition salts. Acid addition salts can be prepared from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, carbonate, bisulfate, hydrogenphosphate, dihydrogenphosphate, bicarbonate, etc.; salts derived from organic acids include formate, acetate, propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, salicylate, lactate, nicotinate, lauryl sulfate, naphthalenesulfonate, camphorsulfonate, gluconate, glucuronate, oleate, palmitate, stearate, pamoate, trifluoroacetate, etc. Base addition salts can be formed with inorganic or organic bases. Salts derived from inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, lithium, barium, aluminum salts and the like; salts derived from organic bases include salts formed with various primary, secondary and tertiary amines, for example, ethylamine, diethylamine, n-propylamine, isopropylamine, diethanolamine, meglumine, lysine, piperazine, piperidine, morpholine, tromethamine, choline and the like.

[0051] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0052] As used herein, the term "delivery system" refers to a formulation or composition that regulates the spatial, temporal, and dosage distribution of a biologically active ingredient in an organism.

[0053] Compound

[0054] In some embodiments, the present invention provides a lipid compound represented by formula (I)

[0055] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1, L2, G1, G2, R1, R2, R3, m, n, p, q, and r are as defined herein.

[0056] In some embodiments, the present invention provides a lipid compound represented by formula (II)

[0057] or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein

[0058] L1, L2, G1, G2, R1, R2, R3, p, q and r are as defined herein.

[0059] In some embodiments, the present invention provides a lipid compound represented by formula (III)

[0060] or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein

[0061] L1, L2, G1, G2, R1, R2, R3, p, q and r are as defined herein.

[0062] In some embodiments, the steroid compound in the steroid compound group of the present invention is selected from naturally occurring steroid compounds or their analogs; preferably, it includes plant sterols and animal sterols, or their analogs; more preferably, it is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucoxosterol, hexahydroluminosterol, hydroxycholesterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, demosterol, cholestanol, cholestanone, choletenone, 3p-[ N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cyclohexyl alcohol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumilastol, cetocalciferol, calcipotriol, coprostol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroautocalciferol, tomatine, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, etc.

[0063] In some embodiments, in the lipid compound provided by the present invention or its stereoisomers, tautomers, and pharmaceutically acceptable salts, the steroid compound in the steroid compound group is selected from cholesterol and cholesterol derivatives.

[0064] In some embodiments, in the lipid compounds provided herein or their stereoisomers, tautomers, and pharmaceutically acceptable salts, the steroidal compound group has the following structure:

[0065] R5 is selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxycarbonyl C1-C 20 alkyl-;

[0066] R6 is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl;

[0067] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0068] In some embodiments, the steroid group is selected from:

[0069] Where R' is C 1-20 alkyl.

[0070] In some embodiments, the present invention provides a lipid compound represented by formula (IV-a), (IV-b), (IV-c), or (IV-d):

[0071] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1, L2, G1, G2, R1, R3, p, q and r are as defined herein.

[0072] In some embodiments, the present invention provides a lipid compound represented by Formula (V-1), (V-2), (V-3), (V-4) or Formula (VI-5):

[0073] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1, L2, G1, G2, R1, R3, p, q and r are as defined herein.

[0074] In some specific embodiments, R1 and / or R2 are selected from optionally substituted C1-C 20 Alkyl; wherein the C1-C 20 One or more -CH2- in the alkyl group may be optionally replaced by O, S, -NR a -, carbocyclic group substituted; or R1 and / or R2 are selected from

[0075] R5 is selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxycarbonyl C1-C 20 alkyl-;

[0076] R6 is selected from hydrogen, halogen, hydroxy, C1-C 20 alkyl.

[0077] In some specific embodiments, R1 and / or R2 are selected from

[0078] In some specific embodiments, R1 and / or R2 are selected from the following structures:

[0079] In some embodiments, the present invention provides a compound selected from the group consisting of:

[0080] or its stereoisomers, tautomers, and pharmaceutically acceptable salts.

