Ionizable sterol derivatives for delivery of therapeutic agents and uses thereof

Abstract

The present disclosure relates to ionizable sterol compounds of Formula A, lipid nanoparticle compositions comprising the same, and medical uses thereof. And Z-L-(M) r A.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese invention patent application 2024113028634, filed on September 18, 2024. Technical Field

[0003] This disclosure relates to ionizable sterol derivatives, lipid nanoparticles comprising the same, compositions thereof, and their pharmaceutical uses. Background Technology

[0004] The efficient and targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids remains an ongoing medical challenge. Furthermore, gene therapy, which uses genetic material to alter the expression of target genes or modify the biological characteristics of living cells to meet therapeutic needs, hinges on the safe and effective delivery of therapeutic drugs into target cells via vectors within the body. Recent advancements in the interpretation of the human genome have made gene therapy a hot topic in the field, with nucleic acids attracting particular attention. Several gene-based products have been approved by regulatory agencies for various applications, such as the use of mRNA vaccines to combat COVID-19.

[0005] Nucleic acids, due to their negative charge, high molecular weight, and hydrophilicity, are relatively unstable and have low cell permeability, making their delivery to the active sites within cells one of the most challenging aspects of gene therapy. RNA delivery, in particular, is problematic due to its short duration of activity and low intracellular retention, requiring frequent administration and creating a trade-off between payload and carrier toxicity. Therefore, further methods are needed to facilitate the delivery of therapeutic and / or preventative drugs, such as nucleic acids, into cells.

[0006] Commonly used nucleic acid delivery vectors include viral vectors and non-viral vectors. Although viral vectors are highly efficient delivery systems for transfection and treatment, they have drawbacks such as containing immunogenic viral proteins, limited target gene loading capacity, and high cost. As a result, lipid nanoparticles (LNPs), as non-viral vectors, have attracted widespread attention due to their advantages such as good in vitro stability, in vivo degradation, and safety.

[0007] However, endosomal escape remains a significant obstacle in LNP drug delivery, and the development of ionizable lipids with high transfection efficiency is a cornerstone of development in this field. Ionizable cationic lipids, which are important components of the current mainstream lipid non-viral vector LNP, are usually composed of a head group with at least one ionizable site, a linker unit, and a tail chain. They exhibit neutrality under physiological conditions, but carry a positive charge under the acidic environment of endosomes, and electrostatically bind to negatively charged hydrophilic nucleic acid molecules. Although a variety of types of ionizable lipids have been developed or reported for use in the delivery of active agents such as nucleic acids, there is still a need for ionizable lipids and LNP delivery systems that have improved safety, delivery efficiency, or specificity.

[0008] To this end, the present disclosure constructs a class of chemical derivatives based on the structure of sterols, in which the sterol structure and its derivatives are used as the tail chain, and the ionizable head group containing amine, guanidine, heterocyclic group, etc. is connected through a biodegradable linker unit at a specific site, thereby providing a new class of cationic lipid compounds, enriching the current types of cationic lipids. The raw materials for preparing the ionizable lipid compounds are easy to obtain, the reaction conditions are mild, the product yield is high, the equipment requirements are low, and the operation is simple. Moreover, the LNP prepared based thereon can deliver and transfect nucleic acids with satisfactory efficacy, and shows excellent stability, thereby meeting the above-mentioned needs.

[0009] Brief description of the disclosure

[0010] The present disclosure provides ionizable sterol-based lipid compounds, and lipid nanoparticle compositions comprising the same. The lipid nanoparticle compositions are suitable for delivering biologically active molecules, especially for delivering nucleic acid molecules with negative charge, and have the advantages of high encapsulation efficiency, low toxicity, high expression, and good stability. The present disclosure also provides methods and uses of using such compositions to deliver active agents to subjects.

[0011] In particular, the first aspect of the present application provides an ionizable sterol compound of the following formula A, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof,

[0012] Z-L-(M)r A

[0013] wherein the variables are as defined herein.

[0014] The above-mentioned compounds and the general compounds and specific compounds disclosed in the context of the present disclosure, as well as their stereoisomers, pharmaceutically acceptable salts, or solvates, are referred to herein as "compounds of the present disclosure".

[0015] In a second aspect, the present disclosure provides a lipid carrier composition, in particular a lipid particle, more particularly a lipid nanoparticle (LNP), comprising a compound of the present disclosure.

[0016] In a third aspect, the present disclosure provides a lipid nanoparticle composition comprising a compound of the present disclosure or a lipid carrier composition of the present disclosure, and one or more therapeutic / prophylactic agents.

[0017] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle composition of the present disclosure and a pharmaceutically acceptable excipient.

[0018] In a fifth aspect, the present disclosure provides the use of a compound, a lipid carrier composition, a lipid nanoparticle composition, or a pharmaceutical composition of the present disclosure for delivering a therapeutic and / or prophylactic agent to an individual, for treating or preventing a disease or a condition, and in the manufacture of a medicament, in particular a medicament for delivering a therapeutic and / or prophylactic agent to an individual, or for treating a disease or a condition in an individual, or for introducing a nucleic acid into a cell.

[0019] In a sixth aspect, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to an individual, the method comprising administering to an individual in need thereof the lipid nanoparticle composition or the pharmaceutical composition.

[0020] In a seventh aspect, the present disclosure provides a method of treating or preventing a disease or a condition, the method comprising administering to an individual in need thereof an effective amount of the lipid nanoparticle composition or the pharmaceutical composition.

[0021] In an eighth aspect, the present disclosure provides a method of introducing a nucleic acid into a cell, the method comprising contacting the cell with the lipid carrier composition; and the use of the lipid carrier composition for introducing a nucleic acid into a cell. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1A A mass spectrometry detection profile of ST-001 is shown ([M+1]+ = 514.6).

[0023] Figure 1B A nuclear magnetic detection profile of ST-001 is shown.

[0024] Figure 2A Particle size and morphology detection results of Example LNP (0 days) are shown.

[0025] Figure 2B Morphology detection results of Example LNP (0 days) are shown.

[0026] Figure 3 Changes in hydration particle size of Example LNP after storage at 100 mM Tris-HCl / 4°C for different times are shown.

[0027] Figure 4 This image shows the morphological changes of the LNP sample after 30 days of storage in 100 mM Tris-HCl at 4 °C, as observed by transmission electron microscopy (TEM).

[0028] Figure 5 The encapsulation efficiency of the LNP in the example is shown after storage in 100mM Tris-HCl at 4°C for different times.

[0029] Figure 6 This image shows an example of LNP transfecting green fluorescent protein (GFP) in cells.

[0030] This disclosure details

[0031] Definitions

[0032] The following words, phrases, and symbols used in this disclosure have the meanings described below, unless the context otherwise requires. Undefined technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0033] As used herein, the terms "comprising" or "including" are intended to indicate the presence of a described feature, ingredient, or step, but do not exclude the presence or addition of one or more other features, ingredients, or steps. Unless otherwise specified, "comprising" or "including" as used herein covers situations consisting of the described features, ingredients, or steps.

[0034] As used in this article, the term “about” means within ±20% of a given value or range, such as within ±10% or ±5%.

[0035] All numerical ranges mentioned herein should be understood as disclosing every numerical value and subset thereof within that range, regardless of whether they are specifically disclosed separately. For example, referring to any numerical range should be considered as referring to every numerical value within that range, such as every integer within that range, such as C in this article. 1-6 This indicates a range including 1, 2, 3, 4, 5, or 6 Cs, and different ranges consisting of different values ​​within that range. This disclosure relates to all values ​​falling within these ranges, all smaller ranges, and upper or lower limits of numerical ranges.

[0036] When used to connect two or more options, the word "and / or" as used in this article should be understood as any one of the options or any combination of two or more of the options.

[0037] As used in this article, "alkyl" refers to a straight-chain or branched saturated hydrocarbon having one or more carbon atoms, for example, 1-22 carbon atoms (C1-C2). 1-12), 6-22 carbon atoms (C 6-22 ), 6-20 carbon atoms (C 6-20 ), 6-18 carbon atoms (C 6-18 ), 6-16 carbon atoms (C 6-16 ), 6-12 carbon atoms (C 6-12 ), 8-12 carbon atoms (C 8-12 ), 5-9 carbon atoms (C 5-9 ), 6-8 carbon atoms (C 6-8 ), 2-10 carbon atoms (C 2-10 ), 2-8 carbon atoms (C 2-8 ), 1-12 carbon atoms (C 1-12 ), 1-6 carbon atoms (C 1-6 ), 1-4 carbon atoms (C 1-4 ), or 1-3 carbon atoms (C 1-3 ) straight or branched chain saturated hydrocarbon groups, e.g., straight chain hydrocarbon groups. Within these groups, the expression C m-n alkyl means an alkyl group having m-n carbon atoms, including alkyl groups having any point value of carbon atoms between m and n, as well as alkyl groups having ranges of carbon atoms comprising any point values between m and n, e.g., "C 1-6 alkyl" means a straight or branched chain alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms; examples of C 6-22 alkyl groups include, but are not limited to, C 6-18 straight or branched chain alkyl groups, C 8-12 straight or branched chain alkyl groups, or C 6-12 straight or branched chain alkyl groups; examples of C 1-6 alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, i-propyl), butyl (e.g., n-butyl, i-butyl, sec-butyl, and t-butyl), pentyl (e.g., n-pentyl, i-pentyl, neopentyl), hexyl, and the like.

[0038] The term "alkenyl" as used herein refers to a straight or branched chain unsaturated hydrocarbon group having two or more carbon atoms, e.g., 2-22 carbon atoms (C 2-22 ), 6-22 carbon atoms (C 6-22 ), 6-20 carbon atoms (C 6-20 ), 6-18 carbon atoms (C 6-18 ), 6-16 carbon atoms (C 6-16 ), 6-12 carbon atoms (C 6-12 ), 8-12 carbon atoms (C 8-12 ), 5-9 carbon atoms (C 5-9 ), 6-8 carbon atoms (C 6-8 ), 2-12 carbon atoms (C2-12 ), 2-6 carbon atoms (C 2-6 ), 2-4 carbon atoms (C 2-4 ), or 2-3 carbon atoms (C 2-3 ) straight or branched chain unsaturated hydrocarbyl groups. C 6-22 Examples of alkenyl groups include, but are not limited to, C 6-18 straight or branched chain alkenyl, C 8-12 straight or branched chain alkenyl, or C 6-12 straight or branched chain alkenyl, C 2-6 Examples of alkenyl groups include, but are not limited to, ethenyl, 1- propenyl, 2-propenyl, 2-butenyl, and the like.

[0039] The term "alkynyl" as used herein refers to a straight or branched chain unsaturated hydrocarbyl group having two or more carbon atoms, for example, 2, 3, or 4 carbon atoms, containing one or more, for example, 1, 2, or 3 carbon-carbon triple bonds (C≡C), for example, 2-22 carbon atoms (C 2-22 ), 6-22 carbon atoms (C 6-22 ), 6-20 carbon atoms (C 6-20 ), 6-18 carbon atoms (C 6-18 ), 6-16 carbon atoms (C 6-16 ), 6-12 carbon atoms (C 6-12 ), 8-12 carbon atoms (C 8-12 ), 5-9 carbon atoms (C 5-9 ), 6-8 carbon atoms (C 6-8 ), 2-12 carbon atoms (C 2-12 ), 2-6 carbon atoms (C 2-6 ), 2-4 carbon atoms (C 2-4 ), or 2-3 carbon atoms (C 2-3 ) straight or branched chain unsaturated hydrocarbyl groups. C 6-22 Examples of alkynyl groups include, but are not limited to, C 6-18 straight or branched chain alkynyl, C 8-12 straight or branched chain alkynyl, or C 6-12 straight or branched chain alkynyl, C 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl, 1- propynyl, 2-propynyl, 2-butynyl, and the like.

[0040] The terms "alkylene" and "alkenylene" as used herein refer to divalent radicals of the alkyl or alkenyl groups defined herein, respectively. Examples of alkylene groups include, but are not limited to, straight or branched chain C 1-12 alkylene, C 2-10 alkylene, C 1-6 alkylene. Examples of alkenylene groups include, but are not limited to, straight or branched chain C 2-12 alkenylene, C 2-10 alkenylene, C 2-6 alkenylene.

[0041] The term "cycloalkyl" as used herein refers to non-aromatic carbocyclic rings and optionally contain one or more double or triple bonds, for example C 3-14 cycloalkyl, C 3-8 cycloalkyl, C 3-6 cycloalkyl. Unless otherwise indicated, the cycloalkyl groups described herein refer to optionally substituted cycloalkyl groups.

[0042] The term "heterocycle" or "heterocyclyl" as used herein refers to a monocyclic or polycyclic non-aromatic system in which at least one ring includes at least 1 heteroatom, such as 1, 2, 3, 4, 5 heteroatoms, which can be, for example, N, O, or S atoms, preferably N atoms. The heterocycle can have 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15 members in the ring, for example, a 3-14 membered heterocycle, a 3-12 membered heterocycle, a 3-8 membered heterocycle, a 3-6 membered heterocycle, a 4-7 membered heterocycle. The heterocycle also optionally includes one or more double or triple bonds, i.e., can be saturated (i.e., heterocycloalkyl) or unsaturated. In some embodiments of the disclosure, the heterocyclyl group can be a 3-8 membered heterocyclyl group comprising at least one nitrogen atom, additionally optionally comprising 1 to 3, for example, 1 to 2 heteroatoms selected from N, O, S. Examples of heterocycles include, but are not limited to: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl (e.g., 1-pyrrolidinyl, 2-pyrrolidinyl, and 3-pyrrolidinyl), tetrahydrofuranyl (e.g., 1-tetrahydrofuranyl, 2-tetrahydrofuranyl, and 3-tetrahydrofuranyl), tetrahydrothiophenyl (e.g., 1-tetrahydrothiophenyl, 2-tetrahydrothiophenyl, and 3-tetrahydrothiophenyl), piperidinyl (e.g., 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, and 4-piperidinyl), tetrahydropyranyl (e.g., 4-tetrahydropyranyl), tetrahydrothiopyranyl (e.g., 4-tetrahydrothiopyranyl), morpholinyl (e.g., morpholino), thiomorpholinyl, dioxanyl, piperazinyl, or azepinyl, diazepinyl, for example, 1,4-diazepinyl, 2-aza- bicyclo[2.2.1]heptyl, 2-aza-bicyclo[2.2.2]octyl, 3,6-diaza-bicyclo[3.1.1]heptyl, 3-aza- bicyclo[3.2.1]octyl, 1,7-dioxa[4.5]decyl, 2-oxa-7-azaspiro[4.4]nonyl, 7-oxaspiro[3.5]nonyl, 5-oxaspiro[2.4]heptyl, octahydropyrrolo[3.4-c]pyrrolyl, octahydro-1H-isoindolyl, octahydrobenzo[b][1.4]dioxine. The atom in the heterocyclyl group that is attached to the rest of the compound can be a carbon atom or a heteroatom, as is chemically feasible. The heterocyclyl groups in the compounds of the disclosure can be unsubstituted or substituted with one or more substituents as defined.

