Lipids conjugated with poly(ethylene oxide) bearing C1-C3-alkyloxymethyl side chains

Abstract

The present invention relates to novel polyoxyalkylene compounds and methods for their preparation, as well as compositions comprising at least one novel polyoxyalkylene compound and at least one active agent. The present invention further relates to the preparation of the compositions of the invention and their use for the treatment of diseases in mammals or humans.

Description

[Technical Field]

[0001] The present invention relates to a novel polyoxyalkylene compound and its preparation method, and to a composition comprising at least one novel polyoxyalkylene compound and at least one active agent.In particular, the compound is suitable as a novel lipid that can be used in a composition suitable for lipid nanoparticles, and optionally contains other lipid components.The composition is particularly suitable as a delivery vehicle for at least one active agent, for facilitating the intracellular delivery of therapeutic nucleic acid.

[0002] Background technology Polyethylene oxide (PEO)-based lipids are an important class of excipients. Early publications on such lipids include Frisch et al., Bioconjugate Chemistry (2004), 15(4), 754–764. They are used in lipid nanoparticle formulations, such as vaccines against COVID-19. The primary function of PEO is its so-called "stealth effect," which allows it to avoid recognition by the patient's reticuloendothelial system. The main concern with PEO is the immune response of the patient's body, which can result in the formation of anti-PEO antibodies, as disclosed, for example, in J. Selva et al., ACS Nano (2022), 16(8), 11769–11780. Anti-PEO antibodies can result in the loss of the desired "stealth effect," loss of drug function, and allergic reactions.

[0003] Therefore, alternative compounds that have a "stealth effect" but have reduced or no antibody formation, ie, immunogenic potential, are needed.

[0004] The present inventors have surprisingly found that the above-mentioned problems can be solved by the specific polyoxyalkylene compounds according to the present invention, which have polyoxyalkylene units different from PEO.Furthermore, the obtained compounds surprisingly show better storage stability than similar common PEO lipids.

[0005] Summary of the Invention Thus, in a first aspect, the present invention provides a compound of formula (I): [ka] For compounds having the formula In the formula, R 1 and R 2 is R 1 and R 2 saturated or unsaturated, branched or linear C3-C with up to three C=C bonds, provided that at least one of the 20 are independently selected from a hydrocarbon group or -H; L is a linker unit, preferably [ka] wherein in L6, X + is hydrogen, an alkali metal cation or ammonium, preferably an alkali metal cation; In L1 to L7, the ends marked with * are bonded to the oxygen atoms of the polyoxyalkylene group A, A is a polyoxyalkylene group having at least one of the following units: [ka] and [ka] and [ka] and at least one unit selected from the group R 3 -H, -OH, -SH, -NH2, -NHR 4 , -NR 4 R 5 , -OR 6 , -SR 6 or a linear, branched or cyclic alkyl group having up to 20 carbon atoms, R 4 ~R6 are independently selected from linear, branched, or cyclic alkyl groups having up to 20 carbon atoms, in which up to 5 carbon atoms may be substituted with oxygen or sulfur atoms; -AR 3 has a molecular weight of 1100 to 7500 g / mol.

[0006] In a second aspect, the present invention relates to a composition comprising at least one compound of formula (I) according to the invention and at least one active agent.

[0007] In a third aspect, the present invention relates to a method for preparing a compound of formula (I) according to the present invention, the method comprising: A and R 3 is defined by formula (I), 3 preparing a i) replacing -H with a leaving group -X capable of undergoing a substitution or coupling reaction; or ii) -H is unit a) [ka] provided that it is bonded to the oxygen atom of [ka] oxidizing this group to form a terminal group; Then, R 1 and R 2 is defined in formula (I), and L is a linker unit HL or H-LR 1 R 2 with the proviso that when step ii) is used, [ka] The remaining terminal group of [ka] is immediately considered to be part of the linker group L, and when HL is used, L may be further chemically modified by an esterification reaction to form -R 1 and -R 2 Introduce.

[0008] In a fourth aspect, the present invention relates to a method for producing a composition according to the present invention, the method comprising the steps of providing at least one compound of formula (I) according to the present invention, at least one active agent and optionally further ingredients, and combining all ingredients to obtain a composition according to the present invention.

[0009] In a fifth aspect, the present invention relates to a composition according to the invention for treating a disease in a human.

[0010] In a sixth aspect, the present invention relates to a composition according to the invention for treating a disease in a mammal.

[0011] These and other aspects, embodiments, features, and advantages of the present invention will become apparent to those skilled in the art through a study of the following detailed description and claims. Any feature from one aspect of the present invention can be used in any other aspect of the invention. Furthermore, it will be readily understood that the examples included herein are intended to explain and illustrate the present invention, but are not intended to limit the invention, and in particular, the present invention is not limited to these examples. [Brief explanation of the drawings]

[0012] [Figure 1a] Agarose gel electrophoresis (AGE) of LNPs formulated with different PEO / GME lipids. M: RNA ladder; 1, 2: free FLuc mRNA; 4: m(8); 5: i(8); 6: h(8); 7: k(8); 8: j(8). [Figure 1b] 1: Agarose gel electrophoresis (AGE) of LNPs formulated with different PEO / GME lipids. 1: Free FLuc mRNA, 2: PEO2k-DMG LNPs, 3: n(11) [Figure 2]Figure 1 shows the transfection efficiency of LNPs formulated with different PEO / GME lipids as assessed by luciferase assay in different cell lines. [Figure 3] FIG. 1 shows an ELISA assay using different N,N-ditetradecylacetamide lipids. [Figure 4] FIG. 1 shows an ELISA assay using different (1,2-dimyristoyl-glycerol) (DMG) lipids.

[0013] MODE FOR CARRYING OUT THE INVENTION As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0014] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises," "comprising," "contain," and "containing," are to be construed in their open and inclusive sense, i.e., "including but not limited to."

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0016] Numerical ranges expressed in the format "x to y" are inclusive of the recited values. When several preferred numerical ranges are expressed in this format, it is clear that all ranges resulting from combining the various endpoints are also included.

[0017] As used herein, "one or more" refers to at least one and includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or more of the referenced species. Similarly, "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more. As used herein with respect to any component, "at least one" refers to the number of chemically distinct molecules, i.e., the number of different types of the referenced species, and not the total number of molecules. For example, "at least one therapeutic agent" means that at least one type of molecule included within the definition of therapeutic agent is used, but also means that two or more different types of therapeutic agents included within this definition can be present, not that only one or more molecules of one type of therapeutic agent are present.

[0018] All percentages given herein with respect to compositions relate to weight percent based on the total weight of the respective composition, unless otherwise specified.

[0019] "Essentially free" according to the present invention in relation to compounds means that the compounds may only be present in amounts that do not affect the properties of the composition, in particular that the respective compound is present in less than 3% by weight, preferably less than 1% by weight, more preferably less than 0.01% by weight relative to the total weight of the composition, or is not present at all.

[0020] The term "nucleic acid" as used herein refers to a compound containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded, double-stranded, or triple-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA (pDNA), linear or circular DNA, PCR product, or vector. RNA can be in the form of self-amplifying RNA (saRNA) or small hairpin RNA (shRNA), small interfering RNA (siRNA), chemically modified or unmodified messenger RNA (mRNA), antisense RNA, circular RNA (circRNA) containing at least one coding sequence, microRNA (miRNA), micRNA, polyvalent RNA, transfer RNA (tRNA), single guide RNA (sgRNA), replicative RNA (repRNA), dicer substrate RNA or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have binding properties similar to those of the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs).Unless specifically limited, this term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid.Unless otherwise indicated, a specific nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequence explicitly indicated.

[0021] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are generally poorly soluble in water but soluble in many organic solvents. They are typically divided into at least three classes: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0022] "Cationic lipid" refers to a lipid that can be positively charged.Exemplary cationic lipids contain one or more amine groups with positive charge.Preferred cationic lipids are ionizable, so that they can exist in positively charged or neutral form depending on pH.The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions.

[0023] The term "neutral lipid" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at selected pH values.

[0024] The term "ionizable lipid" refers to any of several lipid species that exist in either a positively or negatively charged form independent of pH within a useful physiological range, e.g., a pH of about 3 to about 9. Ionizable lipids can be synthetic or naturally derived.

[0025] An "effective amount" or "therapeutically effective amount" of an active agent, such as a nucleic acid, is an amount sufficient to produce a desired effect, such as an increase or inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of nucleic acid.Increased expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of nucleic acid.If the expression product is present at some level before contact with nucleic acid, increased expression is achieved when the fold increase in the value obtained using nucleic acid such as mRNA is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10,000 or more compared to the control. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using a nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% compared to the control. Suitable assays for measuring the expression of a target gene or target sequence include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence, or luminescence of appropriate reporter proteins, as well as phenotypic assays known to those skilled in the art.

[0026] The disclosure disclosed herein is also intended to encompass all pharmaceutically acceptable compounds of the compounds of formula (I), which are their pharmaceutically acceptable salts and / or are isotopically labeled by having one or more atoms replaced by atoms with different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine.

[0027] The embodiments disclosed herein are also meant to encompass the in vivo metabolic products of the compounds according to the invention. Such products may result, for example, from the oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a process comprising administering to a mammal a compound of the present disclosure for a period of time sufficient to yield a metabolic product thereof.

[0028] The compounds of the present invention, including their pharmaceutically acceptable salts, can contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)- for amino acids, or as (D)- or (L)-. The present invention is meant to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When compounds described herein contain an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0029] "Stereoisomer" refers to a compound made up of the same atoms connected by the same bonds, but with different, incompatible three-dimensional structures. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0030] "Tautomer" refers to a proton migration from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any such compounds.

[0031] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0032] "Pharmaceutically acceptable acid addition salts" are those which retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and include those formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gen This refers to salts formed with thicic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0033] The present invention particularly relates to a compound of formula (I) [ka] For compounds having the formula In the formula, R 1 and R 2 is R 1and R 2 saturated or unsaturated, branched or linear C3-C with up to three C=C bonds, provided that at least one of the 20 are independently selected from a hydrocarbon group or -H; L is a linker unit, preferably [ka] wherein in L6, X + is hydrogen, an alkali metal cation or ammonium, In L1 to L7, the ends marked with * are bonded to the oxygen atoms of the polyoxyalkylene group A, A is a polyoxyalkylene group and has at least one unit [ka] and [ka] and [ka] and at least one unit selected from the group R 3 -H, -OH, -SH, -NH2, -NHR 4 , -NR 4 R 5 , -OR 6 , -SR 6 or a linear, branched or cyclic alkyl group having up to 20 carbon atoms, R 4 ~R 6 are independently selected from linear, branched, or cyclic alkyl groups having up to 20 carbon atoms, in which up to 5 carbon atoms may be substituted with oxygen or sulfur atoms; -AR 3 has a molecular weight of 1100 to 7500 g / mol, preferably 1500 to 3500 g / mol, and more preferably 2000 to 3000 g / mol.

[0034] Molecular weight is calculated using the -AR method as used in chemistry. 3 It may be a calculated theoretical molecular weight of, for example, the unit [ka] has a theoretical molecular weight of 44 g / mol and has units [ka] has a theoretical molecular weight of 88 g / mol, rounded to the nearest integer. Alternatively, the molecular weight can be determined by size exclusion chromatography of the precursor HAR 3 The weight-average molecular weight can be determined by measuring the weight-average molecular weight of the polymer and subtracting the theoretical molecular weight of either -H (1 g / mol; method (i)) or -C2H4OH (46 g / mol; method (ii)), taking into account the actual reaction mechanism described below. Size exclusion chromatography can be preferably performed on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column at 50 °C using dimethylformamide (DMF containing 1 g / L LiBr) as the mobile phase (flow rate 1 mL / min). The polymer concentration was 1 mg / mL. Calibration was performed using poly(ethylene glycol) standards (from Polymer Standard Service, Mainz, Germany).

[0035] The unit L is bonded to an oxygen atom of the polyoxyalkylene group A, in particular the oxygen atom is the terminal oxygen atom of the polyoxyalkylene group A. The unit L can be any trivalent unit, preferably suitable for pharmaceutical use, to provide a compound of formula (I) that is a lipid.

[0036] The compounds of formula (I) are suitable as lipids.

[0037] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C4-C alkyl group with up to two -C=C- bonds 20are independently selected from a hydrocarbon group or -H.

