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WO2025010554A1 - Composés lipidiques déstinés à la délivrance d'agents thérapeutiques et leur procédé de préparation et leur utilisation - Google Patents

Composés lipidiques déstinés à la délivrance d'agents thérapeutiques et leur procédé de préparation et leur utilisation Download PDF

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WO2025010554A1
WO2025010554A1 PCT/CN2023/106421 CN2023106421W WO2025010554A1 WO 2025010554 A1 WO2025010554 A1 WO 2025010554A1 CN 2023106421 W CN2023106421 W CN 2023106421W WO 2025010554 A1 WO2025010554 A1 WO 2025010554A1
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lipid
compound
alkyl
formula
alkenyl
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Zijun Wang
Yang GUI
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Yoltech Therapeutics Co Ltd
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Yoltech Therapeutics Co Ltd
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Priority to PCT/CN2023/106421 priority Critical patent/WO2025010554A1/fr
Priority to PCT/CN2024/104304 priority patent/WO2025011532A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • 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
    • C07C219/16—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 at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
    • C07C275/14—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/03—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C311/05—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms to acyclic carbon atoms of hydrocarbon radicals substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C329/00—Thiocarbonic acids; Halides, esters or anhydrides thereof
    • C07C329/02—Monothiocarbonic acids; Derivatives thereof
    • C07C329/04—Esters of monothiocarbonic acids
    • C07C329/06—Esters of monothiocarbonic acids having sulfur atoms of thiocarbonic groups bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C333/02—Monothiocarbamic acids; Derivatives thereof
    • C07C333/04—Monothiocarbamic acids; Derivatives thereof having nitrogen atoms of thiocarbamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table

Definitions

  • the present invention belongs to the field of biopharmaceuticals and, specifically, relates to lipid compounds for delivery of therapeutic agents and preparation method and its use thereof.
  • nucleic acid drugs can prevent and treat cancer, bacterial and viral infections, and treat diseases with inherited pathogenic factor, etc. Since nucleic acid drugs are easily degradable and difficult to enter cells, they usually need to be encapsulated and delivered to target cells with the help of carriers, therefore, the development of safe and efficient delivery carriers is a precondition for the clinical application of gene therapy.
  • Lipid nanoparticle is currently a hot research topic in the field of non-viral gene vectors.
  • FDA approved LNP delivery of patisiran (onpattro) for the treatment of hereditary transthyretin familial amyloidosis and since then the research on the delivery of nucleic acid drugs using LNP technology has shown a burst of growth.
  • LNPs usually contain ionizable lipids which have an impact on the effectiveness of LNP delivery.
  • lipid compound or a pharmaceutically acceptable form thereof e.g., salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug, etc.
  • other lipid compounds e.g., neutral lipids, charged lipids, steroids
  • therapeutic agents e.g., nucleic acid molecules, specifically also including mRNA
  • lipid compounds with specific structures can be used as lipid carriers depending on the organ where the nucleic acid drug needs to be enriched.
  • nucleic acid lipid nanoparticle compositions comprising the above compounds or pharmaceutically acceptable forms thereof or the above lipid carriers.
  • R 1 is selected from -OH and R a (R b ) N-, wherein R a and R b are each independently hydrogen, C1-C 10 alkyl or C1-C 10 haloalkyl;
  • R 2 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 3 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, or R c - (CH 2 ) n-, wherein n is a positive integer of 1-20, preferably, a positive integer of 1-14, more preferably a positive integer of 1-10;
  • R c is selected from the structure as shown below:
  • L1, L2, L3, L4, L5 are absent or each independently selected from the group consisting of:
  • X is absent or -CH-or N
  • R 4 is absent or C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 5 is absent or C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 6 is C 1 -C 30 alkyl, C 2 -C 30 alkenyl, C 2 -C 30 alkynyl;
  • R 7 is C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl;
  • R 8 , R 9 are absent or is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl or -R h -C 1 -C 14 alkyl, -R h -C 2 -C 14 alkenyl, -R h -C 2 -C 14 alkynyl, wherein R h is O or S;
  • R 10 , R 11 is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl, or -R h -C 1 -C 14 alkyl, -R h -C 2 -C 14 alkenyl, -R h -C 2 -C 14 alkynyl, wherein R h is O or S.
  • R a and R b are each independently hydrogen, C1-C 6 alkyl or C1-C 6 haloalkyl.
  • R 2 is selected from - (CH 2 ) m-, wherein m is a positive integer from 1-20, preferably, a positive integer from 1-14, preferably, a positive integer from 1-10, more preferably, a positive integer from 1-6.
  • R 6 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl.
  • R 2 is C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl.
  • R 2 , R 7 is each independently C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl.
  • R 4 , R 5 is each independently - (CH 2 ) q-CH 3 , wherein q is selected from a positive integer of 1-20, preferably, a positive integer of 1-12, more preferably, a positive integer of 1-8.
  • R 4 , R 5 each independently has a structure -R d -R e -;
  • R e is - (CH 2 ) p-CH3, wherein p is selected from a positive integer of 0-20, preferably, p is selected from a positive integer of 0-12, preferably, p is selected from a positive integer of 0-8.
  • R 8 , R 9 is each independently - (CH 2 ) q-CH 3 , wherein q is selected from a positive integer of 1-14, preferably, a positive integer of 1-12, and preferably, a positive integer of 1-8.
  • R 7 is - (CH 2 ) q-CH 3 , wherein q is selected from a positive integer of 1-14, preferably, a positive integer of 1-12, and preferably, a positive integer of 1-8.
  • R 6 has a structure -R f -R g -;
  • X is absent or -CH-.
  • the compound of Formula I has a structure of Formula I-2 as follows:
  • R 1 -R 2 , R 6 -R 7 , R 10 -R 11 , L 1 -L 5 are defined as above;
  • R 3 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, or R c - (CH 2 ) n-, wherein n is a positive integer of 1-20, preferably, a positive integer of 1-14, more preferably a positive integer of 1-10, R c is defined as above;
  • R 5 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 5 is C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl.
  • R 3 is C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl.
  • R 3 is C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl.
  • the compound of Formula I has a structure of Formula I-3 as follows:
  • R 1 -R 3 , R 6 , R 7 , R 10 , R 11 , L 1 -L 5 are defined as above;
  • R 4 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 5 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 8 , R 9 is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl.
  • R 4 is C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl.
  • R 4 is C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl.
  • R 5 is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • R 8 , R 9 is each independently C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl.
  • R 9 is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • the compound of Formula I has a structure of Formula I-4 as follows:
  • R 1 -R 7 , R 10 -R 11 , L 1 -L 3 , L 5 are defined as above.
  • R 1 -R 11 , L 1 -L 5 , X are the specific groups corresponding to each specific compound in the embodiment.
  • the compound is each specific compound prepared in the embodiment, preferably selected from the group consisting of (Table 1) :
  • the compound of Formula I preferably has the structure as shown below:
  • the compound of Formula I preferably has the structure as shown below:
  • the compound of Formula I can be used to prepare a drug delivery system, comprising a lipid nanoparticle (LNP) , liposome, polymer nanoparticle, etc., preferably for preparing a lipid nanoparticle.
  • a drug delivery system comprising a lipid nanoparticle (LNP) , liposome, polymer nanoparticle, etc., preferably for preparing a lipid nanoparticle.
  • lipid carrier comprising a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, as described in the first aspect of the present invention.
  • the lipid carrier further comprises an auxiliary lipid.
