A class of degradable liposomes and lipid nanoparticles for active molecule delivery

Abstract

The present invention provides an ionizable lipid compound having an optimized carbon chain length and an amine head, thereby increasing the delivery efficiency of active molecules, including but not limited to nucleic acids, proteins and small molecule drugs, etc.; the present invention also relates to lipid nanoparticles (lipid nanoparticles, LNPs) comprising the ionizable lipid compound and the active molecule, and a pharmaceutical composition comprising the lipid nanoparticles.

Description

Technical Field

[0001] The present invention provides an ionizable lipid compound having an optimized hydrophobic carbon chain length and an ionizable amine head structure, thereby being capable of efficiently delivering active molecules, including but not limited to nucleic acids, proteins, and small molecule drugs, etc. The present invention also relates to lipid nanoparticles (lipid nanoparticles, LNPs) comprising the ionizable lipid compound and the active molecule, and a pharmaceutical composition comprising the lipid nanoparticles. Background Art

[0002] Biomacromolecules such as nucleic acids and proteins have great advantages and potential in regulating gene expression and preventing and treating malignant diseases. However, biomacromolecules are a type of charged molecules with strong hydrophilicity, which makes it difficult for them to cross the cell membrane and enter the cell on their own. In addition, nucleic acid drugs such as RNA are easily degraded by nucleases, which limits their stability in the body and further restricts the drugability of RNA.

[0003] Lipid nanoparticles (LNPs) are usually composed of four components: cationic lipids or ionizable lipids, phospholipids, cholesterol, and PEG lipids. LNPs can encapsulate nucleic acid drug molecules such as RNA and protect them from nuclease degradation, and have high stability and biocompatibility. Therefore, they have become a promising drug delivery system and have been clinically applied.

[0004] The drug delivery efficiency of LNP is affected by its composition. Among them, the amine head of cationic lipids or ionizable lipids can promote the binding and encapsulation of LNP and biomacromolecules through electrostatic interaction, and improve the efficiency of LNP uptake by cells. Studies have shown that the length of the hydrophobic carbon chain of cationic lipids or ionizable lipids affects the stability of LNP and the drug encapsulation and delivery efficiency. For example, Min Qiu et al. (PNAS 2022, 119, e2116271119) reported that the delivery efficiency of LNP increases with the increase of the length of the lipid hydrophobic carbon chain. For example, lipid molecules containing 16 carbon atoms in the hydrophobic chain have the highest nucleic acid delivery efficiency than lipid molecules containing 12-14 carbon atoms in the hydrophobic chain; Chun Chan et al. (Curr Top Med Chem 2021, 21, 92–114) also reported that lipid molecules with a carbon chain length of 12 to 18 carbon atoms provide sufficient fluidity and rigidity for LNP; Shuai Liu et al. (Nature Mater, 2021, 20, 701–710) showed in this document that the carbon chain length affects the delivery efficiency of nucleic acid drugs, and lipid molecules with carbon chain lengths of 10, 11 and 12 have higher delivery efficiencies.

[0005] In summary, the hydrophobic carbon chain length of cationic lipids or ionizable lipids affects the formation and drug delivery efficiency of LNPs. Therefore, a balance of these factors needs to be considered when designing LNPs to improve their delivery efficiency and stability in vivo. Summary of the invention

[0006] The present invention provides an ionizable lipid compound with an optimized hydrophobic carbon chain length and an amine head. Different from the cationic lipids or ionizable lipid compounds containing a carbon chain length of 12 to 18 carbon atoms in the prior art, the inventors of the present invention have found that for the ionizable lipid compound of the present invention, further reducing the hydrophobic carbon chain length increases the delivery efficiency. For this reason, the inventors of the present invention have completed the present invention.

[0007] In a first aspect, the present invention provides a liposome compound of formula (I),

[0008]

[0009] in

[0010] R 2a and R 3a Each is independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group or Ht;

[0011] t and s are each independently 0 or 1, and when t or s is zero, it indicates that the part is directly a single bond; provided that t and s are not 0 at the same time;

[0012] A1, A2 and A3 are each independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group or a divalent heteroaromatic hydrocarbon group or a combination of the above;

[0013] Ht is independently -R1-X-R2-Y-R3-Z-R4 at each occurrence,

[0014] in

[0015] R1 is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group,

[0016] or a divalent heteroaromatic hydrocarbon group;

[0017] X is independently

[0018] in

[0019] m, n, p, q and r are each independently 1-6;

[0020] W is O, S or NR c ;

[0021] L1, L3, L5, L7 and L9, which are directly connected to R1 or R2 and are each independently a single bond, O, S or

[0022] NR d ;

[0023] L2, L4, L6, L8 and L 10 are each independently a bond, O, S or NR e ;

[0024] V is an aliphatic hydrocarbon group, OR f , SR g or NR h R i ,

[0025] Where R b , R c , R d , R e , R f , R g , R h and R i are each independently hydrogen, hydroxyl, an oxyaliphatic radical, a monovalent aliphatic radical, a monovalent heteroaliphatic radical, a monovalent aromatic radical or a monovalent heteroaromatic radical;

[0026] Y and Z are each independently S or O at each occurrence;

[0027] R2, at each occurrence, is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group;

[0028] R3, at each occurrence, is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group;

[0029] R4 is independently a hydrophobic group at each occurrence selected from -(CH2CH2O) y -C3-C8 alkyl, -(CH2CH2O) y -C3-C8 alkenyl or -(CH2CH2O) y -C3-C8 alkynyl, wherein y is 0 or 1 or 2.

