Material for local drug delivery and application thereof

Abstract

The invention relates to a material for local drug delivery and an application thereof. In particular, provided are nanoparticle compositions comprising a lipid component comprising a compound of formula (I). The nanoparticle compositions of the invention are delivered only at the site of administration.

Description

Technical Field

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

[0002] The success of the new crown mRNA vaccine reveals that mRNA therapy delivered by lipid nanoparticles (LNP) has great potential. Currently, LNPs on the market (BNT162b2 from BioNTech and mRNA-1273 from Moderna) express target proteins in large quantities in the human liver through various routes of administration (intravenous, subcutaneous, and intramuscular injection). Although they can play an important role in the treatment of liver diseases, these delivery systems have potential toxic side effects for extrahepatic lesions, which is very unfavorable for clinical transformation and application. This field still needs low-toxicity specific LNP delivery carriers. Summary of the invention

[0003] The first aspect of the present invention provides a compound of formula (I) or a salt or isomer thereof,

[0004]

[0005] in,

[0006] s is 5 or 8, t is 7 or 8,

[0007] M 1 is -C(O)O-, -C(O)NR-,

[0008] M 2 It is -C(O)O-, -C(O)NR 5 -,

[0009] R 1 It is C 1-18 Alkyl, C 2-18 Alkenyl, or -CHR 2 R 3 ,

[0010] R 2 , R 3 , R 5 Each independently selected from: H, C 1-14 Alkyl and C 2-14 Alkenyl,

[0011] R 4 It is C 1-3 Alkyl, -(CH 2 ) n CHR 6 R 7 or -CHR 6 R 7 , where R 6and R 7 Each is independent (CH 2 ) n Q, where n is 0, 1, 2, 3, or 4, and Q is C 1-3 Alkyl or OR, R is C 1-3 Alkyl or H.

[0012] Another aspect of the present invention provides a compound of formula (Ia) or a salt or isomer thereof,

[0013]

[0014] in,

[0015] s is 5 or 8, t is 7 or 8,

[0016] M 1 is -C(O)O- or -C(O)NR-,

[0017] M 2 is -C(O)O- or -C(O)NR 5 -,

[0018] R 1 It is C 1-18 Alkyl, C 2-18 Alkenyl, or -CHR 2 R 3 ,

[0019] R 2 , R 3 , R 5 Each independently selected from: H, C 1-14 Alkyl and C 2-14 Alkenyl,

[0020] R 4 It is C 1-3 Alkyl, -(CH 2 ) n CHR 6 R 7 or -CHR 6 R 7 , where R 6 and R 7 Each is independent (CH 2 ) n Q, where n is 0, 1, 2, 3, or 4, and Q is C 1-3 Alkyl or OR, R is C 1-3 Alkyl or H.

[0021] In one or more embodiments, M 1 It is -C(O)O- or -C(O)NH-.

[0022] In one or more embodiments, M 2 Yes-C(O)NR 5 -, R 2 Yes H, R 3 It is C 6 Alkyl, R 5 It is C 7 alkyl.

[0023] In one or more embodiments, R 1 It is C 1-14 alkyl.

[0024] In one or more embodiments, Q is OH.

[0025] In one or more embodiments, R 2 , R 3 Each independently selected from C 1-12 alkyl.

[0026] The present invention also provides nanoparticle compositions comprising a lipid component comprising a compound as described in any of the embodiments herein (eg, a compound of formula (I)).

[0027] In one or more embodiments, the lipid component further comprises phospholipids (eg, polyunsaturated lipids), PEG lipids, and structured lipids.

[0028] In one or more embodiments, the nanoparticle composition further comprises a therapeutic and / or prophylactic agent.

[0029] The present invention also provides a pharmaceutical composition comprising the nanoparticle composition described in any embodiment of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition is a local-acting pharmaceutical composition.

[0030] 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 the administration comprises contacting a cell with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the cell.

[0031] In one or more embodiments, the therapeutic and / or prophylactic agent is a polynucleotide.

[0032] In one or more embodiments, the therapeutic and / or prophylactic agent is mRNA or siRNA.

[0033] In one or more embodiments, the cell is a mammalian cell.

[0034] In one or more embodiments, the delivery is local (rather than systemic) delivery.

[0035] In one or more embodiments, the cell is a cell near the site of administration.

[0036] In one or more embodiments, the cell is a cell of an organ or tissue where the administration site is located, such as liver, spleen, lung, muscle or femur.

[0037] In one or more embodiments, the subject is a mammal, such as a human.

[0038] 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, including 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) an mRNA encoding the polypeptide, whereby the mRNA can be translated in the cell to produce the polypeptide.

[0039] In one or more embodiments, the cell is a mammalian cell.

[0040] 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 a prophylactic agent, or for locally taking effect on a therapeutic agent and / or a 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 a prophylactic agent (e.g., mRNA).

[0041] In one or more embodiments, the disease or condition is a disease or condition in which a protein or polypeptide is malfunctioning or abnormal.

[0042] 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.

[0043] The present invention also provides a method for delivering a therapeutic agent and / or a prophylactic agent locally (rather than systemically) to a mammal, the method comprising the step of administering to a subject (e.g., a mammal) a nanoparticle composition, the 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 agent and / or a prophylactic agent (e.g., mRNA), wherein the administration comprises contacting mammalian cells with the nanoparticle composition, thereby delivering the therapeutic agent and / or the prophylactic agent to the local part of the mammal.

[0044] In one or more embodiments, the local area is a target organ or tissue, such as the liver, spleen, lung, muscle or femur.

[0045] The present invention also provides processes for preparing the compounds of formula (I) described herein.

[0046] The present invention also provides a method for preparing the nanoparticle composition described herein, wherein the method comprises: mixing (eg, microfluidizing) the components of the nanoparticle composition.

[0047] In one or more embodiments, the method comprises mixing (e.g., microfluidizing) the components of the nanoparticle composition described herein (e.g., lipids of formula (I), phospholipids, PEG lipids, structural lipids, and RNA) at a weight ratio of lipid component to therapeutic and / or prophylactic agent of about 5:1 to about 60:1.

[0048] 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.

[0049] 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.

[0050] In one or more embodiments, the medicament is one that delivers a therapeutic and / or prophylactic agent locally rather than systemically.

[0051] 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).

[0052] In one or more embodiments, the local area is a target organ or tissue, such as the liver, spleen, lung, muscle or femur. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The H NMR spectrum of compound 1 is shown.

[0054] Figure 2 The mass spectrum of Compound 1 is shown.

[0055] Figure 3 The H NMR spectrum of compound 2 is shown.

[0056] Figure 4 The mass spectrum of Compound 2 is shown.

[0057] Figure 5 The H NMR spectrum of compound 3 is shown.

[0058] Figure 6 The mass spectrum of compound 3 is shown.

[0059] Figure 7 The H NMR spectrum of compound 4 is shown.

[0060] Figure 8 The mass spectrum of compound 4 is shown.

[0061] Fig. 9 The H NMR spectrum of compound 5 is shown.

[0062] Fig.10 The mass spectrum of compound 5 is shown.

[0063] Fig.11 The H NMR spectrum of compound 6 is shown.

[0064] Fig.12 The mass spectrum of compound 6 is shown.