[0038] In one embodiment, R 1 and R 2 is a saturated or unsaturated, straight-chain C4-C alkylene compound with up to two -C=C- bonds. 20 are independently selected from a hydrocarbon group or -H.

[0039] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C4-C alkylene compound with one -C=C- bond. 20 are independently selected from a hydrocarbon group or -H.

[0040] In one embodiment, R 1 and R 2 is a saturated or unsaturated, straight-chain C4-C alkylene compound with one -C=C- bond. 20 are independently selected from a hydrocarbon group or -H.

[0041] In one embodiment, R 1 and R 2 is a saturated, branched or straight chain C4-C 20 are independently selected from a hydrocarbon group or -H.

[0042] In one embodiment, R 1 and R 2 is a saturated straight chain C4-C 20 are independently selected from a hydrocarbon group or -H.

[0043] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C8-C alkylene compound with up to two -C=C- bonds 18 are independently selected from a hydrocarbon group or -H.

[0044] In one embodiment, R 1 and R 2is a saturated or unsaturated, linear C8-C 18 are independently selected from a hydrocarbon group or -H.

[0045] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C8-C alkylene compound having one -C=C- bond. 18 are independently selected from a hydrocarbon group or -H.

[0046] In one embodiment, R 1 and R 2 is a saturated or unsaturated, straight-chain C8-C 18 are independently selected from a hydrocarbon group or -H.

[0047] In one embodiment, R 1 and R 2 is a saturated, branched or straight chain C8-C 18 are independently selected from a hydrocarbon group or -H.

[0048] In one embodiment, R 1 and R 2 is saturated straight chain C8-C 18 are independently selected from a hydrocarbon group or -H.

[0049] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0050] In one embodiment, R 1 and R 2 is a saturated or unsaturated linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0051] In one embodiment, R 1 and R 2 is a saturated or unsaturated, branched or linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0052] In one embodiment, R 1 and R 2 is a saturated or unsaturated linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0053] In one embodiment, R 1 and R 2 is a saturated, branched or linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0054] In one embodiment, R 1 and R 2 is a saturated linear C 12 ~C 17 are independently selected from a hydrocarbon group or -H.

[0055] In one embodiment, R 1 and R 2 is a saturated linear C 12 ~C 14 are independently selected from a hydrocarbon group or -H.

[0056] In one preferred embodiment, R 1 and R 2 is the same.

[0057] In one preferred embodiment, L is [ka] Selected from X +is hydrogen, ammonium, sodium or potassium, preferably sodium or potassium, and the ends marked with * are bonded to the oxygen atoms of the polyoxyalkylene group A.

[0058] In one preferred embodiment, L is [ka] The terminals marked with * are each bonded to an oxygen atom of the polyoxyalkylene group A.

[0059] In one preferred embodiment, L is [ka] and the end marked with * is bonded to the oxygen atom of the polyoxyalkylene group A.

[0060] In one preferred embodiment, L is [ka] and the end marked with * is bonded to the oxygen atom of the polyoxyalkylene group A.

[0061] In one preferred embodiment, L is [ka] and the end marked with * is bonded to the oxygen atom of the polyoxyalkylene group A.

[0062] In one embodiment, the compound of formula (I) is of formula (Ia): [ka] In the formula, R 1 and R 2 are independently saturated linear C 12-18 , preferably C 12-16 , more preferably C 12-14hydrocarbon groups, preferably R 1 and R 2 are the same, and most preferably C 14 is a hydrocarbon group, R 3 HA-OR 6 where R 6 is selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, of which up to 5 carbon atoms may be replaced by oxygen atoms; Preferably, R 3 is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, decanoxy, 2-ethylhexoxy, dodecan-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propan-2-oxy, 1-octadecanoxy, 3-methylbutan-1-oxy, phenylmethanoxy, 3-ethyl-butoxy, and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy; A is defined as in formula (I).

[0063] In one embodiment, the compound of formula (I) is of formula (Ib): [ka] In the formula, R 1 and R 2 are independently saturated linear C 12-18 , preferably C 12-16 , more preferably C 12-14 hydrocarbon groups, preferably R 1 and R 2 are the same, and most preferably C 13 is a hydrocarbon group, R 3 HA-OR 6 where R 6 is selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, of which up to 5 carbon atoms may be replaced by oxygen atoms; Preferably, R 3is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, decanoxy, 2-ethylhexoxy, dodecan-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propan-2-oxy, 1-octadecanoxy, 3-methylbutan-1-oxy, phenylmethanoxy, 3-ethyl-butoxy, and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy; A is defined as in formula (I).

[0064] In one embodiment, the compound of formula (I) is of formula (Ic): [ka] In the formula, R 1 and R 2 are independently saturated linear C 12-18 , preferably C 14-18 , more preferably C 16-17 hydrocarbon groups, preferably R 1 and R 2 are the same, and most preferably C 17 is a hydrocarbon group, R 3 -H, -OH, -SH, -NH2, -NHR 4 , -NR 4 R 5 , -OR 6 , -SR 6 or a linear, branched or cyclic alkyl group having up to 20 carbon atoms, R 4 ~R 6 are independently selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, of which up to 5 carbon atoms may be replaced by oxygen or sulfur atoms; Preferably, R 3 is -NH2 or -OR 6 where R 6 is selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, of which up to 5 carbon atoms may be replaced by oxygen atoms; More preferably, R 3 is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, decanoxy, 2-ethylhexoxy, dodecan-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propan-2-oxy, 1-octadecanoxy, 3-methylbutan-1-oxy, phenylmethanoxy, 3-ethyl-butoxy, and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy; A is defined as in formula (I).

[0065] Base-AR 3 is the precursor HAR, in which -H is attached to the terminal oxygen atom of unit A. 3 Then, HAR 3 is further modified, followed by substitution, coupling or polycondensation reactions to give compounds of formula (I).

[0066] HAR 3 is preferably Ethylene oxide at least one comonomer selected from 2-(methoxymethyl)oxirane (glycidyl methyl ether), 1,2-epoxy-3-ethoxypropane, 1,2-epoxy-3-n-propoxypropane, 1,2-epoxy-3-iso-propoxypropane, preferably at least one comonomer which is 2-(methoxymethyl)oxirane, and -R as defined in the compounds of formula (I) 3 in the presence of a base with an initiator suitable for forming It is obtained by anionic ring-opening copolymerization.

[0067] The base is preferably a base having a pka of at least 16, preferably at least 19, more preferably potassium tert-butoxide.

[0068] In a preferred embodiment, a small amount of pure ethylene oxide is added after the copolymerization step so that 2 to 5 additional units derived from ethylene oxide are present at one or both ends of A.

[0069] HOCH2CH2-AR via anionic ring-opening copolymerization 3 The synthesis of HOCH2CH2-AR is described, for example, in PCT / EP2022 / 062896, which is incorporated by reference, and is also described in the literature as HOCH2CH2-AR 3 is called a polymer.

[0070] The anionic ring-opening copolymerization is carried out at a temperature in the range of preferably -10 to 90°C, more preferably -10 to 70°C, and most preferably -10 to 60°C.

[0071] To obtain the compound of formula (I), the precursor HAR 3 After obtaining i) replacing -H with a leaving group -X capable of undergoing a substitution or coupling reaction, such as tosylate, tosyl, mesylate or carbonyldiimidazole, preferably a tosylate or tosyl group or carbonyldiimidazole, or ii) -H is unit a) [ka] This group is bonded to the oxygen atom of [ka] To form the terminal group, for example, TEMPO / OCl - , 4-acetamido-TEMPO, 4-methoxy-TEMPO group, phenyliodine diacetate, phenyliodine bis(trifluoroacetate), preferably TEMPO / OCl-, Subsequent substitution or coupling (step i) or polycondensation (step ii) reactions yield HL or H-LR 1 R 2is used after step i) or step ii), provided that if step ii) is used, [ka] The remaining terminal group of [ka] is considered herein to be part of the linker group L, and when HL is used, L may be linked to -R, for example by an esterification reaction. 1 and -R 2 provided that it is further chemically modified to contain

[0072] The introduction of the leaving group -X and the oxidation reaction are well known to those skilled in the art.

[0073] The polyoxyalkylene group A comprises unit (a) and at least one of units (b) to (e). In one embodiment, the polyoxyalkylene group A consists essentially of or consists of unit (a) and at least one of units (b) to (e). In particular, the polyoxyalkylene group A is essentially free of or free of residues.

[0074] In a preferred embodiment, the polyoxyalkylene group A comprises or consists of units (a) and (b) and optionally further units selected from (c) to (e). In a preferred embodiment, the polyoxyalkylene group A comprises or consists of units (a) and (b).

[0075] For clarity, a passage containing unit (a) does not mean that only one unit (a) is present, but that at least one unit of (a) is present in the group, i.e., several monomer units derived from ethylene oxide may be present. For example, there may be 1 to 20 units (a) in group A. The same is true for passages containing at least one unit (b) to (e). However, unless explicitly defined otherwise, it is possible for only one (number) unit (a) or only one (number) unit (b) to (e) to be present in group A.

[0076] In a preferred embodiment, units (a) constitute 5-95% of group A, totalling 100% with the other units. In a more preferred embodiment, units (b) are present in up to 70% of group A, more preferably units (b) are present in 30-70% of group A, and most preferably units (a) are present in a further 30-70% to total 100%.

[0077] In a preferred embodiment, the molar ratio of (a) to (b) to (e), preferably (a) to (b), is 1:9 to 9:1, preferably 2:8 to 8:2, more preferably 3:7 to 7:3.

[0078] In a preferred embodiment, -AR 3 The polydispersity index (PDI) of the polymer is 1.15 or less, more preferably 1.10 or less, and most preferably 1.08 or less. Preferably, the weight-average and number-average molecular weights are determined using size-exclusion chromatography as described for Mw above. Size-exclusion chromatography is preferably performed on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column at 50°C using dimethylformamide (DMF containing 1 g / L LiBr) as the mobile phase (flow rate 1 mL / min). The polymer concentration was 1 mg / mL. Calibration was performed using poly(ethylene glycol) standards (Polymer Standard Service, Mainz, Germany).

[0079] In one embodiment, -AR3 has a molecular weight, preferably a weight average molecular weight, of 1500 to 3500 g / mol, preferably 2000 to 3000 g / mol. 3 is 1500g / mol, 1550g / mol, 1600g / mol, 1650g / mol, 1700g / mol, 1750g / mol, 1800g / mol, 1850g / mol, 1900g / mol, 1950g / mol or 2000g / mol, and and having a molecular weight, preferably a weight average molecular weight, within a range having in combination with any upper limit of 2350 g / mol, 2400 g / mol, 2450 g / mol, 2500 g / mol, 2550 g / mol, 2600 g / mol, 2650 g / mol, 2700 g / mol, 2750 g / mol, 2800 g / mol, 2850 g / mol, 2900 g / mol, 2950 g / mol, 3000 g / mol, 3050 g / mol, 3100 g / mol, 3150 g / mol, 3200 g / mol, 3250 g / mol, 3300 g / mol, 3350 g / mol, 3400 g / mol, 3450 g / mol, or 3500 g / mol.

[0080] base R 3 can be achieved by selecting the appropriate initiator and / or precursor HAR 3 The end groups can be modified by chemically modifying the end groups originally formed during the anionic ring-opening copolymerization in the formation of R. Such reactions are well known in the art. 3may be a functional group selected from, for example, acetal (dialkoxy), aldehyde (formyl), amide (carboxamide), azide, carbonate (alkoxycarbonyl)oxy), carboxyl (carboxy), carboxylic anhydride, ester (alkoxycarbonyl), ether, halo, haloformyl (carbonohalidoyl), hemiacetal (alkoxyol), hemiketal (alkoxyol), hydroxy, imide (imido), imine (imino), ketal (dialkoxy), ketone (oil), orthoester (trialkoxy), primary, secondary, and tertiary amino groups, primary, secondary, and tertiary alkoxy groups, sulfhydryl (sulfanyl, HS-), thioether, and combinations thereof. In a preferred embodiment, the terminal group is selected from the group consisting of alkyl, hydrogen, hydroxy, alkoxy, sulfanyl, phthalimide, amide, amine, and combinations thereof. The terminal group has the formula R-(CH2) n -O-, where R is linear, branched or cyclic alkyl or phenyl, and n is equal to 1 to 20.