[0081] Lipid nanoparticles (LNPs)

[0082] In some embodiments, the present invention provides a lipid nanoparticle as a delivery vehicle for therapeutic and / or prophylactic agents (e.g., nucleic acids including DNA, RNA, etc.), comprising a lipid compound as described herein or its stereoisomers, tautomers, and pharmaceutically acceptable salts thereof. In some embodiments, the lipid nanoparticles described herein further comprise one or more phospholipids. In some embodiments, the lipid nanoparticles described herein further comprise one or more PEG lipids. In some embodiments, the lipid nanoparticles described herein further comprise a combination of phospholipids and PEG lipids. The lipid nanoparticles described herein can deliver therapeutic and / or prophylactic agents to target sites of interest (e.g., cells, tissues, organs, etc.). Therefore, the lipid nanoparticles described herein further comprise one or more therapeutic or prophylactic agents (e.g., nucleic acids, particularly therapeutic nucleic acids (TNA)).

[0083] In some embodiments, the lipid nanoparticles have a molar ratio of lipid compound: phospholipid: PEG lipid of 30-90:10-60:0.5-20; preferably, the molar ratio of lipid compound: phospholipid: PEG lipid is 30-80:30-80:0.5-20; more preferably, the molar ratio of lipid compound: phospholipid: PEG-lipid is 40-60:40-60:0.5-5; most preferably, the molar ratio of lipid compound: phospholipid: PEG-lipid is 49.25:49.25:1.5.

[0084] phospholipids

[0085] In some embodiments, the lipid nanoparticles described herein further comprise a phospholipid. Examples of phospholipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DP ... Phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine Ethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), rutoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DEPE) The phospholipids of claim 1 are phospholipids, phosphatidylcholine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexadecyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine or its mixture. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid with a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearyl or oleoyl.

[0086] In some embodiments, the molar percentage of phospholipids in the total lipid of the lipid nanoparticles is about 15% to about 65%, for example, about 20% to about 65%, about 25% to about 65%, about 30% to about 65%, about 35% to about 65%, about 40% to about 65%, about 45% to about 65%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%.

[0087] PEG lipids

[0088] In some embodiments, PEG lipids are incorporated into lipid nanoparticles as described herein to inhibit aggregation of particles, thereby improving the stability of lipid nanoparticles. In some embodiments, PEG lipids described herein are lipids that are covalently or non-covalently linked to one or more polyethylene glycol (PEG) chains. In some embodiments, PEG lipids described herein are lipids that are covalently linked to one or more polyethylene glycol (PEG) chains.

[0089] In some embodiments, the molecular weight of PEG molecules suitable for use in the PEG lipids described herein is from about 500 to about 10,000, from about 1,000 to about 10,000, from about 1,000 to about 5,000, from about 1,000 to about 4,000, from about 1,000 to about 3,000, from about 1,000 to about 2,000, e.g., PEG 2000, PEG 2500, PEG 3000, etc.

[0090] Examples of PEG lipids include, but are not limited to, PEG-diacylglycerols (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEGS-DAG) (e.g., 4-O-(2',3'-di-tetradecanoyloxy) )propyl-1-O-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropylaminoformamide, sodium N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-dilauroyloxypropyl, PEG-dimyristoyloxypropyl, PEG-dipalmitoyloxypropyl, PEG-distearoyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3 -Dimyristoylglycerol-PEG (DMG-PEG), distearoyl-rac-glycerol-PEG (DSG-PEG), PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-distearoylglycerol, PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearoylglyceramide, (1-[8'-(cholest-5-ene-3β-oxy)carboxamido-3', 6′-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecyloxybenzyl-ω-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH).

[0091] In some embodiments, the molar percentage of PEG lipid in the total lipid of the lipid nanoparticle is about 0.1% to about 10%, for example, about 1.0% to about 10%, about 1.5% to about 10%, about 2.0% to about 10%, about 2.5% to about 10%, about 3.0% to about 10%, about 3.5% to about 10%, about 4.0% to about 10%, about 4.5% to about 10%, about 5.0% to about 10%, about 5.5% to about 10%, about 6.0% to about 10%, about 6.5% to about 10%, about 7.0% to about 10%, about 7.5% to about 10%, about 8.0% to about 10%, about 8.5% to about 10%, about 9. 9.0% to about 10%, about 9.5% to about 10%, about 1.0% to about 1.5%, about 1.5% to about 2.0%, about 2.0% to about 2.5%, about 2.5% to about 3.0%, about 3.0% to about 3.5%, about 3.5% to about 4.0%, about 4.0% to about 4.5%, about 4.5% to about 5.0%, about 5.0% to about 5.5%, about 5.5% to about 6.0%, about 6.0% to about 6.5%, about 6.5% to about 7.0%, about 7.0% to about 7.5%, about 7.5% to about 8.0%, about 8.0% to about 8.5%, about 8.5% to about 9.0%, about 9.0% to about 10%.

