Lipid compounds and lipid nanoparticles for delivery

Abstract

The present application relates to a kind of for delivery lipid compound and lipid nanoparticle, disclose a kind of compound, structural formula is as shown in formula I, and its pharmaceutically acceptable salt or its stereoisomer, the present application also discloses a kind of nanoparticle composition comprising above-mentioned compound or its pharmaceutically acceptable salt or its stereoisomer, the nanoparticle of the present application can be efficiently delivered drug, vaccine to cell, exert the treatment or prevention purposes of drug, vaccine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to lipid compounds and lipid nanoparticles for the delivery of active ingredients in drugs, vaccines, and other products. Background Technology

[0002] The efficient and targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids remains an ongoing medical challenge. A key to the success of gene therapy lies in the safe and effective delivery of therapeutic drugs into target cells via vectors within the body. The relative instability of nucleic acids and their low cell permeability make their delivery to cells difficult. Therefore, methods and compositions are needed to facilitate the delivery of therapeutic and / or preventative drugs, such as nucleic acids, to cells. Gene therapy vectors are categorized into viral vectors and non-viral vectors. Although viral vectors serve as highly efficient delivery systems for gene transfection and treatment, their immunogenic viral proteins, limited target gene loading capacity, and high cost have led to increased interest in non-viral vectors such as lipid nanoparticles (LNPs) due to their superior in vitro stability, in vivo degradation, and safety. LNPs are widely used in gene therapy research for both congenital and acquired genetic defects.

[0003] Lipid-containing nanoparticles, lipid nanoparticles, liposomes, and lipid complexes have been proven to be effective transport carriers for bioactive substances such as small molecule drugs, proteins, and nucleic acids that enter and / or enter cells. LNPs are small vesicles formed by one or more lipid components that can effectively compress and deliver various nucleic acid molecules, from DNA and RNA to chromosomes and even cells. LNPs are conducive to large-scale production due to their well-defined construction schemes and ease of modification with targeted ligands.

[0004] Lipid-containing nanoparticles (LNPs) typically comprise one or more cationic lipids and / or amino (ionizable) lipids, phospholipids containing polyunsaturated lipids, structural lipids (such as sterols), and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, lipids containing amines, which can be readily ionized. Various such lipid-containing nanoparticle compositions have been demonstrated in the prior art; for example, patent document WO2017049245A discloses a compound and composition for intracellular delivery of therapeutic agents. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a lipid compound and a lipid nanoparticle composition containing the compound.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] 1. The compound represented by Formula I, or its salt or isomer,

[0008]

[0009] Among them, Y is independently selected from (C=O)O, O(C=O), and (C=O)NH;

[0010] Z is independently selected from (C=O)O and O(C=O);

[0011] R1 is independently a C1-C6 alkyl group, R2 and R3 are independently selected from H or C1-C12 alkyl groups, R4 and R5 are independently selected from C1-C6 alkyl groups, R6 is independently selected from C1-C6 alkyl groups, R7 and R8 are independently selected from C1-C6 alkyl groups, and R9 is independently selected from H or C1-C6 alkyl groups.

[0012] m is independently selected from 2, 3, 4, 5, 6, 7 or 8;

[0013] n is independently selected from 3, 4, 5, 6, 7, 8, or 9;

[0014] q is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8;

[0015] p is independently selected from 1, 2, 3, 4 or 5.

[0016] 2. The compound represented by formula II, or its salt or isomer,

[0017]

[0018] Wherein R1 is independently a C1-C6 alkyl group, R2 and R3 are independently selected from H or C1-C12 alkyl groups, R4 and R5 are independently selected from C1-C6 alkyl groups, R6 is independently selected from C1-C6 alkyl groups, R7 and R8 are independently selected from C1-C6 alkyl groups, and R9 is independently selected from H or C1-C6 alkyl groups.