[0081] In a preferred embodiment, R 3 HA-OR 6 where R 6 is selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, of which up to 5 carbon atoms may be substituted with oxygen atoms, more preferably R 3 is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, decanoxy, 2-ethylhexoxy, dodecane-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propan-2-oxy, 1-octadecanoxy, 3-methylbutan-1-oxy, phenylmethanoxy, 3-ethyl-butoxy, and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy. The end groups are selected from the end groups -R described above. 3The initiator can be an alkyl anion or a hydride anion, such as a metal alkyl or metal hydride compound of the formula (I). However, preferably, the alkoxy and thioalkoxy anions are not tertiary alkoxy anions. The imide anion is preferably a phthalimide anion. The metal counterion is preferably Na. + , K. + or Cs + is.

[0082] In one embodiment, the initiator is MeOCH2CH2O - , MeO(CH2CH2O)2 - , benzyl OCH2CH2O - , BzO(CH2CH2O)2 - , (Bz)2N-CH2CH2O - , (Bz)2N-(CH2CH2O)2 - , phthalimide-CH2CH2O - , phthalimide-(CH2CH2O)2 - where Me is methyl and Bz is benzyl. Most preferred is MeO(CH2CH2O)2 - , BzOCH2CH2O - and (Bz)2N-CH2CH2O - The counter ion is preferably Na + , K. + or Cs + is.

[0083] The initiator may be provided in an inert solvent, preferably an aprotic solvent, most preferably dimethyl sulfoxide (DMSO) or toluene, and the copolymerization reaction is preferably carried out in the same solvent.

[0084] The group -AR of the present invention 3 The end group fidelity of the chromatograms can be determined by MALDI TOF or by MALDI TOF and 1 By combining with H NMR, each precursor HAR 3 The group -AR of the present invention can be determined by known methods.3 preferably has an end group fidelity of at least 95%, more preferably at least 98%.

[0085] The polyoxyalkylene group A of the present invention may be a random copolymer. Such groups offer the lowest immunogenicity because they do not provide a blueprint for the immune system to generate antibodies. They are inherently resistant to immune responses and are therefore a preferred embodiment of the present invention.

[0086] In alternative embodiments, the polyoxyalkylene group A of the present invention may have a block-like structure or a tapered or gradient structure. Methods for preparing such polymers are known to those skilled in the art of polyoxyalkylenes. In such embodiments, it is preferred that no more than 5% of the groups A contain blocks with more than 15 repeating units derived from ethylene oxide, and more preferably no more than 5% of the macromolecules of the polymer contain blocks with more than 8 repeating units derived from ethylene oxide.

[0087] In many cases, crystallization results in a solvate of the compound of the present invention. As used herein, the term "solvate" refers to an aggregate containing one or more molecules of the compound of the present invention and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compound of the present invention may exist as a hydrate, including a monohydrate, a dihydrate, a hemihydrate, a sesquihydrate, a trihydrate, a tetrahydrate, etc., as well as corresponding solvated forms. The solvate of the compound of the present invention may be a true solvate, but in other cases, the compound of the present invention may simply contain incidental water or may be a mixture of water and some incidental solvent.

[0088] The present invention also refers to a composition comprising at least one compound of formula (I) according to the invention and at least one active agent, the at least one active agent preferably being present in an effective amount.

[0089] As used herein, an active agent includes any molecule or compound that can exert a desired effect on a cell, tissue, organ, or subject. Such effects can be biological, physiological, or cosmetic. Active agents can be any type of molecule or compound, including, for example, nucleic acids, nucleic acid analogs, peptides and polypeptides, antibodies such as polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and small molecules, including small organic molecules or compounds.

[0090] In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. The therapeutic agent derivative may be therapeutically active itself or may be a prodrug that becomes active upon further modification.

[0091] In one embodiment, the therapeutic agent includes any therapeutically effective agent or drug, such as anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cytovascular agents, signal transduction inhibitors, cardiovascular agents, such as antiarrhythmic agents, vasoconstrictors, hormones, and steroids.

[0092] In one embodiment, the therapeutic agent is an oncology agent, which may also be referred to as an anti-tumor agent, anti-cancer agent, tumor agent, anti-neoplastic agent, etc. Examples of oncology agents that may be used in accordance with the present invention include, but are not limited to, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, intravenous busulfan, oral busulfan, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporine A, cytarabine, cytosine arabinoside, daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, Goserelin acetate, Hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megestrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen These include mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, Rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that can be used in accordance with the present invention are ellipticine and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecin.

[0093] In a preferred embodiment, the at least one active agent is selected from the group consisting of proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogs, organic molecules with a molecular weight of up to 1000 g / mol, and combinations thereof.

[0094] Any known protein is generally suitable. Illustratively, proteins include glycoproteins and apolipoproteins. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins known to those skilled in the art and their variants and fragments, as well as apolipoprotein agonists, their analogs or fragments, and chimeric constructs of apolipoproteins. The apolipoproteins utilized in the present invention also include recombinant, synthetic, semi-synthetic, or purified apolipoproteins.

[0095] Any known peptide is generally suitable. The term peptide according to the present invention includes peptidomimetics. A peptide or peptidomimetic can be about 5 to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A "cell-penetrating peptide" can penetrate cells, e.g., microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin PI), disulfide-bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T.

[0096] In one embodiment, the targeting peptide tethered to the iRNA agent and / or carrier oligomer can be an amphipathic alpha-helical peptide.

[0097] Peptide and peptidomimetic ligands include naturally occurring or modified peptides, such as D or L peptides, α, β or γ peptides, N-methyl peptides, azapeptides, peptides with one or more amide bonds, i.e., peptides in which a bond has been replaced by one or more urea, thiourea, carbamate or sulfonylurea bonds, or cyclic peptides.

[0098] Any known carbohydrate is generally suitable. Exemplary carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid.

[0099] As described herein, the compositions of the present invention are particularly useful for the delivery of nucleic acid or nucleic acid analogues, including, for example, siRNA molecules, mRNA molecules, plasmids, microRNA, antagomir, aptamers and ribozymes.Therefore, the compositions of the present invention can be used to regulate the expression of target genes and proteins both in vitro and in vivo by contacting cells with the nucleic acid that reduces target gene expression, such as siRNA or microRNA, or the nucleic acid that can be used to increase the expression of desired proteins, such as the mRNA or plasmid that encodes desired proteins.

[0100] Generally, any known nucleic acid and nucleic acid analog or plasmid is suitable.Their preparation methods include but are not limited to chemical synthesis, and the enzymatic chemical cleavage of longer precursor or in vitro transcription.The method of synthesizing DNA and RNA nucleotide is widely used and is well known in the art.

[0101] Nucleic acids or nucleic acid analogs include polymers containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded, double-stranded, or triple-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of linear DNA, circular DNA, plasmid DNA (pDNA), antisense molecules, PCR products, or vectors. RNA can be in the form of chemically modified or unmodified messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA containing at least one coding sequence (circRNA), short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA), viral RNA (vRNA), and combinations thereof. Nucleic acids can contain one or more oligonucleotide modifications.

[0102] Nucleic acids of the present invention can be of various lengths, generally depending on the particular form of the nucleic acid. For example, in certain embodiments, a plasmid or gene can be about 1,000 to 100,000 nucleotide residues in length. In certain embodiments, oligonucleotides can range from about 10 to 100 nucleotides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, and triple-stranded, can range in length from about 10 to about 50 nucleotides in length, from about 20 to about 50 nucleotides in length, from about 15 to about 30 nucleotides in length, or from about 20 to about 30 nucleotides in length.

[0103] The term "circular DNA" includes any DNA that forms a closed loop and has no ends. Examples of circular DNA are plasmid DNA, minicircle DNA, and doggybone DNA (dbDNA™).

[0104] In the case of plasmid DNA, preparation for use in embodiments of the present invention generally utilizes, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art. Plasmid isolation can be performed using a variety of commercially available kits, including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega GmbH, Walldorf, Germany) kits, as well as commercially available reagents.

[0105] In a preferred embodiment, the present invention specifically refers to compositions for delivering mRNA or siRNA molecules.

[0106] In the case of mRNA, the primary methodology for preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for generating long, sequence-specific mRNAs. In vitro transcription describes the process of template-directed synthesis of RNA molecules from engineered DNA templates consisting of an upstream bacteriophage promoter sequence (such as, but not limited to, those derived from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification.

[0107] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits, including, but not limited to, the RiboMax Large Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents containing RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art.

[0108] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0109] Further non-limiting examples of purification procedures that can be used include size exclusion chromatography, silica-based affinity chromatography, and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0110] Furthermore, while reverse transcription can yield large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from incomplete transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extension. It has been demonstrated that these contaminants, which have dsRNA structures, can result in undesired immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce potent immune responses. This, in turn, can dramatically reduce mRNA translation due to reduced protein synthesis during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification. HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0111] A variety of important modifications have been described in the art and are used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP) and enhancing intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5' triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Further modifications include methylation of the last and penultimate 5'-most nucleotides on the 2'-hydroxyl group.

[0112] Several different cap structures can be used to generate the 5' cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally (i.e., capping during in vitro transcription) with a chemical cap analog. For example, the anti-reverse cap analog (ARC A) cap contains a 5'-5'-guanine triphosphate-guanine linkage, in which one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5' cap structure of authentic cellular mRNA, potentially reducing translation and cellular stability. Alternatively, synthetic mRNA molecules can be enzymatically capped after transcription. These may generate more authentic 5' cap structures that structurally or functionally mimic endogenous 5' caps with enhanced cap-binding protein binding, extended half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. A number of synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.

[0113] At the 3' end, a long chain of adenine nucleotides (poly-A tail) is typically added to mRNA molecules during RNA processing. Shortly after transcription, the 3' end of the transcript is cleaved, freeing a 3' hydroxyl, to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly-A tails have been widely shown to enhance both the translation efficiency and stability of mRNA.

[0114] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including, but not limited to, cloning a poly(T) tract into a DNA template or by post-transcriptional addition using poly(A) polymerase. The first approach allows for in vitro transcription of mRNAs with poly(A) tails of defined lengths, depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter approach involves the enzymatic addition of poly(A) tails to in vitro transcribed mRNAs using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA. This approach does not require further manipulation of the DNA template, but results in mRNAs with poly(A) tails of heterogeneous lengths. 5' capping and 3' poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, Poly(A) Polymerase Tailing Kit (Epicenter), mMESSAGE mMACHINE T7 Ultra Kit, and Poly(A) Polymerase Tailing Kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0115] In addition to 5' caps and 3' polyadenylation, other modifications of in vitro transcripts have been reported to offer benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and trigger strong innate immune responses. Because most nucleic acids from natural sources contain modified nucleosides, the ability to distinguish pathogenic DNA and RNA from self-DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications. In contrast, in vitro-synthesized RNA lacks these modifications, making it immunostimulatory, which in turn can inhibit effective mRNA translation, as outlined above. The introduction of modified nucleosides into in vitro-transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesirable immunostimulatory activity and enhancing translational capacity. The modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and procedures known in the art. For example, as disclosed in U.S. Patent No. 2012 / 0251618, a wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA to some degree, either alone or in combination with other modified nucleosides. In vitro synthesis of nucleoside-modified mRNA has been reported to reduce its ability to activate immunosensors while simultaneously enhancing translational capacity.

[0116] Other components of mRNA that can be modified to benefit translatability and stability include 5' and 3' untranslated regions (UTRs).Optimizing UTRs (preferable 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA, both or independently.

[0117] In one embodiment, the RNA is a self-amplifying RNA. When a self-amplifying RNA molecule (replicon) is delivered to a vertebrate cell, even in the absence of protein, it can produce multiple daughter RNAs by transcription from itself (through antisense copies generated from itself). Thus, in certain embodiments, the self-amplifying RNA molecule is a (+)-strand molecule that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA produces multiple daughter RNAs. These daughter RNAs and collinear subgenomic transcripts can be translated themselves to provide in situ expression of the encoded protein, or can be transcribed to provide additional transcripts with the same sense as the delivered RNA, which are translated to provide in situ expression of the protein. The overall result of this series of transcriptions is an amplification of the number of introduced self-amplifying RNAs, and thus the encoded protein becomes the major polypeptide product of the host cell.

[0118] In one embodiment, the RNA is circular RNA (circRNA), which is a type of single-stranded RNA that, unlike linear RNA, forms a covalently closed continuous loop by linking the 3'-end and 5'-end that are normally present in RNA molecules. Similar to mRNA, circRNA can be designed to encode and express proteins. In certain embodiments, the oligonucleotide (or its strand) of the present invention specifically hybridizes or is complementary to a target polynucleotide.