[0030] In some embodiments, t and s are both 0; or t is 0 and s is 1; or t is 01 and s is 0.

[0031] In some embodiments, R1 is C 1-6 (For example, C 1-4 ) a divalent aliphatic hydrocarbon group or C 1-6(For example, C 1-4 ) a divalent heteroaliphatic hydrocarbon group, preferably C 1-4 Divalent alkyl or C 1-4 A divalent heteroalkyl group.

[0032] In some embodiments, X is wherein the variables are as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH. In further embodiments, X is

[0033] Where R d and R e As defined in formula (I). In some embodiments, R d and R e Each independently is H or C 1-4 A monovalent aliphatic hydrocarbon group, preferably H or C 1-4 A monovalent alkyl group.

[0034] In some embodiments, Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O.

[0035] In some embodiments, R2 is independently a single bond or C 1-6 A divalent aliphatic hydrocarbon group (e.g., C 1-4 A divalent aliphatic hydrocarbon group, preferably C 1-4 A divalent alkyl group, more preferably C 1-2 divalent alkyl).

[0036] In some embodiments, R3 is independently a single bond or C 1-6 A divalent aliphatic hydrocarbon group (e.g., C 1-4 A divalent aliphatic hydrocarbon group, preferably C 1-4 In a further embodiment, R3 is independently a single bond or or methylene Preferably

[0037] In some embodiments, R4 at each occurrence is independently C3-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, -(CH2CH2O)-C3-C8 alkyl, -(CH2CH2O)-C3-C8 alkenyl, -(CH2CH2O)-C3-C8 alkynyl, -(CH2CH2O)2-C3-C8 alkyl, -(CH2CH2O)2-C3-C8 alkenyl, or -(CH2CH2O)2-C3-C8 alkynyl. In some embodiments, R4 at each occurrence is independently C4-C8 alkyl, C4-C8 alkenyl, C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkenyl, -(CH2CH2O)2-C4-C8 alkynyl, -(CH2CH2O)2-C4-C8 alkyl, -(CH2CH2O)2-C4-C8 alkenyl, or -(CH2CH2O)2-C4-C8 alkynyl. In some embodiments, the alkyl group in the C4-C8 alkyl, -(CH2CH2O)-C4-C8 alkyl or -(CH2CH2O)2-C4-C8 alkyl is a straight-chain alkyl group; the alkenyl group in the C4-C8 alkenyl, -(CH2CH2O)-C4-C8 alkenyl or -(CH2CH2O)2-C4-C8 alkenyl is a straight-chain alkenyl group; the alkynyl group in the C4-C8 alkynyl, -(CH2CH2O)-C4-C8 alkynyl or -(CH2CH2O)2-C4-C8 alkynyl is a straight-chain alkynyl group. In some embodiments, R4 at each occurrence is independently -(CH2CH2O)-(CH2)3CH3, -(CH2CH2O)2-(CH2)3CH3, -(CH2CH2O)-(CH2)4CH3, -(CH2CH2O)2-(CH2)4CH3, -(CH2CH2O)-(CH2)5CH3, -(CH2CH2O)2-(CH2)5CH3, -(CH2CH2O)2-(CH2)5CH3, -(CH2CH2O)-(CH2)6CH3, -(CH2CH2O)2-(CH2)6CH3, -(CH2CH2O)-(CH2)7CH3, or -(CH2CH2O)2-(CH2)7CH3.

[0038] In some embodiments, Ht is independently at each occurrence wherein Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O; R3 is independently a single bond or or methylene R 44is C4-C8 alkyl, C4-C8 alkenyl or C4-C8 alkynyl; in some embodiments, R4 at each occurrence is independently -(CH2)3CH3, -(CH2CH2O)-(CH2)3CH3, -(CH2CH2O)-(CH2)4CH3, -(CH2CH2O)-(CH2)5CH3, -(CH2CH2O)-(CH2)6CH3 or -(CH2CH2O)-(CH2)7CH3.

[0039] In the present invention, A1, A2 and A3 constitute the amine head of the liposome, which reacts with the hydrophobic lipid tail to obtain a series of liposomes. Since the amine head can be protonated, the liposome nanoparticles are positively charged as a whole, and thus can interact electrostatically with negatively charged mRNA and cell membranes and lysosomal membranes, so that the liposomes can effectively encapsulate and deliver mRNA. In some embodiments, the pKa value of the amine head is greater than 4, preferably greater than 6, and more preferably greater than 8.