[0019] m is independently selected from 2, 3, 4, 5, 6, 7 or 8;

[0020] n is independently selected from 3, 4, 5, 6, 7, 8, or 9;

[0021] q is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8;

[0022] p is independently selected from 1, 2, 3, 4 or 5.

[0023] 3. The compound according to any one of items 1-2, wherein p is independently selected from 2, 3 or 4, preferably 3.

[0024] 4. The compound according to any one of items 1-3, wherein Y is (C=O)O, Z is independently selected from (C=O)O and O(C=O), R1 is a C1-C3 alkyl group, preferably methyl; R2 and R3 are each independently C6-C10 straight-chain alkyl groups, preferably C7-C9 straight-chain alkyl groups; R4 and R5 are each independently C1-C3 alkyl groups, preferably methyl; R6 is independently C1-C3 alkyl groups, preferably methyl; R7 and R8 are each independently C1-C3 alkyl groups, preferably methyl; and R9 is independently H or a C1-C3 alkyl group, preferably H.

[0025] 5. The compound according to any one of items 1-3, wherein Y is (C=O)O, Z is independently selected from (C=O)O and O(C=O), R1 is a C1-C3 alkyl group, preferably methyl; R2 is selected from H or a C1-C3 alkyl group; R3 is independently a C6-C11 straight-chain alkyl group, preferably a C7-C10 straight-chain alkyl group; R4 and R5 are each independently a C1-C3 alkyl group, preferably methyl; R6 is independently a C1-C3 alkyl group, preferably methyl; R7 and R8 are each independently a C1-C3 alkyl group, preferably methyl; and R9 is independently H or a C1-C3 alkyl group, preferably H.

[0026] 6. The compound according to claim 1, wherein the compound of formula I is:

[0027]

[0028]

[0029]

[0030]

[0031] 7. A lipid nanoparticle composition comprising a lipid component, said lipid component comprising any one of claims 1-6, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0032] 8. The lipid nanoparticle composition according to claim 7, wherein the lipid component further comprises phospholipids, structural lipids and / or PEG lipids.

[0033] 9. The lipid nanoparticle composition according to claim 8, wherein the phospholipid is selected from one or more of the following compounds:

[0034] Dilauroyl lecithin (DLPC)

[0035] Dimyristic phosphatidylcholine (DMPC)

[0036] Dioleoyl lecithin (DOPC)

[0037] Dipalmitoylphosphatidylcholine (DPPC)

[0038] Distearate phosphatidylcholine (DSPC)

[0039] Dioleoylphosphatidylcholine (DUPC)

[0040] Palmitoyl oleoyl phosphatidylcholine (POPC)

[0041] 1,2-Di-O-octadecyl-sn-glycerol-3-phosphate choline (18:0 Diether PC)

[0042] 1-Oleoyl-2-cholesterol dimethylsuccinate-sn-glycerol-3-phosphate choline (OChemsPC)

[0043] l-Hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC)

[0044] 1,2-Divinyl-sn-glycerol-3-phosphate choline,

[0045] 1,2-Diarylac-sn-glycerol-3-phosphocholine,

[0046] 1,2-Dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE)

[0047] 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine,

[0048] 1,2-Divinyl-sn-glycerol-3-phosphate ethanolamine,

[0049] 1,2-Divinyl-sn-glycerol-3-phosphate ethanolamine,

[0050] 1,2-Diaryl-sn-glycerol-3-phosphate ethanolamine

[0051] 1,2-Dithiohexaenoic acid-sn-glycerol-3-phosphate ethanolamine,

[0052] 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1-glycerol)sodium salt (DOPG) or sphingomyelin,

[0053] 10. The nanoparticle composition according to item 8, wherein the phospholipid is DOPE.

[0054] 11. The nanoparticle composition according to item 8, wherein the phospholipid is DSPC.

[0055] 12. The nanoparticle composition according to any one of items 7-11, wherein the lipid component further comprises structural lipids.

[0056] 13. The nanoparticle composition according to claim 12, wherein the structural lipid is selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, and stigmasterol.