[0043] The term "aryl" as used herein refers to a monocyclic or fused polycyclic aromatic ring structure having the indicated number of ring atoms. Specifically, the term includes groups comprising, for example, 6 to 10, preferably 6 ring members. Particular aryl groups include phenyl and naphthyl. Aryl groups in the compounds of the disclosure can be unsubstituted or substituted by one or more substituents as defined.

[0044] The term "heteroaryl" as used herein refers to a mono- or bicyclic ring system having 5-10 (5-10 membered), for example 5-9 (5-9 membered), for example 5-6 (5-6 membered) or 8-9 (8-9 membered) ring atoms, wherein at least one ring is an aromatic ring containing a heteroatom selected from N, O, S. In embodiments of the disclosure, the heteroaryl is a 5-10 membered heteroaryl comprising at least one nitrogen atom, for example, the heteroaryl includes one or more, for example 1, 2, 3 or 4, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S (preferably N), and at least one heteroatom is N, the remaining ring atoms being carbon atoms. The heteroaryl groups include, but are not limited to:

[0045] 5-6 membered monocyclic heteroaryl groups including one or more, for example 1, 2 or 3, heteroatoms independently selected from N, O and S (preferably N), and at least one heteroatom is N, for example including 1, 2 or 3 N heteroatoms, and the remaining ring atoms are carbon atoms, for example, but not limited to, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrazinyl, pyrimidinyl, isoxazolyl, triazinyl, oxazolyl, thiadiazolyl, pyridazinyl; and

[0046] 8-10 membered bicyclic heteroaryl groups including one or more, for example 1, 2, 3 or 4, preferably 1, 2 or 3, heteroatoms independently selected from N, O and S (preferably N), and at least one heteroatom is N, the remaining ring atoms being carbon atoms, wherein at least one ring is an aromatic ring; for example 8-9 membered bicyclic heteroaryl groups, for example indolyl, indol-3-yl.

[0047] The terms "cycloalkyl", "heterocyclyl", "aryl" and "heteroaryl" as used herein refer to monovalent radicals of the respective "cycloalkyl", "heterocyclyl", "aryl" and "heteroaryl" groups as defined herein.

[0048] The term "optionally" as used herein means that the event or circumstance subsequently described can or can not occur, and includes instances in which the event or circumstance occurs and instances in which it does not. For example, "optionally substituted" includes both unsubstituted and substituted, in a chemically feasible manner, with one or more of the substituents described. "Optionally comprising" includes both comprising and not comprising.

[0049] Unless otherwise indicated, the optional substituents involved in the "optionally substituted" used in the definition of groups herein are selected from, but not limited to, halogen atoms (e.g., chloro, bromo, fluoro, or iodo groups), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, where X is selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., -OC(O)OR), alkoxys (e.g., -OR), acetals, phosphates, thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfites (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thioaldehydes (e.g., -C(S)H), sulfates, sulfonyls (e.g., -S(O)2-), amides (e.g., -C(O)NR2or -N(R)C(O)R), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, -NRH, or -NH2), carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), sulfonamides, alkyls, alkenyls, and cyclic groups (e.g., cycloalkyls or heterocyclic groups). In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituents themselves can be further substituted with, for example, 1, 2, 3, 4, 5, 6 substituents as defined herein. For example, a C 1-6 An alkyl group can be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0050] As used herein, the following abbreviations have the following meanings: represents a single or double bond as appropriate, and

[0051] As used herein in describing groups of compounds, the expression "separated by" includes instances where the spacer is intermediate the groups described, and also instances where the spacer is terminal to either of the groups described.

[0052] The terms "compound" or "compounds of the disclosure" as used herein refer to all isomers and isotopic variants (e.g., one or more deuterated variants) of the structures described. In addition, pharmaceutically acceptable salts, solvates, and hydrates of the structures described are also included.

[0053] The term "isomer" as used herein refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound can contain one or more chiral centers and / or double bonds and therefore exist as stereoisomers, such as double-bond isomers (i.e., geometric E / Z isomers) or diastereomers. It will also be understood by those skilled in the art that the present disclosure includes all individual stereoisomers (e.g., enantiomers) of the defined compounds, racemic mixtures or mixtures of partially resolved or partially enriched isomers, and where appropriate, individual tautomers thereof.

[0054] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a free acid or base of a compound which is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject for treatment or prevention. For example, acid addition salts include, for example, salts derived from inorganic acids and organic acids, including, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids including, for example, p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. See, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002, for a general description of pharmaceutically acceptable salts.

[0055] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein refers to an excipient or carrier that is compatible with the active ingredients or lipid particles in the composition (in some embodiments, that stabilizes the active ingredients) and that does not cause an undesirable physiological effect in a subject to whom the composition is administered. Suitable pharmaceutically acceptable excipients or carriers are disclosed in standard references in the field (Remington's Pharmaceutical Sciences).

[0056] The term "linking unit" as used herein refers to a linker, i.e., a cleavable group, that covalently links two moieties but is broken to sever the covalent linkage between the two moieties under physiologically relevant conditions. The linking unit can comprise one or more groups, including but not limited to phosphate groups (e.g., phosphates, boronophosphates, thiophosphates, selenophosphates, and phosphonates), alkyl groups, esters, amides, carbonates, sulfonamides, or glycerol. Typically, the cleavable group is severed in the intracellular environment more rapidly than outside the cell, resulting in the preferential release of the loaded drug inside the target cell. Cleavage can be enzymatic or non-enzymatic, and can leave a portion of the linker attached to the loaded drug, or can release the loaded drug without any linker residue.

[0057] The term "lipid component" as used herein is a component of a nanoparticle composition comprising one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, polymer-conjugated lipids such as PEGylated lipids, structural lipids, or other lipids, e.g., phospholipids.

[0058] The term "ionizable lipid" as used herein refers to a lipid having a group or atom that can become charged (ionized) with changes in environmental pH, such that they can exist in a positively charged (e.g., at low pH) or neutral (e.g., at higher pH) form depending on the pH. The compounds of the present disclosure are ionizable lipids. Ionization of ionizable lipids affects the surface charge of the lipid nanoparticle under different pH conditions.

[0059] The term "polymer-conjugated lipid" as used herein refers to a lipid conjugated to a hydrophilic polymer, including but not limited to polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyl oxazoline, polyethyl oxazoline, and the like. In particular embodiments, the hydrophilic polymer-conjugated lipid is a PEG lipid.

[0060] As used herein, "PEG lipid" includes, but is not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid is selected from 1,2-dimyristoyl-sn-glycero methoxypolyethyleneglycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-distearoylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacetylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). The PEG lipid is preferably 1,2-dimyristoyl-sn-glycero methoxypolyethyleneglycol (PEG-DMG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)], for example 1,2-dimyristoyl-sn-glycero methoxypolyethyleneglycol 2000 (PEG-DMG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol 2000)] (PEG-DSPE). 2k -DMG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol 2000)] (PEG 2k -DSPE).

[0061] As used herein, the term "phospholipid" includes a lipid having a phosphate moiety and one or more carbon chains, e.g., saturated or unsaturated fatty acid chains (e.g., having 1 to 20 carbon atoms). The phospholipid can comprise one or more unsaturated bonds (e.g., double or triple bonds). Particular phospholipids can facilitate fusion with a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of the phospholipid with the membrane can allow one or more elements of the lipid-containing composition to pass through the membrane, thereby allowing, for example, delivery of one or more elements to a cell.

[0062] As used herein, the term "structural lipid" refers to a steroid or a lipid containing a steroid moiety, e.g., a sterol and / or a lipid containing a sterol moiety. In particular embodiments, the structural lipid is cholesterol, which can be naturally occurring or synthetic, a compound having a cyclopentanoperhydrophenanthrene skeleton. The structural lipid of the present disclosure can also be a derivative of cholesterol, i.e., a cholesterol molecule having a cyclopentanoperhydrophenanthrene and one or more additional functional groups.

[0063] The term "sterol" as used herein refers to a group of steroid compounds or salts or esters thereof, naturally occurring or synthetic, having a cyclopentanoperhydrophenanthrene skeleton and one or more OH groups. Sterols are generally classified into two categories, phytosterols or animal sterols, including but not limited to cholesterol, beta-sitosterol, coprostanol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, tomatidine, hydrolyzable tomatine, ursolic acid, alpha-tocopherol, and the like.

[0064] The term "delivering" as used herein refers to providing an entity to a target. For example, delivering a therapeutic and / or prophylactic agent to an individual can involve administering a lipid nanoparticle comprising the therapeutic and / or prophylactic agent to the individual (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a lipid nanoparticle to a mammal or mammalian cell can involve contacting one or more cells with the nanoparticle composition.

[0065] The term "specific delivery" or "specific delivery" or "enhanced delivery" as used herein refers to the delivery of more (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) of a therapeutic and / or prophylactic drug by a nanoparticle to a target tissue (e.g., a mammalian liver, spleen, stomach, intestine) compared to a non-target tissue. It will be appreciated that "specific delivery" or "specific delivery" or "enhanced delivery" of a nanoparticle to a target tissue can be determined in a subject being treated, but also in an animal model (e.g., a rat model).

[0066] The term "therapeutic and / or prophylactic agent", "therapeutic agent" or "prophylactic agent", "active substance" or "active agent" as used herein refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject, including but not limited to cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0067] In particular embodiments, the therapeutic and / or prophylactic agent can be selected from ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs), such as natural or modified RNAs or DNAs. For example, the RNA is selected from single-stranded RNA, double-stranded RNA (dsRNA), partially double-stranded RNA, and mixtures thereof, and / or the RNA is selected from circular RNA, linear RNA, and mixtures thereof. In this regard, the therapeutic and / or prophylactic agent includes, but is not limited to, messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antisense RNA, ribozymes, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof. The mRNA, for example, includes one or more of a stem loop, a chain-terminating nucleoside, a poly-A sequence, a polyadenylation signal, and / or a 5’ cap structure. The mRNA can include one or more modified nucleobases, nucleosides, or nucleotides, in which case it can be referred to as “modified mRNA” or “mmRNA.”

[0068] The DNA molecule can be double-stranded DNA, single-stranded DNA (ssDNA), or a molecule that is partially double-stranded DNA, i.e., has portions that are double-stranded and portions that are single-stranded. The DNA molecule can be triple-stranded or partially triple-stranded, i.e., has portions that are triple-stranded and portions that are double-stranded. The DNA molecule can be a circular DNA molecule or a linear DNA molecule. The DNA therapeutic and / or prophylactic agent can be a DNA molecule that is capable of transferring a gene into a cell, e.g., which encodes and can express a transcript. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule produced in vitro. For example, the DNA molecule is a recombinant molecule. Non-limiting example DNA therapeutic and / or prophylactic agents include plasmid expression vectors and viral expression vectors.

[0069] The term “lipid carrier composition” as used herein is a composition comprising one or more lipids, including lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles), and lipoplexes, in particular referring to lipid nanoparticles, which typically have a particle size in the order of microns or less, and can include a lipid bilayer. The lipid carrier composition serves as a carrier for the therapeutic and / or prophylactic agent, delivering the latter to the interior of a cell.

[0070] The term "lipid nanoparticle composition" as used herein is a composition comprising one or more lipids or a lipid carrier composition of the present disclosure, and one or more therapeutic and / or prophylactic agents, preferably a lipid nanoparticle LNP, having a nanometer scale (1-1000 nm) size, a spherical or polyhedral particle with an electron-dense core, due to the presence of ionizable lipids, the LNP can entrap a negatively charged nucleic acid in the core part of the particle by mutual adsorption of positive and negative charges. For example, the diameter of the lipid nanoparticle composition is about 50-500 nm, for example about 50-350 nm. The therapeutic or prophylactic agent, such as a nucleic acid, can be 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 undesired effects such as an adverse immune response induced by mechanisms of the host organism or cell.

[0071] The term "encapsulation efficiency" as used herein refers to the amount of therapeutic and / or prophylactic agent that becomes part of the lipid particle composition relative to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of the lipid particle. For example, if 97 mg of the 100 mg of therapeutic and / or prophylactic agent initially provided to the composition is encapsulated in the lipid particle, an encapsulation efficiency of 97% can be given. As used herein, "encapsulated" can refer to complete, substantial or partial encapsulation, enclosure, surrounding or encapsulation.

[0072] The term "polydispersity index" or "PDI" as used herein is a ratio that describes the uniformity of the particle size distribution of a system. A lower value (e.g. less than 0.3) indicates a narrower particle size distribution.

[0073] The term "particle size" or "average particle size" as used herein describes the average diameter of the lipid particle.

[0074] The term "zeta potential" as used herein describes the electrokinetic potential of the lipids in the particle composition.

[0075] The term "N / P ratio" or "nitrogen / phosphor ratio" as used herein refers to the molar ratio of ionizable nitrogen atoms in the lipids to the phosphate groups in the RNA, for example in a composition comprising lipid particles and RNA, such as mRNA, in the physiological pH range.

[0076] "Expression" of a nucleic acid sequence as used herein refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein. "Transfection" as used herein refers to the introduction of a species (e.g. RNA) into a cell, and transfection can be performed, for example, in vitro, ex vivo or in vivo.

[0077] The term "treatment" as used herein refers to the application of a composition described herein to a subject, e.g., a mammal, e.g., a human, who has a disease or symptoms of a disease, with the aim to cure, alleviate or lessen the disease or symptoms of the disease. Preferably, the treatment is curative or ameliorative.

[0078] The term "prevention" as used herein refers to the application of a composition described herein to a subject, e.g., a mammal, e.g., a human, who is suspected of having or susceptible to a disease, such that the risk of developing the defined disease is reduced, or the onset of the disease is prevented. The term "prevention" includes the use of the compositions of the disclosure prior to the diagnosis or determination of any clinical and / or pathological symptoms.

[0079] The term "effective amount" as used herein refers to the amount of a therapeutic and / or prophylactic agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) to be delivered that is sufficient to have an effect when administered to an individual having or susceptible to an infection, disease, and / or disorder, to treat, alleviate, lessen, ameliorate, inhibit progression of, reduce severity of, diagnose, prevent, and / or delay onset of the infection, disease, and / or disorder.

[0080] The term "administration" as used herein refers to physical introduction of one or more therapeutic and / or prophylactic agents into an individual using a variety of methods known to those of skill in the art and delivery systems of the disclosure, e.g., LNP, including, e.g., oral, intravenous (e.g., infusion, drip, or injection), intramuscular, subcutaneous, intradermal, intraperitoneal, spinal, topical, or other parenteral routes of administration. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and non-limitingly includes intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection as well as infusion techniques, and in vivo electroporation. Accordingly, each active agent in the combination of the disclosure can be formulated into a capsule, tablet, injection (including infusion or injectable solution), syrup, spray, lozenge, liposome, or suppository, etc.