[0040] In some embodiments, t is 1, s is 0, A2 is a single bond, and A1 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in further embodiments, A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group); in further embodiments, A1 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a is hydrogen or a monovalent aliphatic hydrocarbon group, or is hydrogen or a C1-C6 alkyl group or a methyl group. In some embodiments, t is 1, s is 1, A1, A2 and A3 are each independently a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group; in further embodiments, A1, A2 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group); in further embodiments, A1, A2 and A3 are each independently -CH2CH2- or -CH2CH2CH2-, and R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or a methyl group.

[0041] In some embodiments, for Where R 2a and R 3a Each is independently hydrogen or a monovalent aliphatic hydrocarbon group, or hydrogen or a C1-C6 alkyl group or a methyl group.

[0042] In some embodiments, the liposome of formula (I) is

[0043]

[0044] The liposome of formula (I) of the present invention is biodegradable because it contains a ketal response unit (especially a thioketal corresponding unit when Y and Z are both S) in the Ht group. This type of liposome can efficiently bind to mRNA and form a nanostructure through assembly. After entering the diseased cells, it selectively responds to the highly oxidative environment in the cells and degrades, thereby efficiently releasing mRNA and expressing the response protein.

[0045] In a second aspect, the present invention provides a method for preparing a liposome of formula (I) wherein R1 is ethylene, the method comprising:

[0046] The acrylic compound CH2=CH-X-R2-Y-R3-Z-R4 (II) of formula (II) is mixed with a hydrophilic amine in a molar ratio, wherein the variables are defined as in formula (I), wherein the molar ratio of the hydrophilic amine to the acrylic compound of formula (II) is greater than 4, for example, 4.3:1 or 4.5:1, to ensure that the reaction is fully completed; and then the product is optionally purified by column chromatography to obtain the desired liposome of formula (I);

[0047] wherein the variables R2, X, Y, R3, Z and R4 are as defined in formula (I). In some embodiments, X is wherein the variables are as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are connected to R1 and are each independently a single bond, O, S or NH. In further embodiments, X is Where R d and R e As defined in formula (I). In some embodiments, R d and R e Each independently is H or C 1-4 A monovalent aliphatic hydrocarbon group, preferably H or C 1-4 A monovalent alkyl group.

[0048] In some embodiments, in method (a), the product is purified by column chromatography to obtain the desired liposomes of formula (I), wherein the column chromatography purification method is well known in the art.

[0049] In some embodiments, in the present invention, A1, A2 and A3 and their connected N together constitute the amine head of the liposome, which reacts with the hydrophobic lipid tail to obtain a series of liposomes. Since the amine head can be protonated, the liposome nanoparticles are positively charged as a whole, and can thus electrostatically interact with negatively charged mRNA and cell membranes and lysosomal membranes, thereby enabling the liposomes to effectively encapsulate and deliver mRNA.

[0050] In some embodiments, the hydrophilic amine in the method is selected from

[0051] In some embodiments, the ketal-containing acrylate CH2=CH-C(O)O-R2-Y-R3-Z-R4 is The remaining variables are as defined in formula (I).

[0052] In some embodiments, the reaction is an addition reaction, preferably a Michael addition reaction.

[0053] In some embodiments, the reaction is carried out at a temperature of 60-85°C, such as at 70°C, for 24 to 96 hours or longer.

[0054] The acrylate containing ketal of the present invention can be synthesized according to methods known in the art, such as the method disclosed in CN110101665A. The acrylate containing ketal (thio) is an ester compound obtained by esterification reaction of acrylic acid and alcohol compound containing ketal (thio) ester.

[0055] Other liposomes of the present invention can be prepared by other suitable starting materials through the above-mentioned synthetic routes and other routes known in the art. The method listed above can include one or more additional steps to add or remove suitable protecting groups to ultimately allow the liposome to be synthesized. In addition, each synthetic step can be carried out in an alternative order or sequence to obtain the required material. Synthetic chemical transformations and protecting group methods (protection and deprotection) that can be used to synthesize applicable liposomes are known in the art.

[0056] In a third aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutical carrier and lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise the liposomes of formula (I) of the present invention and pharmaceutically active molecules.

[0057] In some embodiments, the pharmaceutically active molecule is a small molecule drug, a protein, a peptide, a nucleic acid, a carbohydrate or a combination thereof. In further embodiments, the pharmaceutically active molecule is mRNA. In further embodiments, the mRNA may be Firefly luciferase mRNA expressing luciferase.

[0058] In some embodiments, the lipid nanoparticles have a particle size distribution of 50 to 500 nm.

[0059] In some embodiments, the liposome is associated with the pharmaceutically active molecule via non-covalent interactions, covalent bonds, or both.

