Ionizable lipid

Abstract

The present invention generally relates to the field of ionizable (also called cationic) lipids and, in particular, provides a novel class of such lipids as represented by any of the formulas disclosed herein. The present invention further provides methods for making such lipids, as well as their use, in particular in the preparation of nanoparticle compositions, more specifically nanoparticle compositions containing nucleic acids. The present invention further provides vaccine formulations and pharmaceutical formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein.

Description

Technical Field

[0001] The present invention generally relates to the field of ionizable (also called cationic) lipids, and in particular provides a novel type of such lipids as represented by any of the formulas disclosed herein. The present invention further provides methods for making such lipids, as well as their use, particularly in the preparation of nanoparticle compositions, more specifically nanoparticle compositions containing nucleic acids. The present invention further provides vaccine formulations and pharmaceutical formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein.

Background Art

[0002] Nucleic acid-based drugs are being explored in an increasing number of therapeutic fields. Nevertheless, due to their negative charge, size and instability, the targeted delivery of nucleic acids, such as plasmid DNA, messenger RNA, small interfering RNA, single guide RNA and microRNA, to tissues and cells poses a major challenge. A number of nanoparticle delivery systems are being explored for encapsulating and delivering nucleic acids. These nanoparticles need to combine efficient and stable encapsulation of nucleic acids during storage and in the extracellular environment, with maximal cellular uptake and efficient release of the payload from endosomes into the cytosol. Lipid-based nanoparticles are clinically used to deliver small interfering RNAs and mRNA vaccines and represent the most advanced class of RNA delivery vehicles. Lipid-based nanoparticles typically consist of a cationic or ionizable lipid that can be protonated at acidic pH, a helper lipid, a PEGylated lipid, and a sterol. Each component has a special function in the stability and activity of the LNP. Sterols and PEGylated lipids are essential for the LNP structure and stability, while phospholipids can contribute to stability and endosomal escape. Cationic or ionizable lipids are then considered the main drivers of activity and tolerability by controlling mRNA encapsulation, cell uptake, and endosomal escape. Although effective nucleic acid delivery vehicles, LNPs can induce dose-limiting toxicities such as complement activation-related pseudoallergic reactions, inflammatory cytokine release, and cytotoxicity due to the accumulation of non-degradable ionizable lipids in the cell membrane.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, in order to improve the effectiveness and safety of LNP-delivered nucleic acid drugs, further improvement of cationic or ionizable lipid chemistry is required. The present invention relates to a new class of ionizable lipids having improved properties over currently available classes of ionizable lipids, as defined by the set of claims of the present invention.

Means for Solving the Problems

[0005] In a first aspect, the present invention provides a lipid, particularly an ionizable lipid represented by formula (I)

Chemical Formula

[0006] In certain embodiments, the present invention provides a lipid, particularly an ionizable lipid represented by any one of formulas (Ia), (Ib), and (Ic) as defined herein:

Chemical formula

[0007] In yet a further embodiment, the present invention provides a lipid, in particular an ionizable lipid represented by any one of formulas (II) or (III) as defined herein:

Chemical formula

[0008] The present invention further provides a lipid, in particular, an ionizable lipid defined herein and represented by formula (IIa);

Chemical formula

[0009] The present invention further provides a lipid, in particular, an ionizable lipid selected from the list defined herein and including the following:

Chemical formula

Chemical formula

[0010] In still further embodiments, the present invention provides an ionizable lipid, in particular, as defined herein; wherein the total number of C atoms in R 1 and R 2 together is at least 14.

[0011] The present invention further provides an ionizable lipid, in particular, as defined herein; wherein the total number of C atoms in R 7 and R 7 " is at least 12.

[0012] In further embodiments, the present invention provides a lipid, in particular, an ionizable lipid as defined herein; wherein m and n are the same and are integers selected from 1, 2, 3, and 4, preferably 2.

[0013] The present invention further provides an ionizable lipid, in particular, as defined herein; wherein R 5 is -NH-.

[0014] In a further aspect, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising a lipid, particularly an ionizable lipid as defined herein. The nanoparticle composition may further comprise a phospholipid, a sterol, and a PEG lipid.

[0015] In yet a further embodiment of the present invention, the lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise an active agent, particularly a nucleic acid, preferably mRNA.

[0016] In a further aspect, the present invention provides the use of a lipid, particularly an ionizable lipid as defined herein, in the manufacture of lipid nanoparticles or lipid nanoparticle compositions.

[0017] In a last aspect, the present invention provides a pharmaceutical composition comprising a lipid nanoparticle or lipid nanoparticle composition as defined herein and a pharmaceutically acceptable agent.

[0018] The present invention also provides a pharmaceutical composition as defined herein for use in human and / or veterinary medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Referring now to the drawings, it is emphasized that the details shown are by way of example and for purposes of illustrative discussion of different embodiments of the present invention only. They are presented as a basis for providing what is considered to be the most useful and readily understood description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention. The description using the drawings makes it apparent to those skilled in the art how some forms of the present invention may be actually implemented.

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0020] Next, the present invention will be further described. In the following sections, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect unless it is clearly shown that it is not suitable. In particular, any feature shown as being preferred or advantageous can be combined with any other feature shown as being preferred or advantageous. Unless the context otherwise indicates, an asterisk is used herein to indicate that a monovalent or divalent radical described in this specification is bonded to the structure of which it forms part in relation to that radical.

[0021] As already described above, in a first aspect, the present invention is a lipid, particularly an ionizable lipid represented by formula (I);

Chemical formula

[0022] Accordingly, the present invention also provides a lipid, particularly an ionizable lipid represented by any one of formulas (Ia), (Ib) and (Ic);

Chemical formula

[0023] When describing the compounds / lipids of the present invention, the terms used should be construed in accordance with the following definitions, unless the context indicates otherwise: The term "alkyl", by itself or as part of another substituent, refers to a group of the formula C x H 2x+1refers to a fully saturated hydrocarbon (where x is a number of 1 or greater). Generally, the alkyl groups of the present invention contain from 1 to 20 carbon atoms. The alkyl group may be linear or branched and may be substituted as shown herein. When a subscript is used herein after a carbon atom, the subscript refers to the number of carbon atoms that the specified group may contain. Thus, for example, -C 1-4 alkyl means alkyl of 1 to 4 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g., n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers, undecyl and its isomers, dodecyl and its isomers, tridecyl and its isomers, tetradecyl and its isomers, pentadecyl and its isomers, hexadecyl and its isomers, heptadecyl and its isomers, octadecyl and its isomers, nonadecyl and its isomers, eicosanyl and its isomers.