[0092] Particle size of lipid nanoparticles

[0093] In some embodiments, the lipid nanoparticles described herein have a particle size range of about 40 nm to about 150 nm, such as about 45 nm to about 150 nm, about 50 nm to about 150 nm, about 55 nm to about 150 nm, about 60 nm to about 150 nm, about 65 nm to about 150 nm, about 70 nm to about 150 nm, about 75 nm to about 150 nm, about 80 nm to about 150 nm, about 85 nm to about 150 nm, about 90 nm to about 150 nm, about 95 nm to about 150 nm, about 100 nm to about 150 nm, about 105 nm to about 150 nm, about 110 nm to about 150 nm, about 115 nm to about 150 nm, about 120 nm to about 150 nm, about 125 nm to about 150 nm, about 130 nm to about 150 nm, about 135 nm to about 150 nm, about 140 nm to about 150 nm, about 145 nm to about 150 nm. In some embodiments, the lipid nanoparticles described herein have a particle size range of about 40 nm to about 120 nm, e.g., about 45 nm to about 120 nm, about 50 nm to about 120 nm, about 55 nm to about 120 nm, about 60 nm to about 120 nm, about 65 nm to about 120 nm, about 70 nm to about 120 nm, about 75 nm to about 120 nm, about 80 nm to about 120 nm, about 85 nm to about 120 nm, about 90 nm to about 120 nm, about 95 nm to about 120 nm, about 100 nm to about 120 nm, about 105 nm to about 120 nm, 110 nm to about 120 nm, 115 nm to about 120 nm. In some embodiments, the lipid nanoparticles described herein have a particle size range of about 40 nm to about 110 nm, for example, about 45 nm to about 110 nm, about 50 nm to about 110 nm, about 55 nm to about 110 nm, about 60 nm to about 110 nm, about 65 nm to about 110 nm, about 70 nm to about 110 nm, about 75 nm to about 110 nm, about 80 nm to about 110 nm, about 85 nm to about 110 nm, about 90 nm to about 110 nm, about 95 nm to about 110 nm, about 100 nm to about 110 nm, about 105 nm to about 110 nm. In some embodiments, the lipid nanoparticles described herein have a particle size range of about 40 nm to about 100 nm, for example, about 45 nm to about 100 nm, about 50 nm to about 100 nm, about 55 nm to about 100 nm, about 60 nm to about 100 nm, about 65 nm to about 100 nm, about 70 nm to about 100 nm, about 75 nm to about 100 nm, about 80 nm to about 100 nm, about 85 nm to about 100 nm, about 90 nm to about 100 nm, about 95 nm to about 100 nm.In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 90 nm, for example, about 45 nm to about 90 nm, about 50 nm to about 90 nm, about 55 nm to about 90 nm, about 60 nm to about 90 nm, about 65 nm to about 90 nm, about 70 nm to about 90 nm, about 75 nm to about 90 nm, about 80 nm to about 90 nm, about 85 nm to about 90 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 85 nm, for example, about 45 nm to about 85 nm, about 50 nm to about 85 nm, about 55 nm to about 85 nm, about 60 nm to about 85 nm, about 65 nm to about 85 nm, about 70 nm to about 85 nm, about 75 nm to about 85 nm, about 80 nm to about 85 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40 nm to about 80 nm, for example, from about 45 nm to about 80 nm, from about 50 nm to about 80 nm, from about 55 nm to about 80 nm, from about 60 nm to about 80 nm, from about 65 nm to about 80 nm, from about 70 nm to about 80 nm, from about 75 nm to about 80 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40 nm to about 70 nm, for example, from about 45 nm to about 70 nm, from about 50 nm to about 70 nm, from about 55 nm to about 70 nm, from about 60 nm to about 70 nm, from about 65 nm to about 70 nm. In some embodiments, the particle size range of the lipid nanoparticles described herein is from about 40 nm to about 60 nm, for example, from about 45 nm to about 60 nm, from about 50 nm to about 60 nm, from about 55 nm to about 60 nm.