[0081] The term "individual," "subject," or "patient" as used herein refers to a mammal and non-mammal that can seek or need treatment, need treatment, is receiving treatment, will receive treatment, or is receiving care for a particular disease. Mammals include, but are not limited to, humans; non-human primates, cattle, horses, sheep, pigs, rabbits, dogs, and cats, etc. An "individual" is not limited to a particular age or gender. Preferably, the individual, subject, or patient is a human. DETAILED DESCRIPTION

[0082] I. Ionizable sterol compounds of the disclosure

[0083] The compound of the present disclosure takes a sterol structure connected at a specific site as a hydrophobic tail chain, takes a tertiary amine structure or a nitrogen-containing heterocycle or heteroaryl as an ionizable head group, and the two are connected through a connecting unit containing a biodegradable chemical bond. The constructed amphiphilic compound can efficiently deliver RNA into cells or tissues when forming a lipid nanoparticle, showing strong transfection efficiency, and the prepared lipid nanoparticle has good stability, and the encapsulation efficiency and particle size remain stable after long-term storage.

[0084] The ionizable sterol compound of the present disclosure not only can be used as an important lipid component for forming a lipid nanoparticle, but also in some embodiments, the ionizable sterol compound itself has a strong cancer cell differentiation and anticancer form, with tumor inhibition, chemoprevention and neuroprotection properties. Therefore, the lipid carrier composition or the lipid nanoparticle composition of the present disclosure is expected to play a dual role of delivering active agents and tumor inhibition / chemoprevention / neuroprotection, providing a better choice for the field of targeted therapy and gene therapy.

[0085] Specifically, the present disclosure provides the following ionizable sterol technical solutions in one aspect:

[0086] The ionizable sterol compound of formula A, or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof,

[0087] Z-L-(M)r formula A

[0088] Wherein:

[0089] Z is the structure described in the following formula B,

[0090]

[0091] Wherein, the wavy line represents the connection point with L;

[0092] R 1a is H, C 1-6 alkyl, -C(O)NR a R b , -C(O)R c , -C(O)OR d , or an amino acid residue Wherein R e is selected from the residue part of L-histidine, L-leucine, L-isoleucine, L-valine, L-phenylalanine, L-tryptophan, L-lysine, L-tyrosine;

[0093] Wherein R a and R b are the same or different from each other, selected from H, -C 1-6 alkyl, -C 2-6 alkenyl and -C2-6 Each alkynyl group may be optionally substituted with one or more groups selected from allyl, carbonyl, and 5- to 10-membered heteroaryl groups comprising 1 to 3 heteroatoms selected from N, O, and S.

[0094] R c and R d Each is independently -C 1-6 Alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl group;

[0095] R 1b It is H, -C 1-6 Alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl group;

[0096] R2 is H, OH, or -OC. 1-6 alkyl;

[0097] R3 is H or CH3;

[0098] When W forms a single bond with the adjacent carbon atom at the * position, W is selected from N and CR. f R4 is H or OH; when W has a double bond with the carbon atom at the adjacent * position, W is C, and R4 does not exist.

[0099] R f It is absent, H, halogen or -C 1-6 alkyl;

[0100] R 5a and R 5b Each can be H, OH, or -OC independently. 1-6 Alkyl, or R 5a and R 5b Together with the ring carbon atoms they are attached to, they form -C(O)-;

[0101] L is a linking unit which is an optionally substituted straight chain or branched alkylene having 3 to 50 skeletal carbon atoms, covalently linking the steroid ring fragment and r ionizable head groups M, wherein at least one skeletal carbon atom of L is replaced by -0-, -S-, -NR6-, -NR6-NR6', -N=N-, -CH=N-, -N=CH-, -CH=CH-, -CºC-, -0-NR6-, -S-S-, -SO-, -S02, -C(O)-, -C(=S)-, -C(=NR6)-, -C(0)0-, -C(0)S-, -OC(O)-, -SC(O)-, -NR6C(0)-, -C(0)NR6-, -NR6S02-, -S02NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -SC(0)NR6-, -NR6C(0)NR6'-, -NR6C(0)S-, -P(0)(OR6)0-, -OP(0)(OR6)-, -OP(0)(OR6)0-, optionally substituted C 3-8 cycloalkylene, optionally substituted 3-8 membered heterocyclylene, optionally substituted C 6-10 arylene or optionally substituted 5-10 membered heteroarylene;

[0102] wherein R6and R6' are each independently H, optionally substituted -C 1-12 alkyl or optionally substituted -C 2-12 alkenyl;

[0103] M is -(CH2) p -M1, wherein M1is selected from NR7R8, optionally substituted saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom and optionally substituted 5-10 membered heteroaryl containing at least one nitrogen atom;

[0104] R7and R8are each independently selected from H, optionally substituted -C 1-6 alkyl, optionally substituted -C 2-6 alkenyl, optionally substituted -C 2-6 alkynyl;

[0105] G1is absent or is

[0106] G2is absent or is

[0107] G3is absent or is

[0108] R 11a and R 11b are each independently H, -OH or C 1-6 alkyl;

[0109] R12 selected from -C 3-10 alkyl, -C 3-10 alkenyl, -C 3-10 cycloalkyl, -C 3-10 cycloalkenyl, -C 6-10 aryl, -3-8 membered heterocyclyl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, -5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, halogen, -OH, -OC 3-10 alkyl, -NR 13a R 13b wherein each of said cycloalkyl, aryl, heterocyclyl, and heteroaryl is optionally substituted with -C 1-6 alkyl;

[0110] R 13a and R 13b each independently is H or -C 1-6 alkyl;

[0111] p is an integer from 0 to 6; and

[0112] q and r are each independently an integer from 1 to 3.

[0113] In one embodiment, the ionizable sterol compound of Formula A is a compound of Formula I below, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof,

[0114]

[0115] wherein:

[0116] L1and L2are each independently absent or a straight or branched -C 1-12 alkylene-, wherein the alkylene is optionally substituted with OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl, and wherein the alkylene is optionally interrupted with one or more -O-, -S-, -NR6-, -CH=CH-, -CºC-, -C(O)-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -OC(O)NR6-, or -OC(O)O-;

[0117] X is selected from the group consisting of absent, -0-, -S-, -NR6-, -NR6-NR6'-, -N=N-, -CH=N-, -N=CH-, -CH=CH-, -CºC-, -0-NR6-, -S-S-, -SO-, -S02, -C(O)-, -C(=S)-, -C(=NR6)-, -C(0)0-, -C(0)S-, -OC(O)-, -SC(O)-, -NR6C(0)-, -C(0)NR6-, -NR6S02-, -S02NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -SC(0)NR6-, -NR6C(0)NR6'-, -NR6C(0)S-, -P(0)(OR6)0-, -OP(0)(OR6)-, -OP(0)(OR6)0-, optionally substituted C 3-8 cycloalkylene, optionally substituted 3-8 membered heterocyclyl, optionally substituted C 6-10 aryl or optionally substituted 5-10 membered heteroaryl, wherein the optional substituents are selected from the group consisting of -OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(0)N(R6R6'), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl;

[0118] R6and R6' are each independently H, -C 1-12 alkyl or -C 2-12 alkenyl, each optionally substituted with -OH, -COC 1-6 alkyl, -COOC 1-6 alkyl, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, oxo, halogen, cyano, nitro, -C(0)NH2, -C(0)NHC 1-6 alkyl, -C(0)N(C 1-6 alkyl)2, -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl substituted;

[0119] M is -(CH2) p-M1, wherein M1is selected from the group consisting of NR7R8, a saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, a 5-10 membered heteroaryl containing at least one nitrogen atom, wherein the heterocyclyl or heteroaryl is optionally substituted by one or more OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), amidine, guanidine, -OC 1-6 alkyl and -C 1-6 alkyl substituted;

[0120] R7and R8are each independently selected from the group consisting of H, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, each optionally substituted by one or more groups selected from OH, -COR6, -COOR6, -NR6R6', -C(O)N(R6R6'), amidine, guanidine, halogen, cyano, nitro, -OC 1-6 alkyl, -(OC 1-6 alkylene) n -OH, a saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, a 5-10 membered heteroaryl containing at least one nitrogen atom, wherein the heterocyclyl or heteroaryl is optionally substituted by one or more OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), amidine, guanidine, -OC 1-6 alkyl and -C 1-6 alkyl substituted;

[0121] n is an integer from 1 to 4;

[0122] the remaining groups are as defined for formula A.

[0123] In further embodiments, the compounds of formula I can have the following subformulae:

[0124]

[0125]

[0126] wherein the individual groups are as defined for formula A and / or formula I, respectively.

[0127] In some embodiments of the above compounds, R 1a is H and R 1b is H.

[0128] In some embodiments of the above compounds, R 1a is -C 1-6 alkyl, in particular -C 1-3 alkyl, such as methyl, ethyl or propyl.

[0129] In some embodiments of the above compounds, R 1a is -C(O)NR a R b wherein R a and R b are the same or different from each other, selected from H, -C 1-6 alkyl, -C 2-6 alkenyl and -C 2-6 alkynyl, each optionally substituted with one or more groups selected from allyl, carbonyl and 5- to 10-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, S; preferably R a and R b are the same or different from each other, selected from H or -C 1-6 alkyl, -C 2-6 alkenyl and -C 2-6 alkynyl optionally substituted with 5- to 10-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, S, for example 1H-imidazol-4-yl.

[0130] In some embodiments of the above compounds, R 1a is -C(O)R c wherein R c is -C 1-6 alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl, preferably -C 1-6 alkyl, more preferably -C 1-3 alkyl, for example methyl, ethyl or propyl.

[0131] In some embodiments of the above compounds, R 1a is -C(O)OR d wherein R d is -C 1-6 alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl, preferably -C 1-6 alkyl, for example ethyl or butyl.

[0132] In some embodiments of the above compounds, R 1a is an amino acid residue In particular wherein R e is selected from the residue moiety of L-histidine, L-leucine, L-isoleucine, L-valine, L-phenylalanine, L-tryptophan, L-lysine, L-tyrosine, in particular

[0133]

[0134] In some embodiments of the above compounds, R1a Selected from H, -C 1-6 Alkyl and -C(O)C 1-6 Alkyl groups, preferably selected from H and -C 1-3 Alkyl and -C(O)C 1-3 alkyl.

[0135] In some embodiments of the above compounds, R 1b It is H.

[0136] In some embodiments of the above compounds, R 1b It is -C 1-6 Alkyl, -C 2-6 alkenyl or -C 2-6 Alkyne group.

[0137] In some embodiments of the above compounds, R2 is H.

[0138] In some embodiments of the above compounds, R2 is OH or -OC. 1-6 Alkyl groups, such as OH or -OC 1-3 alkyl.

[0139] In some embodiments of the above compounds, R3 is CH3.

[0140] In some embodiments of the above compounds, W is N, in which case there is a single bond between W and the adjacent carbon atom, and R4 is H.

[0141] In some embodiments of the above compounds, W is -CR f At this point, W has a single bond with the adjacent carbon atom, R4 is H, and R f It is H.

[0142] In some embodiments of the above compounds, W is -CR f At this point, W has a single bond with the adjacent carbon atom, R4 is OH, and R f It is H; furthermore, the configuration of the ring carbon atom connected to R4 is S configuration.

[0143] In some embodiments of the above compounds, W is -CR f At this point, W has a double bond with the adjacent carbon atom, R4 does not exist, and R f It does not exist.

[0144] In some embodiments of the above compounds, R 5a and R 5b Each is H independently.

[0145] In some embodiments of the above compounds, R 5a and R 5b Each can be H, OH, or -OC independently.1-6 alkyl.

[0146] In some embodiments of the above compounds, R 5a and R 5b Together with the ring carbon atoms they are attached to, they form -C(O)-.

[0147] In some embodiments of the above compounds, L is a linking unit, which is an optionally substituted straight-chain or branched alkylene group having 3-50 skeletal carbon atoms, covalently linked to a steroidal segment and r ionizable head groups M; specifically, it is an optionally substituted straight-chain or branched alkylene group having, for example, 3-40, 3-30, or 3-20 skeletal carbon atoms, more specifically, an optionally substituted straight-chain or branched alkylene group having 2-12 skeletal carbon atoms.

[0148] In some embodiments of the above compounds, at least one skeletal carbon atom of L is converted to -O-, -S-, -NR6-, -NR6-NR6', -N=N-, -CH=N-, -N=CH-, -CH=CH-, -C≡C-, -O-NR6, -SS-, -SO-, -SO2, -C(O)-, -C(=S)-, -C(=NR6)-, -C(O)O-, -C(O)S-, -OC(O)-, - SC(O)-, -NR6C(O)-, -C(O)NR6-, -NR6SO2-, -SO2NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -SC(O)NR6-, -NR6C(O)NR6'-, -NR6C(O)S-, -P(O)(OR6)O-, -OP(O)(OR6)-, -OP(O)(OR6)O-, optional substituted C 3-8 Cycloalkylene, optionally substituted 3-8 membered heterocyclic alkylene, optionally substituted C 6-10 A arylene or optionally substituted 5-10 heteroarylene groups;

[0149] Specifically, at least one skeletal carbon atom is substituted with -O-, -S-, -NR6-, -NR6-NR6', -SS-, -SO-, -SO2, -CH=CH-, -C≡C-, -C(O)-, -C(O)O-, -OC(O-, -NR6C(O-), -C(O)NR6-, -NR6SO2-, -SO2NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -NR6C(O)NR6'-, or optionally C-. 3-6 Cycloalkylene, optionally substituted 5-7 membered heterocyclic alkylene, optionally substituted C 6-10 A arylene or optionally substituted 5-10 heteroarylene groups;

[0150] More specifically, at least one backbone carbon atom of L is replaced by -0-, -S-, -NR6-, -S-S-, -SO-, -SO2, -CH=CH-, -CºC-, -C(O)-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -NR6C(O)NR6’-.

[0151] In some embodiments of the above compounds, the optional substituents on L are selected from OH, -COR6, -COOR6, -NR6R6’, oxo, halogen, cyano, nitro, -C(O)N(R6R6’), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl; in particular selected from OH, -NR6R6’, halogen, -OC 1-6 alkyl, -C 2-6 alkenyl, -C 3-6 cycloalkyl.

[0152] In some embodiments of the above compounds, in view of the fact that the linking unit L itself can be a branched structure, and that its backbone atoms themselves or on the substituents fragments can carry substituted or unsubstituted amino groups, the ionizable sterol compounds of the present disclosure can carry two or more ionizable sites.