[0057] 14. The nanoparticle composition according to item 12, wherein the structural lipid is cholesterol.

[0058] 15. The nanoparticle composition according to any one of claims 7-14, wherein the lipid component further comprises PEG lipids.

[0059] 16. The nanoparticle composition according to claim 15, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol.

[0060] 17. The nanoparticle composition according to any one of claims 7-16, wherein the lipid component further comprises cationic and / or ionizable lipids.

[0061] 18. The nanoparticle composition according to any one of claims 7-17, further comprising a therapeutic agent and / or a preventive agent selected from vaccines or compounds capable of inducing an immune response, nucleic acids, preferably said nucleic acid being RNA, said RNA being selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

[0062] 19. The nanoparticle composition according to any one of claims 7-18, wherein the encapsulation efficiency of the therapeutic agent and / or preventive agent is ≥50%; or ≥80%; or ≥90%.

[0063] 20. The nanoparticle composition according to any one of items 7-19, wherein the average particle size of the nanoparticle composition is 50 nm to 110 nm.

[0064] 21. The nanoparticle composition according to any one of items 7-19, wherein the dispersibility index of the nanoparticle composition is 0.04-0.20.

[0065] 22. Use of the compound of any one of items 1-6 in the preparation of lipid nanoparticle compositions.

[0066] 23. A pharmaceutical composition comprising the nanoparticle composition described in any one of claims 7-21 and a pharmaceutically acceptable carrier.

[0067] 24. A method of delivering a therapeutic and / or preventive agent to mammalian cells, the method comprising administering to a subject the nanoparticle composition of any one of items 7-21 or the pharmaceutical composition of item 23, the administration comprising contacting cells with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or preventive agent to the cells.

[0068] 25. The method according to item 24, wherein the mammalian cell is in a mammal.

[0069] 26. The method according to item 24 or 25, wherein the mammal is a human.

[0070] 27. The method according to any one of items 24-26, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0071] 28. A method for producing a target polypeptide in mammalian cells, the method comprising contacting the cells with a nanoparticle composition according to any one of items 7-21 or a pharmaceutical composition according to item 23 to deliver a therapeutic and / or preventive agent to the cells, wherein the therapeutic and / or preventive agent is mRNA encoding the target polypeptide, thereby enabling the mRNA to be translated in the cells to produce the target polypeptide.

[0072] 29. The method according to item 28, wherein the mammalian cell is in a mammal.

[0073] 30. The method according to any one of items 28 or 29, wherein the mammalian cell is human.

[0074] 31. The method according to any one of items 28-30, wherein the nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0075] 32. A method for specifically delivering a therapeutic and / or preventive agent to a mammalian organ, the method comprising administering to a mammal any of the nanoparticle compositions described in any one of items 7-21 or the pharmaceutical composition described in item 23, the administration comprising contacting the mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or preventive agent to the organ, preferably the kidney.

[0076] 33. The method according to item 32, wherein the mammal is a human.

[0077] 34. The method according to item 32 or 33, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0078] 35. The method according to any one of items 32-34, wherein the mammal is pretreated 24 hours or less prior to the contact or administration step.

[0079] 36. The method according to any one of claims 32-35, wherein the mammal is pretreated for about one hour prior to the contact or administration step.

[0080] The technical effects of this invention are as follows:

[0081] The compounds of this application can be used to prepare lipid nanoparticles. Nanoparticle compositions containing the compounds provided in this application can encapsulate and deliver therapeutic / preventive agents, safely delivering the therapeutic / preventive agents to the target site, achieving high expression, and exerting the therapeutic / preventive effects.

[0082] The lipid nanoparticles prepared in this application have small average particle size, high encapsulation efficiency, and high expression, and have broad application prospects in the field of drug delivery. Attached Figure Description

[0083] Figure 1 The values ​​represent the Luciferase fluorescence intensity of LNPs of compounds 1 and MC3 6 hours after intravenous injection.