[0024] The term "optionally substituted alkyl" refers to an alkyl group that may be substituted at any available point of attachment with one or more substituents (e.g., from 1 to 4 substituents, such as 1, 2, 3, or 4 substituents). Non-limiting examples of such substituents include esters, carboxylic acids, alkyl moieties, alkene moieties, alkyne moieties, and the like. In the context of the present invention, the alkyl, alkene, and alkyne moieties defined herein may further contain one or more heteroatoms in that, for example, a C atom in an alkyl, alkene, or alkyne chain is replaced by a heteroatom selected from N, S, or O.

[0025] As used herein, the term "alkenyl" or "alkene" means, unless otherwise specified, a straight-chain, cyclic, or branched hydrocarbon radical containing at least one carbon-carbon double bond. Examples of alkenyl radicals include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, hexadienyl, etc., which are located at terminal or internal positions, etc. Generally, the alkenyl or alkene moiety of the present invention contains 2 to 20 C atoms. Optionally, the alkenyl which may be substituted refers to an alkenyl which may optionally have one or more substituents (e.g., 1, 2, 3, or 4) selected from those defined above for substituted alkyl.

[0026] As used herein, the term "alkynyl" or "alkyne" means, unless otherwise specified, a straight-chain or branched hydrocarbon radical containing at least one carbon-carbon triple bond. Examples of alkynyl radicals include ethynyl, E- and Z-propynyl, isopropynyl, E- and Z-butynyl, E- and Z-isobutynyl, E- and Z-pentynyl, E,Z-hexynyl, etc. Generally, the alkenyl or alkene moiety of the present invention contains 2 to 20 C atoms. Optionally, the alkynyl which may be substituted refers to an alkynyl which may optionally have one or more substituents (e.g., 1, 2, 3, or 4) selected from those defined above for substituted alkyl.

[0027] The term "cycloalkyl", by itself or as part of another substituent, is a cyclic alkyl group, i.e., a monovalent, saturated, or unsaturated hydrocarbyl group having 1, 2, or 3 cyclic structures. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups containing 1 to 3 rings, including monocyclic, bicyclic, or polycyclic alkyl groups. The cycloalkyl group may contain more than 3 carbon atoms in the ring and generally contains 3 to 15 atoms according to the present invention. Specific examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantanyl, and cyclodecyl, with cyclopropyl being particularly preferred. "Optionally substituted cycloalkyl" refers to a cycloalkyl that may optionally have one or more substituents (e.g., 1 to 3 substituents, e.g., 1, 2, 3, or 4 substituents) selected from those defined above for substituted alkyl.

[0028] When the defined alkyl group is divalent, i.e., has two single bonds for attachment to two other groups, they are referred to as "alkylene" groups. Non-limiting examples of alkylene groups include methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1,2-dimethylethylene, pentamethylene, and hexamethylene. Similarly, when the defined alkenyl group and the defined alkynyl group are each a divalent radical having a single bond for attachment to two other groups, they are each referred to as "alkenylene" and "alkynylene", respectively.

[0029] As used herein, the term "heterocyclic ring" alone or as part of another radical refers to a non-aromatic, fully saturated, or partially unsaturated cyclic radical having at least one heteroatom in at least one carbon atom-containing ring (e.g., a monocyclic ring system of 3 to 13 members, a bicyclic ring system of 7 to 17 members, or a tricyclic ring system of 10 to 20 members, or containing a total of 3 to 10 ring atoms). Each ring of a heterocyclic radical containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms, and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocyclic radical may be bonded to any heteroatom or carbon atom of the ring or ring system where the valence permits. The rings of a polycyclic heterocyclic ring may be fused, bridged, and / or joined via one or more spiro atoms. Optionally, a heterocyclic ring that may be optionally substituted refers to a heterocyclic ring that may optionally have one or more substituents (e.g., 1 to 4 substituents, or e.g., 1, 2, 3, or 4 substituents) selected from those defined above for substituted alkyl. Non-limiting examples of heterocyclic rings include piperidinyl, pyrrolidinyl, azepanyl, morpholinyl, etc.

[0030] As used herein, the term "aryl" (also referred to herein as an aromatic heterocycle) refers to a polyvalent unsaturated aromatic hydrocarbyl group typically containing 6 to 10 atoms, where a single ring (i.e., phenyl) or multiple aromatic rings are fused together (e.g., naphthalene or anthracene) or covalently bonded; here, at least one ring is aromatic. The aromatic ring may optionally contain 1 to 3 additional rings (any of cycloalkyl, heterocyclyl, or heteroaryl) fused thereto. Aryl is also intended to include partially hydrogenated derivatives of the carbocyclic systems listed herein. Non-limiting examples of aryl include phenyl.

[0031] An aryl ring or a heteroaryl ring as defined herein may optionally be substituted at any available point of attachment with one or more substituents (e.g., 1 to 5 substituents, such as 1, 2, 3, or 4). Non-limiting examples of such substituents include halogen, hydroxyl, oxo, nitro, amino, hydrazine, aminocarbonyl, azide, cyano, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkylalkyl, alkylamino, alkoxy, -SO 2 -NH 2 , aryl, heteroaryl, aralkyl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkylaminocarbonyl, heteroarylalkyl, alkylsulfonamide, heterocyclyl, alkylcarbonylaminoalkyl, aryloxy, alkylcarbonyl, acyl, arylcarbonyl, aminocarbonyl, alkylsulfoxide, -SO 2 R a , alkylthio, carboxyl, etc., selected from, where R a is alkyl or cycloalkyl. When a carbon atom in an aryl group is replaced by a heteroatom, the resulting ring is referred to herein as a heteroaryl ring.

