Drug delivery material and application thereof

Abstract

The present invention relates to a drug delivery material and an application thereof. Specifically provided is a nanoparticle composition comprising a lipid component, the lipid component comprising a compound represented by formula (I). The nanoparticle composition of the present invention is delivered only at the site of administration.

Description

Drug delivery materials and their applications Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to drug delivery materials and applications thereof. Background Art

[0002] Lipid materials and their derivatives have been extensively studied for RNA delivery in vivo due to their excellent biocompatibility and high RNA encapsulation efficiency. RNA is safe and effective, and holds great promise for the development of new therapies for genetic diseases, expression of functional proteins and antibodies, vaccines, or gene editing. Currently, commercial lipid delivery vehicles (BioNTech's BNT162b2 and Moderna's mRNA-1273) express target proteins in the liver via various routes of administration (intravenous, subcutaneous, and intramuscular injection), posing potential toxic side effects to extrahepatic lesions and hindering clinical translation and application.

[0003] Therefore, the development of LNP delivery vectors with local expression specificity can provide a new development platform for the treatment of currently intractable or untreatable diseases, which will greatly expand the clinical application of RNA therapy.

[0004] Summary of the Invention

[0005] The first aspect of the present invention provides a compound of formula (I) or a salt or isomer thereof,

[0006] in,

[0007] R1 is H or C1-C8 alkyl,

[0008] R2 is C1-10 alkyl, C3-C18 diene

[0009] R3 is H or R1-R8 alkyl,

[0010] R4 is a C1-10 alkyl group, a C3-C18 diene group,

[0011] R5 is C1-C14 alkyl, C2-C14 alkenyl, or

[0012] M is O or N,

[0013] t is an integer from 3 to 7,

[0014] R6 is H or C1-10 alkyl,

[0015] R7 is a C1-10 alkyl group or a C3-C18 diene group.

[0016] In one or more embodiments, R1 is the same as R3, and R2 is the same as R4.

[0017] In one or more embodiments, R1 is H and R2 is C6-10 alkyl or C14-C18 diene.

[0018] In one or more embodiments, R1 is C4-C8 alkyl and R2 is C4-C8 alkyl.

[0019] In one or more embodiments, R3 is H and R4 is C6-10 alkyl or C14-C18 diene.

[0020] In one or more embodiments, R3 is C4-C8 alkyl and R4 is C4-C8 alkyl.

[0021] In one or more embodiments, R5 is C1-C4 alkyl, C10-C14 alkyl, C10-C14 alkenyl, or

[0022] M is O or N,

[0023] t is 3 or 7,

[0024] R6 is C4-C8 alkyl and R7 is C4-C8 alkyl, or R6 is H and R7 is C14-C18 diene group.

[0025] In one or more embodiments, R1 is H, R2 is C6-10 alkyl, R1 is the same as R3, R2 is the same as R4, and R5 is C10-C14 alkyl, C10-C14 alkenyl, or M is O or N, t is 7, R6 is C4-C8 alkyl and R7 is C4-C8 alkyl.

[0026] In one or more embodiments, R1 is H, R2 is C14-C18 diene, R1 is the same as R3, R2 is the same as R4, and R5 is M is O, t is 3, R6 is H, and R7 is C14-C18 diene group. Preferably, R7 is the same as R2.

[0027] In one or more embodiments, R1 is C4-C8 alkyl, R2 is C4-C8 alkyl, R1 is the same as R3, R2 is the same as R4, R5 is C1-C4 alkyl, M is O, t is 3, R6 is H, and R7 is C14-C18 diene group.

[0028] In one or more embodiments, each of the alkyl groups is a normal alkyl group.

[0029] In one or more embodiments, when R2, R4, and R7 are C14-C18 diene groups, the alkenyl groups of the C14-C18 diene groups are located on any two carbons from carbon 7 to carbon 14. In one or more embodiments, the alkenyl groups are located on carbons P and P+3, with P being 7 to 11. In one or more embodiments, the C14-C18 diene groups are 9,12-diene groups having 14 to 18 carbons.

[0030] In one or more embodiments, the compound of formula (I) is as described by any one of formulas 15, 15A-F.

[0031] The present invention also provides nanoparticle compositions comprising a lipid component comprising a compound as described in any embodiment herein (eg, a compound of Formula (I)).

[0032] In one or more embodiments, the lipid component further comprises phospholipids (eg, polyunsaturated lipids), PEG lipids, and structured lipids.

[0033] In one or more embodiments, the nanoparticle composition further comprises a therapeutic and / or prophylactic agent.

[0034] The present invention also provides a pharmaceutical composition comprising the nanoparticle composition according to any embodiment of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition is a locally effective pharmaceutical composition.

[0035] The present invention also provides a method for delivering a therapeutic and / or prophylactic agent to a cell, the method comprising the step of administering (e.g., injecting) to a subject a nanoparticle composition comprising: (1) a lipid component comprising a phospholipid (e.g., a polyunsaturated lipid), a PEG lipid, a structured lipid, and a compound of formula (I), and (2) a therapeutic and / or prophylactic agent, wherein administering comprises contacting a cell with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the cell.

[0036] In one or more embodiments, the therapeutic and / or prophylactic agent is a polynucleotide.

[0037] In one or more embodiments, the therapeutic and / or prophylactic agent is mRNA.

[0038] In one or more embodiments, the cell is a mammalian cell.

[0039] In one or more embodiments, the delivery is local (rather than systemic) delivery.

[0040] In one or more embodiments, the cells are cells near the site of administration.

[0041] In one or more embodiments, the cells are cells of the same organ or tissue as the administration site, such as the heart, liver, spleen, lung, muscle, or femur.

[0042] In one or more embodiments, the subject is a mammal, such as a human.

[0043] The present invention also provides a method for producing a polypeptide in a cell, the method comprising the step of contacting the cell with a nanoparticle composition, the composition comprising: (1) a lipid component comprising a phospholipid (e.g., a polyunsaturated lipid), a PEG lipid, a structured lipid, and a compound of formula (I) or a salt or isomer thereof as described in any embodiment herein, and (2) mRNA encoding the polypeptide, whereby the mRNA can be translated in the cell to produce the polypeptide.

[0044] In one or more embodiments, the cell is a mammalian cell.

[0045] The present invention also provides a method for treating a disease or condition in a mammal (e.g., a human) by locally (rather than systemically) administering a therapeutic agent and / or prophylactic agent, or for locally effecting a therapeutic agent and / or prophylactic agent, the method comprising the step of locally administering (e.g., injecting) a therapeutically effective amount of a nanoparticle composition to the mammal, the nanoparticle composition comprising: (1) a lipid component comprising a phospholipid (e.g., a polyunsaturated lipid), a PEG lipid, a structured lipid, and a compound of formula (I) or a salt or isomer thereof as described in any embodiment herein, and (2) a therapeutic agent and / or prophylactic agent (e.g., mRNA or siRNA).

[0046] In one or more embodiments, the disease or condition is a disease or condition in which a protein or polypeptide is malfunctioning or abnormal.

[0047] In one or more embodiments, the disease or condition is selected from rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

[0048] The present invention also provides a method for locally (rather than systemically) delivering a therapeutic and / or prophylactic agent to a mammal, the method comprising the step of administering to a subject (e.g., a mammal) a nanoparticle composition comprising: (1) a lipid component comprising a phospholipid, a PEG lipid, a structured lipid, and a compound of formula (I) or a salt or isomer thereof as described in any embodiment herein and (2) a therapeutic and / or prophylactic agent (e.g., mRNA or siRNA), wherein the administration comprises contacting mammalian cells with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the local area of ​​the mammal.

[0049] In one or more embodiments, the local area is a target organ or tissue, such as the heart, liver, spleen, lung, muscle, or femur.

[0050] The present invention also provides methods for preparing the compounds of formula (I) described herein or methods for preparing the nanoparticle compositions described herein.

[0051] Use of a compound of formula (I) or a salt or isomer thereof as described in any embodiment herein, or a nanoparticle composition as described in any embodiment herein in the preparation of a locally acting medicament for treating or preventing a disease or condition.

[0052] In one or more embodiments, the disease or condition is one that would benefit from local (rather than systemic) delivery of a therapeutic and / or prophylactic agent.

[0053] In one or more embodiments, the drug is one that delivers a therapeutic and / or prophylactic agent locally rather than systemically.

[0054] In one or more embodiments, the nanoparticle compositions or nanoparticle compositions formed from compounds of Formula (I) are used to deliver therapeutic and / or prophylactic agents locally (rather than systemically).

[0055] In one or more embodiments, the local area is a target organ or tissue, such as the heart, liver, spleen, lung, muscle, or femur. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the H NMR spectrum of compound 15.

[0057] Figure 2 shows the mass spectrum of compound 15.

[0058] Figure 3 shows the H NMR spectrum of compound 15A.

