CN116589435A - Ionizable lipids and compositions thereof for nucleic acid delivery - Google Patents

Ionizable lipids and compositions thereof for nucleic acid delivery Download PDF

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Publication number
CN116589435A
CN116589435A CN202310287179.2A CN202310287179A CN116589435A CN 116589435 A CN116589435 A CN 116589435A CN 202310287179 A CN202310287179 A CN 202310287179A CN 116589435 A CN116589435 A CN 116589435A
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compound
independently selected
mrna
disease
lipid
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宋相容
魏霞蔚
魏于全
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Chengdu Westin Biomedical Technology Co ltd
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Chengdu Westin Biomedical Technology Co ltd
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Abstract

The present invention relates to ionizable lipids and compositions thereof for nucleic acid delivery, belonging to the field of pharmaceutical chemistry. The invention provides a compound shown as a formula I. The invention also provides the use of the compounds as nucleic acid delivery vehicles. The compounds of the present invention, when used as delivery vehicles for nucleic acid drugs, exhibit greater delivery efficiency and greater safety than conventional materials.

Description

Ionizable lipids and compositions thereof for nucleic acid delivery
Technical Field
The present invention relates to ionizable lipids and compositions thereof for nucleic acid delivery, belonging to the field of pharmaceutical chemistry.
Background
Nucleic acid drugs include DNA, antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), micrornas (mirnas), miRNA micrometers, anti-imirs, ribozymes, mRNA, aptamers, plasmids, CRISPR RNA, and the like. The application of nucleic acid medicine is limited by the instability of its chemical properties, and the nucleic acid medicine is easy to be degraded into single nucleotide in vitro and in vivo, and loses efficacy.
The use of nucleic acid drugs often requires specific delivery vectors, including viral vectors and non-viral vectors. Common viral vectors include retrovirus, lentivirus, adeno-associated virus and the like, and the viral vectors can smoothly invade cells by virtue of natural cell infection activity, and have high transport efficiency, however, the clinical application of the viral vectors is limited by factors such as limited immunogenicity, limited loading capacity, complex production process and the like. The non-viral vector is a type of gene delivery vector with more researches and better application prospect at present, and is mainly loaded with mRNA through the adsorption action of cations formed by delivery materials and mRNA phosphate ions to form structures such as liposome or nanoparticle and the like, so that the non-viral vector is protected from degradation of nuclease and changes the cell entering way, and has the advantages of relatively easy acquisition of the vector, low immunogenicity and higher safety.
Most of traditional non-viral nucleic acid delivery materials are cationic lipids or cationic polymers, and due to their strong electropositivity, the materials are easily absorbed by plasma proteins in vivo and then absorbed by reticuloendothelial systems, so that the loaded nucleic acid drugs are destroyed. Many of the non-viral vectors are lipid nanoparticles based on ionizable lipids, the nanoparticles prepared by the ionizable lipid materials are positively charged in an in-vitro acidic environment, the electrostatic adsorption of nucleic acid realizes the loading of nucleic acid drugs, and the charged nanoparticles are electrically neutral after entering a neutral environment in vivo, so that the adsorption of plasma proteins and the capture of reticuloendothelial system are avoided. Based on this, ionizable lipid nanoparticles have very broad prospects in the field of nucleic acid delivery.
However, the clinical use of ionizable lipid nanoparticles is relatively few, and the core difficulty is the development of safe and effective ionizable lipids. Therefore, the development of ionizable lipid nucleic acid delivery materials with higher delivery efficiency and safer is of great significance for the wide application of nucleic acid drug gene therapy.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide ionizable lipids for nucleic acid delivery.
The present application provides a compound of formula i, or a pharmaceutically acceptable salt, isomer, deuterate or prodrug thereof:
wherein a is 1,2 or 3;
X 1 、X 2 each independently selected from N or C;
L 1 、L 2 、L 3 、L 4 independently selected from-R e CH(OH)-、-R e C(＝O)-、-R e C(＝O)O-、-R e OC(＝O)-、-R e C(＝O)S-、-R e SC(＝O)-、-R e C(＝O)NR a -、-R e NR a C(＝O)-、-R e NR a C(＝O)O-、-R e OC(＝O)NR a -、-R e O-、-R e -O-O-、-R e S-、-R e -S-S-、-R e -S-S-S-、-R e CH(OH)CH 2 O-、-R e CH(OH)CH 2 S-or absent, R e Is thatOr is absent, k is an integer greater than 1, R a -H, substituted or unsubstituted alkyl;
R 1 、R 2 、R 3 、R 4 independently selected from C 1 -C 30 Straight chain alkyl, C 1 -C 30 Branched alkyl, C 2 -C 30 Straight chain alkenyl, C 2 -C 30 Branched alkenyl, C 2 -C 30 Straight chain alkynyl or C 2 -C 30 Branched alkynyl groups;
G 1 、G 2 、G 3 、G 4 independently selected from-R c -、-R c CH(OH)R d -、-R c C(＝O)R d -、-R c C(＝O)OR d -、-R c OC(＝O)R d -、-R c C(＝O)SR d -、-R c SC(＝O)R d -、-R c C(＝O)N(R b )R d -、-R c N(R b )C(＝O)R d -、-R c N(R b )C(＝O)OR d -、-R c OC(＝O)N(R b )R d -、-R c OR d -、-R c -O-O-R d -、-R c SR d -、-R c -S-S-R d -、-R c -S-S-S-R d -or not present, R b is-H, substituted or unsubstituted alkyl, R c 、R d Independently selected fromOr is absent, n is an integer greater than 1;
R 5 、R 6 independently selected from-H, substituted or unsubstituted alkyl;
Q 1 、Q 2 independently selected from O or S.
The lipid nanoparticle formed by the ionizable lipid has positive charges in an in-vitro acidic environment, realizes the load of nucleic acid drugs by electrostatic adsorption of nucleic acid, has neutral charges after entering a neutral environment in the body, and avoids the adsorption of plasma proteins and the capture of reticuloendothelial system. The lipid delivery vehicle formed by the ionizable lipid has stronger targeting to the liver and the lung. The nucleic acid medicine carried by the nucleic acid medicine, such as mRNA, has higher expression in receptor cells, and the antigen expressed by the antigen mRNA has stronger presentation in vivo. Can be used for in vivo delivery of nucleic acid medicines such as mRNA and the like, and achieves the purposes of up-regulating or down-regulating corresponding genes, delivering antigen mRNA to express antigen in vivo and achieve immunotherapeutic effect, delivering mRNA encoding antibody to express antibody in vivo and the like. Meanwhile, the inventor of the application also unexpectedly found that after the lipid carrier formed by the ionizable lipid is loaded with the drug, when the drug is administrated to a mouse intramuscular injection route, the local irritation of the drug is obviously lower than that of a positive control carrier, the drug has no obvious effect on the weight gain of the mouse, and the positive control carrier has an inhibiting effect on the weight gain of the mouse.
Wherein, the writing sequence of the L1, L2, L3 and L4 connecting bonds is from left to right corresponding to the near nitrogen end to the far nitrogen end.
The writing sequence of the G1, G2, G3 and G4 connecting bonds defined above corresponds to the main chain direction of the formula I from left to right.
According to an embodiment of the invention, the X 1 And X 2 And is also N. The inventors found that when X 1 And X 2 And meanwhile, when N is adopted, the lipid nanoparticle formed by the compound has better preparation properties, better encapsulation effect on mRNA, and further, mRNA can be expressed more efficiently in a test animal body.
According to an embodiment of the invention, the compound has a structure represented by formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuteride or prodrug of a structure represented by formula II,
according to an embodiment of the invention, k is 1,2,3,4,5,6,7,8,9 or 10.
According to an embodiment of the invention, k is 1.
