CN120271651A

CN120271651A - Ionizable cationic lipid compounds and compositions for nucleic acid delivery and uses thereof

Info

Publication number: CN120271651A
Application number: CN202411951304.6A
Authority: CN
Inventors: 王正华; 严志红; 李荩; 王浩猛; 代守一; 顾超男; 刘健; 朱涛; 邱东旭; 宇学峰
Original assignee: Kangxinuo Shanghai Biological Research And Development Co ltd; Tianjin Jenkem Technology Co Ltd
Current assignee: Kangxinuo Shanghai Biological Research And Development Co ltd; Tianjin Jenkem Technology Co Ltd
Priority date: 2023-12-29
Filing date: 2024-12-27
Publication date: 2025-07-08
Also published as: WO2025140596A1

Abstract

The present invention provides an ionizable cationic lipid compound having a structure represented by formula (I), which is useful for preparing Lipid Nanoparticles (LNP) for delivering therapeutic and/or prophylactic agents. LNPs prepared using the ionizable cationic lipid compounds of the present invention have superior stability and transfection efficiency, and can efficiently and stably deliver biologically active substances (including nucleic acids, e.g., mRNA) to target cells or organs, thereby eliciting highly specific antibody responses in vivo.

Description

Ionizable cationic lipid compounds and compositions for nucleic acid delivery and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to an ionizable cationic lipid compound and composition for nucleic acid delivery and application thereof.

Background

Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic characteristics and pharmacological activity, can be used for treating tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infection and the like, and after being injected into a human body, the nucleic acid drugs need to be delivered to a lesion site by a high-efficiency and safe drug delivery system which needs to stay for enough time to accurately target the lesion site and avoid damaging normal cells.

Current delivery systems can be divided into viral vectors and non-viral vectors. Viral vectors have found less use in nucleic acid drugs due to their immunogenicity, tumorigenicity, and limited drug loading, and non-viral vectors, such as polymers, lipids (liposome or LNP), which bind nucleic acid drugs to specific ligands to enable their targeting to specific cells, are used in many current nucleic acid drugs. LNP is one of the most widely used delivery systems for current nucleic acid drug research, and LNP delivery systems are capable of safely and effectively delivering nucleic acids. The lipid nanoparticle delivery system has the advantages of high nucleic acid encapsulation efficiency, capability of effectively transfecting cells, strong tissue penetrability, low cytotoxicity and immunogenicity and the like, is favorable for delivering drugs and the like, and has great advantages compared with other delivery systems, so that the lipid nanoparticle delivery system has wide development and application prospects.

In the prior art, LNP delivery systems often consist of four components, namely, an ionizable lipid (cationic lipid), a steroid, a neutral lipid, and a PEG-lipid, for example, AU2020325221A1 discloses a composition for targeted cell delivery LNPs comprising (i) an ionizable lipid, (ii) a sterol or other structural lipid, (iii) a non-cationic auxiliary lipid or phospholipid, (iv) a PEG lipid, and (v) an agent (e.g., a nucleic acid molecule) encapsulated in LNP and/or associated therewith, in specific ratios to effect entrapment and delivery of the nucleic acid drug. Conventional ionizable lipids (or cationic lipids) are essentially composed of branched or straight fatty chains. Cationic-based cholesterol compounds, such as DC-Chol, can also be used to deliver nucleic acids with little structural variability compared to linear fatty chains. Patent US7514099B2And CN112424214ATwo classes of ionizable cholesterol-modified amino lipid compounds are respectively disclosed, and researches show that although the asymmetric cholesterol-based amino lipid compounds can improve the order of lipid nanoparticles, the compounds still need to adopt more than four or even more than five lipid components to construct nano delivery vehicles, and the construction mode is still complicated.

The invention introduces the sterol structure into the cationic lipid compound through the piperazine or piperazine dione structure to form a novel steroid-cationic lipid compound, and the novel steroid-cationic lipid compound and the three-component lipid nanoparticle constructed by auxiliary lipid and PEG lipid can realize the efficient encapsulation and delivery of nucleic acid without the participation of extra cholesterol, and the construction of the novel steroid-cationic lipid compound is simpler than that of the traditional four-component lipid nanoparticle, and the novel steroid-cationic lipid compound has higher delivery efficiency of nucleic acid due to better endosome escape capability.

Disclosure of Invention

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

Or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein,

Each occurrence of L ₁ and L ₂ is independently selected from the group consisting of a bond, an optionally substituted C ₁-C₂₀ alkylene, an optionally substituted C ₂-C₂₀ alkenylene, an optionally substituted C ₂-C₂₀ alkynylene, an optionally substituted C ₁-C₂₀ acyl, an optionally substituted carbocyclylene, an optionally substituted arylene;

Each occurrence of G ₁ and G ₂ is independently selected from the group consisting of bonds 、-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)-;