[0032] The term "heteroaryl," as used herein, either by itself or as part of another group, refers to an aromatic ring of 5 to 12 carbon atoms or a ring system containing 1 to 3 rings, which are fused or covalently bonded to each other, typically a ring system containing 5 to 8 atoms, but not limited thereto, at least one of which is aromatic, and one or more carbon atoms in one or more of these rings can be replaced by oxygen, nitrogen, or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl, or heterocyclyl ring. Non-limiting examples of such heteroaryl include pyridinyl, azepinyl,....

[0033] "Optionally substituted heteroaryl" refers to heteroaryl which optionally has one or more substituents (e.g., 1 to 4 substituents, e.g., 1, 2, 3, or 4) selected from those defined above for substituted aryl.

[0034] As used herein, the term "oxo" refers to the group =O.

[0035] As used herein, the term "alkoxy" or "alkyloxy" refers to a radical of the formula O-R b (wherein R b is alkyl). Preferably, alkoxy is C 1 ~C 10 alkoxy, C 1 ~C 6 alkoxy, or C 1 ~C 4 alkoxy. Non-limiting examples of suitable alkoxys include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy. When the oxygen atom in an alkoxy group is replaced by sulfur, the resulting radical is called thioalkoxy. "Haloalkoxy" is an alkoxy group in which one or more hydrogen atoms in the alkyl group are replaced by halogen. Non-limiting examples of suitable haloalkoxys include fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2-fluoroethoxy, 2-chloroethoxy, 2,2-difluoroethoxy, 2,2,2-trichloroethoxy; trichloromethoxy, 2-bromoethoxy, pentafluoroethyl, 3,3,3-trichloropropoxy, 4,4,4-trichlorobutoxy and the like can be mentioned.

[0036] The terms "carboxy", "carboxyl", or "hydroxycarbonyl" refer to the -CO 2 H group, either by itself or as part of another substituent. Thus, carboxyalkyl is an alkyl group as defined above having at least one substituent which is -CO 2 H.

[0037] The term "alkoxycarbonyl" refers to a carboxy group linked to an alkyl radical, either by itself or as part of another substituent, i.e., -C(=O)OR e is formed, where R e is as defined above for alkyl. The term "alkylcarbonyloxy" refers to -O-C(=O)R e either by itself or as part of another substituent, where R e is as defined above for alkyl.

[0038] When the term "substituted" is used in the present invention, it always means that one or more hydrogens on the atom indicated in the expression using "substituted" are replaced by a selection from the indicated groups, provided that the normal valence of the indicated atom is not exceeded and the substitution results in a chemically stable compound, i.e., a compound that can be isolated to a useful purity from the reaction mixture and is sufficiently robust to achieve formulation into a therapeutic agent while remaining active.

[0039] When a group may optionally be substituted, such group may be substituted one or more times, preferably once, twice or three times. The substituents may be selected from the group including, for example, halogen, hydroxyl, oxo, nitro, amide, carboxy, amino, cyano - haloalkoxy, and haloalkyl.

[0040] As used herein, the terms "alkyl, aryl, or cycloalkyl, each optionally substituted with ~", or "alkyl, aryl, or cycloalkyl, optionally substituted with ~", etc. refer to optionally substituted alkyl, optionally substituted aryl, and optionally substituted cycloalkyl.

[0041] Furthermore, when the group is divalent, i.e., has two single bonds for attachment to two other groups, its presence can be as either in both directions in the molecule even if not specifically shown in the structural formula or the definition of the R group. For example, -(C=N-NH as part of X 2 ) may be represented by X being, for example, -(C=N-NH 2 )-, or alternatively by being the reverse -(NH 2 -C=N)-.

[0042] In the context of the present invention, the term lipid means a chemically defined substance that is insoluble in water but soluble in, inter alia, alcohol, ether, and chloroform. Ionizable or cationic lipids are typically lipids composed of three sections: an amine head group, a linker portion, and a hydrophobic tail. The term "ionizable" (or alternatively cationic) in the context of a compound or lipid means the presence of any uncharged group in said compound or lipid that can dissociate by generating an ion (usually an H + ion), and thus becomes positively charged itself. Alternatively, any uncharged group in said compound or lipid can generate an electron and thus can become negatively charged.

[0043] In the context of the present invention, the linker portion can be selected from a variety of different linkers, but disulfide, ketal, and ether linkers are particularly preferred. Thus, and in order to obtain those lipid characteristics, the compounds of the present invention are R 1 and R 2comprising a lipid tail represented by, wherein the total number of C atoms of the combined two groups is at least 8, for example, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20.

[0044] Thus, in the context of the present invention, R 1 may, for example, contain 3 C atoms, while R 2 may contain 5 C atoms, whereby the total number of C atoms of the combined two groups is at least 8. In any case in the context of the present invention, R 1 and R 2 are not identical to each other.

[0045] Thus, in certain embodiments, the present invention provides a lipid, particularly an ionizable lipid as defined herein, particularly an ionizable lipid defined herein and represented by any one of Formula (II) or (III);

Chemical Formula

[0046] In yet a further embodiment, the present invention provides a lipid, particularly an ionizable lipid defined herein and represented by any one of formulas (IIa), (IIb), or (IIc);

Chemical Formula

[0047] In yet a further embodiment, the present invention provides a lipid, particularly an ionizable lipid defined herein and represented by any one of formulas (IIIa), (IIIb) or (IIIc);

Chemical formula

[0048] In a further specific embodiment, the present invention provides an ionizable lipid, in particular a lipid as defined herein, in particular a lipid as defined herein, represented by any one of formulas (IV) or (V), which is an ionizable lipid as defined herein;

Chemical formula

[0049] In a further embodiment, the present invention provides a lipid, in particular, an ionizable lipid defined herein and represented by any one of formulas (IVa), (IVb), or (IVc);

Chemical formula

[0050] In a further embodiment, the present invention provides a lipid, in particular an ionizable lipid defined herein and represented by any one of formulas (Va), (Vb), or (Vc);

Chemical formula

[0051] In another specific embodiment, the moiety -NR 3 R 4 can be selected from the list including -N-(C 1-6 alkyl) 2 or an N-containing non-aromatic heterocyclic ring using the N atom as a bonding point. In a very specific embodiment, the moiety -NR3 R 4 is -N-(CH 3 ) 2 , -pyrrolidinyl, -piperidinyl, or azepanyl; in particular, it may be selected from the list containing -N-(CH 3 ) 2 , or azepanyl.