In accordance with an embodiment of the present invention, the L1, L2, L3, L4 are independently selected from-CH (OH) -, -C (=O) -, -CH2C (=O) O-, -OC (=O) -, -C (=O) S-, -SC (=O) -, -CH2C (=O) NRa-, -NRaC (=O) O-, -OC (=O) NRa-, -CH2O-, -CH2-O-, -CH2S-, -CH2-S-S-, -CH (OH) CH2O-, -CH (OH) CH2S-, or are absent, ra is-H, substituted or unsubstituted alkyl.
According to an embodiment of the invention, the L 1 、L 2 、L 3 、L 4 Independently selected from-C (=o) -, -C (=o) NR a -、-CH 2 C(＝O)NR a -、-NR a C(＝O)-、-C(＝O)O-、-CH 2 C(＝O)O-、-OC(＝O)-、-CH 2 O-、-O-、-CH 2 S-、-S-、-CH(OH)-、-CH(OH)CH 2 O-、-CH(OH)CH 2 S-or absent, R a is-H or unsubstituted alkyl.
According to an embodiment of the invention, the R a is-H or unsubstituted C 1 ～C 6 An alkyl group.
According to an embodiment of the invention, the R a is-H.
According to an embodiment of the invention, the L 1 、L 2 、L 3 、L 4 Independently selected from-C (=o) -, -C (=o) NH-, -CH 2 C(＝O)NH-、-C(＝O)O-、-CH 2 C(＝O)O-、-CH 2 O-、-CH 2 S-、-CH(OH)-、-CH(OH)CH 2 O-or absent; preferably L 1 、L 2 、L 3 、L 4 Independently selected from-C (=O) NH-, -C (=O) O-, -CH (OH) CH 2 O-or absent.
According to an embodiment of the invention, the L 1 And L 2 Selected from the same groups and or L 3 And L 4 Selected from the same groups.
According to an embodiment of the invention, the L 1 And L 3 Selected from the same groups and or L 2 And L 4 Selected from the same groups.
According to an embodiment of the invention, the L 1 、L 2 、L 3 And L 4 Selected from the same groups.
According to an embodiment of the invention, the R 1 、R 2 、R 3 、R 4 Independently selected from C 1 -C 30 Straight chain alkyl, C 2 -C 30 Straight chain alkenyl, C 2 -C 30 Straight chain alkynyl groups.
According to an embodiment of the invention, the R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 1 ～C 30 A linear alkyl group.
According to an embodiment of the invention, the R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 8 ～C 18 A linear alkyl group.
According to the inventionIn the embodiment of (2), the R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 10 ～C 14 A linear alkyl group.
According to an embodiment of the invention, the R 1 And R is 2 Selected from the same groups and or R 3 And R is 4 Selected from the same groups.
According to an embodiment of the invention, the R 1 And R is 3 Selected from the same groups and or R 2 And R is 4 Selected from the same groups.
According to an embodiment of the invention, the R 1 、R 2 、R 3 And R is 4 Selected from the same groups.
According to an embodiment of the invention, the G 1 、G 2 、G 3 、G 4 Independently selected fromOr absent, n3 is 1,2,3,4,5,6,7,8,9 or 10.
According to an embodiment of the invention, the G 3 、G 4 Is not present.
According to an embodiment of the invention, the G 1 、G 2 Independently selected from-CH 2 -or-CH 2 CH 2 -。
According to an embodiment of the invention, the R 5 、R 6 Independently selected from-H, unsubstituted C 1 ～C 6 C substituted by alkyl or-OH 1 ～C 6 An alkyl group.
According to an embodiment of the invention, the R 5 、R 6 Independently selected from-H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl.
According to an embodiment of the invention, the R 5 、R 6 Selected from the same groups.
According to a specific embodiment of the invention, the compound has one of the following structures:
pharmaceutically acceptable salts, stereoisomers, deuterides or prodrugs thereof.
In another aspect, the invention provides the use of a compound as described hereinbefore, or a pharmaceutically acceptable salt, stereoisomer, deuteride or prodrug thereof, as a drug delivery vehicle.
Further, the active ingredient of the medicament is optionally at least one of nucleic acid, small molecule drug, protein drug, polypeptide.
Specifically, the active ingredient of the drug is selected from nucleic acids.
In particular, the medicament has cardiac, hepatic, spleen, lung or kidney targeting.
Preferably, the drug targets the lung, spleen.
In yet another aspect, the present invention provides a drug delivery vehicle. According to an embodiment of the invention, the drug delivery vehicle comprises a pharmaceutical composition comprising a compound as described above, or a pharmaceutically acceptable salt, stereoisomer, deuterate or prodrug thereof.
In yet another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical complex comprises a carrier and an active ingredient, the carrier being associated with the active ingredient, the carrier comprising a cationic lipid comprising a compound as described hereinbefore, or a pharmaceutically acceptable salt, stereoisomer, deuteride or prodrug thereof.
According to a specific embodiment of the invention, the active ingredient is optionally selected from at least one of nucleic acids, small molecule drugs, protein drugs, polypeptides, antibodies.
According to a specific embodiment of the invention, the active ingredient of the medicament is selected from the group consisting of nucleic acids;
According to a specific embodiment of the invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, aptamer.
According to a specific embodiment of the invention, the nucleic acid is mRNA;
according to a specific embodiment of the invention, the delivery vehicle is ionically linked to the mRNA.
According to a specific embodiment of the invention, the pharmaceutical complex targets the lung, spleen.
According to a specific embodiment of the invention, the drug complex is present in one form, optionally from among lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, polymer nanoparticles.
According to a specific embodiment of the invention, the pharmaceutical complex is in the form of a lipid nanoparticle LNP in the form of a formulation.
According to a specific embodiment of the invention, the pharmaceutical composition further comprises an excipient, optionally comprising at least one selected from the group consisting of neutral phospholipids, steroids, pegylated lipids.
According to a specific embodiment of the invention, the neutral phospholipid is selected from at least one of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, POPC.
According to a specific embodiment of the invention, the neutral phospholipid is DOPE.
According to a specific embodiment of the invention, the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride or a prodrug thereof, to the neutral phospholipid is from 1:10 to 10:1.
According to a specific embodiment of the invention, the steroid is at least one selected from cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, fucosterol.
According to a specific embodiment of the invention, the steroid is cholesterol.
According to a specific embodiment of the invention, the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride or a prodrug thereof, to the steroid is from 1:10 to 10:1.
According to a specific embodiment of the present invention, the pegylated lipid is selected from at least one of DMG-PEG, DSPE-PEG;
preferably, the pegylated lipid is DMG-PEG2000.
According to a specific embodiment of the invention, the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride or a prodrug thereof, to the pegylated lipid is between 5:1 and 1000:1.
According to a specific embodiment of the invention, the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride or a prodrug thereof, to the pegylated lipid is between 10:1 and 20:1.
According to a specific embodiment of the invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, aptamer.
According to a specific embodiment of the invention, the nucleic acid is mRNA.
In another aspect, the present invention provides a lipid nanoparticle. According to an example of the invention, the lipid nanoparticle comprises a lipid nanoshell and a nucleic acid, the nucleic acid being encapsulated within the lipid nanoshell, the lipid nanoshell comprising a cationic lipid comprising a compound comprising the foregoing, or a pharmaceutically acceptable salt, stereoisomer, deuteride, or prodrug thereof.
According to a specific embodiment of the present invention, the lipid nanoparticle has an average particle diameter of 40nm to 500nm, di (90)
The polydispersion coefficient is less than or equal to 500nm and less than or equal to 30 percent.
In another aspect, the invention provides a pharmaceutical composition. According to a specific embodiment of the invention, the aforementioned pharmaceutical complex or the aforementioned lipid nanoparticle is comprised.