R ₁ and R ₂ are each independently selected from the group consisting of a steroid group, an optionally substituted C ₁-C₂₀ alkyl group, an optionally substituted C ₂-C₂₀ alkenyl group, an optionally substituted C ₂-C₂₀ alkynyl group, wherein one or more of the C ₁-C₂₀ alkyl group, C ₂-C₂₀ alkenyl group, C ₂-C₂₀ alkynyl group, -CH ₂ -, may optionally be replaced by-O-, -S-, -NR _a -, carbocyclyl, aryl, heteroaryl, and/or heterocyclyl;

R ₃ is selected from the group consisting of C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl, carbocyclyl, aryl, aralkyl, halogen, C ₁-C₂₀ alkoxy, C ₁-C₂₀ 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;

R ₄ and R ₄' are each independently selected from H, C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and/or heterocyclylalkyl;

R _a and R _b are each independently selected from H, optionally substituted C ₁-C₂₀ alkyl, optionally substituted C ₂-C₂₀ alkenyl, optionally substituted C ₂-C₂₀ alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, wherein one or more of the C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl, -CH ₂ -, 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 R ₁ and R ₂ is selected from steroid groups.

In another aspect, the present invention also provides a lipid nanoparticle, including a lipid compound represented by formula (I), or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

In another aspect, the invention also provides a pharmaceutical composition comprising a lipid nanoparticle as described herein and a pharmaceutically acceptable carrier.

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

In another aspect, the present invention also provides the use of a lipid compound of formula (I) or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof as described herein in the preparation of a therapeutic and/or prophylactic agent delivery system.

The beneficial effects are that:

The three-component lipid nanoparticle prepared by the lipid compound or the stereoisomer, the tautomer and the pharmaceutically acceptable salt thereof has simple process, and simultaneously has better stability and transfection efficiency. The lipid nanoparticle is used for delivering nucleic acid (such as mRNA), can be efficiently and stably delivered to target cells or organs, can cause higher specific antibody response and cellular immune response in experimental animals, and has good safety.

Drawings

FIG. 1 shows the expression levels of firefly luciferase transfected with Luc-mRNA-LNP complex in Hep3B cells.

Figure 2 shows serum antibody titers after mice are immunized with new coronal mRNA-LNP.

Detailed Description

Definition of the definition

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.

As used herein, the term "lipid nanoparticle", or "LNP", refers to particles having a nanometer scale, e.g., 1nm to 1,000nm, that comprise one or more types of lipid molecules.

As used herein, the term "genetic drug" generally consists of a vector or delivery system containing an engineered gene construct whose active ingredient may be DNA, RNA, genetically engineered virus, bacteria or cells, by introducing an exogenous gene into a target cell or tissue to replace, compensate, block, correct a particular gene for the purpose of treating and preventing a disease.

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

As used herein, the term "lipid compound" refers to a group of organic compounds including, but not limited to, esters of fatty acids and is generally characterized as poorly soluble in water but soluble in many organic solvents including, but not limited to, benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, diethyl ether, ethyl acetate, acetone, carbon tetrachloride.

As used herein, the term "alkyl" refers to a monovalent group of a straight or branched saturated hydrocarbon chain having from 1 to 20 carbon atoms (more typically from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms). The term is illustratively a group 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-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, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, and the like.

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

As used herein, the term "alkenyl" refers to a straight 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 a carbon-carbon double bond (e.g., 1,2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond may be present at any stable point along the chain. The term is illustratively a group such as vinyl (i.e., -ch=ch ₂), propen-1-yl (i.e., -ch=chch ₃), propen-3-yl (or allyl, i.e., -CH ₂CH＝CH₂), propen-2-yl (i.e., -C (CH ₃)＝CH₂), butadienyl (including 1, 2-butadienyl and 1, 3-butadienyl), and the like.

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 a carbon-carbon double bond (e.g., 1, 2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond may be present at any stable point along the chain. The term is illustratively a group such as ethenylene, propenylene, butenylene, pentenylene, hexenylene, and the like.

As used herein, the term "alkynyl" refers to a straight 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 a carbon-carbon triple bond (e.g., 1,2, or 3 carbon-carbon triple bonds). The term is illustratively a group such as ethynyl (i.e., -C.ident.CH), propargyl (i.e., -CH ₂ C.ident.CH), propynyl (i.e., -C.ident.CCH ₃), and the like.

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

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

As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. The term is illustratively a group such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

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, heterocyclyl are as defined herein. The term is illustratively a group such as formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-pentanoyl, n-hexanoyl, acryloyloxy, benzoyl, cyclopropylacyl and the like.

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, where alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl are as defined herein. The term is illustratively a group such as formyloxy, acetoxy, propionyloxy, n-butyryloxy, isobutyryloxy, n-pentanoyloxy, n-hexanoyloxy, and the like.

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 illustratively a group such as phenyl, fluorenyl, naphthyl, anthracenyl, 1,2,3, 4-tetrahydronaphthalene (if the point of attachment is through an aryl group), and the like.

As used herein, the term "carbocyclyl" refers to a mono-or partially unsaturated group having 3 to 14 carbon atoms (more typically 3 to 8 carbon atoms, or 3 to 6 carbon atoms) as ring atoms, or multiple fused (condensed) or bridged or spiro rings. Carbocycles or carbocyclyls may be saturated or partially unsaturated and may be fused to another saturated, partially unsaturated or aromatic ring, provided that the ring atom attached to the target molecule is not an aromatic carbon. Examples of carbocycles or carbocyclyls include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene, and the like.