[0052] In very specific embodiments, the present invention provides compounds according to any of formulas (I), (II), (III), (IV), (V) and any of their variants (a), (b), or (c), where one or more of the following apply: R 1 and R 2 are each independently selected from -C 1~20 alkyl, -C 2~20 alkenyl, and -C 2~20 alkynyl; where each of said -C 1~20 alkyl, -C 2~20 alkenyl, and -C 2~20 alkynyl is optionally substituted with one to three -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1~20 alkyl, -C 2~20 alkenyl, and -C 2~20 alkynyl; where the total number of C atoms in R 1 and R 2 is at least 8 together; where R 1 and R 2 are different from each other; R 3 and R 4 are each independently -C 1~6 alkyl; or R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may further optionally contain one or more additional N atoms, and / or -C 1~6One to three substituents selected from alkyl and -OH, optionally substituted; R 7 When each of the following is present, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl is independently selected; wherein -C 1-20 alkyl, -C 2-20 alkynyl, -C 2-20 alkynyl may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; m and n are each independently an integer selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -S-S-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH 2 ), -O-CR 8 R 9 -O-, and -S-CR 8 R 9 -S-; Each R 8 and R 9 is independently selected from -H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 5 is selected from -NH- and -O-; and, R 6 is -C 1-6 alkylene.

[0053] In another very specific embodiment, the present invention provides a compound according to any of formulas (I), (II), (III), (IV), (V) and any variant thereof (a), (b), or (c), wherein one or more of the following apply: R 1 and R 2 are each independently -C 1~20 alkyl, -C 2~20Alkenyl, and -C 2~20 selected from alkynyl; wherein R 1 and R 2 the total number of C atoms in is, together, at least 8; wherein R 1 and R 2 are different from each other; R 3 and R 4 are each independently -C 1~6 alkyl; or, R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered non-aromatic heterocyclic ring; R 7 in each case where it exists, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl are independently selected from; m and n are each independently an integer selected from 1, 2, 3, and 4; X is -O-, -S-S-, -O-CR 8 R 9 -O- selected from; each R 8 and R 9 is independently selected from -H, -C 1-6 alkyl; R 5 is -NH-; and R 6 is -C 1-6 alkylene.

[0054] The present invention further provides a lipid, in particular, an ionizable lipid selected from the list defined herein and including the following:

Chemical formula

Chemical formula

[0055] All lipids defined herein can exist as different isomers / stereoisomers. In particular, lipids defined herein can exist in a trans or cis configuration, such as when they contain double bonds. In preferred embodiments, lipids defined herein exist in a cis configuration. The term "cis" means that the functional groups are on the same side of the plane, and the term "trans" means that the functional groups are on opposite sides.

[0056] In still further embodiments, the invention relates to lipids having a total number of C atoms in R 1 and R 2 that together are at least 14, such as at least 15, at least 17, at least 18, at least 19, or at least 20, and in particular, ionizable lipids as defined herein.

[0057] The invention further provides lipids having a total number of C atoms in R 7 and R 7 that is at least 12, and in particular, ionizable lipids as defined herein.

[0058] In further embodiments, the invention provides lipids, and in particular, ionizable lipids as defined herein, where m and n are the same and are integers selected from 1, 2, 3, and 4; such as 1 or 2 or 3 or 4; preferably 2.

[0059] The invention further provides lipids where R 5 is -NH-, and in particular, ionizable lipids as defined herein.

[0060] In a further aspect, the invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, and in particular, ionizable lipids as defined herein.

[0061] In the context of the present invention, the term lipid nanoparticles (LNP), also referred to as solid lipid nanoparticles, means nanoparticles containing lipids. They are often used as pharmaceutical drug delivery systems or pharmaceutical formulations. LNP as a drug delivery vehicle was first approved in 2018 and is currently used in several candidate RNA-based vaccines. Lipid nanoparticles are typically spherical with an average diameter of 10 to 1000 nanometers and have a lipid core matrix that can solubilize lipophilic molecules. The term lipid is used herein in a broader sense and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (steroids such as cholesterol) and waxes. Biomembrane lipids such as phospholipids, sphingomyelin, bile acids and steroids are typically used as stabilizers in LNP.

[0062] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes it suitable for systemic administration, particularly intravenous administration, and nucleic acids typically have a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, for example 50 - 200 nm, preferably 80 - 160 nm.

[0063] Thus, in the context of the present invention, the nanoparticles disclosed herein further comprise one or more additional lipids, either acting or not acting as stabilizers such as phospholipids, steroids and / or PEG lipids.

[0064] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group. In the context of the present invention, particularly suitable PEG lipids are characterized by being C18 - PEG lipids, C14 - PEG lipids (e.g., DMG - PEG or DMG - PEG2000) or C16 - PEG lipids.

[0065] The C18-PEG lipid contains a polyethylene glycol moiety that defines the molecular weight of the lipid, as well as a fatty acid tail containing 18 carbon atoms. In certain embodiments, the C18-PEG2000 lipid is selected from the list comprising: (distearoyl-based)-PEG2000 lipids, such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoyl-based)-PEG2000 lipids, such as DOG-PEG2000 lipid (1,2-dioleoyl-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]).

Chemical formula

[0066] The C14-PEG lipid contains a polyethylene glycol moiety that defines the molecular weight of the lipid, as well as a fatty acid tail containing 14 carbon atoms. In certain embodiments, the C14-PEG2000 lipid is selected from the list based on dimyristoyl, i.e., having two C14 tails: (dimyristoyl-based)-PEG2000 lipids, such as DMG-PEG2000 lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or 2-dimyristoyl-sn-glycero-3-phosphoethanolamine glycol-2000 (DMPE-PEG2000).