[0119] In one embodiment, the RNA is a hairpin siRNA and has a duplex region equal to or greater than 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region can be 200, 100, or 50 nucleotides or less. In certain embodiments, the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length. The hairpin can have a single-stranded overhang or a terminal unpaired region. In certain embodiments, the overhang is 2 to 3 nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, on the antisense side of the hairpin.

[0120] In one embodiment, the RNA is an siRNA. SiRNAs are RNA duplexes, typically 16-30 nucleotides in length, that can associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Because RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts, siRNAs can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNAs function through a natural mechanism that evolved to control gene expression via non-coding RNA.

[0121] " single-stranded siRNA compound " as used herein refers to the siRNA compound that is composed of a single molecule.It can comprise the double region formed by intrastrand pairing, for example, can be or comprise hairpin or panhandle structure.Single-stranded siRNA compound can be antisense with respect to target molecule.

[0122] Single-stranded siRNA compound can be long enough so that it can enter RISC and participate in the RISC-mediated cleavage of target mRNA.Single-stranded siRNA compound is at least 14 nucleotides long, and in other embodiments, at least 15, 20, 25, 29, 35, 40 or 50 nucleotides long.In certain embodiments, it is less than 200, less than 100 or less than 60 nucleotides long.

[0123] As used herein, a "double-stranded siRNA compound" is an siRNA compound that comprises multiple, optionally two, strands, where inter-strand hybridization is capable of forming a region of duplex structure.

[0124] The antisense strand of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 or more nucleotides in length. It can be 200, 100, or 50 or less nucleotides in length. Ranges can be 17-25, 19-23, and 19-21 nucleotides in length. As used herein, the term "antisense strand" refers to the strand of an siRNA compound that is sufficiently complementary to a target molecule, e.g., a target RNA.

[0125] The sense strand of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 or more nucleotides in length. It can be 200, 100, or 50 or less nucleotides in length. Ranges can be 17-25, 19-23, and 19-21 nucleotides in length.

[0126] The double-stranded portion of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 or more nucleotide pairs in length. It can be 200, 100, or 50 or less nucleotide pairs in length.

[0127] Ranges can be 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0128] In many embodiments, the siRNA compound is large enough that it can be cleaved by an endogenous molecule, eg, Dicer, to yield a smaller siRNA compound, eg, an siRNA agent.

[0129] The sense strand and the antisense strand can be selected so that the double-stranded siRNA compound contains a single-stranded or unpaired region at one or both ends of the molecule.Therefore, the double-stranded siRNA compound can contain a paired sense strand and an antisense strand, such as one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides.The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.Some embodiments have at least one 3' overhang.In one embodiment, both ends of the siRNA molecule have a 3' overhang.In some embodiments, the overhang is 2 nucleotides.

[0130] In certain embodiments, the length of the double-stranded region is 15 to 30, or 18, 19, 20, 21, 22, and 23 nucleotides, for example, within the range of the siRNA compounds described above. The siRNA compounds are similar in length, allowing for the construction of natural Dicer-processed products from long dsiRNAs. Also included are embodiments in which the two strands of the siRNA compound are linked, for example, covalently linked. Hairpins or other single-stranded structures that provide the necessary double-stranded region and 3' overhang are also within the scope of the present invention.

[0131] The siRNA compounds described herein, including double-stranded siRNA compounds and single-stranded siRNA compounds, can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of the gene that codes for protein.For convenience, this mRNA is also referred to herein as the mRNA that is silenced.This gene is also referred to as target gene.Generally, the RNA that is silenced is endogenous gene or pathogenic gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA can also be targeted.

[0132] As used herein, the phrase " mediate RNAi " refers to the ability to silence target RNA in a sequence-specific manner.Without wishing to be bound by theory, it is believed that silencing uses RNAi mechanism or RNAi process and guide RNA, for example, 21-23 nucleotide siRNA compound.

[0133] In one embodiment, siRNA compounds are "sufficiently complementary" to target RNA, for example, target mRNA, so that the siRNA compounds can silence the production of the protein encoded by the target mRNA.In another embodiment, siRNA compounds are "exactly complementary" to target RNA, for example, target RNA and siRNA compounds anneal to form a hybrid that is made only by Watson-Crick base pairing in the exact complementary region.A "sufficiently complementary" target RNA can comprise an internal region (for example, at least 10 nucleotides) that is exactly complementary to the target RNA.Furthermore, in certain embodiments, siRNA compounds specifically discriminate between single base differences.In this case, siRNA compounds can only mediate RNAi when exact complementarity is found in the region (for example, within 7 nucleotides) of a single base difference.

[0134] In addition to conventional siRNAs, dicer substrate siRNAs can be used as a less immunogenic alternative. DsiRNAs are 25–30 nucleotides in length and, after cellular uptake, are further cleaved and processed by the Dicer enzyme to convert them into an active form, which then associates with RISC.

[0135] Antisense RNA is directed against a target polynucleotide. The term "antisense RNA" or simply "antisense" is meant to include RNA complementary to a target polynucleotide sequence. Antisense RNA is a single strand of RNA complementary to a selected sequence, such as a target gene mRNA. Antisense RNA is thought to inhibit gene expression by binding to complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand or by causing degradation of the target mRNA. In certain embodiments, antisense RNA contains about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense RNA that may not be exactly complementary to the desired target gene.

[0136] MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA within the genomes of plants and animals but are not translated into proteins. Processed miRNAs are single-stranded 17–25 nucleotide (nt) RNA molecules that become incorporated into the RNA-induced silencing complex (RISC) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation.

[0137] In one embodiment, the RNA is transfer RNA (tRNA). Transfer RNA is an adaptor molecule, typically composed of RNA 76-90 nucleotides in length, that acts as a physical link between mRNA and the amino acid sequence of a protein. Transfer RNA does this by carrying amino acids to the cell's protein synthesis machinery, called the ribosome. Complementation of a three-nucleotide codon in messenger RNA (mRNA) with a three-nucleotide anticodon in tRNA results in protein synthesis based on the mRNA code. Thus, tRNA is a necessary component of translation, the biological synthesis of new proteins according to the genetic code.

[0138] In one embodiment, the nucleic acid is a single guide RNA applied to direct CRISPR / Cas9-mediated gene editing. The single guide RNA hybridizes to a target sequence in the genome of a cell, complexes with the Cas9 protein at the target site, and initiates single- or double-strand cleavage.

[0139] In one embodiment, the at least one active agent is selected from an antagomir, an aptamer, a ribozyme, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, an miRNA mimic, an antimir or an miRNA inhibitor and a UI adaptor.

[0140] The antagonists are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacological properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, sugars, phosphorothioate backbones, and complete 2'-0-methylation of, for example, cholesterol moieties at the 3' end.

[0141] Aptamers are nucleic acid or peptide molecules that bind to specific molecules of interest with high affinity and specificity. DNA or RNA aptamers have been successfully produced to bind to many different entities, from large proteins to small organic molecules. Aptamers can be RNA- or DNA-based and can include riboswitches. Riboswitches are portions of mRNA molecules that can directly bind to small target molecules, and binding to the target affects gene activity. Aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers," which contain consensus sequences derived from comparing two or more known aptamers to a given target.

[0142] Ribozymes are RNA molecular complexes that contain a specific catalytic domain with endonuclease activity. For example, many ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity results from the requirement that the substrate bind to the ribozyme's internal guide sequence ("IGS") through specific base-pairing interactions prior to chemical reaction.

[0143] Nucleic acids associated with the lipid particles of the present invention may be immunostimulatory and include immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that are capable of inducing an immune response when administered to a subject, which may be a mammal or other patient.

[0144] Because transcription factors recognize their relatively short binding sequences, short oligonucleotides containing the consensus binding sequence of a specific transcription factor can be used as a tool to manipulate gene expression in living cells, even in the absence of surrounding genomic DNA. This strategy requires the intracellular delivery of such "decoy oligonucleotides," which are then recognized and bound by the target factor. Once the DNA-binding site of the transcription factor is occupied by the decoy, the transcription factor is unable to subsequently bind to the promoter region of the target gene.

[0145] Supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or both, or their modifications, and has a nucleotide sequence that is substantially identical to miRNA and antisense to its target. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, and contains at least one non-naturally occurring moiety that functions similarly. Such modified or substituted oligonucleotides are preferred over natural forms due to desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0146] miRNA mimics represent a class of molecules that can be used to mimic the gene silencing ability of one or more miRNAs. Thus, the term "microRNA mimic" refers to synthetic non-coding RNAs that can enter the RNAi pathway and regulate gene expression (i.e., miRNAs that are not obtained by purification from endogenous miRNA sources). miRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimic precursors (e.g., pri- or pre-miRNAs).

[0147] The terms "antimir," "microRNA inhibitor," "miR inhibitor," or "inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the activity of specific miRNAs. Generally, inhibitors are natural or modified nucleic acids, including oligonucleotides containing RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNA), or any combination of the above. Modifications include 2' modifications and internucleotide modifications (e.g., phosphorothioate modifications), which can affect delivery, stability, specificity, intracellular compartmentalization, or potency. Furthermore, miRNA inhibitors may contain conjugates, which can affect delivery, intracellular compartmentalization, stability, and / or potency. Inhibitors can adopt a variety of configurations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally, microRNA inhibitors contain one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand of one or more targeted miRNAs. In addition, miRNA inhibitors may also contain additional sequences located 5' and 3' relative to the sequence that is the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of the sequence adjacent to the mature miRNA in the pri-miRNA from which it is derived, or the additional sequence may be any sequence (having a mixture of A, G, C, or U).

[0148] The Ul adaptor is a bifunctional oligonucleotide that interrupts the poly(A) site and has a targeting domain complementary to a site in the terminal exon of the target gene and an "Ul domain" that binds to the Ul smaller nuclear RNA component of the Ul snRNP. The Ul snRNP is a ribonucleoprotein complex that functions primarily to guide the initial steps of plyosome formation by binding to pre-mRNA exon-intron boundaries. Nucleotides 2-11 at the 5' end of the Ul snRNA base pair with the 5' ss of the pre-mRNA. In one embodiment, the oligonucleotide of the present invention is a Ul adaptor.

[0149] In a preferred embodiment, the at least one active agent is selected from the group consisting of linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA comprising at least one coding sequence (circRNA), short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA), and combinations thereof.

[0150] In one embodiment, at least one active agent is an organic molecule, also called a small molecule in the pharmaceutical field, having a molecular weight of up to 1000 g / mol, preferably the organic molecule is selected from paclitaxel, doxorubicin, irinotecan, vincristine and oxaliplatin.

[0151] The composition according to the present invention may further comprise a compound selected from a lipid different from the compound of formula (I), such as an ionizable lipid, a cationic lipid, a neutral lipid or a structured lipid, a sterol or a sterol derivative, a buffer, a pharmaceutically acceptable salt, a cryoprotectant or any combination thereof.

[0152] Suitable lipids other than the compounds of formula (I) according to the present invention that may be present include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, or mixtures thereof. Lipids with various acyl chain groups of various chain lengths and degrees of saturation are available or can be isolated or synthesized by well-known techniques. In one embodiment, lipids containing saturated fatty acids with carbon chain lengths in the range of C10 to C20 are preferred. In one embodiment, lipids containing mono- or di-unsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used. Furthermore, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Preferred lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC) or any related phosphatidylcholine.

[0153] Further suitable lipids are comprised of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups, such as serine and inositol, and sterols, particularly cholesterol and phytosterols.

[0154] In one embodiment, the further lipid is preferably 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-dioleyloxy-3-dimethylaminopropane, (6Z,9Z,28Z,31Z)-heptatriacon-6,9,28,31-tetraene-19- The ionizable lipid is selected from N,N-dimethyl-2,2-di-(9Z,12Z)-9,12octadecadien-1-yl-1,3-dioxolane-4-ethanamine, [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), 9-heptadecanyl 4-(dimethylamino)butanoate, 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12octadecadien-1-yl-1,3-dioxolane-4-ethanamine, and [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0155] In one embodiment, the further lipid is preferably 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium-propane, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, N 4-cholesteryl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn The cationic lipid is selected from the salts of -glycero-3-ethylphosphocholine, 1,2-dimyristoleyl-sn-glycero-3-ethylphosphocholine, dimethyldioctadecylammonium, 1,2-dimyristoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-stearoyl-3-trimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol.The salt can be any pharmaceutically acceptable salt, preferably a fluoride or chloride salt.