[0059] Figure 4 shows the mass spectrum of compound 15A.

[0060] FIG5 shows the H NMR spectrum of compound 15B.

[0061] Figure 6 shows the mass spectrum of compound 15B.

[0062] FIG7 shows the H NMR spectrum of compound 15C.

[0063] FIG8 shows the mass spectrum of compound 15C.

[0064] FIG9 shows the LNP particle size distribution. The particle size of SM-102LNP-1 is 92.38±1.639nm, PDI0.035±0.022; the particle size of SM-102LNP-2 is 68.53±1.003nm, PDI 0.053±0.027; the particle size of 15LNP-1 is 87.34±2.507nm, PDI 0.054±0.017; the particle size of 15LNP-2 is 64.42±0.7681nm, PDI 0.107±0.037; the particle size of 15A LNP is 73.84±0.94nm, PDI 0.036±0.028; the particle size of 15B LNP is 74.50±2.22nm, PDI0.23±0.02; the particle size of 15C LNP is 64.57±1.26nm, PDI 0.11±0.04.

[0065] Figure 10 shows the fluorescence semi-quantitative results of in vivo characterization of LNPs injected into the gastrocnemius muscle of C57 mice. SM-102LNP-2 exhibited high fluorescence intensity at the muscle injection site and in the liver, while 15LNP-2 exhibited high fluorescence intensity only at the muscle injection site and not in the liver, demonstrating the superior localized expression of 15LNP-2.

[0066] Figure 11 shows the expression results of 15LNP-2 after intramuscular injection into C57 mice. 15LNP-2 was significantly expressed only at the site of intramuscular injection, but not in the liver, indicating that 15LNP-2 has good local expression properties.

[0067] Figure 12 shows the expression activity and local expression area of ​​15LNP-2 after intramuscular injection into C57 mice over time. The expression area decreased over time after intramuscular injection of 15LNP-2, and the expression site was limited to the injection site during the experimental period.

[0068] Figure 13 shows the expression activity and local expression area of ​​15LNP-2 after intramuscular injection into SD rats over time. The expression area decreased over time after intramuscular injection of 15LNP-2, and the expression site was limited to the injection site during the experimental period.

[0069] Figure 14 shows the results of local expression of SM-102LNP-2. SM-102LNP-2 was expressed both at the site of intramuscular injection and in the liver, indicating that SM-102LNP-2 did not have the ability to express locally.

[0070] Figure 15 shows the local expression results of 15A LNP, 15B LNP, and 15C LNP after injection. 15A LNP showed expression both at the intramuscular injection site and in the liver, indicating that 15A LNP lacked local expression. 15B LNP showed weak expression at the intramuscular injection site. 15C LNP showed expression only at the intramuscular injection site, indicating that 15C LNP exhibited superior local expression.

[0071] Figure 16 shows the fluorescence semi-quantitative results of in vivo characterization of LNP myocardial injection in SD rats. SM-102LNP-1, SM-102LNP-2, and 15LNP-1 exhibited high fluorescence intensity values ​​at the cardiac injection site and liver. 15LNP-2 exhibited high fluorescence intensity only at the cardiac injection site and low fluorescence intensity in the liver, demonstrating the superior localized expression of 15LNP-2.

[0072] Figure 17 shows the expression results of SM-102LNP-1 after myocardial injection. SM-102LNP-1 was expressed both at the cardiac injection site and the liver, indicating that SM-102LNP-1 does not have local expression properties.

[0073] Figure 18 shows the expression results of SM-102LNP-2 after myocardial injection. SM-102LNP-2 was expressed both at the cardiac injection site and the liver, indicating that SM-102LNP-2 does not have local expression properties.

[0074] Figure 19 shows the expression results of 15LNP-1 after myocardial injection. 15LNP-1 was expressed both in the heart and in the liver, indicating that 15LNP-1 does not have local expression properties.

[0075] Figure 20 shows the expression results of 15LNP-2 after myocardial injection. 15LNP-2 was significantly expressed only in the heart, but not in the liver, indicating that 15LNP-2 has good local expression properties.

[0076] Figure 21 shows the expression results of 15LNP-2 in the spleen of C57 mice. 15LNP-2 was significantly expressed only at the injection site of the spleen, with no expression observed in other sites, indicating that compound 15LNP-2 has good local expression properties.

[0077] Figure 22 shows the expression results of 15LNP-2 injected into the kidneys of C57 mice. 15LNP-2 was significantly expressed only at the injection site in the kidneys, with no expression observed in other sites, indicating that 15LNP-2 has good local expression properties.

[0078] Figure 23 shows the expression results of 15LNP-2 in the spleen of SD rats. 15LNP-2 was significantly expressed only at the injection site of the spleen, with no expression observed in other sites, indicating that 15LNP-2 has good local expression properties.

[0079] Figure 24 shows the expression results of 15LNP-2 injected into the kidneys of SD rats. 15LNP-2 was significantly expressed only at the injection site in the kidneys, with no expression observed in other sites, indicating that 15LNP-2 has good local expression properties. DETAILED DESCRIPTION

[0080] The inventors synthesized a novel ionizable cationic lipid and used it to prepare LNPs. These LNPs, loaded with mRNA, were injected into the leg muscles, myocardium, spleen, and kidneys of mice. In vivo fluorescence analysis of the small animals demonstrated that this delivery system resulted in mRNA protein expression exclusively at the injection site, with no expression observed in other major organs (heart, liver, spleen, lungs, and kidneys).

[0081] The present disclosure relates to novel lipids and lipid nanoparticle compositions comprising the novel lipids. The present disclosure also provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells, particularly methods for delivering therapeutic and / or prophylactic agents to a local area of ​​a mammal, producing a polypeptide of interest in local mammalian cells, and methods for treating a disease or condition in a mammal.

[0082] The method of delivering a therapeutic and / or prophylactic agent to a mammalian cell or local area involves administering to a subject a nanoparticle composition comprising a therapeutic and / or prophylactic agent, wherein administration involves contacting the cell or local area with the composition, whereby the therapeutic and / or prophylactic agent is delivered to the cell or local area.

[0083] As used herein, "local" refers to cells, tissues, or organs within a certain range of the site of application. In the prior art, special dosage forms are often required to retain the drug at the site of application, such as creams, ointments, sprays, and powder aerosols, and are mostly used on body surfaces or mucosal surfaces. The inventors have discovered that drugs prepared using the compounds of Formula (I) described herein (e.g., the nanoparticle compositions or pharmaceutical compositions described herein) can achieve local administration and local efficacy within organs, tissues, or cells. In some embodiments, the nanoparticle compositions of the present invention are suitable for preparing locally applied, locally acting drugs (LALAPs). As used herein, "locally acting drugs," "locally acting pharmaceutical compositions," "locally administered locally acting drugs," or "locally administered locally acting" refer to drugs that are applied locally and act at the site of application. If such drugs exhibit systemic effects (e.g., liver targeting), these effects are considered unintended drug effects. Local can 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 herein, local refers to only cells, tissues, or organs at the site of application. In some embodiments, when the drug is administered locally to an organ (e.g., heart, liver, spleen, kidney, or lung), the drug is delivered only to the organ at the administration site; when the drug is administered locally to a tissue (e.g., a muscle), the drug is delivered only to the tissue (e.g., the muscle) at the administration site; when the drug is administered locally to cells, the drug is delivered only to cells within the organ or tissue (e.g., the muscle) at the administration site. In some embodiments, when the drug is administered to the gastrocnemius muscle, the drug is delivered only to the gastrocnemius muscle.

[0084] In some embodiments, local can refer to that the LNP composition, therapeutic agent and / or prophylactic agent (mRNA) is primarily maintained in an organ (e.g., heart, liver, spleen, kidney, or lung) within 4-8 hours (e.g., 6 hours) after administration of the LNP composition described herein. In some embodiments, local can refer to that the LNP composition, therapeutic agent and / or prophylactic agent (mRNA) is primarily maintained in a tissue (e.g., muscle or bone) within 4-8 hours (e.g., 4 hours) after administration of the LNP composition described herein; in particular, delivery to the liver is not within 4-8 hours (e.g., 4 hours) after administration.

[0085] Lipid and lipid nanoparticle compositions

[0086] The lipids described herein can be advantageously used in lipid nanoparticle compositions to deliver therapeutic and / or prophylactic agents topically to a mammal. The lipids described herein have little or no immunogenicity.

[0087] In some aspects, the compounds described herein have formula (I) or a salt or isomer thereof. The lipid according to formula (I) may have a positive charge or a partial positive charge at physiological pH. Such lipids may be referred to as cationic lipids.

[0088] As used herein, the term "alkyl" or "alkyl group" refers to a straight or branched chain 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), which is optionally substituted. The notation "C1-14 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise indicated, alkyl groups as described herein refer to both unsubstituted and substituted alkyl groups.