[0065] Fig.13 The H NMR spectrum of compound 7 is shown.

[0066] Fig.14 The mass spectrum of compound 7 is shown.

[0067] Fig.15 The particle size distribution of LNP of compound 1 is shown. The particle size of LNP of compound 1 is 66.47±1.368nm, and the PDI is 0.084±0.045.

[0068] Fig.16 The particle size distribution of SM-102 is shown. The particle size of SM-102 is 70.38±0.468nm, and the PDI is 0.055±0.011.

[0069] Fig.17 The semi-quantitative fluorescence data of intramuscular injection in vivo and in vitro are shown. SM-102 has higher fluorescence intensity values ​​at the intramuscular injection site and the liver site, while compound 1 LNP has higher fluorescence intensity values ​​only at the intramuscular injection site, and no fluorescence intensity values ​​are found at the liver site, indicating that compound 1 LNP has better local expression performance.

[0070] Fig.18The results of intramuscular injection of compound 1 LNP in C57 mice are shown. Compound 1 LNP is only significantly expressed at the site of intramuscular injection, but not in the liver, indicating that compound 1 LNP has good local expression performance.

[0071] Fig.19 The expression activity and local expression area of ​​compound 1 LNP after intramuscular injection into C57 mice are shown. The expression area of ​​compound 1 LNP decreases with time after intramuscular injection, and the expression site is limited to the injection site during the experimental period.

[0072] Fig. 20 The expression activity and local expression area of ​​compound 1 LNP after intramuscular injection into SD rats are shown. The expression area of ​​compound 1 LNP decreases with time after intramuscular injection, and the expression site is limited to the injection site during the experimental period.

[0073] Fig.21 The results of SM-102LNP intramuscular injection in C57 mice are shown. SM-102LNP can be expressed both at the site of intramuscular injection and in the liver, indicating that SM-102LNP does not have local expression properties.

[0074] Fig. 22 The expression results of compound 1 LNP injected into the spleen of C57 mice are shown. Compound 1 LNP is only significantly expressed at the injection site of the spleen, and no expression is observed in other parts, indicating that compound 1 LNP has good local expression performance.

[0075] Fig.23 The expression results of compound 1 LNP injected into the kidney of C57 mice are shown. Compound 1 LNP is only significantly expressed at the injection site of the kidney, and no expression is observed in other parts, indicating that compound 1 LNP has good local expression performance.

[0076] Fig.24 The expression results of compound 1 LNP in SD rat spleen are shown. Compound 1 LNP is only significantly expressed at the spleen injection site, and no expression is observed in other sites, indicating that compound 1 LNP has good local expression performance.

[0077] Fig.25 The expression results of compound 1 LNP injected into SD rat kidneys are shown. Compound 1 LNP was only significantly expressed at the injection site in the kidneys, and no expression was observed in other sites, indicating that compound 1 LNP had good local expression performance. DETAILED DESCRIPTION

[0078] The present invention synthesizes a novel ionizable cationic lipid, and prepares an LNP (lipid nanoparticle) delivery system using the novel lipid. After the novel delivery system is loaded with Luciferase mRNA, it is injected into the leg muscles, spleen, and kidneys of mice. The results of in vivo fluorescence of small animals show that the delivery system can only express the protein of the loaded mRNA at the injection site, while no expression is seen in other major organs (heart, liver, spleen, lung, and kidney). The LNP delivery system provided by the present invention can overcome the defect that the existing LNP has strong liver targeting, and provides an ideal delivery vector for realizing local specific expression through mRNA therapy. The organ-specific LNP delivery vector of the present invention provides a brand-new development platform for treating currently refractory or untreatable diseases, greatly expanding the clinical application of mRNA therapy.

[0079] The present disclosure relates to novel lipids and lipid nanoparticle compositions comprising novel lipids. The present disclosure also provides methods for delivering therapeutic and / or preventive agents to mammalian cells, in particular, delivering therapeutic and / or preventive agents to local parts of mammals, producing target polypeptides in local cells of mammals, and methods for treating diseases or disorders of mammals.

[0080] The method of delivering a therapeutic and / or prophylactic agent to a mammalian cell or local area involves administering a nanoparticle composition comprising a therapeutic and / or prophylactic agent to a subject, 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.

[0081] Herein, "local" refers to cells, tissues or organs within a certain range of the site of application. In the prior art, it is usually necessary to use special dosage forms to keep the drug in the application site, such as creams, ointments, sprays, powder aerosols, and they are mostly used on the body surface or mucosal surface. The inventors have found that the drug prepared using the compound of formula (I) described herein (such as the nanoparticle composition or pharmaceutical composition described herein) can achieve local administration and local effect in organs, tissues or cells. In some embodiments, the nanoparticle composition of the present invention is suitable for preparing locally administered locally effective drugs (locally applied, locally acting products, LALAP). Herein, "locally effective drugs", "locally effective pharmaceutical compositions", "locally administered locally effective drugs" or "locally administered locally effective" refer to drugs that are applied locally and work at the site of application. If such drugs have systemic effects (such as liver targeting), they are considered to be unintended drug effects. Local may include only cells, only organs (such as liver, spleen, kidney or lung), only tissues (such as 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., liver, spleen, kidney, or lung), the drug is delivered only to the organ where the drug is administered; 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) where the drug is administered; when the drug is administered locally to cells, the drug is delivered only to cells in the organ or tissue (e.g., the muscle where the cell is located) where the drug is administered. In some embodiments, when the drug is administered to the gastrocnemius muscle, the drug is delivered only to the gastrocnemius muscle.

[0082] In some embodiments, local may refer to an organ (e.g., liver, spleen, kidney, or lung) within 4-8 hours (e.g., 6 hours) after administration of the LNP composition described herein, the LNP composition, therapeutic agent, and / or prophylactic agent (mRNA) is mainly maintained in the organ. In some embodiments, local may refer to a tissue (e.g., muscle or bone) within 4-8 hours (e.g., 4 hours) after administration of the LNP composition described herein, the LNP composition, therapeutic agent, and / or prophylactic agent (mRNA) is mainly maintained in the tissue; in particular, it is not delivered to the liver within 4-8 hours (e.g., 4 hours) after administration.

[0083] Lipid and lipid nanoparticle compositions

[0084] The lipids described herein can be advantageously used in lipid nanoparticle compositions to deliver therapeutic and / or prophylactic agents locally to mammals. The lipids described herein have little or no immunogenicity.

[0085] 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.

[0086] 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 symbol "C1-14 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise indicated, the alkyl groups described herein refer to unsubstituted and substituted alkyl groups.

[0087] As used herein, the term "alkenyl" or "alkenyl" refers to a straight or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon double bond, which is optionally substituted. The symbol "C2-14 alkenyl" refers to an optionally substituted straight or branched hydrocarbon containing 2-14 carbon atoms and at least one carbon-carbon double bond. The alkenyl group may include 1, 2, 3, 4 or more carbon-carbon double bonds. Unless otherwise indicated, alkenyl as described herein refers to unsubstituted and substituted alkenyl groups.

[0088] As used herein, the term "alkynyl" or "alkynyl" refers to a straight or branched 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 symbol "C2-14 alkynyl" refers to an optionally substituted straight or branched hydrocarbon comprising 2-14 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 described herein refer to unsubstituted and substituted alkynyl groups.