Chemical formula

[0067] In the context of the present invention, the term "phospholipid" means a lipid molecule consisting of two hydrophobic fatty acid tails and a hydrophilic head consisting of a phosphate group. The two components are most often linked together by a glycerol molecule, and thus the phospholipids of the present invention are preferably glycerol phospholipids. Further, the phosphate group is often modified by a simple organic molecule such as choline (i.e., giving phosphocholine) or ethanolamine (i.e., giving phosphoethanolamine).

[0068] Suitable phospholipids within the scope of the present invention are selected from the list comprising; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0069] In more specific embodiments, the phospholipid is selected from the list comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0070] In the context of the present invention, the term "sterol", also known as steroid alcohol, is a subgroup of steroids that occur naturally in plants, animals, and fungi, or can be produced by some bacteria. In the context of the present invention, any suitable sterol selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, androsterol, desmosterol, nicastrerol, sitosterol, and stigmasterol; preferably cholesterol can be used.

[0071] In certain embodiments of the present invention, one or more of the following apply: - The LNP contains about 35 mol% to 65 mol% of the ionizable lipid; - The LNP contains about 5 mol% to 25 mol% of the phospholipid; - The LNP contains about 0.5 mol% to 3.0 mol% of the PEG lipid; The amount of the sterol is balanced.

[0072] In still further embodiments of the invention, the lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise cargo molecules such as pharmaceutically active agents (e.g., small molecules), or biomolecules such as peptides, proteins, or nucleic acids. In certain embodiments, the cargo can be a nucleic acid such as DNA or RNA, preferably mRNA. In another particular embodiment, the cargo can be a TLR agonist, e.g., the TLR3 agonist poly I:C, or the TLR9 agonist CpG.

[0073] Prior to being loaded into the lipid nanoparticles, the cargo molecules may be further modified to induce overall polyanionicity in the molecules. This can be done, for example, by attaching them to the Glu10 moiety, as exemplified in the Examples section. The Glu10 moiety is a portion of 10 glutamic acids that increases the polyanionicity of the molecule to which it is attached.

[0074] Accordingly, the lipid nanoparticles and lipid nanoparticle compositions of the invention are particularly suitable for intracellular delivery of their cargo molecules. Accordingly, the invention provides the use of the lipid nanoparticles and lipid nanoparticle compositions as defined herein for intracellular delivery of cargo molecules.

[0075] In certain embodiments, the lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise a nucleic acid, preferably mRNA.

[0076] In the context of the present invention, "nucleic acid" is deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. Nucleic acids according to the present invention include genomic DNA, cDNA, mRNA, recombinantly produced molecules and chemically synthesized molecules. According to the present invention, the nucleic acid can be single-stranded or double-stranded and can be in the form of a linear or closed covalently circularized molecule. The nucleic acid can be, for example, in the form of RNA prepared by in vitro transcription from a DNA template and can be used for introduction into cells, i.e., transfection of cells. Further, the RNA can be modified by sequence stabilization, capping, and / or polyadenylation prior to application.

[0077] In the context of the present invention, the term "RNA" relates to a molecule containing ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. The term includes isolated RNAs such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNAs that differ from naturally occurring RNAs by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include, for example, the addition of non-nucleotide substances to the end(s) of the RNA or internally at one or more nucleotides of the RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, for example, nucleotides not found in nature, or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs. Nucleic acids can be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial or analogs of naturally occurring RNAs.

[0078] According to the present invention, "RNA" includes "mRNA" which means "messenger RNA", preferably related thereto, can be produced using DNA as a template, and is related to a "transcript" encoding a peptide or protein. mRNA typically includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life both intracellularly and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0079] In a further aspect, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein and a pharmaceutically acceptable agent such as a carrier, excipient, etc. Such a pharmaceutical composition is particularly suitable as a vaccine. Accordingly, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0080] In the context of the present invention, the term "vaccine" as used herein means any preparation intended to provide adaptive immunity (antibody and / or T cell response) against a disease. For this purpose, a vaccine as meant herein contains at least one nucleic acid molecule, for example, an mRNA molecule encoding an antigen against which an adaptive immune response is mounted. This antigen can be present in a weakened or killed form of a microorganism, protein or peptide, or an antigen encoding a nucleic acid. An antigen in the context of the present invention means a protein or peptide that is recognized as foreign by the host's immune system, thereby stimulating the production of antibodies against that antigen, and having the purpose of fighting such an antigen. A vaccine can be prophylactic (e.g., to prevent or ameliorate the effects of future infection by any natural or "wild" pathogen), or therapeutic (e.g., to actively treat or alleviate the symptoms of a progressing disease). Administration of a vaccine is called vaccination.

[0081] The vaccine of the present invention can be used to induce an immune response, particularly an immune response against a disease-related antigen, or an immune response against cells expressing a disease-related antigen, such as cancer. Thus, the vaccine can be used for the prevention and / or therapeutic treatment of diseases related to disease-related antigens or cells expressing disease-related antigens, such as cancer. Preferably, the immune response is a T cell response. In one embodiment, the disease-related antigen is a tumor antigen. The antigen encoded by the RNA contained in the nanoparticles described herein is preferably a disease-related antigen; or induces an immune response against a disease-related antigen or cells expressing a disease-related antigen.

[0082] The present invention also provides LNPs, pharmaceutical compositions and vaccines according to the present invention for use in human or veterinary medicine. The use of LNPs, pharmaceutical compositions and vaccines according to the present invention for human or veterinary medicine is also contemplated. Finally, the present invention provides a method for the prevention and treatment of human and veterinary disorders by administering an LNP, pharmaceutical composition and vaccine according to the present invention to a subject in need thereof. Such pharmaceutical compositions are particularly suitable in various fields such as prophylactic vaccines, therapeutic vaccines, protein replacement therapy, gene editing, gene silencing, small molecule delivery, etc.

[0083] The present invention further provides the use of an LNP, pharmaceutical composition or vaccine according to the present invention for the immunogenic delivery of said one or more nucleic acid molecules.