[0084] Figure 2 The values ​​represent the Luciferase fluorescence intensity of LNPs of compounds 1 and MC3 in the spleen 6 hours after intravenous injection. Detailed Implementation

[0085] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0086] Terms and Definitions

[0087] As used herein, the term "alkyl" refers to a group containing one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which may optionally be substituted. The term "C1-C12 alkyl" refers to a saturated hydrocarbon of 1-12 carbon atoms, either straight-chain or branched. Unless otherwise stated, alkyl as described herein refers to both unsubstituted and substituted alkyl groups.

[0088] Unless otherwise specified, alkyl groups may optionally be substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chlorine, bromine, fluorine, or iodide), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxyl, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyl groups (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, where X is a halide selected from bromides, fluorides, chlorides, and iodides), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals, phosphates, thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfurous acids (e.g., -S(O)R), and sulfurous acids (e.g., -S(O)R). The groups R are: sulfonic acid (e.g., -S(O)2OH), thiol (e.g., -C(S)H), sulfate, sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR2 or -N(R)C(O)R), azide (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyanate (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, -NRH or -NH2), carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH or -OC(O)NH2), sulfonamide, alkyl, alkenyl, and cyclic (e.g., carbocyclic or heterocyclic). In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted with one, two, three, four, five, or six substituents as defined herein. For example, C1-6 alkyl groups can be further substituted with 1, 2, 3, 4, 5 or 6 substituents as described herein.

[0089] As used herein, “encapsulation ratio” refers to the amount of therapeutic and / or preventive agent that is part of the nanoparticle composition, relative to the total amount of therapeutic or preventive agent used in the preparation of the nanoparticle composition. For example, if 97 mg of therapeutic and / or preventive agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or preventive agent initially provided to the composition, the encapsulation ratio can be 97%. As used herein, “encapsulation” can mean complete, substantial, or partial encapsulation, closure, enclosure, or sealing.

[0090] As used in this article, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modifications of the polypeptide or protein.

[0091] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than events that occur within a living organism (e.g., an animal, plant, or microorganism).

[0092] As used herein, the term "in vivo" refers to events that occur within an organism (such as an animal, plant, or microorganism, or its cells or tissues).

[0093] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events can occur in environments with minimal alteration from the natural (e.g., internal) environment.

[0094] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds, and thus may exist in stereoisomeric form, such as double-bonded isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers). This application covers any and all isomers of the compounds described herein. Mixtures of enantiomers and stereoisomers of compounds, and methods for resolving them into their component enantiomers or stereoisomers, are well known.

[0095] As used herein, a "lipid component" is a component of a nanoparticle composition comprising one or more lipids. For example, a lipid component may include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids such as phospholipids.

[0096] As used herein, “modified” means non-natural. For example, RNA can be modified RNA. That is, RNA may include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. A “modified” substance may also be referred to herein as a “modified” substance. Such a substance may be chemically, structurally, or functionally modified or altered. For example, the types of modified nucleobases may include one or more non-naturally occurring substitutions.

[0097] As used herein, a “nanoparticle composition” is a composition comprising one or more lipids. The particle size of a nanoparticle composition is typically on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or smaller.

[0098] As used in this article, “naturally existing” means existing naturally without artificial intervention.

[0099] As used herein, "PEG lipid" or "PEGylated lipid" refers to lipids containing polyethylene glycol.

[0100] The term “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0101] As used herein, the phrase “pharmaceuticalally acceptable excipient” means any component other than the compounds described herein (e.g., a medium capable of suspending, complexing, or dissolving an active compound) and having substantially no toxicity or inflammatory effect on the patient. Excipients may include, for example: anti-adhesives, antioxidants, adhesives, coatings, anti-compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow aids (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydrated water. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (diacid), calcium stearate, crosslinked carboxymethyl cellulose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0102] In this application, for convenience, the structural formulas of the compounds represent certain isomers; however, this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity. Furthermore, the compounds represented by the structural formulas of the compounds described in this application may exhibit crystal polymorphism. Note that any crystal form, mixture of crystal forms, or its anhydrides or hydrates are included within the scope of this application.