[0089] 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 may include one or more carbon-carbon double bonds or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl.

[0090] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring and may or may not include any double or triple bonds. Unless otherwise specified, carbocyclic rings described herein refer to both unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocyclic rings.

[0091] As used herein, the term "heterocycle" or "heterocyclic group" refers to an optionally substituted monocyclic or polycyclic system including one or more rings, wherein at least one ring includes 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 may include one or more double bonds or triple bonds, and may 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, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, pyridyl, piperidyl alkynyl, quinolyl and isoquinolyl.

[0092] 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 specified, the heterocycles described herein refer to both unsubstituted and substituted heterocyclic groups, ie, optionally substituted heterocycles.

[0093] As used herein, "aryl" is an optionally substituted carbocyclic group comprising one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl.

[0094] As used herein, "heteroaryl" is an optionally substituted heterocyclic group, including one or more aromatic rings. Examples of heteroaryl include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. Unless otherwise indicated, aryl or heteroaryl as described herein refers to unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0095] 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)2R', 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) 43+ ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O) 2 OH), thiols (e.g. -C(S)H), sulfates (e.g. S(O) 4 2+ ), sulfonyl (e.g. -S(O) 2 -), amides (e.g. -C(O)NR 2 or -N(R)C(O)R), azido (e.g. -N 3 ), nitro (e.g. -NO 2 ), cyano (e.g. -CN), isocyano (e.g. -NC), acyloxy (e.g. -OC(O)R), amino (e.g. -NR 2 , -NRH or -NH 2 ), carbamoyl (e.g. -OC(O)NR 2 、-OC(O)NRH or -OC(O)NH 2 ), sulfonamides (e.g. -S(O) 2 NR 2 、-S(O) 2 NRH, -S(O) 2 NH 2 、-N(R)S(O) 2 R, -N(H)S(O) 2 R, -N(R)S(O) 2 H or -N(H)S(O) 2 H), alkyl, alkenyl and cyclic groups (eg, carbocyclic or heterocyclic groups). In any of the foregoing, R is an alkyl or alkenyl group as defined herein.

[0096] In some embodiments, the substituents themselves may be further substituted, for example, with 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.

[0097] As used herein, the terms "about" and "approximately" when applied to one or more values ​​of interest refer to values ​​similar to a specified reference value. In certain embodiments, the terms "about" or "approximately" refer to values ​​that fall within 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 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 value. For example, a nanoparticle composition comprising a lipid component having about 40% of a given compound may contain 30-50% of the compound.

[0098] As used herein, the term "compound" is intended to include all isomers and isotopes of the structure shown. "Isotopes" refer to atoms with the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus, such as isotopes of hydrogen including 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.

[0099] 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 biological field. For example, the 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.

[0100] 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). Applying a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with a nanoparticle composition. "Local delivery" means that the drug is mainly delivered to a partial location 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), and an organ, tissue, or cell other than the organ, tissue, or cell is not delivered or is not substantially delivered. For example, when the drug is administered in the kidney, the drug is locally delivered to other parts of the kidney, and is not delivered to organs, tissues, or cells (e.g., through the circulatory system) outside the kidney. When the drug is administered in a muscle (e.g., gastrocnemius), the drug is locally delivered to other parts of the muscle, and is not delivered to organs, tissues, or cells (e.g., through the circulatory system) outside the muscle.

[0101] 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 preventive drugs, they exhibit similar local delivery. In some embodiments, intramuscular injection is superior to intravenous injection.

[0102] As used herein, the terms "specific delivery", "local delivery" refer to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) therapeutic and / or preventive agents to a target cell, tissue or organ (e.g., mammalian muscle) of interest compared to a non-target tissue (e.g., mammalian liver) by nanoparticles. The delivery level 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 preventive 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 preventive amount in the tissue with the total amount of therapeutic and / or preventive 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 receiving treatment, it can be determined in a surrogate such as an animal model (e.g., a rat model).

[0103] 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 may be 97%.

[0104] 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.

[0105] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism). As used herein, the term "in vivo" refers to events that occur in an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). As used herein, the term "ex vivo" refers to events that occur 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.

[0106] 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 may therefore 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 enantiomers or stereoisomer components are well known.

[0107] 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, structural or other lipids, such as phospholipids.

[0108] As used herein, "administering" may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The method of administration may be selected to target delivery (eg, specific delivery) to a particular area or system of the body.

[0109] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides or linkers. RNA also includes any sequence optimization performed on the RNA.

[0110] As used herein, "nanoparticle compositions" are compositions comprising one or more lipids. The size of the nanoparticle compositions is typically micrometer-sized or smaller, and may include a lipid bilayer. The nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle compositions may be liposomes having a lipid bilayer having a diameter of 500 nm or less.

[0111] 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.

[0112] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0113] 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.

[0114] 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 formers or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners and hydration water. 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.

[0115] 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 carbon, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity.

[0116] 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 "crystalline 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 solvents, crystallization rates, storage temperatures and other factors may cause a crystal form to dominate. The crystalline polymorphs of a compound can be prepared by crystallization under different conditions.

[0117] The composition may also include a salt of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salt" refers to a derivative of a disclosed compound in which the parent compound is altered 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.

[0118] 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.

[0119] Representative alkali 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.

[0120] 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.

[0121] The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or in a mixture of the two; typically, 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.

[0122] As used herein, "phospholipid" is a lipid comprising a phosphoric acid moiety and one or more carbon chains such as unsaturated fatty acid chains. Phospholipids may include one or more multiple (e.g., double or triple bonds) bonds (e.g., one or more degrees of unsaturation). Specific phospholipids may promote fusion with membranes. For example, cationic phospholipids may interact with one or more negatively charged phospholipids of membranes (e.g., cell membranes or intracellular membranes). The fusion of phospholipids with membranes 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 cells.

[0123] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, usually linked by peptide bonds, which may be produced naturally (eg, isolated or purified) or synthetically.

[0124] As used herein, "RNA" refers to ribonucleic acid that can be naturally 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 encoding a polypeptide of interest. For example, RNA can be a 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), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA.

[0125] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / once / single route / single contact point, 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 given or prescribed in 24 hours. It can be administered as a single unit dose.

[0126] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0127] As used herein, the term "subject", "object" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and humans) and / or plants. Preferably, the subject described herein suffers from a disease that benefits 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.

[0128] 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.

[0129] As used herein, "target tissue" refers to any one or more tissue types of interest, where delivery of treatment 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).

[0130] As used herein, "muscles" include, but are not limited to, back muscles (including trapezius, latissimus dorsi, levator scapulae, rhomboids, erector spinae, splenius, transverse spinae, interspinal muscles, intertransverse muscles), chest muscles (including pectoralis major, pectoralis minor, serratus anterior, external intercostal muscles, internal intercostal muscles, transverse pectoralis), diaphragm, abdominal muscles (including rectus abdominis, external oblique abdominal muscles, internal oblique abdominal muscles, transverse abdominal muscles, quadratus lumborum), perineal muscles, shoulder girdle muscles (including deltoid, supraspinatus, infraspinatus, teres minor, subscapularis, teres major), upper arm muscles (including biceps, coracobrachialis, brachialis, triceps, anconeus), forearm muscles (including sphincter radialis, 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, gluteus minimus), thigh muscles (including quadriceps femoris, sartorius, tensor fasciae latae, biceps femoris, semitendinosus, semimembranosus, pectineus, adductor longus, adductor brevis, adductor magnus, 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, sternocleidomastoid.