[0094] Lipid / nucleic acid ratio

[0095] In some embodiments, the lipid nanoparticles have a weight or molar ratio of lipid to nucleic acid of about 10:1 to about 100:1, e.g., about 10:1 to about 95:1, about 10:1 to about 90:1, about 10:1 to about 85:1, about 10:1 to about 80:1, about 10:1 to about 75:1, about 10:1 to about 70:1, about 10:1 to about 65:1, about 10:1 to about 60:1, about 10:1 to about 55:1, about 10:1 to about 50:1, about 10:1 to about 45:1, about 10:1 to about 40:1, about 10:1 to about 35:1, about 10:1 to about 30:1, about 10:1 to about 25:1, about 10:1 to about 20:1, about 10:1 to about 15:1.

[0096] In some embodiments, the lipid nanoparticles have an N / P ratio (i.e., the ratio of positively charged lipid amine groups to negatively charged nucleic acid phosphate groups) of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0097] Therapeutic / preventive agents

[0098] In some embodiments, the lipid nanoparticles of the present invention also include therapeutic and / or prophylactic agents. In some embodiments, therapeutic and / or prophylactic agents described herein include organic molecules, inorganic molecules, proteins, polypeptides, nucleic acids, vaccines, immunotherapeutics, etc. In some embodiments, therapeutic and / or prophylactic agents described herein include nucleic acids. In some embodiments, therapeutic and / or prophylactic agents described herein include DNA. In some embodiments, therapeutic and / or prophylactic agents described herein include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA), complement DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplastid DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, or viral DNA. In some embodiments, therapeutic and / or prophylactic agents described herein include RNA. In some embodiments, therapeutic and / or prophylactic agents described herein include small interfering RNA (siRNA). In some embodiments, therapeutic and / or prophylactic agents described herein include messenger RNA (mRNA). In some embodiments, the therapeutic and / or prophylactic agents described herein include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), nuclear heterogeneous RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, and ribozymes.

[0099] Pharmaceutical compositions and preparations

[0100] In some embodiments, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a therapeutic and / or prophylactic agent (e.g., nucleic acid including DNA and RNA, etc.) vaccine comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

[0101] In some embodiments, the pharmaceutically acceptable carriers described herein include diluents, buffers, stabilizers, and the like.

[0102] In some embodiments, the diluent comprises ethylene glycol, glycerol, polyethylene glycol, sucrose, trehalose, or a combination thereof, etc. In some embodiments, the diluent is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0103] In some embodiments, the buffer comprises phosphate, citrate, imidazole, histidine, Tris, HEPES, or a combination thereof, etc. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or more.

[0104] In some embodiments, the stabilizer comprises a salt, including an inorganic metal salt such as sodium chloride, potassium chloride, calcium chloride, etc. In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or more.

[0105] In some embodiments, depending on the route of drug administration, the pharmaceutical composition and / or vaccine of the present invention can be prepared into oral preparations, intramuscular injection preparations, subcutaneous injection preparations, intravenous injection preparations, nebulized inhalation preparations, nasal spray inhalation preparations or dry powder inhalation preparations, and ophthalmic administration preparations.

[0106] Indications

[0107] The lipid nanoparticles and / or pharmaceutical compositions provided by the present invention can be used to prevent and / or treat cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic diseases, oral diseases, musculoskeletal diseases, newborn screening, nutritional diseases, parasitic diseases, skin diseases, etc.

[0108] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions provided by the present invention are mRNA vaccines, which can be used to prevent cancer, viral infections, bacterial infections, fungal infections, etc. The viruses include but are not limited to: norovirus, Ebola virus, coronavirus (including the new coronavirus SARS CoV2), cytomegalovirus, dengue virus, Zika virus, coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus, etc.

