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CN118255678B - Ionizable lipid compound and application thereof - Google Patents

Ionizable lipid compound and application thereof Download PDF

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Publication number
CN118255678B
CN118255678B CN202410369147.1A CN202410369147A CN118255678B CN 118255678 B CN118255678 B CN 118255678B CN 202410369147 A CN202410369147 A CN 202410369147A CN 118255678 B CN118255678 B CN 118255678B
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lipid
compound
peg
ionizable
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CN118255678A (en
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章雪晴
滕以龙
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Rongcan Biomedical Technology Shanghai Co ltd
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    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
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    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C327/22—Esters of monothiocarboxylic acids having carbon atoms of esterified thiocarboxyl groups bound to hydrogen atoms or to acyclic carbon atoms

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Abstract

The invention relates to the technical field of drug delivery, in particular to an ionizable lipid compound and application thereof. The invention provides a novel composite material comprising

Description

Ionizable lipid compound and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to an ionizable lipid compound and application thereof.
Background
Nucleic acid drugs, compared with traditional small molecule drugs and antibody drugs, are theoretically not limited by the possibility of drug formation of target proteins. Along with the advancement of clinic and the maturity of related technologies, the method has wider application scenes.
The nucleic acid medicine is taken as an exogenous medicine, the problems of instability, immunogenicity, low cell uptake efficiency, difficult endosome escape and the like need to be overcome when the nucleic acid medicine enters the body to play a role, and the high-efficiency and safe delivery system is an important guarantee for the nucleic acid medicine to overcome the defects and target to play a stable medicine effect.
At present, the lipid nanoparticle (Lipid Nanoparticle, LNP) has higher delivery efficiency and better safety in vivo by virtue of the unique structure and physical and chemical properties, becomes one of the mainstream delivery systems of nucleic acid medicaments, is verified in a nucleic acid novel crown vaccine, and is gradually applied to the fields of tumor vaccines, cell therapy, gene editing and the like.
The ionizable lipid compound is a key core component in the LNP delivery system, and has the functions of combining with negatively charged nucleic acid, promoting cellular uptake and endosome escape, enhancing nucleic acid drug in-vivo transfection and the like.
Although various ionizable lipid compounds have been disclosed in the prior art, most of the ionizable lipid compounds have problems of low transfection efficiency, poor stability, low safety, etc., and especially for the field of carrying large-dose nucleic acid drugs, besides improving the transfection efficiency, the safety is an important factor to be considered.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide novel ionizable lipid compounds which simultaneously contain- (C=o) O-or-O (c=o) -and hydroxy fragments, this particular structural feature being capable of improving the biocompatibility, mRNA transfection efficiency and safety of LNP as a whole.
To achieve the above and other related objects, a first aspect of the present invention provides an ionizable lipid compound having a chemical structure represented by the following formula (I):
wherein a and b are each independently selected from 0, 1,2, 3, 4, 5, 6, 7 or 8;G 1、G2 are each independently selected from C2-C10 alkylene;
M is CH or N;
G 3 is selected from C1-C10 alkylene, or G 3 is selected from (CH 2)d-O-(CH2)e), wherein d, e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9 and d+e is an integer of 2-10, or G 3 is selected from Wherein g is selected from 1,2, 3 or 4;
L 1 is selected from- (c=o) O-, -O (c=o) -, - (c=o) S-, -S (c=o) -, - (c=o) NR-, -NR (c=o) O-, -O (c=o) NR-, R is independently selected from H, C-C12 hydrocarbyl;
l 2 is selected from- (c=o) O-, -O (c=o) -;
R 1、R2 is independently selected from C1-C20 straight-chain saturated hydrocarbon groups;
r 3、R4 is independently selected from C4-C10 straight-chain saturated hydrocarbon groups.
The second aspect of the present invention provides a lipid nanoparticle comprising a combination of one or more of the above-described ionizable lipid compounds, stereoisomers thereof, tautomers thereof, or pharmaceutically acceptable salts thereof.
In a third aspect, the invention provides the use of the lipid nanoparticle for the preparation of a pharmaceutical composition further comprising the drug carried and/or pharmaceutically acceptable excipients.
Compared with the prior art, the invention has the following beneficial effects:
The ionizable lipid compound provided by the invention takes N atoms as ionizable centers, hydrophilic hydroxyl groups as heads, two hydrophobic alkane chains as tails, and a- (C=O) O-or-O (C=O) -fragment is introduced into one tail, so that certain asymmetry of the two tails is realized, the compound has moderate length (namely, proper hydrophilic and hydrophobic properties are endowed to the ionizable lipid compound), and the ionizable lipid compound of the innovative design can simulate biological phospholipid molecules, promote membrane fusion and improve the escape capability of endosomes of nucleic acids. Compared with the prior art, the nano-carrier formed by self-assembly of the ionizable lipid compound with the structural characteristics not only can improve intracellular delivery efficiency and transfection efficiency, but also has better safety. In particular, compared with the commercial lipid compound ALC0315, the safety of the lipid compound is greatly improved in vitro and in vivo.
Drawings
FIG. 1 is a graph showing the particle size distribution of mRNA-LNP prepared from the ionizable lipid compound J-03 according to the present invention.
Detailed Description
To solve the defects of the prior art, the invention aims to provide a novel composite material containingThe mRNA-LNP prepared by the ionizable lipid compound with a brand new structure can integrally improve the biocompatibility and the mRNA transfection efficiency of the LNP, has extremely high safety, can be applied to the fields of tumor vaccines, cell therapy, gene editing and the like, and has wider clinical application value.
In order to achieve the above object, the present invention adopts the following technical scheme:
an ionizable lipid compound having a chemical structure represented by the following formula (I):
wherein a and b are each independently selected from 0, 1,2, 3, 4, 5, 6, 7 or 8;G 1、G2 are each independently selected from C2-C10 alkylene;
M is CH or N;
G 3 is selected from C1-C10 alkylene, or G 3 is selected from (CH 2)d-O-(CH2)e), wherein d, e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9 and d+e is an integer of 2-10, or G 3 is selected from Wherein g is selected from 1,2, 3 or 4;
L 1 is selected from- (c=o) O-, -O (c=o) -, - (c=o) S-, -S (c=o) -, - (c=o) NR-, -NR (c=o) O-, -O (c=o) NR-, R is independently selected from H, C-C12 hydrocarbyl;
l 2 is selected from- (c=o) O-, -O (c=o) -;
R 1、R2 is independently selected from C1-C20 straight-chain saturated hydrocarbon groups;
r 3、R4 is independently selected from C4-C10 straight-chain saturated hydrocarbon groups.