According to a specific embodiment of the present invention, further comprising pharmaceutically acceptable excipients.
According to a specific embodiment of the invention, the composition is an injection.
According to a specific embodiment of the invention, the composition is suitable for intravenous or intramuscular injection.
In another aspect, the present invention provides the use of the aforementioned pharmaceutical complex or the aforementioned lipid nanoparticle or the aforementioned pharmaceutical composition for the preparation of a medicament for the treatment or prevention of a disease.
According to a specific embodiment of the invention, the disease is a heart, liver, spleen, lung or kidney related disease, preferably the disease is a spleen or lung related disease.
According to a specific embodiment of the invention, the disease is an infectious disease, cancer and proliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, cardiovascular and renal vascular disease, and metabolic disease.
According to a specific embodiment of the invention, the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or various herpes.
According to a specific embodiment of the invention, the cancer is a solid tumor, preferably the cancer is liver cancer or lung cancer.
According to a specific embodiment of the invention, the medicament treats or prevents a disease by presenting an antigen and/or activating an immune response.
The invention provides a novel ionizable lipid, the hydrophilic center of which is composed of 4 tertiary amine effective nitrogen, and the hydrophobic tail is composed of 4 saturated or unsaturated fatty chains. The novel ionizable lipid provided by the invention is positively charged in an acidic environment and is hardly charged in a neutral environment, the nucleic acid medicine can be loaded in an acidic buffer system by utilizing the property, the nucleic acid medicine enters the neutral environment in a body after being loaded, and the lipid nanoparticle shows neutral electricity, so that the adsorption of a nucleic acid medicine compound by plasma proteins can be effectively avoided, thereby realizing higher delivery efficiency of the nucleic acid medicine and obviously improving the safety.
Drawings
FIG. 1 particle size, PDI and potential of LNPs@mRNA prepared in example 4;
FIG. 2 encapsulation efficiency of LNPs@mRNA prepared in example 4;
FIG. 3 expression of LNPs@mRNA cellular level of reporter gene: (a) transfection of lnps@egfp mRNA into HEK293T cells; (B) transfection of LNPs@EGFP mRNA into DC2.4 cells; (C) LNPs@FLuc mRNA transfection of DC2.4 cells;
FIG. 4MTT assay for cytotoxicity of 4N4T-LNPs@FLuc mRNA;
FIG. 5 bioluminescence image of LNPs@FLuc mRNA expression and distribution;
FIG. 6 statistics of expression and distribution of LNPs@FLuc mRNA by intravenous administration;
FIG. 7 activation of BMDC by LNPs@OVA mRNA vaccine:
FIG. 8LNPs@OVA mRNA vaccine treatment of TNF- α cytokine secretion from BMDC.
Detailed Description
The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The invention includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with the use or term used in the present disclosure, the use or term of the present disclosure controls.
The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.
Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative properties such as dosages set forth in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range.
The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
The compounds and derivatives provided in the present invention may be named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.
The term "alkyl" is a radical of a straight or branched saturated hydrocarbon radical. Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6).
The term "alkenyl" refers to a straight or branched hydrocarbon group containing at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl.
The term "alkynyl" refers to a straight or branched hydrocarbon group containing at least one triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl.
The term "pharmaceutically acceptable" means that a carrier, excipient, salt, etc., is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form, and physiologically compatible with the receptor, and in particular, the compound or complex is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or disorder is to be prevented or treated.
The term "subject" or "patient" includes both humans and mammals in the present application.
The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from or having symptoms of a disease, to cure, alleviate, ameliorate or otherwise affect the disease or symptoms of the disease. In the context of the present application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.
The term "pharmaceutically acceptable salts" refers to the acidic and/or basic salts of the compounds of the application with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. Or by mixing the above-mentioned compound with a certain amount of an acid or a base as appropriate (for example, equivalent). These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by lyophilization after reaction in an aqueous medium. The salts of the present application may be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salts of the compounds.
It is further understood that the compound of formula I or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present invention. For example, a compound of formula I or a pharmaceutically acceptable salt thereof may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, and the like).
Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid, chiral malic acid, and the like to form salts with the compounds of the present disclosure, utilizing differences in the physicochemical properties of the products, such as solubility.
The application also includes all suitable isotopic variations of the compounds of the present disclosure. Isotopic variations are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, and 18O, respectively.
A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and disclosure.
In the present application, when the names of the compounds are not identical to the structural formulae, the structural formulae are determined.
It is to be understood that the term "presently disclosed compounds" as used herein may include, depending on the context: a compound of formula I, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.
The term cationic lipid as used herein refers to a lipid that is positively charged at a selected pH.
Cationic liposomes readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in the nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs). LNP is one of the currently mainstream delivery vehicles.
The invention provides a novel ionizable lipid which has a structure shown in a formula (I) or a formula (II), wherein a hydrophilic center of the novel ionizable lipid is composed of 4 tertiary amine effective nitrogen, and a hydrophobic tail is composed of 4 saturated or unsaturated fatty chains. The novel ionizable lipid provided by the invention is positively charged in an acidic environment and is hardly charged in a neutral environment, the nucleic acid medicine can be loaded in an acidic buffer system by utilizing the property, the nucleic acid medicine enters the neutral environment in a body after being loaded, and the lipid nanoparticle shows positive electricity, so that the adsorption of a nucleic acid medicine compound by plasma proteins can be effectively avoided, thereby realizing higher delivery efficiency of the nucleic acid medicine and obviously improving the safety.
Yet another aspect of the present disclosure provides a pharmaceutical complex comprising a carrier comprising a cationic lipid comprising a compound of formula (I) formula (II) or a pharmaceutically acceptable salt or stereoisomer thereof, as described above.
In one embodiment, the complex is a nanoparticle formulation having an average size of 40nm to 500nm, preferably 100nm to 205nm; the nanoparticle formulation has a polydispersity of 50% or less, preferably 30% or less, more preferably 25% or less.
In one embodiment of the complexes/carriers of the present disclosure, the cationic lipid is one or more selected from the compounds of formula (I) or formula (II) above or pharmaceutically acceptable salts or stereoisomers thereof, deuterated. In one embodiment, the cationic lipid is a compound of formula (I) selected from the group consisting of those described above.
In another embodiment of the complex/carrier of the present disclosure, the cationic lipid comprises: (a) One or more selected from the compounds of formula (I) above or deuterated, pharmaceutically acceptable salts or stereoisomers thereof; (b) One or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound may be a commercially available cationic lipid, or a cationic lipid compound reported in the literature.
In one embodiment, the cationic lipid comprises 30% to 70%, such as 35%, 45%, 50%, 55%, 60%, 65% of the carrier by mole.
The carrier may be used to deliver an active ingredient such as a therapeutic or prophylactic agent. The active ingredient may be enclosed within a carrier or may be combined with a carrier.
For example, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a large molecule compound, a polypeptide, an antibody, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), and mixtures thereof.
The carrier may comprise a neutral lipid, such as a neutral phospholipid. Neutral lipids in the present disclosure refer to lipids that are non-charged at a selected pH or that are present as zwitterionic forms that act as a helper. The neutral lipids may modulate nanoparticle mobility into lipid bilayer structures and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 10:1, for example about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5:1.
For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
The carrier component comprising the cationic lipid complex may comprise one or more neutral lipid phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
The neutral lipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2 lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha linolenic acid, erucic acid, phytanic acid, arachic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azide. These reactions can be used to functionalize the lipid bilayer of the complex to facilitate membrane permeation or cell recognition, or to couple the complex with a useful component such as a targeting or imaging moiety (e.g., dye).