As used herein, the term "heteroaryl" refers to an aromatic ring radical comprising a single ring or multiple condensed (fused) rings (e.g., comprising 2 or 3 rings) of 5 to 14 ring atoms (more typically having 5 to 10 ring atoms, or 5 to 6 ring atoms) in the ring, wherein the ring atoms comprise at least one or more heteroatoms selected from oxygen, nitrogen, and/or sulfur in addition to carbon atoms. If the ring is aromatic, the sulfur and nitrogen atoms may also be present in oxidized form. Multiple fused (fused) cycloheteroaryl groups are fused to form multiple fused ring systems from a monocyclic heteroaryl group as defined above with one or more rings selected from heteroaryl groups (to form, for example, naphthyridinyl, such as 1, 8-naphthyridinyl), heterocycles (to form, for example, 1,2,3, 4-tetrahydronaphthyridinyl, such as 1,2,3, 4-tetrahydro-1, 8-naphthyridinyl), carbocycles (to form, for example, 5,6,7, 8-tetrahydroquinolinyl), and aryl groups (to form, for example, indazolyl). It is understood that the point of attachment of the heteroaryl group may be on any suitable atom of the heteroaryl group, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridyl, 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 ] pyridyl, quinazolin-4 (3H) -one, triazolyl, 4,5,6, 7-tetrahydro-1H-indazolyl, and 3b, 4a, 5-tetrahydro-1H-cyclopropane [3,4] cyclopenta [1,2-c ] pyrazolyl.

As used herein, the term "heterocyclyl" refers to a monovalent or divalent saturated or partially unsaturated group having a single ring or multiple condensed (fused) or bridged or spiro rings having from 3 to 14 ring atoms (more typically from 3 to 10 ring atoms, or from 3 to 6 ring atoms) within the ring, wherein the ring atoms contain at least one more nitrogen atom in addition to carbon atoms. Examples of heterocyclyl groups include, but are not limited to, aziridine ring, azetidine ring, tetrahydropyrrole ring, piperidine ring, azepane ring, azacyclooctane ring, tetrahydroimidazole ring, tetrahydropyrazole ring, tetrahydrooxazole ring, tetrahydroisoxazole ring, tetrahydrothiazole ring, tetrahydroisothiazole ring, piperazine ring, morpholine ring, dihydropyridinyl, 4,5,6, 7-tetrahydro-1H-benzo [ d ] imidazole, 4,5,6, 7-tetrahydro-1H-imidazo [4,5-c ] pyridine, and the like. The nitrogen heterocyclic group in the invention is a heterocyclic group containing nitrogen atom in the structure, including but not limited to substituted or unsubstituted aziridinyl, azetidinyl, beta-propiolactamyl, pyrrolyl, piperidyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, caprolactamyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, indolyl, benzimidazolyl, carbazolyl, quinolinyl, isoquinolinyl, pteridinyl, acridinyl, 7H purinyl, phenazinyl, phenothiazinyl or 1H-azepinyl.

As used herein, the term "optionally substituted" means unsubstituted or substituted with one or more groups selected from C ₁-C₆ alkyl, C ₁-C₆ alkoxy, halogen, hydroxy, cyano, nitro, amino, C ₃-C₆ cycloalkyl, oxo.

As used herein, the term "steroid" is an organic compound having a tetracyclic carbon skeleton structure as shown below.

Steroid compounds include naturally occurring or synthetic steroid compounds and analogues thereof. The steroid or analogue thereof includes sterols or analogues thereof of vegetable and/or animal origin. Examples of the steroid compounds described herein include, but are not limited to, oat sterols, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, fecal sterols, dehydrocholesterol, desmosterol, dihydroergocalcitol, cholesterol, dihydrocholesterol, dihydroergosterol, black-sea sterols, epicholesterol, ergosterol, fucosterol, hexahydrolight sterols, hydroxycholesterol, photosterol, algal sterols, sitostanol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurochol, deoxycholic acid, lithocholic acid, ent-cholesterol, epicholesterol, delomosterol, cholestanol, cholestanone, 3p- [ N- (N 'N' -dimethylaminoethyl) carbamoyl cholesterol (DC-Chol) 24 (S) -hydroxycholesterol, 25 (R) -27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lu Misi-tetrol, cetostearyl alcohol, calcipotriol, prostatol, cholecalciferol, lupeol, ergocalcitol, 22-dihydro-autogenous calcitol, lycorine, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, and the like.

As used herein, the term "therapeutically effective amount" refers to an amount sufficient to affect treatment, as defined below, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary with the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration, and the like, which can readily be determined by one of ordinary skill in the art.

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, which cannot be exchanged by single bond rotation. "stereoisomers" include "diastereomers" and "enantiomers. "diastereoisomers" refers to stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. Diastereomeric mixtures can be separated under high resolution analytical procedures such as crystallization, electrophoresis and chromatography. "enantiomer" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other.

