Drug delivery materials and their use

Abstract

This invention relates to a drug delivery material and its use. Specifically, it provides a nanoparticle composition comprising a lipid component containing a compound of formula (I). The nanoparticle composition according to this invention is delivered only to the administration site.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically, to drug delivery materials and their use. [Background technology]

[0002] Lipid-based materials and their derivatives are widely studied and applied for RNA delivery in vivo due to their excellent biocompatibility and good RNA encapsulation rates. RNA is safe and effective, and has promising application prospects in novel therapies for genetic diseases, the expression of functional proteins and antibodies, and the development of vaccines and gene editing. Currently available lipid delivery carriers (BioNTech's BNT162b2 and Moderna's mRNA-1273) express large amounts of target proteins in the liver regardless of the route of administration (intravenous, subcutaneous, intramuscular injection), and have potential toxic side effects when applied to lesions other than the liver, making them unsuitable for clinical application and practical use.

[0003] Therefore, the development of LNP delivery carriers with local expression specificity can provide an entirely new development platform for diseases that are currently difficult or impossible to treat, significantly expanding the clinical application of RNA therapy. [Overview of the project]

[0004] A first aspect of the present invention provides a compound of the following formula (I) or a salt or isomer thereof, [ka] During the ceremony, R1 is H or a C1-C8 alkyl group. R2 is a C1-10 alkyl group, a C3-C18 alkadienyl group, R3 is H or an R1-R8 alkyl group. R4 is a C1-10 alkyl group, a C3-C18 alkadienyl group, R5 is a C1-C14 alkyl group, a C2-C14 alkenyl group, or [ka] And, M is either O or N, t is an integer between 3 and 7. R6 is H or a C1-10 alkyl group. R7 is a C1-10 alkyl group or a C3-C18 alkadienyl group.

[0005] In some embodiments, R1 is the same as R3, and R2 is the same as R4.

[0006] In some embodiments, R1 is H and R2 is a C6-10 alkyl group or a C14-C18 alkadienyl group.

[0007] In some embodiments, R1 is a C4-C8 alkyl group and R2 is a C4-C8 alkyl group.

[0008] In some embodiments, R3 is H and R4 is a C6-10 alkyl group or a C14-C18 alkadienyl group.

[0009] In some embodiments, R3 is a C4-C8 alkyl group, and R4 is a C4-C8 alkyl group.

[0010] In some embodiments, R5 is a C1-C4 alkyl group, a C10-C14 alkyl group, a C10-C14 alkenyl group, or [ka] And, M is either O or N, t is 3 or 7, R6 is a C4-C8 alkyl group and R7 is a C4-C8 alkyl group, or R6 is H and R7 is a C14-C18 alkadienyl group.

[0011] In some embodiments, R1 is H, R2 is a C6-10 alkyl group, R1 is identical to R3, R2 is identical to R4, and R5 is a C10-C14 alkyl group, a C10-C14 alkenyl group or

Chemical Formula

[0012] In some embodiments, R1 is H, R2 is a C14-C18 alkadienyl group, R1 is identical to R3, R2 is identical to R4, and R5 is

Chemical Formula

[0013] In some embodiments, R1 is a C4-C8 alkyl group, R2 is a C4-C8 alkyl group, R1 is identical to R3, R2 is identical to R4, and R5 is a C1-C4 alkyl group,

Chemical Formula

[0014] In some embodiments, each of the alkyl groups is an n-alkyl group.

[0015] In some embodiments, when R2, R4, and R7 are C14-C18 alkadienyl groups, the alkenyl group of the C14-C18 alkadienyl group is located on any two carbons between the 7th and 14th carbons. In some embodiments, the alkenyl group is located on P and P+3 carbons, where P is between 7 and 11. In some embodiments, the C14-C18 alkadienyl group is a 9,12-alkadienyl group having 14 to 18 carbons.

[0016] In some embodiments, the compound of formula (I) is as described in any of formulas 15, 15A-F.

[0017] The present invention further provides a nanoparticle composition comprising a lipid component containing a compound described in any embodiment of this invention (for example, a compound of formula (I)).

[0018] In some embodiments, the lipid component further includes phospholipids (e.g., polyunsaturated lipids), PEG lipids, and structural lipids.

[0019] In some embodiments, the nanoparticle composition further comprises a therapeutic agent and / or a prophylactic agent.

[0020] The present invention further provides a pharmaceutical composition comprising a nanoparticle composition described in any embodiment herein and a pharmaceutically acceptable adjuvant. The pharmaceutical composition is a topical pharmaceutical composition.

[0021] The present invention further provides a method for delivering a therapeutic and / or prophylactic agent to cells, the method comprising administering (e.g., by injection) a nanoparticle composition to a subject, the method comprising (1) a lipid component containing phospholipids (e.g., polyunsaturated lipids), PEG lipids, structural lipids, and compounds of formula (I), and (2) a therapeutic and / or prophylactic agent, wherein the administration comprises delivering the therapeutic and / or prophylactic agent to cells by bringing the cells into contact with the nanoparticle composition.

[0022] In some embodiments, the therapeutic and / or prophylactic agent is a polynucleotide.

[0023] In some embodiments, the therapeutic and / or prophylactic agent is mRNA.

[0024] In some embodiments, the cells are mammalian cells.

[0025] In some embodiments, the delivery is local (not systemic) delivery.

[0026] In some embodiments, the cells are cells located near the administration site.

[0027] In some embodiments, the cells are cells from the same organ or tissue at the administration site. The organ or tissue is, for example, the heart, liver, spleen, lungs, muscle, or femur.

[0028] In some embodiments, the subject is a mammal, such as a human.

[0029] The present invention further provides a method for generating polypeptides in cells, the method comprising contacting cells with a nanoparticle composition comprising (1) a lipid component containing phospholipids (e.g., polyunsaturated lipids), PEG lipids, structural lipids, and a compound of formula (I) or a salt or isomer thereof as described in any embodiment thereof, and (2) mRNA encoding the polypeptide, thereby enabling the mRNA to be translated in cells to generate polypeptides.

[0030] In some embodiments, the cells are mammalian cells.

[0031] The present invention further provides a method for treating a disease or condition in a mammal (e.g., a human) by local (rather than systemic) administration of a therapeutic and / or prophylactic agent, the method comprising the step of locally administering (e.g., by injection) a therapeutically effective amount of nanoparticle composition to a mammal, comprising (1) a lipid component containing a phospholipid (e.g., a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound of formula (I) described in any embodiment thereof, or a salt or isomer thereof, and (2) a therapeutic and / or prophylactic agent (e.g., mRNA or siRNA).

[0032] In some embodiments, the disease or condition is a disease or condition in which a protein or polypeptide becomes dysfunctional or abnormal.

[0033] In some embodiments, the disease or condition is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative disorders, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renovascular diseases, and metabolic diseases.

[0034] The present invention further provides a method for delivering therapeutic and / or prophylactic agents topically (but not systemically) to a mammal, the method comprising administering to a subject (e.g., a mammal) a nanoparticle composition comprising (1) a lipid component containing phospholipids, PEG lipids, structural lipids, and a compound of formula (I) or a salt or isomer thereof as described in any embodiment thereof, and (2) a therapeutic and / or prophylactic agent (e.g., mRNA or siRNA), wherein the administration comprises delivering the therapeutic and / or prophylactic agent topically to the mammal by contacting mammalian cells with the nanoparticle composition.

[0035] In some embodiments, the local area is a target organ or tissue, such as the heart, liver, spleen, lungs, muscle, or femur.

[0036] The present invention further provides a method for producing a compound of formula (I) described herein or a method for producing a nanoparticle composition described herein.

[0037] Use of a compound of formula (I) or a salt or isomer thereof as described in any embodiment of this text, or a nanoparticle composition as described in any embodiment of this text, in the preparation of a topically acting drug for the treatment or prevention of a disease or condition.

[0038] In some embodiments, the disease or condition is one that would benefit from topical (but not systemic) delivery of a therapeutic and / or prophylactic agent.

[0039] In some embodiments, the drug is a drug that delivers a therapeutic and / or prophylactic agent locally (but not systemically).

[0040] In some embodiments, the nanoparticle composition or the nanoparticle composition comprising the compound of formula (I) is intended for the local (but not systemic) delivery of therapeutic and / or prophylactic agents.

[0041] In some embodiments, the local area is a target organ or tissue, such as the heart, liver, spleen, lungs, muscle, or femur. [Brief explanation of the drawing]

[0042] [Figure 1] Figure 1 shows the nuclear magnetic resonance hydrogen spectrum of compound 15.

[0043] [Figure 2] Figure 2 shows the mass spectrum of compound 15.

[0044] [Figure 3] Figure 3 shows the nuclear magnetic resonance hydrogen spectrum of compound 15A.

[0045] [Figure 4] Figure 4 shows the mass spectrum of compound 15A.

[0046] [Figure 5]Figure 5 shows the nuclear magnetic resonance hydrogen spectrum of compound 15B.

[0047] [Figure 6] Figure 6 shows the mass spectrum of compound 15B.

[0048] [Figure 7] Figure 7 shows the nuclear magnetic resonance hydrogen spectrum of compound 15C.

[0049] [Figure 8] Figure 8 shows the mass spectrum of compound 15C.

[0050] [Figure 9-1] Figure 9-1 shows the particle size distribution of LNPs. SM-102 LNP-1 particle size 92.38±1.639 nm, PDI 0.035±0.022;SM-102 LNP-2 particle size 68.53±1.003 nm, PDI 0.053±0.027;15 LNP-1 particle size 87.34±2.507 nm, PDI 0.054±0.017;15 LNP-2 particle size 64.42±0.7681 nm, PDI 0.107±0.037;15A LNP particle size 73.84±0.94 nm, PDI 0.036±0.028;15B LNP particle size 74.50±2.22 nm, PDI 0.23±0.02;15C LNP particle size 64.57±1.26 nm, PDI 0.11±0.04. [Figure 9-2] Continuation of Figure 9-1

[0051] [Figure 10] Figure 10 shows the semi-quantitative fluorescence results of in vivo imaging of small animals in which LNP was injected into the gastrocnemius muscle of C57 mice. High fluorescence intensity was observed for SM-102 LNP-2 at both the intramuscular injection site and the liver, but for 15LNP-2, high fluorescence intensity was observed only at the intramuscular injection site and no fluorescence intensity was observed in the liver, indicating the superior local expression performance of 15LNP-2.

[0052] [Figure 11]Figure 11 shows the expression results after intramuscular injection of 15LNP-2 into C57 mice. Significant expression of 15LNP-2 was observed only at the intramuscular injection site, and no expression was observed in the liver, demonstrating the excellent local expression performance of 15LNP-2.

[0053] [Figure 12] Figure 12 shows the time course of expression activity and local expression area after intramuscular injection of 15LNP-2 into C57 mice. The expression area of ​​15LNP-2 decreased over time after intramuscular injection, and the expression site was limited to the injection site within the experimental cycle.

[0054] [Figure 13] Figure 13 shows the time course of expression activity and local expression area after intramuscular injection of 15LNP-2 into SD rats. The expression area of ​​15LNP-2 decreased over time after intramuscular injection, and the expression site was limited to the injection site within the experimental cycle.