[0084] Thus, the LNPs, pharmaceutical compositions and vaccines of the present invention are very useful for the treatment of several human and veterinary disorders. Accordingly, the present invention provides LNPs, pharmaceutical compositions and vaccines of the present invention for use in the treatment of cancer or infectious diseases.

[0085] The lipid nanoparticles of the present invention can be prepared according to the protocol specified in the Examples section. More generally, LNPs can be prepared using a method comprising: - Preparation of a first alcoholic composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid, and a suitable alcoholic solvent; - Preparation of a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent; - Mixing the first and second compositions in a microfluidic mixing device.

[0086] More specifically, the lipid components are combined at an appropriate concentration in an alcoholic vehicle such as ethanol. In addition, an aqueous composition containing the nucleic acid is added and subsequently the microfluidic mixing device is filled.

[0087] The purpose of microfluidic mixing is to achieve complete and rapid mixing of multiple samples (i.e., the lipid phase and the nucleic acid phase) in a microscale device. Such sample mixing is typically achieved by enhancing the diffusion effect between different species of flows. For example, as outlined in Lee et al., 2011, several microfluidic mixing devices can be used. A particularly suitable microfluidic mixing device according to the present invention is the NanoAssemblr manufactured by PrecisioN-Nanosystems.

[0088] Other techniques suitable for preparing the LNPs of the present invention include dispersing the components in a suitable dispersion medium, such as an aqueous solvent and an alcoholic solvent, and the following methods: ethanol dilution method, simple hydration method, sonication, heating, vortexing, ether injection method, French press method, cholic acid method - Ca 2+ fusion method, freeze-thaw method, reverse phase evaporation method, T-junction mixing, microfluidic focusing, staggered herringbone mixing, etc. One or more of these can be applied.

[0089] The ionizable lipids of the present invention can be prepared according to the reaction schemes provided in the following examples, but those skilled in the art will understand that these are merely illustrative of the present invention, and the compounds of the present invention can be prepared by any of several standard synthetic processes commonly used by those skilled in organic chemistry.

Example

[0090] Preparation of Lipids 1. General Information Unless otherwise specified, all glassware was oven-dried before use, and all reactions were carried out under an argon atmosphere using standard Schlenk techniques. Dry solvents were purchased from Acros O - Organics or Sigma - Aldrich and used without further purification. All reagents were purchased from commercial sources and used without further purification unless otherwise specified. The progress of the reaction was monitored by thin - layer chromatography (TLC) on aluminum plates coated with 0.2 mm thick Kieselgel F254. Visualization was achieved by ultraviolet light (254 nm) or by staining with any of ninhydrin, cerium molybdate, or potassium permanganate. Flash column chromatography was performed using silica gel 60 (230 - 400 mesh, Merck and co.). Mass spectra were obtained using electrospray ionization (ESI) or electron impact ionization (EI) on a Finnigan MAT 8200 (70 eV), Agilent 5973 (70 eV). All 1 1H NMR, 13 13C NMR NMR was recorded on a Bruker AV - 400 in chloroform - d1 or DMSO - d6. Chemical shifts are reported in parts per million (ppm) and referenced to tetramethylsilane using the solvent peak as an internal standard (CDCl 3 3: 1 1H = 7.26 ppm, 13 13C = 77.16 ppm; CD 3 3SOCD 3 6: 1H = 2.50 ppm, 13 The coupling constants for C = 39.52 ppm were reported in Hz. 1 The H NMR splitting patterns were designated as singlet (s), broad (brd), doublet (d), triplet (t), quartet (q), quintet (qu), pentet (p), sextet (se), septet (sep), octet (o), or combinations thereof. Splitting patterns that could not be interpreted were designated as multiplet (m).

[0091] 2. Synthesis of Lipids 2.1 A general route for the synthesis of the lipid represented by the structure of Formula I is shown below. [Chemical formula]

[0092] Synthesis of Compound 3 To a stirred solution of Compound 1 (1.0 equiv) and Alcohol 2 (1.2 equiv) in CH 2 Cl 2 at 0 °C, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.2 equiv), N,N-diisopropylethylamine (4.0 equiv), and DMAP (0.2 equiv) were added under nitrogen (Sabnis et al, 2018). The reaction mixture was stirred at room temperature for 18 h. The reaction was then diluted with dichloromethane and washed with saturated Na 2 CO 3 (aqueous solution), brine, and dried over Na 2 SO 4 . After filtration and concentration in vacuo, the resulting residue was purified by silica gel column chromatography (ethyl acetate / hexane: 1 - 4%) to afford Compound 3.

[0093] Synthesis of Compound 6 To a stirred solution of Compound 4 (1.0 equiv) and Alcohol 5 (1.0 equiv) in CH 2 Cl 2 at 0 °C, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.3 equiv) Et3 N (2 equivalents) and DMAP (0.2 equivalents) were added (Rajappan et al, 2020). The reaction mixture was warmed to room temperature and stirred for 16 hours. The organic phase was successively washed with saturated Na 2 CO 3 (aqueous solution), water, and brine. The organic phase was dried over Na 2 SO 4 and filtered, and concentrated in vacuo. The crude product 6 was used without further purification.

[0094] Synthesis of Compound 7 Compound 6 was dissolved in a mixed solvent of CH 2 Cl 2 / CF 3 COOH (10 mL / 10 mL). After stirring at room temperature for 1 hour, the solvent was removed under reduced pressure and redissolved in CH 2 Cl 2 . The organic phase was first washed with saturated Na 2 CO 3 (aqueous solution). Then it was washed with brine and then dried over Na 2 SO 4 . After filtration and concentration in vacuo, the resulting residue was further purified by silica gel column chromatography (CH 2 Cl 2 / MeOH / NH 4 OH (0.5%): 9 / 1) to obtain Compound 7.

[0095] Synthesis of Compound 8 To a stirred solution of Compound 7 in anhydrous DMF, a solution of anhydrous K 2 CO 3 (3 equivalents) and Compound 3 in DMF were added. The reaction was stirred vigorously at 80 °C for 5 hours under nitrogen. After cooling to room temperature, the solid was removed by filtration and the DMF was removed under reduced pressure. Finally, the crude residue was purified by silica gel column chromatography (hexane:ethyl acetate = 100:0 to 85:15) to obtain Compound 8.