  • the content of the compound of Formula I is in the range of 30-65%molar ratio of the total lipid content.
  • the content of the compound of Formula I is in the range of 40-60%molar ratio of the total lipid content; preferably, 50%molar ratio.
  • the auxiliary lipid comprises one or a combination of more than two of an anionic lipid, a neutral lipid, a steroid and a polymer-bound lipid.
  • the lipid carrier further comprises other cationic or ionizable lipid compounds.
  • the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof of claim 1 and optionally other ionizable lipids, and the second lipid compound comprises one or a combination of more than two of an anionic lipid, a neutral lipid, a steroid and a polymer-bound lipid.
  • the polymer-bound lipid comprises one or a combination of two or more of 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , ⁇ - (3 ′- ⁇ [1, 2-di (myristyloxy) propanoxy] carbonylamino ⁇ propyl) - ⁇ -methoxy, polyoxyethylene (PEG-c-DMG) , PEG-1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA) , 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol) ] (PEG-DSPE) , pegylated phosphatidylethanoloamine (PEG-PE) , PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 55) : (0 to 13) : (5 to 25) : (25 to 51.5) : (0.5 to 15) ; wherein, in the first lipid compound, the compound of Formula I or a pharmaceutically acceptable form thereof such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug and other cationic or ionizable lipids in a molar ratio of (3-4) : (0-5) .
  • the lipid carrier further comprises a bioactive substance encapsulated in the lipid carrier.
  • the bioactive substance is selected from the group consisting of: a nucleic acid, protein, polypeptide, small molecule, and a combination thereof.
  • the nucleic acid comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid, or PNA.
  • the antisense nucleic acid is an antisense oligonucleic acid.
  • the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.
  • the DNA comprises a plasmid.
  • the mRNA comprises a sequence encoding an RNA-directed DNA-binding agent, and more specifically, comprises an mRNA encoding a nuclease or a base editor.
  • the nucleic acid further comprises a guide RNA, specifically, the guide RNA comprises a gRNA nucleic acid.
  • the nucleic acid comprises an mRNA encoding a nuclease or a base editor and a gRNA.
  • the gRNA is a modified or unmodified gRNA.
  • the lipid carrier is highly efficient in encapsulating the bioactive substance and greatly enhances the efficiency of delivery of the bioactive substance in vivo.
  • the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the compound of Formula I according to the first aspect of the present invention and optionally other ionizable lipids, and the second lipid compound comprises one or a combination of more than two of an anionic lipid, a neutral lipid, a steroid and a polymer-bound lipid.
  • the first lipid compound is any of the above compounds or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, reciprocal isomer, solvate, chelate, non-covalent complex or a precursor drug.
  • the first lipid compound is a combination of any of the above compounds or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, reciprocal isomer, solvate, chelate, non-covalent complex or a precursor drug and other cationic or ionizable lipids.
  • the other cationic or ionizable lipid compounds include one or a combination of more than two of 1, 2-diolethoxy-N, N-dimethylaminopropane (DLinDMA) , 1, 2-diolethoxy-N, N-dimethylaminopropane (DODMA) , DLin-MC2-MPZ, 2, 2-diolethoxy-4- (2-dimethylaminoethyl) - [1, 3] -dioxolane pentane (DLin-KC2-DMA) , 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) , 1, 1’- (2- (4- (2- ( (2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol
  • the anionic lipid comprises one or a combination of more than two of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG) , 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) , and dimyristoylphosphatidylglycerol.
  • DOPG dioleoylphosphatidylglycerol
  • DOPS 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine
  • dimyristoylphosphatidylglycerol dimyristoylphosphatidylglycerol.
  • the neutral lipid comprises at least one of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , Dipalmitoylphosphatidylglycerol (DPPG) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , palmitoyloleoyl phosphatidylethanolamine (POPE) , 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE, dipalmitoyl phosphatidylethanolamine (DPPE) Dimyristoylphosphoethanolamine (DMPE)
  • the steroids include one or more of cholesterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, tomatidine, ursolic acid, alpha totaxin, coprosterol, and corticosteroids.
  • the polymer-bound lipid comprises one or a combination of two or more of 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , ⁇ - (3′- ⁇ [1, 2-di (myristyloxy) propanoxy] carbonylamino ⁇ propyl) - ⁇ -methoxy, polyoxyethylene (PEG-c-DMG) , PEG-1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA) , 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol) ] (PEG-DSPE) , pegylated phosphatidylethanoloamine (PEG-PE) , PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 65) : (0 to 20) : (5 to 25) : (25 to 55) : (0.3 to 15) ; exemplarily, the molar ratio may be 20: 20: 5: 50: 5, 30: 5: 25: 30: 10, 20: 5: 5: 55: 15, 65: 0: 9.7: 25: 0.3, etc.; wherein, in the first lipid compound, the compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug thereof and other cationic or ionizable ionizable lipids in a molar ratio of (1 to 10) : (0 to 10) ; exemplarily, the molar ratio may be 1: 1, 1: 2, 1: 5, 1: 7.5, 1: 10, 2: 1, 5:
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 55) : (0 to 13) : (5 to 25) : (25 to 51.5) : (0.5 to 15) ; wherein, in the first lipid compound, the compound of Formula I or a pharmaceutically acceptable form thereof such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug and other cationic or ionizable lipids in a molar ratio of (3-4) : (0-5) .
  • lipid nanoparticle comprising a compound of Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, as described in the first aspect of the present invention.
  • the lipid nanoparticle further comprises an auxiliary lipid.
  • the lipid nanoparticle further comprises a bioactive substance encapsulated in the lipid nanoparticle.
  • a lipid nanoparticle composition comprising a lipid carrier as described in a second aspect of the present invention or a lipid nanoparticle as described in a third aspect of the present invention, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle.
  • the mass ratio of the bioactive substance to the lipid carrier in the composition is 1: (3 to 40) , exemplarily, the mass ratio is 1: 3, 1: 5, 1: 10, 1: 15, 1: 20, 1: 30, 1: 40, etc.
  • the ratio (w/w) of the nuclease or base editor mRNA to gRNA in the bioactive substance is from about 10: 1 to about 1: 10.
  • the ratio (w/w) of the nuclease or base editor mRNA to gRNA of in the bioactive substance is from about 3: 1 to about 1: 3.
  • the ratio (w/w) of the nuclease or base editor mRNA to gRNA in the bioactive substance is about 3: 2.
  • the composition comprises a pharmaceutical composition.
  • a pharmaceutical composition comprising a lipid carrier as described in a second aspect of the present invention or a lipid nanoparticle as described in a third aspect of the present invention, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle, and a pharmaceutically acceptable excipient, carrier or diluent.
  • a pharmaceutical formulation comprising a lipid carrier as described in the second aspect of the present invention or a lipid nanoparticle as described in the third aspect of the present invention, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle, and a pharmaceutically acceptable excipient, carrier or diluent; or the pharmaceutical formulation comprising a lipid nanoparticle composition as described in the fourth aspect of the present invention, and a pharmaceutically acceptable excipient, carrier or diluent.
  • the pharmaceutical formulation has a particle size of 30 to 500 nm, exemplarily, the particle size may be 30 nm, 50 nm, 100 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.
  • the encapsulation efficiency of the bioactive substance in the pharmaceutical formulation is greater than 50%.
  • the encapsulation efficiency may be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.
  • the drug has a hydrated particle size of 50-200 nm, preferably 70-150 nm, more preferably 75-110 nm.