[0103] The nanoparticle compositions of this application may also comprise salts of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound wherein the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; bases or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetates, adipates, alginates, ascorbic acid salts, aspartates, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphor sulfonates, citrates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, glucoheponicates, glucoheponicates, hydrobromide, hydrochloride, hydroiodates, 2-hydroxy-ethanesulfonate, lactates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, palmitates, pectates, 3-phenylpropionates, phosphates, picrates, neopentyl esters, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, valerates, etc.

[0104] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of this application include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this application can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, these salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both. Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typically preferred.

[0105] As used herein, "phospholipid" is a lipid comprising a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may contain one or more (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes can allow one or more elements of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more elements to the cell.

[0106] As used herein, “RNA” refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include cap structures, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a target polypeptide. For example, RNA may be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide, such as in vivo translation of mRNA within mammalian cells, can produce the encoded polypeptide. RNA may be selected from the unrestricted group, including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0107] As used herein, in the context of nanoparticle compositions, “particle size” or “average particle size” refers to the average diameter of the nanoparticle composition.

[0108] As used herein, the terms "subject" or "patient" mean any organism to which the composition according to this application may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0109] The terms "therapeutic agent" or "preventive agent" refer to any pharmaceutical agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces the desired biological and / or pharmacological action. Therapeutic agents are also known as "active agents" or "active components." Such substances include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0110] As used herein, the term "treatment" refers to partial or complete relief, mitigation, improvement, alleviation, delay of its onset, inhibition of its progression, reduction of its severity, and / or reduction of the incidence of one or more of its symptoms or features. A specific infection, disease, symptom, and / or condition. For example, "treatment" of cancer can refer to inhibiting tumor survival, growth, and / or spread. To reduce risk, treatment may be administered to subjects who do not exhibit disease, symptom, and / or condition and / or to subjects who only exhibit early signs of disease, symptom, and / or condition. A pathological development associated with a disease, symptom, and / or condition.

[0111] This application discloses compounds represented by Formula I, or salts thereof, or isomers thereof.

[0112]

[0113] Among them, Y is independently selected from (C=O)O, O(C=O), and (C=O)NH;

[0114] Z is independently selected from (C=O)O and O(C=O);

[0115] R1 is independently a C1-C6 alkyl group, R2 and R3 are independently selected from H or C1-C12 alkyl groups, R4 and R5 are independently selected from C1-C6 alkyl groups, R6 is independently selected from C1-C6 alkyl groups, R7 and R8 are independently selected from C1-C6 alkyl groups, and R9 is independently selected from H or C1-C6 alkyl groups.

[0116] m is independently selected from 2, 3, 4, 5, 6, 7 or 8;

[0117] n is independently selected from 3, 4, 5, 6, 7, 8, or 9;

[0118] q is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8;

[0119] p is independently selected from 1, 2, 3, 4 or 5.

[0120] In a preferred embodiment, R1 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R1 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R1 is methyl.

[0121] In a preferred embodiment, R2 is selected from H or C1-C12 straight-chain alkyl groups, such as H, C2-C12 straight-chain alkyl groups, C3-C12 straight-chain alkyl groups, C4-C12 straight-chain alkyl groups, C5-C12 straight-chain alkyl groups, C6-C12 straight-chain alkyl groups, C7-C12 straight-chain alkyl groups, C8-C12 straight-chain alkyl groups, C9-C12 straight-chain alkyl groups, C10-C12 straight-chain alkyl groups, C11-C12 straight-chain alkyl groups, C6-C11 straight-chain alkyl groups, C6-C10 straight-chain alkyl groups, C6-C9 straight-chain alkyl groups, C7-C11 straight-chain alkyl groups, C7-C10 straight-chain alkyl groups, C7-C9 straight-chain alkyl groups, C6-C8 straight-chain alkyl groups, and C1-C3 straight-chain alkyl groups. For example, R2 can be methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, C6 straight-chain alkyl, C7 straight-chain alkyl, C8 straight-chain alkyl, C9 straight-chain alkyl, C10 straight-chain alkyl, C11 straight-chain alkyl, or C12 straight-chain alkyl.