[0060] In some embodiments, the lipid nanoparticles further contain other lipids selected from cholesterol, dioleoylphosphatidylethanolamine (DOPE, CAS: 4004-05-1) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000, CAS: 147867-65-0) or 1,2-dimyristoyl-RAC-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000, CAS: 160743-62-4) or a combination thereof. At the same time, replacing DOPE with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, CAS: 4235-95-4) or distearoylphosphatidylcholine (DSPC, CAS: 816-94-4) of the same proportion can achieve similar delivery effects. In a further embodiment, the lipid nanoparticles further contain cholesterol, dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-dimyristoyl-RAC-glycero-3-methoxypolyethylene glycol 2000. In the example of the present invention, the molar ratio of liposome: cholesterol: DOPE: DSPE-PEG2000 is 50:38.5:10:1.5.

[0061] In some embodiments, the lipid nanoparticles of the present invention are prepared by the following method: comprising dissolving the ionizable lipid compound of the present invention, cholesterol, DOPE and DSPE-PEG2000 in anhydrous ethanol, respectively, dissolving the active pharmaceutical molecule and 5-20 times the mass thereof (the N / P ratio of liposome to nucleic acid ranges from 5:1 to 20:1) in a 100 mM sodium acetate buffer solution (pH = 5.2). The above ethanol solution and sodium acetate buffer solution are mixed and prepared on a microfluidic machine at a flow rate of 1:3. In a further embodiment, the active pharmaceutical molecule is mRNA.

[0062] definition

[0063] The term "aliphatic" refers to a saturated or unsaturated, linear or branched, acyclic, cyclic or polycyclic hydrocarbon moiety. Examples include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl moieties.

[0064] The term "alkyl" includes hydrocarbon groups selected from straight and branched saturated hydrocarbon groups containing 1 to 30, such as 1 to 24, 1 to 18, such as 1 to 12, further such as 1 to 10, further such as 1 to 8 or 1 to 6 or 1 to 4 carbon atoms. Examples of monovalent alkyl or alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, triacontyl, and the like. Examples of divalent alkyl groups, i.e., alkylene groups, include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, icosylene, triacotylene, and the like.

[0065] A monovalent group is a group formed by removing one hydrogen atom from the corresponding hydrocarbon part. A divalent group is a group formed by removing two hydrogen atoms from the corresponding hydrocarbon part.

[0066] The term "alkenyl" includes hydrocarbon groups selected from straight and branched hydrocarbon groups containing at least one C=C double bond and 2 to 30, such as 2 to 24, 2 to 18, such as 2 to 8, and further such as 2 to 6 carbon atoms. Examples of alkenyl, such as C2-6 alkenyl, include but are not limited to ethenyl / vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, but-1,3-dienyl, 2-methylbut-1,3-dienyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-1,3-dienyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, etc.

[0067] The term "alkynyl" includes a hydrocarbon group selected from straight and branched hydrocarbon groups containing at least one C≡C triple bond and 2 to 30, such as 2 to 24, 2 to 18, such as 2 to 8, and further such as 2 to 6 carbon atoms. Examples of alkynyl, such as C2-6 alkynyl, include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl, 2-butynyl and 3-butynyl.

[0068] The term "cycloalkyl" includes a saturated cycloalkyl selected from monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged or spiral cycloalkyl.Cycloalkyl can include 3 to 30, 3 to 12, such as 3 to 10, further such as 3 to 8, further such as 3 to 6, 3 to 5 or 3 to 4 carbon atoms.Further, for example, cycloalkyl can be selected from a monocyclic group including 3 to 12, such as 3 to 10, further such as 3 to 8, 3 to 6 carbon atoms.The example of monocyclic cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.The term "cycloalkenyl" refers to a non-aromatic, cyclic hydrocarbon moiety containing at least one double bond, such as cyclohexenyl and cyclohexenylene. The term "cycloalkynyl" refers to non-aromatic, cyclic hydrocarbon moieties containing at least one triple bond, cyclooctynyl and cyclooctynylene. Similarly, cycloalkylene, cycloalkenylene and cycloalkynylene are corresponding divalent radicals.