[0096] Synthesis of Compound 12 Compound 10 (1.0 equivalent) (Amano et al, 2017), Compound 8 (2.1 equivalents), DMAP (0.2 equivalent), and Et 3N (5.0 equivalents) was dissolved in DMF at room temperature. The resulting mixture was stirred at room temperature for 24 hours, after which amine 11 (3.5 equivalents) was added. After 5 hours, the solvent DMF was removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na 2 CO 3 (aqueous solution) to turn it white, then washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (CH 2 Cl 2 -CH 3 OH = 30:1 to 10:1) to obtain compound 12. The names of compound 12 are described just below their structures.

[0097] ETG-54

Chemical formula

[0098] ETG-53

Chem.

[0099] ETG-52 [Chemical formula] Yield: 14% - Colorless oil. 1 H-NMR (400 MHz, -Chloroform-d) δ 4.87 (qu, J = 6.2 Hz, 1H, H27), 4.37 - 4.27 (m, 4H, H2 & H7), 4.08 (t, J = 6.8 Hz, 2H, H54), 3.35 - 3.24 (m, 2H, H14), 3.23 - 3.12 (m, 4H, H21 & H22), 3.02 - 2.88 (m, 4H, H3 & H6), 2.50 - 2.40 (m, 2H, H15), 2.35 - 2.24 (m, 10H, H48, H51, H64 & H65), 1.71 - 1.58 (m, 6H, H56, H24 & H47), 1.57 - 1.44 (m, 8H, H16, H18, H29 & H30), 1.42 - 1.21 (m, 48H), 0.89 (t, J = 6.75 Hz, 9H, H37, H49, H63) ppm. 13 C-NMR (100 MHz, -Chloroform-d) δ 173.6 (ester-C=O), 156.4 (carbamate-C=O), 155.8 (carbamate-C=O), 74.1 (C27), 64.4, 62.8, 62.5, 51.8, 45.11, 38.0, 37.9, 34.7, 34.3, 34.1, 31.9, 29.5, 29.2, 29.1, 28.6, 26.7, 25.9, 25.3, 25.1, 24.9, 22.7, 14.1 (C37, -C49, -C63) ppm. LRMS (ESI) (m / z): - Calculated for [M + H] + (C 52 H 101 N 3 O 8 S 2 ) requires 960.7, found: 960.6

[0100] ETG-51

Chem.

[0101] ETG-56

Chem.

[0102] ETG-55

Chem.

[0103] ETG-58

Chem.

[0104] ETG-57

Chem.

[0105] Example: LNP Synthesis mRNA Synthesis: All mRNAs were prepared in vitro by T7-mediated transcription from linearized DNA templates (peTheRNA vectors) incorporating 5' and 3' UTRs and polyA tails. The final mRNAs utilize Cap1 and replace uridine 100% with N1-methylpseudouridine. The ionizable lipids of the present invention were prepared according to the reaction scheme defined above herein. As a comparative example, ETG-23 corresponding to S-Ac7-DOg (see WO2022 / 136641) was used and prepared according to the reaction scheme defined in '641.

[0106] LNP Synthesis: Using a NanoAssemblr Benchtop (PrecisioN-Nanosystems), lipid-based nanoparticles were produced by microfluidically mixing an mRNA solution and a lipid solution in sodium acetate buffer (100 mM, pH 4) at a 2:1 volume ratio and a rate of 16 mL / min. LNPs were generated with a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5 of ionizable lipid / DSPC (Avanti Polar Lipids) / cholesterol (Sigma) / DMG-PEG2000 (Avanti Polar Lipids). eGFP mRNA was encapsulated in all LNPs as a reporter mRNA at an mRNA / ionizable lipid molar ratio of 1 / 10. The LNPs were dialyzed against TBS using a Slide-A-Lyzer dialysis cassette (20K MWCO, 3 mL, ThermoFisher) (a TBS volume more than 10,000 times the LNP volume). Size, polydispersity, and zeta potential were measured using a Zetasizer Nano (Malvern), and mRNA encapsulation was measured by a standard Ribogreen RNA assay (Invitrogen).

[0107] Data Analysis: All raw data were analyzed using Graph Pad Prism version 9 software.

Table 1

[0108] Experimental Example 1: In Vivo Fluorescent mRNA Expression upon IV MRNA LNP Injection LNPs were generated with an ionizable lipid / DSPC / cholesterol / DMG-PEG2000 in a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5. Firefly luciferase (Fluc) mRNA was encapsulated in all LNPs at an mRNA / ionizable lipid molar ratio of 1 / 10. All experimental groups (BALB-C, female, 6- to 8-week-old mice) were intravenously injected once with their respective mRNA LNPs (5 μg mRNA, 100 μL volume; TBS buffer) or control buffer. Fluc mRNA expression was evaluated by in vivo bioluminescence measurements 4 and 24 hours after injection following an intraperitoneal injection of the substrate D-luciferin. mRNA delivery by ETG-based lipids resulted in high expression levels in vivo (Figure 1).

[0109] Experimental Example 2: Evaluation of Antibody Response against Influenza Hemagglutinin HA LNPs were generated with an ionizable lipid / DSPC / cholesterol / DMG-PEG2000 in a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5. Hemagglutinin (HA) mRNA was encapsulated in all LNPs at an mRNA / ionizable lipid molar ratio of 1 / 10. On days 0 and 21, BALB-C mice (female, 6 weeks old) were intramuscularly injected bilaterally with 50 μl of their respective mRNA LNPs (2 μg mRNA, TBS buffer) or control buffer. Blood was collected on day 21 (prime) and day 35 (boost), and then the IgG1 antibody titer against HA was determined using an in-house ELISA assay (Figure 2).