[0131] 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 called "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.

[0132] 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 an infection, disease, disorder, and / or condition.

[0133] As used herein, "transfection" refers to the introduction of a substance (eg, RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0134] 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 may refer to inhibiting the survival, growth, and / or spread of a tumor. Subjects who do not show signs of a disease, condition, and / or disorder and / or subjects who show only early signs of a disease, condition, and / or disorder may be treated in order to reduce the risk of developing a pathology associated with the disease, condition, and / or disorder.

[0135] Another aspect of the present disclosure is a nanoparticle composition comprising a lipid component comprising a compound of formula (I) as described herein.

[0136] Nanoparticle compositions include, for example, lipid nanoparticles (LNP), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition includes 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.

[0137] The nanoparticle composition comprises a lipid component, which includes at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition may include one or more of compounds 1-10. The nanoparticle composition may also include a variety of other components. For example, in addition to the lipid of formula (I), the nanoparticle composition may include one or more cationic lipids.

[0138] The lipid component of nanoparticle composition can comprise the lipid of one or more PEG or PEG modification.Such material can be referred to as pegylated lipid alternatively.PEG lipid is the lipid modified with polyethylene glycol.PEG lipid can be selected from the dialkylamine, diacylglycerol, dialkylglycerol and mixture thereof of the phosphatidylethanolamine, phosphatidic acid, ceramide, PEG-modification of PEG-modification, PEG-modification of PEG-modification.For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0139] The lipid component of nanoparticle composition can include one or more structural lipids.Structural lipids can be selected from but not limited to cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof.In some embodiments, structural lipid is cholesterol.In some embodiments, structural lipid includes cholesterol and corticosteroid (for example prednisolone, dexamethasone, prednisone and hydrocortisone) or its combination.

[0140] The lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. Typically, the phospholipids may include a phospholipid portion and one or more fatty acid portions. For example, the phospholipid may be a lipid according to formula (II):

[0141]

[0142] Wherein Rp represents a phospholipid moiety, and R1 and R2 represent a fatty acid moiety with or without unsaturation, which can be the same or different. The phospholipid moiety can be selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid moiety can 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.

[0143] 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.

[0144] In some embodiments, a nanoparticle composition described herein comprises a compound of Formula (I), a PEG lipid, DSPC, and cholesterol.

[0145] In some embodiments, nanoparticle compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as aluminum hydroxide.

[0146] Therapeutic and / or preventive agents

[0147] The nanoparticle composition may include one or more therapeutic and / or prophylactic agents. The methods of the present invention for delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or condition in a mammal in need thereof, comprise administering to the mammal and / or contacting the mammalian cell with a nanoparticle composition comprising a therapeutic and / or prophylactic agent.

[0148] A therapeutic and / or prophylactic agent can be a substance that, once delivered to a cell or organ, brings about a desired change in a cell, organ, or other body tissue or system. Such species can be used to treat one or more diseases or conditions. In some embodiments, a therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease or condition.

[0149] 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.

[0150] In some embodiments, the therapeutic and / or preventive agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that induces an immune response, and / or another therapeutic and / or preventive 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 its 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.

[0151] Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxazid), alkylating agents (e.g., nitrogen mustard, 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).

[0152] In other embodiments, the therapeutic and / or preventive 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.

[0153] In some embodiments, the therapeutic agent is a polynucleotide or a 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 used 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.

[0154] 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.

[0155] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally 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.

[0156] In other embodiments, the therapeutic and / or preventive agent is siRNA. siRNA may be able to selectively knock down or down-regulate 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 a nanoparticle composition comprising siRNA is used to a subject in need. siRNA may include a sequence complementary to an mRNA sequence encoding a gene of interest or protein. In some embodiments, siRNA may be an immunomodulatory siRNA.

[0157] In some embodiments, the therapeutic and / or preventive agent is shRNA or a vector or plasmid encoding it. shRNA can be produced in the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well known in the relevant art.

[0158] Nucleic acids and polynucleotides used in the present disclosure generally include a first region (e.g., a coding region) encoding a polypeptide of interest connected to nucleosides, a first flanking region (e.g., a 5-UTR) at the 5'-end of the first region, a second flanking region (e.g., a 3-UTR) 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 region of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides).

[0159] Typically, the shortest length of a polynucleotide can be a length of a polynucleotide sequence that is enough to encode a dipeptide. In another embodiment, the length of a polynucleotide sequence is enough to encode a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In some cases, the length of a 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.

[0160] Nucleic acids and polynucleotides may include one or more naturally occurring components, including any typical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) or T (thymidine). In one embodiment, all or substantially all nucleotides comprise (a) 5'-UTR, (b) open reading frame (ORF), (c) 3'-UTR, (d) poly (A) tail, and any combination of a, b, c or d above.

[0161] 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 modifications or changes, such as to nucleobases, sugars, or internucleoside bonds (e.g., to link phosphate / phosphodiester bonds / phosphodiester backbones).

[0162] 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.

[0163] Other components

[0164] 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.

[0165] Nanoparticle compositions can also include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers or other components. Permeability enhancer molecules can be molecules described in, for example, 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, polyethylene imine, 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 poloxamer), mucolytic agents (e.g., acetylcysteine, netiniboxin, and erdosteine), and DNases (e.g., rhDNase). The surface-altering agent may be disposed within the nanoparticle and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent attachment, or other processes).

[0166] The nanoparticle composition may also include one or more functionalized lipids. For example, the lipids may be functionalized with alkynyl groups, which, when exposed to azides under appropriate reaction conditions, may undergo cycloaddition reactions. In particular, the lipid bilayer may be functionalized in this manner with one or more groups that may be used to promote membrane permeation, cell recognition, or imaging.

[0167] 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.

[0168] In addition to these components, the nanoparticle composition can include any substance that can be used in a pharmaceutical composition. For example, the nanoparticle composition 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, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid carriers, adhesives, surfactants, isotonic 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).

[0169] The lipid component of the nanoparticle composition may 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 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 phospholipids can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and / or the structural lipid can be cholesterol.

[0170] The amount of therapeutic and / or preventive agents in the nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition and the properties of the therapeutic and / or preventive agents. For example, the amount of RNA that can be used in the nanoparticle composition may depend on the size, sequence and other characteristics of the RNA. The relative amount of treatment and / or prevention and other components (e.g., lipids) in the nanoparticle composition may also vary. In some embodiments, the weight / weight ratio of the lipid component to the therapeutic and / or preventive agent in the nanoparticle composition may be about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1. For example, the weight / weight ratio of the lipid component to the therapeutic and / or prophylactic agent 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).

[0171] The average size of the nanoparticle composition can be between 10 nanometers and 100 nanometers, 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 nm particle composition may be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In certain embodiments, the average size of the nm particle composition may be from about 70nm to about 100nm. In specific embodiments, the average size may be about 70nm.

[0172] 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.