[0055] [Figure 14] Figure 14 shows the expression results after local injection of SM-102 LNP-2. Since SM-102 LNP-2 was expressed at both the intramuscular injection site and the liver site, it can be seen that SM-102 LNP-2 does not have local expression capabilities.

[0056] [Figure 15] Figure 15 shows the expression results after local injection of 15A LNP, 15B LNP, and 15C LNP. Since 15A LNP was expressed at both the intramuscular injection site and the liver site, it can be seen that 15A LNP does not have local expression capabilities; 15B LNP showed a weak expression effect at the intramuscular injection site; and since 15C LNP was expressed only at the intramuscular injection site, it can be seen that 15C LNP has excellent local expression capabilities.

[0057] [Figure 16]Figure 16 shows the semi-quantitative fluorescence results of in vivo imaging of small animals in which LNPs were injected intracardiacly into SD rats. High fluorescence intensity values ​​were observed at the cardiac injection site and liver site for SM-102 LNP-1, SM-102 LNP-2, and 15 LNP-1. However, high fluorescence intensity values ​​were observed only at the cardiac injection site for 15 LNP-2, and low fluorescence intensity values ​​were observed at the liver site, indicating the superior local expression performance of 15 LNP-2.

[0058] [Figure 17] Figure 17 shows the expression results after intramyocardial injection of SM-102 LNP-1. Since SM-102 LNP-1 was expressed at both the cardiac injection site and the liver site, it can be seen that SM-102 LNP-1 does not have local expression capabilities.

[0059] [Figure 18] Figure 18 shows the expression results after intramyocardial injection of SM-102 LNP-2. Since SM-102 LNP-2 was expressed at both the cardiac injection site and the liver site, it can be seen that SM-102 LNP-2 does not have local expression capabilities.

[0060] [Figure 19] Figure 19 shows the expression results after intramyocardial injection of 15 LNP-1. Since 15 LNP-1 was expressed at both the cardiac injection site and the liver site, it can be seen that 15 LNP-1 does not have local expression capabilities.

[0061] [Figure 20] Figure 20 shows the expression results after intramyocardial injection of 15LNP-2. Significant expression of 15LNP-2 was observed only at the cardiac injection site, and no expression was observed in the liver, demonstrating the excellent local expression performance of 15LNP-2.

[0062] [Figure 21]Figure 21 shows the expression results after 15LNP-2 was injected into the spleen of C57 mice. Significant expression of 15LNP-2 was observed only at the spleen injection site, and no expression was observed at other sites, demonstrating the excellent local expression performance of compound 15LNP-2.

[0063] [Figure 22] Figure 22 shows the expression results after 15LNP-2 was injected into the kidneys of C57 mice. Significant expression of 15LNP-2 was observed only at the kidney injection site, and no expression was observed at other sites, demonstrating the excellent local expression performance of 15LNP-2.

[0064] [Figure 23] Figure 23 shows the expression results after 15LNP-2 was injected into the spleen of SD rats. Significant expression of 15LNP-2 was observed only at the splenic injection site, and no expression was observed at other sites, demonstrating the excellent local expression performance of 15LNP-2.

[0065] [Figure 24] Figure 24 shows the expression results after 15LNP-2 was injected into the kidneys of SD rats. Significant expression of 15LNP-2 was observed only at the kidney injection site, and no expression was observed at other sites, demonstrating the excellent local expression performance of 15LNP-2. [Modes for carrying out the invention]

[0066] The inventors synthesized a novel ionizable cationic lipid and prepared LNPs using this lipid. When these mRNA-encapsulated LNPs were injected into the muscle, cardiac muscle, spleen, and kidney of the mouse thigh, as observed by biofluorescence analysis in small animals, this delivery system expressed the protein from mRNA only at the injection site, and no expression was observed in other major organs (heart, liver, spleen, lungs, kidneys).

[0067] This disclosure relates to novel lipids and lipid nanoparticle compositions comprising novel lipids. This disclosure further provides a method for delivering therapeutic and / or prophylactic agents to mammalian cells, in particular a method for delivering therapeutic and / or prophylactic agents to a mammalian site to generate target peptides in mammalian local cells and to treat a mammalian disease or pathology.

[0068] A method for delivering therapeutic and / or prophylactic agents to mammalian cells or sites relates to administering a nanoparticle composition comprising a therapeutic and / or prophylactic agent to a subject, wherein the administration relates to delivering the therapeutic and / or prophylactic agent to the cells or site by bringing the cells or site into contact with the composition.

[0069] In this text, “local” refers to cells, tissues, or organs within a specified range from the administration site. In the prior art, retaining a drug at the administration site usually requires the use of special dosage forms, such as creams, ointments, sprays, or powder sprays, which are often applied to the body surface or mucous membranes. However, the inventors have found that drugs prepared using the compound of formula (I) described herein (e.g., the nanoparticle compositions or pharmaceutical compositions described herein) can achieve local administration and local action into organs, tissues, or cells. In some embodiments, the nanoparticle compositions according to the present invention are applied to the preparation of locally applied, locally acting products (LALAPs). In this text, “locally acting drug,” “locally acting pharmaceutical composition,” “locally administered, locally acting drug,” or “locally administered, locally acting” refers to a drug that is administered locally and exerts its effect at the administration site. If such a drug exhibits systemic effects (e.g., hepatic targeting), it is considered an unexpected drug action. “Local” may include only cells, only organs (e.g., heart, liver, spleen, kidney, or lung), or only tissues (e.g., muscle, skin, or bone). In some embodiments of this text, "local" refers only to cells, tissues, or organs at the site of administration. In some embodiments, when administered locally to an organ (e.g., heart, liver, spleen, kidney, or lung), the drug is delivered only within the organ where the administration site is located; when administered locally to a tissue (e.g., a muscle), the drug is delivered only within the tissue where the administration site is located (e.g., the muscle); when administered locally to a cell, the drug is delivered only to cells within the organ or tissue where the administration site is located (e.g., the muscle where the cell is located). In some embodiments, when administered to the calf muscle, the drug is delivered only within the calf muscle.

[0070] In some embodiments, local means that the LNP composition, therapeutic agent and / or prophylactic agent (mRNA) is maintained primarily within the organ (e.g., heart, liver, spleen, kidney, or lung) within 4 to 8 hours (e.g., 6 hours) after administration of the LNP composition described herein. In some embodiments, local means that the LNP composition, therapeutic agent and / or prophylactic agent (mRNA) is maintained primarily within the tissue (e.g., muscle or bone) within 4 to 8 hours (e.g., 4 hours) after administration of the LNP composition described herein, and in particular, is not delivered to the liver within 4 to 8 hours (e.g., 4 hours) after administration.

[0071] Lipids and lipid nanoparticle compositions The lipids described herein can be advantageously applied to lipid nanoparticle compositions to deliver therapeutic and / or prophylactic agents topically to mammals. The lipids described herein are little to no immunogenic.

[0072] In some embodiments, the compounds described herein are those having formula (I), or salts or isomers thereof. Lipids relating to formula (I) may be positively charged or partially positively charged at physiological pH. Such lipids are called cationic lipids.

[0073] As used in this text, the term "alkyl" or "alkyl group" refers to an optionally substituted linear or branched saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms). The symbol "C1-14 alkyl group" refers to an optionally substituted linear or branched saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups in this text refer to unsubstituted or substituted alkyl groups.

[0074] As used in this text, the term “alkenyl” or “alkenyl group” refers to an optionally substituted linear or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon double bond. The symbol “C2-14 alkenyl group” refers to an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may contain 1, 2, 3, 4 or more carbon-carbon double bonds. Unless otherwise specified, the term “alkenyl group” in this text refers to an unsubstituted or substituted alkenyl group.

[0075] As used in this text, the term "alkynyl" or "alkynyl group" refers to an optionally substituted linear or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon triple bond. The symbol "C2-14 alkynyl group" refers to an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may contain 1, 2, 3, 5 or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups as used in this text refer to unsubstituted or substituted alkynyl groups.

[0076] As used in this text, the terms “carbocyclic ring” or “carbocyclic group” refer to an optionally substituted monocyclic or polycyclic system containing one or more carbon atom rings. The rings may be multi-membered rings with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more members. The symbol “C3-6 carbocyclic ring” refers to a monocyclic carbocyclic ring having 3 to 6 carbon atoms. The carbocyclic ring may contain one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocyclic rings include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, and naphthyl groups.

[0077] As used in this text, "cycloalkyl group" refers to a non-aromatic carbocyclic group that may or may not contain a double or triple bond. Unless otherwise specified, carbocyclic groups as used in this text refer to unsubstituted or substituted carbocyclic groups, i.e., optionally substituted carbocyclic groups.

[0078] As used in this text, the terms “heterocycle” or “heterocyclyl group” refer to an optionally substituted monocyclic or polycyclic system containing one or more rings, where at least one ring contains at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The rings may be multi-membered rings with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more members. The heterocycle may contain one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocyclylalkyl groups or heteroaryl groups). Examples of heterocyclic groups include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolidinyl, morpholinyl, pyryl, pyridinyl, furanyl, tetrahydrofuranyl, thienyl, pyridyl, piperidinyl alkynyl, quinolyl, and isoquinolyl groups.

[0079] As used in this text, the term "heterocyclylalkyl group" refers to a non-aromatic heterocycle, which may or may not contain double or triple bonds. Unless otherwise specified, heterocycles as used in this text refer to unsubstituted or substituted heterocyclyl groups, i.e., arbitrarily substituted heterocycles.

[0080] As used in this text, "aryl group" refers to an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include the phenyl group and the naphthyl group.

[0081] As used in this text, "heteroaryl group" refers to an optionally substituted heterocyclyl group containing one or more aromatic rings. Examples of heteroaryl groups include pyryl, furanyl, thienyl, imidazolyl, oxazolyl, and thiazolyl groups. Both aryl and heteroaryl groups may be optionally substituted. Unless otherwise specified, aryl or heteroaryl groups as used in this text refer to unsubstituted or substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0082] Unless otherwise specified, alkyl groups, alkenyl groups, and cyclyl groups (e.g., carbocyclic groups and heterocyclyl groups) may be optionally substituted. Optional substituents include halogen atoms (e.g., chloro, bromo, fluoro, or iodo groups), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxyl groups, -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 those represented by C=O), acid 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 (e.g., -C(OR)2R', where each OR may be the same or different alkoxy group, and R' is an alkyl group or alkenyl group), and phosphates (e.g., P(O)4 3+), mercaptans (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thiols (e.g., -C(S)H), sulfates (e.g., S(O)4) 2+ ), sulfonyl groups (e.g., -S(O)2-), amides (e.g., -C(O)NR2 or -N(R)C(O)R), azide groups (e.g., -N3), nitro groups (e.g., -NO2), cyano groups (e.g., -CN), isocyano groups (e.g., -NC), acyloxy groups (e.g., -OC(O)R), amino groups (e.g., -NR2, -NRH or -NH2), carbamoyl groups (e.g., -OC(O)NR2, -OC(O)NRH) It may also be selected from the group consisting of -OC(O)NH2), sulfonamide formulations (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), alkyl groups, alkenyl groups, and cyclyl groups (e.g., carbocyclic groups or heterocyclyl groups), but is not limited to these. In any of the above items, R is an alkyl group or alkenyl group as defined in the text.