As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds, the difference between which is only in the position and electron distribution of one (or more) mobile atoms, such as keto-enol tautomers.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of a given compound, and which is not biologically or otherwise undesirable. The pharmaceutically acceptable salt may be an acid addition salt and/or a base addition salt. Acid addition salts can be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, carbonate, bisulfate, hydrogen phosphate, dihydrogen phosphate, hydrogen carbonate, and the like, and 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, and the like. The base addition salts may 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, and salts derived from organic bases include salts with various primary, secondary, and tertiary amines, such as ethylamine, diethylamine, n-propylamine, isopropylamine, diethanolamine, meglumine, lysine, piperazine, piperidine, morpholine, tromethamine, choline, and the like.

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

As used herein, the term "delivery system" refers to a formulation or composition that modulates the spatial, temporal, and dose distribution of a bioactive ingredient within an organism.

Compounds of formula (I)

In some embodiments, the present invention provides lipid compounds of formula (I)

Or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein L ₁、L₂、G₁、G₂、R₁、R₂、R₃, m, n, p, q, r are as defined herein.

In some embodiments, the present invention provides lipid compounds of formula (II)

Or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein

L ₁、L₂、G₁、G₂、R₁、R₂、R₃, p, q and r are as defined herein.

In some embodiments, the present invention provides lipid compounds of formula (III)

L ₁、L₂、G₁、G₂、R₁、R₂、R₃, p and q and r are as defined herein.

In some embodiments, the steroid in the steroid group of the invention is selected from naturally occurring steroids or analogues thereof; preferably, it includes plant sterols and animal sterols, or analogues thereof; more preferably, the process is carried out, selected from the group consisting of oat sterols, beta-sitosterols, campesterols, ergocalcitol, campesterols, cholestanol, fecal sterols, dehydrocholesterol, chain sterols, dihydroergocalcitol, cholesterol, dihydrocholesterol, dihydroergosterol, black-sea sterols, epicholesterol, ergosterols, fucosterol, hexahydrolight sterols, hydroxycholesterols, photosterol, sitostanol, stigmastanol, stigmasterols, cholic acid, glycocholic acid, taurochol, deoxycholic acid, lithocholic acid, ent-cholesterol, stigmasterol, delumosterol, cholestanol, cholestanone, 3p- [ N- (N' -dimethylaminoethyl) carbamoyl cholesterol (DC-Chol), 24 (S) -hydroxycholesterol, 25 (R) -27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cyclol, 22-ketocholesterol, 20-hydroxysterols, 7-hydroxycholesterol, 19-hydroxycholesterol, 7-hydroxycholesterols, 5-dehydrocholesterol, 5-dehydrogenized, 5-hydroxycholesterols, 25-dehydrocholesterol, cholesterols, 5-dehydrogenized, cholesterols, 5-hydroxycholesterols, 25-hydroxycholesterols, cholesterols, 5-7-hydroxycholesterols, dehydrocholesterol, cholesterols, 7-hydroxycholesterols, dehydrocholesterol, cholesterols, 5, and, 5-hydroxycholesterols, and cholesteatol, 22-dihydro self-ossifying alcohol, lycopersicin, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, etc.

In some embodiments, in the lipid compounds provided herein, or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, the steroid in the steroid group is selected from cholesterol and derivatives of cholesterol.

In some embodiments, in the lipid compounds provided herein, or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, the steroid groups have the following structure:

R ₅ is selected from hydrogen, C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl, C ₁-C₂₀ alkoxycarbonyl C ₁-C₂₀ alkyl-;

R ₆ is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl;

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

In some embodiments, the steroid group is selected from the group consisting of:

Wherein R' is C _1-20 alkyl.

In some embodiments, the present invention provides lipid compounds of formula (IV-a), (IV-b), (IV-c), or (IV-d):

Or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein L ₁、L₂、G₁、G₂、R₁、R₃, p and q and r are as defined herein.

In some embodiments, the present invention provides lipid compounds of formula (V-1), (V-2), (V-3), (V-4), or formula (VI-5):

In some specific embodiments, R ₁ and/or R ₂ are selected from optionally substituted C ₁-C₂₀ alkyl, wherein one or more of the C ₁-C₂₀ alkyl groups, -CH ₂ -, may optionally be replaced by O, S, -NR _a -, carbocyclyl, or R ₁ and/or R ₂ are selected from

R ₅ is selected from hydrogen, C ₁-C₂₀ alkyl, C ₂-C₂₀ alkenyl, C ₂-C₂₀ alkynyl, C ₁-C₂₀ alkoxycarbonyl C ₁-C₂₀ alkyl-, R ₆ is selected from hydrogen, halogen, hydroxy, C ₁-C₂₀ alkyl.