[0089] As used herein, the term "alkenyl" or "alkenyl" refers to a straight or branched chain 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, which is optionally substituted. The symbol "C2-14 alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon containing 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group can include 1, 2, 3, 4 or more carbon-carbon double bonds. Unless otherwise indicated, alkenyl groups as described herein refer to unsubstituted and substituted alkenyl groups.

[0090] As used herein, the term "alkynyl" or "alkynyl" refers to a straight or branched chain hydrocarbon comprising 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, which is optionally substituted. The notation "C2-14 alkynyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may include 1, 2, 3, 5 or more carbon-carbon triple bonds. Unless otherwise indicated, alkynyl groups as described herein refer to both unsubstituted and substituted alkynyl groups.

[0091] As used herein, the term "carbocycle" or "carbocyclic group" refers to an optionally substituted monocyclic or polycyclic system comprising one or more carbon atom rings. The ring can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more rings. The symbol "C3-6 carbocycle" refers to a carbocycle comprising a monocyclic ring with 3-6 carbon atoms. The carbocycle can include one or more carbon-carbon double bonds or triple bonds and can be non-aromatic or aromatic (e.g., cycloalkyl or aryl). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, and naphthyl.

[0092] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring and may or may not include any double or triple bonds. Unless otherwise indicated, carbocyclic rings as described herein refer to both unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocyclic rings.

[0093] As used herein, the term "heterocycle" or "heterocyclic group" refers to an optionally substituted monocyclic or polycyclic ring system comprising one or more rings, wherein at least one ring comprises at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, or sulfur atom. The ring can be a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more ring. The heterocycle can include one or more double or triple bonds and can be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolidinyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, pyridyl, piperidylalkynyl, quinolinyl, and isoquinolinyl.

[0094] As used herein, the term "heterocycloalkyl" refers to a non-aromatic heterocycle and may or may not contain any double or triple bonds. Unless otherwise indicated, heterocycles as described herein refer to both unsubstituted and substituted heterocyclic groups, ie, optionally substituted heterocycles.

[0095] As used herein, "aryl" is an optionally substituted carbocyclic group comprising one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl.

[0096] As used herein, "heteroaryl" is an optionally substituted heterocyclic group comprising one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. Unless otherwise indicated, aryl or heteroaryl groups as described herein refer to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0097] Unless otherwise indicated, alkyl, alkenyl and cyclic groups (e.g., carbocyclic and heterocyclic groups) may be optionally substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, or iodo groups), carboxylic acids (e.g., -C(O)OH), and alcohols (e.g., hydroxyl, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR)R', wherein each OR is an alkoxy group which may be the same or different and R' is an alkyl or alkenyl group), phosphates (e.g., P(O)4 3+), thiols (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 (e.g., -S(O)-), amide (e.g., -C(O)NR or -N(R)C(O)R), azido (e.g., -N), nitro (e.g., -NO), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR, -NRH, or -NH), carbamoyl (e.g., -OC(O)NR, -OC(O)NRH, or -OC(O)NH), sulfonamides (e.g., -S(O)NR, -S(O)NRH, -S(O)NH, -N(R)S(O)R, -N(H)S(O)R, -N(R)S(O)H, or -N(H)S(O)H), alkyl, alkenyl, and cyclic (e.g., carbocyclyl or heterocyclyl). In any of the foregoing, R is alkyl or alkenyl as defined herein.

[0098] In some embodiments, the substituents themselves may be further substituted with, for example, 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C1-6 alkyl may be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0099] As used herein, the terms "about" and "approximately" when applied to one or more values ​​of interest refer to values ​​that are similar to a specified reference value. In certain embodiments, the terms "about" or "approximately" refer to values ​​that are 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) the reference value, unless otherwise specified or obvious from the context (unless the number exceeds 100% of the possible value). For example, when used in the context of the amount of a given compound in a lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value. For example, a nanoparticle composition comprising a lipid component having approximately 40% of a given compound can contain 30-50% of the compound.

[0100] As used herein, the term "compound" is intended to include all isomers and isotopes of the structure shown. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus, for example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present disclosure can be prepared by combining with solvents or water molecules to form solvates and hydrates by conventional methods.

[0101] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means physically connecting the mammalian cell and the nanoparticle. 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 a mammalian cell by different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve different amounts of the nanoparticle composition. In addition, the nanoparticle composition can contact more than one mammalian cell.

[0102] As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a nanoparticle composition comprising a therapeutic and / or prophylactic agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition. "Local delivery" means that the drug is primarily delivered to a portion of the subject, such as an organ, a tissue (e.g., a muscle), or a cell (e.g., an organ to which the drug is administered, a muscle to which the drug is administered, a cell to which the drug is administered), with the exception of the organ, tissue, or cell, which is not delivered or substantially not delivered. For example, when the drug is administered to the kidney, the drug is locally delivered to other parts of the kidney, but not to organs, tissues, or cells outside the kidney (e.g., through the circulatory system). When the drug is administered to a muscle (e.g., the gastrocnemius muscle), the drug is locally delivered to other parts of the muscle, but not to organs, tissues, or cells outside the muscle (e.g., through the circulatory system).

[0103] In certain 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 certain compounds disclosed herein are used for intravenous or intramuscular delivery of therapeutic and / or prophylactic drugs, they exhibit similar local delivery. In some embodiments, intramuscular injection is preferred over intravenous injection.

[0104] As used herein, the terms "specific delivery" and "local delivery" refer to the delivery of more (e.g., at least 1.5 times more, at least 2 times more, at least 3 times more, at least 3 times more, at least 4 times more, at least 5 times more, at least 6 times more, at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more) therapeutic and / or prophylactic agents to target cells, tissues, or organs of interest (e.g., mammalian muscle) as compared to non-target tissues (e.g., mammalian liver) via nanoparticles. The level of delivery of nanoparticles to a specific tissue can be identified by comparing the amount of protein produced in the tissue with the weight of the tissue, the therapeutic and / or prophylactic amount in the tissue with the weight of the tissue, the amount of protein produced in the tissue with the amount of total protein in the tissue, or the therapeutic and / or prophylactic amount in the tissue with the total amount of therapeutic and / or prophylactic in the tissue. It should be understood that the ability of nanoparticles to be specifically delivered to a target tissue does not need to be determined in a subject undergoing treatment; it can be determined in an alternative such as an animal model (e.g., a rat model).

[0105] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition relative to the initial total amount of therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition is encapsulated in the nanoparticle composition, the encapsulation efficiency can be 97%.

[0106] As used herein, "expression" of a nucleic acid sequence includes translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0107] As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism). As used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). Ex vivo events may occur in an environment that is minimally altered compared to the natural (e.g., in vivo) environment.

[0108] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and / or double bonds and, therefore, may exist as stereoisomers. The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms as well as mixtures of enantiomers and stereoisomers, such as racemates. Enantiomers and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0109] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more cationic / ionizable, pegylated, structured or other lipids, such as phospholipids.

[0110] As used herein, "administering" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The method of administration can be selected to target delivery (eg, specific delivery) to a particular area or system of the body.

[0111] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. RNA also includes any sequence optimization performed on the RNA.

[0112] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. The size of the nanoparticle composition is typically on the order of microns or smaller and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle composition can be a liposome having a lipid bilayer having a diameter of 500 nm or less.

[0113] As used herein, "patient" refers to a subject who may seek or need treatment, is in need of treatment, is receiving treatment, will be receiving treatment, or is under the care of a trained professional for a particular disease or condition.

[0114] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0115] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0116] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a carrier capable of suspending, complexing, or dissolving the active compound) and having substantially non-toxic and non-toxic properties. Excipients may include, for example: anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl cellulose methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0117] In this specification, the structural formula of the compound sometimes represents a certain isomer for convenience, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity.

[0118] For the compounds represented by the chemical formula herein, there may be crystalline polymorphism. It should be noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included within the scope of the present disclosure. The term "crystalline polymorph," "polymorph," or "crystal form" refers to a crystal structure in which a compound (or its salt or solvate) can be crystallized in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystal form to dominate. The crystalline polymorphs of a compound can be prepared by crystallization under different conditions.

[0119] The composition may also include one or more salts of the compound. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amino groups; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like.

[0120] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like.

[0121] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0122] The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

[0123] The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts can be found in any edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference in its entirety.

[0124] As used herein, " phospholipid " is a lipid comprising a phosphoric acid moiety and one or more carbon chains such as unsaturated fatty acid chains. Phospholipid may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Specific phospholipid may promote fusion with membrane. For example, cationic phospholipid may interact with one or more negatively charged phospholipids of membrane (e.g., cell membrane or intracellular membrane). The fusion of phospholipid with membrane may allow one or more components containing lipid compositions to pass through the membrane, thereby allowing, for example, one or more components to be delivered to the cell.

[0125] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which can be produced naturally (eg, isolated or purified) or synthetically.