[0083] In some embodiments, the substituent itself may be further substituted by, for example, one, two, three, four, five, or six substituents defined herein. For example, a C1-6 alkyl group may be further substituted by one, two, three, four, five, or six substituents described herein.

[0084] As used in this text, the terms “approximately” and “roughly” refer to a number that is similar to a given reference value when applied to one or more numbers of interest. In some embodiments, the terms “approximately” or “roughly” mean deviating from the reference value by 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (greater or less) in any direction from the reference value, unless otherwise specified or made clear from the context (unless the number exceeds 100% of the possible values). For example, when used in the context of the amount of a specified compound in the lipid component of a nanoparticle composition, “approximately” may represent + / - 10% of the number. For example, a nanoparticle composition containing a lipid component that contains approximately 40% of the specified compound may contain 30-50% of the compound.

[0085] As used in this text, the term “compound” is intended to encompass all isomers and isotopes, including the structures shown. “Isotopes” refer to atoms that have the same atomic number but different mass numbers, resulting from a difference in the number of neutrons in their nuclei; for example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of this disclosure can be prepared by combining them with solvents or water molecules in the usual manner to form solvates or hydrates.

[0086] As used in this text, the term "contact" means establishing a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means physically connecting the mammalian cells with the nanoparticles. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. For example, a nanoparticle composition can be contacted with mammalian cells by different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and may also be in different amounts. Furthermore, the nanoparticle composition may be in contact with one or more types of mammalian cells.

[0087] As used in this text, the term “delivery” means providing an entity to its destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may also mean administering a nanoparticle composition containing the therapeutic and / or prophylactic agent to the subject (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cells may also mean bringing one or more types of cells into contact with the nanoparticle composition. “Local delivery” means that the drug is delivered primarily to a specific location in the subject, for example, to a certain organ, a certain tissue (e.g., a certain muscle), or a certain cell (e.g., the organ to which the drug is administered, the muscle to which the drug is administered, or the cell to which the drug is administered), but not to, or substantially not to, any other organ, tissue, or cell. For example, when a drug is administered to the kidney, the drug is locally delivered to other parts of the kidney, but not to any other organ, tissue, or cell (e.g., via the circulatory system). When a drug is administered to a muscle (e.g., the calf muscle), the drug is delivered locally to other parts of that muscle, but not to other organs, tissues, or cells (e.g., via the circulatory system).

[0088] In some embodiments, nanoparticle compositions comprising compounds according to formula (I) have substantially the same level of local delivery effect regardless of the route of administration. For example, when some of the compounds disclosed herein are used for intravenous or intramuscular delivery of therapeutic and / or prophylactic drugs, they represent similar local delivery. In some embodiments, intramuscular injection is preferred over intravenous injection.

[0089] As used in this text, the terms “specific delivery” and “local delivery” refer to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) of a therapeutic and / or prophylactic agent by nanoparticles to target cells, tissues, or organs of interest (e.g., mammalian muscle) compared to non-target tissues (e.g., mammalian liver). The level of delivery of nanoparticles to a particular tissue can be determined by the following comparisons: the ratio of the amount of protein produced in the tissue to the weight of the tissue; the ratio of the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue; the ratio of the amount of protein produced in the tissue to the total amount of protein in the tissue; or the ratio of the amount of therapeutic and / or prophylactic agent in the tissue to the total amount of therapeutic and / or prophylactic agent in the tissue. It should be understood that the ability of nanoparticles to specifically deliver to target tissues does not need to be determined in the patient receiving treatment, but can be determined using alternatives such as animal models (e.g., rat models).

[0090] As used in this text, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the nanoparticle composition relative to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of the nanoparticle composition. For example, if 97 mg of therapeutic and / or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency may be 97%.

[0091] As used in this text, "expression" of a nucleic acid sequence includes translation from mRNA to polypeptide or protein, and / or post-translational modification of polypeptide or protein.

[0092] As used in this text, the term "in vitro" refers to events occurring in an artificial environment, such as in a test tube, reaction vessel, cell culture, or petri dish, rather than within a living organism (e.g., animal, plant, or microorganism). As used in this text, the term "in vivo" refers to events occurring within a living organism (e.g., animal, plant, microorganism, or their cells or tissues). As used in this text, the term "ex vivo" refers to events occurring outside of a living organism (e.g., animal, plant, microorganism, or their cells or tissues). Ex vivo events may occur in environments that are minimally altered compared to the natural environment (e.g., within a living organism).

[0093] As used herein, the term “isomer” refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may exist as stereoisomers, as they may contain one or more chiral centers and / or double bonds. This disclosure encompasses all isomers of the compounds described herein, including the pure forms of stereoisomers and mixtures of enantiomers and stereoisomers (e.g., racemates). Methods for dividing enantiomers and mixtures of stereoisomers of compounds, as well as their components into enantiomers or stereoisomers, are well known.

[0094] As used in this text, “lipid component” refers to a component of a nanoparticle composition containing one or more types of lipids. For example, the lipid component may include one or more cationic / ionizable lipids, polyethylene glycolated lipids, lipids having one or more structures, or other lipids, such as phospholipids.

[0095] As used in this text, “administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. By selecting a method of administration, targeted delivery (e.g., specific delivery) to a particular area or system of the body can be achieved.

[0096] As used in this text, "modified" refers to something unnatural. For example, RNA may be modified RNA. That is, RNA may contain one or more unnaturally occurring nucleic acid bases, nucleosides, nucleotides, or linkers. RNA also includes any sequence-optimized version of RNA.

[0097] As used in this text, a "nanoparticle composition" is a composition containing one or more types of lipids. The size of a nanoparticle composition is typically on a micrometer scale or smaller, and it may contain a lipid bilayer. A nanoparticle composition may include lipid nanoparticles (LNPs), liposomes (e.g., lipid niosomes), and lipid complexes. For example, a nanoparticle composition may be liposomes having a lipid bilayer with a diameter of 500 nm or less.

[0098] As used in this text, “patient” refers to a subject who may seek or need treatment, a subject who needs treatment, a subject who is receiving treatment, a subject who is scheduled to receive treatment, or a subject who has received professional care for the cause of a particular disease or condition.

[0099] As used in this text, "PEG lipids" or "PEGylated lipids" refer to lipids containing polyethylene glycol components.

[0100] As used in this text, the phrase “pharmaceutically acceptable” means a compound, material, composition and / or dosage form that is suitable for contact with human and animal tissues within reasonable medical judgment and does not cause excessive toxicity, irritation, allergic reactions or other problems or complications, and that is commensurate with a reasonable benefit-risk ratio.

[0101] As used in this text, the phrase “pharmaceutically acceptable auxiliaries” refers to any component other than the compounds described herein (e.g., carriers capable of suspending, complexing, or dissolving active compounds) that is substantially non-toxic and non-toxic in nature. Auxiliaries may include, for example: anti-blocking agents, antioxidants, binders, coatings, tableting aids, disintegrants, dyes (pigments), humectants, emulsifiers, fillers (diluents), film-forming agents and coatings, fragrances, flavorings, fluidizers (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents and dispersants, sweeteners and hydration water. Exemplary adjuvants include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl parahydroxybenzoate, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl parahydroxybenzoate, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn starch), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0102] In this specification, the structural formulas of compounds are shown for convenience as isomers in some situations, but it should be understood that this disclosure includes all isomers, such as geometric isomers, optical isomers based on chiral carbons, stereoisomers, tautomers, etc., and that not all isomers have the same level of activity.

[0103] For compounds represented by the chemical formulas shown herein, polymorphism may occur. It should be noted that all crystalline forms, mixtures of crystalline forms, or their acid anhydrides or hydrates are included within the scope of this disclosure. The terms “polymorph,” “polymorph,” or “crystalline form” refer to crystalline structures in which a compound (or its salt or solvate) crystallizes in different crystalline stacking arrangements, all having the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability, and solubility. One crystalline form may be dominant due to factors such as the recrystallization solvent, crystallization rate, storage temperature, and other factors. Polymorphs of compounds can be prepared by crystallizing them under different conditions.

[0104] The composition may further contain salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salt” means a derivative of the disclosed compound obtained by changing the parent compound by converting an existing acidic or base moiety to the form of its salt (for example, by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of alkaline residues such as amino groups; and alkali metal or organic salts of acidic residues such as carboxylic acids.

[0105] Typical acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, glucarate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfone. This includes acid salts, lactobionates, lactates, laurates, dodecyl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, valersates, and others.

[0106] Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0107] The pharmaceutically acceptable salts of this disclosure include, for example, common non-toxic salts of parent compounds formed with non-toxic inorganic or organic acids.

[0108] The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing alkaline or acidic moieties by conventional chemical methods. Typically, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometrically appropriate base or acid in water, an organic solvent, or a mixture thereof; non-aqueous media (e.g., ether, ethyl acetate, ethanol, isopropanol, or acetonitrile) are usually preferred. A list of suitable salts is found in any edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference in its entirety.

[0109] As used in this text, "phospholipid" refers to a lipid comprising a phosphate group and one or more carbon chains (e.g., an unsaturated fatty acid chain). Phospholipids may also contain one or more multiple bonds (e.g., double or triple bonds) (e.g., one or more degrees of unsaturation). 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). The fusion of phospholipids with a membrane allows one or more components, including the lipid composition, to pass through the membrane, thereby enabling, for example, the delivery of one or more components to a cell.

[0110] As used in this text, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues, usually linked by peptide bonds, which can be produced naturally (e.g., by isolation or purification) or synthetically.

[0111] As used in this text, “RNA” refers to ribonucleic acid, which may be naturally occurring or unnaturally occurring. For example, it may include RNA-modified and / or unnaturally occurring components, such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, a chain termination nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that codes for a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). The coded polypeptide can be produced by translation of mRNA that codes for a specific polypeptide, for example, by in vivo translation of mRNA in mammalian cells. RNA may be selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), and mRNA.

[0112] As used in this text, “single unit dose” refers to the amount of any therapeutic agent administered in a single dose / single dose / single route / single contact point, i.e., in a single administration event. As used in this text, “divided dose” refers to dividing a single unit dose or total daily dose into two or more doses. As used in this text, “total daily dose” refers to the amount administered or specified within a 24-hour period. This may be administered as a single unit dose.

[0113] As used in this text, "size" or "average size" in the context of nanoparticle compositions refers to the average diameter of the nanoparticle composition.

[0114] As used herein, the terms “subject,” “subject,” or “patient” refer to any living organism to which the compositions relating to this disclosure can be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mice, rats, rabbits, non-human primates, and mammals such as humans) and / or plants. Preferably, subjects described herein suffer from diseases that would benefit from local delivery of the drug, for example, lesions of a specific organ or tissue to which it is desired that the drug be delivered only.