[0069] The term "heteroaliphatic" refers to an aliphatic moiety containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge. The heteroaliphatic moieties of the present invention include alkyl, alkenyl or alkynyl moieties containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge, and cycloalkyl, cycloalkenyl or cycloalkynyl moieties containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge. Cyclic heteroaliphatic moieties include 3-7 membered monocyclic heteroaliphatic moieties and 7-12 membered bicyclic heteroaliphatic moieties containing 1-3 or more, for example 1-3 heterocyclic rings selected from oxygen, nitrogen and sulfur. In some embodiments, the cyclic heteroaliphatic hydrocarbon group is a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1-3 heterocyclic rings selected from oxygen, nitrogen and sulfur. Cyclic heteroaliphatic hydrocarbon groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, oxiranyl, aziridine, thioranyl, azetidinyl, oxetanyl, thiol, dithiol, dihydropyridine, tetrahydropyridine, thiomorpholine, homopiperazinyl, homopiperidinyl, azepanyl, oxetanyl, thiol, oxathiol, dioxetanyl, oxathiol, azacycloheptane, dithiol, azathiol, cycloheptane, dioxetanyl ... , diazepanyl, azathiocanyl, oxazepine, diazepine, thiazepine, dihydrothiophenyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolinyl, indolyl, dioxane, dioxolane, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinyl, dioxo-thiomorpholinyl, azabicyclo[3.1.0]hexyl, azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexyl, etc. Typical heteroaliphatic hydrocarbon groups are heteroalkyl groups, i.e., alkyl groups containing at least one heteroatom of N, O or S, such as C containing one N atom. 1-6 Alkyl, C containing one O atom 1-6 Alkyl, or C containing one S atom 1-6 Alkyl; or C containing one nitrogen atom 1-4 Heteroalkyl, C containing one O atom 1-4 Heteroalkyl, or C containing one S atom 1-4 Heteroalkyl.

[0070] The term "oxyaliphatic" refers to an -O-aliphatic hydrocarbon group. Examples of oxyaliphatic groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.

[0071] The term "aryl" refers to a C6 monocyclic, C10 bicyclic, C14 tricyclic, C20 tetracyclic or C24 pentacyclic aromatic ring system. Examples of aryl include phenyl, phenylene, naphthyl, naphthylene, anthracenyl, anthrylene, pyrenyl and pyrenylene.

[0072] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, 11-14 membered tricyclic and 15-20 membered tetracyclic ring system having one or more heteroatoms (such as O, N, S or Se). Examples of heteroaryl include furanyl, furanylene, fluorenyl, fluorenylene, pyrrolyl, pyrrolylene, thienyl, thienylene, oxazolyl, oxazolylene, imidazolyl, imidazolylene, benzimidazolylene, benzimidazolylene, thiazolyl, thiazolylene, pyridinyl, pyridinylene, pyrimidinyl, pyrimidinylene, quinazolinyl, quinazolinylene, quinolyl, quinolylene, isoquinolyl, isoquinolylene, indolyl and indolylene.

[0073] Unless otherwise indicated, the aliphatic hydrocarbon groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbon group oxy groups, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynyl groups, cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenyl groups, cycloalkynyl groups, cycloalkynyl groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenyl groups, aryl groups and heteroaryl groups mentioned in this application include both substituted and unsubstituted parts. Possible substituents on cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenyl groups, cycloalkynyl groups, cycloalkynyl groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenyl groups, aryl groups and heteroaryl groups include, but are not limited to, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, C2-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl, C1-C 10 Alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C1-C 10 Alkylamino, C2-C 20 Dialkylamino, arylamino, diarylamino, C1-C 10 Alkylsulfonylamino, arylsulfonylamino, C1-C 10 Alkyl imino, aryl imino, C1-C 10The substituents of the C1-C10 alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amide, amidino, guanidine, urea, thiourea, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylate. On the other hand, the possible substituents on aliphatic hydrocarbon, heteroaliphatic hydrocarbon, aliphatic hydrocarbonoxy, alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene include all the substituents listed above, but C1-C10 alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amido, amidino, guanidine, urea, thiourea, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylate. 10 The cycloalkyl group, cycloalkylene group, cycloalkenyl group, cycloalkenylene group, heterocycloalkyl group, heterocycloalkylene group, heterocycloalkenyl group, heterocycloalkenylene group, aryl group and heteroaryl group may be condensed with each other.

[0074] The term "pharmaceutically active molecule" refers to any chemical substance intended for medical diagnosis, cure, treatment or prevention of disease, including small molecule drugs and macromolecular drugs, wherein small molecule drugs are such as chemotherapeutic drugs, and macromolecular drugs are such as oligopeptides, oligosaccharides, oligonucleotides, peptides, proteins or nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 a shows whole-body bioluminescence imaging (IVIS) of Firefly luciferase (Fluc) mRNA delivered by LNPs containing lipid compounds with different hydrophobic chain lengths for 6 hours, and mice were administered via the tail vein (dose: 150 μg / kg); Figure 1 b shows the expression level of luciferase and bioluminescence intensity in liver tissue.

[0076] Figure 2 a shows whole body bioluminescence imaging (IVIS) of liposomes containing Firefly luciferase mRNA of different chain lengths after 6 hours, and mice were intramuscularly administered (dose: 250 μg / kg); Figure 2 b shows the expression level of luciferase and bioluminescence intensity at the tissue level.

[0077] Figure 3 The figure shows the expression level and bioluminescence intensity of luciferase in liver tissue 6 hours after YX27 lipid nanoparticles delivered Firefly luciferase mRNA under different N / P conditions. Mice were administered via the tail vein (dose: 150 μg / kg).