[0122] In a preferred embodiment, R3 is selected from C1-C12 straight-chain alkyl groups, such as those selected from C2-C12, C3-C12, C4-C12, C5-C12, C6-C12, C7-C12, C8-C12, C9-C12, C10-C12, C11-C12, C6-C11, C6-C10, C6-C9, C7-C11, C7-C10, C7-C9, and C6-C8. For example, R3 can be methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, C6 straight-chain alkyl, C7 straight-chain alkyl, C8 straight-chain alkyl, C9 straight-chain alkyl, C10 straight-chain alkyl, C11 straight-chain alkyl, or C12 straight-chain alkyl.

[0123] In a preferred embodiment, R4 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R4 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R4 is methyl.

[0124] In a preferred embodiment, R5 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R5 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R5 is methyl.

[0125] In a preferred embodiment, R6 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R6 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R6 is methyl.

[0126] In a preferred embodiment, R7 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R7 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R7 is methyl.

[0127] In a preferred embodiment, R8 is selected from C1-C6 straight-chain alkyl groups, such as C1-C5, C1-C4, C1-C3, and C1-C2 straight-chain alkyl groups. For example, R8 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R8 is methyl.

[0128] In a preferred embodiment, R9 is selected from H or C1-C6 straight-chain alkyl groups, such as H, C1-C5 straight-chain alkyl groups, C1-C4 straight-chain alkyl groups, C1-C3 straight-chain alkyl groups, and C1-C2 straight-chain alkyl groups. For example, R9 is methyl, ethyl, C3 straight-chain alkyl, C4 straight-chain alkyl, C5 straight-chain alkyl, and C6 straight-chain alkyl. In a further preferred embodiment, R9 is H.

[0129] In a preferred embodiment, in the compound of formula (Ⅰ) above, R1, R4, R5, R6, R7, and R8 are all methyl, R9 is H, R2 and R3 are all C8 straight-chain alkyl, Y is (C=O)O, and Z is (C=O)O or O(C=O).

[0130] m is independently selected from 2, 3, 4, 5, 6, 7 or 8;

[0131] n is independently selected from 3, 4, 5, 6, 7, 8, or 9;

[0132] q is independently selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0133] In a preferred embodiment, in the compound of formula (Ⅰ) above, R1, R4, R5, R6, R7, and R8 are all methyl groups, R9 is H, R2 is selected from H or C1-C3 alkyl groups; R3 is a C7-C10 straight-chain alkyl group, Y is (C=O)O, and Z is (C=O)O or O(C=O).

[0134] m is independently selected from 2, 3, 4, 5, 6, 7 or 8;

[0135] n is independently selected from 3, 4, 5, 6, 7, 8, or 9;

[0136] q is independently selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0137] Example

[0138] Example 1: Synthesis of Compound 1

[0139]

[0140] The synthesis steps are as follows:

[0141]

[0142] S1 to compound A (3.00 g, 13.868 mmol) EtOH / DMF (V EtOH =V DMF Compound B (3.91 g, 6.934 mmol) was added to a 30 ml solution and stirred at 70 °C for 16 hours. After the reaction was complete as monitored by TLC, the reaction solution was concentrated and purified by silica gel column chromatography. The solution was eluted with a mixture of dichloromethane and methanol in a volume ratio of 6:1 to give compound C (2.70 g, yield: 27.84%).