[0126] As used herein, "RNA" refers to ribonucleic acid that can be naturally occurring or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA can include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA can have a nucleotide sequence that encodes a polypeptide of interest. For example, RNA can be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA in a mammalian cell, can produce the encoded polypeptide. RNA can be selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA.

[0127] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / at one time / by a single route / at a single point of contact, i.e., a single administration event. As used herein, a "split dose" is a single unit dose or total daily dose divided into two or more doses. As used herein, a "total daily dose" is the amount administered or prescribed over a 24-hour period. It can be administered as a single unit dose.

[0128] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0129] As used herein, the terms "subject," "object," or "patient" refer to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably, the subject described herein suffers from a disease that would benefit from local delivery of a drug, e.g., a lesion in an organ or tissue to which it is desired to deliver the drug only.

[0130] As used herein, "target cell" refers to any one or more cells of interest. The cell can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0131] As used herein, "target tissue" refers to any one or more tissue types of interest to which delivery of therapy and / or prevention will result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In specific applications, the target tissue can be the kidney, lung, spleen, vascular endothelium in a blood vessel (e.g., within a coronary artery or within a femur), or tumor tissue (e.g., by intratumoral injection), or muscle (e.g., by intramuscular injection).

[0132] As used herein, "muscles" include, but are not limited to, back muscles (including the trapezius, latissimus dorsi, levator scapulae, rhomboids, erector spinae, splenius, transverse spinae, interspinal muscles, intertransverse muscles), chest muscles (including the pectoralis major, pectoralis minor, serratus anterior, external intercostal muscles, internal intercostal muscles, and transverse thoracic muscles), diaphragm, abdominal muscles (including the rectus abdominis, external oblique abdominal muscles, internal oblique abdominal muscles, transverse abdominal muscles, and quadratus lumborum), perineal muscles, shoulder girdle muscles (including the deltoid, supraspinatus, infraspinatus, teres minor, subscapularis, and teres major), upper arm muscles (including the biceps, coracobrachialis, brachialis, triceps, and anconeus), forearm muscles (including sphincter brachioradialis, pronator teres, flexor carpi radialis, flexor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor carpi ulnaris), hand muscles, pelvic girdle muscles (including iliopsoas, piriformis, gluteus maximus, gluteus medius, and gluteus minimus), thigh muscles (including quadriceps femoris, sartorius, tensor fasciae latae, biceps femoris, semitendinosus, semimembranosus, pectineus, adductor longus, adductor brevis, adductor magnus, and gracilis), calf muscles (including tibialis anterior, extensor digitorum longus, gastrocnemius, soleus, flexor digitorum longus, flexor digitorum longus, tibialis posterior, peroneus longus, and peroneus brevis), foot muscles, platysma, and sternocleidomastoid.

[0133] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active substances" or "active agents" and include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic drugs, small molecule drugs, proteins, and nucleic acids, such as RNA.

[0134] As used herein, the term "therapeutically effective amount" refers to an amount that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, treats, ameliorates symptoms of, diagnoses, prevents, and / or delays the onset of the infection, disease, disorder, and / or condition.

[0135] As used herein, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0136] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment can be performed on subjects who do not exhibit signs of a disease, condition, and / or disorder and / or subjects who exhibit only early signs of a disease, condition, and / or disorder in order to reduce the risk of developing pathology associated with the disease, condition, and / or disorder.

[0137] Another aspect of the present disclosure is a nanoparticle composition comprising a lipid component comprising a compound of formula (I) as described herein.

[0138] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle compositions are vesicles comprising one or more lipid bilayers. In certain embodiments, the nanoparticle compositions include two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0139] The nanoparticle composition comprises a lipid component comprising at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition can include one or more of compounds 1-10. The nanoparticle composition can also include a variety of other components. For example, in addition to the lipid of formula (I), the nanoparticle composition can include one or more cationic lipids.

[0140] The lipid component of nanoparticle composition can comprise the lipid of one or more PEG or PEG modification.This type of material can alternatively be called PEGylated lipid.PEG lipid is the lipid modified with polyethylene glycol.PEG lipid can be selected from the dialkylamine, diacylglycerol, dialkyl glycerol and its mixture that the phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified that PEG-modifies.For example PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0141] The lipid component of the nanoparticle composition can include one or more structural lipids. The structural lipids can be selected from, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

[0142] The lipid component of the nanoparticle composition can include one or more phospholipids, such as one or more unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. Generally, the phospholipids can include a phospholipid portion and one or more fatty acid portions. For example, the phospholipid can be a lipid according to formula (II):

[0143] wherein Rp represents a phospholipid moiety, and R1 and R2 represent fatty acid moieties with or without unsaturation, which may be the same or different. The phospholipid moiety may be selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from 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.

[0144] Phospholipids useful in the compositions and methods described herein can be selected from 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 includes DSPC and / or DOPE.

[0145] In some embodiments, nanoparticle compositions described herein comprise a compound of Formula (I), PEG lipid, DSPC, and cholesterol.

[0146] In some embodiments, nanoparticle compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as aluminum hydroxide.

[0147] Therapeutic and / or preventive agents

[0148] Nanoparticle compositions can include one or more therapeutic and / or prophylactic agents. The present invention provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing polypeptides of interest in mammalian cells, and treating diseases or conditions in mammals in need thereof, comprising administering to the mammal and / or contacting the mammalian cells with a nanoparticle composition comprising a therapeutic and / or prophylactic agent.

[0149] A therapeutic and / or prophylactic agent can be a substance that, once delivered to a cell or organ, brings about a desired change in the cell, organ, or other body tissue or system. Such substances can be used to treat one or more diseases or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease or condition.

[0150] Examples of drugs that can be used in nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (such as imipramine, amitriptyline, and doxepin), antidepressants (such as phenytoin), antihistamines (such as diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (such as gentamicin, ciprofloxacin, and cefoxitin), antifungals (such as miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0151] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent 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, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansine, for example, maytansinol, rachelmin and their analogs or homologues. Radioactive ions include, but are not limited to, iodine (eg, iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

[0152] Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, raclethiazine, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine alkaloids).

[0153] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles of 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.

[0154] 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 can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), their hybrids, RNAi inducers, one or more of RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0155] In some embodiments, the therapeutic and / or preventive agent is RNA. RNA that can be used for the compositions and methods described herein can 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 certain embodiments, RNA is mRNA or siRNA.

[0156] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0157] In other embodiments, the therapeutic and / or preventive agent is siRNA. siRNA may be able to selectively knock down or lower the expression of a gene of interest. For example, siRNA can be selected to silence the gene associated with a specific disease or condition when administering a nanoparticle composition comprising siRNA to a subject in need. siRNA can comprise a sequence complementary to the mRNA sequence encoding a gene of interest or protein. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0158] In some embodiments, the therapeutic and / or preventive agent is a shRNA or a vector or plasmid encoding the shRNA. The shRNA can be produced in the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with the shRNA are well known in the art.

[0159] The nucleic acids and polynucleotides used in the present disclosure generally include a first region (e.g., a coding region) encoding a polypeptide of interest that is linked to nucleosides, a first flanking region (e.g., a 5-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3-UTR) located at the 3'-end of the first region, at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the nucleic acid or polynucleotide also includes a poly-A region or a Kozak sequence (e.g., in a 5'-UTR). In some cases, the polynucleotide may include one or more intronic nucleotide sequences that can be excised from the polynucleotide. In some embodiments, the polynucleotide or nucleic acid (e.g., mRNA) may include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyadenylic acid sequence, and / or a polyadenylation signal. Any one region of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides).

[0160] In some cases, 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.

[0161] Nucleic acids and polynucleotides may include one or more naturally occurring components, including any of the typical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprise (a) a 5′-UTR, (b) an open reading frame (ORF), (c) a 3′-UTR, (d) a poly(A) tail, and any combination of a, b, c, or d above.

[0162] Polynucleotides and nucleic acids can include one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Nucleic acids and polynucleotides for use in nanoparticle compositions can include any useful modification or alteration, such as to nucleobases, sugars, or internucleoside linkages (e.g., to link phosphate / phosphodiester bonds / phosphodiester backbones).

[0163] In some cases, the nucleic acid does not substantially induce an innate immune response in a cell into which the polynucleotide (eg, mRNA) is introduced.

[0164] Other components

[0165] The nanoparticle composition can include one or more components in addition to those described in the preceding section. For example, the nanoparticle composition can include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

[0166] Nanoparticle compositions can also include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers or other components. Permeability enhancer molecules can be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). Polymers can be included in and / or used to encapsulate or partially encapsulate nanoparticle compositions. Polymers can be biodegradable and / or biocompatible. Polymers can be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile and polyarylates. Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, netinib, and erdosteine), and DNases (e.g., rhDNase). The surface-altering agent can be disposed within the nanoparticle and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent attachment, or other processes).