Neutral lipids useful in these complexes may be selected from the non-limiting group consisting of: 1,2 dioleoyl sn glycerophosphoryl choline (DLPC), 1,2 dimyristoyl sn glycerophosphoryl choline (DMPC), 1,2 dioleoyl sn glycerophosphoryl choline (DOPC), 1,2 dipalmitoyl sn glycerophosphoryl choline (DPPC), 1,2 distearoyl sn glycerophosphoryl choline (DSPC), 1,2 bisundecanoyl sn glycerophosphoryl choline (DUPC), 1 palmitoyl 2 oleoyl sn glycerophosphoryl choline (POPC), 1,2 dioleyl sn glycerophosphoryl choline (18:0 Diether PC), 1 oleoyl 2 cholesteryl hemisuccinyl sn glycerophosphoryl choline (OChemsPC), 1 hexadecyl sn glycerophosphoryl choline (C16 LysoPC), 1,2 dioleoyl sn glycerophosphoryl choline, 1,2 diacetyl sn glycerophosphoryl choline, 1,2 didodecylsn glycerohexanoyl s3, 1,2 didodecylglycerohexanoyl sn 3, 1,2 dioleoyl sn glycerohexanolamine (DOPC), 2-ditartanoyl-sn-glycerol 3-phosphate ethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycerol 3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycerol 3-phosphate ethanolamine, 1, 2-didodecyloyl-sn-glycerol 3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycerol 3-phosphate rac (1-glycerol) sodium salt (DOPG), dipalmitoyl-phosphatidylglycerol (DPPG), palmitoyl-base-oil acyl-phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (dimyristoyl phosphate)
(DMPE), 1 stearoyl 2 oleoyl stearoyl ethanolamine (SOPE), 1 stearoyl 2 oleoyl phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based oil acyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.
In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE.
In some embodiments, the neutral lipid comprises both DSPC and DOPE.
The carrier comprising the complex of cationic lipids may also include one or more structural lipids, such as a steroid. Structured lipids refer in the present disclosure to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.
In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
The structural lipid may be selected from, but is not limited to, the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.
The carrier comprising the complex of cationic lipids may also comprise one or more polymer conjugated lipids, such as pegylated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.
In one embodiment, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The PEG modified PEG molecular weight is typically 350 5000Da.
For example, the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE PEG 2000), dimyristoylglycerol 3 methoxy polyethylene glycol 2000 (DMG PEG 2000) and methoxy polyethylene glycol ditetradecylacetamide (ALC 0159).
In one embodiment of the complex/carrier of the present disclosure, the polymer conjugated lipid is D M G PEG2000.
In one embodiment of the complex/carrier of the present disclosure, the carrier comprises neutral lipids, structural lipids, and polymer conjugated lipids, the molar ratio of the ionizable lipids, the neutral lipids, the structural lipids, and the polymer conjugated lipids being 50:10:38.5:1.
Pharmaceutically active ingredients
Pharmaceutically active ingredients and alternatively referred to as "active agents" may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of the ionizable lipid to the therapeutic or prophylactic agent is from 0.1:1 to 1000:1.
The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
The vectors of the present disclosure may deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a complex comprising a therapeutic and/or prophylactic agent, a pharmaceutical composition, and/or contacting mammalian cells with the complex or pharmaceutical composition.
The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the complex include, but are not limited to, antineoplastic agents (e.g., vincristine (v i n c r i s t i n e), doxorubicin (doxorubicine), mitoxantrone (mitoxantrone), camptothecin (camptothecin), cisplatin (cispratin), bleomycin (b l e o m y c i n), cyclophosphamide (c y c l o p h o s p h a m i d e), methotrexate and streptozotocin), antineoplastic agents (e.g., actinomycin D (actinomycin D), vincristine, vinblastine (vinblaine), cytosine arabinoside (cytosine arabinoside), anthracycline (anthramycin), alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine (dibucaine) and chlorpromazine), beta adrenergic blockers (e.g., propranol (proolol), second and other drugs (prothiolin)), antimuscarin (e.g., benzodiazepinephrine (trimeton), and antihypertensive agents (e.g., trimepraline) and trimethoprim (trimethoprim), and antihypertensive agents (e.g., trimethoprim) and trimethoprim (trimethoprim) Antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics/antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B (amphotericin B)), antiparasitic agents, hormones, hormonal antagonists, immunomodulators, neurotransmitters, anti-glaucoma agents, vitamins, sedatives, and imaging agents.
In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol (taxol), cytochalasin B (cytochalasin B), gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracenedione (dihydroxy anthracin dione), mitoxantrone, mithramycin (mithramycin), actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytanoid) such as maytanol (maytansine), rachimycin (CC 1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and may include mRNA encoding antigens and/or epitopes that are the source of infectious diseases. Vaccines can also include compounds and formulations that direct immune responses against cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes, and/or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, the vaccine and/or compound capable of eliciting an immune response is administered intramuscularly by a composition comprising a compound according to formula (I). Other therapeutic and/or prophylactic agents, but not limited to antimetabolites (e.g., methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine)
And 5 fluorouracil dacarbazine (dacarbazine)), alkylating agents (such as nitrogen mustard (mechlorethamine), thiotepa (thiotepa), chlorambucil (chloramamide), azithromycin (CC 1065), melphalan (melphalan), carmustine (carmustin, BSNU), robustadine (lomustine, CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP), cisplatin), anthracyclines (such as daunorubicin (formerly known as daunomycin (d a u n o m y C i n)) and doxorubicin), antibiotics (such as dactinomycin (dactinomycin) (formerly known as actinomycin), bleomycin, mithramycin (mithramycin) and Amphotericin (AMC)) and antimicrobial (such as vinblastine, neomycin, and taxotere).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles in 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, interferons, heparin, hepatitis b surface antigens, typhoid vaccines, and cholera vaccines.
In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid).
The term "polynucleotide" is intended to include in its broadest sense any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acids (RNAs), including messenger RNAs (mrnas), hybrids thereof; RNAi-inducing factors; RNAi factor; siRNA; shRNA; a miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; an aptamer, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. The RNAs useful in the complexes and methods described herein may be selected from the group consisting of, but not limited to: shortmer, antagomir antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.
In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a complex comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9mRNA may be used as a gene editing tool. For example, sgRNAcas9 complex can affect mRNA translation of cellular genes.
In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.
The complexes/carriers of the present disclosure can deliver active ingredients, including therapeutic or prophylactic agents, to a subject or patient. The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Thus, the complexes of the present disclosure can be used to prepare nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. Because of the wide variety of therapeutic or prophylactic agents described above, the complexes of the present disclosure are useful in the treatment or prevention of a variety of diseases or conditions.
In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.
In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and various herpes.
The complex may include one or more components other than those described in the preceding sections. For example, the complex may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.
The complex may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).
The surface modifying agent may include, but is not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, dyers woad (cleodendrum), bromhexine, carbocisteine, eplerenone (eprazine), mesna (mesna), ambroxol (ambroxol), sobreynol (sobrerol), polyminol)
(domiodol), letostane (letosteine), setronin (stepronin), tiopronin (tiopronin), gelsolin (gelsolin), thymosin beta 4, streptococcal dnase alpha (dornase alfa), netizene and erdosteine (erdosteine) and dnase (e.g. rhdnase). The surface modifying agent may be disposed within and/or on the nanoparticle of the complex (e.g., by coating, adsorption, covalent attachment, or other methods).
The complex may also comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the complex may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
In addition to these components, the complex may further form a pharmaceutical composition. For example, the pharmaceutical composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients, for example starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington' sThe Science and Practice of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).
Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.
In some embodiments, pharmaceutical compositions comprising one or more lipids described herein may also comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The pharmaceutical compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The pharmaceutical compositions of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.
For example, the complexes, pharmaceutical compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.