The "hydrocarbon group" includes a saturated hydrocarbon group and an unsaturated hydrocarbon group, and the preferred saturated hydrocarbon group may be a C1-C20 (i.e., a C1-20) hydrocarbon group, a further preferred C4-C10 hydrocarbon group, and most preferred C5-C8 hydrocarbon group, and the preferred unsaturated hydrocarbon group may be a C2-C20 (i.e., a C2-20) olefin group or a C2-C20 (i.e., a C2-20) alkyne group. The unsaturated hydrocarbon group may be an unsaturated multi-hydrocarbon group or an unsaturated mono-hydrocarbon group.
The "hydrocarbon group" may be of branched or linear structure, preferably of linear structure;
The "hydrocarbon group" may be substituted or unsubstituted, and is preferably unsubstituted.
When describing "C2-C10 hydrocarbylene" it is meant that the group may be hydrocarbylene (e.g., alkylene, alkenylene, alkynylene) having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), the alkylene group may be of a straight or branched chain structure.
In some embodiments of the invention, a and b are each independently selected from 0, 1,2,3, 4 or 5, preferably 0, 1,2 or 3, further preferably a is selected from 2 or 3 and b is selected from 0 or 1.
In some embodiments of the invention, G 1、G2 is each independently selected from C5-C10 hydrocarbylene, preferably G 1 is C5-C9 hydrocarbylene and G 2 is C5-C8 hydrocarbylene.
In some embodiments of the invention, G 3 is selected from C1-C5 hydrocarbylene, preferably C2-C4 hydrocarbylene, further preferably C4 hydrocarbylene, or G 3 is selected from (CH 2)2-O-(CH2)2.
In some embodiments of the invention, each R 1、R2 is independently selected from C6-C10 straight-chain saturated hydrocarbon groups, preferably C8 straight-chain saturated hydrocarbon groups.
In some embodiments of the invention, each R 3、R4 is independently selected from C5-C10 straight-chain saturated hydrocarbon groups, preferably C5-C8 straight-chain saturated hydrocarbon groups.
In some embodiments of the invention, R is independently selected from H, C C1-C4 hydrocarbyl groups, preferably H, -CH 3.
In some embodiments of the invention, the ionizable lipid compound is selected from the group consisting of:
the second aspect of the present invention provides a lipid nanoparticle comprising a combination of one or more of the above-described ionizable lipid compounds, stereoisomers thereof, tautomers thereof, or pharmaceutically acceptable salts thereof.
The term "stereoisomer" refers to an isomer having the same order of attachment of atoms, but different in the spatial arrangement of the atoms.
The term "tautomer" refers to a phenomenon that the structure of a compound undergoes equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.
By "pharmaceutically acceptable salt" is meant an acid addition salt or a base addition salt. All compounds of the invention in free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the invention may be formed by conversion to their free base or acid by standard techniques.
Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorinates, camphorsulphonates, citrates, cyclopentanepropionates, digluconates, citrates, dodecyl sulphates, ethanesulphonates, formates, fumarates, glucoheptanoates, glycerophosphate, gluconate, hemisulphates, heptanoates, caprates, hydroiodinates, 2-hydroxyethanesulphonates, lactates, laurates, lauryl sulphates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulphonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectinates, persulphates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulphates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Where appropriate, additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulphates, phosphates, nitrates, sulphonates and arylsulphonates. Additional pharmaceutically acceptable salts include salts formed from quaternization of amines with suitable electrophiles (e.g., alkyl halides) to form quaternized alkylated amino salts.
The lipid nanoparticle provided by the invention further comprises one or more of a structural lipid, a helper lipid, a PEG-lipid, and a polymer.
In some embodiments of the present invention, the mole percent of the ionizable lipid compound to the auxiliary lipid to the structural lipid to the PEG-lipid is (20-65): 0-60): 0-10, and at least any one of the auxiliary lipid, the structural lipid, and the PEG-lipid is not 0, preferably, the mole percent of the ionizable lipid compound to the auxiliary lipid to the structural lipid to the PEG-lipid is (20-65): 3-50): 15-60): 0.1-10. In other embodiments of the present invention, the molar ratio of the ionizable lipid compound to the polymer in the lipid nanoparticle is 0.5:1-100:1, preferably 10:1-80:1, and more preferably 40:1-80:1.
In some embodiments of the invention, the lipid nanoparticle comprises 20% -65% ionizable lipid compound, 3% -40% helper lipid, 20% -60% structural lipid, 0.1% -10% PEG-lipid, and% refers to mole percent.
In some embodiments of the invention, the lipid nanoparticle comprises 35% -49% of an ionizable lipid compound, 5% -20% of a helper lipid, 35% -50% of a structural lipid, 1% -2% of a PEG-lipid, and% refers to mole percent.
By "structured lipid" is meant a composition containing structures that stabilize the composition, including but not limited to sterols and derivatives thereof and non-sterols and derivatives thereof in combination.
In some embodiments, the structural lipid includes, but is not limited to, sterols and derivatives thereof, non-sterols, sitosterols, ergosterols, cholestanones, cholestenone, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, stigmasterols, alpha-tocopherols, or a combination of one or more corticosteroids. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α -cholesterol, 5α -fecal sterols, cholesteryl- (2 '-hydroxy) ethyl ether, cholesteryl- (4' -hydroxy) butyl ether and 6-ketocholestanol, non-polar analogs such as 5α -cholestane, cholestenone, 5α -cholestenone and decanoic cholesterol esters, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analogue such as cholesteryl- (4' -hydroxy) butyl ether. It is not intended to be exhaustive and any structural lipid may be used in the present invention.
In some embodiments, the structural lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroid, and derivatives thereof.
In some embodiments, the structural lipid is cholesterol.
The "helper lipid" is not limited in kind and preferably comprises a phospholipid lipid including, but not limited to, one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol.
In some embodiments, the helper lipid may be selected from 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphocholine (DUPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphoethanolamine (DSPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPE), 1, 2-dioleoyl-glycero-sn-3-phosphocholine (DPPE), 1, 2-dioleoyl-glycero-3-phosphocholine (DPPE), 1-dioleoyl-oleoyl-2-glycero-3-phosphocholine (DPPC) 1-O-hexadecyl-sn-glycerol-3-phosphocholine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphocholine, 1, 2-di-arachidonoyl-sn-glycerol-3-phosphocholine, 1, 2-di-dodecahexanoyl-sn-glycerol 3-phosphocholine, 1, 2-di-phytoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-stearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-3-phospho-rac- (1-glycerol) sodium salt (DOPG), di-acetyl phosphatidylethanolamine, stearoyl-phosphoethanolamine (pe), lysophosphatidylethanolamine, or a combination of more of these.
In some embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, POPC.
In some embodiments, the helper lipid is phosphatidylcholine, particularly DSPC.