[0156] Additional lipids suitable for the compositions of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids.

[0157] In one embodiment, the additional lipid is selected from phospholipids, aminolipids and sphingolipids.Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus can also be used, such as sphingolipids, glycosphingolipid families, diacylglycerols and β-acyloxyacids.In addition, such amphiphilic lipids can be easily mixed with other lipids, such as triglycerides and sterols.

[0158] In one embodiment, the additional lipid is selected from polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj 52 (polyoxyethylene (40) stearate), and Brij™ S10 (polyoxyethylene (10) stearyl ether), or a combination thereof. These lipids are known in the art as stabilizers and can be present in the compositions of the present invention in addition to Compound (I) of the present invention and any additional lipids described herein.

[0159] A cryoprotectant is an agent that protects a composition from experiencing adverse effects upon freezing and thawing. For example, in the present invention, cryoprotectants such as polyols and / or carbohydrates, among others, can be added to prevent substantial particle aggregation.

[0160] A buffering agent may also be included. Suitable buffering agents are, for example, phosphate, acetate, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, amino acids and other organic compounds, ascorbic acid, and antioxidants including methionine.

[0161] In addition, at least one of the following additives may be further present in the composition: preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides. carbohydrates such as disaccharides, disaccharides, and other sugar compounds such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), vehicles, binders, disintegrants, immune adjuvants such as cell-penetrating peptides, e.g., human lactoferrin protein or fragments thereof, Tat, Ant, Rev, FHV, HSV-1 protein VP22, C6, C6M1, PF20, NAP, POD, polyarginine, polylysine, PTD-5, transportan, MAP, TP10, Pep-7, azurin p18, azurin p28, hCT18-32, Bac 7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, melittin, penetratin, EB1, MPG, CADY, Pep4, preferably human lactoferrin protein or fragments thereof, fillers (diluents), lubricants, flow aids (glidants), compression aids, colorants, sweeteners, suspending / dispersing agents, film formers / coatings, flavors, printing inks.

[0162] In one embodiment, the composition, preferably the lipid nanoparticles, comprises at least one active agent in a weight to weight ratio of 1:0.01 to 1:100 relative to the compound of formula (I).

[0163] In one embodiment, the composition, preferably the lipid nanoparticle, comprises at least one additional compound selected from one or more lipids different from the compound of formula (I), a buffer, a pharmaceutically acceptable salt different from the buffer, a cryoprotectant, or any combination thereof. In a preferred embodiment, the composition, preferably the lipid nanoparticle, further comprises one or more lipids different from the compound of formula (I), more preferably one, two, or three additional lipids different from the compound of formula (I). In a preferred embodiment, the composition, preferably the lipid nanoparticle, consists of the compound of formula (I), at least one active agent, and one or more lipids different from the compound of formula (I), more preferably one, two, or three additional lipids different from the compound of formula (I).

[0164] In one embodiment of the composition, preferably lipid nanoparticles, the compound of formula (I) is present in a ratio of about 0.1 to about 10 mol% based on the total lipid content. In one embodiment, the compound of formula (I) is present in a ratio of more than 10 mol% based on the total lipid content. In one embodiment, the compound of formula (I) is present in a ratio of 0.5 mol% to 5 mol% based on the total lipid content. In some embodiments, the compound of formula (I) is present in a ratio of 1.5 mol%.

[0165] In one embodiment of the composition, preferably lipid nanoparticles, an additional lipid different from the compound of formula (I) is included, and if the additional lipid is a cationic lipid, the cationic lipid is preferably present in a ratio of about 10 to about 80 mol % based on the total lipid content. In one embodiment, the cationic lipid is present in a ratio of about 50 mol % based on the total lipid content.

[0166] In one embodiment of the composition, preferably lipid nanoparticles, an additional lipid different from the compound of formula (I) is included, and if the lipid is an ionizable lipid, the ionizable lipid is preferably present in a ratio of about 10 to about 80 mol % based on the total lipid content. In one embodiment, the ionizable lipid is present in a ratio of about 50 mol % based on the total lipid content.

[0167] In one embodiment of the composition, preferably lipid nanoparticles, where an additional lipid different from the compound of formula (I) is included, which is a structured lipid having a neutral or negative net charge, also known as a "helper lipid," the structured lipid is preferably present in a ratio of about 10 to about 40 mol % based on the total lipid content. In one embodiment, the structured lipid is present in a ratio of about 10 mol % based on the total lipid content.

[0168] In one embodiment of the composition, preferably lipid nanoparticles, when an additional lipid different from the compound of formula (I) is included and is a sterol, such as cholesterol or a phytosterol, the sterol is preferably present in a ratio of about 10 to about 60 mol% based on the total lipid content. In one embodiment, the sterol is present in a ratio of about 35 to about 41 mol% based on the total lipid content. In one embodiment, the sterol is present in a ratio of about 38.5 mol% based on the total lipid content.

[0169] In one embodiment of the composition, preferably lipid nanoparticles, an additional lipid different from the compound of formula (I) is included, and when it is a stabilizer, the stabilizer is preferably present in a ratio of about 0 to about 10 mol %, based on the total lipid content.

[0170] In one embodiment of the composition, preferably lipid nanoparticles, when at least one buffering agent is present, the at least one buffering agent is present in a molar concentration of 0.1 mM to 1000 mM relative to the total volume of the phase in which the composition is dispersed.

[0171] In one embodiment of the composition, preferably lipid nanoparticles, when at least one cryoprotectant is present, the at least one cryoprotectant is present in a mass concentration of 0.1% to 50% by weight relative to the total volume of the phase in which the composition is dispersed.

[0172] The compositions of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.The typical routes of administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal.The term parenteral used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intracisternal injection or infusion techniques.

[0173] The compositions of the present invention, preferably pharmaceutical compositions, are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. In some embodiments, the composition administered to a subject or patient takes the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the compound of formula (I) of the present invention in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. In some embodiments, the composition administered contains a therapeutically effective amount of the compound of formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, for treating the disease or condition of interest according to the teachings of the present disclosure.

[0174] The composition of the present invention, preferably pharmaceutical composition, can be in solid or liquid form.In one embodiment, carrier is particulate, so composition is, for example, in tablet or powder form.Carrier can also be liquid, so composition is, for example, oral syrup, injectable liquid or aerosol, which is useful for, for example, inhalation administration.

[0175] When intended for oral administration, the compositions, preferably pharmaceutical compositions, of the present invention are preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms being included within the forms considered herein to be either solid or liquid.

[0176] As a solid composition for oral administration, the composition, preferably a pharmaceutical composition, can be formulated into powder, granules, compressed tablets, pills, capsules, chewing gum, or wafers. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or orange flavor, and colorants. When the pharmaceutical composition of some embodiments is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as polyethylene glycol or oil.

[0177] The composition of the present invention, preferably pharmaceutical composition, can be in the form of liquid, such as elixir, syrup, solution, emulsion or suspension.Liquid can be for oral administration or for delivery by injection, as two examples.When intended for oral administration, preferred compositions contain one or more of sweeteners, preservatives, dyes / coloring agents and flavor enhancers in addition to the compound of formula (I).The composition intended for administration by injection can contain one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents.

[0178] The liquid compositions of the present invention, preferably liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic monoglycerides or diglycerides that can function as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose; agents acting as cryoprotectants, such as sucrose or trehalose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0179] The composition of the present invention, preferably pharmaceutical composition, can be intended for topical administration, in this case, carrier can suitably comprise solution, emulsion, ointment or gel base.The base can comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluent such as water and alcohol, and emulsifier and stabilizer.Thickener can be present in the pharmaceutical composition for topical administration.When intended for transdermal administration, composition can comprise transdermal patch or iontophoresis device.

[0180] The composition of the present invention, preferably pharmaceutical composition, can be intended for rectal administration, for example, in the form of suppository, which melts in the rectum and releases drug.The composition for rectal administration can contain oily base as suitable non-irritating excipient.Such base includes but is not limited to lanolin, cocoa butter and polyethylene glycol.

[0181] The composition of the present invention, preferably pharmaceutical composition, can contain various materials that modify the physical form of solid or liquid dosage units.For example, the composition can contain materials that form a coating shell around active ingredient.The materials that form the coating shell are usually inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents.

[0182] The compositions of the present invention, preferably pharmaceutical compositions, may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems, ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. The aerosol of the compound of formula (I) of the present invention can be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. The aerosol delivery includes the necessary container, activator, valve, subcontainer, etc., which together can form a kit.

[0183] In a preferred embodiment, the composition is a lipid nanoparticle. In certain embodiments, the active agent is encapsulated in the aqueous interior of the lipid nanoparticle. In other embodiments, the active agent is present within one or more lipid layers of the lipid nanoparticle. In other embodiments, the active agent is bound to the outer or inner lipid surface of the lipid nanoparticle. Lipid nanoparticles include, but are not limited to, liposomes. As used herein, a liposome is a structure having a lipid-containing membrane surrounding an aqueous interior. Liposomes can have one or more lipid membranes. Liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar. When complexed with nucleic acids, the lipid particle can be a lipoplex, consisting of a cationic lipid bilayer sandwiched between DNA layers.

[0184] The lipid nanoparticles of the present invention can be formulated as pharmaceutical compositions, which further comprise a pharmaceutically acceptable diluent, excipient, or carrier, e.g., saline or phosphate buffer, selected according to the route of administration and standard pharmaceutical practice.

[0185] In certain embodiments, the lipid nanoparticles of the present invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier.Generally, saline is used as a pharmaceutically acceptable carrier.Other suitable carriers include, for example, water, buffered water, 0.9% saline, 0.3% glycine, etc., and glycoproteins such as albumin, lipoproteins, and globulins for enhancing stability.In compositions that include saline or other salt-containing carriers, the carrier is preferably added after lipid nanoparticle formation.Therefore, after lipid nanoparticles are formed, the composition can be diluted with a pharmaceutically acceptable carrier such as saline.

[0186] The obtained pharmaceutical preparation can be sterilized by conventional and well-known sterilization techniques.The aqueous solution can then be packaged for use, or filtered under aseptic conditions, lyophilized, and the lyophilized preparation can be combined with a sterile aqueous solution before administration.The composition can contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, isotonicity adjusters, etc., required to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.In addition, the lipid suspension can contain a lipid protectant to protect lipids from free radicals and lipid peroxidation damage during storage.Lipophilic free radical quenchers such as α-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

[0187] The term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer order (e.g., 1-1,000 nm) that contains one or more compounds of Formula (I). In some embodiments, lipid nanoparticles containing at least one compound of Formula (I) are included in formulations that can be used to deliver therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles comprise a compound of Formula (I) and a nucleic acid. In some embodiments, the therapeutic agent, such as a nucleic acid, is encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects elicited by the host organism's or cellular machinery, such as a harmful immune response.

[0188] In various embodiments, the lipid nanoparticles have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 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, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 350 nm, 360 nm, 370 nm, 380 nm, 400 nm, 420 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 500 nm, 520 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 The samples had an average diameter of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nm, and were diluted 1:10 with RNAse-free water, corresponding to an RNA concentration of 5 ng / µL. Preferably, measurements are performed using a Malvern Zetasizer NanoZS.

[0189] Some administration techniques may result in systemic delivery of certain active agents, but not others. Systemic delivery means that a useful, preferably therapeutic amount of the active agent is exposed to most of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0190] "Local delivery" as used herein refers to the direct delivery of active agent to target site in living organism.For example, the agent can be delivered locally by direct injection into disease site such as tumor, other target site such as inflammation site, or target organ such as liver, heart, pancreas, kidney.Local delivery can also include local application or local injection technique, for example, intramuscular, subcutaneous or intradermal injection.Local delivery does not prevent systemic pharmacological effect.

[0191] The composition of the present invention can also be administered simultaneously with, before, or after the administration of one or more other active agents.Such combination therapy includes the administration of the composition of the present invention and one or more additional active agents in a single pharmaceutical dosage formulation, and the administration of the composition of the present invention and each active agent in their own separate pharmaceutical dosage formulation.For example, the composition of the present invention and other active agents can be administered to patients together in a single oral dosage composition such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation.When separate dosage formulations are used, the compound of formula (I) of the present invention and one or more additional active agents can be administered essentially simultaneously, i.e., simultaneously, or separately at different times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0192] The composition of the present invention, preferably pharmaceutical composition, can be prepared by the methodology well known in the pharmaceutical field.For example, the pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a dispersion.Surfactants can be added to promote the formation of a uniform solution or suspension.Surfactants are compounds that interact non-covalently with the compounds of the present disclosure to promote the dissolution or uniform suspension of the compounds in aqueous delivery systems.