[0115] As used in this text, “target cells” refers to any cells of interest, one or more types. The cells may be found in vitro, in vivo, in stew, or in the tissues or organs of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0116] As used in this text, “target tissue” refers to one or more types of tissue that, upon delivery of a therapeutic and / or prophylactic agent, will produce the expected biological and / or pharmacological effects. Examples of target tissues of interest include specific tissues, organs and systems, or combinations thereof. In specific applications, target tissue may be the kidney, lung, spleen, vascular endothelium in blood vessels (e.g., within the coronary arteries or femur), tumor tissue (e.g., by intratumoral injection), or muscle (e.g., by intramuscular injection).

[0117] As used in this text, "muscles" include the back muscles (including the trapezius, latissimus dorsi, levator scapulae, rhomboids, erector spinae, cleavage, transversospinalis, interspinals, and intertransverse muscles), pectoralis muscles (including the pectoralis major, pectoralis minor, serratus anterior, external intercostal muscles, internal intercostal muscles, and transversus thoracis), diaphragm, abdominal muscles (including the rectus abdominis, external oblique, internal oblique, transversus abdominis, and quadratus lumborum), perineal muscles, shoulder girdle muscles (including the deltoid, superior posterior serratus, inferior posterior serratus, teres minor, subscapularis, and teres major), brachialis muscles (including the biceps brachii, coracobrachialis, brachialis, triceps brachii, and anconeus), and forearm muscles (brachioradialis and pronator muscles). This includes, but is not limited to, the muscles of the hand (including the flexor carpi radialis, flexor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, and extensor carpi ulnaris), the muscles of the fingers, the muscles of the pelvic girdle (including the iliopsoas, piriformis, gluteus maximus, gluteus medius, and gluteus minimus), the muscles of the thigh (including the quadriceps femoris, sartorius, tensor fasciae latae, biceps femoris, semitendinosus, semimembranosus, pectineus, flexor longus, flexor brevis, flexor major, and gracilis), the muscles of the lower leg (including the anterior tibialis, extensor digitorum longus, gastrocnemius, ichthyosus, flexor digitorum longus, flexor septum longus, posterior tibialis, longus peroneus, and brevis peroneus), the muscles of the foot, the platysma, and the sternocleidomastoid.

[0118] The term “therapeutic agent” or “preventive agent” refers to any reagent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect, and / or produces an expected biological and / or pharmacological effect. Therapeutic agents are also called “active substances” or “activators,” and include, but are not limited to, cytotoxic substances, radioactive ions, chemotherapeutic drugs, small molecule drugs, proteins, and nucleic acids, such as RNA.

[0119] As used in this text, the term “therapeutic dose” refers to the amount administered to a subject who is suffering from or susceptible to an infection, disease, illness, and / or condition, that treats or improves the symptoms of the infection, disease, illness, and / or condition, and that diagnoses, prevents, and / or delays an outbreak of the infection, disease, illness, and / or condition.

[0120] As used in this text, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.

[0121] As used in this text, the term “treatment” means partially or completely reducing, improving, improving, mitigating, delaying, inhibiting the progression of, reducing the severity of, and / or decreasing the incidence of a particular infection, disease, disorder, and / or condition. For example, “treatment” of cancer may mean inhibiting the survival, growth, and / or spread of the tumor. To reduce risk, treatment of pathological developments associated with a disease, disease, and / or condition may be performed on subjects who are not showing signs of the disease, condition, and / or condition, and / or subjects who are showing only early signs of the disease, condition, and / or condition.

[0122] Another aspect of this disclosure is a nanoparticle composition comprising a lipid component containing the compound of formula (I) described herein.

[0123] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid niosomes, and lipid complexes. In some embodiments, the nanoparticle composition includes niosomes comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition includes two or more concentric bilayers separated by water-containing chambers. The lipid bilayers may be functionalized and / or crosslinked with each other. The lipid bilayers may contain one or more ligands, proteins, or channels.

[0124] The nanoparticle composition contains a lipid component comprising at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition may contain one or more compounds from 1 to 10. The nanoparticle composition may further contain various other components. For example, in addition to the lipid of formula (I), the nanoparticle composition may further contain one or more cationic lipids.

[0125] The lipid component of the nanoparticle composition may contain one or more types of PEG or PEG-modified lipids. Such substances may also be called polyethylene glycolated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0126] The lipid component of the nanoparticle composition may include one or more structural lipids. The structural lipids may be selected from, but are not limited to, the group consisting of cholesterol, fecosterol, glucosterol, ergosterol, liposterol, sosterol, liposterol, tomatine, tomatine, ursodeoxycholic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0127] The lipid component of the nanoparticle composition may include one or more phospholipids, for example, one or more unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. Typically, the phospholipids may consist of a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipids may be lipids according to formula (II): [ka] In the formula, Rp represents the phospholipid portion, and R1 and R2 represent the fatty acid portion with or without a degree of unsaturation, and they may be the same or different. The phospholipid portion may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion may be selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0128] The phospholipids used in the compositions and methods described herein may be selected from the group consisting of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, OChemsPC, DOPG, and sphingomyelin. In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DSPC and / or DOPE.

[0129] In some embodiments, the nanoparticle compositions described herein include a compound of formula (I), a PEG lipid, DSPC, and cholesterol.

[0130] In some embodiments, the nanoparticle composition comprising one or more lipids described herein may further contain one or more adjuvants, such as aluminum hydroxide.

[0131] Therapeutic and / or prophylactic agents The nanoparticle composition may contain one or more therapeutic and / or prophylactic agents. Methods for delivering therapeutic and / or prophylactic agents according to the present invention to mammalian cells or organs, generating polypeptides of interest within mammalian cells, and treating diseases or conditions in mammals where necessary include administering and / or contacting mammalian cells having a nanoparticle composition containing therapeutic and / or prophylactic agents to mammals.

[0132] Therapeutic and / or prophylactic agents may be substances that, upon delivery to cells or organs, cause desirable changes in cells, organs or other body tissues or systems. Such substances can be applied to the treatment of one or more diseases or conditions. In some embodiments, the therapeutic and / or prophylactic agents are small molecule drugs that can be applied to the treatment of specific diseases or conditions.

[0133] Examples of drugs applicable to nanoparticle compositions include antitumor agents (e.g., doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracycline drugs, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs, e.g., methotrexate, purines and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., dibucaine and chlorpromazine), β-adrenergic blockers (e.g., propranolol, timolol and labetalol), antihypertensive drugs (e.g., clonidine and hydralazine), This includes, but is not limited to, antidepressants (e.g., imipramine, amitriptyline, and doxepin), antiepileptic drugs (e.g., phenytoin sodium), antihistamines (e.g., diphenhydramine, chlorpheniramine, promethazine), antibiotics / antimicrobials (e.g., gentamicin, ciprofloxacin, cefoxitin), antifungal drugs (e.g., miconazole, teconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B), antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, anesthetics, and contrast agents.

[0134] In some embodiments, therapeutic and / or prophylactic agents are cytotoxic substances, radioactive ions, chemotherapeutic agents, vaccines, compounds that induce immune responses, and / or other therapeutic and / or prophylactic agents. Cytotoxic substances or cytotoxic agents include any reagent that may be harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, plicamycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, meitansin (e.g., mytansinol), rapamycin and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphates, cobalt, yttrium-90, samarium-153, and praseodymium.

[0135] Other therapeutic and / or prophylactic drugs include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, procarbazine), alkylating agents (e.g., nitrogen mustard, thiotepachlorambucil, rapamycin, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and This includes, but is not limited to, cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracycline drugs (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, plicamycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel and anthamitocin alkaloids).

[0136] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Useful therapeutic proteins in nanoparticles according to the present disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0137] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide” in its broadest sense includes any compound and / or substance that is or may be incorporated into an oligonucleotide chain. Exemplary polynucleotides as used in this disclosure include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (including messenger mRNA (mRNA)), their hybrids, RNAi inducers, one or more RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0138] In some embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA used in the compositions and methods described herein may be selected from, but is not limited to, short chains, antagonists, antisense strands, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA, small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA or siRNA.

[0139] In some embodiments, the therapeutic and / or prophylactic agent is mRNA. mRNA can encode any polypeptide of interest, including any polypeptide that exists naturally or unnaturally, or is otherwise modified. The polypeptide encoded by mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA, when expressed in cells, has a therapeutic effect.

[0140] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. siRNA can selectively reduce or downregulate the expression of genes of interest. For example, by selecting siRNA and administering a nanoparticle composition containing siRNA to a subject in need, genes associated with a specific disease or condition can be silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be immunomodulatory siRNA.

[0141] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or a vector or plasmid encoding it. shRNA can be generated within target cells after a suitable construct is delivered to the cell nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant fields.

[0142] The nucleic acids and polynucleotides used in this disclosure typically include a first region (e.g., coding region) that ligates a nucleoside encoding a polypeptide of interest, a first flanking region (e.g., 5-UTR) located at the 5'-terminus of the first region, a second flanking region (e.g., 3-UTR) located at the 3'-terminus of the first region, and at least one 5'-cap region and a 3'-stabilizing region. In some embodiments, the nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some situations, the polynucleotide may include one or more intronic nucleotide sequences that can be removed from the polynucleotide. In some embodiments, the polynucleotide or nucleic acid (e.g., mRNA) may include a 5' cap structure, a chain termination nucleotide, a stem-loop, a polyadenyle sequence and / or a polyadenylation signal. Any region of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides).

[0143] Typically, the minimum length of a polynucleotide may be a polynucleotide sequence length sufficient to encode a dipeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some situations, the length of the polynucleotide is at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 80, at least 90, at least 100, at least 120, at least 150, at least 180, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 nucleotides.

[0144] Nucleic acids and polynucleotides may contain one or more naturally occurring components (including any typical nucleotides such as A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine)). In one example, all or substantially all nucleotides include (a) 5'-UTR, (b) open reading frame (ORF), (c) 3'-UTR, (d) polyadenylate, and any combination of a, b, c, or d above.

[0145] Polynucleotides and nucleic acids may include one or more modified (e.g., altered or alternative) nucleic acid bases, nucleosides, nucleotides, or combinations thereof. Nucleic acids and polynucleotides used in nanoparticle compositions may include any useful modifications or alterations to the bonds between nucleic acid bases, sugars, or nucleosides (e.g., those linking phosphate / phosphate diester bonds / phosphate diester backbone chains).

[0146] In some situations, nucleic acids do not substantially induce an innate immune response in cells into which polynucleotides (e.g., mRNA) have been introduced.

[0147] Other ingredients In addition to those described above, the nanoparticle composition may contain one or more components. For example, the nanoparticle composition may contain one or more hydrophobic low molecular weight molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0148] The nanoparticle composition may further comprise one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. The permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may comprise monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). Polymers may be included in the nanoparticle composition and / or used to encapsulate or partially encapsulate the nanoparticle composition. The polymers may be biodegradable and / or biocompatible. The polymers may be selected from, but are not limited to, the group consisting of polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimides, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The surface modifier may include, but is not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, e.g., dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucosols (e.g., acetylcysteine, neratinib, and erdosteine), and DNases (e.g., rhDNase). The surface modifier can be positioned within and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent bonding, or other processes).