[0143]

[0144] S2 to compound C (2.70 g, 3.862 mmol) CPME / CH3CN (V CPME =V CH3CN Compound D (1.96 g, 4.248 mmol), K₂CO₃ (2.13 g, 15.448 mmol), and KI (0.06 g, 0.386 mmol) were added to a 10 mL solution, and the mixture was heated to 75 °C for 16 hours. The reaction was monitored by TLC until complete. The reaction solution was filtered, the filtrate was concentrated, and the concentrate was purified by silica gel column chromatography. The concentrate was eluted with a 10:1 mixture of dichloromethane and methanol to give compound E (2.30 g, yield: 55.16%).

[0145]

[0146] S3 added 5 ml of TFA to a dichloromethane (15 ml) solution of compound E (2.30 g, 2.130 mmol), stirred at room temperature for 3 hours, and monitored by TLC until the reaction was complete. The reaction solution was concentrated, dispersed in 50 ml of DCM, washed three times with saturated NaHCO3 solution, dried the organic phase, and concentrated to give compound F (2.10 g, yield: 90.13%).

[0147]

[0148] Compound G (0.22 g, 1.531 mmol) was added to 12 ml of a THF / H₂O mixed solution (5:1 v / v) of compound F (1.00 g, 1.021 mmol). The mixture was heated to 70 °C and reacted for 20 hours. The reaction was monitored by TLC until complete. The reaction solution was dried, filtered, and concentrated. The concentrate was purified by silica gel column chromatography and eluted with a mixture of dichloromethane and methanol (40:1 v / v) to give compound 1 (220 mg, yield: 19.82%). 1H NMR (400MHz, CDCl3) δ5.47–5.38(m,1H),5.00–4.80(m,1H),3.86–3.67(m,2H),3.44(d,J=5.2Hz,1H),3.39(s,3H),3.26–2.81(m,9H), 2.39–2.31(m,8H),1.44–1.39(m,14H),1.30(s,40H),1.07(s,6H),0.99–0.88(m,28H),0.79–0.69(m,6H); MS-ESI(m / z):1089.0(M+H) + .

[0149] Example 2: Synthesis of Compound 2

[0150]

[0151] The preparation method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,2-ethylenediamine can be used instead of compound A as a raw material to prepare compound 2. 1 H NMR (400MHz, CDCl3) δ5.51–5.33(m,1H),5.51–5.33(m,1H),4.75–4.51(m,2H),4.15–4.00(m,2H),3.37(s,8H),3.26–3.0 3(m,6H),2.41–2.27(m,12H),1.30(s,45H),1.06(s,8H),0.92–0.89(m,20H),0.71(s,6H); MS-ESI(m / z):1032.9(M+H)+.

[0152] Example 3: Synthesis of Compound 3

[0153]

[0154] The preparation method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,3-propanediamine can be used instead of compound A as a raw material to prepare compound 3. 1 H NMR (400MHz, CDCl3) δ5.47–5.36(m,1H),4.97–4.84(m,1H),4.70–4.59(m,1H),3.82–3.69(m,3H),3.38–3.31(m,6H),2.39–2 .26(m,12H),1.72–1.63(m,12H),1.29(s,45H),1.06(s,6H),0.92–0.89(m,20H),0.71(s,6H); MS-ESI(m / z):1046.9(M+H)+.

[0155] Experimental Example 1: Lipid Nanoparticle (LNP) Encapsulation

[0156] The mRNA stock solution was dispersed in 20 mM acetic acid solution (pH = 5.3) to a final concentration of 200 μg / mL (aqueous phase). Following the example, the compounds: cholesterol:DSPC:DMG-PEG2000 were mixed in a molar ratio of 50:38.5:10:1.5 to form a lipid mixture (oil phase). The flow rates of the aqueous and oil phases were controlled by T-junction to mix the mRNA with the lipid mixture, yielding mRNA-encapsulated LNPs. The encapsulated LNPs were diluted with buffer, then concentrated by ultrafiltration, and the diluent was replaced until the LNP concentration reached 100 μg / mL. The pH of the LNPs was adjusted to approximately 7-8. Finally, the total and free mRNA content in the LNPs were detected using a Ribogreen kit and 10% OTG as a demulsifier, and the encapsulation efficiency of the LNPs was calculated. The final LNP product was diluted with diluent, 1 mL was added to the particle size distribution chamber, and the particle size was measured using a Malvern ZetaSizer instrument. The results are shown in Table 1.