In some specific embodiments, R ₁ and/or R ₂ are selected from

In some specific embodiments, R ₁ and/or R ₂ are selected from the following structures:

in some embodiments, the invention provides a compound selected from the group consisting of:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

Lipid Nanoparticles (LNP)

In some embodiments, the invention provides a lipid nanoparticle comprising a lipid compound described herein or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, as a delivery vehicle for a therapeutic and/or prophylactic agent (e.g., a nucleic acid comprising DNA, RNA, etc.). In some embodiments, the lipid nanoparticle described herein further comprises 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 a phospholipid and a PEG lipid. The lipid nanoparticles described herein can deliver therapeutic and/or prophylactic agents to a target site of interest (e.g., cell, tissue, organ, etc.). Thus, the lipid nanoparticle described herein further comprises one or more therapeutic or prophylactic agents (e.g., nucleic acids, particularly Therapeutic Nucleic Acids (TNA)).

In some embodiments, the lipid nanoparticle has a lipid compound to PEG lipid molar ratio of 30-90:10-60:0.5-20, preferably a lipid compound to PEG lipid molar ratio of 30-80:30-80:0.5-20, more preferably a lipid compound to PEG-lipid molar ratio of 40-60:40-60:0.5-5, most preferably a lipid compound to PEG-lipid molar ratio of 49.25:49.25:1.5.

Phospholipid

In some embodiments, the lipid nanoparticle described herein further comprises a phospholipid. Examples of phospholipids include, but are not limited to, for example, distearoyl-sn-glycerophosphoryl ethanolamine, distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (e.g., 16-O-monomethylpe), dimethylphospholide (e.g., 16-O-dimethylphospholide), 18-1-trans PE, 1-stearoyl-2-oleoyl Phosphatidylethanolamine (PE), hydrogenated Soybean Phosphatidylethanolamine (HSPC), egg phosphatidylethanolamine (dme), ditolyphosphatidylethanolamine (dme-mal), ditolyphosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DSPE), dimethyl phosphatidylethanolamine (16-O-dimethyl PE), dimethyl phosphatidylethanolamine (spp) Palmitoyl Oleoyl Phosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DEPE), 1, 2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1, 2-biphytoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg Sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexacosyl phosphate, lysophosphatidylcholine, di-linoleoyl phosphatidylcholine, or mixtures thereof. It should be understood that other diacyl phosphatidyl choline and diacyl phosphatidyl ethanolamine phospholipids may also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid having a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

In some embodiments, the mole percent of phospholipids in the total lipid of the lipid nanoparticle is from about 15% to about 65%, such as from about 20% to about 65%, from about 25% to about 65%, from about 30% to about 65%, from about 35% to about 65%, from about 40% to about 65%, from about 45% to about 65%, from about 50% to about 65%, from about 55% to about 65%, from about 60% to about 65%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%.

PEG lipid

In some embodiments, PEG lipids are incorporated into the lipid nanoparticles described herein to inhibit aggregation of the particles, thereby increasing the stability of the lipid nanoparticles. In some embodiments, a PEG lipid described herein is a lipid that is linked to one or more polyethylene glycol (PEG) chains by covalent or non-covalent bonds. In some embodiments, a PEG lipid described herein is a lipid that is linked to one or more polyethylene glycol (PEG) chains by a covalent bond.

In some embodiments, PEG molecules suitable for use in the PEG lipids described herein have a molecular weight of about 500 to about 10000, about 1000 to about 5000, about 1000 to about 4000, about 1000 to about 3000, about 1000 to about 2000, e.g., PEG2000, PEG2500, PEG3000, etc.

Examples of PEG lipids include, but are not limited to, PEG-Diacylglycerol (DAG) (e.g., 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoyl glycerol (PEG-DMG)), PEG-Dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinyl glycerol (PEGS-DAG) (e.g., 4-0- (2 ',3' -di (tetradecanoyloxy) propyl-1-0- (w-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG)), PEG dialkoxypropyl-carbamyl, N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine sodium, PEG-dilauroxypropyl, PEG-dimyristoxypropyl, PEG-dipalmitoxypropyl, PEG-distearoyloxypropyl, 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol-PEG (DMG), distearoyl-PEG-C-PEG, dimyristoyl glycerol (DSG), PEG-dimyristoyl glycerol, PEG-dimyristoyl glycerol (DSG), PEG-dipalmitoyl glyceramide, PEG-distearoyl glyceramide, (1- [8' - (cholest-5-en-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).

In some embodiments, the molar percentage of PEG lipid in the total lipid of the lipid nanoparticle is from about 0.1% to about 10%, for example, from about 1.0% to about 10%, from about 1.5% to about 10%, from about 2.0% to about 10%, from about 2.5% to about 10%, from about 3.0% to about 10%, from about 3.5% to about 10%, from about 4.0% to about 10%, from about 4.5% to about 10%, from about 5.0% to about 10%, from about 5.5% to about 10%, from about 6.0% to about 10%, from about 6.5% to about 10%, from about 7.0% to about 10%, from about 7.5% to about 10%, from about 8.0% to about 10%, from about 8.5% to about 10%, from about 9.0% to about 10%, from about 9.5% to about 10%, from about 5% to about 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%.

Particle size of lipid nanoparticle

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

Lipid/nucleic acid ratio

In some embodiments, the lipid to nucleic acid weight ratio or molar ratio of the lipid nanoparticle is 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.