[0173] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agents encapsulated or otherwise combined with nanoparticle compositions after preparation relative to the initial amount provided. Encapsulation efficiency can be measured, for example, by comparing the therapeutic and / or prophylactic amounts in a solution containing nanoparticle compositions before and after the nanoparticle compositions are broken with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agents (e.g., RNA) in a solution. For nanoparticle compositions as described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents can be at least 50%, such as 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%.

[0174] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. The capsule, film, or tablet of the composition described herein may have any useful size, tensile strength, hardness, or density.

[0175] 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.

[0176] Pharmaceutical composition

[0177] Nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions.A pharmaceutical composition can include one or more nanoparticle compositions.

[0178] For example, a pharmaceutical composition may include one or more nanoparticle compositions, including one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition may 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 that 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 a person skilled in the art as needed.

[0179] The relative amounts of 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 one or more nanoparticle compositions.

[0180] In some embodiments, nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at 4°C or lower, such as a temperature between about -150°C) and between about 0°C or about -80°C and about -20°C (e.g., 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).

[0181] 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 a temperature of 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 of the present disclosure 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 value 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).

[0182] 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, change packaging, etc.) under given manufacturing, preparation, transportation, storage and / or use conditions, for example, when stresses such as shear forces, freeze / thaw stress, etc. are applied.

[0183] 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 or groups thereof.

[0184] Although "composition" primarily refers to compositions suitable for administration to humans, one skilled in the art will appreciate that such compositions are generally suitable for administration to any other mammal.

[0185] Modification of compositions suitable for administration to humans in order to render them suitable for administration to a variety of animals is well known, and such modifications can be designed and / or performed by an ordinarily skilled veterinary pharmacologist with only ordinary, if any, experimentation. Subjects to whom administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cows, pigs, dogs, sheep, cats, dogs, mice, and / or rats.

[0186] 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 include 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 forms. Multiple dosage units.

[0187] 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 the subject and / or a convenient fraction of the dose, such as one-half or one-third of the dose.

[0188] 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.

[0189] 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, isopropanol, 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 inert diluents, oral compositions may include additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and / or aromatics. 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.

[0190] 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 parenteral 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 suspension media. For this purpose, any mild 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, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0191] 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.

[0192] The present disclosure also contemplates the use of transdermal patches, which can be prepared, for example, by dissolving and / or dispersing the compound in an appropriate 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.

[0193] 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 injectors, compressed gas accelerated powder injectors, ballistic powder delivery devices, and the like.

[0194] 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 any atomization and / or atomizing device can be used to apply conveniently.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.

[0195] The formulations described herein that can be used 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 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.

[0196] Methods and uses

[0197] The present disclosure provides a method for producing a target polypeptide in a mammalian cell. The method for producing the polypeptide involves contacting the cell with a nanoparticle composition comprising an mRNA encoding the target polypeptide. When the cell contacts the nanoparticle composition, the mRNA can be absorbed and translated in the cell to produce the target polypeptide.

[0198] 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 cell and / or the amount of the mRNA therein can depend on the type of the contacted cell or tissue, 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.

[0199] The step of contacting the nanoparticle composition comprising mRNA with the cell can involve or cause transfection. The phospholipids included in the lipid component of the nanoparticle composition can promote transfection and / or improve transfection efficiency, for example, by interacting and / or fusing with a cell membrane or intracellular membrane. Transfection can allow mRNA to be translated in the cell.

[0200] 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.

[0201] In certain embodiments, the mRNA included in the nanoparticle composition can encode a recombinant polypeptide that can replace one or more polypeptides that are substantially absent in the cell contacted with the nanoparticle composition. Alternatively, the recombinant polypeptide produced by mRNA translation can antagonize the activity of endogenous proteins in the cell, on the cell surface, or secreted by the cell. In another alternative, the recombinant polypeptide produced by mRNA translation can indirectly or directly antagonize the activity of biological parts in the cell, on the cell surface, or secreted from the cell. The biological parts of antagonism can include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins.

[0202] Methods for delivering therapeutic agents to cells and organs

[0203] The present disclosure provides a method for delivering therapeutic and / or preventive agents to mammalian cells or organs. Therapeutic and / or preventive agents are delivered to cells and relate to administering to the subject a nanoparticle composition comprising the therapeutic and / or preventive agent, wherein the administration of the composition relates to contacting the cell with the composition. For example, protein, cytotoxic agent, radioactive ion, chemotherapeutic agent or nucleic acid (e.g., RNA, e.g., mRNA) can be delivered to cells or organs. In the case where the therapeutic and / or preventive agent is mRNA, when the cell contacts the nanoparticle composition, translatable mRNA can be translated in the cell to produce a polypeptide of interest. However, substantially non-translatable mRNA can also be delivered to the cell.

[0204] In some embodiments, nanoparticle compositions can target specific types of cells (e.g., cells of specific organs or their systems). For example, nanoparticle compositions comprising therapeutic and / or preventive agents of interest can be specifically delivered to mammalian liver, kidney, spleen, femur or lung without being substantially delivered to other types of cells. Specific delivery of specific types of cells, organs or their systems or tissues means that a higher proportion of nanoparticle compositions comprising therapeutic and / or preventive agents is delivered to a destination of interest (e.g., tissue) relative to other destinations. In some embodiments, tissue of interest is selected from liver, kidney, lung, spleen, femur, muscle, tumor tissue (e.g., by intratumoral injection).

[0205] 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 cell targeting specificity to be adjusted. 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, for example. The targeted cell can include but is not limited to hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, pneumocytes, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiomyocytes, 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.

[0206] Methods of treating diseases and conditions

[0207] Nanoparticle compositions can be used to treat diseases or conditions. In particular, such compositions can be used to treat diseases or conditions characterized by protein or polypeptide activity loss or abnormality. For example, nanoparticle compositions containing mRNA encoding missing or abnormal polypeptides can be administered or delivered to cells. Subsequent translation of mRNA can produce polypeptides, thereby reducing or eliminating problems caused by activity loss or abnormal activity caused by polypeptides. The methods and compositions can be used to treat acute diseases or conditions, such as sepsis, stroke, and myocardial infarction. The therapeutic and / or preventive agents contained in the nanoparticle compositions may also be able to change 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), cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0208] The present disclosure provides methods involving the administration of nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents and 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 using effective prevention, treatment, diagnosis or imaging of a disease, disorder and / or condition and / or any reasonable amount and any route of administration. 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.

[0209] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be administered by any route. In some embodiments, compositions, including prophylactic compositions, diagnostic compositions, or imaging compositions, which include 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, such as 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.

[0210] 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. 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 a therapeutic and / or prophylactic agent (e.g., mRNA).

[0211] Dosage can be applied once or multiple times with the same or different amounts every day, to obtain the desired level of mRNA expression and / or treatment, diagnosis, prevention or imaging effect. The 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.

[0212] 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.

[0213] 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 rather is set forth in the appended claims.

[0214] 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.

[0215] The term "comprising" is intended to be open ended and allows 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 therefore encompassed and disclosed. In addition, it should be understood that the order of steps or the order in which certain actions are performed is not important as long as the invention remains operable. Two or more steps or actions may be performed simultaneously.