[0149] The nanoparticle composition may further contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group and can undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, the lipid bilayer may be functionalized in this manner with one or more groups applicable to enhancing membrane penetration, cell recognition, or imaging.

[0150] The surface of the nanoparticle composition may be conjugated with one or more useful antibodies. Functional groups and conjugates used for targeted delivery to cells, imaging, and membrane penetration are well known in this field.

[0151] In addition to these components, the nanoparticle composition may contain any substance applicable to a pharmaceutical composition. For example, the nanoparticle composition may contain, but is not limited to, one or more pharmaceutically acceptable auxiliaries or auxiliary components, such as one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, fluidizers, liquid carriers, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. It may further contain auxiliaries such as waxes, butters, colorants, coatings, flavorings, and fragrances. Pharmaceutically acceptable auxiliaries are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition).

[0152] The lipid component of the nanoparticle composition may include, for example, lipids relating to formula (I), phospholipids (e.g., unsaturated lipids, e.g., DOPE or DSPC), PEG lipids, and structural lipids. Each lipid component may be provided in a specific fraction. In some embodiments, the lipid component of the nanoparticle composition includes about 30 mol% to about 60 mol% of the compound of formula (I), about 0 mol% to about 30 mol% of phospholipids, about 18.5 mol% to about 48.5 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol% to about 55 mol% of the compound of formula (I), about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 40 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids. In certain embodiments, the lipid component comprises about 50 mol% of the compound of formula (I), about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another embodiment, the lipid component comprises about 50 mol% of the compound of formula (I), about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 3.05 mol% of PEG lipids. In yet another specific embodiment, the lipid component comprises about 40 mol% of the compound of formula (I), about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-DMG (e.g., PEG2000-DMG), and / or the structural lipids may be cholesterol.

[0153] Alternatively, in terms of molar parts of the lipid component, for example, the lipid component of the nanoparticle composition includes about 30 to 60 molar parts of the compound of formula (I), about 0 to 30 molar parts of phospholipid, about 18.5 to 48.5 molar parts of structural lipid, and about 0 to 10 molar parts of PEG lipid. In some embodiments, the lipid component of the nanoparticle composition includes about 35 to 55 molar parts of the compound of formula (I), about 5 to 25 molar parts of phospholipid, about 30 to 40 molar parts of structural lipid, and about 0 to 10 molar parts of PEG lipid. In some embodiments, it includes about 50 molar parts of the compound of formula (I), about 10 molar parts of phospholipid, about 38.5 molar parts of structural lipid, and about 1.5 molar parts of PEG lipid. In another embodiment, the lipid component comprises about 50 moles of the compound of formula (I), about 10 moles of phospholipid, about 38.5 moles of structural lipid, and about 3.05 moles of PEG lipid.

[0154] The amount of therapeutic and / or prophylactic agent in the nanoparticle composition may be determined by the size, composition, required target and / or use, or other properties of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA applicable to the nanoparticle composition may be determined by the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition may also vary. In some embodiments, the weight / weight ratio of lipid components to therapeutic and / or prophylactic agent in the nanoparticle composition may be about 5:1 to about 60:1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1. For example, the weight / weight ratio of the lipid component to the therapeutic and / or prophylactic agent may be approximately 10:1 to approximately 40:1. In some examples, the weight / weight ratio is approximately 20:1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, by absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0155] The average size of the nanoparticle composition may be between 10 nm and 100 nm and can be measured, for example, by methods well known in this field. For example, the average size may be approximately 40 nm to approximately 150 nm, for example, approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, from approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, or approximately 90 nm to 100 nm. In some examples, the average size of the nanoparticle composition may be approximately 70 nm to 100 nm. In certain examples, the average size may be approximately 70 nm.

[0156] The nanoparticle composition may be relatively uniform. The polydispersity index can be applied to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) usually indicates a narrow particle size distribution. The nanoparticle composition may have a polydispersity index of about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition may be about 0.05 to about 0.10.

[0157] The encapsulation efficiency of therapeutic and / or prophylactic agents represents the amount of therapeutic and / or prophylactic agent prepared and then encapsulated or otherwise bound to the nanoparticle composition relative to the initial amount provided. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in the nanoparticle composition-containing solution before pulverizing the nanoparticle composition with one or more organic solvents or cleaning agents to the amount thereafter. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some examples, the encapsulation efficiency may be at least 90%.

[0158] The nanoparticle composition may optionally contain one or more coating layers. For example, the nanoparticle composition may be incorporated into coated capsules, films, or tablets. The capsules, films, or tablets of the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0159] The nanoparticle composition according to the present invention may be prepared in various forms applicable to various routes of administration, such as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), topical (including transchuccal and sublingual), transdermal and / or transdermal administration dosage forms (e.g., creams, ointments, pastes, lotions, gels, powders, solutions, sprays, inhalants, and patches), intravaginal administration dosage forms (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), implantable administration dosage forms (e.g., solids, semi-solids, gels), suspensions, powders, and other dosage forms.

[0160] Pharmaceutical composition The nanoparticle composition may be prepared in whole or in part as a pharmaceutical composition. The pharmaceutical composition may contain one or more types of nanoparticle compositions.

[0161] For example, a pharmaceutical composition may include one or more nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition may further include one or more pharmaceutically acceptable adjuvants; for example, general guidance for preparing and manufacturing the pharmaceutical compositions and drugs described herein can be found in Remington's *The Science and Practice of Pharmacy*, 21st edition. Common adjuvants and adjuvants are applicable to any pharmaceutical composition, provided that no common adjuvant or adjuvant could be incompatible with one or more components of the nanoparticle composition. The content of adjuvants in a pharmaceutical composition can be determined as needed by those skilled in the art.

[0162] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable adjuvants, and / or optional additional components in the pharmaceutical composition relating to this disclosure vary depending on the properties, size, and / or the condition of the subject receiving treatment, and are further determined by the route of administration of the composition. For example, the pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more nanoparticle compositions.

[0163] In some embodiments, the nanoparticle compositions and / or pharmaceutical compositions according to the Disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or below, between approximately -150°C, and between approximately 0°C or approximately -80°C and approximately -20°C, for example, approximately -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C).

[0164] For example, the nanoparticle compositions and / or pharmaceutical compositions according to the Disclosure may be stable at a temperature of 4°C or lower (e.g., between approximately 4°C and -20°C) for about 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stable at 4°C for at least 4 weeks. In some embodiments, the pharmaceutical composition according to the Disclosure comprises the nanoparticle composition according to the Disclosure and a pharmaceutically acceptable adjuvant, the adjuvant being one or more selected from the group consisting of Tris, acetates (e.g., sodium acetate), citrates (e.g., sodium citrate), saline solution, PBS, and sucrose. In some embodiments, the pharmaceutical compositions according to the present disclosure have a pH value between about 7 and 8 (for example, between 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5, and between 8 or 7 and 7.8).

[0165] In the context of this disclosure, “stability” and “stable” mean that the nanoparticle compositions and / or pharmaceutical compositions relating to this disclosure are stable against chemical or physical changes (e.g., decomposition, particle size changes, aggregation, changes in encapsulation state, etc.) under specified manufacturing, preparation, transport, storage and / or use conditions, even when subjected to stresses such as shear force and freeze / thaw stress.

[0166] A nanoparticle composition and / or a pharmaceutical composition comprising one or more nanoparticle compositions may be administered to any patient or subject, including cells, tissues, organs or systems, or combinations thereof, that can benefit from the delivery of one or more specific drugs, therapeutic agents and / or prophylactic agents and thereby provide therapeutic effects.

[0167] While the term "composition" primarily refers to compositions suitable for administration to humans, as will be understood by those skilled in the art, such compositions are generally also suitable for administration to any other mammal.

[0168] Modifying a composition suitable for administration to humans to make it suitable for administration to various animals is well known, and a veterinary pharmacologist skilled in the art can design and / or carry out such modifications by ordinary (if any) experiments alone. The intended subjects to whom the composition will be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0169] Pharmaceutical compositions comprising one or more types of nanoparticle compositions can be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods involve combining the active ingredient with an adjuvant and / or one or more other adjuvants, and then, if necessary or as required, dividing, shaping, and / or packaging the product into multiple dose units of one or more forms as needed.

[0170] The pharmaceutical compositions relating to this disclosure may be prepared, packaged and / or sold in bulk, as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” means a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is typically equal to the dose of the active ingredient administered to a subject and / or a discrete fraction of such dose, for example, half or one-third of such dose.

[0171] Pharmaceutical compositions may be prepared in a number of forms applicable to various routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), topical (including transbuccal and sublingual), transdermal and / or transdermal administration dosage forms (e.g., creams, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), intravaginal dosage forms (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams and sprays), implantable dosage forms (e.g., solids, semi-solids, gels), suspensions, powders and other dosage forms.

[0172] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid, and mixtures thereof. In addition to inert diluents, the oral composition may also contain additional therapeutic and / or prophylactic agents, additional reagents, such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavorings, and / or fragrances. In some parenteral administration embodiments, the composition is mixed with a solubilizer, such as alcohol, oil, modified oil, ethylene glycol, polysorbate, cyclodextrin, polymer, and / or a combination thereof.

[0173] Injectable formulations, such as sterile injectable aqueous or oily suspensions, may be prepared by known techniques using appropriate dispersants, wetting agents and / or suspending agents. Sterile injectable formulations may also be sterile injectable solutions, suspensions and / or emulsions in diluents and / or solvents acceptable for non-toxic oral administration. Acceptable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution. Sterile oils may be used as solvents or suspension media. Therefore, any mild non-volatile oil, including synthetic glycerin monoesters or glycerin diesters, may be used. Fatty acids (e.g., oleic acid) may be used in the preparation of injectable formulations. Injectable formulations can be sterilized, for example, by filtration with a bacterial capture filter and / or by a sterilizer in the form of a sterile solid composition, and can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0174] Compositions for rectal or vaginal administration are typically suppositories, which can be prepared by mixing the composition with a suitable non-irritating excipient, the excipient being solid at ambient temperature but liquid at body temperature, and thus dissolving in the rectum or vaginal cavity to release the active ingredient.

[0175] This disclosure further considers the use of transdermal patches, such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the release rate can be controlled by providing a release rate control membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0176] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include all types of liquid or solid injection devices, such as conventional syringes, microneedle syringes, short-needle syringes, liquid-jet syringes, compressed gas-accelerated powder syringes, and ballistic powder delivery devices.

[0177] The pharmaceutical composition may be prepared, packaged and / or sold as a formulation suitable for administration to the lungs via the buccal cavity. Such a formulation may contain dry granules having the active ingredient. Such a composition may also conveniently take the form of a dry powder and be applied to administration using an apparatus equipped with a dry powder storage device and / or a self-propelled solvent / powder dispenser. Such a formulation may further be prepared, packaged and / or sold as an aqueous solution and / or a dilute alcohol solution and / or suspension (optionally sterilized and containing the active ingredient) and can be readily administered using any spray and / or nebulizer. Such a formulation may further contain one or more additional components, including, but not limited to, sweeteners such as sodium saccharin, volatile oils, buffers, surfactants and / or preservatives such as methyl hydroxybenzoate.