[0078] Figure 4The upper figure shows whole-body bioluminescence imaging (IVIS) of YX27, SM102, and D-Lin-MC3-DMA (MC3) lipid nanoparticles 6 hours after delivery, and mice were administered via the tail vein (dose: 150 μg / kg); Figure 4 The figure below shows the expression level of luciferase and bioluminescence intensity in liver tissue.

[0079] Figure 5 The curve showing the changes in the expression level of luciferase and bioluminescence intensity over time in liver tissue of Firefly luciferase mRNA delivered by YX27 lipid nanoparticles.

[0080] Figure 6 The bioluminescence intensity of liver tissue after 6 hours of delivery of Firefly luciferase mRNA by lipid nanoparticles with different molecular structures is shown. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0083] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0084] In the following examples, cholesterol is a product of MacLean, with the product number C10006595, CAS: 57-88-5.

[0085] In the following examples, DOPE (dioleoylphosphatidylethanolamine, CAS: 4004-05-1) and DMG-PEG 2000 (CAS: 147867-65-) are all products of Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0086] In the following examples, Firefly luciferase mRNA is a product of Shanghai Zhaowei Technology Development Co., Ltd.

[0087] In the following examples, Balb / c mice were produced by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0088] Example

[0089] Example 1: Ionizable lipids Synthesis of compounds

[0090] The ionizable lipid compound is synthesized from a corresponding hydrophilic amine compound and a corresponding hydrophobic tail through a Michael addition reaction, wherein the hydrophobic tail is an acrylate containing a ketal, specifically an acrylate containing a thioketal.

[0091] The hydrophobic tail compound TK is synthesized or prepared as follows:

[0092] TK12 is 2-((2-((2-(dodecyloxy)ethyl)thio)propane-2-yl)thio)ethyl acrylate That is, the compound of formula I-1 of Example 2 of CN110101665A, TK-12 1 HNMR: 1 H NMR (300MHz, CDCl3) δ6.18 (s, 1H), 5.97 (d, J = 10.2Hz, 1H), 4.26 (s, 2H), 3.51 (s, 4H), 3.34 ( s,6H),2.87(s,1H),2.74(s,1H),1.55(s,4H),1.50–1.39(m,1H),1.25(s,6H),0.86(s,1H).

[0093] TK2, TK4, TK6, TK8 and TK10 were synthesized by methods similar to those of Examples 1 and 2 of CN110101665A. 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) and the corresponding short-chain alkyl halides Halo-C u H 2u+1 As starting material, prepare Then react with acryloyl halide (such as acryloyl chloride) to obtain the corresponding TK2, TK4, TK6, TK8 and TK10, wherein

[0094] TK10, 2-((2-((2-(decyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK10 1 The H NMR is shown below: 1H NMR(300MHz, CDCl3)δ6.45(d,1H),6.13(m,1H),5.86(d,1H),4.31(t,2H),3.58( t,2H),3.43(t,2H),2.91(t,2H),2.82(t,2H),1.61-1.26(m,23H),0.87(t,3H).

[0095] TK8, 2-((2-((2-(octyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK8 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ6.40(d,1H),6.13(m,1H),5.86(d,1H),4.31(t,2H),3.58( t,2H),3.43(t,2H),2.91(t,2H),2.82(t,2H),1.61-1.27(m,17H),0.87(t,3H).

[0096] TK6, 2-((2-((2-(hexyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK-6 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ6.41(d,1H),6.12(m,1H),5.83(d,1H),4.32(t,2H),3.59(t, 2H),3.46(t,2H),2.92(t,2H),2.82(t,2H),1.61(m,8H),1.31(m,6H),0.88(t,3H).

[0097] TK4, 2-((2-((2-(butyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate TK4 1 The H NMR is shown below: 1H NMR(300MHz, CDCl3)δ6.41(d,1H),6.12(t,1H),5.83(d,1H),4.32(d,2H),3.59( d,2H),3.46(d,2H),2.92(d,2H),2.82(d,2H),1.61–1.38(m,10H),0.89(d,3H).

[0098] The synthesis of the ionizable lipid compound YX of the present invention can be found in CN110101665A and the like.

[0099] YX9, i.e., compound II-10 of Example 5 of CN110101665A, is synthesized from hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine (CAS: 105-83-9) and TK12 in a manner similar to Example 5 of CN110101665A, but wherein the molar ratio of the hydrophilic amine compound to TK12 is 4.3:1. 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.21(t,8H),3.57(t,8H),3.42(t,8H),2.85-2.76(m,24H ),2.44(t,12H),2.29(s,4H),2.18(s,3H),1.59-1.25(m,114H),0.87(t,12H).

[0100] YX8 is synthesized from a hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine and TK10, wherein the hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine and TK10 are mixed at a molar ratio of 4.3:1, heated at 70°C for 72 hours, and the crude product is purified on a silica gel column using dichloromethane / methanol as an eluent to obtain an ionizable lipid compound YX8. 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.21(t,8H),3.57(t,8H),3.42(t,8H),2.85-2.76(m,24H ),2.44(t,12H),2.28(s,4H),2.19(s,3H),1.60-1.25(m,100H),0.87(t,12H).