[0167] Nanoparticle compositions may also include one or more functionalized lipids. For example, lipids can be functionalized with alkyne groups that, when exposed to azide under appropriate reaction conditions, can undergo cycloaddition reactions. In particular, lipid bilayers can be functionalized in this manner with one or more groups that can be used to facilitate membrane permeation, cell recognition, or imaging.

[0168] The surface of the nanoparticle composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates for targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0169] In addition to these components, nanoparticle compositions can include any substance useful in pharmaceutical compositions. For example, nanoparticle compositions can include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulation aids, disintegrants, fillers, glidants, liquid carriers, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. Excipients such as waxes, butters, colorants, coatings, flavorings, and fragrances can also be included. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition).

[0170] The lipid component of the nanoparticle composition can include, for example, lipids according to formula (I), phospholipids (e.g., unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. Each lipid component can be provided in a specific fraction. In certain 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 a specific embodiment, the lipid component includes 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 includes about 50 mol % of formula (I) compound, about 10 mol % of phospholipids, about 38.5 mol % of structural lipids and about 3.05 mol % of PEG lipids. In another specific embodiment, the lipid component includes about 40 mol % of formula (I) compound, about 20 mol % of phospholipids, about 38.5 mol % of structural lipids and about 1.5 mol % of PEG lipids. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG (for example PEG2000-DMG) and / or the structural lipid can be cholesterol.

[0171] Alternatively, the lipid components can be calculated in molar amounts, for example, the lipid component of the nanoparticle composition includes about 30 to about 60 molar parts of the compound of formula (I), about 0 to about 30 molar parts of phospholipids, about 18.5 to about 48.5 molar parts of structural lipids, and about 0 to about 10 molar parts of PEG lipids. In some embodiments, the lipid component of the nanoparticle composition includes about 35 to about 55 molar parts of the compound of formula (I), about 5 to about 25 molar parts of phospholipids, about 30 to about 40 molar parts of structural lipids, and about 0 to about 10 molar parts of PEG lipids. In some embodiments, the lipid component includes about 50 molar parts of the compound of formula (I), about 10 molar parts of phospholipids, about 38.5 molar parts of structural lipids, and about 1.5 molar parts of PEG lipids. In another embodiment, the lipid component includes about 50 molar parts of the compound of formula (I), about 10 molar parts of phospholipids, about 38.5 molar parts of structural lipids, and about 3.05 molar parts of PEG lipids.

[0172] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or application, 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 that can be used in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic and other components (e.g., lipids) in a nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of the lipid component to the therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, e.g., 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, or 60:1. For example, the weight / weight ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the weight / weight ratio is about 20: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0173] The nanoparticle composition can have an average size between 10 nm and 100 nm, for example, as measured by methods well known in the art. For example, the average size can be from about 40 nm to about 150 nm, for example, about 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 from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition may be from about 70 nm to about 100 nm. In a specific embodiment, the average size may be about 70 nm.

[0174] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 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 can be about 0.05 to about 0.10.

[0175] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of the therapeutic and / or prophylactic agent that is encapsulated or otherwise bound to the nanoparticle composition after preparation relative to the initial amount provided. Encapsulation efficiency can be measured, for example, by comparing the therapeutic and / or prophylactic amount in a solution containing the nanoparticle composition before and after the nanoparticle composition is broken up with one or more organic solvents or detergents. 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 the therapeutic and / or prophylactic agent can be at least 50%, e.g., 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 can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0176] The nanoparticle compositions may optionally include one or more coatings. For example, the nanoparticle compositions may be formulated in capsules, films, or tablets having a coating. The capsules, films, or tablets of the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0177] The nanoparticle compositions of the present invention can be prepared into a variety of forms suitable for 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), dosage forms for topical (including buccal and sublingual), transdermal and / or transdermal administration (e.g., creams, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), dosage forms for vaginal administration (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), dosage forms for implant administration (e.g., solids, semisolids, gels), suspensions, powders, and other dosage forms.

[0178] Pharmaceutical composition

[0179] Nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions.A pharmaceutical composition can include one or more nanoparticle compositions.

[0180] For example, a pharmaceutical composition can include one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can also include one or more pharmaceutically acceptable excipients, such as those described herein. General guidance for formulating and manufacturing pharmaceutical compositions and medicaments can be found in Remington's The Science and Practice of Pharmacy, 21st edition. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, except where any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the nanoparticle composition. The amount of excipient in the pharmaceutical composition can be determined by one skilled in the art as needed.

[0181] The relative amounts of the one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the characteristics, size, and / or condition of the subject being treated of the nanoparticles, and further depend on the route of administration of the composition. For example, a pharmaceutical composition may comprise 0.1% to 100% (wt / wt) of the one or more nanoparticle compositions.

[0182] In some embodiments, nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (such as stored at 4°C or lower, such as a temperature between about -150°C) and about 0°C or between about -80°C and about -20°C (such as about -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).

[0183] For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein can be stable for about at least 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, for example, at 4°C or lower (e.g., between about 4°C and -20°C). In one embodiment, the formulation is stable for at least 4 weeks at 4°C. In certain embodiments, the pharmaceutical compositions disclosed herein comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable excipient selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical compositions of the present disclosure have a pH of between about 7 and 8 (e.g., 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 8 or between 7 and 7.8).

[0184] In the context of the present disclosure, "stability" and "stable" refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, altered packaging, etc.) under given conditions of manufacture, preparation, transport, storage, and / or use, for example, when stresses such as shear forces, freeze / thaw stress, etc. are applied.

[0185] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those patient or subject cells, tissues, organs or systems or groups thereof that could benefit from the therapeutic effect provided by the delivery of therapeutic and / or prophylactic agents to one or more specific drug cells, tissues, organs or systems.

[0186] While "composition" primarily refers to compositions suitable for administration to humans, one skilled in the art will understand that such compositions are generally suitable for administration to any other mammal.

[0187] Modifications of compositions suitable for administration to humans in order to render them suitable for administration to various animals are well known, and such modifications can be designed and / or performed by an ordinarily skilled veterinary pharmacologist using only ordinary, if any, experimentation. Contemplated subjects for administration of the compositions include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, dogs, sheep, cats, dogs, mice, and / or rats.

[0188] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known in the art of pharmacology or later developed. Generally, such preparation methods involve combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0189] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of that dose, such as one-half or one-third of that dose.

[0190] Pharmaceutical compositions can be prepared into various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared into 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), dosage forms for topical (including buccal and sublingual), transdermal and / or transdermal administration (e.g., creams, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), dosage forms for vaginal administration (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), dosage forms for implant administration (e.g., solids, semisolids, gels), suspensions, powders, and other dosage forms.

[0191] 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 contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluent, the oral composition may include additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, for example, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0192] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile oils can be used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections. Injectable preparations can be sterilized, for example, by filtering through a bacteria-retaining filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0193] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the composition with a suitable non-irritating excipient which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

[0194] The present disclosure also contemplates the use of transdermal patches, which can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0195] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include any type of liquid or solid injection device, such as conventional syringes, fine-gauge syringes, short-needle syringes, liquid jet syringes, compressed gas-accelerated powder injectors, ballistic powder delivery devices, and the like.

[0196] Pharmaceutical composition can be prepared, packaged and / or sold with the preparation suitable for buccal pulmonary administration.Such preparation can comprise dry particles with active ingredient.Such composition is conveniently in dry powder form, for using the device comprising dry powder reservoir and / or using self-propelled solvent / powder distribution container to apply.Such preparation can also be prepared, packaged and / or sold as aqueous solution and / or dilute alcohol solution and / or suspension (optionally sterile, comprising active ingredient), and can use any atomization and / or atomizing device to apply easily.Such preparation can also comprise one or more additional ingredients, including but not limited to flavoring agent such as saccharin sodium, volatile oil, buffer, surfactant and / or preservative such as methyl hydroxybenzoate.

[0197] The formulations described herein for pulmonary delivery can also be used for intranasal delivery of pharmaceutical compositions. Such formulations are administered by nasal inhalation, i.e., rapid inhalation through the nasal passages from a powder container held near the nose. Pharmaceutical compositions can be prepared, packaged, and / or sold in formulations suitable for oral administration. Such formulations can be in the form of tablets and / or lozenges, for example, prepared using conventional methods.

[0198] Methods and uses

[0199] The present disclosure provides a method for producing a polypeptide of interest in a mammalian cell. The method for producing the polypeptide involves contacting the cell with a nanoparticle composition comprising an mRNA encoding the polypeptide of interest. When the cell contacts the nanoparticle composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest.