[0216] 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 to those skilled in the art. The synthesis of the compounds of the present disclosure will be apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and 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 thedition, John Wiley & Sons: New York, 2001. The synthetic methods described in the examples herein are intended to illustrate but not limit the general procedures for preparing the disclosed compounds. One of ordinary skill in the art will note that during the reaction sequence and synthetic schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups. In the reaction schemes described herein, a variety of stereoisomers may be produced. One of ordinary skill in the art will recognize that the reaction may be optimized to preferentially produce one isomer, or a new scheme may be designed to produce a single isomer. If a mixture is produced, the isomers may be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0217] In one or more embodiments, the compound of formula (I) described herein is compound 1-7 as shown below:

[0218]

[0219]

[0220] Example

[0221] Example 1, Preparation of Compounds

[0222] Experimental Instruments

[0223]

[0224] Experimental reagents

[0225]

[0226] Synthesis process of compound 1:

[0227]

[0228] Synthesis of Intermediate A (8-bromooctanoic acid heptadecan-9-yl ester)

[0229] Take a 100mL dry round-bottom flask equipped with a stirrer, add 8-bromooctanoic acid (5.5mmol, 1.22g), heptadecan-9-ol (5.0mmol, 1.28g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.5mmol, 1.05g), 4-dimethylaminopyridine (0.5mmol, 61mg) and dichloromethane (15mL), and stir at room temperature for 24 hours. After the reaction is completed, the organic solvent is removed by rotary evaporator to obtain a crude product, which is then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain a colorless liquid, which is intermediate A (2.12g, yield 92%).

[0230]

[0231] Synthesis of Intermediate B (6-bromohexanoic acid undecyl ester)

[0232] Take a 100mL dry round-bottom flask equipped with a stirrer, add 6-bromohexanoic acid (5.5mmol, 1.07g), undecanol (5.0mmol, 0.86g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.5mmol, 1.05g), 4-dimethylaminopyridine (0.5mmol, 61mg) and dichloromethane (15mL), and stir at room temperature for 24 hours. After the reaction is completed, the organic solvent is removed by rotary evaporator to obtain a crude product, which is then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain a colorless liquid, which is intermediate B (1.46g, yield 84%).

[0233]

[0234] Synthesis of Intermediate C (8-[(1,3-dihydroxypropan-2-yl)amino]octanoic acid heptadecan-9-yl ester)

[0235] Take a 100mL dry round-bottom flask equipped with a stirrer, add 8-bromooctanoic acid heptadecan-9-yl ester (5.0mmol, 2.3g), 2-aminopropane-1,3-diol (50mmol, 4.5g) and anhydrous ethanol (30mL), and stir at 60°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, which is intermediate C (942mg, yield 40%).

[0236]

[0237] Compound 1 (8-[1-hydroxy-2-(hydroxymethyl)-9-oxydeoxy-3-aza-10-oxahenedecane-3-yl] Synthesis of Octadecan-9-yl Octanoate

[0238] Take a 100mL dry round-bottom flask equipped with a stirrer, add 8-[(1,3-dihydroxypropan-2-yl)amino]octanoic acid heptadecan-9-yl ester (1.5mmol, 706mg), 6-bromohexanoic acid undecyl ester (1.7mmol, 486mg), potassium carbonate (1.8mmol, 248mg), tetrabutylammonium iodide (0.075mmol, 27mg) and anhydrous acetonitrile (8mL), reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless liquid, namely compound 1 (389mg, yield 35%). 1H NMR(600MHz,Chloroform-d)δ4.86(p,J=6.3Hz,1H),4.05(t,J=6.8Hz,2H),3.59(d,J=6.8Hz,4H),2.96(p,J=6.9Hz,1H),2.56(q,J=9.2,8 .6Hz,4H),2.29(dt,J=13.2,7.5Hz,4H),1.66–1.55(m,6H),1.53–1.42(m,8H),1.33–1.21(m,50H),0.91–0.82(m,9H).HRMS(ESI)m / z(M+H) + calculated forC45H90NO6:740.6768,observed:740.6775.

[0239] Synthesis process of compound 2:

[0240]

[0241] Synthesis of Intermediate D (8-[(2,3-dihydroxypropyl)amino]octanoic acid heptadecan-9-yl ester)

[0242] Take a 100mL dry round-bottom flask equipped with a stirrer, add 8-bromooctanoic acid heptadecan-9-yl ester (5.0mmol, 2.3g), 3-aminopropane-1,2-diol (50mmol, 4.5g) and anhydrous ethanol (30mL), and stir at 60°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, which is intermediate d (786mg, yield 33%).

[0243]

[0244] Compound 2 (8-(1,2-dihydroxy-10-oxydeoxy-4-aza-11-oxa-docosapentaenoic acid-4-yl)octanoic acid Synthesis of 9-alkyl esters

[0245] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound D (1.5mmol, 706mg), 6-bromohexanoic acid undecyl ester (1.7mmol, 486mg), potassium carbonate (1.8mmol, 248mg), tetrabutylammonium iodide (0.075mmol, 27mg) and anhydrous acetonitrile (8mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless liquid, namely compound 2 (478mg, yield 43%).

[0246] 1H NMR(600MHz,Chloroform-d)δ4.86(p,J=6.5Hz,1H),4.05(t,J=6.8Hz,2H),3.92–3.85(m,1H),3.75(dd,J=11.5,4.0Hz,1H),3.52(dd,J=11.4,4.2Hz,1H ),2.80–2.73(m,1H),2.73–2.65(m,2H),2.64–2.55(m,3H),2.29(dt,J=18.0 ,7.4Hz,4H),1.68–1.47(m,14H),1.36–1.17(m,50H),0.87(t,J=7.0Hz,9H).

[0247] Synthesis process of compound 3:

[0248]

[0249] Synthesis of Intermediate E (8-(2,6-dioxahept-4-ylamino)octanoic acid heptadecan-9-yl ester)

[0250] Take a 100mL dry round-bottom flask equipped with a stirrer, add 8-bromooctanoic acid heptadecan-9-yl ester (5.0mmol, 2.3g), 2-amino-1,3-dimethoxypropane (50mmol, 5.95g) and anhydrous ethanol (30mL), and stir at 60°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, which is intermediate E (1.45g, yield 58%).

[0251]

[0252] Compound 3 (8-[4-(methoxymethyl)-11-oxydeoxy-5-aza-2,12-dioxatricosan-5-yl] Synthesis of Octadecan-9-yl Octanoate

[0253] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound E (1.5mmol, 749mg), 6-bromohexanoic acid undecyl ester (1.7mmol, 486mg), potassium carbonate (1.8mmol, 248mg), tetrabutylammonium iodide (0.075mmol, 27mg) and anhydrous acetonitrile (8mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless liquid, namely compound 3 (622mg, yield 54%). 1H NMR (600MHz, Chloroform-d) δ4.84 (p, J=6.3Hz, 1H), 4.02 (t, J=6.8Hz, 2H), 3.44 (dd, J= 9.7,5.9Hz,2H),3.35(dd,J=9.7,5.9Hz,2H),3.29(s,6H),2.96(p,J=5.9Hz,1H),2.47(d t,J=9.7,7.2Hz,4H),2.25(dt,J=9.6,7.5Hz,4H),1.64–1.52(m,6H),1.47(q,J=7.0,6. 2Hz,4H),1.43–1.33(m,4H),1.30–1.17(m,48H),0.89–0.82(m,9H).HRMS(ESI)m / z(M+H) + calculated for C47H94NO6:768.7081,observed:768.6803.