  • the pharmaceutical formulation can be applied for the treatment and/or prevention of a disease.
  • the disease is selected from the group consisting of a metabolic disease, genetic disease, cancer, cardiovascular disease, infectious disease, and a combination thereof, preferably the metabolic disease comprises familial hypercholesterolemia (FH) , the genetic disease includes transthyretin amyloidosis (ATTR) , primary hyperoxuria (PH1) , and hereditary angioedema (HAE) , and the infectious disease includes hepatitis B (HEPATITIS B) .
  • FH familial hypercholesterolemia
  • the genetic disease includes transthyretin amyloidosis (ATTR) , primary hyperoxuria (PH1) , and hereditary angioedema (HAE)
  • HEPATITIS B hepatitis B
  • the pharmaceutical formulation is in a dosage form selected from the group consisting of: injection, lyophilized agent, aerosol inhalation, smearing preparation.
  • the pharmaceutical agent is administered by injection, i.e. intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraduodenally or intraperitoneally.
  • the pharmaceutical formulation is administered by inhalation, for example intranasally.
  • the pharmaceutical formulation is administered transdermally, such as transdermal application administration or electrode introduction administration.
  • a seventh aspect of the present invention provides a use of a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof as described in the first aspect of the present invention in the preparation of a lipid nanoparticle.
  • an eighth aspect of the present invention provides a use of a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof described in the first aspect of the present invention or a lipid carrier described in the second aspect of the present invention or a lipid nanoparticle described in the third aspect of the present invention or a lipid nanoparticle composition described in the fourth aspect of the present invention or a pharmaceutical composition described in the fifth aspect of the present invention or a pharmaceutical formulation described in the sixth aspect of the present invention in the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug.
  • a ninth aspect of the present invention provides a method for preparing a lipid nanoparticle composition as described in a fourth aspect of the present invention, comprising:
  • step (c) Mixing the lipid organic phase in step (a) with the aqueous phase in step (b) , thereby obtaining the lipid nanoparticle composition.
  • the organic solvent comprises ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, or a combination thereof.
  • the aqueous solvent is a buffer.
  • the aqueous solvent is a buffer in the pH range of 3-7.
  • the acidic buffer is a citrate buffer of pH 4.0.
  • the volume ratio of the lipid organic phase to the aqueous phase containing the bioactive substance is 1: (2-5) , preferably 1: (3-4) .
  • step (c) the lipid organic phase and the aqueous phase are mixed by means of a microfluidic chip.
  • the method further comprises step (d) : the lipid nanoparticle composition obtained in step (c) is purified and concentrated and filtered for sterilization.
  • a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof as described in the first aspect of the present invention for the preparation of a drug delivery system.
  • the delivery system comprises lipid nanoparticle (LNP) , liposome, polymeric nanoparticle, etc., preferably for the preparation of lipid nanoparticle.
  • LNP lipid nanoparticle
  • the drug delivery system is used to deliver drugs for the treatment and/or prevention of tumors, infectious diseases and rare diseases.
  • an eleventh aspect of the present invention provides a use of a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof as described in a first aspect of the present invention or a lipid carrier as described in a second aspect of the present invention or lipid nanoparticle as described in a third aspect of the present invention or a lipid nanoparticle composition as described in a fourth aspect of the present invention or a pharmaceutical composition as described in a fifth aspect of the present invention or a pharmaceutical formulation as described in a sixth aspect of the present invention for the preparation of drugs for the treatment and/or prevention of a disease.
  • the disease is selected from the group consisting of a metabolic disease, genetic disease, cancer, cardiovascular disease, infectious disease, and a combination thereof, preferably the metabolic disease comprises familial hypercholesterolemia (FH) , the genetic disease includes transthyretin amyloidosis (ATTR) , primary hyperoxuria (PH1) , and hereditary angioedema (HAE) , and the infectious disease includes hepatitis B (HEPATITIS B) .
  • FH familial hypercholesterolemia
  • the genetic disease includes transthyretin amyloidosis (ATTR) , primary hyperoxuria (PH1) , and hereditary angioedema (HAE)
  • HEPATITIS B hepatitis B
  • a method of delivering a therapeutic or prophylactic agent to a cell, tissue or organ in a subject comprising contacting the cell, tissue or organ in the subject with a lipid nanoparticle composition as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention or a pharmaceutical formulation as described in the sixth aspect of the present invention.
  • the therapeutic agent or prophylactic agent is a bioactive substance, preferably mRNA.
  • the cell is selected from the group consisting of: a hepatocyte, lung cell, epithelial cell, hematopoietic cell, endothelial cell, bone cell, stem cell, mesenchymal cell, nerve cell, photoreceptor cell, retinal pigment epithelium cell, secretory cell, heart cell, adipocyte, smooth muscle cell, cardiomyocyte, skeletal muscle cell, ⁇ cell, pituicyte, synovial lining cell, ovarian cell, testicular cell, fibroblast, lymphocyte, B cell, T cell, antigen presenting cell, reticulocyte, leukocyte, granulocyte, and tumor cell.
  • a hepatocyte lung cell, epithelial cell, hematopoietic cell, endothelial cell, bone cell, stem cell, mesenchymal cell, nerve cell, photoreceptor cell, retinal pigment epithelium cell, secretory cell, heart cell, adipocyte, smooth muscle cell, cardiomyocyte, skeletal muscle cell,
  • the tumor cell includes lung cancer cell, colon cancer cell, rectal cancer cell, anal cancer cell, bile duct cancer cell, small intestine cancer cell, stomach cancer cell, esophageal carcinoma cell, gallbladder carcinoma cell, liver cancer cell, pancreatic cancer cell, appendix cancer cell, breast cancer cell, ovarian cancer cell, cervical cancer cell, prostate cancer cell, kidney cancer cell, cancer cell of the central nervous system, glioblastoma tumor cell, skin cancer cell, lymphoma cell, choriocarcinoma tumor cell, head and neck cancer cell, osteogenic sarcoma tumor cell, and blood cancer cell.
  • the tissue or organ is selected from the group consisting of:heart, liver, spleen, lung, kidney, brain, lymph node, muscle, blood, spine, bone, and a combination thereof.
  • the subject is a human or non-human mammal (such as mouse, rat, rabbit, monkey) .
  • a method for producing a target protein or target polypeptide in a subject cell comprising contacting the subject cell with a lipid nanoparticle composition as described in a fourth aspect of the present invention, a pharmaceutical composition as described in a fifth aspect of the present invention, or a pharmaceutical formulation as described in a sixth aspect of the present invention.
  • the pharmaceutical composition or pharmaceutical formulation is mRNA, wherein the mRNA encodes a target protein or polypeptide, whereby the mRNA is capable of being translated in the cell to produce the target protein or target polypeptide.
  • Figure 1 shows a schematic diagram of the delivery strategy for PCSK9 gene base editing in mouse liver cells in the present invention.
  • Figure 2 shows base editing efficiencies for editing of PCSK9 gene in mouse liver cells using lipid nanoparticles (LNPs) containing different compounds.
  • LNPs lipid nanoparticles
  • the present inventors After a long and thorough research, the present inventors have accidentally discovered for the first time an ionizable lipid compound for delivering therapeutic agents, and the ionizable lipid compound can effectively deliver drugs such as nucleic acid molecules and small molecule compounds, and by comparison, the lipid nanoparticles of the present invention have better particle size distribution, high encapsulation efficiency, and the delivery effect can be significantly better than that of the comparison lipid nanoparticles, which can meet the demand for in vivo delivery. On this basis, the present invention is completed.