[0153] In some embodiments, the linker unit L comprises a branched alkylene group, each branch of which can be attached to an M group as defined in the present disclosure, e.g. with two branches and can carry at least two M groups, with three branches and can carry at least 3 M groups.

[0154] In some embodiments, the alkylene group of the linker unit L is a linear alkylene group, whereby the compounds comprise the structural fragment In particular

[0155] In some embodiments, the alkylene group of the linker unit L has two branches at its end, whereby the compounds comprise the structural fragment In particular wherein each p and M1is independently selected.

[0156] In some embodiments, the alkylene group of the linker unit L has three branches at its end, whereby the compounds comprise the structural fragment In particular wherein each p and M1is independently selected.

[0157] In some embodiments of the above compounds, L is -L1-X-L2-, wherein at least one of X, L1, and L2 is present when X is not present.

[0158] In some embodiments of the above compounds, L is -L1-X-L2-, wherein at least one of X and L2 is present when L1 is not present.

[0159] In some embodiments of the above compounds, L is -L1-X-L2-, wherein p is not 0 when L2 is not present.

[0160] In some embodiments of the above compounds, L is -L1-X-L2-, wherein each of L1 and / or L2 is independently absent, or is a straight chain or branched -C 1-12 alkylene-, in particular a straight chain or branched -C 1-6 alkylene-.

[0161] In some embodiments, L1 and / or L2 is a straight chain -C 1-12 alkylene-, the optional substituents and spacers of which are as defined herein in the general or specific or preferred embodiments.

[0162] In some embodiments, L1 and / or L2 is a branched -C 1-12 alkylene-, the optional substituents and spacers of which are as defined herein in the general or specific or preferred embodiments.

[0163] In some embodiments of the above compounds, the alkylene in L1 and / or L2 is optionally substituted with -OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl, in particular optionally substituted with -OH, -NR6R6', halogen, -OC 1-6 alkyl, -C 2-6 alkenyl, -C 3-6 cycloalkyl, more particularly optionally substituted with -OH, -NR6R6', halogen, -OC 1-6 alkyl.

[0164] In some embodiments of the above compounds, the alkylene in L1and / or L2is optionally interrupted by one or more -0-, -S-, -NR6-, -CH=CH-, -CºC-, -C(O)-, -C(0)0-, -OC(O)-, -NR6C(0)-, -C(0)NR6-, -OC(0)NR6-, -NR6C(0)0-, -NR6C(0)NR6’-, or -OC(0)0-; in particular, the alkylene is optionally interrupted by one or more -0-, -NR6-, -C(0)0-, -OC(O)-, -NR6C(0)-, -C(0)NR6-, -NR6C(0)0-, -NR6C(0)NR6’-, -OC(0)NR6-, or -OC(0)0-.

[0165] In some embodiments, the alkylene of L1and / or L2is optionally interrupted by one or more -0-, e.g., L1and / or L2comprises the repeating structural fragment -[(CH2) m -O] t - wherein m is an integer from 1 to 6 and t is an integer from 0 to 6; in one particular embodiment, m is an integer of 2 and t is an integer from 2 to 6; in another particular embodiment, m is an integer from 1 to 6 and t is 1; it is noted that m in each occurrence of -[(CH2) m -O]- is independently an integer from 1 to 6.

[0166] In some embodiments, the alkylene of L1and / or L2is optionally interrupted by one or more -NR6-, e.g., L1and / or L2comprises the repeating structural fragment -[(CH2) m -NR6] t - wherein m is an integer from 1 to 6 and t is an integer from 0 to 6; in one particular embodiment, m is an integer from 2 to 4 and t is an integer from 2 to 6; in another particular embodiment, m is an integer from 1 to 6 and t is 1; it is noted that m in each occurrence of -[(CH2) m -NR6]- is independently an integer from 1 to 6.

[0167] In some embodiments, the alkylene of L1and / or L2is interrupted by one or more -NR6C(0)0-, -NR6C(0)NR6’-, -OC(0)NR6-, or -OC(0)0-; e.g., -C 1-6 alkylene-O-CO-NR6-C 1-6 alkylene-, -C 1-6 alkylene-O-CO-O-C 1-6 alkylene-.

[0168] In some embodiments of the above compounds, L1is absent, or is -C1-6 alkylene- or -C 2-6 Alkenyl-.

[0169] In some embodiments of the above compounds, X is absent.

[0170] In some embodiments of the above compounds, X is selected from -O-, -S-, -NR6-, -NR6-NR6', -N=N-, -CH=N-, -N=CH-, -O-NR6, -SS-, -SO-, -SO2, -C(O)-, -C(=S)-, -C(=NR6)-, -C(O)O-, -C(O)S-, -OC(O-, -SC(O-), -NR6C(O-), -C(O)NR6-, -NR6SO2-, -SO2NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -SC(O)NR6-, -NR6C(O)NR6'-, -NR6C(O)S-, -P(O)(OR6)O-, -OP(O)(OR6-, -OP(O)(OR6)O-, and optionally substituted C 3-8 Cycloalkylene, optionally substituted 3-8 membered heterocyclic alkylene, optionally substituted C 6-10 A 5-10 membered heteroarylene or optionally substituted aryl group, wherein the optional substituent is selected from OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), -OC 1-6 Alkyl, -C 1-6 Alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl;

[0171] Specifically, X is selected from -O-, -S-, -NR6-, -NR6-NR6', -SS-, -SO-, -SO2, -CH=CH-, -C≡C-, -C(O)-, -C(O)O-, -OC(O-), -NR6C(O-), -C(O)NR6-, -NR6SO2-, -SO2NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -NR6C(O)NR6'-, and optionally substituted C. 3-6 Cycloalkylene, optionally substituted 5-7 membered heterocyclic alkylene, optionally substituted C 6-10 Alene or optionally substituted 5-10 heteroaryl groups;

[0172] More particularly, X is selected from -0-, -S-, -NR6-, -S-S-, -SO-, -S02, -CH=CH-, -CºC-, -C(O)-, -C(0)0-, -OC(O)-, -NR6C(0)-, -C(0)NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -NR6C(0)NR6’-;

[0173] More particularly, X is selected from -0-, -S-, -NR6-, -NR6-NR6’-, -0-NR6-, -C(O)-, -C(0)0-, -OC(O)-, -NR6C(0)-, -C(0)NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -NR6C(0)NR6’-;

[0174] More particularly, X is selected from -0-, -NR6-, -OC(O)-, -NR6C(0)-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -NR6C(0)NR6’-.

[0175] In some embodiments of the above compounds, the optional substituents on X are selected from OH, -COR6, -COOR6, -NR6R6’, oxo, halogen, cyano, nitro, -C(0)NR6, -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl; in particular from OH, -NR6R6’, halogen, -OC 1-6 alkyl, -C 2-6 alkenyl, -C 3-6 cycloalkyl.

[0176] In some embodiments of the above compounds, M is -(CH2) p -M1, wherein M1is NR7R8.

[0177] In some embodiments of the above compounds, R7and R8are each independently selected from H, optionally substituted -C 1-6 alkyl, optionally substituted -C 2-6 alkenyl, optionally substituted -C 2-6 alkynyl; in particular from H and optionally substituted -C 1-6 alkyl.

[0178] In some embodiments of the above compounds, R7and R8are each independently selected from H, optionally substituted -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6each alkynyl is optionally substituted with one or more groups selected from -OH, -COR6, -COOR6, -NR6R6', -C(O)N(R6R6'), amidino, guanidino, halo, cyano, nitro, -OC 1-6 alkyl, -(OC 1-6 alkylene) n -OH, saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, 5-10 membered heteroaryl containing at least one nitrogen atom, wherein the heterocyclyl or heteroaryl is optionally substituted with one or more OH, -COR6, -COOR6, -NR6R6', oxo, halo, cyano, nitro, -C(O)N(R6R6'), amidino, guanidino, -OC 1-6 alkyl and -C 1-6 alkyl.

[0179] In some embodiments of the above compounds, the optional substituents on R7and R8are selected from -OH, -COOR6, -NR6R6', halo, cyano, nitro, -OC 1-6 alkyl, saturated or partially unsaturated 4-7 membered heterocyclyl containing at least one nitrogen atom, 5-10 membered heteroaryl containing at least one nitrogen atom, wherein the heterocyclyl or heteroaryl is optionally substituted with one or more OH, -COOR6, -NR6R6', oxo, halo, cyano, nitro, -C(O)N(R6R6'), amidino, guanidino, -OC 1-6 alkyl and -C 1-6 alkyl.

[0180] In some embodiments of the above compounds, the optional substituents on R7and R8are selected from -OH and -NR6R6'.

[0181] In some embodiments of the above compounds, M is -(CH2) p -M1, wherein M1is NR7R8, R7and R8are each independently selected from H and optionally substituted -C 1-6 alkyl, for example -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, wherein the alkyl is optionally substituted with one or more -OH, -COOH, -NR6R6', oxo, halo, cyano, nitro, -OC 1-6 alkyl, in particular optionally substituted with one or more -OH and / or -NR6R6'.

[0182] In some embodiments of the above compounds, M is -(CH2) p -NH2.

[0183] In some embodiments of the above compounds, M is -(CH2) p -M1, wherein M1is -NHC1-6 alkyl or -N(C 1-6 alkyl)2, wherein the alkyl groups are optionally substituted with one or more -OH and / or -NR6R6', wherein R6and R6' are each independently H or -C 1-6 alkyl; for example M is:

[0184] and the corresponding fragment where each terminal alkyl group is substituted with -OH.

[0185] In some embodiments of the above compounds, M is -(CH2) p -M1, wherein M1is an optionally substituted saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, in particular an optionally substituted saturated or partially unsaturated 4-7 membered heterocyclyl containing at least one nitrogen atom, for example, but not limited to each optionally substituted with one or more OH, -COOH, -NH2, oxo, halo, cyano, nitro, -OC 1-6 alkyl and C 1-6 alkyl.

[0186] In some embodiments of the above compounds, M is -(CH2) p -M1, wherein M1is an optionally substituted 5-10 membered heteroaryl containing at least one nitrogen atom, in particular a 5-6 membered heteroaryl, for example, but not limited to each optionally substituted with one or more OH, -COOH, -NH2, oxo, halo, cyano, nitro, -OC 1-6 alkyl and C 1-6 alkyl.

[0187] In some embodiments of the above compounds, M is -(CH2) p -M1, in particular M is M1, wherein M1is selected from -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, wherein -C 1-6 alkyl, heterocyclyl or heteroaryl are each optionally substituted with one or more OH, -COOH, -NH2, oxo, halo, cyano, nitro, -OC 1-6 alkyl and C 1-6 alkyl.

[0188] In some embodiments of the above compounds, R6and R6' are each independently H.

[0189] In some embodiments of the above compounds, R6and R6' are each independently optionally substituted -C 1-12 alkyl; in particular optionally substituted -C 1-6 alkyl.

[0190] In some embodiments of the above compounds, the substituents of R6and R6' are selected from -OH, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2; preferably the substituent is -OH.

[0191] In some embodiments of the above compounds, L1is absent, and / or X is selected from -O-, -S-, -NR6-, -NR6-NR6'- -N=N-, -CH=N-, -N=CH-, -CH=CH-, -C≡C-, -O-NR6-, -S-S-, -SO-, -SO2-, -C(O)-, -C(=S)-, -C(=NR6)-, -C(O)O-, -C(O)S-, -OC(O)-, -SC(O)-, -NR6C(O)-, -C(O)NR6-, -NR6SO2-, -SO2NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -SC(O)NR6-, -NR6C(O)NR6'-, -NR6C(O)S-, -P(O)(OR6)O-, -OP(O)(OR6)-, -OP(O)(OR6)O-, and / or L2is a straight chain or branched -C 1-12 alkylene-, in particular a straight chain or branched -C 1-6 alkylene-, the optional substituents and spacers of which are as defined herein in the general or specific or preferred embodiments, and R6and R6', M are each as defined herein in the general or specific or preferred embodiments;

[0192] In particular, in these embodiments, L1is absent; and / or

[0193] X is selected from -O-, -S-, -NR6-, -NR6-NR6'- -O-NR6-, -C(O)-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -NR6C(O)NR6'-; preferably X is selected from -O-, -NR6-, -OC(O)-, -NR6C(O)-, -OC(O)O-, -NR6C(O)O-, -OC(O)NR6-, -NR6C(O)NR6'-; and / or

[0194] L2is a straight chain or branched -C 1-12alkylene, optionally substituted with -OH, -NR6R6', halogen, -OC 1-6 alkyl, and optionally interrupted with one or more -O-, -NR6-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -NR6C(O)O-, -NR6C(O)NR6'-, -OC(O)O-, or -OC(O)NR6-; and

[0195] wherein R6and R6' are each independently H, -C 1-6 alkyl or -C 2-6 alkenyl; preferably each independently H or -C 1-6 alkyl, optionally substituted with -OH, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, preferably optionally substituted with -OH;

[0196] More specifically, in these embodiments, the alkylene of L2is a straight chain alkylene, whereby the compound comprises the structural fragment Specifically or the alkylene of L2has two branches at the end, whereby the compound comprises the structural fragment Specifically or the alkylene of L2has three branches at the end, whereby the compound comprises the structural fragment Specifically wherein p is an integer from 0 to 6, for example an integer from 1-4 or 1-3, M1is as defined herein in the general or specific or preferred embodiments, preferably M1is -NHC 1-6 alkyl or -N(C 1-6 alkyl)2, wherein the alkyl is substituted with one or more -OH and / or -NR6R6', wherein R6and R6' are each independently H or -C 1-6 alkyl, as exemplified above.

[0197] In some embodiments of the above compounds, the linker L or -L1-X-L2- can have a structure comprising, but not limited to, the following:

[0198]

[0199]

[0200] wherein the asterisk indicates the point of attachment to the steroid ring fragment, a single wavy line indicates the point of attachment to M, multiple wavy lines indicate at least one point of attachment to M and the remaining points of attachment to H or alkyl branches of L2, m is an integer from 1 to 6, and t is an integer from 0 to 6; in a particular embodiment, m is an integer from 2 to 4 and t is an integer from 2 to 6; in another particular embodiment, m is an integer from 1 to 6 and t is 1; it is noted that each occurrence of m in -[(CH2) m -NR6]- or -[(CH2) m -O] is independently an integer from 1 to 6.

[0201] It is noted that the compounds of the disclosure can comprise linking units formed from any combination of the general or specific or preferred definitions of the individual components of the linking units above, and further encompass -L-(M)r fragments formed from any combination of these linking units and any M defined generally or specifically or preferably above, as chemically feasible.