[0254] Synthesis process of compound 4:

[0255]

[0256] Compound 4 (8-[1-hydroxy-2-(hydroxymethyl)-11-octyl-9-oxydeoxy-3-aza-10-oxanonadecane- Synthesis of octanoic acid heptadecan-9-yl ester

[0257] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound D (0.2mmol, 94.2mg), 6-bromohexanoic acid heptadecan-9-yl ester (0.3mmol, 129.6mg), potassium carbonate (0.3mmol, 41.4mg), tetrabutylammonium iodide (0.01mmol, 3.7mg) and anhydrous acetonitrile (4mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol=20:1) to obtain a colorless liquid, namely compound 4 (61mg, yield 37%). 1 H NMR(600MHz,Chloroform-d)δ4.83(p,J=6.3Hz,2H),3.58(d,J=6.8Hz,4H),2.98(p,J=6.8Hz,1H),2.60–2.54(m,4H), 2.25(q,J=7.2Hz,4H),1.59(dt,J=14.4,7.3Hz,4H),1.51–1.41(m,12H),1.31–1.18(m,58H),0.85(t,J=7.0Hz,12H).

[0258] Synthesis process of compound 5:

[0259]

[0260] Synthesis of Intermediate F (6-[(1,3-dihydroxypropan-2-yl)amino]hexanoic acid undecyl ester)

[0261] Take a 100mL dry round-bottom flask equipped with a stirrer, add 6-bromohexanoic acid undecyl ester (5.0mmol, 2.3g), 2-amino-1,3-propanediol (50mmol, 4.55g) and anhydrous ethanol (30mL), and stir at 60°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, which is intermediate F (826mg, yield 46%).

[0262]

[0263] Compound 5 (6-[12-heptyl-1-hydroxy-2-(hydroxymethyl)-11-oxydecan-3,12-diazanonadecan-3- Synthesis of undecyl hexanoate

[0264] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound F (0.2mmol, 71.8mg), 8-bromo-N,N-diheptyloctanamide (0.3mmol, 1025.1mg), potassium carbonate (0.3mmol, 41.4mg), tetrabutylammonium iodide (0.01mmol, 3.7mg) and anhydrous acetonitrile (3mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless liquid, namely compound 5 (61.2mg, yield 44%). 1 H NMR(600MHz,Chloroform-d)δ4.00(t,J=6.8Hz,2H),3.66(d,J=6.5Hz,4H),3.30–3.25(m,1H),3.24–3.20(m,2H),3.19–3. 12(m,2H),2.76(t,J=7.7Hz,4H),2.25(dt,J=15.8,7.5Hz,4H),1.68–1.37(m,16H),1.32–1.17(m,40H),0.88–0.75(m,9H).

[0265] Synthesis process of compound 6:

[0266]

[0267] Synthesis of Intermediate G (2-Hexyldecanoic Acid-8-Bromooctyl Ester)

[0268] Take a 100mL dry round-bottom flask equipped with a stirrer, add 2-hexyldecanoic acid (5.5mmol, 1.41g), 8-bromo-1-octanol (5.0mmol, 1.04g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.5mmol, 1.05g), 4-dimethylaminopyridine (0.5mmol, 61mg) and dichloromethane (15mL), and stir at room temperature for 24 hours. After the reaction is completed, the organic solvent is removed by rotary evaporator to obtain a crude product, which is then purified by column chromatography (petroleum ether / ethyl acetate = 40:1) to obtain a colorless liquid, which is intermediate G (1.87g, yield 84%).

[0269]

[0270] Synthesis of Intermediate H (2-Hexyldecanoic Acid-8-[(1,3-dihydroxypropan-2-yl)amino]octyl Ester)

[0271] A 100 mL dry round-bottom flask equipped with a stirrer was added with compound G (3.0 mmol, 1.34 g), 2-aminopropane-1,3-diol (30 mmol, 3.73 g) and anhydrous ethanol (30 mL), and stirred at 60° C. for 24 hours. After the reaction was completed, the organic solvent was removed by rotary evaporator to obtain a crude product, which was then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, namely intermediate H (576 mg, yield 42%).

[0272]

[0273] Compound 6 (2-hexyldecanoic acid-20-(1,3-dihydroxypropan-2-yl)-9-hexyl-10-oxydeoxy-20-aza- Synthesis of 11-oxaoctacosan-28-yl ester

[0274] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound H (0.2mmol, 91.4mg), compound G (0.3mmol, 134mg), potassium carbonate (0.3mmol, 41.4mg), tetrabutylammonium iodide (0.02mmol, 7.4mg) and anhydrous acetonitrile (3mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless liquid, namely compound 6 (84mg, yield 51%). 1H NMR(600MHz,Chloroform-d)δ4.04(t,J=6.7Hz,4H),3.62(d,J=6.7Hz,4H),3.03(p,J=6.7Hz,1H),2.62(t,J=7.6Hz,4H),2.29 (tt,J=9.4,5.2Hz,2H),1.64–1.52(m,9H),1.51–1.45(m,4H),1.44–1.37(m,4H),1.33–1.17(m,57H),0.86(t,J=6.9Hz,12H).

[0275] Synthesis process of compound 7:

[0276]

[0277] Synthesis of Intermediate I (8-[(1,4-dihydroxybutan-2-yl)amino]octanoic acid heptadecan-9-yl ester)

[0278] A 100 mL dry round-bottom flask equipped with a stirrer was added with compound A (5.0 mmol, 2.3 g), 2-aminobutane-1,4-diol (50 mmol, 5.25 g) and anhydrous ethanol (30 mL), and stirred at 60° C. for 24 hours. After the reaction was completed, the organic solvent was removed by a rotary evaporator to obtain a crude product, which was then purified by column chromatography (dichloromethane / methanol = 10:1) to obtain a colorless liquid, namely intermediate I (800 mg, yield 33%).

[0279]

[0280] Compound 7 (8-[1-hydroxy-3-(hydroxymethyl)-10-oxo-4-aza-11-oxa-docosa-4-yl] Synthesis of Octadecan-9-yl Octanoate

[0281] Take a 100mL dry round-bottom flask equipped with a stirrer, add compound I (0.2mmol, 97mg), compound B (0.3mmol, 104.4mg), potassium carbonate (0.3mmol, 41.4mg), tetrabutylammonium iodide (0.02mmol, 7.4mg) and anhydrous acetonitrile (3mL), and reflux at 70°C for 24 hours. After the reaction is completed, the organic solvent is removed by a rotary evaporator to obtain a crude product, which is then purified by column chromatography (dichloromethane / methanol=20:1) to obtain a colorless liquid, namely compound 7 (78.4mg, yield 52%). 1H NMR(600MHz,Chloroform-d)δ4.85(p,J=6.3Hz,1H),4.05(t,J=6.8Hz,2H),3.75–3.67(m,2H),3.58–3.46(m,2H),3.05–2.92(m,1H) ,2.49(q,J=8.7Hz,4H),2.28(dt,J=14.8,7.5Hz,4H),1.66–1.57(m,6H),1.54–1.42(m,6H),1.34–1.19(m,54H),0.93–0.79(m,9H).