  • references to “some specific/preferred embodiments” , “other specific/preferred embodiments” , “embodiments” , etc. refer to the particular elements (e.g., features, structures, properties, and/or characteristics) described in connection with the embodiment are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Further, it should be understood that the elements described may be combined in any suitable manner in various embodiments.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention that is substantially non-toxic to the organism.
  • a pharmaceutically acceptable salt generally includes, but is not limited to, a salt formed by reaction of a compound of the invention with a pharmaceutically acceptable inorganic/organic acid or inorganic/organic base, which are also referred to as acid addition salts or base addition salts.
  • Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.
  • common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide etc.
  • Common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutal, etc.
  • stereoisomer refers to stable isomers having a vertical plane of asymmetry due to having at least one chiral factor (including a chiral center, a chiral axis, a chiral plane, etc. ) , and thus capable of rotating plane-polarized light. Since there are asymmetric centers as well as other chemical structures in the compounds of the present invention that may lead to stereoisomerism, the present invention also includes these stereoisomer and mixtures thereof. Since the compounds of the present invention and their salts include asymmetric carbon atoms, they can exist as mixtures of single stereoisomer, racemates, enantiomers and diastereoisomers.
  • these compounds can be prepared in the form of racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomer, i.e., single enantiomers or diastereoisomers, or mixtures of single stereoisomer enrichment (Purity ⁇ 98%, purity ⁇ 95%, ⁇ 93%, ⁇ 90%, ⁇ 88%, ⁇ 85%or ⁇ 80%) .
  • Single stereoisomers of compounds were prepared synthetically from spin starting materials containing the desired chiral center, or were prepared by preparing a mixture of enantiomeric products followed by separation or splitting, e.g., conversion to a mixture of diastereoisomers followed by separation or recrystallization, chromatography treatment, use of chiral splitting reagents, or direct separation of the enantiomers on a chiral column.
  • Starting compounds with specific stereochemistry can either be commercially available or can be prepared as described herein and then split by methods well known in the art.
  • tautomer refers to structural isomers with different energies that can be transformed into each other through low energy barriers. If tautomerizm is possible (e.g., in solution) , chemical equilibrium of the tautomer can be achieved.
  • proton tautomer or proton transfer tautomer
  • proton migration include (but are not limited to) interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imidohydrine isomerization, etc. Unless otherwise indicated, all tautomer forms of the compounds of the present invention are within the scope of the present invention.
  • solvate refers to a substance formed by combining a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule by non-covalent intermolecular forces. Common solvate includes (but is not limited to) hydrate, an ethanolate, an acetonide, etc.
  • chelate refers to a complex with a ring structure, obtained by chelating two or more ligands with the same metal ion to form a chelate ring.
  • non-covalent complex is formed by the interaction of a compound with another molecule, where no covalent bond is formed between the compound and the molecule.
  • compounding can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also called ionic bonding) .
  • precursor drug refers to a derivative compound that, when applied to a patient, can directly or indirectly provide a compound of the present invention.
  • Particularly preferred derivative compounds or precursors are compounds that, when administered to a patient, enhance the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or facilitate the delivery of the parent compound to the site of action (e.g., the lymphatic system) .
  • all prodrug forms of the compounds of the present invention are within the scope of the present invention, and the various prodrug forms are well known in the art.
  • each independently means that at least two groups (or ring systems) having the same or similar range of values present in the structure may have the same or different meanings in a particular case.
  • substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl
  • substituent Y when substituent X is hydrogen, substituent Y can be either hydrogen, halogen, hydroxyl, cyano, alkyl or aryl
  • substituent Y when substituent Y is hydrogen, substituent X can be either hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.
  • alkyl refers to a monovalent straight or branched alkyl group consisting solely of carbon and hydrogen atoms, containing no unsaturation, and connected by a single bond to other fragments, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, and tert-butyl.
  • C 1-30 alkyl refers to a saturated monovalent straight or branched chain hydrocarbon group containing 1 to 30 carbon atoms.
  • alkylene refers to a divalent straight or branched alkyl group consisting of only carbon and hydrogen atoms, containing no saturation, and connected by two single bonds to other fragments, respectively, including (but not limited to) methylene, 1, 1-ethylidene and 1, 2-ethylidene.
  • C 1-30 alkylene refers to a saturated, divalent straight or branched alkyl group comprising 1 to 30 carbon atoms.
  • cycloalkyl refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkyl groups may include parallel, bridged or spiro ring systems.
  • C 3-6 cycloalkyl refers to a cycloalkyl group having from 3 to 6 carbon atoms.
  • a cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo [2.2.1] heptyl, etc.
  • cycloalkylene refers to a divalent group obtained by removing the hydrogen atom from a cycloalkyl group as defined above, including (but not limited to) cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, etc.
  • C 3-30 cycloalkylene refers to a divalent group obtained by removing the hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.
  • branched alkyl refers to an alkane radical that is linked to the parent molecule and forms at least two branching structures of its own. For example,
  • alkenyl refers to a monovalent straight or branched alkane group consisting of only carbon and hydrogen atoms, containing at least one double bond, and connected by a single bond to other fragments, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl.
  • alkenylene refers to a divalent straight or branched alkane group consisting of only carbon and hydrogen atoms, containing at least one double bond, and connected by two single bonds to other fragments, respectively, including (but not limited to) a ethenylidene.
  • alkynyl refers to a monovalent straight or branched alkyl group consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected by a single bond to other fragments, including (but not limited to) acetenyl, propinyl, butynyl and pentynyl.
  • C 2-30 alkynyl refers to a monovalent straight or branched chain hydrocarbyl containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.
  • alkynylene refers to a divalent straight or branched alkyl group consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by two single bonds each, including (but not limited to) anethynylene, etc.
  • C 2-30 alkynylene refers to a divalent straight or branched hydrocarbyl comprising 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.
  • cycloalkenyl group refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbyl consisting of only carbon and hydrogen atoms. Cycloalkenyl group may include merged, bridged or spiro ring systems. Examples include cyclopropanyl and cyclobutene.
  • cycloalkenylene refers to a divalent group obtained by removing the hydrogen atom from a cycloalkenyl group as defined above, including (but not limited to) subcyclopropanyl and subcyclobutene, etc.
  • C 3-30 cycloalkenylene refers to a divalent group obtained by removing hydrogen atoms from a cycloalkenyl group comprising 3 to 30 carbon atoms.
  • branched alkenyl refers to an alkenes radical that is linked to the parent molecule and forms at least two branching structures of its own.
  • the heterocyclic group may be attached to the rest of the molecule by any one of the ring atoms if the valence bond requirement is met.
  • the term "3-8-membered heterocyclic group" as used in the present invention refers to a heterocyclic group having from 3 to 8 ring atoms.
  • oxiranyl aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.
  • aryl refers to a monocyclic or dense polycyclic aromatic hydrocarbyl having a conjugated ⁇ -electron system.
  • C 6-10 aryl refers to an aryl group having 6 to 10 carbon atoms.
  • aryl can be phenyl, naphthyl, anthryl, phenanthryl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.
  • heteroaryl refers to a monocyclic or dense polycyclic aromatic group having a conjugated ⁇ -electron system, where the ring atom consists of a carbon atom and at least one heteroatom selected from N, O and S.
  • the heteroaromatic group can be attached to the rest of the molecule through any one of the ring atoms if the valence bonding requirement is met.