[0202] In some embodiments of the above compounds, G1is absent, or is G2is absent, or is G3is absent;

[0203] R 11a and R 11b are each independently H or C 1-6 alkyl, particularly one is H and the other is -CH3;

[0204] R 12 is selected from -C 3-10 cycloalkyl, -C 3-10 cycloalkenyl, -C 6-10 aryl, -3-8 membered heterocyclyl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, -5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, wherein each of said cycloalkyl, aryl, heterocyclyl, and heteroaryl is optionally substituted with -C 1-6 alkyl;

[0205] In particular embodiments, R 12 is optionally substituted with

[0206] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, preferably H or C 1-3 alkyl.

[0207] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, R 12 is selected from -3-8 membered heterocyclyl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, -OH, -OC 3-10 alkyl, -NR 13a R 13b wherein the heterocyclyl is optionally substituted with -C 1-6 alkyl.

[0208] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, R 14a is C 1-6 alkyl, R 14b is selected from H or C 1-6 alkyl.

[0209] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, R 15 is selected from halogen, OH, or C 1-6 alkyl.

[0210] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, R 16a and R 16b are selected from C 1-6 alkyl.

[0211] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11a is H or C 1-6 alkyl, R 17a and R 17b are each independently selected from H, OH, C 1-6 alkyl.

[0212] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 11aH or C 1-6 alkyl, R 18a , R 18b and R 18c are each C 1-6 alkyl.

[0213] In some embodiments of the above compounds, -G1-G2-G3-R 12 is wherein R 19a is selected from the group consisting of H, -C 1-6 alkyl or =CH-C 1-6 alkyl, R 19b and R 19c are each C 1-6 alkyl.

[0214] In some embodiments of the above compounds, especially in the -G1-G2-G3-R 12 schemes, R 11a is selected from the group consisting of H and

[0215] preferably

[0216] In some embodiments of the above compounds, especially in the -G1-G2-G3-R 12 schemes, R 14a , R 14b , R 15 , R 16a , R 16b , R 17a , R 17b , R 18a , R 18b , R 18c , R 19b , R 19c when taken as C 1-6 alkyl can be selected from the group consisting of

[0217] In some embodiments of the above compounds, G1 is wherein R 11a and R 11b are one of H and the other is C 1-3 alkyl, preferably methyl; G2 is wherein q is 2; G3 is absent; and R 12 is selected from the group consisting of -C 3-10 alkyl and -C 3-10 alkenyl, preferably -C 3-6 alkyl and -C 3-6 alkenyl, more preferably -C 4-6 alkyl and -C 4-6alkyl or alkenyl is preferably branched;

[0218] In particular, R 12 having the formula: wherein R 12a is selected from H, -CH3, -CH2CH3, =CHCH3.

[0219] It is noted that the compounds of the present disclosure can comprise Z fragments formed from any combination of the general or specific or preferred definitions of the individual components of Z fragments above, and further encompass ionizable sterol compounds formed from any combination of these Z fragments with the linking units defined generally or specifically or preferably above, any M defined generally or specifically or preferably above, as chemically feasible.

[0220] In some embodiments of the above compounds, wherein Z is, for example but not limited to, the following fragment:

[0221]

[0222]

[0223] and wherein the hydroxyl group at the 3-position of the steroid ring is -O-R 1a as defined herein, preferably wherein R 1a is H, -C 1-6 alkyl and -C(O)C 1-6 alkyl.

[0224] In some embodiments of the above compounds, wherein the ionizable sterol compound is, for example but not limited to, the following compounds:

[0225]

[0226]

[0227] wherein R 1a is as defined generally or specifically or preferably above, in particular H, -C 1-6 alkyl or C 1-6 alkyl-CO-, for example CH3CO-, C2H5CO-, C3H7CO-, -C2H5, -C3H7,

[0228] or a stereoisomer, a pharmaceutically acceptable salt or a solvate thereof.

[0229] In some embodiments, the ionizable sterol compound does not include

[0230] 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -propanoate;

[0231] 5α-Hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl acetate;

[0232] 5α-Hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ethyl carbonate;

[0233] 5α-Hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl butyl carbonate;

[0234] 5α-Hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl 1-H- imidazol-4-ylethylcarbamate;

[0235] 5α-Hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ethylcarbamate;

[0236] L-Histidine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0237] L-Leucine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0238] L-Isoleucine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0239] L-Valine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0240] L-Phenylalanine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0241] L-Tryptophan 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0242] L-Lysine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester;

[0243] L-Tyrosine 5α-hydroxy-6β-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3β-yl ester; and

[0244] L-proline 5a-hydroxy-6b-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3b-yl ester.

[0245] It is to be understood that the present disclosure encompasses the technical solutions of compounds formed by any combination of the general or specific structural fragments defined in the various embodiments described above.

[0246] II. Lipid carrier composition or bioactive substance delivery system

[0247] The ionizable sterol compound of the present disclosure, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, is capable of complexing nucleic acid molecules to form stable lipid nanoparticle structures, which advantageously facilitate the delivery and transfection of nucleic acids and have the ability to specifically target organ tissues, and thus can be used to prepare a delivery carrier or delivery system for nucleic acid drugs. The delivery system can also be advantageously used to deliver other therapeutic and / or prophylactic agents.

[0248] The present disclosure thus provides a lipid carrier composition or bioactive substance delivery system comprising the ionizable sterol compound of the present disclosure, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0249] In further embodiments, the lipid carrier composition or bioactive substance delivery system of the present disclosure further comprises a phospholipid, an (optional) structural lipid, and a polymer-conjugated lipid. In specific embodiments, the lipid carrier composition or bioactive substance delivery system of the present disclosure can be in the form of a lipid nanoparticle, a liposome, a microbubble, a microparticle, a nanoparticle, and a lipoplex. In more specific embodiments, the lipid carrier composition or bioactive substance delivery system of the present disclosure is in the form of a lipid nanoparticle.

[0250] The lipid carrier composition or bioactive substance delivery system of the present disclosure can efficiently deliver bioactive substances into cells, tissues, and organs, achieving efficient regulation of bioactive substances. The bioactive substances can be therapeutic agents, diagnostic agents, prophylactic agents, which can be in the form of nucleic acids, small molecule compounds, proteins or polypeptides, etc., and can be in the form of a liquid, a gas, or a solid.

[0251] In some embodiments of the present disclosure, the lipid carrier composition or bioactive substance delivery system comprises an ionizable sterol compound selected from the specific ionizable sterol compounds shown above, for example, ionizable sterol compounds 1-8 as follows:

[0252]

[0253] or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof.

[0254] For embodiments of the presently disclosed lipid vehicle compositions or biologically active material delivery systems, the mole percent of the ionizable sterol compound included can be about 15% to about 70%, about 15% to about 65%, about 15% to about 6%, about 15% to about 55%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 30% to about 70%, about 30% to about 60%, about 30% to about 55%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, preferably about 30% to about 60%, more preferably about 40% to about 60%, most preferably about 50%.

[0255] For embodiments of the presently disclosed lipid vehicle compositions or biologically active material delivery systems, the phospholipid included can include, but is not limited to, phosphatidylcholine compounds and / or phosphatidylethanolamine compounds, and can also be phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. For example, but not limited to:

[0256] 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),

[0257] 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),

[0258] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0259] 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

[0260] 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

[0261] 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),

[0262] 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),

[0263] 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC),

[0264] 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC),

[0265] 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0266] 1,2-divinyl-sn-glycero-3-phosphocholine,

[0267] 1,2-diarachidonoyl-sn-glycero-3-phosphocholine,

[0268] 1,2-diheneicosanoyl-sn-glycero-3-phosphocholine,

[0269] 1,2-dihomarachidonoyl-sn-glycero-3-phosphocholine,

[0270] 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,

[0271] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0272] 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPPC),

[0273] 1,2-distearyl-sn-glycero-3-phosphocholine,

[0274] 1,2-dilinoleoyl-sn-glycero-3-phosphocholine,

[0275] 1,2-diheneicosanoyl-sn-glycero-3-phosphocholine,

[0276] 1,2-dihomarachidonoyl-sn-glycero-3-phosphocholine,

[0277] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0278] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0279] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0280] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0281] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0282] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

[0283] Dipalmitoylphosphatidylglycerol (DPPG),

[0284] Palmitoyloleoylphosphatidylethanolamine (POPE),

[0285] Distearoyl-phosphatidyl-ethanolamine (DSPE),

[0286] Dipalmitoylphosphatidylethanolamine (DPPE),

[0287] Dimyristic phosphate ethanolamine (DMPE)

[0288] 1-Stearyl-2-oleoyl-phosphatidylethanolamine (SOPE)

[0289] 1-Stearyl-2-oleoyl-phosphatidylcholine (SOPC)

[0290] Sphingomyelin and its mixtures.

[0291] Preferably, in embodiments of the lipid carrier composition or bioactive substance delivery system of this disclosure, the phospholipid contained therein is selected from DSPC, DSPE, DOPE, DMPE, and DMPC. In another embodiment, the phospholipid is selected from DSPC, DOPE, or a combination thereof. In yet another embodiment, the phospholipid is DSPC.

[0292] For embodiments of the lipid carrier composition or bioactive substance delivery system disclosed herein, the molar percentage of phospholipids contained therein to the total lipids may be from about 0% to about 40%, from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 15%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 10% to about 15%, preferably from about 5% to about 20%, more preferably from about 5% to about 15% or from about 10% to about 15%, and most preferably from about 10%.

[0293] For embodiments of the lipid carrier compositions or bioactive substance delivery systems disclosed herein, the structural lipids contained therein may include, when present, steroids or sterols, including but not limited to one or more of cholesterol, coprosterol, sitosterol, ergosterol, stigmasterol, or derivatives thereof with equivalent properties, such as cholesterol succinate. In one embodiment, the structural lipid is cholesterol.

[0294] For embodiments of the presently disclosed lipid vehicle compositions or biologically active material delivery systems, the structural lipid, such as cholesterol, can comprise about 0 to about 60%, about 0 to about 50%, about 0 to about 40%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 30% to about 60%, about 30% to about 50%, about 35% to about 60%, about 35% to about 50%, about 35% to about 45%, preferably about 20% to about 50%, about 25% to about 50%, more preferably about 35% to about 50%, of the total lipid by mole percent.

[0295] For embodiments of the presently disclosed lipid vehicle compositions or biologically active material delivery systems, the polymer-conjugated lipid contained therein is a PEG lipid, comprising a lipid moiety and a PEG-based polymer moiety. The lipid moiety includes one or more of, for example, a phosphatidylethanolamine, a phosphatidic acid, a ceramide, a dialkyl amine, a diacylglycerol, a diacylglycerol amide, a dialkyl glycerol. The PEG polymer moiety can be a copolymer, such as PEG-polyurethane or PEG-polypropylene, or a PEG monomer, the PEG moiety having a molecular weight of 100 to 50,000, preferably 200 to 10,000, further preferably 500 to 3,000, most preferably 1,500 to 2,500. In some embodiments, the PEG is PEG2000, having an average molecular weight of about 2,000 daltons.

[0296] In some embodiments, the PEG lipid molecule can be selected from, but not limited to, 1,2-dimyristoyl-sn-glycero-methoxypolyethyleneglycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-distearoylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacetylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0297] In one embodiment, the PEG lipid is selected from 1,2-dimyristoyl-sn-glycero- methoxypolyethyleneglycol (PEG-DMG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethyleneglycol)] (PEG-DSPE). In one embodiment, the PEG lipid is selected from 1,2-dimyristoyl-sn-glycero-methoxypolyethyleneglycol 2000 (PEG2000-DMG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (PEG2000-DSPE).

[0298] For embodiments of the lipidic carrier composition or bioactive substance delivery system of the present disclosure, the PEG lipid comprised can be in a molar percentage of the total lipids of about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 2.5%, about 1% to about 10%, about 1% to about 5%, about 1% to about 3%, about 1% to about 2.5%, about 1% to about 2%; preferably about 1% to about 5%, about 0.5% to about 2.5%, more preferably about 1% to about 2%.

[0299] In some embodiments, the lipidic carrier composition or bioactive substance delivery system comprises an ionizable sterol compound selected from the ionizable sterol compounds as defined in the present disclosure, for example, ionizable sterol compounds 1-8, for example The phospholipid comprised is selected from DSPC, DOPE or a combination thereof, the structural lipid comprised is cholesterol, and the polymer-conjugated lipid comprised is PEG-DMG and / or PEG-DSPE, in particular PEG2000-DMG and / or PEG2000-DSPE.

[0300] In some embodiments, the molar ratio of the ionizable sterol compound, the phospholipid, the structural lipid and the polymer-conjugated lipid is 15-70:0-30:20-60:0.5-10, for example 20-60:5-30:20-50:0.5-5, 30-60:5-20:25-50:1-5, 40-60:5-15:30-50:1-2.

[0301] In some embodiments, the molar ratio of the ionizable sterol compound, the phospholipid, the structural lipid and the polymer-conjugated lipid is 50:10:38.5:1.5.

[0302] Since the sterol compounds of the present disclosure contain both an ionizable cationic lipid component and a phytosteryl component, they can act as both ionizable lipids and structural lipids, thereby enabling a three-component lipid carrier composition or a bioactive substance delivery system. Thus, in some embodiments, the ionizable sterol compound comprised in the lipid carrier composition or the bioactive substance delivery system is selected from the ionizable sterol compounds 1-8 as defined by the present disclosure, for example The phospholipid comprised is selected from DSPC, DOPE or a combination thereof, and the polymer-conjugated lipid comprised is PEG-DMG and / or PEG-DSPE, in particular PEG2000-DMG and / or PEG2000-DSPE.

[0303] In some embodiments, the molar ratio of the ionizable sterol compound, the phospholipid and the polymer-conjugated lipid is 15-70:5-25:0.5-5, or 30-60:5-40:1-5, or 40-60:5-40:1-2.

[0304] III. Lipid nanoparticle compositions and their preparation

[0305] The present disclosure also provides a lipid nanoparticle composition comprising a therapeutic and / or prophylactic agent, and an ionizable sterol compound of the present disclosure as described above.

[0306] The present disclosure further provides a lipid nanoparticle composition comprising a therapeutic and / or prophylactic agent, and a pharmaceutically acceptable carrier comprising a lipid carrier composition or a bioactive substance delivery system as defined by the present disclosure.

[0307] In some embodiments, the therapeutic and / or prophylactic agent is selected from the group consisting of a nucleic acid, a polypeptide, a protein and a small molecule compound, preferably a nucleic acid, which is embedded in the lipid layer or encapsulated in the inner lumen of the lipid vesicle.

[0308] For the lipid nanoparticle composition of the present disclosure, the nucleic acid comprised therein is selected from one or more of a short interfering RNA (siRNA), a microRNA (miRNA), a pri-miRNA, a messenger RNA (mRNA), a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating RNA (tracrRNA), an antisense RNA, an asymmetric interfering RNA (aiRNA), an RNA interference (RNAi) molecule, a small hairpin RNA (shRNA), a plasmid DNA (pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a Dicer-substrate RNA (dsRNA), a double-stranded DNA (dsDNA), a single-stranded DNA (ssDNA), a single-stranded RNA (ssRNA), a double-stranded RNA (dsRNA), a ribozyme.