In some embodiments, the helper lipid is phosphatidylethanolamine, particularly DOPE.
In some embodiments, the helper lipid is selected from one or more combinations of DOTAP ((1, 2-dioleoxypropyl) trimethylammonium chloride), DOTAP (1, 2-dioleoyl-3-dimethylammonium-propane), 18:1pa (1, 2-DI (cis-9-octadecenoyl) -SN-glycero 3-phosphate sodium salt), HS15 (polyethylene glycol (15) -hydroxystearate), GL67 (N4-argininocarbonamide).
The term "PEG-lipid" as used herein generally refers to a conjugate formed by linking PEG (polyethylene glycol) to a lipid molecule via a chemical bond. Including but not limited to PEG-modified phospholipids and derivatized lipids, exemplified by combinations of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecylamide.
In some embodiments, the PEG-lipid includes, but is not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-DS, chol (cholesterol) -PEG, 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol dimethacrylate (PEG-DMA), PEG distearyl glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearyl, PEG diacyl Gan Xianan, PEG dipalmitoyl phosphatidylethanolamine, PEG-phosphatidylethyldimyristoxypropyl-3-amine, PEG oxypropyl-1, 2-distearoyloxypropyl-3-amine-N [ methoxy (PEG-DSA) ] (PEG-DSA), polyethylene glycol, methoxy-co-Acetyl (ALC), or a combination of one or more of lauric Acid and (ALC) of the group (ALC) of lauric Acid and (ALC) of the above.
In some embodiments, the PEG-lipid is PEG-DMG.
The kind of the "polymer" is not limited, and the polymer may include, but is not limited to, amphiphilic block copolymers, which are block copolymers composed of hydrophobic polymers and hydrophilic compounds, including, but not limited to, polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, poly beta Polyaminoester (PBAE), poly (alpha-hydroxy acid), lactide/glycolide copolymer (PLGA or PLG) (which includes lactide/glycolide copolymer, D-lactide/glycolide copolymer, L-lactide/glycolide copolymer and D, L-lactide/glycolide copolymer), polyglycolide (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), conjugates of poly (alpha-hydroxy acids), polyacetirin (polyaspirins), polyphosphazenes, D-lactide, D, L-lactide-caprolactone, D, L-lactide-glycolide-caprolactone, dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly (N-isopropylenamide), SAIB (sucrose acetate isoparaffinate) hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or salts thereof, carbopol, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl Methacrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer, PEO-PPO-PEO (pluronics)), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLGA, PLA-PLGA, PEG-PLA, PEG-PCL, poloxamer 407, PEG-PLGA-PEG triblock copolymer, PEG-PLA-PEG triblock copolymer, PEG-PCL-PEG triblock copolymer, or block copolymers of these with polyethylene glycol (PEG), or a combination of one or more of the foregoing polymers or copolymers.
In some embodiments of the present invention, the PEG in the PEG-lipid has a weight average molecular weight of 1000 to 10000, for example, 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.
In a third aspect, the invention provides the use of the lipid nanoparticle for the preparation of a pharmaceutical composition further comprising the drug carried and/or pharmaceutically acceptable excipients.
The "drug-loaded" as used herein includes, but is not limited to, any one of nucleic acids, small molecules, proteins, or a combination of a plurality thereof.
The "nucleic acid" according to the present invention may be a nucleotide polymer of any length. Including, but not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNAs (lncRNA), micronon-coding RNAs (miRNA and siRNA), telomerase RNA (Telomerase RNA), small molecule RNAs (snRNA and scRNA), circular RNAs (circRNA), synthetic mirnas (MIRNA MIMICS, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes (ribozyme), asymmetric interfering RNAs (aiRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), guide RNAs (gRNA), small guide RNAs (sgrnas), locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), morpholino antisense oligonucleotides, morpholino oligonucleotides, or combinations of one or more of the biospecific oligonucleotides.
In certain embodiments of the invention, the nucleic acid is mRNA. The mRNA is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to direct protein synthesis. The mRNA may be monocistronic mRNA or polycistronic mRNA.
As used herein, a "small molecule" refers to a compound that is not a protein or nucleic acid molecule. The small molecule may be a small molecule of a therapeutic and/or prophylactic agent, such as an antibiotic, anti-inflammatory, anti-cancer, anti-viral, immunosuppressant, analgesic, anti-fungal, antiparasitic, anticonvulsant, antidepressant, anxiolytic, antipsychotic, or the like.
"Protein" as used herein refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary and/or quaternary structure. The secondary, tertiary and/or quaternary structure of proteins is typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions and/or van der Waals interactions. Additionally, or alternatively, the protein may include disulfide bonds, for example between thiol groups of cysteine residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, and the like.
The pharmaceutical composition of the invention also comprises pharmaceutically acceptable auxiliary materials. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 4 to 8, preferably about 5 to 7, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, intracavity injection, inhalation administration, implant administration, and the like.
By "pharmaceutically acceptable" as used herein is meant that the drug does not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.
The "pharmaceutically acceptable excipients" should be compatible with the active ingredient, i.e. capable of being blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances that may be pharmaceutically acceptable excipients may be sugars, such as glucose, mannitol, sucrose, lactose, trehalose, maltose, and the like, starches, such as corn starch and potato starch, and the like, celluloses and derivatives thereof, such as sodium methyl cellulose, ethyl cellulose, and methyl cellulose, and the like, tragacanth powder, malt, gelatin, talc, solid lubricants, such as stearic acid, magnesium stearate, and the like, calcium sulfate, vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, cocoa butter, and the like, alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol, and the like, alginic acid, emulsifying agents, such as Tween, and the like, wetting agents, such as sodium lauryl sulfate, and the like, surfactants, lyoprotectants, colorants, flavoring agents, compressed tablets, stabilizers, diluents, excipients, antioxidants, preservatives, athermal, isotonic saline solutions, buffers, and the like, and combinations thereof. These substances are used as needed to increase the stability of the formulation or to help increase the activity or its bioavailability or to create an acceptable mouthfeel or odor in the case of oral administration.
The pharmaceutical composition of the present invention can be formulated into inhalable atomized formulations (e.g., dry powder formulations, aerosol formulations, inhalable aerosol droplet formulations, etc.), implantable gel formulations, microneedle formulations, and also can be formulated into injectable forms, for example, using physiological saline or aqueous solutions containing glucose and other adjuvants by conventional methods. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight per day to about 50 milligrams per kilogram of body weight per day.
In some embodiments of the invention, the pharmaceutical composition comprises lipid nanoparticles (Lipid Nanoparticle, LNP) having an average particle size of 60-120 nm, which may be 60-70 nm, 70-80 nm, 80-90 nm, 90-100 nm, 100-110 nm, 110-120 nm, or 60-80 nm, 80-100 nm, 100-120 nm.