In one embodiment, the pharmaceutical composition is administered intravenously.
The complexes, pharmaceutical compositions of the present disclosure are administered in therapeutically effective amounts, which may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attendant physician or clinician.
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 Synthesis of ionizable lipid compounds
1. Synthesis of Compound I-1
The synthetic route is as follows:
(1) Synthesis of Compound 1:
anhydrous piperazine 2a (1.0 equiv.) and anhydrous potassium carbonate (5.0 equiv.) were added to an eggplant-shaped bottle and dissolved in ultra-dry dichloromethane. The acryloyl chloride 1b (3.0 equiv.) was dissolved in ultra-dry dichloromethane and added to the dropping funnel. In ice water bath, the acryloyl chloride solution in the dropping funnel was added dropwise to the eggplant-shaped bottle. After 6h of reaction in an ice-water bath, the reaction was stopped and the reaction solvent was removed by rotary evaporation. The crude product was isolated by column chromatography on silica gel eluting with DCM/meoh=40:1 (V/V) to give the product as a white solid, compound 4, 87% yield. And dissolving a proper amount of the product in deuterated chloroform for nuclear magnetic analysis.
(2) Synthesis of Compound 2:
compound 4 (1.0 equiv.) and tert-butyl 2c (2.0 equiv.) of 2- (methylamino) ethylcarbamate were taken and dissolved in absolute ethanol and reacted under reflux at 80 ℃ in an oil bath for 36h. After the reaction, the reaction solvent was removed by rotary evaporation, and silica gel column chromatography was performed with eluent DCM/meoh=20:1 to 15:1 (V/V) (1% nh3.h2o) to purify the product as a white solid, i.e. compound 5, yield 71%. And dissolving a proper amount of the product in deuterated chloroform for nuclear magnetic analysis.
(3) Synthesis of Compound I-1:
compound 2 (2 mmol,1.0 equiv.) was taken, mixed, 4mL TFA and 4mL DCM and stirred at room temperature for 2h to remove the Boc protecting group. TFA/DCM was removed by rotary evaporation to give a pale yellow oil, which was dissolved in isopropanol, anhydrous potassium carbonate was added, and the remaining TFA was neutralized with stirring at room temperature until the isopropanol solution was basic. 1, 2-epoxytetradecane 3a (12 mmol,6.0 equiv.) is added to the solution and refluxed in an oil bath at 90℃for 36h. After the reaction, the reaction solvent is removed by rotary evaporation, and the mixture is separated and purified by silica gel column chromatography, wherein the eluent is DCM/MeOH=25:1-15:1 (V/V) (1% NH 3H 2O), and the eluent is dried by rotary evaporation to obtain a pale yellow semi-solid, namely the final product I-1, and the yield is 35%.
And dissolving a proper amount of the product in deuterated chloroform for nuclear magnetic analysis. And dissolving a proper amount of the product in deuterated chloroform for nuclear magnetic analysis. The results were as follows: 1H NMR (400 mhz, cdcl 3) δ (ppm) =5.19 (s, 2H), 3.75-3.42 (m, 8H), 3.23 (d, j= 5.0,4H), 2.88-2.16 (m, 30H), 1.47-1.16 (m, 92H), 0.89 (t, j= 6.8,12H). The results showed that I-1 was successfully synthesized.
2. Synthesis of Compound II-1
The synthetic route is as follows:
(1) The synthesis of compound 1 and compound 2 is described in the synthesis section of compound I-1.
(2) Synthesis of Compound II-1: compound 2 (2 mmol,1.0 equiv.) was taken, mixed, 4mL TFA and 4mL DCM and stirred at room temperature for 2h to remove the Boc protecting group. The TFA/DCM was removed by rotary evaporation to give a pale yellow oil which was dissolved in acetonitrile, anhydrous potassium carbonate was added, and the remaining TFA was neutralized with stirring at room temperature until the acetonitrile solution was basic. To the solution was added bromotetradecane 3b (12 mmol,6.0 equiv.) and refluxed in an oil bath at 90 ℃ for 48h. After the reaction, the reaction solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography, with eluent DCM/meoh=25:1-20:1 (V/V) (1% nh3.h2o), and the eluent was dried by rotary evaporation to give a pale yellow semisolid, i.e., the final product II-1, 34% yield.
A proper amount of the product is taken and dissolved in deuterated chloroform for nuclear magnetic analysis, and the result is as follows: 1H NMR (400 mhz, cdcl 3) δ (ppm) =3.57 (dd, j= 48.8,18.3,8H), 2.86-2.21 (m, 30H), 1.55-1.18 (m, 96H), 0.89 (t, j= 6.8,12H). The results showed that II-2 was successfully synthesized.
EXAMPLE 2 Positive control Compound Synthesis
The positive control of the invention is one or more of C12-200 (MW= 1136.96), SM-102 (MW= 710.18), ALC-0315 (MW= 766.29) and DLin-MC3-DMA (MC 3, MW= 642.09). Wherein, C12-200 and MC3 are classical mRNA delivery lipid ionizable lipid, SM-102 is ionizable lipid adopted by Moderna on-market mRNA vaccine products, and ALC-0315 is ionizable lipid adopted by CureVac and BioNTeach on-market mRNA vaccine products.
The structure is as follows:
synthetic routes are reported in the literature, and are either synthesized by a third party company or commercially available.
EXAMPLE 3 Synthesis of other exemplary Compounds of the invention
The compound of the invention has the structure which is divided into a hydrophilic center formed by 4 tertiary amines (N) and a hydrophobic tail formed by 4 saturated fatty chains (T), the electropositivity and the hydrophilic and hydrophobic properties of each ionizable lipid molecule are equivalent, the loading capacity of the ionizable lipid molecule to mRNA is equivalent, and the different structures of the fine structure form the different specific compounds.
The synthesis of the compound of the invention mainly comprises two steps: first, a 4N positive charge center is constructed by an amide formation reaction and Michael addition (Michael addition); second, the 4T hydrophobic tail is attached by an epoxy ring opening reaction, alkylation reaction, or Michael addition reaction. Other exemplary compounds of the present disclosure were prepared from different starting materials by a similar route as described in example 1.
Other compounds of the invention have the structural formula:
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compared with other published ionizable lipid molecules in the field, for example, the literature reports that a relatively dangerous Raney Nickel (Raney Nickel) hydrogenation reduction reaction is needed in a C12-200 synthesis route, the compound synthesis route is simple and easy to implement, the structural characterization is clear, and the compound yield is sufficient.
Example 4 preparation of LNPs@mRNA and examination of the pharmaceutics
Further, the inventors constructed mRNA delivery system lnps@mrna based on the ionizable lipid prepared in example 1, and examined the pharmaceutics properties such as particle size, potential, encapsulation efficiency, TEM, etc., to evaluate the pharmaceutics.
1. Preparation of LNPs@mRNA
(1) Preparing a solution: the ionizable lipid, DOPE (or DSPC), chol, DMG-PEG2000 were dissolved in absolute ethanol to give a concentration of 10mg/mL of ionizable lipid. The mole ratio of the ionizable lipid prepared in example 1 of the present invention to the prescription ionizable lipid of C12-200, MC3, SM-102 and ALC-0315, DSPC, cholesterol (Chol), DMG-PEG2000 is 50:10:38.5:1.5.mRNA was diluted to an appropriate concentration in PBS buffer (RNase-free water). To ensure that the positive control performance is maximized, the C12-200, MC3, SM-102 and ALC-0315 formulations are selected from known literature publications with preferred ratios, as disclosed in the prior art.
(2) LNP preparation: mixing the ionizable lipid solution obtained in step (1) with an mRNA solution. Microfluidic process parameters: the volume ratio of the ethanol phase to the water phase was 1:3, and the flow rate was 9mL/min.