[0109] Treatment

[0110] The present invention provides a method for delivering the lipid nanoparticles described herein in vivo, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need thereof. In some embodiments, the present invention provides a method for delivering the lipid nanoparticles described herein in vivo, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need thereof by pulmonary delivery. In some embodiments, the present invention provides a method for delivering the lipid nanoparticles described herein in vivo, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need thereof by intranasal delivery. In some embodiments, the present invention provides a method for delivering the lipid nanoparticles described herein in vivo, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need thereof by inhalation. In some embodiments, the present invention provides a method for delivering the lipid nanoparticles described herein in vivo, comprising administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need thereof by nebulized inhalation.

[0111] Preparation of lipid nanoparticles

[0112] The lipid nanoparticles of encapsulated therapeutic agent and / or preventive can be prepared using multiple methods known in the art. Typically, first prepare a solution comprising various lipid mixtures, before forming lipid nanoparticles, the solution is mixed with the solution of therapeutic agent and / or preventive, therapeutic agent and / or preventive are encapsulated in the lipid nanoparticles formed by the mixture of various lipids (such as described in WO2016004318, US20160038432). Or, first prepare a solution comprising various lipid mixtures and then form lipid nanoparticles, then the lipid nanoparticles obtained are mixed with therapeutic agent and / or preventive and therapeutic agent and / or preventive are encapsulated in the lipid nanoparticles formed by the mixture of various lipids (such as described in WO2018089801, US20180153822). These methods can effectively encapsulate therapeutic and / or prophylactic agents in lipid nanoparticles, with the encapsulation efficiency generally being not less than about 80%, not less than about 85%, not less than about 90%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, and not less than about 99%.

[0113] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0114] Example

[0115] Example 1 Synthesis of Compound CP01

[0116] Step 1: Synthesis of compound 1a

[0117] Dissolve 3-tert-Butoxycarbonylmethylaminopropionic acid (1.5 g, 7.5 mmol) in 20 mL of dichloromethane, add 1,4-bis(3-aminopropyl)piperazine (3.8 g, 18.7 mmol), HATU (8.5 g, 22.5 mmol), and DIPEA (3.9 g, 30.0 mmol), and stir to dissolve. Stir at room temperature for 16 hours. After the reaction, purify by column chromatography, and concentrate to obtain compound 1a (4.0 g) in a 52% yield.

[0118] MS m / z(ESI):571.8[M+1]

[0119] Step 2: Synthesis of compound 1b

[0120] Compound 1a (4.0 g, 7.0 mmol) was dissolved in 10 mL of dichloromethane at room temperature, and 2N HCl (60 mL) was added. The mixture was stirred at room temperature for 16 h until the reaction was complete. The hydrochloric acid solution was dried and dissolved in isopropanol. The mixture was concentrated to give compound 1b (1.4 g) in a yield of 53%.

[0121] MS m / z(ESI):371.5[M+1]

[0122] Step 3: Synthesis of compound 1c

[0123] 6-Bromohexanol (0.91 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, 4-dimethylaminopyridine (1.22 g, 10 mmol) was added, and p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added in batches. The reaction was stirred at room temperature for 3 h. 2-Hexyldecanoic acid (2.16 g, 5.6 mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight. After TLC showed that the reaction was complete, 20 mL of dichloromethane was added to dilute the mixture, and then washed with 30 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the product 6-bromohexyl 2-hexyldecanoate (1.83 g, light yellow oil) with a yield of 62%.

[0124] At room temperature, compound 1b (924 mg, 2.5 mmol) was dissolved in 6 mL of acetonitrile and 4 mL of tetrahydrofuran. Potassium carbonate (690 mg, 5.0 mmol), potassium iodide (208 mg, 1.25 mmol), and 6-bromohexyl 2-hexyldecanoate (1.0 g, 2.5 mmol) were added. The mixture was heated to 70°C and stirred for 4 h. The product was purified by column chromatography and concentrated to afford compound 1c (1.4 g) in a 79% yield.

[0125] MS m / z(ESI):710.1[M+1]

[0126] Step 4: Synthesis of compound 1d

[0127] Dissolve cholesterol (2.8 g, 7.3 mmol) in 30 mL of dichloromethane, add 4-dimethylaminopyridine (1.7 g, 14.3 mmol), and then add p-nitrophenyl chloroformate (1.6 g, 8.0 mmol) in batches. Stir and react at room temperature for 4 h. TLC indicates complete reaction of the cholesterol, and the reaction mixture is ready for use.