[0200] In general, the step of contacting mammalian cells with a nanoparticle composition comprising an mRNA encoding a polypeptide of interest can be performed in vivo, in vitro, in culture, or in vitro. The amount of the nanoparticle composition contacted with the cells and / or the amount of the mRNA therein can depend on the type of cells or tissues contacted, the mode of administration, the physiochemical characteristics (e.g., size, charge, and chemical composition) of the nanoparticle composition and the mRNA, and other factors. Efficiency indicators may include polypeptide translation (represented by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0201] The step of contacting the nanoparticle composition comprising the mRNA with the cell can involve or cause transfection. The phospholipids contained in the lipid component of the nanoparticle composition can promote transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with the cell membrane or intracellular membrane. Transfection can allow the mRNA to be translated within the cell.

[0202] In some embodiments, the nanoparticle compositions described herein can be used for treatment. For example, the mRNA contained in the nanoparticle composition can encode a therapeutic polypeptide (e.g., in a translatable region) and produce a therapeutic polypeptide after contacting and / or entering (e.g., transfecting) a cell. In other embodiments, the mRNA contained in the nanoparticle composition can encode a polypeptide that improves or increases the immunity of a subject.

[0203] In certain embodiments, the mRNA contained in the nanoparticle composition can encode a recombinant polypeptide that can replace one or more polypeptides that are substantially absent in the cell in contact with the nanoparticle composition. Alternatively, the recombinant polypeptide produced by translation of the mRNA can antagonize the activity of an endogenous protein in the cell, on the cell surface, or secreted by the cell. In another alternative, the recombinant polypeptide produced by translation of the mRNA can indirectly or directly antagonize the activity of a biological part in the cell, on the cell surface, or secreted from the cell. The biological part of the antagonism can include, but is not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins.

[0204] Methods for delivering therapeutic agents to cells and organs

[0205] The present disclosure provides the method for delivering therapeutic and / or preventative to mammalian cells or organs.Therapeutic and / or preventative are delivered to cells and relate to the nanoparticle composition that comprises therapeutic and / or preventative to experimenter's administration, wherein the administration of composition relates to contacting cell with composition.For example, protein, cytotoxic agent, radioactive ion, chemotherapeutic agent or nucleic acid (for example RNA, for example mRNA or siRNA) can be delivered to cell or organ.When therapeutic and / or preventative is mRNA, when cell contacts with nanoparticle composition, translatable mRNA can be translated in cell to produce interested polypeptide.But, substantially non-translatable mRNA also can be delivered to cell.

[0206] In some embodiments, nanoparticle compositions can target specific types of cells (e.g., cells of a specific organ or its system). For example, a nanoparticle composition comprising a therapeutic and / or preventive agent of interest can be specifically delivered to a mammalian heart, liver, kidney, spleen, femur or lung and is not substantially delivered to other types of cells. The specific delivery of a specific type of cell, organ or its system or tissue means that a higher proportion of the nanoparticle composition comprising a therapeutic and / or preventive agent is delivered to the destination of interest (e.g., tissue) relative to other destinations. In some embodiments, the tissue of interest is selected from heart, myocardium, liver, kidney, lung, spleen, femur, muscle, tumor tissue (e.g., by intratumoral injection).

[0207] As another example of targeted or specific delivery, the mRNA of protein binding ligand (for example, antibody or its functional fragment, scaffold protein or peptide) or receptor on the coding cell surface can be included in the nanoparticle composition. In some embodiments, the ligand can be a surface-bound antibody, which can allow the regulation of cell targeting specificity. These methods can improve the affinity and specificity of the targeted interaction. The ligand can be selected by the technicians of the biological field based on the required location or function of the cell. The target cell can include but is not limited to myocardial cells, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, pneumocytes, osteocytes, stem cells, mesenchymal cells, nerve cells, myocardial cells, adipocytes, vascular smooth muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

[0208] Methods of treating diseases and conditions

[0209] Nanoparticle compositions can be used to treat diseases or conditions. In particular, such compositions can be used to treat diseases or conditions characterized by a lack of or abnormal protein or polypeptide activity. For example, a nanoparticle composition comprising mRNA encoding a missing or abnormal polypeptide can be administered or delivered to a cell. Subsequent translation of the mRNA can produce a polypeptide, thereby reducing or eliminating problems caused by the lack of activity or abnormal activity caused by the polypeptide. The methods and compositions can be used to treat acute diseases or conditions, such as sepsis, stroke, and myocardial infarction. The therapeutic and / or prophylactic agents contained in the nanoparticle compositions may also be able to alter the transcription rate of a given species, thereby affecting gene expression. Diseases and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the compositions can be administered include, but are not limited to, rare diseases, infectious diseases (as vaccines and therapeutic agents), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0210] The present disclosure provides methods involving the administration of nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents, as well as pharmaceutical compositions comprising the same. The terms therapeutic and prophylactic are used interchangeably herein. The therapeutic composition or its imaging, diagnostic or prophylactic composition can be administered to a subject in any reasonable amount and by any route of administration to effectively prevent, treat, diagnose or image a disease, disorder and / or condition. The specific amount administered to the subject can vary depending on the species, age and general condition of the subject, the specific ingredients, and the mode of administration. The compositions according to the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage. However, it should be understood that the specific dosage of the compositions of the present disclosure will be determined by the attending physician within the scope of reasonable medical judgment.

[0211] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions, comprising one or more nanoparticle compositions described herein, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, interdermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, mucosal, nasal, oral, intestinal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation, as an oral spray and / or powder, nasal spray and / or aerosol, and / or through a portal vein catheter. Topical administration is preferred, for example, intramuscular, subcutaneous, transdermal or intradermal, interdermal, intraperitoneal, intraocular, subretinal, intravitreal, mucosal, nasal, oral, intratumoral, intranasal. The appropriate route of administration will depend on a variety of factors, including the nature of the nanoparticle composition, the therapeutic and / or prophylactic agent, the condition of the patient, and the like.

[0212] In certain embodiments, the compositions according to the present disclosure may be administered at a level sufficient to deliver a dosage of about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, or about 10 mg / kg to about 10 mg / kg. kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kgmg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 0.05 mg / kg to about 2.5 mg / kg about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg about 0.001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.0001 mg / kg to about 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg of therapeutic and / or prophylactic agent (e.g., mRNA).

[0213] Dosage can be applied once or repeatedly with the same or different amounts every day, to obtain the desired level of mRNA expression and / or treatment, diagnosis, prevention or imaging effect.Desired dosage can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks or every four weeks.In certain embodiments, multiple administrations (for example, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations) can be used to deliver the desired dosage.

[0214] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Each agent will be administered at a dose and / or schedule determined for that agent. The therapeutic, prophylactic, diagnostic, or imaging agents used in combination can be administered together in a single composition or separately in different compositions.

[0215] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the above description, but is instead set forth in the appended claims.

[0216] In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated otherwise or clear from the context.

[0217] The term "comprising" is intended to be open ended and permits, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting essentially of" and "consisting of" are also encompassed and disclosed. Furthermore, it should be understood that the order of steps or the order in which certain actions are performed is not critical, as long as the invention remains operable. Two or more steps or actions may be performed simultaneously.

[0218] The compounds of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates by employing standard synthetic methods and procedures known or becoming known to those skilled in the art. The synthesis of the compounds of the present disclosure will be readily apparent to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and for functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 thed., John Wiley & Sons: New York, 2001. The synthetic methods described in the examples herein are intended to illustrate, but not to limit, general procedures for preparing the disclosed compounds. One of ordinary skill in the art will note that during the reaction sequences and synthetic schemes described herein, the order of certain steps may be varied, such as the introduction and removal of protecting groups. In the reaction schemes described herein, multiple stereoisomers may be produced. One of ordinary skill in the art will recognize that reactions can be optimized to preferentially produce one isomer, or that novel schemes can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0219] In one or more embodiments, the compound of formula (I) described herein is a compound as shown below:

[0220] Example

[0221] Example 1, Preparation of Compounds

[0222] Experimental instruments

[0223] Experimental reagents

[0224] Synthesis process of compound 15:

[0225] Synthesis of Compound A (8-bromooctanoic acid heptadecan-9-yl ester)

[0226] To a 100 mL dry two-necked flask equipped with a stirrer, undecanol (20 mmol, 3.44 g) and dichloromethane (80 mL) were added under nitrogen, and the reaction solution was placed in an ice-water bath. Malonyl chloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were then added to the reaction solution, 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 a colorless liquid, Compound A (7.09 g, 86% yield).

[0227] Synthesis of Compound B (10-(heptadecan-9-yloxy)-10-oxyylidene-2-[(undecyloxy)carbonyl]decanoate)

[0228] To a 100 mL dry two-necked flask equipped with a stirrer, tetrahydrofuran (15 mL), diundecyl malonate (3 mmol, 1.23 g), and NaH (2.6 mmol, 104 mg) were added sequentially under nitrogen and stirred at room temperature for 30 minutes. Then, heptadecyl-9-yl 8-bromooctanoate (2 mmol, 0.92 g) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added under nitrogen and stirred at 70°C for 48 hours. After the reaction, 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 a colorless liquid, Compound B (0.84 g, 53% yield).