In some embodiments of the invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, and the encapsulation rate of the nucleic acid in the lipid nanoparticles is greater than 80%, which can be 80% -85%, 85% -90%, 90% -95%, 95% -97%, 97% -99% or more than 99%.
Before further describing embodiments of the invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is intended to be in the nature of specific embodiments and is not intended to be limiting of the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. The materials and equipment used in the invention are all commercial products.
Example one Synthesis of ionizable lipid Compounds
1. Synthesis of ionizable lipid Compound J-03
The synthetic route is as follows:
(1) Synthesis of Compound J-I01
4, 4-Dimethoxybutyronitrile (10.00 g,77.42 mmol), n-octanol (30.25 g,232.27 mmol) and PTSA (p-toluene sulfonic acid, 1.33g,7.74 mmol) were added to the reaction flask, warmed to 100℃and reacted for 24 hours with nitrogen. After the completion of the reaction by TLC, 300mL of ethyl acetate was added to the reaction solution to dilute the reaction solution, and an equal volume of saturated sodium bicarbonate solution was washed three times, the organic phase was collected, dried over anhydrous sodium sulfate, the reaction solution was filtered, the filtrate was collected, and concentrated, and purified by column separation (silica gel column, eluent: PE: ea=100:1) to give 10.78g of the product in 43% yield.
(2) Synthesis of Compound J-I02
J-I01 (10.00 g,30.72 mmol) was dissolved in 50mL of ethanol, then 25mL of an aqueous solution containing potassium hydroxide (3.45 g,61.44 mmol) was added, and the temperature was raised to 100℃and the reaction was performed under nitrogen protection for 24 hours. After the completion of the TLC monitoring reaction, 200mL of ethyl acetate was added to the reaction solution to dilute the reaction solution, the same volume of saturated sodium bicarbonate solution was washed three times, the organic phase was collected, dried over anhydrous sodium sulfate, the reaction solution was filtered to collect the filtrate, and concentrated, and the filtrate was purified by column separation (silica gel column, eluent: PE: EA=10:1) to give 8.56g of the product in a yield of 81%.
(3) Synthesis of Compound J-I03
J-I02 (5.00 g,14.51 mmol), 6-bromo-N-hexanol (3.15 g,17.41 mmol), 4-dimethylaminopyridine (0.53 g,4.35 mmol) were dissolved in 50mL of dichloromethane, cooled to 0℃and N' N-dicyclohexyldiimine (3.59 g,17.41 mmol) was added, followed by stirring at room temperature for 12h. After the reaction was completed by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and the product was purified by column separation (silica gel column, eluent PE: ea=10:1) to give 6.31g of the product in a yield of 90%.
(4) Synthesis of Compound J-I04
6-Bromohexanoic acid (5.00 g,25.63 mmol), 2-hexyldecanol (6.21 g,25.63 mmol) and DMAP (0.94 g,7.69 mmol) were dissolved in 50mL of dichloromethane, cooled to 0℃and DCC (6.35 g,30.76 mmol) was added and gradually warmed to room temperature and stirred for 12h. After the reaction was completed by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and the product was purified by column separation (silica gel column, eluent PE: ea=50:1) to give 8.31g of the product in 77% yield.
(5) Synthesis of Compound J-I05
J-I04 (2.48 g,5.9 mmol), 4-amino-1-butanol (5.26 g,59.0 mmol) and 5.0mL ethanol were added to the reaction flask, and the reaction mixture was heated to 90℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 200mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 2.52g of product in 89% yield.
(6) Synthesis of ionizable lipid Compound J-03
J-I03 (0.24 g,0.47 mmol), J-I05 (0.2 g,0.47 mmol), DIPEA (73 mg,0.56 mmol) and 2.0mL acetonitrile were added to the reaction flask, the reaction solution was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 50mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE: ea=1:1) to give 271.0mg of product in 68% yield.
By adopting the method, the corresponding initial raw materials are replaced, and the compounds J-01-J-02, J-04, J-06-J-11, J-18-J-20, J-27, J-28, J-30, J-31, J-37-J-41, J-58-J-61 and J-85 can be prepared. For example, J-04 can be prepared by substituting diglycolamine for 4-amino-1-butanol and J-37 can be prepared by substituting 2-hexyldecanoic acid for 2-hexyldecanoamine.
2. Synthesis of ionizable lipid Compound J-46
(1) Synthesis of Compound J-I29
A certain amount of Compound J-I02 (10.0 g,29.02 mmol) was dissolved in 200mL of anhydrous tetrahydrofuran, stirred in an ice bath under nitrogen for 10min, then lithium aluminum hydride tetrahydrofuran solution (11.6 mL,2.5M,29.02 mmol) was added dropwise, and after the addition, the temperature was gradually returned to room temperature and stirred overnight. After completion of the TLC monitoring, 1.1mL of water was added, 1.1mL of 15% aqueous NaOH solution was added, 3.3mL of water was further added, and the solvent was removed by vacuum distillation. Adding proper volume of ethyl acetate, washing with equal volume of saturated salt water for 3 times, drying with anhydrous sodium sulfate, and concentrating. Column separation and purification (silica gel column, eluent: PE: ea=10:1 (volume ratio)) gave 8.31g of colorless oily liquid in 86% yield.
(2) Synthesis of Compound J-I30
Referring to the synthesis method of compound J-I03 of (3), compound J-I27 can be synthesized.
(3) Synthesis of ionizable lipid Compound J-46
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, a compound J-46 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compound J-47-J-57 can be prepared. For example, J-47 can be prepared by substituting 8-bromooctanoic acid for 6-bromohexanoic acid.
3. Synthesis of ionizable lipid Compound J-05
The synthetic route is as follows:
(1) Synthesis of Compound J-I06
9-Heptadecanol (3.85 g,15.0 mmol), N, N-disuccinimidyl carbonate (5.84 g,22.8 mmol) and DMAP (2.75 g,22.5 mmol) were added to DMF (60 mL), heated to 75deg.C, and stirred for 12h. The reaction solution was poured into 300mL of water, extracted 2 times with ethyl acetate, and 150mL of ethyl acetate was used each time. The organic phase was collected and washed three times with an equal volume of saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography on silica gel (column chromatography on silica gel, eluent PE: ea=50:1) to give 1.8g of the product in 42% yield. .
(2) Synthesis of Compound J-I07
J-I06 (1.8 g,4.53 mmol), 6-amino-1-hexanol (636 mg,5.43 mmol) and DMAP (554 mg,4.53 mmol) were added to DMF (30 mL), heated to 60℃and stirred for 12h. The reaction solution was poured into 300mL of water, extracted 2 times with ethyl acetate, and 150mL of ethyl acetate was used each time. The organic phase was collected and washed three times with an equal volume of saturated aqueous sodium chloride solution. The phase was dried over anhydrous sodium sulfate and concentrated to give 1.84g of the product in 100% yield, which was used directly in the next reaction.