[0178] The pulmonary delivery formulations described herein can also be used for intranasal delivery of pharmaceutical compositions. Such formulations are administered by nasal aspiration, i.e., by rapid inhalation through the nasal pathway from a powder container located close to the nasal cavity. The pharmaceutical compositions may be prepared, packaged and / or sold as formulations suitable for oral administration. Such formulations may be in the form of tablets and / or tablets prepared, for example, by conventional methods.

[0179] Methods and Uses This disclosure provides a method for generating a target polypeptide in mammalian cells. The method for generating the polypeptide involves contacting cells with a nanoparticle composition containing mRNA encoding the target polypeptide. Upon contact with the nanoparticle composition, the mRNA can be absorbed and translated within the cells to generate the target polypeptide.

[0180] Generally, the step of contacting mammalian cells with a nanoparticle composition containing mRNA encoding a target polypeptide may be carried out in vivo, ex vivo, in culture, or in vitro. The amount of nanoparticle composition and / or mRNA in it that comes into contact with the cells may be determined by the type of cells or tissue being contacted, the dosage form, the physiological and chemical characteristics of the nanoparticle composition and mRNA (e.g., size, charge, and chemical composition), and other factors. Efficiency indicators may include polypeptide translation (represented by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0181] The step of bringing a nanoparticle composition containing mRNA into contact with cells may also be related to transfection, or may induce transfection. Phospholipids contained in the lipid components of the nanoparticle composition can promote transfection and / or improve transfection efficiency, for example, by interacting with and / or fusing with the cell membrane or intracellular membrane. Transfection enables the translation of mRNA within the cell.

[0182] In some embodiments, the nanoparticle compositions described herein can be applied therapeutically. For example, mRNA contained in a nanoparticle composition may encode a therapeutic polypeptide (e.g., within a translatable region) and, upon contact with and / or entry into cells (e.g., by transfection), may generate the therapeutic polypeptide. In other embodiments, mRNA contained in a nanoparticle composition may encode a polypeptide that improves or enhances the immune function of a subject.

[0183] In some embodiments, mRNA contained in the nanoparticle composition may encode a recombinant polypeptide that replaces one or more polypeptides substantially absent in the cell in contact with the nanoparticle composition. Alternatively, the recombinant polypeptide produced by translation of mRNA may antagonize the activity of endogenous proteins in, on the cell surface, or secreted from the cell. In another alternative, the recombinant polypeptide produced by translation of mRNA may indirectly or directly antagonize the activity of biological parts in, on the cell surface, or secreted from the cell. The biological parts to be antagonized may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and low-molecular-weight toxins.

[0184] Methods for delivering therapeutic agents to cells and organs This disclosure provides a method for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. The delivery of therapeutic and / or prophylactic agents to cells relates to administering a nanoparticle composition containing the therapeutic and / or prophylactic agent to a subject, wherein the administration of the composition relates to bringing cells into contact with the composition. For example, proteins, cytotoxic substances, radioactive ions, chemotherapeutic agents, or nucleic acids (e.g., RNA, e.g., mRNA or siRNA) may be delivered to cells or organs. If the therapeutic and / or prophylactic agent is mRNA, when cells come into contact with the nanoparticle composition, the translatable mRNA can be translated within the cells to produce polypeptides of interest. However, substantially untranslatable mRNA may also be delivered to cells.

[0185] In some embodiments, the nanoparticle composition may target a specific type of cell (e.g., cells of a specific organ or system). For example, a nanoparticle composition containing a therapeutic and / or prophylactic agent of interest may be specifically delivered to the mammalian heart, liver, kidney, spleen, femur, or lung, but not substantially delivered to other types of cells. Specific delivery to a specific type of cell, organ, or system or tissue means that the nanoparticle composition containing the therapeutic and / or prophylactic agent is delivered to the destination (e.g., tissue) at a higher rate than other destinations. In some embodiments, the tissue of interest is selected from the group consisting of the heart, myocardium, liver, kidney, lung, spleen, femur, muscle, and tumor tissue (e.g., by intratumor injection).

[0186] As another example of targeted or specific delivery, mRNA encoding a cell surface protein-binding ligand (e.g., an antibody or its functional fragment, scaffold protein, or peptide) or receptor may be included in the nanoparticle composition. In some embodiments, the ligand is a surface-bound antibody, allowing for modulation of cell target specificity. These methods can improve the affinity and specificity of the target interaction. The ligand may be selected, for example, by those skilled in the field of biology, based on the localization or function required by the cell. Target cells include, but are not limited to, cardiomyocytes, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, skeletal muscle cells, β-cells, pituitary cells, synovial surface cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0187] Methods of treating diseases and symptoms Nanoparticle compositions can be applied to the treatment of diseases or conditions. In particular, such compositions can be applied to the treatment of diseases or conditions characterized by a deficiency or abnormality of protein or polypeptide activity. For example, a nanoparticle composition containing mRNA encoding a defective or abnormal polypeptide may be administered or delivered to cells. Subsequent translation of the mRNA generates the polypeptide, thereby mitigating or eliminating problems caused by the polypeptide's deficiency or abnormal activity. The methods and compositions can be applied to the treatment of acute diseases or conditions such as sepsis, stroke, and myocardial infarction. Therapeutic and / or prophylactic agents contained in nanoparticle compositions can also affect gene expression by altering the transcription rate of a given species. Examples of diseases and / or conditions to which compositions can be administered that are characterized by dysfunction or abnormality of protein or polypeptide activity include, but are not limited to, rare diseases, infectious diseases (as vaccines and therapeutic agents), cancer and proliferative disorders, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal diseases, and metabolic diseases.

[0188] This disclosure provides nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents, and methods for administering pharmaceutical compositions comprising the same. The terms therapeutic and prophylactic may be used interchangeably in this text. The therapeutic composition or its imaging, diagnostic, or prophylactic composition may be administered to a subject in an amount and / or any reasonable amount that effectively prevents, treats, diagnoses, or images a disease, symptom, and / or condition, and via any route of administration. The specific amount administered to a subject may be modified depending on the subject's species, age, general condition, specific components, and method of administration. The compositions relating to this disclosure may be prepared in dose unit form for convenience of administration and uniformity of dosage. However, it should be understood that the specific amount of the composition relating to this disclosure to be used should be determined by the attending physician within the bounds of reasonable medical judgment.

[0189] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents may be administered by any route. In some embodiments, compositions comprising one or more nanoparticle compositions described herein (including prophylactic compositions, diagnostic compositions, or imaging compositions) are administered by one or more of the following routes: oral, intravenous, intramuscular, intraarterial, intramedullary, intrasacral, subcutaneous, intraventricular, percutaneous or intradermal, interdermal, rectal, vaginal, intraperitoneal, intraocular, subretinal, intravitreous, mucosa, nasal cavity, oral cavity, intestinal tract, intratumoral, sublingual, intranasal; intratracheal drip, bronchial drip and / or inhalation, oral spray and / or powder, nasal spray and / or aerosol, and / or via portal vein catheter. Preferably, local administration is used, for example, intramuscular, subcutaneous, percutaneous or intradermal, interdermal, intraperitoneal, intraocular, subretinal, intravitreous, mucosa, nasal cavity, oral cavity, intratumoral, intranasal. The appropriate route of administration is determined by several factors, including the properties of the nanoparticle composition, the therapeutic and / or prophylactic agent, and the patient's condition.

[0190] In some embodiments, the compositions relating to this disclosure may be administered at levels sufficient to deliver the following doses: approximately 0.0001 mg / kg to approximately 10 mg / kg, approximately 0.001 mg / kg to approximately 10 mg / kg, approximately 0.005 mg / kg to approximately 10 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 2 mg / kg to approximately 10 mg / kg, approximately 5mg / kg~about 10mg / kg, about 0.0001mg / kg~about 5mg / kg, about 0.001mg / kg~about 5mg / kg, about 0.005mg / kg~about 5mg / kg, about 0.01mg / kg~about 5mg / kg, about 0.05mg / kg~ Approximately 5 mg / kg approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 2 mg / kg to approximately 5 mg / kg, approximately 0.0001 mg / kg to approximately 2.5 mg / kg mg / kg, approximately 0.001 mg / kg to approximately 2.5 mg / kg, Approximately 0.005mg / kg~approximately 2.5mg / kg, approx. 0.01mg / kg~approximately 2.5mg / kg, approx. 0.05mg / kg~approximately 0.05mg / kg~approximately 2.5mg / kgapproximately 2.5mg / kg, approx. 0.1mg / kg~approximately 2.5mg / kg, approx. 1m g / kg~about 2.5mg / kg, about 2mg / kg~about 2.5mg / kg, about 0.0001mg / kgabout 0.001mg / kg~about 1mg / kg, about 0.001mg / kg~about 1mg / kg, about 0.005mg / kg~about 1mg / kg, about 0 Therapeutic and / or prophylactic agents (e.g., mRNA) in doses of 0.01 mg / kg to approximately 1 mg / kg, approximately 0.05 mg / kg to approximately 1 mg / kg, approximately 0.1 mg / kg to approximately 1 mg / kg, approximately 0.0001 mg / kg to approximately 0.25 mg / kg, approximately 0.001 mg / kg to approximately 0.25 mg / kg, approximately 0.005 mg / kg to approximately 0.25 mg / kg, approximately 0.01 mg / kg to approximately 0.25 mg / kg, approximately 0.05 mg / kg to approximately 0.25 mg / kg, or approximately 0.1 mg / kg to approximately 0.25 mg / kg.

[0191] The dosage may be administered once or multiple times daily in the same or different amounts to obtain the required level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The required dosage may be delivered, for example, three times daily, twice daily, once daily, every other day, every three days, once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the required dosage may be delivered in multiple doses (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more doses).

[0192] A nanoparticle composition containing one or more therapeutic and / or prophylactic agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Each agent is administered according to the dosage and / or administration schedule determined for that agent. The therapeutic, prophylactic, diagnostic, or imaging activators used in combination may be administered together in a single composition or individually in different compositions.

[0193] Those skilled in the art can identify or determine many equivalents of the specific examples based on the disclosures described herein simply by ordinary experimentation. The scope of this disclosure is not limited to the above description, but is set forth in the appended claims.

[0194] In the claims, unless otherwise specified or the context makes clear, articles such as "one," "one," and "the said" may refer to one or more.

[0195] The terms “contains” and “includes” are open, meaning that additional components or steps are permitted but not required. Where the term “includes” is used in the text, the terms “substantially consist of” and “consist of” are also included and disclosed. Furthermore, it should be understood that the order of steps or the order in which specific actions are performed is not important, as long as the invention maintains workability. Two or more steps or actions may be performed simultaneously.