[0101] YX17, YX27 and YX31 were synthesized by mixing the hydrophilic amine compound 3,3'-diamino-N-methyldipropylamine with the corresponding hydrophobic tail compound at a molar ratio of 4.3:1.

[0102] YX17 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ4.21(t,8H),3.58(t,8H),3.43(t,8H),2.85-2.77(m,24 H),2.45(m,12H),2.28(s,4H),2.18(s,3H),1.60-1.28(m,83H),0.87(t,12);

[0103] YX27 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.44(t,8H),2.86-2.77(m,24H ),2.45(t,12H),2.28(t,4H),2.18(s,3H),1.60-1.38(m,45H),0.92(t,12H); and

[0104] YX31 1 The H NMR is shown below: 1 H NMR(300MHz, CDCl3)δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H),2 .45(t,12H),2.28(t,4H),2.18(s,3H),1.60(m,37H),1.29(s,25H),0.88(t,12H).

[0105]

[0106] Ionizable lipid compounds YX28, YX35, YX29 and YX33 were synthesized by a similar method to the above from a hydrophilic amine compound N-methyl-2,2-diaminodiethylamine (CAS No. 4097-88-5) or N,N'-bis(2-aminoethyl)-N,N'-dimethyl-1,3-propanediamine (CAS No. 152829-22-6) and a corresponding hydrophobic tail compound.

[0107] YX28 1 The H NMR is shown below: 1H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.44(t,8H),2.86-2.77(m ,24H),2.55-2.46(m,16H),2.23(s,3H),1.60-1.38(m,41H),0.91(t,12H);

[0108] YX29 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.44(t,8H),2.86-2.77(m,24H),2. 56(t,4H),2.46(t,12H),2.34(t,4H),2.22(s,6H),1.6-1.38(m,50H),0.92(t,12H);

[0109] YX33 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H),2.5 7(s,4H),2.46(t,12H),2.35(s,4H),2.23(s,6H),1.60-1.29(m,60H),0.88(t,12H); and

[0110] YX35 1 The H NMR is shown below: 1 H NMR (300MHz, CDCl3) δ4.22(t,8H),3.58(t,8H),3.43(t,8H),2.86-2.77(m,24H ),2.55-2.46(m,16H),2.23(s,3H),1.60(m,35H),1.29(m,24H),0.89(t,12H).

[0111]

[0112] Example 2: Encapsulation of Firefly luciferase mRNA and preparation of lipid nanoparticles

[0113] In order to achieve animal-level mRNA delivery, the inventors prepared mRNA / lipid nanoparticles. The specific method is as follows: the ionizable lipid compounds (YX8, YX17, YX31, YX27, YX28, YX35, YX29 or YX33), cholesterol, DOPE, and DMG-PEG2000 prepared in Example 1 are respectively configured into 10 mg / mL ethanol solutions. Taking YX27 as an example, when preparing mRNA / LNP complexes once, YX27 solution (1.6 mg) is measured and mixed with cholesterol, DOPE, and DMG-PEG2000 in a molar ratio of 50:38.5:10:1.5, and the total volume of the solution is 450 microliters by adding ethanol. Take 150 micrograms of Firefly luciferase mRNA and add it to sodium acetate buffer solution (50mM, pH=5.2), and the total volume of the solution is 1350 microliters. The ionizable lipid compound solution and the mRNA solution were mixed by introducing into a microfluidic machine (speed: 0.2 mL / min ethanol solution, 0.6 mL / min sodium acetate buffer solution), and lipid nanoparticles encapsulating mRNA were prepared, which were dialyzed for 2 hours with PBS as a buffer, and the obtained samples were directly used for animal experiments. Among them, the ratio of the ionizable lipid compound YX27 to mRNA was calculated according to the molar ratio (N / P) of nitrogen atoms in YX27 to phosphorus atoms in the mRNA phosphate backbone. In this embodiment, N / P is 7.5.

[0114] For lipid nanoparticles encapsulating mRNA, Quant-it TM RiboGreen RNA Assay Kit (ThermoFisher Scientific, USA) determines the concentration of free RNA therein, and on this basis, establishes the determination method of the RNA encapsulation efficiency of the nanoparticle composition. The sample is diluted to a concentration of about 5 μg / mL in TE buffer (Solarbio / Solaibao, T1120, pH 8.0). 50 μL of the diluted sample is transferred to a polystyrene 96-well plate, and 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution (Solarbio / Solaibao, T8200) is added to the well. The plate is incubated at a temperature of 37°C for 10 minutes. The reagent is diluted 1:200 in TE buffer, and 100 μL of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (BIOTEK / Synergy H1) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence value of the lysed sample (caused by the addition of Triton X-100).