(3) Synthesis of Compound J-I08
To a solution of J-I07 (1.84 g,4.53 mmol), DMAP (61 mg,0.5 mmol) and TEA (triethylamine, 2.5mL,18 mmol) in DCM (60 mL) at 0deg.C was slowly added a solution of Ms 2 O (1.57 g,9 mmol) in DCM (9 mL). After stirring the reaction at 0℃for 30min, it was quenched by the addition of water (100 mL). The organic phase was separated, washed once with an equal volume of pure water, washed once with an equal volume of 10% citric acid and once with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to give 2.05g of crude product, which was used directly in the next reaction.
(4) Synthesis of Compound J-I09
J-I08 (2.05 g,4.29 mmol) and LiBr (1.2 g,13.6 mmol) were added to THF (tetrahydrofuran, 25 mL), heated to 45℃and stirred for reaction for 12h. Filtration over silica gel (10 g), collection of the filtrate and concentration, and purification by chromatography on a column of silica gel (column of silica gel, eluent PE: EA=10:1) gave 1.84g of product in 93% yield.
(5) Synthesis of Compound J-I10
J-I09 (5.00 g,10.81 mmol), 4-amino-1-butanol (9.64 g,108.09 mmol) and 10.0mL ethanol were added to the reaction flask, and the reaction mixture was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 300mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 4.72g of the product in 93% yield.
(6) Synthesis of ionizable lipid Compound J-05
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, the compound J-05 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compounds J-12-J-14, J-22-J-23, J-29, J-32, J-44, J-45, J-62, J-63, J-82 and J-86 can be prepared. For example, J-12 may be prepared by substituting 2-hexyldecanol for 9-heptadecanol.
4. Synthesis of ionizable lipid Compound J-15
The synthetic route is as follows:
(1) Synthesis of Compound J-I11
1, 6-Dibromohexane (85.45 g,350.25 mmol) was dissolved in 400mL of tetrahydrofuran, cooled to 0℃and potassium thioacetate (20.00 g,175.13 mmol) was added in six portions over 1h, warmed to room temperature and stirred for reaction for 12h. After the reaction was completed by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column separation (silica gel column, eluent PE: ea=500:1) to give 28.15g of the product in 67% yield.
(2) Synthesis of Compound J-I12
J-I06 (20.00 g,83.62 mmol) and 13.94mL of concentrated hydrochloric acid (12M, 167.24 mmol) were dissolved in 200mL of methanol, raised to 60℃and stirred for reaction for 12h. After completion of the reaction by TLC, the reaction solution was poured into 600mL of ethyl acetate/water (1:1), extracted, the organic phase was collected and washed once with an equal volume of pure water, an equal volume of saturated aqueous sodium bicarbonate solution was washed once, the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column separation (silica gel column, eluent: PE: ea=20:1) to give 13.26g of the product in 80% yield.
(3) Synthesis of Compound J-I13
J-I12 (10.00 g,50.73 mmol), 2-hexyldecanoic acid (13.01 g,50.73 mmol) and DMAP (1.86 g,15.22 mmol) were dissolved in 200mL of dichloromethane, cooled to 0℃and DCC (12.56 g,60.87 mmol) was added, warmed to room temperature and stirred for 12h. After the reaction was completed by TLC, the reaction solution was filtered, and the filtrate was collected and concentrated, and purified by column separation (silica gel column, eluent PE: ea=100:1) to give 17.51g of the product in 79% yield.
(4) Synthesis of Compound J-I14
J-I13 (10.00 g,22.96 mmol), 4-amino-1-butanol (20.47 g,229.60 mmol) and 20.0mL ethanol were added to the reaction flask, the reaction mixture was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 600mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 8.94g of product in 88% yield.
(5) Synthesis of ionizable lipid Compound J-15
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, compound J-15 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compounds J-16-J-17, J-33, J-64-J-67, J-83 and J-87 can be prepared. For example, J-17 can be produced by substituting diglycolamine for 4-amino-1-butanol, and J-64 can be produced by substituting 1, 6-dibromohexane for 1-bromo-2-hexyldecane and substituting 2-hexyldecanoic acid for 6-bromo-hexanoic acid.
5. Synthesis of ionizable lipid Compound J-21
(1) Synthesis of Compound J-I15
J-I09 (1.00 g,2.16 mmol) was added to a suspension of NaH (60% purity, 173mg,4.32 mmol) and MeI (614 mg,4.32 mmol) in THF (80 mL) at 0deg.C. The reaction mixture was warmed to room temperature and stirred for 48 hours. Concentrated, sodium bicarbonate solution (100 mL) was added and extracted three times with ethyl acetate/petroleum ether (20:1). The organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (10% EA in PE) to give 1.01g of the product in 98%.
(2) Synthesis of Compound J-I16
J-I15 (1.00 g,2.10 mmol), 4-amino-1-butanol (1.87 g,20.98 mmol) and 2.0mL ethanol were added to the reaction flask, and the reaction mixture was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 60mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 0.89g of product in 87% yield.
(3) Synthesis of ionizable lipid Compound J-21
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, compound J-21 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compound J-68-J-72 can be prepared. For example, J-72 can be prepared by substituting 8-bromooctanol for 6-bromohexanol.
6. Synthesis of ionizable lipid Compound J-24
(1) Synthesis of Compound J-I17
N-bromohexane (33.00 g,375.59 mmol) was dissolved in DMSO (330 mL), K 2CO3 (51.91 g,375.59 mmol) was added and n-octylamine (48.54 g,375.59 mmol) was reacted at 80℃for 36h. Cooled to room temperature, ethyl acetate (2000 mL) was added, washed three times with an equal volume of saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, DCM: etoh=100:1-40:1) to give 33.05g of product in 41% yield.
(2) Synthesis of Compound J-I18
Triphosgene (202.8 mg, 683.3. Mu. Mol)) was dissolved in DCM (4 mL), a solution of DMAP (758.9 mg,6.2 mmol) in DCM (2 mL) was added dropwise under ice-salt bath, reacted for 30min, 6-bromo-n-hexanol (404.0 mg,2.1 mmol) was added dropwise, J-I17 (500.0 mg,2.1 mmol) was added dropwise, the reaction was slowly warmed to room temperature and allowed to react for 1.5h, water (6 mL) was added to the reaction solution, the aqueous phase was washed with dichloromethane (6 mL. Times.3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product. The crude product was purified by column chromatography (PE: ea=100:1) to give 930.9mg of product in 97.2% yield.