[0309] In particular, the nucleic acid is selected from the group consisting of messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, microRNA (miRNA), antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof; preferably the nucleic acid is RNA selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, or mRNA.

[0310] The kind of the lipid component forming the lipid nanoparticle and its composition in the lipid nanoparticle composition of the present disclosure are as defined above generally or specifically.

[0311] In some embodiments, the lipid nanoparticle composition of the present disclosure comprises mRNA, and any one of the ionizable sterol compounds defined above, for example a phospholipid selected from DSPC, DOPE, or a combination thereof, cholesterol, and PEG-DMG and / or PEG-DSPE, in particular PEG2000-DMG and / or PEG2000-DSPE. Wherein the ionizable sterol compound, phospholipid, structural lipid, and polymer-conjugated lipid are in a molar ratio of 15-70:0-30:20-60:0.5-10, for example 30-60:5-20:25-50:1-5, 40-60:5-15:30-50:1-2, for example 50:10:38.5:1.5.

[0312] For the lipid nanoparticle composition of the present disclosure, the N / P ratio of the lipid carrier composition to the nucleic acid therein is 3-12, for example 3-10, 4-10, 6-10, 6-8, 3-8, 3-6, for example can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, preferably 4-8.

[0313] The amount of therapeutic and / or prophylactic agent in the lipid nanoparticle composition of the present disclosure can depend on the size, composition, desired target and / or application or other properties of the nanoparticle composition and the properties of the therapeutic and / or / and therapeutic agent. For example, the amount of RNA that can be used in the lipid nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic and other elements (e.g. lipids) in the lipid nanoparticle composition can also vary as appropriate.

[0314] In some embodiments, the ratio of lipid to therapeutic and / or prophylactic agent (mass / mass ratio) (e.g. the ratio of lipid to mRNA) is about 10:1 to about 30:1, preferably 12.5:1 to 25:1, more preferably about 15:1.

[0315] The lipid nanoparticle compositions of the present disclosure can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the lipid nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential, and can also be used to determine particle size.

[0316] In some embodiments, the average particle size of the lipid nanoparticle compositions of the present disclosure is 50-200 nm, e.g., 100-200 nm, 170-200 nm, 170-190 nm, e.g., can be 50 nm, 100 nm, 150 nm, 160 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 196 nm, 197 nm, etc.

[0317] The lipid nanoparticle compositions of the present disclosure are relatively uniform. Polydispersity index (PDI) can be used to indicate the uniformity of the particles, i.e., the particle size distribution of the lipid nanoparticle compositions. A smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of the lipid nanoparticle compositions of the present disclosure is 0.1-0.3, e.g., 0.15-0.3, 0.15-0.25, 0.15-0.2, e.g., can be 0.1, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.25, etc.

[0318] The lipid nanoparticle compositions of the present disclosure have an encapsulation efficiency of at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for a therapeutic agent and / or prophylactic agent, e.g., a nucleic acid. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%, or close to 100%.

[0319] In some embodiments, the lipid nanoparticle compositions of the present disclosure have an encapsulation efficiency of at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for a therapeutic agent and / or prophylactic agent, e.g., a nucleic acid. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%, or close to 100%.

[0320] the encapsulation efficiency of the therapeutic agent is ≥ 50%; or ≥ 80%; or ≥ 90%; and / or

[0321] the average particle size of the lipid nanoparticles is 100-200 nm; and / or

[0322] the polydispersity index of the nanoparticle composition is 0.1-0.3.

[0323] The average particle size and encapsulation efficiency of the lipid nanoparticle compositions of the present disclosure show excellent stability, with the highest encapsulation efficiency and the smallest change in particle size after storage at 4°C for different time periods in aqueous media such as 100 mM Tris-HCl, compared to other commonly used ionizable cationic lipid compounds for preparing the lipid nanoparticle compositions, as shown in the examples below.

[0324] The lipid nanoparticle compositions of the present disclosure can also include one or more permeability enhancer molecules, carbohydrates, biodegradable or biocompatible polymers, surface-altering agents, and any substance useful in pharmaceutical compositions, such as one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspending agents, granulating agents, disintegrants, fillers, glidants, liquid carriers, binders, surface active agents, isotonic agents, thickening agents or emulsifiers, buffers, lubricants, oils, preservatives, and other substances.

[0325] In some embodiments, one or more excipients or auxiliary ingredients can comprise greater than 50%, such as 60%, 70%, 80%, 90% or more of the total mass or volume of the lipid nanoparticle composition.

[0326] The present disclosure also provides methods of preparing the lipid nanoparticle compositions, comprising dissolving each lipid molecule defined herein in an organic solvent to form a mixed lipid solution, using the molar ratios described, mixing the oil phase with an aqueous solution of a drug to be delivered, such as mRNA, and using methods including but not limited to spray drying, solvent extraction, phase separation, nanoprecipitation, single or double emulsion solvent evaporation, microfluidics, coacervation, and other methods well known in the art to produce the lipid nanoparticles.

[0327] In some embodiments, the alcohol solution, such as an ethanol solution, of the lipid carrier composition of the present disclosure is prepared into lipid nanoparticles with an aqueous solution of the nucleic acid using a microfluidic system, wherein the flow rate ratio (FRR) of the alcohol solution and the aqueous solution in the microfluidic system is 1:2-5, preferably 1:3, and the total flow rate is 12-20 ml / min.

[0328] IV. Pharmaceutical compositions and formulations

[0329] The lipid nanoparticle compositions of the present disclosure can be formulated, in whole or in part, as a pharmaceutical composition or formulation. The pharmaceutical composition or formulation can include one or more lipid nanoparticle components. For example, the pharmaceutical composition or formulation can include one or more lipid nanoparticle compositions that include one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition or formulation can further include one or more pharmaceutically acceptable, excipient or auxiliary ingredient that is compatible with one or more components of the nanoparticle composition, such as those excipient or auxiliary ingredients and ranges thereof described herein for the lipid nanoparticle compositions.

[0330] The pharmaceutical compositions or formulations of the present disclosure that include one or more lipid nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacy, and can be prepared in various forms suitable for various routes and methods of administration. For example, the pharmaceutical composition or formulation can be administered by injection, orally, subcutaneously, by inhalation, and the like, can be in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, films, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, patches, suppositories, or drops), suspensions, powders, and other forms. The various formulation forms can accordingly include excipient or auxiliary ingredients that are conventional in the art.

[0331] The lipid nanoparticle compositions of the present disclosure and / or the pharmaceutical compositions or formulations that include one or more of the lipid nanoparticle compositions can be administered to any patient or subject, including those that can benefit from the therapeutic effects provided by the delivery of a therapeutic and / or prophylactic agent to one or more particular cells, tissues, organs, or systems or groups thereof (e.g., the renal system). The subject or patient includes humans as well as any other mammal, bovine, porcine, soft tube, ovine, feline, canine, mouse, and / or rat.

[0332] V. Uses and Methods

[0333] In one aspect, the present disclosure provides an ionizable sterol compound of the present disclosure or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, a lipid carrier composition, a lipid nanoparticle composition, a pharmaceutical composition or formulation, for use in delivering a therapeutic and / or prophylactic agent to a mammalian cell, tissue, or organ, for use in producing a polypeptide of interest in a mammalian cell, for use in treating a disease or disorder in an individual, such as a mammal, such as a human, or for use in specifically delivering a therapeutic and / or prophylactic agent to a patient.

[0334] In another aspect, the present disclosure provides the use of an ionizable sterol compound of the present disclosure, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, in the manufacture of a lipid carrier composition, a lipid nanoparticle composition, or a pharmaceutical composition or formulation comprising the lipid nanoparticle composition.

[0335] In another aspect, the present disclosure provides the use of a lipid carrier composition of the present disclosure in the manufacture of a lipid nanoparticle composition, or a pharmaceutical composition or formulation comprising the lipid nanoparticle composition.

[0336] In another aspect, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a mammalian cell, tissue, or organ, a method of producing a polypeptide of interest in a mammalian cell, a method of treating a disease or disorder in a mammal, such as a human, or a method of specifically delivering a therapeutic and / or prophylactic agent to a patient, comprising administering to the mammal or patient a lipid nanoparticle composition of the present disclosure or a pharmaceutical composition or formulation comprising the lipid nanoparticle composition.

[0337] In another aspect, the present disclosure provides the use of a paper nanoparticle composition or a pharmaceutical composition of the present disclosure in the manufacture of a product for delivering a therapeutic and / or prophylactic agent to a mammalian cell, tissue, or organ, in the manufacture of a product for producing a polypeptide of interest in a mammalian cell, in the manufacture of a medicament for treating a disease or disorder in an individual, such as a mammal, such as a human, or in the manufacture of a medicament for specifically delivering a therapeutic and / or prophylactic agent to an individual.

[0338] In some embodiments, the lipid nanoparticle compositions of the present disclosure can be used for therapy. For example, the mRNA comprised in the nanoparticle composition can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact and / or entry (e.g., transfection) into a cell. In other embodiments, the mRNA comprised in the nanoparticle composition can encode a polypeptide that can improve or increase the immune response of a subject.

[0339] For the uses and methods of the present disclosure, the therapeutic and / or prophylactic agent is as defined above, preferably a nucleic acid as defined above, more preferably a natural or modified mRNA.

[0340] In some embodiments, the lipid nanoparticle compositions or pharmaceutical compositions or formulations of the present disclosure can target specific types or classes of cells (e.g., cells of a particular organ or system thereof). For example, the lipid nanoparticle compositions comprising a therapeutic and / or prophylactic agent of interest can be specifically delivered to the liver, kidney, spleen, femur, gastrointestinal tract, eye, or lung, as well as tumor tissue (e.g., by intratumoral injection) of a mammal. In some embodiments, the nanoparticle compositions can target hepatocytes. In some embodiments, the nanoparticle compositions can target extrahepatic cells. In some preferred embodiments, the nanoparticle compositions can target the spleen, stomach, or intestine.

[0341] For uses and methods of the present disclosure, wherein the disease or disorder is characterized by, e.g., a dysregulation or abnormality of a protein or polypeptide activity, is selected from a pathogenic infection, a tumor, an allergy, an autoimmune disease, a genetic disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

[0342] For uses and methods of the present disclosure, the lipid nanoparticle compositions or pharmaceutical compositions or formulations can be administered by any route, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, intradermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosal, nasal, buccal, intestinal, vitreous, intratumoral, sublingual, intranasal, etc.

[0343] For uses and methods of the present disclosure, a dose of 0.0001 mg / kg to 10 mg / kg of a therapeutic and / or prophylactic agent is administered to an individual, such as a mammal, such as a human. For example, a therapeutic and / or prophylactic (e.g., mRNA) dose of a lipid nanoparticle composition can be administered at about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 1 mg / kg, or about 0.05 mg / kg to about 0.25 mg / kg, dosed at a suitable dosing frequency.

[0344] VI. Invention Effects

[0345] The ionizable sterol compounds of the present disclosure can be used in the preparation of lipid carrier compositions, lipid nanoparticles, and pharmaceutical compositions or formulations, the lipid nanoparticle compositions or pharmaceutical compositions containing the ionizable sterol compounds of the present disclosure can achieve the encapsulation, delivery of therapeutic / prophylactic agents, safely deliver therapeutic / prophylactic agents to the targeted location, achieve high expression, and exert the effects of therapeutic / prophylactic agents. Specifically, the technical solutions of the present disclosure can achieve the following technical effects:

[0346] The ionizable sterol compound of the present disclosure can complex nucleic acid molecules to form stable lipid nanoparticle structures;

[0347] The ionizable sterol compound of the present disclosure can carry two or more ionizable sites to achieve a high membrane charge density, promote endosome escape of the lipid nanoparticle composition, and improve nucleic acid transfection efficiency; it can complex multiple nucleic acid molecules, greatly reducing the proportion of ionizable cations in the LNP, thereby reducing the toxicity of the vector;

[0348] The ionizable sterol compound of the present disclosure not only can serve as an important lipid component for forming a lipid nanoparticle, but in some embodiments, the ionizable sterol compound itself has a strong cancer cell differentiation and anticancer form, with tumor inhibition, chemoprevention, and neuroprotection properties, and thus the lipid carrier composition or the lipid nanoparticle composition or the pharmaceutical composition of the present disclosure is expected to exert a dual efficacy of delivering a therapeutic and / or prophylactic agent and tumor inhibition / chemoprevention / neuroprotection, providing a better choice for the field of targeted therapy and gene therapy;

[0349] The sterol compound of the present disclosure contains both ionizable cationic lipid components and sterol components, and thus can serve as both ionizable lipids and structural lipids, thereby enabling a three-component lipid carrier composition or a bioactive substance delivery system, thereby reducing the production cost of the LNP and facilitating clinical translation;

[0350] The lipid nanoparticles of the present disclosure have a small particle size, all within 200 nm, such a particle size can avoid in vivo clearance, prolong the circulation time of the drug in the body, help to avoid early drug clearance, and improve the efficiency of drug targeted delivery; and smaller nanoparticles facilitate sterilization during the preparation process and can be more easily passed through a microporous filter or a microporous membrane;

[0351] The lipid nanoparticles of the present disclosure have a small dispersity index, indicating a uniform particle size distribution, a narrow size distribution, and a small particle size difference, ensuring stable dispersion during application;

[0352] The lipid nanoparticles of the present disclosure have a high encapsulation efficiency, and the particle size and encapsulation efficiency are more stable than those of the lipid nanoparticles prepared from other conventional cationic lipids.

[0353] The various embodiments described in this disclosure, including the examples below, and features of the various embodiments, should be understood to be combinable in any manner, and each of the combinations of the various embodiments are included within the scope of the present application, as if each were specifically and individually listed herein, unless the context clearly indicates otherwise.

[0354] Examples

[0355] The following examples are illustrative of the present disclosure and are not limiting thereof. The data given (e.g., amounts, temperatures, etc.) are believed to be accurate but are not intended to limit the scope of the present disclosure. Unless otherwise indicated, all temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0356] Mass spectrometry data in this application was measured by a mass spectrometer (Waters SYNAPT XS); nuclear magnetic resonance data was measured by a nuclear magnetic resonance spectrometer (Bruker, AVANCE NEO 400M); purification operations used a preparative reverse-phase high-performance liquid chromatograph (HS-D-5800D); preparation of LNP used a refrigerated centrifuge (Beckman), and structure and performance characterization of LNP used a dynamic light scattering instrument (Malvern ZetaSizer), a transmission electron microscope (Hitachi, HT7800), a high-content cell imager (PerkinElmer), and a Spark multifunctional microplate reader (TECAN).

[0357] Unless specifically indicated otherwise, all reagents and materials used in the present disclosure were obtained from commercial sources.