In some embodiments, the lipid nanoparticle has an N/P ratio (i.e., the ratio of positively charged lipid amine groups/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 higher.

Therapeutic/prophylactic agent

In some embodiments, the lipid nanoparticle of the present invention further comprises a therapeutic and/or prophylactic agent. In some embodiments, the therapeutic and/or prophylactic agents described herein include organic molecules, inorganic molecules, proteins, polypeptides, nucleic acids, vaccines, immunotherapeutic agents, and the like. In some embodiments, the therapeutic and/or prophylactic agents described herein include nucleic acids. In some embodiments, the therapeutic and/or prophylactic agents described herein include DNA. In some embodiments, the 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), plastid DNA (kd na), proviral, lysogen, repeat DNA, satellite DNA, or viral DNA. In some embodiments, the therapeutic and/or prophylactic agents described herein include RNA. In some embodiments, the therapeutic and/or prophylactic agents described herein include small interfering RNAs (sirnas). In some embodiments, the 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), heterogeneous nuclear 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 cytosolic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), piwi-interacting RNA (piRNA), polynucleic acids, ribozymes.

Pharmaceutical composition and formulation

In some embodiments, the invention provides a pharmaceutical composition comprising a lipid nanoparticle as described herein and a pharmaceutically acceptable carrier. In some embodiments, the invention provides a therapeutic and/or prophylactic (e.g., nucleic acids including DNA and RNA, etc.) vaccine comprising a lipid nanoparticle as described herein and a pharmaceutically acceptable carrier.

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

In some embodiments, the diluent comprises ethylene glycol, glycerol, polyethylene glycol, sucrose, trehalose, or combinations thereof, and the like. 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%.

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

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

In some embodiments, the pharmaceutical compositions and/or vaccines of the present invention may be formulated into oral formulations, intramuscular formulations, subcutaneous formulations, intravenous formulations, aerosol inhalation formulations, nasal spray inhalation formulations or dry powder inhalation formulations, ocular formulations, depending on the route of administration of the drug.

Indication of disease

The lipid nanoparticle and/or the pharmaceutical composition provided by the invention can be used for preventing and/or treating cancers, inflammations, fibrosis diseases, autoimmune diseases, infections, mental disorders, hematopathy, chromosome diseases, genetic diseases, connective tissue diseases, digestive diseases, otorhinolaryngologic diseases, endocrine diseases, ocular diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic diseases, oral diseases, musculoskeletal diseases, neonatal screening, nutritional diseases, parasite diseases, skin diseases and the like.

In some embodiments, the lipid nanoparticle and/or pharmaceutical composition provided herein is an mRNA vaccine, which can be used to prevent cancer, viral infection, bacterial infection, fungal infection, and the like. Such viruses include, but are not limited to, norovirus, ebola virus, coronavirus (including novel coronavirus SARS CoV 2), cytomegalovirus, dengue virus, zika virus, coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papilloma virus, influenza virus, marburg virus, measles virus, polio virus, rabies virus, rotavirus, and the like.

Therapeutic method

The present invention provides a method of delivering a lipid nanoparticle described herein in vivo comprising administering a lipid nanoparticle or pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the present invention provides a method of delivering a lipid nanoparticle described herein in vivo comprising administering a lipid nanoparticle or pharmaceutical composition described herein to a subject in need thereof by pulmonary delivery. In some embodiments, the present invention provides a method of delivering a lipid nanoparticle described herein in vivo comprising administering a lipid nanoparticle or pharmaceutical composition described herein to a subject in need thereof by intranasal delivery. In some embodiments, the present invention provides a method of delivering a lipid nanoparticle described herein in vivo comprising administering a lipid nanoparticle or pharmaceutical composition described herein to a subject in need thereof by inhalation. In some embodiments, the present invention provides a method of delivering a lipid nanoparticle described herein in vivo comprising administering a lipid nanoparticle or pharmaceutical composition described herein to a subject in need thereof by aerosol inhalation.

Preparation of lipid nanoparticles

Lipid nanoparticles encapsulating therapeutic and/or prophylactic agents may be prepared using a variety of methods known in the art. Typically, a solution comprising a mixture of lipids is first prepared, mixed with a solution of therapeutic and/or prophylactic agents prior to formation of the lipid nanoparticles, and the therapeutic and/or prophylactic agents are encapsulated in the lipid nanoparticles formed from the mixture of lipids (e.g., as described in WO2016004318, US 20160038432). Or first preparing a solution comprising a mixture of lipids to form lipid nanoparticles, and then mixing the resulting lipid nanoparticles with a therapeutic and/or prophylactic agent to encapsulate the therapeutic and/or prophylactic agent in lipid nanoparticles formed from the mixture of lipids (e.g., as described in WO2018089801, US 20180153822). These methods can be effective to encapsulate therapeutic and/or prophylactic agents in lipid nanoparticles, typically at an encapsulation rate of 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%, not less than about 99%.