  • the term "5-10-membered heteroaryl” as used in the present invention refers to a heteroaryl group having 5 to 10 ring atoms.
  • Common heteroaryl groups include (but are not limited to) thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the benzo derivatives thereof, pyrrolopyridyl, pyrrolopyridinyl, pyrazolopyridyl, imidazolopyridyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like.
  • halogen refers to fluorine (F) , chlorine (Cl) , bromine (Br) and iodine (I) .
  • hydroxyl refers to -OH.
  • cyano refers to -CN.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • subject includes both human and non-human animals.
  • Non-human animals include vertebrates, such as mammals, and non-mammals, such as non-human primates, sheep, cats, horses, cattle, chickens, dogs, mice, rats, goats, rabbits, and pigs.
  • the subject is a human.
  • patient or subject are used interchangeably herein except when indicated.
  • nuclease refers to an enzyme that catalyzes the breaking of phosphodiester bonds between nucleotides in a nucleic acid molecule.
  • the nuclease is selected from a broad range of nucleases, zinc-finger nucleases (ZFNs) , TAL-effector DNA-binding domain-nuclease fusion proteins (TALEN) , and RNA-directed nucleases or variants thereof in which the nuclease activity has been reduced or inhibited.
  • the RNA-guided nucleases are naturally occurring CRISPR-Cas proteins or active variants or fragments thereof.
  • CRISPR-Cas systems are classified as either class I or class II systems.
  • Class II systems contain single effector nucleases and include types II, V, and VI.
  • Each category is subdivided into types (types I, II, III, IV, V, VI) , some of which are further subdivided into subtypes (e.g., type II-A, II-B, II-C, V-A, V-B) .
  • type II CRISPR-Cas protein refers to CRISPR-Cas effector proteins that require trans-activated RNA (tracrRNA) and contain two nuclease structural domains (RuvC and HNH) (each of which is responsible for cleaving a single strand of a double-stranded DNA molecule) .
  • the CRISPR-Cas protein is a naturally occurring V-type CRISPR-Cas protein or an active variant or fragment thereof.
  • V-type CRISPR-Cas protein As used herein, the terms "V-type CRISPR-Cas protein, " "V-type CRISPR-Cas effector protein, “ or “Cas12” refer to CRISPR-Cas effector protein that cuts dsDNA and contains a single RuvC nuclease domain or a split RuvC nuclease domain and lacks the HNH domain.
  • the CRISPR-Cas protein is a naturally occurring type VI CRISPR-Cas protein or an active variant or fragment thereof.
  • type VI CRISPR-Cas protein As used herein, the terms "type VI CRISPR-Cas protein, " “type VI CRISPR-Cas effector protein, “ or “Cas13” refer to CRISPR-Cas effector proteins that do not require tracrRNA and contain two HEPN domains that cut RNA.
  • gRNA refers to nucleotide sequences that are sufficiently complementary to the target nucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the associated nuclease to the target nucleotide sequence.
  • the corresponding guide RNA is one or more RNA molecules (typically one or two) that bind the Cas enzyme and direct the Cas enzyme to bind a specific target nucleotide sequence and also cleave the target nucleotide sequence in those cases where the Cas enzyme has a nickase or nuclease activity.
  • C 1 -C 30 alkyl or "C 1 -C 20 alkyl” or “C 1 -C 14 alkyl” or “C 1 -C 10 alkyl” or “C 1 -C 6 alkyl” refers to a straight or branched alkyl group having 1-30, or 1-20, or 1-14, or 1-10 or 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
  • C 2 -C 30 alkenyl or “C 2 -C 20 alkenyl” or “C 2 -C 14 alkenyl” or “C 2 -C 10 alkenyl” or “C 2 -C 6 alkenyl” refer to straight or branched alkenyl having 2-30 or 2-20 or 2-14 or 2-10 or 2-6 carbon atoms, respectively, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.
  • C 2 -C 30 alkynyl or "C 2 -C 20 alkynyl” or “C 2 -C 14 alkynyl” or “C 2 -C 10 alkynyl” or “C 2 -C 6 alkynyl” refer to straight or branched alkynyl having 2-30 or 2-20 or 2-14 or 2-10 or 2-6 carbon atoms, such as an ethynyl, propinyl, or the like.
  • halogenated refers to a group substituted with the same or different one or more of the above halogen atoms, either partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or the like.
  • C 1 -C 10 haloalkyl or "C 1 -C 6 haloalkyl” refers to a straight or branched alkyl group having 1-10 or 1-6 carbon atoms with hydrogen substituted with one or more halogens, for example, a halogenated methyl, halogenated ethyl, halogenated propyl, halogenated isopropyl, or the like, preferably trifluoromethyl.
  • the compounds of the present invention may contain one or more asymmetric centers and thus appear as racemates, racemic mixtures, single enantiomers, diastereoisomeric compounds, and single diastereomers.
  • the asymmetric centers that can exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently give rise to two optical isomers, and all possible mixtures of optical isomers and diastereomers and pure or partially pure compounds are included in the scope of the present invention.
  • the present invention includes all isomeric forms of the compounds
  • ionizable lipid of the present invention and “ionizable cationic lipid of the present invention” are used interchangeably to refer to a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof.
  • Ionizable lipids are protonated and converted to cationic lipids at low pH and then to auxiliary phospholipids at normal physiological pH. Auxiliary phospholipids interact less with the anionic cell membrane of blood cells and improve the biocompatibility of lipid nanoparticles.
  • lipid nanoparticles When lipid nanoparticles are endocytosed, the pH in the endosomes is lower, the lipids are protonated and positively charged, and the stability of the membrane structure becomes less stable or even destroyed, which facilitates the escape of lipid nanoparticle endosomes. In general, it is the pH-sensitive properties of lipids that facilitate the in vivo delivery of lipid nanoparticles encapsulating bioactive components (e.g., mRNA molecules) .
  • bioactive components e.g., mRNA molecules
  • an ionizable lipid having a structure as shown in Formula I:
  • R 1 is selected from -OH and R a (R b ) N-, wherein R a and R b are each independently hydrogen, C1-C 10 alkyl or C1-C 10 haloalkyl;
  • R 2 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 3 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, or R c - (CH 2 ) n-, wherein n is a positive integer of 1-20, preferably, a positive integer of 1-14, more preferably a positive integer of 1-10;
  • R c is selected from the structure as shown below: wherein represents a connection bond
  • L1, L2, L3, L4, L5 are absent or each independently selected from the group consisting of:
  • X is absent or -CH-or N
  • R 4 is absent or C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 5 is absent or C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 6 is C 1 -C 30 alkyl, C 2 -C 30 alkenyl, C 2 -C 30 alkynyl;
  • R 7 is C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl;
  • R 8 , R 9 are absent or is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl or -R h -C 1 -C 14 alkyl, -R h -C 2 -C 14 alkenyl, -R h -C 2 -C 14 alkynyl, wherein R h is O or S;
  • R 10 , R 11 is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl, or -R h -C 1 -C 14 alkyl, -R h -C 2 -C 14 alkenyl, -R h -C 2 -C 14 alkynyl, wherein R h is O or S.
  • the ionizable lipids have the substructures shown in the following structures I-1, I-2, I-3, I-4, respectively:
  • R 1 -R 2 , R 6 -R 11 , L 2 -L 5 are defined as above;
  • R 3 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl.
  • R 1 -R 2 , R 6 -R 7 , R 10 -R 11 , L 1 -L 5 are defined as above;
  • R 3 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, or R c - (CH 2 ) n-, wherein n is a positive integer of 1-20, preferably, a positive integer of 1-14, more preferably a positive integer of 1-10, R c is defined as above;
  • R 5 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 8 , R 9 is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl.