[0128] At room temperature, 2-bromoethylamine (1.5 g, 7.3 mmol) was dissolved in 30 mL of dichloromethane and added to the above reaction solution. 4-Dimethylaminopyridine (1.7 g, 14.3 mmol) and triethylamine (1.1 g, 11.0 mmol) were added, and the reaction was stirred at room temperature for 16 h. After TLC showed the reaction was complete, 20 mL of dichloromethane was added to dilute the mixture, followed by washing with 30 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. Product 1d (872 mg) was obtained by column chromatography in a 58% yield.

[0129] MS m / z(ESI):537.6[M+1]

[0130] Step 5: Synthesis of compound CP01

[0131] Compound 1c (576 mg, 0.8 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 1d (872 mg, 1.6 mmol), potassium carbonate (447 mg, 3.2 mmol), and potassium iodide (269 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 h. The mixture was cooled to room temperature, filtered, and the residue was washed with dichloromethane. Saturated sodium chloride solution was added to the filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the product CP01 (120 mg, light yellow oil) in a 25% yield.

[0132] MS m / z(ESI):1165.3[M+1]

[0133] 1H NMR (300MHz, CDCl3): δ8.30(br,1H),7.90(br,1H),5.68(br,1H),5.39(t,1H,J=5.4Hz),4.59-4.42(m,1H),4. 09(t,2H,J=6.6Hz),3.39-3.22(m,6H),2.71-2.25(m,37H),2.10-1.81(m,6H),1.79-0.85(m,71H),0.70(s,3H)

[0134] Example 2 Synthesis of Compound CP05

[0135] Step 1: Synthesis of compound 5a

[0136] 2-Hexyldecanoic acid (2.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 6-bromohexanol (0.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and stirred to dissolve. A dichloromethane solution of EDC.HCL (1.10 g, 6.0 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, and the mixture was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1a (1.45 g, light yellow oil) with a yield of 70%.

[0137] MS m / z(ESI):419.2[M+1]

[0138] Step 2: Synthesis of compound 5b

[0139] At room temperature, compound 5a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, and piperazine (3.88 g, 45 mmol) was added. The temperature was raised to 50°C and stirred for 8 h. The reaction progress was monitored. After the starting material was completely consumed, the temperature was lowered to room temperature. The ethanol was removed at 45°C, and the crude product was dissolved in dichloromethane and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 5b (1.13 g, light yellow oil) with a yield of 89%.

[0140] MS m / z(ESI):425.4[M+1]

[0141] Step 3: Synthesis of compound 5c

[0142] 4-Bromobutanol (0.77 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 4-dimethylaminopyridine (1.22 g, 10 mmol) was added. Then, p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added in batches. The reaction was stirred at room temperature for 3 h. Cholesterol (2.16 g, 5.6 mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight. After TLC showed that the reaction was complete, 20 mL of dichloromethane was added to dilute the mixture, and the mixture was washed with 30 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give compound 5c (1.83 g, light yellow oil) with a yield of 65%.

[0143] MS m / z(ESI):565.3[M+1]

[0144] Step 4: Synthesis of compound CP05

[0145] Compound 5b (425 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 5c (679 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 h. The mixture was cooled to room temperature, filtered, and the residue was washed with dichloromethane. Saturated sodium bicarbonate solution was added to the filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain the product CP05 (515 mg, light yellow oil) in a 57% yield.

[0146] MS m / z(ESI):909.8[M+1]

[0147] Example 3: Preparation and Characterization of Nucleic Acid-Lipid Nanoparticle Compositions (mRNA-LNPs)

[0148] Table 1 Main experimental consumables

[0149] Table 2 Main experimental equipment

[0150] Table 3 Other main reagents

[0151] 2. Experimental Plan

[0152] Preparation of mRNA-LNPs

[0153] The lipid components were dissolved in ethanol at a molar ratio of 49.25:49.25:1.5. Firefly luciferase mRNA was dissolved in 25 mM sodium acetate buffer, pH 4.0, to a final concentration of 135 ng / µL. The mRNA-LNP complex was prepared using the Ignite microfluidic nanoparticle preparation instrument from Precision Nanosystems at a lipid mixture:mRNA flow rate ratio of 1:3. The prepared mRNA-LNP was dialyzed and concentrated by ultrafiltration into a buffer containing 20 mM Tris, 10.5 mM sodium acetate, and 87% sucrose, pH 7.5. The experimental sample was sterile filtered.