(3) Synthesis of Compound J-I19
J-I18 (5.00 g,11.51 mmol), 4-amino-1-butanol (5.13 g,57.54 mmol) and 10.0mL ethanol were added to the reaction flask, and the reaction mixture was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 200mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 3.96g of product in 80% yield.
(4) Synthesis of ionizable lipid Compound J-24
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, compound J-24 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compounds J-25, J-26, J-73-J-77 and J-84 can be prepared. For example, J-26 can be prepared by substituting diglycolamine for 4-amino-1-butanol.
7. Synthesis of ionizable lipid Compound J-42
(1) Synthesis of Compound J-I22
2-Hexadecanoic acid (4.38 g,17.07 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.34 g,20.48 mmol) was dissolved in 100mL of Dichloromethane (DCM), and after stirring at room temperature for 10min, N-hydroxysuccinimide (2.37 g,20.48 mmol) was added, and after stirring at room temperature for 6h, the reaction solution was washed three times with an equal volume of saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate for 1h, then 6-bromohexanol (2.00 g,17.07 mmol) was added with stirring, and the reaction was stirred at room temperature for 6h. After the completion of the reaction, the solvent was distilled off under reduced pressure using a rotary evaporator. 100mL of ethyl acetate was added, the mixture was washed with an equal volume of saturated sodium bicarbonate solution 2 times, the mixture was washed with an equal volume of saturated sodium chloride solution 1 time, and dried over anhydrous sodium sulfate for 30 minutes, the solvent was distilled off under reduced pressure using a rotary evaporator, and the mixture was purified by column separation (silica gel column, eluent: PE: EA=10:1 (volume ratio)), to obtain 5.16g of colorless liquid, with a yield of 85%.
(2) Synthesis of Compound J-I23
Referring to the synthesis method of compound J-I08 of (3), compound J-I23 can be synthesized.
(3) Synthesis of Compound J-I24
Referring to the synthesis method of compound J-I09 of (4), compound J-I24 can be synthesized.
(4) Synthesis of Compound J-I25
Referring to the synthesis method of compound J-I10 of (5), compound J-I25 can be synthesized.
(5) Synthesis of ionizable lipid Compound J-42
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, the compound J-42 may be synthesized.
By the above method, compound J-78 can be prepared by substituting the corresponding starting materials. J-78 can be prepared by substituting 8-bromooctanol for 6-bromohexanol.
8. Synthesis of ionizable lipid Compound J-43
(1) Synthesis of Compound J-I26
Referring to the synthesis method of compound J-I04 of (4), compound J-I26 can be synthesized.
(2) Synthesis of Compound J-I27
Referring to the synthesis method of compound J-I15 of (1), compound J-I27 can be synthesized.
(3) Synthesis of Compound J-I28
Referring to the synthesis method of compound J-I16 of (2), compound J-I28 can be synthesized.
(4) Synthesis of ionizable lipid Compound J-43
Referring to (6) a method for synthesizing an ionizable lipid compound J-03, the compound J-43 may be synthesized.
By adopting the method, the corresponding initial raw materials are replaced, and the compound J-79-J-81 can be prepared. J-79 can be prepared by replacing 8-bromooctanoic acid with 6-bromohexanoic acid.
9. Synthesis of contrast ionizable lipid Compound j-01
(1) Synthesis of ionizable lipid Compound j-01
J-I03 (0.50 g,0.99 mmol), 4-amino-1-butanol (35.12 mg,0.39 mmol), DIPEA (0.13 g,0.99 mmol) and 2.0mL ethanol were added to a reaction flask, and the reaction mixture was added to reflux and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 50mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE: ea=2:1) to give 265.0mg of product in 71% yield.
10. Synthesis of contrast ionizable lipid Compound j-02
Referring to the synthetic route of J-03, the corresponding raw materials are replaced, and J-02 is prepared.
The structure is as follows:
11. Synthesis of contrast ionizable lipid Compound j-03
(1) Synthesis of Compound j-I03
J-I10 (5.00 g,11.69 mmol), N-Boc-6-bromohexylamine (3.28 g,11.69 mmol), DIPEA (1.81 g,14.03 mmol) and 50.0mL ethanol were added to the reaction flask, and the reaction mixture was heated to 80℃and stirred for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, then poured into 100mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE: ea=3:1) to give 5.12g of product in 70% yield.
(2) Synthesis of Compound j-I04
J-I03 (1.00 g,1.59 mmol), 5mL of methylene chloride and 5mL of trifluoroacetic acid were added to the reaction flask and reacted at room temperature for 2 hours. After TLC monitored completion of the reaction, the reaction solution was concentrated, then poured into 50mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed twice with an equal volume of saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 0.76g of product in 91% yield.
(3) Synthesis of Compound 3- (FMOC-amino) -1-propanal
3- (FMOC-amino) -1-propanol (10.00 g,33.63 mmol), PCC (pyridinium chlorochromate, 8.70g,40.36 mmol) and 100mL of dichloromethane were added to the reaction flask and stirred at room temperature for 12h. After the completion of the TLC monitoring reaction, the reaction mixture was filtered through silica gel and concentrated to give a crude product. The crude product was purified by column chromatography (PE: ea=5:1) to give 4.65g of product in 47% yield.
(4) Synthesis of Compound j-I05
3- (FMOC-amino) -1-propanal (4.00 g,13.44 mmol), n-octanol (5.29 g,40.63 mmol), p-toluenesulfonic acid (0.23 g,1.35 mmol), anhydrous sodium sulfate (5.77 g,40.63 mmol) and 100mL of methylene chloride were added to the reaction flask and stirred at 40℃for 12h. After the completion of the TLC monitoring reaction, the reaction mixture was filtered through silica gel and concentrated to give a crude product. The crude product was purified by column chromatography (PE: ea=50:1) to give 4.65g of product in 62% yield.
(5) Synthesis of Compound j-I06
J-I05 (4.00 g,13.44 mmol), palladium on carbon (0.40 g), 40mL of ethanol were added to the flask, and the mixture was stirred at room temperature for 12h. After the completion of the TLC monitoring reaction, the reaction mixture was filtered through silica gel and concentrated to give a crude product. The crude product was purified by column chromatography (PE: ea=1:5) to give 2.15g of product in 90% yield.