[0229] Synthesis of Compound C (1-[4-(dimethylamino)hexahydropyridin-1-yl]prop-2-en-1-one)

[0230] To a 50 mL dry two-necked flask equipped with a stirrer, under nitrogen, anhydrous dichloromethane (20 mL) and 4-dimethylaminopyridine (6.0 mmol, 768 mg) were added sequentially. The reaction solution was cooled to 0°C, and acryloyl chloride (5 mmol, 455 mg) and triethylamine (7.5 mmol, 758 mg) were added sequentially. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, 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 a light yellow oily liquid, namely compound C (319 mg, 35% yield).

[0231] Synthesis of Compound 15 (2-{3-[4-(dimethylamino)hexahydropyridin-1-yl]-3-oxyylidenepropyl}-10-(heptadecan-9-yloxy)-10-oxyylidene-2-[(undecyloxy)carbonyl]decanoate undecyl ester)

[0232] To a dry 25 mL Schreck tube equipped with a stirrer, under nitrogen, was added toluene (1.5 mL), 10-(heptadecan-9-yloxy)-10-oxyylidene-2-[(undecyloxy)carbonyl]decanoate (0.1 mmol, 79.2 mg), and NaOEt (0.03 mmol, 2 mg). After stirring at room temperature for 10 minutes, 1-[4-(dimethylamino)hexahydropyridin-1-yl]prop-2-en-1-one (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 the crude product, which was then purified by column chromatography (dichloromethane / methanol = 20:1) to afford compound 15 (70.2 mg, 72% yield) as a colorless oil. 1H NMR (600MHz, Chloroform-d) δ4.85 (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 for C60H115N2O7: 975.8704, observed: 975.8708.

[0233] Synthesis of Compound D (Heptadecan-9-yl 3-(Heptadecan-9-yloxy)-3-oxyylidenepropionic acid heptadecan-9-yl ester)

[0234] To a 100 mL dry two-necked flask equipped with a stirrer, 9-heptadecanol (20 mmol, 5.12 g) and dichloromethane (80 mL) were added under nitrogen, and the reaction solution was placed in an ice-water bath. Malonyl chloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were then added to the reaction solution, 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 a colorless liquid, Compound D (5.17 g, 89% yield).

[0235] Synthesis of Compound E (2-[(heptadecan-9-yloxy)carbonyl]-6-{[(10Z,12Z)-octadec-9,12-dienyl]oxy}-6-oxoylidenehexanoic acid heptadecan-9-yl ester)

[0236] To a 100 mL dry two-necked flask equipped with a stirrer, tetrahydrofuran (15 mL), di(9-heptadecayl) malonate (3 mmol, 1.74 g), and NaH (2.6 mmol, 104 mg) were added sequentially under nitrogen and stirred at room temperature for 30 minutes. Then, under nitrogen, 4-bromobutyric acid (10Z,12Z)-octadec-9,12-dien-1-yl ester (2 mmol, 0.83 g) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were added and stirred at 70°C for 48 hours. After the reaction, 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 a colorless liquid, Compound E (1.06 g, 58% yield).

[0237] Synthesis of Compound 15A (2-{3-[4-(dimethylamino)hexahydropyridin-1-yl]-3-oxyylidenepropyl}-2-[(heptadecan-9-yloxy)carbonyl]-6-{[(10Z,12Z)-octadecan-9,12-dienyl]oxy}-6-oxyylidenehexanoic acid heptadecan-9-yl ester)

[0238] To a dry 25 mL Shrek tube equipped with a stirrer, under nitrogen, was added toluene (1.5 mL), compound E (0.1 mmol, 91.5 mg), and NaOEt (0.03 mmol, 2 mg). After stirring at room temperature for 10 minutes, compound C (0.11 mmol, 20.1 mg) was added and stirred at 70°C for 16 hours. After completion of the reaction, 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 afford compound 15A (68.9 mg, 63% yield) as a colorless oil. 1 H NMR (600MHz, Chloroform-d) δ5.42-5.24 (m, 4H), 4.86 (p, J=6.3Hz, 2H), 4.65 (d, J=13.5Hz, 1H), 4.02 ( t, J=6.9Hz, 2H), 3.92 (d, J=13.7Hz, 1H), 2.97 (t, J=12.8Hz, 1H), 2.76 (t, J=7.0Hz, 2H), 2.57-2.51 (m, 1H), 2.34(s, 6H), 2.32-2.26(m, 4H), 2.21(t, J=8.3Hz, 2H), 2.04(q, J=7.2Hz, 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=7.0Hz, 15H).HRMS(ESI)m / z(M+H) +calculated for C 69 H 129 N2O7: 1097.9800, observed: 1097.9334.

[0239] Synthesis of Compound F (3-(Heptadecan-9-yloxy)-3-oxyylidenepropionic acid heptadecan-9-yl ester)

[0240] To a 100 mL dry two-necked flask equipped with a stirrer, (10Z,12Z)-octadeca-9,12-dien-1-ol (20 mmol, 5.32 g) and dichloromethane (80 mL) were added under nitrogen, and the reaction solution was placed in an ice-water bath. Malonyl chloride (10 mmol, 1.40 g) and triethylamine (60 mmol, 6.06 g) were then added to the reaction solution, and the mixture was stirred at room temperature for 6 hours. After the reaction was completed, 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 a colorless liquid, Compound F (4.96 g, 80% yield).

[0241] Synthesis of Compound G (6-{[(10Z,12Z)-octadeca-9,12-dienyl]oxy}-2-({[(10Z,12Z)-octadeca-9,12-dienyl]oxy}carbonyl)-6-oxoylidenehexanoic acid-(10Z,12Z)-octadeca-9,12-dien-1-yl ester)

[0242] To a 100 mL dry two-necked flask equipped with a stirrer, tetrahydrofuran (15 mL), compound F (3 mmol, 1.80 g), and NaH (2.6 mmol, 104 mg) were added sequentially under nitrogen and stirred at room temperature for 30 minutes. Then, under nitrogen, 4-bromobutyric acid-(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 stirred at 70°C for 48 hours. After the reaction, 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 a colorless liquid, compound G (0.88 g, 47% yield).

[0243] Synthesis of Compound 15B (2-{3-[4-(dimethylamino)hexahydropyridin-1-yl]-3-oxyylidenepropyl}-6-{[(10Z,12Z)-octadeca-9,12-dienyl]oxy}-2-({[(10Z,12Z)-octadeca-9,12-dienyl]oxy}carbonyl)-6-oxyylidenehexanoic acid-(10Z,12Z)-octadeca-9,12-dien-1-yl ester)

[0244] To a dry 25 mL Shrek tube equipped with a stirrer, under nitrogen, was added toluene (1.5 mL), compound G (0.1 mmol, 93.5 mg), and NaOEt (0.03 mmol, 2 mg). After stirring at room temperature for 10 minutes, compound C (0.11 mmol, 20.1 mg) was added and stirred at 70°C for 16 hours. After completion of the reaction, 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 afford compound 15B (60.3 mg, 54% yield) as a colorless oil. 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=6.9H z, 2H), 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, 1 H), 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 for C 71 H 125 N2O7:1117.9487.observed:1117.9014.

[0245] Synthesis of Compound H (Heptadecan-9-yl 2-[(Heptadecan-9-yloxy)carbonyl]butyrate)

[0246] To a 100 mL dry two-necked flask equipped with a stirrer, tetrahydrofuran (15 mL), compound D (3 mmol, 1.74 g), and NaH (2.6 mmol, 104 mg) were added sequentially under nitrogen and stirred at room temperature for 30 minutes. Ethyl iodide (2 mmol, 312 mg) and tetrabutylammonium iodide (0.4 mmol, 148 mg) were then added under nitrogen and stirred at 70°C for 48 hours. After the reaction, 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 afford a colorless liquid, compound H (0.83 g, 68% yield).

[0247] Synthesis of Compound 15C (5-[4-(dimethylamino)piperidin-1-yl]-2-ethyl-2-[(heptadecan-9-yloxy)carbonyl]-5-oxopentanoate)

[0248] To a dry 25 mL Shrek tube equipped with a stirrer, under nitrogen, toluene (1.5 mL), compound H (0.1 mmol, 60.9 mg), and NaOEt (0.03 mmol, 2 mg) were added. After stirring at room temperature for 10 minutes, compound C (0.11 mmol, 20.1 mg) was added and stirred at 70°C for 16 hours. After completion of the reaction, 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 afford compound 15C (50.3 mg, 71% yield) as a colorless oil. 1 H NMR (600MHz, Chloroform-d) δ4.85 (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 for C 49 H 95 N2O5: 791.7241, observed: 791.6912.