[0193] The compositions of the present invention are administered in therapeutically effective amounts that vary depending on a variety of factors, including the activity of the particular therapeutic agent used, the metabolic stability and length of action of the therapeutic agent, the patient's age, weight, general health, sex, and diet, the mode and time of administration, the rate of excretion, the drug combination, the severity of the particular disorder or condition, and the subject being treated.

[0194] In a preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating a disease in a human. In a further preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating a disease in a mammal.

[0195] Example Synthesis of polyoxyalkylene copolymers Reagents and Chemicals Reagents and chemicals were purchased from TCI (Tokyo, Japan), Thermo Fisher Scientific (Waltham, MA, USA), Carl Roth GmbH (Karlsruhe, Germany), and Merck KGaA (Darmstadt, Germany) unless otherwise stated. Ethylene oxide (EO) was obtained from Air Liquide (Paris, France). Tetrahydrofuran (THF) was flashed onto basic aluminum oxide prior to use. Glycidyl methyl ether (GME) was dried over calcium hydride (CaH2) and cryotransferred prior to polymerization.

[0196] Example 1: Synthesis of glycidyl methyl ether (GME) [ka] 1-Chloro-3-methoxypropan-2-ol (50.0 g, 401 mmol) was added to a flask equipped with a magnetic stirrer and cooled in an ice bath. Finely ground sodium hydroxide (NaOH, 20.9 g, 522 mmol) was added portionwise under stirring. After complete reaction (TLC control), the crude product was cryogenically transferred from the reaction flask to a vacuum and dried over CaH2 while cooling in an ice bath. After further cryogenic transfer and filtration steps, GME (30.1 g, 85%) was obtained as a colorless liquid.

[0197] Example 2: Synthesis of 1-methoxy-3-(2-methoxyethoxy)propan-2-ol [ka] Ethylene glycol monomethyl ether (8.64 g, 9.00 mL, 113 mmol) was added to a flask equipped with a reflux condenser. NaOH solution (19 M, 3 mL) was added under stirring, and the resulting solution was heated to 55 °C. GME (5.00 g, 5.10 mL, 56.7 mmol) was added, and the solution was stirred overnight. The solution was cooled to room temperature and extracted three times with dichloromethane (DCM, 50 mL). The combined organic phases were dried over magnesium sulfate (MgSO4). After filtration, the solvent was evaporated under reduced pressure. After fractional distillation of the residue, 1-methoxy-3-(2-methoxyethoxy)propan-2-ol (3.74 g, 40%) was obtained as a colorless liquid.

[0198] Example 3: mP(EO 15 -co-GME 15 ) (entry b) [ka] Potassium tert-butoxide (KOtBu, 1.01 g, 8.95 mmol) was dissolved in stabilizer-free THF and a small amount of Millipore water and transferred to a flame-dried, argon-flushed flask equipped with a Teflon stopcock and septum. 1-Methoxy-3-(2-methoxyethoxy)propan-2-ol (1.50 g, 9.14 mmol) from Example 2 was dissolved in benzene and transferred to the flask. High vacuum was applied to the flask, and the solvent was removed under high vacuum. The resulting initiator salt was dried overnight at 55°C under high vacuum. The residue was dissolved in dry dimethyl sulfoxide (DMSO, 86 mL). The resulting solution was frozen at -80°C, and then GME (11.3 g, 11.5 mL, 128 mmol) from Example 1 was added to the flask via syringe. EO (5.63 g, 5.81 mL, 128 mmol) was added to the flask via cryogenic transfer from a graduated ampoule. The cooling bath was removed and the reaction mixture was stirred under high vacuum at 30°C for 2 days with the stopcock closed. The solvent was evaporated under high vacuum at 50°C. Millipore water (100 mL) and acidic ion exchange resin (DOWEX) (1 g) were added to the residue. The resulting suspension was stirred overnight. The suspension was filtered and the resulting solution was freeze-dried. The residue was dissolved in diethyl ether (400 mL). The resulting suspension was filtered and the organic phase was dried over MgSO4. After the filtration step, the solvent was evaporated to give the statistical copolymer mP(EO 15 -co-GME 15 ) (15.2 g, 83%, entry b) was obtained as a viscous liquid.

[0199] Additionally, mP(EO-co-GME) with different stoichiometries were prepared according to the same procedure (Table 1 and Table 2 entries a, c, and d).

[0200] Example 4: mP(EO 15 -co-GME 15 Synthesis of )-b-PEO2 (entry e) [ka] KOtBu (489 mg, 4.36 mmol) was dissolved in stabilizer-free THF and a small amount of Millipore water and transferred to a flame-dried, argon-flushed flask equipped with a Teflon stopcock and septum. 1-Methoxy-3-(2-methoxyethoxy)propan-2-ol (730 mg, 4.45 mmol) was dissolved in benzene and transferred to the flask. High vacuum was applied to the flask, and the solvent was removed under high vacuum. The resulting initiator salt was dried overnight at 55°C under high vacuum. The residue was dissolved in dry DMSO (42 mL). The resulting solution was frozen at -80°C, and then GME (5.48 g, 5.60 mL, 62.2 mmol) from Example 1 was added to the flask via syringe. EO (2.74 g, 2.83 mL, 62.2 mmol) was added to the flask via cryogenic transfer from a graduated ampoule. The cooling bath was removed, and the reaction mixture was stirred under high vacuum at 30°C for 1 day. The solution was cooled to -80°C, and EO (392 mg, 404 μL, 8.89 mmol) was added to the flask by cryogenic transfer from a graduated ampoule. The cooling bath was removed, and the reaction mixture was stirred under high vacuum at 30°C for 1 day. The solvent was evaporated under high vacuum at 50°C. Millipore water (100 mL) and acidic ion exchange resin (DOWEX) (500 mg) were added to the residue. The resulting suspension was stirred overnight. The suspension was filtered, and the resulting solution was lyophilized. The residue was dissolved in diethyl ether (400 mL). The resulting suspension was filtered, and the organic phase was dried over MgSO4. After the filtration step, the solvent was evaporated to give mP(EO 15 -co-GME 15 )-b-PEO2 (7.19 g, 77%, entry e) was obtained as a viscous liquid.

[0201] Furthermore, mP(EO) with different stoichiometries 21 -co-GME 22 )-b-PEO2 was prepared according to the same procedure (Tables 1 and 2, entry f).

[0202] Example 5: Quantifying the ratio of primary and secondary alcohol end groups in polymer samples 20 mg of polymer b(3), e(4), or f(4) was dissolved in deuterated acetonitrile (CD3CN) (1 mL). Trifluoroacetic anhydride (50 μL) was added, and the resulting solution was shaken for 10 min. 0.6 mL of the solution was transferred to an NMR tube. 1 H NMR spectra were measured. The ratio of primary to secondary hydroxyl end groups in the polymer samples was determined by quantitative esterification of the hydroxyl end groups with excess trifluoroacetic anhydride in CD3CN. 1 The H NMR spectrum showed a peak at 5.35(C H OC(O)) and 4.45 ppm(C H The EO (O) end-capping process for polymers e(4) and f(4) efficiently accumulates primary hydroxyl groups at the chain ends.

[0203] Example 6: mPEO 46 Synthesis of (entry g) [ka] KOtBu (289 mg, 2.57 mmol) was dissolved in stabilizer-free THF and a small amount of Millipore water and transferred to a flame-dried, argon-flushed flask equipped with a Teflon stopcock and septum. Triethylene glycol monomethyl ether (431 mg, 2.62 mmol) was dissolved in benzene and transferred to the flask. High vacuum was applied to the flask, and the solvent was removed under high vacuum. The resulting initiator salt was dried overnight at 55°C under high vacuum. The residue was dissolved in dry DMSO (24 mL). The resulting solution was frozen at -80°C, and then EO (4.86 g, 5.01 mL, 110 mmol) was added to the flask from a graduated ampoule via cryogenic transfer. The cooling bath was removed, and the reaction mixture was stirred under high vacuum at 30°C for 1 day. The solvent was evaporated under high vacuum at 50°C. Millipore water (75 mL) and acidic ion exchange resin (DOWEX) (260 mg) were added to the residue. The resulting suspension was stirred overnight. The suspension was filtered and the resulting solution was freeze-dried. The residue was dissolved in chloroform (350 mL). The resulting suspension was filtered and the organic phase was dried over MgSO4. After the filtration step, the solvent was evaporated to give mPEO. 46 (4.04 g, 76%) was obtained as a solid. 1 Determined by 1 H NMR spectroscopy. [Table 1] [Table 2]

[0204] Method for characterizing polyoxyalkylene copolymers 1 H NMR spectra were recorded at 300 MHz on a Bruker Avance III HD 300 spectrometer and were internally referenced to the residual proton signal of the deuterated solvent.

[0205] M for all samples w , M n and dispersity (M w / M nThe PDI (Polarity, Diameter, and Dispersity Index) was determined from the corresponding size-exclusion chromatogram (refractive index (RI) detector, DMF, calibration with PEO standards). Size-exclusion chromatography (SEC) measurements were performed on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column at 50 °C using dimethylformamide (DMF containing 1 g / L lithium bromide (LiBr)) as the mobile phase (flow rate 1 mL / min). The polymer concentration was 1 mg / mL. Calibration was performed using PEO standards (from Polymer Standard Service, Mainz, Germany).

[0206] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF MS) measurements were performed on a Bruker autoflex maX MALDI-TOF / TOF. Potassium trifluoroacetic acid and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) were used as the ionization salt and matrix, respectively.

[0207] Synthesis of mP(EO-co-GME)-N,N-ditetradecylacetamide Reagents and Chemicals Reagents and chemicals were purchased from Carl Roth GmbH, Merck KGaA and Orgentis Chemicals (Gatersleben, Germany) unless otherwise stated. Deionized water was used for all experiments.

[0208] Example 7: Oxidation of mP(EO-co-GME) to mP(EO-co-GME)-acetic acid [ka] mP(EO 21 -co-GME 12) (Entry a from Example 3, 7 g, 3.5 mmol) was charged into a round-bottom flask. Water (42 mL), sodium carbonate (Na2CO3, 1 M, 7 mL), sodium bicarbonate (NaHCO3, 1 M, 7 mL), sodium bromide (NaBr, 0.1 M, 3.5 mL), and 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-TEMPO, 37.3 mg) were added, and the reaction mixture was cooled to 0-5 °C. Bleach (7.5 wt%, 7.2 mL) was then added in four portions within 1 h and stirred for an additional 1 h. Sodium thiosulfate (Na2SO3, 1 M, 3.5 mL) was added to quench the reaction. DCM (35 mL) was then added, and the pH was adjusted to pH = 1-3 by adding HCl (1 M). The organic product phase was separated, and the aqueous phase was extracted with DCM (35 mL). The DCM phases were combined and dried over MgSO4 overnight. After filtration, the organic solvent was removed under vacuum to give crude mP(EO 20 -co-GME 12 )-acetic acid (yield 7.6 g) was obtained as an oil.

[0209] Additionally, different mP(EO-co-GME)-acetic acid compounds were prepared from different polyoxyalkylene copolymer starting materials following the same procedure.

[0210] Example 8: Synthesis of mP(EO-co-GME)-N,N-ditetradecylacetamide [ka] Crude mP(EO) from Example 7 20 -co-GME 12N,N-ditetradecylacetamine (1 g, 3.5 mmol) was dissolved in DCM (70 mL) at room temperature. Carbonyldiimidazole (CDI, 680 mg, 4.2 mmol) was added to the reaction solution, and the reaction mixture was stirred at room temperature for 1-2 hours. N,N-ditetradecylacetamine (1 g, 3.5 mmol) was then added to the reaction, and the reaction mixture was stirred at room temperature for an additional 16 hours. The conversion was quantified in area % (%a) by high-pressure liquid chromatography (HPLC) equipped with a charged particle detector (CAD). Water was added to the reaction mixture, and the two-phase system was stirred for 30 minutes, after which the phases were separated. Additional DCM (70 mL) was added to the aqueous phase for further extraction. After concentrating the organic phase, the product was purified by chromatography. The product-containing fractions were then pooled, and the solvent was removed under vacuum. The desired product, mP(EO), was obtained. 20 -co-GME 12 )-N,N-ditetradecylacetamide (entry h) was isolated in 29% yield. 1 It was characterized by H-NMR spectroscopy (Bruker 600 MHz instrument, CD2Cl2, at 300 K). The purity was determined by HPLC (99.3% a).