[0196] The compounds relating to this disclosure can be prepared in various ways by employing standard synthetic methods and procedures known or hereafter understood to those skilled in the art, using commercially available starting materials, literature-known compounds, or readily prepared intermediates. According to the teachings herein, the synthesis of the compounds relating to this disclosure is obvious to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and the transformation and manipulation of functional groups can be found in the relevant scientific literature or standard textbooks in the art, e.g., Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th This edition is available from John Wiley & Sons: New York, 2001. The synthetic methods described in the examples of this text are intended to illustrate, and not limit, general procedures for preparing the compounds relating to this disclosure. Those skilled in the art should notice that the order of some steps in the reaction sequence and synthetic scheme described herein can be changed, for example, the introduction and removal of protecting groups. Multiple stereoisomers may be produced in the reaction scheme described herein. Those skilled in the art should recognize that the reaction can be optimized to preferentially produce a particular isomer, or a novel scheme can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC may be used to separate the isomers.

[0197] In some embodiments, the compound of formula (I) described herein is the following compound: [Table 1] [Table 2]

[0198] Examples Example 1, Preparation of the compound Experimental equipment [Table 3] Experimental reagents [Table 4] Synthesis scheme for compound 15: [ka] Synthesis of compound A (8-bromooctanoate heptadecane-9-yl ester) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, undecanol (20 mmol, 3.44 g) and dichloromethane (80 mL) were added, and the reaction mixture was placed in an ice bath. Then, malonate dichloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were added to the reaction mixture, and the mixture was stirred at room temperature for 6 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain compound A (7.09 g, yield 86%) as a colorless liquid.

[0199] [ka] Synthesis of compound B (10-(heptadecan-9-yloxy)-10-oxo-2-[(undecyloxy)carbonyl]decyl ester of undecyl ester of undecyl decanoate) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, tetrahydrofuran (15 mL), di(undecyl) malonate (3 mmol, 1.23 g), and NaH (2.6 mmol, 104 mg) were added in sequence, and the mixture was stirred at room temperature for 30 minutes. Then, under the protection of nitrogen gas, heptadecane-9-yl 8-bromooctanoate (2 mmol, 0.92 g) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added, and the mixture was stirred at 70 °C for 48 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 50:1) to obtain compound B (0.84 g, yield 53%) as a colorless liquid.

[0200] [ka] Synthesis of compound C(1-[4-(dimethylamino)hexahydropyridine-1-yl]propa-2-en-1-one) A 50 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, anhydrous dichloromethane (20 mL) and 4-dimethylaminopyridine (6.0 mmol, 768 mg) were added in sequence. After cooling the reaction mixture to 0°C, acrylate chloride (5 mmol, 455 mg) and triethylamine (7.5 mmol, 758 mg) were added in sequence, and the mixture was stirred at room temperature for 8 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (dichloromethane / methanol / triethylamine = 10:1:0.5) to obtain compound C (319 mg, yield 35%) as a pale yellow oily liquid.

[0201] [ka] Synthesis of compound 15 (2-{3-[4-(dimethylamino)hexahydropyridine-1-yl]-3-oxopropyl}-10-(heptadecan-9-yloxy)-10-oxo-2-[(undecyloxy)carbonyl]decyl ester of undecyl ester of undecyl ester of undecyl carbonyl]decanoate) A 25 mL dry Schlenk flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, toluene (1.5 mL), 10-(heptadecan-9-yloxy)-10-oxo-2-[(undecyloxy)carbonyl]decyl undecyl ester ester (0.1 mmol, 79.2 mg), and NaOEt (0.03 mmol, 2 mg) were added. The mixture was stirred at room temperature for 10 minutes, then 1-[4-(dimethylamino)hexahydropyridine-1-yl]propa-2-en-1-one (0.11 mmol, 20.1 mg) was added, and the mixture was stirred at 70°C for 16 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound 15 (70.2 mg, yield 72%) as a colorless oily liquid. 1 ¹H NMR (600MHz, chloroform-d) δ 4.85 (p, J=6.3Hz, 1H), 4.66 (d, J=13.6Hz, 1H), 4.16-4.01 (m, 4H), 3.92 (d, J=13.8Hz, 1H), 3.05-2.96 (m, 1H), 2.62-2.49 (m, 2H), 2.38 (s, 6H), 2.32-2. 23(m,4H),2.20-2.12(m,2H),1.96(d,J=12.7Hz,1H),1.92-1.82(m,3H),1.64-1.55(m, 6H),1.50(q,J=6.1Hz,4H),1.34-1.20(m,66H),0.92-0.81(m,12H).HRMS(ESI)m / z(M+H) + Calculated value C60H115N2O7: 975.8704, measured value: 975.8708.

[0202] [ka] Synthesis of compound D (3-(heptadecane-9-yloxy)-3-oxopropionate heptadecane-9-yl ester) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, 9-heptadecanol (20 mmol, 5.12 g) and dichloromethane (80 mL) were added, and the reaction mixture was placed in an ice bath. Then, malonate dichloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were added to the reaction mixture, and the mixture was stirred at room temperature for 6 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain compound D (5.17 g, yield 89%) as a colorless liquid.

[0203] [ka] Synthesis of compound E (2-[(heptadecane-9-yloxy)carbonyl]-6-{[(10Z,12Z)-octadeca-9,12-alkadienyl]oxy}-6-oxohexanoate heptadecane-9-yl ester) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, tetrahydrofuran (15 mL), di(9-heptadecyl) malonate (3 mmol, 1.74 g), and NaH (2.6 mmol, 104 mg) were added in sequence, and the mixture was stirred at room temperature for 30 minutes. Then, under the protection of nitrogen gas, 4-bromobutyrate-(10Z,12Z)-octadeca-9,12-dien-1-yl (2 mmol, 0.83 g) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added, and the mixture was stirred at 70°C for 48 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 50:1) to obtain compound E (1.06 g, yield 58%) as a colorless liquid.

[0204] [ka] Synthesis of Compound 15A (heptadecan-9-yl 2-{3-[4-(dimethylamino)hexahydropyridin-1-yl]-3-oxopropyl}-2-[(heptadecan-9-yloxy)carbonyl]-6-{[(10Z,12Z)-octadeca-9,12-dienyl]oxy}-6-oxohexanoate) A 25 mL dry Schlenk flask equipped with a stirring bar was taken, and toluene (1.5 mL), compound E (0.1 mmol, 91.5 mg) and NaOEt (0.03 mmol, 2 mg) were added under the protection of nitrogen gas. After stirring at room temperature for 10 minutes, compound C (0.11 mmol, 20.1 mg) was added, and the mixture was stirred at 70°C for 16 hours. After completion of the reaction, the organic solvent was removed using a rotary evaporator to obtain a crude product, which was then purified by column chromatography (dichloromethane / methanol=20:1) to obtain compound 15A (68.9 mg, yield 63%) as a colorless oily liquid. 1 H NMR (600 MHz, chloroform-d) δ 5.42-5.24 (m, 4H), 4.86 (p, J=6.3 Hz, 2H), 4.65 (d, J=13.5 Hz, 1H), 4.02 (t, J=6.9 Hz, 2H), 3.92 (d, J=13.7 Hz, 1H), 2.97 (t, J=12.8 Hz, 1H), 2.76 (t, J=7.0 Hz, 2H), 2.57-2.51 (m, 1H), 2.34 (s, 6H), 2.32-2.26 (m, 4H), 2.21 (t, J=8.3 Hz, 2H), 2.04 (q, J=7.2 Hz, 4H), 1.94-1.84 (m, 4H), 1.62-1.56 (m, 2H), 1.55-1.46 (m, 8H), 1.35-1.20 (m, 69H), 0.87 (t, J=7.0 Hz, 15H). HRMS (ESI) m / z (M+H) + calcd. for C 69 H 129 N₂O₇: 1097.9800, found: 1097.9334.

[0205] [Chemical Formula] Synthesis of Compound F (heptadecan-9-yl 3-(heptadecan-9-yloxy)-3-oxopropionate) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, (10Z,12Z)-octadeca-9,12-dien-1-ol (20 mmol, 5.32 g) and dichloromethane (80 mL) were added, and the reaction mixture was placed in an ice bath. Then, malonic acid dichloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were added to the reaction mixture, and the mixture was stirred at room temperature for 6 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain compound F (4.96 g, yield 80%) as a colorless liquid.

[0206] [ka] Synthesis of compound G(6-{[(10Z,12Z)-octadeca-9,12-alkadienyl]oxy}-2-({[(10Z,12Z)-octadeca-9,12-alkadienyl]oxy}carbonyl)-6-oxohexanoic acid-(10Z,12Z)-octadeca-9,12-dien-1-yl ester) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, tetrahydrofuran (15 mL), compound F (3 mmol, 1.80 g), and NaH (2.6 mmol, 104 mg) were added in sequence, and the mixture was stirred at room temperature for 30 minutes. Then, under the protection of nitrogen gas, 4-bromobutyrate-(10Z,12Z)-octadeca-9,12-dien-1-yl ester (2 mmol, 0.83 g) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added, and the mixture was stirred at 70°C for 48 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 50:1) to obtain compound G (0.88 g, yield 47%) as a colorless liquid.

[0207] [ka] Synthesis of compound 15B (2-{3-[4-(dimethylamino)hexahydropyridine-1-yl]-3-oxopropyl}-6-{[(10Z,12Z)-octadeca-9,12-alkadienyl]oxy}-2-({[(10Z,12Z)-octadeca-9,12-alkadienyl]oxy}carbonyl)-6-oxohexanoic acid-(10Z,12Z)-octadeca-9,12-dien-1-yl ester) A 25 mL dry Schlenk flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, toluene (1.5 mL), compound G (0.1 mmol, 93.5 mg), and NaOEt (0.03 mmol, 2 mg) were added. The mixture was stirred at room temperature for 10 minutes, then compound C (0.11 mmol, 20.1 mg) was added, and the mixture was stirred at 70 °C for 16 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound 15B (60.3 mg, yield 54%) as a colorless oily liquid. 1 H NMR(600MHz,chloroform-d)δ5.43-5.21(m,12H),4.63(d,J=13.5Hz,1H),4.14-4.05(m,4H),4.03(t,J=6.9Hz,2 H),3.93(d,J=13.5Hz,1H),2.99(td,J=13.0,2.6Hz,1H),2.76(t,J=7.0Hz,6H),2.55(td,J=12.9,2.8Hz,1H) ,2.51-2.42(m,1H),2.33(s,6H),2.31-2.26(m,4H),2.23-2.17(m,2H),2.04(q,J=7.2Hz,12H),1.93-1.83( m,4H),1.65-1.57(m,6H),1.56-1.49(m,2H),1.37-1.23(m,50H),0.88(t,J=6.9Hz,9H).HRMS(ESI)m / z(M+H) + Calculated value C 71 H 125 N2O7: 1117.9487, Measured value: 1117.9014.

[0208] [ka] Synthesis of compound H(2-[(heptadecane-9-yloxy)carbonyl]heptadecane-9-yl ester butyrate) A 100 mL dry two-necked flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, tetrahydrofuran (15 mL), compound D (3 mmol, 1.74 g), and NaH (2.6 mmol, 104 mg) were added in sequence, and the mixture was stirred at room temperature for 30 minutes. Then, under the protection of nitrogen gas, ethyl iodide (2 mmol, 312 mg) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added, and the mixture was stirred at 70 °C for 48 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 30:1) to obtain compound H (0.83 g, yield 68%) as a colorless liquid.