[0115] The LNPs comprising YX27 in this example were tested as follows: average particle size 185 nm, PDI 0.148, and Zeta potential -7.87 mV.

[0116] Example 3: Animal delivery experiment of mRNA using lipid nanoparticles of the present invention

[0117] To verify the delivery effect of the above Fluc mRNA / LNP preparation, YX27 LNP encapsulating 150μg / kg Fluc mRNA was injected into 6-8 week old female Balb / c mice by tail vein administration or YX27 LNP encapsulating 250μg / kg Fluc mRNA was injected into the hind legs by intramuscular administration. 6 hours later, 200 microliters of D-Lucifin (mass fraction 30%) were injected intraperitoneally, and the luciferase expression and intensity of the whole body, liver, and muscle administration sites were detected using the PerkinElmer IVIS Lumina III small animal in vivo optical imaging system. In order to study the effect of the hydrophobic chain length of lipid compounds on the mRNA delivery efficiency, lipid compounds YX9, YX8, YX17, and YX31 were used to prepare LNP samples encapsulating Fluc mRNA according to the YX27 experimental operation, and their efficiency of expressing Luciferase in mice was studied. The study found that lipid compounds with 4 and 6 carbon atoms in the hydrophobic carbon chain tail structure are significantly more efficient in delivering mRNA than other lipid molecules.

[0118] The results of in vivo Luciferase live imaging luminescence intensity after delivery of Fluc mRNA using different lipid compounds are shown in the following table and Figure 1 and 2 As shown:

[0119]

[0120] Unit: p / s

[0121] Example 4: Effect of N / P ratio of YX27 formulation on mRNA delivery

[0122] In order to study the effect of the mixing ratio of ionizable lipids and mRNA (N / P ratio) in the LNP component on the mRNA delivery efficiency, YX27 and luciferase mRNA mixed samples with N / P ratios of 7.5 and 9 were prepared according to the operating steps of Example 2, and the mRNA delivery and luciferase expression and intensity in the whole body and liver and other parts were studied by tail vein administration (the delivery results are shown in Figure 3 and in the following table).

[0123]

[0124] Example 5: Comparison of the effects of YX27, SM102 and MC3 on mRNA delivery

[0125] According to the operation steps of Example 2, LNPs containing SM102 or MC3 cationic lipids and luciferase mRNA at an N / P ratio of 7.5 were prepared, wherein the Fluc mRNA dosage was 25 μg / kg, 50 μg / kg or 150 μg / kg, respectively; and then the prepared LNPs were administered via tail vein to study the mRNA delivery and luciferase expression and intensity in the whole body and liver. The delivery results are shown in Figure 4 and in the following table.

[0126]

[0127] Unit: p / s

[0128] As can be seen from the above table, the ionizable lipid YX27 of the present invention exhibits a better delivery effect than the commercial cationic lipids SM102 or MC3 at each mRNA dose.

[0129] Example 6: Time-dependent experiments on mRNA delivery and expression

[0130] To verify the relationship between the expression efficiency of Luciferase and time after administration of Fluc mRNA / LNP, 6-8 week old female Balb / c mice were injected with YX27 solution encapsulating 150μg / kg Fluc mRNA via tail vein administration. 200 μL of D-Lucifin (30% by mass) was injected intraperitoneally 2h, 4h, 6h, 18h and 24h after administration, and the expression and intensity of luciferase in the whole body and liver were detected using the PerkinElmer IVIS Lumina III small animal in vivo optical imaging system. The delivery results are shown in Figure 5 As shown in the table below, the imaging results show that the in vivo expression reaches a peak 6 hours after mRNA delivery.

[0131] Time after administration Average Strength Standard Deviation 2h 1.47E+09 1.32E+08 4h 3.01E+09 1.72E+09 6h 3.51E+09 1.37E+09 18h 1.03E+09 7.34E+08 24h 5.49E+08 2.07E+08

[0132] Unit: p / s

[0133] Example 7 Evaluation of mRNA delivery efficiency of YX lipid compounds with different structures

[0134] According to the operation steps of Example 2, LNPs containing SM102, YX27, YX28, YX29, YX33, YX31, or YX35 as an ionizable lipid compound and luciferase mRNA at an N / P ratio of 7.5 were prepared, wherein the Fluc mRNA administration dose was 150 μg / kg. The in vivo luciferase live imaging luminescence intensity results after using different lipid compounds to deliver Fluc mRNA are as follows Figure 6 As shown in the table below, it can be found that when the carbon number is 4 and 6, liposomes composed of different amine heads can efficiently deliver mRNA, and the efficiency is close to that of the commercial ionizable lipid SM102.

[0135]

[0136]

[0137] Unit: p / s

[0138] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A liposome compound, wherein the liposome compound is 2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and lipid nanoparticles, wherein the lipid nanoparticles comprise the liposome compound of claim 1 and a pharmaceutically active molecule.

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