[0211] Additionally, following the same procedure, different polyoxyalkylene-N,N-ditetradecylacetamide compounds were prepared from different polyoxyalkylene-acetic acid starting materials (entries i–m in Table 3). [Table 3]

[0212] The improved conversion of compound l (8) can be explained by the different end-group fidelity of precursor compound e (4) compared to a (3), b (3), c (3), and d (3). In compound e (4), two repeating units of EO were grafted onto the polymer chain ends. 1H NMR spectroscopy (Example 5) confirmed that this resulted in an increased amount of polymer chains with primary alcohol chain ends (47% for polymer b (3) vs. 87% for polymer e (4)). Only the primary alcohol chain ends were oxidized to carboxylic acid chain ends in the synthesis of mP(EO-co-GME)-acetic acid (Example 7). The secondary alcohol end groups reacted to ketone chain ends, which did not further react in the synthesis of mP(EO-co-GME)-N,N-ditetradecylacetamide (Example 8). A column chromatography purification step removed ketone impurities from the mP(EO-co-GME)-N,N-ditetradecylacetamide product. Thus, increasing the amount of primary alcohol at the polyoxyalkylene copolymer chain ends led to increased conversion over the two-step synthesis route of mP(EO-co-GME)-N,N-ditetradecylacetamide. This is the reason why the conversion by HPLC is higher for compound l (8) (77% a) compared to compound i (8) (26% a).

[0213] Synthesis of mP(EO-co-GME)-CH2CH2-DMG (1,2-dimyristoyl-glycerol) Reagents and Chemicals DCM, triethylamine (NEt), sodium sulfate (NaSO), 1 M hydrochloric acid (HCl) solution, Py, anhydrous tetrahydrofuran (THF, max. 0.05% HO), myristate chloride (MyCl, 97%), 4-dimethylaminopyridine (DMAP), 1,2-isopropylidene-rac-glycerol (IPG) (97%), KOtBu, and acetonitrile (ACN) were obtained from Merck KGaA. p-Toluenesulfonyl chloride (TsCl) was purchased from TCI Chemicals (Tokyo, Japan). Deionized water was used for all experiments.

[0214] Example 9: mP(EO 27 -co-GME 16 Synthesis of -CH2CH2-OTs(p-toluenesulfonyl) [ka] In a 50 mL round-bottom flask, add mP(EO) 28 -co-GME 16 ) (2.46 g, 910 μmol, compound c from Example 3) and DCM (2.46 mL) were charged. The solution was cooled to 0-5 °C under stirring. Then, DMAP (11.2 mg, 91.7 μmol), NEt (166 mg, 1.64 mmol), and p-tolusulfonyl chloride TsCl (263 mg, 1.37 mmol) were added successively. The reaction mixture was stirred at 0-5 °C for an additional 1 h, then heated to 20-25 °C and stirred for 72 h. The conversion was quantified by HPLC. Since the conversion of the starting material was incomplete, NEt (55.3 mg, 546 μmol) and TsCl (87.7 mg, 460 μmol) were added at 20-25 °C, and stirring was continued for an additional 18 h. Then, DCM (22.1 mL) and water (14.8 mL) were added, and the biphasic mixture was vigorously stirred for 5 min. The phases were separated, and the organic layer was mixed with water (4.92 mL) and 1N HCl solution (0.54 mL). After stirring for 10 min, the phases were separated, and the organic layer was washed with water (4.92 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated at 700-5 mbar pressure on a rotary evaporator equipped with a water bath at 60 °C. mP(EO 27 -co-GME 16 )-CH2CH2-OTs was isolated as an orange oil (2.14 g, 749 μmol, 82.3% yield). Purity was determined by HPLC (88.7% a).

[0215] Example 10: mP(EO 27 -co-GME 16 Synthesis of -CH2CH2-DHG (dihydroxyglycerol) [ka] In a 50 mL round bottom flask, add mP(EO) from Example 9 27 -co-GME 16)-CH2CH2-OTs (1.00 g; 350 μmol; 88.7% purity) and anhydrous THF (4 mL) were added and heated to 30 °C. In a separate glass container, IPG (92.6 mg, 700 μmol) and KOtBu (78.6 mg, 700 μmol) were mixed in anhydrous THF (3 mL). The orange suspension was gradually diluted with mP(EO 27 -co-GME 16 The resulting solution was added to the 4-CH2CH2-OTs solution and stirring was continued at 30 °C for 6 h. Water (4 mL) was added and the THF was removed on a rotary evaporator with a water bath temperature of 60 °C at a pressure of 400-150 mbar. The remaining aqueous solution was extracted with DCM (8 mL) and the organic layer was concentrated on a rotary evaporator with a water bath temperature of 60 °C at a pressure of 700-20 mbar. The intermediate mP(EO 27 -co-GME 16 )-CH2CH2-IPG (0.63 g) was isolated as an orange oil.

[0216] Then, mP(EO 27 -co-GME 16 The )-CH2CH2-IPG intermediate was dissolved in water (6.30 mL) and the pH was adjusted to 1.5-1.6 using 0.1 N HCl solution (227 μL). The yellow solution was heated to 40 °C for 75 min, cooled to room temperature, and extracted with DCM (2 × 12.6 mL). The organic phases were combined and concentrated at 700-5 mbar pressure on a rotary evaporator equipped with a water bath at 60 °C. mP(EO 27 -co-GME 16 )-CH2CH2-DHG (430 mg; 160 μmol) was isolated as an orange oil. The product was analyzed by HPLC.

[0217] Example 11: mP(EO 27 -co-GME 16 Synthesis of )-CH2CH2-DMG (entry n) [ka] In a 50 mL round bottom flask, add mP(EO) from Example 10 27 -co-GME 16)-CH2CH2-DHG (430 mg; 160 μmol) and DCM (2.2 mL) were charged. The solution was stirred under ambient conditions for 5 min. Py (61.6 mg; 780 μmol) and MyCl (173 mg; 700 μmol) were added portionwise, and the reaction mixture was stirred at room temperature for 40 h. DCM (3.2 mL) and water (1.7 mL) were added to the crude product mixture, and the pH was adjusted to 1.5 using 1 M HCl solution (0.15 mL). The DCM phase was separated, washed with water (3.9 mL), and the organic solvent was removed on a rotary evaporator at 60 °C water bath temperature under 700 - 5 mbar pressure to give crude mP(EO). 27 -co-GME 16 )-CH2CH2-DMG (359 mg) was obtained as an oil. For purification, the crude product was dissolved in ACN (3.85 mL), heated to 50 °C, and filtered. Further purification was performed by flash column chromatography on a Buchi (Essen, Germany) Pure C-850 FlashPrep system using a 4 g Macherey-Nagel (Dueren, Germany) CHROMABOND Flash RS 4 SiOH cartridge (40 μm-63 μm), a gradient from ACN to iPrOH, and a flow rate of 5 mL / min. Product-containing fractions were combined and concentrated. The material was then purified using a 12 g Buchi FlashPure EcoFlex C18 cartridge (40-60 μm), a gradient from ACN / water (1:1) to iPrOH, and a flow rate of 20 mL / min. Compound n, mP(EO 27 -co-GME 16 )-CH2CH2-DMG (45.6 mg, 14.3 μmol; 8.9%) was isolated as an orange oil. 1 It was characterized by H-NMR spectroscopy and HPLC (purity 67%a).

[0218] Example 12: mP(EO 21 -co-GME 22 Synthesis of )-b-PEO2-1,2-DSPE (distearoyl-sn-glycero-3-phosphoethanolamine) [ka] Reagents and Chemicals 1,1'-Carbonyldiimidazole (CDI) and 4-(dimethylamino)pyridine were obtained from Merck KGaA. Anhydrous toluene was purchased from VWR International GmbH (Radnor, PA, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) was purchased from abcr GmbH (Karlsruhe, Germany).

[0219] Flush the round-bottom flask with nitrogen and add mP(EO 21 -co-GME 22 )-b-PEO2 (0.392 g, 133 μmol, 1.0 eq., compound f from Example 4) was charged, and anhydrous toluene (4 mL) was added. After a few minutes, a clear solution was formed. CDI (22.7 mg, 140 μmol, 1.05 eq.), DSPE (101 mg, 135 μmol, 1.02 eq., 95% purity), and DMAP (10.2 g, 82.7 μmol, 0.62 eq.) were then added to give a cloudy reaction mixture. The vessel was heated to 60 °C, and the reaction was carried out at a constant temperature for 21 h. The solution was analyzed by LC-MS to identify the target compound mP(EO 21 -co-GME 22 )-b-PEO2-DSPE was identified.

[0220] HRMS(ESI):[M deconvoluted ] calc for [C 163 H 324 NO 70 P]:3447.1562;found:3447.1555(ΔM=-0.19 ppm).

[0221] For LC-MS analysis, a Vanquish / Orbitrap Q Exactive HF (Thermo Fischer Scientific) was used. The reaction sample was diluted (1:500) with THF / ACN (8 / 2, v / v), and 10 μL of this solution was injected.

[0222] mP(EO) via cation exchange resin 21-co-GME 22 )-b-PEO2-DSPE can be converted to the corresponding ammonium salt.

[0223] mP(EO) via cation exchange resin 21 -co-GME 22 )-b-PEO2-DSPE can be converted to the corresponding sodium salt.

[0224] Preparation and biological evaluation of lipid nanoparticles (LNPs) Materials for LNP preparation and biological assays CleanCap® Fluc mRNA was obtained from TriLink BioTechnologies (San Diego, CA, USA). D-Lin-MC3-DMA was obtained from MedCHEmExpress (Monmouth Junction, NJ, USA). Cholesterol and PEO2k-DMG were purchased from Merck KGaA, and DSPC was obtained from NOF (White Plains, NY, USA). All cell lines were supplied by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany).

[0225] Example 13: Preparation of LNPs An aqueous phase containing 0.133 g / L of FLuc mRNA and 11 mM acetic acid was mixed with an ethanol phase containing 9.43 mM total lipids (DLin-MC3-DMA 50 mol%, cholesterol 38.5 mol%, DSPC 10 mol%, PEO lipids or mP(EO-co-GME) lipids 1.5 mol%) at a volume ratio of 3:1. The crude LNP colloidal dispersion was dialyzed against phosphate-buffered saline (PBS) for 3 h (with three buffer exchanges). The purified LNPs were stored at 4 °C until further use.

[0226] Example 14: Determination of the mean diameter (z-ave.) and zeta potential (z-pot.) of LNPs Determinations were performed using a Zetasizer NanoZS from Malvern Instruments GmbH (Herenberg, Germany). A DTS 1070 transparent disposable folding capillary cell from Malvern Panalytical GmbH (Kassel, Germany) was used. For particle size measurements, samples were diluted 1:10 with RNAse-free water, corresponding to an RNA concentration of 5 ng / μL. For z-pot measurements, the colloidal LNP dispersion from Example 13 was diluted 1:30 with RNAse-free water, corresponding to an RNA concentration of 1.67 ng / μL. Z-ave, the width of the fitted Gaussian distribution, expressed as the polydispersity index (PDI), as well as the average z-pot value, were calculated from the data of at least 10 runs. [Table 4]

[0227] Example 15: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay MTS assays were performed using HeLa cells. One day before transfection, 10,000 cells were seeded per well in a 96-well plate in 100 μL of medium (containing 10% FBS and 30 μg / mL gentamicin) and cultured at 37°C and 5% CO2 for 24 hours. On day 2, the old medium was removed and 90 μL of fresh medium was added to the cells. For polymer testing, the compounds from Examples 3 and 6 were dissolved in sterile water to the final concentrations shown in Table 5, and the samples were added in 10 μL volumes. For LNP testing, the colloidal LNP dispersion from Example 13 was diluted with ribonuclease-free water to adjust the mRNA concentration to 5–20 ng / μL. 10 μL of each diluted sample was added to cells equivalent to 50–200 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On day 3, cell viability was determined using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) according to the manufacturer's protocol (Promega). The absorbance signal (at 400 nm) was quantified with a multiplate reader (Infinite® 200 PRO, Tecan, Maennedorf, Switzerland). [Table 5] [Table 6]

[0228] Example 16: Characterization of LNPs by Agarose Gel Electrophoresis (AGE) The colloidal LNP dispersion from Example 13 was characterized by AGE. The assay was performed using a Thermo Fisher Scientific E-Gel™ power snap electrophoresis system. A 1% agarose gel with a volume capacity of 20 μL per well was used for sample evaluation. The results are summarized in Figures 1a and 1b. Agarose gel electrophoresis demonstrates complete encapsulation of mRNA for all LNP compositions tested (within the detection range of SYBR Safe staining). Thus, LNPs prepared with mP(EO-co-GME) lipids exhibit similar mRNA encapsulation to a reference prepared with conventional PEO lipids.