  • R 1 -R 3 , R 6 , R 7 , R 10 , R 11 , L 1 -L 5 are defined as above;
  • R 4 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 5 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl;
  • R 8 , R 9 is each independently C 1 -C 14 alkyl, C 2 -C 14 alkenyl, C 2 -C 14 alkynyl or -R h -C 1 -C 14 alkyl, -R h -C 2 -C 14 alkenyl, -R h -C 2 -C 14 alkynyl, wherein R h is O or S;
  • R 1 -R 7 , R 10 -R 11 , L 1 -L 3 , L 5 are defined as above.
  • the ionizable lipid has a structure selected from those shown in Table 1, wherein, preferably, the following compounds of Table 1 are selected:
  • auxiliary lipids refers in lipid nanoparticles, other types of lipids besides ionizable lipids, including one or a combination of more than two of anionic lipids, neutral lipids, steroids, and polymer-bound lipids.
  • auxiliary lipids can be used to improve the properties of lipid nanoparticles, such as enhancing nanoparticle stability, membrane fusion (fusogenicity) , and/or mobility and the like.
  • the anionic lipid comprises one or a combination of more than two of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG) , 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) , and dimyristoylphosphatidylglycerol.
  • DOPG dioleoylphosphatidylglycerol
  • DOPS 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine
  • dimyristoylphosphatidylglycerol dimyristoylphosphatidylglycerol.
  • the neutral lipid comprises at least one of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , Dipalmitoylphosphatidylglycerol (DPPG) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , palmitoyloleoyl phosphatidylethanolamine (POPE) , dipalmitoyl phosphatidylethanolamine (DPPE) Dimyristoylphosphoethanolamine (DMPE) , distearoyl-phosphatidyl-ethanolamine (DSPE) , and 1-ste
  • the steroids include one or more of cholesterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, tomatidine, ursolic acid, alpha totaxin, coprosterol, and corticosteroids.
  • the polymer-bound lipid comprises one or a combination of two or more of 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , ⁇ - (3′- ⁇ [1, 2-di (myristyloxy) propanoxy] carbonylamino ⁇ propyl) - ⁇ -methoxy, polyoxyethylene (PEG-c-DMG) , PEG-1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA) , 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol) ] (PEG-DSPE) , pegylated phosphatidylethanoloamine (PEG-PE) , PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 65) : (0 to 20) : (5 to 25) : (25 to 55) : (0.3 to 15) ; exemplarily, the molar ratio may be 20: 20: 5: 50: 5, 30: 5: 25: 30: 10, 20: 5: 5: 55: 15, 65: 0: 9.7: 25: 0.3, etc.; wherein, in the first lipid compound, the compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug thereof and other cationic or ionizable lipids in a molar ratio of (1 to 10) : (0 to 10) ; exemplarily, the molar ratio may be 1: 1, 1: 2, 1: 5, 1: 7.5, 1: 10, 2: 1, 5: 1, 7.5: 1,
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 55) : (0 to 13) : (5 to 25) : (25 to 51.5) : (0.5 to 15) ; wherein, in the first lipid compound, the compound of Formula I or a pharmaceutically acceptable form thereof such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug and other cationic or ionizable lipids in a molar ratio of (3-4) : (0-5) .
  • the auxiliary lipid is selected from DSPC, cholesterol, and PEG-DMG.
  • the auxiliary lipid comprises DSPC, cholesterol, and PEG-DMG.
  • LNP Lipid Nanoparticles
  • the terms “lipid nanoparticles” , or “LNP” refer to particles with a diameter of about 5 to 500 nm.
  • the lipid nanoparticles contain one or more active agents (bioactive substances) .
  • the lipid nanoparticles comprise nucleic acids.
  • the nucleic acids are condensed in the interior of the nanoparticles with cationic lipids, polymers or multivalent small molecules and an external lipid coating that interacts with the biological environment. Due to the repulsive forces between phosphate groups, nucleic acids are naturally rigid polymers, preferring an elongated conformation.
  • DNA can package itself in appropriate solution conditions with the help of ions and other molecules.
  • DNA condensation is defined as the collapse of an extended DNA strand into a compact and ordered particle containing only one or a few molecules.
  • cationic lipids can condense DNA by neutralizing the phosphate charge and allowing it to stack tightly.
  • the bioactive substance is encapsulated into the LNP.
  • the bioactive substance can be an anionic compound including, but not limited to, DNA (including plasmids) , RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA, etc.
  • oligonucleotides including antisense oligonucleotides, interfering RNAs and small interfering RNAs
  • nuclear proteins nuclear proteins, peptides, nucleic acids, ribozyme, aptamers, immunostimulatory nucleic acids or PNAs
  • DNA-containing nuclear proteins such as complete or partially deproteinized viral particles (viral particles) , oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins) )
  • the bioactive substance may be mixed with an adjuvant.
  • said mRNA comprises sequences encoding RNA-directed DNA binding agents, and more specifically, mRNA encoding a nuclease or base editor.
  • said nucleic acid further comprises a guide RNA, specifically, said guide RNA comprises a gRNA nucleic acid.
  • said nucleic acid comprises an mRNA encoding a nuclease or a base editor; and a gRNA.
  • the bioactive substance is typically contained within the LNP.
  • the bioactive substance comprises nucleic acids.
  • water-soluble nucleic acids are condensed with cationic lipids or polycationic polymers inside the particle, and the surface of the particle is enriched with auxiliary phospholipid or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface, and when entering cytolysosome, ionizable cationic lipids are positively charged by ionization through the acidic environment of the lysosome, interacting with the lysosomal membrane and promoting endosomal escape.
  • ionizable lipids can have different properties or functions. Due to the pKa of the amino group, when the external pH is lower than the pKa of the lipid molecule, it can be protonated and positively charged. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of the nucleic acid, which allows LNP formation and nucleic acid encapsulation, and the LNP has an essentially neutral surface charge in biological fluids of physiological pH (e.g., blood) . High LNP surface charge is associated with rapid clearance of circulating, hemolytic toxicity, including immune activation, by toxic, fixed and free macrophages (Filion et al. Biochim Biophys Acta. 1997 Oct 23; 1329 (2) : 345-56) .
  • pKa can be sufficiently high such that ionizable cationic lipids can adopt a positively charged form at acidic endosomal pH. In this way, cationic lipids can bind to endogenous endosomal anionic lipids to promote membrane cleavage of non-bilayer structures, such as hexagonal HII phases, to produce more efficient intracellular delivery.
  • pKa ranges from 6.2 to 6.5. For example, pKa may be about 6.2, about 6.3, about 6.4, about 6.5. Unsaturated tails also contribute to the ability of lipids to adopt non-bilayer structures. (Jayaraman et al. Angew Chem Int Ed Engl. 2012 Aug 20; 51 (34) : 8529-33) .
  • nucleic acids from LNP formulations can be altered by the presence of polyethylene glycols and/or sterols (e.g., cholesterol) or other potential additives in the LNP, as well as the overall chemical structure, including the pKa of any ionizable cationic lipids that are part of the formulation.
  • polyethylene glycols and/or sterols e.g., cholesterol
  • sterols e.g., cholesterol
  • a lipid nanoparticle comprising a compound of Formula I o or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof as described in a first aspect of the present invention.