[0282] Example 2, detection of local expression activity of mRNA-LNP

[0283] Experimental Materials

[0284]

[0285]

[0286] Experimental methods

[0287] 1. LNP Preparation

[0288] Compound 1-7 was used as a cationic lipid, and the four components of the organic phase lipid were dissolved in anhydrous ethanol to 10 mg / mL, and prepared according to the ratio of Table 1 as the organic phase, with a total lipid concentration of 4.8 mg / mL; 1 mg / mL of Luc mRNA (SEQ ID NO: 1) was prepared into 70 μg / mL as the aqueous phase with 25 mM sodium acetate buffer solution; the two-phase channel of the microfluidic instrument (stainless steel chip) was cleaned with anhydrous ethanol and pure water, respectively, and the aqueous phase was rinsed twice with 25 mM sodium acetate buffer solution. At the beginning of the preparation, the two-phase pipeline was first used to exhaust the air in the pipeline with 300 μL of the configured organic phase component and the aqueous phase component, and then the preparation solution with a volume ratio of 1:3 of the organic phase to the aqueous phase was drawn, and the flow rates of the organic phase and the aqueous phase were 4 mL / min and 12 mL / min, respectively. The LNP was prepared, and the first 0.5 mL of the effluent was discarded, and only the subsequent effluent was collected until the end (the preparation method of SM-102 and compound 1 LNP was the same).

[0289] Table 1

[0290]

[0291] 1.1 LNP ultrafiltration

[0292] Take the prepared LNP stock solution and add it to 5 times the volume of PBS. Use an ultrafiltration tube at 2000-3000rpm for 10 minutes to ultrafilter until the volume is about 1mL. Then add 10 times the volume of PBS to wash the ultrafiltration tube membrane and centrifuge to about 1mL. Add 10 times the volume of PBS to wash the ultrafiltration tube membrane again and centrifuge to about 1mL. After repeatedly rinsing the membrane, take out the above 1mL solution and put it into an enzyme-free EP tube and store it in a 4°C refrigerator for later use.

[0293] 2. Content and encapsulation efficiency determination

[0294] 2.1 Preparation of standard curve

[0295] 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 ELISA plate, and then the operation was performed according to the instructions of the RNA quantification kit (1×TE, 100 μL, 1×Ribo, 100 μL). The ELISA reader was selected with an excitation wavelength of 480 nm and an emission wavelength of 520 nm for detection.

[0296] 2.2 Sample preparation for LNP measurement

[0297] Take the ultrafiltered LNP and dilute it with 1×TE to a theoretical concentration of about 1μg / mL; permeabilization: the diluted LNP is permeabilized with a final concentration of 1% TritonX-100 at room temperature for 10 minutes; non-permeabilization: the diluted LNP is added with an equal volume of DEPC water and placed at room temperature for 10 minutes. Then take 100μL of each sample to a 96-well black ELISA plate, and then add 100μL of 1×Ribo, each sample has 3 replicates, and the ELISA reader selects an excitation wavelength of 480nm and an emission wavelength of 520nm for detection.

[0298] 3. Particle size determination

[0299] Take an appropriate amount of LNP after ultrafiltration, dilute it 10 times with pure water, and then add it to the particle size pool, and measure the particle size on the Malven particle size analyzer.

[0300] 4. Local expression

[0301] Intramuscular injection: Male C57 mice weighing about 20 g were selected. Before injection, the hair in the gastrocnemius muscle of the calf was removed, and then 50 μL LNP (10 μg mRNA) was administered to the gastrocnemius muscle using a fine syringe. 200 μL (15 mg / mL) Luciferase substrate was injected intraperitoneally 4 hours after administration, and in vivo imaging (4 hours) of small animals was performed after isoflurane anesthesia.

[0302] Spleen injection: Male C57 mice (weighing approximately 20 g) were selected. After isoflurane anesthesia, the skin on the right side of the abdomen near the chest was cut open and the spleen was exposed in the supine position. Subsequently, 20 μL of compound 1LNP (4 μg mRNA) was administered to the spleen using a fine syringe. 200 μL (15 mg / mL) of Luciferase substrate was injected intraperitoneally 6 h after administration. After isoflurane anesthesia, in vivo imaging of 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 way.

[0303] Kidney injection: Male C57 mice (weighing approximately 20 g) were selected. After isoflurane anesthesia, the skin on the right side of the abdomen near the chest was cut open and the kidney was exposed in the supine position. Subsequently, 20 μL of compound 1LNP (4 μg mRNA) was administered to the kidney using a fine syringe. 200 μL (15 mg / mL) of Luciferase substrate was injected intraperitoneally 6 hours after administration. After isoflurane anesthesia, in vivo imaging of 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 way.

[0304] Experimental Results

[0305] 1. Particle size, PDI, encapsulation rate %

[0306]

[0307] The particle size distribution of LNP prepared from compound 1 herein is as follows Fig.15 shown.

[0308] SM-102 particle size distribution Fig.16 shown.

[0309] 2. In vivo expression - intramuscular injection

[0310] The results are as follows Figure 17-Figure 25 As shown. It can be seen that after intramuscular injection of compound 1 LNP, the in vivo imaging results of small animals showed that it was only locally expressed at the injection site, and no expression was observed in other organs.

Claims

1. A compound of formula (I) or a salt or isomer thereof, in, s is 5 or 8, t is 7 or 8, M1 is -C(O)O-, -C(O)NR-, M2 is -C(O)O-, -C(O)NR5-, R1 is C 1-18 Alkyl, C 2-18 alkenyl, or -CHR2R3, R2, R3, R5 are each independently selected from: H, C 1-14 Alkyl and C 2-14 Alkenyl, R4 is C 1-3 Alkyl, -(CH2) n CHR6R7 or -CHR6R7, wherein R6 and R7 are each independently (CH2) n Q, where n is 0, 1, 2, 3, or 4, and Q is C 1-3 Alkyl or OR, R is C 1-3 Alkyl or H.

2. The compound or salt or isomer thereof according to claim 1, characterized in that: The compound is as shown in formula (Ia), in, s is 5 or 8, t is 7 or 8, M1 is -C(O)O- or -C(O)NR-, M2 is -C(O)O- or -C(O)NR5-, R1 is C 1-18 Alkyl, C 2-18 alkenyl, or -CHR2R3, R2, R3, R5 are each independently selected from: H, C 1-14 Alkyl and C 2-14 Alkenyl, R4 is C 1-3 Alkyl, -(CH2) n CHR6R7 or -CHR6R7, wherein R6 and R7 are each independently (CH2) n Q, where n is 0, 1, 2, 3, or 4, and Q is C 1-3 Alkyl or OR, R is C 1-3 Alkyl or H.

3. The compound or salt or isomer thereof according to claim 1 or 2, characterized in that: M1 is -C(O)O- or -C(O)NH-, and / or M2 is -C(O)NR5-, R2 is H, R3 is C6 alkyl, R5 is C7 alkyl, and / or R1 is C 1-14 Alkyl, and / or Q is OH, and / or R2 and R3 are each independently selected from C 1-12 alkyl.

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 or a tissue, Preferably, the local area is liver, spleen, kidney, lung, muscle or bone.

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