[0358] In any structural formula in this application, if there is a free valence on any atom, the free valence is actually a hydrogen atom that is not specifically shown for simplicity.

[0359] List of abbreviations used in the following examples:

[0360]

[0361]

[0362] Example 1: Synthesis and characterization of ionizable sterol compounds

[0363] Step 1

[0364]

[0365] Cholesterol (1, Anethole Blue® / QJDHREAR) (1.0 g, 2.59 mmol, 1.00 equiv) was dissolved in dichloromethane (10.0 mL), to which a solution of mCPBA dissolved in dichloromethane (5.0 mL) was added dropwise, stirred at room temperature for 3 hours, washed with 10% aqueous sodium sulfite solution and 5% aqueous sodium bicarbonate solution respectively, the organic phase was dried over anhydrous sodium sulfate, rotary evaporation, to obtain (3S,5aS,6bS,9R,9aR,11aS,11bR-9a,11b)-dimethyl-9-(R)-6-methylheptan-2-yl)hexadecahydrocyclopenta[1,2]phenalene[8a,9-b]oxepin-3-ol (2) (2.18 g, crude, white oily solid), which was directly used in the next step. MS (ESI, m / z): 403 (M+H) + .

[0366] Step 2

[0367]

[0368] (3S,5aS,6bS,9R,9aR,11aS,11bR-9a,11b)-dimethyl-9-(R)-6-methylheptan-2-yl)hexadecahydrocyclopenta[1,2]phenalene[8a,9-b]oxepin-3-ol (2) (2.1 g, 5.22 mol, 1.00 equiv), histamine (McCollin / C14228571) (1.2 g, 10.44 mol, 2.00 equiv), isobutanol (25.0 mL) were stirred at 120 °C for 16 hours, rotary evaporation after cooling to room temperature, purification by reverse phase preparative chromatography (acetonitrile in water (0.5% ammonium bicarbonate), 5%-90%) to obtain ST-001 (DDA) (0.7 g, brown yellow solid, yield 25%). Mass spectrum detection results are shown in Figure 1A , NMR detection results are shown in Figure 1B , MS (ESI, m / z): 514 (M+H) + .

[0369] 1H NMR (500 MHz, DMSO-d6) δ 11.76 (s, 1H), 7.48 (s, 1H), 4.18 (s, 1H), 3.88-3.77 (M, 1H), 3.65 (s, 1H), 2.81-2.69 (m, 1H), 2.61-2.51 (m, 3H), 2.28 (s, 1H), 1.97-1.79 (m, 3H), 1.79-1.71 (m, 1H), 1.61-1.55 (m, 2H), 1.55-1.39 (m, 7H), 1.38-1.22 (m, 11H), 1.21-1.02 (m, 13H), 0.97 (d, J = 12.5 Hz, 6H), 0.88 (d, J = 6.5 Hz, 4H), 0.84 (dd, J = 6.5, 2.5 Hz, 8H), 0.61 (s, 3H).

[0370] In a similar manner, the ionizable sterol compounds 1-8 of the present disclosure were also prepared using the specific sterol fragments described above.

[0371] Example 2: Preparation of LNP

[0372] Lipid nanoparticles (LNP) encapsulating mRNA were prepared as follows using ALC-0315, Dlin-MC3-DMA, SM102, ST-001 as lipids, respectively, and were denoted as LNP1 (containing ALC-0315), LNP2 (containing Dlin-MC3-DMA), LNP3 (containing SM102), LNP4 (containing ST-001), respectively.

[0373] 2.1 Preparation of lipid mixture

[0374] Each lipid (ST-001 was prepared as described in Example 1, and the rest of the lipids were purchased from Avanti Polar Lipids, Inc.) was dissolved in ethanol at the ratio shown in Table 1 below and mixed thoroughly to be clear, and stored at 4°C for later use (lipid phase). The total concentration of all components was configured to be 12 mM (about 7.5 mg / ml).

[0375] Table 1. Each component and its molecular weight in the lipid nanoparticle (LNP)

[0376]

[0377]

[0378] 2.2 Preparation of mRNA

[0379] In this experiment, eGFP-mRNA was used as a model mRNA to prepare LNP and evaluate the transfection efficiency, but the present application is not limited to a specific mRNA.

[0380] The mRNA feeding amount was set by nitrogen to phosphorus ratio (N / P) in this experiment. The nitrogen atom number in ionizable lipid was calculated when calculating the nitrogen to phosphorus ratio. The experiment set 0.5 mol of N in each mol of mixed lipid, and the N / P ratio was 8.

[0381] The average molecular weight of the base of RNA is 324, and each base carries 1 phosphate, so the phosphorus content in RNA is 3.03 nmol / μg. The mass of mRNA that can be loaded per mM of total lipid can be calculated based on the following formula:

[0382]

[0383] Based on the lipid concentration of 12 mM, N / P = 8, and FRR = 3, the required RNA concentration was calculated to be 0.081 μg / μl.

[0384] Use ultrapure water to prepare 100 mM of citric acid monohydrate (C6H8O7·H2O / MW 210.14, 1.05 g) and sodium citrate dihydrate (C6H5Na3O7·2H2O / MW 294.10, 1.47 g) solution, 50 ml each. Take 33.0 ml of citric acid solution and 17.0 ml of sodium citrate solution, mix, then add DEPC, and after standing for 30 minutes, remove DEPC by autoclaving, and after sterilization, use DEPC water (purchased from Solabio) to constant volume to 100 ml to obtain 50 mM pH = 4 citric acid buffer. After determining the concentration of mRNA, dilute to the required concentration using the citric acid buffer according to the lipid concentration (aqueous phase).

[0385] 2.3 Microfluidic mixing preparation of LNP

[0386] The lipid phase lipid ethanol solution and the aqueous phase mRNA-citric acid buffer were filtered and purified through 0.22 micron filter membranes, respectively, the water phase and lipid phase flow rate was controlled (FRR = 3, lipid phase flow rate 4.5 mL / min / water phase flow rate 13.5 mL / min), and the mRNA and lipid mixture was loaded into the microfluidic device (Suzhou Zhongxin Qi Heng standard micro-mixed liposome chip) at a total flow rate of 15 mL / min for mixing. After observing the stable flow rate of the effluent pipe, the collected pipe was used to collect the effluent liquid to obtain LNP loaded mRNA.

[0387] 2.4 Dialysis and storage

[0388] The LNP obtained as described above was placed in a dialysis tube (Slide-A-Lyzer Dialysis Cuvette, 7K MWCO, 20K MWCO, 30K MWCO, 50K MWCO, 100K MWCO, 200K MWCO, 350K MWCO, 0.5L, 2L, 3.5L, 5L, 7L, 10L, 20L, 30L, 50L, 100L, 200L, 500L, 1000L, Thermo Scientific, USA) and dialyzed against 10 mM Tris-HCl buffer (pH 7.4) for 24 hours. TMThe LNP was dialyzed against 14 ml PBS (pH 7.4) on a shaker, and the solution was changed every 2 hours until the end of dialysis at 18 hours. Finally, the LNP was concentrated to a mRNA concentration of 100-200 pg / ml. The LNP was collected and stored in PBS (pH 7.4) containing 2% sucrose at -80°C.

[0389] Example 3: Structure and performance characterization of LNP

[0390] 3.1 Particle size, monodispersity and encapsulation efficiency

[0391] The particle size and monodispersity of each LNP prepared in Example 2 were determined using a dynamic light scattering instrument or a transmission electron microscope (TEM).

[0392] The total and free mRNA content of the obtained LNP-mRNA was determined using the Quant-iT™ PicoGreen® RNA Assay Kit (Thermo Fisher, Cat. No. R11490) with 1% Triton-100 (SIGMA-ALDRICH, T8787-100ML) as a demulsifier and CellCarrier-96 Ultra Microplates (PerkinElmer, 6055308), using a SPARK Multifunctional Microplate Reader (TECAN) to read the fluorescence intensity (excitation 480 nm, emission 520 nm), and the encapsulation efficiency was calculated by the following formula: TM The total and free mRNA content of the obtained LNP-mRNA was determined using the Quant-iT™ PicoGreen® RNA Assay Kit (Thermo Fisher, Cat. No. R11490) with 1% Triton-100 (SIGMA-ALDRICH, T8787-100ML) as a demulsifier and CellCarrier-96 Ultra Microplates (PerkinElmer, 6055308), using a SPARK Multifunctional Microplate Reader (TECAN) to read the fluorescence intensity (excitation 480 nm, emission 520 nm), and the encapsulation efficiency was calculated by the following formula:

[0393] Encapsulation efficiency (%) = (quantification after demulsification - quantification before demulsification) / quantification after demulsification

[0394] The results are shown in Figure 2A and Figure 2B and in Table 2 below:

[0395] Table 2: Particle size, monodispersity and encapsulation efficiency of each LNP

[0396] LNP Encapsulation efficiency Particle size (nm) PDI LNP1 95.8％ 179.8 0.213 LNP2 95.2％ 177.0 0.194 LNP3 97.9％ 189.7 0.187 LNP4 98.1％ 175.1 0.194

[0397] 3.2 Stability

[0398] The prepared LNP was placed in an aqueous phase (100 mM Tris-HCl) at 4°C for different time, and continuously monitored for 2 months. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) observation results showed that the particle stability of each LNP was good, and the LNP4 (ST-001) containing the novel steroidal ionizable lipid had the highest encapsulation efficiency, with little change in hydrated particle size within 30 days of placement, indicating the best particle stability. The results can be seen in Figure 3 .

[0399] Transmission electron microscopy (TEM) observation results also showed that the freshly prepared LNP and the LNP after 30 days of storage at 4°C (medium: 100 mM Tris-HCl) had good stability in morphology, with only a small amount of fusion or enlargement. The results can be seen in Figure 4 .

[0400] Using the above mRNA measurement method, the RNA leakage of the freshly prepared LNP and the LNP after different time of storage at 4°C (100 mM Tris-HCl) was also investigated by gel electrophoresis. The results showed that the RNA leakage rate increased with the placement of the LNP, and the LNP4 (ST-001) containing the novel steroidal ionizable lipid had the highest encapsulation efficiency and the least leakage, with the RNA leakage rate changing from 3% to 9% within 60 days of placement, and the encapsulation efficiency and stability being better than other

[0401] LNP. The results can be seen in Figure 5 .

[0402] 3.3 LNP transfection efficiency evaluation

[0403] Taking mouse monocyte macrophage leukemia cells RAW264.7 cells (Wuhan Pnuo Sai Biological Technology Co., Ltd.) as an example, the prepared LNP encapsulating eGFP-mRNA was used for cell transfection.

[0404] The cells were plated into a 24-well cell culture plate, with a cell density of 2.5×10 4 cells / ml, 1 ml / well, and incubated at 36°C overnight. The eGFP-mRNA LNP (50 μg / ml) was diluted with culture medium to 2.5 μg / mL, and 250 μl of LNP transfection solution was added to each well, with 3 wells for each LNP transfection. After continuous incubation at 36°C for 4, 6, 8, 12, 24, and 48 hours, the green fluorescent protein GFP expression was observed using a fluorescence microscope (Olympus BX43). After 48 hours, the cells were collected, and the GFP signal positive rate was detected by flow cytometry (Beckman Coulter CytoFLEX). GFP expression gradually appeared at 4 hours, and obvious green fluorescence was visible after 8 hours.

[0405] The results show that each LNP achieves different degrees of transfection. The results can be seen in Figure 6 Definitions , and the results show that the transfection efficiency of LNP4 (ST-001) is higher than that of other groups.

Claims

1. An ionizable sterol compound of Formula A, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, Z-L-(M)r Formula A wherein: Z is a structure described by Formula B below, wherein the wavy line indicates the point of attachment to L; R 1a is H, C 1-6 alkyl, -C(O)NR a R b , -C(O)R c , -C(O)OR d , or an amino acid residue wherein R e is selected from the residue moiety of L-histidine, L-leucine, L-isoleucine, L-valine, L-phenylalanine, L-tryptophan, L-lysine, L-tyrosine; wherein R a and R b are the same or different from each other and are selected from the group consisting of H, -C 1-6 alkyl, -C 2-6 alkenyl and -C 2-6 alkynyl, each optionally substituted with one or more groups selected from the group consisting of allyl, carbonyl and 5- to 10-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, S, R c and R d each independently is -C 1-6 alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl; R 1b is H, -C 1-6 alkyl, -C 2-6 alkenyl or -C 2-6 alkynyl; R2is H, OH, or -OC 1-6 alkyl; R3 is H or CH3; W is selected from N and CR when W is a single bond to the carbon atom at the adjacent * position f R4 is H or OH; W is C when W is a double bond to the carbon atom at the adjacent * position, R4 is absent; R f is absent, H, halogen, or -C 1-6 alkyl; R 5a and R 5b each independently is H, OH, or -OC 1-6 alkyl, or R 5a and R 5b together with the ring carbon atom to which they are attached form -C(O)-; L is a linking unit which is an optionally substituted straight chain or branched alkylene having 3-50 skeletal carbon atoms, covalently linking the steroid ring fragment and r ionizable head groups M, wherein at least 1 skeletal carbon atom of L is replaced by -0-, -S-, -NR6-, -NR6-NR6', -N=N-, -CH=N-, -N=CH-, -CH=CH-, -CºC-, -0-NR6-, -S-S-, -SO-, -S02, -C(O)-, -C(=S)-, -C(=NR6)-, -C(0)0-, -C(0)S-, -OC(O)-, -SC(O)-, -NR6C(0)-, -C(0)NR6-, -NR6S02-, -S02NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -SC(0)NR6-, -NR6C(0)NR6'-, -NR6C(0)S-, -P(0)(OR6)0-, -OP(0)(OR6)-, -OP(0)(OR6)0-, optionally substituted C 3-8 cycloalkylene, optionally substituted 3-8 membered heterocyclylene, optionally substituted C 6-10 arylene, or optionally substituted 5-10 membered heteroarylene; wherein each R6and R6' is independently H, optionally substituted -C 1-12 alkyl or optionally substituted -C 2-12 alkenyl; M is -(CH2) p M1is selected from NR7R8, an optionally substituted saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, and an optionally substituted 5-10 membered heteroaryl containing at least one nitrogen atom; R7and R8are each independently selected from H, optionally substituted -C 1-6 alkyl, optionally substituted -C 2-6 alkenyl, optionally substituted -C 2-6 alkynyl; G1is either nothing, or G2 is absent, or is G3 is absent, or is R 11a and R 11b each independently is H, -OH, or C 1-6 alkyl; R 12 selected from -C 3-10 alkyl, -C 3-10 alkenyl, -C 3-10 cycloalkyl, -C 3-10 cycloalkenyl, -C 6-10 aryl, -3-8 membered heterocyclyl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, -5-10 membered heteroaryl containing 1, 2, or 3 heteroatoms independently selected from N, O, S, halogen, -OH, -OC 3-10 alkyl, -NR 13a R 13b wherein each of said cycloalkyl, aryl, heterocyclyl, and heteroaryl is optionally substituted with -C 1-6 alkyl; R 13a and R 13b each independently is H or -C 1-6 alkyl; p is an integer from 0 to 6; and q and r are each independently an integer from 1 to 3.