(6) Synthesis of Compound j-I07
J-I06 (1.00 g,3.03 mmol), triethylamine (0.46 g,4.55 mmol) and 10mL of tetrahydrofuran were added to the flask, cooled to 0deg.C, carbon disulphide (0.28 g,3.64 mmol) was added dropwise with stirring, reacted at 0deg.C for 4h, then Boc anhydride (0.79 g,3.64 mmol) was added, gradually brought to room temperature and reacted for 12h. After the completion of the reaction, the reaction solution was concentrated by TLC, added to 100mL of ethyl acetate/water (1:1), and extracted, and the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE: ea=50:1) to give 0.73g of product in 65% yield
(7) Synthesis of ionizable lipid Compound j-03
J-I07 (0.50 g,1.35 mmol) and 5mL of DMF were added to the flask, and compound j-I04 (0.71 g,1.35 mmol) was added with stirring at 0deg.C and reacted at room temperature for 12h. After completion of the reaction, TLC was followed by addition of 50mL of ethyl acetate/water (1:1) and extraction, the organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE: ea=2:1) to give 0.81g of product in 67% yield.
Table 1 Nuclear magnetic resonance Hydrogen Spectroscopy data comparison Table of exemplary ionizable lipid Compounds J-01-J-33 in example one
Example two preparation and screening of nucleic acid lipid nanoparticles (mRNA-LNP)
1. Preparation of nucleic acid lipid nanoparticles
(1) The ionizable Lipid compounds (Lipid) J-01 to J-87, DSPC, cholesterol, and PEG-Lipid (PEG 2000-DMG) prepared in example I were dissolved in ethanol according to the molar relationship shown by 46.3/9.4/42.7/1.6 (molar ratio), respectively, to prepare different Lipid ethanol solutions (concentration of Lipid 20 mg/mL).
(2) MRNA was prepared at a Lipid Nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1 (15:1 ratio used in this example) and diluted to 0.2mg/mL using citrate or sodium acetate buffer (ph=3 or 5) to obtain an mRNA solution.
(3) The lipid ethanol solution and the mRNA solution were thoroughly mixed at a volume ratio of 1:5 to 1:1 (the ratio used in this example was 1:3). The obtained lipid nanoparticles were purified by ultrafiltration and dialysis, and after sterilization by filtration, the average particle diameter and PDI (polydispersity index) of mRNA-LNP (mRNA-entrapped lipid nanoparticles) were characterized by Malvern Zetasizer Nano ZS, and the encapsulation efficiency of mRNA was determined by using Ribogreen RNA quantitative assay kit. The results are shown in Table 2.
TABLE 2 characterization data for each set of mRNA-LNP
The results show that the ionizable lipid compounds J-01-J-87 provided by the embodiment II of the invention can form stable nano structures, the size distribution of the obtained mRNA-LNP is narrow, the size changes with the structures of different mRNA-LNP, and the size is in the range of 60-110 nm. Under the same conditions, mRNA-LNP was prepared by replacing each of the above-mentioned ionizable lipid compounds J-01 to J-87 with a comparative sample shown in Table 3, commercially available ALC0315, comparative sample J-01 (prepared in example one above), J-02 (prepared in example one above), and J-03 (prepared in example one above), respectively, and the average particle diameter, PDI, and encapsulation efficiency were measured. The structural formulas of ALC0315, j-01, j-02 and j-03 are shown in Table 3. The test results are shown in Table 4.
Table 3 Structure of comparative sample
TABLE 4 characterization data comparing mRNA-LNP of samples
As can be seen from the comparison of the data in tables 2 and 4, the mRNA-LNP provided by the embodiment of the invention can form a stable nano structure, has narrower size distribution, has a size which varies with the structures of different mRNA-LNPs, has an encapsulation rate of more than 95% in the range of 60-100nm, and has excellent physicochemical properties.
2. Low temperature storage stability
The mRNA-LNP provided by J-03 prepared in example II was stored in a dry environment at 4℃and the average particle size, PDI and mRNA encapsulation efficiency of the mRNA-LNP were measured at various time points (0 day, 15 days, 30 days, 60 days and 120 days) and the measurement results are shown in Table 5.
TABLE 5 comparison of Effect at Low temperature for different times
Preservation time (Tian) Average particle diameter (nm) PDI Encapsulation efficiency (%)
0 67.4 0.039 97.2
15 67.1 0.040 97.5
30 67.8 0.043 96.5
60 68.6 0.046 96.9
120 68.8 0.048 96.2
As can be seen from Table 5, the average particle size, PDI and encapsulation efficiency of mRNA-LNP provided by the present example group J-03 were not substantially changed within 120 days, and had good low-temperature storage stability, and the product was easy to store and transport.
MRNA-LNP prepared by the other compounds of this example group was selected according to the method described above, and the measurement results showed substantially no change in average particle size, PDI and encapsulation efficiency for 120 days.
Example III nucleic acid lipid nanoparticle (mRNA-LNP) transfection efficiency and biocompatibility
1. Cell transfection efficiency
HEK293 cells (100. Mu.L, cell density: 6X 10 4/ml) in an exponential growth phase were suspended in 96-well plates, incubated in a cell incubator for 24h, complete medium was changed to 60. Mu.L of DMEM serum-free medium, 0.4. Mu.g or 0.2. Mu.g of prepared Luciferase mRNA LNP formulation was added to each well, and three wells were incubated in a 37℃6% CO 2 incubator for 4h. Then, 60. Mu.L of the complete medium was added thereto, and the culture was continued for 24 hours. The medium was removed, gently washed once with 100. Mu. LPBS added, then 30. Mu.L/well of cell lysate was added, and the cells were lysed by shaking on a mini-shaker at room temperature for 16 min. The lysate was collected and centrifuged at 12000rpm for 6min, and the supernatant was collected. And adding 20 mu L of supernatant into a new black opaque 96-well plate, taking cell lysate as a blank control, adding 100 mu L of firefly luciferase detection solution, incubating for 6min in a dark place, detecting the chemiluminescence intensity RLU by using a multifunctional microplate detector, deducting the RLU of the blank control, and taking an average value. In Table 6E represents the power of scientific counting method 10, for example, "6.41E+04" means 6.41×10 4.
TABLE 6 transfection efficiency comparison Table HEK293 cells
2. Animal transfection efficiency
Male ICR mice (6-8 week, shanghai JieJie laboratory animal Co., ltd.) were kept at 22.+ -. 2 ℃ and a relative humidity of 45-75% for a 12h light/dark cycle. mRNA (luciferase mRNA) encoding luciferase was used as a reporter gene. Luciferase catalyzes luciferin to generate bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence in unit time. Taking luciferase mRNA (purchased from ApexBio Technology) as an example, mRNA-LNP samples J-01 to J-87 obtained in experiment one, commercial comparative samples ALC0315, comparative sample J-01, comparative sample J-02 and comparative sample J-03 were prepared, and the above samples were administered by intramuscular injection at a dose of 100. Mu.g/kg mRNA, two mice per group, and two legs, respectively. At a specific time point, fluorescein (20. Mu.g/mL) was injected into the abdominal cavity of the mice, and after 5 minutes, the mice were placed on a living body imager of the small animals to measure fluorescence intensity, and the final result is represented by average fluorescence intensity, and the experimental results of the fluorescence intensity after the intraperitoneal injection administration of the mice are shown in Table 7.