[0110] Experimental Example 3: In Vivo HEPO Expression after IV MRNA LNP Injection LNP was generated with ionizable lipid / DSPC / cholesterol / DMG-PEG2000 at a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5. Human erythropoietin (hEPO) mRNA was encapsulated in all LNPs at an mRNA / ionizable lipid molar ratio of 1 / 10. BALB-C mice (female, 6-week-old mice) were intravenously injected via the tail vein with 200 μl each of mRNA LNP (10 μg of mRNA, TBS buffer) or control buffer. Blood was collected from the submandibular vein at different time points after determining the hEPO concentration using an hEPO ELISA assay kit (catalog number BMS2035-2-Thermofisher Scientific) according to the manufacturer's instructions. ETG-53 showed higher hEPO expression compared to the ETG-23 control (Figures 3A and B).

Claims

1. An ionizable lipid represented by formula (I): 【Chemical 1】 Wherein R 1 and R 2 each independently is selected from -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl; wherein each of said -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl is optionally substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl; Here, R 1 and R 2 The total number of C atoms in is, together, at least 8; Here, R 1 and R 2 are different from each other; R 3 and R 4 are each independently, -C 1～6 alkyl; or, R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may further optionally contain one or more additional N atoms, and / or be optionally substituted with 1 to 3 substituents selected from -C 1～6 alkyl and -OH; R 7 When each of them exists, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl is independently selected; wherein, -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; m and n are each independently an integer selected from 1, 2, 3, and 4; X is selected from -O-, -S-, -S-S-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH 2 )-, -O-CR 8 R 9 -O-, and -S-CR 8 R 9 -S-; Each R 8 and R 9 is independently selected from -H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 5 is selected from -NH- and -O-; and R 6 is -C 1-6 alkylene.

2. The ionizable lipid according to claim 1, represented by any one of formulas (Ia), (Ib), and (Ic): [Chemical 2] Wherein R 1 and R 2 each independently is selected from -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl; wherein said -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl each may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1～20 alkyl, -C 2～20 alkenyl, and -C 2～20 alkynyl; Here, R 1 and R 2 The total number of C atoms in is, together, at least 8; Here, R 1 and R 2 are different from each other; R 3 and R 4 are each independently, -C 1～6 alkyl; or, R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may further optionally contain one or more additional N atoms, and / or be optionally substituted with 1 to 3 substituents selected from -C 1～6 alkyl and -OH; Each R 7 is independently selected from -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; wherein -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; m and n are each independently an integer selected from 1, 2, 3, and 4; Each R 8 and R 9 is independently selected from -H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 5 is selected from -NH- and -O-; and R 6 is -C 1-6 alkylene.

3. The ionizable lipid according to claim 1, represented by any one of formulas (II) or (III): [Chemical Formula 3] Wherein R 3 and R 4 are each independently -C 1～6 alkyl; or R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may further optionally contain one or more additional N atoms and / or be optionally substituted with 1 to 3 substituents selected from -C 1-6 alkyl and -OH; R 7 and R 7 each exist, then C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, independently, is selected; where the -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 each of alkynyl is optionally substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; R 7 and R 7 the total number of C atoms in "" is at least 6 together; m and n are each independently an integer selected from 1, 2, 3, and 4; o and p are each independently an integer selected from 1 to 10; X is selected from -O-, -S-, -S-S-, -O-(C=O)-, -O-(C=O)-O-, -(C=N-NH 2 )-, -O-CR 8 R 9 -O-, and -S-CR 8 R 9 -S-; Each R 8 and R 9 is independently selected from -H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 5 is selected from -NH- and -O-; R 6 is -C 1-6 alkylene; and, Here, o and p are different from each other; or here, R 7 and R 7 " are different from each other.

4. The ionizable lipid according to claim 1, represented by formula (IIa): 【Chemical Formula 4】 wherein R 3 and R 4 are each independently -C 1～6 alkyl; or R 3 and R 4 together with the N atom to which they are attached form a 5- to 10-membered aromatic or non-aromatic heterocyclic ring; the heterocyclic ring may further optionally contain one or more additional N atoms and is optionally substituted with 1 to 3 substituents selected from -C 1-6 alkyl and -OH; R 7 and R 7 if each of them exists, then C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 is independently selected from alkynyl; wherein said -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 each of alkynyl may optionally be substituted with 1 to 3 -O-(C=O)-R 7 , -(C=O)-O-R 7 , -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl; and the total number of C atoms in R 7 and R 7 " together is at least 6; m and n are each independently an integer selected from 1, 2, 3, and 4; o and p are each independently an integer selected from 1 to 10; R 5 is selected from -NH- and -O-; R 6 is -C 1-6 alkylene; and, Here, o and p are different from each other; and / or here, R 7 and R 7 " are different from each other.

5. An ionizable lipid according to any one of claims 1 to 4, selected from the following list: 【Chemical Formula 5-1】 【Chemical Formula 5-2】

6. R 1 and R 2 The ionizable lipid according to any one of claims 1 or 2, wherein the total number of C atoms in

7. R 7 and R 7 The ionizable lipid according to any one of claims 3 or 4, wherein the total number of C atoms in together is at least 12.

8. The ionizable lipid according to any one of claims 1 to 3 or 5, wherein m and n are the same and are an integer selected from 1, 2, 3, and 4; or m and n are the same and are 2. (Multi-multi)

9. R 5 The ionizable lipid according to any one of claims 1 to 3 or 5, wherein R is -NH-. (Multi-multi)

10. A lipid nanoparticle or lipid nanoparticle composition comprising the ionizable lipid according to any one of claims 1 to 9. (Multi-multi)

11. The lipid nanoparticle or lipid nanoparticle composition according to claim 10, further comprising a phospholipid, a sterol, and / or a PEG lipid.

12. The lipid nanoparticle or lipid nanoparticle composition according to any one of claims 10 to 11, further comprising an active agent, particularly a nucleic acid, preferably mRNA.

13. Use of the ionizable lipid according to any one of claims 1 to 9 in the manufacture of a lipid nanoparticle or lipid nanoparticle composition.

14. A pharmaceutical composition comprising the lipid nanoparticle or lipid nanoparticle composition according to any one of claims 10 to 12 and a pharmaceutically acceptable agent.

15. The pharmaceutical composition according to claim 14 for use in medicine.

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