[0229] Example 17: Characterization of LNPs by RiboGreen assay The colloidal LNP dispersion from Example 13 was characterized by the RiboGreen assay. The Thermo Fischer Quant-iT™ RiboGreen™ RNA Assay Kit was used. The procedure was performed according to the manufacturer's protocol with minor adjustments. Samples were diluted to a theoretical RNA concentration of 0.4 μg / mL using either TE buffer or Triton buffer and added to a 96-well plate in a volume of 100 μl. For LNP dissolution in the presence of Triton buffer, the plate was placed in an incubator at 37°C and 5% CO2 for 10 minutes. 100 μl of dye solution was added to each well, followed by thorough pipetting. Fluorescence signals were measured using an Infinite® 200 PRO microplate reader at excitation / emission values ​​of 480 / 520 nm. All samples and standards were measured in duplicate. [Table 7]

[0230] Example 18: Determination of transfection efficiency of LNPs by luciferase assay Luciferase assays were performed using several immortal cell lines: HeLa, Jurkat, C2C12, HepG2, A549. Cell lines were grown according to standard cell culture conditions.

[0231] Transfection and readout with adherent cells One day before transfection, 10,000 cells were seeded per well in a 96-well plate in 100 μL of medium (containing 10% FBS and 30 μg / mL gentamicin) and cultured at 37°C and 5% CO2 for 24 hours. On day 2, the old medium was removed, and 90 μL of fresh medium (containing no FBS or antibiotics) was added to the cells. The LNP nanodispersion prepared according to Example 13 was adjusted to an mRNA concentration of 10 ng / μL using ribonuclease-free water for dilution. 10 μL of each diluted sample was added to the cells in a total volume of 100 μL, equivalent to 100 ng of mRNA per well. After 4 hours, the old medium containing the remaining sample was removed and replaced with 100 μL of fresh medium (containing 10% FBS and 30 μg / mL gentamicin). The cells were further incubated at 37°C and 5% CO2 for 20 hours. Transfection efficiency was determined using the Dual-Luciferase® Reporter Assay System (Promega) on day 3. Luminescent signals were quantified using an Infinite® 200 PRO multiplate reader.

[0232] Transfection and readout with suspension cells On the day of transfection, 50,000 cells were seeded per well in a 96-well plate in 90 μL of medium (containing 10% FBS and 30 μg / mL gentamicin). RNAse-free water was used for dilution to adjust the sample to an mRNA concentration of 10 ng / μL. Ten μL of each diluted sample was added to cells equivalent to 100 ng of mRNA per well in a total volume of 100 μL. The cells were further incubated at 37°C and 5% CO2 for 24 hours. On day 2, transfection efficiency was determined using the Dual-Luciferase® Reporter Assay System (Promega). Luminescence signals were quantified using an Infinite® 200 PRO multiplate reader.

[0233] For all transfection experiments, jetMessenger was used as a positive control. The reagent was prepared according to the manufacturer's protocol and applied at an RNA dose per well equal to the test samples. The results are summarized in Figure 2.

[0234] Example 19: Determination of the immunogenicity of lipids by competitive enzyme-linked immunosorbent assay (ELISA) The interaction of lipid sample entries h(8), i(8), j(8), k(8), m(8), n(11) and PEO2k-DMG with anti-PEO antibody (APA) was assessed by a competitive PEO ELISA kit using a mouse monoclonal, horseradish peroxidase-conjugated anti-PEO antibody (HRP-anti-PEO, Life diagnostics, West Chester, PA, USA) ranging from 0 to 4600 μg / mL. -1Samples ranging in concentration were prepared in dilution buffer. 50 μL of each sample was dispensed into a PEO-precoated 96-well plate, and 50 μL of HRP-anti-PEO was added to each well. The solution was incubated at 25°C for 1 h on a microplate shaker and then washed six times with 400 μL of wash buffer per well. After removing residual droplets, 100 μL of 3,3',5,51-tetramethylbiphenyl-4,4'-diamine was added to each well, and the solution was mixed on a microplate shaker for 20 min. The reaction was stopped by adding 100 μL of stop solution, and the absorbance at 450 nm was read within 5 min.

[0235] Analysis of ELISA data: The determined absorbance values ​​were normalized to visualize the percentage of maximum binding. Sample concentrations were expressed as log 10 The sigmoid fit was calculated using the following formula, where a is the upper limit, b is the lower limit, c is the inflection point, and d is the slope. y=a+(ba) / 1+10 (c-x)*d

[0236] Figures 3 and 4 show the ELISA results for compounds h(8), i(8), j(8), k(8), m(8), n(11), and PEO2k-DMG. The figures show the logarithm of the polymer lipid concentration in nanograms / mL. 10 Figure 1 shows the normalized absorbance at 450 nm versus the saturation function, thus illustrating the APA interaction with the investigated polymer lipids at various concentrations. The interaction of the polymer lipid with APA reduces adsorption. The ELISA data demonstrate the strong influence of methoxymethyl side chains on the interaction of the polymer lipid with APA (it is important to note that the x-axis has a logarithmic scale). By incorporating the sterically demanding methoxymethyl side groups into the polyether structure, significantly higher polymer lipid concentrations are required to preserve the interaction between the polymer lipid and APA.

Claims

1. Formula (I) below 【Chemistry 1】 A compound having the formula: In the formula, R １ and R ２ is R １ and R ２ saturated or unsaturated, branched or linear C═C— bonds with up to three C═C— bonds, provided that at least one of ３ -C ２０ are independently selected from a hydrocarbon group or —H; L is a linker unit, preferably 【Chemistry 2】 is selected from the group consisting of In the formula, in L6, X ＋ is hydrogen, an alkali metal cation or ammonium, In L1 to L7, the ends marked with * are bonded to the oxygen atom of the polyoxyalkylene group A, A is a polyoxyalkylene group and contains at least one of the following units: 【Transformation 3】 and 【Chemistry 4】 and 【Transformation 5】 and at least one unit selected from the group R ３ is -H, -OH, -SH, -NH ２ , -NHR ４ , -NR ４ R ５ , -OR ６ , -SR ６ or a linear, branched or cyclic alkyl group having up to 20 carbon atoms, R ４ ~R ６ are independently selected from linear, branched, or cyclic alkyl groups having up to 20 carbon atoms, in which up to 5 carbon atoms may be substituted with oxygen or sulfur atoms; -A-R ３ is a compound having a molecular weight of 1100 to 7500 g / mol.

2. R １ and R ２ are independent of each other, i) saturated or unsaturated branched or linear C having up to two -C=C- bonds; ４ -C ２０ a hydrocarbon group or —H, or ii) saturated or unsaturated linear C having up to two -C=C- bonds ４ -C ２０ a hydrocarbon group or —H, or iii) saturated or unsaturated branched or linear C having one -C=C- bond ４ -C ２０ a hydrocarbon group or —H, or iv) saturated or unsaturated linear C having one -C=C- bond ４ -C ２０ a hydrocarbon group or —H, or v) saturated branched or linear C ４ -C ２０ a hydrocarbon group or —H, or vi) saturated linear C ４ -C ２０ a hydrocarbon group or —H, or vii) saturated or unsaturated, branched or linear C having up to two -C=C- bonds; ８ -C １８ a hydrocarbon group or —H, or viii) saturated or unsaturated linear C having up to two -C=C- bonds ８ -C １８ a hydrocarbon group or —H, or ix) saturated or unsaturated branched or linear C having one -C=C- bond ８ -C １８ a hydrocarbon group or —H, or x) saturated or unsaturated linear C having one -C=C- bond ８ -C １８ a hydrocarbon group or —H, or xi) saturated branched or linear C ８ -C １８ a hydrocarbon group or —H, or xii) saturated linear C ８ -C １８ a hydrocarbon group or —H, or xiii) saturated or unsaturated branched or linear C having up to two -C=C- bonds １２ -C １７ a hydrocarbon group or —H, or xiv) saturated or unsaturated linear C having up to two -C=C- bonds １２ -C １７ a hydrocarbon group or —H, or xv) saturated or unsaturated branched or linear C having one -C=C- bond １２ -C １７ a hydrocarbon group or —H, or xvi) saturated or unsaturated linear C having one -C=C- bond １２ -C １７ a hydrocarbon group or —H, or xvii) saturated branched or linear C １２ -C １７ a hydrocarbon group or —H, or xviii) saturated linear C １２ -C １７ a hydrocarbon group or —H, or xix) saturated linear C １２ -C １４ Hydrocarbon group or -H 2. The compound of claim 1 selected from:

3. R １ and R ２ The compound according to claim 1 or 2, wherein

4. L, i) a compound of the formula: 【Transformation 6】 is selected from In L6, X ＋ is hydrogen, sodium, potassium or ammonium, and in L1, L2 and L6, the ends marked with * are bonded to the oxygen atoms of the polyoxyalkylene group A, respectively; or ii) a compound of the formula: 【Transformation 7】 wherein the ends marked with * are each bonded to an oxygen atom of the polyoxyalkylene group A; or iii) a compound of the formula: 【Transformation 8】 and the end marked with * is bonded to an oxygen atom of the polyoxyalkylene group A, or iv) a compound of the formula: 【Chemistry 9】 and the end marked with * is bonded to an oxygen atom of the polyoxyalkylene group A. The compound according to any one of claims 1 to 3.

5. R ３ Ga-OR ６ and R ６ is selected from linear, branched or cyclic alkyl groups having up to 20 carbon atoms, in which up to 5 carbon atoms may be substituted with oxygen atoms.

6. -A-R ３ The compound according to any one of claims 1 to 5, wherein said compound has a molecular weight of 1500 to 3500 g / mol.

7. A composition comprising at least one compound of formula (I) according to any one of claims 1 to 6 and at least one active agent.

8. 8. The composition of claim 7, wherein the at least one active agent is selected from the group consisting of proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogs, organic molecules having a molecular weight of up to 1000 g / mol, and combinations thereof.

9. 9. The composition of claim 8, wherein the at least one active agent is selected from the group consisting of linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA comprising at least one coding sequence (circRNA), short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA), and combinations thereof.

10. 10. The composition of any one of claims 7 to 9, further comprising a compound selected from a lipid different from said compound of formula (I), a buffering agent, a pharmaceutically acceptable salt different from said buffering agent, a cryoprotectant, or any combination thereof.

11. The composition according to any one of claims 7 to 10, which is a lipid nanoparticle.

12. A and R ３ is defined by formula (I), ３ preparing a i) replacing -H with a leaving group -X, which is capable of undergoing a substitution or coupling reaction; or ii) -H is a unit a) 【Chemistry 10】 provided that it is bonded to the oxygen atom of The following end groups: 【Chemistry 11】 oxidizing this group to form Thereafter, step i) is followed by a substitution or coupling reaction, or step ii) is followed by R １ and R ２ is defined in formula (I), and L is a linker unit, H-L or H-LR １ R ２ and / or consisting of carrying out a polycondensation reaction with each of the end groups: 【Chemistry 12】 The remainder of the following end group: 【Chemistry 13】 is immediately considered to be part of the linker group L, and when H-L is used, L may be further chemically modified by an esterification reaction to form -R １ and -R ２ Introduce A process for preparing a compound of formula (I) according to any one of claims 1 to 6.

13. A method for preparing a composition according to any one of claims 7 to 11, comprising the steps of providing at least one compound of formula (I) according to any one of claims 1 to 6, at least one active agent and optionally further ingredients, and combining all ingredients to obtain a composition according to any one of claims 7 to 11.

14. The composition according to any one of claims 7 to 11 for treating a human disease.

15. The composition according to any one of claims 7 to 11 for treating a disease in a mammal.

Images