TABLE 7 fluorescent intensity control Table
Compared with commercial products, the mRNA-LNP prepared by the ionizable lipid compound provided by the invention can improve the level of 1-2 orders of magnitude on the transfection effect of various cell levels, greatly exceeds the expected range, has excellent cell transfection efficiency, and greatly expands the application of the mRNA-LNP in the field of cell therapy. In addition, the transfection efficiency in animals is higher than that of commercial products, and the transfection efficiency in animals is also quite good.
3. Biocompatibility of
Cell viability was determined using the CCK-8 kit. HEK 293 cell suspensions (100. Mu.L, cell density 2X 10 4/mL) in an exponentially growing phase were added to 96-well plates and incubated for 24h in a cell incubator. The cell culture broth was then removed and 100. Mu.L of fresh cell culture broth containing 100. Mu.g/mL of mRNA from example two was added and incubated with the cells for 24h. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and incubation was continued for 24 hours. The supernatant was removed, 100. Mu.L of fresh cell culture medium containing CCK-8 working solution (10. Mu.L/mL) was added, and incubated for 2h. A blank group was set at the same time, using an equivalent amount of CCK-8 working solution to replace mRNA-LNP, and the remaining conditions were identical. Absorbance at 450nm of each well (no bubbles can appear in the well plate during detection) was detected using a multi-functional microplate detector, and the viability of the untreated cells (control group) was set to 100%. The calculation formula of the cell viability of each group is shown as follows, wherein the cell viability (%) = [ A1-A0]/[ A2-A0 ]. Times.100. Wherein, A1 is absorbance of the dosing group, A0 is absorbance of the blank group, and A2 is absorbance of the control group. The experimental results are shown in table 8.
TABLE 8 comparison of cell viability effects
Experimental results show that LNP prepared by the lipid compound has little influence on cell viability, cells keep more than 80% of viability, and the cell viability of a commercial comparison sample ALC0315 is only 55% under the same high concentration, so that the in vitro safety is far lower than that of the lipid compound.
Example IV in vivo safety experiment
To further verify the safety of the lipid compounds of the present invention, we have illustratively selected some of the compounds prepared in example one to conduct related animal experiments.
The preparation method comprises the steps of preparing materials, namely, half female/male Balb/C mice with six weeks of age, keeping the weight of 15-20 g, and feeding 110 mice in an experimental environment with the temperature of 22+/-2 ℃ and the relative humidity of 45-75%, wherein the light/dark period is 12 hours. After the mice are purchased and adapted in animal houses for one week, formal animal tests can be carried out. 110 mice were randomly divided into 11 groups, each group of which was a male and female half, group 1 was intravenous injection of equal volume PBS (negative control group), group 2 was intravenous injection of commercial control sample ALC-0315 (260. Mu.g of mRNA), group 3 was intravenous injection of commercial control sample ALC-0315 (520. Mu.g of mRNA), group 4 was intravenous injection of control sample J-01 (260. Mu.g of mRNA), group 5 was intravenous injection of control sample J-01 (520. Mu.g of mRNA), group 6 was intravenous injection of control sample J-02 (260. Mu.g of mRNA), group 7 was intravenous injection of control sample J-02 (520. Mu.g of mRNA), group 8 was intravenous injection of control sample J-03 (260. Mu.g of mRNA), group 9 was intravenous injection of control sample J-03 (520. Mu.g of mRNA), group 10 was intravenous injection of control sample J-03 (260. Mu.g of mRNA), group 11 was intravenous injection of control sample J-03 (520. Mu.g of mRNA), and the above mRNAs were the full-length mRNAs which were expressed by transcription of luciase in vitro based on an autonomously designed template.
The experimental procedure was to count the number of surviving mice in each group at 0h,24h,48h and 72h, respectively.
Table 10 statistical table of the number of mice surviving
Experimental results show that mRNA-LNP composed of ALC0315 and comparative sample J-03 directly causes different degrees of death (260. Mu.g mRNA) and even all death (520. Mu.g mRNA) of mice when injected intravenously at a large dose, but no death of mice is observed when mRNA-LNP composed of J-03 is injected at a large dose.
For other compounds of this example, which also possess a safety profile similar to that of J-03, exemplary compounds J-01, J-05, J-09, J-48, at higher doses, mice survived almost entirely.
In conclusion, the safety of the ionizable lipid prepared by the invention is extremely high, and the ionizable lipid has wider clinical application value compared with the product ALC0315 and other similar structural compounds on the market.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (12)

1. An ionizable lipid compound, characterized in that said ionizable lipid compound is selected from the group consisting of:
2. The ionizable lipid compound of claim 1, wherein said ionizable lipid compound is selected from the group consisting of:
3. A lipid nanoparticle comprising one or more of the ionizable lipid compounds, stereoisomers thereof, tautomers thereof, or pharmaceutically acceptable salts thereof according to any one of claims 1-2.
4. The lipid nanoparticle of claim 3, further comprising a combination of any one or more of a structural lipid, a helper lipid, a PEG-lipid, a polymer.
5. The lipid nanoparticle of claim 4, wherein the structural lipid is selected from one or more of sterols, non-sterols, or derivatives thereof, and/or the helper lipid is selected from one or more of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, ceramide, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, DOTAP, 18:1pa, HS15, GL67, and/or the PEG-lipid is selected from one or more of PEG-modified phosphatidyl ethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecylamide.
6. The lipid nanoparticle of claim 4, wherein the mole percent of the ionizable lipid compound to the helper lipid to the structural lipid to the PEG-lipid is (20-65): 0-60: (0-10), and at least any one of the helper lipid, the structural lipid, and the PEG-lipid is other than 0.
7. The lipid nanoparticle of claim 4, wherein the mole percent of the ionizable lipid compound, the helper lipid, the structural lipid, and the PEG-lipid is (20-65): 3-50): 15-60): 0.1-10.
8. The lipid nanoparticle of claim 4, wherein the molar ratio of the ionizable lipid compound to the polymer is 0.5:1-100:1.
9. The lipid nanoparticle of claim 4, wherein the molar ratio of the ionizable lipid compound to the polymer is from 10:1 to 80:1.
10. The lipid nanoparticle of claim 4, wherein the molar ratio of the ionizable lipid compound to the polymer is from 40:1 to 80:1.
11. Use of a lipid nanoparticle according to any one of claims 3-10 for the preparation of a pharmaceutical composition further comprising the drug carried and/or pharmaceutically acceptable excipients.
12. The use of claim 11, wherein the drug comprises one or more of a nucleic acid molecule, a small molecule compound, and a protein.
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