[0209] [ka] Synthesis of compound 15C (5-[4-(dimethylamino)hexahydropyridine-1-yl]-2-ethyl-2-[(heptadecane-9-yloxy)carbonyl]-5-oxovalerate heptadecane-9-yl ester) A 25 mL dry Schlenk flask equipped with a stirring bar was taken, and under the protection of nitrogen gas, toluene (1.5 mL), compound H (0.1 mmol, 60.9 mg), and NaOEt (0.03 mmol, 2 mg) were added. The mixture was stirred at room temperature for 10 minutes, then compound C (0.11 mmol, 20.1 mg) was added, and the mixture was stirred at 70°C for 16 hours. After the reaction was complete, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound 15C (50.3 mg, yield 71%) as a colorless oily liquid. 1¹H NMR (600MHz, chloroform-d) δ 4.85 (p, J=6.2Hz, 2H), 4.65-4.53 (m, 1H), 3.93-3.79 (m, 1H), 2.94 (td, J=13.0, 2.6Hz, 1H), 2.52 (td, J=12.9, 2.7Hz, 1H), 2.38 (tt, J=11.2, 3.7Hz, 1H), 2.27(s,6H),2.26-2.23(m,1H),2.20-2.13(m,4H),1.92(q,J=7.5Hz,2H),1.87-1.78(m, 2H),1.54-1.43(m,8H),1.33-1.14(m,52H),0.85(t,J=7.0Hz,12H).HRMS(ESI)m / z(M+H) + Calculated value C 49 H 95 N2O5: 791.7241, Measured value: 791.6912.

[0210] Example 2: Measurement of local expression activity of mRNA-LNPs Experimental materials [Table 5]

[0211] Experimental method 1. Preparation of LNPs The four components of the organic phase lipid were each dissolved in anhydrous ethanol to a concentration of 10 mg / mL, and prepared according to the mixing ratios shown in Table 1 below; 1 mg / mL of Luc mRNA was diluted to 70 μg / mL in 25 mM sodium acetate buffer solution to form the aqueous phase; the two phases of the microfluidic device (stainless steel tip) were washed with anhydrous ethanol and pure water, respectively, and then the aqueous phase was further washed twice with 25 mM sodium acetate buffer solution. At the start of preparation, air was removed from the two phases of the tubing with 300 μL each of the pre-prepared organic and aqueous phase components. Then, the preparation solution was aspirated so that the volume ratio of the organic phase to the aqueous phase was 1:3, and the flow rates of the organic and aqueous phases were set to 4 mL / min and 12 mL / min, respectively, to prepare the LNPs. The first 0.5 mL of effluent was discarded, and only the remaining effluent was collected until completion (the preparation methods for SM-102 LNP and 15 LNP were the same).

[0212] Table 1 [Table 6] Note: The unit is mole percent, and the unit in parentheses is mole parts.

[0213] 1.1 LNP ultrafiltration The prepared LNP stock solution was taken, added to 5 times its volume of PBS, and ultrafiltered at 2000-3000 rpm for 10 minutes using an ultrafiltration tube until the volume was approximately 1 mL. Then, 10 times its volume of PBS was added, the filter of the ultrafiltration tube was blew to wash it, and the solution was centrifuged until it was approximately 1 mL. After repeatedly washing the filter, the 1 mL solution was taken and transferred to an enzyme-free EP tube, and stored in a refrigerator at 4°C for use.

[0214] 2. Measurement of content and encapsulation rate 2.1 Creation of a Calibration Curve mRNA stock solution was taken, diluted to 2 μg / mL with 1×TE, and standard solutions of 0, 2, 10, 25, 50, and 100 μL were placed in a 96-well black microplate. Then, following the instructions of the RNA quantification kit (1×TE, 100 μL; 1×Ribo, 100 μL), measurements were taken using a microplate reader under conditions of excitation wavelength 480 nm and emission wavelength 520 nm.

[0215] 2.2 Preparation of LNP measurement samples Ultrafiltered LNPs were taken and first diluted with 1×TE until the theoretical concentration was approximately 1 μg / mL; with permeabilization: the diluted LNPs were permeabilized with 1% TritonX-100 at room temperature for 10 minutes; without permeabilization: an equal volume of DEPC water was added to the diluted LNPs and left at room temperature for 10 minutes. Then, 100 μL of each sample was placed in a 96-well black microplate, and 100 μL of 1×Ribo was added to each, creating three overlapping wells for each sample. Measurements were then taken using a microplate reader under conditions of excitation wavelength 480 nm and emission wavelength 520 nm.

[0216] 3. Measurement of particle size An appropriate amount of ultrafiltered LNP was taken, diluted 10-fold with pure water, and then added to a particle size cell. The particle size was measured using a Malven particle size analyzer.

[0217] 4. Local expression Intramuscular injection: Male C57 mice weighing approximately 20g / mice were selected. Before injection, hair was first removed from the calf muscle area of ​​the lower leg. Then, using a microneedle syringe, 50 μL of LNP (10 μg of mRNA) was administered to the calf muscle per mouse. Four hours after administration, 200 μL (15 mg / mL) of luciferase substrate was injected intraperitoneally, and small animal in vivo imaging was performed under isoflurane anesthesia.

[0218] Intramyocardial injection: Male SD rats were subjected to microincision and thoracotomy under isoflurane anesthesia, and 40 μL of 15 LNP (12 μg of mRNA) was injected intramyocardially per rat. Six hours after administration, 2 mL (15 mg / mL) of luciferase substrate was administered by intraperitoneal injection, and in vivo imaging was performed under isoflurane anesthesia.

[0219] Splenic injection: Male C57 mice (body weight approximately 20 g) were selected. After anesthesia with isoflurane, the mice were placed in the supine position, the skin near the right abdomen and chest was incised to expose the spleen. Then, using a microneedle syringe, 20 μL of 15 LNP-2 (4 μg of mRNA) per mouse was administered into the spleen. Six hours after administration, 200 μL (15 mg / mL) of luciferin substrate was intraperitoneally injected, and in vivo small animal imaging was performed under isoflurane anesthesia; for male SD rats (body weight approximately 150 g), 30 μL (6 μg of mRNA) per rat was injected, and the same treatment was performed.

[0220] Renal injection: Male C57 mice (body weight approximately 20 g) were selected. After anesthesia with isoflurane, the mice were placed in the supine position, the skin near the right abdomen and chest was incised to expose the kidney. Then, using a microneedle syringe, 20 μL of 15 LNP-2 (4 μg of mRNA) per mouse was administered into the kidney. Six hours after administration, 200 μL (15 mg / mL) of luciferin substrate was intraperitoneally injected, and in vivo small animal imaging was performed under isoflurane anesthesia; for male SD rats (body weight approximately 150 g), 30 μL (6 μg of mRNA) per rat was injected, and the same treatment was performed.

[0221] Experimental Results 1. Particle size, PDI, encapsulation efficiency %

Table 7

[0222] The particle size distribution of SM-102 is shown in Figure 9.

[0223] 2. In vivo expression - Intramuscular injection The results are shown in Figure 11-24. As can be seen, when 15 LNP-2 was injected intramuscularly, small animal in vivo imaging showed that it was locally expressed only at the injection site and not in other organs. When 15 LNP-2 was injected intramyocardially, small animal in vivo imaging showed that it was expressed only in the cardiac organ at the injection site and not in other organs. When 15 LNP-2 was injected into the spleen, small animal in vivo imaging showed that it was expressed only in the splenic organ at the injection site and not in other organs. When 15 LNP-2 was injected into the kidney, small animal in vivo imaging showed that it was expressed only in the renal organ at the injection site and not in other organs.

Claims

1. A compound of formula (I) or its salt or isomer. 【Chemistry 1】 (In the formula, R １ is H or C1-C8 alkyl group, R ２ These are C1-10 alkyl groups and C3-C18 alkadienyl groups. R ３ is H or R １ - R8 alkyl group, R ４ These are C1-10 alkyl groups and C3-C18 alkadienyl groups. R ５ is a C1-C14 alkyl group, a C2-C14 alkenyl group, or 【Chemistry 2】 And, M is either O or N, t is an integer between 3 and 7. R ６ is H or C1-10 alkyl group, R ７ These are C1-10 alkyl groups and C3-C18 alkadienyl groups. Preferably, R １ is R ３ is identical to, R ２ is R ４ is identical to)

2. R １ is H and R ２ is a C6-10 alkyl group or a C14-C18 alkadienyl group; or, R １ is a C4-C8 alkyl group and R ２ is a C4-C8 alkyl group; and / or R ３ is H and R ４ is a C6-10 alkyl group or a C14-C18 alkadienyl group; or, R ３ is a C4-C8 alkyl group and R ４ It is a C4-C8 alkyl group. The compound or a salt or isomer thereof as described in claim 1.

3. R ５ is a C1-C4 alkyl group, a C10-C14 alkyl group, a C10-C14 alkenyl group, or 【Transformation 3】 And, During the ceremony, M is either O or N, t is 3 or 7, R ６ is a C4-C8 alkyl group and R ７ is a C4-C8 alkyl group, or R ６ is H and R ７ The compound according to claim 1 or 2, or a salt or isomer thereof, wherein is a C14-C18 alkadienyl group.

4. A lipid component containing the compound or a salt or isomer thereof as described in any one of claims 1 to 3, Preferably, the lipid component further contains phospholipids, PEG lipids, and structural lipids in the nanoparticle composition.

5. The nanoparticle composition according to claim 4, further comprising a therapeutic agent and / or a preventive agent.

6. A pharmaceutical composition comprising the nanoparticle composition according to claim 4 or 5 and a pharmaceutically acceptable adjuvant.

7. A non-therapeutic method for generating polypeptides in cells, comprising the step of contacting cells with a nanoparticle composition, wherein the nanoparticle composition comprises (1) a lipid component containing phospholipids, PEG lipids, structural lipids, and compounds according to any one of claims 1 to 3 or their salts or isomers, and (2) mRNA encoding the polypeptide, thereby enabling the mRNA to be translated in cells to generate polypeptides, and preferably the cells are mammalian cells.

8. A compound according to any one of claims 1 to 3, or a salt or isomer thereof, The nanoparticle composition according to claim 4 or 5, A method for manufacturing.

9. The use of a compound or a salt or isomer thereof according to any one of claims 1 to 3, or a nanoparticle composition according to claim 4 or 5, in the preparation of a drug for treating or preventing a disease or symptom, Preferably, the drug is a locally acting drug. Preferably, the nanoparticle composition or the nanoparticle composition formed from the compound of formula (I) is for local delivery of therapeutic and / or prophylactic agents. Preferably, the disease or condition is one that would benefit from topical delivery of a therapeutic and / or prophylactic agent.

10. The aforementioned location is an organ, preferably the heart, liver, spleen, kidney, or lung, or The use according to claim 9, wherein the local area is tissue, preferably muscle or bone.

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