(3) Ultrafiltration: diluting the LNP primary preparation by 25 times with PBS buffer solution, ultrafiltering to the initial volume by an ultrafiltration cup to obtain LNP final preparation, and removing ethanol in the primary preparation during ultrafiltration. Ultrafiltration process parameters: the filter membrane has 100kDa, air pressure of 0.2MPa and rotating speed of 100-200rpm.
2. LNPs@mRNA formulation property investigation
Particle size potential measurement: diluting LNPs@mRNA preparation with purified water for 10 times, and detecting the preparation properties such as particle size, particle size distribution, zeta potential and the like by using a Markov nanometer particle size potentiometer.
Characterization by transmission electron microscope: diluting LNPs@mRNA with purified water to make the mass concentration of the nano particles be 2mg/mL, carefully dripping the nano particles onto a copper mesh special for TEM, standing for 2min, sucking excessive liquid by using filter paper, dripping 2% phosphotungstic acid for 3min, sucking excessive dye liquid, drying by using ear washing balls, loading samples, detecting and photographing.
And (3) detecting encapsulation rate: solution preparation: LNPs@mRNA was diluted with RNase-free water to a concentration of 0.1. Mu.g/. Mu.L. And under the condition of avoiding light, a proper amount of RiboGreen dye is diluted 400 times by using 1 xTE buffer solution, and the mixture is preserved in a dark place for later use. Rupture of membranes: to the sample wells were added 25. Mu.L of diluted mRNA preparation and 75. Mu.L of 1 XTE buffer, and to the total mRNA wells were added 25. Mu.L of diluted mRNA preparation, 25. Mu.L of 1 XTE buffer and 50. Mu.L of 1% Triton. The 96-well plate was incubated at 37℃for 10min in the absence of light. Color development: after 10min, 100 μl of diluted RiboGreen dye was added to each well under light-shielding conditions and shaken to homogenize. Detection conditions of the enzyme-labeled instrument: excitation wavelength is 485nm, and emission wavelength is 528nm. The calculation formula is as follows: encapsulation (%) = (1-sample well fluorescence value/total mRNA fluorescence value) ×100%.
The experimental results are shown in fig. 1: the LNP prepared from different ionizable lipids prepared in the embodiment 1 of the present invention has equivalent particle sizes, the average particle size is about 100nm, di (90) is within 500nm, and PDI is below 0.3, which indicates that the LNP has uniform particle size distribution. The potential of the preparation is about 5 mV.
LNPs@mRNA prepared from different ionizable lipids prepared in the embodiment 1 of the invention is in a solid sphere shape, the particle size under TEM is about 100nm, and a fingerprint-like structure is found and is a representative characteristic of an LNP nano structure.
The encapsulation efficiency test results are shown in FIG. 2, and compared with the positive controls MC3, ALC-0315 and SM-102, the encapsulation efficiency of LNPs@mRNA prepared from the ionizable lipid of example 1 of the present invention is significantly higher than that of the positive control.
The test results show that: the LNPs@mRNA prepared from the ionizable lipid has good nano preparation property, and particularly has encapsulation efficiency superior to that of positive controls MC3, ALC-0315 and SM-102.
EXAMPLE 5 cell transfection and cytotoxicity evaluation of LNPs@FLuc mRNA
The preparation property of the ionizable lipid nanoparticle provided by the invention is verified in the previous example, and furthermore, the inventor takes EGFP mRNA and FLuc mRNA as reporter genes, and evaluates the in vitro effect and safety of LNPs@mRNA provided by the invention through transfection experiments and toxicity experiments on HEK293T and DC 2.4.
LNPs@EGFP mRNA transfection experiments: HEK293T and DC2.4 cells were plated. LNPs@EGFP mRNA, positive control preparation MC3-LNPs@EGFP mRNA, ALC-0315-LNPs@EGFP mRNA, SM-102-LNPs@EGFP mRNA were then prepared according to the method described in example 4; the preparation for administration was controlled to have an mRNA concentration of 0.02. Mu.g/. Mu.L, and 50. Mu.L, i.e., 1. Mu.g/well, was administered per well of the 24-well cell plate, and the culture was continued in an incubator at 37℃with 5% CO2 for 24 hours. After 24h, flow cytometry detected GFP positive rate and mean fluorescence intensity (mean fluorescence intensity, MFI).
LNPs@FLuc mRNA transfection experiments: HEK293T and DC2.4 cells were plated. LNPs@FLuc mRNA, positive control preparation MC3-LNPs@FLuc mRNA, ALC-0315-LNPs@FLuc mRNA, SM-102-LNPs@FLuc mRNA were prepared according to the method described in example 4; the preparation for administration was controlled to have an mRNA concentration of 0.02. Mu.g/. Mu.L, and 50. Mu.L, i.e., 1. Mu.g/well, was administered per well of the 24-well cell plate, and the culture was continued in an incubator at 37℃with 5% CO2 for 24 hours. After 24h, 10. Mu.L of substrate fluorescein (potassium fluorescein salt, 15 mg/mL) was added to each well, and after 10min of standing in the dark, the bioluminescence was detected by an ELISA reader.
Cytotoxicity experiment: LNPs@FLuc mRNA and a positive control preparation C12-200-LNPs@FLuc mRNA were selected to examine DC2.4 cell MTT toxicity. The key experimental parameters are as follows: administration of 10 μl per well in a 96-well plate; culturing in an incubator with 5% CO2 at 37 ℃ for 24 hours; 5mg/mL of MTT solution was added to 40. Mu.L of the above MTT solution per well, and incubated at 37℃in an incubator with 5% CO2 for 4 hours.
The results of the cell transfection experiments are shown in figure 3: on HEK-293T and DC2.4, the mRNA expression capacity of the II-1@EGFP provided by the invention is stronger in the cellular level than that of positive controls MC3 and ALC-0315.
The results of the MTT cytotoxicity assay are shown in FIG. 4: incubation of LNPs@FLuc mRNA for 24h was not significantly toxic to DC2.4 cells.
EXAMPLE 6 in vivo expression and distribution of LNPs@mRNA
Further, the ability of lnps@mrna of the invention to deliver mRNA in vivo was examined.
In vivo expression investigation of intravenous route: LNPs@Fluc mRNA preparation and positive Control preparation C12-200@Fluc mRNA were prepared according to the method described in example 4, the mRNA concentration of the preparation was adjusted to 0.05mg/mL with PBS solution (RNase-free water formulation), the osmotic pressure of the preparation was adjusted to be isotonic, 200. Mu.L, i.e.10. Mu.g of Fluc mRNA per mouse was intravenously injected per C57BL/6 mouse, 3 in each group, and PBS was used as negative Control. Mice were kept on normal diet after dosing. After 6h of administration, 200. Mu.L of substrate solution (15 mg/mL, potassium salt of fluorescein) was intraperitoneally injected. Timing is started after substrate injection, euthanasia is performed after 10min, heart, liver, spleen, lung and kidney are rapidly released, and bioluminescence intensity of each isolated organ is detected in an IVIS instrument, and exposure time is 60s. Total flux of each organ was counted after the completion of imaging.