[0154] Characterization of mRNA-LNPs

[0155] The prepared mRNA-LNP experimental sample was diluted 50 times with buffer (final concentration was 2-100 ng / μL), and the average particle size, PDI and ζ potential of the nanoparticles were measured using a Malvern particle size analyzer; the average particle size and PDI were measured using a ZEN0040 DLS sample cell with a sample volume of 200 μL; the ζ potential was measured using a DTS1070 potential cell with a sample volume of 800 μL. The mRNA content and encapsulation efficiency were measured using a Quant-iT TM RiboGreen RNA detection kit, TE buffer to detect free mRNA content C 游离 , 2% Triton buffer was used to detect the total mRNA content C 总 The encapsulation efficiency is calculated by the formula EE=(1-C 游离 / C 总 The experimental results are shown in Table 4.

[0156] Table 4 Physical and chemical parameters of Luc-mRNA-LNP

[0157] Patent CN112424214A compound (3)

[0158] Patented US7514099B2 compound (CLinDMA):

[0159] Patent CN116059170A compound:

[0160] Patented CN10224559B compound:

[0161] Patented CN102421417B compound:

[0162] The results show that the mRNA-LNP formed by the cationic lipid compound of the present invention, phospholipids (DOPE), PEG lipids and mRNA has good physicochemical parameters, an average particle size between about 80 and 105 nm, a PDI of less than 0.15, a good polydispersity coefficient, a zeta potential between about -5 mV and 5 mV, and an LNP encapsulation efficiency of mRNA greater than 89%, which is significantly higher than that of the control group LNP.

[0163] Example 4: mRNA-LNP in vitro cell transfection activity

[0164] Hep3B cells were seeded at a density of 10,000 cells / well in a 96-well white opaque cell culture plate. After 24 hours, each well was transfected with 100 ng of Luc-mRNA-LNP. The plates were then incubated at 37°C in a 5% CO2 incubator. A negative control group was transfected with an equal volume of saline. After 24 hours, bioluminescence was detected using a Promega firefly luciferase assay kit. The results are shown in Figure 1. These results demonstrate that the three-component LNP composition formed using the cationic lipid compound described herein can achieve high intracellular expression of Luc-mRNA, with expression levels significantly superior to those in the control group.