[0157] Table 1: LNP characterization data of the compounds in the examples

[0158] Compound numbering Encapsulation rate / % Particle size / nm PDI Compound 1 98.95 71.24 0.06313

[0159] Experiment 2: Testing the delivery efficiency of luciferase

[0160] S1 encapsulates the Luciferase-expressing mRNA into the LNP formulations of compounds MC3 and 1. The LNP formulation preparation method and the mRNA encapsulation method are as described in Experimental Example 1.

[0161] S2 injected the packaged LNP formulation into Balb / c mice intravenously, 200 μl per mouse, with a dose of 20 μg per mouse, and 5 mice per group.

[0162] S3 detected the fluorescence expression intensity of Luciferase in each mouse at 6 hours.

[0163] S4 fluorescence expression intensity detection: 10 minutes before the detection, each mouse was injected intraperitoneally with sodium D-fluorescein (dosage: 150 mg / kg), and then the mice were anesthetized with isoflurane and placed in the IVIS instrument for bioluminescence detection.

[0164] S5 uses fluorescence intensity results to determine the delivery effectiveness of the compound.

[0165] The Luciferase assay is a primary method for detecting mRNA expression in vivo based on fluorescence intensity. Figure 1The results showed that 6 hours after intravenous injection, both compound 1 and the positive control group compound MC3 could express fluorescent protein and produce fluorescence, but the expression level of compound 1 was significantly better than that of MC3, indicating that it has excellent delivery performance.

[0166] At the same time, according to Figure 2 The results showed that compound 1 had a more significant expression advantage over MC3 in the kidney, which means that compound 1 has potential for kidney-targeted delivery.

[0167] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid compound, or a pharmaceutically acceptable salt thereof, for delivery, characterized in that, The compound is compound 1: Compound 1 .

2. A lipid nanoparticle composition comprising a lipid component, said lipid component comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The lipid nanoparticle composition according to claim 2, wherein the lipid component further comprises phospholipids, structured lipids, and / or PEG lipids; wherein the phospholipids are selected from one or more of the following compounds: dilauroyl lecithin, Dimyristic phosphatidylcholine, Dioleoyl lecithin, Dipalmitoylphosphatidylcholine, Distearate phosphatidylcholine (DSPC) Dioleoylphosphatidylcholine, Palmitoyl oleoyl phosphatidylcholine, 1,2-Di-O-octadecyl-sn-glycerol-3-phosphate choline, 1-Oleoyl-2-cholestyldimethylsuccinate-sn-glycerol-3-phosphocholine, 1-Hexadecyl-sn-glycerol-3-phosphate choline, 1,2-Divinyl-sn-glycerol-3-phosphate choline, 1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-Divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-Divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-Dithiohexaenoic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-Dioleoyl-sn-glycerol-3-phosphate-(1-glycerol) sodium salt or sphingomyelin; The structural lipids are selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, and stigmasterol; The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

4. The lipid nanoparticle composition according to claim 3, characterized in that, The phospholipid is DOPE or DSPC.

5. The lipid nanoparticle composition according to claim 3, characterized in that, The structural lipid is cholesterol.

6. The lipid nanoparticle composition according to claim 3, characterized in that, The PEG lipid is DMG-PEG2000.

7. The lipid nanoparticle composition according to any one of claims 2-6, further comprising a therapeutic agent and / or a preventive agent selected from vaccines or compounds capable of inducing an immune response, nucleic acids.

8. The lipid nanoparticle composition according to claim 7, wherein the nucleic acid is RNA.

9. The lipid nanoparticle composition according to claim 8, wherein the RNA is selected from one or more of siRNA, asymmetric interfering RNA, miRNA, dsRNA, shRNA, or mRNA.

10. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 2-9 and a pharmaceutically acceptable carrier.

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