In vivo expression investigation of intramuscular route: LNP@Fluc mRNA, positive Control preparation MC3-LNPs@Fluc mRNA, ALC-0315-LNPs@Fluc mRNA, SM-102-LNPs@Fluc mRNA were prepared according to the method described in example 4, the mRNA concentration of the preparation was adjusted to 0.1mg/mL with PBS-containing solution (RNase-free water formulation) while the osmotic pressure of the preparation was adjusted to be isotonic, 200. Mu.L, i.e., 20. Mu.g of FLuc mRNA per hind leg muscle of each BALB/c mouse was injected, 3 in each group with PBS as negative Control. Mice were kept on normal diet after dosing. After 8h of administration, 200. Mu.L of substrate solution (15 mg/mL, potassium salt of fluorescein) was intraperitoneally injected, i.e., 3 mg/mouse. Timing is started after the substrate is injected, the mice are put into an air-anaesthesia device after 10min, the bioluminescence intensity of the whole body of a living body is detected in an IVIS instrument after complete anesthesia, and the exposure time is 60s. Normal diet after resuscitation. The above anesthesia and detection were repeated 24 hours and 48 hours after administration. Total flux was counted and luminescence intensity versus time was plotted.
The in vivo expression results of the intravenous route are shown (as shown in fig. 5 and 6): unexpectedly, the lnps@mrna of the invention is unexpectedly highly expressed in the lungs via the intravenous route compared to control C12-200, which would be advantageous over nucleic acid drugs targeting the lungs; meanwhile, the LNPs@mRNA has a certain expression level in the spleen, which suggests that the LNPs@mRNA can be used for nucleic acid medicines in intravenous administration routes.
The test results indicate that the LNPs can be further used for preparing nucleic acid medicaments for targeting lung organs.
EXAMPLE 7 immune anti-tumor Effect of LNPs@OVA mRNA
An E.G7-OVA (ovabumin Ovalbumin) tumor model is further constructed, OVA-mRNA is selected as a model antigen to evaluate the effectiveness of LNPs@mRNA as an mRNA tumor vaccine delivery system, so that the mRNA tumor vaccine delivery system with the efficient tumor inhibiting effect is screened out, and the potential influence of the ionizable lipid chemical structure on the effect of the LNPs@mRNA tumor vaccine is examined.
The E.G7-OVA tumor model building method is as follows: E.G7-OVA cells were cultured to logarithmic phase, cells were collected, washed with sterile PBS, centrifuged to discard the supernatant, resuspended in sterile PBS, and the cell concentration was adjusted to 10 7 /mL. Right side rib of each male C57BL/6 mouse was inoculated subcutaneously with 100. Mu.L of E.G7-OVA cells, 10 6 /only. Observing the growth state of mice and the size of subcutaneous tumor, and forming macroscopic tumor after 5 days of inoculation, thus carrying out immunotherapy experiment. Compared with normal mice, the tumor-bearing mice have no obvious difference in activity, appetite, stool and urine and other reactions. Reference is made to: luo, x.; li, B; zhang, x.; zhao, w.; bratasz, a.; deng, b.; mcComb, d.w.; dong, Y., dual-functional lipid-like nanoparticles for delivery of mRNA and MRI contrast agents, nanoscales 2017,9 (4), 1575-1579, xiong, H.; liu, s; wei, T.; cheng, q.; siegwart, d.j., theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo.j Control Release 2020,325,198-205.
E.G7-OVA cells (mouse T lymphoma cells) were purchased from ATCC and cultured in 1640 complete medium (containing 10% FBS and 1% P/S) at 37℃in an incubator with 5% CO2 at a cell density of 80-90% for passage.
The experimental protocol was as follows: LNPs@OVAmRNA was prepared by a microflow controller technique, and its effect on activation of BMDC in vitro and on cytokine secretion was examined.
BMDC maturation activation investigation method: cell plating. LNPs@OVAmRNA and positive control preparation C12-200-LNPs@OVAmRNA were prepared according to the method described in example 4, and the concentration of mRNA in the preparation to be administered was controlled to be 0.05. Mu.g/. Mu.L. Each well was dosed with 20. Mu.L, i.e.1. Mu.g/well and incubated for 24h at 37℃in an incubator with 5% CO 2. Dyeing and detecting: after the incubation, cells from the 24-well plate were collected into a flow tube and washed twice with pre-chilled 1mL PBS at 1500rpm for 5 min. mu.L of the above cell suspension and 1. Mu.LAnti-mouse CD16/32 were added to each flow tube, and after mixing, incubation was performed at 4℃for 15min to block non-specific binding. mu.L of PE-anti-mouse CD11c, FITC-anti-mouse CD80, APC/Cy7-anti-mouse CD86 and APC-anti-mouse H-2Kb bound to SIINFEKL streaming antibody were added to the corresponding streaming tube, respectively, and incubated at 4℃for 40min. After the incubation, the cells were washed twice by centrifugation at 1500rpm for 5min with 1mL of pre-chilled PBS, and then mixed with 300. Mu.L of PBS, and the BMDC maturation activation state was detected by flow cytometry.
And (3) cytokine detection, namely detecting LNPs@OVA mRNA vaccine to stimulate BMDC maturation and activation d, and collecting cell supernatant after incubation is finished, diluting the cell supernatant by 5 times by using a sample diluent in an ELISA kit, and detecting. The standard substance and the enzyme-labeled antibody are diluted according to the instruction of the kit, a standard working curve of TNF-alpha is established, and the level of BMDC secretion TNF-alpha in cell supernatant is detected by a double antibody sandwich method.
The results show that: the mRNA vaccine based on the piperazine-containing ionizable lipid and the derivative or analogue thereof is superior to positive control C12-200 (shown in figure 8) in terms of BMDC maturation activation (shown in figure 7) and secretion of cytokine TNF-alpha.
In the drawings, ns is not marked as a significant difference unless otherwise specified; * P <0.05; * P <0.01; * P <0.001; * P <0.0001.
It is to be noted that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments described in this specification, as well as the features of the various embodiments, can be combined and combined by one skilled in the art without contradiction.

Claims (21)

1. A compound of formula i, or a pharmaceutically acceptable salt, isomer, deuterate, or prodrug thereof:
wherein a is 1,2 or 3;
X 1 、X 2 each independently selected from N or C;
L 1 、L 2 、L 3 、L 4 independently selected from-R e CH(OH)-、-R e C(＝O)-、-R e C(＝O)O-、-
R e OC(＝O)-、-R e C(＝O)S-、-R e SC(＝O)-、-R e C(＝O)NR a -、-R e NR a C(＝O)-、-
R e NR a C(＝O)O-、-R e OC(＝O)NR a -、-R e O-、-R e -O-O-、-R e S-、-R e -S-S-、-R e -S-S-
S-、-R e CH(OH)CH 2 O-、-R e CH(OH)CH 2 S-or absent, R e Is thatOr is absent, k is an integer greater than 1, R a -H, substituted or unsubstituted alkyl;
R 1 、R 2 、R 3 、R 4 independently selected from C 1 -C 30 Straight chain alkyl, C 1 -C 30 Branched alkyl, C 2 -C 30 Straight chain alkenyl, C 2 -C 30 Branched alkenyl, C 2 -C 30 Straight chain alkynyl or C 2 -C 30 Branched alkynyl groups;
G 1 、G 2 、G 3 、G 4 independently selected from-R c -、-R c CH(OH)R d -、-R c C(＝O)R d -、-
R c C(＝O)OR d -、-R c OC(＝O)R d -、-R c C(＝O)SR d -、-R c SC(＝O)R d -、-
R c C(＝O)N(R b )R d -、-R c N(R b )C(＝O)R d -、-R c N(R b )C(＝O)OR d -、-
R c OC(＝O)N(R b )R d -、-R c OR d -、-R c -O-O-R d -、-R c SR d -、-R c -S-S-R d -、-R c -S-S-S-
R d -or not present, R b is-H, substituted or unsubstituted alkyl, R c 、R d Independently selected fromOr is absent, n is an integer greater than 1;
R 5 、R 6 independently selected from-H, substituted or unsubstituted alkyl;
Q 1 、Q 2 independently selected from O or S.