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Examples

EXAMPLE 1 Synthesis of Compound CP01

Step 1 Synthesis of Compound 1a

3-Boc methyl aminopropionic acid (1.5 g,7.5 mmol) was dissolved in 20mL of dichloromethane, and 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) were added thereto, and the mixture was stirred and dissolved at room temperature for 16 hours. After the completion of the reaction, the mixture was purified by column chromatography and concentrated to give Compound 1a (4.0 g) in a yield of 52%.

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

Step 2 Synthesis of Compound 1b

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

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

Step 3 Synthesis of Compound 1c

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

Compound 1b (924 mg,2.5 mmol) was dissolved in 6mL of acetonitrile and 4mL of tetrahydrofuran at room temperature, potassium carbonate (460 mg,5.0 mmol), potassium iodide (208 mg,1.25 mmol) and 6-bromohexyl 2-hexyldecanoate (1.0 g,2.5 mmol) were added, heated to 70℃and stirred for 4h. Purification by column chromatography and concentration gave compound 1c (1.4 g) in 79% yield.

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

Step 4 Synthesis of Compound 1d

Cholesterol (2.8 g,7.3 mmol) was dissolved in 30mL of methylene chloride, 4-dimethylaminopyridine (1.7 g,14.3 mmol) was added, phenyl p-nitrochloroformate (1.6 g,8.0 mmol) was added in portions, and the reaction was stirred at room temperature for 4 hours. TLC showed complete cholesterol reaction and reaction was ready for use.

2-Bromoethylamine (1.5 g,7.3 mmol) was dissolved in 30mL of methylene chloride at room temperature and added to the above reaction solution. 4-dimethylaminopyridine (1.7 g,14.3 mmol), triethylamine (1.1 g,11.0 mmol) were added, the reaction was stirred at room temperature for 16h, after completion of the reaction by TLC, 20mL of dichloromethane was added, followed by washing with 30mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give product 1d (872 mg) in 58% yield.

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

Step 5 Synthesis of Compound CP01

Compound 1C (576 mg,0.8 mmol) was dissolved in tetrahydrofuran, acetonitrile was added, compound 1d (872 mg,1.6 mmol), potassium carbonate (447 mg,3.2 mmol), potassium iodide (268 mg,2.0 mmol) was stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium chloride solution into the obtained filtrate, extracting with dichloromethane for 2 times, mixing the organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain the product CP01 (120 mg, pale yellow oily substance) with a yield of 25%.

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

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)

EXAMPLE 2 Synthesis of Compound CP05

Step 1 Synthesis of Compound 5a

2-Hexyldecanoic acid (2.12 g,5.0 mmol) was dissolved in 30mL of methylene chloride, and 6-bromo-n-hexanol (0.93 g,5.0 mmol), DMAP (0.21 g,2.0 mmol) and triethylamine (0.62 g,6.0 mmol) were added thereto and dissolved by stirring. EDC. HCL (1.10 g,6.0 mmol) in methylene chloride was added dropwise, and after the completion of the addition, the mixture was stirred at room temperature for 16 hours. Adding water for quenching, adding dilute hydrochloric acid, adjusting the pH value to 1-3, and separating. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 1a (1.45 g, pale yellow oil) in 70% yield.

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

Step 2 Synthesis of Compound 5b

Compound 5a (1.28 g,3 mmol) was dissolved in 20mL of ethanol at room temperature, piperazine (3.88 g,45 mmol) was added, the temperature was raised to 50℃and stirred for 8h, the reaction progress was monitored, the reaction was cooled to room temperature after complete consumption, 45℃was removed, the crude product was dissolved in dichloromethane, 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) in 89% yield.

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

Step 3 Synthesis of Compound 5c

4-Bromobutanol (0.77 g,5.0 mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (1.22 g,10 mmol) was added, phenyl p-nitrochloroformate (1.11 g,5.5 mmol) was added in portions, the reaction was stirred at room temperature for 3h, cholesterol (2.16 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of dichloromethane was added, then washed with 30mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give compound 5c (1.83 g, pale yellow oil) in 65% yield.

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

Step 4 Synthesis of Compound CP05

Compound 5b (425 mg,1.0 mmol) was dissolved in tetrahydrofuran, acetonitrile was added, compound 5C (679 mg,1.2 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, mixing the organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain the product CP05 (515 mg, pale yellow oily substance) with a yield of 57%.

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

Example 3 preparation and characterization of nucleic acid-lipid nanoparticle compositions (mRNA-LNP)

Table 1 major laboratory consumables

Table 2 main equipment for experiment

TABLE 3 other principal reagents

2. Experimental protocol

Preparation of mRNA-LNP

The lipid components were dissolved in ethanol at a molar percentage of 49.25:49.25:1.5, and firefly luciferase mRNA was dissolved in 25mM sodium acetate buffer pH 4.0 at a final concentration of 135ng/uL. The mRNA-LNP composition was prepared by the nanoparticle preparation instrument Ignite of the microfluidic device Precision Nanosystems at a lipid mixture to mRNA flow ratio of 1:3. The prepared mRNA-LNP is dialyzed and ultrafiltered to concentrate into sucrose buffer with the pH of 7.5 to 20mM Tris, 10.5mM sodium acetate and the mass fraction of 87 percent, and an experimental sample is obtained after sterile filtration.