  • said lipid nanoparticles further comprise one or more auxiliary lipids, said auxiliary lipids comprising one or a combination of more than two of anionic lipids, neutral lipids, steroids, and polymer-bound lipids.
  • a pharmaceutical formulation (or lipid nanoparticle composition, or LNP composition) , said pharmaceutical formulation comprising a lipid carrier as described in a third aspect of the present invention, and a bioactive substance encapsulated in said lipid carrier, and a pharmaceutically acceptable carrier.
  • Said pharmaceutical formulation is used to deliver a bioactive substance to cells in a subject in need thereof.
  • the bioactive substance is encapsulated into an LNP.
  • the bioactive substance may be an anionic compound including, but not limited to, DNA (including plasmids) , RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA, etc.
  • the bioactive substance may be mixed with an adjuvant.
  • the LNP composition comprises: a nucleic acid, an ionizable lipid having the structure shown in Formula (I) , optionally an auxiliary phospholipid (e.g., Distearoyl Phosphatidylcholine) .
  • the LNP composition comprises: a nucleic acid; an ionizable lipid having the structure shown in Formula I in an amount of 30-65% (molar ratio) of the total lipid of the composition; and optionally an auxiliary phospholipid (e.g., Distearoyl Phosphatidylcholine) in an amount of 1-10%of the total lipid of the composition.
  • the molar ratio of the first lipid compound (ionizable lipids of the structure shown in Formula (I) and optionally other cationic or ionizable lipids) , the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 65) : (0 to 20) : (5 to 25) : (25 to 55) : (0.3 to 15) ; exemplarily, the molar ratio may be 20: 20: 5: 50: 5, 30: 5: 25: 30: 10, 20: 5: 5: 55: 15, 65: 0: 9.7: 25: 0.3, etc.
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20 to 55) : (0 to 13) : (5 to 25) : (25 to 51.5) : (0.5 to 15) ; wherein, in the first lipid compound, any of the above compounds or a pharmaceutically acceptable form thereof such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor drug and other cationic or ionizable lipids in a molar ratio of (3-4) : (0-5) .
  • the terms “encapsulating” and “encapsulated” mean that the mRNA, DNA, siRNA or other nucleic acid drug is inside the lipid nanoparticle or bound to the lipid nanoparticle.
  • the term “encapsulated” refers to fully or partially encapsulated.
  • mRNA may be selected to treat and/or prevent a disease of interest when administering a lipid nanoparticle composition comprising mRNA to a subject in need thereof.
  • the term "pharmaceutically acceptable carrier” includes, but is not limited to, any adjuvant, carrier, excipient, scintillant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifiers approved by the Food and Drug Administration for use in humans or domestic animals.
  • the present invention also provides a method for in vivo delivery of nucleic acid drugs, comprising administering to a subject in need thereof said lipid nanoparticle composition or said pharmaceutical formulation.
  • said lipid nanoparticle composition or said pharmaceutical formulation is administered by one of the following routes of administration: orally, intranasally, intravenously, intraperitoneally, intramuscularly, intra-articularly, intralesionally, intratracheally, subcutaneously, and intradermally.
  • the above lipid nanoparticle compositions or the above pharmaceutical formulations are administered, for example, via an enteral or parenteral route of administration.
  • said lipid nanoparticle composition or pharmaceutical formulation is administered to said subject at a dose of about 0.001 mg/kg to about 10 mg/kg.
  • a method of preparing a pharmaceutical formulation comprising: (a) mixing a compound of Formula I as described in a first aspect of the present invention and optionally an auxiliary lipid with an organic solvent, thereby obtaining a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent, thereby obtaining an aqueous phase containing the bioactive substance; (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) , thereby obtaining said lipid nanoparticle composition. Further, said method further comprises step (d) : purifying and concentrating and filtering the lipid nanoparticle drug obtained in step (c) to remove bacteria.
  • the organic solvent comprises (but is not limited to) ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or combinations thereof.
  • the lipid organic phase comprises a small percentage of water or pH buffer.
  • the lipid organic phase may comprise up to 60 vol%water, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%by volume.
  • the lipid organic phase comprises between about 0.05%and 60%water by volume, for example, between about 0.05%and 50%, between about 0.05%and 40%, or between about 5%and 20%water by volume.
  • the aqueous solvent is water.
  • the aqueous solvent is an aqueous buffer having a pH between 3 and 8 (e.g., a pH of about 3, about 4, about 5, or about 6, etc. ) .
  • a bioactive substance such as a nucleic acid (e.g., mRNA) is dissolved in said aqueous solvent to obtain an aqueous phase containing the bioactive substance.
  • Said aqueous phase may comprise a small percentage of an aqueous miscible organic solvent.
  • Said aqueous phase may comprise up to 60 volume %of at least one organic solvent miscible with water, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%or any volume in between %of an organic solvent (e.g., an aqueous miscible organic solvent) .
  • an organic solvent e.g., an aqueous miscible organic solvent
  • said aqueous phase comprises between about 0.05%and 60%by volume of an organic solvent, e.g., between about 0.05%and 50%by volume, between about 0.05%and 40%by volume, or between about 5%and 20%by volume of an organic solution (e.g., an aqueous miscible organic solvent) .
  • the aqueous phase buffer may be a citrate buffer, a Tris-Hcl buffer solution, a sodium acetate buffer solution, a PBS buffer solution, or a combination thereof, etc.
  • the aqueous buffer is a citrate buffer with a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6) .
  • the aqueous buffer solution is a citrate buffer with a pH of about 4.
  • a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance can be diluted, the mixture comprising an LNP suspension.
  • the pH of the solution of the mixture of the lipid organic phase comprising the LNP suspension and the aqueous phase containing the bioactive substance can be adjusted.
  • the pH of the LNP suspension can be diluted or adjusted by adding water, an acid, a base, or an aqueous buffer. in some embodiments, no dilution or adjustment of the pH of the LNP suspension is performed. In some embodiments, dilution and adjustment of the pH of the LNP suspension is performed.
  • excess reagents, solvents, unencapsulated nucleic acids may be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., permeation filtration) .
  • TFF tangential flow filtration
  • Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF.
  • the LNP suspension is dialyzed.
  • the LNP suspension is subjected to TFF. in some embodiments, the LNP suspension is subjected to dialysis and TFF.
  • the main advantages of the present invention include:
  • the present invention provides a series of structurally novel compounds of Formula I, which can be used as ionizable lipids to prepare lipid carriers with other lipid compounds with controllable particle size and uniform distribution and high encapsulation efficiency.
  • the lipid compounds of the present invention are synthesized in a simple method with high yield, and can be synthesized rapidly and at low cost.
  • the compounds of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs, enriching the variety of ionizable lipid compounds, which is especially important for the development and application of nucleic acid prophylactic and therapeutic agents.
  • Reagents and materials used in embodiments of the present invention are commercially available products unless otherwise noted.
  • “moderate amount” means that the amount of solvent or drug added is adjustable in a wide range and has a small effect on the synthesis results, so that no specific limitation can be made.
  • the solvents and drugs used are analytically or chemically pure; the solvents are redistilled before use; the anhydrous solvents are treated according to standard methods or literature methods.
  • Compound 2 was prepared according to the procedure of compound 1 from 3- (ethylamino) propan-1-ol.
  • Compound 7 was prepared according to the procedure of compound 6 from N 1 , N 1 -diethylbutane-1, 4-diamine.
  • Compound 8 was prepared according to the procedure of compound 6 from N 1 , N 1 -dimethylpropane-1, 3-diamine.