2. The compound of claim 1, wherein the compound has a structure represented by formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuterate or prodrug of a structure represented by formula II,
3. the compound of claim 1 or 2, wherein k is 1,2,3,4,5,6,7,8,9 or 10;
preferably, k is 1.
4. A compound according to claim 1 or 2, the L1, L2, L3, L4 are independently selected from-CH (OH) -, -C (=O) -, -CH2C (=O) O-, -OC (=O) -, -C (=O) S-, -SC (=O) -, -CH2C (=O) NRa-, -NRaC (=O) O-, -OC (=O) NRa-, -CH2O-, -CH2-O-, -CH2S-, -CH2-S-S-, -CH (OH) CH2O-, -CH (OH) CH2S-, or are absent, ra is-H, substituted or unsubstituted alkyl;
Preferably, the L 1 、L 2 、L 3 、L 4 Independently selected from-C (=o) -, -C (=o) NR a -、-CH 2 C(＝O)NR a -、-NR a C(＝O)-、-C(＝O)O-、-CH 2 C(＝O)O-、-OC(＝O)-、-CH 2 O-、-O-、-CH 2 S-、-S-、-CH(OH)-、-CH(OH)CH 2 O-、-CH(OH)CH 2 S-or absent, R a is-H or unsubstituted alkyl.
5. The compound of any one of claims 1 to 4, wherein R a is-H or unsubstituted C 1 ～C 6 An alkyl group;
preferably, said R a is-H.
6. The compound of claim 1 or 2, wherein L 1 、L 2 、L 3 、L 4 Independently selected from-C (=o) -, -C (=o) NH-, -CH 2 C(＝O)NH-、-C(＝O)O-、-CH 2 C(＝O)O-、-CH 2 O-、-CH 2 S-、-CH(OH)-、-CH(OH)CH 2 O-or absent;
preferably L 1 、L 2 、L 3 、L 4 Independently selected from-C (=O) NH-, -C (=O) O-, -CH (OH) CH 2 O-or absent.
7. The compound of any one of claims 1 to 6, wherein L 1 And L 2 Selected from the same groups and/or L 3 And L 4 Selected from the same groups;
optionally, the L 1 And L 3 Selected from the same groups and/or L 2 And L 4 Selected from the same groups;
preferably, the L 1 、L 2 、L 3 And L 4 Selected from the same groups.
8. The compound of claim 1 or 2, wherein R 1 、R 2 、R 3 、R 4 Independently selected from C 1 -C 30 Straight chain alkyl, C 2 -C 30 Straight chain alkenyl, C 2 -C 30 Straight chain alkynyl;
preferably, said R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 1 ～C 30 A linear alkyl group;
preferably, said R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 8 ～C 18 A linear alkyl group;
preferably, said R 1 、R 2 、R 3 、R 4 Independently selected from unsubstituted C 10 ～C 14 A linear alkyl group.
9. The compound of any one of claims 1 to 8, wherein R 1 And R is 2 Selected from the same groups and/or R 3 And R is 4 Selected from the same groups;
optionally, the R 1 And R is 3 Selected from the same groups and or R 2 And R is 4 Selected from the same groups;
preferably, said R 1 、R 2 、R 3 And R is 4 Selected from the same groups.
10. The compound of claim 1 or 2, wherein G 1 、G 2 、G 3 、G 4 Independently selected fromOr absent, n3 is 1,2,3,4,5,6,7,8,9, or 10;
preferably, the G 1 、G 2 Independently selected from-CH 2 -or-CH 2 CH 2 -；
Preferably, the G 3 、G 4 Is not present.
11. The compound of claim 1 or 2, wherein R 5 、R 6 Independently selected from-H, unsubstituted C 1 ～C 6 C substituted by alkyl or-OH 1 ～C 6 An alkyl group;
preferably, said R 5 、R 6 Independently selected from-H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl, or hydroxypropyl;
preferably, said R 5 、R 6 Selected from the same groups.
12. A compound characterized by one of the following structures;
，
pharmaceutically acceptable salts, stereoisomers, deuterides or prodrugs thereof.
13. Use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, isomer, deuteride or prodrug thereof, in the preparation of a drug delivery vehicle; further, the active ingredient of the medicament is optionally selected from at least one of nucleic acid, small molecule drug, protein drug, polypeptide; preferably, the active ingredient of the medicament is selected from the group consisting of nucleic acids; preferably, the medicament has cardiac, liver, spleen, lung or kidney targeting; preferably, the drug targets the lung, spleen.
14. A drug delivery vehicle comprising a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, stereoisomer, deuterate or prodrug thereof.
15. A pharmaceutical complex comprising a carrier and an active ingredient, the carrier being associated with the active ingredient, the carrier comprising a cationic lipid comprising a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, stereoisomer, deuterate or prodrug thereof; optionally, the active ingredient of the medicament is optionally selected from at least one of nucleic acid, small molecule drug, protein drug, polypeptide, antibody; preferably, the active ingredient of the medicament is selected from the group consisting of nucleic acids; optionally, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, an aptamer; optionally, the nucleic acid is mRNA; optionally, the delivery vehicle is ionically linked to the mRNA; further, the drug targets the lung, spleen;
optionally, the preparation form of the drug complex is selected from one of lipid nanoparticle LNP, PLGA nanoparticle, micelle, liposome, core-shell nanoparticle and polymer nanoparticle;
Preferably, the pharmaceutical complex is in the form of a formulation of lipid nanoparticle LNP.
16. The pharmaceutical composition of claim 15, further comprising at least one excipient selected from the group consisting of neutral phospholipids, steroids, and pegylated lipids;
optionally, the neutral phospholipid is selected from DOPE, DSPC, DOPC, DSPE, DMPC,
DMPE, DPPC, DPPE, DEPC, HSPC, POPC;
preferably, the neutral phospholipid is DOPE;
optionally, the steroid is selected from cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol
At least one of an alcohol and a fucosterol;
preferably, the steroid is cholesterol;
optionally, the pegylated lipid is selected from at least one of DMG-PEG and DSPE-PEG;
preferably, the pegylated lipid is DMG-PEG2000.
17. The pharmaceutical complex of claim 15, wherein the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride, or a prodrug thereof, to the neutral phospholipid is from 1:10 to 10:1.
18. The pharmaceutical complex of claim 15, wherein the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride, or a prodrug thereof, to the pegylated lipid is from 5:1 to 1000:1;
Preferably, the molar ratio of the compound, or a pharmaceutically acceptable salt, an isostere, a deuteride or a prodrug thereof, to the pegylated lipid is from 10:1 to 20:1.
19. The pharmaceutical complex of claim 15, wherein the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, an aptamer;
preferably, the nucleic acid is mRNA.
20. Use of a pharmaceutical complex according to any one of claims 15 to 19 in the manufacture of a medicament for the treatment or prophylaxis of a disease;
optionally, the disease is a heart, liver, spleen, lung or kidney related disease;
preferably, the disease is a spleen or lung related disease;
optionally, the disease is an infectious disease, cancer and proliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, cardiovascular and renal vascular disease, and metabolic disease;
preferably, the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or various herpes;
preferably, the cancer is a solid tumor;
preferably, the cancer is liver cancer or lung cancer.
21. The use according to claim 20, wherein the medicament treats or prevents a disease by presenting an antigen and/or activating an immune response.
CN202310287179.2A 2022-06-20 2023-06-15 Ionizable lipids and compositions thereof for nucleic acid delivery Pending CN116589435A (en)

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WO2018232120A1 (en) * 2017-06-14 2018-12-20 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2019226650A1 (en) * 2018-05-23 2019-11-28 Modernatx, Inc. Delivery of dna
US20230009009A1 (en) * 2018-09-13 2023-01-12 Modernatx, Inc. Polynucleotides encoding glucose-6-phosphatase for the treatment of glycogen storage disease
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