[0165] Example 5: mRNA-LNP Animal Immunization Test

[0166] We used the new coronavirus S protein mRNA to evaluate the immune activity of mRNA-LNP in mice. The LNP formula used was an ionizable cationic lipid compound: DOPE: DMG-PEG2000 = 49.25: 49.25: 1.5 molar ratio for mRNA encapsulation. The specific formula is shown in Table 4. Female BALB / c mice aged 6-8 weeks were randomly divided into 3 groups of 6 / group and immunized by intramuscular injection of the hind legs. Immunizations were performed on day 0 and day 14, with an immunization dose of 5 μg mRNA-LNP / mouse. Blood was collected and serum was separated on the 14th day (before the second immunization) and the 28th day of immunization, and the specific antibody titer against the SARS-CoV2 virus S protein antigen was detected by ELISA (enzyme-linked immunosorbent assay). The antibody titer detection value GMT (95% CI) is shown in Figure 2. The antigen-specific antibody titer induced by the three-component LNP constructed by the lipid compound provided by the present invention after delivering the new coronavirus mRNA was significantly higher than that of the control group.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The lipid compound shown in formula (I): or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein, Each occurrence of L1 and L2 is independently selected from a bond, an optionally substituted C1-C 20 alkylene group, an optionally substituted C2-C 20 alkenylene group, an optionally substituted C2-C 20 alkynylene group, an optionally substituted C1-C 20 acyl group, an optionally substituted carbocyclic group, an optionally substituted arylene group; Each occurrence of G1 and G2 is independently selected from a bond, -O-, -S-, -N(R a )-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-, -N(R a )C(=O)-, -S(=O)-, -S(=O)2-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-, -SP(=O)(OR a )O-, -OP(=S)(OR a )O-, -OP(=O)(SR a )O-, -P(=O)(OR a )(OR a )-, -P(=S)(OR a )(OR a )-, -P(=O)(SR a )(OR a )-, -N(R a )C(=O)O-, -OC(=O)N(R a )-, -S-S-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-; R1 and R2 are each independently selected from a steroid group, an optionally substituted C1-C 20 alkyl group, an optionally substituted C2-C 20 alkenyl group, an optionally substituted C2-C 20 alkynyl group; wherein one or more -CH2- in the C1-C 20 alkyl group, C2-C 20 alkenyl group, C2-C 20 alkynyl group may optionally be replaced by -O-, -S-, -NR a -, a carbocyclic group, an aryl group, a heteroaryl group, and / or a heterocyclic group; R3 is selected from C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl, aralkyl, halogen, C1-C 20 alkoxy, C1-C 20 alkylthio, NR4R4’, R4-C(O)-, R4-S(O)-, R4-S(O)2-, R4-C(O)O-, R4-OC(O)-, R4-NHC(O)-, R4-C(O)NH-, oxo group; R4 and R4’ are each independently selected from H, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic group and / or heterocyclic alkyl; R a and R b each independently selected from H, optionally substituted C1-C 20 alkyl, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclic group, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclic group, optionally substituted heterocyclic alkyl; wherein, said C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 one or more -CH2- in alkynyl may optionally be replaced by -O-, -S-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NH-, -NHC(=O)-, -S(=O)-, -S(=O)2-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, or -C(=S)S-; m, n, p, and q are each independently selected from 1, 2, or 3; r is selected from 0, 1, 2, 3, or 4; provided that at least one of R1 and R2 is selected from a steroid group.

2. The compound according to claim 1, selected from or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein L1, L2, G1, G2, R1, R2, R3, p, q, and r are as defined in claim 1.

3. The compound according to claim 1, selected from or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein L1, L2, G1, G2, R1, R2, R3, p, q, and r are as defined in claim 1.

4. A compound or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof according to any one of the preceding claims; Among them, The steroid compound group has the following structure: R5 is selected from hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C1-C 20 alkoxycarbonyl C1-C 20 alkyl-; R6 is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Preferably, the steroid compound group is selected from: wherein R' is C 1-20 alkyl.

5. The compound according to any one of the preceding claims, selected from: or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein L1, L2, G1, G2, R1, R3, p, q, and r are as defined in claim 1.

6. The compound according to any one of the preceding claims, selected from: or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein L1, L2, G1, G2, R1, R3, p, q, and r are as defined in claim 1.

7. A compound or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof according to any one of the preceding claims; wherein R1 is selected from optionally substituted C1-C 20 alkyl; wherein, One or more -CH2- in the C1-C 20 alkyl group may optionally be replaced by O, S, -NR a -, a carbocyclic group; or R1 is selected from R5 is selected from hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C1-C 20 alkoxycarbonyl C1-C 20 alkyl-; R6 is selected from hydrogen, halogen, hydroxy, C1-C 20 alkyl group.

8. A compound or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof according to any one of the preceding claims; wherein R1 is selected from Preferably, R1 is selected from the following structures:

9. The compound according to claim 1, selected from: or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof.

10. A lipid nanoparticle comprising a lipid compound or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof according to any one of claims 1-9.

11. The lipid nanoparticle according to claim 10, further comprising a phospholipid and / or a polyethylene glycol lipid.

12. The lipid nanoparticle according to claim 11, further comprising a therapeutic agent and / or a prophylactic agent.

13. The lipid nanoparticle according to claim 12, wherein the therapeutic agent and / or prophylactic agent comprises one or more nucleic acids, such as DNA, RNA, etc.

14. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 10-13 and a pharmaceutically acceptable carrier.

15. A method for treating and / or preventing a disease, comprising administering a therapeutically effective amount of the lipid nanoparticle according to any one of claims 10-13 or the pharmaceutical composition according to claim 14 to an individual in need thereof.

16. Use of the compound according to any one of claims 1-9 and / or the lipid nanoparticle according to claims 10-13 in the preparation of a delivery system for a therapeutic agent and / or a prophylactic agent.

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