  • Compound 9 was prepared according to the procedure of compound 6 from N 1 , N 1 -diethylethane-1, 2-diamine.
  • Compound 11 was prepared according to the procedure of compound 6 from 7-chloro-7-oxoheptyl 2-butyloctanoate which was prepared according to the procedure of 6-3.
  • Compound 12 was prepared according to the procedure of compound 6 from 9-chloro-9-oxononyl 2-butyloctanoate which was prepared according to the procedure of 6-3.
  • Compound 13 was prepared according to the procedure of compound 10 from 5-bromopentyl 2-butyloctanoate.
  • Compound 14 was prepared according to the procedure of compound 10 from 7-bromoheptyl 2-butyloctanoate.
  • Example 16 Preparation and characterization of lipid nanoparticles and in vivo editing experiments
  • Cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol.
  • LDLR LDL receptor
  • PCSK9 is a liver-synthesized protease that binds to LDL receptors and promotes LDL receptor entry into hepatocytes, leading to LDL receptor degradation by lysosomes and a decrease in the number of LDL receptors; thus, inhibition of the activity of PSCK9, can increase the number of LDLR, thereby enhancing the uptake and decomposition of cholesterol.
  • FIG 1 illustrates the changes in the number of LDL receptors and the eventual changes in cholesterol metabolism that result before and after site-specific editing of the PCSK9 gene.
  • FIG. 1 The strategy of PCSK9 gene editing delivery to mouse hepatocytes is shown in Figure 1, and the main process is as follows: mRNA and sgRNA encoding ABE8e are targeted to be delivered to mouse hepatocytes by intravenous injection using prepared lipid nanoparticles, and mutations are introduced in the PCSK9 gene in the presence of ABE8e, sgRNA, and base A to G mutations are achieved at specific sites, and the editing efficiency is calculated by sequencing.
  • the single-base editor ABE8e achieves precise A-to-G base substitution without donor template and without causing DSB. Based on this, the first exon of PCSK9 gene was selected as the screening mutation site.
  • the mRNA encoding the base editor ABE8e was translated into protein in the cytoplasm and entered the nucleus after forming a complex with sgRNA, and under the guidance of sgRNA, the base editor ABE8e targeted the first exon of PCSK9 gene to splice the donor site, and deamination of adenine (A) on the template strand of the first exon into inosine (I) , I will be read and replicated as G at the DNA level, eventually achieving an A-to-G substitution, which disrupts the splice donor site allowing early termination of the PCSK9 gene reading frame.
  • A adenine
  • I inosine
  • PCSK9-sgRNA 5'-CCCATACCTTGGAGCAACGG-3' (SEQ ID NO: 1) ;
  • the upstream and downstream sequences were annealed by a preset program (95°C, 5 min; 95°C-85°C at -2°C/s; 85°C-25°C at -0.1°C/s; kept at 4°C) , and the annealed products were ligated to the lenti U6-sgRNA/EF1a-mCherry vector linearized by BbsI (NEB, R3539S) (Addgene, Plasmid, #114199) .
  • the system used in the sgRNA plasmid construction was as follows:
  • the ligation system of sgRNA annealing product and linearized vector was as follows: T4 ligase buffer (NEB: M0202L) 1 ⁇ L, linearized vector 20ng, annealed oligo fragment (10 ⁇ M) 5 ⁇ L, T4 ligase (NEB: M0202L) 0.5 ⁇ L, ddH 2 O was made up to 10 ⁇ L, ligation overnight at 16°C.
  • the ligated vector was transformed into E. coli DH5a competent cells (Weidi Bio, DL1001) .
  • the procedure was as follows: DH5 ⁇ competent cells were taken out from -80°C, quickly inserted into ice, allowed to melt after 5 min, added the ligation product and gently mixed with your hand by tapping the bottom of the centrifuge tube, and left in ice for 25 min. 42°C water bath thermal excitation for 45 s, quickly put back into ice and left for 2 min. Added 700 ⁇ l of sterile LB medium without antibiotics to the centrifuge tube, mixed well and resuscitate at 37°C for 60 min at 200 rpm.
  • ABE8e is the highly efficient base editor ABE8e evolved by David R. Liu's team (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul. 38 (7) : 883-891. doi: 10.1038/s41587-020-0453-z. Epub 2020 Mar 16. erratum in: Nat Biotechnol.
  • Plasmid ABE8e (Plasmid#138489) was obtained from Addgene and the mRNA of ABE8e was expressed and purified by the laboratory for use.
  • Lipid nanoparticles can be prepared by ionizing lipids or compounds of the present invention/DSPC/cholesterol/PEG-lipids in a molar ratio of 50: 10: 38.5: 1.5.
  • DLin-MC3-DMA Dissolve dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, commonly abbreviated as MC3) and compounds 1-compound 10 of the present invention with DSPC, cholesterol, and PEG-DMG, respectively, in anhydrous ethanol in the above molar ratios.
  • the ethanol solutions of the different lipid carriers were mixed with the buffer of mRNA at 1: 3 (v/v) (where the mass ratio of total lipid to mRNA (w/w) was 40:1 and the mass ratio of ABE8e mRNA: sgRNA (w/w) was 3: 2) and passed through the microfluidic nanopharmaceutical manufacturing system at a flow rate of 12 ml/min (NanoAssemblr Ignite, Canada) to obtain nucleic acid lipid nanoparticles 1-10.
  • the obtained nucleic acid lipid nanoparticles were immediately diluted in 40-fold volume into 1 ⁇ DPBS buffer.
  • the diluted nucleic acid lipid nanoparticle solution was passed through an ultracentrifuge tube and concentrated to the target volume. Diluted for DLS particle size measurement and encapsulation efficiency testing.
  • lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode.
  • the encapsulation efficiency of lipid nanoparticles was determined using Quant-it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions, and the test results are shown in Table 2.
  • DLin-MC3-DMA dilinoleylmethyl-4-dimethylaminobutyrate
  • Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated to MC3) encapsulating mRNA and sgRNA of base editor ABE8e were administered to mice of comparable week age and sex groups as positive controls.
  • PBS buffer was also injected caudally intravenously in a similar manner into mice of comparable week age and sex groups as a negative control.
  • mice were tested for editing efficiency one week after administration. Liver tissues were taken from mice after execution, and the genome was extracted after lysis and analyzed for efficiency by deep sequencing.
  • the PCR procedure was as follows: 94°C, 2 min; 98°C 10s, 60°C 30s, 68°C 20s, 34 cycles; 68°C, 5 min. Verified by gel electrophoresis after the completion of PCR, and the right size and single band was selected to determine the correct amplification product, and the obtained PCR product was sent to Nanjing Genscript for sequencing.

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Abstract

L'invention concerne des composés lipidiques destinés à la délivrance d'agents thérapeutiques et leur procédé de préparation et leur utilisation, en particulier, l'invention concerne un composé de formule I ou un sel pharmaceutiquement acceptable, un stéréoisomère, un tautomère, un solvate, un chélate, un complexe non covalent, ou un précurseur de celui-ci. Les composés lipidiques ionisables peuvent délivrer efficacement des molécules d'acide nucléique, des composés à petites molécules et d'autres médicaments, et par comparaison, les nanoparticules lipidiques ont une meilleure distribution granulométrique, une efficacité d'encapsulation élevée, et l'effet de délivrance peut être significativement meilleur qu'avec les nanoparticules lipidiques de comparaison, lequel peut satisfaire la demande de délivrance in vivo (I).
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