2. The ionizable sterol compound according to claim 1, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, having the following formula, wherein: L1and L2are each independently absent or straight-chain or branched -C 1-12 alkylene-, wherein alkylene is optionally substituted with OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl, and wherein alkylene is optionally interrupted with one or more -O-, -S-, -NR6-, -CH=CH-, -CºC-, -C(O)-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -OC(O)NR6- or -OC(O)O-; X is selected from the group consisting of absent, -0-, -S-, -NR6-, -NR6-NR6'-, -N=N-, -CH=N-, -N=CH-, -CH=CH-, -CºC-, -0-NR6-, -S-S-, -SO-, -S02, -C(O)-, -C(=S)-, -C(=NR6)-, -C(0)0-, -C(0)S-, -OC(O)-, -SC(O)-, -NR6C(0)-, -C(0)NR6-, -NR6S02-, -S02NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -SC(0)NR6-, -NR6C(0)NR6'-, -NR6C(0)S-, -P(0)(OR6)0-, -OP(0)(OR6)-, -OP(0)(OR6)0-, optionally substituted C 3-8 cycloalkylene, optionally substituted 3-8 membered heterocyclyl, optionally substituted C 6-10 aryl or optionally substituted 5-10 membered heteroaryl, wherein the optional substituents are selected from the group consisting of -OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(0)N(R6R6'), -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl; R6and R6' are each independently H, -C 1-12 alkyl or -C 2-12 alkenyl, each optionally substituted with -OH, -COC 1-6 alkyl, -COOC 1-6 alkyl, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, oxo, halogen, cyano, nitro, -C(O)NH2, -C(O)NHC 1-6 alkyl, -C(O)N(C 1-6 alkyl)2, -OC 1-6 alkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 3-8 cycloalkyl; M is -(CH2) p -M1, wherein M1 is selected from NR7R8, a saturated or partially unsaturated 3-8 membered heterocyclic group containing at least one nitrogen atom, or a 5-10 membered heteroaryl group containing at least one nitrogen atom, wherein the heterocyclic group or heteroaryl group is optionally surrounded by one or more OH groups, -COR6, -COOR6, -NR6R6', oxo groups, halogens, cyano groups, nitro groups, -C(O)N(R6R6'), amido groups, guanidino groups, or -OC groups. 1-6 Alkyl and -C 1-6 Alkyl substitution; R7and R8are each independently selected from H, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, each optionally substituted with one or more groups selected from OH, -COR6, -COOR6, -NR6R6', -C(O)N(R6R6'), amidine, guanidine, halogen, cyano, nitro, -OC 1-6 alkyl, -(OC 1-6 alkylene) n -OH, saturated or partially unsaturated 3-8 membered heterocyclyl containing at least one nitrogen atom, 5-10 membered heteroaryl containing at least one nitrogen atom, wherein the heterocyclyl or heteroaryl is optionally substituted with one or more OH, -COR6, -COOR6, -NR6R6', oxo, halogen, cyano, nitro, -C(O)N(R6R6'), amidine, guanidine, -OC 1-6 alkyl and -C 1-6 alkyl substituted; n is an integer from 1 to 4.

3. The ionizable sterol compound according to claim 1 or 2, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, which is 4. The ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 3, wherein R 1a is selected from H, -C 1-6 alkyl and -C(O)C 1-6 alkyl.

5. The ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 2, wherein R 1b is H.

6. The ionizable sterol compound according to any one of claims 1 to 2, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein R2 is H.

7. The ionizable sterol compound according to any one of claims 1 to 6, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein R3 is -CH3.

8. The ionizable sterol compound according to any one of claims 1 to 7, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein there is a single bond between W and the * carbon atom, W is CH, and R4 is OH.

9. An ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1, 2, 4 to 8, wherein R 5a and R 5b are each independently H.

10. The ionizable sterol compound according to any one of claims 1 to 9, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein G1is wherein R 11a and R 11b one is H and the other is C 1-3 alkyl, preferably methyl; G2 is wherein q is 2; G3 is absent; and R 12 selected from -C 3-10 alkyl and -C 3-10 alkenyl, preferably -C 3-6 alkyl and -C 3-6 alkenyl.

11. The ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to claim 10, wherein R 12 having the formula: wherein R 12a is selected from H, -CH3, -CH2CH3, =CHCH3.

12. The ionizable sterol compound according to any one of claims 1 to 11, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein Z is selected from the following fragments: wherein R 1a is H, -C 1-6 alkyl and -C(O)C 1-6 alkyl, preferably H, -C 1-3 alkyl and -C(O)C 1-3 alkyl.

13. The ionizable sterol compound according to any one of claims 2 to 12, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein L1 is absent; and / or X is selected from -0-, -S-, -NR6-, -NR6-NR6’-, -0-NR6-, -C(O)-, -C(0)0-, -OC(O)-, -NR6C(0)-, -C(0)NR6-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -NR6C(0)NR6’-; preferably X is selected from -0-, -NR6-, -OC(O)-, -NR6C(0)-, -OC(0)0-, -NR6C(0)0-, -OC(0)NR6-, -NR6C(0)NR6’-; and / or L2is straight-chain or branched -C 1-12 alkylene, optionally substituted with -OH, -NR6R6', halogen, -OC 1-6 alkyl, and optionally interrupted with one or more -O-, -NR6-, -C(O)O-, -OC(O)-, -NR6C(O)-, -C(O)NR6-, -NR6C(O)O-, -NR6C(O)NR6'-, -OC(O)O-, or -OC(O)NR6-; wherein R6and R6' are each independently H, -C 1-6 alkyl or -C 2-6 alkenyl; preferably each independently H or -C 1-6 alkyl, optionally substituted with -OH, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, preferably optionally substituted with -OH.

14. The ionizable sterol compound according to claim 13, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein: L2comprises the repeating structural fragment -[(CH2) m -O] t - wherein m is an integer from 1 to 6 and t is an integer from 0 to 6; for example m is an integer of 2 and t is an integer from 2 to 6; for example m is an integer from 1 to 6 and t is 1 ; and / or L2comprises the repeating structural fragment -[(CH2) m -NR6] t - wherein m is an integer from 1 to 6 and t is an integer from 0 to 6; for example m is an integer from 2 to 4 and t is an integer from 2 to 6; for example m is an integer from 1 to 6 and t is 1 ; and / or alkylene of L2is interrupted by one or more -NR6C(O)O-, -NR6C(O)NR6'-, -OC(O)NR6-, or -OC(O)O-; for example, -C 1-6 alkylene-O-CO-NR6-C 1-6 alkylene-, -C 1-6 alkylene-O-CO-O-C 1-6 alkylene-, -C 1-6 alkylene-NR6-CO-O-C 1-6 alkylene-, -C 1-6 alkylene-NR6-CO-NR6-C 1-6 alkylene-.

15. The ionizable sterol compound according to any one of claims 1 to 12, a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein the linker L or -L1-X-L2- has the following structure: wherein, the asterisk * indicates the point of attachment to the steroid ring, the wavy line alone indicates the point of attachment to M, multiple wavy lines indicate at least one point of attachment to M and the remaining points of attachment to H or alkyl branches of L2, m is an integer from 1 to 6, and t is an integer from 0 to 6.

16. The ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 15, wherein: M is -(CH2) p -M1, wherein M1is NR7R8, R7and R8are each independently selected from H and optionally substituted -C 1-6 alkyl, wherein the alkyl is optionally substituted with one or more -OH, -COOH, -NR6R6', oxo, halogen, cyano, nitro, or -OC 1-6 alkyl, in particular optionally substituted with one or more -OH and / or -NR6R6'; For example, M is selected from: wherein -C 1-6 alkyl is optionally substituted with -OH.

17. An ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 15, wherein M is -(CH2) p M1, wherein M1 is selected from each optionally substituted by one or more OH, -COOH, -NH2, oxo, halo, cyano, nitro, -OC 1-6 alkyl and C 1-6 alkyl.

18. The ionizable sterol compound, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 17, wherein 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -propionic acid; 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid; L-Histidine 5a-Hydroxy-6 -[2-(1 H-imidazol-4-yl)-ethylamino]-cholestane-3 -hexanoic acid.

19. A lipid carrier composition comprising the ionizable sterol compound, or stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 18, and a phospholipid, a structural lipid, and a polymer-conjugated lipid, preferably in the form of a lipid nanoparticle.

20. The lipid carrier composition according to claim 19, wherein the ionizable sterol compound is present in a molar percentage of about 15% to about 65%, preferably about 30% to about 60%, more preferably about 40% to about 60%, most preferably about 50%, relative to the total lipid.

21. The lipid carrier composition according to claim 19 or 20, wherein the phospholipid is selected from: Dilauroylphosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DUPC), Palmitoyloleoylphosphatidylcholine (POPC), 1,2-Di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-Oleoyl-2-cholamidemethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Divinyl-sn-glycero-3-phosphocholine, 1,2-Diarylacyl-sn-glycero-3-phosphocholine, 1,2-Di-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-Di-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Di-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-glycero-3-phosphoethanolamine, 1,2-Diaryl-sn-glycero-3-phosphoethanolamine, 1,2-Dithiahexadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-Di-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diolyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Palmitoyloleoylphosphatidyethanolamine (POPE), Distearoyl-phosphatidyl-ethanolamine (DSPE), Dipalmitoylphosphatidyethanolamine (DPPE), Dimyristoylphosphoethanolamine (DMPE), 1-Stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1-Stearoyl-2-oleoyl-phosphatidylcholine (SOPC), or Sphingomyelin, and mixtures thereof, Preferably the phospholipid is DOPE or DSPC.

22. The lipid vehicle composition according to any one of claims 19 to 21, wherein the phospholipid is present in a molar percentage of the total lipids of about 0% to about 30%, preferably about 5% to about 20%, more preferably about 5% to about 15%, most preferably about 10%.

23. The lipid vehicle composition according to any one of claims 19 to 22, wherein the structural lipid is selected from one or more than two of cholesterol, cholestanol, sitosterol, ergosterol, stigmasterol, preferably cholesterol.

24. The lipid vehicle composition according to any one of claims 19 to 23, wherein the structural lipid is present in a molar percentage of the total lipids of about 20% to about 50%, preferably about 25% to about 50%, more preferably about 35% to about 50%.

25. The lipid carrier composition according to any one of claims 19 to 24, wherein the polymer-conjugated lipid is a PEG-modified lipid selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol, preferably selected from 1,2-dimyristoyl-sn-glycero- methoxypolyethyleneglycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG- dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacetylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE) or PEG-l,2-dimyristyloxypropyl-3- amine (PEG-c-DMA), more preferably selected from 1,2-dimyristoyl-sn-glycero-methoxypoly- ethyleneglycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly- ethyleneglycol)] (PEG-DSPE).

26. The lipid carrier composition according to any one of claims 19 to 25, wherein the polymer-conjugated lipid is present in a molar percentage of about 1% to about 10% of the total lipid, preferably about 1% to about 5%, more preferably about 1% to about 2%.

27. The lipid carrier composition according to any one of claims 19 to 26, wherein the molar ratio of the ionizable sterol compound, the phospholipid, the structural lipid and the polymer- conjugated lipid is 30-60:5-20:25-50:1-5, preferably 40-60:5-15:30-50:1-2.

28. A lipid nanoparticle composition comprising the lipid carrier composition according to any one of claims 19 to 27, and a therapeutic and / or prophylactic agent selected from the group consisting of nucleic acid molecules, polypeptides, proteins and small molecule compounds, preferably nucleic acids.

29. The lipid nanoparticle composition according to claim 28, wherein the nucleic acid is selected from the group consisting of messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antisense RNA, ribozymes, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA) and mixtures thereof.

30. The lipid nanoparticle composition according to claim 28 or 29, wherein the mass ratio of the lipid carrier composition and the therapeutic or prophylactic agent is 10:1-30:1, preferably 12.5:1-25:1, more preferably 15:1, and / or the N / P (nitrogen to phosphor ratio) of the lipid carrier composition and the nucleic acid is 3-12, preferably 6-10, more preferably 4-8.

31. The lipid nanoparticle composition according to any one of claims 28 to 30, wherein the encapsulation efficiency of the therapeutic agent is > 50%; or > 80%; or > 90%; and / or the average particle size of the lipid nanoparticle is 100-200 nm; and / or the nanoparticle composition has a dispersibility index of 0.1-0.

3.

32. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 28 to 31 and a pharmaceutically acceptable excipient.

33. The ionizable sterol compound of any one of claims 1 to 18, or the lipid carrier composition of any one of claims 19 to 27, for use in delivering a therapeutic and / or prophylactic agent to an individual.

34. The lipid nanoparticle composition of any one of claims 28 to 31 or the pharmaceutical composition of claim 32 for use as a medicament, preferably for treating a disease or condition in an individual.

35. Use of the lipid nanoparticle composition of any one of claims 28 to 31 or the pharmaceutical composition of claim 32 in the manufacture of a medicament for treating or preventing a disease or condition in a mammal, e.g., a human, e.g., the disease or condition is characterized by a dysfunction or abnormality in a protein or polypeptide activity; e.g., the disease or condition is selected from the group consisting of an infectious disease, a cancer and a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

36. The lipid nanoparticle composition of any one of claims 28 to 31 or the pharmaceutical composition of claim 32 for use in treating or preventing a disease or condition in a mammal, e.g., a human, e.g., the disease or condition is characterized by a dysfunction or abnormality in a protein or polypeptide activity; e.g., the disease or condition is selected from the group consisting of an infectious disease, a cancer and a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

37. A method of delivering a therapeutic or prophylactic agent to a mammalian cell or organ, comprising administering to a subject the lipid nanoparticle composition of any one of claims 28 to 31 or the pharmaceutical composition of claim 32, and contacting the cell or organ with the lipid nanoparticle or the pharmaceutical composition comprising the same; preferably the mammal is a human; preferably the lipid nanoparticle or the pharmaceutical composition comprising the same is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

38. A method of treating or preventing a disease or condition in a mammal, e.g., a human, the method comprising administering to the mammal a therapeutically or prophylactically effective amount of the lipid nanoparticle composition of any one of claims 28 to 31 or the pharmaceutical composition of claim 32; e.g., the disease or condition is characterized by a dysfunction or abnormality in a protein or polypeptide activity; e.g., the disease or condition is selected from the group consisting of an infectious disease, a cancer and a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease; e.g., the nanoparticle composition or the pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

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