Characterization of mRNA-LNP

The prepared mRNA-LNP experimental sample is diluted by 50 times by using a buffer solution (the final concentration is 2-100 ng/. Mu.L), and the average particle size, PDI and zeta potential of the nano particles are detected by using a Markov particle size potentiometer, wherein the average particle size and PDI are respectively measured by using a ZEN0040 type DLS sample cell, the loading volume is 200 mu.L, the zeta potential is measured by using a DTS1070 potential cell, and the loading volume is 800 mu.L. The detection of mRNA content and encapsulation efficiency was performed using the Quant-iT ^TM riboGreen RNA detection kit, the free mRNA content C _{Free form} was detected with TE buffer, the total mRNA content C _{Total (S)} was detected with 2% Triton buffer, and the encapsulation efficiency was calculated as formula EE= (1-C _{Free form}/C_{Total (S)}). Times.100%. The experimental results are shown in table 4.

TABLE 4 characterization of Luc-mRNA-LNP physicochemical parameters

Patent CN112424214A Compound (3)

Patent US7514099B2 compound (CLinDMA):

patent CN116059170a compound:

Patent CN10224559B compound:

patent CN102421417B compound:

The result shows that the mRNA-LNP formed by the cationic lipid compound, phospholipid (DOPE), PEG lipid and mRNA has good physical and chemical parameters, the average particle size is about 80-105 nm, the PDI is less than 0.15, the cationic lipid compound has good polydispersity, the zeta potential is about-5 mV-5mV, the encapsulation rate of the LNP to the mRNA is more than 89%, and the encapsulation rate is obviously higher than that of the LNP of a control group.

Example 4 in vitro cell transfection Activity of mRNA-LNP

Hep3B cells were seeded at a density of 10000 cells/well into 96-well white opaque cell culture plates, and after 24 hours, 100ng Luc-mRNA-LNP per well were transfected, the cell culture plates were placed in 37 ℃ and 5% CO ₂ cell incubator for culture, and the negative control group was transfected with an equal volume of physiological saline. After 24 hours, bioluminescence assay was performed using the Promega firefly luciferase assay kit, and the results are shown in fig. 1. The results show that the three-component LNP composition formed by using the cationic lipid compound can realize high expression of Luc-mRNA in cells, and the expression level is obviously superior to that of a control group.

EXAMPLE 5 mRNA-LNP animal immunoassay

We used the novel coronavirus S protein mRNA to evaluate mRNA-LNP immune activity in mice using LNP formulations of ionizable cationic lipid compounds to DOPE to DMG-PEG2000 = 49.25:49.25:1.5 molar ratio for mRNA encapsulation, see table 4 for specific formulations. Female BALB/c mice of 6-8 weeks of age were randomly divided into 3 groups of 6 mice/group and immunized by the immunization route of hind leg intramuscular injection. Immunization was performed on day 0 and day 14, respectively, at an immunization dose of 5 μ gmRNA-LNP/dose. Day 14 (before the second immunization) and day 28 of immunization were collected and serum was isolated, and specific antibody titers against the SARS-CoV2 virus S protein antigen were detected by ELISA (enzyme-linked immunosorbent assay). Antibody titer detection value GMT (95% ci) as shown in fig. 2, the antigen-specific antibody titer induced after the three-component LNP constructed by the lipid compound provided by the present invention delivered the new crown mRNA was significantly higher than that of the control group.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims

1. A lipid compound represented by formula (I):

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 R ₁ and R ₂ is selected from steroid groups.

2. The compound according to claim 1, selected from

L ₁、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

L ₁、L₂、G₁、G₂、R₁、R₂、R₃, p and q and r are as defined in claim 1.

4. A compound according to any one of the preceding claims or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof;

Wherein the steroid group has the following structure:

m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, the steroid group is selected from:

Wherein R' is C _1-20 alkyl.

5. A compound according to any one of the preceding claims, selected from:

Or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein L ₁、L₂、G₁、G₂、R₁、R₃, p and q and r are as defined in claim 1.

6. A compound according to any one of the preceding claims, selected from:

7. The compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein

R ₁ is selected from optionally substituted C ₁-C₂₀ alkyl, wherein one or more of the C ₁-C₂₀ alkyl groups-CH ₂ -may optionally be replaced by O, S, -NR _a -, carbocyclyl, or R ₁ is selected from

R ₆ is selected from hydrogen, halogen, hydroxy, C ₁-C₂₀ alkyl.

8. The compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein

R ₁ is selected from

Preferably, the R ₁ is selected from the following structures:

9. a compound according to claim 1, selected from:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

10. A lipid nanoparticle comprising the lipid compound according to any one of claims 1-9, or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof.

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

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

13. The lipid nanoparticle according to claim 12, wherein the therapeutic 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 of treating and/or preventing a disease comprising administering to a subject in need thereof a therapeutically effective amount of a lipid nanoparticle according to any one of claims 10-13 or a pharmaceutical composition according to claim 14.

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