[0249] Example 2, detection of local expression activity of mRNA-LNP

[0250] Experimental Materials

[0251] Experimental methods

[0252] 1. LNP Preparation

[0253] All four lipid components of the organic phase were dissolved in anhydrous ethanol to 10 mg / mL and prepared according to the proportions in Table 1 below. Luc mRNA at 1 mg / mL was diluted to 70 μg / mL in 25 mM sodium acetate buffer as the aqueous phase. The two-phase channels of the microfluidic instrument (stainless steel chip) were cleaned with anhydrous ethanol and purified water, respectively, and the aqueous phase was rinsed twice with 25 mM sodium acetate buffer. At the beginning of the preparation, 300 μL of the prepared organic and aqueous phase components were first injected into the two-phase channels to expel air from the channels. Then, the preparation solution was aspirated at a volume ratio of 1:3. LNPs were prepared at flow rates of 4 mL / min for the organic phase and 12 mL / min for the aqueous phase, respectively. The first 0.5 mL of the effluent was discarded, and only the subsequent effluent was collected until the end of the preparation. (The preparation method for SM-102LNP and 15LNP was the same.)

[0254] Table 1 Note: The unit is mol%, and the unit in brackets is molar part.

[0255] 1.1 LNP ultrafiltration

[0256] The prepared LNP stock solution was added to 5 times the volume of PBS, and ultrafiltration was performed using an ultrafiltration tube at 2000-3000 rpm for 10 min until the volume was about 1 mL. Then, 10 times the volume of PBS was added to wash the ultrafiltration tube membrane, and the solution was centrifuged to about 1 mL. 10 times the volume of PBS was added to wash the ultrafiltration tube membrane again, and the solution was centrifuged to about 1 mL. After repeatedly rinsing the filter membrane, the above 1 mL solution was taken out and placed in an enzyme-free EP tube and stored in a refrigerator at 4°C for later use.

[0257] 2. Content and Encapsulation Efficiency Determination

[0258] 2.1 Preparation of standard curve

[0259] The mRNA stock solution was diluted to 2 μg / mL with 1×TE, and 0, 2, 10, 25, 50, and 100 μL of each concentration standard solution were respectively added to a 96-well black microplate. Then, the operation was performed according to the label of the RNA quantification kit (1×TE, 100 μL, 1×Ribo, 100 μL). The microplate reader was selected to select an excitation wavelength of 480 nm and an emission wavelength of 520 nm for detection.

[0260] 2.2 Sample preparation for LNP measurement

[0261] After ultrafiltration, LNPs were diluted with 1× TE to a theoretical concentration of approximately 1 μg / mL. Permeabilization was performed by permeabilizing the diluted LNPs with a final concentration of 1% Triton X-100 for 10 minutes at room temperature. Unpermeabilization was performed by adding an equal volume of DEPC water to the diluted LNPs and incubating them at room temperature for 10 minutes. 100 μL of each sample was then transferred to a 96-well black microtiter plate, followed by 100 μL of 1× Ribosol. Each sample was plated in triplicate and detected using a microtiter plate reader with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0262] 3. Particle Size Determination

[0263] Take an appropriate amount of LNP after ultrafiltration, dilute it 10 times with pure water, and then add it to the particle size pool. Measure the particle size on a Malven particle size analyzer.

[0264] 4. Local expression

[0265] Intramuscular injection: Male C57 mice weighing approximately 20 g were selected. Prior to injection, the calf gastrocnemius muscle was shaved. Then, 50 μL of LNP (10 μg mRNA) per mouse was administered intramuscularly using a fine syringe. Four hours after administration, 200 μL (15 mg / mL) of Luciferase substrate was injected intraperitoneally. In vivo imaging of the animals was performed under isoflurane anesthesia.

[0266] Myocardial injection: Male SD rats were anesthetized with isoflurane and injected with 40 μL 15LNP (12 μg mRNA) per rat through minimally invasive myocardial incision. Six hours after administration, 2 mL (15 mg / mL) of Luciferase substrate was injected intraperitoneally. The rats were anesthetized with isoflurane and then photographed alive.

[0267] Spleen injection: Male C57 mice (weighing approximately 20 g) were selected and anesthetized with isoflurane. The skin was cut open on the right side of the abdomen near the chest cavity to expose the spleen in the supine position. Subsequently, 20 μL of 15LNP-2 (4 μg mRNA) was administered to the spleen using a fine syringe. 6 hours after administration, 200 μL (15 mg / mL) of Luciferase substrate was injected intraperitoneally. After isoflurane anesthesia, in vivo imaging of the small animals was performed. Male SD rats (weighing approximately 150 g) were injected with 30 μL (6 μg mRNA) each and treated in the same manner.

[0268] Kidney injection: Male C57 mice (weighing approximately 20 g) were selected and anesthetized with isoflurane. The skin on the right side of the abdomen near the chest was incised and the kidneys were exposed. Subsequently, 20 μL of 15LNP-2 (4 μg mRNA) was administered to the kidneys using a fine syringe. 6 hours after administration, 200 μL (15 mg / mL) of Luciferase substrate was injected intraperitoneally. After isoflurane anesthesia, in vivo imaging of the small animals was performed. Male SD rats (weighing approximately 150 g) were injected with 30 μL (6 μg mRNA) each and treated in the same manner.

[0269] Experimental results

[0270] 1. Particle size, PDI, encapsulation rate (%)

[0271] The particle size distribution of LNPs prepared from the compounds herein is shown in FIG9 .

[0272] The particle size distribution of SM-102 is shown in Figure 9.

[0273] 2. In vivo Expression - Intramuscular Injection

[0274] The results are shown in Figures 11-24. As can be seen, after intramuscular injection of 15LNP-2, in vivo imaging results of small animals showed that it was only locally expressed at the injection site, with no expression observed in other organs. After myocardial injection of 15LNP-2, in vivo imaging results of small animals showed that it was only expressed in the heart, at the injection site, with no expression observed in other organs. After spleen injection of 15LNP-2, in vivo imaging results of small animals showed that it was only expressed in the spleen, at the injection site, with no expression observed in other organs. After kidney injection of 15LNP-2, in vivo imaging results of small animals showed that it was only expressed in the kidney, at the injection site, with no expression observed in other organs.

Claims

1. A compound of formula (I) or a salt or isomer thereof, in, R1 is H or C1-C8 alkyl, R2 is C1-10 alkyl, C3-C18 diene R3 is H or R1-R8 alkyl, R4 is a C1-10 alkyl group, a C3-C18 diene group, R5 is C1-C14 alkyl, C2-C14 alkenyl, or M is O or N, t is an integer from 3 to 7, R6 is H or C1-10 alkyl, R7 is a C1-10 alkyl group, a C3-C18 diene group, Preferably, R1 is the same as R3, and R2 is the same as R4.

2. The compound or salt or isomer thereof according to claim 1, characterized in that: R1 is H and R2 is C6-10 alkyl or C14-C18 diene group, or, R1 is C4-C8 alkyl and R2 is C4-C8 alkyl, and / or R3 is H and R4 is C6-10 alkyl or C14-C18 diene group, or, R3 is C4-C8 alkyl and R4 is C4-C8 alkyl.

3. The compound or salt or isomer thereof according to claim 1 or 2, characterized in that: R5 is C1-C4 alkyl, C10-C14 alkyl, C10-C14 alkenyl, or in, M is O or N, t is 3 or 7, R6 is C4-C8 alkyl and R7 is C4-C8 alkyl, or, R6 is H and R7 is C14-C18 diene.

4. A nanoparticle composition comprising a lipid component, wherein the lipid component comprises a compound or a salt or isomer thereof according to any one of claims 1 to 3, Preferably, the lipid component further comprises phospholipids, PEG lipids, and structural lipids.

5. The nanoparticle composition according to claim 4, characterized in that The nanoparticle composition also includes a therapeutic and / or prophylactic agent.

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

7. A non-therapeutic method for producing a polypeptide in a cell, the method comprising the step of contacting the cell with a nanoparticle composition comprising: (1) a lipid component comprising a phospholipid, a PEG lipid, a structured lipid, and a compound or a salt or isomer thereof according to any one of claims 1 to 3, and (2) an mRNA encoding the polypeptide, whereby the mRNA can be translated in a cell to produce the polypeptide, Preferably, the cell is a mammalian cell.

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

9. Use of the compound or salt or isomer thereof according to any one of claims 1 to 3, and / or the nanoparticle composition according to claim 4 or 5 in the preparation of a medicament for treating or preventing a disease or condition, Preferably, the drug is a local acting drug, Preferably, the nanoparticle composition or the nanoparticle composition formed by the compound of formula (I) is used to deliver a therapeutic agent and / or a preventive agent to a local area. Preferably, the disease or condition is one that would benefit from local delivery of a therapeutic and / or prophylactic agent.

10. The use according to claim 9, characterized in that The part is an organ, preferably the heart, liver, spleen, kidney or lung, or The part is tissue, preferably muscle or bone.

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